CD4+ CD25+ FOXP3+ T cell frequency in the peripheral blood is a biomarker that distinguishes intestinal tuberculosis from Crohn's disease.

BACKGROUND: Distinguishing between Crohn's Disease (CD) and Intestinal Tuberculosis (ITB) has been a challenging task for clinicians due to their similar presentation. CD4+FOXP3+ T regulatory cells (Tregs) have been reported to be increased in patients with pulmonary tuberculosis. However, there is no such data available in ITB. The aim of this study was to investigate the differential expression of FOXP3+ T cells in patients with ITB and CD and its utility as a biomarker. METHODS: The study prospectively recruited 124 patients with CD, ITB and controls: ulcerative colitis (UC) and patients with only haemorrhoidal bleed. Frequency of CD4+CD25+FOXP3+ Tregs in peripheral blood (flow cytometry), FOXP3 mRNA expression in blood and colonic mucosa (qPCR) and FOXP3+ T cells in colonic mucosa (immunohistochemistry) were compared between controls, CD and ITB patients. RESULTS: Frequency of CD4+CD25+FOXP3+ Treg cells in peripheral blood was significantly increased in ITB as compared to CD. Similarly, significant increase in FOXP3+ T cells and FOXP3 mRNA expression was observed in colonic mucosa of ITB as compared to CD. ROC curve showed that a value of >32.5% for FOXP3+ cells in peripheral blood could differentiate between CD and ITB with a sensitivity of 75% and a specificity of 90.6%. CONCLUSION: Phenotypic enumeration of peripheral CD4+CD25+FOXP3+ Treg cells can be used as a non-invasive biomarker in clinics with a high diagnostic accuracy to differentiate between ITB and CD in regions where TB is endemic.

Long-term follow-up reveals high incidence of colorectal cancer in Indian patients with inflammatory bowel disease.

BACKGROUND: As the magnitude of sporadic colorectal cancer (CRC) in India is low, magnitude of CRC in ulcerative colitis (UC) is also considered low. As a result, screening for CRC in UC although advocated may not be followed everywhere. We report our data of UC-related CRC from a low-incidence area of sporadic CRC. METHODS: A total of 1012 patients with left-sided colitis/pancolitis having more than one full-length colonoscopy performed at least a year after the onset of symptoms were included in retrospective analysis of prospectively maintained case records. In addition, 136 patients with duration of disease >10 years underwent surveillance white-light colonoscopy prospectively during the study period. RESULTS: A total of 1012 individuals were finally included (6542 person-years of follow-up, 68.5% males, disease duration: 6.4 ± 6.8 years). Twenty (1.97%) patients developed CRC. Two (10%) patients developed CRC during the first decade, 10/20 (50%) during the second and 8/20 (40%) after the second decade of disease. The cumulative risk of developing CRC was 1.5%, 7.2% and 23.6% in the first, second and third decade, respectively. Of 136 high-risk UC cases, five (3.6%) had CRC on screening colonoscopy. Disease duration and increasing age of onset were associated with higher risk of CRC. CONCLUSIONS: Cumulative risk of CRC in Indian UC patients is as high as 23.6% at 30 years. The risk of CRC increases with increasing age of onset and increasing duration of disease. A low risk of sporadic CRC does not confer a low risk of UC-related CRC, and regular screening is warranted.

Evaluation of Xpert MTB/RIF assay performance in the diagnosis of abdominal tuberculosis.

BACKGROUND/AIMS: The use of genetic probes for the diagnosis of pulmonary tuberculosis (TB) has been well described. However, the role of these assays in the diagnosis of intestinal tuberculosis is unclear. We therefore assessed the diagnostic utility of the Xpert Mycobacterium tuberculosis/rifampicin (MTB/RIF) assay, and estimated the prevalence of multidrug-resistant (MDR) TB in the Indian population. METHODS: Of 99 patients recruited, 37 had intestinal TB; two control groups comprised 43 with Crohn's disease (CD) and 19 with irritable bowel syndrome. Colonoscopy was performed before starting any therapy; mucosal biopsies were subjected to histopathology, acid-fast bacilli staining, Lowenstein-Jensen culture, and nucleic acid amplification testing using the Xpert MTB/RIF assay. Patients were followed up for 6 months to confirm the diagnosis and response to therapy. A composite reference standard was used for diagnosis of TB and assessment of the diagnostic utility of the Xpert MTB/RIF assay. RESULTS: Of 37 intestinal TB patients, the Xpert MTB/RIF assay was positive in three of 37 (8.1%), but none had MDR-TB. The sensitivity, specificity, positive predictive value, and negative predictive value of the Xpert MTB/RIF assay was 8.1%, 100%, 100%, and, 64.2%, respectively. CONCLUSIONS: The Xpert MTB/RIF assay has low sensitivity but high specificity for intestinal TB, and may be helpful in endemic tuberculosis areas, when clinicians are faced with difficulty differentiating TB and CD. Based on the Xpert MTB/RIF assay, the prevalence of intestinal MDR-TB is low in the Indian population.

