Multi-tissue proteogenomic analysis for mechanistic toxicology studies in non-model species.

New approach methodologies (NAM), including omics and in vitro approaches, are contributing to the implementation of 3R (reduction, refinement and replacement) strategies in regulatory science and risk assessment. In this study, we present an integrative transcriptomics and proteomics analysis workflow for the validation and revision of complex fish genomes and demonstrate how proteogenomics expression matrices can be used to support multi-level omics data integration in non-model species in vivo and in vitro. Using Atlantic salmon as an example, we constructed proteogenomic databases from publicly available transcriptomic data and in-house generated RNA-Seq and LC-MS/MS data. Our analysis identified ∼80,000 peptides, providing direct evidence of translation for over 40,000 RefSeq structures. The data also highlighted 183 co-located peptide groups that supported a single transcript each, and in each case, either corrected a previous annotation, supported Ensembl annotations not present in RefSeq, or identified novel previously unannotated genes. Proteogenomics data-derived expression matrices revealed distinct profiles for the different tissue types analyzed. Focusing on proteins involved in defense against xenobiotics, we detected distinct expression patterns across different salmon tissues and observed homology in the expression of chemical defense proteins between in vivo and in vitro liver systems. Our study demonstrates the potential of proteogenomic analyses in extending our understanding of complex fish genomes and provides an advanced bioinformatic toolkit to support the further development of NAMs and their application in regulatory science and (eco)toxicological studies of non-model species.

Dietary beauvericin and enniatin B exposure cause different adverse health effects in farmed Atlantic salmon.

The extensive use of plant ingredients in novel aquafeeds have introduced mycotoxins to the farming of seafood. The emerging enniatin B (ENNB) and beauvericin (BEA) mycotoxins have been found in the novel aquafeeds and farmed fish. Little is known about the potential toxicity of ENNs and BEA in farmed fish and their feed-to-organ transfer. Atlantic salmon (Salmo salar) pre-smolt (75.3 ± 8.10 g) were fed four graded levels of spiked chemical pure ENNB or BEA feeds for three months, in triplicate tanks. Organismal adverse health end-point assessment included intestinal function (protein digestibility), disturbed hematology (red blood cell formation), bone formation (spinal deformity), overall energy use (feed utilization), and lipid oxidative status (vitamin E). Both dietary BEA and ENNB had a low (<∼0.01%) transfer to organs (kidney > liver > brain > muscle), with a higher transfer for ENNB compared to BEA. BEA caused a growth reduction combined with a decreased protein digestion and feed conversion rate- ENNB caused a stunted growth, unrelated to feed utilization capacity. In addition, ENNB caused anemia while BEA gave an oxidative stress response. Lower bench-mark dose regression assessment showed that high background levels of ENNB in commercial salmon feed could pose a risk for animal health, but not in the case of BEA.

Prenatal fever and autism risk.

Some studies suggest that prenatal infection increases risk of autism spectrum disorders (ASDs). This study was undertaken in a prospective cohort in Norway to examine whether we could find evidence to support an association of the prenatal occurrence of fever, a common manifestation of infection, with ASD risk. Prospective questionnaires provided maternal exposure data; case status was established from clinical assessments and registry linkages. In a large, prospectively ascertained cohort of pregnant mothers and their offspring, we examined infants born ⩾32 weeks for associations between fever exposure in each trimester and ASD risk using logistic regression. Maternal exposure to second-trimester fever was associated with increased ASD risk, adjusting for presence of fever in other trimesters and confounders (adjusted odds ratio (aOR), 1.40; 95% confidence interval, 1.09-1.79), with a similar, but nonsignificant, point estimate in the first trimester. Risk increased markedly with exposure to three or more fever episodes after 12 weeks' gestation (aOR, 3.12; 1.28-7.63). ASD risk appears to increase with maternal fever, particularly in the second trimester. Risk magnified dose dependently with exposure to multiple fevers after 12 weeks' gestation. Our findings support a role for gestational maternal infection and innate immune responses to infection in the pathogenesis of at least some cases of ASD.

Acute and Chronic Erysipelothrix rhusiopathiae Infection in Lambs.

