Short-term GSNO catabolism by GGT and IL-8 production in serumfree medium Both the IB3-1 cell line and LHC-8 complete medium (i.e., supplemented with serum) present quite low GGT activities, corresponding to 1.270.041 mU/mg protein  and 0.270.07 mU/ml, respectively.
[Show abstract] [Hide abstract] ABSTRACT: S-Nitrosoglutathione (GSNO) is an endogenous nitrosothiol involved in several pathophysiological processes. A role of GSNO has been envisaged in the expression of inflammatory cytokines such as IL-8, however conflicting results have been reported. Gamma-glutamyltransferase (GGT) enzyme activity can hydrolyze the gamma-glutamyl bond present in GSNO molecule thus greatly accelerating the release of bioactive nitric oxide (NO). Expression of GGT is induced by oxidative stress, and activated neutrophils contribute to GGT increase in cystic fibrosis (CF) lung exudates by releasing GGT-containing microvescicles. The present study was aimed to evaluate the effect of GSNO catabolism mediated by GGT on production of IL-8 in CFTR-mutated IB3-1 bronchial cells.
The rapid, GGT-catalysed catabolism of GSNO caused a decrease of both basal and LPS-stimulated IL-8 production in IB3-1 cells, by modulating both NF-κB and ERK1/2 pathways, along with a decrease of cell proliferation. On the contrary, a slow decomposition of GSNO produced a significant increase of both cell proliferation and expression of IL-8, the latter possibly through p38-mediated stabilization of IL-8 mRNA.
Our data suggest that the differential GSNO catabolism mediated by GGT enzyme activity can downregulate the production of IL-8 in CF cells. Hence, the role of GGT activity should be considered when evaluating GSNO for both in vitro and in vivo studies, the more so in the case of GSNO-based therapies for cystic fibrosis.
The cell membrane depolarization assay demonstrated functional response only in monocytes from healthy controls and, to a lesser extent, from obligate CF heterozygotes. Of relevance, monocytes from CF patients showed a completely different pattern of response permitting the correct classification of healthy and diseased subjects . As a reference, the authors measured NPD in selected subjects in parallel to the monocyte assay and always obtained consistent results.
[Show abstract] [Hide abstract] ABSTRACT: In the majority of cases, there is no difficulty in diagnosing Cystic Fibrosis (CF). However, there may be wide variation in signs and symptoms between individuals which encourage the scientific community to constantly improve the diagnostic tests available and develop better methods to come to a final diagnosis in patients with milder phenotypes. This paper is the result of discussions held at meetings of the European Cystic Fibrosis Society Diagnostic Network supported by EuroCareCF. CFTR bioassays in the nasal epithelium (nasal potential difference measurements) and the rectal mucosa (intestinal current measurements) are discussed in detail including efforts to standardize the techniques across Europe. New approaches to evaluate the sweat gland, future of genetic testing and methods on the horizon like CFTR expression in human leucocytes and erythrocytes are discussed briefly.
However, in the airway system, higher expression of HLA-G is associated with P. aeruginosa infection. Moreover, CF cell line and murine model expressed higher HLA-G molecules in the presence of P. aeruginosa, thus suggesting a role of HLA-G in reducing systemic inflammation, thus supporting P. aeruginosa infection . Psoriasis 14 bp del/del genotype of HLA-G gene is more frequent in patients responding to therapy Positive correlation with response to treatment Borghi et al.  Asthma SNPs in 3 í® í° UTR of HLA-G gene modulate the binding of mIRNAs
[Show abstract] [Hide abstract] ABSTRACT: HLA-G is a HLA-class Ib molecule with potent immunomodulatory activities, which is expressed in physiological conditions, where modulation of the immune response is required to avoid allograft recognition (i.e., maternal-fetal interface or transplanted patients). However, HLA-G can be expressed de novo at high levels in several pathological conditions, including solid and hematological tumors and during microbial or viral infections, leading to the impairment of the immune response against tumor cells or pathogens, respectively. On the other hand, the loss of HLA-G mediated control of the immune responses may lead to the onset of autoimmune/inflammatory diseases, caused by an uncontrolled activation of the immune effector cells. Here, we have reviewed novel findings on HLA-G functions in different physiological and pathological settings, which have been published in the last two years. These studies further confirmed the important role of this molecule in the modulation of the immune system.
