Impairment of serine transport across the blood-brain barrier by deletion of Slc38a5 causes developmental delay and motor dysfunction.

Brain L-serine is critical for neurodevelopment and is thought to be synthesized solely from glucose. In contrast, we found that the influx of L-serine across the blood-brain barrier (BBB) is essential for brain development. We identified the endothelial Slc38a5, previously thought to be a glutamine transporter, as an L-serine transporter expressed at the BBB in early postnatal life. Young Slc38a5 knockout (KO) mice exhibit developmental alterations and a decrease in brain L-serine and D-serine, without changes in serum or liver amino acids. Slc38a5-KO brains exhibit accumulation of neurotoxic deoxysphingolipids, synaptic and mitochondrial abnormalities, and decreased neurogenesis at the dentate gyrus. Slc38a5-KO pups exhibit motor impairments that are affected by the administration of L-serine at concentrations that replenish the serine pool in the brain. Our results highlight a critical role of Slc38a5 in supplying L-serine via the BBB for proper brain development.

A new type of blood-brain barrier aminoacidopathy underlies metabolic microcephaly associated with SLC1A4 mutations.

Mutations in the SLC1A4 transporter lead to neurodevelopmental impairments, spastic tetraplegia, thin corpus callosum and microcephaly in children. SLC1A4 catalyses obligatory amino acid exchange between neutral amino acids, but the physiopathology of SLC1A4 disease mutations and progressive microcephaly remain unclear. Here, we examined the phenotype and metabolic profile of three Slc1a4 mouse models: a constitutive Slc1a4-knockout mouse; a knock-in mouse with the major human Slc1a4 mutation (Slc1a4-K256E); and a selective knockout of Slc1a4 in brain endothelial cells (Slc1a4tie2-cre). We show that Slc1a4 is a bona fideL-serine transporter at the blood-brain barrier (BBB) and that acute inhibition or deletion of Slc1a4 leads to a decrease in serine influx into the brain. This results in microcephaly associated with decreased L-serine content in the brain, accumulation of atypical and cytotoxic 1-deoxysphingolipids, neurodegeneration, synaptic and mitochondrial abnormalities and behavioural impairments. Prenatal and early postnatal oral administration of L-serine at levels that replenish the serine pool in the brain rescued the observed biochemical and behavioural changes. Administration of L-serine until the second postnatal week also normalized brain weight in Slc1a4-E256K mice. Our observations suggest that the transport of 'non-essential' amino acids from the blood through the BBB is at least as important as that of essential amino acids for brain metabolism and development. We propose that SLC1A4 mutations cause a BBB aminoacidopathy with deficits in serine import across the BBB, required for optimal brain growth, leading to a metabolic microcephaly, which may be amenable to treatment with L-serine.

Endogenous histidine peptides are physiological antioxidants that prevent oligodendrocyte cell death and myelin loss in vivo.

Histidine dipeptides (HDs) are synthesized in brain oligodendrocytes by carnosine synthase (carns1), but their role is unknown. Using metabolomics and in vivo experiments with both constitutive and oligodendrocyte-selective carns1-KO mouse models, we found that HDs are critical for oligodendrocyte survival and protect against oxidative stress. Carns1-KO mouse models had lower numbers of mature oligodendrocytes, increased lipid peroxidation, and behavioral changes. Cuprizone administration, which increases reactive oxygen species in vivo, resulted in higher oligodendrocyte death, demyelination, axonal alterations, and oxidative damage in the corpus callosum of carns1-KO mice. Gliosis and oxidative damage by cuprizone were prevented by pretreatment with the antioxidant N-acetylcysteine. NADPH levels were increased threefold in the brains of carns1-KO mice as an antioxidant response to oxidative stress through acceleration of the pentose phosphate pathway (PPP). This was due to overexpression of glucose-6-phosphate dehydrogenase, the rate-limiting enzyme of the PPP. Likewise, expression of NAD kinase, the biosynthetic enzyme for NADP+, and NAMPT, which replenishes the NAD+ pool, was higher in carns1-KO mice brains than in controls. Our observations suggest that HDs cell-autonomously protect oligodendrocytes from oxidative stress, with implications for demyelinating diseases.

Attention deficits as a key player in the symptomatology of posttraumatic stress disorder: A review.

