scholarly journals Andean Berry (Vaccinium meridionale Swartz) Juice in Combination With Aspirin Modulated Apoptotic Signaling in Colon Cancer In Vitro and In Vivo

2021 ◽  
Vol 5 (Supplement_2) ◽  
pp. 261-261
Author(s):  
Sandra Arango-Varela ◽  
Ivan Luzardo ◽  
Maria Maldonado-Celis
Keyword(s):  
Colorectal Cancer ◽  
Colon Cancer ◽  
In Silico ◽  
Aberrant Crypt Foci ◽  
Apoptotic Signaling ◽  
Polyphenolic Composition ◽  
Apoptotic Effects ◽  
The Impact

Abstract Objectives This research aimed to assess the impact of Andean Berry (Vaccinium meridionale Swartz) juice (ABJ) in combination with Aspirin in the apoptotic signaling in colon cancer in vitro and in vivo. We hypothesized that ABJ + Aspirin would produce the most effective anti-proliferative and pro-apoptotic effects in vitro and in vivo. Methods The polyphenolic composition of ABJ was carried out by HPLC-DAD. ABJ (0–30% v/v), Aspirin (0–20 mM), and their mixture were evaluated for their pro-apoptotic effects in human SW480 colorectal cancer cells, followed by human apoptosis proteomic and bioinformatic analysis and in silico docking potential between ABJ components and selected pro-apoptotic targets. For the in vivo assays, colorectal cancer was induced with two injections (separated 1 week each) of azoxymethane (AOM: 15 mg/kg body weight, BW), and treatments were evaluated for its chemopreventive and chemoprotective effects. Hence, 30 male and female Balb/c mice were randomly divided in 5 groups: negative control (basal diet, BD); and four AOM-induced groups: positive control (BD), Aspirin (25 mg/kg BW + BD), ABJ (30% v/v in drinking water ABJ + BD), and ABJ + Aspirin (30% v/v ABJ + 25 mg/kg BW Aspirin + BD). Macroscopic and histopathological parameters were evaluated in vivo. Results The mixture displayed the highest antiproliferative effects (+46%), arrested cell cycle at the G2/M phase, decreased cloning efficiency, but reduced Caspase 3/7 activity, suggesting an alternative apoptotic pathway, compared to untreated SW480 cells. Several pro-apoptotic (cytochrome C, TNFRSF1A, Bax, and Bad) and anti-apoptotic (Hsp70/Hsp32) proteins were decreased. ABJ flavonoids (rutin and kaempferol) exhibited the highest in silico affinity with proteins like TRAILR2 or Catalase. Both chemopreventive and chemoprotective approaches showed similar body/liver weight outcomes, but the mixture displayed the strongest aberrant crypt foci reduction in vivo. The chemopreventive approach was more effective in protecting the colon from AOM. Conclusions Results suggested the potential of ABJ to reduce Aspirin use in the alleviation of colorectal cancer markers in vitro and in vivo, modulating alternate pro-apoptotic signaling. Funding Sources The funding provided by COLCIENCIAS and DGAPA-CTIC-UNAM is appreciated.

scholarly journals Farnesyl dimethyl chromanol targets colon cancer stem cells and prevents colorectal cancer metastasis

2021 ◽  
Vol 11 (1) ◽  
Author(s):  
Kazim Husain ◽  
Domenico Coppola ◽  
Chung S. Yang ◽  
Mokenge P. Malafa
Keyword(s):  
Colorectal Cancer ◽  
Colon Cancer ◽  
Cancer Metastasis ◽  
Annexin V ◽  
Cancer Cell Growth ◽  
Ki 67 ◽  
Sox2 Expression ◽  
Tumour Metastasis

