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Duchenne muscular dystrophy (DMD) is a muscle degenerative disease that affects mainly boys, which is caused by the loss or drastic reduction in the expression of dystrophin. Gene therapy for the restoration of a functional shortened form of dystrophin (µ-dystrophin) have provided encouraging results in animal models. Nevertheless, in treated DMD patients the therapeutic benefit of this approach is still questionable, and therefore there is an urgent need for improved approach. Recently, we performed a non-supervised omics study (plasma miRNA profiling) of a large DMD cohort and found that cholesterol metabolism is a therapeutic target. In lysosomal storage diseases, lysosomal damage is often linked to cholesterol excess, and interestingly, previous and more recent investigations identified lysosomal perturbations in DMD. Taken together, these observations support the possible link in DMD between cholesterol excess and lysosomal perturbation. In the present work, we observed upregulation of the lysosomal damage biomarker Galectin-3 (Gal-3) in dystrophic muscle, thus confirmed Gal-3 as a lysosomal damage biomarker. We then treated mdx mice with Adeno Associated Virus (AAV) expressing µ-dystrophin and found that Gal-3 expression pattern was only partially normalized, supporting an incomplete correction of lysosomal damage by µ-dystrophin gene therapy. Consequently, we treated the mdx mouse by the combination of Trehalose, a safe dietary supplement disaccharide to alleviate lysosomal damage, and AAV µ-dystrophin to restore dystrophin expression, as compared to mice treated by only one of these treatments. We found that correction of lysosomal damage by trehalose exceeded lysosomal correction by µ-dystrophin. Importantly, the best correction of lysosomal damage and muscle function parameters was achieved by the combined treatment. Lysosomal damage may play a key role in the pathology of DMD and is therefore a potential therapeutic target. Trehalose and µ-dystrophin combined therapy for DMD holds the potential for improved therapeutic outcome. [ABSTRACT FROM AUTHOR]

Presents a corrected reprint of an article published in the 1999 edition of 'Nature Medicine' about the association of calpain 3 deficiency with myonuclear apoptosis and perturbation of the IkBa/NF-kB pathway in limb-girdle muscular dystrophy.

Physalia spp., or Man-of-War, drifts in tropical and subtropical waters, transported by ocean surface conditions. Its unique drifting behavior, influenced by dimorphism (left or right-handedness), complicates stranding predictions. Specifically, the quantification of the influence of the wind on Physalia spp. movements remains an open question, although essential for accurate Lagrangian tracking models. We investigated the wind effect on Physalia spp. by testing 3D-printed replicas in a controlled wind flume. Our findings reveal that under weak winds, left- and right-handed specimens drift symmetrically apart, aligning with strandings observed near Sydney, Australia. As wind speed rises, the drift angle decreases exponentially from approximately 40°, eventually stabilizing near a downwind direction in high winds. On average, the drift speed is 1.7 % of the wind speed. Variations in body shape impact drift angles and stability, adding stochasticity to paths. The proposed empirical relationship might advance knowledge of Physalia spp. sources, distribution and pathways., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 The Authors. Published by Elsevier Ltd.. All rights reserved.)

Single-molecule localization microscopy (SMLM) can reach sub-50 nm resolution using techniques such as stochastic optical reconstruction microscopy (STORM) or DNA-point accumulation for imaging in nanoscale topography (PAINT). Here we implement two approaches for faster multicolor SMLM by splitting the emitted fluorescence toward two cameras: simultaneous two-color DNA-PAINT (S2C-DNA-PAINT) that images spectrally separated red and far-red imager strands on each camera, and spectral demixing dSTORM (SD-dSTORM) where spectrally close far-red fluorophores appear on both cameras before being identified by demixing. Using S2C-DNA-PAINT as a reference for low crosstalk, we evaluate SD-dSTORM crosstalk using three types of samples: DNA origami nanorulers of different sizes, single-target labeled cells, or cells labeled for multiple targets. We then assess if crosstalk can affect the detection of biologically relevant subdiffraction patterns. Extending these approaches to three-dimensional acquisition and SD-dSTORM to three-color imaging, we show that spectral demixing is an attractive option for robust and versatile multicolor SMLM investigations., Competing Interests: Declaration of interests K.F., A.M., V.C., and N.B. are employees of Abbelight. N.B. and S.L.F. are shareholders of Abbelight., (Copyright © 2023 The Authors. Published by Elsevier Inc. All rights reserved.)