Polyarthritis caused by Erysipelothrix rhusiopathiae is a relatively common infection in lambs characterized by low mortality and high morbidity. E. rhusiopathiae is a ubiquitous Gram-positive bacterium that is both a commensal and a pathogen of vertebrates. The disease was studied during an outbreak in a Norwegian Spæl sheep flock. In the acute phase, 48 of 230 (20%) lambs developed clinical signs and 4 died (1.7%). One acute case was necropsied and E. rhusiopathiae was cultured from all major organs investigated and from joints. There was a fibrinous polyarthritis, increased presence of monocytes in vessels, and necrosis of Purkinje cells. Sixteen of the diseased animals (33%) developed a chronic polyarthritis. Eight of these lambs were necropsied; all had lesions in major limb joints, and 3 of 8 also had lesions in the atlanto-occipital joint. At this stage, E. rhusiopathiae was cultured only from the joints in 7 of 8 (87.5%) lambs, but by real-time polymerase chain reaction, we showed persistence of the bacterium in several organs. Pulsed-field gel electrophoresis typing of the bacterial isolates indicated that the same strain caused the acute and chronic disease. Five of 6 (83%) chronically affected animals had amyloidosis of the spleen, and 6 of 8 (75%) had amyloidosis of the liver. All chronically affected animals had a glomerulonephritis, and 6 of 8 (75%) had sparse degeneration in the brain. Ceruloplasmin and haptoglobin were significantly increased in the chronically diseased lambs. These results show that chronic ovine erysipelas is not restricted to joints but is a multisystemic disease.

Infectious pancreatic necrosis virus in fish by-products is inactivated with inorganic acid (pH 1) and base (pH 12).

The aquaculture industry needs a simple, inexpensive and safe method for the treatment of fish waste without heat. Microbial inactivation by inorganic acid (HCl) or base (KOH) was determined using infectious pancreatic necrosis virus (IPNV) as a model organism for fish pathogens. Salmonella and spores of Clostridium perfringens were general hygiene indicators in supplementary examinations. IPNV, which is considered to be among the most chemical- and heat-resistant fish pathogens, was reduced by more than 3 log in 4 h at pH 1.0 and pH 12.0. Salmonella was rapidly inactivated by the same treatment, whereas spores of C. perfringens were hardly affected. The results indicate that low and high pH treatment could be particularly suitable for fish waste destined for biogas production. pH treatment at aquaculture production sites could reduce the spread of fish pathogens during storage and transportation without disturbing the anaerobic digestion process. The treatment could also be an alternative to the current energy-intensive steam pressure sterilization of fish waste to be used by the bioenergy, fertilizer and soil improver industries.

Bovine respiratory syncytial virus: infection dynamics within and between herds.

The infection dynamics of bovine respiratory syncytial virus (BRSV) were studied in randomly selected Norwegian dairy herds. A total of 134 herds were tested twice, six months apart. The herds were classified as positive for BRSV if at least one animal between 150 and 365 days old tested positive for antibodies against BRSV, thereby representing herds that had most likely had the virus present during the previous year. The prevalence of positive herds at the first and second sampling was 34 per cent and at 41 per cent, respectively, but varied greatly between regions. Negative herds were found in close proximity to positive herds. Some of these herds remained negative despite several new infections nearby. Of the herds initially being negative, 42 per cent changed status to positive during the six months. This occurred at the same rate during summer as winter, but a higher rate of animals in the herds was positive if it took place during winter. Of the herds initially being positive, 33 per cent changed to negative. This indicates that an effective strategy to lower the prevalence and the impact of BRSV could be to employ close surveillance and place a high biosecurity focus on the negative herds.

Salvage of failed prosthetic breast reconstructions by autologous conversion with free tissue transfers.

