Calcium phosphate silicate ceramics for heavy metal immobilization and antibacterial activity in waste water

In the present work we have prepared a hydroxyapatite (HA)–silica porous ceramic material for the immobilization of heavy metals (Pb, Cd, Zn). This material, shaped as Raschig rings, is obtained by extrusion starting from urban and industrial wastes. Additionally, it has been proved that when Ag or Cu is added over the rings surfaces, these ceramics also have a bactericidal activity.     

含纳米银的明胶/壳聚糖纳米纤维的制备及其抗菌性能研究

以浓度为88%的甲酸溶液作为纺丝溶剂,采用静电纺丝和紫外光照射还原的方法制备了含纳米银颗粒的明胶/壳聚糖纳米纤维。研究发现,壳聚糖的加入量低于明胶质量的3/16时可以得到纳米纤维,纤维平均直径随着硝酸银加入量的增大而减小,纤维表面纳米银的平均直径随着硝酸银加入量的增大而增大,在纺丝体系中硝酸银的加入量存在一个极限值。所制得含纳米银的明胶/壳聚糖纳米纤维对金黄色葡萄球菌和绿脓杆菌具有较好的抑菌性能,纺丝时加入1%硝酸银制得纳米纤维膜的抑菌率达到99%以上,这种抗菌型纳米纤维可以应用于医用敷料等领域。     

Preparation and characterizations of antibacterial PET-based hollow fibers containing silver particles

In this work, we have developed a facile differential pressure route to prepare PET-based hollow fibers containing silver particles (Ag/PET hollow fibers). X-ray Diffraction and Scanning Electron Microscopy were used to confirm the existence of silver particles, and the results showed that 0.1-0.5 μm silver particles were incorporated in the inner side-wall of the hollow portion. Inductively Coupled Plasma Atomic Emission Spectrometer was applied to detect the release behavior of silver ions from various Ag/PET hollow fibers. And the results showed that the release behavior of silver ions was depended on time and the length of the fibers, which may be explained by the water uptake property of the Ag/PET hollow fibers.     

Investigation into the antibacterial property and bacterial adhesion of diamond-like carbon films

Diamond-like carbon films have unique properties for biological and medical applications due to their excellent biocompatibility, chemical inertness, and superior mechanical properties. In order to attend biomedical applications, there is an increasing interest in developing antibacterial coatings. In this paper, we investigated the bactericidal properties of diamond-like carbon films produced using plasma enhanced chemical vapor deposition. The films were deposited over 316L stainless steel substrates using a pulsed directly current discharge of methane gas. Diamond-like carbon structural quality was evaluated using Raman scattering spectroscopy. The bacterial adhesion and bactericidal activity of the coating was evaluated against Escherichia coli ATCC 25922, Pseudomonas aeruginosa ATCC 27853, Salmonella typhimurium ATCC 14028 and Staphylococcus aureus ATCC 25923. These tests show diamond-like carbon bactericidal activity ranged between 25 and 55%, depending on the kind of bacteria. The bacterial adhesion on diamond-like carbon surface was influenced by its structure, chemical bonds and hydrogen content. The low surface roughness did not have decisive effect on its antibacterial performance.     

Synthesis and characterization of dextran-capped silver nanoparticles with enhanced antibacterial activity

Dextran-capped silver nanoparticles were synthesized by reducing silver nitrate with NaBH4 in the presence of dextran as capping agent. The characters of silver nanoparticles were investigated using UV-Vis spectrophotometer, nano-grainsize analyzer, X-ray diffraction, and transmission electron microscopy. Results showed that the silver nanoparticles capped with dextran were in uniform shape and narrow size distribution. Moreover, compared with polyvinylpyrrolidone (PVP)-capped silver nanoparticles, the dextran-capped ones possessed better stability. Antibacterial tests of these silver nanoparticles were carried out for Escherichia coli, Staphylococcus aureus, Staphylococcus epidermidis, Pseudomonas aeruginosa, and Klebsiella pneumoniae. Results suggested that the dextran-capped silver nanoparticles had high antibacterial activity against both Gram-positive and Gram-negative bacteria. In addition, the cytotoxicity in vitro of the dextran-capped silver nanoparticles was investigated using mouse fibrosarcoma cells (L929). The toxicity was evaluated by the changes of cell morphology and 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-tetrazolium bromide assay. Results indicated that these silver nanoparticles had slight effect on the survival and proliferation of L-929 cells at their minimal inhibitory concentration (MIC). After modified by dextran, the physiochemical properties of the silver nanoparticles had been improved. We anticipated that these dextran-capped silver nanoparticles could be integrated into systems for biological and pharmaceutical applications.     

