Low expression of Beclin 1, associated with high Bcl-xL,predicts a malignant phenotype and poor prognosis of gastric cancer

Recent studies have suggested that dysregulation of autophagy plays a pivotal role in tumorigenesis. Here, we determined the prognostic value of autophagy-related protein Beclin 1 in gastric cancer. A total of 153 primary gastric cancer patients were subjected to analysis of Beclin 1 expression and survival prognosis. Among them, 68 patients were assigned randomly and used as a training set to generate a cutoff score for Beclin 1 expression by receive operating characteristic (ROC) curve analysis. The ROC-generated cutoff score was subjected to analyze the association of Beclin 1 with clinical characteristics and patient outcome. In a testing set (n = 85) and overall patients (n = 153), both univariate and multivariate analysis found that low expression of Beclin 1 predicted adverse overall survival and progression-free survival for gastric cancer patients. Furthermore, in each stage of gastric cancer patients, Beclin 1 expression was a prognostic indicator in patients with stage II, III and IV. Importantly, a reverse relationship between Beclin 1 and Bcl-xL expression was demonstrated. In patients of elevated Bcl-xL expression, a subset with lower Beclin 1 expression displayed an inferior overall survival and progression-free survival than those with higher Beclin 1 expression. Thus, our data demonstrated that low expression of Beclin 1, associated with high Bcl-xL, played as an independent biomarker, contributing to a more aggressive cancer cell phenotype and poor prognosis for gastric tumor.     

Low expression of Beclin 1, associated with high Bcl-xL, predicts a malignant phenotype and poor prognosis of gastric cancer

Recent studies have suggested that dysregulation of autophagy plays a pivotal role in tumorigenesis. Here, we determined the prognostic value of autophagy-related protein Beclin 1 in gastric cancer. A total of 153 primary gastric cancer patients were subjected to analysis of Beclin 1 expression and survival prognosis. Among them, 68 patients were assigned randomly and used as a training set to generate a cutoff score for Beclin 1 expression by receive operating characteristic (ROC) curve analysis. The ROC-generated cutoff score was subjected to analyze the association of Beclin 1 with clinical characteristics and patient outcome. In a testing set (n = 85) and overall patients (n = 153), both univariate and multivariate analysis found that low expression of Beclin 1 predicted adverse overall survival and progression-free survival for gastric cancer patients. Furthermore, in each stage of gastric cancer patients, Beclin 1 expression was a prognostic indicator in patients with stage II, III and IV. Importantly, a reverse relationship between Beclin 1 and Bcl-xL expression was demonstrated. In patients of elevated Bcl-xL expression, a subset with lower Beclin 1 expression displayed an inferior overall survival and progression-free survival than those with higher Beclin 1 expression. Thus, our data demonstrated that low expression of Beclin 1, associated with high Bcl-xL, played as an independent biomarker, contributing to a more aggressive cancer cell phenotype and poor prognosis for gastric tumor.     

Prognostic significance of lncRNA DANCR expression in human cancers: a systematic review and meta-analysis

Several studies demonstrated that lncRNA differentiation antagonizing non-protein coding RNA (lncRNA DANCR) expression might have the potential capacity to predict the cancer prognosis; however, definite conclusion has not been obtained. The aim of this meta-analysis was to evaluate the prognostic value of lncRNA DANCR expression in cancers. PubMed, Web of Science, Scopus, and Embase were comprehensively searched for relevant studies. Studies meeting all inclusion standards were included into this meta-analysis. The analysis of overall survival (OS), disease-free survival (DFS), or clinicopathological features was conducted. Total 11 studies containing 1154 cancer patients were analyzed in this meta-analysis. The results showed, compared with low lncRNA DANCR expression, high lncRNA DANCR expression was significantly associated with shorter OS (hazard ratio [HR] = 1.85; 95% CI = 1.52–2.26; P<0.01) and DFS (HR = 1.82; 95% CI = 1.43–2.32; P<0.01) in cancers. Besides, high lncRNA DANCR expression predicted deeper tumor invasion (P<0.01), earlier lymph node metastasis (P<0.01), earlier distant metastasis (P<0.01), and more advanced clinical stage (P<0.01) compared with low lncRNA DANCR expression in cancer populations. High lncRNA DANCR expression was associated with worse prognosis compared with low lncRNA DANCR expression in cancers. LncRNA DANCR expression could serve as a prognostic factor of human cancers.     

