Clinical efficacy of chidamide combined with BEAC preconditioning regimen in high-risk or refractory diffuse large B-cell lymphoma receiving autologous hematopoietic stem cell transplantation / 白血病·淋巴瘤

Objective:To investigate the clinical efficacy of chidamide combined with BEAC (camustine+etoposide+ cytarabine+cyclophosphamide) preconditioning regimen in high-risk or refractory diffuse large B-cell lymphoma (DLBCL) receiving autologous stem cell transplantation.Methods:The clinical data of 10 high-risk or refractory DLBCL patients with autologous stem cell transplantation after receiving chidamide combined with BEAC preconditioning regimen who were admitted to Xuzhou Central Hospital from March 2022 to May 2023 were retrospectively analyzed. The related complications during preconditioning and hematopoietic reconstruction process, the time of hematopoietic stem cell reconstruction after transplantation, and the short-term efficacy were summarized.Results:Of the 10 patients, 6 were women and 4 were men; the median age was 58 years old (27-68 years old). Hematopoietic reconstruction was achieved in all 10 patients after transplantation. The median time of neutrophil engraftment was 11 d (range 7-12 d), and the median time of platelet engraftment was 12 d (range 9-16 d) after transplantation. Hematological adverse reactions were described as follows: 2 cases had grade 3 febrile neutropenia, 1 case had grade 4 febrile neutropenia, 3 cases had grade 2 anemia, and 1 case had grade 3 anemia. Non-hematological adverse reactions were described as follows: 1 case had grade 2 nausea with vomiting, and 1 case had diarrhea. Eight patients were followed-up for >3 months after transplantation, 6 patients achieved complete remission, 1 patient achieved partial remission, and 1 patient with TP53 deletion developed disease progression 1 month after transplantation.Conclusions:Autologous hematopoietic stem cell transplantation with chidamide combined with BEAC preconditioning regimen is effective for patients with high-risk or refractory DLBCL, and the adverse reactions are tolerable.

Clinical treatment analysis of newly treated peripheral T-cell lymphoma / 白血病·淋巴瘤

Objective:To investigate the treatment methods of peripheral T-cell lymphoma (PTCL).Methods:The clinical data of 251 newly treated PTCL patients in the First Hospital of Jilin University from August 2011 to October 2021 were retrospectively analyzed, from which 168 patients were intercepted from February 2015 (the first targeted drug of PTCL, chidamide, was launched in China) to October 2021, among which 20 patients received chemotherapy combined with brentuximab vedotin (BV, BV group), 37 patients received chemotherapy combined with chidamide (chidamide group), and 111 patients received non-targeted therapy (non-targeted therapy group); all patients received ≥2 courses of treatment. Ten patients received autologous peripheral blood hematopoietic stem cell transplantation, with non-transplanted patients in the same period as controls. The clinical efficacy and prognosis of patients with different treatment methods were analyzed. Kaplan-Meier method was used for survival analysis and log-rank test was performed.Results:Of all 251 patients with PTCL, 26.7% (67/251) received targeted therapy in combination with chemotherapy. In the chidamide group, the efficacy could be evaluated in 36 cases, with an overall response rate (ORR) of 91.7% (33/36); in the non-targeted therapy group, the efficacy could be evaluated in 88 cases, with an ORR of 71.6% (63/88); in the BV group, 20 cases were evaluable, with an ORR of 75.0% (15/20). The difference in ORR between the non-targeted therapy group and the chidamide group was statistically significant ( χ2 = 5.89, P = 0.015), and the difference in ORR between the non-targeted therapy group and the BV group was not statistically significant ( χ2 = 0.09, P = 0.759). The 1-year progression-free survival (PFS) rates were 79.9%, 88.2% and 64.2%, and the 1-year overall survival (OS) rates were 85.7%, 89.7% and 70.1% in the chidamide, BV and non-targeted therapy groups, respectively; the PFS and OS in the chidamide and BV groups were better than those in the non-targeted therapy group (all P < 0.05), and the adverse effects were mostly tolerable. Among patients treated with chemotherapy combined with BV, the ORR of patients with CD30 expression rate <60% and ≥60% were 54.5% (6/11) and 100.0% (9/9), and the difference was statistically significant ( P = 0.038). In the 10 hematopoietic stem cell transplanted patients and 50 non-transplanted patients, 1-year PFS rates were 87.5% and 59.5%, 1-year OS rates were 90.0% and 67.1%, and the differences were not statistically significant (both P > 0.05). Conclusions:Chemotherapy-based combination therapy is the main treatment methods for PTCL, and chemotherapy combined with chidamide or BV targeted therapy and hematopoietic stem cell transplantation can improve the long-term survival of PTCL patients.

