Nucleoid Size Scaling and Intracellular Organization of Translation across Bacteria.

The scaling of organelles with cell size is thought to be exclusive to eukaryotes. Here, we demonstrate that similar scaling relationships hold for the bacterial nucleoid. Despite the absence of a nuclear membrane, nucleoid size strongly correlates with cell size, independent of changes in DNA amount and across various nutrient conditions. This correlation is observed in diverse bacteria, revealing a near-constant ratio between nucleoid and cell size for a given species. As in eukaryotes, the nucleocytoplasmic ratio in bacteria varies greatly among species. This spectrum of nucleocytoplasmic ratios is independent of genome size, and instead it appears linked to the average population cell size. Bacteria with different nucleocytoplasmic ratios have a cytoplasm with different biophysical properties, impacting ribosome mobility and localization. Together, our findings identify new organizational principles and biophysical features of bacterial cells, implicating the nucleocytoplasmic ratio and cell size as determinants of the intracellular organization of translation.

A constant size extension drives bacterial cell size homeostasis.

Cell size control is an intrinsic feature of the cell cycle. In bacteria, cell growth and division are thought to be coupled through a cell size threshold. Here, we provide direct experimental evidence disproving the critical size paradigm. Instead, we show through single-cell microscopy and modeling that the evolutionarily distant bacteria Escherichia coli and Caulobacter crescentus achieve cell size homeostasis by growing, on average, the same amount between divisions, irrespective of cell length at birth. This simple mechanism provides a remarkably robust cell size control without the need of being precise, abating size deviations exponentially within a few generations. This size homeostasis mechanism is broadly applicable for symmetric and asymmetric divisions, as well as for different growth rates. Furthermore, our data suggest that constant size extension is implemented at or close to division. Altogether, our findings provide fundamentally distinct governing principles for cell size and cell-cycle control in bacteria.

Redefining the bacteriophage mv4 site-specific recombination system and the sequence specificity of its attB and core-attP sites.

Through their involvement in the integration and excision of a large number of mobile genetic elements, such as phages and integrative and conjugative elements (ICEs), site-specific recombination systems based on heterobivalent tyrosine recombinases play a major role in genome dynamics and evolution. However, despite hundreds of these systems having been identified in genome databases, very few have been described in detail, with none from phages that infect Bacillota (formerly Firmicutes). In this study, we reanalyzed the recombination module of Lactobacillus delbrueckii subsp. bulgaricus phage mv4, previously considered atypical compared with classical systems. Our results reveal that mv4 integrase is a 369 aa protein with all the structural hallmarks of recombinases from the Tn916 family and that it cooperatively interacts with its recombination sites. Using randomized DNA libraries, NGS sequencing, and other molecular approaches, we show that the 21-bp core-attP and attB sites have structural similarities to classical systems only if considering the nucleotide degeneracy, with two 7-bp inverted regions corresponding to mv4Int core-binding sites surrounding a 7-bp strand-exchange region. We also examined the different compositional constraints in the core-binding regions, which define the sequence space of permissible recombination sites.

Surprising and novel multivariate sequential patterns using odds ratio for temporal evolution in healthcare.

BACKGROUND: Pattern mining techniques are helpful tools when extracting new knowledge in real practice, but the overwhelming number of patterns is still a limiting factor in the health-care domain. Current efforts concerning the definition of measures of interest for patterns are focused on reducing the number of patterns and quantifying their relevance (utility/usefulness). However, although the temporal dimension plays a key role in medical records, few efforts have been made to extract temporal knowledge about the patient's evolution from multivariate sequential patterns. METHODS: In this paper, we propose a method to extract a new type of patterns in the clinical domain called Jumping Diagnostic Odds Ratio Sequential Patterns (JDORSP). The aim of this method is to employ the odds ratio to identify a concise set of sequential patterns that represent a patient's state with a statistically significant protection factor (i.e., a pattern associated with patients that survive) and those extensions whose evolution suddenly changes the patient's clinical state, thus making the sequential patterns a statistically significant risk factor (i.e., a pattern associated with patients that do not survive), or vice versa. RESULTS: The results of our experiments highlight that our method reduces the number of sequential patterns obtained with state-of-the-art pattern reduction methods by over 95%. Only by achieving this drastic reduction can medical experts carry out a comprehensive clinical evaluation of the patterns that might be considered medical knowledge regarding the temporal evolution of the patients. We have evaluated the surprisingness and relevance of the sequential patterns with clinicians, and the most interesting fact is the high surprisingness of the extensions of the patterns that become a protection factor, that is, the patients that recover after several days of being at high risk of dying. CONCLUSIONS: Our proposed method with which to extract JDORSP generates a set of interpretable multivariate sequential patterns with new knowledge regarding the temporal evolution of the patients. The number of patterns is greatly reduced when compared to those generated by other methods and measures of interest. An additional advantage of this method is that it does not require any parameters or thresholds, and that the reduced number of patterns allows a manual evaluation.