AdCCs of the salivary glands and breast have been associated with the presence of MYB-NFIB fusion, resulting in overexpression of MYB mRNA transcript, which was observed in our case as well (67 times the normal salivary gland control using Illumina microarray methodology) [21, 22, 23, 24, 25]. Pathologically, these cancers are composed of a dual cell population of luminal and myoepithelial cells.
[Show abstract] [Hide abstract] ABSTRACT: Cancer of Cowper's gland is a very rare cancer. This case represents the 9th case in the medical literature. As such, there are no phase II or phase III trials to guide treatment. In this article, we report the successful treatment of a patient over a 7-year period guided solely by molecular profiling. Through multiple cycles to treatment, the cancer was controlled using drugs targeting c-kit, as the cancer steadily increased the expression of c-kit. This report also documents the use of a novel drug combination based on sunitinib that was well tolerated and may warrant testing in other c-kit-dependent cancers.
Non-invasive imaging techniques are increasingly considered in biomedical research and can lead to important insights on disease development on the same mouse model. In this study we combined in vivo gene delivery technology and non-invasive bioluminescence imaging, creating a unique tool to consider CF mice as suitable models in testing anti-inflammatory drugs and in investigating the molecular mechanism linked to cystic fibrosis . Moreover, the possibility to repetitively test a single set of CF animals longitudinally has several advantages as it reduces the number of animals to sacrifice and the costs in WT and CF mice after challenge with intratracheal VR1Sn.
[Show abstract] [Hide abstract] ABSTRACT: Background Experimentally, lung inflammation in laboratory animals is usually detected by the presence of inflammatory markers, such as immune cells and cytokines, in the bronchoalveolar lavage fluid (BALF) of sacrificed animals. This method, although extensively used, is time, money and animal life consuming, especially when applied to genetically modified animals. Thus a new and more convenient approach, based on in vivo imaging analysis, has been set up to evaluate the inflammatory response in the lung of CFTR-deficient (CF) mice, a murine model of cystic fibrosis. Methods Wild type (WT) and CF mice were stimulated with P. aeruginosa LPS, TNF-alpha and culture supernatant derived from P. aeruginosa (strain VR1). Lung inflammation was detected by measuring bioluminescence in vivo in mice transiently transgenized with a luciferase reporter gene under the control of a bovine IL-8 gene promoter. Results Differences in bioluminescence (BLI) signal were revealed by comparing the two types of mice after intratracheal challenge with pro-inflammatory stimuli. BLI increased at 4 h after stimulation with TNF-alpha and at 24 h after administration of LPS and VR1 supernatant in CF mice with respect to untreated animals. The BLI signal was significantly more intense and lasted for longer times in CF animals when compared to WT mice. Analysis of BALF markers: leukocytes, cytokines and histology revealed no significant differences between CF and WT mice. Conclusions In vivo gene delivery technology and non-invasive bioluminescent imaging has been successfully adapted to CFTR-deficient mice. Activation of bIL-8 transgene promoter can be monitored by non-invasive BLI imaging in the lung of the same animal and compared longitudinally in both CF or WT mice, after challenge with pro-inflammatory stimuli. The combination of these technologies and the use of CF mice offer the unique opportunity of evaluating the impact of therapies aimed to control inflammation in a CF background.
Recent applications include the analysis of prostaglandins, cysteinyl leukotrienes, and oxylipins in breath condensates and sputum (Fritscher et al., 2012; Sylvan et al., 2011), as well as studies in asthma, its triggers and the role of oxylipins in aspirin-intolerant asthma (Higashi et al., 2010; Sanak et al., 2011 ). Furthermore, the biochemistry of lung surfactant phospholipids and their role in lung injury therapy has been facilitated by lipidomics (Goss et al., 2013), while altered plasma fatty acids were proved to provide an accurate and alternative diagnostic test for cystic fibrosis (Batal et al., 2007; Risé et al., 2010).
