Percentage differences between measurements were determined through quantitative result assessment. Both methods detected a statistically significant cerebral blood flow increase during DNA-PK inhibitor neuroactivation. Correlation of TCD and SPECT showed statistically significant correlation only for the increase of cerebral blood flow velocity in the right MCA and for the right-sided cerebral blood flow increase, demonstrating

that both methods partially measure similar cerebral blood flow changes that occur during neuroactivation. Comparison of TCD and SPECT showed TCD to be inadequately sensitive method for evaluation of cerebral blood flow during complex activation paradigm.”
“Partial trisomy 4q is a rare chromosomal disease. It involves duplication of a portion (particularly the distal one) of the long arm of chromosome 4. In most cases results from a balanced translocation on one single progenitor. The “de novo” appearance is less common. Depending on the size and location of duplicated genetic

material, patients may have different clinical manifestations. Associated eye pathology has been scarcely informed. We report on a novel case of a male infant with a proximal “de novo” 4q12-q22 duplication and bilateral iris, retinal and optic nerve coloboma.”
“It is argued that a multidimensional approach is necessary for burden assessment. Reducing caregiver burden is a social problem in the ageing Japan society. We examined the combined effect of factors affecting the care burden among community-dwelling handicapped people and their caregivers.\n\nThe DZNeP in vivo participants were 49 handicapped people (aged 53-104 years) who received home-visit rehabilitation, and their 49 caregivers (age

42-85 years). Caregivers were provided questionnaires consisting of questions on social support, subjective well-being, self-efficacy with regard to care continuation, the Motor Fitness CT99021 clinical trial Scale and caregiver burden. Care recipients were assessed using the Bedside Mobility Scale and the Barthel Index.\n\nWe prepared the hypothesis model using structural equation modeling with the bootstrap method within outcome measures. The hypothesis model did not fit the data well. The impact of the Motor Fitness Scale was shifted from the caregiver burden to care self-efficacy and well-being, having a cooperator for care and variable of spouse caregiver or others associated with caregiver well-being in the revised model. The fit of the revised model was acceptable (goodness of fit index, 0.903; comparative fit index, 0.998; root mean square error of approximation, 0.017). In the revised model, the care recipients’ disabled state was associated with caregiver burden. In addition, higher burden and poor motor fitness of caregivers might lead to lower care self-efficacy in providing continuous care and lower caregiver well-being.

A variety of nanoparticles have been shown to induce autophagy, a critical cellular degradation

process, and the elevated autophagy in most of these Selleckchem Barasertib situations promotes cell death. Whether Ag NPs can induce autophagy and how it might affect the anticancer activity of Ag NPs has not been reported. Here we show that Ag NPs induced autophagy in cancer cells by activating the PtdIns3K signaling pathway. The autophagy induced by Ag NPs was characterized by enhanced autophagosome formation, normal cargo degradation, and no disruption of lysosomal function. Consistent with these properties, the autophagy induced by Ag NPs promoted cell survival, as inhibition of autophagy by either chemical inhibitors or ATG5 siRNA enhanced Ag NPs-elicited cancer cell killing. We further demonstrated that wortmannin, a widely used inhibitor of autophagy, significantly enhanced the antitumor effect of Ag NPs in the B16 mouse melanoma cell model. Our results

revealed a novel biological activity of Ag NPs in inducing cytoprotective autophagy, and inhibition of autophagy may be a useful strategy for improving the efficacy of Ag NPs in anticancer therapy.”
“First-generation, E1/E3-deleted adenoviral vectors with diverse transgenes are produced routinely in laboratories worldwide for development ABT-263 clinical trial of novel prophylactics and therapies for a variety of applications, including candidate vaccines against important infectious diseases, such as HIV/AIDS, tuberculosis, and malaria. Here, we show, for two different transgenes (both encoding malarial antigens) inserted at the E1 locus, that rare viruses containing a transgene-inactivating mutation exhibit a selective growth advantage during propagation in E1-complementing HEK293 cells, such that they rapidly become the major or sole species in the viral population. For one of these transgenes, we demonstrate that viral yield and cytopathic effect are enhanced by repression of transgene expression in the producer cell line, using the tetracycline repressor system. In addition to these transgene-inactivating mutations, one of which occurred during propagation of the pre-viral

