MD Rocío N Villar-Quiles

MS Fabio Catervi

PhD Eva Cabet

MD Raul Juntas-Morales

MS Casie A Genetti

MD, PhD Teresa Gidaro

MD Asuman Koparir

MD Adnan Yüksel

MD Sandra Coppens

MD, PhD Nicolas Deconinck

BSc Emma Pierce-Hoffman

PhD Xavière Lornage

Julien Durigneux

PhD Jocelyn Laporte

PharmD, PhD John Rendu

MD, PhD Norma B Romero

MD, PhD Alan H Beggs

MD, PhD Laurent Servais

MD Mireille Cossée

MD, PhD Montse Olivé

PhD Johann Böhm

PhD Isabelle Duband-Goulet
email: isabelle.duband-goulet@univ-paris-diderot.fr

MD, PhD Ana Ferreiro
email: ana.b.ferreiro@gmail.com

ASC1 is a cell cycle regulator associated with severe and mild forms of myopathy

Keywords: 4 (Figures 2, 3, 4, 5

teaching and research institutions in France or abroad, or from public or private research centers.

and/or respiratory failure [START_REF] North | Approach to the diagnosis of congenital myopathies[END_REF][START_REF] Schorling | Congenital Muscular Dystrophies and Myopathies: An Overview and Update [Internet][END_REF][START_REF] Wang | Diagnostic approach to the congenital muscular dystrophies. HHS Public Access[END_REF] and have no specific treatment [START_REF] Jungbluth | Current and future therapeutic approaches to the congenital myopathies[END_REF] . More than 20 genes have been associated with CMs [START_REF] Kaplan | The 2016 version of the gene table of monogenic neuromuscular disorders (nuclear genome)[END_REF][START_REF] Ravenscroft | Pathophysiological concepts in the congenital myopathies: blurring the boundaries, sharpening the focus[END_REF] , most of them encoding proteins involved in (i) skeletal muscle calcium homeostasis [START_REF] Treves | Ca 2+ handling abnormalities in early-onset muscle diseases: Novel concepts and perspectives[END_REF] ; (ii) excitation-contraction coupling 8 ; (iii) membrane remodeling [START_REF] Dowling | Triadopathies: an emerging class of skeletal muscle diseases[END_REF][START_REF] Jungbluth | Pathogenic Mechanisms in Centronuclear Myopathies[END_REF] and (iv) myofibrillar force generation [START_REF] Wallgren-Pettersson | Nemaline Myopathies[END_REF] or thin-thick filament assembly [START_REF] Tajsharghi | Myosinopathies: pathology and mechanisms[END_REF] and interactions [START_REF] Jungbluth | Congenital myopathies: disorders of excitationcontraction coupling and muscle contraction[END_REF] . However, the genetic defect remains unknown in In 2016, we described a form of congenital myopathy caused by recessive mutations in Thyroid Hormone Receptor Interactor 4 (TRIP4), encoding the transcriptional coactivator ASC-1 (Activating Signal Cointegrator-1), in a large consanguineous family. ASC-1 defines the ASC-1 transcriptional cointegrator complex, together with three other subunits (ASCC1, ASCC2 and ASCC3). We termed this novel condition ASC-1 Related Myopathy (ASC1-RM), thus identifying transcriptional coregulation as a novel CM pathomechanism [START_REF] Davignon | The transcription coactivator ASC-1 is a regulator of skeletal myogenesis, and its deficiency causes a novel form of congenital muscle disease[END_REF] . Patients presented with severe neonatal hypotonia, potentially-lethal respiratory failure, muscle weakness with partial or no ambulation, scoliosis, joint hyperlaxity and skin abnormalities. Muscle biopsy revealed the so-far unreported association of multiminicores, caps and dystrophic lesions.

The role of the ASC-1 complex in neuromuscular diseases was confirmed by the simultaneous description of recessive mutations in TRIP4 (three families sharing two mutations) or in ASCC1 (one family) [START_REF] Knierim | Mutations in Subunits of the Activating Signal Cointegrator 1 Complex Are Associated with Prenatal Spinal Muscular Atrophy and Congenital Bone Fractures[END_REF] in patients diagnosed with Spinal Muscular Atrophy (SMA) with arthrogryposis multiplex congenita, respiratory distress and congenital bone fractures. Thereafter, no other patient with TRIP4 mutations has been described, but four additional families with ASCC1 recessive nonsense or frameshift mutations have been recently reported [START_REF] Oliveira | The new neuromuscular disease related with defects in the ASC-1 complex: report of a second case confirms ASCC1 involvement[END_REF][START_REF] Böhm | Novel ASCC1 mutations causing prenatal-onset muscle weakness with arthrogryposis and congenital bone fractures[END_REF] . They showed a similar neonatal phenotype including arthrogryposis, bone fractures and respiratory failure, but muscle biopsies disclosed myopathic features and were not suggestive of motoneuron involvement [START_REF] Böhm | Novel ASCC1 mutations causing prenatal-onset muscle weakness with arthrogryposis and congenital bone fractures[END_REF] .

Transcriptional co-regulators are emerging as key modulators of the functions of nuclear receptors and transcription factors [START_REF] Dasgupta | Nuclear receptor coactivators: master regulators of human health and disease. [Internet][END_REF] . The ASC-1 complex acts as a coactivator through direct binding to transcription factors like AP-1, NF-kappa-B and Serum Response Factor (SRF), involved in cell fate-controlling pathways [START_REF] Jung | Novel Transcription Coactivator Complex Containing Activating Signal Cointegrator 1[END_REF] . ASC-1 is known to interact with basal transcription factors (TBP, TFIIA), transcription integrators (CBP, SRC-1) and nuclear receptors (TR, RXR, ER) in vitro through its highly conserved zinc finger domain [START_REF] Kim | Activating signal cointegrator 1, a novel transcription coactivator of nuclear receptors, and its cytosolic localization under conditions of serum deprivation[END_REF] . Moreover, ASC-1 contains a conserved C-terminal ASCH domain with putative RNA-binding activity predicted in silico to coordinate pre-mRNA processing [START_REF] Auboeuf | Coordinate regulation of transcription and splicing by steroid receptor coregulators[END_REF][START_REF] Iyer | The ASCH superfamily: novel domains with a fold related to the PUA domain and a potential role in RNA metabolism[END_REF] . Interestingly, the ASC-1 complex has been recently implicated in Amyotrophic Lateral Sclerosis (ALS) pathogenesis, being proposed as a common link between ALS and SMA [START_REF] Chi | The neurodegenerative diseases ALS and SMA are linked at the molecular level via the ASC-1 complex[END_REF] . We demonstrated that ASC-1 plays a pivotal role in muscle, its absence leading to defects in human myotube growth [START_REF] Davignon | The transcription coactivator ASC-1 is a regulator of skeletal myogenesis, and its deficiency causes a novel form of congenital muscle disease[END_REF] . However, the precise ASC-1 function and the phenotypical spectrum associated with TRIP4 mutations (including motoneuron versus primary muscle involvement) remained largely unknown.

We report here the first series of patients with TRIP4 mutations, redefine the phenotypical and molecular spectrum of disease and establish the first phenotype-genotype correlations. We also clarify the disease pathophysiology by revealing an unsuspected function of ASC-1 as a cell cycle regulator.

METHODS

Patients and biological samples

Patients were recruited through an international collaboration. We also reassessed evolution of the previously-reported ASC1-RM family [START_REF] Davignon | The transcription coactivator ASC-1 is a regulator of skeletal myogenesis, and its deficiency causes a novel form of congenital muscle disease[END_REF] . Clinical data were systematically retrieved and analyzed retrospectively according to a standardized form. All patients were examined by at least one of the authors in specialized neuromuscular departments.

Diagnostic muscle biopsies from four patients were processed for standard histological and immunochemical studies and fixed for electron microscopy. Primary fibroblasts from patients and agepaired controls were obtained from skin biopsies or surgically-discarded tissues. Muscle tissue was obtained from diagnostic biopsies in two patients and from surgically-discarded tissue in controls.

Informed consent was obtained from all patients or their guardians in agreement with local Ethic

Committees and with the 1964 Helsinki declaration and its later amendments.

Genotyping

TRIP4 mutations were identified on DNA from peripheral blood samples by exome sequencing or next-generation sequencing-based myopathy genes panel, Sanger-confirmed in patients and relatives Exome Aggregation Consortium (ExAC, http://exac.broadinstitute.org/) and Genome Aggregation (gnomAD, http://gnomad.broadinstitute.org/) databases were interrogated to identify previouslyreported mutations and to determine the frequency of each mutation in the general population.

Alamut® Visual (Interactive Biosoftware, North Seattle, WA) was used to predict the effect of the variants.

Cell Culture

All cells were grown at 37°C with 5% CO2. Cells were grown in Dulbecco Modified Eagle Media containing Glutamax (Gibco), fetal calf serum (FCS) 20% and penicillin-streptomycin (P/S) 1% (primary fibroblasts) or FCS 10% and P/S 1% (C2C12).

Transfection: siRNA and Plasmid DNA C2C12 were transfected with Trip4-specific siRNAs or scrambled control siRNA (Origene) 18 hours after seeding according to the manufacturer's instructions using Lipofectamine RNAimax (Invitrogen). The final concentration of siRNAs was 4 nM. Maximum down-regulation was achieved 48h post transfection.

For rescue experiments, mammalian expression vectors (Promega) containing the ORF sequence of the human TRIP4 (pFN21AB7885) or CELF1 (pFN21AB0039) genes fused with a HaloTag were used. Cells were transfected with siRNAs as described above. A second transfection was performed to overexpress TRIP4 or CELF. Plasmids and Lipofectamine 2000 were mixed in Opti-MEM according to the manufacturer's instructions (Invitrogen).

Cell proliferation studies

After seeding at 40000cells/well, the number of primary fibroblasts per optical field was counted from 24 to 96 hours on bright-field images. Proliferation of C2C12 cells was analyzed from 24 to 72 hours after transfection. Cell size measurements C2C12 were imaged from 24 to 96h post-seeding on a Zeiss Axio Observer A1 inverted microscope.

Length and width of cells were measured on bright-field images with Image J (National Institute of Health, Bethesda, MS, USA).

