Phenotype-specific melanoma uptake of fatty acid from human adipocytes activates AXL and CAV1-dependent ß-catenin nuclear accumulation.

Phenotypic diversity of cancer cells within tumors generated through bi-directional interactions with the tumor microenvironment has emerged as a major driver of disease progression and therapy resistance. Nutrient availability plays a critical role in determining phenotype, but whether specific nutrients elicit different responses on distinct phenotypes is poorly understood. Here we show, using melanoma as a model, that only MITF Low undifferentiated cells, but not MITF High cells, are competent to drive lipolysis in human adipocytes. In contrast to MITF High melanomas, adipocyte-derived free fatty acids are taken up by undifferentiated MITF Low cells via a fatty acid transporter (FATP)-independent mechanism. Importantly, oleic acid (OA), a monounsaturated long chain fatty acid abundant in adipose tissue and lymph, reprograms MITF Low undifferentiated melanoma cells to a highly invasive state by ligand-independent activation of AXL, a receptor tyrosine kinase associated with therapy resistance in a wide range of cancers. AXL activation by OA then drives SRC-dependent formation and nuclear translocation of a ß-catenin-CAV1 complex. The results highlight how a specific nutritional input drives phenotype-specific activation of a pro-metastasis program with implications for FATP-targeted therapies.

Role of Human Epicardial Adipose Tissue-Derived miR-92a-3p in Myocardial Redox State.

BACKGROUND: Visceral obesity is directly linked to increased cardiovascular risk, including heart failure. OBJECTIVES: This study explored the ability of human epicardial adipose tissue (EAT)-derived microRNAs (miRNAs) to regulate the myocardial redox state and clinical outcomes. METHODS: This study screened for miRNAs expressed and released from human EAT and tested for correlations with the redox state in the adjacent myocardium in paired EAT/atrial biopsy specimens from patients undergoing cardiac surgery. Three miRNAs were then tested for causality in an in vitro model of cardiomyocytes. At a clinical level, causality/directionality were tested using genome-wide association screening, and the underlying mechanisms were explored using human biopsy specimens, as well as overexpression of the candidate miRNAs and their targets in vitro and in vivo using a transgenic mouse model. The final prognostic value of the discovered targets was tested in patients undergoing cardiac surgery, followed up for a median of 8 years. RESULTS: EAT miR-92a-3p was related to lower oxidative stress in human myocardium, a finding confirmed by using genetic regulators of miR-92a-3p in the human heart and EAT. miR-92a-3p reduced nicotinamide adenine dinucleotide phosphate (NADPH)-oxidase-derived superoxide (O2.-) by targeting myocardial expression of WNT5A, which regulated Rac1-dependent activation of NADPH oxidases. Finally, high miR-92a-3p levels in EAT were independently related with lower risk of adverse cardiovascular events. CONCLUSIONS: EAT-derived miRNAs exert paracrine effects on the human heart. Indeed miR-92a-3p suppresses the wingless-type MMTV integration site family, member 5a/Rac1/NADPH oxidase axis and improves the myocardial redox state. EAT-derived miR-92a-3p is related to improved clinical outcomes and is a rational therapeutic target for the prevention and treatment of obesity-related heart disease.

The MITF regulatory network in melanoma.

Bidirectional interactions between plastic tumor cells and the microenvironment critically impact tumor evolution and metastatic dissemination by enabling cancer cells to adapt to microenvironmental stresses by switching phenotype. In melanoma, a key determinant of phenotypic identity is the microphthalmia-associated transcription factor MITF that promotes proliferation, suppresses senescence, and anticorrelates with immune infiltration and therapy resistance. What determines whether MITF can activate or repress genes associated with specific phenotypes, or how signaling regulating MITF might impact immune infiltration is poorly understood. Here, we find that MITF binding to genes associated with high MITF is via classical E/M-box motifs, but genes downregulated when MITF is high contain FOS/JUN/AP1/ATF3 sites. Significantly, the repertoire of MITF-interacting factors identified here includes JUN and ATF3 as well as many previously unidentified interactors. As high AP1 activity is a hallmark of MITFLow , invasive, slow-cycling, therapy resistant cells, the ability of MITF to repress AP1-regulated genes provides an insight into how MITF establishes and maintains a pro-proliferative phenotype. Moreover, although ß-catenin has been linked to immune exclusion, many Hallmark ß-catenin signaling genes are associated with immune infiltration. Instead, low MITF together with Notch signaling is linked to immune infiltration in both mouse and human melanoma tumors.

