Atmospheric Pressure Cold Plasma Treatment Research Articles

High-efficiency and low-damage modification of engineering metal materials by oxygen-mixing atmospheric pressure cold plasma jets

Engineering metal materials have been widely developed and applied in aviation, aerospace and biomedicine fields. Wettability improvement of these materials can alleviate problems caused by low adhesion and poor bioactivity, thereby facilitating their practical applications. Compared with other methods, atmospheric pressure cold plasma treatment has better versatility and can efficiently improve wettability without causing serious damages, and has therefore been widely used in polymer hydrophilization. However, when treating metal materials, the cold plasma may induce surface breakdown or have low efficiency. In this paper, we developed a new cold plasma jet by mixing oxygen into working gas to achieve high-efficiency and low-damage modification. When oxygen mixing ratio was 0.4 %, the cold plasma jet could modify TC4 titanium alloy surface to be superhydrophilic within 20 s, which was much more efficient than traditional plasma jet (120 s). We then further investigated the mechanism contributing to higher efficiency, and found that the oxygen-mixing plasma jet did not cause serious damages, while generating more polar groups by higher-concentration active particles, as confirmed by surface energy calculation and optical emission spectra. Besides titanium alloy, the oxygen-mixing plasma jet could also achieve rapid hydrophilization of various engineering metal materials, showing promising application prospects.

Changes in Flavor and Volatile Composition of Meat and Meat Products Observed after Exposure to Atmospheric Pressure Cold Plasma (ACP).

Studies on the atmospheric pressure cold plasma (ACP) exposure of meat and meat products mainly determine microbial inactivation, lipid oxidation, and meat color. Some studies include sensory evaluation, but only a few determine the changes in volatile composition due to ACP treatment. The results of sensory evaluation are inconclusive and range from "improvement" to "off-odor". This could be due to differences in the food matrix, especially in processed foods, or different experimental settings, including inadvertent effects such as sample heating. The few studies analyzing volatile composition report changes in alcohols, esters, aldehydes, and other compounds, but not necessarily changes that are novel for meat and meat products. Most studies do not actually measure the formation of reactive species, although this is needed to determine the exact reactions taking place in the meat during ACP treatment. This is a prerequisite for an adjustment of the plasma conditions to achieve antimicrobial effects without compromising sensory quality. Likewise, such knowledge is necessary to clarify if ACP-exposed meat and products thereof require regulatory approval.

Investigation of cold atmospheric plasma effects on functional and physicochemical properties of wheat germ protein isolate

Wheat germ protein isolate (WGPI) was modified by atmospheric pressure cold plasma (ACP) at 25 kV for 5 (T2), 10 (T3), 20 (T4), and 40 (T5) min to investigate the effects on molecular weight (MW), microstructure, and functional attributes (T1 or non-treated WGPI). MW of WGPI showed slight alteration especially when 40 min ACP was exerted and microstructure evaluation confirmed it. The higher the processing time was applied; the higher solubility was obtained. Emulsifying activity and stability, foaming capacity, and stability were improved after 5 min ACP processing time especially when pH was at basic condition. Water holding capacity values were significantly increased by increasing processing time without significant difference among T4 and T5. Conversely, oil holding capacity was significantly reduced by increasing time and without a significant difference between T1 and T2. Consequently, using ACP treatment at 25 kV for 5 min could be suggested for plant-based protein modification.

Changes in structure and emulsifying properties of coconut globulin after the atmospheric pressure cold plasma treatment

Atmospheric pressure cold plasma (ACP) treatment, a non-thermal sterilization technology with great potential in coconut milk, can maximize the retention of the taste and flavour. However, it is unavoidable to have an impact on the coconut milk components. In this study, coconut globulin (CG), a component important for coconut milk stability, was selected to explore the effect of the ACP treatment on the structure and emulsifying properties of CG. After the ACP treatment, the structure of CG unfolded, which was the increase in random coils and reduction in intrinsic fluorescence intensity. In addition, the carbonyl and S–S bond content increased, while the free –SH content decreased. Compared with untreated CG (440.57 nm), the particle size of CG after the ACP treatment decreased, especially at 60 kV 60 s (207.63 nm) and 60 kV 90 s (204.20 nm). Simultaneously, poor modification and excessive modification of CG have similar surface hydrophobicity and interfacial tension to untreated CG. Notably, moderate modification (60 kV 60 s and 60 kV 90 s) resulted in the unfolding of CG molecules and the exposure of hydrophobic groups, increasing their adsorption at the oil-water interface. As expected, the emulsifying ability and emulsifying stability of CG were significantly improved after the ACP treatment at 60 kV 60 s and 60 kV 90 s. In conclusion, moderate ACP treatment could improve the emulsifying properties of CG by modifying its structure. This study provides new insights into the effect of ACP on food components during liquid food sterilization.

