Activation of endogenous neurogenesis and angiogenesis by basic fibroblast growth factor-chitosan gel in an adult rat model of ischemic stroke

Attempts have been made to use cell transplantation and biomaterials to promote cell proliferation,differentiation,migration,and survival,as well as angiogenesis,in the context of brain injury.However,whether bioactive materials can repair the damage caused by ischemic stroke by activating endogenous neurogenesis and angiogenesis is still unknown.In this study,we applied chitosan gel loaded with basic fibroblast growth factor to the stroke cavity 7 days after ischemic stroke in rats.The gel slowly released basic fibroblast growth factor,which improved the local microenvironment,activated endogenous neural stem/progenitor cells,and recruited these cells to migrate toward the penumbra and stroke cavity and subsequently differentiate into neurons,while enhancing angiogenesis in the penumbra and stroke cavity and ultimately leading to partial functional recovery.This study revealed the mechanism by which bioactive materials repair ischemic strokes,thus providing a new strategy for the clinical application of bioactive materials in the treatment of ischemic stroke.

Synthesis of chitosan oligosaccharide selenium and its antitumor activity

Background:Polysaccharides have various biological activities;the complexation of polysaccharides with trace element ions can produce synergistic effects,improving the original biological activities of sugars and trace elements.Methods:The preparation process of chitosan oligosaccharide selenium(COSSe)was optimized by the response surface method,followed by a detailed analysis of the resultant compound’s characteristics.The anti-cancer activity of COSSe was studied using the human ovarian cancer cell line SKOV3 as a cell model.Results:The prepared COSSe response surface was well predicted,indicating successful chitosan oligosaccharide binding with selenium.Response surface method analyses identified the optimal synthesis conditions for COSSe:the reaction time of 5.08 h,the reaction temperature of 71.8°C,and mass ratio(Na2SeO3:chitosan oligosaccharide)of 1.02.Under the optimal conditions,the final product,the selenium content,reached 1.302%.The results of cell experiments showed that COSSe significantly inhibited SKOV3 proliferation in a concentration-dependent manner.RNA-seq results showed that chitosan oligosaccharide and COSSe significantly modulated the expression of genes’DNA metabolic processes and cell cycle in SKOV3 cells.Gene enrichment analysis showed the inhibition of the cell cycle,and the results of flow cytometry showed that SKOV3 cells increased in the S phase and decreased in the G2/M phase,with a noted suppression in the protein expression of cyclin-dependent kinase 2(CDK2)and cyclin A1(CCNA1).Conclusion:COSSe has a stronger effect than chitosan oligosaccharide,leading to the arrest of the cell cycle in the S phase.Thus,COSSe may be an effective candidate for the treatment of ovarian cancer.

Chitosan-based triboelectric materials for self-powered sensing at high temperatures

Although biopolymers have been widely utilized as triboelectric materials for the construction of self-powered sensing systems,the annihilation of triboelectric charges at high temperatures restricts the output signals and sensitivity of the assembled sensors.Herein,a novel chitosan/montmorillonite/lignin(CML)composite film was designed and employed as a tribopositive layer in the assembly of a self-powered sensing system for use under hot conditions(25-70℃).The dense contact surface resulting from the strong intermolecular interaction between biopolymers and nanofillers restrained the volatilization of induced electrons.The optimized CML-TENG delivered the highest open-circuit voltage(V_(oc))of 262 V and maximum instantaneous output power of 429 mW/m^(2).Pristine CH-TENG retained only 39%of its initial Voc at 70℃,whereas the optimized CM_(5)L_(3)-TENG retained 66%of its initial Voc.Our work provides a new strategy for suppressing the annihilation of triboelectric charges at high temperatures,thus boosting the development of self-powered sensing devices for application under hot conditions.