TBX2 controls a proproliferative gene expression program in melanoma.

Senescence shapes embryonic development, plays a key role in aging, and is a critical barrier to cancer initiation, yet how senescence is regulated remains incompletely understood. TBX2 is an antisenescence T-box family transcription repressor implicated in embryonic development and cancer. However, the repertoire of TBX2 target genes, its cooperating partners, and how TBX2 promotes proliferation and senescence bypass are poorly understood. Here, using melanoma as a model, we show that TBX2 lies downstream from PI3K signaling and that TBX2 binds and is required for expression of E2F1, a key antisenescence cell cycle regulator. Remarkably, TBX2 binding in vivo is associated with CACGTG E-boxes, present in genes down-regulated by TBX2 depletion, more frequently than the consensus T-element DNA binding motif that is restricted to Tbx2 repressed genes. TBX2 is revealed to interact with a wide range of transcription factors and cofactors, including key components of the BCOR/PRC1.1 complex that are recruited by TBX2 to the E2F1 locus. Our results provide key insights into how PI3K signaling modulates TBX2 function in cancer to drive proliferation.

TOP1 modulation during melanoma progression and in adaptative resistance to BRAF and MEK inhibitors.

In melanomas, therapy resistance can arise due to a combination of genetic, epigenetic and phenotypic mechanisms. Due to its crucial role in DNA supercoil relaxation, TOP1 is often considered an essential chemotherapeutic target in cancer. However, how TOP1 expression and activity might differ in therapy sensitive versus resistant cell types is unknown. Here we show that TOP1 expression is increased in metastatic melanoma and correlates with an invasive gene expression signature. More specifically, TOP1 expression is highest in cells with the lowest expression of MITF, a key regulator of melanoma biology. Notably, TOP1 and DNA Single-Strand Break Repair genes are downregulated in BRAFi- and BRAFi/MEKi-resistant cells and TOP1 inhibition decreases invasion markers only in BRAFi/MEKi-resistant cells. Thus, we show three different phenotypes related to TOP1 levels: i) non-malignant cells with low TOP1 levels; ii) metastatic cells with high TOP1 levels and high invasiveness; and iii) BRAFi- and BRAFi/MEKi-resistant cells with low TOP1 levels and high invasiveness. Together, these results highlight the potential role of TOP1 in melanoma progression and resistance.

TBX3 Promotes Melanoma Migration by Transcriptional Activation of ID1, which Prevents Activation of E-Cadherin by MITF.

In melanoma, a phenotype switch from proliferation to invasion underpins metastasis, the major cause of melanoma-associated death. The transition from radial to vertical growth phase (invasive) melanoma is characterized by downregulation of both E-cadherin (CDH1) and MITF and upregulation of the key cancer-associated gene TBX3 and the phosphatidylinositol 3 kinase signaling pathway. Yet, whether and how these diverse events are linked remains poorly understood. Here, we show that TBX3 directly promotes expression of ID1, a dominant-negative regulator of basic helix-loop-helix transcription factors, and that ID1 decreases MITF binding and upregulation of CDH1. Significantly, we show that TBX3 activation of ID1 is necessary for TBX3 to enhance melanoma cell migration, and the mechanistic links between TBX3, ID1, MITF, and invasion revealed here are reflected in their expression in human melanomas. Our results reveal that melanoma migration is promoted through a TBX3-ID1-MITF-E-cadherin axis and that ID1-mediated repression of MITF activity may reinforce maintenance of an MITFLow phenotype associated with disease progression and therapy resistance.

Mitochondrial Inhibitor Atovaquone Increases Tumor Oxygenation and Inhibits Hypoxic Gene Expression in Patients with Non-Small Cell Lung Cancer.

