Finding Species-Specific Extracellular Surface-Facing Proteomes in Toxic Dinoflagellates.

As a sequel to our previous report of the existence of species-specific protein/peptide expression profiles (PEPs) acquired by mass spectrometry in some dinoflagellates, we established, with the help of a plasma-membrane-impermeable labeling agent, a surface amphiesmal protein extraction method (SAPE) to label and capture species-specific surface proteins (SSSPs) as well as saxitoxins-producing-species-specific surface proteins (Stx-SSPs) that face the extracellular space (i.e., SSSPsEf and Stx-SSPsEf). Five selected toxic dinoflagellates, Alexandrium minutum, A. lusitanicum, A. tamarense, Gymnodinium catenatum, and Karenia mikimotoi, were used in this study. Transcriptomic databases of these five species were also constructed. With the aid of liquid chromatography linked-tandem mass spectrometry (LC-MS/MS) and the transcriptomic databases of these species, extracellularly facing membrane proteomes of the five different species were identified. Within these proteomes, 16 extracellular-facing and functionally significant transport proteins were found. Furthermore, 10 SSSPs and 6 Stx-SSPs were identified as amphiesmal proteins but not facing outward to the extracellular environment. We also found SSSPsEf and Stx-SSPsEf in the proteomes. The potential functional correlation of these proteins towards the production of saxitoxins in dinoflagellates and the degree of species specificity were discussed accordingly.

Production of Paralytic Shellfish Toxins (PSTs) in Toxic Alexandrium catenella is Intertwined with Photosynthesis and Energy Production.

To investigate the mechanism for the production of paralytic shellfish toxins (PST) in toxic dinoflagellates, with a 2D-gel based approach, we had made two sets of proteomic comparisons: (a) between a toxic Alexandrium catenella (AC-T) and a phylogenetically closely related non-toxic strain (AC-N), (b) between toxic AC-T grown in a medium with 10% normal amount of phosphate (AC-T-10%P) known to induce higher toxicity and AC-T grown in normal medium. We found that photosynthesis and energy production related proteins were up-regulated in AC-T when compared to AC-N. However, the same group of proteins was down-regulated in AC-T-10%P when compared to normal AC-T. Examining the relationship of photosynthesis and toxin content of AC-T upon continuous photoperiod experiment revealed that while growth and associated toxin content increased after 8 days of continuous light, toxin content maintained constant when cells were shifted from continuous light to continuous dark for 3 days. This emphasized the cruciality of light availability on toxin biosynthesis in AC-T, while another light-independent mechanism may be responsible for higher toxicity in AC-T-10%P compared to normal AC-T. Taken all together, it is believed that the interplay between "illumination", "photosynthesis", "phosphate availability", and "toxin production" is much more complicated than what we had previously anticipated.

Chronic Methylmercury Exposure Induces Production of Prostaglandins: Evidence From A Population Study and A Rat Dosing Experiment.

Methylmercury (MeHg) is a well-known environmental neurotoxicant affecting millions worldwide who consume contaminated fishes and other food commodities. Exposure to MeHg has been shown to associate positively with some chronic diseases including cardiovascular diseases, but the mechanism is poorly characterized. MeHg had been shown to affect prostaglandin (PG) regulations in in vitro studies, but neither in vivo nor human studies investigating the effects of MeHg on PG regulations has been reported. Thus, the current study aimed to investigate the association between MeHg exposure and serum PG concentrations in a cross-sectional study among human adults followed by a validation investigation on the cause-effect relationship using a rat model. First, a total of 121 women were recruited from two cities: Wanshan and Leishan in Guizhou, China. Statistical analysis of the human data showed a positive association between blood total mercury (THg) levels and serum concentrations of PGF2α, 15-deoxy-PGJ2, and PGE2 after adjusting for site effects. In the animal study, adult female Sprague-Dawley rats were dosed with 40 µg MeHg/kg body weight/day for 12 weeks. Serum 15-deoxy-PGJ2 and 2,3 d-6-keto-PGF1α concentrations were found to increase significantly after 6 and 10 weeks of MeHg dosing, respectively, while serum PGF2α concentration increased significantly after 12 weeks of MeHg dosing. Combined results of our human and rat studies have shown that chronic MeHg exposure induced dysregulation of PG metabolism. As PGs are a set of mediators with very diverse functions, its abnormal production may serve as the missing mechanistic link between chronic MeHg exposure and various kinds of associated clinical conditions including neurodegeneration and cardiovascular diseases.

