Effect of re-ensiling on the quality of sorghum silage.

The commercialization of silage in many countries, including Brazil, has increased in recent years. Re-ensiling of previously ensiled forage occurs when silage is relocated from one farm to another, where it will be compacted and sealed again. During this process, silage is exposed to oxygen before being ensiled, which may affect its quality. We exposed sorghum silage to air during the anaerobic storage phase to simulate the transportation of silages between farms. Experimental treatments included silage exposed to air for 0 or 12 h, with or without the use of an inoculant containing a mixture of Lactobacillus plantarum and the propionic bacteria Propionibacterium acidipropionici (1 × 106 cfu/g of forage; Biomax corn, Lallemand, Saint-Simon, France), totaling 4 treatments: conventional silage, conventional silage with inoculant use, re-ensilage after exposure to air, and re-ensilage after exposure to air with use of an inoculant. The sorghum was stored in experimental silos containing about 9.0 kg of fresh forage per replicate. Treatments were tested in a factorial 2 × 2 design with 5 replicates each. Chemical composition, in vitro dry matter digestibility, fermentative characteristics, losses (due to gas, effluents, and total dry matter), microorganism counts, and aerobic stability of sorghum silage were evaluated. Dry matter content of sorghum before ensiling was 273.12 g/kg. The 12-h re-ensiling process increased the effluent loss of the silage when compared with conventional silage (456.42 vs. 201.19 g/kg of FM, respectively). In addition, re-ensiled silages presented lower concentrations of lactic acid and higher concentrations of propionic acid than the silages that had not been opened during storage. The aerobic stability of silage was not affected by the re-ensiling process and the use of inoculant. The use of inoculant increased the pH and loss of dry matter of the silages (4.23 vs. 3.98 and 14.05 vs. 7.82%, respectively) and therefore did not provide any benefits in this study.

Comorbidities in overweight children and adolescents: do we treat them effectively?

OBJECTIVE: The aim was to analyze the effectiveness of treatment concerning obesity-associated comorbidities in clinical practice. METHODS: A total of 11,681 overweight children with ≥ 6-month follow-up treated at 175 centers specialized in pediatric obesity care in Central Europe were included in this analysis (mean body mass index (BMI) 29.0 ± 5.6 kg m(-)(2), standard deviation score body mass index (SDS-BMI) 2.48 ± 0.54, 45% boys, age 11.4 ± 2.8 years). The changes of weight status, blood pressure, fasting lipids and glucose, and oral glucose tolerance tests were documented by standardized prospective quality documentation software (APV). RESULTS: After follow-up of in median 1.2 (interquartile range 0.9-2.2) years, a mean reduction of -0.15 SDS-BMI was achieved. The prevalence of prehypertension (37->33%) and hypertension (17->12%) decreased, while prevalences of triglycerides >150 mg dl(-1) (22->21%), low-density-lipoprotein-cholesterol >130 mg dl(-1) (15->14%), impaired fasting glucose (6->6%) and impaired glucose tolerance (9->8%) remained stable. Drug treatment according to cutoffs recommended in European obesity guidelines were not frequently indicated (hypertension: 10%; dyslipidemia: 1%, type 2 diabetes <1%). None of the children with dyslipidemia received lipid-lowering drugs and only 1.4% of the children with hypertension were treated with antihypertensive drugs. CONCLUSIONS: Achieving sufficient weight loss to improve obesity associated comorbidities was difficult in clinical practice. Drug treatment of hypertension, dyslipidemia and type 2 diabetes was rarely performed even if it was indicated only in a minority of the overweight children. Future analyses should identify reasons for this insufficient drug treatment of comorbidities and analyze whether the benchmarking processes of APV improve medical care of childhood obesity.

Fungal contamination and determination of fumonisins and aflatoxins in commercial feeds intended for ornamental birds in Rio de Janeiro, Brazil.