Duchenne muscular dystrophy (DMD) is a yet incurable rare genetic disease that affects the skeletal and cardiac muscles, leading to progressive muscle wasting and premature death. DMD is caused by the lack of dystrophin, a muscle protein essential for the biochemical support and integrity of muscle fibers. Gene replacement strategies for Duchenne muscular dystrophy (DMD) employing the adeno-associated virus (AAV) face the challenge imposed by the limited packaging capacity of AAV, only allowing the accommodation of a short version of dystrophin (µDys) that is still far removed from correcting human disease. The need to develop strategies leading to the expression of a best performing dystrophin variant led to only few studies reporting on the use of dual vectors, but none reported on a method to assess in vivo transgene reconstitution efficiency, the degree of which directly affects the use of safe AAV dosing. We report here on the generation of a dual AAV vector approach for the expression of a larger dystrophin version (quasidystrophin) based on homologous recombination, and the development of a methodology employing a strategic droplet digital PCR design, to determine the recombination efficiency as well as the occurrence of unwanted concatemerization events or aberrant expression from the single vectors. We demonstrated that, upon systemic delivery in the dystrophic D2.B10-Dmd mdx /J (DBA2mdx) mice, our dual AAV approach led to high transgene reconstitution efficiency and negligible Inverted Terminal Repeats (ITR)-dependent concatemerization, with consequent remarkable protein restoration in muscles and improvement of muscle pathology. This evidence supports the suitability of our system for gene therapy application and the potential of this methodology to assess and improve the feasibility for therapeutic translation of multiple vector approaches.

Duchenne muscular dystrophy (DMD) is a severe muscle disease caused by impaired expression of dystrophin. Whereas mitochondrial dysfunction is thought to play an important role in DMD, the mechanism of this dysfunction remains to be clarified. Here we demonstrate that in DMD and other muscular dystrophies, a large number of Dlk1-Dio3 clustered miRNAs (DD-miRNAs) are coordinately up-regulated in regenerating myofibers and in the serum. To characterize the biological effect of this dysregulation, 14 DD-miRNAs were simultaneously overexpressed in vivo in mouse muscle. Transcriptomic analysis revealed highly similar changes between the muscle ectopically overexpressing 14 DD-miRNAs and the mdx diaphragm, with naturally up-regulated DD-miRNAs. Among the commonly dysregulated pathway we found repressed mitochondrial metabolism, and oxidative phosphorylation (OxPhos) in particular. Knocking down the DD-miRNAs in iPS-derived skeletal myotubes resulted in increased OxPhos activities. The data suggest that (1) DD-miRNAs are important mediators of dystrophic changes in DMD muscle, (2) mitochondrial metabolism and OxPhos in particular are targeted in DMD by coordinately up-regulated DD-miRNAs. These findings provide insight into the mechanism of mitochondrial dysfunction in muscular dystrophy., (© 2022 Vu Hong et al.)

The growth and survival of individual trees determine the physical structure of a forest with important consequences for forest function. However, given the diversity of tree species and forest biomes, quantifying the multitude of demographic strategies within and across forests and the way that they translate into forest structure and function remains a significant challenge. Here, we quantify the demographic rates of 1961 tree species from temperate and tropical forests and evaluate how demographic diversity (DD) and demographic composition (DC) differ across forests, and how these differences in demography relate to species richness, aboveground biomass (AGB), and carbon residence time. We find wide variation in DD and DC across forest plots, patterns that are not explained by species richness or climate variables alone. There is no evidence that DD has an effect on either AGB or carbon residence time. Rather, the DC of forests, specifically the relative abundance of large statured species, predicted both biomass and carbon residence time. Our results demonstrate the distinct DCs of globally distributed forests, reflecting biogeography, recent history, and current plot conditions. Linking the DC of forests to resilience or vulnerability to climate change, will improve the precision and accuracy of predictions of future forest composition, structure, and function., (© 2022 John Wiley & Sons Ltd.)