Ambient-Condition Strategy for Production of Hollow Ga_(2)O_(3)@rGO Crystalline Nanostructures Toward Efficient Lithium Storage

Crystallineγ-Ga_(2)O_(3)@rGO core-shell nanostructures are synthesized in gram scale,which are accomplished by a facile sonochemical strategy under ambient condition.They are composed of uniformγ-Ga_(2)O_(3)nanospheres encapsulated by reduced graphene oxide(rGO)nanolayers,and their formation is mainly attributed to the existed opposite zeta potential between the Ga_(2)O_(3)and rGO.The as-constructed lithium-ion batteries(LIBs)based on as-fabricatedγ-Ga_(2)O_(3)@rGO nanostructures deliver an initial discharge capacity of 1000 mAh g^(-1)at 100 mA g^(-1)and reversible capacity of 600 mAh g^(-1)under 500 mA g^(-1)after 1000 cycles,respectively,which are remarkably higher than those of pristineγ-Ga_(2)O_(3)with a much reduced lifetime of 100 cycles and much lower capacity.Ex situ XRD and XPS analyses demonstrate that the reversible LIBs storage is dominant by a conversion reaction and alloying mechanism,where the discharged product of liquid metal Ga exhibits self-healing ability,thus preventing the destroy of electrodes.Additionally,the rGO shell could act robustly as conductive network of the electrode for significantly improved conductivity,endowing the efficient Li storage behaviors.This work might provide some insight on mass production of advanced electrode materials under mild condition for energy storage and conversion applications.

Mass-Based Environmental Factor and Energy Assessment of Microwave-Assisted Synthesized Transition Metal Nanostructures

This paper describes mass-based energy phase-space projection of microwave-assisted synthesis of transition metals (zinc oxide, palladium, silver, platinum, and gold) nanostructures. The projection uses process energy budget (measured in kJ) on the horizontal axes and process density (measured in kJg−1) on the vertical axes. These two axes allow both mass usage efficiency (Environmental-Factor) and energy efficiency to be evaluated for a range of microwave applicator and metal synthesis. The metrics are allied to the: second, sixth and eleventh principle of the twelve principle of Green Chemistry. This analytical approach to microwave synthesis (widely considered as a useful Green Chemistry energy source) allows a quantified dynamic environmental quotient to be given to renewable plant-based biomass associated with the reduction of the metal precursors. Thus allowing a degree of quantification of claimed “eco-friendly” and “sustainable” synthesis with regard to waste production and energy usage.

Constructing a biofunctionalized 3D-printed gelatin/sodium alginate/chitosan tri-polymer complex scaffold with improvised biological andmechanical properties for bone-tissue engineering

Sodium alginate(SA)/chitosan(CH)polyelectrolyte scaffold is a suitable substrate for tissue-engineering application.The present study deals with further improvement in the tensile strength and biological properties of this type of scaffold to make it a potential template for bone-tissue regeneration.We experimented with adding 0%–15%(volume fraction)gelatin(GE),a protein-based biopolymer known to promote cell adhesion,proliferation,and differentiation.The resulting tri-polymer complex was used as bioink to fabricate SA/CH/GEmatrices by three-dimensional(3D)printing.Morphological studies using scanning electron microscopy revealed the microfibrous porous architecture of all the structures,which had a pore size range of 383–419μm.X-ray diffraction and Fourier-transform infrared spectroscopy analyses revealed the amorphous nature of the scaffold and the strong electrostatic interactions among the functional groups of the polymers,thereby forming polyelectrolyte complexes which were found to improve mechanical properties and structural stability.The scaffolds exhibited a desirable degradation rate,controlled swelling,and hydrophilic characteristics which are favorable for bone-tissue engineering.The tensile strength improved from(386±15)to(693±15)kPa due to the increased stiffness of SA/CH scaffolds upon addition of gelatin.The enhanced protein adsorption and in vitro bioactivity(forming an apatite layer)confirmed the ability of the SA/CH/GE scaffold to offer higher cellular adhesion and a bone-like environment to cells during the process of tissue regeneration.In vitro biological evaluation including the MTT assay,confocal microscopy analysis,and alizarin red S assay showed a significant increase in cell attachment,cell viability,and cell proliferation,which further improved biomineralization over the scaffold surface.In addition,SA/CH containing 15%gelatin designated as SA/CH/GE15 showed superior performance to the other fabricated 3D structures,demonstrating its potential for use in bone-tissue engineering.

