[Show abstract][Hide abstract] ABSTRACT: Because of its availability, ease of collection, and correlation with physiology and pathology, urine is an attractive source for clinical proteomics/peptidomics. However, the lack of comparable data sets from large cohorts has greatly hindered the development of clinical proteomics. Here, we report the establishment of a reproducible, high resolution method for peptidome analysis of naturally occurring human urinary peptides and proteins, ranging from 800 to 17,000 Da, using samples from 3,600 individuals analyzed by capillary electrophoresis coupled to MS. All processed data were deposited in an Structured Query Language (SQL) database. This database currently contains 5,010 relevant unique urinary peptides that serve as a pool of potential classifiers for diagnosis and monitoring of various diseases. As an example, by using this source of information, we were able to define urinary peptide biomarkers for chronic kidney diseases, allowing diagnosis of these diseases with high accuracy. Application of the chronic kidney disease-specific biomarker set to an independent test cohort in the subsequent replication phase resulted in 85.5% sensitivity and 100% specificity. These results indicate the potential usefulness of capillary electrophoresis coupled to MS for clinical applications in the analysis of naturally occurring urinary peptides.
[Show abstract][Hide abstract] ABSTRACT: Wine chemical compositions, which result from a complex interplay between environmental factors, genetic factors, and viticultural practices, have mostly been studied using targeted analyses of selected families of metabolites. Detailed studies have particularly concerned volatile and polyphenolic compounds because of their acknowledged roles in the organoleptic and therapeutic properties. However, we show that an unprecedented chemical diversity of wine composition can be unraveled through a nontargeted approach by ultrahigh-resolution mass spectrometry, which provides an instantaneous image of complex interacting processes, not easily or possibly resolvable into their unambiguous individual contributions. In particular, the statistical analysis of a series of barrel-aged wines revealed that 10-year-old wines still express a metabologeographic signature of the forest location where oaks of the barrel in which they were aged have grown.
Proceedings of the National Academy of Sciences 07/2009; 106(23):9174-9. · 9.81 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: The coupled reverse osmosis–electrodialysis (RO/ED) method was used to isolate dissolved organic matter (DOM) from 16 seawater samples. The average yield of organic carbon was 75±12%, which is consistently greater than the yields of organic carbon that have been commonly achieved using XAD resins, C18 adsorbents, and cross-flow ultrafiltration. UV–visible absorbance spectra and molar C/N ratios of isolated samples were consistent with the corresponding properties of DOM in the original seawater samples, indicating that DOM samples can be isolated using the coupled RO/ED method without any bias for/against these two properties. Five of the samples were desalted sufficiently that reliable measurements of their 13C and 1HNMR spectra and their Fourier transform ion cyclotron resonance (FTICR) mass spectra could be obtained. The 13C and 1HNMR spectra of RO/ED samples differed distinctly from those of samples that have been isolated in much lower yields by other methods. In particular, RO/ED samples contained a relatively lower proportion of carbohydrate carbon and a relatively greater proportion of alkyl carbon than samples that have been isolated using cross-flow ultrafiltration. From the FTICR mass spectra of RO/ED samples, samples from the open ocean contained a much lower proportion of unsaturated compounds and a much higher proportion of fatty acids than coastal samples.
