Airway basal stem cells are necessary for the maintenance of functional intraepithelial airway macrophages.

Adult stem cells play a crucial role in tissue homeostasis and repair through multiple mechanisms. In addition to being able to replace aged or damaged cells, stem cells provide signals that contribute to the maintenance and function of neighboring cells. In the lung, airway basal stem cells also produce cytokines and chemokines in response to inhaled irritants, allergens, and pathogens, which affect specific immune cell populations and shape the nature of the immune response. However, direct cell-to-cell signaling through contact between airway basal stem cells and immune cells has not been demonstrated. Recently, a unique population of intraepithelial airway macrophages (IAMs) has been identified in the murine trachea. Here, we demonstrate that IAMs require Notch signaling from airway basal stem cells for maintenance of their differentiated state and function. Furthermore, we demonstrate that Notch signaling between airway basal stem cells and IAMs is required for antigen-induced allergic inflammation only in the trachea where the basal stem cells are located whereas allergic responses in distal lung tissues are preserved consistent with a local circuit linking stem cells to proximate immune cells. Finally, we demonstrate that IAM-like cells are present in human conducting airways and that these cells display Notch activation, mirroring their murine counterparts. Since diverse lung stem cells have recently been identified and localized to specific anatomic niches along the proximodistal axis of the respiratory tree, we hypothesize that the direct functional coupling of local stem cell-mediated regeneration and immune responses permits a compartmentalized inflammatory response.

A nasal cell atlas reveals heterogeneity of tuft cells and their role in directing olfactory stem cell proliferation.

The olfactory neuroepithelium serves as a sensory organ for odors and forms part of the nasal mucosal barrier. Olfactory sensory neurons are surrounded and supported by epithelial cells. Among them, microvillous cells (MVCs) are strategically positioned at the apical surface, but their specific functions are enigmatic, and their relationship to the other specialized epithelial cells is unclear. Here, we establish that the family of MVCs comprises tuft cells and ionocytes in both mice and humans. Integrating analysis of the respiratory and olfactory epithelia, we define the distinct receptor expression of TRPM5+ tuft-MVCs compared with Gɑ-gustducinhigh respiratory tuft cells and characterize a previously undescribed population of glandular DCLK1+ tuft cells. To establish how allergen sensing by tuft-MVCs might direct olfactory mucosal responses, we used an integrated single-cell transcriptional and protein analysis. Inhalation of Alternaria induced mucosal epithelial effector molecules including Chil4 and a distinct pathway leading to proliferation of the quiescent olfactory horizontal basal stem cell (HBC) pool, both triggered in the absence of olfactory apoptosis. Alternaria- and ATP-elicited HBC proliferation was dependent on TRPM5+ tuft-MVCs, identifying these specialized epithelial cells as regulators of olfactory stem cell responses. Together, our data provide high-resolution characterization of nasal tuft cell heterogeneity and identify a function of TRPM5+ tuft-MVCs in directing the olfactory mucosal response to allergens.

Transgenic ferret models define pulmonary ionocyte diversity and function.

Speciation leads to adaptive changes in organ cellular physiology and creates challenges for studying rare cell-type functions that diverge between humans and mice. Rare cystic fibrosis transmembrane conductance regulator (CFTR)-rich pulmonary ionocytes exist throughout the cartilaginous airways of humans1,2, but limited presence and divergent biology in the proximal trachea of mice has prevented the use of traditional transgenic models to elucidate ionocyte functions in the airway. Here we describe the creation and use of conditional genetic ferret models to dissect pulmonary ionocyte biology and function by enabling ionocyte lineage tracing (FOXI1-CreERT2::ROSA-TG), ionocyte ablation (FOXI1-KO) and ionocyte-specific deletion of CFTR (FOXI1-CreERT2::CFTRL/L). By comparing these models with cystic fibrosis ferrets3,4, we demonstrate that ionocytes control airway surface liquid absorption, secretion, pH and mucus viscosity-leading to reduced airway surface liquid volume and impaired mucociliary clearance in cystic fibrosis, FOXI1-KO and FOXI1-CreERT2::CFTRL/L ferrets. These processes are regulated by CFTR-dependent ionocyte transport of Cl- and HCO3-. Single-cell transcriptomics and in vivo lineage tracing revealed three subtypes of pulmonary ionocytes and a FOXI1-lineage common rare cell progenitor for ionocytes, tuft cells and neuroendocrine cells during airway development. Thus, rare pulmonary ionocytes perform critical CFTR-dependent functions in the proximal airway that are hallmark features of cystic fibrosis airway disease. These studies provide a road map for using conditional genetics in the first non-rodent mammal to address gene function, cell biology and disease processes that have greater evolutionary conservation between humans and ferrets.

