[33-36]. Palinauskas et al. [33] infected 5 passerine species with the same generalist Plasmodium relictum (lineage

SGS1) and investigated the parasitaemia selleck screening library and the associated costs for the hosts. While starlings (Sturnus vulgaris) were fully resistant to the infection, the other four species showed a variable pattern of resistance/tolerance. House sparrows (Passer domesticus) were partially resistant because 50% of inoculated birds established a successful infection, whereas 100% of chaffinches (Fringilla coelebs), crossbills (Loxia curvirostra) and siskins (Carduelis spinus) were susceptible to the infection. Within the susceptible species, infection intensity showed huge variation with siskins and crossbills having the highest peak of parasitaemia. However, when looking at the reduction in haematocrit (the proportion of red blood cells, a good proxy of infection-induced fitness cost), only the two species

with experimental highest parasitaemia seemed to suffer from the infection. This study therefore strongly suggests that avian check details hosts exhibit interspecific variation in their propensity to be resistant/tolerant to Plasmodium parasites. The co-infection with two Plasmodium species (Plasmodium relictum and Plasmodium ashfordi) led to a very different outcome depending on the host species [34]. Whereas starlings were again fully resistant to the infection by the two parasites, siskins and crossbills were highly susceptible, with parasitaemia in double-infected birds being higher than in single infected hosts. The two susceptible species appear to differ in terms of tolerance to the infection.

Indeed, even though siskins and crossbills have similar peak parasitaemia, siskins paid a much smaller cost of infection (a smaller reduction in haematocrit values and no infection-induced mortality). This experimental work therefore shows that generalist malaria parasites infecting a large number of host species nevertheless achieve quite different infection dynamics and incur quite different costs for their hosts possibly due to a combination of resistance and tolerance processes. A pending question is what accounts for this interspecific pattern of resistance/tolerance even for closely related host selleck species. Variation in life history traits among species has been suggested to explain specific propensity to invest in costly inflammatory response [20]. However, the species used by Palinauskas et al. [33, 34] have similar paces of life. Immunologically naïve hosts, in particular those that have not coevolved with avian malaria, are predicted to suffer more from infection. The accidental introduction of avian malaria in the Hawaiian archipelago provides a textbook illustration of a rapid evolutionary change in a novel host–parasite association.

Activating KIR show much greater variation in their presence/absence in different populations. For example KIR2DS1 has four populations with greater than 80% frequency (Australia Aborigines, Brazil Amazon, Brazil Rodonia Province Karitiana and Papua New Guinea Nasioi) but three African populations with < 10%; Central Africa Republic Bagandu Biaka, Ghana and Nigeria Enugu Ibo. Similarly, KIR2DS2 has high frequencies (> 70%) in nine populations (e.g. Australia Aborigines, South Africa San and Xhosa and populations from India) but very low frequencies in Japan

(8·5–16·0%), South Korea (16·9%) and China (17·3%). In some of the South American Amerindian populations KIR2DS3 is absent – Argentina Salta Wichis, Mexico Tarahumaras, Venezuela Bari Selleck PLX4032 and Venezuela Yucpa.53,54 The frequency of this gene is also low BGJ398 datasheet in Japan and China. The KIR2DS4 gene is present in seven populations at 100% – either from Africa or African Americans in USA. However, it has also low frequencies – Costa Rica (31%), Australia Aborigines (52%), Taiwan (59·4%). Selection against having KIR3DS1 has been reported

in African populations25 with KIR3DS1 present in San (2·2%), Xhosa (4·0%), Nigeria (3·4% and 6·3%), Senegal (4·0%), Kenya (0·7%), Ghana (4·9%), Central Africa Republic Bagandu Biaka (2·9%). Global phenotype frequencies of KIR3DS1 are shown as an example of how the data can be represented (Fig. 6). Obviously there is a close inverted correspondence between the frequencies of KIR3DL1 and KIR3DS1 in an individual population. A very small percentage of individuals (0·34%) are negative for both KIR3DL1 and KIR3DS1. Such extensive diversity between modern populations may indicate that geographically distinct diseases have exerted recent, or perhaps ongoing, selection on KIR