OBJECTIVE: Implant-based breast reconstructions are conceptually simple but prone to surgical revisions. Additional procedures often fail to address the problems associated with the reconstructive outcome, especially in patients who have received radiotherapy. However, conversion to free flaps may improve symptoms and aesthetic results. We reviewed our experience in the United Kingdom with autologous replacement of failed prosthetic reconstructions with the aims of documenting the indications for "tertiary" reconstructions and comparing our outcomes with those of other centers. METHODS: Patients undergoing salvage surgery for suboptimal prosthetic breast reconstructions between 2000 and 2012 were retrospectively reviewed for their original reconstructive operation, previous radiotherapy, indications for revision, corrective procedures undertaken, and final outcomes. RESULTS: Of 14 patients identified, 7 had delayed and 7 had immediate reconstructions. Twelve had received radiotherapy; 6 before the initial delayed prosthetic reconstructions and 6 after immediate reconstructions. Ten patients presented after undergoing previous revisions of their original reconstructions (average 1.6). Indications for autologous conversion were capsular contracture, persistent pain, and poor cosmetic outcomes (often in combination). Salvage comprised explantation, total capsulectomy, and abdominal free flap reconstruction using deep inferior epigastric artery flaps (9) and transverse rectus abdominis myocutaneous flaps (5). The average interval between initial reconstruction and salvage was 8 years (r = 1-14). All flap transfers were successful with satisfactory aesthetic outcomes (average 21 months follow-up). CONCLUSIONS: We recommend early salvage autologous conversion of implant-based reconstructions once initial prosthetic reconstructions become unsatisfactory, particularly in recipients of radiotherapy. Many of these patients may have been better served by initial autologous reconstruction; the challenge is to identify them prospectively.

Meningococcal omp85 in detergent-extracted outer membrane vesicle vaccines induces high levels of non-functional antibodies in mice.

The vaccine potential of meningococcal Omp85 was studied by comparing the immune responses of genetically modified deoxycholate-extracted outer membrane vesicles, expressing five-fold higher levels of Omp85, with wild-type vesicles. Groups (n = 6-12) of inbred and outbred mouse strains (Balb/c, C57BL/6, OFI and NMRI) were immunized with the two vaccines, and the induced antibody levels and bactericidal and opsonic activities measured. Except for Balb/c mice, which were low responders, the genetically modified vaccine raised high Omp85 antibody levels in all mouse strains. In comparison, the wild-type vaccine gave lower antibody levels, but NMRI mice responded to this vaccine with the same high levels as the modified vaccine in the other strains. Although the vaccines induced strain-dependent Omp85 antibody responses, the mouse strains showed high and similar serum bactericidal titres. Titres were negligible with heterologous or PorA-negative meningococcal target strains, demonstrating the presence of the dominant bactericidal PorA antibodies. The two vaccines induced the same opsonic titres. Thus, the genetically modified vaccine with high Omp85 antibody levels and the wild-type vaccine induced the same levels of functional activities related to protection against meningococcal disease, suggesting that meningococcal Omp85 is a less attractive vaccine antigen.

Inflammatory response to therapeutic gold bead implantation in canine hip joint osteoarthritis.

Inflammatory changes associated with periarticular pure gold bead implants were studied in dogs involved in a clinical trial investigating motor dysfunction and chronic pain owing to hip joint dysplasia and osteoarthritis. Gold beads were percutaneously implanted via a needle into different locations surrounding the greater trochanter of the femur. Nine dogs with implants were necropsied. In all examined animals, characteristic histologic lesions were observed in the tissue surrounding the gold implants--namely, a fibrous capsule composed of concentric fibroblasts intermixed with a variable number of inflammatory cells and a paucicellular innermost layer of collagen with a few fibrocyte-like cells in empty lacunae. Lymphocytes dominated the inflammatory infiltrate, with rarely observed macrophages present in close proximity to the implant site. No giant cells were observed. Immunohistochemistry showed mixed populations of lymphocytes, both CD3 positive (T cells) and CD79a positive (B cells), which in some cases formed lymphoid follicles. Diffuse inflammatory changes were present to a minor extent in the perimysium and surrounding fascia. The inflammation observed in dogs is similar to that observed with gold implants in humans. It is possible that the clinically beneficial effect of gold beads for chronic osteoarthritis depends on sustained localized inflammation with localized release of soluble mediators. The encapsulation of the implant by a paucicellular and poorly vascularized fibrous capsule may help prevent an exaggerated inflammatory reaction by sequestering the gold bead from the surrounding tissue.

Experience with the Wise mammaplasty skin resection pattern in skin-sparing mastectomy and immediate breast reconstruction for large breast volumes.