Study on the Regulation of Chidamide on CD8+T Cells in T-cell Acute Lymphoblastic Leukemia / 中国实验血液学杂志

OBJECTIVE@#To explore the regulatory effect of chidamide on CD8+ T cells in T-cell acute lymphoblastic leukemia.@*METHODS@#The expression levels of CXCL9 and CXCL3 mRNA in Jurkat cells, lymphocytes treated with chidamide and lymphocytes co-cultured with chidamide-treated Jurkat cells were detected by fluorescence quantitative PCR. The proportion of CD8+ T cells in lymphocytes treated with chidamide and lymphocytes co-cultured with chidamide-treated Jurkat cells was determined by flow cytometry.@*RESULTS@#Chidamide upregulated CXCL9 mRNA expression in Jurkat cell line in a dose-dependent manner (r=0.950). The mRNA expression of CXCL9 in chidamide 5 μmol/L group was 164 times higher than that in control group. Chidamide upregulated CXCL9 mRNA expression in lymphocytes, but the up-regulated level was significantly lower than that in Jurkat cell line treated with the same concentration of chidamide. Co-culture with chidamide treated Jurkat cells upregulated the proportion of CD8+ T cells in lymphocytes.@*CONCLUSION@#In T-cell acute lymphoblastic leukemia, chidamide may increase the concentration of CXCL9 in the tumor microenvironment by up-regulating the expression of CXCL9 in tumor cells, leading to an increase in the number of CD8+ T cells.

Chidamide inhibits the NOTCH1-MYC signaling axis in T-cell acute lymphoblastic leukemia / 医学前沿

T-cell acute lymphoblastic leukemia (T-ALL) is one of the most dangerous hematological malignancies, with high tumor heterogeneity and poor prognosis. More than 60% of T-ALL patients carry NOTCH1 gene mutations, leading to abnormal expression of downstream target genes and aberrant activation of various signaling pathways. We found that chidamide, an HDAC inhibitor, exerts an antitumor effect on T-ALL cell lines and primary cells including an anti-NOTCH1 activity. In particular, chidamide inhibits the NOTCH1-MYC signaling axis by down-regulating the level of the intracellular form of NOTCH1 (NICD1) as well as MYC, partly through their ubiquitination and degradation by the proteasome pathway. We also report here the preliminary results of our clinical trial supporting that a treatment by chidamide reduces minimal residual disease (MRD) in patients and is well tolerated. Our results highlight the effectiveness and safety of chidamide in the treatment of T-ALL patients, including those with NOTCH1 mutations and open the way to a new therapeutic strategy for these patients.

The development of folate modified squalene-chidamide prodrug self-assembled nanoparticles to enhance the drug delivery in pancreatic cancer microenvironment / 药学学报

This research aimed at the key issue that chemical drugs and Chinese medicine hydrophilic small molecule anti-tumor drugs were difficult to break through the dense interstitial permeability barrier of pancreatic cancer to achieve the key problem of drug efficacy in the deep part of tumor tissue. To solve this problem, the lipophilic molecule squalene (SQ) and the hydrophilic anti-tumor drug chidamide (CHI) were linked by a trypsin responsive bond to form a prodrug (SQ-CHI) and a folic acid modified prodrug self-assembled nanoparticles (FA-SQ-CHI NPs) were further developed. The feature of prodrug molecules and nanoparticles were characterized. The in vitro release characteristics and cytotoxicity of blank vector were investigated. The efficacy and permeability of the prodrug nanoparticles in the PSN-1 monolayer cell and PSN-1/HSPC co-cultured tumor spheroids model was evaluated. The results showed that SQ-CHI prodrug molecules and FA-SQ-CHI NPs were successfully developed. The nanoparticles were regular spherical, well-dispersed, with a particle size of (173.3 ± 1.5) nm, a drug load of (59.02 ± 0.8) % and showed trypsin responsive release ability. The prodrug nanoparticles can significantly enhance the penetration and anti-proliferation effects of CHI in the PSN-1/HSPC tumor spheroids. In conclusion, the construction of folic acid-modified SQ-CHI prodrug self-assembled nanoparticles can significantly enhance the penetration of CHI in the pancreatic cancer microenvironment in vitro. This research would provide a new idea for the construction of targeted drug delivery system for chemical drugs and Chinese medicine hydrophilic small molecule drugs in the application of anti-pancreatic cancer.

