An evaluation of protein assays for quantitative determination of drugs

We have evaluated the response of six protein assays [the biuret, Lowry, bicinchoninic acid (BCA), Coomassie Brilliant Blue (CBB), Pyrogallol Red-Molybdate (PRM), and benzethonium chloride (BEC)] to 21 pharmaceutical drugs. The drugs evaluated were analgesics (acetaminophen, aspirin, codeine, methadone, morphine and pethidine), antibiotics (amoxicillin, ampicillin, gentamicin, neomycin, penicillin G and vancomycin), antipsychotics (chlorpromazine, fluphenazine, prochlorperazine, promazine and thioridazine) and water-soluble vitamins (ascorbic acid, niacinamide, pantothenic acid and pyridoxine). The biuret, Lowry and BCA assays responded strongly to most of the drugs tested. The PRM assay gave a sensitive response to the aminoglycoside antibiotics (gentamicin and neomycin) and the antipsychotic drugs. In contrast, the CBB assay showed little response to the aminoglycosides and gave a relatively poor response with the antipsychotics. The BEC assay did not respond significantly to the drugs tested. The response of the protein assays to the drugs was further evaluated by investigating the linearity of the response and the combined response of drug plus protein. The results are discussed with reference to drug interference in protein assays and the development of new methods for the quantification of drugs in protein-free solution.     

A technique for isolation of rubella virus-like particles by sucrose gradient ultracentrifugation using Coomassie brilliant blue G crystals

An improved method for the isolation of rubella virus-like particles (RVLP) from cell culture supernatant of transfected Chinese hamster ovary (CHO24S) cells is described. It employs a combination of membrane filtration with sucrose gradient ultracentrifugation. It was found that staining the RVLP band with Coomassie brilliant blue G (CBB) resulted in the CBB crystals adsorbing RVLP. After ultracentrifugation (25,000 rpm, 3h, 4 degrees C) a sharp blue band with crystals (diameter 30-40 microm) was observed (at a density of 1.250 g/ml at 25 degrees C) in a 30-60% sucrose gradient. Using a combination of SDS-PAGE and Western blotting techniques, E1 rubella virus structural protein was detected only in the solutions derived from the sharp blue band. A decrease in crystal concentration a few millimeters above or below the main band was associated with a decrease in protein concentration. By dilution with a saturated ice-cold 30% sucrose solution it was possible to pellet the crystals by centrifugation (15,000 rpm, 10 min). SDS-PAGE showed a much higher concentration of RVLP structural protein in the pellet than in the supernatant. This RVLP-containing material is especially suitable for the preparation of rubella virus immunoblot stripes.     

Precipitation of dilute chromatographic samples (ng/ml) containing interfering substances for SDS-PAGE

SDS-PAGE of chromatographic fractions requires prior removal of salts, detergents, denaturants, or organic solvents which may perturb the electrophoretic separation. Likewise, to successfully visualize minute amounts of protein present in chromatographic fractions, they must often be concentrated before analysis by SDS-PAGE. In this study, we used a dye precipitation procedure for simultaneous removal of interfering substances and concentration of dilute samples (ng/ml) before analysis by SDS-PAGE. Nanogram amounts of protein (143 ng) were effectively precipitated with a pyrogallol red-molybdate reagent from commonly used chromatographic buffers containing various interfering solutes or solvents. Proteins were successfully precipitated from solution in the presence of organic solvents (acetonitrile, methanol, 2-propanol), chaotropic agents (6 M urea, 6 M guanidine-HCl), a protein stabilizer (40% sucrose), metal chelators (30 mM EDTA and 30 mM EGTA), or high salt (1.0 M NaCl). Detergents, at concentrations up to twice their critical micelle concentrations, from the nonionic class (Triton X-100, Tween 20) or from the zwitterionic class (3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate) did not inhibit protein precipitation. Some interference was observed when proteins were precipitated in the presence of ammonium sulfate (0. 5-2.0 M). Proteins did not precipitate in the presence of ionic detergents (SDS and cetyltrimethylammonium bromide). The sensitivity of the combined pyrogallol red-molybdate precipitation/SDS-PAGE procedure is approximately 7 ng. Two other methods of precipitating proteins (trichloroacetic acid and phenol-ether) both exhibited varying degrees of effectiveness, ranging from 714 to 7 ng/ml, in the precipitation of individual proteins. In summary, the pyrogallol red-molybdate protein precipitation procedure facilitates the SDS-PAGE analysis of dilute protein samples (ng/ml) from chromatographic fractions of various compositions. The method is useful for rapid pilot-scale protein fractionation and facilitates the ongoing propensity of researchers to work with minuscule amounts of protein.     