INTRODUCTION: Skin-sparing mastectomy (SSM) and immediate breast reconstruction (IBR) remain technically challenging in very large breasted women, often requiring extensive skin reduction, with a high incidence of post-operative complications. We report our retrospective experience (1999-2007) with SSM and Wise pattern skin reduction in IBR, and examine factors contributing to recipient site post-operative complications. RESULTS: 29 reconstructions comprising 16 free TRAM/DIEPs, 5 pedicled TRAMs, and 8 implant-based ones were undertaken in 25 patients aged 32-70 years (median = 48). Their mean BMI was 30.4 kg/m² (r = 22.3-39.3) and 6/25 were current smokers. There were 14 (48%) associated axillary clearances. All breasts had grade 2 or 3 ptosis. The mastectomies weighed 805 g-1972 g (mean = 1326). Sixteen operations (55%) developed complications; 7 minor, 9 requiring further surgical intervention. Of these 16 cases, 13 involved skin complications and 9 involved culture-proven wound infections. All patients achieved satisfactory aesthetic outcomes. At median cancer follow-up of 24 months (r = 2-98), there have been no local recurrences but two deaths from distant metastases. Mastectomy weight was significantly associated with major skin complications requiring surgery (age-adjusted OR per 100 g = 1.6; CI = 1.1-2.3, p = 0.02). Performance of axillary clearance at the time of skin-sparing mastectomy was significantly associated with risk of post-operative infection (age-adjusted OR = 5.8; CI = 1.3-26.0, p = 0.021). These relationships were maintained after adjusting for patient, surgical and tumour factors. CONCLUSION: Wise pattern skin reduction is a useful technique for managing large, ptotic breasts during SSM and IBR. Increasing breast size and the type of cancer surgery are important predictors of post-operative complications.

The Autism Birth Cohort: a paradigm for gene-environment-timing research.

The reported prevalence of autism spectrum disorders (ASDs) has increased by 5- to 10-fold over the past 20 years. Whether ASDs are truly more frequent is controversial; nonetheless, the burden is profound in human and economic terms. Although autism is among the most heritable of mental disorders, its pathogenesis remains obscure. Environmental factors are proposed; however, none is implicated. Furthermore, there are no biomarkers to screen for ASD or risk of ASD. The Autism Birth Cohort (ABC) was initiated to analyze gene x environment x timing interactions and enable early diagnosis. It uses a large, unselected birth cohort in which cases are prospectively ascertained through population screening. Samples collected serially through pregnancy and childhood include parental blood, maternal urine, cord blood, milk teeth and rectal swabs. More than 107,000 children are continuously screened through questionnaires, referral, and a national registry. Cases are compared with a control group from the same cohort in a 'nested case-control' design. Early screening and diagnostic assessments and re-assessments are designed to provide a rich view of longitudinal trajectory. Genetic, proteomic, immunologic, metagenomic and microbiological tools will be used to exploit unique biological samples. The ABC is a paradigm for analyzing the role of genetic and environmental factors in complex disorders.

Transcriptional effects on glutathione S-transferases in first feeding Atlantic cod (Gadus morhua) larvae exposed to crude oil.

Polycyclic aromatic hydrocarbons (PAHs) and other oil compounds are known to induce stress and impact health of marine organisms. Water-soluble fractions of oil contain components known to induce glutathione S-transferases (GSTs), one of the major classes of phase II detoxifying enzymes present in essentially all eukaryotic organisms. In this study, the transcriptional responses of six GSTs (GST pi, GST mu, GST omega, GST theta, GSY zeta and GST kappa) were examined in early larvae of Atlantic cod Gadus morhua exposed to five concentrations of dispersed oil (containing oil droplets and water-soluble fraction) and water-soluble fractions (WSF) of oil. When Atlantic cod larvae were exposed to WSF (containing 1.31+/-0.31microg summation PAH/L for 4 days), expression of GSTM3 and GSTO1 was significantly increased, whereas no differences in GST expression were observed in larvae exposed to a corresponding 50% lower amount of dispersed oil (containing 0.36+/-0.10 microg summation PAH/L for 4 days). The study suggest that although the oil clearly had severe negative effects on the larvae (i.e. concentration-dependent lethality and growth reduction), only minor effects on GST transcription could be observed using RNA obtained from pooled whole-larvae homogenates. This result indicates that the expression of these important detoxification enzymes is only moderately inducible at such an early developmental stage either reflecting low tolerance of cod larvae to dispersed oil or alternatively that using whole-larvae homogenates may have masked tissue-specific mRNA induction.

Respiratory infections in Norwegian dairy calves.

