Risk stratification in gastric cancer lung metastasis: Utilizing an overall survival nomogram and comparing it with previous staging.

BACKGROUND: Gastric cancer (GC) is prevalent and aggressive, especially when patients have distant lung metastases, which often places patients into advanced stages. By identifying prognostic variables for lung metastasis in GC patients, it may be possible to construct a good prediction model for both overall survival (OS) and the cumulative incidence prediction (CIP) plot of the tumour. AIM: To investigate the predictors of GC with lung metastasis (GCLM) to produce nomograms for OS and generate CIP by using cancer-specific survival (CSS) data. METHODS: Data from January 2000 to December 2020 involving 1652 patients with GCLM were obtained from the Surveillance, epidemiology, and end results program database. The major observational endpoint was OS; hence, patients were separated into training and validation groups. Correlation analysis determined various connections. Univariate and multivariate Cox analyses validated the independent predictive factors. Nomogram distinction and calibration were performed with the time-dependent area under the curve (AUC) and calibration curves. To evaluate the accuracy and clinical usefulness of the nomograms, decision curve analysis (DCA) was performed. The clinical utility of the novel prognostic model was compared to that of the 7th edition of the American Joint Committee on Cancer (AJCC) staging system by utilizing Net Reclassification Improvement (NRI) and Integrated Discrimination Improvement (IDI). Finally, the OS prognostic model and Cox-AJCC risk stratification model modified for the AJCC system were compared. RESULTS: For the purpose of creating the OS nomogram, a CIP plot based on CSS was generated. Cox multivariate regression analysis identified eleven significant prognostic factors (P < 0.05) related to liver metastasis, bone metastasis, primary site, surgery, regional surgery, treatment sequence, chemotherapy, radiotherapy, positive lymph node count, N staging, and time from diagnosis to treatment. It was clear from the DCA (net benefit > 0), time-dependent ROC curve (training/validation set AUC > 0.7), and calibration curve (reliability slope closer to 45 degrees) results that the OS nomogram demonstrated a high level of predictive efficiency. The OS prediction model (New Model AUC = 0.83) also performed much better than the old Cox-AJCC model (AUC difference between the new model and the old model greater than 0) in terms of risk stratification (P < 0.0001) and verification using the IDI and NRI. CONCLUSION: The OS nomogram for GCLM successfully predicts 1- and 3-year OS. Moreover, this approach can help to appropriately classify patients into high-risk and low-risk groups, thereby guiding treatment.

Middle-aged and older people's preference for medical-elderly care integrated institutions in China: a discrete choice experiment study.

BACKGROUND: With the continuing impact of the aging population, medical-elderly care integrated institutions, as a way to bear the pressure of medical and elderly care, effectively ensure the quality of life of the elderly in their later years. OBJECTIVES: To explore the preferences of medical-elderly care integrated institutions among Chinese middle-aged and older people and to provide a reference for establishing elderly-oriented development of medical-elderly care integrated institutions. METHODS: In this study, a discrete choice experiment (DCE) was used to investigate the preferences of people aged 45 years and older in medical-elderly care integrated institutions in China from October 20, 2022, to November 10, 2022. A mixed logit regression model was used to analyze the DCE data. Participants' willingness to pay for each attribute was also calculated. RESULTS: Data from 420 participants who provided valid responses were included in the analysis. In terms of the choice preference, moderate service quality (vs. poor service quality: ß = 1.707, p < 0.001, 95% CI 1.343 ~ 2.071) and high medical technology level (vs. low medical technology level: ß = 1.535, p < 0.001, 95% CI 1.240 ~ 1.830) were the most important attributes to middle-aged and older people, followed by monthly cost, environmental facilities, the convenience of transportation, and entertainment activities. Regarding the willingness to pay, participants were more willing to pay for service quality and medical technology level than for other attributes. They were willing to pay $3156 and $2838 more for "poor service quality" and "low medical technology level," respectively, to receive "moderate service quality " (p = 0.007, 95% CI 963 ~ 5349) and "high medical technology level" (p = 0.005, 95% CI 852 ~ 4824). CONCLUSIONS: The state should attach great importance to the development of medical-elderly care integrated services industry, actively optimize the model of the medical-elderly care integrated service, improve the facilities, and create a healthy environment. At the same time, give full play to the role of medical insurance, long-term care insurance, and commercial insurance, so as to improve the comprehensive quality of life of the elderly. PUBLIC CONTRIBUTION: The design of the experimental selection was guided by 10 experts in the field, 5 Chinese government officials, and interviews and focus group discussions, without whose participation this study would not have been possible.

