These glycoproteins, which include Flo1p, Flo5p, Flo9p, Flo10p and Flo11p, are termed flocculins or adhesins (reviewed in Verstrepen & Klis, 2006; Dranginis et al., 2007; Bauer et al., 2010). On the basis of their sensitivity to sugar inhibition, three distinct flocculation phenotypes have been characterized, which include Flo1-type

[mannose-sensitive (MS)], NewFlo-type [glucose- and mannose-sensitive (GMS)] and a mannose-insensitive (MI)-type (Masy et al., 1992). Both MS- and GMS-types are Ca2+-dependent flocculation phenotypes that can be attributed to FLO1-, FLO5- and FLO9-overexpression in Saccharomyces cerevisiae strains (Guo et al., 2000; Liu et al., 2007; Govender et al., 2008, 2010; Van Mulders et al., 2009). It should also be noted that Flo11p is required for strong Flo1-type flocculation

in Saccharomyces diastaticus strains (Bayly et al., 2005). In contrast, the MI phenotype displays Ca2+-independent flocculation and is yet to www.selleckchem.com/products/ink128.html be ascribed to a particular FLO gene. To meet the demands of a consumer-driven market, wine processing currently involves fining and clarification procedures to produce clear and physicochemical stable wines. Wine fining entails the purposeful addition of an adsorptive compound (bentonite, gelatin or albumin), followed by the settling or precipitation (cold stabilization) of partially soluble components from the wine. Further clarification is usually achieved by sedimentation, racking, centrifugation and filtration (Boulton et al., 1996; Maraviroc in vitro Ribéreau-Gayon et al., 2000; Pretorius & Bauer, 2002). Moreover, studies have shown that filtration alters the aroma and colour of the wine and also removes molecules that would otherwise positively contribute to the impression of body and volume on the palate (Lubbers et al., 1994; Boulton et al., 1996; Moreno & Azpilicueta, 2004; Moreno et al., 2007). Thus, it may be concluded that the fining and clarification of wine are expensive and time-consuming procedures that ultimately negatively impact on

the cost of the finished product. Efficient wine yeast flocculation after primary alcoholic fermentation leads to the formation of compacted sediments (Lahtchev & Pesheva, 1991) or ‘caked’ lees, thereby reducing the handling of wines and minimizing problems DNA Damage inhibitor associated with wine clarification (Pretorius & Bauer, 2002). As such, this ultimately contributes to lower volume loss of the finished wine products. The fact that the natural flocculent ability of certain commercial wine yeast strains is advertised by retailers of active dry wine yeasts further highlights the significance and attractiveness of this trait to the wine industry (http://www.maurivinyeast.com/media/51.pdf, 18 January 2010). Being mindful of this, we showed in a recent study that by placing the native chromosomal copies of two dominant flocculation genes, FLO1 and FLO5 in two nonflocculent commercial S.

Fast nicotinic transmission might play a greater role in cholinergic signaling than previously assumed. We provide a model for the examination of synaptic properties of basal forebrain cholinergic innervation in the brain. “
“Nigral dopamine (DA) neurons in vivo exhibit complex firing patterns consisting of tonic single-spikes and phasic bursts that encode information for certain types of reward-related learning and behavior. Non-linear dynamical analysis has previously demonstrated the presence of a non-linear deterministic structure in complex selleck chemicals llc firing patterns of DA neurons, yet the origin of this non-linear determinism remains unknown. In this study, we hypothesized

that bursting activity is the primary source of non-linear determinism in the firing patterns of DA neurons. To test this hypothesis, we investigated the dimension complexity of inter-spike interval data

recorded in vivo Sotrastaurin cell line from bursting and non-bursting DA neurons in the chloral hydrate-anesthetized rat substantia nigra. We found that bursting DA neurons exhibited non-linear determinism in their firing patterns, whereas non-bursting DA neurons showed truly stochastic firing patterns. Determinism was also detected in the isolated burst and inter-burst interval data extracted from firing patterns of bursting neurons. Moreover, less bursting DA neurons in halothane-anesthetized rats exhibited higher dimensional spiking dynamics than do more bursting DA neurons in chloral hydrate-anesthetized rats. These results strongly indicate that bursting activity is the main source of low-dimensional, non-linear determinism in the firing patterns of DA neurons. This finding furthermore suggests that bursts are the likely carriers of meaningful information in the firing activities of DA neurons. “
“Increasing evidence shows that sensory experience is not necessary for initial patterning of neural circuitry but is essential for maintenance and plasticity. We have Proteases inhibitor investigated the role of visual experience in development and plasticity

