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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.

Duchenne Muscular Dystrophy (DMD) is a lethal muscle disorder, caused by mutations in the DMD gene and affects approximately 1:5000-6000 male births. In this report, we identified dysregulation of members of the Dlk1-Dio3 miRNA cluster in muscle biopsies of the GRMD dog model. Of these, we selected miR-379 for a detailed investigation because its expression is high in the muscle, and is known to be responsive to glucocorticoid, a class of anti-inflammatory drugs commonly used in DMD patients. Bioinformatics analysis predicts that miR-379 targets EIF4G2, a translational factor, which is involved in the control of mitochondrial metabolic maturation. We confirmed in myoblasts that EIF4G2 is a direct target of miR-379, and identified the DAPIT mitochondrial protein as a translational target of EIF4G2. Knocking down DAPIT in skeletal myotubes resulted in reduced ATP synthesis and myogenic differentiation. We also demonstrated that this pathway is GC-responsive since treating mice with dexamethasone resulted in reduced muscle expression of miR-379 and increased expression of EIF4G2 and DAPIT. Furthermore, miR-379 seric level, which is also elevated in the plasma of DMD patients in comparison with age-matched controls, is reduced by GC treatment. Thus, this newly identified pathway may link GC treatment to a mitochondrial response in DMD.

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.

Here, we present a 3D localization-based super-resolution technique providing a slowly varying localization precision over a 1 μm range with precisions down to 15 nm. The axial localization is performed through a combination of point spread function (PSF) shaping and supercritical angle fluorescence (SAF), which yields absolute axial information. Using a dual-view scheme, the axial detection is decoupled from the lateral detection and optimized independently to provide a weakly anisotropic 3D resolution over the imaging range. This method can be readily implemented on most homemade PSF shaping setups and provides drift-free, tilt-insensitive and achromatic results. Its insensitivity to these unavoidable experimental biases is especially adapted for multicolor 3D super-resolution microscopy, as we demonstrate by imaging cell cytoskeleton, living bacteria membranes and axon periodic submembrane scaffolds. We further illustrate the interest of the technique for biological multicolor imaging over a several-μm range by direct merging of multiple acquisitions at different depths.

Daptomycin is a last-resort membrane-targeting lipopeptide approved for the treatment of drug-resistant staphylococcal infections, such as bacteremia and implant-related infections. Although cases of resistance to this antibiotic are rare, increasing numbers of clinical, in vitro , and animal studies report treatment failure, notably against Staphylococcus aureus The aim of this study was to identify the features of daptomycin and its target bacteria that lead to daptomycin treatment failure. We show that daptomycin bactericidal activity against S. aureus varies significantly with the growth state and strain, according to the membrane fatty acid composition. Daptomycin efficacy as an antibiotic relies on its ability to oligomerize within membranes and form pores that subsequently lead to cell death. Our findings ascertain that daptomycin interacts with tolerant bacteria and reaches its membrane target, regardless of its bactericidal activity. However, the final step of pore formation does not occur in cells that are daptomycin tolerant, strongly suggesting that it is incapable of oligomerization. Importantly, membrane fatty acid contents correlated with poor daptomycin bactericidal activity, which could be manipulated by fatty acid addition. In conclusion, daptomycin failure to treat S. aureus is not due to a lack of antibiotic-target interaction, but is driven by its capacity to form pores, which depends on membrane composition. Manipulation of membrane fluidity to restore S. aureus daptomycin bactericidal activity in vivo could open the way to novel antibiotic treatment strategies., (Copyright © 2018 American Society for Microbiology.)