Chitosan/Sodium Alginate Multilayer pH-Sensitive Films Based on Layer-by-Layer Self-Assembly for Intelligent Packaging

The abuse of plastic food packaging has brought about severe white pollution issues around the world.Developing green and sustainable biomass packaging is an effective way to solve this problem.Hence,a chitosan/sodium alginate-based multilayer film is fabricated via a layer-by-layer(LBL)self-assembly method.With the help of superior interaction between the layers,the multilayer film possesses excellent mechanical properties(with a tensile strength of 50 MPa).Besides,the film displays outstanding water retention property(blocking moisture of 97.56%)and ultraviolet blocking property.Anthocyanin is introduced into the film to detect the food quality since it is one natural plant polyphenol that is sensitive to the pH changes ranging from 1 to 13 in food when spoilage occurs.It is noted that the film is also bacteriostatic which is desired for food packaging.This study describes a simple technique for the development of advanced multifunctional and fully biodegradable food packaging film and it is a sustainable alternative to plastic packaging.

Chitosan-based thermosensitive hydrogel with long-term release of murine nerve growth factor for neurotrophic keratopathy

Neurotrophic keratopathy is a persistent defect of the corneal epithelium,with or without stromal ulceration,due to corneal nerve deficiency caused by a variety of etiologies.The treatment options for neurotrophic keratopathy are limited.In this study,an ophthalmic solution was constructed from a chitosan-based thermosensitive hydrogel with long-term release of murine nerve growth factor(CTH-mNGF).Its effectiveness was evaluated in corneal denervation(CD)mice and patients with neurotrophic keratopathy.In the preclinical setting,CTH-mNGF was assessed in a murine corneal denervation model.CTH-mNGF was transparent,thermosensitive,and ensured sustained release of mNGF for over 20 hours on the ocular surface,maintaining the local mNGF concentration around 1300 pg/mL in vivo.Corneal denervation mice treated with CTH-mNGF for 10 days showed a significant increase in corneal nerve area and total corneal nerve length compared with non-treated and CTH treated mice.A subsequent clinical trial of CTH-mNGF was conducted in patients with stage 2 or 3 neurotrophic keratopathy.Patients received topical CTH-mNGF twice daily for 8 weeks.Fluorescein sodium images,Schirmer’s test,intraocular pressure,Cochet-Bonnet corneal perception test,and best corrected visual acuity were evaluated.In total,six patients(total of seven eyes)diagnosed with neurotrophic keratopathy were enrolled.After 8 weeks of CTH-mNGF treatment,all participants showed a decreased area of corneal epithelial defect,as stained by fluorescence.Overall,six out of seven eyes had fluorescence staining scores<5.Moreover,best corrected visual acuity,intraocular pressure,Schirmer’s test and Cochet-Bonnet corneal perception test results showed no significant improvement.An increase in corneal nerve density was observed by in vivo confocal microscopy after 8 weeks of CTH-mNGF treatment in three out of seven eyes.This study demonstrates that CTH-mNGF is transparent,thermosensitive,and has sustained-release properties.Its effectiveness in healing corneal epithelial defects in all eyes with neurotrophic keratopathy suggests CTH-mNGF has promising application prospects in the treatment of neurotrophic keratopathy,being convenient and cost effective.

Smart Colorimetric Corn Starch Films Combined with Anthocyanin-Loaded Glutenin-Carboxymethyl Chitosan Nanocomplexes for Freshness Monitoring of Chilled Pork

In this study,intelligent,pH-responsive colorimetric films were prepared by encapsulating anthocyanins in nanocomplexes prepared from glutenin and carboxymethyl chitosan.These nanocomplexes were added to a corn starch matrix and used in the freshness monitoring of chilled pork.The effects of anthocyanin-loaded nanocomplexes on the physical,structural,and functional characteristics of the films were investigated.The addition of anthocyanin-loaded nanocomplexes increased the tensile strength,elongation at break,hydrophobicity,and light transmittance of the films while decreasing their water vapor permeability.This is because new hydrogen bonds are formed between the film components,resulting in a more homogeneous and dense structure.The colorimetric film has a significant color response to pH changes.These films were used in experiments on the freshness of chilled pork,in which the pH changes with changing freshness states.The results show that the colorimetric film can monitor changes in the freshness of chilled pork in real time,where orange,pink,and green represent the fresh,secondary fresh,and putrefied states of pork,respectively.Therefore,the intelligent colorimetric film developed in this study has good application potential in the food industry.