AbstractThe activation and growth of tumour-initiating cells with stem-like properties in distant organs characterize colorectal cancer (CRC) growth and metastasis. Thus, inhibition of colon cancer stem cell (CCSC) growth holds promise for CRC growth and metastasis prevention. We and others have shown that farnesyl dimethyl chromanol (FDMC) inhibits cancer cell growth and induces apoptosis in vitro and in vivo. We provide the first demonstration that FDMC inhibits CCSC viability, survival, self-renewal (spheroid formation), pluripotent transcription factors (Nanog, Oct4, and Sox2) expression, organoids formation, and Wnt/β-catenin signalling, as evidenced by comparisons with vehicle-treated controls. In addition, FDMC inhibits CCSC migration, invasion, inflammation (NF-kB), angiogenesis (vascular endothelial growth factor, VEGF), and metastasis (MMP9), which are critical tumour metastasis processes. Moreover, FDMC induced apoptosis (TUNEL, Annexin V, cleaved caspase 3, and cleaved PARP) in CCSCs and CCSC-derived spheroids and organoids. Finally, in an orthotopic (cecum-injected CCSCs) xenograft metastasis model, we show that FDMC significantly retards CCSC-derived tumour growth (Ki-67); inhibits inflammation (NF-kB), angiogenesis (VEGF and CD31), and β-catenin signalling; and induces apoptosis (cleaved PARP) in tumour tissues and inhibits liver metastasis. In summary, our results demonstrate that FDMC inhibits the CCSC metastatic phenotype and thereby supports investigating its ability to prevent CRC metastases.

scholarly journals Iminoflavones Combat 1,2-Dimethyl Hydrazine-Induced Aberrant Crypt Foci Development in Colon Cancer

2014 ◽  
Vol 2014 ◽  
pp. 1-7 ◽  
Author(s):  
V. Ganga Prasad ◽  
Shishir Kawade ◽  
B. S. Jayashree ◽  
Neetinkumar D. Reddy ◽  
Albi Francis ◽  
...  
Keyword(s):  
Colon Cancer ◽  
Treated Group ◽  
Aberrant Crypt Foci ◽  
Colonic Crypts ◽  
Antitumor Potential ◽  
Histopathological Images ◽  
Nuclear Changes ◽  
The Imf

The aim of the present study was to evaluate the antitumor potential of iminoflavones inin vitroandin vivoanticancer models. Preliminary screening in various cancer cell lines revealed four potential iminoflavones out of which IMF-8 was taken based on its activity against colon cancer cells. This was further confirmed by observing the nuclear changes in the cells by AO/EB and Hoechst 33342 staining studies.In vivoactivity was assessed by dimethyl hydrazine-(DMH-) induced colon cancer model in rats. Animals were administered DMH (20 mg/kg, b.w. for 10 weeks and 30 mg/kg b.w.,i.p.for 10 weeks) and were supplemented with (IMF-8) iminoflavone-8 (200 mg/kg,p.o.for 14 days). Results showed that DMH induced 100% aberrant crypt foci (ACF) and polyps which were significantly reduced in the IMF-8 treated group. IMF-8 significantly increased the catalase and GSH levels whereas it reduced the TNF-αand IL-6 levels markedly which suggests the antioxidative and anti-inflammatory actions of flavonoids present in IMF-8. The histopathological images of the IMF-8 treated colon showed no signs of mucosal crypt abscess. These findings suggest that the semi-synthetic iminoflavones, IMF-8, effectively inhibit DMH-induced ACFs and colonic crypts by alleviating the oxidative stress and suppressing the inflammation.

Schisandrin B Attenuates Colitis-Associated Colorectal Cancer through SIRT1 Linked SMURF2 Signaling

2021 ◽  
pp. 1-17
Author(s):  
Zhichen Pu ◽  
Weiwei Zhang ◽  
Minhui Wang ◽  
Maodi Xu ◽  
Haitang Xie ◽  
...  
Keyword(s):  
Colorectal Cancer ◽  
Colon Cancer ◽  
Cell Growth ◽  
Protein Expression ◽  
Hct116 Cell ◽  
Enzyme Linked Immunosorbent Assay ◽  
Schisandrin B ◽  
In Vivo Models