Fluorescence Activated Cell Sorting (FACS) Analysis

C2C12 were harvested 48 hours after transfection and primary fibroblasts during exponential growth phase. Cells were fixed in 70% ethanol at -20°C overnight and then stained with 50µg/ml of propidium iodide, 0.1 or 1% triton and 100 or 50 µg/ml RNAse A for fibroblasts or C2C12 respectively. Analysis was performed using Cyan-ADP analyser and FlowJo-software.

Population doubling time and cell phase duration

Doubling time was calculated using the website doubling-time.com/compute.php (Roth, A., 2006,

Doubling time computing) and the formula

The duration of each cell phase was obtained by multiplying each sample doubling time by the percentage of cells in each phase (FACS).

Western Blot

Cells were lysed with cold lysis buffer. Cell extracts (20 μg) were separated using 10% SDS-PAGE gel and transferred on nitrocellulose membranes (Bio-Rad Trans Turbo Transfer system) which were blocked and probed with the primary antibodies anti-ASC-1 (Abcam), anti-tubulin (Sigma), anti-p21 (Santa-Cruz), anti-cyclin D3 (Santa-Cruz), anti-cyclin D1 (Santa-Cruz or Millipore), anti-pRb (BD Pharmingen), anti-MHCe (DSHB) or anti-halotag (Promega). Membranes were incubated with the secondary antibodies goat anti-rabbit HRP conjugate (Thermo Scientific) or goat anti-mouse HRP

Statistical analyses

Results were expressed as mean ± standard deviation. All data were analyzed with the SPSS® Statistics version 22.0 (Armonk, NY: IBM Corp.). Differences were considered significant if p-value was <0.05.

RESULTS

Clinical findings: Expanding the clinical spectrum

We identified six novel patients (two females and four males) from five families with TRIP4 mutations (Table 1). Age at ascertainment ranged from 7 to 63 years. All pedigrees were compatible with autosomal recessive transmission. Consanguinity was confirmed in three families (A, C and E).

Patient CIII.7 had two siblings deceased during the first year of life due to respiratory distress. Patient DII.2 had a brother diagnosed with a congenital myopathy with type I fiber uniformity who died at 5 years due to respiratory failure. These three affected siblings were not included due to lack of genetic confirmation. We also reassessed the three surviving patients from the originally reported family (family F [START_REF] Davignon | The transcription coactivator ASC-1 is a regulator of skeletal myogenesis, and its deficiency causes a novel form of congenital muscle disease[END_REF] ) after three years of additional follow-up.

Overall, the clinical phenotype in the novel patients was marked, like in the original family, by early onset proximal and axial weakness, respiratory failure with variable severity, scoliosis and skin involvement without prominent limb contractures.

None of the patients presented with arthrogryposis or congenital contractures. There were no complications during pregnancy or labor, excepting reduced fetal movements in the most severe patient (CIII.7). First signs appeared before adulthood in all cases. Patient CIII.7 presented at birth with severe neonatal hypotonia, poor antigravity limb movements, dysmorphic features, feeding difficulties, perinatal asphyxia and respiratory distress (Fig. 1A). He never acquired cephalic control. Two patients (AIII.2 and DII.2) presented with neonatal hypotonia and delayed motor milestones. The two siblings from family B presented with delayed gait acquisition, waddling gait and fatigability. The mildest patient (EII.4) presented with difficulties for running and poor sports performance in adolescence. The last five patients acquired independent ambulation, although only one did so before the age of 18 months. In all patients from the six families, muscle weakness and amyotrophy was predominantly axial and proximal (3-4/5, Medical Research Council scale), while distal limb strength was relatively well preserved. Only one patient had marked facial weakness. There was bilateral horizontal ophthalmoparesis in one patient and ophthalmoplegia in the most severe case. Ptosis was not observed.

Muscle weakness was stable or slowly progressive. Excepting the three patients from the original family F, who never acquired independent ambulation, all the adult patients can walk outdoors without support at ages 23-63 years, walking distance being limited by fatigue or dyspnea. Three patients are able to climb stairs with aid. Patient AIII.2, currently 9 years old, is still improving her motor abilities and starting to run.

All the patients who survived beyond childhood, except the mildest patient EII.4, had early-onset progressive scoliosis, associated with lateral trunk deviation and tilted pelvis in two cases (Fig. 1A). This includes the youngest patient of family F (FIII.1), reassessed after arthrodesis at age 12 years.

Scoliosis was associated with spinal rigidity in a total of five cases. Thorax deformities (flat thorax and pectus excavatum) were present in five patients. Five also had dysmorphic craniofacial features with a similar flat face, along with a large and thick neck appearance.

Respiratory failure was present in 9 of the 10 patients since childhood. The most severe patient suffered neonatal respiratory distress requiring tracheostomy and assisted ventilation. He died at 8 months due to respiratory failure, and so did patient FII.5 at 16 months. Five patients needed chronic ventilation (nocturnal non-invasive in three cases, through a tracheostomy in two), while in the Muscle MRI of the lower limbs in two of the novel patients showed a predominant involvement of the posterior compartment of the thigh, notably of gluteus maximus and adductor longus muscles with relative preservation of gracilis, sartorius, and semitendinosus muscles (Fig. 1B). This pattern was comparable to that previously observed in family F [START_REF] Davignon | The transcription coactivator ASC-1 is a regulator of skeletal myogenesis, and its deficiency causes a novel form of congenital muscle disease[END_REF] .

Histopathological phenotype: cores, cytoplasmic bodies, nemaline, centronuclear, and cap lesions

One patient had a quadriceps biopsy in early childhood, reportedly normal, which was not available.

Samples from the remaining four biopsied patients showed congenital myopathy lesions, including 2, Fig. 2). Three patients had multiminicores, often associated with other forms of sarcomere disorganization (cap lesions, rods and cytoplasmic bodies, whorled fibers). The most severe patient showed fibers with centrally located nuclei interspersed with very small, rounded fibers without fascicular distribution, compatible with centronuclear/myotubular myopathy.

Lobulated fibers and rimmed sarcolemma (intense oxidative rims beneath the sarcolemma, Fig. 2e) were -occasionally observed. None of the four biopsies disclosed fiber type grouping, angulated fibers or group atrophy suggestive of denervation.

Genetic studies: Novel TRIP4 mutations

Seven previously unreported mutations in the TRIP4 gene were identified (Fig. 3A-B). Transmission was autosomal recessive in all cases. Tested parents from three families were healthy heterozygous carriers.

The mutations were distributed along the entire length of the gene. These included the first TRIP4 missense variant, affecting a highly-conserved residue in the Zinc finger domain (c.534C>G, p. His178Gln). Two frameshift mutations predicted loss of the ASCH domain (c.1544_1547delACTG, p.Asp515Alafs*34 and homozygous deletion of exons 8 and 9, respectively) (Fig. 3C).

Western blot was performed on primary skin fibroblast cultures (family B) or frozen muscle biopsies (family D and E) from four patients. Family B was compound heterozygous for a missense and a truncating mutation, which led to a reduced level of expression of the full-length protein (10-20% compared to control) (Fig. 3D). Frameshift mutations (family D) or large deletions affecting the Cterminal part of the protein (family E) led to a total absence of full length ASC-1 and no detectable truncated form (Fig. 3E).

None of the patients had mutations in other known neuromuscular genes except for the oldest patient (EII.4), in which exome studies revealed a heterozygous variant of the titin gene (TTN) (c.6379_6380del, p.Tyr2127Leufs*8), also identified in one of his asymptomatic older sisters whose echocardiography was normal. This variant, predicted to lead to a truncated protein, was absent from ExAC and was not associated with any other TTN change (including missense variants) in the patient.

ASC-1 defects alter proliferation of patient fibroblasts and myogenic cells

When amplifying primary fibroblasts for Western blot, we observed a faster growth of patient cells compared to healthy controls. Thus, we quantified proliferation of skin fibroblasts from families B (BIII.1 and BIII.2) and F (FII.2) versus four fibroblast cultures from passage-and age-paired controls (Fig. 4). TRIP4-mutant fibroblasts showed a significantly higher proliferation rate with lower mean doubling time than controls, compatible with a shorter cell cycle length (Fig. 4C). This suggested that ASC-1 might be involved in the control of cell cycle progression.

Consistently, FACS studies in non-synchronous cell cultures showed that, while most (>80%) control fibroblasts were in G0/G1 phase, there were significantly less patient cells in G0/G1 and more in S and G2 phases (Fig. 4D), indicating a significant increase of cycling cells in ASC-1 depleted cultures.

Considering the cell cycle length, the G0/G1 phase was significantly shortened in patient fibroblasts compared to controls (Fig. 4E), suggesting cell cycle acceleration.

To further clarify the role of ASC-1 in myogenic cells, we used our in vitro Trip4 knock-down (Trip4KD) C2C12 model, comparing cells transfected with a siRNA against Trip4 (siTrip4) to those transfected with a control siRNA (Scbl) or non-transfected (NT) (Fig. 5). siTrip4 cells showed a higher proliferation rate with a shorter doubling time compared to Scbl. Moreover, ASC-1 depleted cells were smaller. Consistently, FACS studies confirmed a decrease of cells in G0/G1 phase and a significantly reduced duration of the latter upon ASC-1 depletion. Thus, ASC-1 is involved in cell cycle regulation in both fibroblasts and myogenic cells, its depletion leading to cell cycle acceleration and reduced cell growth due to shortening of the G0/G1 phase.

ASC-1 depletion is associated with altered expression and phosphorylation of cell cycle exit regulators

F o r P e e r R e v i e

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Cell cycle arrest is an important step in myogenic differentiation, which involves transitioning from proliferative myoblasts to post-mitotic myotubes and muscle fibers. D-type cyclins are regulators of one key G1-phase checkpoint, determining cell cycle arrest or progression into S-phase. Quantification of cyclins D1, D3 and the cell cycle exit marker p21 in frozen muscle samples from three TRIP4mutant patients showed significant cyclin D1 decrease and cyclin D3 increase compared to control (Fig. 6A). p21 was significantly reduced in the two older patients. Embryonic myosin heavy chain was undetectable, suggesting that the cyclin changes in patient muscles are unlikely to be non-specifically due to muscle fiber regeneration and probably are directly associated with ASC1-related cell cycle defects in post-mitotic muscles.