PURPOSE: Tumor hypoxia fuels an aggressive tumor phenotype and confers resistance to anticancer treatments. We conducted a clinical trial to determine whether the antimalarial drug atovaquone, a known mitochondrial inhibitor, reduces hypoxia in non-small cell lung cancer (NSCLC). PATIENTS AND METHODS: Patients with NSCLC scheduled for surgery were recruited sequentially into two cohorts: cohort 1 received oral atovaquone at the standard clinical dose of 750 mg twice daily, while cohort 2 did not. Primary imaging endpoint was change in tumor hypoxic volume (HV) measured by hypoxia PET-CT. Intercohort comparison of hypoxia gene expression signatures using RNA sequencing from resected tumors was performed. RESULTS: Thirty patients were evaluable for hypoxia PET-CT analysis, 15 per cohort. Median treatment duration was 12 days. Eleven (73.3%) atovaquone-treated patients had meaningful HV reduction, with median change -28% [95% confidence interval (CI), -58.2 to -4.4]. In contrast, median change in untreated patients was +15.5% (95% CI, -6.5 to 35.5). Linear regression estimated the expected mean HV was 55% (95% CI, 24%-74%) lower in cohort 1 compared with cohort 2 (P = 0.004), adjusting for cohort, tumor volume, and baseline HV. A key pharmacodynamics endpoint was reduction in hypoxia-regulated genes, which were significantly downregulated in atovaquone-treated tumors. Data from multiple additional measures of tumor hypoxia and perfusion are presented. No atovaquone-related adverse events were reported. CONCLUSIONS: This is the first clinical evidence that targeting tumor mitochondrial metabolism can reduce hypoxia and produce relevant antitumor effects at the mRNA level. Repurposing atovaquone for this purpose may improve treatment outcomes for NSCLC.

Paradoxical activation of AMPK by glucose drives selective EP300 activity in colorectal cancer.

Coordination of gene expression with nutrient availability supports proliferation and homeostasis and is shaped by protein acetylation. Yet how physiological/pathological signals link acetylation to specific gene expression programs and whether such responses are cell-type-specific is unclear. AMP-activated protein kinase (AMPK) is a key energy sensor, activated by glucose limitation to resolve nutrient supply-demand imbalances, critical for diabetes and cancer. Unexpectedly, we show here that, in gastrointestinal cancer cells, glucose activates AMPK to selectively induce EP300, but not CREB-binding protein (CBP). Consequently, EP300 is redirected away from nuclear receptors that promote differentiation towards ß-catenin, a driver of proliferation and colorectal tumorigenesis. Importantly, blocking glycogen synthesis permits reactive oxygen species (ROS) accumulation and AMPK activation in response to glucose in previously nonresponsive cells. Notably, glycogen content and activity of the ROS/AMPK/EP300/ß-catenin axis are opposite in healthy versus tumor sections. Glycogen content reduction from healthy to tumor tissue may explain AMPK switching from tumor suppressor to activator during tumor evolution.

ABCB5 is activated by MITF and ß-catenin and is associated with melanoma differentiation.

Defining markers of different phenotypic states in melanoma is important for understanding disease progression, determining the response to therapy, and defining the molecular mechanisms underpinning phenotype-switching driven by the changing intratumor microenvironment. The ABCB5 transporter is implicated in drug-resistance and has been identified as a marker of melanoma-initiating cells. Indeed ongoing studies are using ABCB5 to define stem cell populations. However, we show here that the ABCB5 is a direct target for the microphthalmia-associated transcription factor MITF and its expression can be induced by ß-catenin, a key activator and co-factor for MITF. Consequently, ABCB5 mRNA expression is primarily associated with melanoma cells exhibiting differentiation markers. The results suggest first that ABCB5 is unlikely to represent a marker of de-differentiated melanoma stem cells, and second that ABCB5 may contribute to the non-genetic drug-resistance associated with highly differentiated melanoma cells. To reconcile the apparently conflicting observations in the field, we propose a model in which ABCB5 may mark a slow-cycling differentiated population of melanoma cells.

Lineage-Restricted Regulation of SCD and Fatty Acid Saturation by MITF Controls Melanoma Phenotypic Plasticity.

