BACKGROUND AND PURPOSE: The Registry of Stroke Care Quality (RES-Q) is a worldwide quality improvement data platform that captures performance and quality measures, enabling standardized comparisons of hospital care. The aim of this study was to determine if, and how, RES-Q data are used to influence stroke quality improvement and identify the support and educational needs of clinicians using RES-Q data to improve stroke care. METHODS: A cross-sectional self-administered online survey was administered (October 2021-February 2022). Participants were RES-Q hospital local coordinators responsible for stroke data collection. Descriptive statistics are presented. RESULTS: Surveys were sent to 1463 hospitals in 74 countries; responses were received from 358 hospitals in 55 countries (response rate 25%). RES-Q data were used "always" or "often" to: develop quality improvement initiatives (n = 213, 60%); track stroke care quality over time (n = 207, 58%); improve local practice (n = 191, 53%); and benchmark against evidence-based policies, procedures and/or guidelines to identify practice gaps (n = 179, 50%). Formal training in the use of RES-Q tools and data were the most frequent support needs identified by respondents (n = 165, 46%). Over half "strongly agreed" or "agreed" that to support clinical practice change, education is needed on: (i) using data to identify evidence-practice gaps (n = 259, 72%) and change clinical practice (n = 263, 74%), and (ii) quality improvement science and methods (n = 255, 71%). CONCLUSION: RES-Q data are used for monitoring stroke care performance. However, to facilitate their optimal use, effective quality improvement methods are needed. Educating staff in quality improvement science may develop competency and improve use of data in practice.
The Sprouty family is a highly conserved group of intracellular modulators of receptor tyrosine kinase (RTK)-signaling pathways, which have been recently linked to primary cilia. Disruptions in the structure and function of primary cilia cause inherited disorders called ciliopathies. We aimed to evaluate Sprouty2 and Sprouty4 gene-dependent alterations of ciliary structure and to focus on the determination of its association with Hedgehog signaling defects in chondrocytes. Analysis of the transgenic mice phenotype with Sprouty2 and Sprouty4 deficiency revealed several defects, including improper endochondral bone formation and digit patterning, or craniofacial and dental abnormalities. Moreover, reduced bone thickness and trabecular bone mass, skull deformities, or chondroma-like lesions were revealed. All these pathologies might be attributed to ciliopathies. Elongation of the ciliary axonemes in embryonic and postnatal growth plate chondrocytes was observed in Sprouty2-/- and Sprouty2+/- /Sprouty4-/- mutants compared with corresponding littermate controls. Also, cilia-dependent Hedgehog signaling was upregulated in Sprouty2/4 mutant animals. Ptch1 and Ihh expression were upregulated in the autopodium and the proximal tibia of Sprouty2-/- /Sprouty4-/- mutants. Increased levels of the GLI3 repressor (GLI3R) form were detected in Sprouty2/4 mutant primary fibroblast embryonic cell cultures and tissues. These findings demonstrate that mouse lines deficient in Sprouty proteins manifest phenotypic features resembling ciliopathic phenotypes in multiple aspects and may serve as valuable models to study the association between overactivation of RTK and dysfunction of primary cilia during skeletogenesis. © 2021 American Society for Bone and Mineral Research (ASBMR).
- MeSH
- cilie metabolismus MeSH
- ciliopatie genetika MeSH
- fenotyp MeSH
- membránové proteiny genetika MeSH
- myši transgenní MeSH
- myši MeSH
- protein-serin-threoninkinasy genetika MeSH
- proteiny hedgehog * metabolismus MeSH
- proteiny nervové tkáně genetika MeSH
- signální transdukce * MeSH
- upregulace MeSH
- zvířata MeSH
- Check Tag
- myši MeSH
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
Mutations in genes affecting primary cilia cause ciliopathies, a diverse group of disorders often affecting skeletal development. This includes Jeune syndrome or asphyxiating thoracic dystrophy (ATD), an autosomal recessive skeletal disorder. Unraveling the responsible molecular pathology helps illuminate mechanisms responsible for functional primary cilia. We identified two families with ATD caused by loss-of-function mutations in the gene encoding adrenergic receptor kinase 1 (ADRBK1 or GRK2). GRK2 cells from an affected individual homozygous for the p.R158* mutation resulted in loss of GRK2, and disrupted chondrocyte growth and differentiation in the cartilage growth plate. GRK2 null cells displayed normal cilia morphology, yet loss of GRK2 compromised cilia-based signaling of Hedgehog (Hh) pathway. Canonical Wnt signaling was also impaired, manifested as a failure to respond to Wnt ligand due to impaired phosphorylation of the Wnt co-receptor LRP6. We have identified GRK2 as an essential regulator of skeletogenesis and demonstrate how both Hh and Wnt signaling mechanistically contribute to skeletal ciliopathies.