The purposes of this study were to determine the distribution of total mycobiota, to determine the occurrence of Aspergillus spp., Penicillium spp. and Fusarium spp. and to detect and quantify fumonisin B1 and aflatoxin B1 in birds' feedstuffs. Sixty samples from different commercial feeds were collected. Analysis of the total mycobiota was performed and total fungal counts were expressed as CFU g(-1). The isolation frequency (%) and relative density (%) of fungal genera and species were determined. Mycotoxins determination was carried out using commercial ELISA kits. The 48% of standard, 31% of premium and only 9% of super premium feed samples were found above of recommended limit (1 × 10(4) CFU g(-1)). Aspergillus (82%), Cladosporium (50%) and Penicillium (42%) were the most frequently isolated genera. Aspergillus niger aggregate (35%), Aspergillus fumigatus (28%) and Aspergillus flavus (18%) had the highest relative densities. Contamination with fumonisins was detected in 95% of total samples with levels from 0·92 to 6·68 µg g(-1), and the aflatoxins contamination was found in 40% of total samples with levels between 1·2 and 9·02 µg kg(-1). Feed samples contaminated with fumonisins and aflatoxins are potentially toxic to birds.

Gliotoxin contamination in and pre- and postfermented corn, sorghum and wet brewer's grains silage in Sao Paulo and Rio de Janeiro State, Brazil.

AIMS: The aim of this study was to determine total fungal counts and the relative density of Aspergillus fumigatus and related species in silage samples intended for bovines before and after fermentation as well as to monitor the natural occurrence of gliotoxin in silage samples (pre- and postfermentation). METHODS AND METHODS: The survey was performed in farms located in São Paulo and Rio de Janeiro States in Brazil. In addition, the ability of A. fumigatus strains and related species strains to produce gliotoxin was also evaluated. A total of 300 samples were taken, immediately after opening of the silo (3-5 months) and during the ensiling period. Fungal counts were done by the surface-spread method. Gliotoxin production ability of isolates and natural contamination were determined by HPLC. RESULTS: All postfermented samples had a total number of moulds exceeding 1 × 10(4) CFU g(-1), with Aspergillus sp. as the most prevalent genus. Frequency of strains, among A. fumigatus and related species, was able to produce gliotoxin was similar in pre- and postfermented samples, except for sorghum, which showed differences between both kinds of samples. The highest toxin levels were produced by strains isolated from postfermented samples. More than 50% of the samples showed gliotoxin contamination levels that exceeded concentrations known to induce immunosuppressive and apoptotic effects in cells. CONCLUSIONS: The present data suggest that care should be taken because gliotoxin contamination in feedstuffs could affect productivity and also present a health risk for herds. SIGNIFICANCE AND IMPACT OF THE STUDY: Gliotoxin was found at quite important concentrations levels in pre- and postfermented substrates and its presence could therefore probably affect the productivity and health of herds. Current conservation and management practices do not avoid contamination with A. fumigatus on silage. Therefore, farm workers should be adequately protected during its handling.

Obese boys at increased risk for nonalcoholic liver disease: evaluation of 16,390 overweight or obese children and adolescents.

OBJECTIVE: Comorbidities of childhood obesity challenge health-care systems in Europe. Further, there is a lack of population-specific prevalence data and diagnostic strategies available, especially for obesity-related disturbances of liver function. Therefore, the prevalence of elevated liver enzymes and their relationship to biological parameters were studied in a large pediatric obesity cohort. METHODS: In 111 specialized pediatric obesity centers in Germany, Austria and Switzerland, 16,390 children and adolescents (age 12.4±2.6 years, 58% boys) were categorized as overweight (body mass index (BMI) >90th percentile) and obese (>97th percentile) and studied for related comorbidities, especially nonalcoholic fatty liver disease (NAFLD; as defined by aspartate aminotransferase (AST) and/or alanine aminotransferase (ALT) >50 U l(-1)). Data were collected using a standardized software program (APV) for longitudinal multicenter documentation. Pseudonymized data were transmitted for central statistical analysis. RESULTS: In this pediatric cohort, 16% of the study population was overweight, 46% obese and 35% extremely obese (>99.5th percentile extreme obesity (Xob)). NAFLD was present in 11% of the study population, but predominantly in boys (boys vs girls; 14.4:7.4%; P<0.001), in Xob (obese vs Xob; 9.5:17.0%; P<0.001) and in older age (< 12 vs ≥12 years; 8:12%; P<0.001; adjusted for BMI). ALT >50 U l(-1) was significantly associated with fasting insulin and BMI-SDS. In multiple logistic regression models, Xob and male gender were strongly associated with NAFLD (odds ratio Xob vs normal weight=3.2; boys vs girls OR=2.3). CONCLUSION: In a large cohort of overweight and obese European children and adolescents, markers of nonalcoholic liver disease, especially ALT, are frequent and predicted by Xob and male gender. The results underline the epidemiological dimension of this obesity-related morbidity even in childhood. Therefore, at least ALT is recommended as a screening parameter in basic care.