Cystatin C as a potential cerebrospinal fluid marker for the diagnosis of Creutzfeldt-Jakob disease

The definite diagnosis of Creutzfeldt-Jakob disease (CJD), the most common form of human prion diseases, relies upon neuropathological data usually obtained at autopsy. In living patients, the diagnosis, based on suggestive clinical features and EEG abnormalities, can be aided by the detection of altered levels of isoforms of the 14-3-3 protein in the cerebrospinal fluid (CSF). However, the validity of this test has been recently challenged and the search for other, more reliable biomarkers for CJD remains highly desirable. The present study describes the identification of a new potential surrogate marker in the CSF of CJD-affected patients. A preliminary study employing surface-enhanced laser desorption/ionization-time of flight (SELDI-TOF) technology highlighted a protein at 13.4 kDa in a small group (n = 8) of CJD-affected patients. Further analysis aimed at identifying this protein using cationic exchange chromatography, sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), and liquid chromatography-tandem mass spectrometry (LC-MS/MS) revealed it to be cystatin C. Additional immunoblot assays confirmed that the level of cystatin C was significantly increased (p 相似文献   

DPHL:A DIA Pan-human Protein Mass Spectrometry Library for Robust Biomarker Discovery

To address the increasing need for detecting and validating protein biomarkers in clinical specimens, mass spectrometry (MS)-based targeted proteomic techniques, including the selected reaction monitoring (SRM), parallel reaction monitoring (PRM), and massively parallel data-independent acquisition (DIA), have been developed. For optimal performance, they require the fragment ion spectra of targeted peptides as prior knowledge. In this report, we describe a MS pipeline and spectral resource to support targeted proteomics studies for human tissue samples. To build the spectral resource, we integrated common open-source MS computational tools to assemble a freely accessible computational workflow based on Docker. We then applied the workflow to generate DPHL, a comprehensive DIA pan-human library, from 1096 data-dependent acquisition (DDA) MS raw files for 16 types of cancer samples. This extensive spectral resource was then applied to a proteomic study of 17 prostate cancer (PCa) patients. Thereafter, PRM validation was applied to a larger study of 57 PCa patients and the differential expression of three proteins in prostate tumor was validated. As a second application, the DPHL spectral resource was applied to a study consisting of plasma samples from 19 diffuse large B cell lymphoma (DLBCL) patients and 18 healthy control subjects. Differentially expressed proteins between DLBCL patients and healthy control subjects were detected by DIA-MS and confirmed by PRM. These data demonstrate that the DPHL supports DIA and PRM MS pipelines for robust protein biomarker discovery. DPHL is freely accessible at https://www.iprox.org/page/project.html?id=IPX0001400000.     

Four-dimensional proteomics analysis of human cerebrospinal fluid with trapped ion mobility spectrometry using PASEF

A quadrupole time-of-flight mass spectrometer coupled with a trapped ion mobility spectrometry (timsTOF) operated in parallel accumulation-serial fragmentation (PASEF) mode has recently emerged as a platform capable of providing four-dimensional (4D) features comprising of elution time, collision cross section (CCS), mass-to-charge ratio, and intensity of peptides. The PASEF mode provides ∼100% ion sampling efficiency both in data-dependent acquisition (DDA) and data-independent acquisition (DIA) modes without sacrificing sensitivity. In addition, targeted measurements using PASEF integrated parallel reaction monitoring (PRM) mode have also been described. However, only limited number of studies have used timsTOF for analysis of clinical samples. Although Orbitrap mass spectrometers have been used for biomarker discovery from cerebrospinal fluid (CSF) in a variety of neurological diseases, these Orbitrap-derived datasets cannot readily be applied for driving experiments on timsTOF mass spectrometers. We generated a catalog of peptides and proteins in human CSF in DDA mode on a timsTOF mass spectrometer and used these data to build a spectral library. This strategy allowed us to use elution times and ion mobility values from the spectral library to design PRM experiments for quantifying previously discovered biomarkers from CSF samples in Alzheimer's disease. When the same samples were analyzed using a DIA approach combined with a spectral library search, a higher number of proteins were identified than in a library-free approach. Overall, we have established a spectral library of CSF as a resource and demonstrated its utility for PRM and DIA studies, which should facilitate studies of neurological disorders.     

