Chitosan/Hyaluronic acid/Alginate and an assorted polymers loaded with honey, plant, and marine compounds for progressive wound healing-Know-how.

Biomaterials are being extensively used in regenerative medicine including tissue engineering applications, as these enhance tissue development, repair, and help in the process of angiogenesis. Wound healing is a crucial biological process of regeneration of ruptured tissue after getting injury to the skin and other soft tissue in humans and animals. Besides, the accumulation of microbial biofilms around the wound surface can increase the risk and physically obstruct the wound healing activity, and may even lead to amputation. Hence, in both acute and chronic wounds, prominent biomaterials are required for wound healing along with antimicrobial agents. This review comprehensively addresses the antimicrobial and wound healing effects of chitosan, chitin, cellulose acetate, hyaluronic acid, pullulan, bacterial cellulose, fibrin, alginate, etc. based wound dressing biomaterials fabricated with natural resources such as honey, plant bioactive compounds, and marine-based polymers. Due to their excellent biocompatibility and biodegradability, bioactive compounds derived from honey, plants, and marine resources are commonly used in biomedical and tissue engineering applications. Different types of polymer-based biomaterials including hydrogel, film, scaffold, nanofiber, and sponge dressings fabricated with bioactive agents including honey, curcumin, tannin, quercetin, andrographolide, gelatin, carrageenan, etc., can exhibit significant wound healing process in, diabetic wounds, diabetic ulcers, and burns, and help in cartilage repair along with good biocompatibility and antimicrobial effects. Among the reviewed biomaterials, carbohydrate polymers such as chitosan-based biomaterials are prominent and widely used for wound healing applications followed by hyaluronic acid and alginate-based biomaterials loaded with honey, plant, and marine compounds. This review first provides an overview of the vast natural resources used to formulate different biomaterials for the treatment of antimicrobial, acute, and chronic wound healing processes.

Analytical implications of different methods for preparing plant cell wall material.

Almost all plant cells are surrounded by a wall constructed of co-extensive networks of polysaccharides and proteoglycans. The capability to analyse cell wall components is essential for both understanding their complex biology and to fully exploit their numerous practical applications. Several biochemical and immunological techniques are used to analyse cell walls and in almost all cases the first step is the preparation of an alcohol insoluble residue (AIR). There is significant variation in the protocols used for AIR preparation, which can have a notable impact on the downstream extractability and detection of cell wall components. To explore these effects, we have formally compared ten AIR preparation methods and analysed polysaccharides subsequently extracted using high-performance anion exchange chromatography (HPAEC-PAD) and Micro Array Polymer Profiling (MAPP). Our results reveal the impact that AIR preparation has on downstream detection of cell wall components and the need for optimisation and consistency when preparing AIR.

Herbal Based Polymeric Nanoparticles as a Therapeutic Remedy for Breast Cancer.

BACKGROUND: The currently available anti breast cancer agents as well as conventional drug delivery methods have some limitations. OBJECTIVE: In view of these limitations, researchers used phytochemicals/herbal extracts as anti-breast cancer agents together with the polymeric nanoparticles to provide an effective way of targeted drug delivery with lesser /no side effects. METHODS: The literature for this review was searched during the year 2015 to 2019, using the keywords, ' 'breast cancer', 'breast cancer and its current treatments', 'plants against the breast cancer', 'polymeric nanoparticles', 'herbal based polymeric nanoparticles'. The databases i.e., PubMed, Science Direct, and Google Scholar, were used for collecting the information. RESULTS: In the present review, an attempt was made to summarize the potential of herbal-based nanoformulation as a specific and high efficacy therapeutic strategy in order to pave the way for future research involving screening and use of herbal nanoparticles for the treatment of breast cancer. CONCLUSION: The encapsulation of the herbal extract in the polymeric nanoparticles is the prominent, effective, and emerging way of targeted drug delivery for cancer. It may serve as a safer way of targeted drug delivery and maybe the answer to the complications related to the currently available anti-breast cancer agents as well as limitations of the conventional method of drug delivery.

Pressurized Solvent Extraction of Paulownia Bark Phenolics.

