In this study, we investigated

the oxygen-sensitive regulator FNR in V. fischeri. Vibrio fischeri fnr complemented Palbociclib concentration an E. coli fnr mutant, and like fnr in E. coli, it is required for fumarate- and nitrate-dependent anaerobic respiration. Moreover, our data and another recent bioinformatic analysis (Ravcheev et al., 2007) suggest that the FNR-box recognition site is conserved in V. fischeri. For example, we observed fnr-mediated regulation of reporters for arcA (Fig. 3), dmsA (Dunn & Stabb, 2008), torE (Dunn & Stabb, 2008), and yfiD (data not show), which have predicted FNR boxes upstream. Taken together, FNR’s function in V. fischeri appears to be similar to that in its fellow gammaproteobacterium E. coli. As the first experimental examination of FNR in the Vibrionaceae, this study should underpin future efforts to understand FNR-mediated regulation in this important bacterial family. We initiated this study largely learn more because FNR is cited as an activator of luminescence in V. fischeri (e.g. see Meighen, 1994; Spiro, 1994; Sitnikov et al., 1995; Ulitzur & Dunlap, 1995; Stevens & Greenberg, 1999). However, that paradigm was based on a preliminary study that used the MJ1 lux genes cloned in E. coli (Muller-Breikreutz & Winkler, 1993). Our results appear to contradict that report, showing instead that FNR mediates repression of the luminescence-generating lux system in

V. fischeri under anaerobic conditions (Fig. 2). It is perhaps not surprising that lux regulation should be different in transgenic E. coli than in V. fischeri. For example, LitR, which activates luxR transcription, is absent in E. coli (Fidopiastis et al., 2002). It is also possible that FNR does activate luminescence in V. fischeri under conditions

different from those tested here, and that the discrepancy between our study and previous work simply reflects methodological differences. Repression of the lux genes anaerobically may minimize the production of luciferase when its O2 substrate is unavailable. This is consistent with the finding that luminescence is repressed by the ArcAB two-component regulatory system, which is more active under relatively reduced conditions (Bose et al., 2007). The observation that arcA∷lacZ reporters showed a lower expression in the absence of fnr (Fig. 3) suggests that the effect of FNR on bioluminescence C-X-C chemokine receptor type 7 (CXCR-7) may at least in part be indirect and mediated by FNR’s stimulation of arcA. Consistent with this idea, fnr did not exert much influence on luminescence in arcA mutant backgrounds, although arcA fnr double mutants were noticeably attenuated in anaerobic growth (data not shown). We speculate that FNR may amplify the repressive effect of ArcA on luminescence under reduced conditions. Although we cannot rule out the possibility that FNR exerts a direct effect by binding the lux region, as described above, we believe this model is unlikely.

Numbered sera of BD showed no reactivity to B. henselae proteins (Figs 1, 3 and 4). Table 2 shows the Se and Sp of clinical biomarkers based on the immunoreactivity of the B. henselae proteome against serum samples from CSD and IE patients. For these biomarker proteins, combining both IE and CSD, the sensitivity ranged Enzalutamide in vivo from 21% to 100%. The sensitivity for IE serum samples was higher than that of CSD serum samples, ranging from 43% to 100%, with the lowest and the highest reactivity percentage observed for Pnp and GroEL, respectively. Although ATPD, GroEL and FusA each showed a sensitivity of 100% for CSD serum samples, these proteins were also detected using the control group serum samples as shown in

Fig. 2. The proteins that showed 100% specificity for both diseases were GroES, HbpD, Pap31, PdhD2, SodB, Ppi and two proteins of a nonapplicable locus: BH11510 (OMP) and BH12180 (ABC IDH inhibitor transporter, periplasmic oligopeptide-binding protein). Based on these results, BH11510, BH12180, GroES, Pap31, PdhD2 and SodB were selected as biomarkers for IE, while BH02000 was selected as a biomarker for CSD. The aim of this study was to identify the serodiagnostic markers of CSD and endocarditis due to B. henselae and expand the number of biomarkers selected previously by others (McCool et al., 2008; Eberhardt et al., 2009). Indirectly, our study allowed cross-validation of some

