المؤلفون: Maria Duda, Wilhelm Krek, Holger Moch, Thomas C. Weber, Michael Hell

المصدر: CANCER RESEARCH

مصطلحات موضوعية: Cancer Research, Pathology, medicine.medical_specialty, Cell division, Carcinogenesis, Aneuploidy, Mitosis, Apoptosis, Spindle Apparatus, Biology, urologic and male genital diseases, Mice, Chromosome instability, Chromosomal Instability, Chromosome Segregation, medicine, Animals, Carcinoma, Renal Cell, Cells, Cultured, Cell Proliferation, Mice, Knockout, Kidney, Cell growth, Epithelial Cells, medicine.disease, Phenotype, female genital diseases and pregnancy complications, Kidney Neoplasms, Clear cell renal cell carcinoma, medicine.anatomical_structure, Kidney Tubules, Oncology, Von Hippel-Lindau Tumor Suppressor Protein, Cancer research

الوصف: The von Hippel–Lindau (VHL) tumor suppressor protein pVHL is commonly mutated in clear cell renal cell carcinoma (ccRCC) and has been implicated in the control of multiple cellular processes that might be linked to tumor suppression, including promoting proper spindle orientation and chromosomal stability. However, it is unclear whether pVHL exerts these mitotic regulatory functions in vivo as well. Here, we applied ischemic kidney injury to stimulate cell division in otherwise quiescent mouse adult kidneys. We show that in the short term (5.5 days after surgery), Vhl-deficient kidney cells demonstrate both spindle misorientation and aneuploidy. The spindle misorientation phenotype encompassed changes in directed cell division, which may manifest in the development of cystic lesions, whereas the aneuploidy phenotype involved the occurrence of lagging chromosomes but not chromosome bridges, indicative of mitotic checkpoint impairment. Intriguingly, in the long term (4 months after the ischemic insult), Vhl-deficient kidneys displayed a heterogeneous pattern of ccRCC precursor lesions, including cysts, clear cell–type cells, and dysplasia. Together, these data provide direct evidence for a key role of pVHL in mediating oriented cell division and faithful mitotic checkpoint function in the renal epithelium, emphasizing the importance of pVHL as a controller of mitotic fidelity in vivo. Cancer Res; 74(9); 2422–31. ©2013 AACR.

الوصول الحر: https://explore.openaire.eu/search/publication?articleId=doi_dedup___::feec13eb128f2914089229c876aa6dedTest

المؤلفون: Silvia Casagrande, Markus Rechsteiner, Wilhelm Krek, Laura Morra, Adriana von Teichman, Holger Moch, Peter Schraml, Melanie Ruf, Sonja Brun-Schmid

المصدر: The Journal of Pathology. 229:525-534

مصطلحات موضوعية: Regulation of gene expression, 0303 health sciences, Reporter gene, Tissue microarray, biology, In situ hybridization, urologic and male genital diseases, medicine.disease, Molecular biology, female genital diseases and pregnancy complications, Receptor tyrosine kinase, Pathology and Forensic Medicine, 03 medical and health sciences, Clear cell renal cell carcinoma, 0302 clinical medicine, Downregulation and upregulation, 030220 oncology & carcinogenesis, biology.protein, medicine, Cancer research, Gene silencing, 030304 developmental biology

الوصف: Mass spectrometry analysis of renal cancer cell lines recently suggested that the protein-tyrosine phosphatase receptor type J (PTPRJ), an important regulator of tyrosine kinase receptors, is tightly linked to the von Hippel-Lindau protein (pVHL). Therefore, we aimed to characterize the biological relevance of PTPRJ for clear cell renal cell carcinoma (ccRCC). In pVHL-negative ccRCC cell lines, both RNA and protein expression levels of PTPRJ were lower than those in the corresponding pVHL reconstituted cells. Quantitative RT-PCR and western blot analysis of ccRCC with known VHL mutation status and normal matched tissues as well as RNA in situ hybridization on a tissue microarray (TMA) confirmed a decrease of PTPRJ expression in more than 80% of ccRCCs, but in only 12% of papillary RCCs. ccRCC patients with no or reduced PTPRJ mRNA expression had a less favourable outcome than those with a normal expression status (p = 0.05). Sequence analysis of 32 PTPRJ mRNA-negative ccRCC samples showed five known polymorphisms but no mutations, implying other mechanisms leading to PTPRJ's down-regulation. Selective silencing of HIF-α by siRNA and reporter gene assays demonstrated that pVHL inactivation reduces PTPRJ expression through a HIF-dependent mechanism, which is mainly driven by HIF-2α stabilization. Our results suggest PTPRJ as a member of a pVHL-controlled pathway whose suppression by HIF is critical for ccRCC development.