Prognostic Value of SMAD4 in Pancreatic Cancer: A Meta-Analysis

PURPOSE: The prognostic value of SMAD4 in pancreatic cancer has been evaluated in several studies. However, the conclusions remain controversial. Therefore, we aimed to evaluate the association between SMAD4 expression and the outcome of pancreatic cancer patients by performing a meta-analysis. METHODS: We systematically searched for relevant studies evaluating the relationship between SMAD4 expression and the outcome of pancreatic cancer patients until May 2015. A meta-analysis was performed using STATA 12.0, and pooled hazard ratios (HRs) with 95% confidence intervals (CIs) were used to estimate the strength of the association between SMAD4 expression and the prognosis of pancreatic cancer patients. RESULTS: The analysis included 1762 patients from 14 studies, with 1401 patients from 11 studies and 927 patients from 8 studies included in the univariate and multivariate analyses, respectively. Loss of SMAD4 expression was found to be significantly correlated with poor overall survival, with the combined HR (95% CI) of 1.20 (1.03-1.40). After adjusting for potential confounders using the Cox regression model, the pooled HR (95% CI) was 1.88 (1.31-2.70). In subgroup analysis, study region, number of patients, follow-up duration, and cutoff value were found to affect the significance of the association between loss of SMAD4 expression and poor prognosis. In addition, there was no evidence of publication bias, as suggested by Begg’s and Egger’s test. CONCLUSIONS: Loss of SMAD4 was associated with poor survival and was a negative prognostic indicator in patients with pancreatic cancer.     

HIF1A-AS2 predicts poor prognosis and regulates cell migration and invasion in triple-negative breast cancer

The aberrant expression of hypoxia-inducible factor 1 alpha (HIF1A)-antisense RNA 2 (HIF1A-AS2) was found in various human cancers including breast cancer. The aim of this study was to present more evidence about the role HIF1A-AS2 on triple-negative breast cancer (TNBC). In our results, HIF1A-AS2 was also found to be upregulated in TNBC tissues compared with non-TNBC tissues or adjacent normal tissues. Besides, HIF1A-AS2 expression was also elevated in TNBC cell lines compared with the normal breast epithelial cell line. Moreover, high expression of HIF1A-AS2 was associated with lymph node metastasis, distant metastasis and unfavorable histological grade in TNBC patients. Survival analysis showed a TNBC patient with high HIF1A-AS2 expression had shorter overall survival than patients with low HIF1A-AS2 expression, and HIF1A-AS2 high expression acted as an independent poor prognostic factor for overall survival in TNBC patients. The cell migration and invasion assays suggested inhibition of HIF1A-AS2 obviously depressed TNBC cell migration and invasion. In conclusion, HIF1A-AS2 serves as a novel biomarker for predicting clinical progression and prognosis in TNBC.     

Upregulated Expression of C-X-C Chemokine Receptor 4 Is an Independent Prognostic Predictor for Patients with Gastric Cancer

Aberrant chemokine (C-X-C motif) receptor CXCR4 expressions in malignant tissues have been reported, but its role in gastric cancer prognosis remains unknown. Our studies were designed to investigate the expression and prognostic significance of CXCR4 in patients with gastric cancer. CXCR4 expression was retrospectively analyzed by immunohistochemistry in 97 patients with gastric adenocarcinoma from China. Results were assessed for association with clinical features and overall survival by using Kaplan-Meier analysis. Prognostic values of CXCR4 expression and clinical outcomes were evaluated by Cox regression analysis. A molecular prognostic stratification scheme incorporating CXCR4 expression was determined by using receiver operating characteristic (ROC) analysis. The results show that CXCR4 predominantly localized in the cell membranes and cytoplasm. The protein level of CXCR4 was upregulation in gastric cancer tissues and upregulated expression of CXCR4 was only significantly associated with Lauren classification (P<0.001). Increased CXCR4 expression in gastric cancer tissues was positively correlated with poor overall survival of gastric cancer patients (P<0.001). Further multivariate Cox regression analysis suggested that intratumoral CXCR4 expression was an independent prognostic indicator for the disease. Applying the prognostic value of intratumoral CXCR4 density to TNM stage system showed a better prognostic value in patients with gastric cancer. In conclusion, intratumoral CXCR4 expression was recognized as an independent prognostic marker for the overall survival of patients with gastric cancer. On the basis of TNM stage, detection of CXCR4 expression will be helpful for predicting prognosis for patients with gastric cancer.     

