[Utility of soluble transferrin receptor measurement for early diagnostic of iron deficiency in elderly hospitalized patients]. / Intérêt du dosage du récepteur soluble de la transferrine dans le diagnostic biologique précoce du déficit martial chez les patients âgés hospitalisés.

Diagnosis of iron deficiency is not easily performed in inflammatory situation as usually encountered in elderly hospitalized patients (>70 years old). At first, we determined serum soluble receptor transferrin (RsTf), RsTf/serum ferritin ratio (RsTf-F index) and biochemical and haematological values used to evaluate iron status, in iron-depleted subjects (ferritinemia≤50 µg/L) (group 2) (n=22, 82±7 years) or not (group 1, reference group) (n=18, 82±6 years), without inflammatory diseases. Relevance of the biological parameters to diagnose iron deficiency was evaluated (ROC curve) and a cut-off value of RsTf-F (>1.85) was established. Then, we selected 60 patients (group 3) suspect of iron deficiency as previously validated with an inflammatory syndrome (CRP>12 mg/L). Almost all patients (95%) presented at least one risk factor of iron deficiency (anticoagulant drugs, nutritional or gastrointestinal diseases). In group 3, index RsTf-F values were increased (RsTf-F: 2.69±0.82 versus group 1: 1.25±0.34, p<0.05), in anemic patients (women Hb<120 g/L, men Hb<130 g/L) (n=42) and in non-anemic patients (n=18) (respectively RsTf-F: 2.84±0.87 versus 2.35±0.58, p<0.05). Thus, in elderly patients with inflammatory disorders, RsTf-F index could suspect iron deficiency before appearance of biological anemia.

Neurotensin protects pancreatic beta cells from apoptosis.

The survival of pancreatic beta cells depends on the balance between external cytotoxic and protective molecular systems. The neuropeptide neurotensin (NT) has been shown to regulate certain functions of the endocrine pancreas including insulin and glucagon release. However, the mechanism of action of NT as well as the identification of receptors involved in the pancreatic functions of the peptide remained to be studied. We demonstrate here that NT is an efficient protective agent of pancreatic beta cells against cytotoxic agents. Both beta-TC3 and INS-1E cell lines and the mouse pancreatic islet cells express the three known NT receptors. The incubation of beta cells with NT protects cells from apoptosis induced either by staurosporine or by IL-1beta. In beta-TC3 cells, NT activates both MAP and PI-3 kinases pathways and strongly reduces the staurosporine or the Il-1beta-induced caspase-3 activity by a mechanism involving Akt activation. The NTSR2 agonist levocabastine displays the same protective effect than NT whereas the NTSR1 antagonist is unable to block the effect of NT suggesting the predominant involvement of the NTSR2 in the action of NT on beta cells. These results clearly indicate for the first time that NT is able to protect endocrine beta cells from external cytotoxic agents, a role well correlated with its release in the circulation after a meal.

[Factors of therapeutic imbalance of antivitamins K and their haemorrhagic consequences in elderly]. / Les facteurs de déséquilibre thérapeutique des antivitamines K et leurs conséquences hémorragiques en pratique gériatrique.

Objective. To determinate, for older subjects, specific factors of imbalance of the oral anticoagulant treatments. Method. We conducted an epidemiological and analytical case-control study, during 18 months, in 2 geriatrics centers. Each patient with excessive anticoagulation (INR>4.5) was matched with 2 controls under anticoagulant, whom the INR (international normalized ratio) had stayed in the therapeutic range. Results. One-hundred fifty nine subjects (53 cases and 106 controls) were included. Haemorrhagic complications has been observed in 19.2% of cases, versus 3.9% of the controls. Some medicines frequently prescribed to the old subjects were correlated at a risk of excessive anticoagulation: amiodarone (9.4% versus 0, p<0.004), acetaminophen (18.9% versus 0.9%, p<0.001), tramadol (5.6% versus 0, p<0.04), ofloxacine (11.3% versus 1.9%, p<0.001), and lactulose (11.3% versus 0, p<0.001). Furthermore, several acute states increase the risk of excessive INR to the old subjects: fever (p<0.001), malnutrition (p<0.001), dehydration (p=0.006), and acute diarrhea (p<0.001). Conclusion. Some specific geriatric factors raised may destabilize treatments by anticoagulants.