Synthesis and biological evaluation of resveratrol derivatives with anti-breast cancer activity.

Resveratrol is a natural phytoestrogen produced by plants to protect themselves from injury, UV irradiation, and fungal attack. The main active structure is E-resveratrol, which has many pharmacological activities. As the structure of resveratrol is similar to the natural estrogen 17ß-estradiol and the synthetic estrogen E-diethylstilbestrol, resveratrol is used in reducing the incidence of breast cancer. However, the therapeutic application of resveratrol is limited due to its low bioavailability. To improve its bioavailability and pharmacological activity, some resveratrol derivatives have been designed and synthesized by substitutions of methoxy, hydroxyl, and other functional groups or heterocyclic esterification either on the "A" or "B" ring, and double bonds were replaced by imine bonds and isometric heterocycles such as naphthyl and imidazole, or synthetic resveratrol oligomers. The structures, synthetic routes, and evaluation of the biological activities of these compounds are discussed. These are aimed at providing some references for the study of resveratrol derivatives in anti-breast cancer treatment.

Cryptococcal pleuritis with pleural effusion as the only clinical presentation in a patient with hepatic cirrhosis: A case report and literature review.

RATIONALE: Cryptococcosis is a significant life-threatening fungal infection in worldwide, mainly reported in immunocompromised patients. Pleural effusion presentation of cryptococcal infection as the only clinical presentation is rarely seen in pulmonary cryptococcosis, which may lead to be misdiagnosed, and the study on this subject will provide further insights. PATIENT CONCERNS: A 64-year-old man was hospitalized in our department and diagnosed as hepatic B cirrhosis. A computed tomography (CT) of the thorax showed a massive right pleural effusion without pulmonary parenchymal abnormalities. He was started on empirical treatment for pleural tuberculosis (TB). However, during his hospitalization, a right pleural effusion developed and fever was not controlled. DIAGNOSES: On day 14 admission, pleural fluid cultured positive for Cryptococcus neoformans. The C neoformans isolate belonged to ST5 and molecular type VNI (var. grubii). INTERVENTIONS: The patient was diagnosed with cryptococcal pleuritis, then amphotericin B and fluconazole were administrated. OUTCOMES: Finally, the patient was improved and discharged from our hospital. LESSONS: Similar cases in cryptococcal pleuritis patients with pleural effusion as the only clinical presentation in the literature are also reviewed. Through literature review, we recommend that pleural effusion cryptococcal antigen test should be used to diagnose cryptococcal pleuritis to reduce misdiagnosis. The early administration of antifungal drug with activity to Cryptococcus seemed beneficial in preventing dissemination of cryptococcosis.

Optimal Time and Target for Evaluating Energy Delivery after Adjuvant Feeding with Small Bowel Enteral Nutrition in Critically Ill Patients at High Nutrition Risk.

Small bowel enteral nutrition (SBEN) may improve nutrient delivery to critically ill patients intolerant of gastric enteral nutrition. However, the optimal time and target for evaluating SBEN efficacy are unknown. This retrospective cohort study investigates these parameters in 55 critically ill patients at high nutrition risk (modified NUTRIC score ≥ 5). Daily actual energy intake was recorded from 3 days before SBEN initiation until 7 days thereafter. The energy achievement rate (%) was calculated as follows: (actual energy intake/estimated energy requirement) × 100. The optimal time was determined from the day on which energy achievement rate reached >60% post-SBEN. Assessment results were as follows: median APACHE II score, 27; SOFA score, 10.0; modified NUTRIC score, 7; and median time point of SBEN initiation, ICU day 8. The feeding volume, energy and protein intake, and achievement rate (%) of energy and protein intake increased significantly after SBEN (p < 0.001). An energy achievement rate less than 65% 3 days after SBEN was significantly associated with increased mortality after adjusting for confounding factors (odds ratio, 4.97; 95% confidence interval, 1.44⁻17.07). SBEN improves energy delivery in critically ill patients who are still at high nutrition risk after 1 week of stomach enteral nutrition.