Mycological survey and ochratoxin A natural contamination of swine feedstuffs in Rio de Janeiro State, Brazil.

Mycotoxin contamination of animal feeds represents a hazard to human and animal health due to potential transmission to meat and milk. Barley by-products are alternative feeding supplies for animal production. The aims of this assay were to study the mycobiota of feedstuffs and finished swine feed, to determine the ability of Aspergillus and Penicillium isolates to produce ochratoxin A (OTA) and to evaluate OTA occurrence in these substrates. Corn, brewers' grains and finished swine feed samples were collected from different factories. Fungal counts were higher than 2.8x10(4)CFU g(-1). Fusarium, Aspergillus and Penicillium genera were isolated at high levels. A 23.7% of the isolates produced 9-116 microg kg(-1) of OTA in vitro. Corn samples (44%) were contaminated with 42-224 microg kg(-1) of OTA. Finished feed (31%) and brewers' grains samples (13%) were contaminated with 36-120 microg kg(-1) and 28-139 microg kg(-1) of OTA, respectively. This is the first scientific report on contamination by OTA-producer molds and OTA in swine feedstuffs from Brazil. The presence of OTA in raw materials and finished feed requires periodic monitoring to prevent mycotoxicoses in animal production, reduce economic losses and minimize hazards to human health.

High proportion of large genomic deletions and a genotype phenotype update in 80 unrelated families with juvenile polyposis syndrome.

BACKGROUND: In patients with juvenile polyposis syndrome (JPS) the frequency of large genomic deletions in the SMAD4 and BMPR1A genes was unknown. METHODS: Mutation and phenotype analysis was used in 80 unrelated patients of whom 65 met the clinical criteria for JPS (typical JPS) and 15 were suspected to have JPS. RESULTS: By direct sequencing of the two genes, point mutations were identified in 30 patients (46% of typical JPS). Using MLPA, large genomic deletions were found in 14% of all patients with typical JPS (six deletions in SMAD4 and three deletions in BMPR1A). Mutation analysis of the PTEN gene in the remaining 41 mutation negative cases uncovered a point mutation in two patients (5%). SMAD4 mutation carriers had a significantly higher frequency of gastric polyposis (73%) than did patients with BMPR1A mutations (8%) (p<0.001); all seven cases of gastric cancer occurred in families with SMAD4 mutations. SMAD4 mutation carriers with gastric polyps were significantly older at gastroscopy than those without (p<0.001). In 22% of the 23 unrelated SMAD4 mutation carriers, hereditary hemorrhagic telangiectasia (HHT) was also diagnosed clinically. The documented histologic findings encompassed a wide distribution of different polyp types, comparable with that described in hereditary mixed polyposis syndromes (HMPS). CONCLUSIONS: Screening for large deletions raised the mutation detection rate to 60% in the 65 patients with typical JPS. A strong genotype-phenotype correlation for gastric polyposis, gastric cancer, and HHT was identified, which should have implications for counselling and surveillance. Histopathological results in hamartomatous polyposis syndromes must be critically interpreted.

The mycobiota and toxicity of equine feeds.

Feed contamination can lead to nutrient losses and detrimental effects on animal health and production. The purposes of this study were to investigate the mycobiota in equine mixed feeds and to determine natural contamination with aflatoxin B1 (AFB1) and fumonisin B1 (FB1). Fungal enumeration of equine feed samples was done. A commercially available enzyme-linked immunosorbent assay kit was applied to quantify AFB1 and FB1. A comparison between ELISA and HPLC was carried out. Feed mould counts ranged from <1 x 10(2) to 1 x 10(5) cfu/g. The most frequent genus isolated was Aspergillus (40.54%), followed by Penicillium (18.38%) and Fusarium (16.22%). The most prevalent Aspergillus sp. was A. flavus (36%). AFB1 values ranged between 0.01 and 99.4 microg/kg. FB(1) levels ranged between 0.01 and 7.49 microg/kg. HPLC and ELISA methods showed positive correlation for AFB1 and FB1 determinations (r = 0.9851 and r = 0.9791, respectively). The ELISA analytical method was efficient for AFB1 and FB1 detection. The scarcity of studies on natural fungal contamination and on the presence of AFB1 and FB1 in materials used as equine feed ingredients highlights the value and contribution of this study.