Adding chitosan nanoparticles of green tea extract in diluent and thawing temperatures ameliorate the post-thawed quality of Boer buck semen

Objective:To improve the quality of post-thawing Boer buck semen for artificial insemination by adding green tea extract chitosan nanoparticles to skimmed egg yolk diluent,and the proper thawing temperature.Methods:The ejaculate of Boer buck was added to skimmed egg yolk diluent without(the control group)and with adding 1μg of chitosan nanoparticles of green tea extract per mL of diluent(the treatment group).Then,the diluted semen was filled in French mini straws containing 60×106 live sperm per straw,frozen in a standard protocol,and stored as frozen semen at−196℃for a week.Six replicates from each group were diluted for 30 s at 37℃or 39℃sterile water to evaluate the semen quality.Results:Post-thawing(at 37℃or 39℃)of live sperm,progressive motility,and plasma membrane integrity were lower compared to those of the pre-freezing stage(P<0.05).Thawing at 37℃resulted in no significant difference in live sperm,progressive motility,and plasma membrane between the control group and the treatment group(P>0.05).The live sperm,progressive motility,and plasma membrane of the treatment group in the pre-freezing stage,and post-thawed at 39℃were higher compared to those of the control group(P<0.05).There was no significant difference in malondialdehyde(MDA)concentration,DNA fragmentation,and catalase concentration of thawing at 37℃compared to those of 39℃in the same group.The MDA concentration and DNA fragmentation in thawing at 37℃and 39℃of the treatment group were significantly lower than those of the control group(P<0.05).However,the catalase concentration in thawing at 37℃and 39℃of the treatment group was not significantly different than the control group(P>0.05).Conclusions:Higher quality post-thawing Boer buck semen is achieved by adding 1μg/mL of chitosan nanoparticles of green tea extract to the skimmed egg yolk diluent and thawing at 39℃.

Antoioxidant Activity of Carboxymethyl Chitosan with Different Substituted Degrees

[Objective] In order to study the relations among different positions, degrees of substitution and antioxidant ability. [Method] N, O-carboxymethyl chitosan （NOA, NOB and NOC）with various degrees of substitution （DS）were obtained by etherizing chito-oligosacchaside. Their structure and substituted degree were characterized and their antioxldant activity to·OH was evaluated. [ Result] The IC50 s of NOA ,NOB and NOC were 0.15 ,0. 29 ,0. 23 mg/ml while their DSs of -NH2 position（DSN） were 0.51,0.29 and 0.38 and DSo were 0. 74 ,0. 84 ,0. 97respectively.[ Conclusion] With the increase of DSN ,antioxidant activity of N,O-carboxymethyl chitosan oligosaccharide to·OH was up.

Extraction, Production and Quality Evaluation of Margarine from Oil Extracted from Waste Biomass Peels of Avocado and Virgin Coconut Oil, Using Chitosan from Reared Shells as Preservative

The production and consumption of avocado pears generates tons of wastes, mainly the pear peels which are usually discarded, although they have been reported to contain important phyto-chemicals with biological activities. The adverse health effect associated with the consumption of saturated lipid based foods has ignited research on reformulation of lipid based foods to eliminate Trans Fatty Acids (TFAs). This study was thus aimed at the extraction and characterization of oil from Avocado Peels (APO) and evaluation of the quality of margarine produced from it. Five verities of pear were used for oil extraction by soxhlet method and physiochemical, oxidative, functional and antioxidant characterization was done. Margarines were formulated using a central composite design using oil blends of APO and Virgin Coconut Oil (VCO) with an oil ratio of 10:90, 40:60, 70:30 respectively, varied blending speed, blending time, and chitosan concentration. Samples were characterized and the effect of process parameters on the physiochemical and functional properties of the margarine studied. Optimized conditions were used to produce samples for sensory evaluation. Color, spreadability, aroma, taste and general acceptability was evaluated using ranking difference test. The results showed that the yield, density, and iodine values of APOs oils ranged from 14.91 ± 0.18 to 11.76 ± 0.46;0.93 ± 0.001 to 0.99 ± 0.1;46.63 ± 1.70 to 52.4 ± 0.63, their acid values, TBA and PV values ranged from 1.42 ± 0.39 to 1.97 ± 0.5;0.11 ± 0.002 to 0.18 ± 0.04;and 2.72 ± 0.14 to 4.43 ± 0.36 respectively, with Brogdon avocado peel variety having the overall best properties prepared blends of trans-free APO margarines showed that increase in APO ratio decreased melting point, increased oxidative stability and reduced moisture content of margarine samples. Chitosan addition leads to decrease moisture content and increase functional properties. VCO lead to increase in phenolic and flavonoid content of the margarines. Samples were spreadable and palatable with R20 being most palatable and the most accepted being R26 with a mean score of 7.07 ± 0.70. Decrease in color intensity increased acceptability. This study therefore demonstrated that avocado peel waste biomass can be valorized by using it as raw material for oil extraction, which can serve as good material for the production of trans-free margarines with good oxidative stability, functional and antioxidant properties.