Chidamide monotherapy for the treatment of hepatosplenic T-cell lymphoma: A case report / 中国输血杂志

【Objective】 To generate an efficacious therapeutic plan for patients suffered from Hepatosplenic T-cell lymphoma (HSTCL). 【Methods】 A patient diagnosed as HSTCL in January 2016 was observed, and chidamide monotherapy instead of traditional chemotherapy was applied due to the poor performance and infection complications of the patient. 【Results】 The patient has achieved amazing improvements after chidamide monotherapy. As we followed and evaluated recently, the patient has received partial remission and can keep durable remission for 4 years. 【Conclusion】 The study suggests that chidamide is a novel therapeuticchoice in patients with HSTCL.

Effect of chidamide combined with matrine on proliferation and apoptosis of cutaneous T-cell lymphoma cell lines HH and Hut78 and possible apoptotic mechanisms / 中华皮肤科杂志

Objective To evaluate the effect of chidamide combined with matrine on proliferation and apoptosis of cutaneous T-cell lymphoma (CTCL) cell lines HH and Hut78,and to explore their apoptotic mechanisms.Methods Both HH and Hut78 cells were treated with 0.4 μmol/L chidamide and 0.6 g/L matrine alone or in combination for 24,48 and 72 hours,with those treated with dimethyl sulfoxide (DMSO) serving as control groups.MTS assay was performed to deteet cellular proliferation rates of HH and Hut78 cells at each time point.After 48-hour treatment,flow cytometry was conducted to detect cell apoptosis,and Western blot analysis to determine expression of apoptosis-related proteins in these cells.Statistical analysis was carried out by using repeated measures analysis of variance,one-way analysis of variance,and least significant difference (LSD)-t test for multiple comparisons.Results Compared with DMSO,chidamide and matrine alone or in combination could inhibit the proliferation of HH and Hut78cells to different extents (F =15.88,558.26,P ＜ 0.05,＜ 0.001,respectively).At 48 hours,the apoptosis rate in Hut78 cells was significantly higher in the matrine group (20.98％ ± 1.53％),chidamide group (22.44％ ± 7.74％) and combination group (44.53％ ± 1.85％) than in the control group (8.42％ ± 4.23％;LSD-t =4.76,5.31,13.69 respectively,all P ＜ 0.05),as well as in the combination group than in the matrine group and chidamide group (LSD-t =8.93,8.37 respectively,both P ＜ 0.01);no significant differences were observed in the apoptosis rate of HH cells between the matrine group (13.98％ ± 3.86％)or chidamide group (13.61％ ± 1.62％) and control group (11.44％ ± 1.43％,both P ＞ 0.05),while the combination group (20.94％ ± 0.64％) showed a significantly higher apoptosis rate compared with the control group,matrine group and chidamide group (LSD-t =7.37,5.40,5.69 respectively,all P ＜ 0.05).In the case of HH cells,the combination group showed significantly higher cleaved caspase-3 expression (all P ＜ 0.05),but significantly lower protein expression of E-cadherin,nuclear factor (NF)-κB,phosphorylated-Bad (p-Bad) and Bcl-2 compared with the other 3 groups (all P ＜ 0.05).In the case of Hut78 cells,the expression of E-cadherin,NF-κB,p-Bad and Bcl-2 was significantly lower in the matrine group,chidamide group and combination group than in the control group (all P ＜ 0.05),while cleaved caspase-3 expression was significantly higher in the chidamide group and combination group than in the control group (both P ＜0.05).No matter in HH cells or in Hut78 cells,there were no significant differences in Bad protein expression between the 4 groups (all P ＞ 0.05).Conclusion Chidamide in combination with matrine can inhibit proliferation and induce apoptosis of HH and Hut78 cells,likely by regulating the expression of apoptosis-related proteins E-cadherin,NF-κB,p-Bad,Bcl-2 and cleaved caspase-3.