Infection of the Coffee Berry Borer Hypothenemus hampei (Coleoptera: Scolytidae) by Brazilian Isolates of the Entomopathogenic Fungi Beauveria bassiana and Metarhizium anisopliae (Deuteromycotina: Hyphomycetes)

The virulence of four fungal isolates (three Beauveria bassiana and one Metarhizium anisopliae ) against adult female coffee berry borers (CBB) was investigated. The most virulent isolate from initial bioassays, B. bassiana LPP1, with a LT 50 of 3.4 days, was further investigated by application to berries prior to infestation and to berries already infested with CBB. At the highest concentration applied to berries (1 ×10 7 conidia mL -1 ), CBB mortality was 83% (berries inoculated prior to infestation) and 62% (berries inoculated after infestation).     

水稻可溶性糖蛋白的纯化与鉴定研究

从水稻鲜叶中提取总蛋白,对总蛋白中的蛋白质含量进行了测定;通过硫酸铵沉淀将总蛋白提取液进行分级,从而达到了总蛋白细分和放量的目的。四级份的分级蛋白分别通过ConA-Sepharose 4B 亲和层析进行糖蛋白纯化,按吸收峰收集的各级糖蛋白混合物进行冷冻干燥,得到干粉;结合PAS法染色和考马斯亮蓝R-250染色对四级份的糖蛋白样品鉴定,在其中3个级份中均检测出糖蛋白;由于感度的差异,按考马斯亮蓝R-250染色可检测出近30种糖蛋白(包括部分糖肽),按PAS法染色可检测到7种糖蛋白;对3种含量较高的糖蛋白进行了胶上纯化,3种糖蛋白的PAS法染色均证实了3种样品为单一的糖蛋白或者糖肽,分别命名为RG1、RG2和RG3。     

In-depth analysis of the human CSF proteome using protein prefractionation

The identification of disease markers in human body fluids requires an extensive and thorough analysis of its protein constituents. In the present study, we have extended our analysis of the human cerebrospinal fluid (CSF) proteome using protein prefractional followed by shotgun mass spectrometry. After the removal of abundant protein components from the mixture with the help of immunodepletion affinity chromatography, we used either anion exchange chromatography or sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) to further subfractionate the proteins present in CSFs. Each protein subfraction was enzyme digested and analyzed by tandem mass spectrometry and the resulting data evaluated using the Spectrum Mill software. Different subfractionation methods resulted in the identification of a grant total of 259 proteins in CSF from a patient with normal pressure hydrocephalus. The greatest number of protein, 240 in total, were identified after prefractionating the CSF proteins by immunodepletion and SDS-PAGE. Immuno-depletion combined with anion exchange fractionation resulted in 112 proteins and 74 proteins were found when only immunodepletion of the CSF samples was carried out. All methods used showed a significant increase in the number of identified proteins as compared with nondepleted and unfractionated CSF sample analysis, which yielded only 38 protein identifications. The present work establishes a platform for future studies aimed at a detailed comparative proteome analysis of CSFs from different groups of patients suffering from various psychiatric and neurological disorders.     