protein targets for clinical application. The systemic infection leading to the massive infiltration of bacteria may be explained by the higher IFA titer serum samples obtained from patients suffering from IE compared with samples from CSD patients. The titers for the IE samples ranged from 1 : 400 to 1 : 6400 as reported previously (Fournier et al., 2002; Jacomo et al., 2002). The main problem with IFA is that cross-reactive antibodies between Bartonella species, especially B. henselae and B. quintana, prevent the identification of the bacteria at the species level (La Scola & Raoult 1996) (Table 1). Only serologically

sophisticated methods such Metalloexopeptidase as Western blots with cross-adsorption studies may help to eventually identify the causative agent at the species level (Houpikian & Raoult, 2003). Several diagnostic assays based on whole-cell detection have been used in clinics (Giladi et al., 2001; Herremans et al., 2007, 2009; Vermeulen et al., 2007). According to ELISA assays for B. henselae infection, recombinant proteins rGroES, rRplL, rBepA and rGroEL yielded high sensitivity >70%, but low specificity ≤59% (Table 3). Only the B. henselae r17-kDa protein has high sensitivity and specificity (Loa et al., 2006; McCool et al., 2008; Hoey et al., 2009) (Table 3). The application of an immunoproteomic strategy to identify the antigenic proteins associated with diseases has been used recently for B. quintana (Boonjakuakul et al., 2007) and B.

It is generally assumed that in the developing neuron a filopodium is formed first; following establishment of contact with an afferent fiber, it retracts and becomes a spine (Fiala et al., 1998; Sorra & Harris, 2000). In this case the outcome would be viewed as an increase in the efficacy of synaptic transmission. However, stable synaptic connections leading to spontaneous network activity have also been seen in young neurons (3–4 days in vitro) even before the formation of spines, and these synapses are formed primarily on dendritic shafts (Lauri et al., 2003). Likewise,

it is not entirely clear that the process of conversion of filopodia to spines is a necessary step in an already mature neuron, where filopodia are rare and spines can form and dissolve within hours, Ganetespib price as shown in estrus-cycling female rats (Woolley & McEwen, 1993) and during recovery from hibernation (Popov & Bocharova, 1992; Popov et al., 2007), as well as in time-lapse microscopy in adult mice (Xu et al., 2009; Yang et al., 2009). On the other hand, within hours following activity blockade with tetrodotoxin (TTX), filopodia grow off existing spines, Roxadustat molecular weight indicating that they are being used as a means of searching for glutamate-releasing presynaptic terminals (Richards et al., 2005). Thus, with a few exceptions, it can be concluded that spines can be formed from shaft synapses, and the presence of spines reduces rather

than enhances the impact of an individual synapse on the activity of the parent neuron. A corollary issue is whether a neuron loses its synapses when spines are pruned, just to regain them when the spines reappear, or whether it retains the synapses with its afferent terminals,

which may form shaft synapses? Intuitively, a synapse which is rich in adhesion molecules crossing between pre- and postsynaptic membranes has a bond strong enough to resist mechanical dissociation of the tissue (e.g. during preparation of synaptosomes). Why then should the synapse lose the presynaptic partner just because it retracts by a few micrometers NADPH-cytochrome-c2 reductase only to reappear a day later, as is the case with the estrus cycle? Recent electron-microscopic data indicate that spine-pruned cortical neurons do lose their connection with afferent inputs (Knott et al., 2006). On the other hand, in hibernating animals there is a marked decrease in spine density during hibernation but there is an increase in shaft synapses (Popov et al., 2007; von der Ohe et al., 2006), and when the animals wake up from hibernation they regain the spines and appear to remember tasks learnt before hibernation, indicating that regardless of the persistence of spines, memories are retained (Clemens et al., 2009). In fact, if trained 24 h after arousal from hibernation, they remember better than controls (Weltzin et al., 2006). Likewise, female rats in the estrus phase of the cycle, when their spine density is down by 30%, are not less capable of remembering items learnt previously.