of inhibitory synapses in the retinocollicular pathway of an altricial rodent, the Syrian hamster. We reported previously that visual receptive field (RF) refinement in superior colliculus (SC) occurs with the same time course in long-term dark-reared (LTDR) as in normally-reared hamsters, but RFs in LTDR animals become unrefined in adulthood. Here we provide support for the hypothesis that this failure to maintain refined RFs into adulthood results from inhibitory plasticity at both pre- and postsynaptic levels. Iontophoretic application of gabazine, a GABAA receptor antagonist, or muscimol, a GABAA receptor agonist, had less of an effect on RF size and excitability of adult LTDR animals than in short-term DR animals or normal animals.

3I). These results indicate that Cbln1 bound to NRXs in a manner distinct from NLs or LRRTMs. As

Cbln1 binds to GluD2 at the postsynaptic site, we next examined whether the binding between Cbln1 and GluD2 was affected by extracellular Ca2+ concentrations. Immunocytochemical analyses of the surface HA-Cbln1 revealed that HA-Cbln1 bound to HEK293 expressing GluD2 under low extracellular Ca2+ concentrations (Fig. 3J). Together, these results indicate that, unlike NRX/NL- or NRX/LRRTM-based cell adhesion, trans-synaptic cell adhesion mediated by NRX1β(S4+)/Cbln1/GluD2 is resistant to low extracellular Ca2+ concentrations. Cbln1, which accumulates at the synaptic junction by binding to GluD2, serves as a presynaptic organizer (Matsuda et al., 2010). As NRX is known to recruit selleck synaptic vesicles (Dean et al., 2003), it probably mediates the presynaptic organizing function of Cbln1. To examine this hypothesis, we first examined whether Cbln1 and GluD2 formed a tripartite complex HCS assay with NRXs. Immunocytochemical analyses showed that NRX1β(S4+)-Fc but not NRX1β(S4−)-Fc specifically bound to HEK293 cells expressing GluD2 only when HA-Cbln1 was

added to the culture medium (Fig. 4A). Similarly, when NRX1β(S4+) and GFP were coexpressed in cbln1-null cerebellar granule cells, NRX1β(S4+) accumulated in GFP-positive axons around the beads coated with HA-Cbln1 but not around uncoated beads (Fig. 4B). We expressed NRX1β(S4+)-Flag, in which the region necessary for binding to presynaptic organizing proteins such as calcium/calmodulin-dependent serine protein kinase (CASK) (Hata et al., 1996; Dean et al., 2003) was disrupted by attaching the Flag tag at the extreme Interleukin-3 receptor C-terminus of NRX1β(S4+) (Fairless et al., 2008) in wild-type hippocampal neurons. Importantly, NRX1β(S4+)-Flag also accumulated in axons contacting the beads coated with HA-Cbln1 without recruiting the presynaptic marker synapsin I (Supporting

information Fig. S2A), indicating that accumulation of NRX1β(S4+) was directly caused by HA-Cbln1 and not by other presynaptic molecules that bound to the C-terminus of NRX1β(S4+). In addition, not only overexpressed NRX1β(S4+), but also endogenous NRXs in cbln1-null granule cells preferentially accumulated in axons contacting the beads coated with HA-Cbln1 (Supporting Information Fig. S2B). Furthermore, NRX1β(S4+)-Flag expressed in cbln1-null granule cells accumulated in axons that crossed Purkinje cells only when HA-Cbln1 was added to the culture medium (Supporting Information Fig. S2C), indicating that Cbln1, which was bound to GluD2 on Purkinje cell dendrites, induced clustering of NRX1β(S4+) at presynaptic terminals. Although beads coated with Cbln1 accumulated synapsin I-positive synaptic vesicles in cbln1-null granule cell axons (Matsuda et al., 2010), addition of NRX1β(S4+)-Fc and not NRX1β(S4−)-Fc to the culture medium significantly inhibited Cbln1 presynaptic organizing function (Fig. 4C).

