6% were rearfoot strikers (Table 1). Results of chi-square analyses indicate that observed foot strike frequency distributions differ significantly between barefoot and minimally shod runners (X2 = 13.5, df = 2, p < 0.01). The foot strike frequency distribution for barefoot runners in this study differs significantly from those recorded for traditionally shod Baf-A1 road racers in Larson et al. 3 (X2 = 571.63, df = 2, p < 0.0001) and Kasmer et al. 4 (X2 = 751.86, df = 2, p < 0.0001). The foot strike frequency distribution for minimally

shod runners in this study differs significantly from those recorded for traditionally shod road racers in Larson et al. 3 (X2 = 149.2, df = 2, p < 0.0001) and Kasmer et al. 4 (X2 = 265.88, df = 2, p < 0.0001) . Available published data from road race studies conducted to date indicate that approximately 75%–95% of runners land on their rearfoot when initially contacting the ground1, 2, 3 and 4 (Table 1). It is reasonable to presume that the vast majority

of the runners SAHA HDAC in these studies were habitually shod and wore some type of cushioned running shoe during the race, though exact shoe properties might differ among running populations (e.g., racing flats for elite half-marathoners, conventionally cushioned running shoes for recreational marathoners). In support of this presumption, only two of the 936 runners examined by Larson et al.3 were wearing minimally cushioned running shoes (VFF for both; no runners were barefoot). In contrast to the above studies, Lieberman et al.9 observed that initial contact on the midfoot or forefoot is typical for habitually barefoot Kenyan adolescents on a dirt road (88% of foot strikes) and habitually barefoot

American adults in the laboratory (75% of foot strikes). Incidence of rearfoot striking in this same population of habitually barefoot American adults increased from 25% to 50% when shod, and habitually shod Kenyans and Americans tended to rearfoot strike regardless of whether they were wearing shoes.9 These results suggest Thiamine-diphosphate kinase that footwear may influence foot strike patterns. Foot strike distributions for barefoot runners observed here were significantly different from those observed previously for shod road racers. Larson et al.3 and Kasmer et al.4 observed that less than 10% of runners in their samples were symmetrical forefoot or midfoot strikers. In this study, 79.3% of barefoot runners were forefoot or midfoot strikers. This is fairly close to the percentages observed for habitually barefoot American adults and Kenyan adolescents running without shoes.9 It is also similar to the pattern observed for adult male Hadza hunter-gatherers running in sandals or barefoot.16 However, it differs markedly from habitually barefoot Kenyans of the Daasanach tribe,10 Hadza juveniles, and adult Hadza women.16 It is possible that speed, surface properties, and running experience are confounding variables when it comes to comparing foot strike patterns among studies.

The GABAB receptor antagonist, CGP 54626 (2 μM), inhibited the evoked current, confirming the identity of the GABAB sIPSC (Figure 1), similar to previous studies (Johnson and

North, 1992 and Bonci and Williams, 1996). In DA neurons, the GABAB sIPSC did not significantly change 24 hr following METH, compared to saline injection (Figures 1A and 1B). By contrast, the sIPSC was significantly smaller in GABA neurons (Figures 1D and 1E). Moreover, the sIPSC in GABA neurons remained depressed for at least 7 days (Figure 1E). Examination of the paired-pulse ratio for the fast GABAA-mediated IPSC revealed no difference in either DA or GABA neurons (Figures 1C and 1F), suggesting that the depression KRX-0401 cell line of the sIPSC in GABA neurons was not due to the inability of GABA terminals to release GABA. To investigate the effects of METH on synaptic and extrasynaptic GABAB receptors, the GABAB receptor agonist baclofen was applied to the bath. As described previously (Labouèbe et al., 2007), saturating doses of baclofen (300 μM for DA and 100 μM for GABA) elicited large and desensitizing GABABR-activated GIRK currents in DA neurons and small nondesensitizing currents in GABA neurons (Figure 2). All baclofen-activated currents were inhibited with the inwardly rectifying K+ selleck chemical channel inhibitor Ba2+ or the GABAB

receptor antagonist (CGP 54626, not shown). In contrast to the sIPSC recordings, there was an ∼40% decrease in the GABABR-GIRK currents of DA neurons 24 hr following a METH injection (Figures 2A and 2B). However, this decrease in current was not apparent at 7 days following METH injection (Figure 2B). By contrast, the baclofen-activated

