What Is Chalky? Investigating Consumer Language and Perception of Fine Particles in Beverages Containing Pea and Potato Starch.

Despite its importance as an undesirable food texture, the phenomenon of chalkiness remains understudied. Chalky sensations presumably arise from fine particulates found in foods, but semantic overlap with other common descriptors of small particles, like gritty or sandy, is unclear. Here, we compare the usage of Chalky with related descriptors, and determine the effect of particle size, concentration, and xanthan content on Chalky ratings in a model beverage. A 23 factorial design with starch particle size (D90 = 33.8 and 64.6 µm), starch concentrations (10 and 20% w/v), and xanthan content (0.075 and 0.15% w/v) was used. Participants' salivary flow rate was also assessed. A multi-sip taste test was performed where naïve consumers (n = 82; 39% men, 60% women; age range = 18-79 years) rated the intensity of Chalky, Powdery, Gritty, Sandy, Mouthdrying, and Residual mouthcoating at 0, 30, and 60 s after each of three consecutive sips. All attribute ratings were highly correlated, with Chalky, Powdery, and Residual Mouthcoating being more closely correlated with each other than Gritty or Sandy. Although Chalky was still reported 60 s after consumption, no evidence of build-up was found with repeated sips. A larger size and higher concentration increased Chalky ratings, with the low-salivary-flow group reporting greater ratings for Chalky relative to the high-flow group. Our results suggest consumer percepts of small particles are overlapping but not entirely redundant. This suggests researchers and product developers should carefully distinguish between these descriptors when trying to understand consumer perception of food products containing fine particles.

Sugar reduction in chocolate compound by replacement with flours containing small insoluble starch granules.

Consumer concern about intake of added sugars has increased commercial demand for sugar-reduced chocolates. However, substitution with high-potency sweeteners is not possible as sugar serves as an important bulking agent. Here, we investigated replacement of sucrose in chocolate compound with oat or rice flours, with starch granules smaller than 10 µm, focusing on texture, sweetness, and acceptability. In Study 1, six chocolates were made: a control with 54% sucrose, four sucrose-reduced versions (reductions of 25% or 50%, using either oat or sweet rice flour), and one 54% sucrose chocolate with reduced refining time. These chocolates were compared in a Difference from Control (DFC) test in a within-subject design, with and without nose clips. Particle size distribution, yield stress, and plastic viscosity were measured. Chocolates with 25% sucrose reduction by either sweet rice or oat flour (or reduced refining time) were not significantly different from the blind control (p > 0.05), regardless of nose clip use. In open-ended comments, participants reported differences in rice-flour-containing chocolates were due to a chalkier texture, while oat-flour-containing chocolates were described as smoother, softer, and creamier. DFC scores from the chocolates were positively correlated with plastic viscosity and negatively correlated with yield stress. In Study 2, 25% reduced sugar chocolates made with rice flour were liked significantly less than control, but the oat flour sample did not differ from control. Collectively, these results suggest up to 25% of sucrose in chocolate can be replaced with oat flour without negatively affecting texture or consumer acceptance.

Mycorrhizal status and host genotype interact to shape plant nutrition in field grown maize (Zea mays ssp. mays).

Arbuscular mycorrhizal fungi (AMF) establish symbioses with the major cereal crops, providing plants with increased access to nutrients while enhancing their tolerance to toxic heavy metals. However, not all plant varieties benefit equally from this association. In this study, we used quantitative trait loci (QTL) mapping to evaluate the combined effect of host genotypic variation (G) and AMF across 141 genotypes on the concentration of 20 mineral elements in the leaves and grain of field grown maize (Zea mays spp. mays). Our mapping design included selective incorporation of a castor AMF-incompatibility mutation, allowing estimation of AMF, QTL and QTLxAMF effects by comparison of mycorrhizal and non-mycorrhizal plants. Overall, AMF compatibility was associated with higher concentrations of boron (B), copper (Cu), molybdenum (Mo), phosphorus (P), selenium (Se) and zinc (Zn) and lower concentrations of arsenic (As), iron (Fe), magnesium (Mg), manganese (Mn), potassium (K) and strontium (Sr). In addition to effects on individual elements, pairwise correlation matrices for element concentration differed between mycorrhizal and non-mycorrhizal plants. We mapped 22 element QTLs, including 18 associated with QTLxAMF effects that indicate plant genotype-specific differences in the impact of AMF on the host ionome. Although there is considerable interest in AMF as biofertilizers, it remains challenging to estimate the impact of AMF in the field. Our design illustrates an effective approach for field evaluation of AMF effects. Furthermore, we demonstrate the capacity of the ionome to reveal host genotype-specific variation in the impact of AMF on plant nutrition.