Colon cancer, a common type of malignant tumor, seriously endangers human health. However, due to the relatively slow progress in diagnosis and treatment, the clinical therapeutic technology of colon cancer has not been substantially improved in the past three decades. The present study was designed to investigate the effects and involved mechanisms of schisandrin B in cell growth and metastasis of colon cancer. C57BL/6 mice received AOM and dextran sulfate sodium. Mice in treatment groups were gavaged with 3.75–30 mg/kg/day of schisandrin B. Transwell chamber migration, enzyme-linked immunosorbent assay (ELISA), Western blot analysis, immunoprecipitation (IP) and immunofluorescence were conducted, and HCT116 cell line was employed in this study. Data showed that schisandrin B inhibited tumor number and tumor size in the AOD+DSS-induced colon cancer mouse model. Schisandrin B also inhibited cell proliferation and metastasis of colon cancer cells. We observed that schisandrin B induced SMURF2 protein expression and affected SIRT1 in vitro and in vivo. SMURF2 interacted with SIRT1 protein, and there was a negative correlation between SIRT1 and SMURF2 expressions in human colorectal cancer. The regulation of SMURF2 was involved in the anticancer effects of schisandrin B in both in vitro and in vivo models. In conclusion, the present study revealed that schisandrin B suppressed SIRT1 protein expression, and SIRT1 is negatively correlated with the induction of SMURF2, which inhibited cell growth and metastasis of colon cancer. Schisandrin B could be a leading compound, which will contribute to finding novel potential agents and therapeutic targets for colon cancer.

scholarly journals THE POTENTIAL OF ROBUSTA COFFEE (COFFEA CANEPHORA) AS A COLORECTAL CANCER THERAPY MODALITY: AN IN SILICO STUDY

2021 ◽  
pp. 83-87
Author(s):  
DESSY AGUSTINI ◽  
LEO VERNADESLY ◽  
DELVIANA ◽  
THEODORUS
Keyword(s):  
Colorectal Cancer ◽  
In Silico ◽  
Hydrophobic Interactions ◽  
Binding Energies ◽  
In Vivo Studies ◽  
Discovery Studio ◽  
Robusta Coffee ◽  
In Silico Study

Objectives: This research aims to determine the efficacy of compounds in robusta coffee against colorectal cancer through the inhibition of the T-cell immunoglobulin and immunoreceptor tyrosine-based inhibitory motif domain (TIGIT) receptor. Methods: This in silico study has been conducted in computing platform from June to August 2021. The selected test compounds would go through the Lipinski rule screening through the SwissADME website and the compounds that met these regulations would be docked to the TIGIT protein using AutoDock Tools and AutoDock Vina. The interactions with the highest binding energies were visualized using BIOVIA Discovery Studio 2020. The test compounds then underwent a toxicity profile analysis on the admetSAR 2.0 website. Results: All test compounds complied with the Lipinski rule. The molecular docking results showed the highest binding energy in kahweol and cafestol (−8.1 kcal/mol) compared to OMC (−7.9 kcal/mol), chlorogenic acid (−7.8 kcal/mol), caffeic acid (−6.3 kcal/mol), caffeine (−6.1 kcal/mol), trigonelline (−5.3 kcal/mol), HMF (−5.1 kcal/mol), furfuryl alcohol (−4.4 kcal/mol), and 5-fluorouracil as the comparator drug (−5.3 kcal/mol). Kahweol, cafestol, and 5-fluorouracil revealed the hydrophobic interactions and hydrogen bonds with amino acid residues in TIGIT. Kahweol and cafestol unveiled minimal toxicity prediction Conclusion: Kahweol and cafestol demonstrated the best results in inhibiting the TIGIT protein which played a role in colorectal cancer. In vitro and in vivo studies are needed to strengthen the findings of this research.

Effect of the ADAPT strategy on dormant CD133+ colon cancer stem cells (CSC) and molecular complete remission measured by peripheral blood mononuclear (PBMC) CD133 mRNA.

2012 ◽  
Vol 30 (15_suppl) ◽  
pp. e14153-e14153
Author(s):  
Edward H. Lin ◽  
Yu Xiazhen ◽  
Xi C He ◽  
Xifeng Wu ◽  
Yang Xie ◽  
...  
Keyword(s):  
Colon Cancer ◽  
Median Survival ◽  
Treatment Protocol ◽  
Mrna Levels ◽  
Peripheral Blood Mononuclear ◽  
Colon Cells ◽  
Ca Ix ◽  
The Impact