Analysis of cyclins D1, D3 and p21 expression during proliferation in Trip4KD C2C12 myoblasts confirmed cyclin changes, showing a significant increase in cyclin D1 and p21 (Fig. 6B) without significant changes in cyclin D3 (data not shown). Increased expression of cyclin D1 and p21 in Trip4KD was rescued by expression of human wild-type ASC-1 (hASC-1) (Fig. 6C and data not shown), confirming that this molecular phenotype is not due to off-target effects of Trip4 siRNA.

The Retinoblastoma Protein (pRb) interacts with cyclins and regulates both cell cycle and musclespecific gene expression through its target E2F. Thus, abnormalities of Rb, whose activity is modulated by Cyclin/CDK-mediated phosphorylation, might be an underlying mechanism in ASC1-RM muscle phenotype. Indeed, we found an increase of the hyper-phosphorylated forms of Rb and the hyper-phosphorylated/total Rb ratio in Trip4KD C2C12 compared to controls (Fig. 6D). This, associated with cyclin D1 changes, could explain the rapid progression of Trip4KD cells from G1 to S phase and the myofibrillar abnormalities in patients' muscles, suggesting that ASC-1 is critical for controlling both cell cycle progression and myofibrillogenesis during myogenesis.

DISCUSSION

ASC-1 related disease is emerging as a novel cause of congenital neuromuscular disorders, but its phenotypical spectrum and pathophysiology remained unclear. So far, three TRIP4 mutations have been described in four families with a severe neonatal phenotype (one reported as a CM, the three others as SMA) [START_REF] Davignon | The transcription coactivator ASC-1 is a regulator of skeletal myogenesis, and its deficiency causes a novel form of congenital muscle disease[END_REF][START_REF] Knierim | Mutations in Subunits of the Activating Signal Cointegrator 1 Complex Are Associated with Prenatal Spinal Muscular Atrophy and Congenital Bone Fractures[END_REF] . We report here five additional families with seven novel TRIP4 mutations, expanding the phenotypical spectrum beyond lethal congenital forms to mild ambulatory adult patients.

The clinical phenotype in these families is marked by early-onset axial and proximal weakness, progressive scoliosis sometimes associated with rigid spine, dysmorphic features, cutaneous involvement and respiratory failure. No clinical, electromyographic or histological sign of motor neuron involvement was present. The severity of respiratory involvement correlated with the degree of muscle weakness, unlike in other CMs with minicores (e.g SEPN1-Related Myopathy [START_REF] Ferreiro | Mutations of the selenoprotein N gene, which is implicated in rigid spine muscular dystrophy, cause the classical phenotype of multiminicore disease: reassessing the nosology of early-onset myopathies[END_REF][START_REF] Scoto | SEPN1-related myopathies: Clinical course in a large cohort of patients[END_REF] ). The skin phenotype includes skin hyperlaxity, xerosis and follicular hyperkeratosis. Remarkably, while the first patients identified never reached full ambulation, five of the novel patients are able to walk without support, being limited mainly by respiratory failure and fatigability. In the mildest case, the first referral signs were noticed in adolescence, and he remains able to climb stairs at 63 years.

Interestingly, dilated cardiomyopathy was detected in two patients in the third and fifth decades. We previously showed that TRIP4 expression is relatively high in murine cardiac muscle [START_REF] Davignon | The transcription coactivator ASC-1 is a regulator of skeletal myogenesis, and its deficiency causes a novel form of congenital muscle disease[END_REF] . Moreover, neonatal cardiac involvement (cardiomyopathy, atrial septal defect or patent ductus arteriosus) was reported in two SMA families with TRIP4 mutations [START_REF] Knierim | Mutations in Subunits of the Activating Signal Cointegrator 1 Complex Are Associated with Prenatal Spinal Muscular Atrophy and Congenital Bone Fractures[END_REF] . We confirm here that cardiac disease can be part of the TRIP4-associated phenotype, and reveal that the absence of pediatric cardiac involvement does not preclude the subsequent development of cardiomyopathy. Therefore, cardiac function should be periodically screened in all TRIP4-mutated patients.

Primary myocardial disease is not typical of CM, although it may appear in TTN-, MYH7-or ACTA1related myopathies [START_REF] North | Approach to the diagnosis of congenital myopathies[END_REF] . One of our patients with DCM carries a heterozygous TTN truncating variant (TTNtv). Heterozygous TTNtvs are fairly common in the general population (up to 1%) [START_REF] Ware | Role of titin in cardiomyopathy: from DNA variants to patient stratification[END_REF][START_REF] Franaszczyk | Titin Truncating Variants in Dilated Cardiomyopathy -Prevalence and Genotype-Phenotype Correlations[END_REF][START_REF] Schafer | Titin-truncating variants affect heart function in disease cohorts and the general population[END_REF] and have never been demonstrated to cause congenital skeletal muscle disease by themselves. Indeed, congenital titinopathies are autosomal recessive myopathies associated with the combination of either two truncating or one truncating and one missense TTN changes [START_REF] Savarese | Interpreting Genetic Variants in Titin in Patients With Muscle Disorders[END_REF][START_REF] Chauveau | A rising titan: TTN review and mutation update[END_REF][START_REF] Oates | Congenital Titinopathy: Comprehensive characterization and pathogenic insights[END_REF] . The TTNtv in our patient is carried by an older sister with normal neurological and cardiological examination, and his muscle MRI is comparable to that in the other ASC1-RM patients. Thus, his skeletal muscle phenotype is most likely explained by the large homozygous TRIP4 deletion leading to absence of ASC-1. This TTNtv has never been reported in DCM and does not affect the titin domains most commonly associated with cardiomyopathy [START_REF] Schafer | Titin-truncating variants affect heart function in disease cohorts and the general population[END_REF] . However, a potential digenic contribution to his cardiac phenotype cannot be fully excluded at this point. In the other patient with adult-onset DCM, linkage and exomic studies excluded recessive pathogenic TTN changes [START_REF] Davignon | The transcription coactivator ASC-1 is a regulator of skeletal myogenesis, and its deficiency causes a novel form of congenital muscle disease[END_REF] .

The spectrum of muscle architectural lesions in ASC1-RM is particularly large. Aside from the multiminicores, internalized nuclei, cap lesions and mild dystrophic lesions reported [START_REF] Davignon | The transcription coactivator ASC-1 is a regulator of skeletal myogenesis, and its deficiency causes a novel form of congenital muscle disease[END_REF] , we observed nemaline and cytoplasmic bodies in novel patients. Our findings suggest TRIP4 as a novel culprit gene for nemaline myopathy, as well as for other forms of congenital muscle disease (including cap disease, core, centronuclear or cytoplasmic-body myopathies and congenital muscular dystrophy); therefore, we propose that it should be included in the corresponding diagnostic gene panels. Furthermore, we found in one biopsy rimmed fibers comparable to those associated with ASCC1 mutations [START_REF] Böhm | Novel ASCC1 mutations causing prenatal-onset muscle weakness with arthrogryposis and congenital bone fractures[END_REF] . This suggests that TRIP4 and ASCC1 are implicated in a common pathophysiological pathway which leads to multiple forms of myofibrillar disarray, and thus to an overlap of different histopathological CM lesions.

Our phenotypical findings can be useful for differential diagnosis. The joint hyperlaxity, mild to moderate joint contractures, spinal rigidity and skin phenotype observed in some of our patients raise the question of differential diagnosis with collagen 6-related muscular dystrophies [START_REF] Bönnemann | The collagen VI-related myopathies: muscle meets its matrix[END_REF] , although TRIP4 mutant patients had no hypertrophic scars. Moreover, minicore-like lesions may be occasionally found in patients with collagen VI-related myopathies [START_REF] Bönnemann | The collagen VI-related myopathies: muscle meets its matrix[END_REF][START_REF] Bönnemann | The collagen VI-related myopathies [Internet[END_REF] . Extraocular muscle involvement, previously unreported, may be part of the ASC1-RM spectrum but is typically absent in congenital titinopathies [START_REF] Oates | Congenital Titinopathy: Comprehensive characterization and pathogenic insights[END_REF] .

One of the patients with dilated cardiomyopathy had rigid spine and developed mild elbow contractures with age, features that overlap with Emery Dreifuss muscular dystrophy. However, the conduction defects and marked contractures typical of the latter were absent in ASC1-RM patients. In addition, muscle MRI in three ASC1-RM patients revealed a consistent pattern, with predominant involvement of the adductor longus and posterior thigh compartment and relative preservation of the semitendinosus muscle, sometimes associated to increased subcutaneous adipose tissue [START_REF] Davignon | The transcription coactivator ASC-1 is a regulator of skeletal myogenesis, and its deficiency causes a novel form of congenital muscle disease[END_REF] . This MRI pattern differed from the typical abnormalities associated with collagen 6 defects [START_REF] Bönnemann | The collagen VI-related myopathies: muscle meets its matrix[END_REF][START_REF] Mercuri | Muscle MRI in Ullrich congenital muscular dystrophy and Bethlem myopathy[END_REF] or with other CMs with minicores such as those related to SEPN1 [START_REF] Hankiewicz | Whole-body muscle magnetic resonance imaging in SEPN1-related myopathy shows a homogeneous and recognizable pattern[END_REF] , RYR1 [START_REF] Jungbluth | Magnetic resonance imaging of muscle in congenital myopathies associated with RYR1 mutations[END_REF] or TTN [START_REF] Carlier | Myoimaging in Congenital Myopathies[END_REF] , although its specificity requires confirmation by further studies. This work significantly increases the number of known TRIP4 mutations, confirms autosomal recessivity and defines the first genotype-phenotype correlations. The novel mutations include one large deletion and five frameshift changes predicting a reduction or absence of full-length ASC-1 protein. ASC-1 depletion was confirmed experimentally in three families, including two patients compound heterozygous for the first identified TRIP4 missense mutation. Interestingly, ASC-1 was undetectable by Western blot both in our mildest patient and in the first severe cases reported [START_REF] Davignon | The transcription coactivator ASC-1 is a regulator of skeletal myogenesis, and its deficiency causes a novel form of congenital muscle disease[END_REF] , suggesting other severity modulators. Aditionally, the patients reported as having severe SMA [START_REF] Knierim | Mutations in Subunits of the Activating Signal Cointegrator 1 Complex Are Associated with Prenatal Spinal Muscular Atrophy and Congenital Bone Fractures[END_REF] carried TRIP4 nonsense mutations resulting in exon skipping and upregulation of a shorter isoform containing most ASC-1 functional domains. Although their muscle biopsies showed major reduction of myofiber size compatible with a primary muscle component, our findings suggest that the TRIP4 mutations leading to ASC-1 protein depletion are associated with a primary striated muscle phenotype, while those leading to expression of a truncated protein might be associated with motoneuron disease.