Microscopic assessment of the degradation of millet starch granules by endogenous and exogenous enzymes during mashing.

The high gelatinization temperature (GT) of millet starch prevents the usage of infusion or step mashes as an effective means to generate fermentable sugars (FS) in brewing because the malt amylases lack thermostability at GT. Here, we investigate processing modifications to determine if millet starch can be efficiently degraded below GT. We determined that producing finer grists through milling did not introduce enough granule damage to markedly change gelatinization characteristics, though there was improved liberation of the endogenous enzymes. Alternatively, exogenous enzyme preparations were added to investigate their ability to degrade intact granules. At the recommended dosages (0.625 µL/g malt), significant FS concentrations were observed, although at lower concentrations and with a much-altered profile than possible with a typical wort. When exogenous enzymes were introduced at high (10×) addition rates, significant losses of granule birefringence and granule hollowing were observed well below GT, suggesting these exogenous enzymes can be utilized to digest millet malt starch below GT. The exogenous maltogenic α-amylase appears to drive the loss of birefringence, but more research is needed to understand the observed predominate glucose production.

Salivary α-amylase activity and flow rate explain differences in temporal flavor perception in a chewing gum matrix comprising starch-limonene inclusion complexes.

Starch-guest inclusion complexes (ICs) are a novel, clean-label flavor encapsulation system with the potential to improve stability of aroma volatiles. While amylase has been shown to modulate guest release in vitro, release by sensory perception has not been evaluated. Here, Temporal Check-All-That-Apply (TCATA) and CATA were used to compare flavor perception of starch-limonene ICs to uncomplexed limonene, and the differences in perception were explored as a function of participant salivary α-amylase activity (sAA) and salivary flow rate (sFR). High sFR levels decreased limonene perception while high sAA increased limonene perception, highlighting the potential influence of these physiological factors on flavor perception of foods. Temporal flavor perception of a chewing gum containing starch-limonene ICs and a second chewing gum containing uncomplexed limonene and corn starch (CTL) was evaluated by 99 untrained consumers who assessed taste, texture, and aroma attributes over 17 min by TCATA and CATA. In addition, participants were segmented into three clusters based on their sAA and sFR, and cluster TCATA curves for each sample and attribute were statistically compared. Overall, participants rated Citrus, Sour and Bitter (p < 0.05) significantly higher for the IC sample and rated Sweet higher for the CTL. For Citrus, Sour, and Bitter, significant differences were observed between the three clusters for the IC chewing gum, while the CTL gum showed no significant differences for these three attributes. We demonstrate that flavor perception of starch-guest ICs varies with participants' salivary α-amylase activity and flow rate. Additionally, TCATA and CATA were found to be well suited to characterize flavor release systems over a long period of time as multiple flavor percepts can be simultaneously tracked.

Genome-wide association study suggests an independent genetic basis of zinc and cadmium concentrations in fresh sweet corn kernels.

Despite being one of the most consumed vegetables in the United States, the elemental profile of sweet corn (Zea mays L.) is limited in its dietary contributions. To address this through genetic improvement, a genome-wide association study was conducted for the concentrations of 15 elements in fresh kernels of a sweet corn association panel. In concordance with mapping results from mature maize kernels, we detected a probable pleiotropic association of zinc and iron concentrations with nicotianamine synthase5 (nas5), which purportedly encodes an enzyme involved in synthesis of the metal chelator nicotianamine. In addition, a pervasive association signal was identified for cadmium concentration within a recombination suppressed region on chromosome 2. The likely causal gene underlying this signal was heavy metal ATPase3 (hma3), whose counterpart in rice, OsHMA3, mediates vacuolar sequestration of cadmium and zinc in roots, whereby regulating zinc homeostasis and cadmium accumulation in grains. In our association panel, hma3 associated with cadmium but not zinc accumulation in fresh kernels. This finding implies that selection for low cadmium will not affect zinc levels in fresh kernels. Although less resolved association signals were detected for boron, nickel, and calcium, all 15 elements were shown to have moderate predictive abilities via whole-genome prediction. Collectively, these results help enhance our genomics-assisted breeding efforts centered on improving the elemental profile of fresh sweet corn kernels.

