Fluorescent sensor based on solid-phase extraction with negligible depletion: A proof-of-concept study with amines as analytes.

This paper describes the development and proof-of-concept testing of an easy-to-use trace analysis technique, namely F-SPE, by coupling fluorescent sensor with solid phase extraction (SPE). F-SPE is a two-step methodology that concentrates an analyte from a liquid sample onto a fluorophore-modified membrane and measures the amount of analyte from the extent the extracted analyte quenches the emission of the fluorophore. By applying the principle of negligible depletion (ND) intrinsic to SPE, the procedure of F-SPE for analyzing a sample can be markedly simplified while maintaining the ability to detect analytes at low limits of detection (LOD). The merits of this approach are demonstrated by impregnating a SPE membrane with a perylene diimide (PDI) fluorophore, N,N'-di(nonyldecyl)-perylene-3,4,9,10-tetracarboxylic diimide (C9/9-PDI), for the low-level detection of organic amines (e.g., aniline) and amine-containing drugs (e.g., Kanamycin). The sensing mechanism is based on the donor-acceptor quenching of PDI by amines, which, when coupled with the concentrative nature of SPE, yields LODs for aniline and Kanamycin of 67 nM (∼6 ppb) and 32 nM (∼16 ppb), respectively.

Role of YAP in early ectodermal specification and a Huntington's Disease model of human neurulation.

The Hippo pathway, a highly conserved signaling cascade that functions as an integrator of molecular signals and biophysical states, ultimately impinges upon the transcription coactivator Yes-associated protein 1 (YAP). Hippo-YAP signaling has been shown to play key roles both at the early embryonic stages of implantation and gastrulation, and later during neurogenesis. To explore YAP's potential role in neurulation, we used self-organizing neuruloids grown from human embryonic stem cells on micropatterned substrates. We identified YAP activation as a key lineage determinant, first between neuronal ectoderm and nonneuronal ectoderm, and later between epidermis and neural crest, indicating that YAP activity can enhance the effect of BMP4 stimulation and therefore affect ectodermal specification at this developmental stage. Because aberrant Hippo-YAP signaling has been implicated in the pathology of Huntington's Disease (HD), we used isogenic mutant neuruloids to explore the relationship between signaling and the disease. We found that HD neuruloids demonstrate ectopic activation of gene targets of YAP and that pharmacological reduction of YAP's transcriptional activity can partially rescue the HD phenotype.

A poly(3,4-ethylenedioxythiophene):poly(styrenesulfonate)-based electrochemical sensor for tert.-butylhydroquinone.

Poly(3,4-ethylenedioxythiophene):poly(styrenesulfonate) (PEDOT:PSS) is a readily available copolymer that comes as an aqueous dispersion with good processability. A flexible voltammetric sensor for the widely used food stabilizer tert.-butylhydroquinone (TBHQ) was constructed by using a film of PEDOT:PSS. The electron transfer efficiency of the electrode was enhanced by doping with dimethyl sulfoxide (DMSO), and mass transport at the electrode-electrolyte interface was increased by adding the cationic surfactant cetyltrimethylammonium bromide (CTAB) which acts as a sorbent for TBHQ. SEM, AFM, XPS, UV - vis and electrochemical analysis were conducted to characterize the properties of the electrode. After optimization of the experimental conditions, the electrode operated at a working potential of 0.17 V (vs. SCE) has a linear response in the 0.5-200 µM TBHQ concentration range and a lower detection limit of 0.15 µM (at S/N = 3). It was applied for the determination of TBHQ in spiked real samples, and recoveries ranged between 96.85 and 103.41%. Graphical abstractSchematic representation of an electrochemical flexible electrode for the determination of tert.-butylhydroquinone based on the use of poly(3,4-ethylenedioxythiophene):poly(styrenesulfonate).

Aerogels prepared from polymeric ß-cyclodextrin and graphene aerogels as a novel host-guest system for immobilization of antibodies: a voltammetric immunosensor for the tumor marker CA 15-3.

