The breaking traces measured in the presence of para-OPV3 molecules show a predominant

occurrence of such plateaus as evidenced in Figure 2b by the yellow/orange regions at these conductance values. These conductance plateaus are the signature of the formation of molecular junctions. We have observed that by adding 2 meq of tetrabutylammonium hydroxide (Bu4 NOH) to the solution, the probability of forming such junctions increases. Roughly, we found that the number of traces with plateaus is about two times higher in the presence of this deprotecting agent. We ascribe this observation to the increased reactivity of free thiols to the gold surface with respect to the acetyl-protected SHP099 purchase thiols. To confirm reproducibility, we have performed several measurements for para- and meta-OPV3 molecules during different days and using different

Ro-3306 purchase MCBJ devices. In Figure 3 typical trace histograms [31] and one-dimensional histograms (right panel) built from 1,000 consecutive breaking traces measured in the presence of the molecules are shown. To build the trace histograms, the individual traces (as the ones shown in the inset) were shifted horizontally to fix the rupture of Au-Au contacts at zero electrode displacement. The color scale in the trace histogram indicates the density of data points found at each displacement and conductance value, and, therefore, the colored areas represent the most probable evolution during the breaking process. Figure 3 Two-dimensional trace histogram. Two-dimensional trace histogram constructed from 1,000 consecutive breaking traces measured at room temperature and 0.1 V bias voltage for MCBJ devices exposed to 1 mM solution of (a) para-OPV3 and (b) meta-OPV3 molecules in 1,2-dichlorobenzene. Regions of high counts (blue areas) Flavopiridol (Alvocidib) represent the most probable evolution during the breaking of the contact. The most probable conductance values were extracted by fitting the characteristic peak of the 1D-conductance histograms (right) to a Gaussian function (red dashed curve). The one-dimensional conductance histograms of Figure 3 show broad

peaks centered at 1.1 × 10−4 G 0 and 1.5 × 10−5 G 0 for para-OPV3 and meta-OPV3 molecules, respectively. These values have been obtained from a Gaussian fit (showed as dashed red lines in the 1D conductance histograms). The trace and the 1D conductance histograms show conductance variations around these values. It is well known that the electron transport through a molecule depends on the local environment and the nature of metal/molecule interfaces. They affect the formation and stability of single-molecule junctions, giving rise to variations in the conductance [22]. The dramatic suppression in conductance cannot be explained from a single-barrier tunneling mechanism, because the meta-OPV3 is shorter than the para-OPV3 and therefore should be more conductive.

Membrane and DNA dyes were used concomitantly to visualise the cell periphery and the nucleoid (Figure 1B and

1C). Cells were classified into populations defined according to their number of foci, and the positioning of foci along the length of cells was evaluated for each population (Figures 1C and 2). The distances of the foci to the closest cell pole were scored on a five points scale along the long axis of the cell from the pole to mid-cell (Additional file 1, Figure S1). The ori, right and NS-right loci displayed 2 to 4 foci that mostly found at or near the quarter positions, whereas the ter locus displayed 1 or 2 foci, which were mostly located at mid-cell (Additional file 1, Figure S1). The proportion of mid-cell-located ter foci was lower for cells harbouring a single focus than for cells with two foci, consistent with a progressive migration of the ter region from the new cell MEK inhibitor pole to the GF120918 cell line mid-cell during the cell cycle [7, 8, 21]. These findings are

consistent with previous observations using similar [9, 20] or different detection systems and growth conditions [6, 10]. Positioning of chromosome loci along the cell diameter The position of a fluorescent focus along the width of the cell cannot be directly determined using 2-D wide-field microscopy. Indeed, a focus located near the cell periphery may appear at the centre of the cell diameter or at the edge according to the orientation of the cell cylinder with respect to the focal plan. Nevertheless, since the orientations of the cell cylinder are expected to be random for a population of rod-shaped bacteria deposited on a plane surface, the mean position of particular foci can be calculated from the apparent distributions of foci along the cell diameter. We therefore measured the apparent distance along the cell diameter between foci and the membrane (Figure 1C). The datasets obtained were then compared with distributions calculated for different models of positioning across

the width of the cell (Methods). We defined five slices of equivalent surface in a quarter of Methocarbamol the cell section and calculated the expected distributions of foci according to the various models of positioning (the 2-D apparent foci distributions for various 3-D localisation patterns are shown in Figures 2, 3 and 4). Figure 2 Distributions of foci along the cell diameter. (A) Drawing showing the measurement of the apparent positions of foci along the cell diameter. Distances along the cell diameter between the centres of foci and the nearest membrane were measured. (B) Distributions of foci along the cell diameter for the ori, right and NS-rigth loci in the various cell classes. Distributions are plotted as the percentage of total foci in each cell class (Y-axis). The sample size of the cell classes is given on each graph.