The aims of this study were to estimate the seroprevalence of respiratory agents in Norwegian dairy calves and to identify risk factors for respiratory disease. The participating 135 herds were randomly selected from those in The Norwegian Dairy Herd Recording System with at least 15 cow years. Each herd was followed for 1 yr. Blood samples from calves of >150 d of age (n = 1,348) were analyzed for antibodies against parainfluenza virus 3, bovine coronavirus (BCoV), bovine respiratory syncytial virus (BRSV), and Mycoplasma bovis. Calves reported to have been on pasture (n = 139) were tested for antibodies against Dictyocaulus viviparus. Seroprevalences for parainfluenza virus 3, BCoV, BRSV, and D. viviparus at the calf level were 50.2, 39.3, 31.2, and 4.3%, respectively. No calves were antibody positive for M. bovis. Calves in herds with BCoV-seropositive calves had an increased risk of respiratory disease compared with herds in which BCoV antibodies were not detected [hazard ratio (HR) = 3.9], as had calves in herds in which the majority (>54%) of the sampled calves were seropositive for BRSV (HR = 2.7). Other factors found to increase the risk of respiratory disease in calves were shared housing with cows during the first week of life compared with separate housing (HR = 16.7), a larger herd size (>50 cow years) compared with smaller herds (HR = 8.2), more than an 8-wk age difference between calves housed together in the same group pen compared with having pen mates of a more similar age (HR = 3.9), previous recordings of diarrhea compared with no recorded diarrhea (HR = 3.9), and leaving calves with dams for >24 h after birth compared with earlier separation (HR = 3.5).

Enteropathogens and risk factors for diarrhea in Norwegian dairy calves.

The aims of the current study were to estimate the prevalence of enteropathogens in calves in Norwegian dairy herds, evaluate the clinical consequences of protozoal infections, and identify risk factors for diarrhea. The 135 participating herds were randomly selected from those in The Norwegian Dairy Herd Recording System that had at least 15 cow-years. Each herd was followed for 1 yr. Fecal samples from calves with (n = 68) or without (n = 691) diarrhea were analyzed for the presence of Cryptosporidium, Giardia, and Eimeria species. Diarrheic samples (n = 191) were assayed for rotavirus group A, bovine coronavirus (BCoV), Cryptosporidium, and Escherichia coli F5 by antigen ELISA. Blood samples (n = 1,348) were analyzed for antibodies against BCoV and rotavirus. Potential risk factors for diarrhea were analyzed by using Cox regression analysis adjusted for herd frailty effect. Rotavirus and Cryptosporidium were the most commonly detected enteropathogens in diarrheic samples. A high level of Cryptosporidium shedding or BCoV seropositive calves in a herd was associated with an increased risk of diarrhea. Other factors found to increase the risk of diarrhea were use of slatted concrete floor in group pens versus other floor types [hazard ratio (HR) = 8.9], housing of calves in free-stalls compared with tie-stalls (HR = 3.7), purchasing of calves into the herd versus not purchasing calves (HR = 4.1), and calves being born during winter compared with other seasons of the year (HR = 1.5).

Calf mortality in Norwegian dairy herds.

The aims of this study were to estimate mortality rates in Norwegian dairy calves and young stock up to 1 yr of age, identify risk factors for calf mortality, and evaluate the etiology of calf mortality based on postmortem analyses. The material comprised 3 data sets. The first data set included information on 289,038 offspring in 14,474 dairy herds registered in the Norwegian Dairy Herd Recording System (NDHRS) in 2005. The second included recordings on 5,382 offspring in 125 Norwegian dairy herds participating in a survey on calf health, and the third included results from postmortem analyses of 65 calves from 37 of the survey herds. The calf mortality rate during the first year of life in all herds registered in the NDHRS was 7.8%, including abortion (0.7%) and stillbirth (3.4%). The overall calf mortality rate in liveborn calves in the survey herds was 4.6%. Cows with severe calving difficulties had an odds ratio (OR) of 38.7 of stillbirth compared with cows with no calving difficulties. Twins and triplets showed an increased risk of stillbirth compared with singletons (OR = 4.2 and 46.3, respectively), as did calves born in free stalls compared with tie stalls (OR = 1.9). Respiratory disease increased the risk of death in all age groups with hazard ratios (HR) of 6.4, 6.5, 7.4, and 5.6 during the first week of life, 8 to 30 d of age, 31 to 180 d of age, and 181 to 365 d of age, respectively. Diarrhea increased the risk of death among calves younger than 180 d of age, but the influence was only significant during the first week of life and between 8 to 31 d of age (HR = 2.4 and 2.9, respectively). Calves born during the winter were more likely to die during the first week of life than calves born during the summer (OR = 1.2), and were more likely to die during the first month of life than calves born during the autumn (OR = 1.2). Calf mortality rates in all age groups increased with increasing herd size. Calves housed in a group pen from 2 wk of age were more likely to die during the first month of life than calves housed individually (HR = 1.5). Bronchopneumonia and enteritis were the most frequent postmortem diagnoses, with proportional rates of 27.7 and 15.4%, respectively.