Successful treatment of an acute infective endocarditis secondary to fish bone penetrating into left atrium caused by Granulicatella adiacens and Candida albicans: A case report.

RATIONAL: Infective endocarditis caused by a foreign body of the upper digestive tract is rare. We report a rare case of Granulicatella adiacens and Candida albicans coinfection acute endocarditis combined with systematic embolization caused by a fish bone from the esophagus penetrating into the left atrium. PATIENT CONCERN: A 42-year-old woman was admitted to our hospital because of fever, abdominal pain, headache, and right limb weakness. DIAGNOSES: Clinical examination indicated endocarditis and systemic embolisms secondary to a fish bone from the esophagus penetrating into the left atrium. The emergency surgery confirmed the diagnosis. Cultures of blood and vegetation show G adiacens and C albicans. INTERVENTIONS: Antimicrobial therapy lasted 6 weeks after surgery. OUTCOMES: The patient was discharged with excellent condition7 weeks after hospitalization and was well when followed 6 months later. LESSONS: The successful treatment of this patient combines quick diagnosis, timely surgery, and effective antimicrobial regimen. This rare possibility should be kept up in mind in acute infective endocarditis cases.

A fatal case of Trichosporon asahii fungemia and pneumonia in a kidney transplant recipient during caspofungin treatment.

Trichosporon asahii is an emerging opportunistic pathogen that is life-threatening particularly for immunosuppressed patients. Only a few studies have described Trichosporon infection in kidney transplant recipients. This study reports a 67-year-old male kidney transplant recipient who developed fatal fungemia and pneumonia caused by T. asahii during caspofungin treatment. Although funguria is benign, kidney transplant recipients are still at risk of T. asahii fungemia and invasive T. asahii infection even if they are under antifungal therapy, particularly echinocandins.

The effect of non-pharmacological staged interventions on fatigue and dyspnoea in patients with chronic obstructive pulmonary disease: a randomized controlled trial.

The aim of this study was to examine the effect of non-pharmacological staged interventions on fatigue and dyspnoea in patients with chronic obstructive pulmonary disease. A randomized controlled trial was conducted with 64 patients in a tertiary hospital in China from 2010 to 2011. Patients were randomly assigned to the control group (n = 32), who received routine care, and the intervention group (n = 32), who received additional non-pharmacological staged interventions. The Multidimensional Fatigue Inventory and the five-grade Medical Research Council dyspnoea scale were used to collect data at baseline and after 6 weeks. Compared with the control group, patients in the intervention group had significantly lower scores on general fatigue (P < 0.001), physical fatigue (P < 0.001), reduced activity (P < 0.001) and reduced motivation (P = 0.03) and had better relief of dyspnoea (P = 0.02). Our study showed that non-pharmacological staged interventions were effective in relieving fatigue and dyspnoea in patients with chronic obstructive pulmonary disease.

Indocyanine green clearance in evaluating the recovery of liver reserve function after superselective transarterial chemoembolization.

Transarterial chemoembolization (TACE) may ravage normal liver tissues apart from the neoplastic nodules which offset the anti-tumor effect. This study aimed to evaluate the recovery of liver reserve function (LRF) after TACE by indocyanine green (ICG) clearance test and other routine liver function tests. Forty-six newly diagnosed HCC patients who had undergone TACE as the initial treatment from January 2011 to January 2012 were enrolled in this study. The effects of age, basic ICG clearance rate and interval time between two assessments on the recovery of LRF were analyzed. We found that ICG retention rate at the 15 minutes (ICGR15) was significantly increased after TACE (12.3+/-8.1% vs 16.8+/-12.1%, P<0.01) in all the 46 patients. In particular, the ICGR15 value was increased in older patients (age>55 years, 20.3+/-12.5% vs 13.7+/-7.2%, P<0.01). The interval of ICG test also affected the ICGR15 value (≤47 days, 17.8+/-11.4% after vs 12.1+/-7.1% before TACE, P<0.01). Our data suggested that TACE decreased LRF, especially in older patients. ICG test was more sensitive to evaluate the recovery of LRF after TACE than the Child-Pugh grade and routine liver function tests.

The impact of body weight management in chronic kidney disease patients with obesity.