e14153 Background: The median survival for patients with unresectable metastatic colorectal cancer (CRC) is ~2 years with modern chemotherapy which yields only 5-10% complete responses (CR) including metastasectomy. Recurrences after CR are very common thanks to presence of dormant CSC that are best targeted by our proposed two-step ADAPT strategy: activate from dormancy and potentiate targeting. We examine this strategy in various CRC models and reviewed the impact on stemess including CD133 mRNA, a circulating CSC marker that predict colon cancer relapse. Methods: Different CRC models (in vitro and in vivo) were interrogated similar to clinical ADAPT treatment protocol using capecitabine (or 5FU) plus celecoxib. We also conducted IRB approved retrospective review of unresectable metastatic CRC patients treated ADAPT therapy and in those who also had PBMC CD133 mRNA measured. Results: Contrary to 5FU, which eliminates proliferating CRC cells via apoptosis but also stimulates stemness, celecoxib preferentially deplete CD133+ colon cells and exert potent stemness inhibition via rapid tumor necrosis by perturbing hypoxia and energy metabolism via CA-IX. Following response to first-line chemotherapy, ADAPT strategy plus radiation improved CR or near CR rate to 49/126 (40%) in unresectable CRC patients whose median survival had reached 92.7 months (95% CI, 53.5 months - not reached). Paradoxically, none surgical CR patients (n= 16) enjoyed 100% 5-year relapse free survival compared to 42% of surgical patients (p = 0.04). The PBMC CD133 mRNA in five long-term CR patients were 0.0024, 0.29, 0.5, 0.56, 2.96 respectively, all below previously reported cutoff value of 4.79 for recurrence and far below CD133 mRNA levels (28, 375, 3997, 15662, 83240) in none CR patients. Conclusions: ADAPT plus radiation preferentially targets colon CSC via hypoxia/CA-IX and improves clinical CR rate and molecular CR as measured by PBMC CD133 mRNA. We are actively interrogating the effects of ADAPT strategies in a phase II study funded by Gateway in CRC patients and in genetic CRC animal models.

scholarly journals Targeting AKT with costunolide suppresses the growth of colorectal cancer cells and induces apoptosis in vitro and in vivo

2021 ◽  
Vol 40 (1) ◽  
Author(s):  
Hai Huang ◽  
Song Park ◽  
Haibo Zhang ◽  
Sijun Park ◽  
Wookbong Kwon ◽  
...  
Keyword(s):  
Colorectal Cancer ◽  
Colon Cancer ◽  
Cell Proliferation ◽  
Cell Growth ◽  
The Body ◽  
Migration And Invasion ◽  
Regulation Mechanism ◽  
Mesenchymal Transformation

Abstract Background Colorectal cancer (CRC) is a clinically challenging malignant tumor worldwide. As a natural product and sesquiterpene lactone, Costunolide (CTD) has been reported to possess anticancer activities. However, the regulation mechanism and precise target of this substance remain undiscovered in CRC. In this study, we found that CTD inhibited CRC cell proliferation in vitro and in vivo by targeting AKT. Methods Effects of CTD on colon cancer cell growth in vitro were evaluated in cell proliferation assays, migration and invasion, propidium iodide, and annexin V-staining analyses. Targets of CTD were identified utilizing phosphoprotein-specific antibody array; Costunolide-sepharose conjugated bead pull-down analysis and knockdown techniques. We investigated the underlying mechanisms of CTD by ubiquitination, immunofluorescence staining, and western blot assays. Cell-derived tumour xenografts (CDX) in nude mice and immunohistochemistry were used to assess anti-tumour effects of CTD in vivo. Results CTD suppressed the proliferation, anchorage-independent colony growth and epithelial-mesenchymal transformation (EMT) of CRC cells including HCT-15, HCT-116 and DLD1. Besides, the CTD also triggered cell apoptosis and cell cycle arrest at the G2/M phase. The CTD activates and induces p53 stability by inhibiting MDM2 ubiquitination via the suppression of AKT’s phosphorylation in vitro. The CTD suppresses cell growth in a p53-independent fashion manner; p53 activation may contribute to the anticancer activity of CTD via target AKT. Finally, the CTD decreased the volume of CDX tumors without of the body weight loss and reduced the expression of AKT-MDM2-p53 signaling pathway in xenograft tumors. Conclusions Our project has uncovered the mechanism underlying the biological activity of CTD in colon cancer and confirmed the AKT is a directly target of CTD. All of which These results revealed that CTD might be a new AKT inhibitor in colon cancer treatment, and CTD is worthy of further exploration in preclinical and clinical trials.

In vitro and in vivo activities of KRN330, a fully human monoclonal antibody against colon cancer.