In this

latter case, the use of the small molecule RITA (reactivation of p53 and induction of tumor cell apoptosis) that inhibits MDM2/p53 interaction and induces expression of p53 target genes and massive apoptosis in various tumor cells lines [35], can be useful to counteract HIPK2 degradation and to reactivate p53 apoptotic function [38]. Interestingly, also zinc ions treatment has been shown to relapse the MDM2-induced HIPK2 downregulation, by counteracting the MDM2 E3 ubiquitin ligase activity finally reactivating the HIPK2-induced p53Ser46 phosphorylation and apoptotic activity [39], although the molecular mechanism needs to be elucidated. HIPK2 depletion has been shown to induce cancer cell resistance to different Akt inhibitor anticancer drugs even in p53-null Roscovitine cells, suggesting the involvement of additional HIPK2 targets other than p53. In particular, it has been found that HIPK2 phosphorylates and promotes proteasomal degradation of ΔNp63α, a prosurvival dominant negative (DN) isoform of the p53 family member p63. HIPK2 phosphorylates ΔNp63α at the T397 residue, thus, the nonphosphorylatable

ΔNp63α-T397A mutant is not degraded in spite of either HIPK2 overexpression or ADR treatment. These findings underline ΔNp63α as a novel HIPK2 target in response to genotoxic drugs [33]. These data indicate that HIPK2 has a double commitment, working as activator for proapoptotic factors (i.e., p53) on one hand and inhibitor for antiapoptotic factors (i.e., CtBP, MDM2, ΔNp63α, HIF-1α) on the other hand. IMP dehydrogenase On the opposite side, these considerations would allow to suppose that tumor-associated inhibition of HIPK2 activity might strongly contribute to chemoresistance and tumor progression, in addition to other better-characterized events, such as p53 mutation/inactivation and MDM2 or ΔNp63α overexpression. Mechanisms of HIPK2 inhibition and its impact on both p53 function and tumor progression Several proteins have been shown to target the HIPK2/p53 axis and therefore to inhibit

stress- or drug-induced apoptosis to clear cancer. Recent studies demonstrated that High-mobility group A1 (HMGA1) proteins interact with p53 and inhibit its apoptotic activity [40]. Interestingly, HMGA1 overexpression is responsible for HIPK2 cytoplasmic sequestration and the subsequent inhibition of HIPK2/p53 interaction and apoptosis activation [41]. HMGA1 is frequently overexpressed in tumors and correlates with low apoptotic index in wild-type p53 breast cancer tissues [41]. Thus, immunostaining of breast ductal carcinomas with low HMGA1 expression and with high apoptotic index (not shown) results in HIPK2 nuclear localization (Figure 1A). On the other hand, breast ductal carcinomas with high HMGA1 expression and with low apoptotic index (not shown) show HIPK2 cytoplasmic localization (Figure 1B), meaning likely HIPK2 inactivation [41].