Role of random biopsies in surveillance of dysplasia in ulcerative colitis patients with high risk of colorectal cancer.

BACKGROUND/AIMS: Recent data suggest that the incidence of ulcerative colitis (UC) related colorectal cancer (CRC) in India is similar to that of West. The optimum method for surveillance is still a debate. Surveillance with random biopsies has been the standard of care, but is a tedious process. We therefore undertook this study to assess the yield of random biopsy in dysplasia surveillance. METHODS: Between March 2014 and July 2015, patients of UC attending the Inflammatory Bowel Disease clinic at the All India Institute of Medical Sciences with high risk factors for CRC like duration of disease >15 years and pancolitis, family history of CRC, primary sclerosing cholangitis underwent surveillance colonoscopy for dysplasia. Four quadrant random biopsies at 10 cm intervals were taken (33 biopsies). Two pathologists examined specimens for dysplasia, and the yield of dysplasia was calculated. RESULTS: Twenty-eight patients were included. Twenty-six of these had pancolitis with a duration of disease greater than 15 years, and two patients had associated primary sclerosing cholangis. No patient had a family history of CRC. The mean age at onset of disease was 28.89±8.73 years and the duration of disease was 19.00±8.78 years. Eighteen patients (64.28%) were males. A total of 924 biopsies were taken. None of the biopsies revealed any evidence of dysplasia, and 7/924 (0.7%) were indefinite for dysplasia. CONCLUSIONS: Random biopsy for surveillance in longstanding extensive colitis has a low yield for dysplasia and does not suffice for screening. Newer techniques such as chromoendoscopy-guided biopsies need greater adoption.

Sclerosing mesenteric panniculitis in a young patient : common cause of diagnostic dilemma and treatment refractoriness.

BACKGROUND: Sclerosing mesenteric panniculitis (SMP) is an idiopathic chronic fibroinflammatory disorder of the intra-abdominal fat. CASE PRESENTATION: Herin, we report a case of SMP, involving the omentum, mesentery and peri-colic fat in a 18 year old male, who presented with significant and recurrent abdominal distension for 4.5 years. Computed tomogram revealed ascites, with nodular and irregular omental thickening and foci of calcification. Non-specific radiological and histological features made an accurate diagnosis extremely difficult. After a thorough work up and exclusion of other differentials, diagnosis of a nodular SMP (Weber Christian disease) was given. After showing resistance to chemotherapeutic agents, slow response was noted with cyclophosphamide, followed by rapid symptomatic improvement with mesenterectomy. CONCLUSION: SMP is an uncommon benign mesenteric/ omental inflammation, and is a diagnosis of exclusion. As treatment refractoriness is common, management should be individualized and continued for along period. Surgical omentectomy may be helpful.

An unusual cause of reversible axonal neuropathy and hypertension in a 10-year-old girl.

A 10-year-old girl, who was referred with refractory epilepsy, had 1.5 years of episodic abnormal behavior. On examination, she also had hypertension and peripheral neuropathy. Hypoglycemia with correspondingly high insulin levels was documented during a confusional episode. MRI of the abdomen revealed an islet cell tumor in the body of the pancreas. One year after tumor excision, both the neuropathy and hypertension showed remarkable improvement. A final diagnosis of insulinoma with hypoglycemic axonal neuropathy and hypertension (reversed with tumor excision) was made. Insulinoma is the commonest cause of hyperinsulinemic hypoglycemia in adults, but is rare in childhood. To our knowledge, distal symmetrical motor-sensory axonal neuropathy has been described in only 40 patients, and hypertension has not been reported with insulinoma.

