In vivo evaluation of combination therapy targeting the isoprenoid biosynthetic pathway.

Geranylgeranyl diphosphate synthase (GGDPS), an enzyme in the isoprenoid biosynthetic pathway (IBP), produces the isoprenoid (geranylgeranyl pyrophosphate, GGPP) used in protein geranylgeranylation reactions. Our prior studies utilizing triazole bisphosphonate-based GGDPS inhibitors (GGSIs) have revealed that these agents represent a novel strategy by which to induce cancer cell death, including multiple myeloma and pancreatic cancer. Statins inhibit the rate-limiting enzyme in the IBP and potentiate the effects of GGSIs in vitro. The in vivo effects of combination therapy with statins and GGSIs have not been determined. Here we evaluated the effects of combining VSW1198, a novel GGSI, with a statin (lovastatin or pravastatin) in CD-1 mice. Twice-weekly dosing with VSW1198 at the previously established maximally tolerated dose in combination with a statin led to hepatotoxicity, while once-weekly VSW1198-based combinations were feasible. No abnormalities in kidney, spleen, brain or skeletal muscle were observed with combination therapy. Combination therapy disrupted protein geranylgeranylation in vivo. Evaluation of hepatic isoprenoid levels revealed decreased GGPP levels in the single drug groups and undetectable GGPP levels in the combination groups. Additional studies with combinations using 50% dose-reductions of either VSW1198 or lovastatin revealed minimal hepatotoxicity with expected on-target effects of diminished GGPP levels and disruption of protein geranylgeranylation. Combination statin/GGSI therapy significantly slowed tumor growth in a myeloma xenograft model. Collectively, these studies are the first to demonstrate that combination IBP inhibitor therapy alters isoprenoid levels and disrupts protein geranylgeranylation in vivo as well as slows tumor growth in a myeloma xenograft model, thus providing the framework for future clinical exploration.

Synthesis and preliminary anticancer evaluation of new triazole bisphosphonate-based isoprenoid biosynthesis inhibitors.

The synthesis of a new set of triazole bisphosphonates 8a-d and 9a-d presenting an alkyl or phenyl substituent at the C-4 or C-5 position of the triazole ring is described. These compounds have been evaluated for their antiproliferative activity against MIA PaCa-2 (pancreas), MDA-MB-231 (breast) and A549 (lung) human tumor cell lines. 4-hexyl- and 4-octyltriazole bisphosphonates 8b-c both displayed remarkable antiproliferative activities with IC50 values in the micromolar range (0.75-2.4 µM) and were approximately 4 to 12-fold more potent than zoledronate. Moreover, compound 8b inhibits geranylgeranyl pyrophosphate biosynthesis in MIA PaCa-2 cells which ultimately led to tumor cells death.

Pristane promotes anaerobic glycolysis to facilitate proinflammatory activation of macrophages and development of arthritis.

Pristane-induced arthritis (PIA) could be adoptively transferred by splenic T cells in rats, and innate immunity should play critical roles in T cell activation. However, in pre-clinical stage, the activation mechanism of innate cells like macrophages remains unclear. Here we found that PIA was dependent on macrophages since cell depletion alleviated disease severity. Splenic macrophages of PIA rats showed M1 phenotypic shifting. The quantitative proteomics analysis suggested that macrophages initiated metabolic reprogramming with the conversion of aerobic oxidation to glycolysis in response to pristane in vivo. Notably, macrophages treated with pristane showed mitochondrial dysregulation and increased glycolysis flux and enzyme activity. Additionally, TNFα production, strongly associating with the glycolysis enzyme Ldha/Ldhb, could be reduced as glycolysis was inhibited or be enhanced as citrate cycle was blocked. This work provides detailed insights into the molecular mechanisms of pristane-mediated metabolic reprogramming in macrophages and suggests a new therapeutic strategy for arthritic disorders.

A Metabolic Dependency for Host Isoprenoids in the Obligate Intracellular Pathogen Rickettsia parkeri Underlies a Sensitivity to the Statin Class of Host-Targeted Therapeutics.

