Whole Genome Scan Uncovers Candidate Genes Related to Milk Production Traits in Barka Cattle.

In this study, our primary aim was to explore the genomic landscape of Barka cattle, a breed recognized for high milk production in a semi-arid environment, by focusing on genes with known roles in milk production traits. We employed genome-wide analysis and three selective sweep detection methods (ZFST, θπ ratio, and ZHp) to identify candidate genes associated with milk production and composition traits. Notably, ACAA1, P4HTM, and SLC4A4 were consistently identified by all methods. Functional annotation highlighted their roles in crucial biological processes such as fatty acid metabolism, mammary gland development, and milk protein synthesis. These findings contribute to understanding the genetic basis of milk production in Barka cattle, presenting opportunities for enhancing dairy cattle production in tropical climates. Further validation through genome-wide association studies and transcriptomic analyses is essential to fully exploit these candidate genes for selective breeding and genetic improvement in tropical dairy cattle.

Metagenomics workflow for hybrid assembly, differential coverage binning, metatranscriptomics and pathway analysis (MUFFIN).

Metagenomics has redefined many areas of microbiology. However, metagenome-assembled genomes (MAGs) are often fragmented, primarily when sequencing was performed with short reads. Recent long-read sequencing technologies promise to improve genome reconstruction. However, the integration of two different sequencing modalities makes downstream analyses complex. We, therefore, developed MUFFIN, a complete metagenomic workflow that uses short and long reads to produce high-quality bins and their annotations. The workflow is written by using Nextflow, a workflow orchestration software, to achieve high reproducibility and fast and straightforward use. This workflow also produces the taxonomic classification and KEGG pathways of the bins and can be further used for quantification and annotation by providing RNA-Seq data (optionally). We tested the workflow using twenty biogas reactor samples and assessed the capacity of MUFFIN to process and output relevant files needed to analyze the microbial community and their function. MUFFIN produces functional pathway predictions and, if provided de novo metatranscript annotations across the metagenomic sample and for each bin. MUFFIN is available on github under GNUv3 licence: https://github.com/RVanDamme/MUFFIN.

Ebola Virus Disease and Current Therapeutic Strategies: A Review.

The Ebola virus disease is a severe hemorrhagic fever that affects humans and other primates. Ebola virus, the causative agent of the disease, is transmitted to humans from wild animals and is highly contagious and aggressive with an estimated fatality rate to be around 50%. Since 1976, 11 outbreaks of Ebola virus disease have been reported in total, affecting mostly sub-Saharan Africa, while the most recent ongoing outbreak in the Democratic Republic of the Congo has more than 3000 reported cases and 72 deaths. Although an effective vaccine against Ebola virus disease has become available, no targeted treatment with proven efficacy upon infection is developed. Herein, we review the epidemiology of Ebola virus and the current situation in terms of prevention, diagnosis, and treatment of the disease.

LPS-treatment of bovine endometrial epithelial cells causes differential DNA methylation of genes associated with inflammation and endometrial function.

BACKGROUND: Lipopolysaccharide (LPS) endotoxin stimulates pro-inflammatory pathways and is a key player in the pathological mechanisms involved in the development of endometritis. This study aimed to investigate LPS-induced DNA methylation changes in bovine endometrial epithelial cells (bEECs), which may affect endometrial function. Following in vitro culture, bEECs from three cows were either untreated (0) or exposed to 2 and 8 µg/mL LPS for 24 h. RESULTS: DNA samples extracted at 0 h and 24 h were sequenced using reduced representation bisulfite sequencing (RRBS). When comparing DNA methylation results at 24 h to time 0 h, a larger proportion of hypomethylated regions were identified in the LPS-treated groups, whereas the trend was opposite in controls. When comparing LPS groups to controls at 24 h, a total of 1291 differentially methylated regions (DMRs) were identified (55% hypomethylated and 45% hypermethylated). Integration of DNA methylation data obtained here with our previously published gene expression data obtained from the same samples showed a negative correlation (r = - 0.41 for gene promoter, r = - 0.22 for gene body regions, p < 0.05). Differential methylation analysis revealed that effects of LPS treatment were associated with methylation changes for genes involved in regulation of immune and inflammatory responses, cell adhesion, and external stimuli. Gene ontology and pathway analyses showed that most of the differentially methylated genes (DMGs) were associated with cell proliferation and apoptotic processes; and pathways such as calcium-, oxytocin- and MAPK-signaling pathways with recognized roles in innate immunity. Several DMGs were related to systemic inflammation and tissue re-modelling including HDAC4, IRAK1, AKT1, MAP3K6, Wnt7A and ADAMTS17. CONCLUSIONS: The present results show that LPS altered the DNA methylation patterns of bovine endometrial epithelial cells. This information, combined with our previously reported changes in gene expression related to endometrial function, confirm that LPS activates pro-inflammatory mechanisms leading to perturbed immune balance and cell adhesion processes in the endometrium.

