TP53 loss creates therapeutic vulnerability in colorectal cancer.

TP53, a well-known tumour suppressor gene that encodes p53, is frequently inactivated by mutation or deletion in most human tumours. A tremendous effort has been made to restore p53 activity in cancer therapies. However, no effective p53-based therapy has been successfully translated into clinical cancer treatment owing to the complexity of p53 signalling. Here we demonstrate that genomic deletion of TP53 frequently encompasses essential neighbouring genes, rendering cancer cells with hemizygous TP53 deletion vulnerable to further suppression of such genes. POLR2A is identified as such a gene that is almost always co-deleted with TP53 in human cancers. It encodes the largest and catalytic subunit of the RNA polymerase II complex, which is specifically inhibited by α-amanitin. Our analysis of The Cancer Genome Atlas (TCGA) and Cancer Cell Line Encyclopedia (CCLE) databases reveals that POLR2A expression levels are tightly correlated with its gene copy numbers in human colorectal cancer. Suppression of POLR2A with α-amanitin or small interfering RNAs selectively inhibits the proliferation, survival and tumorigenic potential of colorectal cancer cells with hemizygous TP53 loss in a p53-independent manner. Previous clinical applications of α-amanitin have been limited owing to its liver toxicity. However, we found that α-amanitin-based antibody-drug conjugates are highly effective therapeutic agents with reduced toxicity. Here we show that low doses of α-amanitin-conjugated anti-epithelial cell adhesion molecule (EpCAM) antibody lead to complete tumour regression in mouse models of human colorectal cancer with hemizygous deletion of POLR2A. We anticipate that inhibiting POLR2A will be a new therapeutic approach for human cancers containing such common genomic alterations.

Lyoprotectant Optimization for the Freeze-Drying of Receptor-Targeted Trojan Horse Liposomes for Plasmid DNA Delivery.

Trojan horse liposomes (THLs) are a form of ligand-targeted nanomedicine, where a plasmid DNA is encapsulated in the interior of a 100-150 nm pegylated liposome, and the tips of a fraction of the surface pegylated strands are covalently linked to a receptor-specific monoclonal antibody (MAb) via a thio-ether linkage. The goal of this work was to develop a lyophilization methodology that enables retention of the structure and function of the THLs following the freeze-drying/hydration process. THL fusion and leakage of plasmid DNA were observed with several lyoprotectants, including trehalose, hyaluronic acid, γ-cyclodextrin, or sulfobutylether-ß-cyclodextrin. However, the use of hydroxypropyl-γ-cyclodextrin, at a 40:1 wt/wt ratio relative to the THL phospholipid, eliminated liposome fusion and produced high retention of encapsulated plasmid DNA and THL-mediated gene expression after lyophilization followed by hydration. The freeze-dried THL cake was amorphous without cavitation, and the diameters and functional properties of the THLs were preserved following hydration of cakes stored for at least six months. Intravenous administration of the hydrated freeze-dried THLs in the Rhesus monkey demonstrated the safety of the formulation. Blood plasmid DNA was measured with a quantitative polymerase chain reaction method, which enabled a pharmacokinetics analysis of the blood clearance of the THL-encapsulated plasmid DNA in the primate. The work shows that optimization of the lyoprotectant enables long-term storage of the MAb-targeted DNA encapsulated liposomes in the freeze-dried state.

[Quantitative determination of nine furanocoumarins for quality evaluation of Angelica dahurica from different habitats].

A UPLC method has been developed for simultaneous determination of nine furanocoumarins of Angelica dahurics,and was used for quality evaluation of A. dahurica from different habitats. ACQUITY UPLC BEH C18 chromatographic column was employed,the separation was performed with the mobile phase consisting of acetonitrile and water,and the detection wavelength was set at254 nm. This method was used to simultaneously determine the content of xanthotoxol,oxypeucedaninhydrate,byak-angelicin,psoralen,xanthotoxin,bergapten,oxypeucedanin,imperatorin and isoimperatorin in A. dahurica from different habitats. Then,the further quality assessment of the drug was carried out by similarity evaluation,cluster analysis( CA),principal component analysis( PCA),and orthogonal partial least squares discriminant analysis( OPLS-DA). The content order of measured furanocoumarins from high to low was: oxypeucedanin>imperatorin>isoimperatorin>oxypeucedaninhydrate>bergapten>byak-angelicin>xanthotoxin>xanthotoxol>psoralen,with the mean content 2. 844,1. 277,0. 649 2,0. 216 2,0. 129 8,0. 062 68,0. 052 68,0. 019 30,0. 018 19 mg·g-1,respectively. There were difference between the batches of the drug,and the quality was influenced by smouldering sulphur based on the results of chemical pattern recognition and content determination. Finally,six active ingredients were recognized as the quality makers using OPLS-DA method. The validated UPLC fingerprint combined with chemical pattern recognition method can be used in the quality control and evaluation of A. dahurica.

