Our group previously identified a defective rho mutant (SP3710) in a Tn5 mutagenesis screen of C. crescentus for mutants with

decreased tolerance to NaCl. Tn5 insertion before the first codon in the amino terminal RNA-binding motif results in the expression of a 45-kDa carboxyl-terminal domain of Rho, expressed by a transposon promoter (Italiani et al., 2002; Italiani & Marques, 2005). The sensitivity of the mutant to bicyclomycin suggests that the ATP-binding site of Rho is intact in the 45-kDa truncated protein (Italiani & Marques, 2005). Moreover, the Rho protein in strain SP3710 is functional enough to ensure viability. However, its transcription termination activity is severely impaired, as observed by the lack of autoregulation (Italiani & Marques, 2005). The studies on Rho function in C. crescentus reported here GDC-0973 mouse are based on our observation that rho mutant strain SP3710 shows an unusual distortion in its response to environmental stress (Italiani et al., 2002). Strain SP3710 is sensitive to NaCl, as expected from the screen used for check details its isolation. However, this rho mutant strain is essentially wild type in its response to UV light and alkaline pH and is only moderately sensitive to acid pH and to heat shock. In contrast, strain SP3710 is highly sensitive to exogenously added hydrogen peroxide (H2O2), both in the exponential and

in the stationary phase. Although a variety of cellular phenotypes have been reported for rho mutants, to our knowledge, strain SP3710 is the first rho mutant with such drastic

distortions in its stress response. Thus, strain SP3710 and the partially functional Rho it expresses are new and potentially valuable tools for identifying additional physiological roles of rho. Caulobacter crescentus has several enzymes involved in the oxidative stress response. It was shown to express a cytosolic iron superoxide dismutase (FeSOD), a periplasmic copper–zinc almost superoxide dismutase (CuZnSOD) and a catalase–peroxidase (KatG) (Schnell & Steinman, 1995; Steinman et al., 1997). Caulobacter crescentus contains just one bifunctional catalase–peroxidase, KatG, and evidently lacks monofunctional catalases and thiol peroxidases. In this work, our goal was to identify the determinants of C. crescentus oxidative stress response affected by the rho mutation, based on the oxidative stress phenotype of strain SP3710 cited above and prior studies on the roles and regulation of antioxidant defense enzymes in C. crescentus (Schnell & Steinman, 1995; Steinman et al., 1997; Rava et al., 1999; Alvarez-Martinez et al., 2006). Caulobacter crescentus strain NA1000 (Evinger & Agabian, 1977) was used as the wild type in all the experiments; strain SP3710 has a Tn5 insertion in the rho gene (Italiani et al., 2002; Italiani & Marques, 2005) and strain SGC111 is a katG null mutant (Steinman et al., 1997).

It is possible that a common mechanism produces phase reversals in Rpe65−/−;Opn4−/− mice and in wild-type mice during dim LD cycles. The identification of ‘clock genes’ and the invention of reporter gene technology enabled the assessment of rhythmicity in cultured cells and tissues, such as SCN slice preparations. The technical developments and experimental findings based on assessing the activities of specific genes and proteins within cells and tissues has led to a reconceptualization of the

circadian organization as a hierarchy of oscillators. This vision has brought the circadian timing system to the attention of a very broad clinical and basic research community. Ignoring circadian

effects leads to errors CAL 101 of interpretation in basic research and can result in suboptimal diagnosis and treatments in medicine. Circadian clocks regulate the timing of gene expression in each organ, and the regulated genes are unique to each organ (Akhtar et al., 2002; Duffield et al., 2002; Miller et al., 2007; Hughes et al., 2009; Dibner et al., 2010). Thus, circadian control overlies the normal expression MAPK inhibitor of tissue-specific genes and proteins. Not surprisingly, the maintenance of normal phase relationships among tissues and organs appears to be adaptive. Disrupting the circadian network can produce severe pathology (Litinski et al., 2009; Karatsoreos et al., 2011). Optimizing the circadian timing system for treatment, such as appropriately timing drug administration is a frontier research area (Levi & Schibler, 2007; and see below). Since the discovery of the SCN, and the consistent finding that most circadian rhythms are abolished following its destruction,