A three-dimensional porous network graphene aerogel (GAs) with large specific area and excellent conductivity was loaded with ß-cyclodextrin polymer (Pß-CD) to serve as a support for immobilization of antibodies. A highly sensitive immunosensor for the cancer marker carbohydrate antigen 15-3 (CA15-3) was designed based on the use of Pß-CD/GAs. The large specific area of GAs warrants high loading with antibodies, and their excellent electrical conductivity warrants strong electrical signals. Based on the synergistic effect of GAs and Pß-CD, an immunoassay was designed that is making use of hexacyanoferrate as an electrochemical probe and having a pleasantly low working potential of 0.2 V (vs. SCE). Response is linear in the 0.1 mU mL-1 to 100 U mL-1 activity range, and the lower detection limit is 0.03 mU mL-1 (at S/N = 3). The immunoassay is stable, selective and reproducible. It was applied to the analysis of spiked samples, and results were satisfactory. Graphical abstract Schematic of an electrochemical immunoassay for the carbohydrate antigen 15-3. It is based on the use of ß-cyclodextrin polymer and a graphene aerogel.

p204 Is Required for Canonical Lipopolysaccharide-induced TLR4 Signaling in Mice.

p204, a murine member of an interferon-inducible p200 family, was reported to recognize intracellular viral and bacterial DNAs, however, its role in the innate immunity in vivo remains unknown due to the lack of p204-deficient animal models. In this study we first generated the p204-/- mice. Unexpectedly, p204 deficiency led to significant defect in extracellular LPS signaling in macrophages, as demonstrated by dramatic reductions of LPS-mediated IFN-ß and pro-inflammatory cytokines. The serum levels of IFN-ß and pro-inflammatory cytokines were also significantly reduced in p204-/- mice following LPS challenge. In addition, p204-/- mice were resistant to LPS-induced shock. LPS-activated NF-ĸB and IRF-3 pathways were all defective in p204-deficient macrophages. p204 binds to TLR4 through its Pyrin domain, and it is required for the dimerization of TLR4 following LPS-challenge. Collectively, p204 is a critical component of canonical LPS-TLR4 signaling pathway, and these studies also suggest that p204 could be a potential target to prevent and treat inflammatory and infectious diseases.

Serum progranulin levels in Hispanic rheumatoid arthritis patients treated with TNF antagonists: a prospective, observational study.

Since progranulin (PGRN) is a natural ligand of TNF receptors, we assessed whether serum PGRN levels predict and/or reflect responsiveness of RA patients to TNF-antagonist therapy. TNF-antagonist-naïve RA patients (N = 35) were started on TNF-antagonist therapy. At baseline and at follow-up visits, DAS28-ESR, DAS28-CRP, and CDAI were calculated, and venous blood was collected for serum PGRN determination. Disease activity and clinical response were based on EULAR criteria. Baseline serum PGRN levels varied considerably and correlated with ESR and CRP. DAS28-ESR, DAS28-CRP, and CDAI were greater in "PGRN-high" than in "PGRN-low". Baseline serum PGRN levels did not predict clinical responsiveness to TNF-antagonist therapy. Nevertheless, changes in serum PGRN levels at 274+ days following initiation of TNF-antagonist therapy correlated with changes in ESR, CRP, DAS28-ESR, DAS28-CRP, and CDAI. At this time, DAS28-ESR, DAS28-CRP, and CDAI in PGRN-high and PGRN-low equalized, but serum PGRN levels remained greater in PGRN-high than in PGRN-low. To our knowledge, the present report is the first prospective study to longitudinally assess changes in serum PGRN levels following initiation of TNF-antagonist therapy. Although pre-treatment serum PGRN levels may not predict clinical responsiveness to TNF-antagonist therapy, changes in serum PGRN levels correlate with changes in disease metrics over time. By inference, administration of PGRN may represent an effective therapeutic option for development in RA patients.

Progranulin Recruits HSP70 to ß-Glucocerebrosidase and Is Therapeutic Against Gaucher Disease.

Gaucher disease (GD), the most common lysosomal storage disease, is caused by mutations in GBA1 encoding of ß-glucocerebrosidase (GCase). Recently it was reported that progranulin (PGRN) insufficiency and deficiency associated with GD in human and mice, respectively. However the underlying mechanisms remain unknown. Here we report that PGRN binds directly to GCase and its deficiency results in aggregation of GCase and its receptor LIMP2. Mass spectrometry approaches identified HSP70 as a GCase/LIMP2 complex-associated protein upon stress, with PGRN as an indispensable adaptor. Additionally, 98 amino acids of C-terminal PGRN, referred to as Pcgin, are required and sufficient for the binding to GCase and HSP70. Pcgin effectively ameliorates the disease phenotype in GD patient fibroblasts and animal models. These findings not only demonstrate that PGRN is a co-chaperone of HSP70 and plays an important role in GCase lysosomal localization, but may also provide new therapeutic interventions for lysosomal storage diseases, in particular GD.