Prognostic Value of Survivin in Patients with Gastric Cancer: A Systematic Review with Meta-Analysis

Objective

The prognostic significance of survivin for the survival of patients with gastric cancer remains controversial. Thus, the objective of this study was to conduct a systematic review of the literature evaluating survivin expression in gastric cancer as a prognostic indicator.

Methods

Relevant literature was searched using PubMed, EMBASE, and Chinese biomedicine databases. A meta-analysis of the association between survivin expression and overall survival in patients with gastric cancer was performed. Studies were pooled and summary hazard ratios (HRs) were calculated. Subgroup analyses were also conducted.

Results

Final analysis of 1365 patients from 16 eligible studies was performed. Combined HR suggested that survivin expression had an unfavorable impact on survival of gastric cancer patients (HR=1.39, 95% CI: 1.16-1.68). The unfavorable impact also appeared significant when stratified according to the studies categorized by patients’ ethnicity, detection methods, type of sample, and HR estimate. The combined HR in the English literature showed an inverse effect on survival (HR=1.40, 95% CI: 1.13-1.75), while HR in the non-English literature did not (HR=1.38, 95% CI: 0.93-2.05). When stratified according to the location of survivin expression, combined HR showed that expression in cytoplasm was significantly associated with poor prognosis of gastric cancer patients (HR=1.46, 95% CI: 1.12-1.90). While expression in nucleus was not significantly associated with poor prognosis (HR=1.29, 95% CI: 0.72-2.31), the heterogeneity was highly significant (chi-squared=11.5, I²=74%, p=0.009).

Conclusions

This study showed that survivin expression was associated with a poor prognosis in patients with gastric cancer. Cytoplasmic expression of survivin may be regarded as a prognostic factor for gastric cancer patients. In contrast, survivin expression in nucleus did not have a significant impact on patients’ overall survival.     

Clinical Significance of MiR-137 Expression in Patients with Gastric Cancer After Radical Gastrectomy

The dysregulation of miR-137 plays vital roles in the oncogenesis and progression of various types of cancer, but its role in prognosis of gastric cancer patients remains unknown. This study was designed to investigate the expression and prognostic significance of miR-137 in gastric cancer patients after radical gastrectomy. Quantitative real-time PCR (qRT-PCR) was performed to evaluate the expression of miR-137 in human gastric cancer cell lines and tissues in patients with gastric adenocarcinoma. Results were assessed for association with clinical factors and overall survival by using Kaplan-Meier analysis. Prognostic values of miR-137 expression and clinical outcomes were evaluated by Cox regression analysis. The results exhibited that the expression level of miR-137 was decreased in human gastric cancer cell lines and tissues, and down-regulated expression of miR-137 was associated with tumor cell differentiation, N stage, and TNM stage. Decreased miR-137 expression in gastric cancer tissues was positively correlated with poor overall survival of gastric cancer patients. Further multivariate Cox regression analysis suggested that miR-137 expression was an independent prognostic indicator for gastric cancer except for TNM stage. Applying the prognostic value of miR-137 expression to TNM stage III group showed a better risk stratification for overall survival. In conclusion, the results reinforced the critical role for the down-regulated miR-137 expression in gastric cancer and suggested that miR-137 expression could be a prognostic indicator for this disease. In addition, these patients with TNM stage III gastric cancer and low miR-137 expression might need more aggressive postoperative treatment and closer follow-up.     

Long non-coding RNA FTX predicts a poor prognosis of human cancers: a meta-analysis

