| 1. |
- Acosta, Helena, et al.
(författare)
-
The serpin PN1 is a feedback regulator of FGF signaling in germ layer and primary axis formation.
- 2015
-
Ingår i: Development: For advances in developmental biology and stem cells. - : The Company of Biologists. - 1477-9129. ; 142:6, s. 1146-1158
-
Tidskriftsartikel (refereegranskat)abstract
- Germ layer formation and primary axis development rely on Fibroblast growth factors (FGFs). In Xenopus, the secreted serine protease HtrA1 induces mesoderm and posterior trunk/tail structures by facilitating the spread of FGF signals. Here, we show that the serpin Protease nexin-1 (PN1) is transcriptionally activated by FGF signals, suppresses mesoderm and promotes head development in mRNA-injected embryos. An antisense morpholino oligonucleotide against PN1 has the opposite effect and inhibits ectodermal fate. However, ectoderm and anterior head structures can be restored in PN1-depleted embryos when HtrA1 and FGF receptor activities are diminished, indicating that FGF signals negatively regulate their formation. We show that PN1 binds to and inhibits HtrA1, prevents degradation of the proteoglycan Syndecan 4 and restricts paracrine FGF/Erk signaling. Our data suggest that PN1 is a negative-feedback regulator of FGF signaling and has important roles in ectoderm and head development.
|
|
| 2. |
|
|
| 3. |
- Gupta, Abhishek, et al.
(författare)
-
Human CIDEC transgene improves lipid metabolism and protects against high-fat diet–induced glucose intolerance in mice
- 2022
-
Ingår i: Journal of Biological Chemistry. - : Elsevier BV. - 0021-9258. ; 298:9
-
Tidskriftsartikel (refereegranskat)abstract
- Cell death–inducing DNA fragmentation factor-like effector C (CIDEC) expression in adipose tissue positively correlates with insulin sensitivity in obese humans. Further, E186X, a single-nucleotide CIDEC variant is associated with lipodystrophy, hypertriglyceridemia, and insulin resistance. To establish the unknown mechanistic link between CIDEC and maintenance of systemic glucose homeostasis, we generated transgenic mouse models expressing CIDEC (Ad-CIDECtg) and CIDEC E186X variant (Ad-CIDECmut) transgene specifically in the adipose tissue. We found that Ad-CIDECtg but not Ad-CIDECmut mice were protected against high-fat diet-induced glucose intolerance. Furthermore, we revealed the role of CIDEC in lipid metabolism using transcriptomics and lipidomics. Serum triglycerides, cholesterol, and low-density lipoproteins were lower in high-fat diet-fed Ad-CIDECtg mice compared to their littermate controls. Mechanistically, we demonstrated that CIDEC regulates the enzymatic activity of adipose triglyceride lipase via interacting with its activator, CGI-58, to reduce free fatty acid release and lipotoxicity. In addition, we confirmed that CIDEC is indeed a vital regulator of lipolysis in adipose tissue of obese humans, and treatment with recombinant CIDEC decreased triglyceride breakdown in visceral human adipose tissue. Our study unravels a central pathway whereby adipocyte-specific CIDEC plays a pivotal role in regulating adipose lipid metabolism and whole-body glucose homeostasis. In summary, our findings identify human CIDEC as a potential ‘drug’ or a ‘druggable’ target to reverse obesity-induced lipotoxicity and glucose intolerance.
|
|
| 4. |
- Hou, Shirui, et al.
(författare)
-
The secreted serine protease xHtrA1 stimulates long-range FGF signaling in the early Xenopus embryo
- 2007
-
Ingår i: Developmental Cell. - : Elsevier BV. - 1534-5807. ; 13:2, s. 226-241
-
Tidskriftsartikel (refereegranskat)abstract
- We found that the secreted serine protease xHtrA1, expressed in the early embryo and transcriptionally activated by FGF signals, promotes posterior development in mRNA-injected Xenopus embryos. xHtrA1 mRNA led to the induction of secondary tail-like structures, expansion of mesoderm, and formation of ectopic neurons in an FGF-dependent manner. An antisense morpholino oligonucleotide or a neutralizing antibody against xHtrA1 had the opposite effects. xHtrA1 activates FGF/ ERK signaling and the transcription of FGF genes. We show that Xenopus Biglycan, Syndecan-4, and Glypican-4 are proteolytic targets of xHtrA1 and that heparan sulfate and dermatan sulfate trigger posteriorization, mesoderm induction, and neuronal differentiation via the FGIF signaling pathway. The results are consistent with a mechanism by which xHtrA1, through cleaving proteoglycans, releases cellsurface-bound FGF ligands and stimulates long-range FGF signaling.
|
|
| 5. |
- Khairul, Mohd Fadzli Mustaffa, et al.