Sarcoglycanopathies are rare autosomic limb girdle muscular dystrophies caused by mutations in one of the genes coding for sarcoglycan (α, β, δ, and γ-sarcoglycans). Sarcoglycans form a complex, which is an important part of the dystrophin-associated glycoprotein complex that protects sarcolemma against muscle contraction-induced damages. Absence of one of the sarcoglycan at the plasma membrane induces the disappearance of the whole complex and perturbs muscle fiber membrane integrity. We previously demonstrated that point mutations in the human sarcoglycan genes affects the folding of the corresponding protein, which is then retained in the endoplasmic reticulum by the protein quality control and prematurely degraded by the proteasome. Interestingly, modulation of the quality control using pharmacological compounds allowed the rescue of the membrane localization of the mutated sarcoglycan. Two previously generated mouse models, knock-in for the most common sarcoglycan mutant, R77C α-sarcoglycan, failed in reproducing the dystrophic phenotype observed in human patients. Based on these results and the need to test therapies for these fatal diseases, we decided to generate a new knock-in mouse model carrying the missense mutation T151R in the β-sarcoglycan gene since this is the second sarcoglycan protein with the most frequently reported missense mutations. Muscle analysis, performed at the age of 4 and 9-months, showed the presence of the mutated β-sarcoglycan protein and of the other components of the dystrophin-associated glycoprotein complex at the muscle membrane. In addition, these mice did not develop a dystrophic phenotype, even at a late stage or in condition of stress-inducing exercise. We can speculate that the absence of phenotype in mouse may be due to a higher tolerance of the endoplasmic reticulum quality control for amino-acid changes in mice compared to human.

We propose a straightforward sample-based technique to calibrate the axial detection in 3D single-molecule localization microscopy. Using microspheres coated with fluorescent molecules, the calibration curves of point spread function-shaping or intensity-based measurements can be obtained over the imaging depth range. This experimental method takes into account the effect of the spherical aberration without requiring computational correction. We demonstrate its efficiency for astigmatic imaging in a 1.2 μm range above the coverslip.

Limb Girdle Muscular Dystrophies type 2I (LGMD2I), a recessive autosomal muscular dystrophy, is caused by mutations in the Fukutin Related Protein (FKRP) gene. It has been proposed that FKRP, a ribitol-5-phosphate transferase, is a participant in α-dystroglycan (αDG) glycosylation, which is important to ensure the cell/matrix anchor of muscle fibers. A LGMD2I knock-in mouse model was generated to express the most frequent mutation (L276I) encountered in patients. The expression of FKRP was not altered neither at transcriptional nor at translational levels, but its function was impacted since abnormal glycosylation of αDG was observed. Skeletal muscles were functionally impaired from 2 months of age and a moderate dystrophic pattern was evident starting from 6 months of age. Gene transfer with a rAAV2/9 vector expressing Fkrp restored biochemical defects, corrected the histological abnormalities and improved the resistance to eccentric stress in the mouse model. However, injection of high doses of the vector induced a decrease of αDG glycosylation and laminin binding, even in WT animals. Finally, intravenous injection of the rAAV-Fkrp vector into a dystroglycanopathy mouse model due to Fukutin (Fktn) knock-out indicated a dose-dependent toxicity. These data suggest requirement for a control of FKRP expression in muscles., (© The Author 2017. Published by Oxford University Press. All rights reserved. For Permissions, please email: journals.permissions@oup.com.)

Determining how cells generate and transduce mechanical forces at the nanoscale is a major technical challenge for the understanding of numerous physiological and pathological processes. Podosomes are submicrometer cell structures with a columnar F-actin core surrounded by a ring of adhesion proteins, which possess the singular ability to protrude into and probe the extracellular matrix. Using protrusion force microscopy, we have previously shown that single podosomes produce local nanoscale protrusions on the extracellular environment. However, how cellular forces are distributed to allow this protruding mechanism is still unknown. To investigate the molecular machinery of protrusion force generation, we performed mechanical simulations and developed quantitative image analyses of nanoscale architectural and mechanical measurements. First, in silico modeling showed that the deformations of the substrate made by podosomes require protrusion forces to be balanced by local traction forces at the immediate core periphery where the adhesion ring is located. Second, we showed that three-ring proteins are required for actin polymerization and protrusion force generation. Third, using DONALD, a 3D nanoscopy technique that provides 20 nm isotropic localization precision, we related force generation to the molecular extension of talin within the podosome ring, which requires vinculin and paxillin, indicating that the ring sustains mechanical tension. Our work demonstrates that the ring is a site of tension, balancing protrusion at the core. This local coupling of opposing forces forms the basis of protrusion and reveals the podosome as a nanoscale autonomous force generator.

