AIMS: The objective of this study was to investigate the association and combined prognostic significance of the PD-L1, Smoothened protein and β-catenin expressions in patients with clear cell renal cell carcinoma (ccRCC). METHODS: The PD-L1, Smoothened protein and β-catenin expression were evaluated in 104 ccRCC patients. All studied tumor samples were acquired from nephrectomy specimens of primary tumors and not from biopsies or metastases. An indirect immunohistochemistry using polyclonal rabbit anti-Smoothened antibody, monoclonal mouse anti-human β-catenin-1 antibody, immunohistochemical assay PD-L1 28-8 pharmDx using monoclonal rabbit anti-PD-L1 antibody and anti-VHL (C- terminal) rabbit antibody was used. Immunohistochemistry was scored semiquantitavely. RESULTS: Median overall survival (OS) was significantly better in patients with lower PD-L1 expression (≤5%), Smoothened protein (SMO) expression (<5%) or cytoplasmic β-catenin expression (≤75%) than in patients with higher expressions of these biomarkers (P<0.001, P=0.047, and P<0.001, respectively). Membranous β-catenin showed an opposite effect with its lower expression (≤75%) being associated with longer OS (P=0.020). There was significant association between PD-1 and PD-L1 expression (P=0.007) and significant association of tumor grade (WHO 2016) with membranous β-catenin (P<0.001), cytoplasmic β-catenin (P=0.005), pVHL (P=0.042), PD-L1 (P=0.049) and PD-1 (P=0.028) expression. CONCLUSION: The present study provides the first data on the potential association and combined prognostic significance of frequency of primary cilia, PD-L1, Smoothened protein and β-catenin expression with the outcome in clear cell renal cell carcinoma.

• Klíčová slova
• clear cell renal carcinoma, cytoplasmic β-catenin, membranous β-catenin, primary cilia, programmed cell death protein ligand 1, smoothened protein,
• Publikační typ
• časopisecké články MeSH

Neural stem cells are fundamental to development of the central nervous system (CNS)-as well as its plasticity and regeneration-and represent a potential tool for neuro transplantation therapy and research. This study is focused on examination of the proliferation dynamic and fate of embryonic neural stem cells (eNSCs) under differentiating conditions. In this work, we analyzed eNSCs differentiating alone and in the presence of sonic hedgehog (SHH) or triiodothyronine (T3) which play an important role in the development of the CNS. We found that inhibition of the SHH pathway and activation of the T3 pathway increased cellular health and survival of differentiating eNSCs. In addition, T3 was able to increase the expression of the gene for the receptor smoothened (Smo), which is part of the SHH signaling cascade, while SHH increased the expression of the T3 receptor beta gene (Thrb). This might be the reason why the combination of SHH and T3 increased the expression of the thyroxine 5-deiodinase type III gene (Dio3), which inhibits T3 activity, which in turn affects cellular health and proliferation activity of eNSCs.

• Klíčová slova
• cell differentiation, embryonic neural stem cells, sonic hedgehog, triiodothyronine,
• MeSH
• jodidperoxidasa genetika   metabolismus   MeSH
• kultivované buňky MeSH
• myší embryonální kmenové buňky cytologie   metabolismus   MeSH
• myši inbrední C57BL MeSH
• myši MeSH
• nervové kmenové buňky cytologie   metabolismus   MeSH
• neurogeneze * MeSH
• proteiny hedgehog genetika   metabolismus   MeSH
• receptor Smoothened genetika   metabolismus   MeSH
• trijodthyronin metabolismus   MeSH
• tyreoidální hormony, receptory beta genetika   metabolismus   MeSH
• zvířata MeSH
• Check Tag
• myši MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• Názvy látek
• iodothyronine deiodinase type III MeSH Prohlížeč
• jodidperoxidasa MeSH
• proteiny hedgehog MeSH
• receptor Smoothened MeSH
• Shh protein, mouse MeSH Prohlížeč
• Smo protein, mouse MeSH Prohlížeč
• trijodthyronin MeSH
• tyreoidální hormony, receptory beta MeSH

