The muscle-specific kinase MuSK is one of the key molecules orchestrating neuromuscular junction (NMJ) formation. MuSK interacts with the Wnt morphogens, through its Frizzled-like domain (cysteine-rich domain [CRD]). Dysfunction of MuSK CRD in patients hasbeen recently associated with the onset of myasthenia, common neuromuscular disorders mainly characterized by fatigable muscle weakness. However, the physiological role of Wnt-MuSK interaction in NMJ formation and function remains to be elucidated. Here, we demonstrate that the CRD deletion of MuSK in mice caused profound defects of both muscle prepatterning, the first step of NMJ formation, and synapse differentiation associated with a drastic deficit in AChR clusters and excessive growth of motor axons that bypass AChR clusters. Moreover, adult MuSK⌬CRD mice developed signs of congenital myasthenia, including severe NMJs dismantlement, muscle weakness, and fatigability. We also report, for the first time, the beneficial effects of lithium chloride, a reversible inhibitor of the glycogen synthase kinase-3, that rescued NMJ defects in MuSK⌬CRD mice and therefore constitutes a novel therapeutic reagent for the treatment of neuromuscular disorders linked to Wnt-MuSK signaling pathway deficiency. Together, our data reveal that MuSK CRD is critical for NMJ formation and plays an unsuspected role in NMJ maintenance in adulthood.

Digit length ratios, especially the second-to-fourth digit ratio (2D : 4D), are associated with various pathological and behavioural conditions in many species including humans and are dependent upon prenatal androgen to oestrogen balance. It is unknown whether digit ratios are modified by environmental exposure to ubiquitous endocrine disruptors. We studied the effect on adult male Wistar rat digit ratios of a gestational exposure to the oestrogenic and antiandrogenic compounds bisphenol A (BPA), genistein and vinclozolin, in low doses, and in combination with investigating in parallel a possible sexual dimorphism of this trait. We also investigated the effects on the male progeny not exposed during gestation. X-rays were taken of the left and right forepaws, and 2D-5D proximal to distal phalanx distances were measured by a standardized procedure based on semi-automatic image analysis. We provide evidence that there is a sexual dimorphism of digit ratios in the Wistar rat, and we found that BPA alone or in combination with genistein and vinclozolin significantly feminized digit ratios in male rats. Intriguingly, significant feminization of digit ratios was also found in the unexposed male progeny of males that had been exposed to compound mixtures. In conclusion, prenatal environmental levels of endocrine-active substances permanently disrupt digit ratios. Digit ratio measurement in adults is thus a promising biomarker of prenatal exposure to low-dose endocrine disruptors in rodents, with potential implications for future studies in humans.

Mutations in PHEX (phosphate-regulating gene with homologies to endopeptidases on the X-chromosome) cause X-linked familial hypophosphatemic rickets (XLH), a disorder having severe bone and tooth dentin mineralization defects. The absence of functional PHEX leads to abnormal accumulation of ASARM (acidic serine- and aspartate-rich motif) peptide − a substrate for PHEX and a strong inhibitor of mineralization − derived from MEPE (matrix extracellular phosphoglycoprotein) and other matrix proteins. MEPE-derived ASARM peptide accumulates in tooth dentin of XLH patients where it may impair dentinogenesis. Here, we investigated the effects of ASARM peptides in vitro and in vivo on odontoblast differentiation and matrix mineralization. Dental pulp stem cells from human exfoliated deciduous teeth (SHEDs) were seeded into a 3D collagen scaffold, and induced towards odontogenic differentiation. Cultures were treated with synthetic ASARM peptides (phosphorylated and nonphosphorylated) derived from the human MEPE sequence. Phosphorylated ASARM peptide inhibited SHED differentiation in vitro, with no mineralized nodule formation, decreased odontoblast marker expression, and upregulated MEPE expression. Phosphorylated ASARM peptide implanted in a rat molar pulp injury model impaired reparative dentin formation and mineralization, with increased MEPE immunohistochemical staining. In conclusion, using complementary models to study tooth dentin defects observed in XLH, we demonstrate that the MEPE-derived ASARM peptide inhibits both odontogenic differentiation and matrix mineralization, while increasing MEPE expression. These results contribute to a partial mechanistic explanation of XLH pathogenesis: direct inhibition of mineralization by ASARM peptide leads to the mineralization defects in XLH teeth. This process appears to be positively reinforced by the increased MEPE expression induced by ASARM. The MEPE-ASARM system can therefore be considered as a potential therapeutic target.

Claudin-16 protein (CLDN16) is a component of tight junctions (TJ) with a restrictive distribution so far demonstrated mainly in the kidney. Here, we demonstrate the expression of CLDN16 also in the tooth germ and show that claudin-16 gene (CLDN16) mutations result in amelogenesis imperfecta (AI) in the 5 studied patients with familial hypomagnesemia with hypercalciuria and nephrocalcinosis (FHHNC). To investigate the role of CLDN16 in tooth formation, we studied a murine model of FHHNC and showed that CLDN16 deficiency led to altered secretory ameloblast TJ structure, lowering of extracellular pH in the forming enamel matrix, and abnormal enamel matrix protein processing, resulting in an enamel phenotype closely resembling human AI. This study unravels an association of FHHNC owing to CLDN16 mutations with AI, which is directly related to the loss of function of CLDN16 during amelogenesis. Overall, this study indicates for the first time the importance of a TJ protein in tooth formation and underlines the need to establish a specific dental follow-up for these patients.