Geochimica et Cosmochimica Acta 01/2009; 73(14):4215-4231. · 3.88 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Soils, sediments, freshwaters, and marine waters contain natural organic matter (NOM), an exceedingly complex mixture of organic compounds that collectively exhibit a nearly continuous range of properties (size-reactivity continuum). NOM is composed mainly of carbon, hydrogen, and oxygen, with minor contributions from heteroatoms such as nitrogen, sulfur, and phosphorus. Suwannee River fulvic acid (SuwFA) is a fraction of NOM that is relatively depleted in heteroatoms. Ultrahigh resolution Fourier transform ion cyclotron (FTICR) mass spectra of SuwFA reveal several thousand molecular formulas, corresponding in turn to several hundred thousand distinct chemical environments of carbon even without accountancy of isomers. The mass difference deltam among adjoining C,H,O-molecules between and within clusters of nominal mass is inversely related to molecular dissimilarity: any decrease of deltam imposes an ever growing mandatory difference in molecular composition. Molecular formulas that are expected for likely biochemical precursor molecules are notably absent from these spectra, indicating that SuwFA is the product of diagenetic reactions that have altered the major components of biomass beyond the point of recognition. The degree of complexity of SuwFA can be brought into sharp focus through comparison with the theoretical limits of chemical complexity, as constrained and quantized by the fundamentals of chemical binding. The theoretical C,H,O-compositional space denotes the isomer-filtered complement of the entire, very vast space of molecular structures composed solely of carbon, hydrogen, and oxygen. The molecular formulas within SuwFA occupy a sizable proportion of the theoretical C,H,O-compositional space. A 100 percent coverage of the theoretically feasible C,H,O-compositional space by SuwFA molecules is attained throughout a sizable range of mass and H/C and O/C elemental ratios. The substantial differences between (and complementarity of) the SuwFA molecular formulas that are observed using six different modes of ionization (APCI, APPI, and ESI in positive and negative modus) imply considerable selectivity of the ionization process and suggest that the observed mass spectra represent simplified projections of still more complex mixtures.
[Show abstract][Hide abstract] ABSTRACT: A non-targeted, ultra-high-resolution mass spectrometric, direct analysis of oak-wood extracts from two species (Quercus robur L. and Quercus petraea Liebl.) from three French forests, and of a wine aged in barrels derived therefrom has been performed to identify families of metabolites that could discriminate both the species and the geographical origin of woods. From 12 T ultra-high-resolution Fourier transform ion cyclotron resonance mass spectra of wood extracts, hundreds of mass signals were identified as possible significant biomarkers of the two species, with phenolic and carbohydrate moieties leading the differentiation between Q. robur and Q. petraea, respectively, as corroborated by both FTMS and NMR data. For the first time, it is shown that oak woods can also be discriminated on the basis of hundreds of forest-related compounds, and particular emphasis is put on sessile oaks from the Tronçais forest, for which sugars are significantly discriminant. Despite the higher complexity and diversity of wine metabolites, forest-related compounds can also be detected in wines aged in related barrels. It is only by using these non-targeted analyses that such innovative results, which reveal specific chemodiversities of natural materials, can be obtained.
[Show abstract][Hide abstract] ABSTRACT: The photocatalytic transformation of imazamox, a herbicide of imidazolinone family, is investigated in aqueous solution containing titanium dioxide, hydrogen peroxide or the combination of TiO2/H2O2 under simulated sunlight irradiation. The effect of parameters such as the amount of catalysts, the concentration of herbicide, and the pH were investigated by measurement of the rate constant of degradation. Experimental data obtained under different conditions describe the dependency of degradation rate on the above mentioned parameters. Consequently, kinetic parameters were experimentally determined and a pseudo-first-order kinetic was observed. Mulliken charge distributions calculated by the DFT method B3LYP/6–31+G(d) level of theory for key cationic, anionic and neutral structures of imazamox give interpretation for the dependency of photodegradation rate constant on pH. The degradation rate constants were always higher for the heterogeneous catalysis in reactions (TiO2/UV, TiO2/UV/H2O2) compared to the homogeneous systems (UV alone, H2O2/UV). In parallel, five photoproducts could be tentatively identified using Electrospray ionization Fourier transform ion cyclotron resonance mass spectroscopy based on precise chemical formula assignments.
[Show abstract][Hide abstract] ABSTRACT: Owing to its availability, ease of collection, and correlation with pathophysiology of diseases, urine is an attractive source for clinical proteomics. However, many proteomic studies have had only limited clinical impact, due to factors such as modest numbers of subjects, absence of disease controls, small numbers of defined biomarkers, and diversity of analytical platforms. Therefore, it is difficult to merge biomarkers from different studies into a broadly applicable human urinary proteome database. Ideally, the methodology for defining the biomarkers should combine a reasonable analysis time with high resolution, thereby enabling the profiling of adequate samples and recognition of sufficient features to yield robust diagnostic panels. Capillary electrophoresis coupled to mass spectrometry (CE-MS), which was used to analyze urine samples from healthy subjects and patients with various diseases, is a suitable approach for this task. The database of these datasets compiled from the urinary peptides enabled the diagnosis, classification, and monitoring of a wide range of diseases. CE-MS exhibits excellent performance for biomarker discovery and allows subsequent biomarker sequencing independent of the separation platform. This approach may elucidate the pathogenesis of many diseases, and better define especially renal and urological disorders at the molecular level.