Simulating Illumina metagenomic data with InSilicoSeq.

Motivation: The accurate in silico simulation of metagenomic datasets is of great importance for benchmarking bioinformatics tools as well as for experimental design. Users are dependant on large-scale simulation to not only design experiments and new projects but also for accurate estimation of computational needs within a project. Unfortunately, most current read simulators are either not suited for metagenomics, out of date or relatively poorly documented. In this article, we describe InSilicoSeq, a software package to simulate metagenomic Illumina sequencing data. InsilicoSeq has a simple command-line interface and extensive documentation. Results: InSilicoSeq is implemented in Python and capable of simulating realistic Illumina (meta) genomic data in a parallel fashion with sensible default parameters. Availability and implementation: Source code and documentation are available under the MIT license at https://github.com/HadrienG/InSilicoSeq and https://insilicoseq.readthedocs.io/. Supplementary information: Supplementary data are available at Bioinformatics online.

Utilizing the Dog Genome in the Search for Novel Candidate Genes Involved in Glioma Development-Genome Wide Association Mapping followed by Targeted Massive Parallel Sequencing Identifies a Strongly Associated Locus.

Gliomas are the most common form of malignant primary brain tumors in humans and second most common in dogs, occurring with similar frequencies in both species. Dogs are valuable spontaneous models of human complex diseases including cancers and may provide insight into disease susceptibility and oncogenesis. Several brachycephalic breeds such as Boxer, Bulldog and Boston Terrier have an elevated risk of developing glioma, but others, including Pug and Pekingese, are not at higher risk. To identify glioma-associated genetic susceptibility factors, an across-breed genome-wide association study (GWAS) was performed on 39 dog glioma cases and 141 controls from 25 dog breeds, identifying a genome-wide significant locus on canine chromosome (CFA) 26 (p = 2.8 x 10-8). Targeted re-sequencing of the 3.4 Mb candidate region was performed, followed by genotyping of the 56 SNVs that best fit the association pattern between the re-sequenced cases and controls. We identified three candidate genes that were highly associated with glioma susceptibility: CAMKK2, P2RX7 and DENR. CAMKK2 showed reduced expression in both canine and human brain tumors, and a non-synonymous variant in P2RX7, previously demonstrated to have a 50% decrease in receptor function, was also associated with disease. Thus, one or more of these genes appear to affect glioma susceptibility.

The eBioKit, a stand-alone educational platform for bioinformatics.

Bioinformatics skills have become essential for many research areas; however, the availability of qualified researchers is usually lower than the demand and training to increase the number of able bioinformaticians is an important task for the bioinformatics community. When conducting training or hands-on tutorials, the lack of control over the analysis tools and repositories often results in undesirable situations during training, as unavailable online tools or version conflicts may delay, complicate, or even prevent the successful completion of a training event. The eBioKit is a stand-alone educational platform that hosts numerous tools and databases for bioinformatics research and allows training to take place in a controlled environment. A key advantage of the eBioKit over other existing teaching solutions is that all the required software and databases are locally installed on the system, significantly reducing the dependence on the internet. Furthermore, the architecture of the eBioKit has demonstrated itself to be an excellent balance between portability and performance, not only making the eBioKit an exceptional educational tool but also providing small research groups with a platform to incorporate bioinformatics analysis in their research. As a result, the eBioKit has formed an integral part of training and research performed by a wide variety of universities and organizations such as the Pan African Bioinformatics Network (H3ABioNet) as part of the initiative Human Heredity and Health in Africa (H3Africa), the Southern Africa Network for Biosciences (SAnBio) initiative, the Biosciences eastern and central Africa (BecA) hub, and the International Glossina Genome Initiative.