Paulownia bark is mostly utilized jointly with wood, but the possibility of a separate valorization through the pressurized extraction of bark bioactives has been assessed. Subcritical water extraction and supercritical CO2 extraction are green technologies allowing shorter times than conventional solvent extraction under atmospheric shaken conditions. Subcritical water extraction was carried out at temperatures ranging from 140 to 240 °C and supercritical CO2 extraction was performed at different pressures (10, 20 and 30 MPa), temperatures (35, 45 and 55 °C) and ethanol concentrations (0, 10 and 15% (w/w)). Subcritical water extraction under a non-isothermal operation during heating up to 160 °C (19 min) provided extraction yields up to 30%, and the extracts contained up to 7% total phenolics with an ABTS (2,2'-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid)) radical scavenging capacity equivalent to 35% the activity of Trolox, whereas at 240 °C, the yield decreased to 20%, but the phenolic content reached 21%, and the antiradical activity was equivalent to 85% of Trolox. Supercritical CO2 extraction at 30 MPa, 45 °C and 30 min reached a global yield of 2% after 180 min of extraction, but the product showed very low antiradical capacity. Gallic acid, vanillic acid, vanillin and apigenin were the major phenolic compounds found in the extracts.

Identification and quantification of free and bound phenolic compounds contained in the high-molecular weight melanoidin fractions derived from two different types of cocoa beans by UHPLC-DAD-ESI-HR-MSn.

The aim of the present study was to establish the profiles of soluble free phenolics (SFPs) and bound phenolics (BPs) in high molecular weight (HMW) melanoidin fractions isolated from raw and roasted beans of two Theobroma cacao L. varieties. Samples were prepared using three methods (saline treatment and acidic and alkaline hydrolysis) to obtain different forms of phenolic compounds. A total of fifteen phenolics, including three flavan-3-ols, seven phenolic acids, one phenolic aldehyde, and four N-phenylpropenoyl-L-amino acids (NPAs), were identified using ultra-high-performance liquid chromatography coupled to diode array detection and electrospray ionization high-resolution mass spectrometry (UHPLC-DAD-ESI-HR-MSn). In HMW fractions from both studied cocoa types, the main SFPs were N-caffeoyl-L-Asp and procyanidin B2, whereas the main BPs were catechin, epicatechin, ellagic acid, protocatechualdehyde, and N-caffeoyl-L-Asp. The concentrations of individual BPs were much higher than the content of total SFPs. It was also found that, as compared to alkaline hydrolysis, acid hydrolysis released a significantly higher amount of BPs from HMW melanoidin fractions. A comprehensive quantitative analysis indicated significant variation in the investigated phenolic compounds depending on the cocoa type and roasting conditions. An increase in treatment temperature from 110 to 150 °C led to a decline in SFPs and an increment in BPs. The HMW fractions of unroasted Criollo beans exhibited the highest content of SFPs and the lowest content of BPs. The highest BP concentrations were obtained for both cocoa bean varieties roasted at 150 °C. The present study revealed that HMW melanoidin fractions from cocoa beans of different varieties roasted at higher temperatures are a good source of phenolic compounds that can be released under both acidic and alkaline conditions.

Structural polymeric features that contribute to in vitro immunostimulatory activity of instant coffee.

An instant coffee fraction, rich in arabinogalactans, obtained by ultrafiltration, using 1 and 5kDa membranes, has previously shown in vitro stimulatory activity on BALB/c mice spleen B lymphocytes. The compounds inducing lymphocytic activation were shown to elute, mainly near the void volume by size-exclusion chromatography, using Bio-Gel P6 (1-6kDa). Treatment of the compounds with chymotrypsin, a digestive protease, did not affect the induced B lymphocyte activation. On the contrary, hydrolysis with an α-l-arabinofuranosidase, removing single terminally-linked arabinose residues, decreased the extent of B cell activation. The immunostimulatory activity of these compounds was also observed by in vitro experiments, using bone marrow-derived macrophages and dendritic cells as responders. Altogether, these results show the relevance of single arabinose residues, present at the non-reducing end of polymeric compounds, to the coffee stimulatory activity in cells mediating innate and acquired immunity.

Bioactive sesquiterpene polyol esters from the leaves of Tripterygium wilfordii.