Posttraumatic stress disorder (PTSD) is a debilitating psychiatric disorder characterized by symptoms such as re-experiencing of the psychotrauma and hyperarousal. Although current literature mainly discusses the emotionally related aspects of these symptoms, studies also highlight the relation between re-experiencing, hyperarousability, and attention deficits, which are associated with poorer daily function and reduced quality of life. This review provides a comprehensive analysis of the existing research on attention deficits among adults with PTSD. A systematic search through five databases resulted in the inclusion of 48 peer-reviewed, English-language articles, describing 49 distinct studies. Using a total of 47 different attentional assessment tools, the majority of studies investigated sustained (n = 40), divided (n = 16), or selective (n = 14) attention. A total of 30 studies (61.2%) found significant correlations between PTSD symptoms and attention deficits, and 10 studies (20.4%) found that higher levels of attention deficits were predictive of worse PTSD symptoms. Moreover, neuroimaging results of six (f)MRI and three EEG studies identified various potential neurobiological pathways involved, including (pre)frontal attention networks. Together, the body of research shows that attention deficits in individuals with PTSD are common and occur in surroundings with emotionally neutral stimuli. Nonetheless, current treatment strategies do not target these attentional difficulties. We propose a novel perspective to PTSD diagnosis and treatment strategies based on attention deficits and their relation with top-down regulation of re-experiencing and subsequent other PTSD symptoms.

ASCT1 (Slc1a4) transporter is a physiologic regulator of brain d-serine and neurodevelopment.

d-serine is a physiologic coagonist of NMDA receptors, but little is known about the regulation of its synthesis and synaptic turnover. The amino acid exchangers ASCT1 (Slc1a4) and ASCT2 (Slc1a5) are candidates for regulating d-serine levels. Using ASCT1 and ASCT2 KO mice, we report that ASCT1, rather than ASCT2, is a physiologic regulator of d-serine metabolism. ASCT1 is a major d-serine uptake system in astrocytes and can also export l-serine via heteroexchange, supplying neurons with the substrate for d-serine synthesis. ASCT1-KO mice display lower levels of brain d-serine along with higher levels of l-alanine, l-threonine, and glycine. Deletion of ASCT1 was associated with neurodevelopmental alterations including lower hippocampal and striatal volumes and changes in the expression of neurodevelopmental-relevant genes. Furthermore, ASCT1-KO mice exhibited deficits in motor function, spatial learning, and affective behavior, along with changes in the relative contributions of d-serine vs. glycine in mediating NMDA receptor activity. In vivo microdialysis demonstrated lower levels of extracellular d-serine in ASCT1-KO mice, confirming altered d-serine metabolism. These alterations are reminiscent of some of the neurodevelopmental phenotypes exhibited by patients with ASCT1 mutations. ASCT1-KO mice provide a useful model for potential therapeutic interventions aimed at correcting the metabolic impairments in patients with ASCT1 mutations.

Asc-1 Transporter Regulation of Synaptic Activity via the Tonic Release of d-Serine in the Forebrain.

d-Serine is a co-agonist of NMDA receptors (NMDARs) whose activity is potentially regulated by Asc-1 (SLC7A10), a transporter that displays high affinity for d-serine and glycine. Asc-1 operates as a facilitative transporter and as an antiporter, though the preferred direction of d-serine transport is uncertain. We developed a selective Asc-1 blocker, Lu AE00527, that blocks d-serine release mediated by all the transport modes of Asc-1 in primary cultures and neocortical slices. Furthermore, d-serine release is reduced in slices from Asc-1 knockout (KO) mice, indicating that d-serine efflux is the preferred direction of Asc-1. The selectivity of Lu AE00527 is assured by the lack of effect on slices from Asc-1-KO mice, and the lack of interaction with the co-agonist site of NMDARs. Moreover, in vivo injection of Lu AE00527 in P-glycoprotein-deficient mice recapitulates a hyperekplexia-like phenotype similar to that in Asc-1-KO mice. In slices, Lu AE00527 decreases the long-term potentiation at the Schaffer collateral-CA1 synapses, but does not affect the long-term depression. Lu AE00527 blocks NMDAR synaptic potentials when typical Asc-1 extracellular substrates are present, but it does not affect AMPAR transmission. Our data demonstrate that Asc-1 mediates tonic co-agonist release, which is required for optimal NMDAR activation and synaptic plasticity.