Cleavage of mutant huntingtin (HTT) is an essential process in Huntington's disease (HD), an inherited neurodegenerative disorder. Cleavage generates N-ter fragments that contain the polyQ stretch and whose nuclear toxicity is well established. However, the functional defects induced by cleavage of full-length HTT remain elusive. Moreover, the contribution of non-polyQ C-terminal fragments is unknown. Using time- and site-specific control of full-length HTT proteolysis, we show that specific cleavages are required to disrupt intramolecular interactions within HTT and to cause toxicity in cells and flies. Surprisingly, in addition to the canonical pathogenic N-ter fragments, the C-ter fragments generated, that do not contain the polyQ stretch, induced toxicity via dilation of the endoplasmic reticulum (ER) and increased ER stress. C-ter HTT bound to dynamin 1 and subsequently impaired its activity at ER membranes. Our findings support a role for HTT on dynamin 1 function and ER homoeostasis. Proteolysis-induced alteration of this function may be relevant to disease., (© 2015 The Authors.)

Meeting the nanometre resolution promised by super-resolution microscopy techniques (pointillist: PALM, STORM, scanning: STED) requires stabilizing the sample drifts in real time during the whole acquisition process. Metal nanoparticles are excellent probes to track the lateral drifts as they provide crisp and photostable information. However, achieving nanometre axial super-localization is still a major challenge, as diffraction imposes large depths-of-fields. Here we demonstrate fast full three-dimensional nanometre super-localization of gold nanoparticles through simultaneous intensity and phase imaging with a wavefront-sensing camera based on quadriwave lateral shearing interferometry. We show how to combine the intensity and phase information to provide the key to the third axial dimension. Presently, we demonstrate even in the occurrence of large three-dimensional fluctuations of several microns, unprecedented sub-nanometre localization accuracies down to 0.7 nm in lateral and 2.7 nm in axial directions at 50 frames per second. We demonstrate that nanoscale stabilization greatly enhances the image quality and resolution in direct stochastic optical reconstruction microscopy imaging.

Recombinant adeno-associated virus (rAAV) is currently the best vector for gene delivery into the skeletal muscle. However, the 5-kb packaging size of this virus is a major obstacle for large gene transfer. This past decade, many different strategies were developed to circumvent this issue (concatemerization-splicing, overlapping vectors, hybrid dual or fragmented AAV). Loss of function mutations in the DYSF gene whose coding sequence is 6.2kb lead to progressive muscular dystrophies (LGMD2B: OMIM_253601; MM: OMIM_254130; DMAT: OMIM_606768). In this study, we compared large gene transfer techniques to deliver the DYSF gene into the skeletal muscle. After rAAV8s intramuscular injection into dysferlin deficient mice, we showed that the overlap strategy is the most effective approach to reconstitute a full-length messenger. After systemic administration, the level of dysferlin obtained on different muscles corresponded to 0.5- to 2-fold compared to the normal level. We further demonstrated that the overlapping vector set was efficient to correct the histopathology, resistance to eccentric contractions and whole body force in the dysferlin deficient mice. Altogether, these data indicate that using overlapping vectors could be a promising approach for a potential clinical treatment of dysferlinopathies.

Muscular dystrophies are a group of genetically distinct diseases for which no treatment exists. While gene transfer approach is being tested for several of these diseases, such strategies can be hampered when the size of the corresponding complementary DNA (cDNA) exceeds the packaging capacity of adeno-associated virus vectors. This issue concerns, in particular, dysferlinopathies and titinopathies that are due to mutations in the dysferlin (DYSF) and titin (TTN) genes. We investigated the efficacy of RNA trans-splicing as a mode of RNA therapy for these two types of diseases. Results obtained with RNA trans-splicing molecules designed to target the 3' end of mouse titin and human dysferlin pre-mRNA transcripts indicated that trans-splicing of pre-mRNA generated from minigene constructs or from the endogenous genes was achieved. Collectively, these results provide the first demonstration of DYSF and TTN trans-splicing reprogramming in vitro and in vivo. However, in addition to these positive results, we uncovered a possible issue of the technique in the form of undesirable translation of RNA pre-trans-splicing molecules, directly from open reading frames present on the molecule or associated with internal alternative cis-splicing. These events may hamper the efficiency of the trans-splicing process and/or lead to toxicity.