ZnO Incorporated Acrylamide Grafted Chitosan Based Composite Film for Advanced Wound Healing Applications

This study was carried out to prepare ZnO nanoparticles incorporated acrylamide grafted chitosan composite film for possible biomedical application especially drug loading in wound healing. ZnO nanoparticles were prepared by co-precipitation method from zinc acetate di-hydrate and incorporated in acrylamide grafted chitosan. FT-IR and TGA of the prepared composite film confirmed the successful incorporation of ZnO nanoparticles in the acrylamide-grafted polymer matrix. SEM images showed that the ZnO nanoparticles were homogeneously distributed on the porous matrix of the composite film. Water uptake and buffer uptake analysis revealed that the composite film could hold water and buffer sufficiently, which facilitated the absorption of exudate from the wound site. Amoxicillin was loaded in the prepared composite film and the maximum loading efficiency was found to be 67.33% with drug concentration of 300 ppm. In vitro studies showed greater antimicrobial activity of drug-loaded composite film compared to both pure film and standard antibiotic disc. Finally, the In vivo mouse model showed maximum healing efficiency compared to conventional gauge bandages because the loading of antibiotic in the film produced a synergistic effect and healing time was reduced.

Preparation of Cetyl-Chitosan Nanoparticles as Carriers for Paracetamol

Cetyl-chitosan, prepared by reacting chitosan with chlorocetane under alkaline condition, is soluble and spontaneously forms nanoparticles about 100 nm in diameter. Infrared spectra (IR) revealed that there was a substitution reaction mainly on the amine groups of chitosan (CS). By using paracetamol (PCTM) as a model drug, the balanced release concentration of PCTM in phosphate buffer solution (pH=7.4) can be decreased with the increase of degree of substitution alkyl and can be reduced effectively even under a lower PCTM loading.

Effect of Liposome Double-Coated with Chitosan and Chitosan-EDTA Conjugates on Oral Absorption of Insulin

Aim To evaluate the gastrointestinal uptake of the insulin liposomes double-coated with chitosan （Ch） and chitosan-EDTA conjugates （CEC）, and verify their efficiencies. Methods Insulin-liposomes were prepared by reversed-phase evaporation. The hypoglycemic effects of the insulin liposomes coated with Ch or/and CEC were investigated using the glucose oxidase method after oral administration in diabetic rats, normal rats, and beagle dogs. Serum insulin concentrations in beagle dogs were determined by radioimmunoassay and were assessed by Pkanalyst computer program. Results The animals fed the insulin liposomes coated with Ch or/and CEC were able to regulate better the glucose load than the animals receiving PBS or uncoated insulin liposome, and the regulative effects of the insulin liposomes double-coated with Ch and CEC were better than those of the insulin liposomes coated with Ch or CEC alone. After oral administration of the insulin-liposomes double-coated with Ch and CEC to animals, a significant （P 〈 0. 05 ） blood glucose reduction was observed. Their relative pharmacological bioavailability was higher than 9 % in comparison with subcutaneous injection of insulin. In addition, in comparison with subcutaneous injection of insulin, the relative bioavailability was 12. 67 % calculated by area under the curve of serum insulin concentration versus time profile after oral administration of the insulin-liposomes double-coated with Ch and CEC to beagle dogs. Conclusion The insulin-liposomes double-coated with Ch and CEC were conducive to improving oral bioavailability of insulin.

Preparation of Carboxymethyl Sulfochitosans with Differentially Substituted Regions

Five kinds of carboxymethyl sulfochitosans with different regions such as N-carboxymethyl-O-sulfochitosan, O-carboxymethyl-N-sulfochitosan, O-carboxymethyl chitosan sulfate, N-carboxymethyl chitosan-6-sulfate, and N,O-carboxymethyl -N,O-sulfochitosan were prepared respectively by using differential carboxymethylation and sulfation methods, and their IR spectrum and 13C-NMR spectrum were measured.