Limb-girdle muscular dystrophy type R3 (LGMD R3) is a rare genetic disorder characterized by a progressive proximal muscle weakness and caused by mutations in the SGCA gene encoding alpha-sarcoglycan (α-SG). Here, we report the results of a mechanistic screening ascertaining the molecular mechanisms involved in the degradation of the most prevalent misfolded R77C-α-SG protein. We performed a combinatorial study to identify drugs potentializing the effect of a low dose of the proteasome inhibitor bortezomib on the R77C-α-SG degradation inhibition. Analysis of the screening associated to artificial intelligence-based predictive ADMET characterization of the hits led to identification of the HDAC inhibitor givinostat as potential therapeutical candidate. Functional characterization revealed that givinostat effect was related to autophagic pathway inhibition, unveiling new theories concerning degradation pathways of misfolded SG proteins. Beyond the identification of a new therapeutic option for LGMD R3 patients, our results shed light on the potential repurposing of givinostat for the treatment of other genetic diseases sharing similar protein degradation defects such as LGMD R5 and cystic fibrosis., Competing Interests: Authors NM, JD, MG, DP, and NS were employed by the company KANTIFY. The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Hoch, Bourg, Degrugillier, Bruge, Benabides, Pellier, Tournois, Mahé, Maignan, Dawe, Georges, Papazian, Subramanian, Simon, Fanen, Delevoye, Richard and Nissan.)

Physalia physalis, the bluebottle in Australia, are colonial siphonophores that live at the surface of the ocean, mainly in tropical and subtropical waters. P. physalis are sometimes present in large swarms, and with tentacles capable of intense stings, they can negatively impact public health and commercial fisheries. P. physalis, which does not swim, is advected by ocean currents and winds acting on its gas-filled sail. While previous studies have attempted to model the drift of P. physalis, little is known about its sources, distribution, and the timing of its arrival to shore. In this study, we present a dataset with four years of daily P. physalis beachings and stings reports at three locations off Sydney's coast in Australia. We investigate the spatial and temporal variability of P. physalis presence (beachings and stings) in relation to different environmental parameters. This dataset shows a clear seasonal pattern where more P. physalis beachings occur in the Austral summer and less in winter. Cold ocean temperatures do not hinder the presence of P. physalis and the temperature seasonal cycle and that observed in P. physalis presence/absence time-series are out of phase by 3-4 months. We identify wind direction as the major driver of the temporal variability of P. physalis arrival to the shore, both at daily and seasonal time-scales. The differences observed between sites of the occurrence of beaching events is consistent with the geomorphology of the coastline which influences the frequency and direction of favorable wind conditions. We also show that rip currents, a physical mechanism occurring at the scale of the beach, can be a predictor of beaching events. This study is a first step towards understanding the dynamics of P. physalis transport and ultimately being able to predict its arrival to the coast and mitigating the number of people who experience painful stings and require medical help., Competing Interests: The authors have declared that no competing interests exist.

Duchenne muscular dystrophy (DMD) is the most common and cureless muscle pediatric genetic disease, which is caused by the lack or the drastically reduced expression of dystrophin. Experimental therapeutic approaches for DMD have been mainly focused in recent years on attempts to restore the expression of dystrophin. While significant progress was achieved, the therapeutic benefit of treated patients is still unsatisfactory. Efficiency in gene therapy for DMD is hampered not only by incompletely resolved technical issues, but likely also due to the progressive nature of DMD. It is indeed suspected that some of the secondary pathologies, which are evolving over time in DMD patients, are not fully corrected by the restoration of dystrophin expression. We recently identified perturbations of the mevalonate pathway and of cholesterol metabolism in DMD patients. Taking advantage of the mdx model for DMD, we then demonstrated that some of these perturbations are improved by treatment with the cholesterol-lowering drug, simvastatin. In the present investigation, we tested whether the combination of the restoration of dystrophin expression with simvastatin treatment could have an additive beneficial effect in the mdx model. We confirmed the positive effects of microdystrophin, and of simvastatin, when administrated separately, but detected no additive effect by their combination. Thus, the present study does not support an additive beneficial effect by combining dystrophin restoration with a metabolic normalization by simvastatin.

Arbuscular mycorrhizal (AM) and ectomycorrhizal (EcM) associations are critical for host-tree performance. However, how mycorrhizal associations correlate with the latitudinal tree beta-diversity remains untested. Using a global dataset of 45 forest plots representing 2,804,270 trees across 3840 species, we test how AM and EcM trees contribute to total beta-diversity and its components (turnover and nestedness) of all trees. We find AM rather than EcM trees predominantly contribute to decreasing total beta-diversity and turnover and increasing nestedness with increasing latitude, probably because wide distributions of EcM trees do not generate strong compositional differences among localities. Environmental variables, especially temperature and precipitation, are strongly correlated with beta-diversity patterns for both AM trees and all trees rather than EcM trees. Results support our hypotheses that latitudinal beta-diversity patterns and environmental effects on these patterns are highly dependent on mycorrhizal types. Our findings highlight the importance of AM-dominated forests for conserving global forest biodiversity.