Each NASA-TLX dimension was presented as a visual analog scale with a title and a bipolar descriptor (very low/very high) at each end. Numerical values were not displayed, but values ranging from 0 to 8 (9 points) were assigned to scale

the position RG7422 in vitro during data analysis. The SAM uses a nine-point scale to rate the perceived valence (i.e. level of happiness) and arousal. Values range between 1 and 9, with higher scores indicating higher valence/arousal. Eye position was acquired binocularly and non-invasively with a fast video-based eye tracker at 500 Hz (desktop configuration of the EyeLink 1000, SR Research, instrument noise 0.01 deg RMS). First, we discarded the eye position data corresponding to the time periods in which participants entered their answers on the keypad. Then, we identified and removed blink periods as portions of the raw data where pupil information was missing. We also removed portions of data where very fast decreases and increases in pupil area occurred (> 50 units/sample, such periods are probably semi-blinks where the pupil is never fully occluded; Troncoso et al., 2008). We added 200 ms before and after each blink/semi-blink to eliminate the initial and final parts where the pupil was still partially selleck chemicals occluded (Troncoso et al.,

2008). We identified saccades with a modified version of the algorithm developed by Engbert and Kliegl (2003; Laubrock et al., 2005; Engbert,

ifenprodil 2006a; Rolfs et al., 2006) with λ = 6 (used to obtain the velocity threshold) and a minimum saccadic duration of 6 ms. To reduce the amount of potential noise, we considered only binocular saccades, that is, saccades with a minimum overlap of one data sample in both eyes (Laubrock et al., 2005; Engbert, 2006a,b; Rolfs et al., 2006). Additionally, we imposed a minimum intersaccadic interval of 20 ms so that potential overshoot corrections might not be categorized as new saccades (Møller et al., 2002). Microsaccades were defined as saccades with magnitude < 2 deg in both eyes (Martinez-Conde et al., 2006, Martinez-Conde et al., 2009; Troncoso et al., 2008; McCamy et al., 2013b). To calculate microsaccade properties, such as magnitude and peak velocity, we averaged the values for the right and left eyes (McCamy et al., 2012; Costela et al., 2013). Figure 2 shows the microsaccadic peak velocity–magnitude relationship (main sequence), and the corresponding microsaccade magnitude and peak velocity distributions. We defined fixations as those time periods during which subjects were not blinking or making saccades larger than 2 deg (Otero-Millan et al., 2008). We assumed a linear relationship between microsaccade magnitude and peak velocity rather than a power law one because the value of r2 was always higher for the linear fits (r2: linear 0.908; power law 0.906).

There is a need to develop standardized guidelines that can be easily adopted and replicated in resource-poor settings. Many different protocols are available, and with a concerted effort by interested parties such as medical schools, academic residency programs, the CDC, and professional societies such as the Infectious Diseases Society of America, consensus guidelines could be developed. There is also a need for further research on strategies to improve the comprehension of risk of traveling

medical trainees, how they actually use the medications for PEP, how to improve their adherence to the regimen while based overseas, and what should be done with the medications after the end of the rotation. The authors state they have no conflicts of interest to declare. “
“Up to 65% of travelers to less developed countries report health problems while traveling. International Bafetinib clinical trial travel is an increasing concern for health practitioners.

To date, there have not been any published analyses of mortality amongst foreign nationals visiting Thailand. Our objectives are to examine the magnitude and characterize the deaths among foreign nationals in Chiang Mai, a popular tourist province in Thailand. The study commenced with a review of the Thai death registration. Death certificates were retrieved, reviewed, and classified by the causes of death. Basic statistics and proportionate mortality ratio (PMR) were used to describe the pattern of deaths. Standardized mortality ratio (SMR) was used to assess the excess mortality risk among foreign Entinostat nationals. Between January 1, 2010 and May 31, 2011, there were 1,295 registered deaths in Chiang Mai City, of which 102 records (7.9%) were foreign nationals. Median age of decedents was 64 years (range 14–102 y). Female–to–male ratio was 1 : 5.4. The highest mortality Aurora Kinase was among Europeans (45.1%). Most of the deaths were natural causes (89.2%) including 36 cardiac diseases (PMR = 35.3) and 20 malignancy diseases (PMR = 19.6). Deaths due to external causes were low. The SMRs range

between 0.15 and 0.30. Communicable diseases and injuries were not the leading causes of death among foreign nationals visiting Chiang Mai, Thailand. It is essential that travelers are aware of mortality risk associated with their underlying diseases and that they are properly prepared to handle them while traveling. As overseas travel becomes more affordable, the number of people traveling outside their home countries has increased. According to data from the United Nations World Tourism Organization, approximately 880 million travelers visited foreign countries in 2009.[1] The number increased by 7% in 2010, to 940 million travelers.[1] The numbers of international travelers visiting Southeast Asia has also increased significantly; by 2010, this region hosted 69.6 million travelers.[1] Thailand hosted approximately 15.