Evaluation of epithelial proliferation in paediatric and adult cholesteatomas using the Ki-67 proliferation marker.

INTRODUCTION: The aggressiveness of cholesteatoma in children compared with adults is well known. However, the factors influencing the poorer prognosis of paediatric cholesteatoma are not well understood. This study compared the proliferative potential of paediatric cholesteatoma with that of adult cholesteatoma, using Ki-67 as a proliferation marker. METHODS: A prospective study of 67 patients with aural cholesteatoma was performed. Thirty-eight adult and 29 paediatric cases were evaluated using clinical parameters including bone erosion, complications and extent of disease. A surgical specimen underwent histological evaluation and measurement of the proliferation index using Ki-67 labelling. Normal epithelium from a control group was also examined. RESULTS: Cholesteatoma epithelium has a greater rate of proliferation than normal skin. There were however no statistical differences between the paediatric and adult cholesteatoma groups in terms of clinical behaviour or proliferation potential. Paediatric cholesteatoma was similar to adult cholesteatoma in terms of complications, bone erosion and disease spread. CONCLUSION: Cholesteatoma is a disorder of epithelial proliferation. Although postulated to be more aggressive in children than adults, this study found no clinicopathological differences between paediatric and adult cases.

Liver and portal histopathological correlation with age and survival in extra hepatic biliary atresia.

AIM: To grade histopathological parameters in biliary atresia (BA) and correlate it with advancing age and outcome. METHOD: Liver and porta hepatis biopsies of 18 BA cases were graded for cholestasis (C), hepatocellular degeneration (HD), bile duct proliferation (BDP), bile duct fibrosis (BDF), bile duct inflammation (BDI), portal edema (PE), portal inflammation (PI) and portal fibrosis (PF) with a total histopathological score (THS) of 25. Confidence intervals were calculated. Image analysis at porta hepatis for largest subhepatic bile ductule diameter (BDD) and area was correlated with liver histopathology. Patients were followed up to at least 1 year after surgery. Four cases of neonatal autopsies were taken as control. RESULTS: Mean age at Kasai was 95.5 + 32.03 (51-172) days. Mean THS was 16.83 + 2.57 (13-23) with no significant difference in relation to age <90; >90 days (p = 0.4410) or age <60; 61-90; 91-120; >121 days (p = 0.6549). There was no significant difference with relation to age in grading of parameters; C (p = 1.000) HD (p = 0.638), BDP (p = 0.530), BDF (p = 0.342), BDI (p = 0.395), PE (p = 0.476), PI (p = 0.342), PF (p = 0.530), BDD (p = 0.1097) and area (p = 0.1097). There was no significant difference between survival and age at operation (p = 0.4178) or THS (p = 0.3025). CONCLUSION: Advanced histopathological findings even at younger age suggests the need to consider BA as an emergency.

Dissipative quantum systems and the heat capacity.

We present a detailed study of the quantum dissipative dynamics of a charged particle in a magnetic field. Our focus of attention is the effect of dissipation on the low- and high-temperature behaviors of the specific heat at constant volume. After providing a brief overview of two distinct approaches to the statistical mechanics of dissipative quantum systems, viz., the ensemble approach of Gibbs and the quantum Brownian motion approach due to Einstein, we present exact analyses of the specific heat. While the low-temperature expressions for the specific heat, based on the two approaches, are in conformity with power-law temperature dependence, predicted by the third law of thermodynamics, and the high-temperature expressions are in agreement with the classical equipartition theorem, there are surprising differences between the dependencies of the specific heat on different parameters in the theory, when calculations are done from these two distinct methods. In particular, we find puzzling influences of boundary confinement and the bath-induced spectral cutoff frequency. Further, when it comes to the issue of approach to equilibrium, based on the Einstein method, the way the asymptotic limit (t-->infinity) is taken seems to assume significance.

Menstruation and suicide: a histopathological study.