Non-uniform illumination limits quantitative analyses of fluorescence imaging techniques. In particular, single molecule localization microscopy (SMLM) relies on high irradiances, but conventional Gaussian-shaped laser illumination restricts the usable field of view to around 40 µm × 40 µm. We present Adaptable Scanning for Tunable Excitation Regions (ASTER), a versatile illumination technique that generates uniform and adaptable illumination. ASTER is also highly compatible with optical sectioning techniques such as total internal reflection fluorescence (TIRF). For SMLM, ASTER delivers homogeneous blinking kinetics at reasonable laser power over fields-of-view up to 200 µm × 200 µm. We demonstrate that ASTER improves clustering analysis and nanoscopic size measurements by imaging nanorulers, microtubules and clathrin-coated pits in COS-7 cells, and β2-spectrin in neurons. ASTER's sharp and quantitative illumination paves the way for high-throughput quantification of biological structures and processes in classical and super-resolution fluorescence microscopies.

We demonstrate subwavelength axial sectioning on biological samples with a stimulated emission depletion (STED) microscope combined with supercritical angle fluorescence (SAF) detection. SAF imaging is a powerful technique for imaging the membrane of the cell based on the direct exploitation of the fluorophore emission properties. Indeed, only when fluorophores are close to the interface can their evanescent near-field emission become propagative and be detected beyond the critical angle. Therefore, filtering out the SAF emission from the undercritical angle fluorescence (UAF) emission in the back focal plane of a high-NA objective lens permits nanometer axial sectioning of fluorescent emitters close to the coverslip. When combined with STED microscopy, a straightforward gain in axial resolution can be reached without any alteration of the STED beam path. Indeed, STED-SAF implementation only requires a modification in the detection path of the STED microscope and thus could be widely implemented., (Copyright © 2019 Elsevier Inc. All rights reserved.)

Limb-girdle muscular dystrophy type 2A (LGMD2A or LGMDR1) is a neuromuscular disorder caused by mutations in the calpain 3 gene ( CAPN3 ). Previous experiments using adeno-associated viral (AAV) vector-mediated calpain 3 gene transfer in mice indicated cardiac toxicity associated with the ectopic expression of the calpain 3 transgene. Here, we performed a preliminary dose study in a severe double-knockout mouse model deficient in calpain 3 and dysferlin. We evaluated safety and biodistribution of AAV9-desmin-hCAPN3 vector administration to nonhuman primates (NHPs) with a dose of 3 × 10 13 viral genomes/kg. Vector administration did not lead to observable adverse effects or to detectable toxicity in NHP. Of note, the transgene expression did not produce any abnormal changes in cardiac morphology or function of injected animals while reaching therapeutic expression in skeletal muscle. Additional investigation on the underlying causes of cardiac toxicity observed after gene transfer in mice and the role of titin in this phenomenon suggest species-specific titin splicing. Mice have a reduced capacity for buffering calpain 3 activity compared to NHPs and humans. Our studies highlight a complex interplay between calpain 3 and titin binding sites and demonstrate an effective and safe profile for systemic calpain 3 vector delivery in NHP, providing critical support for the clinical potential of calpain 3 gene therapy in humans., (Copyright © 2019 The Authors, some rights reserved; exclusive licensee American Association for the Advancement of Science. No claim to original U.S. Government Works.)

Limb-girdle muscular dystrophy type 2D (LGMD2D) is characterized by a progressive proximal muscle weakness. LGMD2D is caused by mutations in the gene encoding α-sarcoglycan (α-SG), a dystrophin-associated glycoprotein that plays a key role in the maintenance of sarcolemma integrity in striated muscles. We report here on the development of a new in vitro high-throughput screening assay that allows the monitoring of the proper localization of the most prevalent mutant form of α-SG (R77C substitution). Using this assay, we screened a library of 2560 FDA-approved drugs and bioactive compounds and identified thiostrepton, a cyclic antibiotic, as a potential drug to repurpose for LGMD2D treatment. Characterization of the thiostrepton effect revealed a positive impact on R77C-α-SG and other missense mutant protein localization (R34H, I124T, V247M) in fibroblasts overexpressing these proteins. Finally, further investigations of the molecular mechanisms of action of the compound revealed an inhibition of the chymotrypsin-like activity of the proteasome 24 h after thiostrepton treatment and a synergistic effect with bortezomib, an FDA-approved proteasome inhibitor. This study reports on the first in vitro model for LGMD2D that is compatible with high-throughput screening and proposes a new therapeutic option for LGMD2D caused by missense mutations of α-SG.