[Show abstract][Hide abstract] ABSTRACT: Many soil microorganisms antagonistic to soil borne plant pathogens are well known for their ability to control diseases in situ. A variety of substances, like lytic enzymes, siderophores and antibiotics, produced by these organisms have the potential to protect roots against pathogens. Understanding the ecology and a functional assessment of antagonistic microbial communities in soil requires in-depth knowledge of the mechanisms involved in these interactions, a challenging task in complex systems if low-resolution methods are applied. We propose an information-rich strategy of general relevance, composed of adequate preconcentration in conjunction with ultrahigh resolution ion cyclotron resonance Fourier transform mass spectrometry (ICR-FT/MS) and nuclear magnetic resonance (NMR) spectroscopy to identify any bioactive substances in complex systems. This approach is demonstrated on the specific example of substance identification considered responsible for in vitro antagonism of an actinobacterial antagonist isolated from European beech (Fagus sylvatica) rhizosphere soil against the oomycetous root rot pathogen Phytophthora citricola. The isolate belonging to the genus Kitasatospora exhibited strong antibiosis against the oomycete in vitro. The bioactive substance was observed to exhibit a molar mass of 281.1699 g/mol in positive electrospray ionization mass spectra, and the high mass accuracy of the ICR-FT/MS measurements allowed a precise assignment of a molecular formula that was found identical to the macrolide polyketide cycloheximide C(15)H(23)NO(4)+H(+); its identity was then unequivocally confirmed by the information-rich atomic signature of proton NMR spectroscopy. In conclusion, the combination of the near orthogonal methods (pre)fractionation, ultrahigh-resolution ICR-FT mass spectrometry (yielding molecular and MS(n) fragment signatures) and nuclear magnetic resonance spectroscopy (providing atomic signatures) has been found capable of identifying a biocontrol active compound of Kitasatospora active against Phytophthora citricola expediently, quickly, and accurately. This straightforward approach is of general applicability to elucidate biocontrol mechanisms in any complex system with improved efficiency.
Journal of Microbiological Methods 07/2008; 75(2):188-95. · 2.16 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: The biogeography of prokaryotes and the effect of geographical barriers as evolutionary constraints are currently subjected to great debate. Some clear-cut evidence for geographic isolation has been obtained by genetic methods but, in many cases, the markers used are too coarse to reveal subtle biogeographical trends. Contrary to eukaryotic microorganisms, phenotypic evidence for allopatric segregation in prokaryotes has never been found. Here we present, for the first time, a metabolomic approach based on ultrahigh resolution mass spectrometry to reveal phenotypic biogeographical discrimination. We demonstrate that strains of the cosmopolitan extremophilic bacterium Salinibacter ruber, isolated from different sites in the world, can be distinguished by means of characteristic metabolites, and that these differences can be correlated to their geographical isolation site distances. The approach allows distinct degrees of discrimination for isolates at different geographical scales. In all cases, the discriminative metabolite patterns were quantitative rather than qualitative, which may be an indication of geographically distinct transcriptional or posttranscriptional regulations.
The ISME Journal 04/2008; 2(3):242-53. · 8.95 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: This chapter presents the technique of capillary electrophoresis coupled to mass spectrometry (CE/MS). The introductory section is targeted mainly at CE/MS beginners and notes briefly the theoretical background of electrospray ionization (ESI), the most commonly used ionization mode in CE/MS. The specifics of CE/MS are described--also in comparison with more classic methods like LC/MS. Important caveats to be taken into consideration for successful CE/MS operation are noted in the interest of avoiding pitfalls. CE/MS is illustrated with three representative examples, which might serve as starting points for more in-detail experiments: (1) partial-filling micellar electrokinetic chromatography (MEKC) of neutral bacterial signaling molecules (N-acylhomoserine lactones) extracted from culture supernatants, (2) capillary zone electrophoresis (CZE) of their anionic degradation products, and finally (3) CZE separation of cationic hydroxy-s-triazines.