A better understanding of the molecular mechanisms of ASC-1 related disease should help to clarify this point.

On this note, we report a pathophysiological pathway associated with ASC-1 defects which contributes to clarify its so-far undefined role and the disease mechanism. Consistently with a muscle and skin phenotype, in the absence of ASC-1 proliferation is accelerated both in fibroblasts and myogenic cells. This increased proliferation is mainly due to a reduction of the G0/G1 phase and is associated with cell size reduction. Shortening of the G1 phase has been associated with cell size and growth reduction in mammalian cells [START_REF] Jiang | Overexpression of cyclin D1 in rat fibroblasts causes abnormalities in growth control, cell cycle progression and gene expression[END_REF][START_REF] Cadart | Size control in mammalian cells involves modulation of both growth rate and cell cycle duration[END_REF] and may contribute to explain the myotube growth defect in ASC-1 depleted patient-and C2C12 cells [START_REF] Davignon | The transcription coactivator ASC-1 is a regulator of skeletal myogenesis, and its deficiency causes a novel form of congenital muscle disease[END_REF] . This cell cycle phenotype is associated with altered cyclin expression. In C2C12, we found that Trip4KD leads to an increase of cyclin D1 and p21, regulators of the G0/G1 phase, and of several downstream targets. Over-expression of D-type cyclins has been shown to shorten the G0/G1 phase [START_REF] Buttitta | Mechanisms controlling cell cycle exit upon terminal differentiation[END_REF] .

Furthermore, the high levels of p21 and cyclin D1 in the absence of ASC-1 suggest an increased formation of the cyclin D1 and CDK4/6-complex [START_REF] Labaer | New functional activities for the p21 family of CDK inhibitors[END_REF] . This complex phosphorylates proteins such as the tumor suppressor pRb [START_REF] Ezhevsky | Differential regulation of retinoblastoma tumor suppressor protein by G(1) cyclin-dependent kinase complexes in vivo[END_REF] , known to control cell cycle. Hypophosphorylated pRb inhibits E2F transcription factors required for S phase entry [START_REF] Leshem | Phosphorylation of pRb is required for HGF-induced muscle cell proliferation and is p27kip1-dependent[END_REF] . Indeed, in Trip4KD cells we found a consistent tendency to the reduction of the hypophosphorylated forms of pRb in favour of hyperphosphorylation, which indicates loss of pRb-mediated inhibition of the G0/G1-S transition. Interestingly, pRb activity is also necessary for late muscle development, promoting myofibrillogenesis and muscle cell growth by activating transcription of myogenic and metabolic genes [START_REF] Zappia | Rbf Activates the Myogenic Transcriptional Program to Promote Skeletal Muscle Differentiation[END_REF] . Defects in this pathway could contribute to explain the various myofibrillar defects in patients' biopsies. Further experiments are required to corroborate pRb abnormalities and to clarify the latter point.

Cyclin D3 is a critical regulator of the proliferation/differentiation balance of myogenic progenitors in skeletal muscle [START_REF] Cenciarelli | Critical Role Played by Cyclin D3 in the MyoD-Mediated Arrest of Cell Cycle during Myoblast Differentiation[END_REF][START_REF] Santa | pRb-dependent cyclin D3 protein stabilization is required for myogenic differentiation[END_REF] . Under non-pathological conditions, this protein is not expressed in adult muscle fibers, as it is more important for the establishment than the maintenance of terminal myogenic differentiation [START_REF] Bartkova | Cyclin D3: requirement for G1/S transition and high abundance in quiescent tissues suggest a dual role in proliferation and differentiation[END_REF] . Cyclin D3 increase in ASC-1 depleted patients' muscles suggests a dysregulation of its expression that could be attributed to inappropriate activation by MyoD or to improper stabilization by pRb [START_REF] Cenciarelli | Critical Role Played by Cyclin D3 in the MyoD-Mediated Arrest of Cell Cycle during Myoblast Differentiation[END_REF][START_REF] Santa | pRb-dependent cyclin D3 protein stabilization is required for myogenic differentiation[END_REF] . Cyclin D3 is also involved in adipogenesis through its interaction with the nuclear receptor PPAR [START_REF] Sarruf | Cyclin D3 promotes adipogenesis through activation of peroxisome proliferator-activated receptor gamma[END_REF] . This could be related to the subcutaneous adiposity observed in some patients. In addition, cyclin D1 represses PPAR and inhibits adipocyte differentiation [START_REF] Fu | Cyclin D1 Inhibits Peroxisome Proliferator-activated Receptor γ-mediated Adipogenesis through Histone Deacetylase Recruitment[END_REF] . Drastically reduced cyclin D1 in postnatal patient muscles might contribute to this adipose phenotype via PPAR derepression.

In conclusion, our work expands the clinical, histological and molecular spectrum of ASC1-RM and contributes to disentangle its pathophysiological mechanisms. We propose that TRIP4 mutations should be considered in any patient with histopathological CM lesions and a non-contractile myopathy, even in adult ambulant patients without clear neonatal signs, particularly if the phenotype includes respiratory insufficiency, joint hyperlaxity, skin abnormalities or cardiomyopathy. Moreover, we reveal that ASC-1 plays a key role not only in late stages of myogenic differentiation and myotube growth [START_REF] Davignon | The transcription coactivator ASC-1 is a regulator of skeletal myogenesis, and its deficiency causes a novel form of congenital muscle disease[END_REF] but also as a novel cell-cycle regulator in several cell types, controlling cell proliferation via regulation of key cell-cycle proteins. This work confirms transcriptional co-regulation defects as an emerging mechanism in inherited neuromuscular disease. Based on the identification of common histopathological lesions in TRIP4-and ASCC1-mutant patients, we propose the term 'ASC1-related myopathies' to include pathologies associated with both genes and with potential defects of any other protein of the ASC-1 complex, whose relevance in muscle and/or motoneuron pathophysiology is likely to increase in the future.

002). (E)

The duration of cell cycle phases G0/G1 (blue), S (yellow) and G2M (green) for each sample was determined as the percentage of cells in each phase multiplied by the doubling time corresponding to each sample.

INTRODUCTION

  Congenital myopathies (CMs) are genetically and clinically heterogeneous inherited disorders. They usually present during the first years of life with delayed motor development, muscular weakness and hypotonia and have a stable or slowly progressive course. CMs are sometimes associated with cardiac

  to Human Genome Variation Society recommendations (http://varnomen.hgvs.org).

  Scientific). Signals were detected with Enhanced chemiluminescence (ECL, Bio-Rad) and quantified using Image J software.

  only respiratory physiotherapy was prescribed. The mildest patient, currently aged 63, has a FVC of 80% but is treated with CPAP due to sleep apnea/hypopnea syndrome.No patient had congenital or childhood cardiomyopathy. However, patient EII.4 developed a severe dilated cardiomyopathy (DCM) in the fifth decade, requiring a preventive automated implantable cardioverter defibrillator. Follow-up of family F revealed DCM from the beginning of the third decade in patient FII.2. All the other patients have normal cardiac evaluations at ages 9-35 years.Limb contractures, mild to moderate, were present in five patients, mainly affecting Achilles tendon and elbows. Prominent and generalized joint hyperlaxity was observed in five. Deep tendon reflexes were universally diminished or absent.Extramuscular involvement included skin abnormalities (hyperelasticity, follicular hyperkeratosis, and xerosis with scratch lesions but without keloids, N=7) and body composition changes. Three of the novel patients were underweight (<4 th percentile) and one was overweight with abundant adipose tissue in lower limbs, resembling the female patients from family F[START_REF] Davignon | The transcription coactivator ASC-1 is a regulator of skeletal myogenesis, and its deficiency causes a novel form of congenital muscle disease[END_REF] . None of the novel patients had intellectual disability or signs of CNS abnormalities.CK level was normal or mildly elevated (<3xN) in all patients. Electromyography showed a myopathic pattern without denervation or abnormalities in nerve conduction studies (N=6) or repetitive stimulation (N=3).

  size variability, type 1 fiber predominance (and frequent hypotrophy) and internalized nuclei, along with other changes in the muscle fiber architecture (Table

Fig. 1 :

 1 FIGURE LEGENDS

Fig. 2 :

 2 Fig. 2: Spectrum of histopathological lesions. Skeletal muscle biopsies from patients CIII.7 (a),

Fig. 3 :

 3 Fig. 3: TRIP4 mutations in the novel families. (A) Pedigree of novel families. (B) Summary of the

Fig. 4 :

 4 Fig. 4: Accelerated proliferation and reduced G0/G1 phase of the cell cycle in patient fibroblasts.

Fig. 5 :

 5 Fig. 5: Accelerated proliferation and G0/G1 phase reduction and reduced growth in Trip4KD

Fig. 6 :

 6 Fig. 6: Altered expression of cell cycle proteins in patient muscles and in Trip4KD myogenic

  patient DII.2 and control muscle samples excluded that this might be due to active muscle regeneration (including the presence of immature myofibers) in patients (lower panel, C2C12 used as a positive control). (B) Increased cyclin D1 and p21 expression in Trip4KD C2C12 with <20% ASC-1 residual expression (data not shown) compared to non-transfected cells (NT) or cells transfected with siRNA control (Scbl) (left and right panel, normalized to Scbl transfected cells; Mann-Whitney U, *pvalue <0.05). (C) Rescue of the altered levels of cyclin D1 in Trip4KD C2C12 by wild-type human ASC-1. Western blot of protein extracts from Scbl (lanes 1 and 4), siTrip4 (lanes 2 and 5) or ctl C2C12 (lane 3) double-transfected with hASC-1 (lanes 3-5) 24h after endogenous ASC-1 silencing (upper panel). Quantification of the relative expression of cyclin D1 over tubulin (normalized for Scbl) in ctl, Scbl and siTrip4 cells in the presence or absence of hASC-1. A vector expressing hCelf1 was used as a non-specific control (lower panel). mASC-1= murine endogenous ASC-1; hASC-1 = human ectopic ASC-1; hCelf1 = human ectopic Celf1. (D) Western blot analysis of pRb phosphorylation state in NT, Scbl and siTrip4 cells revealed a fast migrating band corresponding to the hypo-phosphorylated form of Rb (pRb, 110 kDa) and slower migrating bands corresponding to hyper-phosphorylated forms of Rb (ppRb, 116kDa) (left panel). Red ponceau (redP) staining of the membrane showed similar loading in each condition. In all conditions, the hyper-phosphorylated forms of Rb were predominant compared to the hypo-phosphorylated form as expected for cycling cells. Although variability precluded reaching statistical significance, quantification of the ratio ppRb/total pRb for each condition (N=4) showed an increase by nearly 50% of hyper-phosphorylated forms in siTrip4 cells (right panel).