Tripterygium wilfordii, a member of Celastraceae family, has been used as a traditional plant insecticide and a medicinal plant. Phytochemical investigation of the leaves of T. wilfordii has resulted in the isolation of eight sesquiterpene polyol esters triptersinines M-T (1-8) and one sesquiterpene pyridine alkaloid (9). The structures of the compounds were elucidated on the basis of spectroscopic data analyses, including UV, IR, MS, and NMR experiments. The inhibitory effects on nitric oxide production in LPS-induced macrophages of 1-9 were also evaluated.

The marine sponge-derived inorganic polymers, biosilica and polyphosphate, as morphogenetically active matrices/scaffolds for the differentiation of human multipotent stromal cells: potential application in 3D printing and distraction osteogenesis.

The two marine inorganic polymers, biosilica (BS), enzymatically synthesized from ortho-silicate, and polyphosphate (polyP), a likewise enzymatically synthesized polymer consisting of 10 to >100 phosphate residues linked by high-energy phosphoanhydride bonds, have previously been shown to display a morphogenetic effect on osteoblasts. In the present study, the effect of these polymers on the differential differentiation of human multipotent stromal cells (hMSC), mesenchymal stem cells, that had been encapsulated into beads of the biocompatible plant polymer alginate, was studied. The differentiation of the hMSCs in the alginate beads was directed either to the osteogenic cell lineage by exposure to an osteogenic medium (mineralization activation cocktail; differentiation into osteoblasts) or to the chondrogenic cell lineage by incubating in chondrocyte differentiation medium (triggering chondrocyte maturation). Both biosilica and polyP, applied as Ca²âº salts, were found to induce an increased mineralization in osteogenic cells; these inorganic polymers display also morphogenetic potential. The effects were substantiated by gene expression studies, which revealed that biosilica and polyP strongly and significantly increase the expression of bone morphogenetic protein 2 (BMP-2) and alkaline phosphatase (ALP) in osteogenic cells, which was significantly more pronounced in osteogenic versus chondrogenic cells. A differential effect of the two polymers was seen on the expression of the two collagen types, I and II. While collagen Type I is highly expressed in osteogenic cells, but not in chondrogenic cells after exposure to biosilica or polyP, the upregulation of the steady-state level of collagen Type II transcripts in chondrogenic cells is comparably stronger than in osteogenic cells. It is concluded that the two polymers, biosilica and polyP, are morphogenetically active additives for the otherwise biologically inert alginate polymer. It is proposed that alginate, supplemented with polyP and/or biosilica, is a suitable biomaterial that promotes the growth and differentiation of hMSCs and might be beneficial for application in 3D tissue printing of hMSCs and for the delivery of hMSCs in fractures, surgically created during distraction osteogenesis.

Extraction and characterization of Aegle marmelos derived polymer as a pharmaceutical excipient.

BACKGROUND: Natural polymers have been used as pharmaceutical excipients. They are easily available, cheap, less toxic andbiodegradable. Many of them have been identified and research is ongoing regarding their characterization. OBJECTIVE: The present study depicts the extraction and characterization of Aegle marmelos derived polymer which can be used as a pharmaceutical excipient. MATERIAL AND METHODS: A water based extraction method was used to extract Aegle marmelos derived polymer. Its yield was found to be 15.07%. Characterization was based on various parameters such as a test for carbohydrates, test for purity, organoleptic properties, ash value, solubility behavior, pH, swelling index, surface tension, viscosity, particle size, loss on drying, bulk density, bulkiness, powder flow behavior, etc. RESULT: The polymer was yellowish-brown and showed poor flow (angle of repose 19.28 degrees ± 0.883) with neutral pH, i.e. 7, and bulkiness depicting the heaviness of polymer. The extracted polymer showed solubility in warm water and insolubility in organic solvents. CONCLUSIONS: The results easily predict the fact that the yield of the polymer was quite good, so it can be used as a commercial source of mucilage. The isolated polymer can be used as a pharmaceutical excipient in different dosage forms. 2 /

Lanthanide-IMAC enrichment of carbohydrates and polyols.

