쿠싱증후군 환자에서 당 대사 이상 정도에 따른 인슐린 감수성과 인슐린 저항성의 변화

정인경,김성훈,정재훈,민용기,이명식,이문규,유형준,안규정,노정현,김동준,김광원 대한내분비학회 2003 Endocrinology and metabolism Vol.18 No.4

연구배경 당질 코르티코이드는 당 대사에 매우 중요한 호르몬으로 내인성 당질 코르티코이드 과다상태인 쿠싱증후군에서는 말초조직에서 인슐린 저항이 증가하고 이를 보상하고자 인슐린 분비의 증가로 고인슐린혈증이 동반된다고 보고되고 있다. 하지만 생체 내에서와 달리 시험관내에서는 췌도세포에 당질 코르티코이드를 장시간 처리하면, 인슐린 분비 및 생합성이직접적으로 억제됨이 확인된 바 있어 쿠싱증후군 환자에서 당뇨병의 원인으로는 아마도 말초조직에서 증가된 인슐린 저항성 뿐 아니라 이를 충분히 보상하지 못하는 췌장에서의 인슐린 분비 저하가 같이 동반되어있지 않을까 하는 가설을 세우게 되었고, 아직까지 당질코르티코이드가 당대사 이상을 일으키는 기전에 대해 쿠싱증후군을 당대사 정도에 따라 인슐린 감수성과 분비능을 분석한 연구는 없었기에 이를 알아보고자 하였다. 방법: 삼성서울병원에서 쿠싱증후군으로 진단 받은 환자 15명을 대상으로 하였다. 이에 대한 대조군으로는 쿠싱증후군 환자와 같은 성별 그리고 체질량지수를 갖은 15명의 건강한 성인을 대상으로 비교 하였다 쿠싱증후군 환자를 대상으로 경구당부하 검사를 통해 당대사 정도를 정상군, 내당능장애군, 그리고 당뇨병군으로 나눈 후 정맥 당부하 검사를 시행하여 각군의 인슐린 저항성과 인슐린 분비능의 지표를 비교하고, 수술 후 쿠싱증후군이 완치된 상태에서 수술 전후의 당대사 지표의 변화를 조사하였다. 결과: 1) 쿠싱증후군 환자 중 정상인은 20%, 내당능 장애는 27%, 그리고 당뇨병은 53%였다. 체질량지수, 나이, 그리고 발병 기간은 세 군간에 의미 있는 차이가 없었으나, 24시간 소변검사의 코르티솔 농도는 당뇨병군에서 의미있게 높았다. 2) 정맥당부하 검사 결과, 인슐린 감수성 지표인 Sl는쿠싱증추린」서 1.58±0.10[×10^(-4)(min^(-1)(μU/mL)^(-1)]로 정상 대조군의 3.37±0.49[×10^(-4)(min^(-1)(μU/mL)^(-1)]에 비해 의미있게 낮았으나(P=0.024), 쿠싱증후군 환자 중 NGT, IGT, DM 군간에 서로 통계적인 차이는 없었다. 3) SG는 정상 대조군과 쿠싱증후군 환자간에는 의미있는 차이가 없었고, 쿠싱 증후군에 있어서 당대사가 악화될수록 감소하는 경향을 보였으나 의미있는 차이는 없었다. 4) 인슐린 분비능의 지표인 AIRg는 정상인에 비해 전체 쿠싱증후군 환자의 경우 증가하는 경향을 보였으나 의미있는 차이는 없었다. 하지만 쿠싱증후군 환자중에서 당대사 상태에 따라 NGT군은 1299 (1297∼1310)(mu/g/min ×10^(-2))로 정상 대조군(368.9±98.6[mu/g/min ×10^(-2)]) 보다도 의미있게 높았고, DM군{202.2 (91.1~371.4) [mu/g/min ×10^(-2)}은 NGT군에 비해 의미있게 낮았다(P=0.0031). 5) 15명중 현재 완치 상태에 있는 6명에 대해 수술전과 후로 비교하였다. 수술 전 당대사 상태가 1명은정상, 1명은 내당능 장애, 그리고 4명은 당뇨병이었으나 수술 후 시행한 경구 당부하 검사상 모두 정상 당대사 상태를 보였다. 6) 수술 후 완치된 환자 6명에 있어 인슐린 감수성지표인 Sl는 수술전에 중앙값이 1.22[×10^(-4)(min^(-1)(μU/mL)^(-1)]로 대조군에 비해 의미있게 감고』어 있었으나(p.0.05), 수술후 10.95 [×10^(-4)(min^(-1)(μU/mL)^(-1)]로 정상 수준으로 회복되었고(P=0.0022), 인슐린 분비능을 나타내는 AIRg [mu/g/min ×10^(-2)] 값도 정상수준으로 회복되었다. 특히 인슐린 분비능의 회복양상은 혈당농도에 따라 판이하게 나타나서, 정상과 내당능장애 상태에 있던 2명은 수술전에 1201 [mu/g/min ×10^(-2)]로 증가되어 있던 AIRg 값이 수술 후 정상 수준으로 감소하였고, 수술 전에 당뇨병 상태에 있던 4명의 경우 245.9 [mu/g/min ×10^(-2)]로 인슐린 분비능이 감고il어 있었는데 이들은 수술 후 모두 정상 수준으로 증가되었다 (P=0.0286). 결론: 쿠싱증후군 환자에서 당대사 이상은 80%로 높은 유병률을 보였다. 모든 쿠싱증후군환자에서 인슐린 감수성은 정상인에 비해 저하되어 있어 말초조직의 인슐린 저항이 선행됨을 시사하며, 인슐린 분비능은 당대사의 정도에 따라 다르게 나타났는데, 정상 당대사군에서는 인슐린의 저항성을 극복할 만큼 정상 대조군보다 더 많은 양의 인슐린 분비를 하다가 고코르티솔혈증이 심할수록 인슐린 분비능의 감소로 당뇨병으로 진행됨을 확인할 수 있었고, 이런 인슐린 저항성과 인슐린 분비장애는 수술 후 다시 회복되는 가역적인변화를 보였다. Background: Glucocorticoid plays an important role in the control of carbohydrate metabolism. Patients with Cushing's syndrome have been reported to have an increased incidence of carbohydrate intolerance due to peripheral insulin resistance and hyperinsulinemia, although the exact incidence