Single-cell transcriptomics of a dynamic cell behavior in murine airways.

Despite advances in high-dimensional cellular analysis, the molecular profiling of dynamic behaviors of cells in their native environment remains a major challenge. We present a method that allows us to couple the physiological behaviors of cells in an intact murine tissue to deep molecular profiling of individual cells. This method enabled us to establish a novel molecular signature for a striking migratory cellular behavior following injury in murine airways.

Health care professionals' perceptions of unprofessional behaviour in the clinical workplace.

BACKGROUND: Unprofessional behaviour undermines organizational trust and negatively affects patient safety, the clinical learning environment, and clinician well-being. Improving professionalism in healthcare organizations requires insight into the frequency, types, sources, and targets of unprofessional behaviour in order to refine organizational programs and strategies to prevent and address unprofessional behaviours. OBJECTIVE: To investigate the types and frequency of perceived unprofessional behaviours among health care professionals and to identify the sources and targets of these behaviours. METHODS: Data was collected from 2017-2019 based on a convenience sample survey administered to all participants at the start of a mandatory professionalism course for health care professionals including attending physicians, residents and advanced practice providers (APPs) working at one academic hospital in the United States. RESULTS: Out of the 388 participants in this study, 63% experienced unprofessional behaviour at least once a month, including failing to respond to calls/pages/requests (44.3%), exclusion from decision-making (43.0%) and blaming behaviour (39.9%). Other monthly experienced subtypes ranged from 31.7% for dismissive behaviour to 4.6% for sexual harassment. Residents were more than twice as likely (OR 2.25, p<0.001)) the targets of unprofessional behaviour compared to attending physicians. Female respondents experienced more discriminating behaviours (OR 2.52, p<0.01). Nurses were identified as the most common source of unprofessional behaviours (28.1%), followed by residents from other departments (21%). CONCLUSIONS: Unprofessional behaviour was experienced frequently by all groups, mostly inflicted on these groups by those outside of the own discipline or department. Residents were most frequently identified to be the target and nurses the source of the behaviours. This study highlights that unprofessional behaviour is varied, both regarding types of behaviours as well as targets and sources of such behaviours. This data is instrumental in developing training and remediation initiatives attuned to specific professional roles and specific types of professionalism lapses.

Mapping emergency department asthma visits to identify poor-quality housing in New Haven, CT, USA: a retrospective cohort study.

BACKGROUND: Housing conditions are a key driver of asthma incidence and severity. Previous studies have shown increased emergency department visits for asthma among residents living in poor-quality housing. Interventions to improve housing conditions have been shown to reduce emergency department visits for asthma, but identification and remediation of poor housing conditions is often delayed or does not occur. This study evaluates whether emergency department visits for asthma can be used to identify poor-quality housing to support proactive and early intervention. METHODS: We conducted a retrospective cohort study of children and adults living in and around New Haven, CT, USA, who were seen for asthma in an urban, tertiary emergency department between March 1, 2013, and Aug 31, 2017. We geocoded and mapped patient addresses to city parcels, and calculated a composite estimate of the incidence of emergency department use for asthma for each parcel (Nv × Np/log2[P], where Nv is the estimated mean number of visits per patient, Np is the number of patients, and P is the estimated population). To determine whether parcel-level emergency department use for asthma was associated with public housing inspection scores, we used regression analyses, adjusting for neighbourhood-level and individual-level factors contributing to emergency department use for asthma. Public housing complex inspection scores were obtained from standardised home inspections, which are conducted every 1-3 years for publicly funded housing. We used a sliding-window approach to estimate how far in advance of a failed inspection the model could identify elevated use of emergency departments for asthma, using the city-wide 90th percentile as a cutoff for elevated incidence. FINDINGS: 11 429 asthma-related emergency department visits from 6366 unique patients were included in the analysis. Mean patient age was 32·4 years (SD 12·8); 3836 (60·3%) patients were female, 2530 (39·7%) were male, 3461 (57·2%) were Medicaid-insured, and 2651 (41·6%) were Black. Incidence of emergency department use for asthma was strongly correlated with lower housing inspection scores (Pearson's r=-0·55 [95% CI -0·70 to -0·35], p=3·5 × 10-6), and this correlation persisted after adjustment for patient-level and neighbourhood-level demographics using a linear regression model (r=-0·54 [-0·69 to -0·33], p=7·1 × 10-6) and non-linear regression model (r=-0·44 [-0·62 to -0·21], p=3·8 × 10-4). Elevated asthma incidence rates were typically detected around a year before a housing complex failed a housing inspection. INTERPRETATION: Emergency department visits for asthma are an early indicator of failed housing inspections. This approach represents a novel method for the early identification of poor housing conditions and could help to reduce asthma-related morbidity and mortality. FUNDING: Harvard-National Institute of Environmental Health Sciences (NIEHS) Center for Environmental Health.