Background: Several studies have assessed the relationship between long non-coding RNA five prime to Xist (FTX) expression, clinicopathological features, and survival outcomes in patients with cancer with conflicting results. This meta-analysis synthesized existing data to clarify the association between FTX with cancer prognosis.Methods: PubMed, Embase, Cochrane library, Web of Science, Chinese CNKI, and the Chinese WanFang databases were used to search for relevant studies. The role of FTX in cancers was evaluated by pooled odds ratios (ORs) and hazard ratios (HRs) with 95% confidence intervals (CIs).Results: Eleven studies comprising 1210 participants including colorectal cancer (CRC), hepatocellular carcinoma (HCC), gastric cancer (GC), renal cell carcinoma (RCC), osteosarcoma (OSC), and glioma were enrolled in this analysis. The meta-analysis showed that high FTX expression was significantly associated with several clinicopathological characteristics, including lymph node metastasis in patients with CRC, GC, HCC, and RCC, distant metastasis in patients with CRC, GC, HCC, and OSC, larger tumor size in patients with CRC, GC, HCC, RCC, and OSC, and subsequently TNM/clinical stage in patients with CRC, GC, HCC, OSC, and glioma. The pooled results from the survival analysis revealed a significant correlation between high FTX expression and shorter OS in patients with HCC, CRC, GC, OSC, and glioma. Further, FTX overexpression could be an independent predictive marker for shorter OS in patients with CRC, HCC, OSC, and glioma.Conclusions: FTX may be a potential oncogene, with high FTX expression being associated with a poorer prognosis in patients with CRC, HCC, OSC, and glioma.     

Long noncoding RNA Breast cancer antiestrogen resistance 4 is associated with cancer progression and its significant prognostic value

Breast cancer antiestrogen resistance 4 (BCAR4) is a novel long noncoding RNA. It was originally identified in a screen for genes responsible for the development of resistance to antiestrogens in breast cancer cells and plays a major role in various tumors. However, the clinical diagnostic role of BCAR4 in tumors is not completely understood. This current meta-analysis aimed to comprehensively explore the potential role of BCAR4 as a prognostic biomarker in a number of cancers. Five public databases PubMed, EMBASE, Web of Science, Wiley Online Library, and Medline were used to search for articles. Nine studies comprising 1,293 patients were included in this meta-analysis. The results of analysis showed that BCAR4 expression in human cancer was significantly associated with poor overall survival (hazard ratio [HR] = 1.98, confidence interval [CI]: [1.71–2.29]), p < 0.00001, and high BCAR4 expression was associated with clinical stage (OR and its 95% CI was 3.30 [1.99–5.46], p < 0.00001), distant metastasis (OR = 3.83, 95% CI: 2.15–6.82, p < 0.00001), and lymph node metastasis (OR and its 95% CI was 2.91 [1.62–5.25], p = 0.0004) in patients with cancer. Furthermore, the results revealed the prognostic significance of BCAR4 in gastrointestinal malignancy, breast cancer, and osteosarcoma (HR and its 95% CI were 2.05 [1.56–2.68], p < 0.00001; 1.78 [1.46–2.16], p < 0.00001; and 2.47 [1.41–4.34], p < 0.00001, respectively). This meta-analysis indicated the potential value of BCAR4 as a biomarker for predicting a poor prognosis in patients with cancer.     

Osteopontin in metastatic lesions as a prognostic marker in ovarian cancers

Summary Osteopontin (OPN) is expressed in various human cancers and associated with tumor progression, invasion and metastasis in many manners. The purpose of this study is to investigate the clinical significance of OPN expression in metastatic lesions of ovarian cancers, since the prognosis of the patients with peritoneal dissemination is extremely poor. In primary tumors and peritoneal metastatic lesions from 40 patients with stage III ovarian cancers, the protein levels of OPN and histoscores were determined by enzyme immunoassay and immunohistochemistry, respectively. Immunohistochemical staining revealed OPN was distributed in the cytoplasm and nuclear compartments of the cancer and stromal cells within and around the tumor. The OPN level was significantly (p < 0.05) increased in 32 of 40 metastatic lesions of ovarian cancers. The OPN increased cases identified by immunohistochemical staining were consistent with those identified by the sandwich immunoassay. The prognosis of the 32 patients with significant increase of OPN in ovarian cancers was extremely poor, whereas the 36-month survival rate of the 8 patients with no increase of OPN was 75%. Multivariate analysis revealed that the levels of OPN were independent predictors of prognosis from clinical characteristics (age, lesion size, histological types). OPN might be associated with peritoneal metastasis and its advancement, and that the OPN level in metastatic lesion may be a prognostic indicator in ovarian cancers.     