The GOBLET training portal: a global repository of bioinformatics training materials, courses and trainers.

SUMMARY: Rapid technological advances have led to an explosion of biomedical data in recent years. The pace of change has inspired new collaborative approaches for sharing materials and resources to help train life scientists both in the use of cutting-edge bioinformatics tools and databases and in how to analyse and interpret large datasets. A prototype platform for sharing such training resources was recently created by the Bioinformatics Training Network (BTN). Building on this work, we have created a centralized portal for sharing training materials and courses, including a catalogue of trainers and course organizers, and an announcement service for training events. For course organizers, the portal provides opportunities to promote their training events; for trainers, the portal offers an environment for sharing materials, for gaining visibility for their work and promoting their skills; for trainees, it offers a convenient one-stop shop for finding suitable training resources and identifying relevant training events and activities locally and worldwide. AVAILABILITY AND IMPLEMENTATION: http://mygoblet.org/training-portal.

GOBLET: the Global Organisation for Bioinformatics Learning, Education and Training.

In recent years, high-throughput technologies have brought big data to the life sciences. The march of progress has been rapid, leaving in its wake a demand for courses in data analysis, data stewardship, computing fundamentals, etc., a need that universities have not yet been able to satisfy--paradoxically, many are actually closing "niche" bioinformatics courses at a time of critical need. The impact of this is being felt across continents, as many students and early-stage researchers are being left without appropriate skills to manage, analyse, and interpret their data with confidence. This situation has galvanised a group of scientists to address the problems on an international scale. For the first time, bioinformatics educators and trainers across the globe have come together to address common needs, rising above institutional and international boundaries to cooperate in sharing bioinformatics training expertise, experience, and resources, aiming to put ad hoc training practices on a more professional footing for the benefit of all.

The genomes of three stocks comprising the most widely utilized live sporozoite Theileria parva vaccine exhibit very different degrees and patterns of sequence divergence.

BACKGROUND: There are no commercially available vaccines against human protozoan parasitic diseases, despite the success of vaccination-induced long-term protection against infectious diseases. East Coast fever, caused by the protist Theileria parva, kills one million cattle each year in sub-Saharan Africa, and contributes significantly to hunger and poverty in the region. A highly effective, live, multi-isolate vaccine against T. parva exists, but its component isolates have not been characterized. Here we sequence and compare the three component T. parva stocks within this vaccine, the Muguga Cocktail, namely Muguga, Kiambu5 and Serengeti-transformed, aiming to identify genomic features that contribute to vaccine efficacy. RESULTS: We find that Serengeti-transformed, originally isolated from the wildlife carrier, the African Cape buffalo, is remarkably and unexpectedly similar to the Muguga isolate. The 420 detectable non-synonymous SNPs were distributed among only 53 genes, primarily subtelomeric antigens and antigenic families. The Kiambu5 isolate is considerably more divergent, with close to 40,000 SNPs relative to Muguga, including >8,500 non-synonymous mutations distributed among >1,700 (42.5 %) of the predicted genes. These genetic markers of the component stocks can be used to characterize the composition of new batches of the Muguga Cocktail. CONCLUSIONS: Differences among these three isolates, while extensive, represent only a small proportion of the genetic variation in the entire species. Given the efficacy of the Muguga Cocktail in inducing long-lasting protection against infections in the field, our results suggest that whole-organism vaccines against parasitic diseases can be highly efficacious despite considerable genome-wide differences relative to the isolates against which they protect.

Best practices in bioinformatics training for life scientists.

The mountains of data thrusting from the new landscape of modern high-throughput biology are irrevocably changing biomedical research and creating a near-insatiable demand for training in data management and manipulation and data mining and analysis. Among life scientists, from clinicians to environmental researchers, a common theme is the need not just to use, and gain familiarity with, bioinformatics tools and resources but also to understand their underlying fundamental theoretical and practical concepts. Providing bioinformatics training to empower life scientists to handle and analyse their data efficiently, and progress their research, is a challenge across the globe. Delivering good training goes beyond traditional lectures and resource-centric demos, using interactivity, problem-solving exercises and cooperative learning to substantially enhance training quality and learning outcomes. In this context, this article discusses various pragmatic criteria for identifying training needs and learning objectives, for selecting suitable trainees and trainers, for developing and maintaining training skills and evaluating training quality. Adherence to these criteria may help not only to guide course organizers and trainers on the path towards bioinformatics training excellence but, importantly, also to improve the training experience for life scientists.

