Fetal alcohol spectrum disorders: an overview of current evidence and activities in the UK.

Estimates for the UK suggest that alcohol consumption during pregnancy and prevalence of fetal alcohol spectrum disorder (FASD)-the most common neurodevelopmental condition-are high. Considering the significant health and social impacts of FASD, there is a public health imperative to prioritise prevention, interventions and support. In this article, we outline the current state of play regarding FASD knowledge and research in the UK, which is characterised by a lack of evidence, a lack of dedicated funding and services, and consequently little policy formulation and strategic direction. We highlight progress made to date, as well as current knowledge and service gaps to propose a way forward for UK research.

Decreased behavioural and neurochemical effects of angiotensin IV following prenatal alcohol exposure in the mouse.

Angiotensin IV (ang IV) is known to improve learning and memory in animal models but the mechanism is unclear. We have previously demonstrated sex differences in the pro-cognitive effects of ang IV, and that prenatal alcohol exposure (PAE) abolishes these effects. This study aimed to explore a possible mechanism underlying the sex differences and the effects of PAE in male mice. Mouse breeding harems received 5% ethanol in drinking water throughout pregnancy and lactation in a two-bottle schedule. The effects of ang IV were assessed in offspring at 4 months of age using the open field test, novel object recognition test and elevated plus maze. Aminopeptidase activity of brain insulin-regulated aminopeptidase (IRAP), a putative target of ang IV, was determined. As seen in a previous similar study, ang IV administered immediately after the second training trial significantly improved novel object recognition 24 h later in male mice but not female. PAE abolished this pro-cognitive effect in males. PAE also increased anxiety-like behaviour in male but not female offspring. Ang IV decreased the aminopeptidase activity of brain IRAP in control male, but not female, mice; PAE abolished this inhibitory effect. Ang IV improved memory consolidation in male but not female mice and PAE abolished this effect in the males. While the effects of PAE may be related to increased anxiety; ang IV decreased the aminopeptidase activity in male but not female mice and PAE abolished this inhibitory effect. The results therefore suggest that improvements in learning and memory induced by peripheral administration of ang IV correlate with a reduction of the enzyme activity of IRAP. This is the first demonstration that ang IV administered peripherally can induce long-term (24 h) changes in IRAP function which are probably not simple competitive inhibition and the first demonstration that PAE alters IRAP activity.

Changing status in health care: community and hospital pharmacists' perceptions of pharmacy practice.

OBJECTIVES: This study aimed to explore experienced community and hospital pharmacists' perceptions of how their pharmacy practice and status in health care are affected by others' views of them. METHODS: A qualitative collective case study was conducted. The primary data were 20 in-depth semistructured interviews of community and hospital pharmacists in England that were audio-recorded, transcribed and analysed thematically. KEY FINDINGS: Thematic analysis of the data identified four themes: (1) ambiguities about being professionals, (2) internal divisions, (3) medicines experts and (4) shopkeepers as healthcare providers. CONCLUSIONS: Pharmacists want to be recognised as medicines experts in health care. They are aware that their status is assessed by the public based on their practice, which is dispensing of medicines, and that the public's image of all pharmacists is that of 'a typical community pharmacist' working in a retail shop while having little experience of pharmacists in other healthcare settings. Pharmacists consider that the public does not view them as registered healthcare professionals. They mainly associate being registered professionals with being controlled from afar by their professional regulator, instead of utilising this as an enabling strategy to support their reprofessionalisation efforts. Pharmacists remain the hidden healthcare profession and need to act in practice as healthcare professionals, so the public is aware of their place and contributions in health care to maintain or enhance their status. Internal divisions between community and hospital pharmacists appear to be due to differences in practice, knowledge and aspirations having the potential to adversely affect the pharmacy profession's status.

Prenatal alcohol exposure reduces 5-HT concentration in mouse intestinal muscle and mucosa.

