Prediction of Pharmacokinetic Profile of Valsartan in Humans Based on in vitro Uptake‐Transport Data

The aim of this study was to evaluate a physiologically based pharmacokinetic (PBPK) model for predicting PK profiles in humans based on a model refined in rats and humans in vitro uptake‐transport data using valsartan as a probe substrate. Valsartan is eliminated unchanged, mostly through biliary excretion, both in humans and rats. It was, therefore, chosen as model compound to predict in vivo elimination based on in vitro hepatic uptake‐transport data using a fully mechanistic PBPK model. Plated rat and human hepatocytes, and cell lines overexpressing human OATP1B1 and OATP1B3 were used for in vitro uptake experiments. A mechanistic two‐compartment model was used to derive the active and passive transport parameters, namely uptake Michaelis–Menten parameters (V_max and K_m,u) together with passive diffusion (P_dif). These transport parameters were then used as input in a whole body physiologically based pharmacokinetic (PBPK) model. The uptake rate of valsartan was higher for rat hepatocytes (K_m,u=28.4±3.7 μM , V_max=1320±180 pmol/mg/min, and P_dif =1.21±0.42 μl/mg/min) compared to human hepatocytes (K_m,u=44.4±14.6 μM , V_max=304±85 pmol/mg/min, and P_dif=0.724±0.271 μl/mg/min). OATP1B1 and ‐1B3 parameters were correlated to human hepatocyte data, using experimentally established relative activity factors (RAF). Resulting PBPK simulations were compared for plasma‐ (humans and rats) and bile‐ (rats) concentration–time profiles following iv bolus administration of valsartan. Plasma clearances (CL_P) for rats and humans were predicted within twofold relative to predictions based on respective in vitro data. The simulations were extended to simulate the impact of either OATP1B1 or ‐1B3 inhibition on plasma profile. The limited data set indicates that the mechanistic model allowed for accurate evaluation of in vitro transport data; and the resulting hepatic uptake transport kinetic parameters enabled the prediction of in vivo PK profiles and plasma clearances, using PBPK modelling. Moreover, the interspecies difference in elimination rate observed in vivo was correctly reflected in the transport parameters determined in vitro.     

Expression of Lysophosphatidic Acid Receptor 1 and Relation with Cell Proliferation,Apoptosis, and Angiogenesis on Preneoplastic Changes Induced by Cadmium Chloride in the Rat Ventral Prostate

Background

Lysophosphatidic acid (LPA) is a phospholipid growth factor involved in cell proliferation, differentiation, migration, inflammation, angiogenesis, wound healing, cancer invasion, and survival. This study was directed to evaluate the immunoexpression of LPA-1, cell proliferation, apoptosis, and angiogenesis markers in preneoplastic lesions induced with cadmium chloride in rat prostate.

Methods

The following parameters were calculated in ventral prostate of normal rats and rats that received Cd in drinking water during 24 months: percentages of cells immunoreactive to LPA-1 (LI_LPA1), PCNA (LI_PCNA), MCM7 (LI_MCM7), ubiquitin (LI_UBI), apoptotic cells (LI_APO), and p53 (LI_p53); volume fraction of Bcl-2 (V_FBcl-2); and length of microvessels per unit of volume (L_VMV/mm3). Data were analyzed using Student''s t-test and Pearson correlation test.

Results

The LI_LPA1 in dysplastic lesions and normal epithelium of Cd-treated rats was significantly higher than those in the control group. Markers of proliferation were significantly increased in dysplastic lesions, whereas some apoptotic markers were significantly decreased. No significant differences between groups were found in V_FBcl-2. Dysplastic lesions showed a significant increase of LI_p53. The length of microvessels per unit of volume was elevated in dysplastic acini. Statistically significant correlations were found only between LI_LPA1 and LI_UBI.

Conclusions

Our results suggest that LPA-1 might be implicated in dysplastic lesions induced by cadmium chloride development. More studies are needed to confirm its potential contribution to the disease.     