Figure 1 .Figure 2 .Figure 3 .

 123 Figure 1. ASC-1 Related Myopathy phenotypical spectrum. (A) Clinical findings in patients CIII.7 (a), DII.2 (b-f) and EII.4 (g,h). (a) Congenital presentation with neonatal hypotonia (frog position), poor limb movements and respiratory distress requiring tracheostomy and assisted ventilation. He had skin lesions, dysmorphic facial features (i.e flat face; not shown) and tapering fingers. (b-f) Patient DII.2, still ambulant at age 35 years: note severe scoliosis with dorsal lordosis and unbalanced hips (b,d), thoracic deformities (pectus excavatum) and elbow contractures (b) contrasting with prominent joint hyperlaxity (f). Dysmorphic features included large, thick neck and retrognathism and low-set ears (not shown). The skin phenotype was marked by follicular hyperkeratosis, xerosis with scratch lesions, prominent scars (but not keloid) and skin hyperelasticity (c,e). (g,h): The mildest patient (EII.4), still ambulant at 63 years, has proximal amyotrophy, pectus excavatum and dysmorphic facial features (flat face, thick neck, retrognathia). (B) Muscle imaging in two patients revealed predominant involvement of posterior thigh compartment with relative preservation of the semitendinosus muscle. (a-c): Lower limb MRI from a mild patient (BIII.1), still ambulant at age 19 years. Axial T1-weighted images showed mild muscle atrophy and fatty infiltration of glutei, iliopsoas and posterior thigh muscles with major involvement of adductor longus and relative

Figure 4 .

 4 Figure 4. Accelerated proliferation and reduced G0/G1 phase of the cell cycle in patient fibroblasts. (A) Bright-field pictures of control (Ctl1) and patient (BIII.2) fibroblasts cultures from 24 to 96 hours after seeding (B) proliferation curves from four control (blue) and three patient fibroblasts cultures (red). (C) Differences between the proliferation curves from control and patient cells were significant (ANOVA 3DF, *p-value<0.001). Mean population doubling time of control and patient cells was calculated for cells in exponential proliferation between 48 and 72h. (D) FACS analysis of cell cycle progression in nonsynchronous proliferative control (Ctl1) or patient (BIII.1; FII.2) fibroblasts. Histogram showing the mean percentage of cells per phase in the four controls and three patients fibroblast samples (ANOVA 2DF, p-value <0.001; Tukey HSD *p-value 0.01 **p-value 0.002). (E) The duration of cell cycle phases G0/G1 (blue), S (yellow) and G2M (green) for each sample was determined as the percentage of cells in each phase multiplied by the doubling time corresponding to each sample.

Figure 5 .Figure 6 .

 56 Figure 5. Accelerated proliferation and G0/G1 phase reduction and reduced growth in Trip4KD myogenic cells. (A) Bright-field pictures of Trip4KD C2C12 (siTrip4) compared to not treated (NT) or scramble (Scbl) transfected controls from 24 to 72 hours post siRNA transfection. (B) Proliferation curves of siTrip4, Scbl and NT cells. Overall, the proliferation curve of siTrip4 cells was higher than that from Scbl-transfected controls (ANOVA, **p-value <0.001), maintaining values comparable to non-transfected cells. Values are the mean of four independent measurements for each time per condition. Mean population doubling time (hours) was calculated for cells in exponential proliferation between 24 and 48h after transfection: NT 16.6±1, Scbl 20.1±4.4, siTrip4 15.1±1.5. (C) SiTrip4 cells exhibited a smaller cell surface (μm2) compared to NT or Scbl cells after 24, 48 or 72h of culture (Kruskal-Wallis, *p-value< 0.05). A strong ASC-1 reduction (>80%) of ASC-1 in Trip4KD cells was observed 48h after transfection. This was also observed within 48 hours with three independent siRNAs against Trip4 (data not shown). (D) FACS analysis of cell cycle in nonsynchronous NT, Scbl and siTrip4 cells. Mean cell cycle distributions measured in three experiments showed a decrease in the percentage of cells in G0/G1 phase in siTrip4 condition compared to Scbl and NT (respectively 51±3.3; 54.3±6.1; 60.1±4.7), an increase in S phase (respectively 36.4±2.3; 35.6±3.9; 29.1±3.7) and in G2M (respectively 10.8±1.3; 9.64±1.1; 9.74±2.3). The duration of the cell cycle phases (±SEM), G0/G1 (blue), S (yellow) and G2M (green), was determined as mentioned in Fig. 4 (ANOVA *pvalue <0.05). A parallel increase in the number of cells in S phase in both Scbl and siTrip4 samples could be explained by the stress induced by transfection.

F

Table 1 .

 1 Summarized clinical findings. F: female; M: male; m: months; y: years; UK: unknown; NA: non applicable; NH: neonatal hypotonia; DMM: Delayed motor milestones; RF: respiratory failure; WCB: wheelchair-bound; CPAP: Continuous Positive Airway Pressure; DCM: Dilated cardiomyopathy; LVEF: Left ventricular ejection fraction; FH: follicular hyperkeratosis; LL: lower limbs; # Proband BOS1248-1 in The Manton Center for Orphan Disease Research database.

			o r
			P
		No	No	e e r No	No	DCM (LVEF	DCM in the
						25%) in the	3 rd decade
	Joint contractures Skin involvement Dysmorphic features	Yes (Achilles) No Flat face, retrognathia	No No No	5 th decade Yes (elbow, mild) Yes (xerosis, FH) Flat face, retrognathia. Flat thorax, pectus excavatum. w Yes (elbows, R achilles). Joint hyperlaxity LL e (hyperelasticity) No Yes Yes (hyperelasticity, v FH) i Flat face, prominent Elongated face, low-set ears, e venous markings, tapering fingers retrognathia. Flat thorax, pectus excavatum.	(n=1) Yes (n=2); Hyperlaxity (n=4) Yes (hyper-elasticity, xerosis, FH) Flat face (n=2), flat thorax (n=3), funnel thorax (n=1), pectus excavatum
							(n=2), valgus
							feet (n=2),
							high arched
							palate (n=1)
	Other	Underweight	Right-eye	Severe	Underweight	Mild	LL
	features	(<P3)	keratoconus	ophthalmo-	(<P3). Myopia	ophthalmo-	lipodystrophy
		Learning and	(III.1)	plegia		paresis.	(n=2),
		writing				Myopia	learning
		difficulties	Underweight				difficulties
			(<P4) (III.2)				(n=2),
							language
							delay(n=1),
							testicular
							ectopia and
							delayed
							puberty (n=1)

Table 2 . Main histological findings in skeletal muscle biopsies from five patients

 2

. IN: internalized nuclei. FSV: abnormally increased fiber size variation.

ACKNOWLEDGMENTS

The authors wish to thank the patients and their families for their cooperation. We also want to thank Dr Miguel Iniesto (Université Paris Sud) for assistance in statistical analysis. We acknowledge the ImagoSeine core facility of the Institut Jacques Monod, member of the France BioImaging (ANR-10-INSB-04) and the support of La ligue contre le Cancer (R03/75-79). Microscopy analyses were performed on the 'Plateau imagerie' facility of the Functional and Adaptive Biology Unit (BFA), Université de Paris/CNRS, Sorbonne Paris Cité, France. We thank MYOBANK-AFM (Institut de Myologie, BB-0033-00012) and the Banque de Cellules et d'ADN (Genethon, Evry, France) for the disposal of biological samples and their contribution to human cell cultures, respectively. This work was funded by the Association Française contre les Myopathies (AFM, Grants #20923 and #21267), the Institut National de la Santé et la Recherche Médicale (Inserm), the Centre National de la Recherche Scientifique (CNRS) and the Université de Paris, France. R.N.V.Q. is the recipient of a research grant from the Alfonso Martín Escudero Foundation (Beca de investigación en universidades o centros en el extranjero, Convocatoria 2017). M.O. is supported by the "Instituto de Salud Carlos III" through the project PI14/00738 (co-funded by European Regional Development Fund ERDF, a way to build Europe). WES and analysis of Family C were supported by National Institutes of Health (USA) grants UM1 HG008900 and R01 HD075802 and MDA602235 from the Muscular Dystrophy Association (USA). Sanger confirmation for family C was performed by the Molecular Genetics Core of the Boston Children's Hospital IDDRC funded by National Institutes of Health grant U54 HD090255.

AUTHOR CONTRIBUTIONS

AF, IDG and RNVQ conceived and designed this study. Acquisition and analysis of data were performed by RNVQ, FC, RJM, CAG, TG, AK, AY, SC, EPH, JR, NBR, AHB, LS, MC, MO, XL, JD, JB, IDG and AF. ND and JL contributed to discussions. RNVQ, IDG, AF were responsible for drafting the manuscript and figure preparation. All authors read and approved the final manuscript.


R Mitteau

S Berrebi

Ph P Chappuis

Darses

A Dufayet

L Garampon

D Guilhem

M Lipa

V Martin

H Roche

Non destructive testing of actively cooled plasma facing components by means of thermal transient excitation and infrared imaging

SATIR is a new test-bed installed at Tore Supra to perform non destructive examination of actively cooled plasma facing components. Hot and cold water flow successively in the cooling tube of the component and the surface temperature is recorded with an infrared camera. Defects are detected by a slower temperature response above unbrazed areas. The connection between temperature differences and defect sizes is the main difficulty. It is established by tests of standard defects and thermal transient calculations of defective geometries. SATIR has been in use for two years and has proved to be very valuable to test industrial components as well as prototypes.

