On the excessive use of coefficient of variation as a metric of quantitation quality in proteomics.

The coefficient of variation (CV) is often used in proteomics as a proxy to characterize the performance of a quantitation method and/or the related software. In this note, we question the excessive reliance on this metric in quantitative proteomics that may result in erroneous conclusions. We support this note using a ground-truth Human-Yeast-E. coli dataset demonstrating in a number of cases that erroneous data processing methods may lead to a low CV which has nothing to do with these methods' performances in quantitation.

IQMMA: Efficient MS1 Intensity Extraction Pipeline Using Multiple Feature Detection Algorithms for DDA Proteomics.

One of the key steps in data dependent acquisition (DDA) proteomics is detection of peptide isotopic clusters, also called "features", in MS1 spectra and matching them to MS/MS-based peptide identifications. A number of peptide feature detection tools became available in recent years, each relying on its own matching algorithm. Here, we provide an integrated solution, the intensity-based Quantitative Mix and Match Approach (IQMMA), which integrates a number of untargeted peptide feature detection algorithms and returns the most probable intensity values for the MS/MS-based identifications. IQMMA was tested using available proteomic data acquired for both well-characterized (ground truth) and real-world biological samples, including a mix of Yeast and E. coli digests spiked at different concentrations into the Human K562 digest used as a background, and a set of glioblastoma cell lines. Three open-source feature detection algorithms were integrated: Dinosaur, biosaur2, and OpenMS FeatureFinder. None of them was found optimal when applied individually to all the data sets employed in this work; however, their combined use in IQMMA improved efficiency of subsequent protein quantitation. The software implementing IQMMA is freely available at https://github.com/PostoenkoVI/IQMMA under Apache 2.0 license.

Wide-scale identification of novel/eliminated genes responsible for evolutionary transformations.

BACKGROUND: It is generally accepted that most evolutionary transformations at the phenotype level are associated either with rearrangements of genomic regulatory elements, which control the activity of gene networks, or with changes in the amino acid contents of proteins. Recently, evidence has accumulated that significant evolutionary transformations could also be associated with the loss/emergence of whole genes. The targeted identification of such genes is a challenging problem for both bioinformatics and evo-devo research. RESULTS: To solve this problem we propose the WINEGRET method, named after the first letters of the title. Its main idea is to search for genes that satisfy two requirements: first, the desired genes were lost/emerged at the same evolutionary stage at which the phenotypic trait of interest was lost/emerged, and second, the expression of these genes changes significantly during the development of the trait of interest in the model organism. To verify the first requirement, we do not use existing databases of orthologs, but rely purely on gene homology and local synteny by using some novel quickly computable conditions. Genes satisfying the second requirement are found by deep RNA sequencing. As a proof of principle, we used our method to find genes absent in extant amniotes (reptiles, birds, mammals) but present in anamniotes (fish and amphibians), in which these genes are involved in the regeneration of large body appendages. As a result, 57 genes were identified. For three of them, c-c motif chemokine 4, eotaxin-like, and a previously unknown gene called here sod4, essential roles for tail regeneration were demonstrated. Noteworthy, we established that the latter gene belongs to a novel family of Cu/Zn-superoxide dismutases lost by amniotes, SOD4. CONCLUSIONS: We present a method for targeted identification of genes whose loss/emergence in evolution could be associated with the loss/emergence of a phenotypic trait of interest. In a proof-of-principle study, we identified genes absent in amniotes that participate in body appendage regeneration in anamniotes. Our method provides a wide range of opportunities for studying the relationship between the loss/emergence of phenotypic traits and the loss/emergence of specific genes in evolution.

[The social and medical aspects of falls in elderly and senile age under eyesight deficiency].

The aging of population is accompanied by simultaneous increasing of rate of age-associated ophthalmic diseases resulting in vision decreasing. However, visual impairment in elderly and senile age is rarely considered in the epidemiology of falls in these groups. The purpose of the study is to investigate medical social aspects of falls in older age groups with visual impairment. The retrospective methodology was applied to study falls in 4832 elderly and senile patients with visual impairment due to cataract glaucoma, diabetic retinopathy and age-related macular degeneration. The high incidence of falls in men and women aged 80 and older, amounting to 82.6 and 125.7 cases per 1000 of population of corresponding age respectively was established. The falls in elderly patients with low vision is more often registered in case of diabetic retinopathy than of glaucoma, cataract and age-related macular degeneration without significant differences at the age 50-59 years and 60-69 years. The diabetic retinopathy is the most common cause of falls requiring hospitalization in all age groups. To In reducing prevalence of falls and resulted hospitalization, to optimize traumatological care of patients of older age groups, the priority is for early identification and treatment of people with diabetic retinopathy.