Metabolic shifts toward glutamine regulate tumor growth, invasion and bioenergetics in ovarian cancer.

Glutamine can play a critical role in cellular growth in multiple cancers. Glutamine-addicted cancer cells are dependent on glutamine for viability, and their metabolism is reprogrammed for glutamine utilization through the tricarboxylic acid (TCA) cycle. Here, we have uncovered a missing link between cancer invasiveness and glutamine dependence. Using isotope tracer and bioenergetic analysis, we found that low-invasive ovarian cancer (OVCA) cells are glutamine independent, whereas high-invasive OVCA cells are markedly glutamine dependent. Consistent with our findings, OVCA patients' microarray data suggest that glutaminolysis correlates with poor survival. Notably, the ratio of gene expression associated with glutamine anabolism versus catabolism has emerged as a novel biomarker for patient prognosis. Significantly, we found that glutamine regulates the activation of STAT3, a mediator of signaling pathways which regulates cancer hallmarks in invasive OVCA cells. Our findings suggest that a combined approach of targeting high-invasive OVCA cells by blocking glutamine's entry into the TCA cycle, along with targeting low-invasive OVCA cells by inhibiting glutamine synthesis and STAT3 may lead to potential therapeutic approaches for treating OVCAs.

Exploiting synthetic biology platforms for enhanced biosynthesis of natural products in Yarrowia lipolytica.

With the rapid development of synthetic biology, researchers can design, modify, or even synthesize microorganisms de novo, and microorganisms endowed with unnatural functions can be considered "artificial life" and facilitate the development of functional products. Based on this concept, researchers can solve critical problems related to the insufficient supply of natural products, such as low yields, long production cycles, and cumbersome procedures. Due to its superior performance and unique physiological and biochemical characteristics, Yarrowia lipolytica is a favorable chassis cell used for green biomanufacturing by numerous researchers. This paper mainly reviews the development of synthetic biology techniques for Y. lipolytica and summarizes the recent research progress on the synthesis of natural products in Y. lipolytica. This review will promote the continued innovative development of Y. lipolytica by providing theoretical guidance for research on the biosynthesis of natural products.

First proteomic exploration of protein-encoding genes on chromosome 1 in human liver, stomach, and colon.

The launch of the Chromosome-Centric Human Proteome Project provides an opportunity to gain insight into the human proteome. The Chinese Human Chromosome Proteome Consortium has initiated proteomic exploration of protein-encoding genes on human chromosomes 1, 8, and 20. Collaboration within the consortium has generated a comprehensive proteome data set using normal and carcinomatous tissues from human liver, stomach, and colon and 13 cell lines originating in these organs. We identified 12,101 proteins (59.8% coverage against Swiss-Prot human entries) with a protein false discovery rate of less than 1%. On chromosome 1, 1,252 proteins mapping to 1,227 genes, representing 60.9% of Swiss-Prot entries, were identified; however, 805 proteins remain unidentified, suggesting that analysis of more diverse samples using more advanced proteomic technologies is required. Genes encoding the unidentified proteins were concentrated in seven blocks, located at p36, q12-21, and q42-44, partly consistent with correlation of these blocks with cancers of the liver, stomach, and colon. Combined transcriptome, proteome, and cofunctionality analyses confirmed 23 coexpression clusters containing 165 genes. Biological information, including chromosome structure, GC content, and protein coexpression pattern was analyzed using multilayered, circular visualization and tabular visualization. Details of data analysis and updates are available in the Chinese Chromosome-Centric Human Proteome Database ( http://proteomeview.hupo.org.cn/chromosome/ ).

Qualitative and quantitative expression status of the human chromosome 20 genes in cancer tissues and the representative cell lines.