repertoires. The differences in frequencies therefore make the choice of controls for disease studies very important for all populations. We linked the published data by analysing all populations submitted to the website that had data for 13 KIR genes (excluding KIR2DP1 and KIR3DP1).55 Glutamate dehydrogenase The 56 populations analysed, using neighbour-joining dendrograms and correspondence analysis, grouped with a few exceptions according to a geographical gradient. Subsequently, we selected 38 of the 56 populations that we considered to be well defined in the anthropological sense. We found that based on KIR haplotype B genes (i.e. genes mainly encoding activating KIR) the populations were related to geography like a good anthropological marker such as HLA or Y chromosome. However, the results based on the KIR haplotype A (i.e. genes mainly encoding inhibitory KIR) did not show such a correlation.56 There has been an increase in the number of known alleles from 87 in the first KIR nomenclature report in 2002 to 335 in the latest release on the IPD-KIR database, where the sequence of all KIR alleles is kept.

Different techniques

have been used: DNA probes targeting the 18S ribosomal subunit, DNA sequencing after PCR with pan-fungal primers, 18S-targeted seminested PCR as well as real-time PCR targeting cytochrome b gene.[28, 50, 51] Although the molecular methods can render diagnosis much easier; yet, there is no standardised method available.[52] PF-02341066 in vitro Serologic tests are not widely used. Kaufman et al. [53]; reported an immunodiffusion test for detection of both Basidiobolus and Conidiobolus. The assay was 100% sensitive and specific. Moreover, Khan et al. [11]; detected B. ranarum antibodies using immunodiffusion and ELISA tests. Treatment for entomophthoromycosis is usually both medical and surgical. Systemic prolonged antifungal therapy coupled with surgical debridement is the cornerstone treatment.[6] Potassium iodide,

co-trimoxazole, amphotericin-B, imidazoles, hyperbaric oxygen and combinations of these agents have all been used with varying success.[54, 55] However, treatment with potassium iodide and nitrogen heterocyclic ring azole compounds (particularly itraconazole) is considered the baseline.[22] The role of newer azoles (e.g. posaconazole) in the treatment of entomophthoromycosis is not yet known.[39] As the organisms exhibit relative resistance to antifungals, higher doses than usual are required for effective treatment, and prolonged daily therapy, for months, is usually recommended. Considering these factors, patients Ivacaftor clinical trial often do not comply due to the adverse effects and drug cost.[46] In case of GIB, resection of the affected bowel is required, followed RAS p21 protein activator 1 by prolonged antifungal therapy with itraconazole.[56] Facial reconstructive surgery may be necessary in case of conidiobolomycosis, as the extensive fibrosis persists after eradication of the fungus.[39] Cryotherapy has been tried with limited success.

Relapses are common, even after successful treatment.[40] Knowledge of uncommon fungi such as entomophthoromycotina is of great clinical value. Entomophthoromycosis includes basidiobolomycosis (subcutaneous zygomycosis) and condidiobolomycosis (rhinofacial zygomycosis). These fungal infections not only affect the immunocompromised, but immunocompetent hosts may be also involved. Although the disease is uncommon; yet, seen predominantly in tropical and subtropical regions, physicians should be aware of it, given the rise in global travel. Keeping a high index of suspicion for the predominant disease manifestations can aid in early diagnosis and implementation of adequate therapy. Despite the characteristic clinical feature, the disease requires biopsy for diagnosis. The histopathologic picture is the same for Basidiobolus and Conidiobolus and is marked by typical zygomycotic hyphae, often surrounded by eosinophilic Splendore–Hoeppli material.

In a subanalysis (data not shown), the differences in attack frequency did not appear to be accountable to differences in prescribing of attenuated androgens. When attack frequency at the main three sites was compared for types I and II HAE, no differences were observed. The variation in attack frequency ranged

from 30% of patients who had had no attacks during the year to others with daily attacks. Information on the employment FK228 datasheet status of 213 patients shows that 76% were in employment (full- or part-time), were homemakers or students. Seven percent were unemployed and the remainder retired. The percentage in paid employment (full- and part-time) was 48% (Fig. 7). Information Proteasome inhibitor on days lost from work/school or where activities of daily living could not be performed was available on 116 patients, with an annual mean of 9 days per patient [standard deviation (s.d.) 24]; however, this is almost certainly an underestimate, as it was not