الوصول الحر: https://explore.openaire.eu/search/publication?articleId=doi_________::4ec14debd8e560e1fe0787d6e500d4c0Test
https://doi.org/10.1002/path.4107Test

المؤلفون: Holger Moch, Tullio Sulser, Sarah M. Mühl, Peter Schraml, Rafal Pawłowski, Wilhelm Krek

المساهمون: University of Zurich, Schraml, Peter

المصدر: International journal of cancer

مصطلحات موضوعية: Cancer Research, Pathology, medicine.medical_specialty, Tumor suppressor gene, Gene Expression, 610 Medicine & health, Context (language use), Kaplan-Meier Estimate, Biology, urologic and male genital diseases, PBRM1, 03 medical and health sciences, 0302 clinical medicine, SETD2, 10049 Institute of Pathology and Molecular Pathology, Cell Line, Tumor, medicine, Humans, 1306 Cancer Research, Carcinoma, Renal Cell, 030304 developmental biology, Neoplasm Staging, 0303 health sciences, BAP1, Nuclear Proteins, medicine.disease, Kidney Neoplasms, female genital diseases and pregnancy complications, 3. Good health, Chromatin, DNA-Binding Proteins, 10062 Urological Clinic, Clear cell renal cell carcinoma, Oncology, Tissue Array Analysis, Von Hippel-Lindau Tumor Suppressor Protein, 030220 oncology & carcinogenesis, Mutation, Cancer research, Disease Progression, 2730 Oncology, Clear cell, Transcription Factors

الوصف: Although von Hippel-Lindau (VHL) tumor suppressor gene alterations dominate the genetic landscape of clear cell renal cell carcinoma (ccRCC), recent studies have identified new ccRCC genes, including SETD2, KDM6A, KDM5C, BAP1 and PBRM1. Strikingly, all these genes fall into a category of histone/chromatin regulators. Polybromo-1 (PBRM1) is the second most frequently mutated gene after VHL; however, the clinical relevance of its loss in ccRCC has not yet been reported. Here, we analyzed the expression of PBRM1, the product encoded by PBRM1, in ccRCC cell lines and in more than 300 RCC tumor samples. The data were correlated with clinicopathological parameters and VHL mutation status. We found that a significant number of ccRCC cancer cell lines lack detectable PBRM1 expression. Loss of PBRM1 was predominant in the clear cell subtype of RCC (∼ 70%) and correlated with advanced tumor stage (p < 0.0001), low differentiation grade (p = 0.0002) and worse patient outcome (p = 0.025), but not with the VHL mutation status. Our results indicate a critical role for PBRM1 in the suppression of ccRCC progression. Moreover, the results suggest that functional inactivation of PBRM1 in the context of pVHL loss-of-function may represent a key event in facilitating the development of key aspects of an aggressive tumor behavior. Given the role of PBRM1 in chromatin modification, the gene expression pathways disrupted by the inactivation of this protein may lead to new treatment strategies for ccRCC.

وصف الملف: Pawlowski_iJC_2012.pdf - application/pdf

الوصول الحر: https://explore.openaire.eu/search/publication?articleId=doi_dedup___::572b0e0065ae66d078b505d34c17d303Test