7 ± 5.4 †*# 65.6 ± 9.6 †  60 min 69.4 ± 6.2

* 72.9 ± 7.2 †*# 62.7 ± 8.2 †  80 min 70.1 ± 7.0 * 72.6 ± 8.0 †* 60.7 ± 7.8 †‡ Exercise mean 70.0 ± 7.0 * 74.4 ± 6.4 *# 65.1 ± 8.7   % energy from Fat  20 min 27.5 ± 9.1   21.8 ± 4.9 *# 28.7 ± 9.1    40 min 31.9 ± 5.5   26.3 ± 5.4 *# 34.4 ± 9.6    60 min 30.6 ± 6.2 * 27.1 ± 7.2 *# 37.3 ± 8.2 †  80 min 29.9 ± 7.0 * 27.4 ± 8.0 *# 39.3 ± 7.8 † Exercise mean 30.0 ± 7.0 * 25.6 ± 6.4 *# 34.9 ± 8.7   Values are means ± SD for 11 men. *, significantly different from water; #, significantly different from raisins; †, significantly different from 20 min; ‡, significantly different from 40 min RPE, rate of perceived exertion; CHO, carbohydrate. Figure 1 Respiratory exchange ratio (RER) during 80-min of running at 75% VO 2 max. Values are means ± SD for 11 men. *, significantly different from water and #, significantly different from raisin for (c) Entospletinib cell line chews at 20 and 40-min and for (r) raisins and (c) chews at 60 and 80-min (p ≤ 0.05). Blood parameters Hematocrit was not different between treatments before exercise (44 ± 3, 44 ± 3, 44 ± 2%, for raisin, chews and water respectively). Hematocrit increased from pre-exercise values for all treatments during the first 20-min, but remained the same for the rest of the sub-maximal exercise bout. Thus, we just report the average

for the entire 80-min sub-maximal exercise bout. Hematocrit averaged 47 ± 3, 47 ± 3, 47 ± 3% for raisin, chews and Baricitinib water respectively, with no difference between treatments. Metabolic responses averaged over the 80-min of exercise at 75%VO2max are presented in Table 3. Blood glucose P5091 mw was similar pre-exercise between treatments and only increased from rest at 40-min of the 80-min sub-maximal exercise bout in the chews treatment and at 80-min for the raisin treatment. Blood lactate was similar pre-exercise for all treatments and did not increase significantly above rest for the 80-min sub-maximal exercise bout

for any treatment. Serum free fatty acid (FFA) concentrations (Figure 2) remained at pre-exercise levels for the entire 80-min sub-maximal exercise bout for the chews treatment, but increased significantly at 80-min compared to all time points for the water only treatment. The 20-min FFA was significantly lower than 60- and 80-min and the 40-min FFA was lower than the 60-min value for the raisin treatment. FFA was significantly higher with the water treatment compared to chews at 40-and 60-min of the 80-min sub-maximal exercise bout. Raisin was higher than chews at 60-min of sub-maximal exercise. Water had higher FFA than both raisin and chews at 80-min of sub-maximal exercise. Serum glycerol concentrations (Table 3) were not different at rest between treatments (0.09 ± 0.06, 0.11 ± 0.06, 0.12 ± 0.07 mmol·L-1 for raisin, chews and water respectively). Values increased for all treatments during exercise, but there were no differences between treatments.

There is no consensus as to the period of vulnerability, but it may be in the order of 2 weeks [32]. When to proceed and when to defer? A good rule of thumb when considering whether to proceed with operative treatment is to determine whether there are conditions present that may be detrimental or even life-threatening that require medical treatment in its own right in the absence of surgery.

Such conditions may include dehydration with acute renal impairment, severe electrolyte abnormalities Selleckchem MK-8931 (a sodium or potassium level outside the range of 120 to 150 mmol/L and 2.8 to 6.0 mmol/L, respectively), symptomatic anaemia and uncontrolled diabetes with risk of developing dehydration from polyuria or hyperosmolar coma. In addition, one would consider delaying surgery for unfasted patients and to correct any correctable coagulopathy and anaemia. The level at which this occurs should ideally be individualised, but transfusion 4SC-202 should be considered when preoperative haemoglobin level is between 7 and 10 g/dL. Operation should only be deferred if there is

a reasonable likelihood of improving the conditions that are precluding surgery. To optimize, as defined by the Oxford Dictionary, is to “make the best or most effective use of a situation or resource”. Optimization is what we hope to achieve for every preoperative patient; however, there are times when the best a patient can achieve still places him or her in a high-risk category, despite having achieved certain objective criteria. If there are no further improvements possible without subjecting the patient to other stressful procedures, a decision has to be made to either proceed with operative or conservative treatment. Prolonged or repeated fasting orders during periods of decisional uncertainty can only cause further harm to patients. Many intervening factors, medical or non-medical, may wade into the decision to operate

or not. Ultimately, each case BCKDHA have to be considered on its own merit and communication between surgeons, anaesthesiologists, physicians, intensive care physicians and the patient is paramount in decision making. Why then does last minute cancellation occur? Last minute cancellation or undue delay of an operation due to medical reasons is frustrating to all concerned as it is mostly avoidable and is costly to both the patient and the health care system. It frequently occurs consequent to expectation differences and breakdown in communication between the physicians from different disciplines involved. The development of institutional guidelines on the management of fractured hip patients (see Fig. 1) that is followed from the time the diagnosis is first suspected would bypass much of the uncertainty regarding expectations of what need to be achieved for the patient before surgery is considered.