Identification of peptides released from hot water insoluble fraction of edible bird's nest under simulated gastro-intestinal conditions.

For centuries, edible bird's nest (EBN) has been consumed as a Chinese delicacy. In the past decades, numerous studies reported that water soluble extract of the EBN not only possessed epidermal growth factor, but also associated with a wide range of health-promoting effects. However, based on the traditional Chinese way of EBN preparation and consumption, the bioactive components should be originated from both its hot water soluble and insoluble fractions. Nevertheless, information on the hot water insoluble fraction (HWIF) of EBN is not currently available. In this study, peptides released from the HWIF of EBN under simulated gastro-intestinal conditions were identified for the first time by de novo sequencing using a combination of MALDI TOF/TOF MS and ESI-ion-trap MS/MS. The released peptides were found to share very high similarities to mucin, NADH dehydrogenase, acidic mammalian chitinase-like protein, immunoglobulin, proline-rich protein, von Willebrand factor and epidermal growth factor domain-containing protein.

Chronic exposure of adult rats to low doses of methylmercury induced a state of metabolic deficit in the somatosensory cortex.

Because of the ever-increasing bioaccumulation of methylmercury (MeHg) in the marine food chain, human consumers are exposed to low doses of MeHg continually through seafood consumption. Epidemiological studies strongly suggest that chronic prenatal exposure to nanomolar of MeHg has immense negative impacts on neurological development in neonates. However, effects of chronic exposure to low doses (CELDs) of MeHg in adult brains on a molecular level are unknown. The current study aims to investigate the molecular effects of CELD of MeHg on adult somatosensory cortex in a rat model using proteomic techniques. Young adult rats were fed with a low dose of MeHg (40 µg/kg body weight/day) for a maximum of 12 weeks. Whole proteome expression of the somatosensory cortex (S1 area) of normal rats and those with CELD to MeHg were compared. Levels of MeHg, total calcium, adenosine triphosphate (ATP), and pyruvate were also measured. Comparative proteomic studies of the somatosensory cortexes revealed that 94 proteins involved in the various metabolic processes (including carbohydrate metabolism, generation of precursors for essential metabolites, energy, proteins, cellular components for morphogenesis, and neurotransmission) were down-regulated. Consequently, levels of important end products of active metabolism including ATP, pyruvate, and total calcium were also found to be significantly reduced concomitantly. Our results showed that CELD of MeHg induced a state of metabolic deficit in the somatosensory cortex of adult rats.

Daily changes in the phosphoproteome of the dinoflagellate Lingulodinium.

The dinoflagellate Lingulodinium has a large number of daily rhythms, many of which have no biochemical correlates. We examined the possibility that changes in protein phosphorylation may mediate some of the rhythmic changes by comparing proteins prepared from midday (LD6) and midnight (LD18) cultures. We used two different methods, one a 2D gel protocol in which phosphoproteins were identified after staining with ProQ Diamond, and the other an LC-MS/MS identification of tryptic phosphopeptides that had been purified by TiO(2) chromatography. Two differentially phosphorylated proteins, a light harvesting complex protein and Rad24, were identified using the 2D gel protocol. Six differentially phosphorylated proteins, a polyketide synthase, an uncharacterized transporter, a LIM (actin binding) domain and three RNA binding domain proteins, were identified using the phosphopeptide enrichment protocol. We conclude that changes in protein phosphorylation may underlie some of the rhythmic behavior of Lingulodinium.

Mercury species of sediment and fish in freshwater fish ponds around the Pearl River Delta, PR China: human health risk assessment.