Massive Proteogenomic Reanalysis of Publicly Available Proteomic Datasets of Human Tissues in Search for Protein Recoding via Adenosine-to-Inosine RNA Editing.

The proteogenomic search pipeline developed in this work has been applied for reanalysis of 40 publicly available shotgun proteomic datasets from various human tissues comprising more than 8000 individual LC-MS/MS runs, of which 5442 .raw data files were processed in total. This reanalysis was focused on searching for ADAR-mediated RNA editing events, their clustering across samples of different origins, and classification. In total, 33 recoded protein sites were identified in 21 datasets. Of those, 18 sites were detected in at least two datasets, representing the core human protein editome. In agreement with prior artworks, neural and cancer tissues were found to be enriched with recoded proteins. Quantitative analysis indicated that recoding the rate of specific sites did not directly depend on the levels of ADAR enzymes or targeted proteins themselves, rather it was governed by differential and yet undescribed regulation of interaction of enzymes with mRNA. Nine recoding sites conservative between humans and rodents were validated by targeted proteomics using stable isotope standards in the murine brain cortex and cerebellum, and an additional one was validated in human cerebrospinal fluid. In addition to previous data of the same type from cancer proteomes, we provide a comprehensive catalog of recoding events caused by ADAR RNA editing in the human proteome.

Identification of Alternative Splicing in Proteomes of Human Melanoma Cell Lines without RNA Sequencing Data.

Alternative splicing is one of the main regulation pathways in living cells beyond simple changes in the level of protein expression. Most of the approaches proposed in proteomics for the identification of specific splicing isoforms require a preliminary deep transcriptomic analysis of the sample under study, which is not always available, especially in the case of the re-analysis of previously acquired data. Herein, we developed new algorithms for the identification and validation of protein splice isoforms in proteomic data in the absence of RNA sequencing of the samples under study. The bioinformatic approaches were tested on the results of proteome analysis of human melanoma cell lines, obtained earlier by high-resolution liquid chromatography and mass spectrometry (LC-MS). A search for alternative splicing events for each of the cell lines studied was performed against the database generated from all known transcripts (RefSeq) and the one composed of peptide sequences, which included all biologically possible combinations of exons. The identifications were filtered using the prediction of both retention times and relative intensities of fragment ions in the corresponding mass spectra. The fragmentation mass spectra corresponding to the discovered alternative splicing events were additionally examined for artifacts. Selected splicing events were further validated at the mRNA level by quantitative PCR.

Proteomics-based scoring of cellular response to stimuli for improved characterization of signaling pathway activity.

Omics technologies focus on uncovering the complex nature of molecular mechanisms in cells and organisms, including biomarkers and drug targets discovery. Aiming at these tasks, we see that information extracted from omics data is still underused. In particular, characteristics of differentially regulated molecules can be combined in a single score to quantify the signaling pathway activity. Such a metric can be useful for comprehensive data interpretation to follow: (1) developing molecular responses in time; (2) potency of a drug on a certain cell culture; (3) ranking the signaling pathway activity in stimulated cells; and (4) integration of the omics data and assay-based measurements of cell viability, cytotoxicity, and proliferation. With recent advances in ultrafast mass spectrometry for quantitative proteomics allowing data collection in a few minutes, proteomics score for cellular response to stimuli can become a fast, accurate, and informative complement to bioassays. Here, we utilized an interquartile-based selection of differentially regulated features and a variety of schemes for quantifying cellular responses to come up with the quantitative metric for total cellular response and pathway activity. Validation was performed using antiproliferative and virus assays and label-free proteomics data collected for cancer cells subjected to drug stimulation.

[Allostatic load in patients with diabetic retinopathy]. / Allostaticheskaya nagruzka u patsientov s diabeticheskoi retinopatiei.