- MeSH
- Ellisův-van Creveldův syndrom * MeSH
- kinasa 2 receptorů spřažených s G-proteiny genetika MeSH
- lidé MeSH
- mutace MeSH
- proteiny hedgehog * genetika MeSH
- signální dráha Wnt MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
- Research Support, N.I.H., Extramural MeSH
Vertebrate primary cilium is a Hedgehog signaling center but the extent of its involvement in other signaling systems is less well understood. This report delineates a mechanism by which fibroblast growth factor (FGF) controls primary cilia. Employing proteomic approaches to characterize proteins associated with the FGF-receptor, FGFR3, we identified the serine/threonine kinase intestinal cell kinase (ICK) as an FGFR interactor. ICK is involved in ciliogenesis and participates in control of ciliary length. FGF signaling partially abolished ICK's kinase activity, through FGFR-mediated ICK phosphorylation at conserved residue Tyr15, which interfered with optimal ATP binding. Activation of the FGF signaling pathway affected both primary cilia length and function in a manner consistent with cilia effects caused by inhibition of ICK activity. Moreover, knockdown and knockout of ICK rescued the FGF-mediated effect on cilia. We provide conclusive evidence that FGF signaling controls cilia via interaction with ICK.
- MeSH
- buňky NIH 3T3 MeSH
- cilie metabolismus MeSH
- CRISPR-Cas systémy MeSH
- fibroblastové růstové faktory metabolismus MeSH
- fosforylace MeSH
- HEK293 buňky MeSH
- interakční proteinové domény a motivy MeSH
- lidé MeSH
- modely u zvířat MeSH
- myši knockoutované MeSH
- myši MeSH
- protein-serin-threoninkinasy genetika metabolismus MeSH
- proteiny hedgehog metabolismus MeSH
- proteomika MeSH
- receptor fibroblastových růstových faktorů, typ 1 metabolismus MeSH
- receptor fibroblastových růstových faktorů, typ 3 genetika metabolismus MeSH
- receptor fibroblastových růstových faktorů, typ 4 metabolismus MeSH
- receptory fibroblastových růstových faktorů genetika metabolismus MeSH
- signální transdukce MeSH
- simulace molekulového dockingu MeSH
- zvířata MeSH
- Check Tag
- lidé MeSH
- myši MeSH
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
- Research Support, N.I.H., Extramural MeSH
Receptor tyrosine kinases (RTKs) form multiprotein complexes that initiate and propagate intracellular signals and determine the RTK-specific signalling patterns. Unravelling the full complexity of protein interactions within the RTK-associated complexes is essential for understanding of RTK functions, yet it remains an understudied area of cell biology. We describe a comprehensive approach to characterize RTK interactome. A single tag immunoprecipitation and phosphotyrosine protein isolation followed by mass-spectrometry was used to identify proteins interacting with fibroblast growth factor receptor 3 (FGFR3). A total of 32 experiments were carried out in two different cell types and identified 66 proteins out of which only 20 (30.3%) proteins were already known FGFR interactors. Using co-immunoprecipitations, we validated FGFR3 interaction with adapter protein STAM1, transcriptional regulator SHOX2, translation elongation factor eEF1A1, serine/threonine kinases ICK, MAK and CCRK, and inositol phosphatase SHIP2. We show that unappreciated signalling mediators exist for well-studied RTKs, such as FGFR3, and may be identified via proteomic approaches described here. These approaches are easily adaptable to other RTKs, enabling identification of novel signalling mediators for majority of the known human RTKs.