INTRODUCTION

The in situ maintenance of plasma facing components (PFC) is very difficult and a high level of reliability has to be reached. Therefore, non destructive examination (NDE) is systematically applied to test components manufactured by industry. Thermal techniques present the benefit to test what really matters : the thermal transfer of the bonds. Such techniques have already been in use at Tore Supra as well as in other laboratories [START_REF] Schlosser | Fusion Engineering[END_REF][START_REF] Falter | Fusion Technology[END_REF]. At Tore Supra, the necessity for a permanent NDE testbed for PFCs led to the development of SATIR (figure 1). SATIR is an acronym for Station d'Acquisition et de Traitement InfraRouge. This equipment and its use are described in the following pages.

TEST -BED DESCRIPTION

The majority of Tore Supra's PFCs are actively cooled. It gives the opportunity to use their cooling channels to heat or cool the components with hot and cold water. An open water circuit is set up (figure 2). Cold water comes from the commercial network and three heating tanks totalling 600 l are installed to deliver hot water. Two elements can be installed in parallel. Various types of connections can be used. This thermal excitation is highly efficient. For a temperature difference of 80 °C between the component and the water, and with an heat transfer coefficient to 17000 W/m²K, the wall heat flux reaches 1.4 MW/m². The surface temperature of the elements is measured with an infrared camera (type Inframetrix 600). The video signal is numerised and stored in a PC. Safety copy of the film may be stored with a VCR. The PC works with a PTR-based software (CEDIP) which both remote controls the acquisition sequence as well as it does the thermal analysis. The PC has a 486 processor and 64 Mo RAM which enable to record up to 12.5 images per second. The data are safeguarded on 5 Go cartridges.

TEST PROCEDURE

The choice of hot and cold water durations and sampling frequency depends on the element being tested. The inner first wall whose thermal time constant is 15 seconds was tested with 60 seconds fronts and a sampling frequency of an image out of 6 (roughly 2 images per second) [START_REF] Lipa | Development and fabrication of improved CFC-brazed components for the inner first wall of Tore Supra, 19th SOFT[END_REF].

Mock-up aimed at developments are tested individually and get a customised analysis. Elements from large fabrication series are tested simultaneously with a sound reference element, which is chosen after test in a high heat flux test-bed. The subsequent analysis relies on the comparison between the two. the 6 lines are merged The thermal analysis is based on the difference between the time response of each couple of points (test and reference, figure 3). These points are located at the same relative positions on the tiles to allow comparison. The temperatures are extracted from the film, usually from a 3*3 pixel matrix. Figure 3 does not permit to distinguish differences in the surface temperature. One has to display the differences to see the differences (figure 4.a). Because of emissivity differences at the surface of the tiles, stable hot and cold temperatures measured by the camera may vary from point to point. In order to calculate the real temperature difference between the points, the temperature curves are normalised linearly (figure 4.b).

Let Tc and Th be the cold and hot stable temperature as measured by the infrared camera, Tc and Th the cold and hot stable temperatures averaged on all curves. The normalised temperature is given by :

    T = (T -T ) T -T T -T T normalized measured c h c h c c  
~~M aximum temperature differences are then deduced from the normalised curves and displayed on the screen (see example table 1). In the case of the inner first wall, the temperature differences were also corrected from the various wall thicknesses measured on the stainless steel heat sink.

A maximal temperature difference is authorized. When an element shows a temperature lag that exceeds the limit, it is more thoroughly investigated and can be rejected. The standard defects are either fabricated (e.g. during the brazing cycle by forbidding the braze to wet the armour material using stop-off fluid, figure 5) or created on sound elements (by drilling or grinding the joint with narrow tools). The elements are then tested and the temperature differences plotted against defect sizes.

T

Figure 5 : Standard defects

Finite elements calculations of faulty geometries are also extensively performed. Both 2D and 3D calculations are made. They are compared to the results of the standard defects. The calculations showed that the temperature difference on the surface of the tile is better correlated to the braze void extension rather than to the braze void area. When no boundary is present, the braze void extension is the radius of the largest circle that can be inscripted in the defect (figure 6).

Figure 6 : illustration of braze void extension

However, with boundaries, a more complicated definition has to be used. If we consider the shortest path between a point of the braze void and all points of the brazed area, the braze void extension is the longest of the those paths. This can be mathematically written by the following expression :

d max          M braze void N brazed area MN ( ) ( ) min ( )
Overheating of the tile's surface under heat flux is governed by the same parameter, so that setting an acceptance limit on the test bed is equivalent to accept a limited overheating under heat flux. For the inner first wall, those considerations led to a limit of 6°C ([3], figure5).

TWO YEARS EXPERIENCE

The experience was gained mainly on the inner first wall, which elements were tested after delivery and after the assembly steps. Testing 1 m² takes approximately 1 month. The test-bed led to the rejection of three elements. One tile had an 11°C lag (figure 7).

Figure 7 : Defect on a PPI element X-ray testing of this element had shown no defect, but cuts through the element confirmed the presence of a large defect. This example proved the usefulness of SATIR and its complementarity to Xray examination.

Many mock-up were also individually tested (fingers, macroblocs, bolted tiles, metallic mock-ups, see table 2). They showed that, as in other NDE techniques, experience is vital and allows to have a better sensibility.

FUTURE PROSPECTS

The software is currently being improved to analyse highest and lowest temperatures on surfaces rather than on points. The aim is to detect surface temperature's differences within single tiles. By so doing, the analysis will be independent from temperature lag caused by conductivity or thickness differences between the measured and reference tiles. This will allow to reduce the acceptance limit close to the level of the signal's noise. A comparison to a reference element will however be maintained, to avoid the risk of generalized defects. These techniques will be used to test the high heat flux fingers that are developed for the Tore Supra's Toroidal pump limiter. A 3°C limit is foreseen, which would guaranty a steady state temperature smaller than 1500 °C (in the worst case, figure 8). This value is correlated to a 6 mm defect. Beside this improvement, other techniques could be used to increase both the sensibility of the testbed and its capacity :  Going towards higher water pressure, velocity and temperature. However, this would require a stronger water loop and stronger connections.  Cycling the excitation and measure the phase shift.  Narrowing the temperature range of the camera to increase the precision of the measure. Correcting the emissivity differences on the surface of the tile would require to store a map of the surface's emissivity.

CONCLUSION

Figure 2 : 1 :

 21 Figure 2 : Test-bed diagram Depending on the pressure drop of the elements, the hot water flow rate amounts up to 2.5 m 3 /h and the cold water flow rate up to 4.3 m 3 /h. These flow rates are sufficient to have a water transit duration Figure 1 : View of the test-bed

Figure 3 :

 3 Figure 3 : Surface temperature of 6 points :the 6 lines are merged The thermal analysis is based on the difference between the time response of each couple of points (test and reference, figure3). These points are located at the same relative positions on the tiles to allow comparison. The temperatures are extracted from the film, usually from a 3*3 pixel matrix. Figure3does not permit to distinguish differences in the surface temperature. One has to display the differences to see the differences (figure 4.a).

Figure 4 :

 4 Figure 4 : Temperature difference with and without normalisation

  ) under 8 MW/m² T (°C) on SATIR Points : standard defects Curves : Finite Element calculations

Figure 8 :

 8 Figure 8 : Setting the limit for the fingers

Table 1 : Results of the thermal analysis

 1 Setting the limit is the greatest difficulty. Two methods are employed : test of standard defects and finite element calculations.

	up	T down	defect size
	pair N°1 1.3 °C	1.3 °C	2.8 mm
	pair N°2 1.1 °C	1.6 °C	3.2 mm
	pair N°3 1.3 °C	1.5 °C	3.2 mm

Table 2 : Experience gained during two years of operation

 2 SATIR has proved to be a very valuable testbed to test fabrication series as well as prototypes. In comparison to other thermal techniques used in NDE, SATIR presents the advantage of being quantitative. It helps judgement when elements are defective but might be accepted.

	component	Nb Nb tiles area	tests	results	remarks
				(cm²)
	Tore Supra	88 1506 19500 2/3	3 elements rejected	60° toroïdal extension
	inner first wall (IFW)						6 months
	Tore Supra IFW	1	4	24	1 T function of void percentage	correlated to FE
	standard defects						calculations
	LPT short fingers	8	56	350 2 defaults hardly visible before	studies are under progress
						FE200, easily visible after	to improve the analysis and
						damage on FE200	lower the acceptance limit
	LPT long fingers	4	84	500 2	idem	idem
	HIP copper -	1 no tile	100 1	non homogenous copper	task T8
	Stainless Steel (ITER)					emissivity
	rheocast (ITER)	1 1 copper	30	1	very large defect	deteriorated during tests at
			tile				FE200
	macrobloc monotube	3 1 large	30	1	evidence of braze voids	each face has to be tested
							separately
	macrobloc multitube	2 1 large 300 3
	macrobloc	2 1 large 300 1	to be tested	(recessed hole)
	ergodic divertor	1	B4C	100 3	the thickness of the B4C	no thermographic test for
	neutraliser		coating			coating prevails	this fabrication
	ergodic divertor	1 32 bolted	430 1	the loosest tiles lag	semi-inertial element
	front face		CFC				time constant 300 s


Antonia Machlouzarides-Shalit
email: antonia.machlouzarides-shalit@inria.fr

Guillermo Gallardo

Demian Wassermann

Keywords:

published or not. The documents may come

We took the significant sulcal relations and constructed dependency paths per lobe (see Fig. 2), whereby any sulcus can be found based on previously found sulci.

We applied the relative position terms to 589 subjects (53% female, age 29.3±3.28).

[1]

We have shown how primary and secondary sulci relate to each other and have quantified our findings as being consistent in a large population, thus providing additional insight into cortical mapping.

Anteroposterior axis

Superoinferior axis

Figure 2 :Figure 1 :

 21 Figure 2: Sulcal dependency paths, where each sulcus of the frontal lobe is organised in relation to previously identified sulci, beginning with the primary sulci, in this case the lateral fissure and the central sulcus.