Atypical low-copy number plasmid segregation systems, all in one?

Plasmid families harbor different maintenances functions, depending on their size and copy number. Low copy number plasmids rely on active partition systems, organizing a partition complex at specific centromere sites that is actively positioned using NTPase proteins. Some low copy number plasmids lack an active partition system, but carry atypical intracellular positioning systems using a single protein that binds to the centromere site but without an associated NTPase. These systems have been studied in the case of the Escherichia coli R388 and of the Staphylococcus aureus pSK1 plasmids. Here we review these two systems, which appear to be unrelated but share common features, such as their distribution on plasmids of medium size and copy number, certain activities of their centromere-binding proteins, StbA and Par, respectively, as well as their mode of action, which may involve dynamic interactions with the nucleoid-packed chromosome of their hosts.

The use of networks in spatial and temporal computational models for outbreak spread in epidemiology: A systematic review.

OBJECTIVES: To examine recent literature in order to present a comprehensive overview of the current trends as regards the computational models used to represent the propagation of an infectious outbreak in a population, paying particular attention to those that represent network-based transmission. METHODS: a systematic review was conducted following the PRISMA (Preferred Reporting Items for Systematic Reviews and Meta-Analyses) guidelines. Papers published in English between 2010 and September 2021 were sought in the ACM Digital Library, IEEE Xplore, PubMed and Scopus databases. RESULTS: Upon considering their titles and abstracts, 832 papers were obtained, of which 192 were selected for a full content-body check. Of these, 112 studies were eventually deemed suitable for quantitative and qualitative analysis. Emphasis was placed on the spatial and temporal scales studied, the use of networks or graphs, and the granularity of the data used to evaluate the models. The models principally used to represent the spreading of outbreaks have been stochastic (55.36%), while the type of networks most frequently used are relationship networks (32.14%). The most common spatial dimension used is a region (19.64%) and the most used unit of time is a day (28.57%). Synthetic data as opposed to an external source were used in 51.79% of the papers. With regard to the granularity of the data sources, aggregated data such as censuses or transportation surveys are the most common. CONCLUSION: We identified a growing interest in the use of networks to represent disease transmission. We detected that research is focused on only certain combinations of the computational model, type of network (in both the expressive and the structural sense) and spatial scale, while the search for other interesting combinations has been left for the future.

Meaningful time-related aspects of alerts in Clinical Decision Support Systems. A unified framework.

Alerts are a common functionality of clinical decision support systems (CDSSs). Although they have proven to be useful in clinical practice, the alert burden can lead to alert fatigue and significantly reduce their usability and acceptance. Based on a literature review, we propose a unified framework consisting of a set of meaningful timestamps that allows the use of state-of-the-art measures for alert burden, such as alert dwell time, alert think time, and response time. In addition, it can be used to investigate other measures that could be relevant as regards dealing with this problem. Furthermore, we provide a case study concerning three different types of alerts to which the framework was successfully applied. We consider that our framework can easily be adapted to other CDSSs and that it could be useful for dealing with alert burden measurement thus contributing to its appropriate management.

A Novel Introgression Line Library Derived from a Wild Melon Gives Insights into the Genetics of Melon Domestication, Uncovering New Genetic Variability Useful for Breeding.

