A Deoxyribonucleic Acid Decoy Trapping DUX4 for the Treatment of Facioscapulohumeral Muscular Dystrophy

Facioscapulohumeral dystrophy (FSHD) is characterized by a loss of repressive epigenetic marks leading to the aberrant expression of the DUX4 transcription factor. In muscle, DUX4 acts as a poison protein though the induction of multiple downstream genes. So far, there is no therapeutic solution for FSHD. Because DUX4 is a transcription factor, we developed an original therapeutic approach, based on a DNA decoy trapping the DUX4 protein, preventing its binding to genomic DNA and thereby blocking the aberrant activation of DUX4’s transcriptional network. In vitro, transfection of a DUX4 decoy into FSHD myotubes reduced the expression of the DUX4 network genes. In vivo, both double-stand DNA DUX4 decoys and adeno-associated viruses (AAVs) carrying DUX4 binding sites reduced transcriptional activation of genes downstream of DUX4 in a DUX4-expressing mouse model. Our study demonstrates, both in vitro and in vivo, the feasibility of the decoy strategy and opens new avenues of research

Transiently expressed CRISPR/Cas9 induces wild-type dystrophin in vitro in DMD patient myoblasts carrying duplications

Among the mutations arising in the DMD gene and causing Duchenne Muscular Dystrophy (DMD), 10-15% are multi-exon duplications. There are no current therapeutic approaches with the ability to excise large multi-exon duplications, leaving this patient cohort without mutation-specific treatment. Using CRISPR/Cas9 could provide a valid alternative to achieve targeted excision of genomic duplications of any size. Here we show that the expression of a single CRISPR/Cas9 nuclease targeting a genomic region within a DMD duplication can restore the production of wild-type dystrophin in vitro. We assessed the extent of dystrophin repair following both constitutive and transient nuclease expression by either transducing DMD patient-derived myoblasts with integrating lentiviral vectors or electroporating them with CRISPR/Cas9 expressing plasmids. Comparing genomic, transcript and protein data, we observed that both continuous and transient nuclease expression resulted in approximately 50% dystrophin protein restoration in treated myoblasts. Our data demonstrate that a high transient expression profile of Cas9 circumvents its requirement of continuous expression within the cell for targeting DMD duplications. This proof-of-concept study therefore helps progress towards a clinically relevant gene editing strategy for in vivo dystrophin restoration, by highlighting important considerations for optimizing future therapeutic approaches

RIPK3-mediated cell death is involved in DUX4-mediated toxicity in facioscapulohumeral dystrophy

BACKGROUND: Facioscapulohumeral dystrophy (FSHD) is caused by mutations leading to the aberrant expression of the DUX4 transcription factor in muscles. DUX4 was proposed to induce cell death, but the involvement of different death pathways is still discussed. A possible pro-apoptotic role of DUX4 was proposed, but as FSHD muscles are characterized by necrosis and inflammatory infiltrates, non-apoptotic pathways may be also involved. METHODS: We explored DUX4-mediated cell death by focusing on the role of one regulated necrosis pathway called necroptosis, which is regulated by RIPK3. We investigated the effect of necroptosis on cell death in vitro and in vivo experiments using RIPK3 inhibitors and a RIPK3-deficient transgenic mouse model. RESULTS: We showed in vitro that DUX4 expression causes a caspase-independent and RIPK3-mediated cell death in both myoblasts and myotubes. In vivo, RIPK3-deficient animals present improved body and muscle weights, a reduction of the aberrant activation of the DUX4 network genes, and an improvement of muscle histology. CONCLUSIONS: These results provide evidence for a role of RIPK3 in DUX4-mediated cell death and open new avenues of research

Observation of magnetic circular dichroism in Fe L_{2,3} x-ray-fluorescence spectra

We report experiments demonstrating circular dichroism in the x-ray-fluorescence spectra of magnetic systems, as predicted by a recent theory. The data, on the L_{2,3} edges of ferromagnetic iron, are compared with fully relativistic local spin density functional calculations, and the relationship between the dichroic spectra and the spin-resolved local density of occupied states is discussed