BACKGROUND: Previous research, albeit limited, has reported mixed findings on the impact of menstruation cycle on suicidal behavior. The contribution of menstruation to completed suicide is also controversial; the studies are, in fact, very limited and are not carefully designed. AIMS: To examine whether the menstruation cycle impacts on suicide. METHODS: In order to explore this relationship, 56 autopsies on completed suicides in females were performed and matched to a control group of 44 females who had died from other causes, at the All India Institute of Medical Sciences in New Delhi. Histopathological examination, a method of collecting tissue from the uterus through biopsy, was used to determine the stage of the menstrual cycle. RESULTS: The results show that 25% of women who had died by suicide were menstruating at the time, compared to 4.5% of the control group; this is statistically (chi2) significant at the p < .002 level. CONCLUSIONS: Menstruation in the women who completed suicide, compared to a control group, appeared to have an association, though more research is warranted. Not only there are serious methodological problems in the study of menstruation and suicide (largely because of problematic tissue storage and examination), but also because of the need to understand the impact within a larger psychological, social, and cultural frame.

Evaluation of ultrastructural changes by electron microscopy in neonatal cholestasis.

BACKGROUND: Biliary atresia (BA) and idiopathic neonatal hepatitis (NH) account for 50-70% of all cases with neonatal cholestasis. The treatment of the former is early surgical intervention, while the latter requires non-surgical supportive care. Failure to differentiate the two conditions may result in avoidable surgery in NH, which may significantly increase morbidity. The lack of differentiating clinical features, biochemical markers and other specific investigations to distinguish the two is still a major problem. AIM: This study was thus initiated to evaluate electron microscopic changes in the liver in patients with NH and BA, to correlate these with changes on light microscopy and look for specific differentiating features between the two. METHODS: Ten patients with neonatal cholestasis whose liver specimens were available for electron microscopic analysis were included in the study. There were 6 patients with BA and 4 patients with NH. RESULTS: Among the biochemical parameters, serum alkaline phosphatase and gamma glutamyl transpeptidase were significantly higher in BA than in patients with NH. On light microscopy, giant cell transformation was seen in 75% patients with NH and 33.3% of patients with BA. Even in BA, intracellular cholestasis was more prominent than ductular cholestasis (100% vs. 50%). Ductular proliferation was seen in 50% of NH patients and all patients of BA. Electron microscopy revealed prominent endoplasmic changes in all patients with NH and to a milder degree in BA. Changes in mitochondria and glycogen content were similar in both groups. CONCLUSION: Ultrastructural changes in neonatal cholestasis seen through electron microscopy are largely non-specific and do not differentiate BA from NH.

Potential of magnetic resonance spectroscopic imaging in predicting absence of prostate cancer in men with serum prostate-specific antigen between 4 and 10 ng/ml: a follow-up study.

OBJECTIVES: Screening for prostate cancer using serum prostate-specific antigen (PSA) determination has a positive predictive value of only 30% to 42% for a PSA level between 4 and 10 ng/mL. Magnetic resonance spectroscopic imaging (MRSI), which identifies cancer on the basis of changes in cellular metabolite levels, might be able to identify patients with noncancerous PSA elevation and help avoid unnecessary biopsies. We tested this hypothesis by evaluating the incidence of prostate cancer in men with a PSA level of 4 to 10 ng/mL and a negative MRSI study. METHODS: A total of 155 men underwent a three-dimensional proton MRSI of the prostate before transrectal ultrasound-guided biopsy for clinical indications. MRSI was performed using an endorectal coil on a 1.5-T magnetic resonance scanner. Patients with no voxels positive for malignancy underwent standard sextant biopsy, and additional MRSI-targeted biopsies were obtained in men with suspicious or malignant voxels. Patients with a biopsy negative for cancer underwent repeat serum PSA estimation every 6 months for a minimum of 18 months. RESULTS: Of the 155 men, 36 (mean PSA level of 6.47 ng/mL, range 4.25 to 9.9) had no malignant voxels on MRSI. None of them were positive for cancer on biopsy. Of these 36 men, 26 completed at least 18 months (mean 26.9, range 18 to 44) of follow-up. Four patients required repeat biopsies and one, with a persistently elevated PSA level was diagnosed with prostate cancer 29 months after the initial MRSI. CONCLUSIONS: The results of our study have shown that prostate biopsy can be deferred in patients with an increased serum PSA of 4 to 10 ng/mL if their MRSI does not show any malignant voxels.

Clinical significance of 14-3-3 zeta in human esophageal cancer.