Inhibition of gibberellin biosynthesis by paclobutrazol in cell-free homogenates ofCucurbita maxima endosperm andMalus pumila embryos

The plant growth retardant paclobutrazol, (PP333) (2RS, 3RS)-1-(4-chlorophenyl)-4,4-dimethyl-2-(1,2,4-triazol-1-yl)pentan-3-ol, inhibits specifically the three steps in the oxidation of the gibberellin-precursorent-kaurene toent-kaurenoic acid in a cell-free system fromCucurbita maxima endosperm. The KI₅₀ for this inhibition is 2×10^–8 M. The KI₅₀ values for the separated2S, 3S, and2R, 3R enantiomers of paclobutrazol in this system are 2×10^–8 M and 7×10^–7 M, respectively. A cell-free preparation from immatureMalus pumila embryos convertsent-kaurene to gibberellin A₉, whereas no conversion occurs in a similar preparation fromMalus endosperm. The conversion ofent-kaurene by the embryo preparation is inhibited by paclobutrazol with KI₅₀ values for the2S,3S and2R,3R enantiomers of 2×10^–8 M and 6×10^–8 M, respectively.     

TDFCAM: A method for estimating stable isotope trophic discrimination in wild populations

1. Stable isotope mixing models (SIMMs) are widely used for characterizing wild animal diets. Such models rely upon using accurate trophic discrimination factors (TDFs) to account for the digestion, incorporation, and assimilation of food. Existing methods to calculate TDFs rely on controlled feeding trials that are time-consuming, often impractical for the study taxon, and may not reflect natural variability of TDFs present in wild populations.
2. We present TDF_CAM as an alternative approach to estimating TDFs in wild populations, by using high-precision diet estimates from a secondary methodological source—in this case nest cameras—in lieu of controlled feeding trials, and provide a framework for how and when it should be applied.
3. In this study, we evaluate the TDF_CAM approach in three datasets gathered on wild raptor nestlings (gyrfalcons Falco rusticolus; peregrine falcons Falco perigrinus; common buzzards Buteo buteo) comprising contemporaneous δ¹³C & δ¹⁵N stable isotope data and high-quality nest camera dietary data. We formulate Bayesian SIMMs (BSIMMs) incorporating TDFs from TDF_CAM and analyze their agreement with nest camera data, comparing model performance with those based on other relevant TDFs. Additionally, we perform sensitivity analyses to characterize TDF_CAM variability, and identify ecological and physiological factors contributing to that variability in wild populations.
4. Across species and tissue types, BSIMMs incorporating a TDF_CAM outperformed any other TDF tested, producing reliable population-level estimates of diet composition. We demonstrate that applying this approach even with a relatively low sample size (n < 10 individuals) produced more accurate estimates of trophic discrimination than a controlled feeding study conducted on the same species. Between-individual variability in TDF_CAM estimates for ∆¹³C & ∆¹⁵ N increased with analytical imprecision in the source dietary data (nest cameras) but was also explained by natural variables in the study population (e.g., nestling nutritional/growth status and dietary composition).
5. TDF_CAM is an effective method of estimating trophic discrimination in wild animal populations. Here, we use nest cameras as source dietary data, but this approach is applicable to any high-accuracy method of measuring diet, so long as diet can be monitored over an interval contemporaneous with a tissue's isotopic turnover rate.

     

滨海湿地植被地上生物量遥感反演

盐沼植被生物量是滨海湿地生态系统碳循环研究的重要参数,是湿地生态系统健康评价、资源可持续利用的关键指标,开展盐沼植被地上生物量监测方法研究具有重要意义。目前,遥感技术在湿地生物量监测领域已经得到广泛应用,但反演方法仍以统计模型为主,模型构建需要实测数据支撑,时空拓展性不强。选择江苏盐城丹顶鹤保护区为研究区,基于冠层辐射(PROSAIL)传输模型,通过局部和全局敏感性分析,对模型参数本地化,构建了互花米草地上生物量半经验反演模型,应用于Landsat 8 OLI遥感影像,获得了互花米草地上生物量的时空分布。研究结果表明,利用PROSAIL模型模拟互花米草冠层反射率,叶面积指数(LAI)、叶片干物质含量(Cm)、叶倾角分布参数(LIDF)、等效水厚度(Cw)、叶绿素含量(Cab)、叶片结构参数(N)为高敏感性参数,类胡萝卜素含量(Car)、土壤参数(P_soil)为低敏感性参数;利用不同时刻的遥感影像反演了地上生物量,遥感反演结果与实测数据对比,拟合度R²为0.83,均方根误差(RMSE)为0.43kg/m²,平均相对误差(MRE)为15.7%,精度较高,模型具有较好的时空普适性。研究发展了盐沼植被地上生物量遥感反演方法,解决了以往过于依赖现场实测数据构建反演模型的局限性,该方法可以为研究滨海湿地生态系统碳循环以及准确估算其碳汇潜力提供技术支持。     