Mask disinfection using atmospheric pressure cold plasma

ObjectivesMask usage has increased over the last few years due to the COVID-19 pandemic, resulting in a mask shortage. Furthermore, their prolonged use causes skin problems related to bacterial overgrowth. To overcome these problems, atmospheric pressure cold plasma was studied as an alternative technology for mask disinfection. MethodsDifferent microorganisms (Pseudomonas aeruginosa, Escherichia coli, Staphylococcus spp.), different gases (nitrogen, argon, and air), plasma power (90-300 W), and treatment times (45 seconds to 5 minutes) were tested. ResultsThe best atmospheric pressure cold plasma treatment was the one generated by nitrogen gas at 300 W and 1.5 minutes. Testing of breathing and filtering performance and microscopic and visual analysis after one and five plasma treatment cycles, highlighted that these treatments did not affect the morphology or functional capacity of the masks. ConclusionConsidering the above, we strongly believe that atmospheric pressure cold plasma could be an inexpensive, eco-friendly, and sustainable mask disinfection technology enabling their reusability and solving mask shortage.

COLD ATMOSPHERIC PLASMA ON TREATMENT CHRONIC SKIN LESSIONS IN DIABETES PATIENT: A CASE REPORT

Diabetes mellitus has been being a global health problem that leading to increase in diabetesrelatedcomplications, including chronic skin lesions. Many studies have shown that atmosphericpressure cold plasma treatment has many positive results in the treatment of acute and chronicwounds. In a case of a patient with Diabetes type 2 with a chronic wound at multiple skin sitesand complicated infection. We treated with the PlasmaMed device, a dose of 30 seconds/cm2 ofwound area per day. Patient was cared for ulcers according to standard procedures, wound size,infection status and sensation during treatment. After 4 weeks, the wounds reduced inflammation,necrosis and not abnormal sensations at the irradiated area.

Regulation of cellular redox homeostasis in Arabidopsis thaliana seedling by atmospheric pressure cold plasma-generated reactive oxygen/nitrogen species

Atmospheric pressure cold plasma (APCP) holds great potential as an efficient, economical and eco-friendly approach for improving crop production. Although APCP-induced plant growth promotion is undisputedly attributed to the reactive oxygen and nitrogen species (RONS), how these RONS regulate the intracellular redox state and plant growth is still largely unknown. This study systematically investigates the regulation mechanism of APCP-generated RONS on intracellular redox homeostasis in Arabidopsis thaliana seedling by measuring the RONS compositions in APCP-treated solutions and intracellular RONS and antioxidants in Arabidopsis seedlings. The results show that APCP exhibited a dual effect (stimulation or inhibition) on Arabidopsis seedling growth dependent on the treatment time. APCP-generated RONS in liquids increased in a time-dependent manner, leading to an increase of conductivity and oxidation reduction potential (ORP) and decrease of pH. APCP caused an enrichment of intracellular RONS and most of them increased with APCP treatment time. Meanwhile, APCP treatment accelerated malondialdehyde (MDA) generation, and the level of intracellular antioxidants were enhanced by low-dose APCP treatment while decreased at high doses. The results of correlation analysis showed that the extracellular RONS produced by APCP were positively correlated with the intracellular RONS and negatively correlated with the antioxidants. These results demonstrate that the improved antioxidant capacity induced by moderate APCP-generated RONS plays an important role in the growth promotion of Arabidopsis seedlings, which may be a promising alternative for fertilizers in agricultural production.