The alanine-serine-cysteine-1 (Asc-1) transporter controls glycine levels in the brain and is required for glycinergic inhibitory transmission.

Asc-1 (SLC7A10) is an amino acid transporter whose deletion causes neurological abnormalities and early postnatal death in mice. Using metabolomics and behavioral and electrophysiological methods, we demonstrate that Asc-1 knockout mice display a marked decrease in glycine levels in the brain and spinal cord along with impairment of glycinergic inhibitory transmission, and a hyperekplexia-like phenotype that is rescued by replenishing brain glycine. Asc-1 works as a glycine and L-serine transporter, and its transport activity is required for the subsequent conversion of L-serine into glycine in vivo. Asc-1 is a novel regulator of glycine metabolism and a candidate for hyperekplexia disorders.

Methylphenidate and environmental enrichment ameliorate the deleterious effects of prenatal stress on attention functioning.

Either pre- or post-natal environmental factors seem to play a key role in brain and behavioral development and to exert long-term effects. Increasing evidence suggests that exposure to prenatal stress (PS) leads to motor and learning deficits and elevated anxiety, while enriched environment (EE) shows protective effects. The dopaminergic system is also sensitive to environmental life circumstances and affects attention functioning, which serves as the preliminary gate to cognitive processes. However, the effects of methylphenidate (MPH) on the dopaminergic system and attentional functioning, in the context of these life experiences, remain unclear. Therefore, we aimed to examine the effects of EE or PS on distinct types of attention, along with possible effects of MPH exposure. We found that PS impaired selective attention as well as partial sustained attention, while EE had beneficial effects. Both EE and MPH ameliorated the deleterious effects of PS on attention functioning. Considering the possible psychostimulant effect of MPH, we examined both anxiety-like behavior as well as motor learning. We found that PS had a clear anxiogenic effect, whereas EE had an anxiolytic effect. Nevertheless, the treatment with both MPH and/or EE recovered the deleterious effects of PS. In the motor-learning task, the PS group showed superior performance while MPH led to impaired motor learning. Performance decrements were prevented in both the PS + MPH and EE + MPH groups. This study provides evidence that peripubertal exposure to EE (by providing enhanced sensory, motor, and social opportunities) or MPH treatments might be an optional therapeutic intervention in preventing the PS long-term adverse consequences.

Intrusive trauma recollections is associated with impairment of interference inhibition and psychomotor speed in PTSD.

BACKGROUND: Intrusive cognitions that enter consciousness involuntarily are prominent symptoms of posttraumatic stress disorder (PTSD). The present study aimed to identify neuropsychological mechanisms involved. METHOD: Fifty PTSD outpatients and 50 healthy controls were tested using Finger Tapping, Simple and Choice Reaction Times and Stroop Tasks, to measure motor, psychomotor speed, response selection, and interference inhibition ability respectively. RESULTS: PTSD patients performed poorly in all tests, presumably owing to their generalized slowness of information processing and motor reaction. Psychomotor speed was a predictor of slowness and high error rate during the Stroop. Impaired inhibition, as measured by the interference index of the Stroop task, explained 9.7% of the predicated variance in frequency of re-experiencing PTSD symptoms and 23.5% of the predicated variance in augmentation of the interference response time. CONCLUSION: Impaired interference control may be related to internal (re-experiencing) and external (sensory) stimuli that leads to cognitive deficits in PTSD patients.

Establishing hospital admission criteria of pediatric Henoch-Schonlein purpura.

The current study aimed to define evidence-based admission criteria of pediatric Henoch-Schonlein purpura (HSP). In addition, we aimed to better characterize epidemiological and clinical features of pediatric HSP in Israel. We performed a retrospective cohort study of all children with HSP admitted during a 15 years period to a single pediatric department. We strictly collected the clinical data of all HSP cases. Each case was categorized as either "necessary admission" or "unnecessary admission." We compared the two groups, using initially Chi square (χ(2)) and student "t" tests, and thereafter, we employed logistic stepwise regression analysis. One hundred and sixty-three children with HSP were included. A set of six clinical criteria of which the presence of minimum one predicts the need for hospitalization were identified including: orchitis, moderate or severe abdominal pain, arthritis involving more than two joints, proteinuria, clear evidence of gastrointestinal bleeding, and inability to ambulate. In conclusion, we suggest a predictive model for the admission of pediatric patients with acute HSP. The implementation of this model can significantly reduce unnecessary admissions.