possible to analyse non-numerical data (e.g. yes: +++, plenty, very few, etc.) in 11 patients. The impact of HAE on quality of life was assessed by asking patients to rate the overall impact of their disease on their quality of life as either none, mild, moderate or severe. Information was available on 223 patients and the impact was noted to be moderate or severe in 37% of adult patients (Fig. 8a). While this approach

is Amylase straightforward, detail and sensitivity is likely to be improved significantly using a validated disease-specific quality-of-life tool for HAE [22]. Swellings are generally rare before the age of 2 years and are relatively uncommon before adolescence, with a mean age of onset of swellings of 8–12 years [23]. The reported impact on quality of life in children available in 29 patients was rated as moderate in 14%, with no patients in the severe category. The annual frequency of swellings in children was peripheral, six; abdominal, six; and airway, 0·2 (Fig. 8b). Interpretation of attack frequency and impact upon quality of life in children is complicated by the fact that this information is reported by the parents. Furthermore, as there may be an increase in swellings during adolescence, consideration of childhood as less than 16 years of age may not capture important information. The questions on family history included one on deaths in the family related directly to an HAE attack. When multiple entries for the same family members (and two entries stating ‘uncertain’ or ‘possible’) were excluded there was a total of 55 deaths in 33 families, ranging from one to three deaths per family. This clearly underpins the lethal potential of this condition.

10,11 In contrast, both B/PBSM/FVIII and B/FVIIIM/FVIII mice had residual maternal anti-FVIII IgG. At 8 weeks of age, the different groups of mice were treated with 1 IU FVIII once a week for 4 weeks. Blood was collected 5 days after the fourth FVIII administration, and the anti-FVIII IgG titres were measured (Fig. 3b). As expected, B/FVIIIM/FVIII were protected against the development of FVIII inhibitors. The B/PBSM/FVIII demonstrated anti-FVIII IgG titres (51·4 ± 84·8 μg/ml) that were lower than those measured in the case of B/FVIIIM/PBS (133·1 ± 44·0 μg/ml) and B/PBSM/PBS,

without reaching statistical significance. This suggests that optimal protection is conferred when maternal IgG are transferred during both fetal PS 341 life and lactation. Furthermore, residual levels of anti-FVIII IgG in B/FVIIIM/FVIII mice at 11 weeks of age (third injection of FVIII) were identical to the theoretical values of clearance rates of IgG (Supporting information Fig. S2). Residual levels of anti-FVIII IgG were however higher than the theoretical value in the case of B/PBSM/FVIII mice. This suggests that B/PBSM/FVIII mice were in the process of developing

novel anti-FVIII IgG, whereas B/FVIIIM/FVIII mice were not. At 12 weeks of age (fourth injection), the experimental values of IgG levels were systematically higher than the theoretical ones. We then reconstituted naive FVIII-deficient mice with IgG pools from either FVIII-treated mice that contain anti-FVIII IgG (‘inhibitor+’, Fig. 3c, selleck compound 131·9 ± 24·1 μg/ml) or naive mice (‘inhibitor−’). Three days later, mice were treated with exogenous FVIII. Naive mice reconstituted with anti-FVIII IgG developed significantly lower titres of anti-FVIII IgG than control mice (Fig. 3d, P < 0·05). The protective effect of the presence of anti-FVIII IgG was confirmed by a χ2 analysis of the pooled data on pre-treatment anti-FVIII IgG titres and anti-FVIII IgG titres

measured after the fourth FVIII administration from Figs 2 and 3 (odds ratio = 7·2; 95% confidence interval, 1·64–31·54, P < 0·01). In this work, we have shown that maternally transferred anti-FVIII IgG can delay the development of the anti-FVIII immune response. 3-oxoacyl-(acyl-carrier-protein) reductase The offspring from FVIII-treated mothers, who received maternal anti-FVIII IgG in utero and during lactation, developed lower levels of inhibitory anti-FVIII IgG, and demonstrated reduced FVIII-specific proliferative T-cell responses. The reduced capacity of the immune system of the offspring to mount an anti-FVIII immune response was transient as the effect diminished if these offspring were nursed by naive mothers. However, the suppression of the anti-FVIII response could be reproduced upon reconstitution of naive mice with ‘purified’ anti-FVIII IgG, so replicating the classic studies of Bystryn et al.13 which demonstrated that passive antibody can inhibit the subsequent immune response.