Effect of atmospheric pressure cold plasma (ACP) treatment on the technological characteristics of quinoa flour

Atmospheric pressure cold plasma (ACP) is considered as non-thermal treatment with potential microbial inactivation efficiencies. This study is aimed to determine the effect of ACP treatment on technological characteristics of quinoa flour using Fourier Transform Infrared Spectroscopy, flour hydration characteristics, thermal properties, rheological measurement and morphological characterization. Whole quinoa grains were subjected to a dielectric barrier discharge plasma reactor for 5 min at 50 kV, 10 min at 50 kV, 5 min at 60 kV and 10 min at 60 kV known as S1, S2, S3 and S4 respectively. Untreated sample is named as control sample. Results indicated the significant impact of ACP treatment on rheological, thermal, hydration and morphological characteristics of quinoa flour depending on the exposed time and voltage. For example, while a voltage dependent decrease (p < 0.05) has been found in enthalpy with values equal to 743.6 ± 0.5, 1395 ± 1, 635.6 ± 0.6 and 804.3 ± 0.9 J/g for S1, S2, S3 and S4 respectively, it is positively influenced by increasing the exposure time at constant voltage. Consequently, it seems that the influence of ACP treatment on technological characteristics which is mainly induced by time- and/or voltage-dependent changes in proteins and starch structures needs to be optimized regarding the desired behavior characteristics.

Antithetic superhydrophobic/superhydrophilic surfaces formation by simple gas switching in an atmospheric-pressure cold plasma treatment

The wettability control of surfaces is a significant issue in many industrial fields such as encapsulation, water repellency enhancement, oil recovery, etc. From a practical point of view, to reach the intended surface wettability, it is important for a surface modification process to be low-cost, easily implemented and fast. Therefore, in this work, a facile plasma deposition method is suggested to form surfaces with antithetic wettabilities, i.e. superhydrophobic and superhydrophilic, via simple introduction of additive gases in an atmospheric-pressure plasma generated with argon and hexamethyldisilazane (HMDS). The hydrophobic surface (WCA: 115°) modified by Ar/HMDS plasma is easily switched to superhydrophobic surface (WCA: 160°) by N2 addition and to superhydrophilic surface (WCA: 10°) by O2 addition. From physical and chemical analyses of plasma treated surfaces, a small amount of N2 or O2 gas is shown to dramatically change the surface morphology and chemical composition. Besides, by finely tuning the additive gases flow rates, the surface wettability could be continuously modulated in the whole ∼0° to 160° water contact angle range.

Influence of atmospheric-pressure cold plasma-induced oxidation on the structure and functional properties of egg white protein

This study examined the effects of atmospheric-pressure cold plasma (ACP) on the structure and functional properties of egg white protein (EWP). Egg white solution was treated with ACP for 0, 10, 20, 30, and 40 min. The structural unfolding of EWP was confirmed as the exposed free SH group content and surface hydrophobicity increased during the 30 min treatment; however, 40 min of ACP treatment showed the opposite tendency, suggesting the formation of protein aggregates. The emulsion stability of egg white emulsions was the highest at 30 min but decreased again at 40 min. In addition, the proportion of large droplets decreased as the treatment time increased up to 30 min. As a result, moderate oxidation was induced during 30 min of ACP treatment, which could contribute to the enhancement of functional properties of EWP. However, excessive oxidation caused by further ACP treatment negatively affected the enhanced functional properties. Industrial relevanceEgg whites are used as functional ingredients for a variety of food products, so the quality and functionality of egg whites can directly affect the productivity of the final products. The present study suggests the moderate oxidation of egg white protein induced by atmospheric-pressure cold plasma, which could contribute to the enhancement of functional properties. These applied studies can serve as a cornerstone for the establishment of advanced oxidation techniques that can enhance the functionality of egg whites without chemical additives, and consequently contribute to the improvement of food quality and productivity.

Effect of atmospheric pressure cold plasma treatment time and composition of feed gas on properties of skim milk

Effect of cold plasma generated using Nitrogen–Oxygen and Nitrogen as feed gases on the properties of skim milk treated for 1, 2, 3, 4 and 5 min was evaluated. Cold plasma treatment with N2–O2 plasma (0.9 mol N2 & 0.1 mol O2 per mol feed gas) significantly decreased the pH and increased the titratable acidity of milk. Furthermore, skim milk treated with N2–O2 plasma showed higher browning intensity at the end of 5 min. Also, the particle size of milk significantly increased with time in case of N2–O2 plasma treatment , indicating aggregation of proteins. SDS PAGE results showing changes in the electrophoretic bands of casein as well as whey proteins also signified denaturation and aggregation of proteins and indicated the formation of insoluble protein aggregates. Apparently, the resultant protein aggregation led to a significant decrease in the surface hydrophobicity of skim milk. FTIR spectroscopy used to study the changes in the secondary structure of milk proteins revealed an overall increase in α-helix and aggregated β-sheet. The results showed that the composition of feed gas had a significant effect on physico-chemical properties, particle characteristics and surface hydrophobicity of skim milk as well as the molecular weight of milk proteins.

