Double layer bacterial nanocellulose - poly(hydroxyoctanoate) film activated by prodigiosin as sustainable, transparent, UV-blocking material.

Synthetic materials alternatives are crucial for reaching sustainable development goals and waste reduction. Biomaterials and biomolecules obtained through bacterial fermentation offer a viable solution. Double-layer active UV-blocking material composed of bacterial nanocellulose as an inner layer and poly(hydroxyoctanoic acid) containing prodigiosin as an active compound was produced by layer-by-layer deposition. This study referred the new material consisted of the three components produced in sustainable manner, by bacterial activity: bacterial bio-pigment prodigiosin, bacterial nanocellulose and poly(hydroytoctanoate) - biopolymer obtained by microbial fermentations. Prior the final double layer film was produced, PHO films containing different PG concentrations as a layer in charge of the bioactivity (0.2, 0.5 and 1 wt%) was casted and systematically characterized (FTIR, DSC, XRD, wettability, SEM, transparency, mechanical tests) to optimize their properties. The formulation with the best UV-blocking properties and less toxicity effect tested using MRC5 cells was chosen as an outer layer in double-layer films production. Water contact angle measurements confirmed that hydrophilic - hydrophobic double layer film was obtained with the improved mechanical properties in comparison to the native BNC. Migration test indicated release of PG in all tested media as a consequence of bilayer formulation, while the PG release from PHO in 10 % ethanol was not detected. All findings from the study suggested this activated, UV-blocking material as a candidate with excellent potential in packaging industry.

Preparation, Physicochemical, and Cyto- and Genotoxic Characterisation of Polysaccharide Composites Containing Carbon Quantum Dots.

Rapid industrial growth is associated with an increase in the production of environmentally harmful waste. A potential solution to significantly reduce pollution is to replace current synthetic materials with readily biodegradable plastics. Moreover, to meet the demands of technological advancements, it is essential to develop materials with unprecedented properties to enhance their functionality. Polysaccharide composites demonstrate significant potential in this regard. Polysaccharides possess exceptional film-forming abilities and are safe for human use, biodegradable, widely available, and easily modifiable. Unfortunately, polysaccharide-based films fall short of meeting all expectations. To address this issue, the current study focused on incorporating carbon quantum dots (CQDs), which are approximately 10 nm in size, into the structure of a starch/chitosan biocomposite at varying concentrations. This modification has improved the mechanical properties of the resulting nanocomposites. The inclusion of nanoparticles led to a slight reduction in solubility and an increase in the swelling degree. The optical characteristics of the obtained films were influenced by the presence of CQDs, and the fluorescence intensity of the nanocomposites changed due to the specific heavy metal ions and amino acids used. Consequently, these nanocomposites show great potential for detecting these compounds. Cellular viability assessments and comet assays confirm that the resulting nanocomposites do not exhibit any cytotoxic properties based on this specific analytic method. The tested nanocomposites with the addition of carbon quantum dots (NC/CD II and NC/CD III) were characterised by greater genotoxicity compared to the negative control. The positive control, the starch/chitosan composite alone, was also characterised by a greater induction of chromatin damage in mouse cells compared to a pure mouse blood sample.

Linseed Oil-Based Oleogel Vehicles for Hydrophobic Drug Delivery-Physicochemical and Applicative Properties.

In this study, a methodology for synthesizing oleogels based on linseed oil and emulsifiers, such as beeswax and Tween 20 and Tween 80, was developed. Linseed oil served as the main oil phase, while beeswax acted as a gelling and emulsifying agent. Tween compounds are non-ionic surfactants composed of hydrophobic and hydrophilic parts, allowing for the formation of a stable system with promising properties. Surface wetting analysis of the obtained oleogels, FT-IR spectroscopy, and determination of relative and absolute humidity over time, as well as optical microscope analysis and rheological analysis of the obtained oleogels, were conducted as part of the research. The results indicate that increasing the amount of Tween 20 decreases the hydrophilicity of the oleogel, while Tween 80 exhibits the opposite effect. Surface energy analysis suggests that a higher content of Tween 20 may lead to a reduction in the surface energy of the oleogels, which may indicate greater material stability. Changes in relative humidity and FT-IR spectral analysis confirm the influence of emulsifiers on the presence of characteristic functional groups in the structure of the oleogels. Additionally, microscopic analysis suggests that an emulsifier with a longer hydrophobic tail leads to a denser material structure.