Amino acid sulfur as a source of sulfate for sulfated proteoglycans produced by Swiss mouse 3T3 cells.

The uptake of sulfate by Swiss mouse 3T3 cells is blocked in the presence of 1 mM 4-isothiocyano-4'-acetamido-stilbene-2,2-disulfonic acid (SITS). In the absence of an exogenous source of sulfate, glycosaminoglycans produced by cells in the presence of the inhibitor are sulfated to the same extent as those produced by cells grown in its absence. The sulfate utilized in the absence of medium sulfate has been identified as that produced by the oxidation of the sulfur present in the amino acids cysteine and methionine. This finding indicates that, under conditions of restricted exogenous sulfate, caution is needed in the interpretation of data obtained with the use of [35S]methionine and/or [35S]cysteine as a general protein label, since both tyrosine and a variety of types of protein-linked carbohydrate chains may be modified by sulfation.

The C-terminus of type I collagen is a major binding site for heparin.

The binding of collagens and fragments of type I collagen to heparin was studied by gel electrophoresis and affinity chromatography. Samples bound in 150 mM NaCl/10 mM Hepes (pH 6.5) were eluted with 2 M NaCl, 6 M urea, or a linear gradient of 0.15-1.0 M NaCl. The triple-helical conformation was shown to be essential for binding. The vertebrate collagenase-generated C-terminal fragment, TCB, was shown to have greater binding affinity for heparin than the N-terminal TCA fragment. Both type II collagen and the NC1 domain of type IV collagen bound to heparin, whereas pepsin-solubilized tetrameric type IV failed to bind.

Metabolism of glycosyl ureides by human intestinal brush border enzymes.

13C-labeled glycosyl ureides were recently proposed as a new marker of the orocecal transit time: after passing the small bowel the sugar-urea bond is split by bacterial allantoicase. Further degradation results in 13CO2 which can be measured in the exhaled breath. The aim of this study was to detect an eventual allantoicase-like activity in the human gut and to elucidate the metabolism of glycosyl ureides by human intestinal brush border enzymes. Biopsies of 15 duodenal specimen and 6 colon specimen were homogenised and incubated with several disaccharides and their corresponding disaccharide ureides under various experimental conditions. Hydrolysis of the sugar-urea bond could not be observed neither in the small bowel nor in the colon. However, the conjugation between the two sugars was split. In a modified Dahlqvist assay lactase showed the same kinetics with lactose and lactose ureide as substrates whereas maltose showed a significantly 2.6-fold higher affinity to maltase than maltose ureide (P < 0.001). No major difference between these two substrates could be detected when total maltase activity was inhibited by acarbose. In summary, the human gut tissue possesses no allantoicase-like activity. Therefore, glycosyl ureides seem to be appropriate substances to test the orocecal transit time.

Discovery of TBC11251, a potent, long acting, orally active endothelin receptor-A selective antagonist.

Previously we reported the discovery of amidothiophenesulfonamides as endothelin receptor-A antagonists with high potency and selectivity. Replacement of an amide group in this class of compounds with an acetyl group maintained the in vitro binding affinity and in vivo activity while providing a compound with oral bioavailability and longer duration of action. The optimal compound discovered during these studies, 15q (TBC11251), binds competitively to human ETA receptors with a Ki of 0.43 +/- 0.03 nM and an IC50 of 1.4 nM (IC50 for ETB = 9800 nM). This compound inhibits ET-1-induced stimulation of phosphoinositide turnover with a Ki of 0.686 nM and a pA2 of 8.0. The compound has a serum half-life in the rat and the dog of 6-7 h and 60-100% oral bioavailability. This compound is one of the most selective ETA antagonists reported and therefore is suitable for additional pharmacological and clinical investigation of the role of ETA receptors in diseases.

Rational design and synthesis of small molecule, non-oligosaccharide selectin inhibitors: (alpha-D-mannopyranosyloxy)biphenyl-substituted carboxylic acids.