pylori is no longer detected, even when seropositivity suggests prior infection [136]. These observations have led to the proposal of an alternative model for H. pylori carcinogenesis in which the deterioration of the gastric niche, driven by long-term H. pylori host interaction, causes

dysbiosis with the expansion of cancer-provoking oropharyngeal and intestinal Raf targets pathobionts [137]. Dysbiosis of the intestinal microbiota or its physical interaction with hematopoietic cells following barrier damage can both regulate inflammation (reviewed in [132, 133] and be a cause of cancer [138-140]. IL-18 has been shown to mediate mucosal protective mechanisms [141]. In particular, mice that are unable to produce, process, or respond to IL-18 (e.g., deficient in IL-18, IL-18R, MyD88, inflammasomes, or inflammasome signaling molecules) are characterized by intestinal dysbiosis and elevated susceptibility to chemically induced colon carcinogenesis

and nonalcoholic steatohepatatis [141-143]. The intestinal dysbiosis in these mice is characterized by the proportional expansion of the bacterial phyla Bacteroidetes (Prevotellaceae) and TM7, and colon carcinogenesis can be transferred to healthy mice by cohousing or fecal transfer [143]. SCFAs, such as butyrate, which are bacterial products derived from the fermentation of dietary fibers in the colon, have been shown to induce IL-18 production in intestinal epithelial cells by activating the GPR109a receptor, and also to act directly on DCs, macrophages, and T cells [44]. SCFAs have also been Opaganib purchase shown to induce the expansion of Treg cells, producing the anti-inflammatory cytokine IL-10, thus

suppressing colonic inflammation and carcinogenesis [44, 46]. IL-18 in turn favors mucosal tissue repair by regulating the production and availability of IL-22 [144]. IL-22 is produced by innate lymphoid cells in the intestinal lamina propria and, through activation of STAT3, induces epithelial cell proliferation and production of antibacterial peptides [145]. Thus, IL-22 favors epithelial repair and, depending on the extent of mucosal damage in the different experimental models, it may be pro- or anticarcinogenic [144-147]. Furthermore, in addition to having decreased IL-18 production by enterocytes, mice deficient for the NOD-like receptor-related Florfenicol protein 6 inflammasome have also been shown to have defective autophagy in goblet cells and abrogated mucus secretion into the large intestinal lumen [148]. These mice are therefore unable to clear enteric pathogens from the mucosal surface and are susceptible to persistent infection. Mice genetically deficient for other immunologically relevant genes, such as Tlr5, Il10, Tbx1, and Rag2, also show susceptibility to colitis and colon carcinogenesis due to gut dysbiosis, which can be transferred to healthy mice [149]. Many individual microbes have been associated with colorectal cancer either in human studies or in experimental animals.

Interaction with non-pathogenic E. coli HB101 did not induce localization of TLR5 on the cell surface (Figure S2). These results are consistent with the FACS experiments, where almost all TLR5 was located in intracellular compartments. In contrast, in cells infected with EPEC strains, E2348/69 and E22, TLR5 was clearly detected on the cell

surface (Figure S2). These results confirmed that EPEC infection induces TLR5 re-localization towards the cell surface. Infection with any of the E22 mutant was unable to provoke TLR5 detection on the epithelial cell surface (Figure S2). These results indicate that EPEC T3SS and flagellum participate in the re-localization of TLR5 towards the cellular surface. Notably, in these assays intimin appeared to be necessary Omipalisib supplier for the re-localization of TLR5, a more obvious result than the one obtained with FACS. To know if the localization of another receptor besides TLR5 is altered during EPEC infection, we inquired about TLR4 subcellular

distribution in non-infected cells and in cells infected with E2348/69 during 4 h by examination of immunofluorescent preparations (Figure S3). In mock cells, we found TLR4 equivalent signal intensity and distribution selleck inhibitor in permeabilized and in non-permeabilized cells (total and surface TLR4). This indicates that TLR4 is mainly located at the surface of HT-29 cells, which was also true for E2348/69 cells. Therefore, EPEC infection does not affect TLR4 distribution, unlike TLR5 recruitment to the cell surface that was induced by EPEC infection. ERK1/2 signalling pathway (phosphorylation and nuclear translocation) is an important activator of cellular proinflammatory responses. ERK1/2 phosphorylation during EPEC infection (at 2 or 4 h) was detected by WB. Phosphorylated ERK1/2 was not detected in mock-treated cells (normalized band intensity value of 0.026 ± 0.045). HB101 interaction