This study conformed to The Venetoclax in vivo Code of Ethics of the World Medical Association (Declaration of Helsinki), printed in the British

Medical Journal (18 July 1964). The experimental procedure was approved by the Institutional Review Board of the University of Southern California. All participants signed the written informed consent. The primary task used in this study was a four-element finger sequence task. The participant positioned the four fingers (5th, 4th, middle and index fingers) of his or her non-dominant hand on four keys (Z, X, C and V) of a standard computer keyboard. The four-element sequence was displayed on a computer monitor positioned in front of the participant (Fig. 1, top). Each of the four numbers on the computer screen was embedded within a square box. The participant was told that the relative position of their four fingers on the keyboard corresponded to the relative position of the four squares on the computer screen. Thus, when performing the task with the left hand, the box furthest to the left on the computer screen corresponded to the fifth finger and the box furthest to the right corresponded to the index finger.

The participant was additionally told that the sequence with which the keys were pressed should follow the numerical sequence of 1-2-3-4. Thus, for the sequence displayed on the computer screen (4-1-3-2), the correct order of key presses was 4th finger–index finger–middle finger–5th finger when using the left hand to perform the task. At the beginning of each trial, the sequence 4-1-3-2 was displayed Selleckchem HSP inhibitor on the computer monitor for 600 ms, followed by a ‘go’ signal.

Participants were instructed to start the movement as soon as they saw the ‘go’ signal and finish the sequence as fast as possible. Feedback about performance (accuracy and the time taken to finish all four key presses) was given after each trial (Fig. 1, top). Participants practiced the same sequence throughout the experiment. The secondary task (probe task) was a two-choice audio–vocal RT task. Participants heard either a high pitch (1000 Hz) or a low pitch (500 Hz) sound via headphones and were required to make a vocal response by saying ‘high’ or ‘low’ correspondingly into a microphone. The audio stimulus was presented 100 ms after the sequence was displayed ADP ribosylation factor on the computer monitor (prior to movement onset), at which time the primary task was assumed to engage planning processes (Fig. 1, top). Participants were assigned to groups based on whether they performed the secondary probe task (control vs. probe) and whether they received the rTMS manipulation (No rTMS vs. dPM rTMS). This resulted in four experimental groups: Control–NoTMS, Probe–NoTMS, Control–dPM, and Probe–dPM with a sample size of 10 for each group. The last group of participants (Probe–M1, n = 10) served as the TMS site control. The experiment took place over two consecutive days.

The replacement of ethanol by formate reversed the domination in selleckchem favor of the vibrio morphotype and eventually resulted in the isolation

of the extremely natronophilic SRB strain AHT8 described previously as Desulfonatronospira thiodismutans (Sorokin et al., 2008). The curved rods were purified from single colonies on the original medium with ethanol and thiosulfate and the resulting strain was designated AHT5. A similar strain, ASP-p, was obtained from another Kulunda lake enrichment at 2 M Na+ and pH 10 with formate/acetate as an electron donor/carbon source and thiosulfate as an electron acceptor. In that case, the enrichment was dominated by the SRB Desulfonatronovibrio sp., while the curved rods became dominant after the replacement of formate/acetate with pyruvate. According to 16S rRNA gene sequence analysis, both rod-shaped

isolates were very close to each other and to Tindallia magadiensis (99% sequence similarity), which was found previously in the hypersaline soda lake Magadi (Kenya) and described as an obligately heterotrophic haloalkaliphilic acetogen preferentially fermenting amino acids (Kevbrin et al., 1998). DNA–DNA hybridization showed that the novel isolates were nearly identical (around 95% DNA similarity) and belonged to T. magadiensis species (85% DNA hybridization HKI-272 mouse value with the type strain). However, despite this