genomic clone in JAK inhibitor bacteria, and the other after viral reconstitution in HEK293 cells, we describe two other types of mutation, a small deletion and a gross rearranging duplication, in one of the transgenes studied. These were of uncertain origin, and the effects on transgene expression and viral growth were not fully characterized. We demonstrate that, together with minor protocol modifications, repression of transgene expression in HEK293 cells during viral propagation enables production of a genetically stable chimpanzee adenovirus vector expressing a malarial antigen which had previously been impossible to derive. These results have important implications for basic and pre-clinical studies using adenoviral vectors and for derivation of adenoviral vector products destined for large-scale amplification during biomanufacture. Biotechnol. Bioeng.

Participants reported that 2-3.7% of patches

fell off and rated skin irritation as absent or mild for 92 95% of patches, according to site. Conclusion: Investigator- and participant-rated assessments of LNG/EE patch adhesiveness and irritation demonstrated a low incidence of patch detachment, skin irritation and pruritus. Implications statement: This secondary analysis of a phase 3 clinical trial of a new weekly low-dose LNG and EE contraceptive patch, which used assessment by both investigators and participants, observed a low incidence of skin irritation, pruritus and patch detachment. (C) 2015 Elsevier Inc. All rights reserved.”
“Sepsis is a leading cause of death. Rapid and accurate identification of pathogens and antimicrobial resistance directly from blood ON-01910 culture could improve patient outcomes. The FilmArray (R) (FA; Idaho Technology, Salt Lake City. Selleck Adriamycin UT, USA) Blood Culture (BC) panel can identify >25 pathogens and 4 antibiotic resistance genes from positive blood cultures in 1 h. We compared a development version of the panel to conventional culture and susceptibility testing on 102 archived blood cultures from adults and children with bacteremia. Of 109 pathogens identified by culture, 95% were identified by FA. Among 111 prospectively collected blood cultures, the FA identified 84 (91%) of 92 pathogens covered by the panel. Among 25

Staphylococcus aureus and 21 Enterococcus species detected, FA identified all culture-proven methicillin-resistant S. aureus and vancomycin-resistant enterococci. The FA BC panel is an accurate method for the rapid identification of pathogens and resistance genes from blood culture. (C) 2012 Elsevier Inc. All rights reserved.”
“The transcriptional co-activator PGC-1 alpha and the NAD(+)-dependent deacetylase SIRT1 are considered important inducers of mitochondrial biogenesis because in the nucleus they regulate transcription DMXAA order of nucleus-encoded mitochondrial genes. We demonstrate that PGC-1 alpha and SIRT1 are

also present inside mitochondria and are in close proximity to mtDNA. They interact with mitochondrial transcription factor A (TFAM) as assessed by confocal microscopy analysis and by blue native-PAGE. Nucleoid purification allowed us to identify SIRT1 and PGC-1 alpha as proteins associated with native and cross-linked nucleoids, respectively. After mtDNA immunoprecipitation analysis, carried out on mitochondrial extracts, we found that PGC-1 alpha is present on the same D-loop region recognized by TFAM. Finally, by oligonucleotide pulldown assay, we found PGC-1 alpha and SIRT1 associated with the TFAM consensus sequence (human mitochondrial transcription factor-binding site H). The results obtained suggest that in mitochondria PGC-1 alpha and SIRT1 may function as their nuclear counterparts and represent the genuine factors mediating the cross-talk between nuclear and mitochondrial genome.

The proposed new design of saturation pulse train can saturate effectively, and with this method first-pass myocardial perfusion imaging is feasible in humans at 7T. Magn

Reson Med 73:1450-1456, 2015. (c) 2014 The Authors. Magnetic Resonance in Medicine Published by Wiley Periodicals, Inc. on behalf of International Society of Medicine in Resonance.”
“OBJECTIVE. The objective of our study was to estimate the mortality benefit-to-risk ratio of pulmonary CT angiography (CTA) by setting (ambulatory [emergency department or outpatient] or inpatient), age, and sex.\n\nMATERIALS AND METHODS. A retrospective evaluation of 1424 consecutive pulmonary Selleck GDC-973 CTA examinations was performed and the following information was recorded: examination setting, patient age, patient sex, pulmonary CTA interpretation for pulmonary embolus (PE), and CT radiation exposure (dose-length product). We estimated mortality benefit of pulmonary CTA by multiplying the rate of positive pulmonary CTA examinations by published estimates of mortality of untreated PE in ambulatory and inpatient settings. We estimated the lifetime attributable Milciclib price risk of cancer mortality due to radiation from pulmonary CTA by calculating the estimated effective dose and using sex-specific polynomial equations derived from the Biological Effects of Ionizing Radiation VII report. We calculated benefit-to-risk ratios by dividing