Dysferlinopathy refers to a group of autosomal recessive muscular dystrophies due to mutations in the dysferlin gene causing deficiency of a membrane-bound protein crucially involved in plasma membrane repair. The condition is characterized by marked clinical heterogeneity, the different phenotypes/modes of presentation being unrelated to the genotype. For unknown reasons, patients are often remarkably active before the onset of symptoms. Dysferlin deficiency-related persistence of mechanically induced sarcolemma disruptions causes myofiber damage and necrosis. We postulate that limited myodamage may initially remain hidden with well-preserved resistance to physical strains. By subjecting dysferlin-deficient B6.A/J-Dysf(prmd) mice to long-term swimming exercise, we observed that concentric/isometric strain improved muscle strength and alleviated muscular dystrophy by limiting the accumulation of membrane lesions. By contrast, eccentric strain induced by long-term running in a wheel worsened the dystrophic process. Myofiber damage induced by eccentric strain increased with age, reflecting the accumulation of non-necrotic membrane lesions up to a critical threshold. This phenomenon was modulated by daily spontaneous activity. Transposed to humans, our results may suggest that the past activity profile shapes the clinical phenotype of the myopathy and that patients with dysferlinopathy should likely benefit from concentric exercise-based physiotherapy., (Copyright © 2013 American Society for Investigative Pathology. Published by Elsevier Inc. All rights reserved.)

Mutations in the dysferlin gene are the cause of Limb-girdle Muscular Dystrophy type 2B and Miyoshi Myopathy. The dysferlin protein has been implicated in sarcolemmal resealing, leading to the idea that the pathophysiology of dysferlin deficiencies is due to a deficit in membrane repair. Here, we show using two different approaches that fulfilling membrane repair as asseyed by laser wounding assay is not sufficient for alleviating the dysferlin deficient pathology. First, we generated a transgenic mouse overexpressing myoferlin to test the hypothesis that myoferlin, which is homologous to dysferlin, can compensate for the absence of dysferlin. The myoferlin overexpressors show no skeletal muscle abnormalities, and crossing them with a dysferlin-deficient model rescues the membrane fusion defect present in dysferlin-deficient mice in vitro. However, myoferlin overexpression does not correct muscle histology in vivo. Second, we report that AAV-mediated transfer of a minidysferlin, previously shown to correct the membrane repair deficit in vitro, also fails to improve muscle histology. Furthermore, neither myoferlin nor the minidysferlin prevented myofiber degeneration following eccentric exercise. Our data suggest that the pathogenicity of dysferlin deficiency is not solely related to impairment in sarcolemmal repair and highlight the care needed in selecting assays to assess potential therapies for dysferlinopathies.

A multiprotein complex encompassing a transcription regulator, cardiac ankyrin repeat protein (CARP), and the calpain 3 protease was identified in the N2A elastic region of the giant sarcomeric protein titin. The present study aimed to investigate the function(s) of this complex in the skeletal muscle. We demonstrate that CARP subcellular localization is controlled by the activity of calpain 3: the higher the calpain 3, the more important the sarcomeric retention of CARP. This regulation would occur through cleavage of the N-terminal end of CARP by the protease. We show that, upon CARP over-expression, the transcription factor nuclear factor NF-κB p65 DNA-binding activity decreases. Taken as a whole, CARP and its regulator calpain 3 appear to occupy a central position in the important cell fate-governing NF-κB pathway. Interestingly, the expression of the atrophying protein MURF1, one of NF-κB main targets, remains unchanged in presence of CARP, suggesting that the pathway encompassing calpain 3/CARP/NF-κB does not play a role in muscle atrophy. With NF-κB also having anti-apoptotic effects, the inability of calpain 3 to lower CARP-driven inhibition of NF-κB could reduce muscle cell survival, hence partly accounting for the dystrophic pattern observed in limb girdle muscular dystrophy 2A, a pathology resulting from the protease deficiency., (© 2010 The Authors Journal compilation © 2010 FEBS.)

Deficiency of the dysferlin protein presents as two major clinical phenotypes: limb-girdle muscular dystrophy type 2B and Miyoshi myopathy. Dysferlin is known to participate in membrane repair, providing a potential hypothesis to the underlying pathophysiology of these diseases. The size of the dysferlin cDNA prevents its direct incorporation into an adeno-associated virus (AAV) vector for therapeutic gene transfer into muscle. To bypass this limitation, we split the dysferlin cDNA at the exon 28/29 junction and cloned it into two independent AAV vectors carrying the appropriate splicing sequences. Intramuscular injection of the corresponding vectors into a dysferlin-deficient mouse model led to the expression of full-length dysferlin for at least 1 year. Importantly, systemic injection in the tail vein of the two vectors led to a widespread although weak expression of the full-length protein. Injections were associated with an improvement of the histological aspect of the muscle, a reduction in the number of necrotic fibers, restoration of membrane repair capacity and a global improvement in locomotor activity. Altogether, these data support the use of such a strategy for the treatment of dysferlin deficiency.