and nature of this disorder have remained unclear. Few results have been published about insulin resistance and insulin secretion according to the level of glucose concentration, or about the reversibility of such defects in patients with Cushing's syndrome. Methods: To assess the effect of glucocorticoid on the insulin sensitivity and insulin secretion in Cushing's syndrome, 15 patients with Cushing's syndrome were classified into 3 groups (normal glucose tolerance: NGT, impaired glucose tolerance: IGT, diabetes: DM) according to the degree of glucose tolerance based on the oral glucose tolerance test (OGTT). Insulin modified, frequently sampled, intravenous glucose tolerance test (FSIGT) was performed before and after curative surgery on these patients and on 15 healthy control subjects. Data were evaluated by non-parametric statistical analysis. Results: 1) Among the 15 patients with Cushing's syndrome, 3 (20%) were NGT, 4 (27%) IGT, and 8 (53%) DM, based on OGTT. Twenty-four hour urinary free cortisol (UFC) was significantly higher in the DM group. 2) Insulin sensitivity index (SI) of Cushing's syndrome was significantly lower than that of the control group p=0.0024), but was not significantly different among the three Cushing's syndrome groups of NGT, IGT and DM. 3) Glucose mediated glucose disposal (SG) (Ed- confirm this abbreviation; it does not seem to match the definition) of Cushing's syndrome was not significantly different from that of the control group. 4) Insulin secretion (AIRg) of Cushing's syndrome tended to be high, but it was not significantly different from that of control. However, according to the level of glucose concentration there was significant difference in AlRg among the three Cushing's syndrome groups p=0.0031); AIRg of DM was significantly lower than that of NGT. 5) After surgical treatment, parameters of insulin sensitivity and insulin secretion were normalized in 6 cured patients; 1 with NGT, 1 with IGT, and 4 with DM, preoperatively. Median SI of all 6 patients was significantly improved up to the normal range postoperatively p=0.0022). Median AIRg of these 6 patients was balanced around that of normal control postoperatively p=0.0286). Conclusion: Eighty percent of patients with Cushing's syndrome had abnormality of carbohydrate metabolism. Insulin sensitivity was significantly decreased in Cushing's syndrome. Insulin secretion was significantly higher only in the NGT and IGT groups of Cushing's syndrome. As the hypercortisolemia is exacerbated, insulin secretion is significantly decreased and causes DM, suggesting that glucocorticoid has a direct or indirect toxic effect on the pancreatic beta cell (J Kor SOC Endocrinol 18:392-403, 2003).