the mortality benefit of preventing a fatal PE by the mortality risk of a radiation-induced cancer.\n\nRESULTS. Pulmonary CTA diagnosed PE in 188 of 1424 patients (13.2%). Both inpatients (101/723, 14.0%) and emergency department patients (74/509, 14.5%) had significantly higher rates of PE than outpatients

(13/192 [6.8%]). Males received significantly (p = 0.02451) higher radiation dose (9.7 mSv) than females (8.4 mSv), but males had a significantly (p < 0.0001) lower lifetime attributable risk of cancer mortality than females. Assuming an untreated PE mortality rate Ulixertinib datasheet of 5% for ambulatory patients and 30% for inpatients, the benefit-to-risk ratio ranged from 25 for ambulatory patients to 187 for inpatients. Ambulatory women had the lowest benefit-to-risk ratio.\n\nCONCLUSION. The benefit-to-risk ratio of pulmonary CTA in patients with suspected PE ranges from 25 to 187 and can be increased by optimizing the radiation dose.”
“A type of pH-responsive nano multi-drug delivery systems (nano-MDDSs) with uniform particle size (100 +/- 13 nm) and excellent monodispersity was developed by in situ co-self-assembly among water-insoluble anti-cancer drug (doxorubicin, DOX), surfactant micelles (CTAB) as chemosensitiver and silicon species forming drugs/surfactant micelles-co-loaded mesoporous silica nanoparticles (drugs@micelles@MSNs or DOX@CTAB@MSNs) via a micelles-MSNs self-assembly mechanism.

This study compared the diagnostic usefulness of p16 FISH and GLUT-1 immunohistochemical analysis in the diagnosis of mesothelial S3I-201 proliferations in 158 cases with a diagnosis of benign (45.4%), atypical (10.4%), or malignant/mesothelioma (44.2%). Of the 70 benign cases, none had a deletion of p16 and 5 cases (7%) were positive for GLUT-1. Of the 68 mesotheliomas, 40 (59%) had a deletion of p16 (sensitivity, 59%; specificity, 100%) and 27 (40%) were positive for GLUT-1 (sensitivity, 40%; specificity, 93%). GLUT-1 showed lower sensitivity in pleural (56% vs 70%) and peritoneal (29% vs 51%) mesotheliomas (P = .004).

Our results demonstrate that p16 FISH is a more sensitive and specific

test VS-6063 Angiogenesis inhibitor than GLUT-1 immunohistochemical analysis and can be a more reliable ancillary tool to support the diagnosis of mesothelioma.”
“Hepatocellular carcinoma (HCC) is one of the leading causes of cancer death worldwide, with similar to 70% of cases resulting from hepatitis B and C viral infections, aflatoxin exposure, chronic alcohol use or genetic liver diseases. The remaining similar to 30% of cases are associated with obesity, type 2 diabetes and related metabolic diseases, although a direct link between these pathologies and HCCs has not been established. We tested the long-term effects of high-fat and low-fat diets on males of two inbred strains of mice and discovered that C57BL/6J but not A/J males were susceptible to non-alcoholic steatohepatitis (NASH) and HCC on a high-fat Tariquidar clinical trial but not low-fat diet. This strain-diet interaction represents an important model for genetically controlled, diet-induced HCC. Susceptible mice showed morphological characteristics of NASH (steatosis, hepatitis, fibrosis and cirrhosis), dysplasia and HCC. mRNA profiles of HCCs versus tumor-free liver showed involvement of two signaling networks, one centered on Myc and the other on NF kappa B, similar to signaling described for the two major classes of HCC in humans. miRNA profiles revealed dramatically increased

expression of a cluster of miRNAs on the X chromosome without amplification of the chromosomal segment. A switch from high-fat to low-fat diet reversed these outcomes, with switched C57BL/6J males being lean rather than obese and without evidence for NASH or HCCs at the end of the study. A similar diet modification may have important implications for prevention of HCCs in humans.”
“Objective: Mutations in COL6A1, COL6A2, and COL6A3, the genes that encode the extracellular matrix component collagen VI, lead to Bethlem myopathy (BM) and Ullrich congenital muscular dystrophy (UCMD). Unlike UCMD, BM is difficult to diagnose because of its clinical overlap with other contractural phenotypes and the lack of sensitivity of standard muscle biopsy immunohistochemical diagnostic techniques.