Under the guidance of the Chromosome-centric Human Proteome Project (C-HPP), (1, 2) we conducted a systematic survey of the expression status of genes located at human chromosome 20 (Chr.20) in three cancer tissues, gastric, colon, and liver carcinoma, and their representative cell lines. We have globally profiled proteomes in these samples with combined technology of LC-MS/MS and acquired the corresponding mRNA information upon RNA-seq and RNAchip. In total, 323 unique proteins were identified, covering 60% of the coding genes (323/547) in Chr.20. With regards to qualitative information of proteomics, we overall evaluated the correlation of the identified Chr.20 proteins with target genes of transcription factors or of microRNA, conserved genes and cancer-related genes. As for quantitative information, the expression abundances of Chr.20 genes were found to be almost consistent in both tissues and cell lines of mRNA in all individual chromosome regions, whereas those of Chr.20 proteins in cells are different from tissues, especially in the region of 20q13.33. Furthermore, the abundances of Chr.20 proteins were hierarchically evaluated according to tissue- or cancer-related distribution. The analysis revealed several cancer-related proteins in Chr.20 are tissue- or cell-type dependent. With integration of all the acquired data, for the first time we established a solid database of the Chr.20 proteome.

Three-stage fermentation of the feed and the application on weaned piglets.

Numerous studies have demonstrated that soybean meal (SBM) contains high levels of anti-nutritional factors, which interrupt gastrointestinal homeostasis or metabolism normally of the weaned piglets. Here, the mixed probiotics, including Bacillus licheniformis (B. licheniformis, CGMCC 8147), Saccharomyces cerevisiae H11 (S. cerevisiae H11) and Lactobacillus casei (L. casei, CGMCC 8149) were applied to the three-stage fermentation of functional feed. Our research investigated the optimum ratio of inoculation, optimal time of inoculation, combination of substrates, and nutritional value of the fermented feed. The optimal microbial combination was B. licheniformis: S. cerevisiae: L. casei = 2:2:1, inoculating at 0, 12 and 24 h, respectively. The results revealed that crude protein and acid-soluble protein were remarkably improved and had lower pH. Trypsin inhibitor, glycine and ß-glycine were reduced by 79.86, 77.18, and 69.29%, respectively. Moreover, animal trials further evaluated the growth-promoting effects of the fermented feed. It was noted that the average daily gain of weaned piglets was significantly higher, and the ratio of feed with weight, diarrhea incidence and mortality were lower significantly. The concentrations of serum immunoglobulin G(IgG), IgA, IgM, Complement C3 and interferon-γ (IFN-γ), and lysozyme activity were all increased. The relative abundance of fecal microbiota improved, especially lactobacillus, which increased the abundance of fecal dominant probiotics. Overall, the fermented feed may be conducive to the growth and health of weaned piglets by improving nutritional value, immunity properties, relative abundance of fecal microflora, and decreasing anti-nutritional factors of feed, thereby making them viable and usable feedstuffs for potential use in livestock industries.

Corrigendum: Three-stage fermentation of the feed and the application on weaned piglets.

[This corrects the article DOI: 10.3389/fvets.2023.1123563.].

CircSCAPER contributes to IL-1ß-induced osteoarthritis in vitro via miR-140-3p/EZH2 axis.

