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Piezo proteins are mechanically activated ion channels, which are required for mechanosensing functions in a variety of cell types. While we and others have previously demonstrated that the expression of Piezo1 in osteoblast lineage cells is essential for bone-anabolic processes, there was only suggestive evidence indicating a role of Piezo1 and/or Piezo2 in cartilage. Here we addressed the question if and how chondrocyte expression of the mechanosensitive proteins Piezo1 or Piezo2 controls physiological endochondral ossification and pathological osteoarthritis (OA) development. Mice with chondrocyte-specific inactivation of Piezo1 (Piezo1Col2a1Cre), but not of Piezo2, developed a near absence of trabecular bone below the chondrogenic growth plate postnatally. Moreover, all Piezo1Col2a1Cre animals displayed multiple fractures of rib bones at 7 days of age, which were located close to the growth plates. While skeletal growth was only mildly affected in these mice, OA pathologies were markedly less pronounced compared to littermate controls at 60 weeks of age. Likewise, when OA was induced by anterior cruciate ligament transection, only the chondrocyte inactivation of Piezo1, not of Piezo2, resulted in attenuated articular cartilage degeneration. Importantly, osteophyte formation and maturation were also reduced in Piezo1Col2a1Cre mice. We further observed increased Piezo1 protein abundance in cartilaginous zones of human osteophytes. Finally, we identified Ptgs2 and Ccn2 as potentially relevant Piezo1 downstream genes in chondrocytes. Collectively, our data do not only demonstrate that Piezo1 is a critical regulator of physiological and pathological endochondral ossification processes, but also suggest that Piezo1 antagonists may be established as a novel approach to limit osteophyte formation in OA. [ABSTRACT FROM AUTHOR]

Mental traumatization is associated with long-bone growth retardation, osteoporosis and increased fracture risk. We revealed earlier that mental trauma disturbs cartilage-to-bone transition during bone growth and repair in mice. Trauma increased tyrosine hydroxylase-expressing neutrophils in bone marrow and fracture callus. Here we show that tyrosine hydroxylase expression in the fracture hematoma of patients correlates positively with acknowledged stress, depression, and pain scores as well as individual ratings of healing-impairment and pain-perception post-fracture. Moreover, mice lacking tyrosine hydroxylase in myeloid cells are protected from chronic psychosocial stress-induced disturbance of bone growth and healing. Chondrocyte-specific β2-adrenoceptor-deficient mice are also protected from stress-induced bone growth retardation. In summary, our preclinical data identify locally secreted catecholamines in concert with β2-adrenoceptor signalling in chondrocytes as mediators of negative stress effects on bone growth and repair. Given our clinical data, these mechanistic insights seem to be of strong translational relevance. Authors present both preclinical data in mice and clinical data from humans in support of the hypothesis that stress negatively affects bone growth and repair. These effects are mediated by neutrophil-derived catecholamines inhibiting cartilage-to-bone transition via β2-adrenoceptor signaling in chondrocytes. [ABSTRACT FROM AUTHOR]

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Vitamin D measurements in biological fluids by mass spectrometry are challenging at very low concentration levels. As a result, chemical derivatization is often employed to enhance the ionization properties of low abundant vitamin D compounds. Cookson-type reagents such as 4-phenyl-1,2,4-triazoline-3,5-dione (PTAD) or similar derivatives work well but require careful, water-free experimental conditions, as traces of water inactivate the reagent and inhibit or stop the derivatization reactions, thus making quantitative measurements in aqueous samples impossible. We describe a novel electrospray liquid chromatography-tandem mass spectrometry (LC-MS/MS) assay for determining 25-hydroxyvitamin D (25(OH)D) directly in aqueous cellular systems using a new derivatization reagent, the ionic liquid 12-(maleimidyl)dodecyl-tri- n-butylphosphonium bromide (MDBP). The proof-of-concept for the MDBP assay was demonstrated by measuring the levels of 25(OH)D in four different human cell types, namely T cells, helper T cells, B cells, and macrophages. In addition to the ability to determine the levels of 25(OH)D directly in aqueous samples, the cellular integrity was maintained in our application. We show the time-dependent uptake of 25(OH)D into the investigated cells to demonstrate the applicability of the new label. Furthermore, the MDBP derivatization technique may be equally useful in imaging mass spectrometry, where it could be used for response enhancements of spatially localized vitamin D metabolites on wet tissue surfaces, without destroying the integrity of the tissue surface. [ABSTRACT FROM AUTHOR]

In this study, 98 families with 101 mutations were analyzed in depth in which a mutation had been observed at one of the four loci D3S1358, FGA, ACTBP2, and VWA. To determine the origin (male/female) of the mutation, five to seven polymorphic flanking markers were selected for each locus concerned and used to construct family-specific haplotypes. Additionally, all alleles of the STR system concerned were sequenced. With this duplicate approach, it was possible to identify the mutated structure and/or mutation event in the vast majority of cases. The ratio of one-step to two-step mutations was 100:1. The ratio of paternal to maternal mutations was 76:8. The ratio of gains to losses was 47:50. Also, the mutation rates in two systems, ACTBP2 and VWA, were clearly higher than those given in the literature. [ABSTRACT FROM AUTHOR]