In this study a new type of immobilized metal ion affinity chromatography resin for the enrichment of carbohydrates and polyols was synthesized by radical polymerization reaction of vinyl phosphonic acid and 1,4-butandiole dimethacrylate using azo-bis-isobutyronitrile as radical initiator. Interaction between the chelated trivalent lanthanide ions and negatively charged hydroxyl groups of carbohydrates and polyols was observed by applying high pH values. The new method was evaluated by single standard solutions, mixtures of standards, honey and a more complex extract of Cynara scolymus. The washing step was accomplished by acetonitrile in excess volumes. Elution of enriched carbohydrates was successfully performed with deionized water. The subsequent analysis was carried out with matrix-free laser desorption/ionization-time of flight mass spectrometry involving a TiO2 -coated steel target, especially suitable for the measurement of low-molecular-weight substances. Quantitative analysis of the sugar alcohol xylitol as well as the determination of the maximal loading capacity was performed by gas chromatography in conjunction with mass spectrometric detection after chemical derivatization. In a parallel approach quantum mechanical geometry optimizations were performed in order to compare the coordination behavior of various trivalent lanthanide ions.

How does roasting affect the antioxidants of a coffee brew? Exploring the antioxidant capacity of coffee via on-line antioxidant assays coupled with size exclusion chromatography.

During coffee roasting major changes occur in coffee bean composition. Among others dark coloured melanoidins are formed, which are high molecular weight Maillard reaction products. A new approach is presented here to monitor the influence of roasting conditions on the antioxidant capacity of melanoidins and chlorogenic acids (CGAs) in a coffee brew. Validated Folin-Ciocalteu (FC) and ABTS assays were used as on-line antioxidant assays coupled (post-column) with high performance size-exclusion chromatography (HPSEC). HPSEC enabled the separation of melanoidins from CGAs and the determination of the antioxidant capacity of each fraction, within a total elution time of 25 min. Besides the on-line assay measurements, both assays were also applied off-line with flow injection analysis (FIA). The maximum antioxidant capacity was determined to be at a light-to-medium roast degree, measured with both ABTS-FIA and FC-FIA assays as well as on-line ABTS assay. With FC on-line assay the maximum was found to be at a very light roast degree. Based on the peak areas obtained with the new coupled technique the roasting effects on the variability of melanoidin and CGA contents in coffee brews were studied. The majority of melanoidins are already formed in the early stage of the roasting process and the relative contribution of melanoidins to the total antioxidant capacity increases towards darker roasts, mainly because CGAs degrade during roasting. A new parameter, the ratio of melanoidin to CGA peak area, was introduced as a possible predictor of the roast degree.

[Screening and identification of microorganisms for decolorization of molasses spent wash].

Microorganisms were screened from the natural environment for decolorization of molasses spent wash, and the isolated strains were then employed in the treatment of actual wastewater. The primary screening was carried out on agar plates supplemented with synthesized melanoidin as the target substrate, since melanoidin is one of the most refractory pigments in wastewater. Promising microorganisms were further selected through secondary screening by decolorization of untreated actual wastewater in shaking flask cultures. Gel filtration chromatography was used to determine the molecular weight distribution of pigments in molasses spent wash before and after decolorization. A strain named A5P1 was isolated from the soil samples collected, showing a good ability of decolorizing molasses spent wash, and was later identified as Aspergillus flavus by morphology and ITS sequence analysis. Experimental study of factors affecting the decolorization performance of strain A5P1 gave the optimal conditions as follows: 4.3 x 10(4) mL(-1) of inoculum size, medium with initial pH of 4.5 and cultivation at 39 degrees C. It could decolorize 53.0% of the pigments in the untreated molasses spent wash and decreased 80% of chemical oxygen demand after four-day incubation. The result of gel filtration chromatography demonstrated that both the large and small molecular weight fractions of pigments in the molasses spent wash could be removed by strain A5P1. Based on the measurement of enzyme activities, at least three different kinds of enzymes, i. e. the enzyme with H2O2-producing activity, laccase and manganese peroxidase were involved in the decolorization process. Therefore, the decolorization mechanism of strain A5P1 was preliminarily considered to be mainly biodegradation, with bioadsorption as a minor reaction.

Two new tocopherol polymers from the seeds of Euryale ferox.

Two new tocopherol polymers, chroman-type dimer named ferotocodimer A (1) and spiro-type trimer named ferotocotrimer E (2), were isolated from the seeds of Euryale ferox. Their structures were elucidated on the basis of spectroscopic methods including HR-ESI-MS and 1D and 2D NMR. The absolute configurations of 1 and 2 were determined by CD and ROESY experiments.

Structure, fluorescence quenching and antioxidant activity of a carbohydrate polymer from Eugenia jambolana.