AIMS: Circular RNA (circRNA) S-phase cyclin A-associated protein in the endoplasmic reticulum (ER) (circSCAPER, ID: hsa_circ_0104595) has been found to be highly expressed in osteoarthritis (OA) patients and has been associated with the severity of OA. Hence, the role and mechanisms underlying circSCAPER in OA were investigated in this study. METHODS: In vitro cultured human normal chondrocyte C28/I2 was exposed to interleukin (IL)-1ß to mimic the microenvironment of OA. The expression of circSCAPER, microRNA (miR)-140-3p, and enhancer of zeste homolog 2 (EZH2) was detected using quantitative real-time polymerase chain reaction and Western blot assays. The extracellular matrix (ECM) degradation, proliferation, and apoptosis of chondrocytes were determined using Western blot, cell counting kit-8, and flow cytometry assays. Targeted relationships were predicted by bioinformatic analysis and verified using dual-luciferase reporter and RNA immunoprecipitation (RIP) assays. The levels of phosphoinositide 3-kinase (PI3K)/protein kinase B (AKT) pathway-related protein were detected using Western blot assays. RESULTS: CircSCAPER was highly expressed in OA cartilage tissues and IL-1ß-induced chondrocytes. Knockdown of circSCAPER reduced IL-1ß-evoked ECM degradation, proliferation arrest, and apoptosis enhancement in chondrocytes. Mechanistically, circSCAPER directly bound to miR-140-3p, and miR-140-3p inhibition reversed the effects of circSCAPER knockdown on IL-1ß-induced chondrocytes. miR-140-3p was verified to target EZH2, and overexpression of miR-140-3p protected chondrocytes against IL-1ß-induced dysfunction via targeting EZH2. Additionally, we confirmed that circSCAPER could regulate EZH2 through sponging miR-140-3p, and the circSCAPER/miR-140-3p/EZH2 axis could activate the PI3K/AKT pathway. CONCLUSION: CircSCAPER promoted IL-1ß-evoked ECM degradation, proliferation arrest, and apoptosis enhancement in chondrocytes via regulating miR-140-3p/EZH2 axis, which gained a new insight into the pathogenesis of OA. Cite this article: Bone Joint Res 2022;11(2):61-72.

Endothelial p130cas confers resistance to anti-angiogenesis therapy.

Anti-angiogenic therapies, such as anti-VEGF antibodies (AVAs), have shown promise in clinical settings. However, adaptive resistance to such therapies occurs frequently. We use orthotopic ovarian cancer models with AVA-adaptive resistance to investigate the underlying mechanisms. Genomic profiling of AVA-resistant tumors guides us to endothelial p130cas. We find that bevacizumab induces cleavage of VEGFR2 in endothelial cells by caspase-10 and that VEGFR2 fragments internalize into the nucleus and autophagosomes. Nuclear VEGFR2 and p130cas fragments, together with TNKS1BP1 (tankyrase-1-binding protein), initiate endothelial cell death. Blockade of autophagy in AVA-resistant endothelial cells retains VEGFR2 at the membrane with bevacizumab treatment. Targeting host p130cas with RGD (Arg-Gly-Asp)-tagged nanoparticles or genomic ablation of vascular p130cas in p130casflox/floxTie2Cre mice significantly extends the survival of mice with AVA-resistant ovarian tumors. Higher vascular p130cas is associated with shorter survival of individuals with ovarian cancer. Our findings identify opportunities for new strategies to overcome adaptive resistance to AVA therapy.

Improvements in estrogen deficiency-induced hypercholesterolemia by Hypericum perforatum L. extract are associated with gut microbiota and related metabolites in ovariectomized (OVX) rats.

Hypericum perforatum L. (HP), a well-known natural medicine, has a potential effect on menopausal hypercholesterolemia. However, the effect of HP extract on gut microbiota and related metabolites, which play vital roles in metabolic disease occurrence, in the context of estrogen deficiency have not yet been reported. The aims of the present study were to investigate the effects of HP extract on gut microbial composition and related metabolite profiles in ovariectomized (OVX) rats and reveal the relationships between pathological indicators and alterations in both gut microbial composition at the genus level and metabolites. Body weight, serum parameters, liver lipids and histomorphology were determined. Microbial composition was analyzed using 16S rRNA sequencing. Fecal short-chain fatty acids (SCFAs) and serum bile acids were quantitatively measured. Correlations between pathological indicators and alteration in gut microbiota and metabolites were investigated using Spearman's rank correlation test. Gene expression of 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase, cholesterol 7α-hydroxylase (CYP7A1) and cholesterol 27-hydroxylase (CYP27A1) in the liver and G protein-coupled receptors (GPCRs; GPR43 and GPR41), ZO-1 and occludin in the cecum were determined by PCR. Microbial composition and metabolite profiles were significantly changed in OVX rats compared with sham rats. Twelve bacterial genera, 5 SCFAs and 12 bile acids were identified as differential biomarkers. Differential genera, SCFAs and bile acids were closely associated with weight, total cholesterol (TC) and low-density lipoprotein cholesterol (LDL-C). In OVX rats, HP administration can significantly reverse the pathological symptoms of body weight gain, serum lipid disorders and hepatic steatosis, at the meanwhile, reestablish gut microbial composition and metabolite profiles. Moreover, HP administration significantly upregulated the levels of CYP7A1, GPR43 and GPR41. In conclusion, HP can ameliorate estrogen deficiency-induced hypercholesterolemia. The underlying mechanism may be associated with improvements in gut microbiota composition and the profile of related metabolites as well as increases in bile acid secretion.