Morphine and pain-related stimuli enhance cell surface availability of somatic delta-opioid receptors in rat dorsal root ganglia.

The present study demonstrates that perikaryaldelta-opioid receptors (deltaORs) in rat dorsal root ganglion (DRG) neurons bind and internalize opioid ligands circulating in the CSF. Using confocal and electron microscopy, we found that prolonged morphine treatment increased the cell surface density of these perikaryal deltaORs and, by way of consequence, receptor-mediated internalization of the fluorescent deltorphin (DLT) analog omega-Bodipy 576/589 deltorphin-I 5-aminopentylamide (Fluo-DLT) in all three types of DRG neurons (small, medium, and large). In contrast, chronic inflammatory pain induced by the injection of complete Freund's adjuvant (CFA) into one hindpaw selectively increased Fluo-DLT internalization in small and medium-sized DRG neurons ipsilateral to the inflammation. Based on our previous studies in the spinal cord of mu-opioid receptor (muOR) knock-out mice, it may be assumed that the enhanced membrane recruitment of deltaORs observed after sustained morphine is attributable to stimulation of muORs. However, the selectivity of the effect induced by inflammatory pain suggests that it involves a different mechanism, namely a modality-specific and pain-related activation of C and Adelta fibers. Indeed, stimulation by capsaicin of transient receptor potential vanilloid 1 receptors, which are selectively expressed by small diameter (< 600 microm2) DRG neurons, increased Fluo-DLT internalization exclusively in this cell population. The present results, therefore, demonstrate that DRG neurons express perikaryal deltaORs accessible to CSF-circulating ligands and that the density and, hence, presumably also the responsiveness, of these receptors may be modulated by both pain-related stimuli and sustained exposure to muOR agonists.

Functional roles of the NTS2 and NTS3 receptors.

Neurotensin exerts its actions in the central nervous system and the periphery through three identified receptors. Two of them, the NTS2 and NTS3, display unusual properties either because of their complex signal transduction mechanisms (NTS2) or because of their structural composition as a non-G-protein-coupled receptor (NTS3). Here, we review the transduction mechanisms, cellular trafficking, and potential physiological roles of these two unconventional receptors.

Internalization and recycling properties of neurotensin receptors.

The targeting, internalization and recycling of membrane receptors in response to extracellular ligands involve a series of molecular mechanisms which are beginning to be better understood. The receptor-dependent internalization of neurotensin has been widely investigated using endogenous or heterologous receptor expression systems. This review focuses on the general properties of neurotensin sequestration and on the characterization of the receptors involved in this process.

Involvement of the neurotensin receptor-3 in the neurotensin-induced migration of human microglia.

Microglia motility plays a crucial role in response to lesion or exocytotoxic damage of the cerebral tissue. We used two in vitro assays, a wound-healing model and a chemotaxis assay, to show that the neuropeptide neurotensin elicited the migration of the human microglial cell line C13NJ by a mechanism dependent on both phosphatidylinositol 3-kinase (PI 3-kinase) and mitogen-activated protein (MAP) kinase pathways. The effect of neurotensin on cell migration was blocked by the neurotensin receptor-3 propeptide, a selective ligand of this receptor. We demonstrate, by using RT-PCR, photoaffinity labeling, and Western blot analysis, that the type I neurotensin receptor-3 was the only known neurotensin receptor expressed in these microglial cells and that its activation led to the phosphorylation of both extracellular signal-regulating kinases 1/2 and Akt. Furthermore, the effect of neurotensin on cell migration was preceded by a profound modification of the F-actin cytoskeleton, particularly by the rapid formation of numerous cell filopodia. Both the motility and the filopodia appearance induced by neurotensin were totally blocked by selective inhibitors of MAP kinases or PI 3-kinase pathways. This demonstrates that the neurotensin receptor-3 is functional and mediates the migratory actions of neurotensin.

In vivo and in vitro neurotoxicity of the human prion protein (PrP) fragment P118-135 independently of PrP expression.