A Comparative Experimental Study of the, In-vitro Efficiency of Hypertonic, Saline-Enhanced Hepatic Bipolar and, Monopolar Radiofrequency Ablation

Lee, Jeong Min,Han, Joon Koo,Kim, Se Hyung,Sohn, Kyu Li,Lee, Kyoung Ho,Ah, Su Kyung,Choi, Byung Ihn Korean Radiological Society 2003 Korean Journal of Radiology Vol.4 No.3

Optimization of Electric Field-assisted Ultrasonication for Disintegration of Waste Activated Sludge

( Kyung-won Jung ),( Tae-un Jeong ),( Min-jung Cha ),( Kyu-hong Ahn ) 한국폐기물자원순환학회(구 한국폐기물학회) 2014 한국폐기물자원순환학회 심포지움 Vol.2014 No.1

In the present study, an electric field is applied in order to disintegrate waste activated sludge (WAS). As a preliminary step, feasibility tests are investigated using different applied voltages of 10 to 100 V for 60 min. As the applied voltage increases, the disintegration degrees (DD) are gradually enhanced, thereby the soluble N, P, and carbohydrate concentrations increase simultaneously due to the protein decomposition. Subsequently, an optimization process is conducted using response surface methodology with a Box-Behnken design (BBD). The total solid concentration, applied voltage, and reaction time are selected as independent variables, while the DD is selected as the response variable. The overall results demonstrate that the BBD with an experimental design can be useed effectively in the optimization of the electric field treatment of WAS. In the confirmation test, a DD of 10.26±0.14% is recorded, which corresponds to 99.1% of the predicted response value under the statistically optimized conditions. Finally, the statistic optimization of the combined treatment (electric field + ultrasonication) showed that even though this is limited to highly disintegrated WAS when being applied individually, a high DD of 47.28±0.20% was recorded where the TS concentration was 6,780 mg/l, the strength of ultrasonication was 8.0 W, the applied voltage was 68.4 V, and the reaction time was 44 min. E-SEM images clearly revealed that the application of the electric field is a significant alternative method for the combined treatment of WAS, and this is first attempt to increase disintegration using electric field for combined treatment with ultrasonication.

부추 추출물에 의한 Escherichia coli 및 Staphylococcus aureus 의 생육 저해효과

이민경(Min Kyung Lee),이정아(Jeong-ah Lee),박인식(Inshik Park) 한국식품영양과학회 2001 한국식품영양과학회지 Vol.30 No.1

식품오염의 지표로 이용되는 Escherichia coli 와 식중독을 일으키는 Staphylococcus aureus에 대한 부추(Allium tuberosum) 추출물의 항미생물 활성을 검토하고, 그 활성의 열안정성 및 pH 안정성을 조사하였다. 부추 추출물은 미생물의 생육을 저해하였으며 특히 Staphylococcus aureus에서 생육 저해환의 면적이 크게 나타났다. 그리고 paper disc에 50 μL의 부추 추출물을 흡수시켰을 때는 Escherichia coli와 Staphylococcus aureus는 각각 440 ㎟, 609 ㎟의 저해면적을 나타내었으며 부추 추출물의 농도가 높을수록 생육 저해가 현저하였다. 또한 부추 추출물을 68℃에서 30분 또는 98℃ 20분간 열처리 후에도 부추 추출물에 의하여 미생물의 생육은 저해되었다. 그리고 부추 추출물을 pH 2.0에서 3시간 보관 후에도 항미생물활성은 비교적 안정하였다. 그러나 부추 추출물의 항미생물 활성은 투석에 의하여 완전히 활성이 없어졌으며, 따라서 부추 추출물에 존재하는 항미생물 활성은 저분자 물질인 것으로 사료된다. The growth retardation of Escherichia coli and Staphylococcus aureus by heat or acid treated leek (Allium tuberosum) extract was observed. Antimicrobial activity of the leek was the most effective when fresh leek extract was used, but it was stable after heat treatment at 68℃ for 30 min or 98℃ for 20 min. It was also relatively stable after incubated at pH 2.0 for 3 hrs. The antimicrobial activity in leek was not detected after dialysis with molecular weight cutoff of 12,000. Therefore it seems to be small molecule with molecular weight lower than 12,000.