Association Between Progranulin and Gaucher Disease.

BACKGROUND: Gaucher disease (GD) is a genetic disease caused by mutations in the GBA1 gene which result in reduced enzymatic activity of ß-glucocerebrosidase (GCase). This study identified the progranulin (PGRN) gene (GRN) as another gene associated with GD. METHODS: Serum levels of PGRN were measured from 115 GD patients and 99 healthy controls, whole GRN gene from 40 GD patients was sequenced, and the genotyping of 4 SNPs identified in GD patients was performed in 161 GD and 142 healthy control samples. Development of GD in PGRN-deficient mice was characterized, and the therapeutic effect of rPGRN on GD analyzed. FINDINGS: Serum PGRN levels were significantly lower in GD patients (96.65±53.45ng/ml) than those in healthy controls of the general population (164.99±43.16ng/ml, p<0.0001) and of Ashkenazi Jews (150.64±33.99ng/ml, p<0.0001). Four GRN gene SNPs, including rs4792937, rs78403836, rs850713, and rs5848, and three point mutations, were identified in a full-length GRN gene sequencing in 40 GD patients. Large scale SNP genotyping in 161 GD and 142 healthy controls was conducted and the four SNP sites have significantly higher frequency in GD patients. In addition, "aged" and challenged adult PGRN null mice develop GD-like phenotypes, including typical Gaucher-like cells in lung, spleen, and bone marrow. Moreover, lysosomes in PGRN KO mice exhibit a tubular-like appearance. PGRN is required for the lysosomal appearance of GCase and its deficiency leads to GCase accumulation in the cytoplasm. More importantly, recombinant PGRN is therapeutic in various animal models of GD and human fibroblasts from GD patients. INTERPRETATION: Our data demonstrates an unknown association between PGRN and GD and identifies PGRN as an essential factor for GCase's lysosomal localization. These findings not only provide new insight into the pathogenesis of GD, but may also have implications for diagnosis and alternative targeted therapies for GD.

Extracellular matrix protein 1, a direct targeting molecule of parathyroid hormone-related peptide, negatively regulates chondrogenesis and endochondral ossification via associating with progranulin growth factor.

Chondrogenesis and endochondral ossification are precisely controlled by cellular interactions with surrounding matrix proteins and growth factors that mediate cellular signaling pathways. Here, we report that extracellular matrix protein 1 (ECM1) is a previously unrecognized regulator of chondrogenesis. ECM1 is induced in the course of chondrogenesis and its expression in chondrocytes strictly depends on parathyroid hormone-related peptide (PTHrP) signaling pathway. Overexpression of ECM1 suppresses, whereas suppression of ECM1 enhances, chondrocyte differentiation and hypertrophy in vitro and ex vivo In addition, target transgene of ECM1 in chondrocytes or osteoblasts in mice leads to striking defects in cartilage development and endochondral bone formation. Of importance, ECM1 seems to be critical for PTHrP action in chondrogenesis, as blockage of ECM1 nearly abolishes PTHrP regulation of chondrocyte hypertrophy, and overexpression of ECM1 rescues disorganized growth plates of PTHrP-null mice. Furthermore, ECM1 and progranulin chondrogenic growth factor constitute an interaction network and act in concert in the regulation of chondrogenesis.-Kong, L., Zhao, Y.-P., Tian, Q.-Y., Feng, J.-Q., Kobayashi, T., Merregaert, J., Liu, C.-J. Extracellular matrix protein 1, a direct targeting molecule of parathyroid hormone-related peptide, negatively regulates chondrogenesis and endochondral ossification via associating with progranulin growth factor.

Progranulin suppresses titanium particle induced inflammatory osteolysis by targeting TNFα signaling.

