Gastrodin alleviates the deterioration of depressive-like behavior and glucolipid metabolism promoted by chronic stress in type 2 diabetic mice.

The growing burden of psychological stress among diabetes patients has contributed to a rising incidence of depression within this population. It is of significant importance to conduct research on the impact of stress on diabetes patients and to explore potential pharmacological interventions to counteract the stress-induced exacerbation of their condition. Gastrodin is a low molecular weight bioactive compound extracted from the rhizome of Gastrodiae elata Blume, and it may be a preventive strategy for diabetes and a novel treatment for depression symptoms. However, its relevant pharmacological mechanisms for protecting against the impacts of psychological stress in diabetic patients are unclear. In this study, we performed 5 weeks CUMS intervention and simultaneously administered gastrodin (140 mg/kg, once daily) on T2DM mice, to investigate the potential protective effects of gastrodin. The protective effect of gastrodin was evaluated by behavioral tests, biochemical analysis, histopathological examination, RT-qPCR and gut microbiota analysis. We found that the depressive-like behavior and glucolipid metabolism could be deteriorated by chronic stress in type 2 diabetic mice, while gastrodin showed a protective effect against these exacerbations by regulating HPA hormones, activating FXR and Cyp7a1, reducing inflammatory and oxidative stress responses, and regulating ileal gut microbiota abundance. Gastrodin might be a potential therapeutic agent for mitigating the deterioration of diabetes conditions due to chronic stress.

Peptide- and Protein-Level Combined Strategy for Analyzing Newly Synthesized Proteins by Integrating Tandem Orthogonal Proteolysis with Cleavable Bioorthogonal Tagging.

A newly synthesized proteome reflects perturbations sensitively and maintains homeostasis in cells. To investigate the low abundant newly synthesized proteins (NSPs) from a complex background proteome, an enrichment process with high selectivity and reliability is essential. Here, we have developed a strategy to realize comprehensive analysis of NSPs by integrating tandem orthogonal proteolysis (TOP) with cleavable bioorthogonal tagging (CBOT) called TOP-CBOT. A solid-phase-conjugated probe with a clickable moiety and a protease-cleavable site was designed, which allowed NSPs to be covalently captured along with tandem release by trypsin and orthogonal tobacco etch virus (TEV) protease. Our method has integrated the advantages of protein-level and peptide-level enrichment. Trypsin digests larger number of peptides from the recovered proteins for NSPs identification and quantification, while the specific tag-contained peptides from TEV data set enabled further NSPs confirmation. Integrating information from two complementary data sets, the reliability in NSPs identification and quantitation were remarkably enhanced. A total of 3699 proteins were recovered in the trypsin data set. Additionally, 1931 proteins were confirmed as NSPs with 5019 identified peptides in the TEV data set, over 90% of which were overlapped with the tryptic data set. Our strategy was further applied to profile NSP degradation kinetics during rapamycin-induced macroautophagy. The newly synthesized proteome displayed varied alteration of degradation rates among stimulation and more than half of NSPs showed decreased half-lives during autophagy.

Enhancing Comprehensive Analysis of Newly Synthesized Proteins Based on Cleavable Bioorthogonal Tagging.

Protein synthesis and degradation responding to environmental cues is critical for understanding the mechanisms involved. Chemical proteomics introducing bioorthogonal tagging into proteins and isolation by biotin affinity purification is applicable for enrichment of newly synthesized proteins (NSPs). Current enrichment methods based on biotin-streptavidin interaction lack efficiency to release enriched NSPs under mild conditions. Here we designed a novel method for enriching newly synthesized peptides by click chemistry followed by release of enriched peptides via tryptic digestion based on cleavable bioorthogonal tagging (CBOT). CBOT-modified peptides can further enhance identification in mass spectrometry analysis and provide a confirmation by small mass shift. Our method achieved an improvement in specificity (97.1%) and sensitivity for NSPs in cell lysate, corresponding to profiling at a depth of 4335 NSPs from 2 mg of starting materials in a single LC-MS/MS run. In addition, the CBOT strategy can quantify NSPs when coupling a pair of isotope-labeled azidohomoalanine (AHA/hAHA) with decent reproducibility. Furthermore, we applied it to analyze newly synthesized proteomes in the autophagy process after 6 h rapamycin stimulation in cells, 2910 NSPs were quantified, and 337 NSPs among them were significantly up- and down-regulated. We envision CBOT as an effective and alternative approach for bioorthogonal chemical proteomics to study stimuli-sensitive subsets.