- MeSH
- adaptorové proteiny signální transdukční genetika metabolismus MeSH
- buňky NIH 3T3 MeSH
- cyklin-dependentní kinasy genetika metabolismus MeSH
- elongační faktor 1 genetika metabolismus MeSH
- endozomální třídící komplexy pro transport genetika metabolismus MeSH
- fosfatidylinositol-3,4,5-trisfosfát-5-fosfatasy genetika metabolismus MeSH
- fosfoproteiny genetika metabolismus MeSH
- fosforylace MeSH
- HEK293 buňky MeSH
- homeodoménové proteiny genetika metabolismus MeSH
- lidé MeSH
- mapování interakce mezi proteiny MeSH
- myši MeSH
- protein-serin-threoninkinasy genetika metabolismus MeSH
- proteomika metody MeSH
- receptor fibroblastových růstových faktorů, typ 3 genetika metabolismus MeSH
- regulace genové exprese * MeSH
- signální transdukce genetika MeSH
- stanovení celkové genové exprese MeSH
- vazba proteinů MeSH
- zvířata MeSH
- Check Tag
- lidé MeSH
- myši MeSH
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
Sustained activation of extracellular signal-regulated kinase (ERK) drives pathologies caused by mutations in fibroblast growth factor receptors (FGFRs). We previously identified the inositol phosphatase SHIP2 (also known as INPPL1) as an FGFR-interacting protein and a target of the tyrosine kinase activities of FGFR1, FGFR3, and FGFR4. We report that loss of SHIP2 converted FGF-mediated sustained ERK activation into a transient signal and rescued cell phenotypes triggered by pathologic FGFR-ERK signaling. Mutant forms of SHIP2 lacking phosphoinositide phosphatase activity still associated with FGFRs and did not prevent FGF-induced sustained ERK activation, demonstrating that the adaptor rather than the catalytic activity of SHIP2 was required. SHIP2 recruited Src family kinases to the FGFRs, which promoted FGFR-mediated phosphorylation and assembly of protein complexes that relayed signaling to ERK. SHIP2 interacted with FGFRs, was phosphorylated by active FGFRs, and promoted FGFR-ERK signaling at the level of phosphorylation of the adaptor FRS2 and recruitment of the tyrosine phosphatase PTPN11. Thus, SHIP2 is an essential component of canonical FGF-FGFR signal transduction and a potential therapeutic target in FGFR-related disorders.
- MeSH
- adaptorové proteiny signální transdukční genetika metabolismus MeSH
- aktivace enzymů MeSH
- extracelulárním signálem regulované MAP kinasy genetika metabolismus MeSH
- fosfatidylinositol-3,4,5-trisfosfát-5-fosfatasy genetika metabolismus MeSH
- fosforylace MeSH
- HEK293 buňky MeSH
- lidé MeSH
- MAP kinasový signální systém * MeSH
- membránové proteiny genetika metabolismus MeSH
- nádorové buněčné linie MeSH
- receptory fibroblastových růstových faktorů genetika metabolismus MeSH
- skupina kinas odvozených od src-genu genetika metabolismus MeSH
- tyrosinfosfatasa nereceptorového typu 11 genetika metabolismus MeSH
- vazba proteinů MeSH
- zvířata MeSH
- Check Tag
- lidé MeSH
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
- Research Support, N.I.H., Extramural MeSH
The blocking of specific protein-protein interactions using nanoparticles is an emerging alternative to small molecule-based therapeutic interventions. However, the nanoparticles designed as "artificial proteins" generally require modification of their surface with (bio)organic molecules and/or polymers to ensure their selectivity and specificity of action. Here, we show that nanosized diamond crystals (nanodiamonds, NDs) without any synthetically installed (bio)organic interface enable the specific and efficient targeting of the family of extracellular signalling molecules known as fibroblast growth factors (FGFs). We found that low nanomolar solutions of detonation NDs with positive ζ-potential strongly associate with multiple FGF ligands present at sub-nanomolar concentrations and effectively neutralize the effects of FGF signalling in cells without interfering with other growth factor systems and serum proteins unrelated to FGFs. We identified an evolutionarily conserved FGF recognition motif, ∼17 amino acids long, that contributes to the selectivity of the ND-FGF interaction. In addition, we inserted this motif into a de novo constructed chimeric protein, which significantly improved its interaction with NDs. We demonstrated that the interaction of NDs, as purely inorganic nanoparticles, with proteins can mitigate pathological FGF signalling and promote the restoration of cartilage growth in a mouse limb explant model. Based on our observations, we foresee that NDs may potentially be applied as nanotherapeutics to neutralize disease-related activities of FGFs in vivo.
- MeSH
- aminokyselinové motivy MeSH
- buněčné linie MeSH
- chrupavka fyziologie MeSH
- embryo savčí MeSH
- fibroblastové růstové faktory metabolismus MeSH
- lidé MeSH
- ligandy MeSH
- myši MeSH
- nanodiamanty chemie MeSH
- proliferace buněk MeSH
- receptory fibroblastových růstových faktorů metabolismus MeSH
- signální transdukce MeSH
- techniky tkáňových kultur MeSH
- tibie fyziologie MeSH
- vazba proteinů MeSH
- viabilita buněk MeSH
- zvířata MeSH
- Check Tag
- lidé MeSH
- myši MeSH
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
Cilia project from almost every cell integrating extracellular cues with signaling pathways. Constitutive activation of FGFR3 signaling produces the skeletal disorders achondroplasia (ACH) and thanatophoric dysplasia (TD), but many of the molecular mechanisms underlying these phenotypes remain unresolved. Here, we report in vivo evidence for significantly shortened primary cilia in ACH and TD cartilage growth plates. Using in vivo and in vitro methodologies, our data demonstrate that transient versus sustained activation of FGF signaling correlated with different cilia consequences. Transient FGF pathway activation elongated cilia, while sustained activity shortened cilia. FGF signaling extended primary cilia via ERK MAP kinase and mTORC2 signaling, but not through mTORC1. Employing a GFP-tagged IFT20 construct to measure intraflagellar (IFT) speed in cilia, we showed that FGF signaling affected IFT velocities, as well as modulating cilia-based Hedgehog signaling. Our data integrate primary cilia into canonical FGF signal transduction and uncover a FGF-cilia pathway that needs consideration when elucidating the mechanisms of physiological and pathological FGFR function, or in the development of FGFR therapeutics.