, Helicobacter pylori, etc. (Cichewicz & Thorpe, 1996; Jones et al., 1997). A recent study selleck inhibitor has shown that ginger (Zingiber officinale) can inhibit fluid accumulation in mice ileal loop by blocking

the binding of the heat-labile enterotoxin of E. coli to the cell surface receptor, GM1 (Chen et al., 2007). However, there is no report on the effect of red chilli or its active compound, capsaicin, against the virulence gene transcription of V. cholerae or any other diarrheagenic agents without affecting their growth or viability. In this study, we examined whether a methanol extract of red chilli can affect the virulence gene expression of V. cholerae. We also examined the effect of capsaicin on the production of CT by V. cholerae strains belonging to various serogroups. Furthermore, the possible mechanism of virulence gene regulation by capsaicin was investigated using a real-time quantitative reverse transcription-PCR (qRT-PCR) CP-868596 mw assay. A total of 23 clinical toxigenic V. cholerae strains used in this study are described in Table 1. All V. cholerae strains were grown at 37 °C in AKI medium, pH 7.4 (Iwanaga et al., 1986; Mukhopadhyay et al., 1996). The ctxB genotyping was carried out by a mismatch amplification mutation PCR assay according to Morita et al. (2008). Dried red chilli was purchased from

a retail market in Osaka, Japan, and was used for this study. Red chilli was ground using a homogenizer to a fine powder and extracted with 99.9% methanol. The methanol was evaporated using a vacuum dryer. Glycogen branching enzyme Crude methanol extract of red chilli was preserved at 4 °C. Natural capsaicin was purchased from

LKT laboratories Inc. (MN). Red chilli methanol extract and capsaicin were dissolved in 99.9% methanol during use. A single colony of V. cholerae strains was inoculated in AKI medium at 37 °C. After 12 h of growth, OD600 nm was adjusted to 1.0. Subsequently, cultures were 100-fold diluted with AKI medium and incubated with and without red chilli methanol extract or capsaicin. Because red chilli methanol extract and capsaicin were dissolved in methanol, the final concentrations were always adjusted to 0.2% methanol in cultures. The culture condition was followed according to Iwanaga et al. (1986), with slight modifications. Briefly, cultures were kept under a stationary condition for an initial 4 h and then shifted to a shaking condition at 180 r.p.m. for another 4 h. A cell-free supernatant (CFS) was prepared by centrifugation of a bacterial culture at 12 000 g for 10 min, followed by filtration through a 0.22-μm filter (Iwaki, Tokyo, Japan). The CFS was diluted 10, 100 and 500 times with phosphate-buffered saline (PBS, pH 7.0) and dilutions of purified CT (Uesaka et al., 1994) of known concentrations were used to estimate the amount of CT in cultures by a bead-ELISA according to Oku et al. (1988).

0 × 101 to SB203580 3.0 × 10−2 ng μL−1 of 15-ADON strain DNAs for Tox5-1/2 primer set). Values of the threshold cycles (Ct) were recorded and obtained by the opticon monitor™ software version 3.1 (Bio-Rad Laboratories). Standard curves for different primer sets were constructed by plotting the Ct value vs. the logarithm (log10) of the concentration of 10-fold serial-diluted

F. graminearum DNAs as described above. Amplifications with different primer sets on the genomic DNAs of two F. graminearum chemotypes were run in triplicate to obtain the mean and SD of each 10-fold serial dilution. Real-time PCR amplifications on total genomic DNA extracted from the sampling zones (as described above) were performed using MiniOpticon (Bio-Rad Laboratories). All real-time PCR reactions were performed utilizing

the real-time PCR MJ white tubes (Bio-Rad Laboratories) in a total volume of 25 μL. The reaction mixture for all real-time PCR assays were: 12.5 μL of IQ Supermix (Bio-Rad Laboratories), 1 μL of each 10 μM forward/reverse primers (Invitrogen), 9.5 μL of sterilized UltraPure Millipore water and 1 μL of DNA template. Real-time PCR conditions for the Fg16NF/R primer set used are outlined in Nicholson et al. (1998) with melting curve analysis at 60–95 °C. Parameters for the Tox5-1/2 primer set are as described Galunisertib nmr in Schnerr et al. (2001). Ascospore germination of S. mycoparasitica was not normally distributed. Therefore, differences between suspensions of six different Fusarium filtrates and water control were analyzed using the Kruskal–Wallis test (SPSS, 1990). Differences between linear mycelial growth