it was generally assumed that the SCN was the only locus capable of independent circadian rhythm generation. In turn, all circadian rhythms throughout the brain Arachidonate 15-lipoxygenase and body were thought to be driven by downstream communication from the SCN. This notion was challenged following the observation that cultured fibroblasts exhibit circadian rhythms in gene expression following a serum shock (Balsalobre et al., 1998). With this experiment, it became clear that the ability to oscillate was a general property of tissues throughout the central nervous system and periphery (Damiola et al., 2000; Yamazaki et al., 2000; Yoo et al., 2004). The discovery that the SCN is not alone in the capacity to express endogenous oscillation was the beginning of a reconceptualization of the internal timekeeping system (Balsalobre et al., 1998). It is now known that the circadian system is composed of multiple individual cellular oscillators located throughout the body and most of its organs and glands. For example, a role for intrinsic rhythmicity in other tissues has been demonstrated.

p.m. Different nucleosides

were assayed at 30 °C and pH 7. Reaction mixtures contained 1 × 1010 CFU, 2.5 mM 5-fluorouracil and 10 mM uridine, thymidine, 2′-deoxyuridine, 2′-deoxycytidine or 2′,3′-dideoxyuridine. Reactions were performed at different 5-fluorouracil and thymidine ratios (1 : 1, 4 : 1 and 1 : 4) at 30 °C, pH 7, and 200 r.p.m. All assays were performed BMS-777607 mouse three times in 1 mL of reaction medium. Subsequent to system characterization with 5-fluorouracil, the incorporation of other halogenated pyrimidine bases was tested using 5-chlorouracil and 5-bromouracil. Reactions were performed in a 1 : 4 ratio (2.5 mM halogenated base and 10 mM thymidine) in potassium phosphate buffer (30 mM, pH 7) at 30 °C. 1 × 1010 CFU were mixed with 3 mL of 1, 2, and 3% (w/v) agar or agarose. The mixture was then added dropwise to stirred sunflower oil (20 mL) at 25 °C. The resulting gel beads were cooled, filtered, washed with hexane and then with physiological solution to obtain solvent-free beads. 1 × 1010 CFU were mixed with 3 mL of phosphate buffer (30 mM, pH 7) containing 15, 20, and 25% (w/v) acrylamide/bis-acrylamide,

subsequently 50 μL of 10% (w/v) ammonium persulfate (APS) and 14 μL of N, N, N′, N′tetramethylethylenediamine (TEMED). The resulting gel was cut into small cubic pieces (1.0 × 1.0 × 0.2 cm). The biosynthesis of nucleoside analogues www.selleckchem.com/products/DAPT-GSI-IX.html was qualitatively evaluated by TLC Merck Silica gel 60 F254 in chloroform/methanol (80 : 20, v/v) as mobile phase. The quantitative analysis was performed by HPLC (Gilson) equipped with a UV detector (254 nm) using a Nucleodur 100-5 C18 column (5 μm, 125 × 5 mm). The isocratic mobile phase used was water/methanol (95 : 5, v/v) at room temperature and at a flow rate of 1.2 mL min−1. Retention times of substrates and products were as follows: Floxuridine biosynthesis: (1) uracil (1.0 min), 5-fluorouracil (1.4 min), 2′-deoxyuridine (2.0 min), floxuridine (3.0 min); (2) cytosine (1.1 min), 5-fluorouracil (1.4 min),

2′-deoxycytidine (2.2 min), floxuridine (3.0 min); (3) 5-fluorouracil (1.4 min), thymine (2.6 min), Aldehyde dehydrogenase floxuridine (3.0 min), thymidine (4.2 min). 5-fluorouridine biosynthesis: uracil (1.0 min), 5-fluorouracil (1.4 min), uridine (1.8 min), 5-fluorouridine (2.8 min). 5-chloro-2′-deoxyuridine biosynthesis: (1) uracil (1.0 min), 2′-deoxyuridine (2.0 min), 5-chlorouracil (4.8 min), 5-chloro-2′-deoxyuridine (6.0 min); (2) cytosine (1.1 min), 2′-deoxycytidine (2.2 min), 5-chlorouracil (4.8 min), 5-chloro-2′-deoxyuridine (6.0 min); (3) thymine (2.6 min), thymidine (4.2 min), 5-chlorouracil (4.8 min), 5-chloro-2′-deoxyuridine (6.0 min). Product identification was performed by MS-HPLC (See Supporting Information, Data S1). 5-fluoro-2′-deoxyuridine biosynthesis from thymidine and 5-fluorouracil was used as reaction test for the screening (Fig. 1).