The over-usage of antimicrobial compounds has led to an increase of H. pylori resistance to antibiotics and consequent failure in treatment therapy [1, 2]. In accordance with the Maastricht Consensus in Europe, the recommended therapy for H. pylori eradication in the stomach mucosa is the use of a proton pump inhibitor associated with two antibiotics, such as metronidazole, amoxicillin or clarithromycin for a 7-14 days period [3]. This therapy, although highly effective, is unselectively proposed to all patients and can imply serious discomfort to patients due to Combretastatin A4 chemical structure side effects of the antibiotics. Clarithromycin resistance is one of the most prevalent and can reach up

to 20% in Southern European countries [1]. The resistance is associated with point mutations in the peptidyltransferase region encoded in domain V of the H. pylori 23S rRNA gene [2, 4, 5]. The three most prevalent point mutations are the transitions A2142G and A2143G and the transversion A2142C [1, 2, 4]. Until

now, the antibiotic susceptibility has been detected in clinical laboratories by several phenotypical methods such as the agar dilution method, as recommended by the National Committee for Clinical Laboratory Standards (NCCLS) [6], or the alternative E-test that is considered to be more simple [7–10]. However, these methods are fastidious, MK0683 order time consuming [11], and fail to give any information about the point mutation within the sample [3]. Therefore, molecular methods for the detection of clarithromycin resistance in H. pylori have been developed during the last several years in order to overcome these shortcomings. Polymerase chain reaction (PCR) followed by sequencing or reverse hybridization, Real-Time PCR and fluorescence in situ hybridization (FISH) with DNA probes are some examples [2, 9,

12, 13]. When compared to PCR-based methods, the FISH technique presents some advantages since it is not so easily affected by DNA contamination, and allows for direct visualization of bacteria in the gastric biopsy specimens [1, 2]. Recently, peptide nucleic acid (PNA) probes using Selleckchem Docetaxel FISH have been designed and optimized for the detection of several bacteria, such as Enterobacter sakazakii, Pseudomonas aeruginosa and Eschericia coli [14, 15]. PNA molecules are DNA mimics that have the negatively charged sugar-phosphate backbone replaced by an achiral, neutral polyamide backbone formed by repetitive N-(2-aminoethyl) glycine units [16, 17]. Although PNA lacks pentoses, specific hybridization between the PNA sequences and nucleic acid complementary sequences still occur according to the Watson-Crick rules [18, 19]. The neutral PNA molecule characteristic is responsible for a higher thermal stability (high Tm) between PNA/DNA or PNA/RNA bonding, compared with the traditional DNA probes [17].

No significant differences between the numbers

of colonies recovered on plates with or without antibiotics were observed (data not shown). Histology of chinchilla bullae Following sacrifice, the chinchilla ears were dissected, fixed with 10% neutral buffered formalin, and decalcified with 5% formic acid. Each ear was cut at the midline in the sagittal plane, and both halves were processed and paraffin-embedded. Step sections of the distal halves were performed and the resulting slides were stained with hematoxylin-eosin www.selleckchem.com/products/Temsirolimus.html (H&E) for analysis. One of us (A.N.W.), a Board-certified pathologist, scored randomized and blinded sections from the same step-sections of each ear for the relative level of the inflammatory response, with the control (buffer only) ears being scored as 0 (no inflammation), with the most inflammation being designated as 4+. Ribonuclease (RNase) activity assays Varying amounts of purified VapD, VapX, or Cat (chloramphenicol acetyltransferase) proteins were incubated at 37°C for 30 minutes with 25 pmol of RNaseAlert substrate (Integrated DNA Technologies, CHIR-99021 mw Coralville, IA) using the manufacturer’s buffer in a final volume of 25 μl. The RNaseAlert