(författare)
-
Fluoxetine potentiates chloroquine and mefloquine effect on multidrug-resistant Plasmodium falciparum in vitro
- 2006
-
Ingår i: Japanese Journal of Infectious Diseases. - 1344-6304. ; 59:5, s. 329-331
-
Tidskriftsartikel (refereegranskat)abstract
- Fluoxetine (FLX), a P-glycoprotein inhibitor with antimalarial activity, is promising candidate for reversing chloroquine/mefloquine (CQ/MQ) resistance. The Dd2 strain of CQ- and MQ-resistant Plasmodium falciparum was synchronized and assayed with various concentrations of CQ/MQ individually and in combination with FLX or verapamil (VPL). Our results indicated the 50% inhibitory concentration values of CQ and MQ were greatly lowered when FLX was used simultaneously. Isobolograms indicated that CQ-FLX combinations are more synergistic (mean fractional inhibitory concentration [FIC] index 0.55) than MQ-FLX (mean FIC index 0.64), and their synergistic effect was better than or at par with CQ-VPL (mean FIC index 0.88) and MQ-VPL (mean FIC index 0.60) combinations. We conclude that the FLX potentiates the CQ and MQ response on multidrug-resistant P. falciparum at clinically achievable concentrations.
|
|
| 6. |
- Liu, Yang, et al.
(författare)
-
Successful gene therapy of Diamond-Blackfan anemia in a mouse model and human CD34+ cord blood hematopoietic stem cells using a clinically applicable lentiviral vector
- 2022
-
Ingår i: Haematologica. - : Ferrata Storti Foundation (Haematologica). - 1592-8721 .- 0390-6078. ; 107:2, s. 446-456
-
Tidskriftsartikel (refereegranskat)abstract
- Diamond-Blackfan anemia (DBA) is an inherited bone marrow failure disorder in which pure red blood cell aplasia is associated with physical malformations and a predisposition to cancer. Twentyfive percent of patients with DBA have mutations in a gene encoding ribosomal protein S19 (RPS19). Our previous proof-of-concept studies demonstrated that DBA phenotype could be successfully treated using lentiviral vectors in Rps19-deficient DBA mice. In our present study, we developed a clinically applicable single gene, self-inactivating lentiviral vector, containing the human RPS19 cDNA driven by the human elongation factor 1a short promoter, which can be used for clinical gene therapy development for RPS19-deficient DBA. We examined the efficacy and safety of the vector in a Rps19-deficient DBA mouse model and in human primary RPS19-deficient CD34+ cord blood cells. We observed that transduced Rps19-deficient bone marrow cells could reconstitute mice long-term and rescue the bone marrow failure and severe anemia observed in Rps19-deficient mice, with a low risk of mutagenesis and a highly polyclonal insertion site pattern. More importantly, the vector can also rescue impaired erythroid differentiation in human primary RPS19-deficient CD34+ cord blood hematopoietic stem cells. Collectively, our results demonstrate the efficacy and safety of using a clinically applicable lentiviral vector for the successful treatment of Rps19-deficient DBA in a mouse model and in human primary CD34+ cord blood cells. These findings show that this vector can be used to develop clinical gene therapy for RPS19-deficient DBA patients.
|
|
| 7. |
- Luo, Lifang, et al.