The Hedgehog (Hh) signaling pathway is crucial for vertebrate embryonic development, tissue homeostasis and regeneration. Hh signaling is upregulated in basal cell carcinoma and medulloblastoma and Hh pathway inhibitors targeting the Smoothened (SMO) protein are in clinical use. However, the signaling cascade is incompletely understood and novel druggable proteins in the pathway are in high demand. We describe the discovery of the Hh-pathway modulator Pipinib by means of cell-based screening. Target identification and validation revealed that Pipinib selectively inhibits phosphatidylinositol 4-kinase IIIβ (PI4KB) and suppresses GLI-mediated transcription and Hh target gene expression by impairing SMO translocation to the cilium. Therefore, inhibition of PI4KB and, consequently, reduction in phosphatidyl-4-phosphate levels may be considered an alternative approach to inhibit SMO function and thus, Hedgehog signaling.

• Klíčová slova
• Hedgehog signaling, PI4KB, biological activity, inhibitors,
• MeSH
• buněčné linie MeSH
• cilie metabolismus   MeSH
• exprese genu účinky léků   MeSH
• fosfotransferasy s alkoholovou skupinou jako akceptorem antagonisté a inhibitory   genetika   metabolismus   MeSH
• lidé MeSH
• malá interferující RNA metabolismus   MeSH
• morfoliny farmakologie   MeSH
• myši MeSH
• osteogeneze účinky léků   MeSH
• proteiny hedgehog antagonisté a inhibitory   genetika   metabolismus   MeSH
• protinádorové látky chemie   farmakologie   MeSH
• puriny farmakologie   MeSH
• receptor Smoothened genetika   metabolismus   MeSH
• RNA interference MeSH
• signální transdukce účinky léků   MeSH
• thiofeny chemie   farmakologie   MeSH
• vedlejší histokompatibilní antigeny genetika   metabolismus   MeSH
• viabilita buněk účinky léků   MeSH
• vztahy mezi strukturou a aktivitou MeSH
• zvířata MeSH
• Check Tag
• lidé MeSH
• myši MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• přehledy MeSH
• Research Support, N.I.H., Extramural MeSH
• Názvy látek
• fosfotransferasy s alkoholovou skupinou jako akceptorem MeSH
• malá interferující RNA MeSH
• morfoliny MeSH
• phosphatidylinositol phosphate 4-kinase MeSH Prohlížeč
• proteiny hedgehog MeSH
• protinádorové látky MeSH
• puriny MeSH
• purmorphamine MeSH Prohlížeč
• receptor Smoothened MeSH
• thiofeny MeSH
• vedlejší histokompatibilní antigeny MeSH

Silymarin is the standardized extract from the fruits of Silybum marianum (L.) Gaertn., a well-known hepatoprotectant and antioxidant. Recently, bioactive compounds of silymarin, i.e., silybins and their 2,3-dehydro derivatives, have been shown to exert anticancer activities, yet with unclear mechanisms. This study combines in silico and in vitro methods to reveal the potential interactions of optically pure silybins and dehydrosilybins with novel protein targets. The shape and chemical similarity with approved drugs were evaluated in silico, and the potential for interaction with the Hedgehog pathway receptor Smoothened (SMO) and BRAF kinase was confirmed by molecular docking. In vitro studies on SMO and BRAF V600E kinase activity and in BRAF V600E A-375 human melanoma cell lines were further performed to examine their effects on these proteins and cancer cell lines and to corroborate computational predictions. Our in silico results direct to new potential targets of silymarin constituents as dual inhibitors of BRAF and SMO, two major targets in anticancer therapy. The experimental studies confirm that BRAF kinase and SMO may be involved in mechanisms of anticancer activities, demonstrating dose-dependent profiles, with dehydrosilybins showing stronger effects than silybins. The results of this work outline the dual SMO/BRAF effect of flavonolignans from Silybum marianum with potential clinical significance. Our approach can be applied to other natural products to reveal their potential targets and mechanism of action.