Prognostic Role of microRNA-21 in Colorectal Cancer: a Meta-Analysis

Background

To date, many studies have shown that microRNAs (miRNA) exhibit altered expression in various cancers and may play an important role as prognostic biomarker of cancers. The present meta-analysis summarizes the recent advances in the use of microRNA-21 (miR-21) in the assessment of colorectal cancer and analyzes the prognostic role of miR-21 for survival outcome.

Methodology/Principal Findings

The present meta-analysis was performed by searching PubMed through multiple search strategies. Data were extracted from studies comparing overall survival (OS) in patients with colorectal cancer who showed higher expression of miR-21 than similar patients. Pooled hazard ratios (HRs) of miR-21 for survival and 95% confidence intervals (CI) were calculated. Seven studies with a total of 1174 patients were included this meta-analysis. For overall survival (OS), the pooled hazard ratio (HR) of higher miR-21 expression in colorectal cancer was 1.76 (95% CI: 1.34–2.32, P=0.000). After elimination of heterogeneity, the pooled HR was 2.32 (95% CI: 1.82–2.97, P=0.000), which was found to significantly predict poorer survival. The subgroup analysis suggested that elevated miR-21 level and patients’ survival correlated with III/IV stage (HR=5.35, 95% CI: 3.73–7.66).

Conclusions/Significance

The present findings suggest that high expression of miR-21 might predict poor prognosis in patients with colorectal cancer.     

Long Noncoding RNA HOTAIR as an Independent Prognostic Marker in Cancer: A Meta-Analysis

Background

HOTAIR, a newly discovered long intergenic noncoding RNA (lincRNA), has been reported to be aberrantly expressed in many types of cancers. This meta-analysis summarizes its potential role as a biomarker in malignancy.

Methods

A quantitative meta-analysis was performed through a systematic search in Pubmed, Medline and Web of Science for eligible papers on the prognostic impact of HOTAIR in cancer from inception to Feb. 28, 2014. Pooled hazard ratios (HRs) with 95% confidence interval (95% CI) were calculated to summarize the effect.

Results

Nineteen studies were included in the study, with a total of 2033 patients. A significant association was observed between high HOTAIR expression and poor overall survival (OS) in patients with cancer (pooled HR 2.22, 95% CI: 1.68–2.93). Place of residence (Asian or Western countries), type of cancer (digestive or non-digestive disease), sample size (more or less than 100), and paper quality (score more or less than 85%) did not alter the significant predictive value of HOTAIR in OS from various kinds of cancer but preoperative status did. By combining HRs from Cox multivariate analyses, we found that HOTAIR expression was an independent prognostic factor for cancer patients (pooled HR 2.26, 95% CI: 1.62–3.15). Subgroup analysis showed that HOTAIR abundance was an independent prognostic factor for cancer metastasis (HR 3.90, 95% CI: 2.25–6.74). For esophageal carcinoma, high HOTAIR expression was significantly associated with TNM stage (III/IV vs. I/II: OR 6.90, 95% CI: 2.81–16.9) without heterogeneity. In gastric cancer, HOTAIR expression was found to be significantly associated with lymph node metastases (present vs. absent: OR 4.47, 95% CI: 1.88–10.63) and vessel invasion (positive vs. negative: OR 2.88, 95% CI: 1.38–6.04) without obvious heterogeneity.

Conclusions

HOTAIR abundance may serve as a novel predictive factor for poor prognosis in different types of cancers in both Asian and Western countries.     

Jab1/Cops5 contributes to chemoresistance in breast cancer by regulating Rad51

Jab1 overexpression correlates with poor prognosis in breast cancer patients, suggestting that targeting the aberrant Jab1 signaling in breast cancer could be a promising strategy. In the current study, we investigate the hypothesis that Jab1 positively regulates the DNA repair protein Rad51 and, in turn, the cellular response of breast cancer to chemotherapy with adriamycin and cisplatin. High-throughput mRNA sequencing (RNA-Seq) data from 113 normal and 1109 tumor tissues (obtained from TCGA) were integrated to our analysis to give further support to our findings. We found that Jab1 was overexpressed in adriamycin-resistant breast cancer cell MCF-7R compared with parental MCF-7 cells, and that knockdown of Jab1 expression conferred cellular sensitivity to adriamycin and cisplatin both in vivo and in vitro. By contrast, exogenous Jab1 expression enhanced the resistance of breast cancer cells to adriamycin and cisplatin. Moreover, we discovered that Jab1 positively regulated Rad51 in p53-dependent manner and that overexpression of Rad51 conferred cellular resistance to adriamycin and cisplatin in Jab1-deficient cells. Data from TCGA further validated an correlation between Jab1 and Rad51 in breast cancer, and elevated Jab1 and Rad51 associated with poor survival in breast cancer patients. Our findings indicate that Jab1 association with Rad51 plays an important role in cellular response to chemotherapy in breast cancer.     