Enhancing effect of chidamide on the sensibility of human chronic myeloid leukemia K562/ADM cells to daunorubicin / 解剖学报

Objective: To investigate whether chidamide (CDM) could influence the sensibility of human chronic myeloid leukemia K562/ADM cells to daunorubicin (DNR) and its possible mechanism. Methods: The K562 and K562/ADM cells were cultured in vitro and treated with CDM and(or) DNR for 48 hous, and then the cell viability was measured by cell counting kit-8(CCK-8) assay. The proliferation, cell cycle and apoptosis were analyzed by flow cytometry. Western blotting was performed to measure the protein levels of histon 2AX (H2AX), γH2AX (Serl39), ataxia telangiectasia mutated gene (ATM), p-ATM (Serl981), breast cancer susceptibility protein 1(BRCAl), and p-BRCAl (Serl524). Results: DNR remarkably inhibited the cell activity of K562/ADM cells in dose-dependent manner with a half maximal inhibitory concentration(IC50) value of 11.76 μmol/L, and the resistant factor was 18.09. Co-treatment with CMD and DNR produced a synergistic effect confidence interval(GI) (CI<1) with a reversal fold of 8.11. DNR remarkably inhibited proliferation (P<0.05), induced G2/M phase arrest and apoptosis (P<0.05), these effects were enhanced under non-toxic concentration of CMD (P<0.05). K562/ADM cells had a significantly higher protein levels of ATM and BRCA1 than K562 cells (P<0.05). DNR significantly up-regulated the protein levels of γH2AX, p-ATM and p-BRCAl (P<0.05), and the protein level of γH2AX appeared higher in the combination group compared to DNR alone (P<0.05); however, the co-treatment with CMD and DNR induced a decreased expression of p-ATM and p-BRCAl than the DNR alone (P< 0.05). Conclusion: CDM may enhance the sensibility of K562/ADM cells to DNR by up-regulating the protein level of γH2AX, and down-regulating the protein levels of p-ATM and p-BRCAl.

Chidamide enhances inhibitory effect on colon cancer cells by modulating tumorassociated macrophage / 中国肿瘤生物治疗杂志

@# Objective: To investigate the effect of tumor-associated macrophage (TAM) on the anti-tumor function of chidamide and to explore the mechanism. Methods: Mouse macrophage cell linesAna1 and Raw264.7 were cultured in vitro and induced into TAM with tumor supernatant. HDAC enzyme activity was detected after TAM treated with chidamide. The mRNA expressions of cytokines, such as IL-6, IL-12,TNF and IL-1β, in TAM were detected by qPCR. The protein expression of NF-κB and STAT3 in TAM treated with chidamide were detected by Wb. The mixture of TAM and colon cancer CT26 cells was inoculated into nude mice to construct the subcutaneous xenograft model; and the efficacy of chidamide (3.87 mg/kg) on the growth of CT26 xenograft tumors was observed. The protein expressions of PCNA, F4/80, Arg1 and iNos were detected by immunohistochemistry. Results: Chidamide inhibited the proliferation of CT26 cells. In the in vivo experiment, the inhibition rate of chidamide alone on CT26 xenograft was about 18.7%; however, the inhibition rate was up to 57.2% with the presence of TAM. Chidamide could inhibit the activity of HDAC enzyme in TAM, and further increase the Histone acetylation level. Chidamide could affect the expression of nuclear transcription factor NF-κB, inhibit the expressions of Arg1, IL-6 and IL-12, but up-regulate the expressions of iNOS, TNF and IL-1β in TAM. Conclusion: Chidamide can enhance its inhibitory effect on colon cancer CT26 cells via regulating the expression of cytokines and inhibiting the activity of HDAC in TAM.