GIRK (IBaclofen) currents in GABA neurons were significantly depressed by ∼55% 24 hr following a single METH see more injection and the reduced IBaclofen persisted for 7 days (Figures 2C and 2D). We next examined whether METH altered GABABR-GIRK signaling in other brain regions. There was no significant change in the sIPSC or IBaclofen in CA1 hippocampal pyramidal or GABAergic neurons 24 hr following METH (Figure S1 available online). We also measured the sIPSC and IBaclofen in pyramidal and GABAergic neurons of the prelimbic cortex, a target region of VTA DA cells, and observed no significant changes in GABAB-GIRK currents in METH-injected mice (Figure S1). Thus, a single exposure to METH triggered a profound and long-lasting depression in both the sIPSC and IBaclofen in GABA neurons of the VTA. In addition to postsynaptic GABAB receptors, presynaptic GABAB receptors are also involved in reducing GABA release, typically through inhibition of voltage-gated Ca2+ channels (Padgett and Slesinger, 2010).

2 M glycine (pH 2.4), neutralized by Tris-HCl (pH 8.6) after elution. Eluted proteins were

precipitated with 20% TCA overnight at 4°C, washed with cold acetone, resuspended in sample buffer, and analyzed by SDS-PAGE gel followed by Sypro-ruby stain. Appropriate bands present in the LRRTM4-Fc eluate but not in the Fc eluate from the 0.5 M NaCl elution were excised and analyzed by LC-MS mass spectrometry. All animal experiments were compliant with government and institutional guidelines. The targeting vector was generated by the recombineering method (Liu et al., 2003) from a 129S7 bMQ BAC clone, specifically bMQ30G17 from the Sanger Institute (Adams et al., 2005), and was www.selleckchem.com/btk.html confirmed by sequencing. The targeting vector contains a LoxP site and a neomycin cassette flanked by

Frt sites, replacing the major coding exon, Exon2, and a herpes simplex virus thymidine kinase expression cassette for negative selection. Targeting vector linearized by NotI was electroporated into 129/Ola embryonic stem (ES) cells for homologous recombination (Augustin et al., 1999 and Thomas and Capecchi, 1987). Positive selection of ES cells was in the presence of G418 and negative selection against random integration by Gancyclovir. Homologous recombination was verified by Southern blot analysis with a probe 5′ to the targeting vector. Positive ES cells were further expanded and a single Neo cassette insertion was verified by Southern blotting against a probe for the Neo cassette after two independent restriction digestions, by EcoRV and BglII. Positive clones were injected into C57BL/6J blastocysts, which were buy Epacadostat implanted into surrogate female mice. Founder chimeras were backcrossed six to seven generations with C57BL/6J mice. Genotypes were regularly ascertained by PCR analysis. Neurons, COS7 cells, and cocultures were fixed for 12–15 min with warm 4% formaldehyde and 4% sucrose in PBS (pH 7.4) followed by permeabilization with 0.25% Triton X-100, except where live staining or staining of unpermeabilized fixed already cultures was used. Live stainings were followed by fixation with warm 4% formaldehyde/4% sucrose

in PBS (pH 7.4). Fixed cultures were then blocked in 10% BSA in PBS for 30 min at 37°C and primary antibodies applied in 3% BSA in PBS. After overnight incubation at 4°C, the coverslips were washed with PBS and incubated in secondary antibodies in 3% BSA in PBS for 1 hr at 37°C. The coverslips were then washed and mounted in elvanol (Tris-HCl, glycerol, and polyvinyl alcohol, with 2% 1,4-diazabicyclo[2,2,2]octane). Immunofluorescence studies on coronal cryostat sections 20 μm thick at hippocampal level were performed on 6-week-old perfused LRRTM4−/− and littermate wild-type male mice. Fresh frozen sections were fixed by incubating in cold methanol for 10 min or for 7 min in 4% formaldehyde/4% sucrose and then blocked for 1 hr, followed by successive incubations with primary and secondary antibodies.