Diabetic retinopathy (DR) occupies a special place among the causes of progressive decline and loss of visual acuity, it significantly impairs the quality of life and viability of elderly patients, and allostatic load is considered its integral indicator. However, the allostatic load in patients suffering from diabetic retinopathy, as well as in other ophthalmological diseases, has not been extensively studied, so biomarkers characterizing the allostatic load of patients with diabetic retinopathy remain unknown. PURPOSE: This study investigates the allostatic load in patients with diabetic retinopathy and attempts to identify the biomarkers that determine it to the fullest extent. MATERIAL AND METHODS: Allostatic load was studied in 78 elderly patients with diabetic retinopathy and type 2 diabetes mellitus, and in 62 patients with type 2 diabetes mellitus without diabetic retinopathy. Allostatic load was evaluated by analyzing systolic and diastolic blood pressure, body mass index, glycated hemoglobin, total cholesterol, triglycerides, albumins, C-reactive protein, homocysteine in the blood and glomerular filtration rate. RESULTS: It was found that in patients with diabetic retinopathy the most pronounced and statistically significant excess was in the content of glycated hemoglobin in the blood up to 10.2% versus 7.4%, and homocysteine up to 15.5 mmol/L versus 7.9 mmol/L compared to patients with diabetes mellitus without diabetic retinopathy, respectively. The value of the allostatic index was significantly higher in patients with diabetic retinopathy, amounting to 4.6±0.4 points, versus 2.9±0.3 points in patients with diabetes mellitus without the studied ophthalmic pathology (p<0.001). Factor analysis made it possible to identify biomarkers of allostatic load in patients with diabetic retinopathy - glycated hemoglobin, homocysteine, triglycerides and albumins. CONCLUSION: The identified biomarkers can be used for assessing the viability and the effectiveness of rehabilitation measures carried out in patients with diabetic retinopathy.

Proteomic Analysis of Zebrafish Protein Recoding via mRNA Editing by ADAR Enzymes.

RNA editing by adenosine deaminases of the ADAR family can lead to protein recoding, since inosine formed from adenosine in mRNA is complementary to cytosine; the resulting codon editing might introduce amino acid substitutions into translated proteins. Proteome recoding can have functional consequences which have been described in many animals including humans. Using protein recoding database derived from publicly available transcriptome data, we identified for the first time the recoding sites in the zebrafish shotgun proteomes. Out of more than a hundred predicted recoding events, ten substitutions were found in six used datasets. Seven of them were in the AMPA glutamate receptor subunits, whose recoding has been well described, and are conserved among vertebrates. Three sites were specific for zebrafish proteins and were found in the transmembrane receptors astrotactin 1 and neuregulin 3b (proteins involved in the neuronal adhesion and signaling) and in the rims2b gene product (presynaptic membrane protein participating in the neurotransmitter release), respectively. Further studies are needed to elucidate the role of recoding of the said three proteins in the zebrafish.

DirectMS1Quant: Ultrafast Quantitative Proteomics with MS/MS-Free Mass Spectrometry.

Recently, we presented the DirectMS1 method of ultrafast proteome-wide analysis based on minute-long LC gradients and MS1-only mass spectra acquisition. Currently, the method provides the depth of human cell proteome coverage of 2500 proteins at a 1% false discovery rate (FDR) when using 5 min LC gradients and 7.3 min runtime in total. While the standard MS/MS approaches provide 4000-5000 protein identifications within a couple of hours of instrumentation time, we advocate here that the higher number of identified proteins does not always translate into better quantitation quality of the proteome analysis. To further elaborate on this issue, we performed a one-on-one comparison of quantitation results obtained using DirectMS1 with three popular MS/MS-based quantitation methods: label-free (LFQ) and tandem mass tag quantitation (TMT), both based on data-dependent acquisition (DDA) and data-independent acquisition (DIA). For comparison, we performed a series of proteome-wide analyses of well-characterized (ground truth) and biologically relevant samples, including a mix of UPS1 proteins spiked at different concentrations into an Echerichia coli digest used as a background and a set of glioblastoma cell lines. MS1-only data was analyzed using a novel quantitation workflow called DirectMS1Quant developed in this work. The results obtained in this study demonstrated comparable quantitation efficiency of 5 min DirectMS1 with both TMT and DIA methods, yet the latter two utilized a 10-20-fold longer instrumentation time.

[Prognostic value of matrix metalloproteinases in the development of diabetic retinopathy in the elderly].