Chemotherapy after chidamide pretreatment in high-risk and refractory lymphoid malignancy: report of 16 cases / 白血病·淋巴瘤

Objective To analyze the therapeutic effects of chemotherapy after chidamide pretreatment in 16 cases of high-risk and refractory lymphoid malignancy. Methods The efficacy and adverse reactions of 16 patients with high-risk and refractory lymphoid malignancy who received chidamide combined with chemotherapy after 3 days pretreatment of chidamide were analyzed. Results Sixteen patients included 6 males and 10 females, and the median age was 49.5 years old (23-88 years old). The median course of previous systemic chemotherapy was 4 (range 0-22). Among 14 patients who received induction chemotherapy, 7 patients achieved complete remission (CR), 7 patients achieved partial remission (PR). Fourteen patients had achieved clinical efficacy, and the overall response rate (ORR) was 100 %. After 2 cases had remission , the patients who entered this regimen for consolidation chemotherapy also had durable CR. The median follow-up time was 13 months (range 2-24 months) until December 2017. Nine cases had overall survival (OS), 7 cases died and 9 cases had progression-free survival. Common adverse effects of the chemotherapy included mild and controllable gastrointestinal reactions after chidamide. Conclusion Chemotherapy after chidamide pretreatment may improve the effect and prognosis of high-risk or refractory lymphoid malignancy.

Regulation of energy metabolism in colon cancer cells by chidamide / 中国药理学与毒理学杂志

OBJECTIVE To observe the regulation effect of chidamide on energy metabolism in HCT-8 and HT-29 cells. METHODS HCT-8 and HT-29 cells were treated with chidamide 5,10 and 20 μmol · L-1. Morphological changes of these cells were observed under an ordinary optical microscope. Cell proliferation was detected by MTT. ATP production was determined by CellTiter-Glo? assay kit. Metabolic changes were tested by glycolytic stress kit. The mRNA level of lactate dehydrogenase A (LDH-A)was analyzed by real-time quantitative PCR,whereas the protein level of LDH-A was analyzed by Western blotting. RESULTS Compared with control group,cell morphology of HCT-8 and HT-29 cells in chidamide treated group was irregular,accompanied by deformation,shrinkage and cell debris, and the inhibitory rate of proliferation increased(P<0.05). There was no significant difference in ATP total content between chidamide 5 and 10 μmol · L-1 16 h treatment groups,but in chidamide 20 μmol · L-1 treatment group it was decreased(P<0.05). Chidamide 20μmol · L-1 had no effect on oxygen consumption rate, but glycolysis ATP generation rate was reduced by 30.7% and 37.9%(P<0.05),respectively. Chidamide 20μmol · L-1 had no effect on LDH-A mRNA level,but it decreased the protein level of LDH-A(P<0.01). CONCLUSION Chidamide can abate the respiratory metabolic ability of HCT-8 and HT-29 cells. The mechanism may be related to the down-regulation of LDH-A.

Effects of chidamide combined with curcumin on human cutaneous T-cell lymphoma cell line Hut78 and their molecular mechanisms / 中华皮肤科杂志

Objective To evaluate the inhibitory effect of chidamide combined with curcumin on the proliferation of cutaneous T-cell lymphoma (CTCL) cell line Hut78,as well as their promotive effect on its apoptosis,and to explore their therapeutic mechanisms in CTCL.Methods Some Hut78 cells were treated with different concentrations of chidamide (0.3,0.6,1.2,2.4 μmol/L) and 10 μ mol/L curcumin alone or the combination of 1.2 μ mol/L chidamide and 10 μ mol/L curcumin for 24,48 and 72 hours separately.MTS assay was conducted to estimate cell viability at each time point.After selection of chidamide concentrations,some Hut78 cells were treated with chidamide (0.6 and 1.2 μmol/L)and curcumin (10 μmol/L) alone or in combination (1.2 μmol/L chidamide and 10 μmol/L curcumin) for 24 hours,then,flow cytometry was performed to detect cell apoptosis and analyze cell cycle,real-time (RT)-PCR and Western-blot analysis were conducted to quantify the mRNA and protein expressions of apoptosis-associated genes Fas,caspase 8,nuclear factor (NF)-κB p65 as well as cell cycle-associated genes P21,CDK2 and cyclin E respectively.Statistical analysis was carried out by repeated-measures analysis of variance,one-way analysis of variance and the least significant difference (LSD)-t test.Results Chidamide could significantly inhibit the proliferation of Hut78 cells in a dosedependent and time-dependent manner (F =266.558,564.966,respectively,both P ＜ 0.001).After 48-and 72-hour culture,the combination of 1.2 μmol/L chidamide and 10 μmol/L curcumin showed significantly stronger inhibitory effect on cell proliferation compared with 1.2 μmol/L chidamide or 10 μmol/L curcumin alone (all P ＜ 0.001).As flow cytometry showed,the percentage of apoptotic cells was significantly higher in the combined treatment group than in the 0.6-,1.2-μmol/L chidamide groups and 10-μmol/L curcumin group (all P ＜ 0.001).Compared with the 0.6-μmol/L chidamide group and 10-μmol/L curcumin group,the combined treatment group and 1.2-μmol/L ehidamide group both showed significantly increased proportion of cells at G0/G1 phase and mRNA expressions of Fas,caspase 8 and P21,but decreased proportion of cells at S phase or G2/M phase and mRNA expressions of NF-κB p65,CDK2 and cyclin E (P ＜0.05 for proportion of cells at different phases,P ＜ 0.001 for mRNA expressions of different genes).Furthermore,the mRNA expression of Fas was significantly higher in the combined treatment group than in the 1.2-μmol/L chidamide group (P ＜ 0.001),while no significant differences were observed in the mRNA expressions of caspase 8,P21,NF-κB p65,CDK2 and cyclin E or the proportion of cells at any phase between the combined treatment group and 1.2-μmol/L chidamide group (all P ＞ 0.05).Western-blot analysis showed that protein expressions of Fas,caspase 8 and P21 significantly increased,but those of NF-κB p65,CDK2 and cyclin E significantly decreased in the combined treatment group compared with the 0.6-,1.2-μmol/L chidamide groups and blank control group receiving no treatment,which were in accordance with the above changes in mRNA expressions of these genes.Conclusion Chidamide can inhibit the growth of the CTCL cell line Hut78 by directly decelerating cell proliferation and inducing cell apoptosis,and the combibation with curcumin can markedly enhance the inhibitory effect of chidamide on the growth of Hut78 cells.