[Show abstract][Hide abstract] ABSTRACT: Root-associated or endophytic diazotrophs, like Azospirillum, Herbaspirillum or Burkholderia spp., are of key interest in plant-growth promotion and biological nitrogen-fixation research. Recently, new species of the
genera Herbaspirillum and Burkholderia were described (Reis et al., 2004; Rothballer et al., 2006). To analyze the diazotrophic or endophytic life style of bacteria,
culture dependent methods, like isolation of bacteria from surface sterilized roots in semisolid N-free media, are not sufficient.
Most important for the in situ characterization of the endophytic life style are microscopic techniques. For this purpose, species-specific phylogenetic
fluorescently labeled oligonucleotide probes (Stoffels et al., 2001) during FISH analysis and the use of GFP-tagged strains
in combination with confocal laser scanning microscopy (CLSM) were used. With orthogonal views of confocal image stacks prepared
with digital-image analysis tools, the endophytic colonization of plant cells can easily be followed (Rothballer et al., 2003).
TEM-based investigations after staining with monospecific polyclonal antibodies were also successfully used (Rothballer et
al., unpublished data, 2007).
[Show abstract][Hide abstract] ABSTRACT: Direct degradation of imazapic, an herbicide of the imidazoline family, has been investigated in aqueous solution at different concentrations, pH values, and temperatures. The efficiency of the photodegradation process has been evaluated through degradation rate constants that could be fitted best with pseudo-first-order kinetics ( Ct = C0 e(- kt )). Ultrahigh resolution mass spectrometry (FTICR/MS) was used in electrospray ionization mode as a tool to study the photolysis process on a molecular level, whereas UV-vis and high-performance liquid chromatography/mass spectrometry analysis were used to follow, by time, the evolution of the intermediates. Taking advantage of the high resolving power of FTICR/MS to perform precise formula assignments taking account of the natural abundance of isotopes, we herein propose and demonstrate an approach using 2D-derived van Krevelen visualization (O/C, H/C, m/z) to confirm the formation of imazapic intermediates. Such an approach allows a qualitative analysis of intermediates and elucidates the plausible reaction pathways of the photolysis process. More than eight photoproducts were separated and identified as a phototransformation of the imidazole ring. A mechanistical pathway was proposed.
Journal of Agricultural and Food Chemistry 12/2007; 55(24):9936-43. · 2.91 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: The photodecomposition of imazamox, a herbicide of the imidazolinone family, was investigated in pure water. The main photoproducts from the photolysis were followed over time by liquid chromatography mass spectrometry and structures were proposed from exact mass determinations obtained by electrospray ionization Fourier transform ion cyclotron resonance mass spectrometry. The method comprised exact mass determination with better than 0.2 ppm mass accuracy and a corresponding structural visualization taking care of respective isotopes with an adapted van Krevelen diagram that enabled a systematic approach to the characterisation of the elementary composition of each photoproduct. By taking advantage of the high resolving power of FT-ICR MS to make precise formula assignments, the derived 2D van Krevelen diagram (O/C; H/C; m/z) enabled one to structurally differentiate the formed photoproducts and to propose a degradation pathway for imazamox.