Mechanically damaged plasma membrane undergoes rapid calcium-dependent resealing that appears to depend, at least in part, on calpain-mediated cortical cytoskeletal remodeling. Cells null for Capns1, the non-catalytic small subunit present in both m- and mu-calpains, do not undergo calcium-mediated resealing. However, it is not known which of these calpains is needed for repair, or whether other major cytosolic proteinases may participate. Utilizing isozyme-selective siRNAs to decrease expression of Capn1 or Capn2, catalytic subunits of mu- and m-calpains, respectively, in a mouse embryonic fibroblast cell line, we now show that substantial loss of both activities is required to compromise calcium-mediated survival after cell scrape-damage. Using skeletal myotubes derived from Capn3-null mice, we were unable to demonstrate loss of sarcolemma resealing after needle scratch or laser damage. Isolated muscle fibers from Capn3 knockout mice also efficiently repaired laser damage. Employing either a cell line expressing a temperature sensitive E1 ubiquitin ligase, or lactacystin, a specific proteasome inhibitor, it was not possible to demonstrate an effect of the proteasome on calcium-mediated survival after injury. Moreover, several cell-permeant caspase inhibitors were incapable of significantly decreasing survival or inhibiting membrane repair. Taken together with previous studies, the results show that m- or mu-calpain can facilitate repair of damaged plasma membrane. While there was no evidence for the involvement of calpain-3, the proteasome or caspases in early events of plasma membrane repair, our studies do not rule out their participation in downstream events that may link plasma membrane repair to adaptive remodeling after injury.

Limb-girdle muscular dystrophy type 2A is an autosomal recessive disorder generated by inactivating mutations in the gene coding for the muscle specific protease calpain-3. It is mainly expressed in skeletal muscle as a monomeric multidomain protein characterized by three unique insertion sequences (NS, IS1, IS2). It is unstable, and undergoes very rapid autolysis in solution, therefore, its heterologous expression and purification have been difficult. So far, calpain-3 substrates have been only identified in vitro and with indirect approaches. We have therefore decided to perform a comprehensive study of the substrates of the protease by comparing the 2D electrophoretic profile of myotubes from obtained from calpain-3 knockout and wild type mice. Digestion of differentially expressed spots was followed by mass spectrometry analysis. We could identify 16 proteins which differed in knockout and wild type mice. Among them: desmin, nestin, spectrin and PDLIM1 were of particular interest. In vitro experiments have then revealed that only PDLIM1 is cleaved directly by the protease, and that a fragment of about 8 kDa is released from the C-terminal portion of the protein.

In an attempt to identify potential therapeutic targets for the correction of muscle wasting, the gene expression of several pivotal proteins involved in protein metabolism was investigated in experimental atrophy induced by transient or definitive denervation, as well as in four animal models of muscular dystrophies (deficient for calpain 3, dysferlin, alpha-sarcoglycan and dystrophin, respectively). The results showed that: (a) the components of the ubiquitin-proteasome pathway are upregulated during the very early phases of atrophy but do not greatly increase in the muscular dystrophy models; (b) forkhead box protein O1 mRNA expression is augmented in the muscles of a limb girdle muscular dystrophy 2A murine model; and (c) the expression of cardiac ankyrin repeat protein (CARP), a regulator of transcription factors, appears to be persistently upregulated in every condition, suggesting that CARP could be a hub protein participating in common pathological molecular pathway(s). Interestingly, the mRNA level of a cell cycle inhibitor known to be upregulated by CARP in other tissues, p21(WAF1/CIP1), is consistently increased whenever CARP is upregulated. CARP overexpression in muscle fibres fails to affect their calibre, indicating that CARP per se cannot initiate atrophy. However, a switch towards fast-twitch fibres is observed, suggesting that CARP plays a role in skeletal muscle plasticity. The observation that p21(WAF1/CIP1) is upregulated, put in perspective with the effects of CARP on the fibre type, fits well with the idea that the mechanisms at stake might be required to oppose muscle remodelling in skeletal muscle.

