Novel copy number variations and phenotypes of infantile epileptic spasms syndrome.

We summarize the copy number variations (CNVs) and phenotype spectrum of infantile epileptic spasms syndrome (IESS) in a Chinese cohort. The CNVs were identified by genomic copy number variation sequencing. The CNVs and clinical data were analyzed. 74 IESS children with CNVs were enrolled. 35 kinds of CNVs were identified. There were 11 deletions and 5 duplications not reported previously in IESS, including 2 CNVs not reported in epilepsy. 87.8% were de novo, 9.5% were inherited from mother and 2.7% from father. Mosaicism occurred in one patient with Xq21.31q25 duplication. 16.2% (12/74) were 1p36 deletion, and 20.3% (15/74) were 15q11-q13 duplication. The age of seizure onset ranged from 17 days to 24 months. Seizure types included epileptic spasms, focal seizures, tonic seizures, and myoclonic seizures. All patients displayed developmental delay. Additional features included craniofacial anomaly, microcephaly, congenital heart defects, and hemangioma. 29.7% of patients were seizure-free for more than 12 months, and 70.3% still had seizures after trying 2 or more anti-seizure medications. In conclusion, CNVs is a prominent etiology of IESS. 1p36 deletion and 15q duplication occurred most frequently. CNV detection should be performed in patients with IESS of unknown causes, especially in children with craniofacial anomalies and microcephaly.

Red light-upregulated MPK11 negatively regulates red light-induced stomatal opening in Arabidopsis.

Stomatal movements allow the uptake of CO2 for photosynthesis and water loss through transpiration, therefore play a crucial role in determining water use efficiency. Both red and blue lights induce stomatal opening, and the stomatal apertures under light are finetuned by both positive and negative regulators in guard cells. However, the molecular mechanisms for precisely adjusting stomatal apertures under light have not been completely understood. Here, we provided evidence supporting that Arabidopsis thaliana mitogen-activated protein kinase 11 (MPK11) plays a negative role in red light-induced stomatal opening. First, MPK11 was found to be highly expressed in guard cells, and MPK11-GFP signals were detected in both nuclear and cytoplasm of guard cells. The transcript levels of MPK11 in guard cells were upregulated by white light, and the stomata of mpk11 opened wider than that of wild type under white light. Consistent with the larger stomatal aperture, mpk11 mutant exhibited higher stomatal conductance and CO2 assimilation rate under white light. The transcript levels of the genes responsible for osmolytes increases were higher in guard cells of mpk11 than that of wild type, which may contribute to the larger stomatal aperture of mpk11 under white light. Furthermore, MPK11 transcript levels in guard cells were upregulated by red light, and mpk11 mutant showed a larger stomatal aperture under red light. Taken together, these results demonstrate that red light-upregulated MPK11 plays a negative role in stomatal opening, which finetuning the stomatal opening apertures and preventing excessive water loss by transpiration under light.

DYNC1H1-related epilepsy: Genotype-phenotype correlation.

AIM: To explore the phenotypic spectrum and refine the genotype-phenotype correlation of DYNC1H1-related epilepsy. METHOD: The clinical data of 15 patients with epilepsy in our cohort and 50 patients with epilepsy from 24 published studies with the DYNC1H1 variants were evaluated. RESULTS: In our cohort, 13 variants were identified from 15 patients (seven males, eight females). Twelve variants were de novo and seven were new. Age at seizure onset ranged from 3 months to 4 years 5 months (median age 1 year). Common seizure types were epileptic spasms, focal seizures, tonic seizures, and myoclonic seizures. Mild-to-severe developmental delay was present in all patients. Six patients were diagnosed with West syndrome and one was diagnosed with epileptic encephalopathy with continuous spikes and waves during slow sleep (CSWS). Collectively, in our cohort and published studies, 17% had ophthalmic diseases, 31% of variants were located in the stalk domain, and 92% patients with epilepsy had a malformation of cortical development (MCD). INTERPRETATION: The phenotypes of DYNC1H1-related epilepsy included multiple seizure types; the most common epileptic syndrome was West syndrome. CSWS is a new phenotype of DYNC1H1-related epilepsy. One-third of the variants in patients with epilepsy were located in the stalk domain. Most patients had a MCD and developmental delay. WHAT THIS PAPER ADDS: Nearly 40% of patients with DYNC1H1 variants had epilepsy. Ninety-two percent of patients with DYNC1H1-related epilepsy had malformation of cortical development. More than 10% of patients with DYNC1H1-related epilepsy were diagnosed with West syndrome. Continuous spikes and waves during slow sleep could be a new phenotype of DYNC1H1 variants. One-third of the variants in patients with epilepsy were located in the stalk domain.