Genomic characterization of Wenzhou mammarenavirus detected in wild rodents in Guangzhou City, China.

Wenzhou mammarenavirus (WENV) is a zoonotic pathogen newly discovered in east and southeast Asia. WENV has been found in wild rodent animals around the world while its standing is barely understood in Guangzhou city, where is known as a region of outbreak hotspot for zoonotic emerging infectious diseases. To investigate the prevalence and genomic characteristics of mammarenavirus in Guangzhou City, lung tissue samples from wild rodent species were collected from five districts of Guangzhou City in the year 2015 and 2016. The viral RNA was extracted and then subjected to mammarenavirus-specific PCR. The result revealed approximately 1.0% (3/306) nucleic acid positivity for lung tissue samples obtained from three rodent species: Mus musculus, Rattus flavipectus, and Rattus norvegicus. Viral metagenomic sequencing of three samples was then carried out and two full segment L and three full segment S sequences were obtained. Phylogenetics analysis indicated the sequences of the new mammarenavirus strain have 76.2% - 94.4% similarity to known WENV encoded genes, with the highest similarity to the WENV 9-24 strain. Population structure analysis grouped all known WENV into seven lineages, and this WENV Guangzhou strain was grouped with WENV 9-24 as well. Though the seroprevalence result was not available, our data provides the first nucleic acid evidence of circulating WENV in Guangzhou city, and it suggested WENV had a broader host tropism than previously known.

Specific Analysis of α-2,3-Sialylated N-Glycan Linkage Isomers by Microchip Capillary Electrophoresis-Mass Spectrometry.

Sialylated N-glycan isomers with α-2,3 and α-2,6 linkages play crucial and distinctive roles in diverse physiological and pathological processes. Changes of α-2,3-linked sialic acids in sialylated N-glycans are especially important in monitoring the initiation and progression of diseases. However, the specific analysis of α-2,3-sialylated N-glycan linkage isomers remains challenging due to their extremely low abundance and technical limitations in separation and detection. Herein, we designed an integrated strategy that combines linkage-specific derivatization and a charge-sensitive separation method based on microfluidic chip capillary electrophoresis-mass spectrometry (microchip CE-MS) for specific analysis of α-2,3-sialylated N-glycan linkage isomers for the first time. The α-2,6- and α-2,3-sialic acids were selectively labeled with methylamine (MA) and N,N-dimethylethylenediamine (DMEN), respectively, which selectively makes α-2,3-sialylated N-glycans positively charged and realizes online purification, concentration, and discrimination of α-2,3-sialylated N-glycans from other N-glycans in microchip CE-MS. This new approach was demonstrated with standard multisialylated N-glycans, and it was found that only the α-2,3-sialylated N-glycans migrated and were detected in order according to the number of α-2,3-sialic acids. Finally, this strategy was successfully applied in highly sensitive profiling and reproducible quantitation of the serum α-2,3-sialylated N-glycome from ovarian cancer (OC) patients, where 7 of 33 detected α-2,3-sialylated N-glycans significantly changed in the OC group compared with healthy controls.

Comparative analysis of intact glycopeptides from mannose receptor among different breast cancer subtypes using mass spectrometry.

Breast cancer is a highly heterogeneous disease, encompassing a number of biologically distinct entities with specific pathologic features and biological behaviors. In the preliminary experiments, we identified several glycosylation sites of mannose receptors in different breast cancer subtypes and showed that the mannose receptors could be a potential marker for breast cancer. However, the glycan composition on each site is still unknown because the glycan was removed by PNGase F in previous work. Analysis of intact glycopeptides can provide the information of both the glycan composition and the glycosylation site, which can further help to reveal the difference of glycosylation in the four subtypes of breast cancer. In this work, we analyzed the intact glycopeptides of the mannose receptors in serum from breast cancer patients using isobaric tags for relative and absolute quantitation (iTRAQ) and LC-MS/MS. In total, 7 glycosylation sites and 12 glycan types corresponding to 26 intact glycopeptides were characterized from the four subtypes of breast cancer. Among them, 11 glycopeptides can be used to differentiate the subtypes of breast cancer, which further supported the previous conclusion that mannose receptor can be used as a potential marker for the identification of breast cancer subtypes.