A collection of 30 melon introgression lines (ILs) was developed from the wild accession Ames 24297 (TRI) into 'Piel de Sapo' (PS) genetic background. Each IL carried an average of 1.4 introgressions from TRI, and the introgressions represented 91.4% of the TRI genome. Twenty-two ILs, representing 75% of the TRI genome, were evaluated in greenhouse (Algarrobo and Meliana) and field (Alcàsser) trials, mainly to study traits related to domestication syndrome such as fruit weight (FW) and flesh content (FFP), as well as other fruit quality traits as fruit shape (FS), flesh firmness (FF), soluble solid concentration (SSC), rind color and abscission layer. The IL collection showed an impressive variation in size-related traits, with FW ranging from 800 to 4100 g, reflecting the strong effect of the wild genome on these traits. Most of the ILs produced smaller fruits compared with PS; however, unexpectedly, the IL TRI05-2 produced bigger fruits, likely due to new epistatic interacions with the PS genetic background. In contrast, the genotypic effect for FS was smaller, and few QTLs with notable effects were detected. Interestingly, variability was also observed for FFP, FF and SSC, rind color and abscission layer formation. Genes in these introgressions are candidates for having been involved in melon domestication and diversification as well. These results confirm that the TRI IL collection is a very powerful tool for mapping traits of agronomic interest in melon, allowing the confirmation of previously reported QTLs and the identification of new ones to better understand the domestication process of this crop.

A cryptic variation in a member of the Ovate Family Proteins is underlying the melon fruit shape QTL fsqs8.1.

KEY MESSAGE: The gene underlying the melon fruit shape QTL fsqs8.1 is a member of the Ovate Family Proteins. Variation in fruit morphology is caused by changes in gene expression likely due to a cryptic structural variation in this locus. Melon cultivars have a wide range of fruit morphologies. Quantitative trait loci (QTL) have been identified underlying such diversity. This research focuses on the fruit shape QTL fsqs8.1, previously detected in a cross between the accession PI 124112 (CALC, producing elongated fruit) and the cultivar 'Piel de Sapo' (PS, producing oval fruit). The CALC fsqs8.1 allele induced round fruit shape, being responsible for the transgressive segregation for this trait observed in that population. In fact, the introgression line CALC8-1, carrying the fsqs8.1 locus from CALC into the PS genetic background, produced perfect round fruit. Following a map-based cloning approach, we found that the gene underlying fsqs8.1 is a member of the Ovate Family Proteins (OFP), CmOFP13, likely a homologue of AtOFP1 and SlOFP20 from Arabidopsis thaliana and tomato, respectively. The induction of the round shape was due to the higher expression of the CALC allele at the early ovary development stage. The fsqs8.1 locus showed an important structural variation, being CmOFP13 surrounded by two deletions in the CALC genome. The deletions are present at very low frequency in melon germplasm. Deletions and single nucleotide polymorphisms in the fsqs8.1 locus could not be not associated with variation in fruit shape among different melon accessions, what indicates that other genetic factors should be involved to induce the CALC fsqs8.1 allele effects. Therefore, fsqs8.1 is an example of a cryptic variation that alters gene expression, likely due to structural variation, resulting in phenotypic changes in melon fruit morphology.

Chronodisruption and Ambulatory Circadian Monitoring in Cancer Patients: Beyond the Body Clock.

PURPOSE OF REVIEW: Circadian rhythms impose daily rhythms a remarkable variety of metabolic and physiological functions, such as cell proliferation, inflammation, and DNA damage response. Accumulating epidemiological and genetic evidence indicates that circadian rhythms' disruption may be linked to cancer. The integration of circadian biology into cancer research may offer new options for increasing cancer treatment effectiveness and would encompass the prevention, diagnosis, and treatment of this disease. RECENT FINDINGS: In recent years, there has been a significant development and use of multi-modal sensors to monitor physical activity, sleep, and circadian rhythms, allowing, for the very first time, scaling accurate sleep monitoring to epidemiological research linking sleep patterns to disease, and wellness applications providing new potential applications. This review highlights the role of circadian clock in tumorigenesis, cancer hallmarks and introduces the state-of-the-art in sleep-monitoring technologies, discussing the eventual application of insights in clinical settings and cancer research.

Business Process Model and Notation and openEHR Task Planning for Clinical Pathway Standards in Infections: Critical Analysis.