Gram-negative bacteria in the order Rickettsiales have an obligate intracellular growth requirement, and some species cause human diseases such as typhus and spotted fever. The bacteria have evolved a dependence on essential nutrients and metabolites from the host cell as a consequence of extensive genome reduction. However, it remains largely unknown which nutrients they acquire and whether their metabolic dependency can be exploited therapeutically. Here, we describe a genetic rewiring of bacterial isoprenoid biosynthetic pathways in the Rickettsiales that has resulted from reductive genome evolution. Furthermore, we investigated whether the spotted fever group Rickettsia species Rickettsia parkeri scavenges isoprenoid precursors directly from the host. Using targeted mass spectrometry, we found that infection caused decreases in host isoprenoid products and concomitant increases in bacterial isoprenoid metabolites. Additionally, we report that treatment of infected cells with statins, which inhibit host isoprenoid synthesis, prohibited bacterial growth. We show that growth inhibition correlates with changes in bacterial size and shape that mimic those caused by antibiotics that inhibit peptidoglycan biosynthesis, suggesting that statins lead to an inhibition of cell wall synthesis. Altogether, our results describe a potential Achilles' heel of obligate intracellular pathogens that can potentially be exploited with host-targeted therapeutics that interfere with metabolic pathways required for bacterial growth.IMPORTANCE Obligate intracellular pathogens, which include viruses as well as certain bacteria and eukaryotes, are a subset of infectious microbes that are metabolically dependent on and unable to grow outside an infected host cell because they have lost or lack essential biosynthetic pathways. In this study, we describe a metabolic dependency of the bacterial pathogen Rickettsia parkeri on host isoprenoid molecules that are used in the biosynthesis of downstream products, including cholesterol, steroid hormones, and heme. Bacteria make products from isoprenoids, such as an essential lipid carrier for making the bacterial cell wall. We show that bacterial metabolic dependency can represent a potential Achilles' heel and that inhibiting host isoprenoid biosynthesis with the FDA-approved statin class of drugs inhibits bacterial growth by interfering with the integrity of the cell wall. This work supports the potential to treat infections by obligate intracellular pathogens through inhibition of host biosynthetic pathways that are susceptible to parasitism.

A Promiscuous CYP706A3 Reduces Terpene Volatile Emission from Arabidopsis Flowers, Affecting Florivores and the Floral Microbiome.

Flowers are essential but vulnerable plant organs, exposed to pollinators and florivores; however, flower chemical defenses are rarely investigated. We show here that two clustered terpene synthase and cytochrome P450 encoding genes (TPS11 and CYP706A3) on chromosome 5 of Arabidopsis (Arabidopsis thaliana) are tightly coexpressed in floral tissues, upon anthesis and during floral bud development. TPS11 was previously reported to generate a blend of sesquiterpenes. By heterologous coexpression of TPS11 and CYP706A3 in yeast (Saccharomyces cerevisiae) and Nicotiana benthamiana, we demonstrate that CYP706A3 is active on TPS11 products and also further oxidizes its own primary oxidation products. Analysis of headspace and soluble metabolites in cyp706a3 and 35S:CYP706A3 mutants indicate that CYP706A3-mediated metabolism largely suppresses sesquiterpene and most monoterpene emissions from opening flowers, and generates terpene oxides that are retained in floral tissues. In flower buds, the combined expression of TPS11 and CYP706A3 also suppresses volatile emissions and generates soluble sesquiterpene oxides. Florivory assays with the Brassicaceae specialist Plutella xylostella demonstrate that insect larvae avoid feeding on buds expressing CYP706A3 and accumulating terpene oxides. Composition of the floral microbiome appears also to be modulated by CYP706A3 expression. TPS11 and CYP706A3 simultaneously evolved within Brassicaceae and form the most versatile functional gene cluster described in higher plants so far.plantcell;31/12/2947/FX1F1fx1.

Synthesis and Biological Evaluation of Novel Triazole Derivatives as Strigolactone Biosynthesis Inhibitors.

Strigolactones (SLs) are one of the plant hormones that control several important agronomic traits, such as shoot branching, leaf senescence, and stress tolerance. Manipulation of the SL biosynthesis can increase the crop yield. We previously reported that a triazole derivative, TIS108, inhibits SL biosynthesis. In this study, we synthesized a number of novel TIS108 derivatives. Structure-activity relationship studies revealed that 4-(2-phenoxyethoxy)-1-phenyl-2-(1 H-1,2,4-triazol-1-yl)butan-1-one (KK5) inhibits the level of 4-deoxyorobanchol in roots more strongly than TIS108. We further found that KK5-treated Arabidopsis showed increased branching phenotype with the upregulated gene expression of AtMAX3 and AtMAX4. These results indicate that KK5 is a specific SL biosynthesis inhibitor in rice and Arabidopsis.