Fumaric acid와 mild heat의 병합 처리에 따른 시금치의 저장 중 미생물 제어 효과

손현정 ( Hyeon Jeong Son ),강지훈 ( Ji Hoon Kang ),오덕환 ( Deog Hwan Oh ),민세철 ( Sea Cheol Min ),송경빈 ( Kyung Bin Song ) 한국응용생명화학회 2016 Journal of Applied Biological Chemistry (J. Appl. Vol.59 No.1

시금치에 fumaric acid와 mild heat의 병합처리를 통해 병원성 미생물 제어효과를 규명하고자 시금치에 E. coli O157:H7, L. mon℃ytogenes 를 접종한 후 각 단일처리 후 미생물 수 변화를 측정하였다. Fumaric acid (0.1, 0.3, 0.5%)와 mild heat (40, 50, 60℃)의 각 단일처리 실험 결과를 토대로, 병합처리를 위한 fumaric acid의 최적농도는 0.5%, mild heat 처리조건으로 50℃ 에서 5 min으로 선정하였고, 병합처리 시 L. mon℃ytogenes, E. coli O157:H7의 수는 대조구에 비해 각각 2.53, 2.62 log CFU/g 감소하였다. 그리고 신선한 시금치에 병합처리 후 4℃에서 12일간 저장하면서 미생물 수 감소 및 품질 변화를 조사하였다. 시금치의 초기 미생물 수에 있어서 대조구와 비교하여, 병합 처리구에서 총 호기성 균을 2.77 log CFU/g 감소시켰다. 특히, 저장 12일 후 병합 처리구의 총 호기성 균 수는 4.84 log CFU/g으로 대조구와 비교하여 1.82 log CFU/g의 감균 효과를 가졌다. 또한 시금치의 저장 중 Hunter 색도 값 및 비타민 C 함량에 있어서 처리구 간의 유의적인 차이를 보이지 않았다. 따라서 본 연구 결과, fumaric acid와 mild heat의 병합처리가 시금치의 미생물학적 안전성 유지에 효과적인 처리라고 판단된다. The objective of this study was to examine the combined effect of fumaric acid with mild heat on the inactivation of microorganisms on spinach. Spinach leaves were in℃ulated with Escherichia coli O157:H7 and Listeria mon℃ytogenes. Based on the results of single treatment of fumaric acid (0.1, 0.3, and 0.5%) or mild heat (40, 50, and 60℃) regarding the inactivation of the in℃ulated bacteria, the optimal condition for the combined treatment was suggested to be 0.5% fumaric acid and mild heat treatment at 50℃ for 5 min. The combined treatment of fumaric acid with mild heat caused 2.53 and 2.62 log reductions of the populations of L. mon℃ytogenes and E. coli O157:H7, respectively. In addition, during storage of fresh spinach at 4℃ for 12 d, the combined treatment reduced initially the populations of total aerobic bacteria by 2.77 log CFU/g compared with the control. In particular, after 12 d of storage, the population of total aerobic bacteria for the combined treatment sample was 4.84 log CFU/g, whereas the control sample had 6.66 log CFU/g. Color and vitamin C content of spinach samples were not altered significantly by the combined treatment during storage. These results indicate that the combined treatment of fumaric acid with mild heat is an effective method to control microorganisms on spinach during storage.

헬스케어용 가다랑어(Katsuwonus pelamis) 통조림의 제조 및 특성

김현정 ( Hyeon Jeong Kim ),김민지 ( Min Ji Kim ),김기현 ( Ki Hyun Kim ),지성준 ( Seung Jun Ji ),임경훈 ( Kyung Hun Lim ),박권현 ( Kwon Hyun Park ),신준호 ( Joon Ho Shin ),허민수 ( Min Soo Heu ),김진수 ( Jin Soo Kim ) 한국수산과학회 2012 한국수산과학회지 Vol.45 No.3