9 versus 38.4 years, p = .002). The majority (74%) of the patients were males with significantly more males among the MDQ positives (83%) compared to the MDQ negatives (70%) (p = .005). There were no significant differences regarding education level, employment status and EuropASI severity scores regarding medical condition, alcohol, family and social relations and mental problems. However, there was a significant difference on the EuropASI severity rating drugs (p = .000) between the MDQ positive

and negative patients ( Table 1). Patients with check details an assessment at T1 (N = 170, 45%) did not differ significantly from patients without an assessment (N = 205, 55%) in terms of age, gender, and employment status. However, MDQ positives at T0 with an assessment at T1 (N = 111) were less educated than

those without an assessment (N = 50). Moreover, MDQ negatives at T0 with an assessment at T1 had a higher mean section A score (0–13) than MDQ negatives without an assessment (8.87; SD ± 2.63 Selleck Panobinostat versus 5.42; SD ± 3.25, p < .01). The severity of alcohol or drug use (ASI score) did not differ between these groups (data not shown). Of the 170 patients with a SCID at T1, 35 patients (20.6%) met criteria for a lifetime diagnosis of BD (BD-I N = 8, BD-II N = 25 and BD-NOS, N = 2), 72 patients (42.4%) had a lifetime major depressive disorder, 10 patients (5.9%) a lifetime depressive disorder NOS, 10 patients (5.9%) met criteria for a substance-induced mood disorder with depressed features, 1 patient (0.6%) had a substance-induced mood disorder with manic features, and 1 patient (0.6%) a mood disorder due to a somatic condition. Forty-one patients (24.1%) did not meet criteria for any mood disorder. Fifty-eight (34.1%) patients had one lifetime SUD diagnosis, 108 patients (63.5%)

had two or more SUD diagnoses, and 4 patients (2.4%) had no lifetime SUD at all. Fifty-nine patients (34.7%) had a current diagnosis of AUD, 31 patients (18.2%) of cocaine or stimulant dependence, 26 patients (15.2%) of cannabis dependence, 8 patients (4.7%) of opiate dependence, and 5 patients (2.9%) of benzodiazepine dependence. Forty-one (24.1%) patients were problems users of alcohol and/or drugs but did not meet criteria of any current SUD. Table 2 shows that 23 of the 35 patients (65.7%) with BD had a positive MDQ score and 47 of the 135 patients through (34.8%) without BD had a negative MDQ score resulting in a weighted sensitivity of .43 and a weighted specificity of .57, a weighted LR+ of 1.00, a weighted LR− of 1.00, a PPV of .21, and a NPV of .80 (Table 3). As expected based on the LR+ and the LR−, the area under the curve (AUC) was .50 (95% CI .41–.61). Omission of the impairment criterion (section C), increases the number of patients with a positive MDQ score (N = 111) and BD from 23 to 32 and decreases the number of patients with a negative MDQ score (N = 59) and BD from 12 to 3, resulting in increased sensitivity of .85 at the expense of a decreased specificity of .

, 2010). Whether similar developmental changes also take place at much smaller cortical synapses is unclear. Experiments on acute hippocampal and neocortical slices suggested that short-term plasticity and Pr at small synapses develop similar to the calyx (Bolshakov and Siegelbaum, 1995; Feldmeyer and Radnikow, 2009; Reyes and Sakmann, 1999). In dissociated culture, on the other hand, endocytosis has been reported to be stable over time (Armbruster and Ryan, 2011), and the vesicle retrieval rate at saturating stimulation intensity (Sankaranarayanan and Ryan, 2000), that is, endocytic

capacity, remains low compared to the mature calyx (Renden and von Gersdorff, 2007; Wu et al., 2009). The total number of vesicles increases during development (Mozhayeva et al., S3I-201 solubility dmso 2002), but it is unclear if partitioning into functional and nonfunctional pools is also developmentally regulated. We set out to study the maturation of presynaptic function at Schaffer collateral (SC) synapses

in slice cultures of rat hippocampus, a preparation that closely recapitulates postnatal development (De Simoni et al., 2003). To measure the fraction of released and recycling vesicles, we developed a dual-color release indicator (ratio-sypHy) for use in intact tissue. In immature slice Galunisertib cultures, after 5–7 days in vitro (DIV 5–7), a sizable fraction of vesicles could not be released. In 2–4 week cultures, however, essentially all vesicles were mobilized in response to either high-frequency AP trains or typical CA3 cell place field activity. The rate of vesicle retrieval increased about 7-fold during maturation. Chronic depolarization induced a sizable resting pool at mature SC boutons. Therefore, SC boutons are capable