Correction: Kudlacik-Kramarczyk et al. Exploring the Potential of Royal-Jelly-Incorporated Hydrogel Dressings as Innovative Wound Care Materials. Int. J. Mol. Sci. 2023, 24, 8738.

The authors wish to make the following corrections to this paper [...].

Biomaterials in Drug Delivery: Advancements in Cancer and Diverse Therapies-Review.

Nano-sized biomaterials are innovative drug carriers with nanometric dimensions. Designed with biocompatibility in mind, they enable precise drug delivery while minimizing side effects. Controlled release of therapeutic substances enhances efficacy, opening new possibilities for treating neurological and oncological diseases. Integrated diagnostic-therapeutic nanosystems allow real-time monitoring of treatment effectiveness, which is crucial for therapy personalization. Utilizing biomaterials as nano-sized carriers in conjunction with drugs represents a promising direction that could revolutionize the field of pharmaceutical therapy. Such carriers represent groundbreaking drug delivery systems on a nanometric scale, designed with biocompatibility in mind, enabling precise drug delivery while minimizing side effects. Using biomaterials in synergy with drugs demonstrates significant potential for a revolutionary impact on pharmaceutical therapy. Conclusions drawn from the review indicate that nano-sized biomaterials constitute an innovative tool that can significantly improve therapy effectiveness and safety, especially in treating neurological and oncological diseases. These findings should guide researchers towards further studies to refine nano-sized biomaterials, assess their effectiveness under various pathological conditions, and explore diagnostic-therapeutic applications. Ultimately, these results underscore the promising nature of nano-sized biomaterials as advanced drug carriers, ushering in a new era in nanomedical therapy.

Advanced Drug Carriers: A Review of Selected Protein, Polysaccharide, and Lipid Drug Delivery Platforms.

Studies on bionanocomposite drug carriers are a key area in the field of active substance delivery, introducing innovative approaches to improve drug therapy. Such drug carriers play a crucial role in enhancing the bioavailability of active substances, affecting therapy efficiency and precision. The targeted delivery of drugs to the targeted sites of action and minimization of toxicity to the body is becoming possible through the use of these advanced carriers. Recent research has focused on bionanocomposite structures based on biopolymers, including lipids, polysaccharides, and proteins. This review paper is focused on the description of lipid-containing nanocomposite carriers (including liposomes, lipid emulsions, lipid nanoparticles, solid lipid nanoparticles, and nanostructured lipid carriers), polysaccharide-containing nanocomposite carriers (including alginate and cellulose), and protein-containing nanocomposite carriers (e.g., gelatin and albumin). It was demonstrated in many investigations that such carriers show the ability to load therapeutic substances efficiently and precisely control drug release. They also demonstrated desirable biocompatibility, which is a promising sign for their potential application in drug therapy. The development of bionanocomposite drug carriers indicates a novel approach to improving drug delivery processes, which has the potential to contribute to significant advances in the field of pharmacology, improving therapeutic efficacy while minimizing side effects.

Exploring the Potential of Royal-Jelly-Incorporated Hydrogel Dressings as Innovative Wound Care Materials.

The development of multifunctional dressing materials with beneficial properties for wound healing has become a recent focus of research. Many studies are being conducted to incorporate active substances into dressings to positively impact wound healing processes. Researchers have investigated various natural additives, including plant extracts and apiproducts such as royal jelly, to enhance the properties of dressings. In this study, polyvinylpyrrolidone (PVP)-based hydrogel dressings modified with royal jelly were developed and analyzed for their sorption ability, wettability, surface morphology, degradation, and mechanical properties. The results showed that the royal jelly and crosslinking agent content had an impact on the physicochemical properties of the hydrogels and their potential for use as innovative dressing materials. This study investigated the swelling behavior, surface morphology, and mechanical properties of hydrogel materials containing royal jelly. The majority of the tested materials showed a gradual increase in swelling ratio with time. The pH of the incubated fluids varied depending on the type of fluid used, with distilled water having the greatest decrease in pH due to the release of organic acids from the royal jelly. The hydrogel samples had a relatively homogeneous surface, and no dependence between composition and surface morphology was observed. Natural additives like royal jelly can modify the mechanical properties of hydrogels, increasing their elongation percentage while decreasing their tensile strength. These findings suggest possible future applications in various fields requiring high flexibility and elasticity.