An additional analysis directly compared the effect of mOFC and ACCg lesions on the same social valuation test (Rudebeck et al., 2006). Figure 5A illustrates the intended lesion

location for the mOFC and ACCg animals. In a comparison of the two groups’ responses to the fear-inducing stimuli no differences between the effects of the two lesions were seen. Specifically, there were no interactions involving group (fear stimuli × group, F1,5 = 1.04, P = 0.355, fear stimuli × session × group, F3,15 = 0.72, P = 0.513) nor main effects of group (F1,5 = 4.38, P = 0.090). The only main effect of interest related to the identity of the fear stimuli (F1,5 = 11.70, P = 0.019). This implies neither the mOFC nor the ACCg have fundamentally critical roles in guiding this type of behaviour. In contrast, a comparison of group responses towards this website the social stimuli (pictures of other monkeys) revealed selleck compound that the ACCg was the critical region for social valuation (Fig. 3D). There was a significant linear main effect of the identity of the social monkey stimuli on responsiveness

(F1,7 = 7.37, P = 0.030), confirming that the monkeys whose behaviour was investigated concurred with one another in their valuations of the videos of other monkeys. There was a significant interaction of social monkey stimulus, session and group (ACCg vs. mOFC) on the log-transformed reaching latencies (F12,60 = 2.45, P = 0.016), in addition to a significant main effect of the identity of the social monkey stimuli (F4,20 = 3.83, P = 0.029). An analysis that compared the two lesion groups’ responses to the human stimuli found no significant group differences (F1,5 = 1.54, P = 0.269) or interaction with the stimulus identity

(F1,5 = 0.058, P = 0.819). Similarly, there were no significant group differences in an analysis of the neutral stimuli (F1,5 = 0.36, P = 0.573) or interactions between group and stimulus identity (F1,5 = 2.10, P = 0.207). A main effect of neutral stimuli was noted (F1,5 = 13.78, P = 0.014); it was a result of longer reaching latencies towards the moving pattern stimuli that the neutral Nintedanib (BIBF 1120) static objects (paired-samples t-test: preoperative, t3, = −3.15, P = 0.051; postoperative, t3 = −3.06, P = 0.055). Not only did Rudebeck et al. (2006) demonstrate that performance in the social valuation task was altered by ACCg lesions but they also reported that lesions of ventrolateral and lateral orbital prefrontal cortex (PFv+o) did not alter monkeys’ reaching latencies in response to social stimuli but that they did affect responsiveness to fear-inducing stimuli (Rudebeck et al., 2006).

Consistent with the Fos data, D-AP5 in the DMS, but not in the DLS, prevented the inhibition of dorsal raphe nucleus

5-HT release normally produced by ES. Furthermore, D-AP5 administered into the DMS before ES, but not into the DLS, increased anxiety 24 h later, leading to levels similar to those produced by IS. These results suggest that, as with appetitive act/outcome contingency learning, the protective effects of behavioral control over a stressor require the DMS. “
“Amphetamine withdrawal in both humans and rats is associated with increased anxiety states, which are thought to contribute to drug relapse. Serotonin in the ventral hippocampus mediates affective behaviors, and reduced serotonin levels in this region are observed in rat models of high anxiety, including during withdrawal from chronic

amphetamine. This goal of this study was to understand AZD8055 chemical structure the mechanisms by which reduced ventral hippocampus serotonergic neurotransmission occurs during amphetamine withdrawal. Serotonin synthesis (assessed by accumulation of serotonin precursor as a measure of the capacity of in vivo tryptophan hydroxylase activity), expression of serotonergic transporters, learn more and in vivo serotonergic clearance using in vivo microdialysis were assessed in the ventral hippocampus in adult male Sprague Dawley rats at 24 h withdrawal from chronic amphetamine. Overall, results showed