The influence of prenatal alcohol exposure on the serotoninergic system in the brain has been well studied, however its influence on the serotoninergic system in the gastrointestinal system remains unknown. The objective of the study was to use a mouse model of prenatal alcohol exposure to investigate the effects on serotonin and its metabolites and precursors in colonic tissue. This study used treatment of mouse breeding harems with 5% ethanol with saccharin via drinking water throughout pregnancy and compared the results with a saccharin control group. Tryptophan, serotonin (5-HT) and 5- hydroxyindoleacetic acid (5-HIAA) concentrations were measured in the longitudinal muscle myenteric plexus (LMMP) and mucosa of intestinal tissue by high-performance liquid chromatography (HPLC). Decreased 5-HT concentrations in mucosa and LMMP (females only) were observed in prenatally exposed mice compared to controls. Increases in mucosal and LMMP tryptophan concentration were only observed in prenatally exposed female mice. In conclusion, prenatal alcohol exposure causes a decrease in conversion of tryptophan to 5-HT in both muscle and mucosa although the effect is more pronounced in females. The observed sex difference may be related to changes associated with the estrous cycle.

The Anatomical Society core anatomy syllabus for pharmacists: outcomes to create a foundation for practice.

The Anatomical Society has developed a series of learning outcomes that 'experts' within the field would recommend as core knowledge outputs for a Master's Degree Programme in Pharmacy (MPharm) within the UK. Using the Anatomical Society core gross anatomy syllabus for medical anatomy as a foundation, a modified Delphi technique was used to develop outcomes specific to pharmacy graduates. A Delphi panel consisting of medical practitioners, pharmacists and anatomists (n = 39) was created and involved 'experts' representing 20 UK Higher Education Institutions. The output from this study was 49 pharmacy-specific learning outcomes that are applicable to all pharmacy programmes. The new MPharm anatomy syllabus offers a basic anatomical framework upon which pharmacy educators can build the necessary clinical practice and knowledge. These learning outcomes could be used to develop anatomy teaching within an integrated curriculum as per requirements of the General Pharmaceutical Council (GPhC).

Low-dose chronic prenatal alcohol exposure abolishes the pro-cognitive effects of angiotensin IV.

Prenatal ethanol exposure (PAE) in humans results in a spectrum of disorders including deficits in learning and memory. Animal models to date have typically used high ethanol doses but have not identified the biochemical changes underlying the cognitive deficit. This study used treatment of mouse breeding harems with 5% ethanol via drinking water throughout pregnancy and lactation and explored the behavioural consequences in the progeny at 3-6 months of age using the open field test, novel object recognition test and elevated plus maze to measure anxiety and memory consolidation. The effects of angiotensin IV on behaviour of the progeny were also determined. The results indicated that PAE increased anxiety-like behaviour as determined in the open field test in male but not female progeny. In control animals, angiotensin IV enhanced memory consolidation in males, but this effect was abolished by PAE. The abolition of the pro-cognitive effect of angiotensin IV was not a consequence of increased anxiety, and there was some evidence of a long-lasting anxiolytic effect of angiotensin IV in the male PAE progeny. These results suggest that PAE may act via alteration of the actions of the brain renin-angiotensin system to impair memory consolidation, but these effects may be partially sex-dependent.

Impaired renal function and biomarkers of vascular disease in Alzheimer's disease.

Renal disease is a risk factor for vascular diseases and for dementia, and renal insufficiency can be a feature of Alzheimer's disease (AD). Evidence has suggested that vascular mechanisms mediate the link between renal disease and dementia. Our study sought to test this hypothesis by examining renal and vascular functioning in AD by investigating estimated glomerular filtration rates (eGFR), calculated from serum creatinine concentrations, and established biomarkers of vascular functioning, asymmetrical dimethylarginine (ADMA) and plasma homocysteine (Hcy), in individuals with mild to moderate AD (n = 34) and a group of older adult controls (n = 34). We found significantly reduced eGFR, indicative of impaired renal functioning, in individuals with AD (M = 62.9, SD = 15.2) compared with controls (M = 73.6, SD = 11.8). However, concentrations of ADMA and Hcy did not differ between patient and control groups (ADMA: M = 0.47; M = 0.50; Hcy: M = 17.2; M = 14.9; patients and controls). The criteria for a mediation analysis were not met, as concentrations of ADMA and Hcy did not predict AD, indicating that these biomarkers of vascular functioning did not mediate a relationship between renal functioning and AD. This study indicated that renal insufficiency may independently contribute to AD pathology, and other vascular mechanisms may influence a relationship between renal impairment and AD.

Blood pro-inflammatory cytokines in Alzheimer's disease in relation to the use of acetylcholinesterase inhibitors.