Effects of Cationic Substitutions on Delayed Rectifier Current in Type I Vestibular Hair Cells

The resting potassium current (I _KI ) in gerbil dissociated type I vestibular hair cells has been characterized under various ionic conditions in whole cell voltage-clamp. When all K⁺ in the patch electrode solution was replaced with Na⁺, (Na⁺) _in or Cs⁺, (Cs⁺) _in , large inward currents were evoked in response to voltage steps between −90 and −50 mV. Activation of these currents could be described by a Hodgkin-Huxley-type kinetic scheme, the order of best fit increasing with depolarization. Above ∼−40 mV currents became outward and inactivated with a monoexponential time course. Membrane resistance was inversely correlated with external K⁺ concentration. With (Na⁺) _in , currents were eliminated when K⁺ was removed from the external solution or following extracellular perfusion of 4-aminopyridine, indicating that currents flowed through I _KI channels. Also, reduction of K⁺ entry through manipulation of membrane potential reduced the magnitude of the outward current. Under symmetrical Cs⁺, 0 K⁺ conditions I _KI is highly permeable to Cs⁺. However, inward currents were reduced when small amounts of external K⁺ were added. Higher concentrations of K⁺ resulted in larger currents indicating an anomalous mole fraction effect in mixtures of external Cs⁺ and K⁺. Received: 23 June 1999/Revised: 27 September 1999     

Spatio-temporal modelling of particulate matter concentrations using satellite derived aerosol optical depth over coastal region of Chennai in India

The present study primarily focuses on describing aerosol optical depth (AOD), its distribution pattern and seasonal variation, and modelling Particulate Matter Concentrations in Chennai. The frequency distribution of AOD and PM_2.5 demonstrates that AOD can be used as a proxy for estimating PM_2.5 in the study region as the occurrence of AOD almost resonates with that of PM_2.5. The seasonal variation of AOD and PM_2.5 revealed that during the winter (October–January) and summer (February–May) seasons, AOD reasonably followed the trend of PM_2.5. However, during the monsoon period, AOD showed random variations. Different models like linear and non-linear regression models and machine learning models such as random forest (RF) have been developed for PM_2.5 estimation. The model's performance in different stations and seasons has been assessed. The effect of meteorology and other factors in the model has also been assessed. From linear and non-linear model analysis, AOD was a significant parameter in estimating PM_2.5. The Random Forest model was the stable model for the study region, with a model R² of 0.53 and an RMSE of 15.89 μg/m³. The inclusion of meteorological parameters like relative humidity, wind speed, and wind direction decreased the error in prediction by 17.45 μg/m³. The seasonal and spatial analysis indicates that the prediction capability of models varies with stations and seasons. The best performing model was found to be Model RF, and the model could explain about 53.14% of the variability in PM_2.5 concentration occurrence in the study region with a prediction error of 15.89 μg/m³.     

Screening Level Assessment of Risks Associated with Dietary Exposure to Selected Heavy Metals,Polycyclic Aromatic Hydrocarbons,and Radionuclides in Kuwait

To assess the health risks associated with dietary exposure of the Kuwait population, two market baskets comprising 146 food items, which represented the diets of various age groups of the Kuwaiti population, were collected twice, once in the winter of 1997 and again in the summer of 1998, to account for seasonal variation. These food items were analyzed for their heavy metals, polycyclic aromatic hydrocarbons (PAHs) and selected radionuclides contents. Probabilistic risk assessments using the Monte Carlo simulation technique were conducted for Cd, Pb, PAHs, ¹³⁷Cs, ⁹⁰Sr, and ²²⁶Ra. The data obtained showed that high Cd and Pb exposures, which exceeded the World Health Organization's (WHO's) standards are encountered among young children. Exposures to PAHs of the different age groups of the Kuwaiti population were not unusually high at the higher percentiles. On the other hand, dietary exposures to ⁹⁰Sr were all below WHO standards for all percentile and age groups. Exposures to ¹³⁷Cs were slightly above the WHO standard at the 90^th percentile for most age groups, and ²²⁶Ra exposures were all well above WHO standards at the upper percentiles for all age groups, but still below the United States Food and Drug Administration's (USFDA's) intervention levels.     