This study investigated total mercury (THg) and methylmercury (MeHg) concentrations in five species of freshwater fish and their associated fish pond sediments collected from 18 freshwater fish ponds around the Pearl River Delta (PRD). The concentrations of THg and MeHg in fish pond surface sediments were 33.1-386 ng g(-1) dry wt and 0.18-1.25 ng g(-1) dry wt, respectively. The age of ponds affected the surface sediment MeHg concentration. The vertical distribution of MeHg in sediment cores showed that MeHg concentrations decreased with increasing depth in the top 10 cm. In addition, a significant correlation was observed between %MeHg and DNA from Desulfovibrionacaea or Desulfobulbus (p<0.05) in sediment cores. Concentrations of THg and MeHg in fish muscles ranged from 7.43-76.7 to 5.93-76.1 ng g(-1) wet wt, respectively, with significant linear relationships (r=0.97, p<0.01, n=122) observed between THg and MeHg levels in fish. A significant correlation between THg concentrations in fish (herbivorous: r=0.71, p<0.05, n=7; carnivorous: r=0.77, p<0.05, n=11) and corresponding sediments was also obtained. Risk assessment indicated that the consumption of largemouth bass and mandarin fish would result in higher estimated daily intakes (EDIs) of MeHg than reference dose (RfD) for both adults and children.

Identification of two plastid proteins in the dinoflagellate Alexandrium affine that are substantially down-regulated by nitrogen-depletion.

The formation of harmful algal blooms (HABs) by dinoflagellates has been correlated with the nitrogen load in coastal waters. Nitrogen is implicated as an important factor in the initiation and maintenance of phytoplankton blooms. To characterize the cellular response to nitrogen, 2DE was used to compare protein expressions from dinoflagellates grown under nitrogen depleted and nitrogen replete conditions. A total of 17 differentially expressed protein spots were found, nine of which showed a roughly 16-fold decrease in N-depleted conditions. Five of these nine spots were all identified as isoforms of the plastid Form II ribulose-1,5 bisphosphate carboxylase/oxygenase (Rubisco II), while an additional four protein spots with a molecular weight of 50 kDa were identified as isoforms of a novel protein named nitrogen-associated protein 50 (NAP50). NAP50 was located in the plastids as shown by the presence of an N-terminal plastid targeting leader sequence and by immunohistochemistry. Levels of both Rubisco II and NAP50 decrease sharply between 24 and 36 h following nitrogen depletion and the decrease can be blocked if the N source is replenished before degradation occurs. Both proteins are rapidly resynthesized if the nitrogen source is replenished after degradation has occurred. These results are a first step in the dissection of the behavior of the dinoflagellate proteome under nitrogen stress conditions and may provide new insights into the relationship between dinoflagellate blooms and the nitrogen budget.

Pain Controlling and Cytokine-regulating Effects of Lyprinol, a Lipid Extract of Perna Canaliculus, in a Rat Adjuvant-induced Arthritis Model.

Using an adjuvant-induced arthritis rat model, we investigated the effects of a lipid extract of Perna canaliculus (Lyprinol(R)) on pain. Radiological examinations, as well as levels of pro- and anti-inflammatory (AI) cytokines, were measured aiming to provide independent objective data to the pain controlling investigation. We confirmed the ability of Lyprinol(R) to control pain at the initial phase of its administration; with similar efficacy to that observed with Naproxen. The pain scores slowly increased again in the group of rats treated with Lyprinol(R) after day 9-14. The Naproxen-treated rats remained pain-free while treated. Both Naproxen and Lyprinol(R) decreased the levels of the pro-inflammatory cytokines TNF-alpha and IFN-gamma, and increased that of IL-10. Extra-virgin olive oil was ineffective on cytokine secretion. Rats treated with Lyprinol(R) were apparently cured after 1 year. This study confirms the AI efficacy of this lipid extract of P. canaliculus, its initial analgesic effect, its perfect tolerance and its long-term healing properties.

Detecting nitrated proteins by proteomic technologies.

Nitration is a posttranslational modification of tyrosine residues of proteins mediated by peroxynitrite (ONOO(-)). It commonly occurs in neurological and pathological disorders, which involve nitric oxide (NO)-mediated oxidative stress. Nitration of tyrosine or tyrosyl groups of a protein modulates protein function and initiates signal transduction pathways, which lead to alternation of cellular metabolism and functions. Because of its apparent significance, there is an increasing urge to identify nitrated proteins as a bridge to expand our understanding of their involvement in different biological processes. This chapter describes strategies that could be used for rapid screening and detection of nitrated proteins, subsequent resolution, and identification of nitrated proteins and peptides using proteomic technologies. These include two-dimensional gel electrophoresis coupled with Western blotting and matrix-assisted laser desorption/ionization time of flight mass spectrometry, as well as liquid chromatography-linked tandem mass spectrometry.