Aseptic loosening is a major complication of prosthetic joint surgery, characterized by chronic inflammation, pain, and osteolysis surrounding the bone-implant interface. Progranulin (PGRN) is known to have anti-inflammatory action by binding to Tumor Necrosis Factor (TNF) receptors and antagonizing TNFα. Here we report that titanium particles significantly induced PGRN expression in RAW264.7 cells and also in a mouse air-pouch model of inflammation. PGRN-deficiency enhanced, whereas administration of recombinant PGRN effectively inhibited, titanium particle-induced inflammation in an air pouch model. In addition, PGRN also significantly inhibited titanium particle-induced osteoclastogenesis and calvarial osteolysis in vitro, ex vivo and in vivo. Mechanistic studies demonstrated that the inhibition of PGRN on titanium particle induced-inflammation is primarily via neutralizing the titanium particle-activated TNFα/NF-κB signaling pathway and this is evidenced by the suppression of particle-induced IκB phosphorylation, NF-κB p65 nuclear translocation, and activity of the NF-κB-specific reporter gene. Collectively, these findings not only demonstrate that PGRN plays an important role in inhibiting titanium particle-induced inflammation, but also provide a potential therapeutic agent for the prevention of wear debris-induced inflammation and osteolysis.

Progranulin knockout accelerates intervertebral disc degeneration in aging mice.

Intervertebral disc (IVD) degeneration is a common degenerative disease, yet much is unknown about the mechanisms during its pathogenesis. Herein we investigated whether progranulin (PGRN), a chondroprotective growth factor, is associated with IVD degeneration. PGRN was detectable in both human and murine IVD. The levels of PGRN were upregulated in murine IVD tissue during aging process. Loss of PGRN resulted in an early onset of degenerative changes in the IVD tissue and altered expressions of the degeneration-associated molecules in the mouse IVD tissue. Moreover, PGRN knockout mice exhibited accelerated IVD matrix degeneration, abnormal bone formation and exaggerated bone resorption in vertebra with aging. The acceleration of IVD degeneration observed in PGRN null mice was probably due to the enhanced activation of NF-κB signaling and ß-catenin signaling. Taken together, PGRN may play a critical role in homeostasis of IVD, and may serve as a potential molecular target for prevention and treatment of disc degenerative diseases.

Progranulin protects against osteoarthritis through interacting with TNF-α and ß-Catenin signalling.

OBJECTIVE: Progranulin (PGRN) was previously isolated as an osteoarthritis (OA)-associated growth factor. Additionally, PGRN was found to play a therapeutic role in inflammatory arthritis mice models through antagonising tumour necrosis factor α (TNF-α). This study was aimed at investigating the role of PGRN in degradation of cartilage and progression of OA. METHODS: Progression of OA was analysed in both spontaneous and surgically induced OA models in wild type and PGRN-deficient mice. Cartilage degradation and OA were evaluated using Safranin O staining, immunohistochemistry and ELISA. Additionally, mRNA expression of degenerative factors and catabolic markers known to be involved in cartilage degeneration in OA were analysed. Furthermore, the anabolic effects and underlying mechanisms of PGRN were investigated by in vitro experiments with primary chondrocytes. RESULTS: Here, we found that deficiency of PGRN led to spontaneous OA-like phenotype in 'aged' mice. Additionally, PGRN-deficient mice exhibited exaggerated breakdown of cartilage structure and OA progression, while local delivery of recombinant PGRN protein attenuated degradation of cartilage matrix and protected against OA development in surgically induced OA models. Furthermore, PGRN activated extracellular signal-regulated kinases (ERK) 1/2 signalling and elevated the levels of anabolic biomarkers in human chondrocyte, and the protective function of PGRN was mediated mainly through TNF receptor 2. Additionally, PGRN suppressed inflammatory action of TNF-α and inhibited the activation of ß-Catenin signalling in cartilage and chondrocytes. CONCLUSIONS: Collectively, this study provides new insight into the pathogenesis of OA, and also presents PGRN as a potential target for the treatment of joint degenerative diseases, including OA.

PGRN protects against colitis progression in mice in an IL-10 and TNFR2 dependent manner.