FluoroTRAQ: Quantitative Analysis of Protein S-Nitrosylation through Fluorous Solid-Phase Extraction Combining with iTRAQ by Mass Spectrometry.

S-Nitrosylation is an important post-translational modification that occurs on cysteine amino acid and regulates signal transduction in diverse cell processes. Dysregulation of protein nitrosylation has shown close association with cardiovascular and neurological diseases, thus demanding further precise and in-depth understanding. Mass spectrometry-based proteomics has been the method of choice for analyzing S-nitrosylated (SNO-) proteins. However, due to their extremely low expression level and rapid turnover rate, quantitative analysis of the S-nitrosylation at the proteomic level remains challenging. Herein, we developed a novel approach termed FluoroTRAQ, which combined the fluorous solid-phase extraction of SNO-peptides and iTRAQ labeling for the quantitative analysis of the SNO-proteome with high sensitivity and specificity. This new analytical strategy was subsequently applied to examine the dynamic SNO-proteome changes of human umbilical vein endothelial cells upon in vitro S-nitrosoglutathione induction. Our data identified a number of novel SNO-proteins and revealed their temporal modulation as validated by biotin switch assay. Our study offered a practical approach for quantitative analysis of protein S-nitrosylation.

Rapid and Easy Enrichment Strategy for Naturally Acetylated N Termini Based on LysN Digestion and Amine-Reactive Resin Capture.

Protein N-terminal acetylation (Nα-acetylation) is one of the most common modifications in both eukaryotes and prokaryotes. Although studies have shown that Nα-acetylation plays important roles in protein assembly, stability, and location, the physiological role has not been fully elucidated. Therefore, a robust and large-scale analytical method is important for a better understanding of Nα-acetylation. Here, an enrichment strategy was presented based on LysN digestion and amine-reactive resin capture to study naturally acetylated protein N termini. Since LysN protease cleaves at the amino-terminus of the lysine residue, all resulting peptides except naturally acetylated N-terminal peptides contain free amino groups and can be removed by coupling with AminoLink Resin. Therefore, the naturally acetylated N-terminal peptides were left in solution and enriched for further liquid chromatography-tandem mass spectrometry (LC-MS/MS) analysis. The method was very simple and fast, which contained no additional chemical derivatization except protein reduction and alkylation necessarily needed in bottom-up proteomics. It could be used to study acetylated N termini from complex biological samples without bias toward different peptides with various physicochemical properties. The enrichment specificity was above 99% when it was applied in HeLa cell lysates. Neo-N termini generated by endogenous degradation could be directly distinguished without the use of stable-isotope labeling because no chemical derivatization was introduced in this method. Furthermore, this method was highly complementary to the traditional analytical methods for protein N termini based on trypsin only with ArgC-like activity. Therefore, the described method was beneficial to naturally acetylated protein N termini profiling.

Stable Isotope Sequential Derivatization for Linkage-Specific Analysis of Sialylated N-Glycan Isomers by MS.

Sialylated N-glycans play pivotal role in several important biological and pathological processes. Their sialyl-linkage isomers, mostly α-2,3- and α-2,6-linked, act differently during the cellular events and several diseases. While mass spectrometry (MS) technology is a powerful tool in N-glycome analysis, it still suffers from an inability to distinguish linkage isomers of native N-glycans. Herein, we described a sequential selective derivatization method, by which α-2,6- and α-2,3-linked sialic acids are sequentially labeled with methylamide incorporated with a different stable isotope. Isobaric labeling avoids inducing bias in ionization efficiency and chromatographic behavior. In optimized reaction conditions, high derivatization selectivity (∼99%) was achieved for both α-2,3- and α-2,6-linked sialic acid. High accuracy of quantitation within a dynamic range of 2 orders of magnitude and high reproducibility (CV < 20%, n = 3) were demonstrated using standard glycans and multisialylated N-glycans. Finally, this method was applied in profiling the N-glycome of serum from CRC patients, where a level of six sialyl-linkage isomers were found to be altered significantly compared with that from healthy individuals.

In-Depth Analysis of C Terminomes Based on LysC Digestion and Site-Selective Dimethylation.