4-O-Methylascochlorin inhibits the prolyl hydroxylation of hypoxia-inducible factor-1α, which is attenuated by ascorbate.

4-O-Methylascochlorin (MAC), a methylated derivative of ascochlorin, was previously shown to promote the accumulation of hypoxia-inducible factor (HIF)-1α in human breast adenocarcinoma MCF-7 cells. In the present study, we further investigated the effects of MAC on the expression and function of HIF-1α in human fibrosarcoma HT-1080 cells. MAC promoted the accumulation of the HIF-1α protein without affecting its constitutive mRNA expression and augmented the transcriptional activation of HIF target genes. Ascorbate, but not N-acetylcysteine, attenuated MAC-mediated HIF-1α accumulation. MAC-induced increases in HIF-1α transcriptional activity were also attenuated by ascorbate. MAC inhibited the hydroxylation of HIF-1α at the proline 564 residue, while it was reversed by ascorbate. MAC slightly decreased the intracellular concentration of ascorbate. The present results demonstrated that MAC promoted the accumulation of HIF-1α by preventing prolyl hydroxylation, and ascorbate attenuated the MAC-mediated inhibition of HIF-1α prolyl hydroxylation.

MEPicides: potent antimalarial prodrugs targeting isoprenoid biosynthesis.

The emergence of Plasmodium falciparum resistant to frontline therapeutics has prompted efforts to identify and validate agents with novel mechanisms of action. MEPicides represent a new class of antimalarials that inhibit enzymes of the methylerythritol phosphate (MEP) pathway of isoprenoid biosynthesis, including the clinically validated target, deoxyxylulose phosphate reductoisomerase (Dxr). Here we describe RCB-185, a lipophilic prodrug with nanomolar activity against asexual parasites. Growth of P. falciparum treated with RCB-185 was rescued by isoprenoid precursor supplementation, and treatment substantially reduced metabolite levels downstream of the Dxr enzyme. In addition, parasites that produced higher levels of the Dxr substrate were resistant to RCB-185. Notably, environmental isolates resistant to current therapies remained sensitive to RCB-185, the compound effectively treated sexually-committed parasites, and was both safe and efficacious in malaria-infected mice. Collectively, our data demonstrate that RCB-185 potently and selectively inhibits Dxr in P. falciparum, and represents a promising lead compound for further drug development.

Functional analysis of the zebrafish ortholog of HMGCS1 reveals independent functions for cholesterol and isoprenoids in craniofacial development.

There are 8 different human syndromes caused by mutations in the cholesterol synthesis pathway. A subset of these disorders such as Smith-Lemli-Opitz disorder, are associated with facial dysmorphia. However, the molecular and cellular mechanisms underlying such facial deficits are not fully understood, primarily because of the diverse functions associated with the cholesterol synthesis pathway. Recent evidence has demonstrated that mutation of the zebrafish ortholog of HMGCR results in orofacial clefts. Here we sought to expand upon these data, by deciphering the cholesterol dependent functions of the cholesterol synthesis pathway from the cholesterol independent functions. Moreover, we utilized loss of function analysis and pharmacological inhibition to determine the extent of sonic hedgehog (Shh) signaling in animals with aberrant cholesterol and/or isoprenoid synthesis. Our analysis confirmed that mutation of hmgcs1, which encodes the first enzyme in the cholesterol synthesis pathway, results in craniofacial abnormalities via defects in cranial neural crest cell differentiation. Furthermore targeted pharmacological inhibition of the cholesterol synthesis pathway revealed a novel function for isoprenoid synthesis during vertebrate craniofacial development. Mutation of hmgcs1 had no effect on Shh signaling at 2 and 3 days post fertilization (dpf), but did result in a decrease in the expression of gli1, a known Shh target gene, at 4 dpf, after morphological deficits in craniofacial development and chondrocyte differentiation were observed in hmgcs1 mutants. These data raise the possibility that deficiencies in cholesterol modulate chondrocyte differentiation by a combination of Shh independent and Shh dependent mechanisms. Moreover, our results describe a novel function for isoprenoids in facial development and collectively suggest that cholesterol regulates craniofacial development through versatile mechanisms.