BACKGROUND: Clinical pathways (CPs) are usually expressed by means of workflow formalisms, providing health care personnel with an easy-to-understand, high-level conceptual model of medical steps in specific patient conditions, thereby improving overall health care process quality in clinical practice. From a standardized perspective, the business process model and notation (BPMN), a widely spread general-purpose process formalism, has been used for conceptual modeling in clinical domains, mainly because of its easy-to-use graphical notation, facilitating the common understanding and communication of the parties involved in health care. However, BPMN is not particularly oriented toward the peculiarities of complex clinical processes such as infection diagnosis and treatment, in which time plays a critical role, which is why much of the BPMN clinical-oriented research has revolved around how to extend the standard to address these special needs. The shift from an agnostic, general-purpose BPMN notation to a natively clinical-oriented notation such as openEHR Task Planning (TP) could constitute a major step toward clinical process improvement, enhancing the representation of CPs for infection treatment and other complex scenarios. OBJECTIVE: Our work aimed to analyze the suitability of a clinical-oriented formalism (TP) to successfully represent typical process patterns in infection treatment, identifying domain-specific improvements to the standard that could help enhance its modeling capabilities, thereby promoting the widespread adoption of CPs to improve medical practice and overall health care quality. METHODS: Our methodology consisted of 4 major steps: identification of key features of infection CPs through literature review, clinical guideline analysis, and BPMN extensions; analysis of the presence of key features in TP; modeling of relevant process patterns of catheter-related bloodstream infection as a case study; and analysis and proposal of extensions in view of the results. RESULTS: We were able to easily represent the same logic applied in the extended BPMN-based process models in our case study using out-of-the-box standard TP primitives. However, we identified possible improvements to the current version of TP to allow for simpler conceptual models of infection CPs and possibly of other complex clinical scenarios. CONCLUSIONS: Our study showed that the clinical-oriented TP specification is able to successfully represent the most complex catheter-related bloodstream infection process patterns depicted in our case study and identified possible extensions that can help increase its adequacy for modeling infection CPs and possibly other complex clinical conditions.

Correlation tests between relative light unit and colony forming unit for improving adenosine triphosphate bioluminescence analysis in bacterial consolidation treatments on palaeontological heritage.

In this article bacterial carbonate mineralization treatments are proposed as a novel strategy for decayed fossils and palaeontological heritage conservation; specifically, by means of inoculation of Myxococcus xanthus, a bacterium of proven effectiveness in ornamental stone bioconsolidation. Bioconsolidation treatments can be very effective, stable, nontoxic, environmentally friendly, and chemically compatible with fossil heritage. The method reproduces what nature has been doing for millennia with fossils that have been permineralized by bacterial calcium carbonate precipitation. There is, however, some concern that bacterial inoculation could lead to the growth of undesirable microbiota, which could subsequently damage the fossil substrate. Because of this, the use of bacteria on heritage items must be meticulously monitored and analysis strategies should be carried out to detect bacteria viability during and after treatments. For this purpose, adenosine triphosphate assay is proposed in this article as a fast, affordable, portable, and easy-to-use system for conservators. as ATP assay results are relative and difficult to relate to colony forming unit, this study aims to improve their applicability by examining the correlation between ATP analysis and total viable bacteria count in the specific case of M. xanthus. This research provides reference and correlatable data to obtain an approximate estimation of M. xanthus viable bacterial colonies based on relative light unit data.

Replication fork passage drives asymmetric dynamics of a critical nucleoid-associated protein in Caulobacter.

In bacteria, chromosome dynamics and gene expression are modulated by nucleoid-associated proteins (NAPs), but little is known about how NAP activity is coupled to cell cycle progression. Using genomic techniques, quantitative cell imaging, and mathematical modeling, our study in Caulobacter crescentus identifies a novel NAP (GapR) whose activity over the cell cycle is shaped by DNA replication. GapR activity is critical for cellular function, as loss of GapR causes severe, pleiotropic defects in growth, cell division, DNA replication, and chromosome segregation. GapR also affects global gene expression with a chromosomal bias from origin to terminus, which is associated with a similar general bias in GapR binding activity along the chromosome. Strikingly, this asymmetric localization cannot be explained by the distribution of GapR binding sites on the chromosome. Instead, we present a mechanistic model in which the spatiotemporal dynamics of GapR are primarily driven by the progression of the replication forks. This model represents a simple mechanism of cell cycle regulation, in which DNA-binding activity is intimately linked to the action of DNA replication.

Genomewide phenotypic analysis of growth, cell morphogenesis, and cell cycle events in Escherichia coli.