Analysis of protein C termini is very important for functional annotations of proteomes, while proteome-wide C termini analysis still poses substantial challenges. Here we described a simple and robust strategy for specific isolation of protein C termini based on LysC digestion and site-selective dimethylation to deplete N-terminal and internal peptides by scavenger materials. The performance of LysC digestion and conditions of site-selective dimethylation and resin coupling were discussed in detail. Then the strategy was successfully applied to the characterization of protein C termini of HeLa cells. A total of 781 protein C termini were identified with a 300 µg digest in our study, among which 38.9% were actually not identifiable using current trypsin digestion-based methods due to their inappropriate peptide length for MS analysis, indicating that our method was highly complementary to the existing methods. The enrichment procedure was rapid and easy to operate and could afford a very good identification efficiency by obtaining the largest C termini data set of the human proteome with the least sample loading. This method was without bias toward physicochemical properties of peptides. Moreover, a peptide-centric database was first introduced to analyze protein C termini, which effectively improved the accuracy and speed of the database search. Therefore, our method can be used to effectively and selectively isolate protein C termini and contributes to the global annotation of C terminomes.

Combining near infrared spectroscopy with predictive model and expertise to monitor herb extraction processes.

Albeit extensively utilized, herb extraction process (HEP) is hard to be monitored because of its batch nature and the fluctuating quality of raw materials. Process analytical tools like near infrared spectroscopy (NIRS) can offer nondestructive examinations and collect abundant data of the process, which in principle contain the information about the quality of both the product and the process itself. However, extra effort is often required for the data mining of such process measurements, and extracting knowledge of the quality of process can be even harder. In this study, we take the extraction process of licorice as a typical HEP instance, and combine NIRS with classical partial least squared regression (PLSR) and expertise for its on-line monitoring. We show that our scheme effectively extracts information with clear physical meanings, through which we can even uncover the process fault that does not induce evident abnormalities in the product quality. Moreover, the constructed model can continuously evolve with more process data from daily operations, and the idea of the whole framework can be directly generalized to other HEP.

Site-Specific Quantification of Protein Ubiquitination on MS2 Fragment Ion Level via Isobaric Peptide Labeling.

Proteome-wide quantitative analysis of protein ubiquitination is important to gain insight into its various cellular functions. However, it is still challenging to monitor how ubiquitination at each individual lysine residue is independently regulated, especially the whereabouts of peptides containing more than one ubiquitination site. In recent years, isobaric peptide termini labeling has been considered a promising strategy in quantitative proteomics, benefiting from its high accuracy by quantifying with a series of b, y fragment ion pairs. Herein, we extended the concept of isobaric peptide termini labeling to large-scale quantitative analysis of protein ubiquitination. A novel MS2 fragment ion based quantitative approach was developed, allowing the quantification of ubiquitination at site level via isobaric K-ε-GG peptide labeling, which combined metabolic labeling, K-ε-GG immunoaffinity enrichment, and site-selective N-terminus dimethylation. The feasibility of this proposed strategy was demonstrated through the ubiquitin proteome analysis of differently labeled MCF-7 cell digests. As a result, 2970 unique K-ε-GG peptides of 1383 proteins containing 2874 ubiquitinated sites were confidently quantified with high accuracy and sensitivity. In addition, we demonstrated that quantification on MS2 fragment ion level makes it possible to precisely quantify each individual ubiquitinated lysine residue in 39 K-ε-GG peptides bearing two ubiquitination sites by the use of specific ubiquitinated b, y ion pairs. It is expected that this proposed approach will serve as a powerful tool to quantify ubiquitination at the site level, especially for those multiubiquitinated peptides.

Highly Selective and Large Scale Mass Spectrometric Analysis of 4-Hydroxynonenal Modification via Fluorous Derivatization and Fluorous Solid-Phase Extraction.