• Klíčová slova
• BRAF kinase, Smoothened, cytotoxicity, in silico methods, silybins,
• Publikační typ
• časopisecké články MeSH

OBJECTIVES: Hedgehog signalling plays a critical role during the pathogenesis of fibrosis in systemic sclerosis (SSc). Besides canonical hedgehog signalling with smoothened (SMO)-dependent activation of GLI transcription factors, GLI can be activated independently of classical hedgehog ligands and receptors (so-called non-canonical pathways). Here, we aimed to evaluate the role of non-canonical hedgehog signalling in SSc and to test the efficacy of direct GLI inhibitors that target simultaneously canonical and non-canonical hedgehog pathways. METHODS: The GLI inhibitor GANT-61 was used to inhibit canonical as well as non-canonical hedgehog signalling, while the SMO inhibitor vismodegib was used to selectively target canonical hedgehog signalling. Furthermore, GLI2 was selectively depleted in fibroblasts using the Cre-LoxP system. The effects of pharmacological or genetic of GLI2 on transforming growth factor-β (TGF-β) signalling were analysed in cultured fibroblasts, in bleomycin-induced pulmonary fibrosis and in mice with overexpression of a constitutively active TGF-β receptor I. RESULTS: TGF-β upregulated GLI2 in a Smad3-dependent manner and induced nuclear accumulation and DNA binding of GLI2. Fibroblast-specific knockout of GLI2 protected mice from TBRact-induced fibrosis. Combined targeting of canonical and non-canonical hedgehog signalling with direct GLI inhibitors exerted more potent antifibrotic effects than selective targeting of canonical hedgehog signalling with SMO inhibitors in experimental dermal and pulmonary fibrosis. CONCLUSIONS: Our data demonstrate that hedgehog pathways and TGF-β signalling both converge to GLI2 and that GLI2 integrates those signalling to promote tissue fibrosis. These findings may have translational implications as non-selective inhibitors of GLI2 are in clinical use and selective molecules are currently in development.

• Klíčová slova
• Fibroblasts, Systemic Sclerosis, Treatment,
• MeSH
• anilidy farmakologie   MeSH
• dospělí MeSH
• fibroblasty účinky léků   metabolismus   MeSH
• fibróza MeSH
• genový knockout MeSH
• inhibitor aktivátoru plazminogenu 1 genetika   MeSH
• kolagen typu I genetika   MeSH
• kultivované buňky MeSH
• kůže účinky léků   patologie   MeSH
• lidé středního věku MeSH
• lidé MeSH
• messenger RNA metabolismus   MeSH
• mladý dospělý MeSH
• myši knockoutované MeSH
• myši transgenní MeSH
• myši MeSH
• plicní fibróza chemicky indukované   metabolismus   MeSH
• protein Gli2 s motivem zinkových prstů MeSH
• protein Smad3 metabolismus   MeSH
• protein-serin-threoninkinasy antagonisté a inhibitory   genetika   MeSH
• proteiny hedgehog metabolismus   MeSH
• pteridiny farmakologie   MeSH
• pyridiny farmakologie   MeSH
• pyrimidiny farmakologie   MeSH
• receptor Smoothened antagonisté a inhibitory   MeSH
• receptory transformujícího růstového faktoru beta antagonisté a inhibitory   genetika   MeSH
• rekombinantní proteiny farmakologie   MeSH
• růstový faktor pojivové tkáně genetika   MeSH
• senioři MeSH
• signální transdukce účinky léků   MeSH
• systémová sklerodermie genetika   metabolismus   MeSH
• TGF-beta receptor I. typu MeSH
• transformující růstový faktor beta metabolismus   farmakologie   MeSH
• transkripční faktory Krüppel-like antagonisté a inhibitory   genetika   metabolismus   MeSH
• zvířata MeSH
• Check Tag
• dospělí MeSH
• lidé středního věku MeSH
• lidé MeSH
• mladý dospělý MeSH
• mužské pohlaví MeSH
• myši MeSH
• senioři MeSH
• ženské pohlaví MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• Názvy látek
• anilidy MeSH
• GANT 61 MeSH Prohlížeč
• Gli2 protein, mouse MeSH Prohlížeč
• HhAntag691 MeSH Prohlížeč
• inhibitor aktivátoru plazminogenu 1 MeSH
• kolagen typu I MeSH
• messenger RNA MeSH
• protein Gli2 s motivem zinkových prstů MeSH
• protein Smad3 MeSH
• protein-serin-threoninkinasy MeSH
• proteiny hedgehog MeSH
• pteridiny MeSH
• pyridiny MeSH
• pyrimidiny MeSH
• receptor Smoothened MeSH
• receptory transformujícího růstového faktoru beta MeSH
• rekombinantní proteiny MeSH
• růstový faktor pojivové tkáně MeSH
• SD-208 MeSH Prohlížeč
• TGF-beta receptor I. typu MeSH
• transformující růstový faktor beta MeSH
• transkripční faktory Krüppel-like 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.