Analytical and Bioanalytical Chemistry 12/2007; 389(5):1459-67. · 3.66 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: This perspective article provides an assessment of the state-of-the-art in the molecular-resolution analysis of complex organic materials. These materials can be divided into biomolecules in complex mixtures (which are amenable to successful separation into unambiguously defined molecular fractions) and complex nonrepetitive materials (which cannot be purified in the conventional sense because they are even more intricate). Molecular-level analyses of these complex systems critically depend on the integrated use of high-performance separation, high-resolution organic structural spectroscopy and mathematical data treatment. At present, only high-precision frequency-derived data exhibit sufficient resolution to overcome the otherwise common and detrimental effects of intrinsic averaging, which deteriorate spectral resolution to the degree of bulk-level rather than molecular-resolution analysis. High-precision frequency measurements are integral to the two most influential organic structural spectroscopic methods for the investigation of complex materials-NMR spectroscopy (which provides unsurpassed detail on close-range molecular order) and FTICR mass spectrometry (which provides unrivalled resolution)-and they can be translated into isotope-specific molecular-resolution data of unprecedented significance and richness. The quality of this standalone de novo molecular-level resolution data is of unparalleled mechanistic relevance and is sufficient to fundamentally advance our understanding of the structures and functions of complex biomolecular mixtures and nonrepetitive complex materials, such as natural organic matter (NOM), aerosols, and soil, plant and microbial extracts, all of which are currently poorly amenable to meaningful target analysis. The discrete analytical volumetric pixel space that is presently available to describe complex systems (defined by NMR, FT mass spectrometry and separation technologies) is in the range of 10(8-14) voxels, and is therefore capable of providing the necessary detail for a meaningful molecular-level analysis of very complex mixtures. Nonrepetitive complex materials exhibit mass spectral signatures in which the signal intensity often follows the number of chemically feasible isomers. This suggests that even the most strongly resolved FTICR mass spectra of complex materials represent simplified (e.g. isomer-filtered) projections of structural space.
Analytical and Bioanalytical Chemistry 12/2007; 389(5):1311-27. · 3.66 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Since highly sensitive on-line coupling of UPLC with FTICR-MS is technically infeasible due to their different scan rates, at-line coupling of these techniques was developed for rapid analysis. To enable cutting of one peak of the chromatogram into one fraction, several conditions and relationships were investigated, e.g. the optimum volume of the inserted delay loop, the relationship between retention time, loop outlet drop speed, individual drop volume versus mobile phase composition under constant speed, and linear solvent strength gradient elution modes. Good and reproducible results were achieved applying UPLC as an efficient separation and fast fractionation tool before the FTICR-MS measurements. A chip-based nanoelectrospray ionization system was employed which was perfectly suited to handling the small-volume fractions and was thus chosen for the at-line coupling. The method was initially applied to spiked extracts of cell-free bacterial culture supernatants in which bacterial signalling compounds, namely N-acyl homoserine lactones (AHL), were detected. Good reproducibility and high recovery was observed. Afterwards, a culture supernatant of Erwinia sp. JX3.2, a putative AHL producer, was investigated and N-hexanoyl-homoserine lactone was determined as a possible signalling molecule. More reliable assignments were achieved by use of at-line coupling of UPLC and FTICR-MS compared with off-line measurements.
Analytical and Bioanalytical Chemistry 12/2007; 389(5):1439-46. · 3.66 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Derivatives of N-acylhomoserine lactones (HSLs) with different alkanoyl side chains occur as quorum or diffusion sensing molecules in gram-negative bacteria and their quantitative chemical analysis became important as a possible way to follow regulation processes of their pathogenicity towards plants and animals. The lactone-ring of HSLs is chemically and biologically not stable: the corresponding serines can be formed in alkaline conditions and these may presumably behave inactive for the biological system. A fast and MS compatible liquid chromatographic method applying high pressure (ultra performance liquid chromatography) with diode array detection was optimized for the rapid quantitative determination of HSLs and their corresponding hydrolysis products. The technique was used to follow and model the hydrolysis reactions of HSLs as function of pH under controlled conditions. Moreover, the method could be triggered to allow a confirmation in the assignment of the potential HSLs in real samples by analysis of the real samples before and after hydrolysis. Quantitative performance characteristics and the character of the hydrolysis reaction were studied as well. The optimized method was successfully applied to a bacterial culture supernatant real sample containing HSLs.
Journal of Chromatography A 09/2007; 1160(1-2):184-93. · 4.61 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: We have established and validated a protocol for the peptidomic analysis of rat urine using CE coupled to MS (CE-MS). In the first experiments, the reproducibility of the CE-MS set-up and of the established preparation procedure were assessed. To establish a first rat urinary peptidome map, samples were also analyzed using CE-FT-ICR. The subsequent analysis of independent samples from two different strains (WISTAR and CD) indicated strain-specific differences, which were validated in a blinded assessment. MS/MS revealed the presence of specific fragments from well-known urinary rat peptides, such as collagens, alpha-1-antitrypsin, and serum albumin. The CE-MS-based peptidomics platform may provide novel insights into body fluids of animal models, such as rat or mice. Together with peptide identification, the technology appears to be an excellent, complimentary, and non-invasive tool to analyze toxicological or other (patho)physiological effects of pharmaceutical compounds in animal models.