[Show abstract] [Hide abstract] ABSTRACT: Lipids are the fundamental components of biological membranes as well as the metabolites of organism. Lipids play diverse and important roles in biological system including composing membrane bilayer, storing energy, producing signal transduction, providing functional implementations of membrane proteins and their interactions, etc. The lipid imbalance was found to be closely associated with numerous human lifestyle-related diseases, such as atherosclerosis, obesity, diabetes and Alzheimer’s disease. Lipidomics or lipid profiling is a system-based study of all lipids aiming at comprehensive analysis of lipids in biological system. Lipidomics has been accepted as a lipid-related research tool in lipid biochemistry, clinical biomarker discovery, disease diagnosis and understanding disease pathology. Lipidomics will not only provide insights into the specific functions of lipid species in health and disease, but will also identify potential biomarkers for establishing preventive or therapeutic programs for human disease. This review presents an overview of lipidomics followed by in-depth discussion of its application to the study of human disease, including extraction methods of lipids, analytical technologies, data analysis and clinical research in cancer, neuropsychiatric disease, cardiovascular disease, kidney disease and respiratory disease. We describe the current status of the identification of metabolic biomarkers in different diseases. We also discuss the lipidomics for the future perspectives and their potential problems. The application of lipidomics in clinical studies provides researchers the opportunity to gain new insights into lipid profiling and pathophysiological mechanisms.
Alveolar macrophages (AM) and neutrophils have been shown by some groups to express CFTR in WT mice or normal individuals (Di et al., 2006; Deriy et al., 2009), but some investigators failed to demonstrate this, either for one or for both cell types (van de Weert-van Leeuwen et al., 2013; Zhou et al., 2013). Functionally, some studies have shown that mutated CFTR has a bearing on the phagocytic function of these cells, e.g. through a modification in endosomal acidification and/or decreased killing (Di et al., 2006; Deriy et al., 2009; Yoshimura et al., 1991; Bruscia et al., 2009; Del Porto et al., 2011; Sorio et al., 2011; Su et al., 2011). Interestingly, a recent study showed that reduced phagocytosis in CF monocytes relied on C3b-dependent opsonization of P.a (van de Weert-van Leeuwen et al., 2013).
Contradictory results about the effects of macrolides on inflammation have been reported by several authors and could be due to the choice of the cell model. Our experimental model has been appropriate for reproducing anti-inflammatory effects of AZM described in vivo [27,34] . On the contrary in primary airway cell cultures pro-inflammatory effects have been described .
[Show abstract] [Hide abstract] ABSTRACT: We aimed at identifying molecular mechanisms for anti-inflammatory effects of azithromycin (AZM) suggested by clinical evidences. IL-8 expression and DNA binding activity of two key pro-inflammatory transcription factors (TF), NF-kappaB and AP-1, were investigated in cystic fibrosis (CF) and isogenic non-CF airway epithelial cell lines. AZM reduced about 40% of IL-8 mRNA and protein expression (n=9, p=0.02, and n=4, p=0.00011) in CF cells reaching the levels of non-CF cells. In the presence of AZM we found about 50% and 70% reduction of NF-kappaB and AP-1 DNA binding, respectively (n=3, p=0.01, and n=3, p=0.0017), leading to levels of non-CF cells. The relevance of NF-kappaB and AP-1 in regulating IL-8 promoter transcriptional activity was demonstrated by gene reporter assays (n=4, p=8.54x10(-7), and n=4, p=6.45x10(-6)). Our data support the anti-inflammatory effects of AZM in CF cells, indicating inhibition of transcription of pro-inflammatory genes as possible mechanism, thus providing a rationale for the possible use of specific TF inhibitors for therapy.
For instance, the presence of MZF1 binding site(s) in all the Pu27 homologous sequences  favors a common mechanism especially in regards to the fact that this transcription factor is involved in differentiation of hematopoietic cells . MZF1 may be involved in an early stage of hematopoiesis and plays a role in terminal differentiation–especially of granulocyte lineage , it has also been shown to regulate c-MYC expression in lung adenocarcinoma  . Furthermore, NHEIII 1 requirement for c-MYC transcription silencing is dependent upon formation of Gquadruplex structures [9, 40, 86].