In vitro anti-proliferation effect of a histone deacetylase inhibitor,chidamide,on a malignant melanoma cell line,A375 / 中华皮肤科杂志

Objective To investigate the in vitro anti-proliferation effect of a histone deacetylase inhibitor,chidamide,on a cutaneous malignant melanoma cell line,A375.Methods Cultured A375 cells were treated with different concentrations of chidamide(5,10,50,100,500 μmol/L)and aichostatin A (TSA)(0.1,0.25,0.5,1.0 μmol/L),respectively,for various durations(24,48,72,96,120 hours).Subsequently,cell proliferation,apoptosis and cell cycle were detected by MTT assay,annexin Vfluorescein isothiocyanate and propidium iodide double staining,and DNA ploid analysis,respectively.Results The proliferation of A375 cells was inhibited in a dose-dependent manner by chidamide of 5-500μmol/L and TSA of 0.1-1 μmol/L,and in a time-dependent manner from 0 to 120 hours after the beginning of trealment with ehidamide of 5-500μmol/L and TSA of 0.25-1μmol/L.The 48-hour 50% growth inhibition concentration(IC50)of ehidamide and TSA on A375 cells was about 250 μmol/L and 0.7μmol/L,respectively.After 48-hour treatment,the apoptosis mte was 80.27%±3.06%,79.53%±5.70%,83.13%±6.90%in A375 cells treated with chidamide of 62.5,125,250 μmol/L,respectively,16.27%±2.46%,28.83%±2.55%,83.40%±8.65%in those treated with TSA of 0.175,0.35,0.7 μmol/L,respectively,10.43%±0.96%in ontreated cells;a statistical increase was noticed in chidamide-treated cells and TSA-treated cells vs.untreated cells(all P＜0.001).A positive correlation was observed between the apoptosis rate and concentrations of TSA(r=0.955,P=0.000).Cell cycle analysis indicated that treatment with chidamide induced cell cycle arrest in G0/G1 phase,with the cell proportion in G0/G1 phase being 76.30%±6.06%,82.79%±0.74%,88.91%±5.29%in A375 cells treated with chidamide of 62.5,125,250μmol/L,respectively,versus 38.73%±3.36%in untreated cells.While after 48-hour treatment with TSA of 0.35 and 0.7 μmol/L,the proportion of cells in G2/M phases was 25.15%±2.71%and 58.71%±3.45%,respectively,compared to 15.73%±0.23%in untreated cells(P＜0.01).Conclusion Chidamide and TSA could induce cell cycle arrest and apoptosis,as well as inhibit the growth of A375 ceils in vitro.