OBJECTIVE: Chronic kidney disease (CKD) and obesity are important public health concerns. Because obesity may initiate and/or accelerate kidney damage, weight control may benefit CKD patients. DESIGN AND METHODS: We examined the influence of dietary management and physical exercise in 38 obese CKD patients with or without target reduction of body weight 3% or more from baseline. RESULTS: After a 2-month lifestyle intervention program, those with target body weight control had significant improvement of blood pressure control, as well as reduced lipid profiles, serum creatinine level (1.1 ± 0.3 vs. 0.8 ± 0.3; P < .001), estimated glomerular filtration rate (75.9 ± 21.2 vs. 104.9 ± 38.1; P < .001), and proteinuria (76.3% vs. 50.0%; P = .02). They had greater improvement in cardiorespiratory endurance in an 800-m running test (375.1 ± 64.7 vs. 327.1 ± 84.0 seconds; P = .001), better abdominal muscle strength and endurance in a timed sit-up test (13.6 ± 9.1 vs. 19.9 ± 9.2 number/minute; P = .005), and greater flexibility in a sit-and-reach test (18.8 ± 10.9 vs. 27.8 ± 10.9 cm; P < .001) comparing baseline and postintervention values. CONCLUSIONS: A combination of dietary management and exercise were associated with improvements in health-related physical fitness, cardiovascular risk factors (blood pressure and lipid control), and renal profiles in obese CKD patients. Supportive individualized programs for lifestyle change could exert beneficial effects, but long-term research with a larger patient population is needed to elucidate the optimal effective combination of dietary management and exercise.

Quantification of cytomegalovirus glycoprotein Bn DNA in hematopoietic stem cell transplant recipients by real-time PCR.

Based on sequence variation in the N-terminus of the UL55 gene, which encodes glycoprotein B (gB), human cytomegalovirus (CMV) can be classified into four gBn genotypes. We assessed the distribution of CMV gBn genotypes and the correlation between CMV gBn DNA (detected by real-time PCR) and CMV-positive pp65 cells (identified by immunohistochemical staining) in a cohort of hematopoietic stem cell transplant patients. The distribution of gB genotypes was as follows: gBn1, 60% of patients; gBn2, 13.3%; mixed gBn1 and gBn3 infection, 26.7%; and gBn4 and other mixed infections, 0%. CMV gBn1 was the most common genotype. The detected level of CMV gB DNA correlated well with the number of CMV-positive pp65 cells detected by immunostaining (r = 0.585).

Bitter melon seed oil-attenuated body fat accumulation in diet-induced obese mice is associated with cAMP-dependent protein kinase activation and cell death in white adipose tissue.

The aim of this study was to investigate the antiadiposity effect of bitter melon seed oil (BMSO), which is rich in the cis-9, trans-11, trans-13 isomer of conjugated linolenic acid. In Expt. 1, C57BL/6J mice were fed a butter-based, high-fat diet [HB; 29% butter + 1% soybean oil (SBO)] for 10 wk to induce obesity. They then continued to receive that diet or were switched to an SBO-based, high-fat diet alone (HS; 30% SBO) or containing bitter melon seed oil (BMSO) (HBM; 15% SBO + 15% BMSO) for 5 wk. The body fat percentage was significantly lower in mice fed the HBM diet (21%), but not the HS diet, compared with mice fed the HB diet. In Expt. 2, mice were fed an SBO-based, high-fat diet containing 0 (HS), 5 (LBM), 10 (MBM), or 15% (HBM) BMSO for 10 wk. In the LBM, MBM, and HBM groups, the body fat percentage was significantly lower by 32, 35, and 65%, respectively, compared with the HS control. The reduction in the HBM group was significantly greater than that in the LBM or MBM group. BMSO administration increased phosphorylation of acetyl-CoA carboxylase, cAMP-activated protein kinase (PKA), and signal transducer and activator of transcription 3 in the white adipose tissue (WAT), suggesting that PKA and leptin signaling might be involved in the BMSO-mediated reduction in lipogenesis and increase in thermogenesis and lipolysis. However, compared with the HS control, the HBM group had a significantly higher TNFα concentration in the WAT accompanied by TUNEL-positive nuclei. We conclude that BMSO is effective in attenuating body fat accumulation through mechanisms associated with PKA activation and programmed cell death in the WAT, but safety concerns need to be carefully addressed.