[Show abstract] [Hide abstract] ABSTRACT: G-quadruplex forming sequences are particularly enriched in the promoter regions of eukaryotic genes, especially of oncogenes. One of the most well studied G-quadruplex forming sequences is located in the nuclease hypersensitive element (NHE) III1 of the c-MYC promoter region. The oncoprotein c-MYC regulates a large array of genes which play important roles in growth regulation and metabolism. It is dysregulated in >70% of human cancers. The silencer NHEIII1 located upstream of the P1 promoter regulates up-to 80% of c-MYC transcription and includes a G-quadruplex structure (Pu27) that is required for promoter inhibition. We have identified, for the first time, a family of seventeen G-quadruplex-forming motifs with >90% identity with Pu27, located on different chromosomes throughout the human genome, some found near or within genes involved in stem cell maintenance or neural cell development. Notably, all members of the Pu27 family interact specifically with NHEIII1 sequence, in vitro. Crosslinking studies demonstrate that Pu27 oligonucleotide binds specifically to the C-rich strand of the NHEIII1 resulting in the G-quadruplex structure stabilization. Pu27 homologous sequences (Pu27-HS) significantly inhibit leukemic cell lines proliferation in culture. Exposure of U937 cells to the Pu27-HS induces cell growth inhibition associated with cell cycle arrest that is most likely due to downregulation of c-MYC expression at the RNA and/or protein levels. Expression of SOX2, another gene containing a Pu27-HS, was affected by Pu27-HS treatment as well. Our data suggest that the oligonucleotides encoding the Pu27 family target complementary DNA sequences in the genome, including those of the c-MYC and SOX2 promoters. This effect is most likely cell type and cell growth condition dependent. The presence of genomic G-quadruplex-forming sequences homologous to Pu27 of c-MYC silencer and the fact that they interact specifically with the parent sequence suggest a common regulatory mechanism for genes whose promoters contain these sequences.
(i) the personal nature of interaction, dealt with cystic fibrosis  and three studies dealt with prenatal counseling [39, 42, 57]. Thirty-one of the thirty-nine studies were randomized controlled trials.
[Show abstract] [Hide abstract] ABSTRACT: Background. Advances in genetic science and biotechnology accumulated huge knowledge of genes and various genetic tests and diagnostic tools for healthcare providers including nurses. Genetic counseling became important to assist patients making decisions about obtaining genetic testing or preventive measures. Method. This review was conducted to describe the counseling topics, various interventions adopted in genetic counseling, and their effectiveness. Experimental studies (
) published between 1999 and 2012 were synthesized. Results. The most frequently covered topic was benefits and limitations of genetic testing on breast cancer ovarian and colorectal cancers. Most of researchers focused on evaluating cognitive aspect and psychological well-being. Conclusion. No single intervention was consistently reported to be effective. Decision aids enhanced with information technologies have potential to improve the outcomes of genetic counseling by providing tailored information and facilitating active engagement of patients in information uptake. Clinical Implication. When nurses are familiar with topics and interventions of genetic counseling, they are well positioned to provide genetic/genomic information to the patient and families.
CAR homodimerization is regulated by MAPK activation triggered by HAdV-C5 interaction (Fig. 3-v). However, FK Ad5 is not able to activate p42/p44 MAPK (Farmer et al., 2009), in contrast to what was demonstrated in lung epithelial cells (Tamanini et al., 2006). Here again though, HAdV-C5 transduction is not perturbed by the lack of CAR ICD (Farmer et al., 2009).
[Show abstract] [Hide abstract] ABSTRACT: The coxsackievirus and adenovirus receptor (CAR) belongs to the immunoglobulin superfamily and acts as a receptor for some adenovirus types and group B coxsackieviruses. Its role is best described in epithelia where CAR participates to tight junction integrity and maintenance. Recently, several studies aimed to characterize its potential interaction with intracellular signaling pathways and highlighted several features linking CAR to gene expression. In addition, the molecular mechanisms leading to CAR-specific membrane targeting via the secretory pathway in polarized cells and its internalization are starting to be unraveled. This chapter discusses the interaction between membrane dynamics, intracellular trafficking, and signaling of CAR.
The c-myb proto-oncogene is a nuclear transcription factor that is homologous to the transforming gene product of avian myeloblastosis virus, which plays an important role in the regulation of cell growth and differentiation . c-myb functions in normal and malignant hematopoiesis by regulating the G1/S transition in cycling hematopoietic cells , and by serving as a transactivator of important cellular genes such as the Kit receptor  and CD34 . Amplification of c-myb in acute myeloid leukemia and c-myb overexpression in the 6q-syndrome have been reported  , suggesting that c-myb may play a role in leukemogenesis.