“
“B-cell translocation gene 1 and 2 (BTG1 and BTG2) are members of the BTG/Tob antiproliferative protein family, which

is able to regulate the cell cycle and cell proliferation. We previously reported that BTG1, BTG2, Tob, and Tob2 are degraded via the ubiquitin-proteasome pathway. In this SNX-5422 Cytoskeletal Signaling inhibitor study, we investigated the mechanism of polyubiquitination of BTG1 and BTG2. Since the Skp1-Cdc53/Cullin 1-F-box protein (SCF) complex functions as one of the major ubiquitin ligases for cell cycle regulation, we first examined interactions between BTG proteins and components of the SCF complex, and found that BTG1 and BTG2 were capable of interacting with the SCF complex containing Cullin-1 (a scaffold protein) and Skp1 (a linker protein). As the SCF complex can ubiquitinate various target proteins by substituting different F-box proteins as subunits that recognize different

target proteins, we next examined which F-box proteins could bind the two BTG proteins, and found that Skp2, beta-transducin repeat-containing protein 1 (beta TrCP1), and beta TrCP2 were able to associate PLX4032 clinical trial with both BTG1 and BTG2. Furthermore, we obtained evidence showing that beta TrCP1 enhanced the polyubiquitination of both BTG1 and BTG2 more efficiently than Skp2 did, and that an F-box truncated mutant of beta TrCP1 had a dominant negative effect on this polyubiquitination.

Thus, we propose that BTG1 and BTG2 are subjected to polyubiquitination, more efficiently when it is mediated by SCF beta TrCP than by SCFSkp2.”
“Coenzyme Q10 is an essential cofactor in the mitochondrial electron transport pathway and is endowed for its LY2835219 clinical trial potent antioxidant capacity characters that endorse its implication in several clinical practices and as a food supplement Nevertheless its potential gastro-protective effect, in acute models has never been assessed which is the objective of this study Since indomethacin mediated gastropathy is multifaceted including mitochondrial dysfunction and generation of reactive oxygen species thus the indomethacin induced gastric injury serves as a convenient animal model for this work.

Results showed that optimum growth of the selected Bacillus spp. occurred at 30 degrees C, pH 7.5, and 1.5% NaCl, and they secreted protease, amylase, and lipase. Vibrio spp. were also inhibited by 3 Bacillus spp. In addition, the selected Bacillus spp. had no pathogenic effect on shrimp postlarvae (PL) and were able to reduce TAN. They promoted better SRT2104 in vitro growth and survival in shrimp PL without water exchange. This study was a systematic approach undertaken for the selection of suitable Bacillus

spp. as bioremediators for a Penaeus monodon culture system.”
“The current study investigated the immediate neurophysiological effects of different types of massage in healthy adults using functional magnetic resonance imaging (fMRI). Much attention has been given to the default mode network, a set of brain regions showing greater activity in the resting state. These regions (i.e. insula, posterior and anterior cingulate, inferior parietal and medial prefrontal cortices) have been postulated to be involved in the neural correlates of consciousness, specifically in arousal and

awareness. We posit that massage would modulate these same regions given the benefits and pleasant affective properties of touch. To this end, healthy participants were randomly assigned to one of four conditions: 1. Swedish massage, 2. reflexology, 3. massage with an object or 4. a resting control condition. The right

foot was massaged while each participant performed a cognitive association PXD101 mouse task in the scanner. We found that the Swedish massage treatment SBE-β-CD concentration activated the subgenual anterior and retrosplenial/posterior cingulate cortices. This increased blood oxygen level dependent (BOLD) signal was maintained only in the former brain region during performance of the cognitive task. Interestingly, the reflexology massage condition selectively affected the retrosplenial/posterior cingulate in the resting state, whereas massage with the object augmented the BOLD response in this region during the cognitive task performance. These findings should have implications for better understanding how alternative treatments might affect resting state neural activity and could ultimately be important for devising new targets in the management of mood disorders.”
“The recently described novel gyroviruses may infect chickens and/or humans; however, their pathogenic potential is unknown. In our metagenomic investigation, we detected many of the novel gyroviruses in the fecal viromes of ferrets with lymph node and organ enlargement. The complete genomic sequences of selected gyrovirus strains showed 90.7-99.4 % similarity to homologous reference gyrovirus strains.