2011 ◽  
Vol 29 (4_suppl) ◽  
pp. 432-432 ◽  
Author(s):  
N. Sawada ◽  
E. Taguchi ◽  
M. Takahashi
Keyword(s):  
Colorectal Cancer ◽  
Monoclonal Antibody ◽  
Colon Cancer ◽  
Western Blot ◽  
Human Colon ◽  
Human Colorectal Cancer ◽  
Antitumor Activities ◽  
Reducing Condition

432 Background: KRN330 is a novel recombinant human IgG1 monoclonal antibody (mAb) targeting A33 surface differentiation antigen that is uniformly expressed on the surface of 95% of colorectal cancer (CRC) cells. In this study, we characterized the activity of KRN330 for its in vitro properties, as well as for its in vivo antitumor activity. Methods: A kinetic analysis of the interaction between KRN330 and recombinant human A33 was conducted using a Biacore 3000. Western blot analysis was conducted using A33 expressing COLO205 lysates under reducing and non-reducing conditions. Binding of KRN330 to human colorectal cancer tissues were investigated using FITC-labeled KRN330. We also developed more conventional staining methods of A33 and investigated A33 expression using human colon cancer tissue microarray (TMA). ADCC and CDC activities of KRN330 were assessed using a standard 51Cr release assay. A33 expression levels of 14 CRC cell lines were analyzed using flow cytometer. In vivo antitumor activities of KRN330 alone or in combination with chemotherapeutic agents against subcutaneous or intraperiotoneal human CRC (COLO205 and LS174T) models were investigated using mice and rats xenograft model. Results: A kinetic analysis revealed that KRN330 showed a high binding affinity to A33. Western blot analysis also showed that antibody recognized not any protein under reducing condition, but non-reducing condition. A33 staining of TMA with 204 different samples revealed the majority of tumor expressed A33. KRN330 exhibited ADCC activity against A33 expressing human colorectal cancer cell lines which include both K-ras wild and mutated types. KRN330 showed dose-dependent antitumor activities in vivo. KRN330 also significantly prolonged survival of human colon tumor bearing mice. In addition, combination treatment of KRN330 with irinotecan showed increased antitumor activitiy and prolongation of survival, compared to either irinotecan or KRN330 alone. Conclusions: These results suggest that KRN330 is a promising candidate of novel therapy for CRC. The phase I/II study of KRN330 plus irinotecan in patients with second line metastatic CRC is ongoing. [Table: see text]

Contribution of FOXC1 to the progression and metastasis and prognosis of human colon cancer.

2015 ◽  
Vol 33 (3_suppl) ◽  
pp. 636-636 ◽  
Author(s):  
Dawei Li ◽  
Qingguo Li ◽  
Changhua Zhuo ◽  
Ye Xu ◽  
Sanjun Cai
Keyword(s):  
Colon Cancer ◽  
Lymph Node ◽  
Cancer Cells ◽  
Distant Metastasis ◽  
Human Colon ◽  
Colon Cancer Cells ◽  
Human Colon Cancer ◽  
The Impact

636 Background: Distant metastasis remains the most common causes to death of colon cancer. Thus it is crucial to identify the molecular markers associated with the progression and metastasis of this disease. Recent evidence for overexpression of FOXC1 in several types of human cancer suggests that it might play a key role in tumor biology. However, the clinical significance of FOXC1 signaling in human colon cancer pathogenesis remains unknown. Methods: We investigated FOXC1 expression in 203 cases of primary colon cancer and matched normal colon tissue and lymph node matastasis in a tissue array. The underlying mechanisms of altered FOXC1 expression and the impact of this altered expression on colon cancer growth and metastasis was explored both in vitro and in vivo. Results: We found elevated expression of FOXC1 protein in cancereous tissue and lymph node metastases than adjacent normal colonic tissues. Overexpression of FOXC1 was associated with higher clinical stage, T stage, lymph node metastasis and presence of distant metastasis. FOXC1 served as an independent prognostic marker whose expression levels correlated with poorer metastasis-free survival (MFS) and poorer overall survival (OS). A Cox proportional hazards model revealed that FOXC1 expression was an independent prognostic factor in multivariate analysis. Experimentally, FOXC1 silencing significantly inhibited the growth and metastasis of colon cancer cells in vitro and in vivo. FOXC1 transcriptionally regulates SNAIL1, contributing to epithelial-to-mesenchymal transition and metastasis in colon cancer cells. Conclusions: Dysregulated expression of FOXC1 may play a critical role in colon cancer progression and metastasis. Thus, FOXC1 may serve as a candidate prognostic biomarker and therapeutically targeted.