Dietary oxidised frying oil causes oxidative damage of pancreatic islets and impairment of insulin secretion, effects associated with vitamin E deficiency.

We previously reported that, in rodents, a diet with a high oxidised frying oil (OFO) content leads to glucose intolerance associated with a reduction in insulin secretion. The present study aimed at investigating the impairment of pancreatic islets caused by dietary OFO. C57BL/6J mice were divided into three groups to receive a low-fat basal diet containing 5 g/100 g of fresh soyabean oil (LF group) or a high-fat diet containing 20 g/100 g of either fresh soyabean oil (HF group) or OFO (HO group). After 8 weeks, mice in the HO group showed glucose intolerance and hypoinsulinaemia, and their islets showed impaired glucose-stimulated insulin secretion (P < 0·05; HO group v. LF and HF groups). Significantly higher oxidative stress and a lower mitochondrial membrane potential were observed in the islets in the HO group compared with the LF and HF groups. Immunoblots showed that the reduction in insulin levels in HO islets was associated with activation of the c-Jun NH2-terminal kinase and a reduction in levels of pancreatic and duodenal homeobox factor-1. In a second study, when dietary OFO-induced tissue vitamin E depletion was prevented by large-dose vitamin E supplementation (500 IU(1·06 mmol all-rac-α-tocopherol acetate)/kg diet; HO+E group), the OFO-mediated reduction in islet size and impairment of glucose tolerance and insulin secretion were significantly attenuated (P < 0·05; HO group v. HO+E group). We conclude that a high level of dietary OFO ingestion impairs glucose metabolism by causing oxidative damage and compromising insulin secretion in pancreatic islets, and that these effects can be prevented by vitamin E supplementation.

Clinical significance of C-reactive protein values in antibiotic treatment for pyogenic liver abscess.

AIM: To investigate the clinical significance of C-reactive protein (CRP) values in determining the endpoint of antibiotic treatment for liver abscess after drainage. METHODS: The endpoints of antibiotic treatment in 46 patients with pyogenic liver abscess after complete percutaneous drainage were assessed by performing a retrospective study. After complete percutaneous drainage, normal CRP values were considered as the endpoint in 18 patients (experimental group), and normal body temperature for at least 2 wk were considered as the endpoints in the other 28 patients (control group). RESULTS: The duration of antibiotic treatment after complete percutaneous drainage was 15.83 ± 6.45 d and 24.25 ± 8.18 d for the experimental and the control groups, respectively (P = 0.001), being significantly shorter in the experimental group than in the control group. The recurrence rate was 0% for both groups. However, we could not obtain the follow-up data about 3 patients in the control group. CONCLUSION: CRP values can be considered as an independent factor to determine the duration of the antibiotic treatment for pyogenic liver abscess after complete percutaneous drainage.

[Association of post transplantation diabetes mellitus to active human cytomegalovirus infection and preoperative hepatitis B virus infection: analysis of 75 cases].

OBJECTIVE: To investigate the relationship of post transplantation diabetes mellitus (PTDM) to active human cytomegalovirus (HCMV) infection and to preoperative hepatitis B virus (HBV) infection respectively. METHODS: The clinical data of 75 patients with liver transplantation was collected to analyze the association of PTDM with active HCMV infection and preoperative HBV infection. RESULTS: The incidence of PTDM was 17.3% (13/75). The incidence of PTDM in the patients with active HCMV infection after liver transplantation was 23.1% (12/52) significantly higher than that of the patients without active HCMV infection (4.3%, 1/23, P < 0.05). The incidence of PTDM in the patients with preoperative HBV infection was 21.1%, not significantly different from that of the patients without infection (5.6%, P > 0.05). CONCLUSION: PTDM may be related to active HCMV infection after liver transplantation, and unrelated to preoperative HBV infection.

Abnormal cellular translocation of alpha-internexin in spinal motor neurons of Dystonia musculorum mice.