This study was aimed to determine the role and regulation of progranulin (PGRN) in the pathogenesis of inflammatory bowel diseases (IBD). Dextran sulfate sodium (DSS)-, picrylsulfonic acid (TNBS)-induced, bone marrow chimera and CD4+CD45Rb(hi) T cell transfer colitis model were established and analyzed in wild-type and several genetically-modified mice, including PGRN, IL-10 and TNFR2 deficient mice. Elevated levels of PGRN were found in colitis samples from human IBD patients and mouse colitis models in comparison to the corresponding controls. PGRN-deficient mice became highly susceptible to DSS- and TNBS-induced colitis, whereas recombinant PGRN ameliorated the pathology and reduced the histological score in both DSS and TNBS colitis models. In addition, hematopoietic-derived PGRN was critical for protection against DSS-induced colitis, and lack of PGRN signaling in CD4+ T cells also exacerbated experimental colitis. PGRN-mediated protective effect in colitis was compromised in the absence of IL-10 signaling. In addition, PGRN's effect was also largely lost in the TNFR2-deficient colitis model. Collectively, these findings not only provide the new insight into PGRN's anti-inflammatory action in vivo, but may also present PGRN and its derivatives as novel biological agent for treating IBD.

Three TNFR-binding domains of PGRN act independently in inhibition of TNF-alpha binding and activity.

PGRN was previously reported to bind to TNF receptors (TNFR) and is therapeutic against inflammatory arthritis. Here we present further evidences demonstrating the PGRN inhibition of TNF-alpha binding and activity, and clarifying the distinct mechanisms underlying TNF-alpha inhibition between PGRN and classic TNF-alpha-binding inhibitors. In addition, we present evidences indicating that three TNFR binding domains of PGRN act independently in binding to TNFR. Furthermore, changing the order of three TNFR-binding domains in Atsttrin, a PGRN-derived molecule composed of these TNFR-binding domains, does not affect its anti-inflammatory and anti-TNF activities in both collagen-induced inflammatory arthritis and human TNF-alpha transgenic mouse model. Taken together, these findings provide the additional molecular basis underlying PGRN/TNFR interaction and PGRN-mediated anti-inflammatory activity in various inflammatory diseases and conditions.

A solid-phase assay for studying direct binding of progranulin to TNFR and progranulin antagonism of TNF/TNFR interactions.

The discovery that TNF receptors (TNFR) serve as the binding receptors for progranulin (PGRN) reveals the significant role of PGRN in inflammatory and autoimmune diseases, including inflammatory arthritis. Herein we describe a simple, antibody-free analytical assay, i.e., a biotin-based solid-phase binding assay, to examine the direct interaction of PGRN/TNFR and the PGRN inhibition of TNF/TNFR interactions. Briefly, a 96-well high-binding microplate is first coated with the first protein (protein A), and after blocking, the coated microplate is incubated with the biotin-labeled second protein (protein B) in the absence or presence of the third protein (protein C). Finally the streptavidin conjugated with a detecting enzyme is added, followed by a signal measurement. Also discussed in this chapter are the advantages of the strategy, key elements to obtain reliable results, and discrepancies among various PGRN proteins in view of the binding activity with TNFR.

FGFR3 induces degradation of BMP type I receptor to regulate skeletal development.

Fibroblast growth factors (FGFs) and their receptors (FGFRs) play significant roles in vertebrate organogenesis and morphogenesis. FGFR3 is a negative regulator of chondrogenesis and multiple mutations with constitutive activity of FGFR3 result in achondroplasia, one of the most common dwarfisms in humans, but the molecular mechanism remains elusive. In this study, we found that chondrocyte-specific deletion of BMP type I receptor a (Bmpr1a) rescued the bone overgrowth phenotype observed in Fgfr3 deficient mice by reducing chondrocyte differentiation. Consistently, using in vitro chondrogenic differentiation assay system, we demonstrated that FGFR3 inhibited BMPR1a-mediated chondrogenic differentiation. Furthermore, we showed that FGFR3 hyper-activation resulted in impaired BMP signaling in chondrocytes of mouse growth plates. We also found that FGFR3 inhibited BMP-2- or constitutively activated BMPR1-induced phosphorylation of Smads through a mechanism independent of its tyrosine kinase activity. We found that FGFR3 facilitates BMPR1a to degradation through Smurf1-mediated ubiquitination pathway. We demonstrated that down-regulation of BMP signaling by BMPR1 inhibitor dorsomorphin led to the retardation of chondrogenic differentiation, which mimics the effect of FGF-2 on chondrocytes and BMP-2 treatment partially rescued the retarded growth of cultured bone rudiments from thanatophoric dysplasia type II mice. Our findings reveal that FGFR3 promotes the degradation of BMPR1a, which plays an important role in the pathogenesis of FGFR3-related skeletal dysplasia.