Reversible modulations of neuronal plasticity by VEGF.

Neurons, astrocytes, and blood vessels are organized in functional "neurovascular units" in which the vasculature can impact neuronal activity and, in turn, dynamically adjust to its change. Here we explored different mechanisms by which VEGF, a pleiotropic factor known to possess multiple activities vis-à-vis blood vessels and neurons, may affect adult neurogenesis and cognition. Conditional transgenic systems were used to reversibly overexpress VEGF or block endogenous VEGF in the hippocampus of adult mice. Importantly, this was done in settings that allowed the uncoupling of VEGF-promoted angiogenesis, neurogenesis, and memory. VEGF overexpression was found to augment all three processes, whereas VEGF blockade impaired memory without reducing hippocampal perfusion or neurogenesis. Pertinent to the general debate regarding the relative contribution of adult neurogenesis to memory, we found that memory gain by VEGF overexpression and memory impairment by VEGF blockade were already evident at early time points at which newly added neurons could not yet have become functional. Surprisingly, VEGF induction markedly increased in vivo long-term potentiation (LTP) responses in the dentate gyrus, and VEGF blockade completely abrogated LTP. Switching off ectopic VEGF production resulted in a return to a normal memory and LTP, indicating that ongoing VEGF is required to maintain increased plasticity. In summary, the study not only uncovered a surprising role for VEGF in neuronal plasticity, but also suggests that improved memory by VEGF is primarily a result of increasing plasticity of mature neurons rather than the contribution of newly added hippocampal neurons.

Prepubertal chronic stress and ketamine administration to rats as a neurodevelopmental model of schizophrenia symptomatology.

Increased vulnerability to psychiatric disorders, such as schizophrenia, has been associated with higher levels of stress. In the early development of the central nervous system, changes in function of glutamatergic N-Methyl-D-aspartate (NMDA) receptors can possibly result in the development of psychosis, cognitive impairment and emotional dysfunction in adulthood. Thus, in this study we examined the behavioural consequences of the exposure of male rats to chronic stress (postnatal days 30-60) and ketamine administration (postnatal days 41-45); both during a sensitive developmental time window. We found that the locomotor activity of both ketamine and ketamine+chronic stress groups was significantly higher compared with that of the control rats. In contrast, the locomotor activity of the chronic stress group was significantly lower compared with all other groups. Examining anhedonia in the sucrose preference test we found a significantly decreased sucrose intake in both ketamine+chronic stress and the chronic stress groups compared with the control rats. No significant differences were observed in sucrose intake between the control and the ketamine group. The object recognition test revealed that the attention to the novel object was significantly impaired in the ketamine+chronic stress group. Similarly, the ketamine+chronic stress group showed the poorest learning ability in the eight-arm radial maze, starting on the 8th day. Finally, throughout the different pre-pulse intensities, the ketamine+chronic stress group showed impaired PPI compared with all other groups. The results indicate that the combination of prepubertal onset of chronic stress and ketamine may serve as a valid novel animal model for schizophrenia-like symptoms.

Emotional and sensory dysregulation as a possible missing link in attention deficit hyperactivity disorder: A review.

Attention Deficit Hyperactivity Disorder (ADHD) is a common developmental disorder affecting 5-7% of adults and children. We surveyed the literature to examine ADHD through three pillars: developmental characteristics, symptomatology, and treatment strategies. Firstly, in terms of developmental characterstics, early life stress may increase the risk of developing ADHD symptoms according to animal models' research. Secondly, the current core symptoms of ADHD are comprised of inattention, hyperactivity, and impulsivity. However, the up-to-date literature indicates individuals with ADHD experience emotional and sensory dysregulation as well, which early-life stress may also increase the risk of. Finally, we discuss the therapeutic benefits of methylphenidate on both the current core ADHD symptoms and the sensory and emotional dysregulation found in those with ADHD. In summation, we surveyed the recent literature to analyze (i) the potential role of early-life stress in ADHD development, (ii) the involvement of emotional and sensory dysregulation in ADHD symptomatology and finally, (iii) the therapeutic intervention with methylphenidate, aiming to reduce the potential effect of early life stress in ADHD, and mainly emotional and sensory dysregulation. The apparent but currently less recognized additional symptoms of emotional and sensory dysregulation in ADHD call for further investigation of these possible causes and thus increasing treatments efficacy in individuals with ADHD.