(författare)
-
BCAT1 decreases the sensitivity of cancer cells to cisplatin by regulating mTOR-mediated autophagy via branched-chain amino acid metabolism
- 2021
-
Ingår i: Cell Death and Disease. - : Springer Science and Business Media LLC. - 2041-4889. ; 12:2
-
Tidskriftsartikel (refereegranskat)abstract
- Cisplatin is one of the most effective chemotherapy drugs and is widely used in the treatment of cancer, including hepatocellular carcinoma (HCC) and cervical cancer, but its therapeutic benefit is limited by the development of resistance. Our previous studies demonstrated that BCAT1 promoted cell proliferation and decreased cisplatin sensitivity in HCC cells. However, the exact role and mechanism of how BCAT1 is involved in cisplatin cytotoxicity remain undefined. In this study, we revealed that cisplatin triggered autophagy in cancer cells, with an increase in BCAT1 expression. The cisplatin-induced up-regulation of BCAT1 decreased the cisplatin sensitivity by regulating autophagy through the mTOR signaling pathway. In addition, branched-chain amino acids or leucine treatment inhibited cisplatin- or BCAT1-mediated autophagy and increased cisplatin sensitivity by activating mTOR signaling in cancer cells. Moreover, inhibition of autophagy by chloroquine increased cisplatin sensitivity in vivo. Also, the knockdown of BCAT1 or the administration of leucine activated mTOR signaling, inhibited autophagy, and increased cisplatin sensitivity in cancer cells in vivo. These findings demonstrate a new mechanism, revealing that BCAT1 decreases cisplatin sensitivity in cancer cells by inducing mTOR-mediated autophagy via branched-chain amino acid leucine metabolism, providing an attractive pharmacological target to improve the effectiveness of chemotherapy.
|
|
| 8. |
- Strate, Ina, et al.
(författare)
-
Retinol dehydrogenase 10 is a feedback regulator of retinoic acid signalling during axis formation and patterning of the central nervous system.
- 2009
-
Ingår i: Development: For advances in developmental biology and stem cells. - : The Company of Biologists. - 1477-9129. ; 136:3, s. 461-472
-
Tidskriftsartikel (refereegranskat)abstract
- Retinoic acid (RA) is an important morphogen that regulates many biological processes, including the development of the central nervous system (CNS). Its synthesis from vitamin A (retinol) occurs in two steps, with the second reaction - catalyzed by retinal dehydrogenases (RALDHs) - long considered to be crucial for tissue-specific RA production in the embryo. We have recently identified the Xenopus homologue of retinol dehydrogenase 10 (XRDH10) that mediates the first step in RA synthesis from retinol to retinal. XRDH10 is specifically expressed in the dorsal blastopore lip and in other domains of the early embryo that partially overlap with XRALDH2 expression. We show that endogenous RA suppresses XRDH10 gene expression, suggesting negative-feedback regulation. In mRNA-injected Xenopus embryos, XRDH10 mimicked RA responses, influenced the gene expression of organizer markers, and synergized with XRALDH2 in posteriorizing the developing brain. Knockdown of XRDH10 and XRALDH2 by specific antisense morpholino oligonucleotides had the opposite effects on organizer gene expression, and caused a ventralized phenotype and anteriorization of the brain. These data indicate that the conversion of retinol into retinal is a developmentally controlled step involved in specification of the dorsoventral and anteroposterior body axes, as well as in pattern formation of the CNS. We suggest that the combinatorial gene expression and concerted action of XRDH10 and XRALDH2 constitute a ;biosynthetic enzyme code' for the establishment of a morphogen gradient in the embryo.
|
|
| 9. |
- Tan Grahn, Hooi Min
(författare)
-
Regulation of morphogen signalling during neural patterning in the Xenopus embryo
- 2011
-
Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- Morphogens such as Hedghog, Wnt, FGF, and retinoic acid are important signals whose concentrations need to be tightly regulated in the vertebrate embryo to ensure body axis development and formation of the central nervous system. We first show that the intracellular cytoplasmic protein XSufu acts as a dual regulator of Hedgehog (Hh) and Wnt signals during neural induction and patterning in the Xenopus embryo. We further reveal an essential role of XSufu in the crosstalk of the two pathways, in which β-catenin activates Hh signalling upon overexpression of Gli1, and Gli inhibits Wnt signalling upon overexpression of β-catenin. A biphasic model for the role of XSufu in anteroposterior patterning of the neural plate is presented suggesting that XSufu suppresses anterior Gli and posterior β-catenin transcription factors in a dose-dependent manner. Then we introduce the secreted serine protease xHtrA1 as feedforward stimulator of long-range FGF signalling. Fibroblast growth factor (FGF) signals activate transcription of xHtrA1, and xHtrA1 stimulates FGF4 and FGF8 gene activities, allowing positive feedback regulation. We also show that xHtrA1 triggers proteolytic cleavage of xBiglycan, xSyndecan-4, and xGlypican-4, suggesting a model, in which xHtrA1 through cleaving proteoglycans releases FGF/proteoglycan complexes that act as long-range messages during anteroposterior patterning, mesoderm induction, and neuronal differentiation. Third, we present Xenopus retinol dehydrogenase-10 (XRDH10) as a critical enzyme for embryonic vitamin A metabolism and retinoic acid (RA) synthesis in the developing embryo. We show that XRDH10, which oxidizes vitamin A to retinal, is transcriptionally inhibited by RA, suggesting negative feedback regulation at the first step of RA biosynthesis. XRDH10 cooperates with XRALDH2, which further oxidizes retinal to bioactive RA, in Spemann’s organizer during dorsoventral patterning of the embryo. We also show that the nested gene expression and cooperate action of XRDH10 and XRALDH2 form a biosynthetic enzyme code that establishes RA gradients along the anteroposterior neuraxis.