Targeting bactoprenol-coupled cell envelope precursors.

Targeting the bactoprenol-coupled cell wall precursor lipid II is a validated antibacterial strategy. In this review, selected prototype lipid II-binding antibiotics of different chemical classes are discussed. Although these compounds attack the same molecular target, they trigger nuanced and diverse cellular effects. Consequently, the mechanisms of antibacterial resistance and the likelihood of resistance development may vary substantially.

Metabolomics-Based Screening of the Malaria Box Reveals both Novel and Established Mechanisms of Action.

High-throughput phenotypic screening of chemical libraries has resulted in the identification of thousands of compounds with potent antimalarial activity, although in most cases, the mechanism(s) of action of these compounds remains unknown. Here we have investigated the mode of action of 90 antimalarial compounds derived from the Malaria Box collection using high-coverage, untargeted metabolomics analysis. Approximately half of the tested compounds induced significant metabolic perturbations in in vitro cultures of Plasmodium falciparum In most cases, the metabolic profiles were highly correlated with known antimalarials, in particular artemisinin, the 4-aminoquinolines, or atovaquone. Select Malaria Box compounds also induced changes in intermediates in essential metabolic pathways, such as isoprenoid biosynthesis (i.e., 2-C-methyl-d-erythritol 2,4-cyclodiphosphate) and linolenic acid metabolism (i.e., traumatic acid). This study provides a comprehensive database of the metabolic perturbations induced by chemically diverse inhibitors and highlights the utility of metabolomics for triaging new lead compounds and defining specific modes of action, which will assist with the development and optimization of new antimalarial drugs.

Untargeted Metabolomics To Ascertain Antibiotic Modes of Action.

Deciphering the mode of action (MOA) of new antibiotics discovered through phenotypic screening is of increasing importance. Metabolomics offers a potentially rapid and cost-effective means of identifying modes of action of drugs whose effects are mediated through changes in metabolism. Metabolomics techniques also collect data on off-target effects and drug modifications. Here, we present data from an untargeted liquid chromatography-mass spectrometry approach to identify the modes of action of eight compounds: 1-[3-fluoro-4-(5-methyl-2,4-dioxo-pyrimidin-1-yl)phenyl]-3-[2-(trifluoromethyl)phenyl]urea (AZ1), 2-(cyclobutylmethoxy)-5'-deoxyadenosine, triclosan, fosmidomycin, CHIR-090, carbonyl cyanidem-chlorophenylhydrazone (CCCP), 5-chloro-2-(methylsulfonyl)-N-(1,3-thiazol-2-yl)-4-pyrimidinecarboxamide (AZ7), and ceftazidime. Data analysts were blind to the compound identities but managed to identify the target as thymidylate kinase for AZ1, isoprenoid biosynthesis for fosmidomycin, acyl-transferase for CHIR-090, and DNA metabolism for 2-(cyclobutylmethoxy)-5'-deoxyadenosine. Changes to cell wall metabolites were seen in ceftazidime treatments, although other changes, presumably relating to off-target effects, dominated spectral outputs in the untargeted approach. Drugs which do not work through metabolic pathways, such as the proton carrier CCCP, have no discernible impact on the metabolome. The untargeted metabolomics approach also revealed modifications to two compounds, namely, fosmidomycin and AZ7. An untreated control was also analyzed, and changes to the metabolome were seen over 4 h, highlighting the necessity for careful controls in these types of studies. Metabolomics is a useful tool in the analysis of drug modes of action and can complement other technologies already in use.

Statin treatment and new-onset diabetes: a review of proposed mechanisms.