Design aspects of vaginal applicators that influence acceptance among target users.

Although sensory-guided product design is most traditionally used by food and beverage companies, the approach has widespread application for many other products, including pharmaceuticals and medical devices. Previously, our team used sensory methods to explore preclinical optimization of soft-gel vaginal microbicides. Past clinical trials suggest vaginal microbicides may be an effective means for women to protect themselves from HIV and other sexually transmitted infections, but these microbicides will not work if they are not used due to poor acceptability. Our prior work suggests properties like firmness, size, and shape all influence women's willingness to try soft-gel vaginal suppositories. As product insertion is part of the overall experience of using vaginal microbicides, understanding the features of vaginal applicators that appeal to women, and incorporating these insights into vaginal drug delivery systems, may also improve user adherence. Despite widespread use of vaginal applicators, there is minimal public data on women's perceptions of and preferences for physical applicator features. Other work suggests women want vaginal applicators that are single use, pre-filled, made of plastic, and easy to use, store, and discard. Applicator attributes that may be important to women, such as length, color, or visual appeal, have not been investigated previously. The objective of this research was to understand what physical applicator attributes are appealing to women. Here, 18 commercially available applicators were evaluated by a convenience sample of women (n = 102) for overall liking and perceptions of various attributes (perceived length and width, ease-of-grip, expected ease-of-use, expected comfort inside the body, visual appeal, color liking, and environmental friendliness). Preference mapping using both liking data and attribute data showed attributes such as color, visual appeal, ease of grip, expected ease of use, and expected comfort inside the body drove higher liking ratings for applicators, while perceived length negatively affected liking. In general, plastic tampon applicators contained more positive features and were better liked relative to a cardboard tampon applicator or applicators for insertion of medicated gels or suppositories. Incorporating more desirable features into applicators meant for insertion of vaginal microbicides or other vaginal medications may improve the user experience, and possibly user adherence.

Crystal and molecular structure of V-amylose complexed with ibuprofen.

Rectangular V-amylose single crystals were prepared by adding racemic ibuprofen to hot dilute aqueous solutions of native and enzymatically-synthesized amylose. The lamellar thickness increased with increasing degree of polymerization of amylose and reached a plateau at about 7 nm, consistent with a chain-folding mechanism. The CP/MAS NMR spectrum as well as base-plane electron and powder X-ray diffraction patterns recorded from hydrated specimens were similar to those of V-amylose complexed with propan-2-ol. Amylose was crystallized in an orthorhombic unit cell with parameters a = 2.824 ± 0.001 nm, b = 2.966 ± 0.001 nm, and c = 0.800 ± 0.001 nm. A molecular model was proposed based on structural analogies with the Vpropan-2-ol complex and on assumptions on the stoichiometry of ibuprofen. The unit cell would contain four antiparallel 7-fold amylose single helices with ibuprofen molecules distributed inside and between the helices.

Electrospinning of Octenylsuccinylated Starch-Pullulan Nanofibers from Aqueous Dispersions.

We aimed to develop a greener process for dry-electrospinning food-grade modified starch through the elimination of organic solvents. The rheological properties and electrospinnability of aqueous dispersions of commercial octenylsuccinylated (OS) starches with various molecular weight (Mw) were investigated, yet only nanofibers with beads or defects could be obtained from OS starch with the highest Mw, i.e., Purity Gum@ Ultra (PGU). Further improvement in the fiber morphology was achieved by adding pullulan (PUL) as a minor component in the spinning dope. Smooth, continuous, and bead-free nanofibers (147-250 nm) were obtained from the PGU-PUL dispersions. Shown on an electrospinnability map, the successful electrospinning of 12%, 15%, and 20% (w/v) aqueous PGU dispersions required a minimum addition of 6%, 5%, and 3% (w/v) of PUL, respectively. The addition of PUL contributed to establishing sufficient molecular entanglement for electrospinning. This study provides a promising green process to produce starch-based nanofibers for use in various applications, e.g., drug delivery, wound dressing, and tissue engineering.