Review of the influencing factors of secondary organic aerosol formation and aging mechanism based on photochemical smog chamber simulation methods.

The formation and aging mechanism of secondary organic aerosol (SOA) and its influencing factors have attracted increasing attention in recent years because of their effects on climate change, atmospheric quality and human health. However, there are still large errors between air quality model simulation results and field observations. The currently undetected components during the formation and aging of SOA due to the limitation of current monitoring techniques and the interactions among multiple SOA formation influencing factors might be the main reasons for the differences. In this paper, we present a detailed review of the complex dynamic physical and chemical processes and the corresponding influencing factors involved in SOA formation and aging. And all these results were mainly based the studies of photochemical smog chamber simulation. Although the properties of precursor volatile organic compounds (VOCs), oxidants (such as OH radicals), and atmospheric environmental factors (such as NOx, SO2, NH3, light intensity, temperature, humidity and seed aerosols) jointly influence the products and yield of SOA, the nucleation and vapor pressure of these products were found to be the most fundamental aspects when interpreting the dynamics of the SOA formation and aging process. The development of techniques for measuring intermediate species in SOA generation processes and the study of SOA generation and aging mechanism in complex systems should be important topics of future SOA research.

Data quality evaluation and calibration of on-road remote sensing systems based on exhaust plumes.

In recent years, with rapid increases in the number of vehicles in China, the contribution of vehicle exhaust emissions to air pollution has become increasingly prominent. To achieve the precise control of emissions, on-road remote sensing (RS) technology has been developed and applied for law enforcement and supervision. However, data quality is still an existing issue affecting the development and application of RS. In this study, the RS data from a cross-road RS system used at a single site (from 2012 to 2015) were collected, the data screening process was reviewed, the issues with data quality were summarized, a new method of data screening and calibration was proposed, and the effectiveness of the improved data quality control methods was finally evaluated. The results showed that this method reduces the skewness and kurtosis of the data distribution by up to nearly 67%, which restores the actual characteristics of exhaust diffusion and is conducive to the identification of actual clean and high-emission vehicles. The annual variability of emission factors of nitric oxide decreases by 60% - on average - eliminating the annual drift of fleet emissions and improving data reliability.

Developing China's roadmap for air quality improvement: A review on technology development and future prospects.

Air pollution control policies in China have been experiencing profound changes, highlighting a strategic transformation from total pollutant emission control to air quality improvement, along with the shifting targets starting from acid rain and NOx emissions to PM2.5 pollution, and then the emerging O3 challenges. The marvelous achievements have been made with the dramatic decrease of SO2 emission and fundamental improvement of PM2.5 concentration. Despite these achievements, China has proposed Beautiful China target through 2035 and the goal of 2030 carbon peak and 2060 carbon neutrality, which impose stricter requirements on air quality and synergistic mitigation with Greenhouse Gas (GHG) emissions. Against this background, an integrated multi-objective and multi-benefit roadmap is required to provide decision support for China's long-term air quality improvement strategy. This paper systematically reviews the technical system for developing the air quality improvement roadmap, which was integrated from the research output of China's National Key R&D Program for Research on Atmospheric Pollution Factors and Control Technologies (hereafter Special NKP), covering mid- and long-term air quality target setting techniques, quantitative analysis techniques for emission reduction targets corresponding to air quality targets, and pathway optimization techniques for realizing reduction targets. The experience and lessons derived from the reviews have implications for the reformation of China's air quality improvement roadmap in facing challenges of synergistic mitigation of PM2.5 and O3, and the coupling with climate change mitigation.

Comprehensive evaluation method of urban air quality statistics based on environmental monitoring data and its application.