The calcium dependent E-selectin/sialyl Lewisx (sLex) interaction plays a key role in inflammation where it mediates the rolling of leukocytes prior to firm adhesion and extravasation from the vasculature. A model of E-selectin/sLex binding, along with previously reported structure-activity relationships of sLex-related oligosaccharide, was used in the rational design of non-oligosaccharide inhibitors of this pivotal interaction. A palladium-mediated biaryl-coupling (Suzuki) reaction was used as the key step to prepare a number of substituted biphenyls which were assayed for their ability to inhibit the binding of E-, P-, and L-selectin-IgG fusion proteins to sLex expressed on the surface of HL60 cells. Some of the compounds developed had greater in vitro potency than the parent sLex tetrasaccharide and are currently being evaluated in in vivo models of inflammation to select a candidate for clinical development.

Acyl substitution at the ortho position of anilides enhances oral bioavailability of thiophene sulfonamides: TBC3214, an ETA selective endothelin antagonist.

Sitaxsentan (3, TBC11251) (Wu et al. J. Med. Chem. 1997, 40, 1690) is an orally active ET(A) selective endothelin antagonist that attenuates pulmonary vascular hypertension and cardiac hypertrophy in rats (Tilton et al. Pulm. Pharmacol. Ther. 2000, 13, 87). It has demonstrated efficacy in a phase II clinical trial for congestive heart failure (Givertz et al. Circulation 2000, 101, 2922). During the discovery of 3, we observed several structure-oral bioavailability relationships. To investigate whether there is any generality in these trends, we synthesized some similar pairs of compounds in the latest series (Wu et al. J. Med. Chem. 1999, 42, 4485) and evaluated their oral properties. In both series, an acyl group at the 2-position of the anilide of these thiophene sulfonamides improved oral bioavailability. As a result of this exercise, TBC3214 (17) was identified as a sitaxsentan follow-on candidate. It is very potent (IC(50) for ET(A) = 40 pM) and highly selective for ET(A) vs ET(B) receptors (400 000-fold), with a half-life of >4 h and oral bioavailability of 25% in rats, 42% in cats, and 70% in dogs.

Differences in the clinical and radiologic patterns of rotavirus and non-rotavirus necrotizing enterocolitis.

We analyzed retrospectively 32 successive infants who developed necrotizing enterocolitis (NEC), 13 with rotavirus (RV) infection (RV+) and 19 RV-negative (RV-). All patients showed at least pneumatosis intestinalis. All patients except one had risk factors for perinatal asphyxia. Our study demonstrated significant differences between RV+ NEC and RV- NEC cases: RV+ NEC infants had a higher birth weight and were born at a later gestational age. Oral feeding was started earlier and symptoms developed later and more insidiously in RV+ patients than in RV- NEC babies. Radiology revealed a less severe and more distal colon involvement in RV+ NEC infants, whereas the RV- NEC patients mostly had small intestinal or ileocecal changes and more frequent complications of pneumoportogram and intestinal perforations. These latter infants often had a rapidly deteriorating clinical course; 84% needed surgical treatment. In conclusion RV may be a cause of NEC in susceptible infants. Historic and clinical data and a more distal colonic pneumatosis allow a differentiation of RV+ NEC from other forms of NEC.

[The diagnostic value of the plain abdominal radiogram in differentiating rotavirus-negative and rotavirus-positive necrotizing enterocolitis]. / Aussagefähigkeit der Abdomenübersichtsaufnahme in der Differenzierung der Rotavirus-negativen und Rotavirus-positiven nekrotisierenden Enterokolitis.

Clinical data and radiographic findings of 32 newborn suffering from necrotising enterocolitis were analysed [12 patients with rotavirus-positive necrotising enterocolitis (RV + NEC), 20 patients with rotavirus-negative necrotising enterocolitis (RV-NEC)]. The presence and degree of pneumatosis intestinalis, portal venous gas and pneumoperitoneum on abdominal radiographs were graded after Kosloske et al. according to "mild, moderate, and severe". Pneumatosis intestinalis occurred twice as often in the ascending colon in RV-NEC compared to RV + NEC, whereas the transverse colon was involved nearly as frequently as the descending colon in both groups. Portal venous gas was present in 10% of the cases with RV-NEC and was absent in RV + NEC. Pneumoperitoneum only occurred in 8% of RV + NEC but in 20% of RV-NEC. Radiographic findings are helpful in the differentiation between both groups of NEC. Conservative therapy is preferable especially in mainly distal colon distribution of pneumatosis intestinalis without pneumoperitoneum.