Study on the structure and antibacterial activity of silver-carried zirconium phosphate

The structure and antibacterial activity of silver-carried zirconium phosphate (AgZrP) were investigated by X-ray photoelectron spectroscopy (XPS), X-ray diffraction (XRD) and viable cell counting method. There existed the exchange of sodium ions with silver ions in carrier, and the silver in AgZrP was in ionic state. Moreover, no significant changes in crystal structure of carrier were found by the exchange of silver ions except for its crystal space distance and crystallinity. The antibacterial tests showed that 100 mg l^− 1 of AgZrP possessed high antibacterial activity and was capable of killing all the Escherichia coli (E. coli) and more than 99.9% of the Staphylococci aureus (S. aureus) within 8.0 h of contact.     

Synthesis of oleic acid-stabilized silver nanoparticles and analysis of their antibacterial activity

The development of new and simple green chemical methods for synthesizing colloidal solutions of functional nanoparticles is desirable for environment-friendly applications. In the present work, we report a feasible method for synthesizing colloidal solutions of silver nanoparticles (Ag NPs) based on the modified Tollens technique. The Ag NPs were stabilized by using oleic acid as a surfactant and were produced for the first time by the reduction of silver ammonium complex [Ag(NH₃)₂]⁺_(aq) by glucose with UV irradiation treatment. A stable and nearly monodisperse aqueous Ag NPs solution with average-sized particles (~ 9–10 nm) was obtained. The Ag NPs exhibited high antibacterial activity against both Gram-negative Escherichia Coli (E. coli) and Gram-positive Staphylococcus aureus bacteria. Electron microscopic images and analyses provided further insights into the interaction and bactericidal mechanism of the Ag NPs. The proposed method of synthesis is an effective way to produce highly bactericidal colloidal solutions for medical, microbiological, and industrial applications.     

银缓释载银杀菌活性炭的低温水热炭化法制备

采用低温水热炭化法制备了载银活性炭(Ag/AC),以大肠杆菌(E.coli)为实验菌种,考察了不同载银量活性炭的灭菌性能和抗银流失性能。利用X射线衍射(XRD)、扫描电子显微镜(SEM)以及低温液氮吸附对载银活性炭进行表征。结果表明,负载银对活性炭的石墨微晶结构无影响。随着载银量的增加,负载到活性炭上的银晶粒变大,比表面积、孔容积和孔径均变小,抗菌性能提高,银流失量变化不明显,抗流失天数增多。经分析,载银量为1.54%(质量分数)Ag/AC3样品,比表面积为813.35m2/g,100min之内杀灭107 CFU/mL的大肠杆菌,9d内银流失量约为2.9%,可见,在保持较高比表面积的基础上,具备良好的抗菌性能和抗银流失性能。     

高透明低方阻ITO—Ag—ITO柔性镀膜技术

高透明低方阻ITO－Ag-ITO柔性薄膜具有广泛的用途，本文介绍了膜系设计，工艺参及控制，对设备结构也做了简要叙述，测试结果表明，ITO－Ag-ITO多层膜采用连续卷绕镀的方法生产，性能优异，工艺稳定，系统可靠。     

Preparation, characterization, and antibacterial activity of shell waste loaded with silver

A green inorganic antibacterial material was prepared using shell waste as a carrier material loaded with silver. It was characterized and analyzed by X-ray diffraction (XRD), scanning electron microscopy equipped with an energy dispersive spectrometer (EDS), X-ray photoelectronic spectroscopy (XPS) and N₂ adsorption isotherms. The antibacterial activity was evaluated using Staphylococcus aureus and Escherichia coli as sensitive indicator strains. The antibacterial mechanism was probed and discussed as well. Silver carbonate was detected in the prepared material through XRD analysis. XPS measurement and EDS analysis also confirmed the loading of silver onto the carrier. The antibacterial test demonstrated that the prepared material had good antibacterial property, especially against E. coli. Based on the silver ion release and pH test, as well as comparatively analyzing the characteristics of carrier material and prepared material, we proposed that the antibacterial mechanism mainly involved the antibacterial activity of silver ion, slightly higher pH value and supplementary photocatalytic antibacterial activity of silver carbonate.     