Effects of N-Alkanol Adsorption on Bubble Acceleration and Local Velocities in Solutions of the Homologous Series from Ethanol to N-Decanol.

The influence of n-alkanol (C2-C10) water solutions on bubble motion was studied in a wide range of concentrations. Initial bubble acceleration, as well as local, maximal and terminal velocities during motion were studied as a function of motion time. Generally, two types of velocity profiles were observed. For low surface-active alkanols (C2-C4), bubble acceleration and terminal velocities diminished with the increase in solution concentration and adsorption coverage. No maximum velocities were distinguished. The situation is much more complicated for higher surface-active alkanols (C5-C10). In low and medium solution concentrations, bubbles detached from the capillary with acceleration comparable to gravitational acceleration, and profiles of the local velocities showed maxima. The terminal velocity of bubbles decreased with increasing adsorption coverage. The heights and widths of the maximum diminished with increasing solution concentration. Much lower initial acceleration values and no maxima presence were observed in the case of the highest n-alkanol concentrations (C5-C10). Nevertheless, in these solutions, the observed terminal velocities were significantly higher than in the case of bubbles moving in solutions of lower concentration (C2-C4). The observed differences were explained by different states of the adsorption layer in the studied solutions, leading to varying degrees of immobilization of the bubble interface, which generates other hydrodynamic conditions of bubble motion.

Synthesis and Investigation of Physicochemical and Biological Properties of Films Containing Encapsulated Propolis in Hyaluronic Matrix.

The dynamic development of nanotechnology has enabled the development of innovative and novel techniques for the production and use of nanomaterials. One of them is the use of nanocapsules based on biodegradable biopolymer composites. Closing compounds with antimicrobial activity inside the nanocapsule cause the gradual release of biologically active substances into the environment, and the effect on pathogens is regular, prolonged and targeted. Known and used in medicine for years, propolis, thanks to the synergistic effect of active ingredients, has antimicrobial, anti-inflammatory and antiseptic properties. Biodegradable and flexible biofilms were obtained, the morphology of the composite was determined using scanning electron microscopy (SEM) and particle size was measured by the dynamic light scattering (DLS) method. Antimicrobial properties of biofoils were examined on commensal skin bacteria and pathogenic Candida isolates based on the growth inhibition zones. The research confirmed the presence of spherical nanocapsules with sizes in the nano/micrometric scale. The properties of the composites were characterized by infrared (IR) and ultraviolet (UV) spectroscopy. It has been proven that hyaluronic acid is a suitable matrix for the preparation of nanocapsules, as no significant interactions between hyaluronan and the tested compounds have been demonstrated. Color analysis and thermal properties, as well as the thickness and mechanical properties of the obtained films, were determined. Antimicrobial properties of the obtained nanocomposites were strong in relation to all analyzed bacterial and yeast strains isolated from various regions of the human body. These results suggest high potential applicability of the tested biofilms as effective materials for dressings to be applied on infected wounds.

Formation and Investigation of Physicochemical and Microbiological Properties of Biocomposite Films Containing Turmeric Extract Nano/Microcapsules.

In the era of growing plastic consumption, food waste by consumers and overproduction caused by economic reasons, the global goal is to decrease these phenomena. Biocomposite films investigated in the past years are creating a promising future toward ecological, intelligent and active packaging. Due to their unique properties, they can be used in many areas of our life and reduce the constantly increasing pollution of our planet. The aim of our study was to obtain innovative and flexible biopolymer films based on sodium alginate and chitosan, as well as to develop methods for generating nanocapsules with turmeric extract in them. Bionanocomposites were analyzed using UV-VIS, FTIR, photoluminescence spectroscopy and SEM microscopy, while contact angles, surface free energy, particle size (DLS) and zeta potential were determined. The mechanical and colorimetric properties of the produced films were investigated, and the water content, solubility and water absorption were determined. Microbiological tests were carried out to analyze the influence of the produced films on the development of microorganisms. The results of the performed analyses allowed us to confirm the presence of curcumin nano- and microcapsules in the alginate-chitosan composite. Moreover, studies have shown that the structure of polysaccharides does not change during capsule manufacturing. The film with the highest concentration of the capsules showed better parameters in tests of solubility, water content, degree of swelling and mechanical properties. The obtained properties of the developed films allow them to be used as active and intelligent packaging materials, or as their parts.