Mutations of Ca(2+)-activated proteases (calpains) cause muscular dystrophies. Nevertheless, the specific role of calpains in Ca(2+) signalling during the onset of dystrophies remains unclear. We investigated Ca(2+) handling in skeletal cells from calpain 3-deficient mice. [Ca(2+)](i) responses to caffeine, a ryanodine receptor (RyR) agonist, were decreased in -/- myotubes and absent in -/- myoblasts. The -/- myotubes displayed smaller amplitudes of the Ca(2+) transients induced by cyclopiazonic acid in comparison to wild type cells. Inhibition of L-type Ca(2+) channels (LCC) suppressed the caffeine-induced [Ca(2+)](i) responses in -/- myotubes. Hence, the absence of calpain 3 modifies the sarcoplasmic reticulum (SR) Ca(2+) release, by a decrease of the SR content, an impairment of RyR signalling, and an increase of LCC activity. We propose that calpain 3-dependent proteolysis plays a role in activating support proteins of intracellular Ca(2+) signalling at a stage of cellular differentiation which is crucial for skeletal muscle regeneration.

Limb-girdle muscular dystrophy type 2A (LGMD2A) is a recessive genetic disorder caused by mutations in the cysteine protease calpain 3 (CAPN3) that leads to selective muscle wasting. We previously showed that CAPN3 deficiency is associated with a profound perturbation of the NF-kappaB/IkappaB alpha survival pathway. In this study, the consequences of altered NF-kappaB/IkappaB alpha pathway were investigated using biological materials from LGMD2A patients. We first show that the antiapoptotic factor cellular-FLICE inhibitory protein (c-FLIP), which is dependent on the NF-kappaB pathway in normal muscle cells, is down-regulated in LGMD2A biopsies. In muscle cells isolated from LGMD2A patients, NF-kappaB is readily activated on cytokine induction as shown by an increase in its DNA binding activity. However, we observed discrepant transcriptional responses depending on the NF-kappaB target genes. IkappaB alpha is expressed following NF-kappaB activation independent of the CAPN3 status, whereas expression of c-FLIP is obtained only when CAPN3 is present. These data lead us to postulate that CAPN3 intervenes in the regulation of the expression of NF-kappaB-dependent survival genes to prevent apoptosis in skeletal muscle. Deregulations in the NF-kappaB pathway could be part of the mechanism responsible for the muscle wasting resulting from CAPN3 deficiency.

Calpains are Ca(2+)-dependent cysteine proteases known to be important for the regulation of cell functions and which aberrant activation causes cell death in a number of degenerative disorders. To provide a tool for monitoring the status of calpain activity in vivo under physiological and pathological conditions, we created a mouse model that expresses ubiquitously a fluorescent reporter consisting of eCFP and eYFP separated by a linker cleavable by the ubiquitous calpains. We named this mouse CAFI for calpain activity monitored by FRET imaging. Our validation studies demonstrated that the level of calpain activity correlates with a decrease in FRET (fluorescence resonance energy transfer) between the two fluorescent proteins. Using this model, we observed a small level of activity after denervation and fasting, a high level of activity during muscle regeneration and ischemia, and local activity in damaged myofibers after exercise. Finally, we crossed the CAFI mouse with the alpha-sarcoglycan-deficient model, demonstrating an increase of calpain activity at the steady state. Altogether, our results present evidence that CAFI mice could be a valuable tool in which to follow calpain activity at physiological levels and in disease states.

Constant efforts are ongoing for the development of new imaging methods that allow the investigation of molecular processes in vivo. Protein-protein interactions, enzymatic activities and intracellular Ca2+ fluxes, have been resolved in cultured cells using a variety of fluorescence resonance energy transfer (FRET) detection methods. However, FRET has not been used so far in conjunction with 3D intravital imaging. We evaluated here a combination of multiphoton microscopy (MPM), method of choice for non-destructive living tissue investigation, and FRET imaging to monitor calpain proteolytic activity in living mice muscle. We show that kinetics of ubiquitous calpains activation can be efficiently and quantitatively monitored in living mouse tissues at cellular level with a FRET-based indicator upon calcium influx. The ability to visualize calpain activity in living tissue offers a unique opportunity to challenge remaining questions on the biological functions of calpains and to evaluate the therapeutic potential of calpain inhibitors in many degenerative conditions.