[3D-TSE MR cholangiopancreatography with breath triggering to clarify unclear hepatopathies in children]. / 3D-TSE MR-Cholangiopankreatikographie mit Atemtriggerung zur Abklärung von unklaren Hepatopathien bei Kindern.

PURPOSE: Evaluation of 3D-TSE MR-cholangiography with respiratory triggering in the work up of hepatopathies in infants and young children. PATIENTS AND METHOD: 16 infants (4-16 years) with increased transaminases, two with recurrent pancreatitis, were examined at 1.5 T (ACS-NT II, Philips Medical Systems) using a 3D-TSE MRCP with respiratory triggering in addition to a regular MRI of the liver. The MRCP was compared to ERCP. Two radiologists and one gastroenterologist evaluated the technical quality, visualization of the pancreaticobiliary system, and the diagnostic value of the examinations. RESULTS: Technically feasible were 14/16 MRCPs and 13/16 ERCPs. Two MRCP were not of diagnostic value due to motion artifacts and in three ERCP cannulation of the papilla was not possible. 14/16 ERCP required general anaesthesia, while MRCP needed i.v. sedation in two patients only. Extrahepatic ducts/cystic duct/pancreatic duct were visualized in 14/12/8 patients using MRCP, and in 13/10/3 patients using ERCP, both without adverse effects or complications. Intrahepatic ducts were better delineated with MRCP. In 10 patients with histologically proven periportal fibrosis (n = 7) and liver fibrosis (n = 1) or antineutrophil cytoplasmatic antibodies and associated inflammatory bowel disease, MRCP and ERCP revealed pathological results. CONCLUSION: MRCP using a 3D-TSE sequence with respiratory triggering is a good non-invasive technique for delineation of the biliary tract in infants and young children for the work up to hepatopathies.

Reduced prevalence of obesity in children with primary fructose malabsorption: a multicentre, retrospective cohort study.

BACKGROUND: Studies in animals and in man have demonstrated that excessive consumption of fructose can cause all components of the metabolic syndrome. OBJECTIVE: To investigate the impact of a condition resulting in decreased absorption of fructose, on obesity. METHODS: In a multicentre study, we analyzed a cohort of paediatric patients with suspected primary fructose malabsorption (FM). Patients with chronic intestinal diseases were excluded. The final cohort comprised 628 patients. RESULTS: 302 patients were diagnosed with primary FM (48.1%). The proportion of obese patients was lower among FM patients, compared to non-FM patients (2.3 vs. 6.1%, P = 0.029). Logistic regression analysis with inclusion of various covariates showed that FM was negatively associated with obesity (OR 0.35, 95% CI [0.13; 0.97]). We discuss several mechanisms involving the metabolic, endocrine and gastrointestinal system. CONCLUSIONS: Our data indicate that primary FM is negatively associated with childhood obesity.

Production of citreoviridin by Penicillium citreonigrum strains associated with rice consumption and beriberi cases in the Maranhão State, Brazil.

The aim of this study was to determine the levels of Penicillium citreonigrum and citreoviridin present in rice samples from Maranhão State, Brazil, where an outbreak of beriberi was reported and 32 deaths occurred (7% of the notified cases died in 2006). The ability of P. citreonigrum to produce citreoviridin was assessed, and a total of 420 samples of 21 different kinds of rice were collected. Mycobiota isolation and identification, the ability of citreoviridin strains to produce toxin, and the natural occurrence of citreoviridin were established. Rice samples were found to have high fungal counts and showed increasing levels from 2004 to 2007 harvest years. The most frequent genus was Aspergillus followed by Penicillium and Cladosporium. Ten out of eleven strains of P. citreonigrum were able to produce citreoviridin. Three rice samples had levels of citreoviridin ranging from 12 to 96.7 ng g(-1), and two bran samples had levels of 128 and 254 ng g(-1). These samples contaminated with P. citreonigrum and citreoviridin were involved in the beriberi cases from Maranhão State. Monitoring rice for mycotoxins in areas where this substrate is the basic food is crucial to prevent outbreaks like the one reported in this study, to improve management practice, and to diminish exposure risk of humans to these harmful toxins.