The use of Trizol reagent (phenol/guanidine isothiocyanate) for producing high quality two-dimensional gel electrophoretograms (2-DE) of dinoflagellates.

Two-dimensional gel electrophoresis (2-DE) is one of the most efficient ways of resolving complex protein mixtures based on the isoelectric point (pI) and molecular mass (M(r)). Although it has been used extensively in proteomic studies on samples from the animal and plant kingdoms, there is limited information on its use on algae, such as dinoflagellates. The preparation of high-quality samples from dinoflagellate cells for 2-DE is difficult due to high endogenous levels of salts, nucleic acids, polysaccharides, phenolic compounds, pigments, and other interfering compounds. Desalting and concentrating steps are usually required for the preparation of dinoflagellate protein sample prior to 2-DE and these steps can be lengthy and complicated. In this study, we report the use of Trizol (a monophasic solution of phenol and guanidine isothiocyanate) for the extraction of proteins from dinoflagellate cells for 2-DE. The method is simple and fast. 2-DE profiles obtained with Trizol treatment are of very high quality in terms of resolution, spot number and spot intensity. This method greatly simplifies protein extraction procedures on dinoflagellate samples for obtaining a high quality and reproductive 2-DE profile. This methodology is generally applicable to algal samples.

Discovery of diagnostic serum biomarkers of gastric cancer using proteomics.

Gastric cancer has significant morbidity and mortality worldwide and locally. Good prognosis relies on an early diagnosis. However, this remains a challenge due to the lack of specific and sensitive serum biomarkers for early detection. Hence, there is a constant search for these biomarkers for screening purposes. Proteomic profiling enables a new approach to the discovery of biomarkers in disease. This review presents recent attempts in search of gastric cancer serum biomarker using proteomics. Different methodologies and different types of samples were employed by different groups of researchers. Major difficulties were encountered in the discovery processes, including interference from abundant proteins and continuous changing serum proteomes from different individuals.

Sodium nitroprusside affects the level of photosynthetic enzymes and glucose metabolism in Phaseolus aureus (mung bean).

Nitric oxide (NO) is an important signaling molecule in plants. The present study aims to investigate the downstream signaling pathways of NO in plants using a proteomic approach. Phaseolus aureus (mung bean) leaf was treated with sodium nitroprusside (SNP), which releases nitric oxide in the form of nitrosonium cation (NO+) upon light irradiation. Changes in protein expression profiles of the SNP treated mung bean leaf were analyzed by two-dimensional gel electrophoresis (2-DE). Comparison of 2-DE electropherograms revealed seven down-regulated and two up-regulated proteins after treatment with 0.5 mM SNP for 6 h. The identities of these proteins were analyzed by a combination of peptide mass fingerprinting and post-source decay using a matrix-assisted-laser-desorption-ionisation-time-of-flight (MALDI-TOF) mass spectrometer. Six out of these nine proteins found are involved in either photosynthesis or cellular metabolism. We have taken our investigation further by studying the effect of NO+ on glucose contents in mung bean leaves. Our results clearly demonstrated that NO+ rapidly and drastically decrease the amount of glucose in mung bean leaves. Moreover, four out of nine of these proteins are chloroplastic isoforms. These results suggested that chloroplasts might be one of the main sub-cellular targets of NO in plants.

Use of two-dimensional gel electrophoresis to differentiate morphospecies of Alexandrium minutum, a paralytic shellfish poisoning toxin-producing dinoflagellate of harmful algal blooms.