We recently demonstrated that the 118-135 putative transmembrane domain of prion protein (PrP) exhibited membrane fusogenic properties and induced apoptotic neuronal cell death of rat cortical neurons, independently of its aggregation state. The aim of the present study was to analyze the in vivo neurotoxicity of the prion fragment P118-135 and to evaluate the potential role of the physiological isoform of PrP in the P118-135-induced cell death. Here, we demonstrate that the nonfibrillar P118-135 is cytotoxic to retinal neurons in vivo as monitored by intravitreal inoculation and recording of the electrical activity of retina and tissue examination. Moreover, knock-out PrP gene mice exhibit similar sensitivity to the nonfibrillar P118-135-induced cell death and electrical perturbations, strongly suggesting that cell death occurs independently of PrP expression. Interestingly, a variant nonfusogenic P118-135 peptide (termed P118-135theta) had no effects on in vivo neuronal viability, suggesting that the P118-135-induced cell death is mediated by its membrane destabilizing properties. These data have further been confirmed in vitro. We show that the fusogenic peptide P118-135 induces death of cultured neurons from both wild-type and knock-out PrP gene mice via an apoptotic-mediated pathway, involving early caspase activation and DNA fragmentation. Altogether these results emphasize the neurotoxicity of the fusogenic nonfibrillar PrP transmembrane domain and indicate that fibril formation and PrP expression are not obligatory requirements for neuronal cell death. The use of synthetic prion peptides could provide insights into the understanding of neuronal loss mechanisms that take place during the development of the various types of spongiform encephalopathies.

Morphine-induced changes in delta opioid receptor trafficking are linked to somatosensory processing in the rat spinal cord.

An in vivo fluorescent deltorphin (Fluo-DLT) internalization assay was used to assess the distribution and regulation of pharmacologically available delta opioid receptors (deltaORs) in the rat lumbar (L4-5) spinal cord. Under basal conditions, intrathecal injection of Fluo-DLT resulted in the labeling of numerous deltaOR-internalizing neurons throughout dorsal and ventral horns. The distribution and number of Fluo-DLT-labeled perikaryal profiles were consistent with that of deltaOR-expressing neurons, as revealed by in situ hybridization and immunohistochemistry, suggesting that a large proportion of these cells was responsive to intrathecally administered deltaOR agonists. Pretreatment of rats with morphine for 48 hr resulted in a selective increase in Fluo-DLT-labeled perikaryal profiles within the dorsal horn. These changes were not accompanied by corresponding augmentations in either deltaOR mRNA or (125)I-deltorphin-II binding levels, suggesting that they were attributable to higher densities of cell surface deltaOR available for internalization rather than to enhanced production of the receptor. Unilateral dorsal rhizotomy also resulted in increased Fluo-DLT internalization in the ipsilateral dorsal horn when compared with the side contralateral to the deafferentation or to non-deafferented controls, suggesting that deltaOR trafficking in dorsal horn neurons may be regulated by afferent inputs. Furthermore, morphine treatment no longer increased Fluo-DLT internalization on either side of the spinal cord after unilateral dorsal rhizotomy, indicating that microOR-induced changes in the cell surface availability of deltaOR depend on the integrity of primary afferent inputs. Together, these results suggest that regulation of deltaOR responsiveness through microOR activation in this region is linked to somatosensory information processing.

Geriatric Nutritional Risk Index: a new index for evaluating at-risk elderly medical patients.

BACKGROUND: Patients at risk of malnutrition and related morbidity and mortality can be identified with the Nutritional Risk Index (NRI). However, this index remains limited for elderly patients because of difficulties in establishing their normal weight. OBJECTIVE: Therefore, we replaced the usual weight in this formula by ideal weight according to the Lorentz formula (WLo), creating a new index called the Geriatric Nutritional Risk Index (GNRI). DESIGN: First, a prospective study enrolled 181 hospitalized elderly patients. Nutritional status [albumin, prealbumin, and body mass index (BMI)] and GNRI were assessed. GNRI correlated with a severity score taking into account complications (bedsores or infections) and 6-mo mortality. Second, the GNRI was measured prospectively in 2474 patients admitted to a geriatric rehabilitation care unit over a 3-y period. RESULTS: The severity score correlated with albumin and GNRI but not with BMI or weight:WLo. Risk of mortality (odds ratio) and risk of complications were, respectively, 29 (95% CI: 5.2, 161.4) and 4.4 (95% CI: 1.3, 14.9) for major nutrition-related risk (GNRI: <82), 6.6 (95% CI: 1.3, 33.0), 4.9 (95% CI: 1.9, 12.5) for moderate nutrition-related risk (GNRI: 82 to <92), and 5.6 (95% CI: 1.2, 26.6) and 3.3 (95% CI: 1.4, 8.0) for a low nutrition-related risk (GNRI: 92 to < or =98). Accordingly, 12.2%, 31.4%, 29.4%, and 27.0% of the 2474 patients had major, moderate, low, and no nutrition-related risk, respectively. CONCLUSION: GNRI is a simple and accurate tool for predicting the risk of morbidity and mortality in hospitalized elderly patients and should be recorded systematically on admission.