Polymeric nanocarriers for nose-to-brain drug delivery in neurodegenerative diseases and neurodevelopmental disorders.

Neurodegenerative diseases are progressive conditions that affect the neurons of the central nervous system (CNS) and result in their damage and death. Neurodevelopmental disorders include intellectual disability, autism spectrum disorder, and attention-deficit/hyperactivity disorder and stem from the disruption of essential neurodevelopmental processes. The treatment of neurodegenerative and neurodevelopmental conditions, together affecting ∼120 million people worldwide, is challenged by the blood-brain barrier (BBB) and the blood-cerebrospinal fluid barrier that prevent the crossing of drugs from the systemic circulation into the CNS. The nose-to-brain pathway that bypasses the BBB and increases the brain bioavailability of intranasally administered drugs is promising to improve the treatment of CNS conditions. This pathway is more efficient for nanoparticles than for solutions, hence, the research on intranasal nano-drug delivery systems has grown exponentially over the last decade. Polymeric nanoparticles have become key players in the field owing to the high design and synthetic flexibility. This review describes the challenges faced for the treatment of neurodegenerative and neurodevelopmental conditions, the molecular and cellular features of the nasal mucosa and the contribution of intranasal nano-drug delivery to overcome them. Then, a comprehensive overview of polymeric nanocarriers investigated to increase drug bioavailability in the brain is introduced.

Brain-Targeted Liposomes Loaded with Monoclonal Antibodies Reduce Alpha-Synuclein Aggregation and Improve Behavioral Symptoms in Parkinson's Disease.

Monoclonal antibodies (mAbs) hold promise in treating Parkinson's disease (PD), although poor delivery to the brain hinders their therapeutic application. In the current study, it is demonstrated that brain-targeted liposomes (BTL) enhance the delivery of mAbs across the blood-brain-barrier (BBB) and into neurons, thereby allowing the intracellular and extracellular treatment of the PD brain. BTL are decorated with transferrin to improve brain targeting through overexpressed transferrin-receptors on the BBB during PD. BTL are loaded with SynO4, a mAb that inhibits alpha-synuclein (AS) aggregation, a pathological hallmark of PD. It is shown that 100-nm BTL cross human BBB models intact and are taken up by primary neurons. Within neurons, SynO4 is released from the nanoparticles and bound to its target, thereby reducing AS aggregation, and enhancing neuronal viability. In vivo, intravenous BTL administration results in a sevenfold increase in mAbs in brain cells, decreasing AS aggregation and neuroinflammation. Treatment with BTL also improve behavioral motor function and learning ability in mice, with a favorable safety profile. Accordingly, targeted nanotechnologies offer a valuable platform for drug delivery to treat brain neurodegeneration.

Exposure to static magnetic field facilitates selective attention and neuroplasticity in rats.

Static magnetic fields (SMF) have neuroprotective and behavioral effects in rats, however, little is known about the effects of SMF on cognition, motor function and the underlying neurochemical mechanisms. In this study, we focused on the effects of short-term (5-10d) and long-term (13-38d) SMF exposure on selective attention and motor coordination of rats, as well as associated alterations in expression level of neuroplasticity-related structural proteins and cryptochrome (CRY1) protein in the cortex, striatum and ventral midbrain. The results showed that 6d SMF exposure significantly enhanced selective attention without affecting locomotor activity in open field. All SMF exposures non-significantly enhanced motor coordination (Rotarod test). Neurochemical analysis demonstrated that 5d SMF exposure increased the expression of cortical and striatal CRY1 and synapsin-1 (SYN1), striatal total synapsins (SYN), and synaptophysin (SYP), growth associated protein-43 (GAP43) and post-synaptic density protein-95 (PSD95) in the ventral midbrain. Exposure to SMF for 14d increased PSD95 level in the ventral midbrain while longer SMF exposure elevated the levels of PSD95 in the cortex, SYN and SYN1 in all the examined brain areas. The increased expression of cortical and striatal CRY1 and SYN1 correlated with the short-lasting effect of SMF on improving selective attention. Collectively, SMF's effect on selective attention attenuated following longer exposure to SMF whereas its effects on neuroplasticity-related structural biomarkers were time- and brain area-dependent, with some protein levels increasing with longer time exposure. These findings suggest a potential use of SMF for treatment of neurological diseases in which selective attention or neuroplasticity is impaired.