Unequal Housing Conditions And Code Enforcement Contribute To Asthma Disparities In Boston, Massachusetts.

Housing quality is a primary determinant of asthma disparities by race and social class in the US. We sought to assess how housing code enforcement systems in Boston, Massachusetts, address tenants' reports of asthma triggers. After adjustment for income and other neighborhood characteristics, racial demographics were significantly associated with asthma trigger incidence. For each 10 percent decrease in neighborhood proportion of White residents, trigger incidence increased by 3.14 reports per thousand residents. These disparities persisted during the study period (from 2011 through 2021), and for mold, which is an established asthma trigger, regressions showed that racial disparities are widening. The municipal response also demonstrated disparities: In neighborhoods with the fewest White residents compared to neighborhoods with the most White residents, adjusted models showed a 17 percent (3.51 days) slower median time until cases (tenant requests for inspections to the Inspectional Services Department) were closed, a 14 percent higher probability of being flagged as overdue, and a 54.4 percent lower probability of a repair. We found evidence that in Boston, despite several healthy housing initiatives, current regulatory systems are insufficient to address disparities in access to healthy housing. To reduce disparities in asthma burden, stronger inspectional standards and further enforcement policies to increase landlords' accountability and support tenants' rights to have repairs made are essential.

Tuft cell-produced cysteinyl leukotrienes and IL-25 synergistically initiate lung type 2 inflammation.

Aeroallergen sensing by airway epithelial cells triggers pathogenic immune responses leading to type 2 inflammation, the hallmark of chronic airway diseases such as asthma. Tuft cells are rare epithelial cells and the dominant source of interleukin-25 (IL-25), an epithelial cytokine, and cysteinyl leukotrienes (CysLTs), lipid mediators of vascular permeability and chemotaxis. How these two mediators derived from the same cell might cooperatively promote type 2 inflammation in the airways has not been clarified. Here, we showed that inhalation of the parent leukotriene C4 (LTC4) in combination with a subthreshold dose of IL-25 led to activation of two innate immune cells: inflammatory type 2 innate lymphoid cell (ILC2) for proliferation and cytokine production, and dendritic cells (DCs). This cooperative effect led to a much greater recruitment of eosinophils and CD4+ T cell expansion indicative of synergy. Whereas lung eosinophilia was dominantly mediated through the classical CysLT receptor CysLT1R, type 2 cytokines and activation of innate immune cells required signaling through CysLT1R and partially CysLT2R. Tuft cell­specific deletion of Ltc4s, the terminal enzyme required for CysLT production, reduced lung inflammation and the systemic immune response after inhalation of the mold aeroallergen Alternaria; this effect was further enhanced by concomitant blockade of IL-25. Our findings identified a potent synergy of CysLTs and IL-25 downstream of aeroallergen-trigged activation of airway tuft cells leading to a highly polarized type 2 immune response and further implicate airway tuft cells as powerful modulators of type 2 immunity in the lungs.

Airway basal stem cells generate distinct subpopulations of PNECs.