The development of diabetic retinopathy is associated with matrix metalloproteinases, but they are rarely used to predict this pathology. The aim of the study was to predict the development of non-proliferative diabetic retinopathy in old age by the level of matrix metalloproteinases in blood plasma. The main study group consisted of 63 patients aged 60-74 years with type 2 diabetes mellitus and non-proliferative diabetic retinopathy, the control was 56 patients of the same age with type 2 diabetes mellitus and the absence of diabetic retinopathy and other ophthalmopathology at present and in the anamnesis. Examination of patients of both groups included: tonometry, visiometry, standard fundus photoregistration, optical coherence tomography, optical coherence tomography-A, fluorescent angiography. Determination of matrix metalloproteinases was carried out by the method of solid-phase enzyme immunoassay. There was a statistically significant increase in matrix metalloproteinase-9 in the main group of patients to 55,7±2,6 ng/ml versus 40,2±1,9 ng/ml in the age control, matrix metalloproteinase-2 to 269,8±4,2 ng/ml versus 221,9±3,6 ng/ml, respectively. Based on the level of matrix metalloproteinases-2 (X1) and -9 (X2) in the blood, a regression model was created by the regression method to predict the development of diabetic retinopathy, having the form Y=28,315+3,892·X1+2,453·X2, which will allow detecting the disease at an early stage.

[Effect of combined diabetic retinopathy and cataract on instrumental functional activity of patients.]

A potential factor that significantly reduces the social activity of older people is visual impairment due to age-associated eye diseases - cataracts and diabetic retinopathy, which dominate in this age cohort. The aim of the study was to study the social activity of elderly patients with diabetic retinopathy and cataracts. According to a special questionnaire, 115 people 60-74 years old with combined cataract and diabetic retinopathy and 102 people 60-74 years old without ophthalmological diseases were questioned based on the Tambov branch of the MNTK «Academician S.N.Fedorov Eye Microsurgery¼. It was found that the average score of social activity in patients with cataract and diabetic retinopathy is statistically significantly lower, amounting to 3,10±0,08 points, versus 5,38±0,09 points in people without ophthalmopathology. The main reasons that reduce social activity in patients with visual impairment are problems with making various purchases and using public or personal transport. Factor analysis confirmed that the contribution of these reasons to the decrease in social activity is the highest and amounts to 0,876 and 0,708, respectively. Therefore, it is recommended to increase social assistance to patients with cataracts and diabetic retinopathy in solving the problems of using transport and purchasing various goods.

THE ROLE OF LACTOBACILLI IN THE HUMAN MICROBIOME AND METHODS OF THEIR CULTIVATION AND PRESERVATION.

The extremely important role of the microbiome for human life and health has long been known. Many studies around the world are devoted to studying the mechanisms of action and functions of various bacteria that are permanent residents of our body. Connections between the bacteria of our microbiome and all organs and systems of the human body (intestine, brain, nervous and cardiovascular systems) have been identified. However, the effect of bacteria can be positive or negative, which affects the emergence and development of diseases or promotes healing. Genus Lactobacillus is one of the most numerous populations of bacteria in the human body. Moreover, they have a significant positive effect on health. Scientists are actively researching methods of cultivating and using bacteria of this genus in the pharmaceutical and industrial fields. Most probiotics contain lactobacilli strains. Therefore, the study of methods of cultivation and storage of lactobacilli in order to find ways to improve their viability and functionality and, at the same time, the invention of options to protect cell culture from various harmful factors is extremely important. In our review, we considered the importance of the microbiome for human health and the role of bacteria of the genus Lactobacillus as its component. Scientific works on studying the mechanisms of influence of lactobacilli on the functional capacity of human organs and systems have been studied. Much of the review is devoted to the study of lactobacilli cultivation methods, the diversity of culture media, and the importance of their components to improve the viability of lactobacilli culture because they are quite demanding and vulnerable. Attention is also paid to the development of methods of storage of grown cultures of bacterial cells and their improvement in order to obtain functional and suitable for further use in the pharmacological and industrial areas of bacterial strains.

Identification of mutant gene for Black crystal coat and non-allelic gene interactions in Neogale vison.