Characterization of Novel CSF Tau and ptau Biomarkers for Alzheimer’s Disease

Cerebral spinal fluid (CSF) Aβ42, tau and p181tau are widely accepted biomarkers of Alzheimer’s disease (AD). Numerous studies show that CSF tau and p181tau levels are elevated in mild-to-moderate AD compared to age-matched controls. In addition, these increases might predict preclinical AD in cognitively normal elderly. Despite their importance as biomarkers, the molecular nature of CSF tau and ptau is not known. In the current study, reverse-phase high performance liquid chromatography was used to enrich and concentrate tau prior to western-blot analysis. Multiple N-terminal and mid-domain fragments of tau were detected in pooled CSF with apparent sizes ranging from <20 kDa to ~40 kDa. The pattern of tau fragments in AD and control samples were similar. In contrast, full-length tau and C-terminal-containing fragments were not detected. To quantify levels, five tau ELISAs and three ptau ELISAs were developed to detect different overlapping regions of the protein. The discriminatory potential of each assay was determined using 20 AD and 20 age-matched control CSF samples. Of the tau ELISAs, the two assays specific for tau containing N-terminal sequences, amino acids 9-198 (numbering based on tau 441) and 9-163, exhibited the most significant differences between AD and control samples. In contrast, CSF tau was not detected with an ELISA specific for a more C-terminal region (amino acids 159-335). Significant discrimination was also observed with ptau assays measuring amino acids 159-p181 and 159-p231. Interestingly, the discriminatory potential of p181 was reduced when measured in the context of tau species containing amino acids 9-p181. Taken together, these results demonstrate that tau in CSF occurs as a series of fragments and that discrimination of AD from control is dependent on the subset of tau species measured. These assays provide novel tools to investigate CSF tau and ptau as biomarkers for other neurodegenerative diseases.     

In vivo influences on cerebrospinal fluid amino acid levels

The concentration of 19 amino acids and ethanolamine in two independent groups (n = 36, n = 19) of normal human cerebrospinal fluid (CSF) samples were measured by high performance liquid chromatography. Age, sex, time of lumbar puncture, position during lumbar puncture, protein and glucose content of the CSF were monitored and the influence of these parameters on CSF amino acid levels was determined. Hypotheses formulated after observing measurements from the first group of CSF samples were tested against the second group of CSF samples using conservative statistics. The main finding was a positive correlation between CSF glucose and CSF glutamate levels.     

Identification of brain cell death associated proteins in human post-mortem cerebrospinal fluid

Following any form of brain insult, proteins are released from damaged tissues into the cerebrospinal fluid (CSF). This body fluid is therefore an ideal sample to use in the search for biomarkers of neurodegenerative disorders and brain damage. In this study, we used human post-mortem CSF as a model of massive brain injury and cell death for the identification of such protein markers. Pooled post-mortem CSF samples were analyzed using a protocol that combined immunoaffinity depletion of abundant CSF proteins, off-gel electrophoresis, SDS-PAGE and protein identification by LC-MS/MS. A total of 299 proteins were identified, of which 172 proteins were not previously described to be present in CSF. Of these 172 proteins, more than 75% have been described as intracellular proteins suggesting that they were released from damaged cells. Immunoblots of a number of proteins were performed on individual post-mortem CSF samples and confirmed elevated concentrations in post-mortem CSF compared to ante-mortem CSF. Interestingly, among the proteins specifically identified in the post-mortem CSF, several have been previously described as biochemical markers of brain damage.     

Prion-Seeding Activity in Cerebrospinal Fluid of Deer with Chronic Wasting Disease

Transmissible spongiform encephalopathies (TSEs), or prion diseases, are a uniformly fatal family of neurodegenerative diseases in mammals that includes chronic wasting disease (CWD) of cervids. The early and ante-mortem identification of TSE-infected individuals using conventional western blotting or immunohistochemistry (IHC) has proven difficult, as the levels of infectious prions in readily obtainable samples, including blood and bodily fluids, are typically beyond the limits of detection. The development of amplification-based seeding assays has been instrumental in the detection of low levels of infectious prions in clinical samples. In the present study, we evaluated the cerebrospinal fluid (CSF) of CWD-exposed (n=44) and naïve (n=4) deer (n=48 total) for CWD prions (PrP^d) using two amplification assays: serial protein misfolding cyclic amplification with polytetrafluoroethylene beads (sPMCAb) and real-time quaking induced conversion (RT-QuIC) employing a truncated Syrian hamster recombinant protein substrate. Samples were evaluated blindly in parallel with appropriate positive and negative controls. Results from amplification assays were compared to one another and to obex immunohistochemistry, and were correlated to available clinical histories including CWD inoculum source (e.g. saliva, blood), genotype, survival period, and duration of clinical signs. We found that both sPMCAb and RT-QuIC were capable of amplifying CWD prions from cervid CSF, and results correlated well with one another. Prion seeding activity in either assay was observed in approximately 50% of deer with PrP^d detected by IHC in the obex region of the brain. Important predictors of amplification included duration of clinical signs and time of first tonsil biopsy positive results, and ultimately the levels of PrP^d identified in the obex by IHC. Based on our findings, we expect that both sPMCAb and RT-QuIC may prove to be useful detection assays for the detection of prions in CSF.     