Pulmonary neuroendocrine cells (PNECs) have crucial roles in airway physiology and immunity by producing bioactive amines and neuropeptides (NPs). A variety of human diseases exhibit PNEC hyperplasia. Given accumulated evidence that PNECs represent a heterogenous population of cells, we investigate how PNECs differ, whether the heterogeneity is similarly present in mouse and human cells, and whether specific disease involves discrete PNECs. Herein, we identify three distinct types of PNECs in human and mouse airways based on single and double positivity for TUBB3 and the established NP markers. We show that the three PNEC types exhibit significant differences in NP expression, homeostatic turnover, and response to injury and disease. We provide evidence that these differences parallel their distinct cell of origin from basal stem cells (BSCs) or other airway epithelial progenitors.

Single-cell meta-analysis of SARS-CoV-2 entry genes across tissues and demographics.

Angiotensin-converting enzyme 2 (ACE2) and accessory proteases (TMPRSS2 and CTSL) are needed for severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) cellular entry, and their expression may shed light on viral tropism and impact across the body. We assessed the cell-type-specific expression of ACE2, TMPRSS2 and CTSL across 107 single-cell RNA-sequencing studies from different tissues. ACE2, TMPRSS2 and CTSL are coexpressed in specific subsets of respiratory epithelial cells in the nasal passages, airways and alveoli, and in cells from other organs associated with coronavirus disease 2019 (COVID-19) transmission or pathology. We performed a meta-analysis of 31 lung single-cell RNA-sequencing studies with 1,320,896 cells from 377 nasal, airway and lung parenchyma samples from 228 individuals. This revealed cell-type-specific associations of age, sex and smoking with expression levels of ACE2, TMPRSS2 and CTSL. Expression of entry factors increased with age and in males, including in airway secretory cells and alveolar type 2 cells. Expression programs shared by ACE2+TMPRSS2+ cells in nasal, lung and gut tissues included genes that may mediate viral entry, key immune functions and epithelial-macrophage cross-talk, such as genes involved in the interleukin-6, interleukin-1, tumor necrosis factor and complement pathways. Cell-type-specific expression patterns may contribute to the pathogenesis of COVID-19, and our work highlights putative molecular pathways for therapeutic intervention.

A Synthesis Concerning Conservation and Divergence of Cell Types across Epithelia.

Advances in single-cell RNA-seq (scRNA-seq) and computational analysis have enabled the systematic interrogation of the cellular composition of tissues. Combined with tools from developmental biology, cell biology, and genetics, these approaches are revealing fundamental aspects of tissue geometry and physiology, including the distribution, origins, and inferred functions of specialized cell types, and the dynamics of cellular turnover and differentiation. By comparing different tissues, such studies can delineate shared and specialized features of cell types and their lineage. Here, we compare two developmentally related murine epithelia, the airway and the small intestinal epithelia, which are both derived from the embryonic endodermal gut tube. We examine how airway and intestine generate and functionalize common archetypal cell types to fulfill similar shared physiologic functionalities. We point to cases in which similar cell types are repurposed to accommodate each tissue's unique physiologic role, and highlight tissue-specific cells whose specializations contribute to the distinct functional roles of each organ. We discuss how archetypal and unique cell types are incorporated within a cellular lineage, and how the regulation of the proportions of these cell types enables tissue-level organization to meet functional demands and maintain homeostasis.

The Taste Receptor TAS1R3 Regulates Small Intestinal Tuft Cell Homeostasis.

Tuft cells are an epithelial cell type critical for initiating type 2 immune responses to parasites and protozoa in the small intestine. To respond to these stimuli, intestinal tuft cells use taste chemosensory signaling pathways, but the role of taste receptors in type 2 immunity is poorly understood. In this study, we show that the taste receptor TAS1R3, which detects sweet and umami in the tongue, also regulates tuft cell responses in the distal small intestine. BALB/c mice, which have an inactive form of TAS1R3, as well as Tas1r3-deficient C57BL6/J mice both have severely impaired responses to tuft cell-inducing signals in the ileum, including the protozoa Tritrichomonas muris and succinate. In contrast, TAS1R3 is not required to mount an immune response to the helminth Heligmosomoides polygyrus, which infects the proximal small intestine. Examination of uninfected Tas1r3-/- mice revealed a modest reduction in the number of tuft cells in the proximal small intestine but a severe decrease in the distal small intestine at homeostasis. Together, these results suggest that TAS1R3 influences intestinal immunity by shaping the epithelial cell landscape at steady-state.