Air quality monitoring is effective for timely understanding of the current air quality status of a region or city. Currently, the huge volume of environmental monitoring data, which has reasonable real-time performance, provides strong support for in-depth analysis of air pollution characteristics and causes. However, in the era of big data, to meet current demands for fine management of the atmospheric environment, it is important to explore the characteristics and causes of air pollution from multiple aspects for comprehensive and scientific evaluation of air quality. This study reviewed and summarized air quality evaluation methods on the basis of environmental monitoring data statistics during the 13th Five-Year Plan period, and evaluated the level of air pollution in the Beijing-Tianjin-Hebei region and its surrounding areas (i.e., the "2+26" region) during the period of the three-year action plan to fight air pollution. We suggest that air quality should be comprehensively, deeply, and scientifically evaluated from the aspects of air pollution characteristics, causes, and influences of meteorological conditions and anthropogenic emissions. It is also suggested that a three-year moving average be introduced as one of the evaluation indexes of long-term change of pollutants. Additionally, both temporal and spatial differences should be considered when removing confounding meteorological factors.

Prospect of China's ambient air quality standards.

Ambient air quality standards are the core strategic goal of ambient air quality management. Countries worldwide have given importance to research on the development of ambient air quality standards. To understand the history of the development of China's ambient air quality standards, this study analyzed the background associated with all previous formulations and revisions of the standards, classification of functional areas, standard grading, pollutants, and evolution of the standard limits over the past 40 years. The results show that since the initial release of the "Ambient Air Quality Standard" by China in 1982, it has been supplemented once, revised twice, and modified twice. The first ambient air quality standard specified the standard limits of six pollutants commonly found in ambient air. With the development of ambient air quality management, the number of pollutants has increased to ten. Since the release of the "Ambient Air Quality Standards" in 2012, the ambient air quality in China has significantly improved. However, the proportion of cities meeting these standards is still low. At present, China is suitably positioned to make the standards associated with 24 hr SO2 concentrations more stringent such that it meets the values defined in the World Health Organization (WHO) interim target-2 and the WHO air quality guideline (AQG). We further suggested that the SO2 standard should be revised promptly. Simultaneously, regions with a relatively high proportion of cities meeting the standard are encouraged to introduce more stringent interim target limits in due course to manage the local ambient air quality.

Evaluating cost and benefit of air pollution control policies in China: A systematic review.

China has put great efforts into air pollution control over the past years and recently committed to its most ambitious climate target. Cost and benefit analysis has been widely used to evaluate the control policies in terms of past performance, future reduction potential, and direct and indirect impacts. To understand the cost and benefit analysis for air pollution control in China, we conducted a bibliometric review of more than 100 studies published over the past two decades, including the current research progress, most commonly adopted methods, and core findings. The control target in cost and benefit analysis has shifted in three stages, from individual and primary pollution control, moving to joint prevention of multiple and secondary pollutants, and then towards synergistic control of air pollution and carbon. With the expansion of the research scope, the integrated assessment model has gradually demonstrated the necessity for long-term ex-anti policy simulation, especially for dealing with complex factors. To ensure long-term air quality, climate, public health, and sustainable economic development, substantial evidence from published studies has suggested that China needs to continue its efforts in the upstream adjustment of the energy system and industrial structure with multi-regional and -sector collaboration. This cost and benefit review paper provides decision-makers with the fundamental information and knowledge gaps in air pollution control strategies in China, and direction for facing future challenges.

Genotype-phenotype correlation of CACNA1A variants in children with epilepsy.

AIM: To explore the genotypes and phenotypes of CACNA1A variants in children with epilepsy. METHOD: Eighteen children (six males, 12 females) with CACNA1A variants were identified using next-generation sequencing. RESULTS: There were 14 missense variants, two nonsense variants, one frameshift variant, and one splice site variant. Sixteen variants were de novo. Age at seizure onset ranged from 1 day to 8 years; median age was 8 months. Multiple seizure types were observed, including focal, generalized tonic-clonic, myoclonic, and absence seizures, as well as epileptic spasms and tonic seizures. Focal motor status epilepticus occurred in 10 individuals and generalized motor status epilepticus occurred in two individuals. All 18 children showed developmental delay. Focal motor status epilepticus resulted in cerebral atrophy in five individuals, mainly on the contralateral side. Interictal electroencephalogram showed focal discharges in 12 individuals, whereas five individuals had generalized discharges. Three individuals were seizure-free, whereas 15 still had seizures and five had recurrent status epilepticus at last follow-up. INTERPRETATION: Most children with epilepsy and CACNA1A variants had early seizure onset and developmental delay. Focal seizure was the most common seizure type. Most patients experienced status epilepticus. Unilateral cerebral atrophy could occur after focal motor status epilepticus. Patients with CACNA1A variants located in the transmembrane region may be at high risk of status epilepticus.