Design of Carbon Nanocomposites Based on Sodium Alginate/Chitosan Reinforced with Graphene Oxide and Carbon Nanotubes.

The aim of this study was to use a simple, low-cost and environmentally friendly synthesis method to design nanocomposites. For this purpose, carbon nanostructures were used to reinforce the chitosan/alginate bond in order to improve the mechanical, solubility, water absorption and barrier (protection against UV radiation) properties of the chitosan/alginate structure. Scanning electron microscopy (SEM), Fourier-transform infrared spectroscopy (FTIR), ultraviolet and visible light absorption spectroscopy (UV-VIS) and color analysis were utilized, and the thickness and mechanical properties of the obtained films were determined. The tests that were carried out showed an equal distribution of nanostructures in the composite material and the absence of chemical interactions between nanoparticles and polymers. It was also proven that the enrichment of the polysaccharide composite with graphene oxide and carbon nanotubes positively affected its absorption, mechanical capabilities and color.

The Influence of the Surface Chemistry of Cellulose Nanocrystals on Ethyl Lauroyl Arginate Foam Stability.

Guanidine-based surfactant ethyl lauroyl arginate (LAE) and cellulose nanocrystals (CNCs) form complexes of enhanced surface activity when compared to pure surfactants. The LAE-CNC mixtures show enhanced foaming properties. The dynamic thin-film balance technique (DTFB) was used to study the morphology, drainage and rupture of LAE-CNC thin liquid films under constant driving pressure. A total of three concentrations of surfactant and the corresponding mixtures of LAE with sulfated (sCNC) and carboxylated (cCNC) cellulose nanocrystals were studied. The sCNC and cCNC suspension with LAE formed thin films, with stability increasing with surfactant concentration and with complex rheological properties. In the presence of LAE, the aggregation of CNC was observed. While the sCNC aggregates were preferentially present in the film volume with a small fraction at the surface, the cCNC aggregates, due to their higher hydrophobicity, were preferentially located at film interfaces, forming compact layers. The presence of both types of aggregates decreased the stability of the thin liquid film compared to the one for the LAE solution with the same concentration. The addition of CNC to LAE was critical for foam formation, and foam stability was in qualitative agreement with the thin films' lifetimes. The foam volume increased with the LAE concentration. However, there was an optimum surfactant concentration to achieve stable foam. In particular, the very resistant foam was obtained with cCNC suspensions that formed the interfaces with a complex structure and rheology. On the other hand, at high LAE concentrations, the aggregates of CNC may exhibit antifoaming properties.

Preparation of Nano/Microcapsules of Ozonated Olive Oil in Hyaluronan Matrix and Analysis of Physicochemical and Microbiological (Biological) Properties of the Obtained Biocomposite.

Hydrogels, based on natural polymers, such as hyaluronic acid, are gaining an increasing popularity because of their biological activity. The antibacterial effect of ozone is widely known and used, but the instability the gas causes, severely limits its application. Ozone entrapment in olive oil by its reaction with an unsaturated bond, allows for the formation of stable, therapeutically active ozone derivatives. In this study, we obtained an innovative hydrogel, based on hyaluronic acid containing micro/nanocapsules of ozonated olive oil. By combination of the biocompatible polymer with a high regenerative capacity and biologically active ingredients, we obtained a hydrogel with regenerative properties and a very weak inhibitory effect against both bacterial commensal skin microbiota and pathogenic Candida-like yeasts. We assessed the stability and rheological properties of the gel, determined the morphology of the composite, using scanning electron microscopy (SEM) and particle size by the dynamic light scattering (DLS) method. We also performed Attenuated total reflectance Fourier transform infrared (FTIR-ATR) spectroscopy. The functional properties, including the antimicrobial potential were assessed by the microbiological analysis and in vitro testing on the HaCat human keratinocyte cell line. The studies proved that the obtained emulsions were rheologically stable, exhibited an antimicrobial effect and did not show cytotoxicity in the HaCat keratinocyte model.

Surface Properties of Saponin-Chitosan Mixtures.