substrate is a single stranded RNA with a fluorophore (FAM) on one end and a quencher on the other. When cleaved, the substrate fluoresces brightly. This sensitive assay allows us to monitor RNase activity in real time. Negative controls consisted of the MagneHis protein elution buffer with no protein, and 0.6 μg of the Cat and VapX proteins. The reactions were placed in a Bio-Rad white 48-well PCR plate, covered with optical film and incubated in a MiniOpticon thermocycler at 37°C. Plate reads were taken every 5 minutes for 30 minutes. The average

relative fluorescence units (RFU) from two independent assays are reported. All solutions used were nuclease free or treated with diethyl pyrocarbonate. Statistical analysis Data are presented as the mean ± standard 3-mercaptopyruvate sulfurtransferase deviation (SD). Differences among multi-group treatments were determined by one-way ANOVA using the VassarStats website for statistical computation (http://​faculty.​vassar.​edu/​lowry/​VassarStats.​html). P values of ≤0.05 were considered significant, with significant differences further analyzed using a Tukey HSD post hoc test. Acknowledgements This study was funded by a National Institutes of Health grant DC010187 from the National Institute on Deafness and Other Communication Disorders (NIDCD) to D.A.D. NIDCD had no role in the design, collection, analysis, and interpretation of the data, nor any role in the writing of the manuscript or in the decision to submit the manuscript for publication. We are grateful to Wenzhou Hong, Medical College of Wisconsin, for sharing his expertise in the chinchilla model; to Shirley A.

However, the present interpretation system for CT has not kept up with

the modality’s technological development, FHPI and real-time interpretation by radiologists is not available in many institutions in Japan because of a nationwide shortage of radiologists. Many EPs, therefore, must make decisions regarding trauma treatment plans without radiological support. Hunter et al. reported that only wet reading was available in the majority of medical institutions surveyed and that emergency CT was usually supported only by radiology residents even in university hospitals [15]. Torreggiani et al. reported that real-time interpretation by radiologists was not available in many institutions and that, in some, radiologist interpretation took more than 48 hours to prepare [16]. They also reported that EPs and radiologists felt very differently about whether the interpretation system was adequate. Many EPs complained of a deficiency in the current interpretation system. Such problems are likely to continue into the long term unless effective

measures are taken. Our hope is that this study may provide an effective CT interpretation system for EPs to use in blunt trauma cases. In this study, EPs misinterpreted 40 of 1606 cases (2.5%) in the first period. Seven of the 365 total patients (1.9%) were most likely placed at a disadvantage by a major misinterpretation; these patients were categorized as gravity level 2 or 3, and they required additional treatments (such as emergency surgery). Chung et al. studied the accuracy of 4768 https://www.selleckchem.com/products/azd3965.html interpretation reports of torso CT performed by a radiology resident [9]. In this study, serious misdiagnosis occurred in 2.0% of the cases, and changes in treatment were required in 0.3%. Petinaux et al. reported major discrepancies between the interpretations

from EPs and radiologists in 3% of cases (for plain chest and abdominal X-rays) [17]. Most of the discrepancies were considered misdiagnoses, and changes for in treatment were required in 0.05% of the cases. Gray comprehensively surveyed the occurrence of diagnostic mistakes in the ED [18] and found that 79.7% of mistakes were associated with bone trauma and that most misdiagnoses could likely be avoided by careful interpretation. There were no large differences in the number and level of diagnostic mistakes between these studies and our study. However, even a small misinterpretation by the EP may lead to irrelevant treatment or a potentially fatal delay in appropriate treatment. This must be avoided wherever possible, but is difficult to achieve in actuality. One solution is to further train EPs to improve their interpretations of CT results. However, a high level of skill is required to interpret CT results, and we believe that it would be almost impossible to improve interpretation ability with unsystematic short-term training. Keijzers et al.

As a result, ρ xx ~ ρ xy can occur on both sides of B c as seen clearly in Figure 2d. Interestingly, in the crossover from SdH oscillations to the QH state, we observe additional T-independent points, labeled by circles in Figure 2 for each V g, other than the one corresponding to the onset of strong localization. As shown in Figure 2a APR-246 concentration for V g = −0.125 V, the resistivity peaks at