Altering microbial community for improving soil properties and agricultural sustainability during a 10-year maize-green manure intercropping in Northwest China.

Maize is a crop that is cultivated worldwide. The Hexi Oasis is one of the most important areas for high-yield maize seed production in China. Green manure, a plant fertilizer, has great potential for increasing crop yield and agricultural sustainability. However, the role of microorganisms in soil health and the microbiological mechanism of green manure in improving soil fertility and crop production in the Hexi Oasis area remain unknown. The effects of maize-green manure intercropping on the soil microbial community structure and diversity and the mechanism of soil improvement were investigated in a 10-year field experiment. The study revealed that microbial phylotypes were grouped into four major ecological clusters. Module #2 is a soil core ecological cluster enriched with many plant growth-promoting rhizobacteria and arbuscular mycorrhizal fungi. The application of green manure led to significantly increased soil pH, nutrient contents, and enzyme activities, and significantly reduced the relative abundance of potential plant pathogens compared with monocropping, which should ensure high and stable maize yield under long-term continuous cropping. It also increased the economic benefits by 56.39% compared with monocropping, owing to the additional products produced by the green manure. These improvements were associated with changes in the microbial community structure and activity, consistent with the structural equation model results. Therefore, soil microorganisms are the key drivers of the potential benefits of maize-green manure on agricultural sustainability.

Modeling of spatial and temporal variations of ozone-NOx-VOC sensitivity based on photochemical indicators in China.

Comprehensive air quality model with extensions (CAMx)-decoupled direct method (DDM) was used to simulate ozone-NOx-VOCs sensitivity of for May-November in 2016-2018 in China. Based on the relationship between the simulated ozone (O3) sensitivity values and the ratio of formaldehyde (HCHO) to NO2 (FNR) and the ratio of production rate of hydrogen peroxide (H2O2) to production rate of nitric acid (HNO3) ( [Formula: see text] ), the localized range of FNR and [Formula: see text] thresholds in different regions in China were obtained. The overall simulated FNR values are about 1.640-2.520, and [Formula: see text] values are about 0.540-0.830 for the transition regime. Model simulated O3 sensitivities or region specific FNR or [Formula: see text] thresholds should be applied to ensure the accurate local O3 sensitivity regimes. Using the tropospheric column FNR values from ozone monitoring instrument (OMI) satellite data as an indicator with the simulated threshold values, the spatial distributions of O3 formation regimes in China are determined. The O3 sensitivity regimes from eastern to central China are gradually from VOC-limited, transition to NOx-limited spatially, and moving toward to transition or NOx-limited regime from 2005 to 2019 temporally.

Transmission characteristics of SMN from 227 spinal muscular atrophy core families in China.

To define the relationship between the survival motor neuron 1 gene (SMN1) and SMN2, and explore the variability of these two genes within the generations, SMN1 and SMN2 copy numbers were determined for 227 SMA families. The association analysis indicated that there was a negative correlation between the copy number of SMN1 and SMN2 (Spearman = -0.472, P < 0.001) in 227 SMA children and 454 of their parents. The average SMN copies from father and mother in each SMA family were used to represent the copy number in the parent's generation. Subsequently, SMN transmission analysis showed that the similar distribution trend of SMN1 and SMN2 copy number was not only in the SMA children and their parents' generation but also in the non-SMA families. Moreover, when the SMN2 copy number was one in the parent's generation, 75% of their SMA children had type I and 25% of them had type II/III. However, when the SMN2 copies were three in the parent's generation, all of their SMA children were type II/III. Therefore, the diversity of SMN copies was mostly inherited and the SMN2 copy number in the parent's generation could predict the disease severity of SMA children to some extent.

Salinity decreases Cd translocation by altering Cd speciation in the halophytic Cd-accumulator Carpobrotus rossii.