close relation, anaerobic respiration has not been demonstrated previously in the genus Tindallia, except for the ability to reduce ferric iron, which was not coupled to growth in T. magadiensis and Tindallia texcoconensis (Kevbrin et al., 1998; Alazard et al., 2007). Examination of the type species T. magadiensis confirmed the absence of growth by thiosulfate respiration in this organism. There were two ecologically important differences of Tindallia sp. strains AHT5 and ASP-p from the previously described acetogenic Tindallia tuclazepam species. First, they both grew lithoautotrophically with H2 and formate. Second, they were capable of true anaerobic respiration with H2, formate, pyruvate, lactate and glycerol as electron donors using thiosulfate, sulfur or fumarate as an electron acceptor (Table 1). Interestingly, formate was detected as a product of anaerobic H2 metabolism instead of acetate, which is expected for an acetogen. It might be speculated that formate in this case is a product of reversed formate lyase reaction, but the significance of its formation is not clear and needs further investigation. Recently, a possibility of anaerobic growth by the opposite reaction (conversion of formate to H2) has been demonstrated for a thermophilic archaeon (Kim et al., 2010) and for syntrophic cultures of acetogens and methanogens (Dolfing et al., 2008).

However, compliance with pre-travel advice on personal hygiene measures was limited, since half of the participants experienced one or more episodes of diarrhea, indicating exposure to feco-oral infection, as was demonstrated in another study conducted in the same cohort.8 These results suggest that personal hygiene measures were of limited contribution to the low seroconversion to anti-HEV. Pre-travel, we found an anti-HEV seroprevalence of 2.0% (24 out of 1206) which is comparable to the seroprevalence in the general Dutch population (0.5–2%).9,10 No risk factors for previous HEV infection

were identified. Despite the limitations of this study we conclude that the risk for short-term travelers Belinostat clinical trial to acquire a hepatitis E infection is very low. The authors state they have no conflicts of interest to declare. “
“It is well known that animals show a stress response when confronted with a novel environment. The aim of the this study was to investigate whether humans show a similar response by studying the reaction to a travel-related transitory change of residence. Forty-eight individuals (32 women, 16 men, age 40–83 years) traveling to a health resort approximately 120 km from their home town participated in the study. Individuals

monitored their blood pressure (BP) twice a day 3 weeks before AZD5363 cost (baseline) and during the stay and filled out a diary stating their mood and sleep. The change of the variables relative to baseline on the day before departure, the travel day, and the day after arrival as well as 5 days after arrival were determined. Systolic and diastolic BPs were increased on the day before travel and diastolic BP remained increased on the travel day and the day after arrival. Sleep was poorer during the first night at the new residence. All three variables had returned to baseline level 5 days into the stay. Mood was not affected by the

change of residence. The aminophylline results indicate that not only the change of residence but also its anticipation affects individuals in a transient way. The findings are relevant not only for the basic understanding of the reaction to novel environments but also to travel, tourism as well as rehabilitation, and spa-research. Humans as well as animals are sensitive to changes in their environment. The most prominent feature is the so-called orienting response, a short-term psychophysiological reaction improving information uptake and attention and potentially preparing for fight or flight when confronted with a novel stimulus.[1-3] Typically, however, the individual will get used to the stimuli after repeated presentations or prolonged exposure and habituate, thereby ceasing to show any further response.[4, 5] In animals, a commonly used paradigm for the study of more enduring reactions is “environmental novelty” used to explore, among others, stress, fear, and exploration.

91 (95% CI 0.83–1.00); P = 0.040]. The proportional hazards assumption was not violated. Overall, 495 patients were diagnosed with TB in the first year after ART initiation during 5728 person-years of follow-up. The overall incidence rate was 8.6/100 PYAR (95% CI 7.9–9.4 PYAR). The incidence

Erismodegib datasheet rate fell from 8.2/100 PYAR (95% CI 7.0–9.5 PYAR) in 2005 to 6.5/100 PYAR (95% CI 5.3–8.1 PYAR) in 2007, and then rose in 2008 (9.5/100 PYAR (95% CI 7.6–11.9 PYAR)] and 2009 [15.6/100 PYAR (95% CI 12.4–19.7 PYAR)]; the log-rank test for equality of survivor functions was P = 0.003. The cumulative incidence of TB in the first year after ART initiation is depicted in Figure 3. The proportional hazards assumption of the multivariable Cox Talazoparib price proportional hazards model was violated. We therefore stratified our analysis for the years 2005, 2006 and 2007 versus 2008 and 2009, based on the difference in TB incidence. The two models showed the same covariates to be significantly associated with the outcome with similar HR estimates, and visual inspection of the curves also showed a great similarity between the two periods. A multivariable