This work acknowledges the support of ANR NeuroRef and ERC-StG NeuroLang How do we address this? 2


Lucile Orcin-Chaix

Stéphane Campidelli

Loïc Rondin

Fabien Bretenaker

Yannick Chassagneux

Christophe Voisin

Jean-Sébastien Lauret
email: lauret@ens-paris-saclay.fr

Photostability of single-walled carbon nanotubes/polymer core-shell hybrids as Telecom Wavelength Emitters

Keywords: Carbon nanotubes, Hybrid nanomaterials, Functionalization, Luminescence, Single emitter 1.Introduction

In the spirit of the core-shell approach that made it possible to greatly enhance the photostability of semiconductor nanoparticles, we study the emission stability of single carbon nanotubes emitting at telecommunication wavelengths protected by a double shell of polymer. Two types of structures with dierent polymer layers have been studied at cryogenic temperatures, whereas nanotubes wrapped with poly(9,9-di-n-octyluorenyl-2,7diyl) and embedded in a bulk polystyrene matrix were used as a reference. A Mandel parameter description applied to the emission intensity and an Allan deviation analysis of the spectral diusion show quantitatively that the polymer shells stabilize the emission of single carbon nanotubes by more than one order of magnitude in comparison with micelle wrapped nanotubes deposited on a SiO 2 surface. This represent an additional step towards the use of single-walled carbon nanotubes as quantum emitters at telecommunication wavelengths.

Photonics and quantum photonics using singlewalled carbon nanotubes (CNT) as building blocks are developing strongly. Carbon nanotubes can be used as light sources, optical modulators or detectors. 110 Strategies based both on lms or on single nanotubes are currently being developed depending on the application. In particular, single nanotubes are promising sources of single photons emitting in the telecommunication window. [START_REF] He | Carbon nanotubes as emerging quantumlight sources[END_REF][START_REF] He | Tunable room-temperature single-photon emission at telecom wavelengths from sp3 defects in carbon nanotubes[END_REF][START_REF] Luo | Carbon Nanotube Color Centers in Plasmonic Nanocavities: A Path to Photon Indistinguishability at Telecom Bands[END_REF] In this context, the emission stability of single nanotubes is a key issue towards their use in quantum devices. Indeed, nanotubes being made only of surface atoms their properties are very sensitive to their local environment. While this extreme sensitivity is an asset for sensing applications, [START_REF] Battie | Gas sensors based on thick lms of semi-conducting single walled carbon nanotubes[END_REF] in optics it gives rise to the broadening of the emission peaks and to the instability of the emission spectrum including the socalled spectral jitter and blinking phenomena. [START_REF] Ai | Suppression of Blinking and Enhanced Exciton Emission from Individual Carbon Nanotubes[END_REF] These two processes are detrimental to the use Acquisition time:30s, excitation power: ∼150µW. (e)(f) Histograms of heights of CNT@PFO/PS and CNT@PS/PS core-shell structures deduced from AFM measurements. of nanotubes for application as single emitters for quantum technologies. In this context, the search for strategies improving the stability of the nanotubes' emission is an important question. For instance, it has been shown that embedding nanotubes in a polymer matrix can improve their optical performances. [START_REF] Ai | Suppression of Blinking and Enhanced Exciton Emission from Individual Carbon Nanotubes[END_REF]15 Nevertheless, once nanotubes are capped or embedded in a polymer layer, they can not be handled anymore. Recently, Noé et al showed that the emission of nanotubes deposited on an h-BN substrate is much more stable than when a SiO 2 substrate is used. [START_REF] Noé | Environmental Electrometry with Luminescent Carbon Nanotubes[END_REF] Nevertheless, in some cases, one would need to choose more common substrates such as Si/SiO 2 wafers in the view of building devices. Here, our strategy is to assemble core-shell structures where a polymer shell wraps the active core made of a CNT. This approach is inspired by the great improvement of the emission stability of semiconductor nanocrystals when they are capped with a protective shell. [START_REF] Chen | Compact high-quality CdSe-CdS core-shell nanocrystals with narrow emission linewidths and suppressed blinking[END_REF][START_REF] Ji | Non-blinking quantum dot with a plasmonic nanoshell resonator[END_REF] The objective is to combine the advantages of being able to choose any desired substrate and to perform postprocessing with a high emission stability.

Previously, we reported the synthesis of core-shell structures with HiPCo nanotubes. [START_REF] Orcin-Chaix | Single-Walled Carbon Nanotube/Polystyrene Core-Shell Hybrids: Synthesis and Photoluminescence Properties[END_REF] Briey, a rst layer of polymer is wrapped around the nanotubes through a non-covalent interaction. Non-covalent chemistry is used to prevent any damaging of the nanotube surface. The non-covalent layer showing poor stability, a second layer of polymer is build on the top of the rst one by a radical polymerisation of polystyrene in order to lock the structure. These core-double-shell structures are very stable and can be solubilized in usual organic solvents making them easy to handle and process. [START_REF] Orcin-Chaix | Single-Walled Carbon Nanotube/Polystyrene Core-Shell Hybrids: Synthesis and Photoluminescence Properties[END_REF] The intended utility of the shell is both to protect the nanotube from being damaged during a device fabrication and to isolate the nanotube from its local environment to stabilize its emission.

In this paper, we study in particular the inuence of the double shell structure on the luminescence stability of single nanotubes at cryogenic temperatures. Two types of structures have been synthesized.

In addition to the combination of polystyrene and crosslinked polystyrene previously reported, [START_REF] Orcin-Chaix | Single-Walled Carbon Nanotube/Polystyrene Core-Shell Hybrids: Synthesis and Photoluminescence Properties[END_REF] a new combination of poly(9,9-di-n-octyluorenyl-2,7diyl) (PFO) as inner shell with cross-linked polystyrene as outer shell has been synthesized. Moreover, in the view of using these structures as quantum emitters for quantum communications, the synthesis procedure has been applied to single-walled carbon nanotubes synthesized by laser ablation emitting in the C band of telecommunication. Furthermore, in order to benchmark these hybrids, we used PFO wrapped nanotubes in a bulk polystyrene matrix as a control sample. The stability of the luminescence intensity is analyzed using a Mandel parameter description and the spectral jitter is studied by means of the Allan deviation.

Quantitative comparison is done with the control sample in the polystyrene matrix and with other reports in the literature. We nd that the protective shells stabilize the emission by more than one order of magnitude in comparison with bare nanotubes deposited on a SiO 2 surface [START_REF] Noé | Environmental Electrometry with Luminescent Carbon Nanotubes[END_REF] and are nearly as good as the bulk polystyrene embedded CNTs, with the key advantage of remaining processable for their integration in devices. This demonstrates that the core-shell approach is promising towards the use of CNT as quantum emitters.

Experimental

Sample preparation. The nanotubes were synthesized by laser ablation. Their mean diameter is of 1.1 nm, leading to an absorption peak at ∼1.55 µm. [START_REF] Lauret | Bandgap Photoluminescence of Semiconducting Single Wall Carbon Nanotubes[END_REF][START_REF] Nguyen | Elastic Exciton-Exciton Scattering in Photoexcited Carbon Nanotubes[END_REF] The synthesis of the core-double-shell structures has been reported previously [START_REF] Orcin-Chaix | Single-Walled Carbon Nanotube/Polystyrene Core-Shell Hybrids: Synthesis and Photoluminescence Properties[END_REF] and is described in the Supplementary information (SI). Here, the rst shell non-covalently attached to the nanotube is composed either of PFO or polystyrene. The second layer used to lock the structure is made of crosslinked polystyrene. In the following, these two structures are respectively named CNT@PFO/PS and CNT@PS/PS. PFO has been chosen since it is a widely used polymer in nanotubes sorting, [START_REF] Graf | Large scale , selective dispersion of long single-walled carbon nanotubes with high photoluminescence quantum yield by shear force mixing[END_REF] and in CNT based photonics devices. [START_REF] Gaufrès | Optical microcavity with semiconducting single-wall carbon nanotubes[END_REF][START_REF] Graf | Electrical pumping and tuning of excitonpolaritons in carbon nanotube microcavities[END_REF] Moreover, polystyrene has proven its eciency in stabilizing the luminescence of carbon nanotubes when used as a matrix. 15 Finally, the combination of PFO nanotubes with a polystyrene matrix was shown to lead to a very stable emission of nanotubes at low temperature. [START_REF] Jeantet | Widely Tunable Single-Photon Source from a Carbon Nanotube in the Purcell Regime[END_REF][START_REF] Jeantet | Interplay of spectral diusion and phonon-broadening in individual photo-emitters: the case of carbon nanotubes[END_REF] Our approach tends to benet from these advantages in an integrated system much simpler to handle experimentally. After the synthesis, the core-shell structures are dispersed in THF and then spin-coated on the at surface of a solid immersion lens (SIL) to enhance the collection of photons in photoluminescence experiments (see gure 1(a)).

Likewise, PFO wrapped nanotubes in toluene are mixed with a polystyrene solution and then spin-coated on the at surface of a SIL to form the bulk reference sample named CNT@Matrix.

Sample characterization. In order to perform structural characterizations, part of a solution of core-shell structures is deposited on a silicon substrate. Figure S1 displays images of dierent core-shell structures taken with a Hitachi S-4500 Scanning Electron Microscope (SEM). It shows tubular structures compatible with laser nanotubes wrapped with a polymer layer. Likewise, atomic force microscopy (AFM) measurements has been performed using either a Veeco Dimension 3100 AFM or a Multimode AFM equipped with Nanoscope IIIa controllers. Figure 1(b) and (c) display typical AFM images of CNT@PFO/PS and CNT@PS/PS structures. The deposition of the core-shell structures leads to well separated objects making a thorough height analysis possible. Note that this is not the case when bare nanotubes are wrapped in usual surfactant. This highlights the advantage of using organic dispersions, which is made possible with the present polymer capping. Figure 1(e) and (f) show histograms of heights for both structures. In order to build these histograms, the height is measured several times along the same object and the median value is reported. In the case of CNT@PS/PS hybrids, the polymer is almost homogenously wrapped around the nanotube. The histogram is peaked and can be tted with a Gaussian distribution centred at ∼3.5 nm for the full height of the objects. The histogram of CNT@PFO/PS hybrids shows a peaked distribution, centred around 6.5 nm, together with counts above 10 nm. These are due to the presence of big clusters of polymer attached to the nanotubes. Therefore, one can deduce an average value of the shell thickness for both structures: ∼1.2 nm for CNT@PS/PS and ∼2.7 nm for CNT@PFO/PS ones when big clusters are excluded.