Zooplankton as a compound mineralising and synthesizing system: phosphorus excretion

Data on phosphate excretion rates of zooplankton are based on measurements using the pelagic crustacean zooplankton of Lake Vechten and laboratory-cultured Daphnia galeata. In case of Daphnia sp we measured the effects of feeding on P-rich algae and P-poor algae (Scenedesmus) as food on the P-excretion rates at 20°C. The excretion rates of the natural zooplankton community, irrespective of the influence of the factors mentioned, varied by an order of magnitude: 0.025–0.275µg PO₄-Pmg^–1C in zooplankton (C _zp ) h^–1. The temperature accounted for about half the observed variation in excretion rates. The mean excretion rates in the lake, computed for 20°C, varied between 0.141 and 0.260 µg Pmg^–1C _zp h^–1. Based on data of zooplankton biomass in the lake the P-regeneration rates by zooplankton covered between 22 and 239% of the P-demand of phytoplankton during the different months of the study period.In D. galeata, whereas the C/P ratios of the Scenedesmus used as food differed by a factor 5 in the experiments, the excretion rates differed by factor 3 only. Despite the higher P-excretion rates (0.258± 0.022 µg PO₄-P mg^–1 C h^–1) of the daphnids fed with P-rich food than those fed with P-poor food (0.105 ± 0.047 µg PO₄-P mg^–1 C h^p–1), both the categories of the animals were apparently conserving P. A survey of the literature on zooplankton excretion shows that in Daphnia the excretion rates vary by a factor 30, irrespective of the species and size of animals and method of estimation and temperature used.About two-thirds of this variation can be explained by size and temperature. A major problem of comparability of studies on P-regeneration by zooplankton relates to the existing techniques of P determination, which necessitates concentrating the animals several times above the in situ concentration (crowding) and prolonged experimental duration (starving), both of which manifest in marked changes that probably lead to underestimation of the real rates.     

Application of Physiologically Based Absorption Modeling to Formulation Development of a Low Solubility,Low Permeability Weak Base: Mechanistic Investigation of Food Effect

Physiologically based pharmacokinetic (PBPK) modeling has been broadly used to facilitate drug development, hereby we developed a PBPK model to systematically investigate the underlying mechanisms of the observed positive food effect of compound X (cpd X) and to strategically explore the feasible approaches to mitigate the food effect. Cpd X is a weak base with pH-dependent solubility; the compound displays significant and dose-dependent food effect in humans, leading to a nonadherence of drug administration. A GastroPlus Opt logD Model was selected for pharmacokinetic simulation under both fasted and fed conditions, where the biopharmaceutic parameters (e.g., solubility and permeability) for cpd X were determined in vitro, and human pharmacokinetic disposition properties were predicted from preclinical data and then optimized with clinical pharmacokinetic data. A parameter sensitivity analysis was performed to evaluate the effect of particle size on the cpd X absorption. A PBPK model was successfully developed for cpd X; its pharmacokinetic parameters (e.g., C _max, AUC_inf, and t _max) predicted at different oral doses were within ±25% of the observed mean values. The in vivo solubility (in duodenum) and mean precipitation time under fed conditions were estimated to be 7.4- and 3.4-fold higher than those under fasted conditions, respectively. The PBPK modeling analysis provided a reasonable explanation for the underlying mechanism for the observed positive food effect of the cpd X in humans. Oral absorption of the cpd X can be increased by reducing the particle size (<100 nm) of an active pharmaceutical ingredient under fasted conditions and therefore, reduce the cpd X food effect correspondingly.     

Absorption, tissue distribution, metabolism and excretion of ormetoprim and sulphadimethoxine in cod (Gadus morhua) after oral administration of Romet30

Uptake, tissue distribution, metabolism and elimination of sulphadimethoxine (SDM) and ormetoprim (OMP) were examined in Atlantic cod (Gadus morhua) following oral administration of Romet³⁰ at a dose of 5 mg OMP and 25 mg SDM per kilogram fish. Absorption and elimination was rapid for OMP and slow for SDM characterized by peak plasma concentrations (C_max) of 1.44 and 20.93 μg ml^?1 after 7 and 59 h, respectively, and plasma elimination halflives (t_1/2β) of 7 h for OMP and 56 h for SDM. High concentrations of SDM were found in the bile indicating high biliary excretion of this drug whereas moderate concentrations of N‐acetylated SDM were found in tissues and bile indicating N‐acetylation to be a less important metabolic pathway for SDM in cod. The biliary concentration of OMP was low.     