The surface properties of saponin and saponin-chitosan mixtures were analysed as a function of their bulk mixing ratio using vibrational sum-frequency generation (SFG), surface tensiometry and dilational rheology measurements. Our experiments show that saponin-chitosan mixtures present some remarkable properties, such as a strong amphiphilicity of the saponin and high dilational viscoelasticity. We believe this points to the presence of chitosan in the adsorption layer, despite its complete lack of surface activity. We explain this phenomenon by electrostatic interactions between the saponin as an anionic surfactant and chitosan as a polycation, leading to surface-active saponin-chitosan complexes and aggregates. Analysing the SFG intensity of the O-H stretching bands from interfacial water molecules, we found that in the case of pH 3.4 for a mixture consisting of 0.1 g/L saponin and 0.001 g/L chitosan, the adsorption layer was electrically neutral. This conclusion from SFG spectra is corroborated by results from surface tensiometry showing a significant reduction in surface tension and effects on the dilational surface elasticity strictly at saponin/chitosan ratios, where SFG spectra indicate zero net charge at the air-water interface.

Synergy in Aqueous Systems Containing Bioactive Ingredients of Natural Origin: Saponin/Pectin Mixtures.

Biocompatible and biodegradable ingredients of natural origin are widely used in the design of foam and emulsion systems with various technological applications in the food, cosmetics and pharmaceutical industries. The determination of the precise composition of aqueous solution formulations is a key issue for the achievement of environmentally-friendly disperse systems with controllable properties and reasonable stability. The present work is focused on the investigation of synergistic interactions in aqueous systems containing Quillaja saponins and Apple pectins. Profile analysis tensiometer (PAT-1) is applied to study the surface tension and surface dilational rheology of the adsorption layers at the air/solution interface. The properties and the foam films (drainage kinetics, film thickness, disjoining pressure isotherm, critical pressure of rupture) are investigated using the thin-liquid-film (TLF) microinterferometric method of Scheludko-Exerowa and the TLF-pressure-balance technique (TLF-PBT). The results demonstrate that the structure and stability performance of the complex aqueous solutions can be finely tuned by changing the ratio of the bioactive ingredients. The attained experimental data evidence that the most pronounced synergy effect is registered at a specific saponin:pectin ratio. The obtained information is essential for the further development of aqueous solution formulations intended to achieve stable foams based on mixtures of Quillaja saponins and Apple pectins in view of future industrial, pharmaceutical and biomedical applications.

Hydrophobisation of Silica Nanoparticles Using Lauroyl Ethyl Arginate and Chitosan Mixtures to Induce the Foaming Process.

We studied silica suspensions with chitosan and biodegradable synthetic surfactant lauroyl ethyl arginate (LAE). Hydrophilic and negatively charged silica nanoparticles were neutralised due to the coating with chitosan. That presence of LAE led to the partial hydrophobisation of their surface, which favoured their attachment to the surface of a thin foam film. It was found that the presence of small and medium-sized (6-9 nm) hydrophobic particles in the interfacial layer of lamella foam film inhibited the coalescence and coarsening processes, which prolonged the life of the foam. Furthermore, hydrophobising of 30 nm particles allowed the formation of large aggregates precipitating from the mixture under steady-state conditions. These aggregates, however, under the conditions of the dynamic froth flotation process in the foam column, were floated into the foam layer. As a result, they were trapped in the foam film and Plateau borders, effectively preventing liquid leakage out of the foam. These results demonstrate the efficiency of using chitosan-LAE mixtures to remove silica nanoparticles from aqueous phase by foaming and flotation.

The Composites of Polyamide 12 and Metal Oxides with High Antimicrobial Activity.

The lack of resistance of plastic objects to various pathogens and their increasing activity in our daily life have made researchers develop polymeric materials with biocidal properties. Hence, this paper describes the thermoplastic composites of Polyamide 12 mixed with 1-5 wt % of the nanoparticles of zinc, copper, and titanium oxides prepared by a twin-screw extrusion process and injection moulding. A satisfactory biocidal activity of polyamide 12 nanocomposites was obtained thanks to homogenously dispersed metal oxides in the polymer matrix and the wettability of the metal oxides by PA12. At 4 wt % of the metal oxides, the contact angles were the lowest and it resulted in obtaining the highest reduction rate of the Escherichia coli (87%), Candida albicans (53%), and Herpes simplex 1 (90%). The interactions of the nanocomposites with the fibroblasts show early apoptosis (11.85-27.79%), late apoptosis (0.81-5.04%), and necrosis (0.18-0.31%), which confirms the lack of toxicity of used metal oxides. Moreover, the used oxides affect slightly the thermal and rheological properties of PA12, which was determined by oscillatory rheology, thermogravimetric analysis, and differential scanning calorimetry.