Sable (Martes zibellina) and American mink (Neogale vison) are valuable species characterized by a variety of coat colour produced on fur farms. Black crystal fur phenotype is Mendelian codominant trait: heterozygous animals (Cr/ +) have white guard hairs scattered predominantly on the spine and the head, while homozygous (Cr/Cr) minks have coats resembling the Himalayan (ch/ch) or white Hedlund (h/h) types. It is one of the most recent of more than 35 currently known phenotypic traits of fur colour in American mink. Black crystal fur phenotype was first described in 1984 in the Russian population of mink, which had undergone selection for domestic defensive response to humans. Here, we performed whole-genome sequencing of American mink with Cr/Cr phenotype. We identified a missense mutation in the gene encoding the α-COP subunit of the COPI complex (COPA). The COPI complex mediates retrograde trafficking from the Golgi system to the endoplasmic reticulum and sorting of transmembrane proteins. We observed an interaction between a newly identified mutation in the COPA gene and a mutation in the microphthalmia-associated transcription factor (MITF), the latter mutation led to the formation of the white Hedlund (h/h) phenotype. Double heterozygotes for these mutations have an entirely white coat and a black-eyed phenotype similar to the phenotype of Cr/Cr or h/h minks. Our data could be useful for tracking economically valuable fur traits in mink breeding programs to contribute to global fur production.

Validating Amino Acid Variants in Proteogenomics Using Sequence Coverage by Multiple Reads.

Mass spectrometry-based proteome analysis implies matching the mass spectra of proteolytic peptides to amino acid sequences predicted from genomic sequences. Reliability of peptide variant identification in proteogenomic studies is often lacking. We propose a way to interpret shotgun proteomics results, specifically in the data-dependent acquisition mode, as protein sequence coverage by multiple reads as it is done in nucleic acid sequencing for calling of single nucleotide variants. Multiple reads for each sequence position could be provided by overlapping distinct peptides, thus confirming the presence of certain amino acid residues in the overlapping stretch with a lower false discovery rate. Overlapping distinct peptides originate from miscleaved tryptic peptides in combination with their properly cleaved counterparts and from peptides generated by multiple proteases after the same specimen is subject to parallel digestion and analyzed separately. We illustrate this approach using publicly available multiprotease data sets and our own data generated for the HEK-293 cell line digests obtained using trypsin, LysC, and GluC proteases. Totally, up to 30% of the whole proteome was covered by tryptic peptides with up to 7% covered twofold and more. The proteogenomic analysis of the HEK-293 cell line revealed 36 single amino acid variants, seven of which were supported by multiple reads.

A Comprehensive Evaluation of Consensus Spectrum Generation Methods in Proteomics.

Spectrum clustering is a powerful strategy to minimize redundant mass spectra by grouping them based on similarity, with the aim of forming groups of mass spectra from the same repeatedly measured analytes. Each such group of near-identical spectra can be represented by its so-called consensus spectrum for downstream processing. Although several algorithms for spectrum clustering have been adequately benchmarked and tested, the influence of the consensus spectrum generation step is rarely evaluated. Here, we present an implementation and benchmark of common consensus spectrum algorithms, including spectrum averaging, spectrum binning, the most similar spectrum, and the best-identified spectrum. We have analyzed diverse public data sets using two different clustering algorithms (spectra-cluster and MaRaCluster) to evaluate how the consensus spectrum generation procedure influences downstream peptide identification. The BEST and BIN methods were found the most reliable methods for consensus spectrum generation, including for data sets with post-translational modifications (PTM) such as phosphorylation. All source code and data of the present study are freely available on GitHub at https://github.com/statisticalbiotechnology/representative-spectra-benchmark.

Genomics of Ancient Pathogens: First Advances and Prospects.

Paleogenomics is one of the urgent and promising areas of interdisciplinary research in the today's world science. New genomic methods of ancient DNA (aDNA) analysis, such as next generation sequencing (NGS) technologies, make it possible not only to obtain detailed genetic information about historical and prehistoric human populations, but also to study individual microbial and viral pathogens and microbiomes from different ancient and historical objects. Studies of aDNA of pathogens by reconstructing their genomes have so far yielded complete sequences of the ancient pathogens that played significant role in the history of the world: Yersinia pestis (plague), Variola virus (smallpox), Vibrio cholerae (cholera), HBV (hepatitis B virus), as well as the equally important endemic human infectious agents: Mycobacterium tuberculosis (tuberculosis), Mycobacterium leprae (leprosy), and Treponema pallidum (syphilis). Genomic data from these pathogens complemented the information previously obtained by paleopathologists and allowed not only to identify pathogens from the past pandemics, but also to recognize the pathogen lineages that are now extinct, to refine chronology of the pathogen appearance in human populations, and to reconstruct evolutionary history of the pathogens that are still relevant to public health today. In this review, we describe state-of-the-art genomic research of the origins and evolution of many ancient pathogens and viruses and examine mechanisms of the emergence and spread of the ancient infections in the mankind history.