Chitosan-DNA介导关节基因转移的体外研究

目的 :探索体外Chitosan对关节软骨细胞、滑膜细胞的转染。方法 :分离培养兔正常滑膜细胞、软骨细胞 ,使用荧光质粒 pEGFP -C3作为报道基因 ,制备Chitosan -DNA超微颗粒转染兔正常软骨细胞、滑膜细胞 ,荧光显微镜观察绿色荧光蛋白的表达。结果 :Chitosan能将DNA带入胞内 ,该DNA超微颗粒随时间的延长可缓慢进入胞内 ,其包被的DNA也可以被缓慢释放 ,且对软骨细胞的转染能力强。结论 :Chitosan在体外可介导关节软骨细胞、滑膜细胞基因转移 ,为今后关节疾病非病毒基因转移的研究提供实验依据。

Adsorption of Cr(VI) by modified chitosan from heavy-metal polluted water of Xiangjiang River, China

Methacrylic acid was used together with a molecular imprinting technique to modify chitosan. In addition, the adsorption kinetics and adsorption isotherms were recorded and the results were analyzed to investigate reparative adsorption for Cr（VI） from the polluted Xiangjiang River water. A comparative X-ray analysis shows that the degree of crystallization in the imprinted polymer was significantly weakened, the area of the non-crystalline region was larger. There were more adsorption sites in the imprinted polymer, and the adsorption capacity towards Cr（VI） was increased. The adsorption capacity of the imprinted polymer towards Cr（VI） increased with time and reaches saturation after 8 h. The optimal adsorption time was 4-8 h after the adsorption starting and the optimal pH value for the solution was in the range of 4.5-7.5. When the chitosan reaches saturation, the adsorption capacity achieves a state of equilibrium, and the maximum Cr（VI） extraction rate reaches 33.7%. Moreover, the adsorption capacity of the imprinted polymer towards Cr（VI） increases with increasing chitosan concentration. In this situation, the Cr（VI） extraction rate shows little variation, and the maximum removal rate can reach 98.3%. Furthermore, the Cr（VI） extraction rate increases with an increase in the degree of deacetylation in the chatoyant and chitosan, with the best adsorption effect corresponding to 90% deacetylation. Fitting the adsorption data to the quasi first- and second-order kinetic models yields correlation coefficients of 0.9013 and 0.9875, respectively. The corresponding rate constants for the two models are 0.0091 min-1 and 7.129 g/（mg.min）, respectively. Hence, the adsorption using Cr（VI）-imprinted chitosan is more consistent with the second-order kinetics. Comparing the data to Freundlich and Langrnuir adsorption isotherms shows that the latter has a better linear fit and a maximum adsorption capacity of 15.784 mg/g.

Effects of Chitosan and Salicylic Acid on Cold Resistance of Litchi under Low Temperature

This paper focused on the effect of spraying chitosan and salicylic acid to Litchi under low temperature stress conditions.The physiology and biochemistry of litchi were studied as well.Results showed that the appropriate concentration of chitosan and salicylic acid treatment could effectively reduce injury caused by low temperature to litchi,compared with water control,chlorophyll,proline,soluble protein content of litchi after treatment and the activity of protective enzyme increasing significantly.However,the accumulation of resistance could significantly be improved.Furthermore,when 1 000 mg/L chitosan combined with 50 mg/L salicylic acid,the litchi acquired the best cold resistance capability.

ADSORPTION SELECTIVITY FOR Cu^(2+),Ni^(2+),Co^(2+)IONS USING CROSSLINKING CHITOSAN RESINS IMPRINTED BY METAL IONS

Metal ion-imprintedly crosslinked chitosan resin 1 and resin 2 were prepared by theuse of Cu2+ and Ni2+ as template ions and glutaraldehyde as crosslinking agent, respectively.Through investigation on the adsorption capacities and binding constants for Cu2+, Ni2+andCo2+ ions on chitosan resins, resin 1 and resin 2 exhibit the adsorption selectivity for themixture solution of 1:1 Cu2+ and Ni2+ ions. The adsorption selectivity of metal ion-imprintedresins for their template ions is much higher than that of uncrosslinked chitosan resin.