• Klíčová slova
• GRK2, Wnt, asphyxiating thoracic dystrophy, hedgehog, smoothened,
• 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
• Názvy látek
• GRK2 protein, human MeSH Prohlížeč
• kinasa 2 receptorů spřažených s G-proteiny MeSH
• proteiny hedgehog * MeSH

BACKGROUND: Cancer is the second most common cause of death in the Czech Republic. The treatment of this disease is very exhausting for the patients and the treatment has often limited success only. The disease often relapses after a period of remission. Moreover, metastases often appear in lungs, liver or other organs and worsen patients prognosis and probability of survival. The Hedgehog (Hh) signaling pathway is one of the important pathways that affects initiation and maintenance of various types of tumours. When aberrantly activated, Hh signaling pathway helps cells escape apoptosis, disturbs cell energy metabolism, influences the process of epithelial-mesenchymal transition, helps to escape immune system, maintains cancer stem cells and supports metastasis. The role of Hh signaling cascade in tumour initiation, maintenance and progression is intensively studied. Several types of inhibitors of this pathway were developed. The most intensively studied were inhibitors of the receptor Smoothened. Due to commonly occurring resistance, the research of other groups of inhibitors is in the centre of interest. These new drugs do not target receptor Smoothened but proteins standing downstream of Smoothened (inhibition of final Gli transcription factors). The drugs could give new hope to patients whose treatment fails. PURPOSE: This review summarizes the findings about the role of Hh signaling pathway in tumour development and describes the progress in the development of targeted inhibitors of this pathway.

• Klíčová slova
• Hedgehog signaling pathway, apoptosis, cancer stem cells, drug resistance, epithelial-mesenchymal transition, metastasis, molecular targeted therapy,
• MeSH
• lidé MeSH
• nádory metabolismus   patologie   MeSH
• progrese nemoci MeSH
• protein Gli1 metabolismus   MeSH
• proteiny hedgehog metabolismus   MeSH
• zvířata MeSH
• Check Tag
• lidé MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• přehledy MeSH
• Názvy látek
• protein Gli1 MeSH
• proteiny hedgehog MeSH

Primary cilia are cellular surface projections enriched in receptors and signaling molecules, acting as signaling hubs that respond to stimuli. Malfunctions in primary cilia have been linked to human diseases, including retinopathies and ocular defects. Here, we focus on TMEM107, a protein localized to the transition zone of primary cilia. TMEM107 mutations were found in patients with Joubert and Meckel-Gruber syndromes. A mouse model lacking Tmem107 exhibited eye defects such as anophthalmia and microphthalmia, affecting retina differentiation. Tmem107 expression during prenatal mouse development correlated with phenotype occurrence, with enhanced expression in differentiating retina and optic stalk. TMEM107 deficiency in retinal organoids resulted in the loss of primary cilia, down-regulation of retina-specific genes, and cyst formation. Knocking out TMEM107 in human ARPE-19 cells prevented primary cilia formation and impaired response to Smoothened agonist treatment because of ectopic activation of the SHH pathway. Our data suggest TMEM107 plays a crucial role in early vertebrate eye development and ciliogenesis in the differentiating retina.