Internalization and trafficking of neurotensin via NTS3 receptors in HT29 cells.

The neurotensin receptor-3, originally identified as sortilin, is unique among neuropeptide receptors in that it is a single trans-membrane domain, type I receptor. To gain insight into the functionality of neurotensin receptor-3, we examined the neurotensin-induced intracellular trafficking of this receptor in the human carcinoma cell line HT29, which expresses both neurotensin receptor-1 and -3 sub-types. At steady state, neurotensin receptor-3 was found by sub-cellular fractionation and electron microscopic techniques to be predominantly associated with intracellular elements. A small proportion (approximately 10%) was associated with the plasma membrane, but a significant amount (approximately 25%) was observed inside the nucleus. Following stimulation with neurotensin (NT), neurotensin/neurotensin receptor-3 complexes were internalized via the endosomal pathway. This internalization entailed no detectable loss of cell surface receptors, suggesting compensation through either recycling or intracellular receptor recruitment mechanisms. Internalized ligand and receptors were both sorted to the pericentriolar recycling endosome/Trans-Golgi Network (TGN), indicating that internalized neurotensin is sorted to this compartment via neurotensin receptor-3. Furthermore, within the Trans-Golgi Network, neurotensin was bound to a lower molecular form of the receptor than at the cell surface or in early endosomes, suggesting that signaling and transport functions of neurotensin receptor-3 may be mediated through different molecular forms of the protein. In conclusion, the present work suggests that the neurotensin receptor-3 exists in two distinct forms in HT29 cells: a high molecular weight, membrane-associated form responsible for neurotensin endocytosis from the cell surface and a lower molecular weight, intracellular form responsible for the sorting of internalized neurotensin to the Trans-Golgi Network.

Beta-arrestin is involved in the desensitization but not in the internalization of the somatostatin receptor 2A expressed in CHO cells.

The interaction of beta-arrestin-1 with the somatostatin receptor type 2A (sst2A) was monitored using both biochemical and confocal imaging approaches. We show that, using transient transfection of either beta-arrestin-1 or its dominant negative Delta-arrestin-1 in CHO cells stably transfected with the sst2A, beta-arrestin-1 is colocalized with the receptor in endosomal vesicles after somatostatin-induced sequestration. However, this interaction leads to a role of beta-arrestin-1 in the desensitization of the sst2A rather than in the internalization process of the receptor-ligand complex.

Risk factors for deep vein thrombosis in inpatients aged 65 and older: a case-control multicenter study.

OBJECTIVES: To identify independent risk factors of symptomatic deep vein thrombosis (DVT) in geriatric inpatients and to define high-risk patients likely to benefit from preventive treatment. DESIGN: Hospital-based case-control multicenter study with prospective data collection. SETTINGS: Geriatric university hospitals with long-, intermediate-, and short-term care facilities. PARTICIPANTS: All patients aged 65 and older in 19 geriatric departments were submitted to clinical surveillance over a 16-month period. MEASUREMENTS: Twenty-three potential risk factors of phlebitis were screened for. Comparison using logistic regression of 310 consecutive patients with symptomatic DVT versus 310 randomly selected controls was performed. The risk for symptomatic DVT in geriatrics was then scored from the clinical risk factors identified using multivariate analysis. This score is defined by the sum of the odds ratio (OR) of each risk factor present. RESULTS: Six factors were identified as independently related to the development of DVT: restriction of mobility (from OR=1.73, limited mobility without immobilization, to OR=5.64, bedridden during <15 days), aged 75 and older (OR=1.5/10 years), history of DVT or pulmonary embolism (OR=3.38), acute heart failure (OR=2.52), chronic edema of the lower limbs (OR=2.51), and paresis or paralysis of a lower limb (OR=2.06). The defined score of 8 or higher corresponded to an 88.7% probability of having symptomatic DVT. CONCLUSION: Treatments to prevent symptomatic DVT in hospitalized elderly should be evaluated on patients with these factors.