|
|
| 10. |
- Tan Grahn, Hooi Min, et al.
(författare)
-
Roxithromycin potentiates the effects of chloroquine and mefloquine on multidrug-resistant Plasmodium falciparum in vitro
- 2007
-
Ingår i: Experimental Parasitology. - : Elsevier BV. - 0014-4894. ; 115:4, s. 387-392
-
Tidskriftsartikel (refereegranskat)abstract
- Chloroquine (CQ) and mefloquine (MQ) are no longer potent antimalarial drugs due to the emergence of resistant Plasmodium falciparum. Combination therapy has become the standard for many regimes in overcoming drug resistance. Roxithromycin (ROM), a known p-glycoprotein inhibitor, is reported to have antimalarial activity and it is hoped it will potentiate the effects of both CQ/MQ and reverse CQ/MQ-resistance. We assayed the effects of CQ and MQ individually and in combination with ROM on synchronized P. falciparum (Dd2 strain) cultures. The IC(50) values of CQ and MQ were 60.0+/-5.0 and 16.0+/-3.0 ng/ml; these were decreased substantially when combined with ROM. Isobolograms indicate that CQ-ROM combinations were relatively more synergistic (mean FICI 0.70) than MQ-ROM (mean FICI 0.85) with their synergistic effect at par with CQ-verapamil (VRP) (mean FICI 0.64) and MQ-VRP (mean FICI 0.60) combinations. We conclude that ROM potentiates the CQ/MQ response on multidrug-resistant P. falciparum.
|
|
| 11. |
- Tan Grahn, Hooi Min, et al.
(författare)
-
S100A6 is a critical regulator of hematopoietic stem cells.
- 2020
-
Ingår i: Leukemia. - : Springer Science and Business Media LLC. - 0887-6924 .- 1476-5551.
-
Tidskriftsartikel (refereegranskat)abstract
- The fate options of hematopoietic stem cells (HSCs) include self-renewal, differentiation, migration, and apoptosis. HSCs self-renewal divisions in stem cells are required for rapid regeneration during tissue damage and stress, but how precisely intracellular calcium signals are regulated to maintain fate options in normal hematopoiesis is unclear. S100A6 knockout (KO) HSCs have reduced total cell numbers in the HSC compartment, decreased myeloid output, and increased apoptotic HSC numbers in steady state. S100A6KO HSCs had impaired self-renewal and regenerative capacity, not responding to 5-Fluorouracil. Our transcriptomic and proteomic profiling suggested that S100A6 is a critical HSC regulator. Intriguingly, S100A6KO HSCs showed decreased levels of phosphorylated Akt (p-Akt) and Hsp90, with an impairment of mitochondrial respiratory capacity and a reduction of mitochondrial calcium levels. We showed that S100A6 regulates intracellular and mitochondria calcium buffering of HSC upon cytokine stimulation and have demonstrated that Akt activator SC79 reverts the levels of intracellular and mitochondrial calcium in HSC. Hematopoietic colony-forming activity and the Hsp90 activity of S100A6KO are restored through activation of the Akt pathway. We show that p-Akt is the prime downstream mechanism of S100A6 in the regulation of HSC self-renewal by specifically governing mitochondrial metabolic function and Hsp90 protein quality.
|
|
| 12. |
- Tan Grahn, Hooi Min, et al.