induced phosphorylation of ERK1/2 (0.673 ± 0.108) but only until 4 h post-interaction. However, in EPEC-infected cells, p-ERK1/2 was clearly detected (Fig. 2A). At 2 h post-infection, both EPEC strains caused equivalent phosphorylation of ERK1/2 (0.737 ± 0.246 for E2348/69 and 0.741 ± 0.064 for E22 infection). However, at 4 h, p-ERK1/2 was stronger during E22 infection (E2348/69: 0.643 ± 0.089 and E22: Y-27632 2HCl 1.01 ± 0.126). Therefore, we confirmed that ERK1/2 phosphorylation in epithelial cells is caused by EPEC E2348/69 infection and found that it was also true for E22. To understand the role of EPEC virulence factors on the phosphorylation of ERK1/2, we performed WB analysis of lysates from cells infected for 4 h with the isogenic EPEC mutants E22 Δeae, ΔescN, ΔespA or ΔfliC (Fig. 2B). Cells infected with T3SS mutants induced ERK1/2 phosphorylation at levels not significantly different than the ones produced by WT infection (1.01 ± 0.126); normalized band intensity values were 1.186 ± 0.207 for E22ΔescN and 1.025 ± 0.209 for E22ΔespA.

Thus, it should be cautioned that the KHQ might not completely represent this important area and it is recommended that it could be supplemented with a short measure

of sexual functioning, such as the Brief Sexual Function Inventory,19 to enhance clinical assessment of HR-QoL. Our results support the usefulness of the traditional Chinese version of the KHQ for assessment in men with LUTS and it is hoped that the KHQ and the IPSS may help health providers in primary care settings recognize the impact of LUTS and further improve HR-QoL. Nonetheless, some limitations must be noted. First, we did not analyze other medical problems (e.g. hypertension, diabetes, or others), or background variables for HR-QoL in this study. We assumed that the influence of the other factors was minimal to the disease-specific KHQ. Second, the responsiveness, Venetoclax which assesses whether a questionnaire can detect changes in a patient’s condition after treatment, and the test-retest reproducibility are also considered to be important indicators of validity. However, we could not perform such evaluations in this cross-sectional study. Third, the possibility of participants failing to correctly recall the information requested in this self-report survey might make a recall bias. Fourth, our findings are potentially

limited by the selection of participants on a convenience basis, leading to difficulties in generalization to other populations in Taiwan. Furthermore, the IPSS-QOL score is a single

Leukotriene-A4 hydrolase question to measure the quality of life for lower urinary tract dysfunction especially related to BPH. selleck chemicals llc However, in this study we did not use IPSS-QOL for analysis. In conclusion, LUTS produced a substantial impact on different domains of HR-QoL measured by the traditional Chinese KHQ. The traditional Chinese KHQ has suitable reliability and validity, which could be used as an assessment tool for HR-QoL in men with general LUTS for future studies. There are no financial or commercial interests for the authors of the present paper. “
“To evaluate the inter-observer, intra-observer and intra-individual reliability of uroflowmetry and post-void residual urine (PVR) tests in adult men. Healthy volunteers aged over 40 years were enrolled. Every participant underwent two sets of uroflowmetry and PVR tests with a 2-week interval between the tests. The uroflowmetry tests were interpreted by four urologists independently. Uroflowmetry curves were classified as bell-shaped, bell-shaped with tail, obstructive, restrictive, staccato, interrupted and tower-shaped and scored from 1 (highly abnormal) to 5 (absolutely normal). The agreements between the observers, interpretations and tests within individuals were analyzed using kappa statistics and intraclass correlation coefficients. Generalizability theory with decision analysis was used to determine how many observers, tests, and interpretations were needed to obtain an acceptable reliability (> 0.80).