Aqueous synthesis of silver nanoparticle embedded cationic polymer nanofibers and their antibacterial activity

This paper describes the one-pot, aqueous synthesis of cationic polymer nanofibers with embedded silver nanoparticles. Poly[2-(tert-butylaminoethyl) methacrylate] (PTBAM) was used as a cationic polymer substrate to reinforce the antimicrobial activity of the embedded silver nanoparticles. Electron microscope analyses revealed that the as-synthesized nanofibers had diameters of approximately 40 nm and lengths up to about 10 μm. Additionally, silver nanoparticles of approximately 8 nm in diameter were finely embedded into the prepared nanofibers. The embedded silver nanoparticles had a lower tendency to agglomerate than colloidal silver nanoparticles of comparable size. In addition, the nanofibers with embedded silver nanoparticles exhibited excellent antibacterial performance against Gram-negative Escherichia coli and Gram-positive Staphylococcus aureus. Interestingly, the prepared nanofibers exhibited enhanced bactericidal performance compared with the silver-embedded poly(methyl methacrylate) (PMMA) nanofibers, presumably because of the antibacterial properties of the PTBAM substrate.     

Porcine skin gelatin–silver nanocomposites: synthesis,characterisation, cell cytotoxicity,and antibacterial properties

Silver nanoparticles (AgNPs) were synthesised with hydrothermal autoclaving technique by using AgNO₃ salt (silver precursor) at different concentrations (0.01, 0.1, 0.55, 1.1, 5.5, and 11 mM) and porcine skin (1% (w/v)) gelatin polymeric matrix (reducing and stabiliser agent). The reaction was performed in an autoclave at 103 kPa and 121°C and the hydrothermal autoclaving exposure time and AgNO₃ molar concentration were varied at a constant porcine skin gelatin concentration. The as‐prepared AgNPs were characterised by UV–visible spectroscopy, transmission electron microscopy, and Fourier transform infrared spectroscopy. The antibacterial properties of AgNPs were tested against gram‐positive and gram‐negative bacteria. Furthermore, 3‐(4,5‐dimethylthiazol‐2‐yl) 2,5‐diphenyltetrazolium bromide and 2,2‐diphenyl‐1‐picrylhydrazyl assays were used to test whether the synthesised AgNPs can be potentially applied in cancer therapy or used as an antioxidant. This approach is a promising simple route for synthesising AgNPs with a smaller average particle 10 nm diameter. Furthermore, AgNPs exhibited a good cytotoxicity activity, reducing the viability of the liver cancer cell line HepG2 with a moderate IC₅₀; they also showed a low‐to‐fair antioxidant activity. In addition, AgNPs had a remarkable preferential antibacterial activity against gram‐positive bacteria than gram‐negative bacteria. Therefore, these fabricated AgNPs can be used as an antibacterial agent in curative and preventive health care.Inspec keywords: gelatin, silver, nanoparticles, nanocomposites, nanobiotechnology, biomedical materials, antibacterial activity, microorganisms, Fourier transform infrared spectra, ultraviolet spectra, visible spectra, transmission electron microscopy, cancer, cellular biophysicsOther keywords: porcine skin gelatin–silver nanocomposites, cell cytotoxicity, antibacterial properties, silver nanoparticles, hydrothermal autoclaving technique, gelatin polymeric matrix, UV–visible spectroscopy, transmission electron microscopy, Fourier transform infrared spectroscopy, gram‐positive bacteria, gram‐negative bacteria, 3‐(4,5‐dimethylthiazol‐2‐yl) 2,5‐diphenyltetrazolium bromide assays, 2,2‐diphenyl‐1‐picrylhydrazyl assays, cancer therapy, antioxidant, liver cancer cell line HepG2, Ag     

Calcium phosphate formation on plasma immersion ion implanted low density polyethylene and polytetrafluorethylene surfaces