of reducing their output by removing vesicles from the recycling pool, but this homeostatic mechanism seems to be activated only during periods of pathologically high activity. We conclude that synapses in CA1 undergo a pronounced refinement of vesicle use and recycling during early postnatal development. Our indicator is based on a fusion protein of the pH-sensitive GFP variant superecliptic pHluorin (Miesenböck et al., 1998) with the synaptic vesicle protein synaptophysin I (sypI), a these combination known as sypHy (Granseth et al., 2006). To create a dual emission indicator suitable for ratiometric two-photon microscopy, we fused the extraluminal C terminus of sypHy with the dimeric red fluorescent protein tdimer2 (ratio-sypHy; Figure 1A). The C-terminal tdimer2-tag faces the cytoplasm, providing a red fluorescence signal proportional to the total amount of ratio-sypHy present at a synapse. Because of the fixed stoichiometry, the green-to-red fluorescence ratio of ratio-sypHy was independent of expression level and depth of the synapse in the tissue and could be compared across cells.

Biochemical analysis revealed that loss of Mmd2 in the chick spinal cord results in decreased activity of respiratory chain complexes

II and IV, thus correlating the proliferation of glial progenitors with energy metabolism. Indeed, electron transport chain function has previously been linked to cell cycle regulators and proliferation; therefore, it will be important to decipher the relationship between complex II/IV, cell proliferative mechanisms, and glial precursor biology ( Mandal et al., 2010 and Schauen et al., 2006). Moreover, that Mmd2 appears to regulate energy metabolism via complex II/IV and is induced just after glial specification suggests that glial precursors have unique energy and/or metabolic requirements that are distinct from neural stem cells and committed neuronal learn more progenitors. It is likely that each of these cell populations have unique metabolic Birinapant solubility dmso profiles that reflect their biology and/or impending lineage commitments; indeed, neurons, astrocytes, and oligodendrocytes each have distinct metabolic requirements. Interestingly, the timing of cardiac myocyte differentiation has been linked to mitochondria maturation and function, indicating that metabolic function participates in lineage development

( Hom et al., 2011). Therefore, in the future it will be important to identify distinct metabolic features of these precursor populations and to further delineate how these processes are coordinated with transcriptional cascades that specify their identity. Apcdd1 is a membrane-bound glycoprotein that can inhibit canonical Wnt signaling through association with Wnt receptor complexes, though its exact role during spinal cord development remains undefined ( Shimomura et al., 2010). These previous studies revealed a mild effect of Apcdd1-L9R on proliferation and specification during neurogenesis, enough phenotypes that we did

not observe during gliogenesis ( Figures 7 and S8), probably reflecting stage-specific effects of Apcdd1-L9R. Our studies indicate that Apcdd1 plays a key role in the migration of ASP populations, probably through an association with Rho-GTPases. The observation that Apcdd1 can influence Wnt receptor complexes, coupled with the role of noncanonical Wnt signaling in cell migration and regulation of Rho-GTPases, suggest a model whereby Apcdd1 could function to promote ASP migration via noncanonical Wnt signaling ( Schlessinger et al., 2009). That L9R overexpression does not effect the generation of ASP populations in the VZ suggests that Apcdd1 is either not necessary for the generation of these populations or functions through other mechanisms. Alternatively, the epithelial to mesenchymal transition (EMT) has been shown to promote migration and the acquisition of progenitor-like states ( Mani et al., 2008 and Acloque et al., 2009).

Such experiments will improve our understanding of how IEG expression is related to cells’ physiological properties. The cell-type specificity of TRAP is Protein Tyrosine Kinase inhibitor a limitation for some applications. For instance, we found that, after visual stimulation, GABAergic cells were underrepresented among the TRAPed population (Figure S4). This is consistent

with prior work using Fos immunostaining in cats and rats (Mainardi et al., 2009; Van der Gucht et al., 2002). TRAPing of GABAergic cells is likely to be dependent on the stimulus and brain region, and we observed robust TRAPing of some inhibitory neuron types, such as olfactory bulb granule cells and striatal medium spiny neurons (Figure 2). Thus, much of TRAP’s cell type specificity is derived from the cell-type specificity of IEG expression. Additional factors, such as the displacement of regulatory elements during gene targeting, cell-type differences in the accessibility