Phenotypic and metabolic heterogeneity within tumors is a major barrier to effective cancer therapy. How metabolism is implicated in specific phenotypes and whether lineage-restricted mechanisms control key metabolic vulnerabilities remain poorly understood. In melanoma, downregulation of the lineage addiction oncogene microphthalmia-associated transcription factor (MITF) is a hallmark of the proliferative-to-invasive phenotype switch, although how MITF promotes proliferation and suppresses invasion is poorly defined. Here, we show that MITF is a lineage-restricted activator of the key lipogenic enzyme stearoyl-CoA desaturase (SCD) and that SCD is required for MITFHigh melanoma cell proliferation. By contrast MITFLow cells are insensitive to SCD inhibition. Significantly, the MITF-SCD axis suppresses metastasis, inflammatory signaling, and an ATF4-mediated feedback loop that maintains de-differentiation. Our results reveal that MITF is a lineage-specific regulator of metabolic reprogramming, whereby fatty acid composition is a driver of melanoma phenotype switching, and highlight that cell phenotype dictates the response to drugs targeting lipid metabolism.

E2F1 proteolysis via SCF-cyclin F underlies synthetic lethality between cyclin F loss and Chk1 inhibition.

Cyclins are central engines of cell cycle progression in conjunction with cyclin-dependent kinases (CDKs). Among the different cyclins controlling cell cycle progression, cyclin F does not partner with a CDK, but instead forms via its F-box domain an SCF (Skp1-Cul1-F-box)-type E3 ubiquitin ligase module. Although various substrates of cyclin F have been identified, the vulnerabilities of cells lacking cyclin F are not known. Thus, we assessed viability of cells lacking cyclin F upon challenging them with more than 180 different kinase inhibitors. The screen revealed a striking synthetic lethality between Chk1 inhibition and cyclin F loss. Chk1 inhibition in cells lacking cyclin F leads to DNA replication catastrophe. Replication catastrophe depends on accumulation of the transcription factor E2F1 in cyclin F-depleted cells. We find that SCF-cyclin F controls E2F1 ubiquitylation and degradation during the G2/M phase of the cell cycle and upon challenging cells with Chk1 inhibitors. Thus, Cyclin F restricts E2F1 activity during the cell cycle and upon checkpoint inhibition to prevent DNA replication stress. Our findings pave the way for patient selection in the clinical use of checkpoint inhibitors.

MITF controls the TCA cycle to modulate the melanoma hypoxia response.

In response to the dynamic intra-tumor microenvironment, melanoma cells adopt distinct phenotypic states associated with differential expression of the microphthalmia-associated transcription factor (MITF). The response to hypoxia is driven by hypoxia-inducible transcription factors (HIFs) that reprogram metabolism and promote angiogenesis. HIF1α indirectly represses MITF that can activate HIF1α expression. Although HIF and MITF share a highly related DNA-binding specificity, it is unclear whether they co-regulate subset of target genes. Moreover, the genomewide impact of hypoxia on melanoma and whether melanoma cell lines representing different phenotypic states exhibit distinct hypoxic responses is unknown. Here we show that three different melanoma cell lines exhibit widely different hypoxia responses with only a core 23 genes regulated in common after 12 hr in hypoxia. Surprisingly, under hypoxia MITF is transiently up-regulated by HIF1α and co-regulates a subset of HIF targets including VEGFA. Significantly, we also show that MITF represses itself and also regulates SDHB to control the TCA cycle and suppress pseudo-hypoxia. Our results reveal a previously unsuspected role for MITF in metabolism and the network of factors underpinning the hypoxic response in melanoma.

BRN2 suppresses apoptosis, reprograms DNA damage repair, and is associated with a high somatic mutation burden in melanoma.

Whether cell types exposed to a high level of environmental insults possess cell type-specific prosurvival mechanisms or enhanced DNA damage repair capacity is not well understood. BRN2 is a tissue-restricted POU domain transcription factor implicated in neural development and several cancers. In melanoma, BRN2 plays a key role in promoting invasion and regulating proliferation. Here we found, surprisingly, that rather than interacting with transcription cofactors, BRN2 is instead associated with DNA damage response proteins and directly binds PARP1 and Ku70/Ku80. Rapid PARP1-dependent BRN2 association with sites of DNA damage facilitates recruitment of Ku80 and reprograms DNA damage repair by promoting Ku-dependent nonhomologous end-joining (NHEJ) at the expense of homologous recombination. BRN2 also suppresses an apoptosis-associated gene expression program to protect against UVB-, chemotherapy- and vemurafenib-induced apoptosis. Remarkably, BRN2 expression also correlates with a high single-nucleotide variation prevalence in human melanomas. By promoting error-prone DNA damage repair via NHEJ and suppressing apoptosis of damaged cells, our results suggest that BRN2 contributes to the generation of melanomas with a high mutation burden. Our findings highlight a novel role for a key transcription factor in reprogramming DNA damage repair and suggest that BRN2 may impact the response to DNA-damaging agents in BRN2-expressing cancers.