Background and Aims: Salt has been shown to affect Cd translocation and accumulation in plants but the associated mechanisms are unclear. This study examined the effects of salt type and concentration on Cd uptake, translocation and accumulation in Carpobrotus rossii. Methods: Plants were grown in nutrient solution with the same Cd concentration or Cd2+ activity in the presence of 25 mm NaNO3, 12.5 mm Na2SO4 or 25 mm NaCl for ≤10 d. Plant growth and Cd uptake were measured and the accumulation of peptides and organic acids, and Cd speciation in plant tissues were analysed. Key Results: Salt addition decreased shoot Cd accumulation by >50 % due to decreased root-to-shoot translocation, irrespective of salt type. Synchrotron-based X-ray absorption spectroscopy revealed that, after 10 d, 61-94 % Cd was bound to S-containing ligands (Cd-S) in both roots and shoots, but its speciation was not affected by salt. In contrast, Cd in the xylem sap was present either as free Cd2+ or complexes with carboxyl groups (Cd-OH). When plants were exposed to Cd for ≤24 h, 70 % of the Cd in the roots was present as Cd-OH rather than Cd-S. However, NaCl addition decreased the proportion of Cd-OH in the roots within 24 h by forming Cd-Cl complexes and increasing the proportion of Cd-S. This increase in Cd-S complexes by salt was not due to changes in glutathione and phytochelatin synthesis. Conclusions: Salt addition decreased shoot Cd accumulation by decreasing Cd root-to-shoot translocation due to the rapid formation of Cd-S complexes (low mobility) within the root, without changing the concentrations of glutathione and phytochelatins.

Absorption of foliar-applied Zn in sunflower (Helianthus annuus): importance of the cuticle, stomata and trichomes.

Background and Aims: The pathways whereby foliar-applied nutrients move across the leaf surface remain unclear. The aim of the present study was to examine the pathways by which foliar-applied Zn moves across the sunflower (Helianthus annuus) leaf surface, considering the potential importance of the cuticle, stomata and trichomes. Methods: Using synchrotron-based X-ray florescence microscopy and nanoscale secondary ion mass spectrometry (NanoSIMS), the absorption of foliar-applied ZnSO4 and nano-ZnO were studied in sunflower. The speciation of Zn was also examined using synchrotron-based X-ray absorption spectroscopy. Key Results: Non-glandular trichomes (NGTs) were particularly important for foliar Zn absorption, with Zn preferentially accumulating within trichomes in ≤15 min. The cuticle was also found to have a role, with Zn appearing to move across the cuticle before accumulating in the walls of the epidermal cells. After 6 h, the total Zn that accumulated in the NGTs was approx. 1.9 times higher than in the cuticular tissues. No marked accumulation of Zn was found within the stomatal cavity, probably indicating a limited contribution of the stomatal pathway. Once absorbed, the Zn accumulated in the walls of the epidermal and the vascular cells, and trichome bases of both leaf sides, with the bundle sheath extensions that connected to the trichomes seemingly facilitating this translocation. Finally, the absorption of nano-ZnO was substantially lower than for ZnSO4, with Zn probably moving across the leaf surface as soluble Zn rather than nanoparticles. Conclusions: In sunflower, both the trichomes and cuticle appear to be important for foliar Zn absorption.

Manganese distribution and speciation help to explain the effects of silicate and phosphate on manganese toxicity in four crop species.

Soil acidity and waterlogging increase manganese (Mn) in leaf tissues to potentially toxic concentrations, an effect reportedly alleviated by increased silicon (Si) and phosphorus (P) supply. Effects of Si and P on Mn toxicity were studied in four plant species using synchrotron-based micro X-ray fluorescence (µ-XRF) and nanoscale secondary ion mass spectrometry (NanoSIMS) to determine Mn distribution in leaf tissues and using synchrotron-based X-ray absorption spectroscopy (XAS) to measure Mn speciation in leaves, stems and roots. A concentration of 30 µM Mn in solution was toxic to cowpea and soybean, with 400 µM Mn toxic to sunflower but not white lupin. Unexpectedly, µ-XRF analysis revealed that 1.4 mM Si in solution decreased Mn toxicity symptoms through increased Mn localization in leaf tissues. NanoSIMS showed Mn and Si co-localized in the apoplast of soybean epidermal cells and basal cells of sunflower trichomes. Concomitantly, added Si decreased oxidation of Mn(II) to Mn(III) and Mn(IV). An increase from 5 to 50 µM P in solution changed some Mn toxicity symptoms but had little effect on Mn distribution or speciation. We conclude that Si increases localized apoplastic sorption of Mn in cowpea, soybean and sunflower leaves thereby decreasing free Mn2+ accumulation in the apoplast or cytoplasm.