LncRNA TP73-AS1 predicts poor prognosis and functions as oncogenic lncRNA in osteosarcoma

TP73 antisense RNA 1 (TP73-AS1), a novel long noncoding RNA (lncRNA), has been suggested to be deregulated in various human cancers and serve as a tumor suppressor or promoter, depending on tumor types. The role of TP73-AS1 in osteosarcoma is still unknown. In our results, TP73-AS1 was highly expressed in osteosarcoma tissue samples and cell lines compared with matching adjacent nontumor tissue specimens and a normal human osteoblast cell line, respectively. Moreover, high expression of TP73-AS1 was statistically associated with advanced Enneking stage, large tumor size, present distant metastasis, and poor histological grade, while exhibiting no statistical association with age, sex, and tumor site. The survival analyses showed that patients with osteosarcoma with high expression of TP73-AS1 obviously had lower overall survival than osteosarcoma patients with low expression of TP73-AS1, and high expression of TP73-AS1 was an independent poor prognostic factor for osteosarcoma patients. The experiments in vitro indicated that inhibition of TP73-AS1 expression depressed osteosarcoma cell viability, migration, and invasion, and arrested cell cycle. In conclusion, TP73-AS1 serves as oncogenic lncRNA participated in osteosarcoma progression.     

A systematic review and meta-analysis of higher expression of folate receptor alpha (FOLR1) predicts poor cancer prognosis

Abstract

Folate receptor alpha (FOLR1), a glycosylphosphatidylinositol-linked protein, is a well characterized folate transporter. However, the prognostic power of FOLR1 in cancer remains controversial. We conducted a meta-analysis to assess the prognostic roles of FOLR1 on different cancers. Twelve studies involving 4471 patients were included in this meta-analysis. The pooled analysis indicated that high FOLR1 significantly predicted poor overall survival (OS) (pooled hazard ratio (HR)?=?0.78, 95% confidence interval (CI)?=?0.64–0.94, p?=?0.009) and the disease-free survival (DFS) (HR?=?1.25, 95% CI?=?1.07–1.47, p?=?0.005). Subgroup analyses based on tumour type found that high FOLR1 level was associated with poor OS in breast cancer (HR?=?2.66, 95% CI?=?1.54–4.59, p?=?0.0005) and endometrial carcinoma (HR?=?1.30, 95% CI?=?1.05–1.61, p?=?0.02). However, FOLR1 has relatively weakly correlation with gender, tumour size and chemotherapy. Additionally, overexpression of FOLR1 was correlated with grade, FIGO stage, vital status and nodule status. The present meta-analysis indicated that the high expression of FOLR1 is associated with the poor survival of cancer patients, which is helpful for the clinical decision-making process.     

MMP14在非小细胞肺癌中的表达及其与预后的关系

肺癌5年生存率低,侵润和转移是导致其死亡的主要原因.探讨MMP14(matrix mettallo proteinase 14)在非小细胞肺癌(non-small cell lung cancer,NSCLC)组织中的表达,及其与浸润转移和预后的关系.通过采用免疫组化检测92例NSCLC组织及25例癌旁正常组织中MMP14蛋白的表达,并分析其与临床病理学特征及预后的关系.免疫组化结果显示,MMP14蛋白在NSCLC组织中表达的阳性率为64.1%(59/92),显著高于癌旁正常组8%(2/25)(P<0.001).MMP14阳性率与NSCLC的分化程度、T分期、淋巴结转移和TNM分期相关(P<0.05),而与性别、年龄、吸烟及组织学类型无关(P>0.05).Kaplan-meier生存曲线显示,MMP14阳性表达组的患者5年生存期显著低于阴性表达组(P=0.004).Cox多因素回归分析显示,MMP14不是NSCLC的独立预后因素(P>0.05).MMP14在肺癌的分化、浸润和转移中扮演着重要的作用,并对生存期有一定的影响.