around B = 0.73 and 1.03 T appear to move with increasing T, a feature of the scaling behavior [7] of standard QH theory around the crossing points B = 0.70 and 0.96 T, respectively. Therefore, survival of the SdH theory for 0.46 T ≤ B ≤ 1.03 T reveals that semiclassical metallic transport may coexist with quantum localization. The superimposed background MR may be the reason for this coexistence, which is demonstrated by the upturned deviation from the parabolic dependence as shown in Figure 2a [45]. Therefore, it is reasonable to attribute the overestimated μ′ shown by the blue symbols in Figure 5a to the influence of the background MR. Similar behavior can also be found for V g Selleckchem CP673451 = −0.145 V even though spin splitting is unresolved, indicating that the contribution of background MR mostly comes from semiclassical effects. However, such a crossing point cannot be observed for V g = −0.165 V since there is no clear separation between extended and localized

states with strong disorder. Only a single T-independent point corresponding to the onset of strong localization occurs at B = 1.12 T. In order to check the validity of our present results, further experiments were performed on a device (H597) with nominally T-independent Hall slope at different applied gate voltages [27]. As shown in Figure 7a for V g = −0.05 V, weakly insulating

behavior occurs as B < 0.62 T ≡ B c, which corresponds to the direct I-QH transition since there is no evidence of the ν = 1 or ν = 2 QH state near B c. The crossing of ρ xx and ρ xy is found to occur at B ~ 0.5 T which is smaller than B c. As we decrease V g to −0.1 V, thereby increasing the effective amount of disorder in the 2DES, the relative positions between these two fields remain the same as shown in Figure 7b. Nevertheless, it can be observed that ρ xy tends to move closer to ρ xx with decreasing Parvulin V g. This may be quantified by defining the ratio ρ xy/ρ xx at B c, whose value is 1.57 and 1.31 for V g = −0.05 and −0.1 V, respectively. Figure 7 ρ xx and ρ xy as functions of B at various T ranging from 0. 3 to 2 K. For (a) V g = −0.05 V and (b) V g = −0.1 V. The interaction-induced parabolic NMR can be observed at both gate voltages. This result, together with the negligible T dependence of the Hall slope as shown in Figure 8a, implies that the ballistic part of the e-e interactions dominates as mentioned above.

The RAPD fingerprints obtained from colonies processed in this way were identical to those produced from conventionally extracted high molecular weight DNA (Fig. 4). However, it was found that consistent profiles were only obtained if the RAPD HSP activation PCR was set up immediately after the boiling and chilling cycles of the colony extraction procedure. The amplified PCR fingerprints deteriorated after subsequent frozen storage of the Chelex® resin extracted DNA. To overcome this potential problem, we examined if prolonged frozen storage (-20°C) of the resuspended colony in Chelex® resin prior to full extraction by boiling was possible. This procedure did

not affect the quality of the RAPD profiles (Fig. 4). The ability to fingerprint from frozen stored colony material

provided a high throughput strategy that could be used to systematically screen the multiple colony types isolated from human faeces as part of a Lactobacillus strain feeding study (see below). Figure 4 Reproducibility of single colony RAPD fingerprints. The polymorphismsamplified by primer 272 from conventionally extracted DNA compared to single colony Chelex® extracted DNA are shown for two LAB strains as follows: lane 1, L. rhamnosus strain MW standard DNA extraction; lanes 2 to 4, single colonies of strain MW that were picked into Chelex® resin, stored frozen and then extracted immediately prior to PCR; lane 5, L. acidophilus strain LMG 8151 standard DNA extraction; lanes 6 to Selonsertib ic50 8, single colonies of strain LMG 8151 that were processed with Chelex® as described. The size of relevant molecular size markers (lane M) are shown

in bp. Lactobacillus species feeding study design A small scale proof-of-principle human feeding study was performed to evaluate if the colony-fingerprint strategy could be used to track specific LAB strains from ingestion as capsule recovery from faeces. A capsule for oral administration was formulated to commercial Flavopiridol (Alvocidib) standards which contained two Lactobacillus species isolates: L. salivarius strain NCIMB 30211 (1.8 × 1010 colony forming units [cfu] per capsule) and L. acidophilus strain NCIMB 30156 (5.6 × 109 mean cfu per capsule). Twelve volunteers participated in a feeding study where the capsule was taken daily for 14 days; faecal samples were provided on days before, during and after consumption as described in the Methods. The volunteers were not advised to change their diets in any way other than to take the capsule once a day with some food on each of the trial days. At each faecal sampling point, LAB were plated as described below, enumerated and multiple colonies genotyped by RAPD.