Optical experiments. SIL are attached to the top of the cold nger of a cryostation from Montana Instruments. All experiments are performed at ∼5 K. The nanotubes are excited at 905 nm, approximately in resonance with their second excitonic transition (S 22 ), through a Mitutoyo objective (NA=0.7). The photoluminescence is collected with the same objective, dispersed in an Acton SP2300i (Princeton Instruments) spectrometer and detected with a nitrogen cooled InGaAs array (OMA V, Princeton Instruments). Figure 1(d) displays typical photoluminescence spectra of single core-shell structures and nanotubes in matrix. The luminescence energy is in the range of 0.8 eV i.e. at telecommunication wavelengths.

Results and Discussion

Figure 2(a), (b) and (c) displays PL spectra as a function of time for CNT@PFO/PS, CNT@PS/PS and CNT@Matrix respectively. As an example, we display in the upper panel the typical spectrum for a nanotube considered as unstable for each of the samples. On the contrary, in the bottom panel we display our observation for a typically stable CNT. If all samples could present blinking and spectral diusion, it seems that this eect is stronger for CNT@PFO/PS (gure 2(a)), and that CNT@PS/PS is qualitativelly more stable (gure 2(b)) and even compare well with the CNT@Matrix sample (gure 2(c)). In order to get quantitative information on the blinking phenomenon we use a Mandel analysis, calculating Mandel parameters:

Q = < n 2 > -< n > 2 < n > -1 (1)
where n is the number of counts per time bins. This parameter describes the deviation of the emission statistics from a Poissonian distribution. When Q∼0, the emission is shot noise limited. This parameter has been used for instance to characterize the emission stability of CdSe nanocrystals. [START_REF] Margolin | Photon Counting Statistics for Blinking CdSe-ZnS Quantum Dots: A Lévy Walk Process[END_REF] When no particular care is taken to prevent blinking, these nanocrystals can show Mandel parameters Q up to 10 5 in recording conditions similar to the present study. [START_REF] Margolin | Photon Counting Statistics for Blinking CdSe-ZnS Quantum Dots: A Lévy Walk Process[END_REF] The CNT@PFO/PS structures show a large dispersion of Mandel parameters ranging from few unities to few hundreds. The mean value of < Q P F O/P S > parameter is ∼115.

On the contrary, distribution of Mandel parameter of CNT@PS/PS structures is much less dispersed, with a mean value < Q P S/P S >∼ 9.

Therefore, the CNT@PS/PS structure is, in average, one order of magnitude more stable in terms of blinking than the PS/PFO structures. The complete statistics of Mandel parameters is reported on gure S4. For comparison, the luminescence of micelle wrapped CoMoCat nanotubes deposited on polylysine, reported few years ago, shows Q factors of the order of few hundreds (see gure S2). This shows that the PS/PS shells indeed protect, at least partially, the nanotube from environmental uctuations.

In the following, we investigate the emission stability in terms of spectral diusion. The calculated standard deviation of the central energy of the emission line, calculated over 200 s with an integration time of 1 s, of CNT@PFO/PS structures ranges from ∼90 µeV to ∼250 µeV. For CNT@PS/PS structures it ranges from ∼60 µeV to ∼200 µeV. A gure of merit for the emission stability is the ratio of the amplitude of the frequency shift versus the emitter linewidth. Here, for the best core-shell structures this gure of merit is about 1/10 of their linewidth which signies a good luminescence stability. The results of spectral stability can be compared with ones recorded for micelle wrapped CoMoCat nanotubes (see SI). In this case the standard deviation of the central energy of the emission line can be of the order of 1.7 to 2 meV, therefore, one order of magnitude larger than for both core-shell structures. This again conrms that the shell structure pro-tects the carbon nanotube. Finally, we can compare the results obtained on the core-shell structures to the ones recorded on nanotubes embedded in the polystyrene matrix. As mentioned in the introduction, this conguration has been studied before and used in CQED experiments thanks to the emission stability of nanotubes. [START_REF] Jeantet | Widely Tunable Single-Photon Source from a Carbon Nanotube in the Purcell Regime[END_REF] Therefore, this represents a relevant control sample. In optimal fabrication conditions (see SI), the nanotubes emission is very stable both for blinking and spectral diffusion (see gure 2(c)). Indeed, the mean value of the Mandel parameter measured on this sample is < Q matrix >∼ 14 which is quite comparable with what we obtained on the CNT@PS/PS core-shell structure. This comparison demonstrates that the core-shell approach is as ecient as the inclusion of nanotubes in a matrix to prevent blinking, while keeping the possibility of handling and processing the objects. In addition, the nanotubes embedded in the PS matrix show very limited spectral diusion. Indeed, the calculated standard deviation is typically of the order of few tenth of µeV for an emission linewidth which is of the order of few hundreds of µeV. Here the mean value of the standard deviation is ∼ 30 µeV. The comparison shows that the best values of the core-shell structures reach the one of the nanotubes in the matrix. This demonstrates again the suitability of the core-shell approach.

In order to go further in the quantitative study of the spectral diusion, we performed a Allan deviation analysis. This method is commonly used to investigate the frequency stability of oscillators over time [START_REF] Kozlova | Caractérisation d'une horloge à piégeage cohérent de population dans une vapeur thermique de césium. Principaux eets pouvant aecter la stabilité de fréquence á moyen-long terme[END_REF] and has been recently applied to carbon nanotubes. [START_REF] Noé | Environmental Electrometry with Luminescent Carbon Nanotubes[END_REF] One of the advantages of this analysis method is to infer the type of noise. In fact, the slope of the Allan deviation depends on the origin of the noise. For instance, a frequency drift leads to a τ +1 slope whereas a icker noise leads to a τ 0 slope. The Allan deviation is dened as:

σ 2 e (τ ) = 1 2 < (e k+1 -e k ) 2 >= 1 2 lim N →∞ 1 N N k=1 (e k+1 -e k ) 2 (2)
where e k is the mean value of the energy e(t) of the line on the time interval τ . Figure 3 displays the Allan deviation as a function of the interval τ for six CNT@PFO/PS (green curves) and six CNT@PS/PS (blue curves) structures. The red stars in gure 3 are the average values of the Allan deviations of nanotubes in matrix. The whole statistics of CNT@Matrix is available in SI (gure S5). Except for one CNT@PS/PS, all the core-shell structures have their Allan deviation between few 10 -5 few 10 -4 eV, slightly above the average value of CNT in matrix. For comparison purpose, Figure S6 shows several time traces and their Allan deviation curves for dierent degree of stability. Moreover, the variation of the Allan deviation with time is relatively at. Therefore, we can assume that the Allan deviation in both CNT@PFO/PS and CNT@PS/PS shows a close to τ 0 variation. In this framework, this would mean that spectral diusion of core-shell structures is a icker noise. In electronics this type of noise can be observed, for instance, when charges are randomly emitted by impurities. If one assumes that charges in the environment of the nanotubes are trapped and can jump randomly from one trap to another, the random variation of the charge distribution to nanotube distance would account for this type of noise of the luminescence frequency due to Stark eect. Finally, the Allan deviation analysis allows to compare the performances of our core-shell structures to other congurations reported in the literature. In fact, Noé et al recently reported the same analysis for bare nanotubes on a SiO 2 surface and on a h-BN substrate (see guides for the eye in gure 3). [START_REF] Noé | Environmental Electrometry with Luminescent Carbon Nanotubes[END_REF] First, the Allan deviations measured for our core-shell structures are one or two orders of magnitude lower than the one reported for a nanotube on a SiO 2 surface that reached ∼ 10 -3 in average. This comparison is in agreement with what we measured for Co-MoCat nanotubes on polylysine (see SI). Moreover it strongly supports the fact that the double shell structure is indeed protecting, at least partially, the nanotube from the charge uctuations of the substrate. Moreover, the Allan deviations of the core-shell structures displayed on gure 3 are of the same order of magnitude (few 10 -5 to few 10 -4 ) as the one reported for a nanotube on the h-BN substrate (few 10 -5 ), which is considered as a material of choice to stabilize quantum emitters luminescence. This comparison shows that the core-shell structures can reach the best performance reported in the literature, while oering the possibility to be handle and to process the single emitters on any substrate of interest.

Conclusion

In conclusion, we report on a quantitative study of blinking and spectral diusion processes of core-double shell structures based on single carbon nanotubes emitting at the telecommunication wavelength. The double shell structure is shown to protect the nanotube from the uctuations of its local environment leading to a strong reduction of both blinking and spectral diusion. The quantitative comparison with litera-ture highlights that these structures can reach the performance of single nanotubes laid on h-BN while being easy to handle and to process on any substrate of interest. These results show that the core-shell approach is relevant in the context of the use of single-walled carbon nanotubes in applications needing quantum emitters at telecommunication wavelengths.

Supporting

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Figure 1 :

 1 Figure 1: (a) Description of the samples deposited on the surface of a Solid Immersion Lens (SIL). (b)(c) Typical AFM images of CNT@PFO/PS and CNT@PS/PS core-shell structures respectively. (d) Typical PL spectra of single core-shell structures excited at 1.37 eV (905 nm).Acquisition time:30s, excitation power: ∼150µW. (e)(f) Histograms of heights of CNT@PFO/PS and CNT@PS/PS core-shell structures deduced from AFM measurements.

Figure 2 :

 2 Figure 2: Temporal evolution of the spectra of single nanotubes recorded at 5 K with a 1 s integration time. (a) CNT@PFO/PS core-shell structures. (b) CNT@PS/PS core-shell structures. (c) CNT@Matrix. The top traces correspond to an example of typically unstable nanotube for each of the samples. The bottom traces correspond to typically stable ones.

A l l a n d e v i a t i oFigure 3 :

 3 Figure3: Allan deviation of CNT@PS/PS (blue), CNT@PFO/PS (green) and mean value of CNT@Matrix (red). All the Allan curves for CNT@Matrix are depicted on gure S5. The dotted black lines are guide for the eye showing values of Allan deviation for CoMoCat CNT on SiO 2 and h-BN extracted from ref.[START_REF] Noé | Environmental Electrometry with Luminescent Carbon Nanotubes[END_REF]

Table 1 :

 1 Summary of average values of Mandel parameters for the dierent type of samplesCNT@PFO/PS CNT@PS/PS CoMoCat@SiO 2 CNT@Matrix

	Q	115	9	few hundreds	14

Acknowledgement The authors are grateful to A. Högele for sharing raw data of single carbon nanotubes on SiO 2 and h-BN. J.S. Lauret is member of "Institut Universitaire de France".