Cell size, cell growth, and cell cycle events are necessarily intertwined to achieve robust bacterial replication. Yet, a comprehensive and integrated view of these fundamental processes is lacking. Here, we describe an image-based quantitative screen of the single-gene knockout collection of Escherichia coli and identify many new genes involved in cell morphogenesis, population growth, nucleoid (bulk chromosome) dynamics, and cell division. Functional analyses, together with high-dimensional classification, unveil new associations of morphological and cell cycle phenotypes with specific functions and pathways. Additionally, correlation analysis across ~4,000 genetic perturbations shows that growth rate is surprisingly not predictive of cell size. Growth rate was also uncorrelated with the relative timings of nucleoid separation and cell constriction. Rather, our analysis identifies scaling relationships between cell size and nucleoid size and between nucleoid size and the relative timings of nucleoid separation and cell division. These connections suggest that the nucleoid links cell morphogenesis to the cell cycle.

TERT promoter mutations are associated with poor prognosis in cutaneous squamous cell carcinoma.

BACKGROUND: Telomerase reverse transcriptase gene (TERT) promoter (TERTp) mutations have been reported as potential predictors of poor prognosis in several cancers, but the prognostic value of TERTp mutations for cutaneous squamous cell carcinoma (cSCC) has not been determined. OBJECTIVE: To evaluate the frequency of TERTp mutations and correlate it with clinicopathologic features and patient outcome. METHODS: We performed genetic profiling of TERTp mutations in a retrospective series of cSCCs. The predictive value of TERTp mutations and clinicopathologic parameters were assessed by using logistic regression models. RESULTS: A total of 152 cSCCs from 122 patients were analyzed for TERTp mutations; the mutation rate was 31.6% (48 of 152), and it was higher in invasive cSCC (42 of 121 [34.7%]) than in in situ cSCC (6 of 31 [19.4%]). Age older than 75 years (odds ratio [OR], 14.84; P = .013] and TERTp mutation (OR, 8.11; P = .002) were independent predictors of local recurrence. TERTp mutation (OR, 15.89; P = .022) was independently associated with higher risk of lymph node metastasis. LIMITATIONS: The restricted number of metastatic cases. CONCLUSION: TERTp mutations may prove to be a molecular biomarker with prognostic significance in invasive cSCC, but larger studies are needed.

Impact of expert knowledge on the detection of patients at risk of antimicrobial therapy failure by clinical decision support systems.

Antimicrobial Susceptibility Tests (ASTs) are performed in hospitals to detect whether an infectious agent is resistant or susceptible to a set of antimicrobials. When AST results are available, the evaluation of the patient's antimicrobial therapy is a critical task to ensure its effectiveness against the found microorganism. Since not all the available antimicrobials can be tested in ASTs, clinicians rely on their expert knowledge to complement AST results and prescribe the most appropriate antimicrobials for each infection. Our goal is to help physicians in this task by improving the detection of antimicrobial therapies at risk of failure by Clinical Decision Support Systems (CDSSs). With this aim, we have incorporated the EUCAST expert rules in antimicrobial susceptibility testing into a CDSS to improve the results of ASTs. In order to achieve this, we have combined both ontologies and production rules. Furthermore, we have evaluated the impact of EUCAST expert rules on the detection of antimicrobial therapies at risk of failure. We performed a retrospective study with one year of clinical data, obtaining a total of 148 alerts from which 62 (41.9%) were based on the additional expert knowledge. Furthermore, the evaluation of the clinical relevance of 27 alerts resulted in 8 of them (29.7%) being clinically relevant. Of these, 6 were based on expert knowledge. Finally, an alarm fatigue study suggests that waiting between 48 and 72 h from the reception of the AST results can significantly reduce the number of alerts that are unnecessary in our CDSS because they are already being addressed in the hospital's daily workflow. In conclusion, we demonstrate that the incorporation of expert knowledge improves the capabilities of CDSSs as regards detecting the risk of antimicrobial therapy failure, which may improve the institutional outcomes in antimicrobial stewardship.

DNA-relay mechanism is sufficient to explain ParA-dependent intracellular transport and patterning of single and multiple cargos.