Plasmid DNA gene therapy of the Niemann-Pick C1 mouse with transferrin receptor-targeted Trojan horse liposomes.

Niemann-Pick C1 (NPC1) is a lysosomal cholesterol storage disorder, that severely affects the brain, and is caused by mutations in the NPC1 gene, which encodes an intracellular membrane transporter of non-esterified cholesterol. Therapeutic options for NPC1 are few, and classical enzyme replacement therapy with the recombinant protein is not possible as the NPC1 gene product is an insoluble membrane protein, which increases the need for development of gene therapy for NPC1. While viral based gene therapy is under development, it is important to investigate alternative approaches to brain gene therapy without viral vectors. The present work develops a plasmid DNA approach to gene therapy of NPC1 using Trojan horse liposomes (THLs), wherein the plasmid DNA is encapsulated in 100 nm pegylated liposomes, which are targeted to organs with a monoclonal antibody against the mouse transferrin receptor. THLs were encapsulated with a 8.0 kb plasmid DNA encoding the 3.9 kb human NPC1 open reading frame, under the influence of a 1.5 kb platelet derived growth factor B (PDGFB) promoter. THLs were administered weekly beginning at 6-7 weeks in the NPC1-/- null mouse, and delivery of the plasmid DNA, and NPC1 mRNA expression in brain, spleen, and liver were confirmed by quantitative PCR. THL treatment reduced tissue inclusion bodies in brain, and peripheral organs, but did not prolong lifespan in these mice. The work suggests that early treatment after birth may be required to reverse this disease model with NPC1 gene replacement therapy.

Preparation of a Copper Polyphosphate Kinase Hybrid Nanoflower and Its Application in ADP Regeneration from AMP.

In this research article, we reported a self-assembly approach to prepare a copper polyphosphate kinase 2 hybrid nanoflower and established a cofactor ADP regeneration system from AMP using the nanoflower. First, the structure of the hybrid nanoflower was confirmed by scanning electron microscopy, X-ray diffraction, Fourier transform infrared spectroscopy, and X-ray photoelectron spectroscopy, which indicated the successful loading of the enzyme in the hybrid nanoflower. Moreover, compared to the free enzyme, the hybrid nanoflower exhibited a better performance in ADP production and possessed wider catalytic pH and temperature ranges as well as improved storage stability. The hybrid nanoflower also exhibited well reusability, preserving 71.7% of initial activity after being used for ten cycles. In addition, the phosphorylation of glucose was conducted by utilizing ADP-dependent glucokinase coupled with the ADP regeneration system, in which the hybrid nanoflower was used for regenerating ADP from AMP. It was observed that the ADP regeneration system operated effectively at a very small amount of AMP. Thus, the hybrid nanoflower had great application potential in industrial catalytic processes that were coupled with ADP-dependent enzymes.

Research on the mechanism of Chinese herbal medicine Radix Paeoniae Rubra in improving chronic pelvic inflammation disease by regulating PTGS2 in the arachidonic acid pathway.

Radix Paeoniae Rubra (RPR) is a traditional Chinese medicine with anti-inflammatory effects that has been used in chronic pelvic inflammation disease (CPID) therapy. However, research on the mechanism of RPR in CPID therapy is lacking. Here, we used a network pharmacology method to screen targets and found that the PTGS2 target in the arachidonic acid (AA) pathway was significantly related to CPID. Then, regarding the molecular mechanism, it was further confirmed that RPR may reduce the development of CPID by regulating the PTGS2 target. The CPID rat model was established by mixed bacterial infection. We verified the expression of PTGS2 by immunohistochemical analysis, western blotting assays to detect the expression of PTGS2 protein, and polymerase chain reaction detection of PTGS2 mRNA expression. It was observed that the PTGS2 target decreased significantly after RPR administration at different doses. It is suggested that RPR can reverse the abnormal expression of PTGS2 in CPID rats. We believe that RPR is effective in the treatment of CPID, and RPR can reduce the inflammatory symptoms of CPID by regulating the level of PTGS2 in the AA pathway.