RASSF1A uncouples Wnt from Hippo signalling and promotes YAP mediated differentiation via p73.

Transition from pluripotency to differentiation is a pivotal yet poorly understood developmental step. Here, we show that the tumour suppressor RASSF1A is a key player driving the early specification of cell fate. RASSF1A acts as a natural barrier to stem cell self-renewal and iPS cell generation, by switching YAP from an integral component in the ß-catenin-TCF pluripotency network to a key factor that promotes differentiation. We demonstrate that epigenetic regulation of the Rassf1A promoter maintains stemness by allowing a quaternary association of YAP-TEAD and ß-catenin-TCF3 complexes on the Oct4 distal enhancer. However, during differentiation, promoter demethylation allows GATA1-mediated RASSF1A expression which prevents YAP from contributing to the TEAD/ß-catenin-TCF3 complex. Simultaneously, we find that RASSF1A promotes a YAP-p73 transcriptional programme that enables differentiation. Together, our findings demonstrate that RASSF1A mediates transcription factor selection of YAP in stem cells, thereby acting as a functional "switch" between pluripotency and initiation of differentiation.

Spatiotemporally restricted arenavirus replication induces immune surveillance and type I interferon-dependent tumour regression.

Immune-mediated effector molecules can limit cancer growth, but lack of sustained immune activation in the tumour microenvironment restricts antitumour immunity. New therapeutic approaches that induce a strong and prolonged immune activation would represent a major immunotherapeutic advance. Here we show that the arenaviruses lymphocytic choriomeningitis virus (LCMV) and the clinically used Junin virus vaccine (Candid#1) preferentially replicate in tumour cells in a variety of murine and human cancer models. Viral replication leads to prolonged local immune activation, rapid regression of localized and metastatic cancers, and long-term disease control. Mechanistically, LCMV induces antitumour immunity, which depends on the recruitment of interferon-producing Ly6C+ monocytes and additionally enhances tumour-specific CD8+ T cells. In comparison with other clinically evaluated oncolytic viruses and to PD-1 blockade, LCMV treatment shows promising antitumoural benefits. In conclusion, therapeutically administered arenavirus replicates in cancer cells and induces tumour regression by enhancing local immune responses.

Translation reprogramming is an evolutionarily conserved driver of phenotypic plasticity and therapeutic resistance in melanoma.

The intratumor microenvironment generates phenotypically distinct but interconvertible malignant cell subpopulations that fuel metastatic spread and therapeutic resistance. Whether different microenvironmental cues impose invasive or therapy-resistant phenotypes via a common mechanism is unknown. In melanoma, low expression of the lineage survival oncogene microphthalmia-associated transcription factor (MITF) correlates with invasion, senescence, and drug resistance. However, how MITF is suppressed in vivo and how MITF-low cells in tumors escape senescence are poorly understood. Here we show that microenvironmental cues, including inflammation-mediated resistance to adoptive T-cell immunotherapy, transcriptionally repress MITF via ATF4 in response to inhibition of translation initiation factor eIF2B. ATF4, a key transcription mediator of the integrated stress response, also activates AXL and suppresses senescence to impose the MITF-low/AXL-high drug-resistant phenotype observed in human tumors. However, unexpectedly, without translation reprogramming an ATF4-high/MITF-low state is insufficient to drive invasion. Importantly, translation reprogramming dramatically enhances tumorigenesis and is linked to a previously unexplained gene expression program associated with anti-PD-1 immunotherapy resistance. Since we show that inhibition of eIF2B also drives neural crest migration and yeast invasiveness, our results suggest that translation reprogramming, an evolutionarily conserved starvation response, has been hijacked by microenvironmental stress signals in melanoma to drive phenotypic plasticity and invasion and determine therapeutic outcome.

NFIB Mediates BRN2 Driven Melanoma Cell Migration and Invasion Through Regulation of EZH2 and MITF.