of the effector http://www.selleckchem.com/products/Docetaxel(Taxotere).html locus for recombination, and cell-type differences in the regulation and trafficking of CreERT2 could potentially contribute. Nonetheless, we show that most cell types in the brain can be TRAPed with the current version of the method. Future modifications, such as the development of CreERT2 knockin alleles for IEGs that are expressed in different neuronal types and that are sensitive to different features of neuronal activity (Schoenenberger et al., 2009; Worley et al., 1993), could extend the approach to cell types that currently cannot be robustly TRAPed. Another concern is that our CreERT2 knockin alleles are expected to be null for Fos and Arc. We did not observe any abnormalities in ArcTRAP or FosTRAP mice, and we are not aware of any severe phenotypes in previously generated Arc and Fos heterozygous knockout mice ( Johnson et al., 1992; Paylor et al., 1994; Wang et al., 2006; Wang et al., 1992).

However, some subtle phenotypes of Arc or Fos haploinsufficiency have been reported. These include a low penetrance Cell press of increased seizure susceptibility in Arc+/− mice ( Peebles et al., 2010), and, for Fos+/− mice, increased susceptibility to drug-induced neurotoxicity ( Deng et al., 1999) and attenuated morphological changes associated with kindling stimuli in an epilepsy model ( Watanabe et al., 1996). Although these phenotypes are unlikely to affect many TRAP experiments, alternative knockin or transgenic strategies that do not produce null alleles could mitigate such concerns. Given that considerable recombination is induced in many brain areas that process sensory information even under homecage conditions, the use of sensory deprivation is useful for improving TRAP specificity (Figure 2).

Functional connectivity analysis during a perceptual attention task revealed that visual cortical areas that process target information coupled with right MFG and bilateral IFJ during enhancement and with mPFC and PCC during suppression (Chadick and Gazzaley,

2011). The differences between enhancement and suppression in connectivity suggest that on-task perceptual attention contributed to enhancement effects and that off-task, self-referential attention (activating the “default network” [see below]) contributed to suppression effects. Additional studies are needed to compare such network effects for perception and reflection. Perceptual attention is controlled by two orienting systems (Corbetta and Shulman, 2002 and Corbetta et al., 2008). A dorsal system includes the frontal eye fields (FEF) and intraparietal cortex (IPS, SPL) and is involved in goal-directed,

top-down attention to stimuli. A ventral network Torin 1 concentration includes inferior frontal cortex and IPL (TPJ) and is specialized for bottom-up detection of salient or unexpected events. The ventral network has a right hemisphere bias and learn more mediates the ability to “reorient” quickly to salient events that are potentially rewarding or dangerous to an observer. Reorienting involves interruption and resetting of ongoing activity in the dorsal network, which otherwise suppresses the ventral network during focused and sustained attention to an ongoing task. One would expect that perceptual attention would be important for encoding events for long-term memory (LTM) and indeed, this is the case. For example, Uncapher et al. (2011) found that cuing top-down perceptual attention to an upcoming target location engaged the IPS and was associated with better subsequent memory, while cuing participants to an invalid Calpain nontarget location engaged TPJ and was associated with poorer subsequent memory. Presumably, activity in TPJ reflected perceptual capture and/or reorienting necessary when the cued location did not contain a target. These

findings provide important evidence of the role during encoding of top-down and bottom-up perceptual attention, but the study did not compare perceptual and reflective attention. Whether a simple dorsal/ventral distinction applies to remembering is a subject of current debate. Lateral parietal activity is commonly found to be associated with correct recognition memory for old items ( Vilberg and Rugg, 2008). It has been proposed that the dorsal/ventral distinction in perceptual attention may generalize to the kind of reflective attention processes engaged during remembering. Cabeza et al. (2008) and Ciaramelli et al. (2008) suggested that superior parietal cortex supports retrieval search, monitoring, and verification, similar to its role in the top-down, voluntary control of perceptual attention, and that inferior parietal cortex is active when there is clear and more detailed recollection, similar to the exogenous capture of attention by salient, bottom-up perceptual events.