Calpain 3 (Capn3) is known as the skeletal muscle-specific member of the calpains, a family of intracellular nonlysosomal cysteine proteases. This enigmatic protease has many unique features among the calpain family and, importantly, mutations in Capn3 have been shown to be responsible for limb girdle muscular dystrophy type 2A. Here we demonstrate that the Capn3 activation mechanism is similar to the universal activation of caspases and corresponds to an autolysis within the active site of the protease. We undertook a search for substrates in immature muscle cells, as several lines of evidence suggest that Capn3 is mostly in an inactive state in muscle and needs a signal to be activated. In this model, Capn3 proteolytic activity leads to disruption of the actin cytoskeleton and disorganization of focal adhesions through cleavage of several endogenous proteins. In addition, we show that titin, a previously identified Capn3 partner, and filamin C are further substrates of Capn3. Finally, we report that Capn3 colocalizes in vivo with its substrates at various sites along cytoskeletal structures. We propose that Capn3-mediated cleavage produces an adaptive response of muscle cells to external and/or internal stimuli, establishing Capn3 as a muscle cytoskeleton regulator.

Objective: Mutations in the skeletal muscle gene dysferlin cause two autosomal recessive forms of muscular dystrophy: Miyoshi myopathy (MM) and limb girdle muscular dystrophy type 2B (LGMD2B). The purpose of this study was to define the genomic organization of the dysferlin gene and conduct mutational screening and a survey of clinical features in 21 patients with defined molecular defects in the dysferlin gene., Methods: Genomic organization of the gene was determined by comparing the dysferlin cDNA and genomic sequence in P1-derived artificial chromosomes (PACs) containing the gene. Mutational screening entailed conformational analysis and sequencing of genomic DNA and cDNA. Clinical records of patients with defined dysferlin gene defects were reviewed retrospectively., Results: The dysferlin gene encompasses 55 exons spanning over 150 kb of genomic DNA. Mutational screening revealed nine novel mutations associated with MM. The range of onset in this patient group was narrow with a mean of 19.0 +/- 3.9 years., Conclusion: This study confirms that the dysferlin gene is mutated in MM and LGMD2B and extends understanding of the timing of onset of the disease. Knowledge of the genomic organization of the gene will facilitate mutation detection and investigations of the molecular biologic properties of the dysferlin gene.

Calpain 3 is known as the skeletal muscle-specific member of the calpains, a family of intracellular nonlysosomal cysteine proteases. It was previously shown that defects in the human calpain 3 gene are responsible for limb girdle muscular dystrophy type 2A (LGMD2A), an inherited disease affecting predominantly the proximal limb muscles. To better understand the function of calpain 3 and the pathophysiological mechanisms of LGMD2A and also to develop an adequate model for therapy research, we generated capn3-deficient mice by gene targeting. capn3-deficient mice are fully fertile and viable. Allele transmission in intercross progeny demonstrated a statistically significant departure from Mendel's law. capn3-deficient mice show a mild progressive muscular dystrophy that affects a specific group of muscles. The age of appearance of myopathic features varies with the genetic background, suggesting the involvement of modifier genes. Affected muscles manifest a similar apoptosis-associated perturbation of the IkappaBalpha/nuclear factor kappaB pathway as seen in LGMD2A patients. In addition, Evans blue staining of muscle fibers reveals that the pathological process due to calpain 3 deficiency is associated with membrane alterations.

The autosomal progressive muscular dystrophies which are grouped together under the term limb girdle muscular dystrophies (LGMD) are diseases characterized by a progressive impairment of the proximal limb muscles and myopathic changes on electromyogram and muscle biopsy. Eight independent purely recessive genetic entities have been recognized in this group of diseases by genetic localization or causative gene identification. We have developed fluorescent genetic markers bracketing six of these loci (LGMD2A-LGMD2F). The marker loci were genotyped in 96 LGMD2 families leading to genetic definition of 25 of them either with a high likelihood or with a suggested localization (7 LGMD2A, 5 LGMD2B, 4 LGMD2C, 4 LGMD2D, 2 LGMD2E and 3 LGMD2F). In addition, 18 families were excluded for all six tested loci; for 45 of the 53 remaining families at least one exclusion could be demonstrated. This kit, which makes the rapid genetic testing of LGMD2 families possible, may be useful in a diagnostic process.