Natural products provide an excellent source for novel antioxidants. Herein, we have studied the water-extracted carbohydrate polymer (WE) of Eugenia jambolana using chemical, chromatographic, and spectroscopic methods. A 116 kDa arabinogalactan containing p-coumaric and ferulic acids in monomeric and dimeric forms has been isolated. Cellulase generated oligomeric fragments containing ester linked phenolic acids were also characterized. The antioxidant capacity of this carbohydrate polymer is comparable to butylated hydroxy anisole and butylated hydroxy toluene. Interaction of WE with bovine serum albumin (BSA) was studied by fluorescence quenching measurement. Conformational change of BSA at high carbohydrate polymer concentration was indicated.

Carboxymethyl gum kondagogu: synthesis, characterization and evaluation as mucoadhesive polymer.

The objective of the study was to modify gum kondagogu by carboxymethylation and to evaluate it for potential pharmaceutical applications. Carboxymethylation of gum kondagogu was carried out by reacting gum kondagogu with monochloroacetic acid under alkaline conditions. The results of characterization studies revealed that carboxymethylation of gum kondagogu increases its degree of crystallinity and surface roughness, reduces its viscosity and improves its mucoadhesive properties. Further, carboxymethyl gum kondagogu was explored for pharmaceutical applications by formulating ionotropically gelled beads using metformin as the model drug and calcium chloride as cross-linking agent. Ex vivo bioadhesion study conducted using isolated chick-ileum by wash-off test revealed bioadhesion of >80% over a period of 24 h. It was observed that increasing the concentration of cross-linking agent increases the % drug entrapment and reduces the release rate. The beads were found to release the drug by Fickian-diffusion mechanism and following zero-order release kinetics.

High molecular weight coffee melanoidins are inhibitors for matrix metalloproteases.

High molecular (above 10 kDa) melanoidins isolated from coffee beans of varying roasting degree were found to be efficient inhibitors for the zinc-containing matrix metalloproteases MMP-1, MMP-2, and MMP-9 with IC(50) values ranging between 0.2 and 1.1 mg/mL in vitro. The inhibitory potential increased with roasting degree. No or only slight inhibition of other zinc-containing peptidases closely related to MMPs, namely, Clostridium histolyticum collagenase and angiotensin converting enzyme, was found, indicating specific structural features of melanoidins to be responsible for the interaction with MMPs. A continuous increase on the apparent molecular weight of melanoidins as well as incorporation of phenolic substances into the melanoidin structure with progress of roasting was observed, concomitant with a significant increase in the carbon/nitrogen of the melanoidins. This suggests that the melanoidins are mainly formed by incorporation of carbohydrates and phenolic compounds onto a proteinaceous backbone. As MMP-1, MMP-2, and MMP-9 play a pivotal role in pathogenesis of colorectal cancer, studies on possible physiological effects of melanoidins are mandatory.

Antiproliferative activity of melanoidins isolated from heated potato fiber (potex) in glioma cell culture model.

Potex constitutes a potato fiber preparation widely used as an ingredient to meat and bakery products which thermal treatment results in creation of new compounds. Melanoidins are high molecular weight brown end products of Maillard reaction, and few data presenting tumor cell growth inhibiting activity of melanoidins have been reported. Thus, in present study we utilized water extract of Potex roasted (180 °C for 2 h), whose chemical characterization revealed the presence of melanoidin complexes. Heated Potex extract inhibited C6 glioma cell proliferation in a dose-dependent manner measured by MTT method. High molecular weight components present in initial extract were responsible for stronger antiproliferative effect compared with low molecular weight fraction. Impaired MAPK (mitogen-activated protein kinase) and Akt signaling was found in cells treated with the extract. Moreover, flow cytometry analyses revealed the extract to induce G1/S arrest in glioma cells. Simultaneously, Western blot analysis showed elevated levels of p21 protein with concomitant decrease of cyclin D1. In conclusion, observed antiproliferative activity of melanoidins present in heated Potex was linked to disregulated MAPK and Akt signaling pathways, as well as to cell cycle cessation. These results suggest potential application of Potex preparation as a functional food ingredient and chemopreventive agent.

Coffee reduces liver damage in a rat model of steatohepatitis: the underlying mechanisms and the role of polyphenols and melanoidins.