OBJECTIVE: A potential anti-inflammatory role for acetylcholinesterase inhibitors (AChEIs) has been supported by animal studies. As very limited data exist from individuals with Alzheimer's disease (AD), the aim of this study was to assess the potential influence of AChEIs on blood pro-inflammatory cytokines. We hypothesized that pro-inflammatory cytokine concentrations were lower in individuals with AD stabilized on AChEIs. METHODS: Blood interleukin-1 beta, interleukin-6, and tumor necrosis factor-alpha concentrations were assessed using specific enzyme-linked immunosorbent assays in three groups of participants: patients with AD stabilized on a therapeutic dose of an AChEI (n = 42); AChEIs drug naïve patients (n = 24); and a cognitively unimpaired control group (n = 35). Patients in the AChEIs group had received medication for an average of one year. RESULTS: Patients stabilized on an AChEI did not differ significantly from drug naïve patients in relation to the concentrations of interleukin-1 beta, interleukin-6, and tumor necrosis factor-alpha (p = 0.874, 0.225, and 0.978, respectively). Within the group taking AChEIs, the levels of cytokines did not differ between those taking donepezil, rivastigmine, or galantamine (p = 0.368, 0.851, and 0.299, respectively). CONCLUSIONS: Results from animal studies suggesting a modulatory anti-inflammatory role for AChEIs was not advanced in this study. In individuals with AD, very limited evidence currently exists to support the hypothesis that AChEIs may influence inflammatory blood markers and function beyond the enhancement of neuronal transmission. However, further studies assessing a wider range of inflammatory markers and processes are still needed before this hypothesis can be ruled out.

Blockade of pro-cognitive effects of angiotensin IV and physostigmine in mice by oxytocin antagonism.

Low doses of oxytocin enhance learning and memory in animal models. Angiotensin IV inhibits cysteine aminopeptidase, also known as insulin-regulated aminopeptidase and oxytocinase, and enhances memory in animals. The mechanism of this effect of angiotensin IV is unknown. This study explored the role of oxytocin in the cognitive effects of angiotensin IV with physostigmine as a control and used isolated smooth muscle to assess the pharmacological selectivity of the observed antagonism. Using novel object recognition in male mice, the effects of angiotensin IV (4.7 µg/kg), oxytocin (0.1 ng/kg) or physostigmine (200 µg/kg) administered subcutaneously immediately after the second training trial, were assessed in the presence and absence of 10 µg/kg ß-mercapto-ß-ß-cyclopenta-methylenepropionyl; O-Me-Tyr², Orn8-oxytocin, an oxytocin antagonist; n=8 in all cases. The effects of the antagonist on angiotensin IV, oxytocin and acetylcholine-induced contractions of rat isolated uterus were also determined. Oxytocin, angiotensin IV and physostigmine significantly enhanced consolidation of learning (P=0.04, 0.004 and 0.008 respectively), and there were no significant effects on locomotor activity. The oxytocin antagonist similarly not only significantly improved novel object recognition (P=0.03) but also significantly increased locomotor activity (P=0.04). In the learning paradigm the oxytocin antagonist prevented the effects of oxytocin, angiotensin IV and physostigmine but in the uterus, contractions induced by angiotensin IV and acetylcholine were unaffected whilst effects of oxytocin were significantly reduced. These results suggest that the pro-cognitive effects of angiotensin IV may be mediated by accumulation of endogenous oxytocin although the mechanisms underlying the observed interaction between the oxytocin antagonist and physostigmine are unclear.

Healthy children's identification and risk perception of medicines in England.

BACKGROUND: Children's understanding of medicines has an impact on their behavior toward those medicines, and yet there has been a paucity of studies exploring this area. OBJECTIVES: To assess children's ability to identify and to explore their risk perceptions of medicines. METHODS: One hundred eighty-two children aged 4 to 11 years at 2 primary schools in England completed a worksheet containing photos of foods and pharmaceutical products. Children were asked to identify what the picture showed and classify it as "good for them," "bad for them," or "sometimes good/sometimes bad for them." Responses were marked as correct if they identified an item without the need for exact identification. Where an item was correctly identified, risk perception was analyzed. RESULTS: Children correctly identified 5 of the 7 pictures as a form of medicine (mean=5.10, standard deviation=1.51), and identification was positively correlated with age (ρ=0.59, P<.001). A greater percentage of children correctly identified bicolored capsules (86.3% correct, 95% confidence interval [CI]=81.3-91.3) as medicines than either white (71.4% correct, 95% CI=64.9-78) or pink tablets (33.5% correct, 95% CI=26.7-40.4). There was a significant shift with age in the perceptions of the children as they changed from reporting that medicines were good for them to reporting that they were sometimes good and sometimes bad for them. This held for all medicines (χ(2) tests, P<.05) except for the cream and the inhaler. CONCLUSIONS: As children get older, they become better at identifying medicines, and they become more likely to see their potential risks.