The flexible structure of polymers has enabled them to be useful in a wide variety of medical applications due to the possibility to tailor their properties to suit desired applications. For a long time, there has been an increasing interest in utilizing polymers as matrices for calcium phosphate-based composites with applications in hard tissue implants. On the other side, polymers with application as heart valves, urea catheters and artificial vessels present a case where the formation of minerals (namely calcification) should be avoided. The modification of polymer surfaces by various ion beam treatments for reducing the calcification, as for example plasma immersion ion implantation (PIII), is well known and has a long time effect. This work is part of a wider investigation of the ability of plasma immersion ion implanted polymers to induce calcium phosphate formation from an aqueous solution resembling the human blood plasma. In the experiment described in this paper, topographical and chemical changes were inserted on the surfaces of two conventional polymers (low density polyethylene and polytetrafluorethylene) by PIII with nitrogen ions, and under conditions mimicking the natural mineral formation processes. The effect of the plasma modification on the calcium phosphate nucleation and growth from the aqueous solution was ambiguous. We suppose that the complex combination of surface characteristics influenced the ability of the plasma treated polymer films to induce the formation of a calcium phosphate layer.     

Green synthesis of silver nanoparticles using bovine skin gelatin and its antibacterial effect on clinical bacterial isolates

Nanoparticles are being increasingly used in day‐to‐day life. Therefore, concerns have been raised regarding their interactions with the surrounding environment. This study focused on a simple green method for synthesizing silver nanoparticles (Ag‐NPs) in an autoclave at 15 psi (103 kPa) and 121°C. An aqueous solution of AgNO₃ as a precursor of Ag‐NPs and gelatin (type B) reducing and/or stabilizing (capping) agent were used. The effect of various AgNO₃ concentrations of certain gelatin concentration and various gelatin concentrations at constant AgNO₃ concentration, and autoclaving time, was studied. UV‐Vis spectra ascribed that the presence of localized surface plasmon resonance (SPR) of the synthesized Ag‐NPs. TEM images and the selected area of electron diffraction confirmed, the formation of Ag‐NPs with a diameter of approximately 5 ±0.35 nm. Furthermore, FT‐IR revealed that a gelatin polymer matrix stabilized the synthesized Ag‐NPs. The Well diffusion assay was used to test the effect of Ag‐NPs on six clinical bacterial isolates, where Gram positive bacteria were more susceptible to Ag‐NPs than Gram negative bacteria. Therefore, Ag‐NPs capped by gelatin have remarkable potential effect as an antibacterial agent, and they not only have various medical applications but can also be used in biological, pharmaceutical and industrial fields.Inspec keywords: silver, nanoparticles, nanomedicine, antibacterial activity, microorganisms, nanofabrication, skin, gelatin, ultraviolet spectra, visible spectra, surface plasmon resonance, transmission electron microscopy, electron diffraction, Fourier transform infrared spectra, polymers, biomedical materialsOther keywords: green synthesis, silver nanoparticles, bovine skin gelatin, antibacterial effect, clinical bacterial isolates, autoclave, reducing agent, stabilising agent, ultraviolet‐visible spectra, localised surface plasmon resonance, transmissions electron microscope images, electron diffraction, Fourier transform infrared spectroscopy, gelatin polymer matrix, well diffusion assay, gram negative bacteria, Escherichia coli, Pseudomonas aeruginosa, Klebsiella pneumoniae, gram positive bacteria, Bacillus megaterium, Streptococcus pyogenes, Staphylococcus aureus, temperature 121 degC, Ag     

Green synthesized silver nanoparticle from Allium ampeloprasum aqueous extract: Characterization,antioxidant activities,antibacterial and cytotoxicity effects

In the current study for the first time, silver nanoparticles (AgNPs) were biosynthesized by reducing agents from hot water extract of Allium ampeloprasum, an antibacterial and anti-inflammatory edible plant. UV–vis. spectroscopy, Fourier transform infrared spectroscopy, dynamic light scattering, X-ray diffractometric, and transmission electron microscopy (TEM) analyses have been applied to confirm the formation of biosynthesized AgNPs. Total phenol content and antioxidant activities of AgNPs and extract together with their antibacterial and cytotoxic properties, were evaluated. According to TEM, AgNPs were spherical with a diameter of 8–50 nm. Total phenolic compounds were 15.58 μg/mL, and 10.94 μg/mL at a concentration of 150 μg/mL for the A. ampeloprasum extract and the biosynthesized AgNPs, respectively. Biosynthesized AgNPs showed significant antioxidant activity (81%) as compared to A. ampeloprasum extract (32%) and were active on multi-drug resistant P. aeruginosa. Besides, the cytotoxic activity response was also demonstrated that AgNPs were more potent than the A. ampeloprasum extract and showed high activity against Hela cell line with an IC₅₀ value of less than 25 µg/mL. In conclusion, AgNPs synthesized by A. ampeloprasum extract with excellent antioxidant and antibacterial effects and acceptable cytotoxicity on cervical cancer cells have the potential to be used in biological applications.     