Contamination of shellfish with paralytic shellfish poisoning toxins (PST) produced by toxic harmful algal blooms (HABs) have been negatively affecting the shellfish and aquaculture industries worldwide. Therefore, accurate and early identification of toxic phytoplankton species is crucial in HABs surveillance programs that allow fish-farmers to take appropriate preventive measures in shellfish harvesting and other aquaculture activities to overcome the negative impacts of HABs on human health. The identification of toxic dinoflagellates present in the water is currently a time-consuming operation since it requires skillful taxonomists and toxicologists equipped with optical and scanning electron microscopes as well as sophisticated equipment, for example, high-performance liquid chromotography-fluorescence detection. In this paper, a two-dimensional gel electrophoresis (2-DE)-based proteomic approach was applied to discriminate between toxic and nontoxic strains of Alexandrium minutum. Variation in morphological features between toxic and nontoxic strains was minimal and not significant. Also, variation in 2-DE protein patterns within either toxic or nontoxic strains was low, but pronounced differences were detected between toxic and nontoxic strains. The most notable differences between these strains were several abundant proteins with pIs ranging from 4.8 to 5.3 and apparent molecular masses between 17.5 and 21.5 kDa. Groups of proteins, namely NT1, NT2, NT3, and NT4, were consistently found in all nontoxic strains, while T1 and T2 were prominent in the toxic strains. These specific protein spots characteristic for toxic and nontoxic strains remained clearly distinguishable irrespective of the various growth conditions tested. Therefore, they have the potential to serve as "taxonomic markers" to distinguish toxic and nontoxic strains within A. minutum. Initial studies revealed that the expression pattern of T1 was tightly correlated to toxin biosynthesis in the examined alga and may be used to serve as a potential toxin indicator.

Identification of the heterogeneous nuclear ribonucleoprotein A2/B1 as the antigen for the gastrointestinal cancer specific monoclonal antibody MG7.

MG7 is an early gastrointestinal cancer specific monoclonal antibody. It can detect gastric cancer with high sensitivity and specificity. However, the target antigen for MG7 has not been identified. Western blot analysis revealed that the MG7 antibody reproducibly recognized two approximately 35 kDa proteins in the total cell lysates of human gastric carcinoma cell lines KATO III and MKN-45. Using a proteomic approach, we identified these MG7 immunoreactive proteins as the human heterogeneous nuclear ribonucleoprotein A2/B1 (hnRNP A2/B1). Western blot analysis of nuclear and cytosolic fraction of KATO III cells using either MG7 or hnRNP A2/B1 antibodies confirmed that the target antigen is located exclusively in the nucleus. With the use of archival samples, we also found that the level of hnRNP A2/B1 protein was increased in gastric cancer tissues (4 out of 5 patients), when compared to their corresponding matching normal stomach tissue.

Proteomic study of a model causative agent of harmful algal blooms, Prorocentrum triestinum II: the use of differentially expressed protein profiles under different growth phases and growth conditions for bloom prediction.

Simultaneous comparison of differentially expressed protein profiles of Prorocentrum triestinum grown under different growth phases and growth conditions indicated the presence of phase-specific and stress-responsive proteins, respectively. Correlation studies on these proteins in relation to cell division phasing patterns and to models of phytoplankton growth inferred the possible functions. Most notable among these proteins were groups of proteins thought to trigger or mediate cells through specific phases of division of this alga, e.g., BP1, BP2, PB1, PB2, and PB3. Other proteins (e.g., group 1 proteins) thought to be responsible for maintaining and supporting cell concentration under adverse conditions were found. Furthermore, another group of proteins (group 2 proteins) thought to be stress-responsive were also detected. Taken overall, these differentially expressed proteins provided important information for uncovering various protective and adaptive mechanisms in the dinoflagellate's life cycle. These proteins have the potential to serve as "indicator proteins" for rapid assessment of the nutritional or metabolic status of these phytoplankton cells,and monitoring the differential expression of these phase-specific proteins and stress-specific proteins could be an important biomarker for bloom prediction.

Use of two-dimensional gel electrophoresis proteome reference maps of dinoflagellates for species recognition of causative agents of harmful algal blooms.

The sample preparation procedures established for Prorocentrum triestinum were adapted to cover both thecate and athecate dinoflagellates. Further, whether trichloroacetic acid (TCA) precipitation can be used to fix and preserve the harmful or nuisance species from local waters that they infest was tested. Optimized technical procedures developed were used to generate proteome reference maps for eight other local causative species of harmful algal blooms (HABs): Prorocentrum micans, Prorocentrum minimum, Prorocentrum sigmoides, Prorocentrum dentatum, Scrippsiella trochoidea, Karenia longicanalis, Karenia digitata and Karenia mikimotoi; together with one American species Karenia brevis (Florida, USA). These proteome maps were used to test their ability for species recognition in a mixed culture of dinoflagellates and whether such investigations will provide a comparative view at a global level. Comparisons of proteome profiles were made (i). between closely related species within the same family; (ii). between distantly related species belonging to different types, i.e., gymnodinioids, prorocentroids or peridinioids, or (iii). between different groups, i.e., thecate (armored) dinoflagellate cells against athecate (naked or unarmored) dinoflagellate cells. Species-specific two-dimensional electrophoresis (2-DE) protein profiles were observed in all ten species and it was possible to distinguish between even closely related species within the same family. To demonstrate the extent of reproducibility and usefulness of these 2-DE reference maps, 2-DE has been used to analyze three geographically distinct isolates of Prorocentrum dentatum, and to distinguish species composition in a mixed culture. Application of 2-D PAGE analysis to differentiate between taxonomically confused strains of a single species could be a powerful taxonomic tool.