The records at our stem-cell transplantation centre were reviewed to identify the patients who underwent autologous HSCT between April 2009 and December 2010. Patients

were classified as having proven invasive aspergillosis (IA), probable IA, or possible IA on the basis of the criteria established by the European Organization for Research and Treatment of Cancer and Mycoses Study Group (independent of the BDG results). During the study period, the patients were screened for BDG twice a week from transplant (day 0) until engraftment. Three patients were diagnosed with probable IA and five were diagnosed with possible IA. A total of 354 serum FK506 samples from79 patients who met the study inclusion criteria were used for statistical analysis. At the cut-off value of 80 pg ml−1, the sensitivity was 27.2% [95% confidence interval (CI); 7.3–60.6]; specificity, 94.4% (95% CI; 91.3–96.5); positive predictive value, 6.2%; and negative predictive, 93.7%. The clinical contribution Selleck BYL719 of the BDG assay as a screening test was relatively limited in this cohort of patients undergoing autologous HSCT. “
“Centre for Microbial Biotechnology,

Panjab University, Chandigarh, India Mucormycosis remains a devastating invasive fungal infection, with high mortality rates even after PDK4 active management. The disease is being reported at an alarming frequency over the past decades from India. Indian mucormycosis has certain unique features. Rhino-orbito-cerebral presentation associated with uncontrolled diabetes is the predominant characteristic. Isolated renal mucormycosis has emerged as a new clinical entity. Apophysomyces elegans and Rhizopus

homothallicus are emerging species in this region and uncommon agents such as Mucor irregularis and Thamnostylum lucknowense are also being reported. This review focuses on these distinct features of mucormycosis observed in India. Fungi belonging to the class Zygomycetes and order Mucorales often cause devastating angioinvasive fungal infections, primarily in patients with underlying risk factors.[1] These moulds gain entry into the human body via respiratory tract or skin, and less commonly through the gastrointestinal tract, eliciting an acute inflammatory response.[2] Under favourable conditions such as those in immunocompromised hosts, they invade the blood vessels, causing extensive vessel thrombosis and ischaemic tissue necrosis.[2, 3] Most of these infections are rapidly progressive and exhibit high mortality (~50%) even after active management; the mortality rates approach nearly 100% among patients with disseminated disease.

To accurately determine gene expression during other developmental phases, we suggest a similar approach as described in the present study. We thank Drs Hans Wolf-Watz and Betty Guo for critical reading of the manuscript. J.J. received fundings from the Wenner-Gren Foundation, Umeå

University, the Swedish Research Council (grant no. 621-2006-4450), and the European Union (BacRNA 2005 contract no. 018618); S.B. received funds from the Swedish Research Council (grant no. 07922). P.E. and L.B. contributed equally to this work. “
“Dengue disease is a mosquito-borne infection caused by Dengue virus. Infection may be asymptomatic or variably manifest as mild Dengue fever (DF) to the most Cetuximab in vivo severe form, Dengue haemorrhagic fever (DHF). Mechanisms that

influence disease severity are not understood. Complement, an integral RG7422 component of the immune system, is activated during Dengue infection and the degree of activation increases with disease severity. Activation of the complement alternative pathway is influenced by polymorphisms within activation (factor B rs12614/rs641153, C3 rs2230199) and regulatory [complement factor H (CFH) rs800292] proteins, collectively termed a complotype. Here, we tested the hypothesis that the complotype influences disease severity during secondary Dengue infection. In addition to the complotype, we also assessed two other disease-associated CFH polymorphisms (rs1061170, rs3753394) and a structural polymorphism within the CFH protein family. We did not detect any significant association between the examined polymorphisms and Dengue infection

severity in the Thai population. However, the minor allele frequencies of the factor B and C3 polymorphisms were less than 10%, so our study was not sufficiently Methocarbamol powered to detect an association at these loci. We were also unable to detect a direct interaction between CFH and Dengue NS1 using both recombinant NS1 and DV2-infected culture supernatants. We conclude that the complotype does not influence secondary Dengue infection severity in the Thai population. “
“Whitehead Institute, Cambridge, MA, USA Maurus Curti, Viollier AG, Basel, Switzerland Autoimmune diseases develop when self-specific T cells that escaped negative selection initiate a harmful immune response against self. However, factors, which influence the initiation and progression of an autoimmune response remain incompletely understood. By establishing a double-transgenic BALB/c mouse system in which different amounts of a cell-surface neo-self-antigen are expressed under the CD11c promoter, we demonstrate that antigen dose dramatically influences T-cell tolerance mechanisms. Moderate antigen expression in both hematopoietic and nonhematopoietic cells favors the development of antigen-specific Treg cells and the establishment of a tolerogenic environment.