UNLABELLED: Epidemiological data associate coffee consumption with a lower prevalence of chronic liver disease and a reduced risk of elevated liver enzyme levels (γ glutamyl transpeptidase and alanine aminotransferase), advanced liver disease and its complications, and hepatocellular carcinoma. Knowledge of the mechanisms underlying these effects and the coffee components responsible for these properties is still lacking. In this study, 1.5 mL/day of decaffeinated coffee or its polyphenols or melanoidins (corresponding to approximately 2 cups of filtered coffee or 6 cups of espresso coffee for a 70-kg person) were added for 8 weeks to the drinking water of rats who were being fed a high-fat, high-calorie solid diet (HFD) for the previous 4 weeks. At week 12, HFD + water rats showed a clinical picture typical of advanced nonalcoholic steatohepatitis compared with control rats (normal diet + water). In comparison, HFD + coffee rats showed: (1) reduced hepatic fat and collagen, as well as reduced serum alanine aminotransferase and triglycerides; (2) a two-fold reduced/oxidized glutathione ratio in both serum and liver; (3) reduced serum malondialdehyde (lipid peroxidation) and increased ferric reducing antioxidant power (reducing activity); (4) reduced expression of tumor necrosis factor α (TNF-α), tissue transglutaminase, and transforming growth factor ß and increased expression of adiponectin receptor and peroxisome proliferator-activated receptor α in liver tissue; and (5) reduced hepatic concentrations of proinflammatory TNF-α and interferon-γ and increased anti-inflammatory interleukin-4 and interleukin-10. CONCLUSION: Our data demonstrate that coffee consumption protects the liver from damage caused by a high-fat diet. This effect was mediated by a reduction in hepatic fat accumulation (through increased fatty acid ß-oxidation); systemic and liver oxidative stress (through the glutathione system); liver inflammation (through modulation of genes); and expression and concentrations of proteins and cytokines related to inflammation.

Safety evaluation of proanthocyanidin polymer-rich fraction obtained from stem bark of Stryphnodendron adstringens (BARBATIMAO) for use as a pharmacological agent.

The widespread use of medicinal plants among the Brazilian population warrants an assessment of the potential risks associated with their intake. Stryphnodendron adstringens (barbatimão) is one of the most frequently used medicinal plants in Brazil, and the risks associated with its use have yet to be investigated. This study evaluated the genotoxic safety of the use of the proanthocyanidin polymer-rich fraction (F2) of stem bark of S. adstringens. The micronucleus test with 750, 1500, and 2250 mg kg(-1) of F2 administered in Mus musculus (Swiss) outbred mice, showed respectively, 5.0±0.8 (Mean±S.D.), 9.1±1.7, and 10.6±1.9 micronucleated polychromatic erythrocytes (MNPCE). A positive control with cyclophosphamide resulted in 21.0±3.8 MNPCE. Antimutagenicity was also evaluated, by adding 750 mg kg(-1) to cyclophosphamide; the result of 8.7±1.4 showed a protective cytotoxic effect. For the Artemia salina test, 10, 100, and 1000 mg L(-1) of F2 showed, respectively, 8.7±0.6, 7.7±0.6, and 5.7±1.2 survival, i.e., F2 did not inhibit 50% of the population when compared to the control (9.7±0.6). These results indicated that F2 obtained from stem bark of S. adstringens has no genotoxic activity.

Polyacetylene diols with antiproliferative and driving Th1 polarization effects from the marine sponge Callyspongia sp.

A screening of 30 crude extracts of marine sponges against human promyelocytic leukemia cells (HL-60) yielded an EtOAc extract of the sponge Callyspongia sp. (Callyspongiidae) with significant activity. Further bioassay-guided fractionation of the EtOAc extract led to the isolation of three polyacetylene metabolites: a new polyacetylene diol, callyspongidiol (1), along with two known compounds, siphonodiol (2) and 14,15-dihydrosiphonodiol (3). Their structures were determined by a combination of spectroscopic analyses. Compounds 1-3 exhibited antiproliferative activity against HL-60 with IC(50) values of 6.5, 2.8, and 6.5 microg/ml, respectively. These metabolites induce apoptosis in HL-60 cells. Dendritic cells (DC) differentiated with 1-3 enhance the differentiation of naïve T cells towards the Th1 type.