Assessment of Midline Lingual Point-Pressure Somatosensation Using Von Frey Hair Monofilaments.

Detection and discrimination threshold estimates for oral point pressure are assessed using Von Frey Hair monofilaments. Consistent with previously published protocols, threshold estimates are determined using a two-interval forced choice (2-IFC) paradigm with a three down/one up approach. Detection threshold estimates determine the mean force in which a participant can identify the presence of pressure. During the detection threshold procedure, the participant is instructed to choose which of two sequentially presented observation intervals contained the tactile test stimulus. If the participant performs three correct detections in a row (i.e., 3 'hits'), the researcher decreases the stimulus to the next lower target force level. With one incorrect detection (a 'miss'), the researcher increases the force delivered to the next higher level. This threshold estimation approach is known as a 3-down/1-up adaptive staircase. Reponses are recorded on a paper ballot, and a participant's estimated threshold is defined as the geometric mean of five reversals. During the discrimination threshold procedure, the participant is asked to make a choice between two serially presented stimuli as to which is the "harder" or "stronger" pressure. The same scoring of 'hits', 'misses', and stopping points are used. Detection and discrimination testing for oral point pressure at tongue midline takes approximately 20 min to complete. Using these commercially-available clinical tools, individual touch sensation profiles for the midline tongue can be achieved in a relatively time and cost effective means.

Are workers on board vessels involved with chemicals from treated ballast water sufficiently protected? - A decadal perspective and risk assessment.

Ballast Water Managements Systems (BWMS) installed on vessels may use Active Substances (AS) to inactivate organisms. This paper provides new insights in the global issue of noxious Disinfection By-Products (DBP) produced with primarily oxidant-based BWMS, and the risk assessment for workers, including port State control officers, while performing tasks on a vessel that involve exposure to treated ballast water. The Joint Group of Experts on the Scientific Aspects of Marine Environmental Protection - Ballast Water Working Group (GESAMP-BWWG) plays a role in the certification process of BWMS that make use of AS evaluating potential negative effects. All BWMS that passed GESAMP-BWWG Final Approval until mid 2019 were analyzed providing an overview of chemicals in the treated ballast water before and after neutralization. The ballast tank cleaning scenario, the sampling scenario, and the ballast tank inspection scenario all showed elevated human health risks using the Derived Minimal Effect Levels approach. The most critical exposure occurs in the ballast tank cleaning scenario through the inhalation of volatile DBP, such as tribromomethane. This substance may cause acute effects such as headache, dizziness and also has carcinogenic properties. The two risk reducing options available in the GESAMP-BWWG Tier 2 calculations were compared, one being mitigation measures such as protective gloves and coveralls, the other option is taking into account a time correction factor. The results showed that the trihalomethanes in air are most problematic, however, there is a possibility that the calculated values may be overestimated as generally worst case assumptions were used.

Formation of Brominated Organic Compounds and Molecular Transformations in Dissolved Organic Matter (DOM) after Ballast Water Treatment with Sodium Dichloroisocyanurate Dihydrate (DICD).

Estuarine water treated with a ballast water management system (BWMS) using a solution of dissolved dichloroisocyanurate dihydrate (DICD) resulted in the formation of newly described brominated disinfection byproducts (Br-DBPs). Analysis of dissolved organic matter (DOM) in untreated water with ultrahigh resolution Fourier transform ion cyclotron resonance mass spectrometry (FT-ICR MS) identified 3897 m/z ions and their exact molecular formulas. After DICD treatment, a total of 213 halogenated molecular ions with relative abundance of at least 1% were assigned and confirmed using isotope simulation. Halogenated ions were assigned in four DBP elemental groups including CHOBr (180), CHONBr (13), CHOCl (16), and CHOBrCl (4). Forty-nine of the 197 brominated formulas have not been previously reported. We also were able to tentatively assign possible structures to the formula C3HBr3N2 due to very limited isomeric possibilities. The tentatively assigned compound found at 6.4% relative abundance was identified as either tribromoimidazole or tribromopyrazole. Our results show the formation of complex halogenated DBPs that are formed in the treatment of water with a novel BWMS that employs granular DICD as a biocide. The toxicological and mutagenic properties as well as the fate of these newly identified brominated DBPs are unknown.

Perseorangin: A natural pigment from avocado (Persea americana) seed.