that diminished extracellular serotonin at 24 h withdrawal from chronic amphetamine was not accompanied by a change in capacity for serotonin synthesis (in vivo tryptophan hydroxylase Epothilone B (EPO906, Patupilone) activity), or serotonin transporter expression or function in the ventral hippocampus, but instead was associated with increased expression and function of organic cation transporters (low-affinity, high-capacity serotonin transporters). These findings suggest that 24 h withdrawal from chronic amphetamine reduces the availability of extracellular serotonin in the ventral hippocampus by increasing organic cation transporter-mediated serotonin clearance, which may represent a future pharmacological target for reversing anxiety states during drug withdrawal. “
“Compulsive drug use and a persistent vulnerability to relapse are key features of addiction. Imaging studies have suggested that these features may result from deficits in prefrontal cortical structure and function, and thereby impaired top-down inhibitory control over limbic–striatal mechanisms of drug-seeking behaviour. We tested the hypothesis that selective damage to distinct subregions of the prefrontal cortex, or to the amygdala, after a short history of cocaine taking would: (i) result in compulsive cocaine seeking at a time when it would not usually be displayed; or (ii) facilitate relapse to drug seeking after abstinence.

Main themes were: relating, maintaining selleck compound library and moving. Community pharmacists work as isolated healthcare practitioners, but see more patients than other NHS care settings. The Department of Health White Paper (2008)1 and the 2013 NHS England consultation ‘Pharmacy

call to action’ can be viewed as re-professionalisation of community pharmacists in consolidating and expanding their professional practice. There is limited published research on how pharmacists perceive their roles. A qualitative research approach was used to provide insight into how community pharmacists perceive their roles. This qualitative case study consisted of five community pharmacists recruited in 2012 using purposive sampling. Only pharmacists registered for 5 years or more, who had worked in community pharmacy for at least 2 years and provided written consent, were entered. Data were obtained from one in-depth individual semi-structured interview using a guide covering how they viewed their role, contribution and future and how other healthcare professionals viewed their role. Each pharmacist was asked to complete a diary for 5 days to include any positive contributions or frustrations experienced.

The data were analysed using inductive thematic analysis2. Data were coded and themes identified. Ethics approval was obtained. This study is part of a larger new study. The preliminary thematic analysis of the qualitative data led to the identification of three main themes: relating, maintaining and moving, each with three or four sub-themes of how pharmacists perceive Selumetinib their roles. Relating: building and maintaining relationships with GPs practices, policing

and preventing GPs from making mistakes and caring and helping patients. Maintaining: working as isolated practitioners, finding strategies to keep up-to-date, feeling skills are underutilised and lacking opportunities for post-graduate education and training. Moving: struggling to move away from dispensary work, striving to free up GP time, being a healthcare professional that patients can easily access and being seen as a shop-keeper. The findings highlighted that having good working relationships with GPs was important to pharmacists but took a long time to build, whereas getting hold of some GPs was like accessing ‘Fort Knox’. They viewed their role as freeing-up GP time and believed there was more potential for this. They also viewed undertaking Medicines Use Reviews as supporting GPs but felt this was not particularly valued. Pharmacists worked as isolated practitioners both in terms of not being integrated with healthcare teams, including having no access to patients’ medical records and few interactions or peer-review of their practice by other pharmacists.

“
“Sensorimotor integration is important

for motor learning. The inferior parietal lobe, through its connections with the frontal lobe and cerebellum, has been associated LY2157299 cell line with multisensory integration and sensorimotor adaptation for motor behaviors other than speech. In the present study, the contribution of the inferior parietal cortex to speech motor learning was evaluated using repetitive transcranial magnetic stimulation (rTMS) prior to a speech motor adaptation task. Subjects’ auditory feedback was altered in a manner consistent with the auditory consequences of an unintended change in tongue position during speech production, and adaptation performance was used to evaluate sensorimotor plasticity and short-term learning. Prior to the feedback alteration, rTMS or sham stimulation was applied over the left

supramarginal gyrus (SMG). Subjects who underwent the sham stimulation exhibited a robust adaptive response to the feedback alteration whereas subjects who underwent rTMS exhibited a diminished adaptive response. The results suggest that the inferior parietal region, in and around SMG, plays a role in sensorimotor adaptation for speech. The interconnections of the inferior parietal cortex with inferior frontal cortex, cerebellum and primary sensory areas suggest that this region may be an important component in learning and adapting sensorimotor patterns for speech. “
“The article by Bell, De Lorme, Figueira, Kashy and Sisk describes two very interesting experiments demonstrating that during sexual maturation in the male hamster, stimuli