This study was conducted to prepare canned skipjack tuna Katsuwonus pelamis in the mixture of isotonic beverage and tomato paste (ST-S) for use as a health food. An analysis of the time-temperature profile and viable cells showed that a reasonable F0 value for ST-S preparation was 4 min. The proximate composition of ST-S was 76.8% moisture, 20.2% crude protein, 0.8% crude lipid, 1.7% ash and 0.5% carbohydrate. The calorie content of ST-S was 94.8 kcal, which is 47.4% lower than that of commercial canned skipjack tuna in oil (ST-O) and 2.2% lower than that of commercial chicken breast in water (CB-B). The total amino acid content of ST-S was 18.54 g/100 g, which is 31.4% lower than that of ST-O and 7.9% lower than that of CB-B. The major amino acids in ST-S were aspartic acid and glutamic acid. An enrichment effect due to such minerals as phosphorus, potassium and iron would be expected on consuming 100 g of ST-S. The major fatty acids in ST-S were 16:0 (27.4%), 18:1n-9 (14.3%) and 22:6n-3 (27.8%), which are different from those in ST-O and CB-B. The major free amino acids in ST-S were glutamic acid (8.1%), histidine (38.6%) and its related dipeptide, such as anserine (15.7%). In an evaluation of taste, flavor and color, ST-S was found to be superior to ST-O and CB-B.

인체혈장 중 에탐부톨의 HPLC 분석법의 검증 및 단일용량 투여에 의한 약물동태 연구

곽혜선,박경호,최준식,송진아,성민경,장정옥,이화정 한국약제학회 2005 Journal of Pharmaceutical Investigation Vol.35 No.2

An HPLC method was employed for the determination of ethambutol in human plasma. After addition of internal standard (IS, octylamine, 2 μg/mL) and alkalinization of the plasma with 5 M sodium hydroxide, the drug and IS were extracted into the mixture of chloroform and diethyl ether (40:60, v/v). Following a 15-min vortex-mixing and a 10-min centrifugation, the organic phase was spiked with 100 pL of phenylethylisocyanate (2000 μg/mL) for chemical derivatization, mixed for 5 min and evaporated to dryness under a stream of nitrogen. The residue was reconstituted with 100 μL of mobile phase and 20 pL was injected into Cl8 column with a mobile phase consisting of methanol:water (70:30, v/v). The samples were detected utilizing an ultraviolet detector at 200 nm. The method was specific and validated with a limit of 0.15 μg/mL. Infra- and inter-day precision and accuracy were acceptable for all quality control samples including the lower limit of quantification. The applicability of this method was demonstrated by analysis of human plasma after oral administration of a single 1200-mg dose to 20 healthy subjects. From the plasma ethambutol concentration vs. time curves, the mean AUC was 9.61 ± 1.64 μg hr/mL. and Cmax of 2.68 μg/mL reached 2.73 hr after administration. The mean biological half-life of ethambutol was 3.46 ± 1.21 hr. Based on the results, this simple and validated assay could readily be used in any pharmacokinetic studies using humans.

단신 : 실리콘의 염소화반응에 의한 사염화규소 제조

박균영 ( Kyun Young Park ),이미선 ( Mi Sun Lee ),김민철 ( Min Cheol Kim ),이찬희 ( Chan Hee Lee ),박회경 ( Hoey Kyung Park ),강태원 ( Tae Won Kang ),정해성 ( Hae Seong Jeong ),한경아 ( Kyoung Ah Han ),허원회 ( Weon Hoe Huh ),유지 한국화학공학회 2013 Korean Chemical Engineering Research(HWAHAK KONGHA Vol.51 No.3