High consumer demand has led global food color manufacturers and food companies to dramatically increase the development and use of natural colors. We have previously reported that avocado (Persea americana) seeds, when crushed in the presence of air, develop a red-orange color in a polyphenol oxidase-dependent reaction. The objective of this study was to identify the major colored compound in colored avocado seed extract (CASE). Column chromatography and high performance liquid chromatography were used to isolate the most abundant colored compound in CASE. This compound, henceforth referred to as perseorangin, was a yellow-orange solid. Structural analysis was performed using high-resolution mass spectrometry, and infrared and nuclear magnetic resonance spectroscopy. We determined that perseorangin is a glycosylated benzotropone-containing compound with a molecular formula of C29H30O14. Liquid chromatography with electrospray ionization mass spectrometry-based metabolomic analysis of CASE and uncolored avocado seed extract showed that perseorangin was unique to CASE.

In Vitro Antioxidant and Cancer Inhibitory Activity of a Colored Avocado Seed Extract.

Avocado (Persea americana) seeds have been used traditionally for a number of health-related indications. Because of its high polyphenol content, we investigated the potential antioxidant and anticancer effects of a colored avocado seed extract (CASE). CASE exhibited an oxygen radical acceptance capacity value of 2012 ± 300 trolox equivalents/mg. CASE reduced lipid hydroperoxide formation in an oil-in-water emulsion (33% reduction at 500 µg/mL). CASE dose-dependently reduced the viability of human breast (MCF7), lung (H1299), colon (HT29), and prostate (LNCaP) cancer cells in vitro. The half maximal inhibitory concentrations ranged from 19 to 132 µg/mL after treatment for 48 h. CASE treatment downregulated the expression of cyclin D1 and E2 in LNCaP cells. This was associated with cell G0/G1 phase cycle arrest. CASE also dose-dependently induced apoptosis in LNCaP cells. CASE reduced nuclear translocation of nuclear factor κB, a prosurvival signal. Further studies are needed to examine these effects in in vivo models.

Electrospun nanofiber mats from aqueous starch-pullulan dispersions: Optimizing dispersion properties for electrospinning.

A green method to fabricate starch-based nanofibers is provided. High-temperature (≈162 °C) was used to destructure high-amylose starch in water. Sodium palmitate was added to enhance the stability of high-amylose starch in water at room temperature and increase the conductivity of the electrospinning dope. Flow properties and zeta potential of starch-palmitate dispersions were characterized by rheometer and dynamic light scattering, respectively. Pullulan was mixed in as a minor component of the starch-palmitate complex (starch:pullulan at a ca. 2:1 ratio) and the mixture electrospun. Pullulan hindered starch association and modified the dispersion properties, promoting molecular entanglement without gelation. The presence of sodium palmitate-starch inclusion complexes in the fiber was confirmed by differential scanning calorimetry and X-ray diffraction. Tensile strength of the nanofiber composite was found to be weaker than that of micro-sized pure starch fiber mats. This method provides future industry with lower cost by eliminating the use of organic chemicals.

Oral somatosensatory acuity is related to particle size perception in chocolate.

Texture affects liking or rejection of many foods for clinically relevant populations and the general public. Phenotypic differences in chemosensation are well documented and influence food choices, but oral touch perception is less understood. Here, we used chocolate as a model food to explore texture perception, specifically grittiness perception. In Experiment 1, the Just Noticeable Difference (JND) for particle size in melted chocolate was ~5 µm in a particle size range commonly found in commercial chocolates; as expected, the JND increased with particle size, with a Weber Fraction of ~0.17. In Experiment 2, individual differences in touch perception were explored: detection and discrimination thresholds for oral point pressure were determined with Von Frey Hairs. Discrimination thresholds varied across individuals, allowing us to separate participants into high and low sensitivity groups. Across all participants, two solid commercial chocolates (with particle sizes of 19 and 26 µm; i.e., just above the JND) were successfully discriminated in a forced-choice task. However, this was driven entirely by individuals with better oral acuity: 17 of 20 of more acute individuals correctly identified the grittier chocolate versus 12 of 24 less acute individuals. This suggests phenotypic differences in oral somatosensation can influence texture perception of foods.

Synthetic lipids as a biocide candidate for disinfection of ballast water.