On the Papapetrou field in vacuum

In this paper we study the electromagnetic fields generated by a Killing vector field in vacuum space-times (Papapetrou fields). The motivation of this work is to provide new tools for the resolution of Maxwell's equations as well as for the search, characterization, and study of exact solutions of Einstein's equations. The first part of this paper is devoted to an algebraic study in which we give an explicit and covariant procedure to construct the principal null directions of a Papapetrou field. In the second part, we focus on the main differential properties of the principal directions, studying when they are geodesic, and in that case we compute their associated optical scalars. With this information we get the conditions that a principal direction of the Papapetrou field must satisfy in order to be aligned with a multiple principal direction of the Weyl tensor in the case of algebraically special vacuum space-times. Finally, we illustrate this study using the Kerr, Kasner and pp waves space-times.Comment: 24 pages, LaTeX2e, IOP style. To appear in Classical and Quantum Gravit

General approach to the study of vacuum space-times with an isometry

In vacuum space-times the exterior derivative of a Killing vector field is a 2-form (named here as the Papapetrou field) that satisfies Maxwell's equations without electromagnetic sources. In this paper, using the algebraic structure of the Papapetrou field, we will set up a new formalism for the study of vacuum space-times with an isometry, which is suitable to investigate the connections between the isometry and the Petrov type of the space-time. This approach has some advantages, among them, it leads to a new classification of these space-times and the integrability conditions provide expressions that determine completely the Weyl curvature. These facts make the formalism useful for application to any problem or situation with an isometry and requiring the knowledge of the curvature.Comment: 24 pages, LaTeX2e, IOP style. To appear in Classical and Quantum Gravit

Consequences of a Killing symmetry in spacetime's local structure

In this paper we discuss the consequences of a Killing symmetry on the local geometrical structure of four-dimensional spacetimes. We have adopted the point of view introduced in recent works where the exterior derivative of the Killing plays a fundamental role. Then, we study some issues related with this approach and clarify why in many circumstances its use has advantages with respect to other approaches. We also extend the formalism developed in the case of vacuum spacetimes to the general case of an arbitrary energy-momentum content. Finally, we illustrate our framework with the case of spacetimes with a gravitating electromagnetic field.Comment: 20 pages, LaTeX2e, IOP style. Revised version accepted for publication in Classical and Quantum Gravit

Selection of Reference Genes for Transcriptional Analysis of Edible Tubers of Potato (Solanum tuberosum L.)

Potato (Solanum tuberosum) yield has increased dramatically over the last 50 years and this has been achieved by a combination of improved agronomy and biotechnology efforts. Gene studies are taking place to improve new qualities and develop new cultivars. Reverse transcriptase quantitative polymerase chain reaction (RT-qPCR) is a bench-marking analytical tool for gene expression analysis, but its accuracy is highly dependent on a reliable normalization strategy of an invariant reference genes. For this reason, the goal of this work was to select and validate reference genes for transcriptional analysis of edible tubers of potato. To do so, RT-qPCR primers were designed for ten genes with relatively stable expression in potato tubers as observed in RNA-Seq experiments. Primers were designed across exon boundaries to avoid genomic DNA contamination. Differences were observed in the ranking of candidate genes identified by geNorm, NormFinder and BestKeeper algorithms. The ranks determined by geNorm and NormFinder were very similar and for all samples the most stable candidates were C2, exocyst complex component sec3 (SEC3) and ATCUL3/ ATCUL3A/CUL3/CUL3A (CUL3A). According to BestKeeper, the importin alpha and ubiquitin-associated/ts-n genes were the most stable. Three genes were selected as reference genes for potato edible tubers in RT-qPCR studies. The first one, called C2, was selected in common by NormFinder and geNorm, the second one is SEC3, selected by NormFinder, and the third one is CUL3A, selected by geNorm. Appropriate reference genes identified in this work will help to improve the accuracy of gene expression quantification analyses by taking into account differences that may be observed in RNA quality or reverse transcription efficiency across the samples

Dichroism in X-ray Fluorescence

We discuss circular dichroism in the x-ray fluorescence spectra of magnetic systems in the light of a recent theory [1]. Fully relativistic local spin density calculations on the L2,3 edges of ferromagnetic iron are compared with very recent experimental data, and the relationship between the dichroism spectra and the spin-resolved local density of occupied states is discussed