Absorption of foliar-applied Zn fertilizers by trichomes in soybean and tomato.

The present study investigated the role of trichomes in absorption of foliar-applied zinc fertilizers in soybean and tomato. Using synchrotron-based X-ray fluorescence microscopy for in situ analyses of hydrated leaves, we found that upon foliar application of ZnSO4, Zn accumulated within 15 min in some non-glandular trichomes in soybean, but not in tomato. However, analyses of cross-sections of soybean leaves did not show any marked accumulation of Zn in tissues surrounding trichomes. Furthermore, when near-isogenic lines of soybean differing 10-fold in trichome density were used to compare Zn absorption, it was found that foliar Zn absorption was not related to trichome density. Therefore, it is suggested that trichomes are not part of the primary pathway through which foliar-applied Zn moves across the leaf surface in soybean and tomato. However, this does not preclude trichomes being important in other plant species, as they are known to be highly diverse. We also compared the absorption of Zn when supplied as either ZnSO4, nano-ZnO, or bulk-ZnO, and found that absorption from ZnSO4 was about 10-fold higher than from nano- and bulk-ZnO, suggesting that it was mainly absorbed as soluble Zn. This study improves our understanding of the absorption of foliar-applied nutrients.

Sodium chloride decreases cadmium accumulation and changes the response of metabolites to cadmium stress in the halophyte Carpobrotus rossii.

Background and Aims: Salinity affects the bioavailability of cadmium (Cd) in soils and Cd accumulation in plants, but the associated mechanisms remain unclear. This study aimed to assess the metabolic response to NaCl and Cd and the relationship between metabolites and Cd accumulation in the halophyte Carpobrotus rossii, which has potential for Cd phytoextraction. Methods: Plants were grown in nutrient solution with 0-400 mm NaCl in the presence of 5 or 15 µm Cd, with varied or constant solution Cd2+ activity. Plant growth and Cd uptake were measured, and the accumulation of peptides, and organic and amino acids in plant tissues were assessed. Key Results: The addition of NaCl to Cd-containing solutions improved plant growth along with 70-87 % less shoot Cd accumulation, resulting from decreases in Cd root uptake and root-to-shoot translocation irrespective of Cd2+ activity in solutions. Moreover, Cd exposure increased the concentration of phytochelatins, which correlated positively with Cd concentrations in plants regardless of NaCl addition. In comparison, Cd inhibited the synthesis of organic acids in shoots and roots in the absence of NaCl, but increased it in shoots in the presence of NaCl. While Cd increased the concentrations of amino acids in plant shoots, the effect of NaCl on the synthesis of amino acids was inconsistent. Conclusions: Our data provide the first evidence that NaCl decreased Cd shoot accumulation in C. rossii by decreasing Cd root uptake and root-to-shoot translocation even under constant Cd2+ activity. The present study also supports the important role of peptides and organic acids, particular of phytochelatins, in Cd tolerance and accumulation although the changes of those metabolites was not the main reason for the decreased Cd accumulation.

Ambient volatile organic compounds pollution in China.

Owing to rapid economic and industrial development, China has been suffering from degraded air quality and visibility. Volatile organic compounds (VOCs) are important precursors to the formation of ground-level ozone and hence photochemical smog. Some VOCs adversely affect human health. Therefore, VOCs have recently elicited public concern and given new impetus to scientific interest. China is now implementing a series of polices to control VOCs pollution. The key to formulating policy is understanding the ambient VOCs pollution status. This paper mainly analyzes the species, levels, sources, and spatial distributions of VOCs in ambient air. The results show that the concentrations of ambient VOCs in China are much higher than those of developed countries such as the United States and Japan, especial benzene, which exceeds available standards. At the same time, the ozone formation potential (OFP) and secondary organic aerosol formation potential (SOAFP) of various VOCs are calculated. Aromatics and alkenes have much higher OFPs, while aromatics have higher SOAFP. The OFPs of ambient VOCs in the cities of Beijing, Guangzhou and Changchun are very high, and the SOAFP of ambient VOCs in the cities of Hangzhou, Guangzhou and Changchun are higher.