(författare)
-
The dual regulator Sufu integrates Hedgehog and Wnt signals in the early Xenopus embryo
- 2011
-
Ingår i: Developmental Biology. - : Elsevier BV. - 1095-564X .- 0012-1606. ; 358:1, s. 262-276
-
Tidskriftsartikel (refereegranskat)abstract
- Hedgehog (Hh) and Wnt proteins are important signals implicated in several aspects of embryonic development, including the early development of the central nervous system. We found that Xenopus Suppressor-of-fused (XSufu) affects neural induction and patterning by regulating the Hh/Gli and Wnt/beta-catenin pathways. Microinjection of XSufu mRNA induced expansion of the epidermis at the expense of neural plate tissue and caused enlargement of the eyes. An antisense morpholino oligonucleotide against XSufu had the opposite effect. Interestingly, both gain- and loss-of-function experiments resulted in a posterior shift of brain markers, suggesting a biphasic effect of XSufu on anteroposterior patterning. XSufu blocked early Wnt/beta-catenin signaling, as indicated by the suppression of XWnt8-induced secondary axis formation in mRNA-injected embryos, and activation of Wnt target genes in XSufu-MO-injected ectodermal explants. We show that XSufu binds to XG*i1 and X beta-catenin. In Xenopus embryos and mouse embryonic fibroblasts, Gli1 inhibits Wnt signaling under overexpression of beta-catenin, whereas beta-catenin stimulates Hh signaling under overexpression of Glil. Notably, endogenous Sufu is critically involved in this crosstalk The results suggest that XSufu may act as a common regulator of Hh and Wnt signaling and contribute to intertwining the two pathways. (C) 2011 Elsevier Inc. All rights reserved.
|
|
| 13. |
|
|
| 14. |
- Warsi, Sarah, et al.
(författare)
-
BMP signaling is required for postnatal murine hematopoietic stem cell self-renewal
- 2021
-
Ingår i: Haematologica. - : Ferrata Storti Foundation (Haematologica). - 0390-6078 .- 1592-8721. ; 106:8, s. 2203-2214
-
Tidskriftsartikel (refereegranskat)abstract
- Life-long production of blood from hematopoietic stem cells (HSC) is a process of strict modulation. Intrinsic and extrinsic signals govern fate options like self-renewal – a cardinal feature of HSC. Bone morphogenetic proteins (BMP) have an established role in embryonic hematopoiesis, but less is known about its functions in adulthood. Previously, SMAD-mediated BMP signaling has been proven dispensable for HSC. However, the BMP type-II receptor (BMPR-II) is highly expressed in HSC, leaving the possibility that BMP function via alternative pathways. Here, we establish that BMP signaling is required for self-renewal of adult HSC. Through conditional knockout we show that BMPR-II deficient HSC have impaired self-renewal and regenerative capacity. BMPR-II deficient cells have reduced p38 activation, implying that non-SMAD pathways operate downstream of BMP in HSC. Indeed, a majority of primitive hematopoietic cells do not engage in SMAD-mediated responses downstream of BMP in vivo. Furthermore, deficiency of BMPR-II results in increased expression of TJP1, a known regulator of self-renewal in other stem cells, and knockdown of TJP1 in primitive hematopoietic cells partly rescues the BMPR-II null phenotype. This suggests TJP1 may be a universal stem cell regulator. In conclusion, BMP signaling, in part mediated through TJP1, is required endogenously by adult HSC to maintain self-renewal capacity and proper resilience of the hematopoietic system during regeneration.
|
|
| 15. |
|
|
| 16. |
- Zhang, Sheng-Chu, et al.