Calf health monitoring in Norwegian dairy herds.

The aims of this study were to estimate the incidence of calf diseases in Norwegian dairy herds, improve calf health recordings in the Norwegian Cattle Health Recording System (NCHRS), and investigate different methods for validation of calf health data. A longitudinal, cross-sectional survey of calf health in Norway was performed between September 1, 2004, and January 31, 2007. The participating dairy herds were randomly selected from among herds registered in the Norwegian Dairy Herd Recording System as having at least 15 cow-years. Each herd participated for 1 yr. Diseases and treatments of calves of up to 180 d of age in 135 dairy herds were reported using the NCHRS. In total, 6,668 calves were born in the participating dairy herds during the project period. A total of 573 (29.6%) of the 1,936 calf health recordings reported were recordings of diseases and 1,363 (70.4%) were events of preventive therapy, dehorning, or castration. The recorded incidence of diarrhea and respiratory disease was 3.8% and 2.9%, respectively. The median age of occurrence of diarrhea and respiratory disease was 17 and 37 d, respectively. Three different methods, based on sampling of diseased calves, dehorning as an indicator of a well-functioning recording system, or feedback on degree of commitment to calf health recording, were tested to assess validation of the calf health records. The 3 methods indicated an underestimation of calf health records in the NCHRS of approximately 40% and an estimated "true" incidence of diarrhea and respiratory disease of 5.5 and 4.1%, respectively. The results from this study demonstrate the importance of encouraging farmers to conduct calf health recordings. They also indicate that finding a standardized method for validation of health data is a considerable challenge.

Risk factors associated with colostrum quality in Norwegian dairy cows.

The objectives of the present study were to evaluate colostrum quality in Norwegian dairy cows based on IgG content, and to identify associations between possible risk factors and low colostral IgG. A longitudinal cross-sectional survey on calf health in Norway was performed between June 2004 and December 2006. The participating dairy herds were randomly selected among herds registered in the Norwegian Dairy Herd Recording System as having at least 15 cow years. The participating farmers were requested to sample 10 mL of colostrum from the first milking after calving from 12 cows that had calved during the defined project period of 365 d. Colostrum samples from 1,250 cows from 119 herds were collected. The material consisted of 451, 337, 213, and 249 samples collected from cows in their first, second, third, and fourth parity or more, respectively. Analysis was performed on IgG content by using single radial immunodiffusion. Mixed models with herd as a cluster were fit by using grams of IgG per liter of colostrum as the dependent variable for the statistical analyses. The IgG content in the colostrum sampled ranged from 4 to 235 g/L, with a median of 45.0 g of IgG/L, with the 10th, 25th, 75th, and 90th percentiles being 23.1, 31.4, 63.6, and 91.6 g of IgG/L, respectively. Altogether, 57.8% of the samples contained less than the desired 50 g of IgG/L of colostrum. Cows in their fourth parity or more were found to have significantly higher levels of IgG per liter of colostrum than cows in their first or second parity. Colostrum from cows in their second parity had the lowest level of IgG. Cows calving during the winter months (December, January, and February) produced colostrum with a significantly lower IgG content compared with cows calving in any other season of the year. Somatic cell count, measured after calving, was significantly higher in cows producing colostrum of inferior quality compared with those producing high-quality colostrum. Of the total variation in colostrum quality, 13.7% could be explained by cluster effects within herd. The variation in IgG content in colostrum produced by Norwegian dairy cows indicates a need for improved colostrum quality control and subsequent adjustment of the colostrum feeding regimen to ensure a protective immunological status for newborn calves.