Physiological variables affecting surface film formation by native lamellar body-like pulmonary surfactant particles

Pulmonary surfactant (PS) is a surface active complex of lipids and proteins that prevents the alveolar structures from collapsing and reduces the work of breathing by lowering the surface tension at the alveolar air–liquid interface (A_LI). Surfactant is synthesized by the alveolar type II (AT II) cells, and it is stored in specialized organelles, the lamellar bodies (LBs), as tightly packed lipid bilayers. Upon secretion into the alveolar lining fluid, a large fraction of these particles retain most of their packed lamellar structure, giving rise to the term lamellar body like-particles (LBPs). Due to their stability in aqueous media, freshly secreted LBPs can be harvested from AT II cell preparations. However, when LBPs get in contact with an A_LI, they quickly and spontaneously adsorb into a highly organized surface film. In the present study we investigated the adsorptive capacity of LBPs at an A_LI under relevant physiological parameters that characterize the alveolar environment in homeostatic or in pathological conditions. Adsorption of LBPs at an A_LI is highly sensitive to pH, temperature and albumin concentration and to a relatively lesser extent to changes in osmolarity or Ca^2 + concentrations in the physiological range. Furthermore, proteolysis of LBPs significantly decreases their adsorptive capacity confirming the important role of surfactant proteins in the formation of surface active films.     

Using a BP Neural Network for Rapid Assessment of Populations with Difficulties Accessing Drinking Water Because of Drought

Accurately predicting the populations with difficulties accessing drinking water because of drought and taking appropriate mitigation measures can minimize economic loss and personal injury. Taking the 2013 Guizhou extreme summer drought as an example, on the basis of collecting meteorological, basic geographic information, socioeconomic data, and disaster effect data of the study area, a rapid assessment model based on a backpropagation (BP) neural network was constructed. Six factors were chosen for the input of the network: the average monthly precipitation, Digital Elevation Model (DEM), river density, population density, road density, and gross domestic product (GDP). The population affected by drought was the model's output. Using samples from 50 drought-affected counties in Guizhou Province for network training, the model's parameters were optimized. Using the trained model, the populations in need were predicted using the other 74 drought-affected counties. The accuracy of the prediction model, represented by the coefficient of determination (R²) and the normalized root mean square error (N-RMSE), yielded 0.7736 for R² and 0.0070 for N-RMSE. The method may provide an effective reference for rapid assessment of the population in need and disaster effect verification.     

土壤铅胁迫对苘麻光合特性及生物量的影响

为明确不同浓度铅污染对苘麻(Abutilon theophrasti)光合特性及生长的影响程度,揭示苘麻对铅胁迫的光合适应能力,在铅污染土壤区域提供潜在物种。该研究设置五个铅浓度梯度(0、500、1 000、1 500、2 000 mg·kg~(-1)),分析了在不同浓度铅污染胁迫下,苘麻的铅积累、气体交换参数、叶绿素荧光特性及生物量的变化。结果表明:当铅浓度在1 000 mg·kg~(-1)以下时,净光合速率(P_n)和气孔导度(G_s)、光化学效率(F_v/F_m)、电子传递速率(ETR)的值分别与空白组的P_n、G_s、F_v/F_m、ETR值相比均不显著(P0.05),表明铅浓度小于1 000 mg·kg~(-1)时,苘麻的光合活性未受到损伤,有很强的光合适应能力。苘麻生物量在500、1 000 mg·kg~(-1)铅胁迫下为空白组的88.6%、106%,苘麻能够正常生长,说明苘麻能够适应铅污染的胁迫。当铅浓度低于1 000 mg·kg~(-1)时,苘麻地上部与地下部对铅富集与转移的效果较好,表明在铅污染区苘麻也具有较强的铅积累能力。根据铅污染土壤区域的实际情况,结合苘麻在低浓度铅污染修复效率高的特点,认为苘麻可以作为铅污染地区修复的潜在物种。     