• MeSH
• lidé MeSH
• membránové proteiny genetika   metabolismus   MeSH
• myši MeSH
• polycystická choroba ledvin * genetika   MeSH
• poruchy ciliární motility * genetika   metabolismus   MeSH
• retina metabolismus   MeSH
• retinopathia pigmentosa * metabolismus   MeSH
• těhotenství MeSH
• zvířata MeSH
• Check Tag
• lidé MeSH
• myši MeSH
• těhotenství MeSH
• ženské pohlaví MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• membránové proteiny MeSH
• TMEM107 protein, human MeSH Prohlížeč
• Tmem107 protein, mouse MeSH Prohlížeč

The spring-loaded camming device (SLCD), also known as "friend", is a simple mechanism used to ensure the safety of the climber through fall prevention. SLCD consists of two pairs of opposing cams rotating separately, with one (single-axle SLCD) or two (dual-axle SLCD) pins connecting the opposing cams, a stem, connected to the pins, providing the attachment of the climbing rope, springs, which simultaneously push cams to a fully expanded position, and an operating element controlling the cam position. The expansion of cams is thus adaptable to allow insertion or removal of the device into/from a rock crack. While the pins, stem, operating element, and springs can be considered optimal, the (especially internal) shape of the cam allows space for improvement, especially where the weight is concerned. This paper focuses on optimizing the internal shape of the dual-axle SLCD cam from the perspective of the weight/stiffness trade-off. For this purpose, two computational models are designed and multi-step topology optimization (TOP) are performed. From the computational models' point of view, SLCD is considered symmetric and only one cam is optimized and smoothened using parametric curves. Finally, the load-bearing capacity of the new cam design is analyzed. This work is based on practical industry requirements, and the obtained results will be reflected in a new commercial design of SLCD.

• Klíčová slova
• ANSYS, FEM, MATLAB, NURBS, SIMP, SLCD, cam, friend, topology optimization,
• Publikační typ
• časopisecké články MeSH

Ultra-high molecular polyethylene (UHMWPE) is one of the most used materials of the acetabular liners in total tip arthroplasty (THA). Polyethylene has good tribological properties and biocompatibility. However, the lifetime of polyethylene implants is limited by wear related complications. Polyethylene material released into the periprosthetic environment induces osteolysis that can be followed by implant loosening. Wear of cup is influenced mainly by orientation of the cup in pelvis, by initial geometry before the material degradation and by tribological parameters. Aim of this study is to focus on the run-in-phase of the liner which is predictive for future life cycles of liner. Creep deformations of liners for 30°, 45°, 60° inclination angles surgically recommended for the positioning in pelvis were analyzed by the optical scanning method. Load tests were performed for 50,000 cycles. Creep deformations and surface changes were analyzed at each 10,000 cycles. The results showed that liners with 60° inclination angle had higher creep deformations. Penetration of femoral head was 0.04-0.05 mm and occupied bearing area was around 77%. The smallest creep was measured for the 45° angle. However, deformation in the superior quadrant of acetabular rim, which is vulnerable for potential fracture of a liner, was identified in this case. Topography of the surface bearing was also observed during the run-in-phase. The surface was smoothened and showed multidirectional scratches caused by the influence of third body particles. This phase was followed by early delamination. Flakes sized approximately 5-20 µm were observed on the UHMWPE surface. This is similar to the'flake' shape wear debris extracted in vivo. Detailed analysis of run-in phase of loading of modern polyethylene implants can help to distinguish between their creep deformation and true degradation. The latter contributes strongly to the development of wear related complications associated with THAs limiting substantially their time in service.

• Klíčová slova
• Creep deformation, Inclination angle, Optical scanning, Run-in-phase, Scanning Electron Microscopy, UHMWPE,
• MeSH
• acetabulum * MeSH
• časové faktory MeSH
• mechanické jevy * MeSH
• náhrada kyčelního kloubu * MeSH
• polyethyleny * MeSH
• povrchové vlastnosti MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• polyethyleny * MeSH
• ultra-high molecular weight polyethylene MeSH Prohlížeč