Distinct Tissue-Specific Roles for the Disease-Associated Autophagy Genes ATG16L2 and ATG16L1.

The clear role of autophagy in human inflammatory diseases such as Crohn disease was first identified by genome-wide association studies and subsequently dissected in multiple mechanistic studies. ATG16L1 has been particularly well studied in knockout and hypomorph settings as well as models recapitulating the Crohn disease-associated T300A polymorphism. Interestingly, ATG16L1 has a single homolog, ATG16L2, which is independently implicated in diseases, including Crohn disease and systemic lupus erythematosus. However, the contribution of ATG16L2 to canonical autophagy pathways and other cellular functions is poorly understood. To better understand its role, we generated and analyzed the first, to our knowledge, ATG16L2 knockout mouse. Our results show that ATG16L1 and ATG16L2 contribute very distinctly to autophagy and cellular ontogeny in myeloid, lymphoid, and epithelial lineages. Dysregulation of any of these lineages could contribute to complex diseases like Crohn disease and systemic lupus erythematosus, highlighting the value of examining cell-specific effects. We also identify a novel genetic interaction between ATG16L2 and epithelial ATG16L1. These findings are discussed in the context of how these genes may contribute distinctly to human disease.

Ketone Body Signaling Mediates Intestinal Stem Cell Homeostasis and Adaptation to Diet.

Little is known about how metabolites couple tissue-specific stem cell function with physiology. Here we show that, in the mammalian small intestine, the expression of Hmgcs2 (3-hydroxy-3-methylglutaryl-CoA synthetase 2), the gene encoding the rate-limiting enzyme in the production of ketone bodies, including beta-hydroxybutyrate (ßOHB), distinguishes self-renewing Lgr5+ stem cells (ISCs) from differentiated cell types. Hmgcs2 loss depletes ßOHB levels in Lgr5+ ISCs and skews their differentiation toward secretory cell fates, which can be rescued by exogenous ßOHB and class I histone deacetylase (HDAC) inhibitor treatment. Mechanistically, ßOHB acts by inhibiting HDACs to reinforce Notch signaling, instructing ISC self-renewal and lineage decisions. Notably, although a high-fat ketogenic diet elevates ISC function and post-injury regeneration through ßOHB-mediated Notch signaling, a glucose-supplemented diet has the opposite effects. These findings reveal how control of ßOHB-activated signaling in ISCs by diet helps to fine-tune stem cell adaptation in homeostasis and injury.

Intra- and Inter-cellular Rewiring of the Human Colon during Ulcerative Colitis.

Genome-wide association studies (GWAS) have revealed risk alleles for ulcerative colitis (UC). To understand their cell type specificities and pathways of action, we generate an atlas of 366,650 cells from the colon mucosa of 18 UC patients and 12 healthy individuals, revealing 51 epithelial, stromal, and immune cell subsets, including BEST4+ enterocytes, microfold-like cells, and IL13RA2+IL11+ inflammatory fibroblasts, which we associate with resistance to anti-TNF treatment. Inflammatory fibroblasts, inflammatory monocytes, microfold-like cells, and T cells that co-express CD8 and IL-17 expand with disease, forming intercellular interaction hubs. Many UC risk genes are cell type specific and co-regulated within relatively few gene modules, suggesting convergence onto limited sets of cell types and pathways. Using this observation, we nominate and infer functions for specific risk genes across GWAS loci. Our work provides a framework for interrogating complex human diseases and mapping risk variants to cell types and pathways.

Streamlined Protocol for Deep Proteomic Profiling of FAC-sorted Cells and Its Application to Freshly Isolated Murine Immune Cells.

Proteomic profiling describes the molecular landscape of proteins in cells immediately available to sense, transduce, and enact the appropriate responses to extracellular queues. Transcriptional profiling has proven invaluable to our understanding of cellular responses; however, insights may be lost as mounting evidence suggests transcript levels only moderately correlate with protein levels in steady state cells. Mass spectrometry-based quantitative proteomics is a well-suited and widely used analytical tool for studying global protein abundances. Typical proteomic workflows are often limited by the amount of sample input that is required for deep and quantitative proteome profiling. This is especially true if the cells of interest need to be purified by fluorescence-activated cell sorting (FACS) and one wants to avoid ex vivo culturing. To address this need, we developed an easy to implement, streamlined workflow that enables quantitative proteome profiling from roughly 2 µg of protein input per experimental condition. Utilizing a combination of facile cell collection from cell sorting, solid-state isobaric labeling and multiplexing of peptides, and small-scale fractionation, we profiled the proteomes of 12 freshly isolated, primary murine immune cell types. Analyzing half of the 3e5 cells collected per cell type, we quantified over 7000 proteins across 12 key immune cell populations directly from their resident tissues. We show that low input proteomics is precise, and the data generated accurately reflects many aspects of known immunology, while expanding the list of cell-type specific proteins across the cell types profiled. The low input proteomics methods we developed are readily adaptable and broadly applicable to any cell or sample types and should enable proteome profiling in systems previously unattainable.