Differential Impact of Work Overload on Physicians' Attention: A Comparison Between Residential Fields.

OBJECTIVES: Medical errors cause tens of thousands of deaths annually and have a major impact on quality of care and management; however, it receives scant research and public awareness. This study aimed to examine the relation between workload-induced lack of sleep and attention failure, as indications for medical errors risk, among young residents. METHODS: We performed an evaluation of young physicians by the Test of Variables of Attention, before and after a 24-hour shift. RESULTS: Workload was manifested by 13% overall attention impairment at baseline, which increased to 34% with deficiencies below the normal range after the shift. Attention measures differed between physicians of each residential field at baseline, but to greater extent after the shift. CONCLUSIONS: Traditional working schedule is strongly associated with attention failure. Based on the literature linking attention failures to medical errors, we suggest a regulatory change regarding residents' shift duration to decrease preventable errors.

Astrocytes support hippocampal-dependent memory and long-term potentiation via interleukin-1 signaling.

Recent studies indicate that astrocytes play an integral role in neural and synaptic functioning. To examine the implications of these findings for neurobehavioral plasticity we investigated the involvement of astrocytes in memory and long-term potentiation (LTP), using a mouse model of impaired learning and synaptic plasticity caused by genetic deletion of the interleukin-1 receptor type I (IL-1RI). Neural precursor cells (NPCs), derived from either wild type (WT) or IL-1 receptor knockout (IL-1rKO) neonatal mice, were labeled with bromodeoxyuridine (BrdU) and transplanted into the hippocampus of either IL-1rKO or WT adult host mice. Transplanted NPCs survived and differentiated into astrocytes (expressing GFAP and S100ß), but not to neurons or oligodendrocytes. The NPCs-derived astrocytes from WT but not IL-1rKO mice displayed co-localization of GFAP with the IL-1RI. Four to twelve weeks post-transplantation, memory functioning was examined in the fear-conditioning and the water maze paradigms and LTP of perforant path-dentate gyrus synapses was assessed in anesthetized mice. As expected, IL-1rKO mice transplanted with IL-1rKO cells or sham operated displayed severe memory disturbances in both paradigms as well as a marked impairment in LTP. In contrast, IL-1rKO mice transplanted with WT NPCs displayed a complete rescue of the impaired memory functioning as well as partial restoration of LTP. These findings indicate that astrocytes play a critical role in memory functioning and LTP, and specifically implicate astrocytic IL-1 signaling in these processes. The results suggest novel conceptualization and therapeutic targets for neuropsychiatric disorders characterized by impaired astrocytic functioning concomitantly with disturbed memory and synaptic plasticity.

Shyness and social phobia in Israeli Jewish vs Arab students.

BACKGROUND: Social anxiety disorder (SAD) has been repeatedly shown to be very prevalent in the Western society with prevalence rates of 10% or above. However, very few studies have been performed in the Middle East and in Arab countries. METHODS: A total of 300 Israeli students participated in our study and were administered the Liebowitz Social Anxiety Scale (LSAS), the Cheek and Buss Shyness Questionnaire (CBSQ), and a sociodemographic questionnaire. RESULTS: A total of 153 Jewish and 147 Arab students participated in the survey. Social anxiety disorder was found in 12.33% of the sample, according to the LSAS cutoff score of more than 60. The 2 subsamples had similar LSAS and CBSQ scores and similar SAD-positive rates (LSAS >60). Females had higher scores on the LSAS, as were those without a spouse and those who had been in psychological treatment. Based on a regression analysis, the significant predictors of the LSAS score were the CBSQ score and female sex. A very high correlation was found between the LSAS and the CBSQ scores. CONCLUSIONS: Although our sample is not representative of the whole Israeli population, we conclude that SAD and shyness were similarly prevalent in Jewish and Arab students in Israel. Social anxiety disorder scores were higher among females, those without a spouse, and those who received psychological treatment. Further studies on the clinical and cultural characteristics of SAD in Israeli subcultures would add to the growing body of knowledge on SAD in various cultures.