International audienceWhile calcium oxalate monohydrate (whewellite: CaC₂ O ₄ H ₂ O) kidney stones are related to hyperoxaluria, calcium oxalate dihydrate (weddellite: CaC ₂ O ₄(2 + x)H ₂ O) ones are mainly associated with hypercalciuria. Crystalline conversion from weddellite to whe-wellite introduces a contradiction between Fourier transform infrared (FTIR) spectra which indicate the presence of whewellite and the fact that bipyramid crystallites, a morphology specific of weddellite, can be observed. It constitutes thus a major problem for clinicians as hyperoxaluria and hypercalciuria are associated with very different aetiologies and treatments. In this contribution, the complete set of data including neutron diffraction experiments, observations through a last generation field emission scanning electron microscope as well as 3D and 2D cross-section visualisations derived from the mComputed Tomography measurements seems to explain some particular features observed on FTIR spectra related to the crystalline conversion from weddellite to whewellite. The structural hypothesis which is proposed is related to the formation of amorphous whewellite, a new polymorph recently synthesised. Thus, in such cases, special attention should be paid to the stone morphology (and not FTIR spectrum), which is a major element for clinical diagnosis as already underlined for other types of urinary calculi

Although ultrasonography is a non-invasive, inexpensive and painless diagnostic tool for soft tissue imaging, this technique is not currently used for oral exploration. Therefore, we developed a 25-MHz high-frequency ultrasound probe, specially designed for intraoral applications. This paper aims to present clinical intraoral ultrasound images actually interpretable, in order to identify the relevant applications of this novel tool and to design future oral studies. Two independent radiologists performed ultrasound examinations on three healthy volunteers. All the teeth were explored on the lingual and buccal sides (162 samples) to evaluate the ergonomics of the system and the visualisation of anatomic structures. Osseointegrated dental implants and a mucocele were also scanned. At the gingivodental junction of the maxillary and mandibular teeth, the device clearly identifies the tooth surfaces, the alveolar bone reflection with its surrounding subepithelial connective tissue of the gingiva and the gingival epithelia. The bone level and the thickness of soft tissue around the implant are measurable on the buccal and lingual sides. Therefore, intraoral ultrasonography provides additional morphological information that is not accessible by conventional dental x-rays. We propose a novel diagnostic tool that explores the biological width and is able to define the thin or thick nature of the gums. Moreover, intraoral ultrasonography may help to monitor precancerous lesions. This promising device requires large-scale clinical studies to determine whether it should remain a research tool or be used as a diagnostic tool for daily dental practice.

Since Matrix metalloproteinases (MMPs) have been suggested to contribute to dentin caries progression, the hypothesis that MMP inhibition would affect the progression of dentin caries is clinically relevant. Grape seed extracts (GSE) have been previously reported to be natural inhibitors of MMPs.Objective: To evaluate the capacity of a GSE mouthrinse to prevent the degradation of demineralized dentin matrix by MMP-3 (stromelysin-1).Materials and Methods: Standardized blocks of dentin obtained from sound permanent teeth extracted for orthodontic reasons were demineralized with Ethylenediaminetetraacetic acid (EDTA) and pretreated either with (A) GSE (0.2% w/v), (B) amine fluoride (AmF) (20% w/v), (C) a mouthrinse which contains both, (D) placebo, (E) sodium fluoride (0.15 mg.ml−1), (F) PBS, (G) Chlorhexidine digluconate (CHX), or (H) zinc chloride (ZnCl2). The dentin blocks were then incubated with activated recombinant MMP-3. The supernatants were analyzed by Western Blot for several dentin matrix proteins known to be MMP-3 substrate. In parallel, scanning electron microscopy (SEM) was performed on resin replica of the dentin blocks.Results: Western blot analysis of the supernatants revealed that MMP-3 released from the dentin matrix small proteoglycans (decorin and biglycan) and dentin sialoprotein (DSP) in the AmF, sodium fluoride, PBS and placebo pretreated groups, but not in the GSE and mouthrinse pretreated groups. SEM examination of resin replica showed that the mouthrinse and its active components not only had an anti-MMP action but also modified the dentin surface accessibility.Conclusion: This study shows that GSE either alone or combined with AmF as in the evaluated mouthrinse limits dentin matrix degradation. This association may be promising to prevent the progression of caries within dentin. However, the procedure should be adapted to clinically relevant durations.

Over the last decade, biomedical 3D-imaging tools have gained widespread use in the analysis of prehistoric bone artefacts. While initial attempts to characterise the major categories used in osseous industry (i.e. bone, antler, and dentine/ivory) have been successful, the taxonomic determination of prehistoric artefacts remains to be investigated. The distinction between reindeer and red deer antler can be challenging, particularly in cases of anthropic and/or taphonomic modifications. In addition to the range of destructive physicochemical identification methods available (mass spectrometry, isotopic ratio, and DNA analysis), X-ray micro-tomography (micro-CT) provides convincing non-destructive 3D images and analyses. This paper presents the experimental protocol (sample scans, image processing, and statistical analysis) we have developed in order to identify modern and archaeological antler collections (from Isturitz, France). This original method is based on bone microstructure analysis combined with advanced statistical support vector machine (SVM) classifiers. A combination of six microarchitecture biomarkers (bone volume fraction, trabecular number, trabecular separation, trabecular thickness, trabecular bone pattern factor, and structure model index) were screened using micro-CT in order to characterise internal alveolar structure. Overall, reindeer alveoli presented a tighter mesh than red deer alveoli, and statistical analysis allowed us to distinguish archaeological antler by species with an accuracy of 96%, regardless of anatomical location on the antler. In conclusion, micro-CT combined with SVM classifiers proves to be a promising additional non-destructive method for antler identification, suitable for archaeological artefacts whose degree of human modification and cultural heritage or scientific value has previously made it impossible (tools, ornaments, etc.).