Strain differences and the role of AT(1) receptor expression in anxiety.

This study investigated strain specific differences to the anxiolytic response to losartan focusing on genetic variation that may influence such responses. This included: AT(1) receptor sequence variation, angiotensin II receptor associated protein (ATRAP) and receptor expression between strains. Sequencing of exon 3 of AT(1a)R revealed no differences between BKW mice (n=6) and C57 and DBA(2) strains (n=3). Comparisons of AT(1) expression do show significant differences, whereby BKW mice showed the highest levels of expression and DBA(2) mice intermediate levels when compared to the C57 strain. Sequencing of sections of the Angiotensin receptor associated protein (ATRAP) identified a non-synonymous point mutation- (T/C) transversion (position 109-161) (SNP id = rs13467517) resulting in a Valine → Alanine (V157A) amino acid change in the BKW and DBA(2) strains. Our results indicate that the previously reported strain dependent effects are not due to variation in AT(1a) receptor sequence. Differences in AT(1)gene expression levels between strains, which mirror their anxiety phenotype, are observed. This is coupled with a non-synonymous single nucleotide polymorphism in ATRAP, a negative regulator of AT(1) signalling.

Assessment of biological activity of novel peptide analogues of angiotensin IV.

OBJECTIVES: Angiotensin IV (Ang IV) is a metabolite of angiotensin II which acts on specific AT(4) receptors identified as the enzyme insulin regulated aminopeptidase (IRAP). The transduction process of these receptors is unresolved, but Ang IV inhibits the aminopeptidase activity. Ang IV improves cognition in animal models thus there is a desire to develop metabolically stable analogues for further development. METHODS: Peptide analogues of Ang IV were obtained commercially or synthesised. Each peptide was tested in vitro for its ability to inhibit the aminopeptidase activity (IRAP) of mouse brain homogenates and for its effects on isolated rat uterine smooth muscle. KEY FINDINGS: [Des-Val(1) ]-Ang IV, acetylated-Ang IV-amide, Ang IV-amide and [des-His(4) ]-Ang IV all inhibited IRAP. [Sar(1) , Ile(8) ]-Angiotensin II (10 µm) had an effect greater than that of Ang IV or any of the other analogues studied. In isolated uterine smooth muscle, angiotensins II and IV induced contractions, which could be antagonised by an AT(1) -receptor antagonist. None of the novel peptides induced uterine smooth muscle contractions, but [Sar(1) , des Arg(2) -Gly(8) ]-angiotensin II showed significant antagonism of the contractile effects of angiotensin II and carboxyamide-terminated Ang IV-NH(2) showed antagonism of Ang IV-induced contractions. CONCLUSIONS: This study provides five novel inhibitors of IRAP worthy of assessment in behavioural models of learning and memory. The analogues are devoid of AT(1) receptor agonist properties, and the carboxyamide analogue presents an opportunity to elucidate the mechanism of action of Ang IV as, like Ang IV, it inhibits IRAP, but antagonises the effects of Ang IV on isolated smooth muscle.

Non-adherence to antihypertensive medication and impaired cognition: which comes first?