The objective of this study is to propose the use of specific synthetic lipid as an active substance (biocide) in the control of harmful aquatic microorganisms, such as pathogens and non-indigenous species, transported in ships' ballast water. The biocide candidate, without metal or halogen components, was produced from a sub-product of the edible oil industry, the lecithin. Laboratory assays were conducted with phytoplankton, zooplankton, and marine bacteria to evaluate the efficiency of the biocide. The study also considers specific biocide's characteristics related to environmental risks, such as chemical composition, persistence, bioaccumulation, and toxicity. Results showed that, in the first 24 h of treatment, the biocide effectively reduced the concentration of the planktonic micro-organisms to very low levels. Additionally, a preliminary risk evaluation pointed that biocide candidate has a low residual toxicity, also a low potential for persistence and bioaccumulation in the environment.

Qualitative exploration of intrinsic and extrinsic factors that influence acceptability of semisoft vaginal suppositories.

BACKGROUND: Vaginal microbicides are a promising means to prevent the transmission of HIV and other sexually transmitted infections, by empowering women to initiate use prophylactically when they perceive themselves to be at risk. However, in clinical trials, microbicides have shown mixed results, with the consistent finding that effectiveness varies substantially as a function of user adherence. METHODS: Based on the assumption that adherence is driven, at least in part, by product properties that influence acceptability, we used softgel technology to develop vaginal drug delivery systems in the intermediate texture space between solids and liquids to overcome potential shortcomings of current dosage forms. Here, we used focus groups and surveys to determine women's initial reactions (i.e., acceptance and willingness-to-try) for semisoft vaginal suppositories intended for HIV and STI prevention, with a specific focus on how perception of and preferences for vaginal suppositories may be influenced by product characteristics such as size, shape, and firmness. RESULTS: Via focus groups, we identified intrinsic and extrinsic factors relevant to acceptability of semisoft suppository prototypes. Willingness-to-try depended on factors like intended functionality, anticipated leakage, type of sex, recommended frequency of use, type of sexual partner, and perceived risk. When handled ex vivo, shape, size, and firmness of suppositories communicated information about ease of imagined insertion and handling, perceived effectiveness, anticipated awareness and comfort of the product in the body. These impressions were partly based on prior experience with vaginal products. CONCLUSIONS: Sensory attributes appear to play a substantial role in women's preferences and willingness to try the semisoft suppositories. Using these methods during preclinical development should help efficiently optimize a final product that is both biologically efficacious and preferred by women, toward a goal of enhancing adherence and effectiveness.

Long-term algal toxicity of oxidant treated ballast water.

National and international regulations require that ships' ballast water is treated to minimize the risk of introducing potentially invasive species. A common approach employed by commercial ballast water management systems is chlorination. This study presents the algal toxicity findings for three chlorination-based BWMS and their implications to environmental safety of port waters receiving treated ballast water from ships. Discharged treated ballast water from all three BWMS was toxic to algae with IC25s (25% growth inhibition) ranging from 9.9% to 17.9%, despite having total residual oxidant concentrations below 0.02 mg/l, based on Whole Effluent Toxicity assays. When held at 4 °C, some of the ballast water samples continued to exhibit toxic effects with no observed effect concentrations as low as 18% after a 134 day holding time. Thirteen individual disinfection by-products were measured above the detected limit at the time of discharge. No correlation between DBPs and algal toxicity was observed.

Inclusion complex formation between high amylose corn starch and alkylresorcinols from rye bran.

The formation of high amylose corn starch (HACS)-alkylresorcinol (AR) inclusion complexes was demonstrated using HACS and a crude AR extract from rye bran, and was confirmed by complementary characterization techniques. Inclusion complex was extracted using hot 2-propanol/water (3:1), and thirteen different AR homologs were identified in the 2-propanol/water extract. However, this extraction regime was insufficient to remove all guests from the inclusion complexes, but did result in annealing of the V-type crystallinity, yielding an endotherm with higher onset and peak temperatures and dissociation enthalpy. The remaining guest compounds were recovered by disruption of the inclusion complexes with DMSO followed by liquid-liquid extraction. A total of nine AR homologs were recovered and two saturated long alkyl chain AR homologs (C21:0 and C19:0) were obviously enriched in the complexes compared to the crude AR extract. The formation of HACS-AR inclusion complexes may have the potential to inhibit staling in baked goods.