- MeSH
- achondroplazie genetika patofyziologie MeSH
- buňky NIH 3T3 MeSH
- chondrocyty metabolismus MeSH
- chrupavka metabolismus MeSH
- cilie patologie fyziologie MeSH
- ciliopatie genetika patofyziologie MeSH
- fenotyp MeSH
- fibroblastové růstové faktory metabolismus MeSH
- lidé MeSH
- myši MeSH
- primární buněčná kultura MeSH
- receptor fibroblastových růstových faktorů, typ 3 genetika metabolismus MeSH
- růstová ploténka metabolismus MeSH
- signální transdukce fyziologie MeSH
- thanatoforní dysplazie genetika patofyziologie MeSH
- zvířata MeSH
- Check Tag
- lidé MeSH
- myši MeSH
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
- Research Support, N.I.H., Extramural MeSH
Many tyrosine kinase inhibitors (TKIs) have failed to reach human use due to insufficient activity in clinical trials. However, the failed TKIs may still benefit patients if their other kinase targets are identified by providing treatment focused on syndromes driven by these kinases. Here, we searched for novel targets of AZD1480, an inhibitor of JAK2 kinase that recently failed phase two cancer clinical trials due to a lack of activity. Twenty seven human receptor tyrosine kinases (RTKs) and 153 of their disease-associated mutants were in-cell profiled for activity in the presence of AZD1480 using a newly developed RTK plasmid library. We demonstrate that AZD1480 inhibits ALK, LTK, FGFR1-3, RET and TRKA-C kinases and uncover a physical basis of this specificity. The RTK activity profiling described here facilitates inhibitor repurposing by enabling rapid and efficient identification of novel TKI targets in cells.
- Publikační typ
- časopisecké články MeSH
Tyrosine kinase inhibitors are being developed for therapy of malignancies caused by oncogenic FGFR signaling but little is known about their effect in congenital chondrodysplasias or craniosynostoses that associate with activating FGFR mutations. Here, we investigated the effects of novel FGFR inhibitor, ARQ 087, in experimental models of aberrant FGFR3 signaling in cartilage. In cultured chondrocytes, ARQ 087 efficiently rescued all major effects of pathological FGFR3 activation, i.e. inhibition of chondrocyte proliferation, loss of extracellular matrix and induction of premature senescence. In ex vivo tibia organ cultures, ARQ 087 restored normal growth plate architecture and eliminated the suppressing FGFR3 effect on chondrocyte hypertrophic differentiation, suggesting that it targets the FGFR3 pathway specifically, i.e. without interference with other pro-growth pathways. Moreover, ARQ 087 inhibited activity of FGFR1 and FGFR2 mutants associated with Pfeiffer, Apert and Beare-Stevenson craniosynostoses, and rescued FGFR-driven excessive osteogenic differentiation in mouse mesenchymal micromass cultures or in ex vivo calvarial organ cultures. Our data warrant further development of ARQ 087 for clinical use in skeletal disorders caused by activating FGFR mutations.
- MeSH
- aniliny farmakologie terapeutické užití MeSH
- bezbuněčný systém MeSH
- buněčná diferenciace * účinky léků MeSH
- buněčné kultury MeSH
- chinazoliny farmakologie terapeutické užití MeSH
- chondrocyty účinky léků metabolismus patologie MeSH
- extracelulární matrix účinky léků metabolismus MeSH
- fibroblastový růstový faktor 2 farmakologie MeSH
- končetinové pupeny patologie MeSH
- kraniosynostózy farmakoterapie genetika patologie MeSH
- krysa rodu rattus MeSH
- kur domácí MeSH
- lebka patologie MeSH
- mutace genetika MeSH
- myši MeSH
- orgánové kultury - kultivační techniky MeSH
- proliferace buněk účinky léků MeSH
- receptory fibroblastových růstových faktorů genetika MeSH
- signální transdukce * MeSH
- stárnutí buněk účinky léků MeSH
- tibie účinky léků patologie MeSH
- zvířata MeSH
- Check Tag
- krysa rodu rattus MeSH
- myši MeSH
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