New-onset diabetes has been observed in clinical trials and meta-analyses involving statin therapy. To explain this association, three major mechanisms have been proposed and discussed in the literature. First, certain statins affect insulin secretion through direct, indirect or combined effects on calcium channels in pancreatic ß-cells. Second, reduced translocation of glucose transporter 4 in response to treatment results in hyperglycemia and hyperinsulinemia. Third, statin therapy decreases other important downstream products, such as coenzyme Q10, farnesyl pyrophosphate, geranylgeranyl pyrophosphate, and dolichol; their depletion leads to reduced intracellular signaling. Other possible mechanisms implicated in the effect of statins on new-onset diabetes are: statin interference with intracellular insulin signal transduction pathways via inhibition of necessary phosphorylation events and reduction of small GTPase action; inhibition of adipocyte differentiation leading to decreased peroxisome proliferator activated receptor gamma and CCAAT/enhancer-binding protein which are important pathways for glucose homeostasis; decreased leptin causing inhibition of ß-cells proliferation and insulin secretion; and diminished adiponectin levels. Given that the magnitude of the risk of new-onset diabetes following statin use remains to be fully clarified and the well-established beneficial effect of statins in reducing cardiovascular risk, statins remain the first-choice treatment for prevention of CVD. Elucidation of the mechanisms underlying the development of diabetes in association with statin use may help identify novel preventative or therapeutic approaches to this problem and/or help design a new generation statin without such side-effects.

A whole-cell phenotypic screening platform for identifying methylerythritol phosphate pathway-selective inhibitors as novel antibacterial agents.

Isoprenoid biosynthesis is essential for survival of all living organisms. More than 50,000 unique isoprenoids occur naturally, with each constructed from two simple five-carbon precursors: isopentenyl diphosphate (IPP) and dimethylallyl diphosphate (DMAPP). Two pathways for the biosynthesis of IPP and DMAPP are found in nature. Humans exclusively use the mevalonate (MVA) pathway, while most bacteria, including all Gram-negative and many Gram-positive species, use the unrelated methylerythritol phosphate (MEP) pathway. Here we report the development of a novel, whole-cell phenotypic screening platform to identify compounds that selectively inhibit the MEP pathway. Strains of Salmonella enterica serovar Typhimurium were engineered to have separately inducible MEP (native) and MVA (nonnative) pathways. These strains, RMC26 and CT31-7d, were then used to differentiate MVA pathway- and MEP pathway-specific perturbation. Compounds that inhibit MEP pathway-dependent bacterial growth but leave MVA-dependent growth unaffected represent MEP pathway-selective antibacterials. This screening platform offers three significant results. First, the compound is antibacterial and is therefore cell permeant, enabling access to the intracellular target. Second, the compound inhibits one or more MEP pathway enzymes. Third, the MVA pathway is unaffected, suggesting selectivity for targeting the bacterial versus host pathway. The cell lines also display increased sensitivity to two reported MEP pathway-specific inhibitors, further biasing the platform toward inhibitors selective for the MEP pathway. We demonstrate development of a robust, high-throughput screening platform that combines phenotypic and target-based screening that can identify MEP pathway-selective antibacterials simply by monitoring optical density as the readout for cell growth/inhibition.

Floral odor bouquet loses its ant repellent properties after inhibition of terpene biosynthesis.

In their natural environment, plants are synchronously confronted with mutualists and antagonists, and thus benefit from signals that contain messages for both functional groups of interaction partners. Floral scents are complex blends of volatiles of different chemical classes, including benzenoids and terpenoids. It has been hypothesized that benzenoids have evolved as pollinator attracting signals, while monoterpenoids serve as defensive compounds against antagonists. In order to test this hypothesis, we reduced terpene emission in flowers of Phlox paniculata with specific biosynthetic inhibitors and compared the responses of Lasius niger ants to natural and inhibited floral scent bouquets. While the natural odors were strongly repellent to ants, the bouquets with a reduced emission rate of terpenoids were not. The loss of the flowers' ability to repel ants could be attributed predominantly to reduced amounts of linalool, a monoterpene alcohol. Flying flower visitors, mainly hoverflies, did not discriminate between the two types of flowers in an outdoor experiment. Since individual compounds appear to be capable of either attracting pollinators or defending the flower from enemies, the complexity of floral scent bouquets may have evolved to allow flowers to respond to both mutualists and antagonists simultaneously.

Isoprenoid precursor biosynthesis offers potential targets for drug discovery against diseases caused by apicomplexan parasites.