[Show abstract][Hide abstract] ABSTRACT: N-Acylated homoserine lactones (AHLs) are produced by Gram-negative bacteria as communication signals and are frequently studied as mediators of the "quorum sensing" response of bacterial communities. Several reports have recently been published on the identification of AHLs from different species and attempts have been made to study their role in natural habitats, for example the surface of plant roots in the rhizosphere. In this article, different analytical methods, including bacterial biosensors and chromatographic techniques, are reviewed. A concept for assignment of the structures of AHLs is also presented. The retention behaviour of derivatives of AHLs containing beta-keto or hydroxyl groups and/or double bonds has been evaluated in relation to the separation behaviour of AHLs with saturated and unsubstituted alkanoyl chains. Samples have also been analysed by high resolution mass spectrometry (Fourier-transform ion-cyclotron-resonance mass spectrometry, FTICR-MS), nano liquid chromatography-electrospray ionization ion trap mass spectrometry (nano-LC-MS) and by the aid of a biosensor. The results obtained from ultra performance liquid chromatography (UPLC), FTICR-MS, nano-LC-MS, and bioassays have been compared to attempt structural characterisation of AHL without chemical synthesis of analytical standards. The method was used to identify the major AHL compound produced by the rhizosphere bacterium Acidovorax sp. N35 as N-(3-hydroxydecanoyl)homoserine lactone.
Analytical and Bioanalytical Chemistry 02/2007; 387(2):455-67. · 3.66 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: A robust method based on solid-phase extraction (SPE) followed by ultra high pressure liquid chromatography (with trade name of Ultra Performance Liquid Chromatography: UPLC; Waters, Milford, MA, USA) is proposed for the determination of five derivatives of N-acylhomoserine lactones (AHLs) that play a biological role as signal molecules of several gram-negative bacteria. Different commercial SPE cartridges were tested for sample extraction, clean-up and preconcentration. Since the sample matrix was a complex growth media, careful optimization of the SPE with respect to washing procedure, elution solvent and sample solvent was necessary. No sample loss was observed when up to 100 mL spiked full media was added onto the cartridge. Applying UPLC for the determination of AHLs, the performance characteristics of the method showed good separation efficiency and high speed. In order to demonstrate the applicability of the method, supernatants with the known AHL producer Burkholderia cepacia LA3 grown in different media were investigated. Additionally, the method was successfully used for the degradation/uptake study of AHLs from a liquid matrix in which barley was grown under controlled condition.
Journal of Chromatography A 12/2006; 1134(1-2):186-93. · 4.61 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Metal working fluids (MWFs) are widely used as lubricants and coolants for different industrial operations. Biocides are ingredients of MWFs to control the microbial growth; derivatives of hexahydrotriazines and oxazolidines are generally used. Because of the lack of appropriate characterization, an existing capillary electrophoretic method for their quantification was improved. During the process of optimization, it became clear that hydrolysis products, derivatives of amino alcohols, severely interfere with the separation procedure. Since indirect-UV detection lacked the required selectivity, mass-selective detection was employed. NMR and MS established the absence of amino alcohols in the original educts. The aqueous solutions of the biocides stored for extended time remained amino alcohol-free, suggesting that these amino alcohols are formed from the biocides during the capillary electrophoretic separation. The observation of narrow and symmetric peaks indicated hydrolysis, and the polarity of the products implied favorable conditions for capillary electrophoretic separation. Methods were optimized for the analysis of the amino alcohols, the hydrolytic products of the formaldehyde releasers, using indirect-UV and MS detection. This method was extended to other likely solutes used as alkaline-reserve ingredients. The analytes were separated within 9 min with a high precision of migration times (the RSDs were below 1.5%). When quantifying from mobility scale, the calibration curves produced linearity with regression coefficients in the range of 0.990-0.999. The detection limit was lower than 1 mg/L in the case of MS detection. The influence of water-based MWF was also investigated, and no matrix effect on the migration of the analytes and on the peak areas was observed.