A case report of an obesity hypoventilation syndrome associated with obstructive sleep apnea due to a carotid body paraganglioma.

BACKGROUND AND PURPOSE: To the best of our knowledge, the association between an obstructive sleep apnea syndrome (OSAS) due to a neck mass and an obesity hypoventilation syndrome (OHS) has not been reported. PATIENTS AND METHODS: We report the case of a patient with obesity hypoventilation syndrome (OHS) in whom OSAS caused by a carotid body paraganglioma contributed to recurrent bouts of severe alveolar hypoventilation. RESULTS AND CONCLUSIONS: The complete surgical excision of the paraganglioma resulted in the cure of the OSAS and contributed to a clear improvement of the clinical symptoms of OHS.

Atrial fibrillation in the elderly: facts and management.

Although atrial fibrillation is not widely known by the general public, in developed countries it is the most common arrhythmia. The incidence increases markedly with advancing age. Thus, with the growing proportion of elderly individuals, atrial fibrillation will come to represent a significant medical and socioeconomic problem. The consequences of atrial fibrillation have the greatest impact. The risk of thromboembolism is well known; other outcomes of atrial fibrillation are less well recognised, such as its relationship with dementia, depression and death. Such consequences are responsible for diminished quality of life and considerable economic cost. Atrial fibrillation is characterised by rapid and disorganised atrial activity, with a frequency between 300 and 600 beats/minute. The ventricles react irregularly, and may contract rapidly or slowly depending on the health of the conduction system. Clinical symptoms are varied, including palpitations, syncope, dizziness or embolic events. Atrial fibrillation may be paroxysmal, persistent or chronic, and a number of attacks are asymptomatic. Suspicion or confirmation of atrial fibrillation necessitates investigation and, as far as possible, appropriate treatment of underlying causes such as hypertension, diabetes mellitus, hypoxia, hyperthyroidism and congestive heart failure. In the evaluation of atrial fibrillation, cardiac exploration is invaluable, including electrocardiogram (ECG) and echocardiography, with the aim of detecting cardiac abnormalities and directing management. In elderly patients (arbitrarily defined as aged >75 years), the management of atrial fibrillation varies; it requires an individual approach, which largely depends on comorbid conditions, underlying cardiac disease, and patient and physician preferences. This management is essentially based on pharmacological treatment, but there are also nonpharmacological options. Two alternatives are possible: restoration and maintenance of sinus rhythm, or control of ventricular rate, leaving the atria in arrhythmia. Pharmacological options include antiarrhythmic drugs, such as class III agents, beta-blockers and class IC agents. These drugs have some adverse effects, and careful monitoring is necessary. The nonpharmacological approach to atrial fibrillation includes external or internal direct-current cardioversion and new methods, such as catheter ablation of specific foci, an evolving science that has been shown to be successful in a very select group of atrial fibrillation patients. Another serious challenge in the management of chronic atrial fibrillation in older individuals is the prevention of stroke, its primary outcome, by choosing an appropriate antithrombotic treatment (aspirin or warfarin). Several risk-stratification schemes have been validated and may be helpful to determine the best antithrombotic choice in individual patients.

[Morning stiffness]. / Raideur matinale.

STIFFNESS AND UNSTRETCHING: Morning stiffness in the elderly is a painful entity often ignored, but frequent in institutions. Unstretching in the morning is part of the criteria in the diagnosis of inflammatory pathologies, but morning stiffness in old patients appears different from that described in rheumatological diseases and arthrosis. FROM AN AETIOLOGICAL POINT OF VIEW: Various affections can be accompanied by morning stiffness in an elderly patient: arthrosis, but also various consequential affections, static disorders, deformity. We felt it was important to define the precise criteria that individualise this pathology so as to improve its management. MANAGEMENT: Non-medicinal measures often reduce the intensity and duration of this symptom and implicate all those attending to the patient. Medicinal treatment has a more limited place in the fragile elderly patient, but its role remains to be assessed.