Estimation of food composition of Hodotermes mossambicus (Isoptera: Hodotermitidae) based on observations and stable carbon isotope ratios

Abstract The diet of the harvester termite Hodotermes mossambicus was investigated at two sites with distinct dietary components: C₄ grasses (δ¹³C isotope values, ?13.8‰ to ?14.0‰) and C₃ plants (δ¹³C isotope values, ?25.6‰ to ?27.1‰). By comparing observations of food items carried into the colony by the termites and carbon isotope ratios of whole termites (that determined assimilated carbon), the relative proportion of the C₃ and C₄ plant food components of the termite diet was estimated. There was agreement between the observational data and stable carbon isotopic data, with grass representing approximately 93% of the diet of H. mossambicus at two study sites (urban and rural) on the South African highveld. However, when correcting for mass of food items, that is, C₃ and C₄, carried by termites, the proportion of grass (C₄) in the diet may be underestimated.     

Modeling of Pb(II) adsorption by a fixed-bed column

Removal of Pb(II) from an aqueous environment using biosorbents is a cost-effective and environmentally benign method. The biosorption process, however, is little understood for biosorbents prepared from plant materials. In this study, the biosorption process was investigated by evaluating four adsorption models. A fixed-bed column was prepared using a biosorbent prepared from the aquatic plant Hydrilla verticillata. The effect of bed height and flow rate on the biosorption process was investigated. The objective of the study was to determine the ability of H. verticillata to biosorb Pb(II) from an aqueous environment and to understand the process, through modeling, to provide a basis to develop a practical biosorbent column. Experimental breakthrough curves for biosorption of 50 mg L^?1 aqueous Pb(II) using a fixed-bed column with 1.00 cm inner diameter were fitted to the Thomas, Adams-Bohart, Belter, and bed depth service time (BDST) models to investigate the behavior of each model according to the adsorption system and thus understand the adsorption mechanism. Model parameters were evaluated using linear and nonlinear regression methods. The biosorbent removed 65% (82.39 mg g^?1 of biosorbent) of Pb(II) from an aqueous solution of Pb(NO₃)₂ at a flow rate of 5.0 ml min^?1 in a 10 cm column. Na₂CO₃ was used to recover the adsorbed Pb(II) ions as PbCO₃ from the biosorbent. The Pb(II) was completely desorbed at a bed height of 10.0 cm and a flow rate of 5.0 ml min^?1. Fourier transform infrared (FT-IR) analysis of the native biosorbent and Pb(II)-loaded biosorbent indicated that the hydroxyl groups and carboxylic acid groups were involved in the metal bonding process. The FT-IR spectrum of Pb(II)-desorbed biosorbent showed an intermediate peak shift, indicating that Pb(II) ions were replaced by Na⁺ ions through an ion-exchange process. Of the four models tested, the Thomas and BDST models showed good agreement with experimental data. The calculated bed sorption capacity N₀ and rate constant k_a were 31.7 g L^?1 and 13.6 × 10^?4 L mg^?1 min^?1 for the C_t/C₀ value of 0.02. The BDST model can be used to estimate the column parameters to design a large-scale column.     

Inhibition of gibberellin biosynthesis by paclobutrazol in cell-free homogenates ofCucurbita maxima endosperm andMalus pumila embryos

The plant growth retardant paclobutrazol, (PP333) (2RS, 3RS)-1-(4-chlorophenyl)-4,4-dimethyl-2-(1,2,4-triazol-1-yl)pentan-3-ol, inhibits specifically the three steps in the oxidation of the gibberellin-precursorent-kaurene toent-kaurenoic acid in a cell-free system fromCucurbita maxima endosperm. The KI₅₀ for this inhibition is 2×10^?8 M. The KI₅₀ values for the separated2S, 3S, and2R, 3R enantiomers of paclobutrazol in this system are 2×10^?8 M and 7×10^?7 M, respectively. A cell-free preparation from immatureMalus pumila embryos convertsent-kaurene to gibberellin A₉, whereas no conversion occurs in a similar preparation fromMalus endosperm. The conversion ofent-kaurene by the embryo preparation is inhibited by paclobutrazol with KI₅₀ values for the2S,3S and2R,3R enantiomers of 2×10^?8 M and 6×10^?8 M, respectively.