The limb-girdle muscular dystrophies are a genetically heterogeneous group of inherited progressive muscle disorders that affect mainly the proximal musculature, with evidence for at least three autosomal dominant and eight autosomal recessive loci. The latter mostly involve mutations in genes encoding components of the dystrophin-associated complex; another form is caused by mutations in the gene for the muscle-specific protease calpain 3. Using a positional cloning approach, we have identified the gene for a form of limb-girdle muscular dystrophy that we previously mapped to chromosome 2p13 (LGMD2B). This gene shows no homology to any known mammalian gene, but its predicted product is related to the C. elegans spermatogenesis factor fer-1. We have identified two homozygous frameshift mutations in this gene, resulting in muscular dystrophy of either proximal or distal onset in nine families. The proposed name 'dysferlin' combines the role of the gene in producing muscular dystrophy with its C. elegans homology.

The sarcoglycan complex is involved in the etiology of four autosomal recessive limb-girdle muscular dystrophies (LGMD2C-F). A missense mutation (T151R) in the beta-sarcoglycan gene on chromosome 4q12 has been shown to cause a mild form of LGMD2E in 11 families from a Southern Indiana Amish community sharing a common haplotype. We now report that two sibs from another Amish family with mild LGMD2E are compound heterozygotes for chromosome 4q12 markers. In order to characterize the genetic defect in this new family, we determined the genomic organization of the beta-sarcoglycan gene. A second missense mutation (R91C) has now been identified in this LGMD2E Amish family. This mutation is also present in the homozygous state in another family of probable Amish ancestry. Finally, analysis of all the components of the dystrophin-glycoprotein complex was performed for the first time on a biopsy from a patient homozygous for the beta-sarcoglycan mutation (T151R). Interestingly, in addition to the loss of the entire sarcoglycan complex, we detected a reduction of alpha-dystroglycan which suggests a role for the sarcoglycan complex in stabilizing alpha-dystroglycan at the sarcolemma.

The autosomal recessive forms of limb-girdle muscular dystrophies are encoded by at least five distinct genes. The work performed towards the identification of two of these is summarized in this report. This success illustrates the growing importance of genetics in modern nosology.

beta-Sarcoglycan, a 43 kDa dystrophin-associated glycoprotein, is an integral component of the dystrophin-glycoprotein complex. We have cloned human beta-sarcoglycan cDNA and mapped the beta-sarcoglycan gene to chromosome 4q12. Pericentromeric markers and an intragenic polymorphic CA repeat cosegregated perfectly with autosomal recessive limb-girdle muscular dystrophy in several Amish families. A Thr-to-Arg missense mutation was identified within the beta-sarcoglycan gene that leads to a dramatically reduced expression of beta-sarcoglycan in the sarcolemma and a concomitant loss of adhalin and 35 DAG, which may represent a disruption of a functional subcomplex within the dystrophin-glycoprotein complex. Thus, the beta-sarcoglycan gene is the fifth locus identified (LGMD2E) that is involved in autosomal recessive limb-girdle muscular dystrophy.

A gene for a recessive form of limb-girdle muscular dystrophy (LGMD2A) has been localized to chromosome 15. A physical map of the 7-cM candidate 15q15.1-q21.1 region has been constructed by means of a 10-12-Mb continuum of overlapping YAC clones. New microsatellite markers developed from these YACs were genotyped on large, consanguineous LGMD2A pedigrees from different origins. The identification of recombination events in these families allowed the restriction of the LGMD2A region to an estimated 1-cM interval, equivalent to approximately 3-4 Mb. Linkage disequilibrium data on genetic isolates from the island of Réunion and from the Amish community suggest a preferential location of the LGMD2A gene in the proximal part of this region. Analysis of the interrelated pedigrees from Réunion revealed the existence of at least six different carrier haplotypes. This allelic heterogeneity is incompatible with the presumed existence of a founder effect and suggests that multiple LGMD2A mutations may segregate in this population.