OBJECTIVE: Antihypertensive medications are important in the prevention of serious consequences of hypertension, such as stroke and heart failure. Up to one-third of elderly hypertensive patients, however, do not adhere to their medication. Adherence to medication decreases with increasing age, and with decreasing cognitive ability, thus elderly, cognitively-impaired patients have poorer control of blood pressure. Good control of blood pressure is associated with decreased prevalence of dementia and Alzheimer's disease. This study assessed the evidence that antihypertensive medications have effects on the prevalence or severity of mild cognitive impairment, dementia or Alzheimer's disease. METHODS: The ISI Web of Knowledge database was searched; including replicates, the nine searches identified 14400 publications since 1952, of which 9.9% had been published in 2009. This review considers the 18 studies meeting the set criteria published in 2009 or later. KEY FINDINGS: Not all antihypertensive medications are equivalent in their positive cognitive effects, with brain-penetrating angiotensin-converting-enzyme inhibitors and possibly angiotensin receptor antagonists being the most effective. CONCLUSIONS: Based on evidence of blood-pressure control and cost, UK National Institute for Health and Clinical Excellence guidelines recommend calcium-channel blockers or thiazide-type diuretics for the treatment of hypertension in patients over 55 years. These guidelines take no account of the potential cognitive effects of the antihypertensive therapies, consideration of which might lead to a review. There may be benefit in stressing that adherence to antihypertensive medication not only decreases the risk of cardiovascular disease and death, but may also decrease the risk or severity of mild cognitive impairment, dementia and Alzheimer's disease.

Strain differences in the effects of angiotensin IV on mouse cognition.

Angiotensin IV has been shown to improve learning and memory in rodents. Strain dependent variation in murine behaviour, aminopeptidase activity and inhibitory effect of Angiotensin IV, structural variation in insulin regulated aminopeptidase (IRAP) and aminopeptidase N (ApN) and expression of the encoding genes were explored. Strain differences in the behavioural response to Angiotensin IV were observed, where CD mice were refractory. All strains showed inhibition of aminopeptidase activity by Angiotensin IV but CD mice displayed reduced endogenous aminopeptidase activity. No differences in the coding sequence of IRAP or ApN were found. RT-PCR analysis showed no difference in IRAP expression between strains but an increased expression of ApN was observed in CD mice. The lack of cognitive response of CD mice to Angiotensin IV cannot be explained through variation within IRAP sequence nor expression but the results highlight a potential role for ApN in the effects of Angiotensin IV.

Implications of the angiotensin converting enzyme gene insertion/deletion polymorphism in health and disease: a snapshot review.

This review considers the 250+ papers concerning the association of the angiotensin converting enzyme (ACE) gene insertion/deletion polymorphism (rs1799752) and various disease conditions published in 2009. The deletion allele occurs in approximately 55% of the population and is associated with increased activity of the ACE enzyme. It might be predicted that the D allele, therefore, might be associated with pathologies involving increased activity of the renin-angiotensin system. The D allele was seen to be associated with an increased risk of hypertension, pre-eclampsia, heart failure, cerebral infarct, diabetic nephropathy, encephalopathy, asthma, severe hypoglycaemia in diabetes, gastric cancer (in Caucasians) and poor prognosis following kidney transplant. On the positive side, the D allele appears to offer protection against schizophrenia and chronic periodontitis and confers greater up-per-body strength in old age. The I allele, meanwhile, offers improved endurance/athletic performance and aerobic capacity as determined by lung function tests, although it does increase the risk of oral squamous cell carcinoma and obstructive sleep apnoea in hypertensives.

Hip subluxation and dislocation in cerebral palsy - a prospective study on the effectiveness of postural management programmes.

BACKGROUND AND PURPOSE: Hip subluxation and dislocation are common sequelae in children with bilateral cerebral palsy and are currently managed by surgical interventions. This study aimed to investigate the effectiveness of early postural management programmes on hip subluxation and dislocation at five years, and the need for treatment in children with bilateral cerebral palsy, and to compare these findings with a historical control group. METHODS: A prospective cohort study followed 39 children who commenced using postural management equipment under 18 months of age. Levels of ability, type and amount of equipment use and treatments were recorded every three months. At 30 and 60 months, the hips were X-rayed and the hip migration percentage was measured. The results were compared with the historical control group. RESULTS: Children who used equipment at recommended and moderate levels had significantly less chance of both hips being subluxed than those using equipment at minimal levels (two-tailed Fisher's exact chi(2) p = 0.024). The frequency of children with hip problems was significantly less in the intervention group in comparison to the historical control group at five years (chi(2) = 11.53, df = 2, p = 0.006). The frequency of children receiving bilateral or unilateral treatments, i.e. surgery, use of a hip and spinal orthosis and/or botulinum toxin injections, in the intervention group was significantly less compared to the historical control group (two-tailed Fisher's exact p = 0.001). CONCLUSION: The early provision of postural management equipment has a role to play in reducing the number of hip problems and therefore the need for treatment of hip subluxation/dislocation in cerebral palsy at five years of age.