Photocatalytic and antibacterial properties of a TiO2/nylon-6 electrospun nanocomposite mat containing silver nanoparticles

Silver-impregnated TiO(2)/nylon-6 nanocomposite mats exhibit excellent characteristics as a filter media with good photocatalytic and antibacterial properties and durability for repeated use. Silver nanoparticles (NPs) were successfully embedded in electrospun TiO(2)/nylon-6 composite nanofibers through the photocatalytic reduction of silver nitrate solution under UV-light irradiation. TiO(2) NPs present in nylon-6 solution were able to cause the formation of a high aspect ratio spider-wave-like structure during electrospinning and facilitated the UV photoreduction of AgNO(3) to Ag. TEM images, UV-visible and XRD spectra confirmed that monodisperse Ag NPs (approximately 4 nm in size) were deposited selectively upon the TiO(2) NPs of the prepared nanocomposite mat. The antibacterial property of a TiO(2)/nylon-6 composite mat loaded with Ag NPs was tested against Escherichia coli, and the photoactive property was tested against methylene blue. All of the results showed that TiO(2)/nylon-6 nanocomposite mats loaded with Ag NPs are more effective than composite mats without Ag NPs. The prepared material has potential as an economically friendly photocatalyst and water filter media because it allows the NPs to be reused.     

Green synthesis of silver nanoparticle by cauliflower extract: characterisation and antibacterial activity against storage

Green synthesis of silver nanoparticles (AgNPs) was accomplished using different volumes of cauliflower extract and 0.001 M silver nitrate solution at 80°C for 15 min. A brownish‐red solution of AgNPs formed was tested by ultraviolet–visible absorption spectroscopy, Fourier‐transform infrared (FTIR), scanning electron microscopy (SEM), and X‐ray diffraction (XRD). Surface plasmon resonance of AgNPs appeared at 416 nm. Also, the kinetic of AgNPs formation was studied and follows a sigmoidal pattern. Storing time was studied for the freshly prepared AgNPs after 60 days. FTIR analysis shows the adsorption of active components on AgNPs surface, and these components are responsible for reduction besides working as a stabiliser like a capping agent, also FTIR analysis of AgNPs after storage showed no change in peaks location. The SEM exhibited a globular shape of AgNPs, and the particle size ranged from 25 to 100 nm, while the XRD particle size calculation was 25 nm with cubic phase lattice. The antibacterial activity was tested against Gram‐positive and ‐negative bacteria showed an inhibition zone of 16–27 mm and the antibacterial activity tested for the same bacteria after storage for about 10 months showed an inhibition zone of 6–10 mm.Inspec keywords: microorganisms, reduction (chemical), nanofabrication, surface plasmon resonance, silver, transmission electron microscopy, nanoparticles, particle size, visible spectra, ultraviolet spectra, adsorption, antibacterial activity, scanning electron microscopy, X‐ray diffraction, Fourier transform infrared spectra, nanomedicineOther keywords: antibacterial activity, green synthesis, silver nanoparticle, brownish‐red solution, surface plasmon resonance, FTIR analysis, active components, silver nitrate solution, ultraviolet‐visible absorption spectroscopy, AgNP surface, cauliflower extract, Fourier‐transform infrared spectroscopy, scanning electron microscopy, SEM, X‐ray diffraction, XRD, sigmoidal pattern, storing time, adsorption, stabiliser, capping agent, globular shape, particle size, cubic phase lattice, Gram‐positive bacteria, Gram‐negative bacteria, inhibition zone, reduction, time 60.0 d, temperature 80.0 degC, time 15.0 min, wavelength 416.0 nm, Ag     

Biogenic nano silver: Synthesis,characterization, antibacterial,antibiofilms, and enzymatic activity