Cold Plasma Irradiation Regulates Inflammation and Oxidative Stress in Human Bronchial Epithelial Cells and Human Non-Small Cell Lung Carcinoma.

Atmospheric pressure cold plasma has shown multiple biological effects of anti-bacteria and anti-cancer. In this study, the effect of atmospheric pressure cold plasma on respiratory inflammation and oxidant stress is explored. Tunicamycin was used to stimulate human bronchial epithelial cells (HBECs) and A549 cells for inflammatory response and oxidative stress, followed by atmospheric pressure cold plasma treatment. For HBECs and A549 cells, atmospheric pressure cold plasma was able to alleviate tunicamycin-induced cell proliferation inhibition, inflammation and oxidant stress, and enhance nuclear factor-erythroid-2-related factor 2 (NRF2) pathway activation. Moreover, NRF2/ARE (anti-oxidant response elements) pathway was involved in the regulation of atmospheric pressure cold plasma on tunicamycin-induced oxidative stress. These results suggest the positive effect of atmospheric pressure cold plasma on inflammation and oxidant stress of respiratory system, indicating the therapeutic potential of atmospheric pressure cold plasma for respiratory diseases.

Comparative transcriptome analysis of atmospheric pressure cold plasma enhanced early seedling growth in Arabidopsis thaliana

The stimulatory effects of atmospheric pressure cold plasma (APCP) on plant growth have attracted much attention due to its great potential as a new approach to increase crop growth and production. However, the transcriptome changes of plants induced by APCP treatment are unknown. Herein, the comparative transcriptome analysis was performed to identify the transcriptional response of Arabidopsis thaliana seedlings to APCP. Results showed that APCP exhibited a dual effect (stimulation or inhibition) on Arabidopsis seedling growth dependent on the treatment time and the maximum stimulatory effects were achieved by 1 min APCP treatment. The metabolic analysis of amino acid, glutathione (GSH) and phytohormone demonstrated that 1 min APCP treatment decreased most amino acids concentrations in Arabidopsis seedling, while the accumulations of GSH, gibberellins and cytokinin were significantly increased. The RNA-Seq analysis showed that a total of 218 differentially expressed genes (DEGs) were identified in 1 min APCP-treated seedlings versus the control, including 20 up-regulated and 198 down-regulated genes. The DEGs were enriched in pathways related to GSH metabolism, mitogen-activated protein kinase (MAPK) signaling transduction and plant resistance against pathogens. Moreover, most of the DEGs were defense, stimuli or stress-responsive genes and encoded proteins with oxidoreductase activity. Expression determination of six randomly selected DEGs by quantitative real-time PCR demonstrated similar pattern with the RNA-Seq data. These results indicated that the moderate APCP treatment may regulate the expression of stimuli/stress-responsive genes involved in GSH, phytohormone/amino metabolism and plant defense against pathogens via MAPK signal transduction pathway, accordingly enhance Arabidopsis seedling growth. This study provides a theoretical basis for the application of APCP in agriculture.

Cold Plasma Effects on Changes in Physical, Nutritional, Hydration, and Pasting Properties of Pearl Millet (Pennisetum Glaucum)