\n\nMethods: Digital images of 204 patients were analyzed using Photoshop CS3 Extended software (Adobe Systems, San Jose, California). The left eye was superimposed over the right eye using layers in the software program, and the lower eyelid margin and axis were outlined to obtain a diagram with the superimposed

outlines of both eyes. Several measurements (ie, lateral canthal height, lower eyelid margin length selleck kinase inhibitor and axis angle, and lower palpebral fissure surface area) were obtained for each patient. Differences between the right and left eyelids were recorded, and these data were compared between patients.\n\nResults: Of the 204 patients studied, 184 (90.2%) had some degree of asymmetry. Most (118; 64.13%) presented with the right lower eyelid at a more inferior position than the left (R-), while 66 (35.87%) presented with the right lower eyelid at a higher position than the left (R+), a difference that was highly significant (P < .001).\n\nConclusions: This software is a powerful and precise tool to evaluate and measure eyelid asymmetries. The use of this method showed a large degree of eyelid asymmetry preoperatively (more than 90%), which proves

the importance of detecting and adequately analyzing this condition prior to surgery. The authors’ simple method may be an important adjunct to obtaining optimal results in patients who seek eyelid surgery.”
“Objective To develop an in-utero PP2 cell line stent placement training model.\n\nMethods The in-utero stent task trainer was constructed using a formalin-preserved gravid pig uterus. Altering the size of the uterine segment, changing the fluid level in the uterus and addition of a large Ziploc freezer bag variably filled with differing amounts of ultrasound gel can vary the procedural skill required.\n\nResults Thoracoamniotic and PD-1/PD-L1 assay vesicoamniotic shunts can be

simulated using this life-like model. The cost of eight to 10 learning stations is approximately US $60. Fetal position, maternal size and amniotic fluid status can be altered rapidly to increase the complexity of the procedure.\n\nConclusions This low-cost and realistic task trainer can provide the opportunity to practice in-utero shunt procedures in a non-clinical environment. This model should enhance learning and reinforce acquired skills. Copyright (C) 2009 ISUOG. Published by John Wiley & Sons, Ltd.”
“MHC class II molecules influence antigen-specific CD4(+) T lymphocyte responses primed by immunization and infection. CD4(+) T cell responses are important for controlling infection by many bacterial pathogens including Anaplasma marginale and are observed in cattle immunized with the protective A. marginale outer membrane (OM) vaccine. Immunogenic proteins that comprise the protective OM vaccine include type IV secretion system (T4SS) proteins VirB9-1, VirB9-2 and VirB10, candidates for inclusion in a multiepitope vaccine.

To understand these fluxes, we quantified the annual N2O emissions from three tropical mountain rainforests (primary mountain rainforest, PMR; secondary mountain rainforest, SMR; and Podocaipus imbricatus plantation,

PIP) in the Jian-fengling National Natural Reserve on Hainan Island, China. The average of N2O emissions in this area was 2.52 +/- 033 kg N-N2O ha(-1) yr(-1) (3.52 kg N-N2O ha(-1) yr(-1) in the wet season and 1.62 kg N-N2O ha(-1) yr(-1) in the dry season) during our study period, with highly seasonal variations. The mean N2O emission rates were significantly higher during the wet season (68% of the total average) than the dry season (32% of the total average) (P smaller than 0.05). PIP had the highest N2O emission rate at 3.49 +/- 0.61 kg N-N2O ha(-1) yr(-1) (4.74 kg N-N2O ha(-1) yr(-1) in the wet season and 232 kg N-N2O ha(-1) yr(-1) in the dry season), followed by SMR selleck chemicals llc at 3.03 +/- 0.64 kg N-N2O ha(-1) yr(-1) (4.16 kg N-N2O ha(-1) yr(-1) in the PFTα wet season and 1.97 kg N-N2O ha(-1) yr(-1) in the dry season), and then PMR at 1.53 +/- 0.49 kg N-N2O ha(-1) yr(-1) (2.21 kg N-N2O ha(-1) yr(-1) in the wet season and 0.94 kg N-N2O ha(-1) yr(-1)