Powder technology covers many fields from medicine to material science. Especially easy synthesis of nano- and micro-sized particles and low-cost synthesis with non-toxic chemicals are essential. The ability to design nanoparticles according to the application area and desired features is an advantage for many applications. Nanoparticles biosynthesis is under research due to broad biomedical practise. To synthesize of silver nanoparticle from the plant extract, Corylus avellana (Hazelnut) leaves were utilized for bioreduction of silver nitrate. The plant extract is thoroughly mixed with silver nitrate solution, studied the synthesis of nanoparticles using UV–Vis spectroscopy. The nanoparticles were characterized by SEM and XRD-Pattern. XRD analysis proved that the size range of the nanoparticles was 32 nm. Scanning microscope images also demonstrate nanoparticles having spherical shape and a mean size between 9 nm and 50 nm. The antibacterial effect of AgNPs obtained from hazelnut leaves demonstrated antibacterial effect against all tested bacteria strains. The results demonstrated that the plant extract acts as a good bioreductant for the production of silver nanoparticles. This compound was good inhibitor of the α-glycosidase, human carbonic anhydrase I and II isoforms, and cholinesterase enzymes. The results were obtained with Ki values of 12.84 ± 0.41 µM for α-glycosidase, 27.94 ± 3.77 µM for hCA I, 36.12 ± 6.03 µM for hCA II, 47.05 ± 6.83 µM for BChE, and 53.94 ± 7.55 µM for AChE, respectively.     

Preparation of MRI-visible gadolinium methacrylate nanoparticles with low cytotoxicity and high magnetic relaxivity

A new convenient and easy-scalable one-step synthetic strategy to achieve metal-containing polymer nanoparticles for applications as magnetic resonance imaging contrast agent is reported. In this study, a novel contrast agent based on poly(gadolinium methacrylate) (poly(Gd(MAA)₃)) nanoparticles was prepared by one-step aqueous dispersion polymerization of gadolinium methacrylate monomer (Gd(MAA)₃), whereby stable particles were obtained due to the association of Gd^III with the polymer carboxylate anions, which provided latent crosslinking of the polymer nanoparticles without the addition of further crosslinking reagents. The morphology and final composition of the corresponding nanoparticles was thoroughly characterized and their cytotoxicity as well as their potential use in MRI was evaluated in vitro on HEK 293T cells by using the CCK-8 assay. The presented results demonstrated, that the poly(Gd(MAA)₃) nanoparticles had a spherical morphology with mesoporous substructure, a sufficiently low cytotoxicity and an exceptionally high longitudinal relaxivity of r ₁ = 12.613 mM^?1 s^?1, making these nanoparticles excellent candidates for in vivo imaging systems. Herein described poly(Gd(MAA)₃) nanoparticles can be valuable in a wide range of biomedical applications with simultaneous bioconjugation, drug delivery as well as imaging capabilities for the early detection of lesions of the brain and the central nervous system, for assessing cardiac function, and for detecting tumors.     

Calcium phosphate coating on magnesium alloy by biomimetic method: Investigation of morphology, composition and formation process

Magnesium alloy has similar mechanical properties with natural bone and can degrade via corrosion in the electrolytic environment of the human body. Calcium phosphate has been proven to possess bioactivity and bone inductivity. In order to integrate both advantages, calcium phosphate coating was fabricated on magnesium alloy by a biomimetic method. Supersaturated calcification solutions (SCSs) with different Ca/P ratio and Cl⁻ concentration were used as mimetic solutions. The morphology, composition and formation process of the coating were studied with scanning electron microscopy (SEM), energy dispersive spectrometer (EDS), Fourier transformed infrared spectroscopy (FTIR) and X-ray diffraction (XRD). The results show that a uniform calcium phosphate coating was observed on magnesium alloy, the properties of which could be adjusted by the SCSs with different Ca/P ratio. The formation process of the coating was explored by immersing magnesium alloy in SCSs with different Cl⁻ concentration which could adjust the hydrogen production. According to SEM results, the hydrogen bubbles were associated with the formation of grass-like and flower-like coating morphologies. In conclusion, the biomimetic method was effective to form calcium phosphate coating on magnesium alloy and the morphology and composition of the coating could be accommodated by the Ca/P ratio and Cl⁻ concentration in SCSs.