Adaptive responses of 25-hydroxyvitamin D3 1-alpha hydroxylase expression to dietary phosphate restriction in young and adult rats.

Regulation of vitamin D metabolism alters with age. The present study is undertaken to investigate if the loss of renal 1,25-dihydroxyvitamin D(3) (1,25(OH)(2)D(3)) production in response to dietary phosphate (P) restriction in adult rats is due to an alteration in the renal expression of 25-hydroxyvitamin D(3) 1-alpha hydroxylase (1-OHase). Young (4-6 weeks old) and adult (12-14 weeks old) male Sprague Dawley rats were fed either normal P (NPD) or low P diet (LPD) for 0-5 days. Basal expression of 1-OHase protein was higher in adult rats. Young rats, but not adult rats, significantly increased 1-OHase protein and mRNA expressions in response to LPD in a time-dependent manner. To determine if the stability of renal 1-OHase protein changes with LPD feeding, young and adult rats fed either NPD or LPD for 5 days were injected intravenously with cycloheximide (CHX), a protein synthesis inhibitor. CHX decreased 1-OHase protein expression in young rats fed NPD. However, CHX did not alter 1-OHase protein expression in young rats fed LPD nor in adult rats fed either diet. The results indicate that the stability of renal 1-OHase protein increased with age and that LPD increased its stability only in young rats.

"Two-in-one" gel for spot matching after two-dimensional electrophoresis.

Two-dimensional electrophoresis (2-DE) is one of the most commonly used techniques in proteomic investigations. However, due to the complex interplay of incidence including significant biological sample variations, lengthy steps involved in performing 2-DE as well as exposure time with silver staining, it is sometimes difficult to differentiate authentic differences caused by drug treatment with those artifacts caused by sample variations, running conditions of 2-DE as well as treatment time in silver staining etc. If we can compare pooled samples of control and treatment groups run in a single gel and stained together, we would be more comfortable with our findings. We propose here a low cost and highly effective method for locating differentially expressed proteins before and after drug treatment. This "two-in-one gel" technique might partially solve the problems mentioned above.

Proteomic identification of plant proteins probed by mammalian nitric oxide synthase antibodies.

Several studies suggest that a mammalian-like nitric oxide synthase (NOS) exists in plants. Researchers have attempted to verify its presence using two approaches: (i) determination of NOS functional activity and (ii) probing with mammalian NOS antibodies. However, up to now, neither a NOS-like gene nor a protein has been found in plants. While there is still some controversy over whether the NOS functional activity seen is due to nitrate reductase, using the mammalian NOS antibodies in western blot analysis, several groups have reported the presence of immunoreactive protein bands in plant homogenates. Based on these results, immunohistochemical studies using these antibodies have also been used to localize NOS in plant tissues. However, plant NOS has never been positively identified or characterized. Thus, we used a proteomic approach to verify the identities of plant proteins that cross-reacted with the mammalian NOS antibodies. Proteins extracted from maize (Zea mays L.) embryonic axes were separated by two-dimensional gel electrophoresis and subjected to western blot analysis with the mammalian neuronal NOS and inducible NOS antibodies. Twenty immunoreactive protein spots recognized on a corresponding Coomassie blue-stained two-dimensional gel were subjected to tryptic digestion, followed by identification using matrix-assisted laser desorption/ionization-time of flight mass spectrometry. Fifteen proteins were successfully identified and they have described functions that are unrelated to NO metabolism. The remaining five proteins could not be identified. The amino acid sequences of these identified proteins and those used to raise the antibodies were aligned. However, no homologous region could be found. Our results demonstrate that the mammalian NOS antibodies recognize many NOS-unrelated plant proteins. Therefore, it is inappropriate to infer the presence of plant NOS using this immunological technique.