of F. graminearum (3- and 15-ADON) and controls, S. mycoparasitica coinoculated, and S. mycoparasitica preinoculated treatments for 5 days of incubation were analyzed using anova−least significant difference (SPSS, 1990). Differences between S. mycoparasitica-infected (penetrated) or -noninfected (nonpenetrated) F. graminearum (3- and 15-ADON) host cell diameters were analyzed utilizing the t-test (SPSS, 1990). For comparison between different F. graminearum DNA concentrations (with Tox5-1/2 or Fg16NF/R primer set) in different Sclareol treatments, the t-test was employed to analyze the differences between them. Log10 transformations were carried out whenever required to meet the anova requirements (Lehmann, 1975). Sphaerodes mycoparasitica spore germination suspended in both F. graminearum chemotype 3-ADON and 15-ADON filtrates was lower compared with F. avenaceum for the first incubation day, and compared with both F. avenaceum and F. oxysporum for the remaining incubation days (P=0.05; with Kruskal–Wallis test) (Fig. 1). No significant differences in germination of F. graminearum, F. proliferatum and F. sporotrichioides filtrate treatments were observed for the first two incubation days. However, treatments with F. graminearum filtrates showed significantly higher germination rate of S. mycoparasitica compared with F.

Arterial calcification can also make interpretation of the images more difficult, although the information may be beneficial in planning some forms of intervention. Angiography.

Conventional SGI-1776 clinical trial angiography has traditionally been the ‘Gold standard’ and has the added advantage that it can be combined with simultaneous intervention. Diagnostic angiography alone is rarely performed as it is an invasive procedure that requires cannulation of the femoral vessels to inject intra-arterial contrast. The management of CLI in patients with diabetes should be planned within the MDFT, including diabetes and vascular specialists, along with the patient. Amputation rates do vary considerably across England and could in part be due to variations in Y-27632 mouse care delivery.1 MDFTs have been shown to reduce amputation rates.26,27 Multidisciplinary

working with integrated pathways of care has been increasingly emphasised over recent years for optimal care of the diabetes patient with foot disease.22 General management should include a review of metabolic control, assessment and management of cardiovascular risk factors, and antiplatelet therapy instigated (unless contraindicated). It is of vital immediate importance to treat any associated foot infection early on as this can cause a rapid deterioration in an ischaemic or neuroischaemic foot.28 If surgical drainage of the foot is needed, then this should not be delayed. The combination of PAD and infection has a significant negative impact on ulcer healing.16 Historically, the treatment for CLI has relied on bypass surgery, amputation or conservative measures. The role of surgery as the

primary treatment Resveratrol strategy has changed with the development of minimally invasive endovascular techniques (angioplasty, with or without stenting). Endovascular treatment is less invasive practically and physiologically, and so is an attractive option; however, both surgical and endovascular treatments are not mutually exclusive, and can be performed together (‘hybrid’ techniques) to simultaneously manage multi-level arterial disease. Patients with diabetes often have arterial disease involving the below knee vessels which are more complex to treat due to their small calibre and lower blood flows.12 Fortunately, the majority of patients with CLI can still be offered some form of revascularisation in the form of endovascular intervention or open surgery including distal revascularisation.15 Revascularisation techniques, either initially angioplasty or open surgery, have tended to show similar medium-term outcomes although, in patients who survive for more than two years following intervention, surgery may be more effective.

7%), one-to-one consultation skills (n = 60, 73.2%), advice on weight-loss products (n = 52, 63.4%), measurement of blood cholesterol (n = 51, 63%) and advice on weight-loss drugs (n = 49, 60.5%). Conclusions  Community pharmacies could be an ideal setting for the provision of HWM services. ABT-888 mouse The barriers to service provision need to be addressed. Furthermore, the development of appropriate undergraduate and postgraduate training is required to equip pharmacists and their staff with appropriate knowledge and skills to deliver these services effectively. “
“Appropriate household storage and use of drug products can reduce drug wastage and unnecessary hazards. We aimed to quantify the amounts

and types of medications that were stored in Jordanian households and the extent of drug wastage in terms of the amount and cost of these medications. The setting was households in Amman, Jordan. This was a cross-sectional survey study using a pre-piloted questionnaire. Family members were interviewed in person about use of drug products, Selleckchem GSK458 and where drug products were stored. The main outcomes were types, storage methods, cost and quantities of drug products in every household. Two hundred and forty-three households were approached, out of which 219 agreed to participate. A total of 2393 (mean 10.9, SD 5.2) drug products were recorded from the 219 households

surveyed. A significant positive correlation was noted between the number of drug products in a household and family size (r = 0.19, P < 0.01), the level of the mother's education (r = 0.24, P < 0.01), the level of the father's education (r = 0.28, P < 0.01) and income (r = 0.14, P = 0.034). Eighty nine (40.6%) households had at least one child younger than 6 years of age, and 1122 (46.9%) drug products were stored in unsafe places in the houses, within the reach of children. More than a quarter of drug products (1509,