المؤلفون: Ian J. Frew, Wilhelm Krek, Claudio R. Thoma, Zlatko Smole

المساهمون: University of Zurich, Krek, Wilhelm

المصدر: EUROPEAN JOURNAL OF CANCER

مصطلحات موضوعية: Male, Protein isoform, Cancer Research, urologic and male genital diseases, Microtubules, law.invention, Mice, 0302 clinical medicine, law, RNA interference, Protein Isoforms, 1306 Cancer Research, Cells, Cultured, Mice, Knockout, 0303 health sciences, Reverse Transcriptase Polymerase Chain Reaction, NF-kappa B, female genital diseases and pregnancy complications, Cell biology, Oncology, Hypoxia-inducible factors, Von Hippel-Lindau Tumor Suppressor Protein, 10076 Center for Integrative Human Physiology, 030220 oncology & carcinogenesis, Female, RNA Interference, 2730 Oncology, Gene isoform, Transgene, Blotting, Western, Molecular Sequence Data, Mice, Transgenic, 610 Medicine & health, Biology, 03 medical and health sciences, Microtubule, Sequence Homology, Nucleic Acid, Animals, Amino Acid Sequence, Transcription factor, 030304 developmental biology, Base Sequence, Fibroblasts, Embryo, Mammalian, Hypoxia-Inducible Factor 1, alpha Subunit, Molecular biology, Mice, Inbred C57BL, Microscopy, Fluorescence, 570 Life sciences, biology, Suppressor

الوصف: The von Hippel-Lindau tumour suppressor protein (pVHL) controls distinct cellular responses ranging from targeting hypoxia inducible factor α (HIFα) subunits for degradation and promotion of chromosomal stability to the regulation of microtubule dynamics. pVHL is produced in mammalian cells as a long and a short isoform, derived from alternate translational initiation sites in a single Vhl mRNA. However, it is unclear whether these isoforms have different cell biological activities that may represent different tumour suppressor activities of pVHL. Through a knock-in strategy to mutate the first translational initiation site from methionine to leucine (M1L) we have genetically deleted the pVHL long protein isoform in mice, allowing dissection of isoform-specific functions of pVHL. Vhl(M1L/M1L) mice exhibit no obvious phenotypic abnormalities. While numerous pVHL-mediated activities, including degradation of HIFα transcription factors, are unaffected, microtubule dynamics are altered in primary cells derived from Vhl(M1L/M1L) mice to an extent similar to that seen following complete loss of pVHL function. We conclude that the microtubule-regulating function and the HIFα-regulating function of pVHL are separable activities mediated by different protein isoforms.

الوصول الحر: https://explore.openaire.eu/search/publication?articleId=doi_dedup___::31a6bbdf05ef29208e78cb1f32d8b23cTest

المؤلفون: Gaudenz Danuser, Wilhelm Krek, Alexandre Matov, Zlatko Smole, Katrin L. Gutbrodt, Christian R. Hoerner, Claudio R. Thoma

المصدر: The Journal of cell biology
The Journal of Cell Biology

مصطلحات موضوعية: von Hippel-Lindau Disease, endocrine system diseases, Tumor suppressor gene, Recombinant Fusion Proteins, Green Fluorescent Proteins, Molecular Sequence Data, Mutant, Regulator, urologic and male genital diseases, Microtubules, Article, 03 medical and health sciences, Imaging, Three-Dimensional, 0302 clinical medicine, Tubulin, Microtubule, Live cell imaging, Cluster Analysis, Humans, Point Mutation, cardiovascular diseases, Amino Acid Sequence, neoplasms, Research Articles, 030304 developmental biology, 0303 health sciences, biology, Hydrolysis, Nocodazole, Cilium, Cell Biology, Phenotype, Molecular biology, female genital diseases and pregnancy complications, Cell biology, Von Hippel-Lindau Tumor Suppressor Protein, 030220 oncology & carcinogenesis, biology.protein, Guanosine Triphosphate, Microtubule-Associated Proteins

الوصف: The product of the von Hippel-Lindau tumor suppressor gene stabilizes microtubules by inhibiting GTPase activity.
Von Hippel-Lindau (VHL) tumor suppressor gene mutations predispose carriers to kidney cancer. The protein pVHL has been shown to interact with microtubules (MTs), which is critical to cilia maintenance and mitotic spindle orientation. However, the function for pVHL in the regulation of MT dynamics is unknown. We tracked MT growth via the plus end marker EB3 (end-binding protein 3)-GFP and inferred additional parameters of MT dynamics indirectly by spatiotemporal grouping of growth tracks from live cell imaging. Our data establish pVHL as a near-optimal MT-stabilizing protein: it attenuates tubulin turnover, both during MT growth and shrinkage, inhibits catastrophe, and enhances rescue frequencies. These functions are mediated, in part, by inhibition of tubulin guanosine triphosphatase activity in vitro and at MT plus ends and along the MT lattice in vivo. Mutants connected to the VHL cancer syndrome are differentially compromised in these activities. Thus, single cell–level analysis of pVHL MT regulatory function allows new predictions for genotype to phenotype associations that deviate from the coarser clinically defined mutant classifications.