A proteomics sample metadata representation for multiomics integration and big data analysis.

The amount of public proteomics data is rapidly increasing but there is no standardized format to describe the sample metadata and their relationship with the dataset files in a way that fully supports their understanding or reanalysis. Here we propose to develop the transcriptomics data format MAGE-TAB into a standard representation for proteomics sample metadata. We implement MAGE-TAB-Proteomics in a crowdsourcing project to manually curate over 200 public datasets. We also describe tools and libraries to validate and submit sample metadata-related information to the PRIDE repository. We expect that these developments will improve the reproducibility and facilitate the reanalysis and integration of public proteomics datasets.

AA_stat: Intelligent profiling of in vivo and in vitro modifications from open search results.

Characterization of post-translational modifications is among the most challenging tasks in tandem mass spectrometry-based proteomics which has yet to find an efficient solution. The ultra-tolerant (open) database search attempts to meet this challenge. However, interpretation of the mass shifts observed in open search still requires an effective and automated solution. We have previously introduced the AA_stat tool for analysis of amino acid frequencies at different mass shifts and generation of hypotheses on unaccounted in vitro modifications. Here, we report on the new version of AA_stat, which now complements amino acid frequency statistics with a number of new features: (1) MS/MS-based localization of mass shifts and localization scoring, including shifts which are the sum of modifications; (2) inferring fixed modifications to increase method sensitivity; (3) inferring monoisotopic peak assignment errors and variable modifications based on abundant mass shift localizations to increase the yield of closed search; (4) new mass calibration algorithm to account for partial systematic shifts; (5) interactive integration of all results and a rated list of possible mass shift interpretations. With these options, we improve interpretation of open search results and demonstrate the utility of AA_stat for profiling of abundant and rare amino acid modifications. AA_stat is implemented in Python as an open-source tool available at https://github.com/SimpleNumber/aa_stat. SIGNIFICANCE: Mass spectrometry-based PTM characterization has a long history, yet most of the methods rely on a priori knowledge of modifications of interest and do not provide a whole proteome modification landscape in a blind manner. The open database search is an efficient attempt to address this challenge by identifying peptides with mass shifts corresponding to possible modifications. Then, interpreting these mass shifts is required. Therefore, development of bioinformatics software for post-processing of the open search results, which is capable of detection and accurate annotation of new or unexpected modifications, from characterization of sample preparation efficiency and quality control to discovery of rare post-translational modifications, is of high importance.

Methylation of the PTENP1 pseudogene as potential epigenetic marker of age-related changes in human endometrium.

The processed pseudogene PTENP1 is involved in the regulation of the expression of the PTEN and acts as a tumor suppressor in many types of malignances. In our previous study we showed that PTENP1 methylation is present not only in tumor, but also in normal endometrium tissues of women over 45 years old. Here we used methylation-specific PCR to analyze methylation status of CpG island located near promoter region of PTENP1 in malignant and non-malignant endometrium tissues collected from 236 women of different age groups. To confirm our results, we also analyzed RNA sequencing and microarray data from 431 women with endometrial cancer from TCGA database. We demonstrated that methylation of PTENP1 is significantly increased in older patients. We also found an age-dependent increase in the level of PTENP1 expression in endometrial tissue. According to our data, PTENP1 methylation elevates the level of the pseudogene sense transcript. In turn, a high level of this transcript correlates with a more favorable prognosis in endometrial cancer. The data obtained suggested that PTENP1 methylation is associated with age-related changes in normal and hyperplastic endometrial tissues. We assumed that age-related increase in PTENP1 methylation and subsequent elevation of its expression may serve as a protective mechanism aimed to prevent malignant transformation of endometrial tissue in women during the perimenopause, menopause, and postmenopause periods.