Two, simple, C5 compounds, dimethylally diphosphate and isopentenyl diphosphate, are the universal precursors of isoprenoids, a large family of natural products involved in numerous important biological processes. Two distinct biosynthetic pathways have evolved to supply these precursors. Humans use the mevalonate route whilst many species of bacteria including important pathogens, plant chloroplasts and apicomplexan parasites exploit the non-mevalonate pathway. The absence from humans, combined with genetic and chemical validation suggests that the non-mevalonate pathway holds the potential to support new drug discovery programmes targeting Gram-negative bacteria and the apicomplexan parasites responsible for causing serious human diseases, and also infections of veterinary importance. The non-mevalonate pathway relies on eight enzyme-catalyzed stages exploiting a range of cofactors and metal ions. A wealth of structural and mechanistic data, mainly derived from studies of bacterial enzymes, now exists for most components of the pathway and these will be described. Particular attention will be paid to how these data inform on the apicomplexan orthologues concentrating on the enzymes from Plasmodium spp. these cause malaria, one the most important parasitic diseases in the world today.

[Screening of potential antibiotics, inhibitors of the nonmevalonate pathway of isoprenoid biosynthesis--2-C-methyl-D-erythritol-2,4-cyclodiphosphate derivatives].

The recently discovered nonmevalonate pathway of isoprenoid biosynthesis is a prospective target in screening of new antibiotics. Because of the absence of the pathway in the animal cells, the specific inhibitors of the pathway will be a new class of antibiotics against many pathogens (which cause, e.g., malaria, tuberculosis, etc), combining high efficiency and low toxicity. Several derivatives of 2-C-methyl-D-erythritol-2,4-cyclodiphosphate (MEC) were synthesized. 4-Phospho-methyl-D-erythritol-1,2-cyclophosphate, benzyl ether and benzyliden derivative of MEC inhibited the 14C-MEC incorporation into isoprenoids of chromoplasts from red pepper with IC50 of 1.7-5 MM. Some inhibition (about 10%) was also observed with the use of dimethyl ether and isopropyliden derivative of MEC.

Novel terpenoids, trichoderonic acids A and B isolated from Trichoderma virens, are selective inhibitors of family X DNA polymerases.

Trichoderonic acids A (1) and B (2), novel terpenoids, and (+)-heptelidic acid (3) isolated from cultures of a fungus, Trichoderma virens, and their structures were identified by spectroscopic analysis. These compounds selectively and competitively inhibited the activities of mammalian DNA polymerases beta, lambda (pols beta, lambda), and terminal deoxynucleotidyl transferase (TdT) in family X of pols, and compound 2 was a stronger inhibitor than compound 1 or 3. On the other hand, compounds 1-3 did not influence the activities of the other families (A-, B-, and the Y-families) of the mammalian pols tested, and showed no effect on the activities of plant pol alpha, fish pol delta, prokaryotic pol, or the other DNA metabolic enzymes tested. Compound 2 suppressed the growth of two human cancer cell lines, cervix carcinoma cells (HeLa) and breast carcinoma cells (MCF-7). The results suggest that these compounds identified inhibition among the families of mammalian pols.

Differential activities of thalidomide and isoprenoid biosynthetic pathway inhibitors in multiple myeloma cells.

Thalidomide has emerged as an effective agent for treating multiple myeloma, however the precise mechanism of action remains unknown. Agents known to target the isoprenoid biosynthetic pathway (IBP) can have cytotoxic effects in myeloma cells. The interactions between thalidomide and IBP inhibitors in human multiple myeloma cells were evaluated. Enhanced cytotoxicity and induction of apoptosis were observed in RPMI-8226 cells. Examination of intracellular levels of farnesyl pyrophosphate (FPP) and geranylgeranyl pyrophosphate (GGPP) revealed a wide variance in basal levels and response to IBP inhibitors. These findings provide a mechanism for the differential sensitivity of myeloma cells to pharmacologic manipulation of the IBP.

The isoprenoid pathway and transcriptional response to its inhibitors in the yeast Saccharomyces cerevisiae.

This review presents new insights into the regulation of the isoprenoid pathway in the yeast Saccharomyces cerevisiae, in particular the short-term transcriptional response to two inhibitors, lovastatin and zaragozic acid (ZA). Whereas lovastatin blocks whole isoprenoid pathway, ZA only blocks the sterol branch. Consequently, their effects on the cellular level of farnesyl diphosphate (FPP) are different. Lovastatin decreases the FPP level, whereas ZA, by inhibiting the main FPP-consuming enzyme, increases FPP availability in the cell. We discuss the role of genes whose expression is affected by both inhibitors and consider possible association of these genes with the regulation of the isoprenoid pathway.