الوصول الحر: https://explore.openaire.eu/search/publication?articleId=doi_dedup___::359f07bcec62d458e88de4424dd21ed1Test
https://doi.org/10.1083/jcb.201006059Test

المؤلفون: Ian J. Frew, Claudio R. Thoma, Wilhelm Krek

المصدر: Cell Cycle. 6:1809-1813

مصطلحات موضوعية: medicine.medical_specialty, Cell, Biology, urologic and male genital diseases, Microtubules, Kidney cysts, Cancer syndrome, Glycogen Synthase Kinase 3, Microtubule, Internal medicine, medicine, Animals, Humans, Cilia, Molecular Biology, GSK3B, Glycogen Synthase Kinase 3 beta, Cysts, Cilium, Cell Biology, medicine.disease, female genital diseases and pregnancy complications, Clear cell renal cell carcinoma, Endocrinology, medicine.anatomical_structure, Hypoxia-inducible factors, Von Hippel-Lindau Tumor Suppressor Protein, Cancer research, medicine.symptom, Developmental Biology

الوصف: Amongst other clinical manifestations, patients with the von Hippel-Lindau (VHL) cancer syndrome are predisposed to develop kidney cysts, which are considered to be precursor lesions of clear cell renal cell carcinoma (ccRCC). Recent evidence has highlighted an unexpected function of the VHL tumor suppressor protein (pVHL) in maintaining the structural integrity of the primary cilium, a microtubule-based cellular antenna important for suppression of uncontrolled proliferation of kidney epithelial cells and cyst formation. Intriguingly, this function of pVHL is directly linked to its capacity to regulate the microtubule cytoskeleton independent of its well-characterized role in the degradation of hypoxia inducible factor alpha (HIFalpha) subunits. However, loss of pVHL alone does not suffice for a cell to lose the primary cilium. Other pathways need to be additionally inactivated, including one involving glycogen synthase kinase 3 beta (GSK3beta). These new findings draw attention to a primary cilium maintenance network as new territory for pVHL tumor suppressive activity and have implications for understanding the development of kidney pathology in the setting of VHL disease.

الوصول الحر: https://explore.openaire.eu/search/publication?articleId=doi_dedup___::5c3a21bae1c744485f49fb3eefa2b217Test
https://doi.org/10.4161/cc.6.15.4518Test

المؤلفون: Joanna Lisztwan, Claudio R. Thoma, Christiane Wirbelauer, Alexander Hergovich, Robert E. Barry, Wilhelm Krek

المصدر: Molecular and Cellular Biology. 26:5784-5796

مصطلحات موضوعية: Tumor suppressor gene, Molecular Sequence Data, macromolecular substances, Plasma protein binding, Biology, urologic and male genital diseases, Microtubules, Cell Line, law.invention, Serine, Glycogen Synthase Kinase 3, Mice, law, GSK-3, Casein Kinase I, Basic Helix-Loop-Helix Transcription Factors, Animals, Humans, Protein Isoforms, Amino Acid Sequence, Phosphorylation, Molecular Biology, Articles, Cell Biology, Peptide Fragments, female genital diseases and pregnancy complications, Cell biology, Biochemistry, Von Hippel-Lindau Tumor Suppressor Protein, Mutation, Suppressor, Signal transduction, Sequence Alignment, Protein Binding, Signal Transduction

الوصف: Inactivation of the von Hippel-Lindau (VHL) tumor suppressor gene is linked to the development of tumors of the eyes, kidneys, and central nervous system. VHL encodes two gene products, pVHL30 and pVHL19, of which one, pVHL30, associates functionally with microtubules (MTs) to regulate their stability. Here we report that pVHL30 is a novel substrate of glycogen synthase kinase 3 (GSK3) in vitro and in vivo. Phosphorylation of pVHL on serine 68 (S68) by GSK3 requires a priming phosphorylation event at serine 72 (S72) mediated in vitro by casein kinase I. Functional analysis of pVHL species carrying nonphosphorylatable or phosphomimicking mutations at S68 and/or S72 reveals a central role for these phosphorylation events in the regulation of pVHL's MT stabilization (but not binding) activity. Taken together, our results identify pVHL as a novel priming-dependent substrate of GSK3 and suggest a dual-kinase mechanism in the control of pVHL's MT stabilization function. Since GSK3 is a component of multiple signaling pathways that are altered in human cancer, our results further imply that normal operation of the GSK3-pVHL axis may be a critical aspect of pVHL's tumor suppressor mechanism through the regulation of MT dynamics.