Dystonia musculorum (dt) is a mutant mouse with hereditary neuropathy where the dysfunction is mainly found in the dorsal root ganglia (DRG) neurons but not in the spinal motor neurons. However, the accumulation of intermediate filament (IF) proteins in the swelling axons of spinal motor neurons could be found in dt/dt mice. In order to understand the pathological role of neuronal IFs in the swelling axons of spinal motor neurons from dt/dt mice, we extensively examined the distribution of neuronal IF proteins. By immunofluorescence staining, our results indicated that alpha-internexin was a major component in the swelling axon and showed abnormal translocation in the nuclei of spinal motor neurons in dt/dt mice. This abnormal translocation of alpha-internexin in the nuclei of spinal motor neurons was also confirmed by Western blotting and immunoelectron microscopy. Instead of the 10-nm filamentous structure, a diffuse immunopositive pattern of alpha-internexin was observed in the nucleus of spinal motor neurons in dt/dt mutants. We further examined the cell death of spinal motor neurons by TUNEL assay, and no TUNEL-positive cells could be identified from spinal motor neurons in dt/dt mice. From these observations we suggest that abnormal accumulation of neuronal IFs in the swelling axons and abnormal translocation of alpha-internexin in the nuclei of the spinal motor neurons from dt/dt mice may not directly cause cell death of the spinal motor neurons.

Association of cytomegalovirus infection with human leukocyte antigen genotypes in recipients after allogeneic liver transplantation.

BACKGROUND: Cytomegalovirus (CMV) infection is the important cause affecting the survival rate and function of the transplanted organ after transplantation. The occurrence of CMV infection after liver transplantation (LT) is associated with many factors. Lots of studies suggest that genetic mutation between hosts and CMV may play a role in the occurrence and development of CMV infection. CMV exists in an incubative state, affect or destroy the expression of human leukocyte antigen (HLA) molecules in the host cell surface, and interfere antigen's submission. This mechanism is the key of CMV to avoid immune defense mechanism of the host. To detect HLA and CMV antibody (CMV-Ab), CMV antigen (CMV-Ag) of transplantation recipients, we evaluated the association of CMV infection and the particular HLA genotypes in recipients after LT. METHODS: 277 blood samples were collected from 39 LT recipients. CMV antibody and antigen were detected by ELISA or immunohistochemical methods. The HLA types of the recipients were determined by PCR. To analyze the association of HLA alleles and the occurrence of CMV antigenemia in the patients, relative risk degree (RR) was used as the parameter for the Chi-square test. RESULTS: The LT recipients were serum CMV IgG positive (100%), but none of them was CMV IgM positive (0%). Thirty-three LT recipients (84.6%) were CMV antigenic positive with 1-50 positive leukocytes per 50,000 leukocytes in extent and 7.2+/-4.2 positive leukocytes per 50,000 leukocytes on average. Thirteen patients developed CMV pneumonia, with CMV antigenic positive (100%) and 17.7+/-5.5 positive leukocytes per 50,000 leukocytes on average. Some HLA alleles were associated with the occurrence and extent of CMV antigenemia. HLA-A2 was the higher frequency allele for patients with antigenemia (P<0.05), and 7 patients carrying HLA-DR11 allele developed antigenemia (P<0.05). In the lower antigenemia group, HLA-A11 was higher in frequency than others (P<0.05). Besides, none of the patients carrying HLA-B16 allele developed clinical symptoms of CMV infection (P<0.05). CONCLUSIONS: The variability of HLA alleles might modulate immune response to CMV infection. HLA examination before transplantation should be made for prevention and treatment of CMV infection after operation.

Peroxisome proliferation in liver of rats fed oxidized frying oil.

Oxidized frying oil (OFO) activates peroxisome proliferator-activated receptor a (PPAR alpha) in vitro and in vivo. As most PPARalpha activators are also peroxisome proliferators (PP), this study was aimed at exploring whether OFO induces peroxisome proliferation in the liver of rats. Four groups of male weanling Sprague-Dawley rats were fed the following diets for 6 wk: a basal diet containing 5 g/100 g fresh soybean oil (LSB), high-fat diets containing 20 g/100 g of fresh soybean oil (HSB as a control). OFO (HO) or fish oil (HF, as a positive control). Hepatomegaly and peroxisome proliferation in the liver of the HO group of rats were higher than those of the HF group. In addition, the acyl-CoA oxidase (ACO) activity, as well as cytochrome P450 4A (CYP4A) protein content in the livers of the HO group were 6 fold those of the HSB group, but were 2.5 fold in those of the HF group. These results indicated that dietary OFO induced typical responses to PPARalpha signaling. Moreover. as a dietary source, the OFO prepared under our frying conditions appears to be a more potent peroxisome proliferator than fish oil.