While invasion and metastasis of tumour cells are the principle factor responsible for cancer related deaths, the mechanisms governing the process remain poorly defined. Moreover, phenotypic divergence of sub-populations of tumour cells is known to underpin alternative behaviors linked to tumour progression such as proliferation, survival and invasion. In the context of melanoma, heterogeneity between two transcription factors, BRN2 and MITF, has been associated with phenotypic switching between predominantly invasive and proliferative behaviors respectively. Epigenetic changes, in response to external cues, have been proposed to underpin this process, however the mechanism by which the phenotypic switch occurs is unclear. Here we report the identification of the NFIB transcription factor as a novel downstream effector of BRN2 function in melanoma cells linked to the migratory and invasive characteristics of these cells. Furthermore, the function of NFIB appears to drive an invasive phenotype through an epigenetic mechanism achieved via the upregulation of the polycomb group protein EZH2. A notable target of NFIB mediated up-regulation of EZH2 is decreased MITF expression, which further promotes a less proliferative, more invasive phenotype. Together our data reveal that NFIB has the ability to promote dynamic changes in the chromatin state of melanoma cells to facilitate migration, invasion and metastasis.

In Silico Designing and Screening of Antagonists against Cancer Drug Target XIAP.

X-linked inhibitor of apoptosis (XIAP) is a member of inhibitor of apoptosis (IAP) family and involved in the suppression of apoptosis in cancer cells. This property makes it a therapeutic target for the cancer therapy. In the present study, we have developed QSAR models using chemical descriptors, fingerprints, principal components, docking energy parameters and similarity-based approach against XIAP. We have achieved correlation (R) of 0.803 with R(2) value of 0.645 at 10-fold cross validation using SMOreg algorithm. We have evaluated these models on independent dataset to ascertain its robustness and achieved correlation (R) of 0.793 with R(2) value of 0.628. Further, we have used these models for the screening of FDA approved drugs and drug-like molecules from ZINC database and prioritized them on the basis of their predicted pIC50 values. Docking studies of top hits with XIAP-BIR3 domain shows that Iodixanol (DB01249) and ZINC68678304 have higher binding affinities than well-known tetrapeptide inhibitor, AVPI. We have integrated these models in a web server named as "XIAPin". We hope that this web server will contribute in the designing of nifty antagonists against XIAP.

An in silico platform for predicting, screening and designing of antihypertensive peptides.

High blood pressure or hypertension is an affliction that threatens millions of lives worldwide. Peptides from natural origin have been shown recently to be highly effective in lowering blood pressure. In the present study, we have framed a platform for predicting and designing novel antihypertensive peptides. Due to a large variation found in the length of antihypertensive peptides, we divided these peptides into four categories (i) Tiny peptides, (ii) small peptides, (iii) medium peptides and (iv) large peptides. First, we developed SVM based regression models for tiny peptides using chemical descriptors and achieved maximum correlation of 0.701 and 0.543 for dipeptides and tripeptides, respectively. Second, classification models were developed for small peptides and achieved maximum accuracy of 76.67%, 72.04% and 77.39% for tetrapeptide, pentapeptide and hexapeptides, respectively. Third, we have developed a model for medium peptides using amino acid composition and achieved maximum accuracy of 82.61%. Finally, we have developed a model for large peptides using amino acid composition and achieved maximum accuracy of 84.21%. Based on the above study, a web-based platform has been developed for locating antihypertensive peptides in a protein, screening of peptides and designing of antihypertensive peptides.

AHTPDB: a comprehensive platform for analysis and presentation of antihypertensive peptides.

AHTPDB (http://crdd.osdd.net/raghava/ahtpdb/) is a manually curated database of experimentally validated antihypertensive peptides. Information pertaining to peptides with antihypertensive activity was collected from research articles and from various peptide repositories. These peptides were derived from 35 major sources that include milk, egg, fish, pork, chicken, soybean, etc. In AHTPDB, most of the peptides belong to a family of angiotensin-I converting enzyme inhibiting peptides. The current release of AHTPDB contains 5978 peptide entries among which 1694 are unique peptides. Each entry provides detailed information about a peptide like sequence, inhibitory concentration (IC50), toxicity/bitterness value, source, length, molecular mass and information related to purification of peptides. In addition, the database provides structural information of these peptides that includes predicted tertiary and secondary structures. A user-friendly web interface with various tools has been developed to retrieve and analyse the data. It is anticipated that AHTPDB will be a useful and unique resource for the researchers working in the field of antihypertensive peptides.