低毒高效的SDS-PAGE考马斯亮蓝染色方法比较

目的:比较SDS-PAGE的4种考马斯亮蓝染色方法。方法:以牛血清白蛋白为材料进行SDS-PAGE,分别比较考马斯亮蓝G-250(CBB G-250)盐酸法、CBB G-250-Al2(SO4)3法、Bio-Rad公司的Bio-Safe染色液及传统法等4种染色方法的灵敏度和操作性,并将上述4种染色方法应用于蛋白Markers检测。结果:CBB G-250-Al2(SO4)3法和Bio-Safe法的检测灵敏度都可达19.2 ng,而CBB G-250盐酸法和传统法的检测灵敏度则为28.9 ng。结论:CBB G-250盐酸法可作为快速、低毒、高效的染色方法,CBB-Al2(SO4)3法则可用于灵敏度要求较高的检测。     

Measurement of 150 kDa protein of Taenia solium metacestodes by antibody-sandwich ELISA in cerebrospinal fluid of neurocysticercosis patients.

An antigenic protein in cystic fluid of Taenia solium metacestodes (CF) of 150 kDa was measured by antibody-sandwich ELISA in serum and cerebrospinal fluid (CSF) of neurocysticercosis patients. Capture antibodies were rabbit antisera against CF (RACF) and a monoclonal antibody (MAb) against 150 kDa protein in CF. Lower limit of antibody-sandwich ELISA was 8 ng/ml of the protein. Except CF, no tested helminths extracts reacted. Levels of the protein in 351 sera from 255 patients (55 surgery confirmed and 202 antibody and CT/MRI confirmed) were below sensitivity of the assay. Of 276 CSF from 212 patients, 31 samples (11.2%) showed positive findings. This assay, therefore, was not sensitive enough to be a diagnostic. Instead, the 150 kDa protein appeared in CSF in such situations as in 2 days after praziquantel treatment, or as in a patient infected with a racemose cysticercus with degenerated cyst wall. Of cases whose follow-up CSF were assayed, 2 cases showed that the protein appeared intermittently. These results suggest strongly that appearance of free 150 kDa protein is associated with cyst wall rupture. In CSF which contained the 150 kDa protein over 61 ng/ml, the protein was recognized in SDS-PAGE before and after immunoprecipitation.     

Persistence of Perchlorate and the Relative Numbers of Perchlorate- and Chlorate-Respiring Microorganisms in Natural Waters, Soils, and Wastewater

Cell numbers of perchlorate (PRM)- and chlorate (CRM)-reducing microorganisms and the persistence of perchlorate were determined in samples of soils, natural waters, and wastewater incubated under laboratory conditions. Complete perchlorate reduction in raw wastewater and creek water was achieved in 4 to 7 days and 8 to 29 days, respectively, depending on the individual growth substrate (acetate, lactate, citric acid, or molasses) employed. Perchlorate persisted in most mixed cultures developed with 2 g of “pristine” soil, but declined in mixed cultures developed with 100 g of soil. Less than seven days were required to completely reduce perchlorate in cultures started with 10 g of a perchlorate-contaminated soil obtained from a site in Texas. The concentration of PRM was estimated using a 5-tube most probable number (MPN) procedure. To account for discrepancies due to differences in the total number of bacteria (per mass of sample) in the samples, difficulty in removing bacteria from soil samples, and the lack of an unequivocal method to measure total viable cells in these different systems, we normalized our MPN results on the basis of 10⁶ or 10⁹ total bacteria counted using acridine orange direct counts (AODC). There were more PRM in wastewater samples on a per-cell basis (15 to 350 PRM/10⁶-AODC) than in water samples (0.02 to 0.4 PRM/10⁶-AODC). There were also more PRM in soils from sites exhibiting direct evidence of perchlorate contamination (100 to 200 PRM/10⁹-AODC) than from other sites (nondetectable to 0.77 PRM/10⁹-AODC). These results demonstrate that perchlorate-reducing bacteria are present at perchlorate-contaminated sites, and that perchlorate can be degraded by these microorganisms through the addition of different electron donors, such as acetate and lactate.     