الوصول الحر: https://explore.openaire.eu/search/publication?articleId=doi_dedup___::8361020470edb40c884ab95910a2af38Test
https://doi.org/10.1128/mcb.00232-06Test

المؤلفون: Sarah Muehl, Klaus-Peter Knobeloch, Albino Troilo, Wilhelm Krek, Daniela Jaramillo, Irina Alexander

المصدر: EMBO reports

مصطلحات موضوعية: Tumor suppressor gene, Vesicular Transport Proteins, urologic and male genital diseases, Biochemistry, law.invention, Deubiquitinating enzyme, Mice, Ubiquitin, law, Ciliogenesis, Endopeptidases, Genetics, Animals, Humans, Cilia, Molecular Biology, biology, Endosomal Sorting Complexes Required for Transport, Protein Stability, Cilium, HEK 293 cells, Scientific Reports, Ubiquitination, Hypoxia-Inducible Factor 1, alpha Subunit, female genital diseases and pregnancy complications, Cell Hypoxia, Endocytosis, Cell biology, Oxygen, Protein Transport, HEK293 Cells, Von Hippel-Lindau Tumor Suppressor Protein, biology.protein, Suppressor, Ubiquitin Thiolesterase, Deubiquitination, Protein Binding

الوصف: Loss of primary cilia is a key feature of von Hippel-Lindau tumor suppressor (VHL)-associated pathology. Although VHL-deficiency predisposes cells to precipitous cilia disassembly in response to growth factor cues, it does not affect ciliogenesis. Here, using a siRNA-based screen to find genes that are essential for ciliogenesis only in the presence of the VHL tumor suppressor gene product pVHL, we identify ubiquitin-specific protease (USP)8. The pVHL-dependency of USP8 for ciliogenesis is directly linked to its function as a HIF1α deubiquitinating enzyme. By counteracting pVHL-mediated ubiquitination of HIF1α, USP8 maintains a basal expression of HIF1α and HIF transcriptional output in normoxia, including the repression of Rabaptin5, which is essential for endosome trafficking-mediated ciliogenesis.

الوصول الحر: https://explore.openaire.eu/search/publication?articleId=doi_dedup___::0c03041cc58a28e7530d68187d32c8d1Test
https://europepmc.org/articles/PMC4303451Test/

المؤلفون: Christiane Wirbelauer, Georges Imbert, Joanna Lisztwan, Matthias Gstaiger, Wilhelm Krek

المصدر: Genes & Development. 13:1822-1833

مصطلحات موضوعية: Cell Extracts, Tumor suppressor gene, Ubiquitin-Protein Ligases, Molecular Sequence Data, Mutant, urologic and male genital diseases, law.invention, Ligases, Ubiquitin, law, Tumor Cells, Cultured, Genetics, Humans, Genes, Tumor Suppressor, Amino Acid Sequence, Ubiquitins, Gene, DNA Primers, chemistry.chemical_classification, DNA ligase, Base Sequence, Sequence Homology, Amino Acid, biology, Cell growth, Tumor Suppressor Proteins, Proteins, Precipitin Tests, Molecular biology, Kidney Neoplasms, Recombinant Proteins, female genital diseases and pregnancy complications, Cell biology, Ubiquitin ligase, chemistry, Von Hippel-Lindau Tumor Suppressor Protein, Mutation, biology.protein, Suppressor, Research Paper, Developmental Biology

الوصف: pVHL, the product of the VHL tumor suppressor gene, plays an important role in the regulation of cell growth and differentiation of human kidney cells, and inactivation of the VHL gene is the most frequent genetic event in human kidney cancer. The biochemical function of pVHL is unknown. Here we report that pVHL exists in vivo in a complex that displays ubiquitination-promoting activity in conjunction with the universally required components E1, E2, and ubiquitin. pVHL-associated ubiquitination activity requires, at a minimum, pVHL to bind elongin C and Cul-2, relatives of core components of SCF (Skp1–Cdc53/Cul-1–F-box protein) E3 ligase complexes. Notably, certain tumor-derived mutants of pVHL demonstrate loss of associated ubiquitination promoting activity. These results identify pVHL as a component of a potential SCF-like E3 ubiquitin–protein ligase complex and suggest a direct link between pVHL tumor suppressor and the process of ubiquitination.