Modification of proteins with 4-hydroxynonenal (HNE) is known to alter the function of proteins and regulate the associated biological processes in eukaryotic cells. The development of mass spectrometry (MS) makes high-throughput analysis of HNE modification accessible. However, the identification of HNE modification is still hampered by the low frequency of this modification. Therefore, only a limited number of HNE modification sites have been identified. The enrichment of HNE-modified peptides is critical for the MS analysis of this modification because of its low abundance. Herein, we explored a novel strategy for specifically extracting the HNE-modified peptides using fluorous derivatization through oxime click chemistry combined with following fluorous solid-phase extraction (FSPE). This oxime click chemistry-based derivatization is highly efficient (with a yield of almost 100%) and fast (30 min). Because of the hydrophobicity of the fluorous tag, the signal of fluorous-derivatized HNE-modified peptides was greatly enhanced, making the detection of HNE-modified peptides sensitive. The FSPE further allowed the selective enrichment of fluorous-derivatized HNE-modified peptides from salt solutions and complex mixtures with specificity. Finally, 673 HNE modification sites (607 histidine sites, 60 cysteine sites, 5 lysine sites, and 1 arginine site) on 661 HNE-modified peptides mapped to 432 proteins were successfully identified using this novel approach, which presented the largest data set of HNE modification in MCF-7 cells. Three identified proteins were validated by Western blotting.

Far infrared-assisted encapsulation of filter paper strips in poly(methyl methacrylate) for proteolysis.

Filter paper strips were enclosed between two poly(methyl methacrylate) plates to fabricate paper-packed channel microchips under pressure in the presence of far infrared irradiation. After the enclosed paper strip was oxidized by periodate, trypsin was covalently immobilized in them to fabricate microfluidic proteolysis bioreactor. The feasibility and performance of the unique bioreactor were demonstrated by digesting BSA and lysozyme. The results were comparable to those of conventional in-solution proteolysis while the digestion time was significantly reduced to ∼18 s. The suitability of the microfluidic paper-based bioreactors to complex proteins was demonstrated by digesting human serum.

Immobilization of trypsin on miniature incandescent bulbs for infrared-assisted proteolysis.

A novel efficient proteolysis approach was developed based on trypsin-immobilized miniature incandescent bulbs and infrared (IR) radiation. Trypsin was covalently immobilized in the chitosan coating on the outer surface of miniature incandescent bulbs with the aid of glutaraldehyde. When an illuminated enzyme-immobilized bulb was immersed in protein solution, the emitted IR radiation could trigger and accelerate heterogeneous protein digestion. The feasibility and performance of the novel proteolysis approach were demonstrated by the digestion of hemoglobin (HEM), cytochrome c (Cyt-c), lysozyme (LYS), and ovalbumin (OVA) and the digestion time was significantly reduced to 5 min. The obtained digests were identified by MALDI-TOF-MS with the sequence coverages of 91%, 77%, 80%, and 52% for HEM, Cyt-c, LYS, and OVA (200 ng µL(-1) each), respectively. The suitability of the prepared bulb bioreactors to complex proteins was demonstrated by digesting human serum.

Low temperature preparation of a graphene-cobalt microsphere hybrid by borohydride-initiated reduction for enriching proteins and peptides.

A graphene-cobalt microsphere hybrid was prepared by the chemical reduction of a mixture containing graphene oxide and cobalt chloride. It was found that a little amount of potassium borohydride or sodium borohydride could initiate the hydrazine-reduction of the mixture at a low temperature of 80 °C. The structure of the material was investigated by scanning electron microscopy, transmission electron microscopy, energy dispersive spectroscopy, vibrating sample magnetometery, Fourier transform infrared spectroscopy, Raman spectroscopy, and Brunauer-Emmett-Teller (BET) techniques. The average size of the cobalt microspheres on graphene sheets was measured to be ∼590 nm. Magnetic investigations indicated that the graphene-cobalt microsphere hybrid exhibited ferromagnetic behavior at room temperature. In addition, the magnetic hybrid was successfully employed in the enrichment and identification of low-abundance proteins and peptides in combination with mass spectrometry.

Immobilization of trypsin via graphene oxide-silica composite for efficient microchip proteolysis.

In this report, trypsin was covalently immobilized in the graphene oxide (GO)-silica composite coating on the channel wall of poly(methyl methacrylate) (PMMA) microchips to fabricate microfluidic bioreactors for highly efficient proteolysis. A mixture solution containing GO nanosheets and silica gel was injected into the channels to form coating. 1-Ethyl-3-(3-dimethylaminopropyl)carbodiimide and N-hydroxysuccinimide were used as carboxyl activating agents to crosslink the primary amino groups of trypsin to the carboxyl groups of the entrapped GO sheets in the composite to realize covalent immobilization. The feasibility and performance of the novel GO-based microfluidic bioreactors were demonstrated by the digestion of hemoglobin (HEM), cytochrome c (Cyt-c), myoglobin (MYO), and ovalbumin (OVA) and the digestion time was significantly reduced to 5s. The obtained digests were identified by MALDI-TOF MS with the sequence coverages of 95%, 76%, 69%, and 55% for HEM, Cyt-c, MYO, and OVA, respectively. The suitability of the prepared bioreactors to complex proteins was demonstrated by digesting human serum.