ADAMTS-7 forms a positive feedback loop with TNF-α in the pathogenesis of osteoarthritis.

OBJECTIVE: To examine the expression of ADAMTS-7 during the progression of osteoarthritis (OA), defining its role in the pathogenesis of OA, and elucidating the molecular events involved. METHODS: ADAMTS-7 expression in cartilage of a rat OA model was assayed using immunohistochemistry. Cartilage-specific ADAMTS-7 transgenic mice and ADAMTS-7 small interfering (si)RNA knockdown mice were generated and used to analyse OA progression in both spontaneous and surgically induced OA models. Cartilage degradation and OA was evaluated using Safranin-O staining, immunohistochemistry, ELISA and western blotting. In addition, mRNA expression of tumour necrosis factor (TNF)-α and metalloproteinases known to be involved in cartilage degeneration in OA was analysed. Furthermore, the transactivation of ADAMTS-7 by TNF-α and its downstream NF-κB signalling was measured using reporter gene assay. RESULTS: ADAMTS-7 expression was elevated during disease progression in the surgically induced rat OA model. Targeted overexpression of ADAMTS-7 in chondrocytes led to chondrodysplasia characterised by short-limbed dwarfism and a delay in endochondral ossification in 'young mice' and a spontaneous OA-like phenotype in 'aged' mice. In addition, overexpression of ADAMTS-7 led to exaggerated breakdown of cartilage and accelerated OA progression, while knockdown of ADAMTS-7 attenuated degradation of cartilage matrix and protected against OA development, in surgically induced OA models. ADAMTS-7 upregulated TNF-α and metalloproteinases associated with OA; in addition, TNF-α induced ADAMTS-7 through NF-κB signalling. CONCLUSIONS: ADAMTS-7 and TNF-α form a positive feedback loop in the regulation of cartilage degradation and OA progression, making them potential molecular targets for prevention and treatment of joint degenerative diseases, including OA.

Progranulin directly binds to the CRD2 and CRD3 of TNFR extracellular domains.

We previously reported that PGRN directly bound to TNF receptors (TNFR) in vitro and in chondrocytes (Tang, et al., Science, 2011). Here we report that PGRN also associated with TNFR in splenocytes, and inhibited the binding of TNFα to immune cells. Proper folding of PGRN is essential for its binding to TNFR, as DTT treatment abolished its binding to TNFR. In contrast, the binding of PGRN to Sortilin was enhanced by DTT. Protein interaction assays with mutants of the TNFR extracellular domain demonstrated that CRD2 and CRD3 of TNFR are important for the interaction with PGRN, similar to the binding to TNFα. Taken together, these findings provide the molecular basis underlying PGRN/TNFR interaction and PGRN-mediated anti-inflammatory activity in various autoimmune diseases and conditions.

The promotion of bone healing by progranulin, a downstream molecule of BMP-2, through interacting with TNF/TNFR signaling.

Endochondral ossification plays a key role in the bone healing process, which requires normal cartilage callus formation. Progranulin (PGRN) growth factor is known to enhance chondrocyte differentiation and endochondral ossification during development, yet whether PGRN also plays a role in bone regeneration remains unknown. In this study we established surgically-induced bone defect and ectopic bone formation models based on genetically-modified mice. Thereafter, the bone healing process of those mice was analyzed through radiological assays including X-ray and micro CT, and morphological analysis including histology and immunohistochemistry. PGRN deficiency delayed bone healing, while recombinant PGRN enhanced bone regeneration. Moreover, PGRN was required for BMP-2 induction of osteoblastogenesis and ectopic bone formation. Furthermore, the role of PGRN in bone repair was mediated, at least in part, through interacting with TNF-α signaling pathway. PGRN-mediated bone formation depends on TNFR2 but not TNFR1, as PGRN promoted bone regeneration in deficiency of TNFR1 but lost such effect in TNFR2 deficient mice. PGRN blocked TNF-α-induced inflammatory osteoclastogenesis and protected BMP-2-mediated ectopic bone formation in TNF-α transgenic mice. Collectively, PGRN acts as a critical mediator of the bone healing process by constituting an interplay network with BMP-2 and TNF-α signaling, and this represents a potential molecular target for treatment of fractures, especially under inflammatory conditions.