scholarly journals IMCA Induces Ferroptosis Mediated by SLC7A11 through the AMPK/mTOR Pathway in Colorectal Cancer

2020 ◽  
Vol 2020 ◽  
pp. 1-14 ◽  
Author(s):  
Lei Zhang ◽  
Wen Liu ◽  
Fangyan Liu ◽  
Qun Wang ◽  
Mengjiao Song ◽  
...  
Keyword(s):  
Colorectal Cancer ◽  
Cancer Cells ◽  
Screening Tests ◽  
Therapeutic Drug ◽  
Solute Carrier Family ◽  
Colorectal Cancer Cells ◽  
Solute Carrier ◽  
The Impact

Ferroptosis, implicated in several diseases, is a new form of programmed and nonapoptotic cell death triggered by iron-dependent lipid peroxidation after inactivation of the cystine/glutamate antiporter system xc–, which is composed of solute carrier family 7 membrane 11 (SLC7A11) and solute carrier family 3 membrane 2 (SLC3A2). Therefore, inducing ferroptosis through inhibiting the cystine/glutamate antiporter system xc– may be an effective way to treat cancer. In previous screening tests, we found that the benzopyran derivative 2-imino-6-methoxy-2H-chromene-3-carbothioamide (IMCA) significantly inhibited the viability of colorectal cancer cells. However, the impact of IMCA on ferroptosis remains unknown. Hence, this study investigated the effect of IMCA on ferroptosis and elucidated the underlying molecular mechanism. Results showed that IMCA significantly inhibited the cell viability of colorectal cancer cells in vitro and inhibited tumor growth with negligible organ toxicity in vivo. Further studies showed that IMCA significantly induced the ferroptosis of colorectal cancer cells. Mechanistically, IMCA downregulated the expression of SLC7A11 and decreased the contents of cysteine and glutathione, which resulted in reactive oxygen species accumulation and ferroptosis. Furthermore, overexpression of SLC7A11 significantly attenuated the ferroptosis caused by IMCA. In addition, IMCA regulated the activity of the AMPK/mTOR/p70S6k signaling pathway, which is related to the activity of SLC7A11 and ferroptosis. Collectively, our research provided experimental evidences on the activity and mechanism of ferroptosis induced by IMCA and revealed that IMCA might be a promising therapeutic drug for colorectal cancer.

scholarly journals Vitamin D as a Primer for Oncolytic Viral Therapy in Colon Cancer Models

2020 ◽  
Vol 21 (19) ◽  
pp. 7326
Author(s):  
Sang-In Kim ◽  
Shyambabu Chaurasiya ◽  
Anthony K. Park ◽  
Seonah Kang ◽  
Jianming Lu ◽  
...  
Keyword(s):  
Colon Cancer ◽  
Vitamin D ◽  
Human Colon ◽  
Oncolytic Viruses ◽  
Human Colon Cancer ◽  
Oncolytic Viral Therapy ◽  
Viral Therapy ◽  
The Impact

Oncolytic viroimmunotherapy is an exciting modality that can offer lasting anti-tumor immunity for aggressive malignancies like colon cancer. The impact of oncolytic viruses may be extended by combining them with agents to prime a tumor for viral susceptibility. This study investigates vitamin D analogue as an adjunct to oncolytic viral therapy for colon cancer. While vitamin D (VD) has historically been viewed as anti-viral, our in vitro investigations using human colon cancer cell lines showed that VD does not directly inhibit replication of recombinant chimeric poxvirus CF33. VD did restrict growth in HT29 but not HCT116 human colon cancer cells. In vivo investigations using HCT116 and HT29 xenograft models of colon cancer demonstrated that a VD analogue, calcipotriol, was additive with CF33-based viral therapy in VD-responsive HT29 but not in HCT116 tumors. Analyses of RNA-sequencing and gene expression data demonstrated a downregulation in the Jak-STAT signaling pathway with the addition of VD to viral therapy in HT29 models suggesting that the anti-inflammatory properties of VD may enhance the effects of viral therapy in some models. In conclusion, VD may prime oncolytic viral therapy in certain colon cancers.

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