that HER2 inhibitor were previously unrewarding acquire rewarding properties, independent of experience with the stimuli. The idea that puberty is a time of dramatic changes in the brain is not new, ask any parent. What is new here is the clear demonstration that a sexually relevant stimulus can become an unconditioned reward without any social or sexual experience with the stimulus, simply as a Tangeritin consequence of sexual maturation. Puberty has been described as a time of raging hormones, where the increase in hormone secretions from the adrenal glands and gonads activates moods and other behaviors (Buchanan et al., 1992). This view assumes for the most part that sexual differentiation of the brain occurs during early sensitive periods, and then the onset of hormone secretions at puberty activates the previously differentiated brain. The idea is that the brain is organized early in life and then specific behaviors are activated by hormones with puberty onset. The problem with this interpretation of hormone-behavior relations for scientists, clinicians and parents has been that even though there is a documented increase in the onset of mood disorders, use of illicit drugs and other psychiatric conditions at puberty, the direct effects of hormones on these behaviors have not been clear. The amount of circulating hormone does not correlate with changes in behavior.

In subcortical brain parenchyma, Cx26-positive puncta were often co-localized with astrocytic Cx43, and some were localized along astrocyte cell bodies and processes immunolabelled for glial fibrillary acidic

protein. Cx26-positive puncta were also co-localized with punctate labelling of Cx47 around Navitoclax datasheet oligodendrocyte somata. Comparisons of Cx26 labelling in rodent species revealed a lower density of Cx26-positive puncta and a more restricted distribution in subcortical regions of mouse compared with rat brain, perhaps partly explaining reported difficulties in detection of Cx26 in mouse brain parenchyma using antibodies or Cx26 gene reporters. These results support our earlier observations of Cx26 expression in astrocytes Nutlin-3 mw and its ultrastructural localization in individual

gap junction plaques formed between astrocytes as well as in heterotypic gap junctions between astrocytes and oligodendrocytes. “
“Ghrelin is an orexigenic hormone produced by the stomach. Ghrelin, however, may also be a modulator of the circadian system given that ghrelin receptors are expressed in the master clock, the suprachiasmatic nucleus (SCN) and several outputs of this region. To investigate this, we performed analyses of running wheel activity and neuronal activation in wild type (WT) and growth hormone secretagogue receptor-knockout (GHSR-KO) mice under various lighting conditions. GHSR-KO and WT mice were maintained under constant dark (DD) or constant light (LL) with ad libitum access to food before being placed

on a schedule of temporally restricted access to food (4 h/day) for 2 weeks. There were no differences between KO and WT mice in free-running period under DD, but GHSR-KO mice required more days to develop a high level of food anticipatory activity, and this was lower than that observed in WT mice. Under LL, GHSR-KO mice showed greater activity overall, lengthening of their circadian period, and more resistance to the disorganisational Pyruvate dehydrogenase lipoamide kinase isozyme 1 effects of LL. Furthermore, GHSR-KO mice showed greater activity overall, and greater activity in anticipation of a scheduled meal under LL. These behavioral effects were not correlated with changes in the circadian expression of the Fos, Per1 or Per2 proteins under any lighting conditions. These results suggest that the ghrelin receptor plays a role in modulating the activity of the circadian system under normal conditions and under restricted feeding schedules, but does so through mechanisms that remain to be determined. The circadian system controls daily rhythms of rest and activity, hormones, and motivated behaviors like feeding. Light is the primary synchroniser of the master circadian clock, the suprachiasmatic nucleus (SCN) (Reppert & Weaver, 2002). However, feeding also synchronises circadian rhythms (Stephan, 2002).

In multiple labeling experiments, however, this value changed by < 2% points between labeling reactions, suggesting that the unlabeled populations are stable. The results do not rule out the possibility of the other hypotheses. Resolving Opaganib mouse which mechanism is predominant remains an unresolved question. However, dockerin replacement may explain the surprising result that cells with and without the cipA gene showed similar levels of fluorescence after labeling with the SNAP-XDocII fusion protein, because the necessity of displacing