[Show abstract] [Hide abstract] ABSTRACT: c-myb plays an important role in the regulation of cell growth and differentiation, and is highly expressed in immature hematopoietic cells. The human chronic myelogenous leukemia cell K562, highly expresses IGF-I, IGF-II, IGF-IR, and IGF-induced cellular proliferation is mediated by IGF-IR. To characterize the impact of c-myb on the IGF-IGFBP-3 axis in leukemia cells, we overexpressed c-myb using an adenovirus gene transfer system in K562 cells. The overexpression of c-myb induced cell proliferation, compared to control, and c-myb induced cell growth was inhibited by anti-IGF-IR antibodies. c-myb overexpression resulted in a significant increase in the expression of IGF-I, IGF-II, and IGF-IR, and a decrease in IGFBP-3 expression. By contrast, disruption of c-myb function by DN-myb overexpression resulted in significant reduction of IGF-I, IGF-II, IGF-IR, and elevation of IGFBP-3 expression. In addition, exogenous IGFBP-3 inhibited the proliferation of K562 cells, and c-myb induced cell growth was blocked by IGFBP-3 overexpression in a dose-dependent manner. The growth-promoting effects of c-myb were mediated through two major intracellular signaling pathways, Akt and Erk. Activation of Akt and Erk by c-myb was completely blocked by IGF-IR and IGFBP-3 antibodies. These findings suggest that c-myb stimulates cell growth, in part, by regulating expression of the components of IGF-IGFBP axis in K562 cells. In addition, disruption of c-myb function by DN-myb may provide a useful strategy for treatment of leukemia.
Mutations in CFTR gene cause defective Cl -secretion and Na + hyperabsorption by airway epithelia cells . CFTR is also found on cells of the immune system, such as neutrophils , monocytes , and T cells , where loss of CFTR function leads to abnormal immune cellular function. Platelets from CF patients are affected by the molecular defect of CFTR and may play a role in the failure of resolution of inflammation in CF .
[Show abstract] [Hide abstract] ABSTRACT: Background
Cystic fibrosis (CF) is an autosomal recessive genetic disorder that affects multiple organs, including the lungs, pancreas, liver and intestine. Mutations in the cystic fibrosis transmembrane conductance regulator (CFTR) locus lead to defective proteins and reduced Cl⁻ secretion and Na⁺ hyperabsorption in the affected organs. In addition, patients suffering from CF display chronic inflammation that contributes to the pathogenesis of CF. Recent work suggests that CF patients have a reduced capacity to biosynthesize specialized pro-resolving lipid mediators (SPMs), which contributes to the development and duration of the unwanted inflammation. Alterations in the metabolism of arachidonic acid (AA) and docosahexaenoic acid (DHA) to specialized pro-resolving mediators (SPMs), like lipoxins (LXs), maresins (MaRs), protectins (PDs) and resolvins (Rvs), may play a major role on clinical impact of airway inflammation in CF.
In this study, our aims were to detect and quantitate Resolvin D1 (RvD1) in sputum and plasma from patients with CF and compare levels of RvD1 with biomarkers of inflammation and lung function. We studied 27 CF patients aged 6 to 55 years (median 16 years) in a prospective approach.
DHA can be found in the plasma of our CF patients in the milligram range and is decreased in comparison to a healthy control group. The DHA-derived pro-resolving mediator Resolvin D1 (RvD1) was also present in the plasma (286.4 ± 50 pg/ mL, mean ± SEM) and sputum (30.0 ± 2.6 pg/ mL, mean ± SEM) samples from our patients with CF and showed a positive correlation with sputum inflammatory markers. The plasma concentrations of RvD1 were ten times higher than sputum concentrations. Interestingly, sputum RvD1/ IL-8 levels showed a positive correlation with FEV1 (rs = 0.3962, p< 0.05).
SPMs, like RvD1, are well known to down-regulate inflammatory pathways. Our study shows that the bioactive lipid mediator RvD1, derived from DHA, was present in sputum and plasma of CF patients and may serve as a representative peripheral biomarker of the lung resolution program for CF patients.
Since neither binding nor cellular uptake of[ 3 H]feG has been observed with rat leukocytes or neutrophilic transformed HL60 cells (unpublished), we are currently determining if feG may act as a high affinity, low avidity allosteric regulator of integrins and associated co-stimulatory molecules, in a manner similar to a regulation of CD11a/CD18 affinity for counter ligands by a conformational switch in the I domain of this integrin. Since engagement of integrins contributes to increases in vascular permeability and superoxide production[64,65], this mechanism of action may account for the observed properties of feG.