الوصول الحر: https://explore.openaire.eu/search/publication?articleId=doi_dedup___::51d737a63948ec51139ce024d571ccd1Test
https://doi.org/10.1101/gad.13.14.1822Test

المؤلفون: Van-Duc Luu, Barbara Ingold-Heppner, Daniel P. Stiehl, Niklaus Fankhauser, Anna M. Nowicka, Igor Cima, Holger Moch, Tullio Sulser, Wilhelm Krek, Damaris Bausch-Fluck, Gunther Boysen, Bernd Wollscheid, Claudio R. Thoma, Thomas Hermanns, Adriana von Teichman, Roland H. Wenger, Peter Krek

المساهمون: University of Zurich, Moch, Holger

المصدر: Neoplasia, 14 (6)
Neoplasia: An International Journal for Oncology Research, Vol 14, Iss 6, Pp 535-546 (2012)

مصطلحات موضوعية: Proteomics, Cancer Research, Cell, 610 Medicine & health, urologic and male genital diseases, lcsh:RC254-282, Cell Line, 10052 Institute of Physiology, Stable isotope labeling by amino acids in cell culture, 10049 Institute of Pathology and Molecular Pathology, Transcriptional regulation, medicine, Basic Helix-Loop-Helix Transcription Factors, Humans, 1306 Cancer Research, Carcinoma, Renal Cell, neoplasms, Glycoproteins, chemistry.chemical_classification, Papillary renal cell carcinomas, Chemistry, Membrane Proteins, Reproducibility of Results, lcsh:Neoplasms. Tumors. Oncology. Including cancer and carcinogens, medicine.disease, Molecular biology, Kidney Neoplasms, female genital diseases and pregnancy complications, Gene Expression Regulation, Neoplastic, Clear cell renal cell carcinoma, 10062 Urological Clinic, medicine.anatomical_structure, 10022 Division of Surgical Research, Cell culture, Von Hippel-Lindau Tumor Suppressor Protein, Cancer research, 570 Life sciences, biology, Neprilysin, Glycoprotein, Research Article

الوصف: The identification of cell surface accessible biomarkers enabling diagnosis, disease monitoring, and treatment of renalcell carcinoma (RCC) is as challenging as the biology and progression of RCC is unpredictable. A hallmark of most RCCis the loss-of-function of the von Hippel–Lindau (pVHL) protein by mutation of its gene (VHL). Using the cell surfacecapturing (CSC) technology, we screened and identified cell surface N-glycoproteins in pVHL-negative and positive786-O cells. One hundred six cell surface N-glycoproteins were identified. Stable isotope labeling with amino acidsin cell culture–based quantification of the CSC screen revealed 23 N-glycoproteins whose abundance seemed tochange in a pVHL-dependent manner. Targeted validation experiments using transcriptional profiling of primary RCCsamples revealed that nine glycoproteins, including CD10 and AXL, could be directly linked to pVHL-mediated transcrip-tional regulation. Subsequent human tumor tissue analysis of these cell surface candidate markers showed a correla-tion between epithelial AXL expression and aggressive tumor phenotype, indicating that pVHL-dependent regulation ofglycoproteins may influence the biologic behavior of RCC. Functional characterization of the metalloprotease CD10 incell invasion assays demonstrated a diminished penetrating behavior of pVHL-negative 786-O cells on treatment withthe CD10-specific inhibitor thiorphan. Our proteomic surfaceome screening approach in combination with transcrip-tional profiling and functional validation suggests pVHL-dependent cell surface glycoproteins as potential diagnosticmarkers for therapeutic targeting and RCC patient monitoring.
Neoplasia, 14 (6)
ISSN:1522-8002
ISSN:1476-5586

وصف الملف: Boysen_Neoplasia_2012.pdf - application/pdf; application/application/pdf

الوصول الحر: https://explore.openaire.eu/search/publication?articleId=doi_dedup___::5e0010f48cb141b0fd2185578b844c42Test