직경 25 mm의 파이렉스 튜브 내에서 실리콘의 유동층 염소화 반응이 수행되었다. 반응기에 공급되는 질소 유량0.8~1.0 L/min, 염소 유량 0.2 L/min, 반응온도 450℃, SiCl4 응축기의 냉매온도는 -5℃로 설정하였다. 반응기에 도입되는 가스 내 염소의 몰분율이 증가하면 SiCl4의 수율이 증가하였다. 반응가스 중 염소의 몰분율 0.2의 조건에서 SiCl4의 수율은 28% 이었다. 염소의 몰분율 증가는 반응열 상승에 의해 반응온도 상승을 가져옴으로써 안전을 고려하여 염소의 몰분율을 0.2 이상으로 올리지 못했다. 실리콘의 유동층 염소화 반응에 의한 사염화실리콘의 제조 가능성이 입증되 었으며, 향후 보다 가혹한 조건에서의 실용화 연구를 위한 기초로 활용될 수 있을 것으로 기대된다. The chlorination of a metallurgical-grade silicon was carried out in a fluidized bed reactor, 25 mm in diameter. The flow rate of the chlorine admitted into the reactor was 0.2 L/min and that of the carrier nitrogen was 0.8~1.0 L/ min. The reactor temperature was maintained at 450℃ and the temperature of the coolant at the SiCl4 condenser was at -5℃. The SiCl4 yield increased with increasing the mole fraction of chlorine in the feed gas, exhibiting 28% at the mole fraction of 0.2. Further increase of the chlorine mole fraction was not attempted in a worry that the reactor might be failed due to the high exothermicity of the reaction. The production of SiCl4 from silicon by fluidized bed chlorination was demonstrated on a laboratory scale, which is a stepping stone for future studies under more severe conditions toward industrial application.

개의 췌도분리에서 췌장의 팽창과 콜라겐 분해요소

이상훈,김성주,박정환,김종성,오승훈,한진수,정인경,양태영,김동준,김광원,이문규,민용기,이명식,정재훈,함종렬 대한당뇨병학회 2000 Diabetes and Metabolism Journal Vol.24 No.2

Backgrounds: One of the main problems conditioning the outcome of islet transplantation is the ability to separate a sufficient number of viable islets with preserved function. Islet purification is critically affected by all of the isolation stages. Thus, it is necessary to set up the standard isolation method that islets are separate well from acinar without compromising islet yield and viability. Methods: Twenty three adult mongrel dogs were used for the experiment of total pancreatectomy with islet isolation. The islets were properly isolated by a modified Recordi method. The obtained islets were further purified by centrifugation on discontinuous gradients using cell separation system (Model 2991, Cobe, Lakewood Colo). We evaluated islet number (islet equivalent number, 150 um equivalents/kg of recipient body weight, IEq/kg), purity, cell volume, viabilty, recovery rate, and comparison of outcome according to the isolation conditions. Results: 1) The mean of islet numbers before purification were 13543±9431Eq/kg, digestion times were 13.8±2.6 min., digestion temperature was 37.4±0.2℃, purity was 59.7?.0%, viability was 90.0±2.1%, cell volume was 4.7±1.1 mL, islet number after purification were 4064±361 IEq/kg, and recovery rate was 29±2.9%. 2) Isolated islet numbers were different according to the degree of pancreas distension with collagenase, digestion temperature, and digestion time. 3) The best conditions for islet isolation were above 37.5℃ in temperature at recirculation of collagenase, within 12 min in digestion time and well distended pancreas with collagenase. 4) According to multiple regression adjusted by variable factors, the degree of pancreas distension with collagenase and digestion time were independently associated factors for successful islet isolation. Conclusions: In this study, we concluded that the degree of pancreas distension with collagenase and digestion time were independent factors for successful islet isolation and the best conditions for islet isolation were above 37.5℃ in temperature at recirculation of collagenase, within 12 min in digestion time and well distended pancreas with collagenase.

멀티미디어를 기반으로 하는 정보통신 분야 자격시험 학습 자료 개발

민경일,정기석,조용석,황재효 永同工科大學校 1996 硏究論叢 Vol.2 No.1

저작 도구를 사용하여 정보통신분야 1, 2급 멀티미디어 CD 타이틀을 제작하였다. 유제 문제는 STC 교과목 학습을 통하여 엄선하여 데이터 베이스화 하였다. 또한, 정보통신공학을 전공한 학생들이 CD 타이틀 제작에 참여하므로 멀티미디어 시스템 실무 능력을 배양하는 계기가 되었고, 전공에 대한 실질적인 학습 효과가 있었다. 학생들이 제작된 CD 타이틀을 이용하면 자기 학습과 반복 학습이 가능하므로 학습 능률이 극대화되어 기사 자격 시험을 준비하는 학습을 효과적으로 할 수 있을 것이다. A multimedia CD title has constructed for qualifying test of information and communication engineering. Similar problems have been selected during STC lectures and have been databased. Furthermore, the students majored in information and communication engineering participated in the process of constructing the CD title, and as the results, they could obtain practical abilities on the multimedia systems. Using the CD title, they can be able to study for the qualifying test efficiently.