Delta9-THC determination by the EU official method: evaluation of measurement uncertainty and compliance assessment of hemp samples.

Hemp cultivation is living a period of renewed interest worldwide after long years of opposition and abandonment. The European Union (EU) allows and subsidizes the growing of fiber and oilseed cultivars of Cannabis sativa L. with respect to the THC content limit of 0.2%. The EU method for the quantitative determination of Δ9-tetrahydrocannabinol (THC) content in hemp varieties provides to apply a tolerance of 0.03 g of THC per 100 g of sample concerning compliance assessment to that limit. However, the method does not report any precision data, especially useful as a function of THC content to evaluate measurement uncertainty and therefore to establish the conformity of hemp at different THC legal limits. Measurement uncertainty of the method by both bottom-up and top-down approach, besides repeatability and reproducibility, was investigated and estimated in the THC concentration range 0.2-1.0%, which includes the different legal limits set out for hemp around the world. We proposed Decision Rules for conformity of hemp showing that a non-compliant declaration beyond reasonable doubt should be stated when the THC content, as a mean result on a duplicate analysis, exceeds the limit by at least 11-15%, depending on THC limit. We highlighted other issues concerning practical aspects of hemp analysis, from sampling to evaluation of results, as well as the need to carry out collaborative studies on the EU method.

Unburned Tobacco Cigarette Smoke Alters Rat Ultrastructural Lung Airways and DNA.

INTRODUCTION: Recently, the Food and Drug Administration authorized the marketing of IQOS Tobacco Heating System as a Modified Risk Tobacco Product based on an electronic heat-not-burn technology that purports to reduce the risk. METHODS: Sprague-Dawley rats were exposed in a whole-body mode to IQOS aerosol for 4 weeks. We performed the chemical characterization of IQOS mainstream and we studied the ultrastructural changes in trachea and lung parenchyma of rats exposed to IQOS stick mainstream and tissue pro-inflammatory markers. We investigated the reactive oxygen species amount along with the markers of tissue and DNA oxidative damage. Moreover, we tested the putative genotoxicity of IQOS mainstream through Ames and alkaline Comet mutagenicity assays. RESULTS: Here, we identified irritating and carcinogenic compounds including aldehydes and polycyclic aromatic hydrocarbons in the IQOS mainstream as sign of incomplete combustion and degradation of tobacco, that lead to severe remodelling of smaller and largest rat airways. We demonstrated that IQOS mainstream induces lung enzymes that activate carcinogens, increases tissue reactive radical concentration; promotes oxidative DNA breaks and gene level DNA damage; and stimulates mitogen activated protein kinase pathway which is involved in the conventional tobacco smoke-induced cancer progression. CONCLUSIONS: Collectively, our findings reveal that IQOS causes grave lung damage and promotes factors that increase cancer risk. IMPLICATIONS: IQOS has been proposed as a safer alternative to conventional cigarettes, due to depressed concentration of various harmful constituents typical of traditional tobacco smoke. However, its lower health risks to consumers have yet to be determined. Our findings confirm that IQOS mainstream contains pyrolysis and thermogenic degradation by-products, the same harmful constituents of traditional cigarette smoke, and, for the first time, we show that it causes grave lung damage and promotes factors that increase cancer risk in the animal model.

The pursuit of good microbiological conditions in domestic softeners: a new improvement.

Effective resin disinfection is mandatory to ensure the microbiological quality of water treated by domestic softeners. The wet and sometimes warm environment inside the softener is ideal for bacteria growth. Our research was focused on the evaluation of the microbial quality of water from softeners sanitized by chlorine solutions or by electrolytic systems. We employed the heterotrophic plate count and specific tests to monitor the presence of opportunistic and pathogenic bacteria (Pseudomonas aeruginosa, Escherichia coli, enterococci, and coliforms). Completely new devices were equipped with a commercially available electrolytic system or with a newly patented one or sanitized by automatic or manual addition of chlorine solutions. In all cases, the contamination was reduced, not completely avoided. In particular, the patented electrolytic system significantly reduced bacterial proliferation in strongly contaminated devices. Our data confirm the difficulties encountered to solve the problem of microbiological quality of softened water and offer encouraging information on new possible solutions.

Bisphenol A determination in baby bottles by chemiluminescence enzyme-linked immunosorbent assay, lateral flow immunoassay and liquid chromatography tandem mass spectrometry.