Cognitive-enhancing effects of angiotensin IV.

Angiotensin IV is a derivative of the potent vasoconstrictor angiotensin II and it has been shown to enhance acquisition, consolidation and recall in animal models of learning and memory when administered centrally or peripherally. Whether changes in angiotensin IV activity underlie the cognitive effects of those cardiovascular drugs designed to disrupt the peripheral renin-angiotensin system in humans remains undetermined, but angiotensin IV appears to be a worthy candidate for consideration in drug development programmes. The mechanism of action of angiotensin IV is still debated, although its AT4 receptor has been convincingly identified as being insulin-regulated amino peptidase, which is also known as oxytocinase and placental leucine aminopeptidase. It is speculated that angiotensin IV may interact with insulin-regulated amino peptidase to enhance neuronal glucose uptake, prevent metabolism of other neuroactive peptides, induce changes in extracellular matrix molecules, or induce release of acetylcholine and/or dopamine. All of these things may be responsible for the beneficial effects on cognition, but none of them are yet proven. Importantly, strain differences in murine responses to angiotensin IV suggest that some individuals may benefit from drugs targeted to the AT4 receptor whilst others may be refractory. At present it thus appears that those individuals with the poorest baseline cognition may receive greatest benefit, but possible genetic differences in responses to angiotensin IV cannot be ruled-out.

Reduction of paraquat-induced renal cytotoxicity by manganese and copper complexes of EGTA and EHPG.

Superoxide anion generation plays an important role in the development of paraquat toxicity. Although superoxide dismutase mimetics (SODm) have provided protection against organ injury involving generation of superoxide anions, they often suffer problems, e.g., regarding their bioavailability or potential pro-oxidant activity. The aim here was to investigate and compare the therapeutic potential of two novel SODm, manganese(II) and copper(II) complexes of the calcium chelator ethylenebis(oxyethylenenitrilo)tetraacetic acid (EGTA) and of the contrast agent ethylenebis(hydroxyphenylglycine) (EHPG), against paraquat-induced renal toxicity in vitro. Incubation of renal NRK-52E cells with paraquat (1 mM) for 24 h produced submaximal, yet significant, reduction in cellular viability and cell death and produced significant increases in superoxide anion and hydroxyl radical generation. Manganese and copper complexes of EGTA (10-100 microM) and EHPG (30-100 microM) reduced paraquat-induced renal cell toxicity and reduced superoxide anion and hydroxyl radical generation significantly. Manganese complexes displayed greater efficacy than copper complexes and, at equivalent concentrations, manganese complexed with EHPG provided the greatest protection. Furthermore, these metal complexes did not interfere with the uptake of [methyl-(14)C]paraquat into NRK-52E cells, suggesting that they provided protection against paraquat cytotoxicity via intracellular mechanisms. These complexes did not display cytotoxicity at the concentrations examined. Together, these results suggest that manganese and copper complexes of EGTA and EHPG, and especially the manganese-EHPG complex, could provide benefit against paraquat nephrotoxicity.

Interactions of angiotensin IV and oxytocin on behaviour in mice.

INTRODUCTION: Angiotensin (Ang) IV enhances learning and memory in rats but there are strain differences in its effects in mice. Oxytocin (OT) also influences learning and memory in rats and mice and, in the light of the proposed effects of Ang IV on oxytocinase, the hypothesis that the effects of Ang IV on cognition in mice involve OT was tested. MATERIALS AND METHODS: The effects of Ang IV and OT, alone and combined, were determined in rat isolated uterine smooth muscle and in object recognition and forced swim tests in BKW mice. RESULTS: Ang potentiated the contractile effects of OT in the uterus. Neither peptide had any effect on object recognition nor locomotor activity. Ang IV had no effect in the forced swim test but abolished the effects of OT. CONCLUSIONS: Ang IV influences the actions of OT in vitro and in vivo, possibly by inhibition of oxytocinase, but the lack of effect of Ang IV on object recognition in BKW mice is unlikely to be a consequence of a deficiency endogenous OT. Unlike OT, Ang IV alone has no effect on learned helplessness in the forced swim test, an effect often used to predict potential antidepressant efficacy in humans.