in the dry season). We observed a significant Gaussian relationship between the N2O fluxes and soil temperature for SMR and PIP but no significant relationship in PMR. There was a significant exponential relationship between the N2O fluxes and water filled pore space (WFPS) in SMR and PIP but not in PMR. (C) 2014 Elsevier Ltd. All rights reserved.”
“Understanding how genetic diversity is structured on oceanic island taxa requires the integration of physical, biological and anthropomorphic factors. Founder effects coupled with limited dispersal over sea barriers typically result in low levels of genetic variation in island populations. In widespread species, restriction in gene flow across large areas leads to patterns of isolation by distance (IBD),

selleck but recent population-based studies indicate that genetic structure on islands can be complex even at local scales. Here, we investigated the patterns of genetic variation in a widespread island palm (Phoenix canariensis) displaying reproductive syndromes associated with extensive dispersal (wind pollination and zoochory). Genetic variation was assessed at eight nuclear microsatellite loci in 330 individuals of 15 Canarian populations. Our results showed that levels of within-population genetic diversity in P. canariensis depend on the island considered, with a strong decreasing pattern from the easternmost and oldest island to the westernmost and youngest islands. A Mantel test supported a stepping-stone model of differentiation across the archipelago that fits the sequence of island emergence, and results from ABC and clustering analyses also corresponded with this progression rule.

Of the GSK1904529A cost eight foliage

plants, only Cissus rhombifolia displayed distinct foliar injuries within a few days after initial exposure. The severeness of the symptoms such as leaf necrosis and distortion of mesophyll cells was positively correlated with ozone treatment period. No significant differences were observed in the chlorophyll content and chlorophyll fluorescence (Fv/Fm) between control plants and ozone treated plants. Ozone treatment resulted in significant decreases in photosynthetic rate in Cissus Dieffenbachia, Pachira aquatica, and Scindapsus aureus. There were significant differences in carbon fixation among the indoor plants used in this study, Dieffenbachia, and Pachira aquatica had ozone tolerant carbon fixation systems that did not exhibit changes in photosynthetic rate with increasing CO, concentration. Cissus rhombifolia was considered the most sensitive species to ozone among the eight foliage plants due to severe visual injury Dieffenbachia, Pachira aquatica, and Scindapsus aureus were

classified as ozone sensitive species due to their inhibition of photosynthesis by ozone. The remaining species (Spathiphyllum wallisii, Ficus benjamina and Hedera helix) were more tolerant to ozone and thereby potentially better suited for indoor air phytoremediation.”
“Deregulation of HGF/c-Met signaling and its driven neoplastic phenotype are associated with a variety of human BMS-777607 in vitro malignancies. We herein reported SOMCL-863 as a novel selective c-Met inhibitor which effectively abrogated c-Met signaling pathways, thereby leading to substantial impairment of c-Met-dependent cell proliferation, migration, invasion, selleck chemical cell scattering and invasive growth. In EBC-1 and NCI-H1993 xenografts, SOMCL-863 exerted significant anti-tumor efficacy through anti-proliferative effects and antiangiogenic mechanisms, including reduction of tumor cell proliferation and reductions

of microvessel density and secretion of proangiogenic factor IL-8. Together with the optimal pharmacokinetic properties, SOMCL-863 is a promising candidate worthy for further evaluation as a treatment of c-Met-driven human cancers. (C) 2014 Elsevier Ireland Ltd. All rights reserved.”
“Purpose: To investigate whether patients are prescribed rehabilitation early in a new sick leave period, and whether this prescription is associated with sex, age, diagnosis, description of functioning and affiliation of certifying physician. Method: A cross-sectional study using data from sickness certificates issued during a total sick leave period, collected consecutively during 2 weeks in 2007 in Ostergotland County, Sweden. Rehabilitation prescribed in the first certificate or within 28 days after the start of sick leave was defined as early rehabilitation. Results: Musculoskeletal diseases (MSD) were the largest diagnostic group, followed by mental disorders (MD). The mean duration of sick leave was 94 days (SD 139), longest for MD patients.