Atmospheric pressure cold plasma treatment on pearl millet (Pennisetum glaucum) with air as feed gas at an input voltage of 40 and 45 kV for 5, 10, and 15-min exposure time was studied. The influence of cold plasma on the physical, nutritional, hydration, and pasting properties of pearl millet was assessed. There was no significant difference in loose and tapped bulk density, whereas the color intensity (ΔE) and the delta chroma (ΔC) have significantly differed with the treatment. The nutritional properties, such as crude fat (3.46% ± 0.25%-4.71% ± 0.07%) were increased, whereas crude fiber (2.21% ± 0.02%-1.32% ± 0.02%), protein (10.064% ± 0.43%-8.89% ± 0.50%), and moisture (11.92% ± 0.10%-10.25% ± 0.03%) had significantly diminished. The results of hydration properties, such as swelling capacity (0.213 ± 0.003-0.320 ± 0.008 g/g), solubility (183% ± 1.00%-212% ± 2.00%), and water absorption (1.11 ± 0.02-1.51 ± 0.01 g/mL) have shown significant increment with treatment time and voltage. Besides, the pasting properties of the treated millet were significantly improved. The infrared thermal imaging was used and observed a maximum of 7 °C rise in the plasma treatment zone's temperature throughout the treatment time. It can be concluded that the plasma treatment ameliorates hydration and pasting properties of pearl millet, paving the path for their usage in a wide range of convenient foods.

Application of atmospheric pressure cold plasma to sanitize oak wine barrels

Oak wood barrel sanitization is crucial in terms of food safety, since barrels enrich the wine in aromas and taste sensation and provide physico-chemical stability. The most used method is barrel sulphuring; however, there is a European prohibition (Directive 98/8/CE2) that does not allow the use of this method. For this reason, the aim of the present work was to study an alternative technology, the atmospheric pressure cold plasma (APCP) to sanitize oak wood staves. Three wine spoilage microorganisms, two bacteria (Pediococcus pentosaceus and Acetobacter pasteurianus) and a yeast (Brettanomyces bruxellensis), were inoculated in the wood staves. Then, they were exposed to APCP device at a 12-passes treatment with different plasma powers (90 W and 500 W) and plasma gases (air, nitrogen and argon). A total inactivation of the yeast was achieved after the air and nitrogen plasma treatments, being the most sensitive microorganism to the APCP. In terms of bacteria, P. pentosaceus and A. pasteurianus showed a lower inactivation with respect to their controls. The reactive oxygen and nitrogen species generated during the plasma generation process seem to play the main role on the microbial inactivation. Finally, no morphological modifications on the wood surface were identified after APCP treatments.

Degradation of Deoxynivalenol by Atmospheric-Pressure Cold Plasma and Sequential Treatments with Heat and UV Light

Atmospheric-pressure cold plasma (ACP) technology is a novel non-thermal method with the potential to be used as a post-harvest treatment for mycotoxin mitigation. Deoxynivalenol (DON), a mycotoxin that occurs predominantly in grains, is a threat to human health and to the agricultural economy worldwide. In this study, the effects of the treatment time, and the presence of ACN/water on the DON degradation efficacy during ACP treatment were evaluated. ACP treatment for 5 min resulted in 100% degradation in DON in solution (20 μg/ml ACN/water (20/80, v/v)), while the DON degradation was only 75.9% after 60 min of treatment, in dry condition (2 μg DON). Thin-layer chromatography (TLC) and Fourier transform infrared spectroscopy (FTIR) were used to determine structural changes in DON after ACP treatment. FTIR analysis revealed major changes in DON functional groups after ACP treatment in solution form. Sequential treatments involving ACP and heat treatment at 80 °C and ACP and light-emitting diode (LED) treatments were tested to reduce DON concentrations in dry form. Heat treatment alone was not effective in reducing DON, while sequential treatment of 30-min ACP with 25-min heat reduced DON by 64.6%, which was not significantly different from the DON reduction (63%) by 30-min ACP alone. LED treatments for 30 and 60 min reduced DON concentrations by 10.7% and 36.7%, respectively; while sequential treatment of 30-min ACP with 30-min LED reduced DON by 68.8%. This study shows that the ACP technology has the potential to degrade DON.

Improved physicochemical properties of peanut protein isolate glycated by atmospheric pressure cold plasma (ACP) treatment

This study investigated the effects of the atmospheric pressure cold plasma (ACP) treatment on the glycation of peanut protein isolate (PPI) and dextran (Dex). The results showed a rapid conjugation between PPI and dextran with a degree of grafting (DG) of 21.62% after 1.5 min of treatment. The solubility and emulsion stability of PPI-Dex conjugates increased remarkably. SDS-PAGE analysis confirmed the coupling of PPI with dextran to form conjugates. After ACP treatment, the amino acid content analysis revealed a decrease in the content of lysine and phenylalanine. FTIR spectroscopy showed an increase in the absorption of the characteristic hydroxyl peaks at 3500-3000 cm−1 and 1260-1000 cm−1 of the PPI-Dex conjugates, a decrease in the content of α-helix and β-sheets, and an increase in the content of β-turns. These alterations were due to the ACP treatment that changed the structure of PPI from compact and hydrophobic to loose and hydrophilic.