We recently found 14-3-3 zeta to be overexpressed in esophageal squamous cell carcinomas (ESCCs) by differential display. In the present study we determined the clinical significance of 14-3-3 zeta in esophageal tumorigenesis. Immunohistochemical analysis was carried out in 61 ESCCs, 33 dysplasia samples, 14 hyperplasia samples and 7 matched histologically normal esophageal tissues and correlated with clinicopathological parameters. Cytoplasmic expression of 14-3-3 zeta protein was observed in 95% of ESCCs; 63% of tumors also showed nuclear localization. All hyperplastic and dysplastic tissues distant from ESCCs as well as dysplastic endoscopic biopsies showed cytoplasmic immunopositivity for 14-3-3 zeta, while nuclear localization was observed in 58% of dysplasia and 36% of hyperplasia samples. Matched distant histologically normal epithelia either showed basal cytoplasmic expression of 14-3-3 zeta or no detectable nuclear expression of the protein. Interestingly, immunopositivity observed in normal esophageal tissues and early hyperplasia was confined to cytoplasm only, though significant nuclear expression was detected in dysplasia and ESCC. Immunoblotting and RT-PCR analyses further confirmed 14-3-3 zeta expression in dysplasia and ESCC. To our knowledge, this is the first report demonstrating overexpression of 14-3-3 zeta in esophageal hyperplasia, dysplasia and squamous cell carcinoma, suggesting that alteration in its expression occurs in early stages and is associated with esophageal tumorigenesis.

Probing single jumps of surface atoms.

Jumps of single atoms can be followed on their time and space scale (nanoseconds and Angstroms) by applying nuclear resonance scattering of synchrotron radiation. Here we develop the theory for jump diffusion in two-dimensional systems. Two types of phenomena are noteworthy: apparent acceleration of the nuclear decay and relaxation of hyperfine interactions, in particular, electric quadrupole interactions. The latter effect becomes for the first time one of significance and well measurable due to the inherent anisotropy of the surface. We show how, by way of motional narrowing, to distinguish between the motion of the probe atom itself and the motion of adatoms.

Stem cells as a therapeutic modality in pediatric malformations.

AIM: The aim of this study was to explore stem cell use in congenital anomalies. PATIENTS AND METHODS: During July 2005 through July 2006, autologous stem cells were used in 29 patients: 12 with liver cirrhosis and 17 with meningomyelocele. Stem cells were injected into the hepatic artery and the portal vein or into the hepatobiliary radicals for liver cirrhosis, or into the spinal cord and caudal space for meningomyelocele. Preoperative status served as the control condition. OBSERVATIONS AND RESULTS: The ages of patients with liver cirrhosis ranged between 1.5 and 9 months (mean, 4.12 months). The etiology was extra hepatic biliary atresia (EHBA) versus neonatal cholestasis and choledochal cyst in 8; 2 and 2 patients, respectively. Five patients died due to ongoing cirrhosis. Follow-up at 3 to 12 months (N = 7) showed absence of cholangitis (4/7), yellow stools (5/7), decreased liver firmness (3/7), improved liver function (6/7), and better appetite (6/7). Hepatobiliary scan was excretory in 6 of 7 with improved uptake in 4 of 7. Histopathology demonstrated comparative improvement in fibrosis among 3 patients. Meningomyelocele patients were between 0 and 1 month, 1-5 months, and 1-4 years in 5, 8, and 2 cases respectively. Five had a history of rupture. Three had undergone meningocele repair in the past with neurological deficits. Redo surgery for a tethered cord was performed in 1 patient. Follow-up at 3 to 11 months in 14 cases showed improved power in 7 with dramatic recovery in 3 (22%) and status quo in 7 (50%). CONCLUSION: Initial stem cell use in liver cirrhosis and meningomyelocele has suggested beneficial results. However, long-term evaluation in randomized controlled trials is essential to draw further conclusions.

Viral hepatitis in India.