Analysis of soluble interleukin 6 receptor in cerebrospinal fluid in inflammatory and non-inflammatory conditions

The objective of this study was to investigate the pathophysiological roles of soluble interleukin 6 receptor (sIL-6R) in cerebrospinal fluid (CSF). CSF was obtained from patients suspected with meningitis. Eight patients without any meningeal signs or symptoms were enrolled as controls. An additional 34 CSF samples were collected to measure both biologically active and immunoreactive sIL-6R. All CSF samples were proven to be aseptic. IL-6 and sIL-6R were measured using specific ELISAs. Patients were divided into three groups on the basis of cell number in CSF; inflammatory group (cell number >5 microl, mean 241+/-363.1, n=61); non-inflammatory group (cell number < or =5 microl, mean=2.1+/-1.7, n=12) and controls (cell number < or =5 microl, mean=0.3+1.7, n=8). Among these three groups, the differences in protein (F (2,78)=8.274, P<0.0001) and IL-6 concentration (F (2,78)=6.475, P<0.001) were statistically significant but those of sIL-6R concentration were not. There were only weak correlations between log (sIL-6R) versus log (cell number) (r=0.23, P=0.0375), log (protein) (r=0.239, P=0.0358) and log (IL-6) (r=0.27, P=0.0167). Amounts of immunoreactive and biologically active sIL-6R were closely correlated (r=0.62, n=34, P<0.005). It was concluded that sIL-6R is present constitutively in CSF and its level may not increase significantly in inflammatory conditions; infiltrating cells in CSF are not the main source of sIL-6R; and sIL-6R in CSF can bind IL-6.     

Isolation and characterization of human urinary colony-stimulating factor

CSF-1 was isolated from a large volume of human normal urine (10,000 l), using the following 5 stages of purification: concentration by dialysis, silica gel adsorption, hydrophobic chromatography on phenyl-Sepharose CL-6B, fast protein liquid chromatography (FPLC) and finally preparative electrophoresis on polyacrylamide gels. We have isolated 8 mg of purified CSF-1 which migrated as a single band under non-reducing conditions in SDS-PAGE (staining with Coomassie Blue and the sensitive silver techniques). But in the presence of dithiothreitol, the SDS-PAGE pattern revealed a minor second band with a molecular mass of 50,000 Da. CSF-1 was purified 100,000-fold and has a specific activity of 2.16 X 10(7) units/mg protein. Its apparent molecular mass is 57,000 Da with an isoelectric point, pI = 5.8-6.0. The amino-acid composition is reported and compared with that of murine CSF-1. The carbohydrate content (sialic acid, sulphate groups, N-acetylglucosamine, N-acetylgalactosamine) was also determined, and it shows that CSF is a glycoprotein.     

Detection of GDNF secretion in glial cell culture and from transformed cell implants in the brains of live animals

Current ex vivo gene therapy for Parkinson's disease using glial cell line-derived neurotrophic factor (GDNF) is limited by the lack of a monitoring mechanism to determine the expression of GDNF once the cells or other vehicles are transferred into animal models. The purpose of this study was to test whether a Renilla luciferase (RUC)-GDNF fusion protein secreted by the genetically engineered glial cell line RG-1 could be measured photometrically in cerebrospinal fluid (CSF). RG-1 was constructed by permanent transformation with a plasmid DNA construct that contains a GDNF cDNA (gdnf) fused to a RUC cDNA (ruc). The fusion protein secreted by RG-1 was shown to retain both GDNF and RUC activity. The concentration of GDNF determined by enzyme-linked immunoadsorbent assay (ELISA) was correlated with the light emission detected by assaying for RUC bioluminescence in RG-1 culture medium, indicating that RUC can be used as a reporter for GDNF in vitro. The cells were then implanted into rat brain (n=20), and the cisternal CSF was analyzed. Bioluminescence was successfully detected in the CSF samples, and was quantified over a period of 25 days, while Western blotting and ELISA failed to detect GDNF in CSF, presumably because the concentration of the RUC-GDNF fusion was too low. This study demonstrates that the transformed glial cell line RG-1 offers a sensitive self-reporting assay for GDNF expression.