Brachyspira suanatina sp. nov., an enteropathogenic intestinal spirochaete isolated from pigs and mallards: genomic and phenotypic characteristics.

BACKGROUND: The genus Brachyspira currently encompasses seven valid species that colonize the intestines of mammals and birds. In a previous study a group of strongly haemolytic isolates from pigs and mallards was provisionally described as a new species within genus Brachyspira, "B. suanatina", and enteropathogenic properties were demonstrated in a porcine challenge model. METHODS: In the current study characterization of B. suanatina was performed on the basis of cell morphology, growth characteristics, enzyme profiles, DNA-DNA hybridization (DDH) and whole genome comparisons. The draft genome sequence of B. suanatina strain AN4859/03 was determined and compared with the available genomes of all valid species of Brachyspira. RESULTS: According to morphological traits, growth characteristics and enzymatic profiles, B. suanatina was similar to the type strain of B. hyodysenteriae, but using the recommended threshold value of 70% similarity by DDH it did not belong to any of the recognized Brachyspira species (range 16-64% similarity). This was further supported by average nucleotide identity values. Phylogenetic analysis performed using housekeeping genes and core genomes of all valid Brachyspira sp. and "B. hampsonii" revealed that B. suanatina and B. intermedia formed a clade distinct from B. hyodysenteriae. By comparing the genomes of the three closely related species B. intermedia, B. hyodysenteriae and B. suanatina similar profiles of general genomic features and distribution of genes in different functional categories were obtained. However, the genome size of B. hyodysenteriae was smallest among the species, suggesting the possibility of reductive evolution in the divergence of this species. A bacteriophage region and a putative plasmid sequence were also found in the genome of B. suanatina strain AN4859/03. CONCLUSIONS: The results of our study suggest that despite being similar to B. hyodysenteriae phenotypically, B. suanatina should be regarded as a separate species based on its genetic characteristics. Based on characteristics presented in this report we propose that strains AN4859/03, AN1681:1/04, AN2384/04 and Dk12570-2 from pigs in Sweden and Denmark, and strains AN3949:2/02 and AN1418:2/01 isolated from mallards in Sweden, represent a unique species within genus Brachyspira. For this new species we propose the name B. suanatina for which the type strain is AN4859/03T (=ATCC® BAA-2592™=DSM 100974T).

Integrated Bio-Search: challenges and trends for the integration, search and comprehensive processing of biological information.

Many efforts exist to design and implement approaches and tools for data capture, integration and analysis in the life sciences. Challenges are not only the heterogeneity, size and distribution of information sources, but also the danger of producing too many solutions for the same problem. Methodological, technological, infrastructural and social aspects appear to be essential for the development of a new generation of best practices and tools. In this paper, we analyse and discuss these aspects from different perspectives, by extending some of the ideas that arose during the NETTAB 2012 Workshop, making reference especially to the European context. First, relevance of using data and software models for the management and analysis of biological data is stressed. Second, some of the most relevant community achievements of the recent years, which should be taken as a starting point for future efforts in this research domain, are presented. Third, some of the main outstanding issues, challenges and trends are analysed. The challenges related to the tendency to fund and create large scale international research infrastructures and public-private partnerships in order to address the complex challenges of data intensive science are especially discussed. The needs and opportunities of Genomic Computing (the integration, search and display of genomic information at a very specific level, e.g. at the level of a single DNA region) are then considered. In the current data and network-driven era, social aspects can become crucial bottlenecks. How these may best be tackled to unleash the technical abilities for effective data integration and validation efforts is then discussed. Especially the apparent lack of incentives for already overwhelmed researchers appears to be a limitation for sharing information and knowledge with other scientists. We point out as well how the bioinformatics market is growing at an unprecedented speed due to the impact that new powerful in silico analysis promises to have on better diagnosis, prognosis, drug discovery and treatment, towards personalized medicine. An open business model for bioinformatics, which appears to be able to reduce undue duplication of efforts and support the increased reuse of valuable data sets, tools and platforms, is finally discussed.

Relationship between genome and epigenome--challenges and requirements for future research.