[Show abstract] [Hide abstract] ABSTRACT: The D-isomeric form of the tripeptide FEG (feG) is a potent anti-inflammatory agent that suppresses type I hypersensitivity (IgE-mediated allergic) reactions in several animal species. One of feG's primary actions is to inhibit leukocyte activation resulting in loss of their adhesive and migratory properties. Since activation of neutrophils is often associated with an increase in respiratory burst with the generation of reactive oxygen species (ROS), we examined the effect of feG on the respiratory burst in neutrophils of antigen-sensitized rats. A role for protein kinase C (PKC) in the actions of feG was evaluated by using selective isoform inhibitors for PKC.
At 18 h after antigen (ovalbumin) challenge of sensitized Sprague-Dawley rats a pronounced neutrophilia occurred; a response that was reduced in animals treated with feG (100 microg/kg). With antigen-challenged animals the protein kinase C (PKC) activator, PMA, significantly increased intracellular ROS of circulating neutrophils, as determined by flow cytometry using the fluorescent probe dihydrorhodamine-123. This increase was prevented by treatment with feG at the time of antigen challenge. The inhibitor of PKCdelta, rottlerin, which effectively prevented intracellular ROS production by circulating neutrophils of animals receiving a naïve antigen, failed to inhibit PMA-stimulated ROS production if the animals were challenged with antigen. feG treatment, however, re-established the inhibitory effects of the PKCdelta inhibitor on intracellular ROS production. The extracellular release of superoxide anion, evaluated by measuring the oxidative reduction of cytochrome C, was neither modified by antigen challenge nor feG treatment. However, hispidin, an inhibitor of PKCbeta, inhibited the release of superoxide anion from circulating leukocytes in all groups of animals. feG prevented the increased expression of the beta1-integrin CD49d on the circulating neutrophils elicited by antigen challenge.
feG reduces the capacity of circulating neutrophils to generate intracellular ROS consequent to an allergic reaction by preventing the deregulation of PKCdelta. This action of feG may be related to the reduction in antigen-induced up-regulation of CD49d expression on circulating neutrophils.
Meanwhile, reactive oxygen species produced by neutrophils including hypochlorous acid (bleach), a byproduct of the enzyme MPO exocytosed from primary granules at the same time as NE, can quickly and profoundly oxidize the lung microenvironment. Finally, neutrophils can contribute to extracellular GSH catabolism, by expressing at their surface the GSH-metabolizing enzyme gamma-glutamyltransferase. Another example is that of arginine, which neutrophils can deplete from the CF airway lumen by releasing arginase-1from their granules.
[Show abstract] [Hide abstract] ABSTRACT: Introduction
The pathological course of several chronic inflammatory diseases, including cystic fibrosis, chronic obstructive pulmonary disease, and rheumatoid arthritis, features an aberrant innate immune response dominated by neutrophils. In cystic fibrosis, neutrophil burden and activity of neutrophil elastase in the extracellular fluid have been identified as strong predictors of lung disease severity.
Although neutrophils are generally considered to be rigid, pre-programmed effector leukocytes, recent studies suggest extensive plasticity in how neutrophil functions unfold upon recruitment to peripheral tissues, and how they choose their ultimate fate. Indeed, upon migration to cystic fibrosis airways, neutrophils display dysregulated lifespan, metabolic activation, and altered effector and regulatory functions, consistent with profound adaptation and phenotypic reprogramming. Licensed by signals present in cystic fibrosis airway microenvironment to survive and develop these novel functions, neutrophils orchestrate, in partnership with the epithelium and with the resident microbiota, the evolution of a pathological microenvironment. This microenvironment is defined by altered proteolytic, redox, and metabolic balance and the presence of stable luminal structures in which neutrophils and microbes coexist.
The elucidation of molecular mechanisms driving neutrophil plasticity in vivo will open new treatment opportunities designed to modulate, rather than block, the crucial adaptive functions fulfilled by neutrophils. This review aims to outline emerging mechanisms of neutrophil plasticity and their participation in the building of pathological microenvironments in the context of cystic fibrosis and other diseases with similar features.