Viral hepatitis is a major public health problem in India, which is hyperendemic for HAV and HEV. Seroprevalence studies reveal that 90%-100% of the population acquires anti-HAV antibody and becomes immune by adolescence. Many epidemics of HEV have been reported from India. HAV related liver disease is uncommon in India and occurs mainly in children. HEV is also the major cause of sporadic adult acute viral hepatitis and ALF. Pregnant women and patients with CLD constitute the high risk groups to contract HEV infection, and HEV-induced mortality among them is substantial, which underlines the need for preventive measures for such groups. Children with HAV and HEV coinfection are prone to develop ALF. India has intermediate HBV endemicity, with a carrier frequency of 2%-4%. HBV is the major cause of CLD and HCC. Chronic HBV infection in India is acquired in childhood, presumably before 5 years of age, through horizontal transmission. Vertical transmission of HBV in India is considered to be infrequent. Inclusion of HBV vaccination in the expanded programme of immunization is essential to reduce the HBV carrier frequency and disease burden. HBV genotypes A and D are prevalent in India, which are similar to the HBV genotypes in the West. HCV infection in India has a population prevalence of around 1%, and occurs predominantly through transfusion and the use of unsterile glass syringes. HCV genotypes 3 and 2 are prevalent in 60%-80% of the population and they respond well to a combination of interferon and ribavirin. About 10%-15% of CLD and HCC are associated with HCV infection in India. HCV infection is also a major cause of post-transfusion hepatitis. HDV infection is infrequent in India and is present about 5%-10% of patients with HBV-related liver disease. HCC appears to be less common in India than would be expected from the prevalence rates of HBV and HCV. The high disease burden of viral hepatitis and related CLD in India, calls for the setting up of a hepatitis registry and formulation of government-supported prevention and control strategies.

Spin dynamics in a dissipative environment: from quantal to classical.

We address the problem of spin dynamics in the presence of a thermal bath, by solving exactly the appropriate quantum master equations with continued-fraction methods. The crossover region between the quantum and classical domains is studied by increasing the spin value , and the asymptote for the classical absorption spectra is eventually recovered. Along with the recognized relevance of the coupling strength, we show the critical role played by the structure of the system-environment interaction in the emergence of classical phenomenology.

Efficacy and tolerability of rifampicin-based rescue therapy for Helicobacter pylori eradication failure in peptic ulcer disease.

In vitro activity of rifampicin has been shown against H. pylori. It has also been reported that the prevalence of H. pylori is low in patients with tuberculosis treated with rifampicin. Clinical trials are required to establish the efficacy of rifampicin as a salvage therapy for eradication of H. pylori. We aimed to evaluate the efficacy of rifampicin-based salvage therapy for eradication of H. pylori in patients with peptic ulcer disease. Twenty-eight patients with peptic ulcer disease who either had failed eradication of H. pylori or had a recurrence of H. pylori following successful eradication were included in the prospective study. The inclusion criteria included one or more failed attempts at eradication and presence of H. pylori infection as evidenced by positivity of at least two of three tests: rapid urease test (RUT), 14C urea breath test (UBT), and histology. The subjects were treated with a 10-day regimen consisting of rifampicin, 450 mg od, tetracycline, 1 g bd, and esomeprazole, 40 mg bd. Four weeks after completion of therapy, H. pylori status was assessed by RUT, 14C, UBT, and histology. Liver function tests were done before and at the end of therapy. The study subjects included 25 males and 3 females with a mean age of 33.7+/-8.92 years (range: 22-65 years). The median duration of symptoms was 42 months, with a range of 1-180 months. The median number of eradication attempts was two, with one prior attempt in 6 (21.4%), two attempts in 19 (67.9%), and three attempts in 3 (10.7%) patients. Successful H. pylori eradication as defined by concomitant negativity of RUT, UBT, and histology with special stains was achieved in 32.1% (9/28) of patients by intention-to-treat and 33.3% (9/27) of patients by per-protocol analysis. This pilot study suggests that rifampicin-based regimes have no role as salvage eradication therapy in refractory cases of H. pylori infection with peptic ulcer disease.

Etiopathogenesis of acute hepatic failure: Eastern versus Western countries.

Etiopathogenesis of acute hepatic failure (AHF) in Eastern and Western countries is distinct. In the East hepatitis viruses cause AHF in more than 95% of such cases, while causes of AHF in the West are quite heterogenous. Hepatitis E virus is the major etiological agent of AHF in countries like India where the virus is hyperendemic. Occult HBV infection may also be causing AHF in a sizable proportion of cases in areas where chronic HBV infection frequency is high. Paracetamol causes AHF in about 70% cases in the UK and about 20% cases in USA, whereas in France and Denmark, non-steroidal anti-inflammatory drugs are more frequently associated with AHF. Hepatitis B virus causes AHF in about one-third of cases in the latter two countries.