27.2%) were not in their original containers, 360 (15%) were unused since dispensing, 261 (10.9%) had expired and 44 (1.8%) had no clear expiry many date. We estimated that the cost of drug wastage in the 219 households was US$5414. Paracetamol (202, 8.4%), diclofenac (98, 4.1%) and amoxicillin (79, 3.3%) were the most commonly reportedly stored individual drugs. Drug products are stored in large quantities in Jordanian households. Unsafe storage practices have the potential to pose safety hazards, especially to children. “
“Clopidogrel and statins have been commonly coprescribed to patients with atherosclerotic diseases. Clopidogrel–statin interaction was initially described by ex vivo studies, but was not well supported by studies examining health outcomes. This personal view article aims to discuss methodological issues of these studies, especially the retrospective studies assessing health outcomes.

, 2001). Activation of this area is associated with the selection among competing responses (Petrides, 2005), and the more superior portion activated here is especially involved in the spatial domain (Volle et al., 2008). During imitation, this region may serve to maintain a representation of the observed goal in short-term working memory for later execution (Chaminade et al., 2002). Co-activation of the superior frontal gyrus and posterior inferior frontal gyrus

may thus reflect Naïve reliance on kinematic simulation and top-down direction of attention to task-relevant spatial cues. When combined with the anterior inferior parietal and ventral prefrontal activations observed across all groups, these Naïve activations match the general EPZ015666 cell line expectations of a simulation model of novel action understanding (Buccino et al., 2004; Vogt et al., 2007). No activations exclusive to Trained subjects were observed in the Acheulean–Oldowan contrast. Comparison with the numerous activations observed

in the contrast of Toolmaking–Control for Trained subjects (Table 2; Fig. 2) indicates that this result derives from the presence of similar responses to Oldowan and Acheulean stimuli rather than from the absence of significant differences from Control. This is corroborated by the observation of similar activations in separate contrasts of Oldowan–Control and Acheulean–Control (Supporting Information Figs S3 and S4; Tables S1 and S2). The Trained response to both Oldowan Sirolimus nmr and Acheulean stimuli includes: (i) clusters in the anterior insula, lateral premotor cortex, frontal eye field and supplementary eye field likely related to attentional and affective engagement with the stimuli; and (ii) ventral prefrontal clusters likely associated with parsing of observed action

sequences. Insular activations Liothyronine Sodium unique to Trained subjects are in an anterior region associated with interoception, subjective feeling and perceptual awareness (Kikyo et al., 2002; Ploran et al., 2007; Craig, 2009). Activations of the left medial frontal cortex (close to y = 0) and posterior middle frontal gyrus appear to fall within the supplementary and frontal eye fields (Tehovnik et al., 2000), functional regions associated with saccades, visual attention and visual learning (Tehovnik et al., 2000; Grosbras et al., 2005). Together with activation of the precentral gyrus, a region commonly recruited during action observation (Grezes & Decety, 2001; Caspers et al., 2010), these activations likely indicate intense engagement by Trained subjects with the Toolmaking stimuli. These effects of training were not predicted, but are consistent with the pragmatic social and motivational context created by the training programme. Also unique to Trained subjects were inferior frontal gyrus activations of bilateral pars opercularis, left pars triangularis and right pars orbitalis.

The present study aimed to investigate whether the implicit selleck products system underlying vMMN was capable of registering vertical mirror symmetry as a perceptual category. Several behavioral studies have shown that the visual system is particularly sensitive to various forms of symmetry (for a review, see Treder (2010)). According to Carmody et al. (1977) and Tyler et al. (1995), stimulus duration in the 40–80-ms range is long enough for the recognition of symmetric patterns. Other behavioral studies have shown that symmetry can be detected automatically (Baylis & Driver, 1994; Wagemans, 1995; Huang