Nuclear proteome profile of C57BL/6J mouse liver.

The liver proteome can serve as a reference to better understand both disease mechanisms and possible therapeutics, since the liver is an important organ in the body that performs a large number of tasks. Here we identify the organelle proteome of C57BL/6J mouse liver nuclei as a promising strategy to enrich low abundance proteins, in the sense that analysis of whole liver cells is rather complex for current techniques and may not be suitable for proteins with low abundance. Evaluation of nucleus integrity and purity was performed to demonstrate the effectiveness of the optimized isolation procedure. The extracted nuclear proteins were identified by 2-DE MS analyses, and a total of 748 proteins were identified. Bioinformatic analyses were performed to demonstrate the physicochemical properties, cellular locations and functions of the proteins.

Efficient proteolysis strategies based on microchip bioreactors.

In proteome research, proteolysis is an important procedure prior to the mass spectrometric identification of proteins. The typical time of conventional in-solution proteolysis is as long as several hours to half a day. To enhance proteolysis efficiency, a variety of microchip bioreactors have been developed for the rapid digestion and identification of proteins in the past decade. This review mainly focuses on the recent advances and the key strategies of microchip bioreactors in protein digestion. The subjects covered include microchip proteolysis systems, the immobilization of proteases in microchannels, the applications of microchip bioreactors in highly efficient proteolysis, and future prospects. It is expected that microchip bioreactors will become powerful tools in protein analysis and will find a wide range of applications in high-throughput protein identification.

14-3-3ε mediates the cell fate decision-making pathways in response of hepatocellular carcinoma to Bleomycin-induced DNA damage.

BACKGROUND: Lack of understanding of the response of hepatocellular carcinoma (HCC) to anticancer drugs causes the high mortality of HCC patients. Bleomycin (BLM) that induces DNA damage is clinically used for cancer therapy, while the mechanism underlying BLM-induced DNA damage response (DDR) in HCC cells remains ambiguous. Given that 14-3-3 proteins are broadly involved in regulation of diverse biological processes (BPs)/pathways, we investigate how a 14-3-3 isoform coordinates particular BPs/pathways in BLM-induced DDR in HCC. METHODOLOGY/PRINCIPAL FINDINGS: Using dual-tagging quantitative proteomic approach, we dissected the 14-3-3ε interactome formed during BLM-induced DDR, which revealed that 14-3-3ε via its associations with multiple pathway-specific proteins coordinates multiple pathways including chromosome remodeling, DNA/RNA binding/processing, DNA repair, protein ubiquitination/degradation, cell cycle arrest, signal transduction and apoptosis. Further, "zoom-in" investigation of the 14-3-3ε interacting network indicated that the BLM-induced interaction between 14-3-3ε and a MAP kinase TAK1 plays a critical role in determining cell propensity of apoptosis. Functional characterization of this interaction further revealed that BLM triggers site-specific phosphorylations in the kinase domain of TAK1. These BLM-induced changes of phosphorylations directly correlate to the strength of the TAK1 binding to 14-3-3ε, which govern the phosphorylation-dependent TAK1 activation. The enhanced 14-3-3ε-TAK1 association then inhibits the anti-apoptotic activity of TAK1, which ultimately promotes BLM-induced apoptosis in HCC cells. In a data-dependent manner, we then derived a mechanistic model where 14-3-3ε plays the pivotal role in integrating diverse biological pathways for cellular DDR to BLM in HCC. CONCLUSIONS: Our data demonstrated on a systems view that 14-3-3ε coordinates multiple biological pathways involved in BLM-induced DDR in HCC cells. Specifically, 14-3-3ε associates with TAK1 in a phosphorylation-dependent manner to determine the cell fate of BLM-treated HCC cells. Not only individual proteins but also those critical links in the network could be the potential targets for BLM-mediated therapeutic intervention of HCC.