Author Correction: Comprehensive comparative analysis of 5'-end RNA-sequencing methods.

The original version of this paper contained an incorrect primer sequence. In the Methods subsection "Rampage libraries," the text for modification 3 stated that the reverse primer used for library indexing was 5'-CAAGCAGAAGACGGCATACGAGATXXXXXXXXGTGACTGGAGT-3'. The correct sequence of the oligonucleotide used is 5'-CAAGCAGAAGACGGCATACGAGATXXXXXXXXGTGACTGGAGTTCAGACGTGTGCTCTTCCGATCT-3'. This error has been corrected in the PDF and HTML versions of the paper.

T Helper Cell Cytokines Modulate Intestinal Stem Cell Renewal and Differentiation.

In the small intestine, a niche of accessory cell types supports the generation of mature epithelial cell types from intestinal stem cells (ISCs). It is unclear, however, if and how immune cells in the niche affect ISC fate or the balance between self-renewal and differentiation. Here, we use single-cell RNA sequencing (scRNA-seq) to identify MHC class II (MHCII) machinery enrichment in two subsets of Lgr5+ ISCs. We show that MHCII+ Lgr5+ ISCs are non-conventional antigen-presenting cells in co-cultures with CD4+ T helper (Th) cells. Stimulation of intestinal organoids with key Th cytokines affects Lgr5+ ISC renewal and differentiation in opposing ways: pro-inflammatory signals promote differentiation, while regulatory cells and cytokines reduce it. In vivo genetic perturbation of Th cells or MHCII expression on Lgr5+ ISCs impacts epithelial cell differentiation and IEC fate during infection. These interactions between Th cells and Lgr5+ ISCs, thus, orchestrate tissue-wide responses to external signals.

A revised airway epithelial hierarchy includes CFTR-expressing ionocytes.

The airways of the lung are the primary sites of disease in asthma and cystic fibrosis. Here we study the cellular composition and hierarchy of the mouse tracheal epithelium by single-cell RNA-sequencing (scRNA-seq) and in vivo lineage tracing. We identify a rare cell type, the Foxi1+ pulmonary ionocyte; functional variations in club cells based on their location; a distinct cell type in high turnover squamous epithelial structures that we term 'hillocks'; and disease-relevant subsets of tuft and goblet cells. We developed 'pulse-seq', combining scRNA-seq and lineage tracing, to show that tuft, neuroendocrine and ionocyte cells are continually and directly replenished by basal progenitor cells. Ionocytes are the major source of transcripts of the cystic fibrosis transmembrane conductance regulator in both mouse (Cftr) and human (CFTR). Knockout of Foxi1 in mouse ionocytes causes loss of Cftr expression and disrupts airway fluid and mucus physiology, phenotypes that are characteristic of cystic fibrosis. By associating cell-type-specific expression programs with key disease genes, we establish a new cellular narrative for airways disease.

Comprehensive comparative analysis of 5'-end RNA-sequencing methods.

Specialized RNA-seq methods are required to identify the 5' ends of transcripts, which are critical for studies of gene regulation, but these methods have not been systematically benchmarked. We directly compared six such methods, including the performance of five methods on a single human cellular RNA sample and a new spike-in RNA assay that helps circumvent challenges resulting from uncertainties in annotation and RNA processing. We found that the 'cap analysis of gene expression' (CAGE) method performed best for mRNA and that most of its unannotated peaks were supported by evidence from other genomic methods. We applied CAGE to eight brain-related samples and determined sample-specific transcription start site (TSS) usage, as well as a transcriptome-wide shift in TSS usage between fetal and adult brain.