Two immunoassays, a Lateral Flow ImmunoAssay (LFIA) based on colloidal gold nanoparticle labels and an indirect competitive chemiluminescence enzyme-linked immunosorbent assay (CL-ELISA), were developed and a high performance liquid chromatography-tandem mass spectrometry (LC-MS/MS) method was optimized to assess the possible release of bisphenol A (BPA, 4,4'-isopropylidenediphenol) from different plastic baby bottles treated with simulating solutions. Coating conjugate concentration, anti-BPA antibody dilution, incubation time of the primary and secondary antibodies, and tolerance to different organic solvents were optimized to obtain the best performance of the ELISA with chemiluminescent end-point detection. The influence of different buffers on LFIA performance was also evaluated. Both methods showed good repeatability (mean CV value around 13%) and sensitivity. Reproducibility tests for CL-ELISA gave a mean CV value of about 25%. The IC50 and Limit of Detection (LOD) values of CL-ELISA were 0.2 and 0.02 ng mL(-1), respectively. The LOD of LFIA was 0.1 µg mL(-1). A LC-MS/MS method was also optimized. The separation was performed in a C18 column with a triple-quadrupole mass spectrometer with electrospray ionisation interface. The method showed a good linearity in the range 2 to 500 ng mL(-1), with a regression coefficient of 0.998. In the simulating solutions the detection and quantification limits, calculated by the signal to noise level of 3 (S/N = 3), were 5.8 ng mL(-1) and 17.4 ng mL(-1), respectively. This limit of quantification was about 3 and 35 times lower than the permitted limits set by the official method CEN/TS 13130-13 (0.05 µg mL(-1)) and by the Directive 2004/19/EC (0.6 µg mL(-1)), respectively. The methods were applied to determine BPA release from baby bottles, performing repeated procedures according to EU and national regulations. The results demonstrated that no BPA migration from the tested plastic materials occurred with only one exception. The migrated amount, above the regulatory limits, was detected by all the mentioned assays.

Competitive amperometric immunosensor based on covalent linking of a protein conjugate to dendrimer-functionalised nanogold substrate for the determination of 2,4,6-trinitrotoluene.

A new amperometric immunosensor for 2,4,6-trinitrotoluene based on the working principle of competitive enzyme-linked immunosorbent assay was developed and characterised. An electrodeposited nanogold substrate was functionalised by deposition of self-assembled monolayers of 2-aminoethanethiol as linkers for the subsequent immobilisation of polyamidoaminic dendrimers. Our approach makes use of those dendrimers to anchor a trinitrobenzene-ovalbumin conjugate on the electrode surface. The immunosensor was tested and validated for the determination of 2,4,6-trinitrotoluene showing high selectivity with respect to other nitroaromatic compounds, a limit of detection of 4.8 ng/mL and a limit of quantitation of 6 ng/mL. The immunosensor was tested for the quantification of the analyte in spiked soils and in a real sample of post-blast soil, evidencing a good recovery rate (113 %).

Study of non-covalent interactions of luotonin A derivatives and the DNA minor groove as a first step in the study of their analytical potential as DNA probes.

The interaction between DNA and several newly synthesized derivatives of the natural anticancer compound luotonin A has been studied. The results from our work reveal an effective and selective alkaloid/double-stranded DNA (ds-DNA) interaction. In the presence of increasing amounts of ds-DNA, a noticeable fluorescence quenching of the luotonin A derivatives under study was observed. However, this effect did not take place when single-stranded DNA (ss-DNA) was employed. The association constant alkaloids/ds-DNA was calculated by quantitation of such a quenching effect. The influence of other quenchers, namely Co(2+) and Br(-) on the native fluorescence of luotonin A and derivatives was also studied, and a remarkable quenching effect was observed for both ions. We have also investigated how by binding DNA the alkaloids could get protected from the external Co(2+) and Br(-) quenchers. The Stern-Volmer constants (K (SV)) for Co(2+) and Br(-) quenching effect on the studied alkaloids were considerably reduced (10-50%) after incubation of the compounds in the presence of DNA with regard to the K (SV) values in absence of DNA. An increase in the fluorescence anisotropy values of luotonins was also produced only in the presence of ds-DNA but not in the case of ss-DNA. To better characterize the nature of that interaction, viscosimetry assays and ethidium bromide displacement studies were conducted. With regard to DNA reference solutions, the viscosity of solutions containing DNA and luotonin A derivatives was reduced or not significantly increased. It was also observed that the studied compounds were unable to displace the intercalating agent ethidium bromide. All of these results, together with the obtained association constants values (K (ass) = 2.2 × 10(2) - 1.3 × 10(3)), support that neither covalent nor intercalating interactions luotonin A derivatives/ds-DNA are produced, leading to the conclusion that these alkaloids bind ds-DNA through the minor groove. The specific changes in the fluorescence behavior of luotonin A and derivatives distinguishing between ss-DNA and ds-DNA binding, lead us to propose these compounds as attractive turn-off probes to detect DNA hybridization.