Polysaccharides Composite Materials as Carbon Nanoparticles Carrier.

Nanotechnology is a dynamically developing field of science, due to the unique physical, chemical and biological properties of nanomaterials. Innovative structures using nanotechnology have found application in diverse fields: in agricultural and food industries, where they improve the quality and safety of food; in medical and biological sciences; cosmetology; and many other areas of our lives. In this article, a particular attention is focused on carbon nanomaterials, especially graphene, as well as carbon nanotubes and carbon quantum dots that have been successfully used in biotechnology, biomedicine and broadly defined environmental applications. Some properties of carbon nanomaterials prevent their direct use. One example is the difficulty in synthesizing graphene-based materials resulting from the tendency of graphene to aggregate. This results in a limitation of their use in certain fields. Therefore, in order to achieve a wider use and better availability of nanoparticles, they are introduced into matrices, most often polysaccharides with a high hydrophilicity. Such composites can compete with synthetic polymers. For this purpose, the carbon-based nanoparticles in polysaccharides matrices were characterized. The paper presents the progress of ground-breaking research in the field of designing innovative carbon-based nanomaterials, and applications of nanotechnology in diverse fields that are currently being developed is of high interest and shows great innovative potential.

Improvement of La0.8Sr0.2MnO3-δ Cathode Material for Solid Oxide Fuel Cells by Addition of YFe0.5Co0.5O3.

The high efficiency of solid oxide fuel cells with La0.8Sr0.2MnO3-δ (LSM) cathodes working in the range of 800-1000 °C, rapidly decreases below 800 °C. The goal of this study is to improve the properties of LSM cathodes working in the range of 500-800 °C by the addition of YFe0.5Co0.5O3 (YFC). Monophasic YFC is synthesized and sintered at 950 °C. Composite cathodes are prepared on Ce0.8Sm0.2O1.9 electrolyte disks using pastes containing YFC and LSM powders mixed in 0:1, 1:19, and 1:1 weight ratios denoted LSM, LSM1, and LSM1, respectively. X-ray diffraction patterns of tested composites reveal the presence of pure perovskite phases in samples sintered at 950 °C and the presence of Sr4Fe4O11, YMnO3, and La0.775Sr0.225MnO3.047 phases in samples sintered at 1100 °C. Electrochemical impedance spectroscopy reveals that polarization resistance increases from LSM1, by LSM, to LSM2. Differences in polarization resistance increase with decreasing operating temperatures because activation energy rises in the same order and equals to 1.33, 1.34, and 1.58 eV for LSM1, LSM, and LSM2, respectively. The lower polarization resistance of LSM1 electrodes is caused by the lower resistance associated with the charge transfer process.

Physicochemical Characteristics of Chitosan-Based Hydrogels Modified with Equisetum arvense L. (Horsetail) Extract in View of Their Usefulness as Innovative Dressing Materials.

This work focused on obtaining and characterizing hydrogels with their potential application as dressing materials for chronic wounds. The research included synthesizing chitosan-based hydrogels modified with Equisetum arvense L. (horsetail) extract via photopolymerization, and their characteristics determined with regard to the impact of both the modifier and the amount of crosslinker on their properties. The investigations included determining their sorption properties and tensile strength, evaluating their behavior in simulated physiological liquids, and characterizing their wettability and surface morphology. The release profile of horsetail extract from polymer matrices in acidic and alkaline environments was also verified. It was proved that hydrogels showed swelling ability while the modified hydrogels swelled slightly more. Hydrogels showed hydrophilic nature (all contact angles were <77°). Materials containing horsetail extract exhibited bigger elasticity than unmodified polymers (even by 30%). It was proved that the extract release was twice as effective in an acidic medium. Due to the possibility of preparation of hydrogels with specific mechanical properties (depending on both the modifier and the amount of crosslinker used), wound exudate sorption ability, and possibility of the release of active substance, hydrogels show a great application potential as dressing materials.