Cox proportional hazards model was therefore run on all data and showed lower baseline CD4 cell count and male sex at ART initiation to be significantly associated with TB incidence in the first year (Table 3). Patients initiating ART later were more likely to be diagnosed with TB in the first year of ART initiation

[HR per year of later ART initiation, 1.13 (95% CI 1.05–1.21); P = 0.001]. This is one of the first studies to relate decreasing mortality rates in ART initiators to changing patient characteristics and improved TB case finding selleck chemical after the rapid scale-up of ART in East Africa. In our large urban HIV-infected cohort, baseline CD4 cell counts increased significantly over time, which was associated with a decrease in mortality. A later year of ART initiation was independently associated with improved survival. Our findings show that major programmatic changes are possible in resource-limited settings and that these are associated with a measurable effect on all-cause mortality. There are some published data on changing CD4 cell counts over time since the roll-out of large-scale antiretroviral therapy in the developing world. The ART-LINC of IeDEA (ART in Lower Income Countries collaboration of the International Databases to Evaluate AIDS) cohort study, reporting on 17 ART programmes and 36 715 patients initiating ART in 12 countries in sub-Saharan Africa, South America and Asia, showed increasing median baseline CD4 counts between 2001 and 2006, with the lowest CD4 counts for the sub-Saharan African region (60 cells/μL in 2001 increasing to 122 cells/μL in 2006) [19]. In studies specifically looking at sub-Saharan Africa, most data originate from South Africa, where this trend has also been noted [20, 21].

Starting ART early in severely immunosuppressed HIV-positive patients presenting with TB is associated with decreased Dasatinib mortality and a lowering of the rates of disease progression but rates of IRD are high. Patients with HIV and a CD4 cell count >350 cells/μL have a low risk of HIV disease progression or death during the subsequent 6 months of TB treatment, depending on age and VL [6]. They should have their CD4 cell count monitored regularly and ART can be

withheld during the short-course of TB treatment. One study performed in HIV-associated TB meningitis in the developing world, where 90% of the patients were male, the majority drug users, many with advanced disease and the Crizotinib in vivo diagnosis being made clinically, showed no difference in mortality starting ART early or late [7]. We recommend EFV in combination with TDF and FTC as first-line ART in TB/HIV coinfection 1B We recommend that when rifampicin is used with EFV in patients over 60 kg, the EFV dose is increased to 800 mg daily. Standard doses of EFV are recommended if the patient weighs <60 kg 1C We recommend that rifampicin is not used with either NVP or PI/r 1C We recommend that where effective ART necessitates the use

of PI/r, that rifabutin is used instead of rifampicin 1C Proportion of patients with active TB on anti-TB therapy started on ART containing EFV, TDF and FTC. HIV-related TB should be treated with a regimen, including rifamycin for the full course of TB treatment, unless there is rifamycin resistance or intolerance. Rifamycins frequently interact with ARV medications and can lead to similar toxicities, notably rash and hepatitis. We recommend EFV as the preferred therapy for ART Protein kinase N1 because of its confirmed potency when used in TB/HIV coinfection [8-10], and its efficacy in RCT. We recommend that EFV be given with TDF and FTC due to the availability

of a once-daily co-formulation, a reduced risk of rash compared with NVP and improved efficacy at higher HIV VLs (commonly occurring in this setting). ABC-3TC is an alternative acceptable NRTI backbone in patients with lower HIV VLs and that are HLA-B*57:01 negative (see Section 5.3 Which NRTI backbone). There is significant variability in the effect that rifampicin has on EFV concentrations because of liver enzyme induction, especially of CYP450 3A4 [8,11–13]. Subtherapeutic EFV concentrations may occur among patients who weigh more than 60 kg who are taking standard dose EFV together with rifampicin, and increasing the dose of EFV from 600 mg daily to 800 mg daily may be necessary; however, there is a risk of increasing adverse effects.