The Effect of Atmospheric Pressure Cold Plasma Application on Titanium Barriers: A Vertical Bone Augmentation.

The aim of the present, microcomputed tomographic (μCT) and histological study, was to evaluate the effect of surface modification by atmospheric pressure cold plasma (APCP) on vertical guided bone regeneration in a rabbit calvaria model. The experimental study was conducted on 12 male New Zealand rabbits with healing periods of 45 and 90 days. Following surgical exposure of the calvarium, 4 customized titanium cylindricalders were fixed. Surface modification was achieved by application of APCP on 2 of cylinders (P+) in each calvarium and other cylinders were set as control (P-). In both experimental and control groups, one of the cylinders was filled with bone graft (G+) while the other one was left empty (G-). To evaluate short term effects, randomly selected 6 animals were sacrificed at the end of 45 days and remaining 6 animals were left for observing long term effects. Histological and μCT evaluations were used to examine new bone formation. In μCT imaging; the bone volume was greater (P < 0.05) in grafted groups than nongrafted groups in both short and long term. The bone height values were significantly different in (P-G-) group than other groups (P < 0.05) in both evaluation periods. The histological evaluations revealed significant differences between P+G+ group and other groups but in long term both plasma treated groups revealed more bone formation than non plasma treated groups. Modification of the surfaces of titanium cylinders by APCP treatment, accelerated the bone regeneration either bone graft used or not in a rabbit calvaria model.

Effect of Atmospheric Pressure Cold Plasma on Biophysical Properties and Aggregation of Natural Actomyosin from Threadfin Bream (Nemipterus bleekeri)

Biophysical properties and aggregation of natural actomyosin (NAM) from threadfin bream (Nemipterus bleekeri) subjected to atmospheric pressure cold plasma (APCP) treatment with different contact times were determined. Protein solubility, turbidity, Ca2+-ATPase activity, total sulfhydryl (SH) group content, protein carbonyl content, and SDS-PAGE were monitored. With increasing APCP contact times (from 0 to 30 min), total SH group content (5.74–3.77 mol/105 g protein), Ca2+-ATPase activity (0.32–0.23 μmol Pi/mg protein/min), and solubility (100–92%) of NAM decreased, while protein carbonyl content (1.43–36.47 μmol/g protein) increased to a higher extent. During APCP treatment, cross-linking via disulfide bond as visualized by SDS-PAGE along with increased turbidity of NAM was observed. Therefore, APCP treatment markedly induced biophysical changes and aggregation of NAM which can be used as a possible approach to strengthen the gel formation of threadfin bream surimi.

Behavioral Solubilization of Peanut Protein Isolate by Atmospheric Pressure Cold Plasma (ACP) Treatment

The solubilization of peanut protein isolate (PPI) powders modified by atmospheric pressure cold plasma (ACP) treatment was studied by scanning electron microscopy (SEM), energy dispersive X-ray spectroscopy (EDS), low-field nuclear magnetic resonance (low-field NMR) spectroscopy, and FTIR spectroscopy. Significant improvements in both the solubility and water holding capacity (WHC) of the PPI gel were observed after treatment with ACP. The PPI solubility reached a maximum value after 7 min of treatment, with a 12.17% increase over the values observed for the untreated samples. In addition, the WHC increased by 17.90% after 3 min of treatment. The SEM and EDS data revealed that following the 7-min treatment, the PPI surface was rougher and more loosely bound than that of the untreated sample. This indicated an increase in the PPI specific surface area and exposed protein–water binding sites on the treated PPI surface as well as a marked increase in its oxygen content, suggesting an increase in the hydrophilic groups on the PPI surface. The low-filed NMR measurements revealed that the trend in the T21 peak area of the relaxation time was consistent with the data observed for the WHC. The FTIR results revealed a decrease in the proportion of β-sheets and an increase in that of the β-turns within 3 min of treatment, suggesting that the polarity and hydrophilicity of the protein surface were enhanced. The protein structure changed from a compact folding to a loose unfolding configuration after ACP treatment.