Chemiluminescent fingerprints from airborne particulate matter: A luminol-based assay for the characterization of oxidative potential with kinetical implications.

In this study, a new chemiluminescent method based on the dependence of luminol light emission induced by free radicals in airborne particulate matter (PM) is proposed as a screening assay for the rapid characterization of samples from different sources based on their redox properties. This parameter is considered critical for assessing particulate matter toxicity and its impacts on human health. We propose a cell-free, luminescent assay to evaluate the redox potential of particulate matter directly on the filters employed to collect it. A joint chemometric approach based on Principal Component Analysis and Hotelling Analysis was applied to quickly sort out ambient particulate samples with a significantly different light emission profile caused by Luminol reaction. Based on Spearman correlation analysis, the association of the samples light emission intensity with their chemical composition and emission sources was attempted. The overall methodology was tested with certified reference materials and applied to two series of particulate matter samples previously subjected to thorough chemical speciation and subsequent source apportionment. The results show the effectiveness of the luminescent method, allowing the quick assessment of particulate matter oxidative potential, but providing further evidence on the complexity of the oxidative potential determination in this kind of samples. The chemometric processing of the whole dataset clearly highlights the distinct behavior among the two series of samples, the certificate standard reference materials, and the blank controls, supporting the suitability of the approach.

Liquid chromatographic analysis of the anticancer alkaloid luotonin A and some new derivatives in human serum samples.

The quantitation of the natural cytotoxic and anti-inflammatory alkaloid luotonin A and five recently synthesized derivatives is described, constituting the first report of a HPLC method for the analysis of these compounds in human serum samples. The conditions for the chromatographic separation were optimized and the method was validated for the analysis of these compounds in biological samples according to international guidelines. An RP-HPLC method with fluorimetric detection and a C(18) stationary phase was applied. Different ACN/water mobile phases were assayed, including 0-4% of a mobile phase modifier such as tetrahydrofuran, dioxane or tert-butyl methyl ether. Isocratic and gradient elution conditions are compared. The influence of pH on the efficiency and resolution of the separation was also considered. The developed method was applied to the determination of luotonins in pooled human serum samples by gradient elution RP-HPLC using a simple cleanup procedure. The proposed chromatographic method exhibits satisfactory analytical figures of merit, with LOD from 1.0 x 10(-10) to 2.0 x 10(-10) M, intraday and interday precision below 6% except for the concentration level closest to LOD, and good agreement between experimental and theoretical concentrations. Therefore, the developed method is suitable, reliable, rapid, and simple.

Persistence and distribution of pesticide residues in fresh agricultural food consumed in the province of Bologna.

The presence of pesticide residues in fresh vegetables and fruit have been qualitatively and quantitatively determined at the laboratories of the Regional Agency for Environmental Protection (ARPA), Division of the Province of Bologna. More than 1,700 samples have been tested by routine analyses. The possible risks for consumers have been evaluated by various parameters. The most important ones were: the amount of each residue; the respective ADI (Acceptable Daily Intake) limit; the contemporary presence of different residues; an estimation of the daily intake, based on the amount of fruit and vegetables consumed per person. It has been possible to evaluate that the daily intake of pesticide residues in the province of Bologna during the period 2003-06 resulted lower than the ADI limits concerning the vegetables. According to the information on fruit consumption the daily intake of omethoate (O,O-dimethyl S-methylcarbamoylmethyl phosphorothioate) resulted higher than its ADI limit, of dicofol (2,2,2-trichloro-1,1-bis(trichloromethyl)benzenemethanol) very close to the admitted limit, under the respective limits for all the other residues.