Understanding the links between genetic, epigenetic and non-genetic factors throughout the lifespan and across generations and their role in disease susceptibility and disease progression offer entirely new avenues and solutions to major problems in our society. To overcome the numerous challenges, we have come up with nine major conclusions to set the vision for future policies and research agendas at the European level.

Studies of plant colonisation by closely related Bacillus amyloliquefaciens biocontrol agents using strain specific quantitative PCR assays.

Certain strains of Bacillus amyloliquefaciens can colonize plants and improve growth and stress management. In order to study these effects, bacterial growth dynamics on plants and in the rhizosphere are of interest calling for specific analytical tools. For that purpose, quantitative real-time PCR (qPCR) assays were developed in order to differentiate among three closely related B. amyloliquefaciens subsp. plantarum strains (UCMB5033, UCMB5036, UCMB5113) and to determine their levels with high accuracy. Oligonucleotide primers were designed for strain unique gene sequences and used for SYBR green based qPCR analysis. Standard curves covered a wide linear range (10(6)) of DNA amounts with the lowest detection level at 50 fg. Post-reaction melting curve analysis showed only a single product. Accurate threshold cycles were obtained, even in the presence of high excess of related Bacillus strains and total bacterial DNA from soil. Analysis of Bacillus colonisation after seed treatment of two oilseed rape cultivars (Oase and Ritz) grown on agar support showed a time dependent effect but that the bacteria mostly were found on root tissues and little on green tissues. The colonisation on plants grown in soil varied among the Bacillus strains where Oase seemed to house more bacteria than Ritz. Applied as a mixture, all three Bacillus strains co-existed on the roots of plants grown in soil. The qPCR assay in combination with other techniques will be a powerful tool to study plant interactions of these B. amyloliquefaciens biocontrol agents to further understand the requirements for successful interactions and improvement of plant properties.

A novel unstable duplication upstream of HAS2 predisposes to a breed-defining skin phenotype and a periodic fever syndrome in Chinese Shar-Pei dogs.

Hereditary periodic fever syndromes are characterized by recurrent episodes of fever and inflammation with no known pathogenic or autoimmune cause. In humans, several genes have been implicated in this group of diseases, but the majority of cases remain unexplained. A similar periodic fever syndrome is relatively frequent in the Chinese Shar-Pei breed of dogs. In the western world, Shar-Pei have been strongly selected for a distinctive thick and heavily folded skin. In this study, a mutation affecting both these traits was identified. Using genome-wide SNP analysis of Shar-Pei and other breeds, the strongest signal of a breed-specific selective sweep was located on chromosome 13. The same region also harbored the strongest genome-wide association (GWA) signal for susceptibility to the periodic fever syndrome (p(raw) = 2.3 × 10⁻6, p(genome) = 0.01). Dense targeted resequencing revealed two partially overlapping duplications, 14.3 Kb and 16.1 Kb in size, unique to Shar-Pei and upstream of the Hyaluronic Acid Synthase 2 (HAS2) gene. HAS2 encodes the rate-limiting enzyme synthesizing hyaluronan (HA), a major component of the skin. HA is up-regulated and accumulates in the thickened skin of Shar-Pei. A high copy number of the 16.1 Kb duplication was associated with an increased expression of HAS2 as well as the periodic fever syndrome (p < 0.0001). When fragmented, HA can act as a trigger of the innate immune system and stimulate sterile fever and inflammation. The strong selection for the skin phenotype therefore appears to enrich for a pleiotropic mutation predisposing these dogs to a periodic fever syndrome. The identification of HA as a major risk factor for this canine disease raises the potential of this glycosaminoglycan as a risk factor for human periodic fevers and as an important driver of chronic inflammation.

SNP and Structural Study of the Notch Superfamily Provides Insights and Novel Pharmacological Targets against the CADASIL Syndrome and Neurodegenerative Diseases.