CipA protein in the wild type and cipA* strains did not reduce fluorescence intensity. We have shown that the SNAP-tag system can be used to fluorescently label C. thermocellum via the cohesin–dockerin interaction. Previous studies have visualized cellulosomes by transmission electron microscopy (Bayer et al., 1985); however, the ability to specifically label the cellulosome in aqueous solution could lead to the ability to observe cellulosome operation in-vivo. Although much is known about the interaction between free dockerins and free cohesins, the interaction between free dockerins and bound cohesin–dockerin pairs has been less well studied. Dockerin exchange suggests a mechanism for compositional change of the cellulosome. Clostridium thermocellum is known to

release cellulosomes in the late-stationary selleck chemicals phase of growth, as well as optimize the composition of cellulosomes attached to its surface in response to substrate changes (Bayer & Lamed, 1986; Raman et al., 2009). It has been suggested that detachment of intact cellulosomes in these processes is achieved

by proteolytic cleavage of the cohesin-II containing anchor proteins (Raman et al., 2009). The results of this study suggest an alternate or complementary mechanism, wherein the mere production of CipA molecules can effect turnover by dockerin exchange. Similar experiments could be used to probe interactions between type I cohesins and dockerins. In this study, we have demonstrated displacement of bound dockerin-containing proteins with free dockerin-containing proteins. This result sheds light on a possible mechanism for the natural Branched chain aminotransferase turnover and reordering of cellulosome subunits within the polycellulosome. Furthermore, the methods of this article have established the SNAP-tag system as a valuable tool for labeling components and sub-components of the cellulosome. The authors would like to thank G.W. for assistance with flow cytometry studies and K.O. for microscopy research. This research was supported by the BioEnergy Science Center, Oak Ridge National Laboratory, a Department of Energy Bioenergy Research Center supported by the Office of Biological and Environmental Research in the Department of Energy Office of Science, and a Dartmouth College Dean of Faculty Undergraduate Research Grant. We would like to declare one competing interest. L.R.L.

The mlrA of EMS may have been obtained from one or more of

the Sphingopyxis species. Microcystin-degrading bacteria, which possess mlr genes, may play an important role in decreasing microcystin in Lake Taihu and other water bodies. Because mlrB is probably silent, the mlrA gene is a better molecular probe than mlrB for detecting or monitoring dynamics of microcystin-degrading bacteria. This research was supported by the State Key Basic Research and Development Plan of China (2008CB418002), the National Water Science and Technology Projects (2009ZX07101-013-02) and the Talent Scientist Program of the Chinese Academy of Sciences (082303-1-501). Fig. S1. Neighbor-joining trees constructed from the 16S rRNA gene (left) and the mlrA gene sequences (right) of microcystin-degrading Tamoxifen bacteria. Bootstrap values are indicated at nodes. Please note: Wiley-Blackwell is not responsible for the content BMN 673 in vivo or functionality of any supporting materials supplied by the authors. Any queries (other than missing material) should be directed to the corresponding author for the article. “
“The transition metal iron is an important element for the sustenance of life – it can function

either as an electron acceptor or as a donor and serves as a cofactor in many enzymes activities. The cytoplasmic NAD(P)H-dependent ferric reductase in Thermus scotoductus SA-01 shares high sequence and structural similarity to prokaryotic thioredoxin reductases. Here Cobimetinib we report the sequence of the ferric reductase (which is typically annotated as a thioredoxin reductase-like protein) and a comparative

kinetic study with the thioredoxin reductase from SA-01. Structurally, the most noteworthy difference, immediately apparent from the protein sequence, is the absence of the disulphide redox centre in the ferric reductase. This is the first report relating the attributes of such a redox protein to its ability to reduce a ferric substrate. The transition metal, iron, is an important element for most organisms and is required for various physiological functions such as transport of molecular oxygen, involvement in electron transport and a cofactor for enzymes, and functions either as an electron donor or as an acceptor in microbial energy conservation. The dissimilatory reduction of ferric iron is considered the oldest form of respiration, thus providing an electron sink while the earth’s atmosphere was still anoxic (Vargas et al., 1998). Ironically, with the arrival of oxygen, iron posed a new threat to aerobically respiring organisms. Various redox-active biomolecules have been implicated in the cytotoxic effect of iron in aerobic respiring organisms by reducing the cellular ferric iron, which can then participate in the Fenton reaction. The successive univalent reduction of molecular oxygen, during aerobic respiration, generates superoxide (O2·−), hydrogen peroxide (H2O2) and hydroxyl (HO·) radicals, with the latter being most cytotoxic.