This state estimate is transformed into a forward prediction of the acoustic consequences of the motor command. We also assume that a forward prediction of the somatosensory consequences of the motor command is generated, although we will not discuss the role of this system here. The forward auditory prediction, in turn, supports two functions as noted above. One is a rapid internal monitoring function, which calculates whether the current motor commands are likely to hit their intended sensory targets (this implies that the targets are known independently of the forward predictions, see below) and provides corrective feedback

if necessary. Needless to say, the usefulness of this internal feedback depends on how accurate the internal model/forward prediction is. Therefore it is important selleckchem to use actual sensory feedback to update and tune the internal model to ensure it is making accurate predictions. This is the second (slower, external monitoring) function of the forward predictions: to compare predicted with actual sensory consequences and use prediction error to generate a corrective

signal to update the internal model, which in turn provides input to the motor controller. Of course, if internal feedback monitoring fails to catch an error in time, external feedback can be used to correct movements as well. BI6727 As noted above, an internal feedback loop that generates a forward prediction of the sensory consequence of an action is useless if the intended most sensory target is not known. This raises an interesting issue because unlike in typical visuomanual paradigms where actions are often directed at external sensory targets, in most speech acts there is no immediate externally provided sensory target (unless one is repeating heard speech). Instead the sensory goal of a speech act is an internal representation (e.g., a sequence of speech sounds) called up from memory on the basis of a higher-level goal, namely, to express a concept via a word or phrase that corresponds

to that concept. This, in turn, implies that speech production involves the activation of not only motor speech representations but also internal representations of sensory speech targets that can be used to compare against both predicted and actual consequences of motor speech acts. Psycholinguistic models of speech production typically assume an architecture that is consistent with the idea that speech production involves the activation of a sensory target. For example, major stages of such models include the activation of a lexical-conceptual representation and access to the corresponding phonological representation followed by articulatory coding (Dell et al., 1997 and Levelt et al.

, 1994, Feldmeyer and Sakmann, 2000 and Markram et al., 1997). If the goal, however, is to eventually reveal most, or all, connections within a given area, this method is limited given the few connections

that can be tested in one experiment. To circumvent this problem, we designed an optical technique, a modification of one-photon photostimulation (Callaway and Katz, 1993), that reveals synaptic connections in large numbers and can provide a map of most connections to a neuron in a local area, with single-cell resolution (Nikolenko et al., GDC 0068 2007). Using two-photon uncaging of glutamate in brain slices, one can sequentially activate hundreds of potential presynaptic cells, one by one, and quickly test whether they are connected to a given postsynaptic neuron (Nikolenko et al., 2007). We were interested to apply this two-photon mapping technique to inhibitory circuits and determine the functional structure of inhibitory networks in the neocortex. The cortex has numerous types of GABAergic interneurons (Fairen et al., 1984), which play a determinant role in the regulation of the excitability of pyramidal cells (PCs) and the activity of cortical microcircuits,

by controlling different parts of the axo-dendritic arborization of the PCs check details (Somogyi et al., 1998). One can distinguish many subtypes of inhibitory neurons in neocortical circuits morphologically and physiologically (Gupta et al., 2000, Ascoli et al., 2008 and Yuste, 2005). For this study, we focused on dendritic targeting inhibitory cells,

the somatostatin-expressing interneurons (Kawaguchi and Kubota, 1997 and Wang et al., 2004), which represent approximately 30% of the neocortical interneurons in mouse (Gonchar and Burkhalter, 1997). Somatostatin-positive cells are composed of several subtypes, of which Martinotti neurons are the predominant type (Halabisky et al., 2006 and McGarry et al., 2010). They generally, although not always (Gonchar et al., 2002), contact apical and tufted dendrites of PCs (Kawaguchi and Kubota, 1997 and Wang very et al., 2004). Somatostatin-positive interneurons display low-threshold spiking (Kawaguchi, 1995), generating a global dendritic calcium spike (Goldberg et al., 2004), and can fire spontaneously in a pacemaker fashion, in the absence of any synaptic input (LeBon-Jego and Yuste, 2007). Within neuronal circuits, they control local synaptic inputs of PCs (Murayama et al., 2009 and Silberberg and Markram, 2007) and are recruited by network activity (Kapfer et al., 2007), to the point that they can be activated by a single PC (Kozloski et al., 2001), mediating a strong disynaptic inhibition between PCs (Silberberg and Markram, 2007). These specific morphological and physiological properties suggest that somatostatin-positive interneurons implement a specific function in the microcircuit.