(författare)
-
Lentiviral vector-mediated down-regulation of IL-17A receptor in hepatic stellate cells results in decreased secretion of IL-6
- 2012
-
Ingår i: World Journal of Gastroenterology. - : Baishideng Publishing Group Inc.. - 1007-9327. ; 18:28, s. 3696-3704
-
Tidskriftsartikel (refereegranskat)abstract
- AIM: To investigate the mechanism of interleukin (IL)-6 secretion through blocking the IL-17A/IL-17A receptor (IL-17RA) signaling pathway with a short hairpin RNA (shRNA) in hepatic stellate cells (HSCs) in vitro. METHODS: HSCs were derived from the livers of adult male Sprague-Dawley rats. IL-6 expression was evaluated using real-time quantitative polymerase chain reaction and enzyme linked immunosorbent assay. The phosphorylation activity of p38 mitogen activated protein kinases (MAPK) and extracellular regulated protein kinases (ERK) 1/2 upon induction by IL-17A and suppression by IL-17RA shRNA were examined using Western blotting. RESULTS: IL-6 expression induced by IL-17A was significantly increased compared to control in HSCs (P < 0.01 in a dose-dependent manner). Suppression of IL-17RA using lentiviral-mediated shRNA inhibited IL-6 expression induced by IL-17A compared to group with only IL-17A treatment (1.44 +/- 0.17 vs 4.07 +/- 0.43, P < 0.01). IL-17A induced rapid phosphorylation of p38 MAPK and ERK1/2 after 5 min exposure, and showed the strongest levels of phosphorylation of p38 MAPK and ERK1/2 at 15 min in IL-17A-treated HSCs. IL-6 mRNA expression induced by IL-17A (100 ng/mL) for 3 h exposure was inhibited by preincubation with specific inhibitors of p38 MAPK (SB-203580) and ERK1/2 (PD-98059) compared to groups without inhibitors preincubation (1.67 +/- 0.24, 2.01 +/- 0.10 vs 4.08 +/- 0.59, P < 0.01). Moreover, Lentiviral-mediated IL-17RA shRNA 1 inhibited IL-17A-induced IL-6 mRNA expression compared to random shRNA in HSCs (1.44 +/- 0.17 vs 3.98 +/- 0.68, P < 0.01). Lentiviral-mediated IL-17RA shRNA 1 inhibited phosphorylation of p38 MAPK and ERK1/2 induced by 15 min IL-17A (100 ng/mL) exposure. CONCLUSION: Down-regulation of the IL-17RA receptor by shRNA decreased IL-6 expression induced by IL-17A via p38 MAPK and ERK1/2 phosphorylation in HSCs. Suppression of IL-17RA expression may be a strategy to reduce the inflammatory response induced by IL-17A in the liver. (C) 2012 Baishideng. All rights reserved.
|
|
| 17. |
- Zheng, Yi Hu, et al.
(författare)
-
BCAT1, a key prognostic predictor of hepatocellular carcinoma, promotes cell proliferation and induces chemoresistance to cisplatin
- 2016
-
Ingår i: Liver International. - : Wiley. - 1478-3223. ; 36:12, s. 1836-1847
-
Tidskriftsartikel (refereegranskat)abstract
- Background & Aims: BCAT1 initiates the catabolism of branched-chain amino acids. Here, we investigated the function of BCAT1 and its transcriptional regulatory mechanism in hepatocellular carcinoma (HCC). Methods: RNASeq was used to evaluate BCAT1 mRNA levels in HCC and normal matched specimens. After the exogenous expression of BCAT1 in BEL-7404 cells and the suppression of endogenous BCAT1 expression with shRNA in HepG2 cells, the cell proliferation, clone-forming ability and cell-cycle changes were measured with MTT assay, colony-forming assay and flow cytometry respectively. A xenograft model was used to investigate the effect of BCAT1 on cancer growth in vivo. Chromatin immunoprecipitation and luciferase reporter technologies were used to confirm the transcriptional regulation of the BCAT1 gene by MYC. The expression of the BCAT1 and MYC proteins in 122 HCC tissues was determined with an immunohistochemical analysis. Results: BCAT1 mRNA was clearly increased in HCC tissues and hepatomas. The ectopic expression of BCAT1 in BEL-7404 cells enhanced their proliferation, clone formation, tumourigenic properties, S-G2/M phase transition and chemoresistance to cisplatin. The suppression of BCAT1 expression in HepG2 cells significantly inhibited their proliferation, clone formation, and S-G2/M phase transition and caused their chemosensitization to cisplatin. MYC affected the transcriptional regulation of BCAT1. Clinical data showed that BCAT1 expression correlated with a significantly poorer prognosis. Conclusion: BCAT1 plays a pathogenic role in HCC by causing cell proliferation and chemoresistance. The MYC transcription factor is involved in regulating the transcriptional activity of BCAT1. BCAT1 expression has prognostic significance for the survival of patients with HCC.
|
|