Spatial ordering of macromolecular components inside cells is important for cellular physiology and replication. In bacteria, ParA/B systems are known to generate various intracellular patterns that underlie the transport and partitioning of low-copy-number cargos such as plasmids. ParA/B systems consist of ParA, an ATPase that dimerizes and binds DNA upon ATP binding, and ParB, a protein that binds the cargo and stimulates ParA ATPase activity. Inside cells, ParA is asymmetrically distributed, forming a propagating wave that is followed by the ParB-rich cargo. These correlated dynamics lead to cargo oscillation or equidistant spacing over the nucleoid depending on whether the cargo is in single or multiple copies. Currently, there is no model that explains how these different spatial patterns arise and relate to each other. Here, we test a simple DNA-relay model that has no imposed asymmetry and that only considers the ParA/ParB biochemistry and the known fluctuating and elastic dynamics of chromosomal loci. Stochastic simulations with experimentally derived parameters demonstrate that this model is sufficient to reproduce the signature patterns of ParA/B systems: the propagating ParA gradient correlated with the cargo dynamics, the single-cargo oscillatory motion, and the multicargo equidistant patterning. Stochasticity of ATP hydrolysis breaks the initial symmetry in ParA distribution, resulting in imbalance of elastic force acting on the cargo. Our results may apply beyond ParA/B systems as they reveal how a minimal system of two players, one binding to DNA and the other modulating this binding, can transform directionally random DNA fluctuations into directed motion and intracellular patterning.

Bullous lupus erythematosus with an erythema gyratum repens-like pattern.

Bullous lupus erythematosus is a rare clinical form of lupus. The diagnosis is challenging and involves the exclusion of other subepidermal bullous dermatoses. We present a 21-year-old woman with erythematosus, polycyclic plaques with vesiculobullae along the periphery, creating an erythema gyratum repens-like pattern on acral regions. The cutaneous biopsy, analytical, and autoimmune studies support the diagnosis of systemic lupus erythematosus. Dapsone and glucocorticosteroids were given with prompt resolution of the lesions within two weeks. To our knowledge this is the first case of bullous lupus erythematosus with this atypical acral presentation.

A decision support system for antibiotic prescription based on local cumulative antibiograms.

BACKGROUND: Local cumulative antibiograms are useful tools with which to select appropriate empiric or directed therapies when treating infectious diseases at a hospital. However, data represented in traditional antibiograms are static, incomplete and not well adapted to decision-making. METHODS: We propose a decision support method for empiric antibiotic therapy based on the Number Needed to Fail (NNF) measure. NNF indicates the number of patients that would need to be treated with a specific antibiotic for one to be inadequately treated. We define two new measures, Accumulated Efficacy and Weighted Accumulated Efficacy in order to determine the efficacy of an antibiotic. We carried out two experiments: the first during which there was a suspicion of infection and the patient had empiric therapy, and the second by considering patients with confirmed infection and directed therapy. The study was performed with 15,799 cultures with 356,404 susceptibility tests carried out over a four-year period. RESULTS: The most efficient empiric antibiotics are Linezolid and Vancomycin for blood samples and Imipenem and Meropenem for urine samples. In both experiments, the efficacies of recommended antibiotics are all significantly greater than the efficacies of the antibiotics actually administered (P < 0.001). The highest efficacy is obtained when considering 2 years of antibiogram data and 80% of the cumulated prevalence of microorganisms. CONCLUSION: This extensive study on real empiric therapies shows that the proposed method is a valuable alternative to traditional antibiograms as regards developing clinical decision support systems for antimicrobial stewardship.

Oufti: an integrated software package for high-accuracy, high-throughput quantitative microscopy analysis.

With the realization that bacteria display phenotypic variability among cells and exhibit complex subcellular organization critical for cellular function and behavior, microscopy has re-emerged as a primary tool in bacterial research during the last decade. However, the bottleneck in today's single-cell studies is quantitative image analysis of cells and fluorescent signals. Here, we address current limitations through the development of Oufti, a stand-alone, open-source software package for automated measurements of microbial cells and fluorescence signals from microscopy images. Oufti provides computational solutions for tracking touching cells in confluent samples, handles various cell morphologies, offers algorithms for quantitative analysis of both diffraction and non-diffraction-limited fluorescence signals and is scalable for high-throughput analysis of massive datasets, all with subpixel precision. All functionalities are integrated in a single package. The graphical user interface, which includes interactive modules for segmentation, image analysis and post-processing analysis, makes the software broadly accessible to users irrespective of their computational skills.