PTGER3 induces ovary tumorigenesis and confers resistance to cisplatin therapy through up-regulation Ras-MAPK/Erk-ETS1-ELK1/CFTR1 axis.

BACKGROUND: Inflammatory mediator prostaglandin E2-prostaglandin E2 receptor EP3 (PTGER3) signaling is critical for tumor-associated angiogenesis, tumor growth, and chemoresistance. However, the mechanism underlying these effects in ovarian cancer is not known. METHODS: An association between higher tumoral expression of PTGER3 and shorter patient survival in the ovarian cancer dataset of The Cancer Genome Atlas prompted investigation of the antitumor effects of PTGER3 downmodulation. PTGER3 mRNA and protein levels were higher in cisplatin-resistant ovarian cancer cells than in their cisplatin-sensitive counterparts. FINDINGS: Silencing of PTGER3 via siRNA in cancer cells was associated with decreased cell growth and less invasiveness, as well as cell-cycle arrest and increased apoptosis, mediated through the Ras-MAPK/Erk-ETS1-ELK1/CFTR1 axis. Furthermore, sustained PTGER3 silencing with multistage vector and liposomal 2'-F-phosphorodithioate-siRNA-mediated silencing of PTGER3 combined with cisplatin resulted in robust antitumor effects in cisplatin-resistant ovarian cancer models. INTERPRETATION: These findings identify PTGER3 as a potential therapeutic target in chemoresistant ovarian cancers expressing high levels of this oncogenic protein. FUND: National Institutes of Health/National Cancer Institute, USA.

MYC Targeted Long Noncoding RNA DANCR Promotes Cancer in Part by Reducing p21 Levels.

The MYC oncogene broadly promotes transcription mediated by all nuclear RNA polymerases, thereby acting as a positive modifier of global gene expression. Here, we report that MYC stimulates the transcription of DANCR, a long noncoding RNA (lncRNA) that is widely overexpressed in human cancer. We identified DANCR through its overexpression in a transgenic model of MYC-induced lymphoma, but found that it was broadly upregulated in many human cancer cell lines and cancers, including most notably in prostate and ovarian cancers. Mechanistic investigations indicated that DANCR limited the expression of cell-cycle inhibitor p21 (CDKN1A) and that the inhibitory effects of DANCR loss on cell proliferation could be partially rescued by p21 silencing. In a xenograft model of human ovarian cancer, a nanoparticle-mediated siRNA strategy to target DANCR in vivo was sufficient to strongly inhibit tumor growth. Our observations expand knowledge of how MYC drives cancer cell proliferation by identifying DANCR as a critical lncRNA widely overexpressed in human cancers.Significance: These findings expand knowledge of how MYC drives cancer cell proliferation by identifying an oncogenic long noncoding RNA that is widely overexpressed in human cancers. Cancer Res; 78(1); 64-74. ©2017 AACR.

Inhibiting Nuclear Phospho-Progesterone Receptor Enhances Antitumor Activity of Onapristone in Uterine Cancer.

Although progesterone receptor (PR)-targeted therapies are modestly active in patients with uterine cancer, their underlying molecular mechanisms are not well understood. The clinical use of such therapies is limited because of the lack of biomarkers that predict response to PR agonists (progestins) or PR antagonists (onapristone). Thus, understanding the underlying molecular mechanisms of action will provide an advance in developing novel combination therapies for cancer patients. Nuclear translocation of PR has been reported to be ligand-dependent or -independent. Here, we identified that onapristone, a PR antagonist, inhibited nuclear translocation of ligand-dependent or -independent (EGF) phospho-PR (S294), whereas trametinib inhibited nuclear translocation of EGF-induced phospho-PR (S294). Using orthotopic mouse models of uterine cancer, we demonstrated that the combination of onapristone and trametinib results in superior antitumor effects in uterine cancer models compared with either monotherapy. These synergistic effects are, in part, mediated through inhibiting the nuclear translocation of EGF-induced PR phosphorylation in uterine cancer cells. Targeting MAPK-dependent PR activation with onapristone and trametinib significantly inhibited tumor growth in preclinical uterine cancer models and is worthy of further clinical investigation. Mol Cancer Ther; 17(2); 464-73. ©2017 AACR.