The evolutionary conserved Notch signaling pathway functions as a mediator of direct cell-cell communication between neighboring cells during development. Notch plays a crucial role in various fundamental biological processes in a wide range of tissues. Accordingly, the aberrant signaling of this pathway underlies multiple genetic pathologies such as developmental syndromes, congenital disorders, neurodegenerative diseases, and cancer. Over the last two decades, significant data have shown that the Notch signaling pathway displays a significant function in the mature brains of vertebrates and invertebrates beyond neuronal development and specification during embryonic development. Neuronal connection, synaptic plasticity, learning, and memory appear to be regulated by this pathway. Specific mutations in human Notch family proteins have been linked to several neurodegenerative diseases including Alzheimer's disease, CADASIL, and ischemic injury. Neurodegenerative diseases are incurable disorders of the central nervous system that cause the progressive degeneration and/or death of brain nerve cells, affecting both mental function and movement (ataxia). There is currently a lot of study being conducted to better understand the molecular mechanisms by which Notch plays an essential role in the mature brain. In this study, an in silico analysis of polymorphisms and mutations in human Notch family members that lead to neurodegenerative diseases was performed in order to investigate the correlations among Notch family proteins and neurodegenerative diseases. Particular emphasis was placed on the study of mutations in the Notch3 protein and the structure analysis of the mutant Notch3 protein that leads to the manifestation of the CADASIL syndrome in order to spot possible conserved mutations and interpret the effect of these mutations in the Notch3 protein structure. Conserved mutations of cysteine residues may be candidate pharmacological targets for the potential therapy of CADASIL syndrome.

Whole genome sequences of 70 indigenous Ethiopian cattle.

Indigenous animal genetic resources play a crucial role in preserving global genetic diversity and supporting the livelihoods of millions of people. In Ethiopia, the majority of the cattle population consists of indigenous breeds. Understanding the genetic architecture of these cattle breeds is essential for effective management and conservation efforts. In this study, we sequenced DNA samples from 70 animals from seven indigenous cattle breeds, generating about two terabytes of pair-end reads with an average coverage of 14X. The sequencing data were pre-processed and mapped to the cattle reference genome (ARS-UCD1.2) with an alignment rate of 99.2%. Finally, the variant calling process produced approximately 35 million high-quality SNPs. These data provide a deeper understanding of the genetic landscape, facilitate the identification of causal mutations, and enable the exploration of evolutionary patterns to assist cattle improvement and sustainable utilization, particularly in the face of unpredictable climate changes.

Use of in silico approaches, synthesis and profiling of Pan-filovirus GP-1,2 preprotein specific antibodies.

Intermolecular interactions of protein-protein complexes play a principal role in the process of discovering new substances used in the diagnosis and treatment of many diseases. Among such complexes of proteins, we have to mention antibodies; they interact with specific antigens of two genera of single-stranded RNA viruses belonging to the family Filoviridae-Ebolavirus and Marburgvirus; both cause rare but fatal viral hemorrhagic fever in Africa, with pandemic potential. In this research, we conduct studies aimed at the design and evaluation of antibodies targeting the filovirus glycoprotein precursor GP-1,2 to develop potential targets for the pan-filovirus easy-to-use rapid diagnostic tests. The in silico research using the available 3D structure of the natural antibody-antigen complex was carried out to determine the stability of individual protein segments in the process of its formation and maintenance. The computed free binding energy of the complex and its decomposition for all amino acids allowed us to define the residues that play an essential role in the structure and indicated the spots where potential antibodies can be improved. Following that, the study involved targeting six epitopes of the filovirus GP1,2 with two polyclonal antibodies (pABs) and 14 monoclonal antibodies (mABs). The evaluation conducted using Enzyme Immunoassays tested 62 different sandwich combinations of monoclonal antibodies (mAbs), identifying 10 combinations that successfully captured the recombinant GP1,2 (rGP). Among these combinations, the sandwich option (3G2G12* - (rGP) - 2D8F11) exhibited the highest propensity for capturing the rGP antigen.

Fingerprinting Breast Milk; insights into Milk Exosomics.

Breast milk, often referred to as "liquid gold," is a complex biofluid that provides essential nutrients, immune factors, and developmental cues for newborns. Recent advancements in the field of exosome research have shed light on the critical role of exosomes in breast milk. Exosomes are nanosized vesicles that carry bioactive molecules, including proteins, lipids, nucleic acids, and miRNAs. These tiny messengers play a vital role in intercellular communication and are now being recognized as key players in infant health and development. This paper explores the emerging field of milk exosomics, emphasizing the potential of exosome fingerprinting to uncover valuable insights into the composition and function of breast milk. By deciphering the exosomal cargo, we can gain a deeper understanding of how breast milk influences neonatal health and may even pave the way for personalized nutrition strategies.