Airborne particulate matter biotoxicity estimated by chemometric analysis on bacterial luminescence data.

In this work, PM10 samples previously subjected to thorough chemical speciation and receptor modelling, have been investigated for their bio-toxicity using an inhibition test based on bacterial luminescence modulation when in contact with airborne particulate samples. The variation of light emission intensity from a luminescent bacteria strain, the Photobacterium phosphoreum, is proposed as an efficient proxy for the quantification of bio-toxic effects induced by airborne particulate matter. PM10 samples characterized by definite levels of pollutants from the pertaining air shed were found to induce a decrease in the bacterial bioluminescence intensity, expressed as percentage of Inhibition Ratio (IR%). This behaviour suggests the decay of this energy-consuming activity because of a toxic effect. Cluster analysis on chemical composition and IR% data provides evidence of a statistically significant association between the adverse effects on living cells and the range of specific chemical species in PM10.

Microbial processes associated to the decontamination and detoxification of a polluted activated sludge during its anaerobic stabilization.

Xenobiotic compounds accumulate in activated sludge resulting from wastewater treatment plants serving both civil and industrial areas. The opportunity to use anaerobic digestion for the decontamination and beneficial disposal of a contaminated activated sludge was investigated in mesophilic and thermophilic microcosms monitored through an integrated chemical, microbiological and ecotoxicological procedure. The 10 months anaerobic sludge incubation at 35 degrees C resulted in an extensive production of a methane-rich biogas, a marked reduction of pathogenic cultivable bacteria and, importantly, a marked biodegradation of the sludge-carried organic pollutants, including some polychlorinated biphenyls and polycyclic aromatic hydrocarbons, along with a relevant sludge detoxification. The sludge decontamination seemed to occur mostly under methanogenic conditions and was not significantly affected by the addition of yeast extract or molasses. Lower bioremediation and biomethanization yields were observed under thermophilic conditions.

Field detection capability of immunochemical assays during criminal investigations involving the use of TNT.

The capability to collect timely information about the substances employed on-site at a crime scene is of fundamental importance during scientific investigations in crimes involving the use of explosives. TNT (2,4,6-trinitrotoluene) is one of the most employed explosives in the 20th century. Despite the growing use of improvised explosives, criminal use and access to TNT is not expected to decrease. Immunoassays are simple and selective analytical tests able to detect molecules and their immunoreactions can occur in portable formats for use on-site. This work demonstrates the application of three immunochemical assays capable of detecting TNT to typical forensic samples from experimental tests: an indirect competitive ELISA with chemiluminescent detection (CL-ELISA), a colorimetric lateral flow immunoassay (LFIA) based on colloidal gold nanoparticles label, and a chemiluminescent-LFIA (CL-LFIA). Under optimised working conditions, the LOD of the colorimetric LFIA and CL-LFIA were 1 µg mL(-1) and 0.05 µg mL(-1), respectively. The total analysis time for LFIAs was 15 min. ELISA proved to be a very effective laboratory approach, showing very good sensitivity (LOD of 0.4 ng mL(-1)) and good reproducibility (CV value about 7%). Samples tested included various materials involved in controlled explosions of improvised explosive devices (IEDs), as well as hand swabs collected after TNT handling tests. In the first group of tests, targets covered with six different materials (metal, plastic, cardboard, carpet fabric, wood and adhesive tape) were fixed on top of wooden poles (180 cm high). Samples of soil from the explosion area and different materials covering the targets were collected after each explosion and analysed. In the second group of tests, hand swabs were collected with and without hand washing after volunteers simulated the manipulation of small charges of TNT. The small amount of solution required for each assay allows for several analyses. Results of immunoassays confirmed that they were suitable to detect post-blast residues in soil and target materials and post transfer residues on hands, allowing further confirmation by more selective techniques. ELISA and LFIAs results obtained from the same solution were consistently in good agreement, and were confirmed by gas chromatography coupled to mass spectrometry (GC-MS). The reported immunoassays data demonstrates the suitability of LFIAs as on-site rapid and effective assays to detect TNT traces. The CL-ELISA proved useful in obtaining very sensitive detection in forensic investigations and testing, while CL-LFIA had performances in between LFIA and CL-ELISA.