et al., 2004; Machilsen et al., 2009). Vertical mirror symmetry is a salient feature of living objects, and has obvious biological significance (Tyler & Hardage, 1996). However, so far, no ERP study has analysed the level of processing that is sensitive to symmetry and the automaticity of sensitivity to symmetry. Few studies have investigated the processing of symmetric stimuli on the basis of event-related brain activity. Jacobsen & Höfel (2003) and Höfel & Jacobsen (2007) reported a posterior negative wave elicited by symmetric patterns. In these studies, symmetry as such was task-irrelevant; participants made aesthetic judgements, performed a detection task, or contemplated the beauty of the stimuli. The negativity emerged

in the 380–890-ms poststimulus latency range, so this effect may not be a correlate of elementary perceptual processes. However, in a sequence of alternatively presented random and symmetric dot-patterns, the symmetric patterns elicited a sustained posterior negativity with ~ 220-ms Ibrutinib price onset, whereas random patterns elicited positivity with earlier onset (~ 130 ms) (Norcia et al., 2002). Such activities Guanylate cyclase 2C were considered to be correlates of the appearance of global forms, i.e. an activity more general than a specific symmetry effect. In the present study, we tested

whether the system underlying vMMN is sensitive to symmetry as a perceptual category. If this is so, the regular presentation of stimuli belonging to the same perceptual category (symmetry) will establish a mental representation containing the sequential rule of the stimulation. Irregular stimuli (which do not belong to this category) will violate the prediction that derives from mental representation, and therefore elicit the vMMN component. For this reason, we infrequently embedded symmetric patterned stimuli (deviants) in a series of random patterned stimuli (standards), whereas, in another condition, random deviants appeared in the context of symmetric standards. Thus, we could compare the ERPs elicited by categorically identical standard and deviant stimuli. We expect an ERP difference between the deviant and standard random pattern; we hypothesise that the ERP difference is a vMMN, i.e. a posterior negativity within the 100–300-ms latency range.

The present study aimed to investigate whether the implicit click here system underlying vMMN was capable of registering vertical mirror symmetry as a perceptual category. Several behavioral studies have shown that the visual system is particularly sensitive to various forms of symmetry (for a review, see Treder (2010)). According to Carmody et al. (1977) and Tyler et al. (1995), stimulus duration in the 40–80-ms range is long enough for the recognition of symmetric patterns. Other behavioral studies have shown that symmetry can be detected automatically (Baylis & Driver, 1994; Wagemans, 1995; Huang

et al., 2004; Machilsen et al., 2009). Vertical mirror symmetry is a salient feature of living objects, and has obvious biological significance (Tyler & Hardage, 1996). However, so far, no ERP study has analysed the level of processing that is sensitive to symmetry and the automaticity of sensitivity to symmetry. Few studies have investigated the processing of symmetric stimuli on the basis of event-related brain activity. Jacobsen & Höfel (2003) and Höfel & Jacobsen (2007) reported a posterior negative wave elicited by symmetric patterns. In these studies, symmetry as such was task-irrelevant; participants made aesthetic judgements, performed a detection task, or contemplated the beauty of the stimuli. The negativity emerged

in the 380–890-ms poststimulus latency range, so this effect may not be a correlate of elementary perceptual processes. However, in a sequence of alternatively presented random and symmetric dot-patterns, the symmetric patterns elicited a sustained posterior negativity with ~ 220-ms Trichostatin A onset, whereas random patterns elicited positivity with earlier onset (~ 130 ms) (Norcia et al., 2002). Such activities Uroporphyrinogen III synthase were considered to be correlates of the appearance of global forms, i.e. an activity more general than a specific symmetry effect. In the present study, we tested

whether the system underlying vMMN is sensitive to symmetry as a perceptual category. If this is so, the regular presentation of stimuli belonging to the same perceptual category (symmetry) will establish a mental representation containing the sequential rule of the stimulation. Irregular stimuli (which do not belong to this category) will violate the prediction that derives from mental representation, and therefore elicit the vMMN component. For this reason, we infrequently embedded symmetric patterned stimuli (deviants) in a series of random patterned stimuli (standards), whereas, in another condition, random deviants appeared in the context of symmetric standards. Thus, we could compare the ERPs elicited by categorically identical standard and deviant stimuli. We expect an ERP difference between the deviant and standard random pattern; we hypothesise that the ERP difference is a vMMN, i.e. a posterior negativity within the 100–300-ms latency range.