[Headaches in the elderly]. / Céphalées du sujet âgé

ACCORDING TO AGE: It is generally thought that the prevalence of headaches decreases with ageing. However recent studies, with stricter epidemiology and methodology, clearly indicate that this decreases is less obvious than that perceived. PRIMARY AND SECONDARY HEADACHES: In elderly patients, primary headaches and notably migraine (often with altered presentation) are less frequent, even though new authentic cases may appear. However the other types of headache are clearly present. In particular, the incidence and prevalence of secondary headaches slightly increases and they represent up to 30% of all the headaches observed, compared with less than 10% in young or adult patients. The causes of such symptomatic headaches are multiple and it is important to be able to identify them since an aetiological treatment is often possible. REGARDING TREATMENT: The symptomatic treatment of headaches in the elderly follows the same principles as that of younger patients. However, the side effects and drug interactions related to pharmacological treatments can be disastrous in fragilised patients. It is therefore important to emphasize the interest of physical methods of analgesia, particularly adapted to the elderly. Some of these methods have demonstrated their efficacy and are recommended by international consensuses.

[Impact of nasal colonization by methicillin-resistant Staphylococcus aureus among geriatric intermediate care facility patients]. / Impact de la colonisation nasale par le Staphylococcus aureus résistant à la méticilline dans une unité de soins de suite gériatrique.

OBJECTIVE: To evaluate the impact of nasal carriage of Methicillin Resistant Staphylococcus aureus (MRSA) on antibiotic cost, infection morbidity, mortality and length of stay in a geriatric population. METHODS: 341 consecutive elderly patients (mean age 83.4 +/- 8.7 years) admitted to an intermediate care facility were prospectively include between November 1998 and October 1999. Nasal swab cultures were taken on admission. RESULTS: In sixty patients (17.6%) no nasal swab was taken. Among the 281 patients screened, 52 were identified as MRSA carriers. The principle predictive factors were: diabetes (p=0,046), sores (p=0,03), malnutrition (p=0,02), polypathology (p=0,02) and prolongation of previous hospitalisation (p=0,09). CONCLUSION: Nasal carriage of MRSA on admission to the facility was not a deleterious prognostic factor regarding duration of stay, infectious morbidity and antibiotic cost, but was associated with higher mortality risk.

Impact of protein pulse feeding on lean mass in malnourished and at-risk hospitalized elderly patients: a randomized controlled trial.

BACKGROUND & AIMS: Aging is associated with a blunted anabolic response to dietary intake, possibly related to a decrease in systemically available amino acids (AAs), which in turn may stem from increased splanchnic AA metabolism. This splanchnic sequestration can be saturated by pulse feeding (80% of daily protein intake in a single meal), enabling increased protein synthesis. This study aimed to evaluate the efficacy of a new nutritional strategy, termed protein pulse feeding. METHODS: This prospective randomized study (ClinicalTrials.gov registration number NCT00135590) enrolled 66 elderly malnourished or at-risk patients in an inpatient rehabilitation unit. All were given a controlled diet for 6 weeks. In a spread diet (SD) group (n = 36), dietary protein was spread over the four daily meals. In a pulse diet (PD) group (n = 30), 72% of dietary protein (1.31 g/kg weight/d on average) was consumed in one meal at noon. The patients were evaluated at admission and at 6 weeks for body composition [lean mass (LM), appendicular skeletal muscle mass (ASMM), and body cell mass (BCM) indices, measured by X-ray absorptiometry combined with bioelectrical impedance analysis] (primary outcome), hand grip strength, and activities of daily living (ADL) score. RESULTS: Protein pulse feeding was significantly more efficacious than protein spread feeding in improving LM index (mean changes from baseline for PD group: +0.38 kg/m(2); 95% confidence interval (CI), [0; 0.60]; for SD group: -0.21 kg/m(2); 95% CI, [-0.61; 0.20]; p = 0.005 between the two groups), ASMM index (+0.21 kg/m(2); 95% CI, [0; 0.34] and -0.11 kg/m(2); 95% CI, [-0.20; 0.09]; p = 0.022), BCM index (+0.44 kg/m(2); 95% CI, [0.08; 0.52] and -0.04 kg/m(2); 95% CI, [-0.09; 0.10]; p = 0.004). There was no significant effect for hand-grip strength or ADL score. CONCLUSIONS: This study demonstrates for the first time that protein pulse feeding has a positive, clinically relevant effect on lean mass in malnourished and at-risk hospitalized elderly patients.