Development of chemiluminescent ELISAs to DDT and its metabolites in food and environmental samples.

Two enzyme-linked immunosorbent assays (ELISA) with chemiluminescent (CL) detection for the insecticide DDT and the group of DDT-related compounds have been optimized and characterized. Both conjugate-coated ELISAs are based on monoclonal antibodies (MAbs) of different specificity and homologous protein conjugates. Effects of several physicochemical factors (ionic strength, pH, Tween-20 and Bovine serum albumin (BSA) concentrations) and solvents (methanol, ethanol, acetone and N,N'-dimethylformamide) on the performance of the assays were studied and optimized. For the DDT-selective assay, the sensitivity, estimated as the I(50) value, was 0.6 microg/l, with a linear working range between 0.1 and 2 microg/l and a limit of detection of 0.06 microg/l. For the DDT group-selective assay, the sensitivity was 0.2 microg/l, with a linear working range between 0.07 and 1 microg/l and a limit of detection of 0.04 microg/l. CL-ELISAs were four times more sensitive compared to colorimetric ELISAs. Finally, both immunoassays were applied to the detection of DDT and group of DDT-related compounds in spiked real water, soil and food samples.

Environmental monitoring for microbial contamination by batch and flow bioluminescent systems.

A study was performed to optimise a rapid method to determine the microbial content on surfaces by means of flow and batch microplate ATP bioluminescent assay. Sampling, ATP extraction and testing of these specimens were considered. The data obtained gave a general picture of the state of hygiene and cleanliness of the surfaces examined, mainly classrooms.

Development of a chemiluminescence-based quantitative lateral flow immunoassay for on-field detection of 2,4,6-trinitrotoluene.

Simple, rapid and highly sensitive assays, possibly allowing on-site analysis, are required in the security and forensic fields or to obtain early signs of environmental pollution. Several bioanalytical methods and biosensors based on portable devices have been developed for this purpose. Among them, Lateral Flow ImmunoAssays (LFIAs) offer the advantages of rapidity and ease of use and, thanks to the high specificity of antigen-antibody binding, allow greatly simplifying and reducing sample pre-analytical treatments. However, LFIAs usually employ colloidal gold or latex beads as labels and they rely on the formation of colored bands visible by the naked eye. With this assay format, only qualitative or semi-quantitative information can be obtained and low sensitivity is achieved. Recently, the use of enzyme-catalyzed chemiluminescence detection in LFIA has been proposed to overcome these problems. In this work, we describe the development of a quantitative CL-LFIA assay for the detection of 2,4,6-trinitrotoluene (TNT) in real samples. Thanks to the use of a portable imaging device for CL signal measurement based on a thermoelectrically cooled CCD camera, the analysis could be performed directly on-field. A limit of detection of 0.2 µg mL(-1) TNT was obtained, which is five times lower than that obtained with a previously described colloidal gold-based LFIA developed employing the same immunoreagents. The dynamic range of the assay extended up to 5 µg mL(-1) TNT and recoveries ranging from 97% to 111% were obtained in the analysis of real samples (post blast residues obtained from controlled explosion).

Optimal conditions for stability of photoemission and freeze drying of two luminescent bacteria for use in a biosensor.

Bioluminescent bacteria have been used for many years for biotoxicological analysis. One of the main concerns with this microorganism is the low experimental repeatability when subjected to external factors. The aim of the present study was to obtain accurate, sensitive, and repeatable measurements with stable signals (during the detection and over days) for application in a water-analysis device for the detection of pollutants. Growth conditions were tested and optimized. An optimal freeze-drying procedure for the constitutive bioluminescent bacteria Vibrio fischeri and Photobacterium phosphoreum was developed. The luminescence stability after rehydration was also investigated. Freeze drying was found to be a critical process in survival and signal stability of luminescent bacteria; for this reason, different suspension fluids and various bacterial pellet/suspension fluid ratios (g/ml) were evaluated. The toxicity of heavy metals and organic compounds in water was determined to investigate the applicability of a test based on bacteria obtained in this way, comparing the data with legal limits. A scale-up process was developed with industrial technology: freeze-dried bacteria that emitted a stable luminous signal after rehydration were obtained. Moreover, the median effective concentration (EC50) was calculated with these bacteria.

Chemiluminescent ELISA for the BTEX determination in water and soil.

An indirect competitive chemiluminescence enzyme-linked immunosorbent assay (CL-ELISA) for the determination of benzene, toluene, ethylbenzene, and ortho-, meta-, para-xylenes (BTEX) in soil and water was developed. The assay was optimized concerning the coating conjugate concentration, anti-BTEX antiserum dilution, incubation time effect on primary and secondary antibody incubation, and temperature effect on the competitive step and tolerance to different organic solvents. The IC(50) and lower limit of the detection (LDD(90)) values were 4.6 and 0.5 microg mL(-1), respectively. While water samples could be analyzed directly, soil has to be extracted and diluted prior to immunochemical measurements. BTEX could be recovered from spiked soil with a yield higher than 60% using 5-min ultrasonication with methanol. Finally, the assay was applied to soil and water samples collected at a contaminated site in Italy, and was compared to GC-MS.

Monitoring of environmental pollutants by bioluminescent bacteria.

This review deals with the applications of bioluminescent bacteria to the environmental analyses, published during the years 2000-2007. The ecotoxicological assessment, by bioassays, of the environmental risks and the luminescent approaches are reported. The review includes a brief introduction to the characteristics and applications of bioassays, a description of the characteristics and applications of natural bioluminescent bacteria (BLB), and a collection of the main applications to organic and inorganic pollutants. The light-emitting genetically modified bacteria applications, as well as the bioluminescent immobilized systems and biosensors are outlined. Considerations about commercially available BLB and BLB catalogues are also reported. Most of the environmental applications, here mentioned, of luminescent organisms are on wastewater, seawater, surface and ground water, tap water, soil and sediments, air. Comparison to other bioindicators and bioassay has been also made. Various tables have been inserted, to make easier to take a rapid glance at all possible references concerning the topic of specific interest.

Chemiluminescent determination of total antioxidant capacity during winemaking.

The total antioxidant capacity (TAC) of five different wines (four red and one white) was determined in five different steps of winemaking carried out in a commercial wine cellar by a chemiluminescence (CL) assay. The CL method is suitable to determine the antioxidant capacity of beverages, and preliminary trials showed that the TAC immediately after the bottle was opened was greater than the day after (about 25% decrease). Immediate analysis or a correct sample storage is therefore necessary. The wines were characterized by different levels of total phenolics and TAC: these differences were related to grape composition and winemaking technologies. The TAC values were the highest immediately after devatting. The TAC suffered the highest decrease (30-50%) after the clarification procedure, which may be due to the fining agents used and to oxygen contact, then remained more or less constant in the subsequent steps.

Plasma antioxidant capacity determination: comparative evaluation of chemiluminescent and spectrophotometric assays.

All aerobic organisms have developed different mechanisms for neutralising the free radicals, mostly produced by the monoelectronic reduction of O(2), and preventing the severe damages these can provoke. The efficiency of these mechanisms can be assessed, in different matrices, by a simple and direct chemiluminescent assay (CL) based on luminol oxidation catalysed by horseradish peroxidase. Light emission is mediated by the production of free radicals and it is inhibited after a sample addition in a way that is directly proportional to the sample total content of molecules displaying antioxidant activity. The performances of this chemiluminescent assay were compared with those of two spectrophotometric methods already applied in clinical practice. First spectrophotometric method measures, like CL assay, the total antioxidant capacity, whereas the second one determines free thiol groups content. The chemiluminescent assay has a linearity interval between 0.60 and 9.46 mumol l(-1) of Trolox (y=34.91x+3.10; r=0.999; n=5) with an imprecision, expressed as CV, of 3.8% and an inaccuracy, expressed as percentage recovery, of 109%. The first spectrophotometric method, based on the same reference standard, the Trolox molecule, has a linearity interval between 0.2 and 2.5 mmol l(-1) of Trolox (y=-0.01x+4.54; r=0.95; n=5); the thiol groups assay has a linearity interval between 0.1 and 1 mmol l(-1) of l-cysteine (y=1.68x-47.09; r=0.998; n=5). Different clinical samples of plasma from healthy individuals, obese subjects and patients with liver diseases were tested. Interesting correlations were obtained among the three methods, but no significant correlations emerged between antioxidant capacity and clinical parameters. Significant differences were there only between men and women among obese subjects and between drinkers and non-drinkers among liver disease patients.