胃癌和癌前病变中幽门螺杆菌及环氧化酶-2和p53的表达及相关性研究

[目的]检测慢性胃炎、胃癌前病变及胃癌（GGa）的胃黏膜组织中幽门螺杆菌（Hp）,环氧化酶-2（COX-2）和突变型p53的表达,探讨Hp感染在胃癌发生过程中与COX-2、p53动态表达的相关性.[方法]选择经胃镜检查及病理组织学证实为慢性浅表性胃炎（CSG）、慢性萎缩性胃炎（CAG）、肠上皮化生（IM）、不典型增生（Dys）及GCa患者各100例,快速尿素酶试验（HPUT法）和组织学改良Giemsa染色联合检测Hp,通过免疫组化检测Hp感染组和非感染组患者胃黏膜COX-2、p53.[结果]①Hp、COX-2阳性率随病变进展呈上升趋势,Hp阳性率在CAG、IM、Dys、GCa各组中显著高于CSG组（P＜0.05）;COX-2在IM、Dys、GCa各组中与慢性胃炎比较有统计学意义（P＜0.05）;②Hp感染阳性率和COX-2蛋白表达阳性率在胃癌前病变组织中存在相关性（P＜0.05）;③p53阳性率在GCa与CSG、CAG相比差异有统计学意义（P＜0.01）;④在GCa组中,Hp阳性组p53的阳性表达明显高于Hp阴性组（P＜0.05）.[结论]GCa的形成与Hp感染、突变型p53、COX-2等多种因素及其相互作用有关,可视为GCa发生的危险预警信号之一;在GCa高危人群的追踪观察和随访中,进行Hp、p53、COX-2的联合检测,对发现胃癌前病变和GCa有一定临床意义.     

幽门螺杆菌对肠化生胃黏膜Cdx2和肿瘤坏死因子-α蛋白表达的影响

目的探讨幽门螺杆菌对慢性浅表性胃炎伴肠化生胃黏膜尾型同源异型核基因(Cdx)2和肿瘤坏死因子(TNF)-α蛋白表达的影响。方法选择30例慢性浅表性胃炎、30例轻度肠化生,35例中度肠化生和35例重度肠化生患者进行幽门螺杆菌(Hp)检查,并检测各组在不同Hp感染情况、Hp阳性Hp根除前后Cdx2和TNF-α蛋白的表达情况。结果 Cdx2、TNF-α蛋白在慢性浅表性胃炎组(胃炎组)、轻、中、重度肠化生组的表达阳性率逐渐升高,且Cdx2表达在不同组间比较差异均有统计学意义(P0.05);TNF-α表达在胃炎组及不同程度肠上皮化生组间比较差异均有统计学意义(P0.05),随着肠化生加重,Hp感染率呈增加趋势,不同Hp感染情况时各组内Cdx2表达差异均无统计学意义(P0.05),TNF-α表达差异均有统计学意义(P0.05); Hp阳性经Hp根除后Cdx2阳性表达率下降,但仅有轻度肠化生组差异有统计学意义(P0.05);Hp阳性经Hp根除后TNF-α蛋白表达率下降,轻度肠化生组、中度肠化生组差异均有统计学意义(P0.05),重度肠化生组差异无统计学意义(P0.05)。结论监测Cdx2、TNF-α蛋白表达水平有助于判断肠上皮化生程度,胃黏膜轻度肠化生根除Hp后可逆转,中度肠化生胃黏膜根除Hp后胃黏膜炎症可减退。     

幽门螺杆菌感染在胃黏膜癌变过程中与生长抑素的关系

[目的]分析幽门螺杆菌(Hp)感染在胃黏膜癌变过程中对血清及胃黏膜生长抑素(SS)水平的影响,探讨Hp与SS在胃癌(GC)中的关系。[方法]90例患者随机分为3组,I组为慢性浅表性胃炎(CSG);Ⅱ组为中重度慢性萎缩性胃炎(CAG),伴或不伴肠上皮化生(IM)、轻度至中度不典型增生(ATP);Ⅲ组为GC或重度ATP。采用快速尿素酶试验、Gimsa染色、ELISA法检测Hp,放免法和免疫组化法检测3组血清和胃黏膜组织中SS水平和表达的变化。[结果]Ⅰ、Ⅱ、Ⅲ组血清SS水平分别为(62.28±5.97)、(49.63±5.29)(、25.87±2.64)pg/300μl,3组比较差异有统计学意义(P<0.01)。但各组内Hp感染阳性组与阴性组血清SS水平比较无显著性差异(P>0.05)。免疫组化显示3组SS的阳性表达率比较有统计学意义(P<0.05)。SS在Hp感染阳性组的表达逐渐降低,Ⅲ组中的表达远低于Ⅰ、Ⅱ组,且明显低于Hp阴性组(P<0.05)。[结论]SS在胃黏膜癌变过程表达下降,可能是Hp致胃黏膜癌变的机制之一。     

胃癌组织中P53、IL-1β、NF-κB的表达及与幽门螺杆菌感染的相关性分析

[目的]探讨胃癌组织中p53、IL-1β、NF-κB的表达及与幽门螺杆菌(Hp)感染的关系。[方法]选取2016年2月～2018年5月收治的97例胃癌患者为研究对象,根据Hp检查结果分组,Hp阳性组81例,Hp阴性组16例,另选97例体检健康者为对照组,对所有受检者胃癌组织中的p53、IL-1β、NF-κB表达情况进行观察。[结果]胃癌组患者的p53、IL-1β、NF-κB表达量均明显较对照组高,差异有统计学意义(P0.05);Hp阳性组患者的p53、IL-1β、NF-κB表达量明显高于Hp阴性组患者,差异有统计学意义(P0.05);Hp感染密度3级患者的p53、IL-1β、NF-κB表达量明显高于2级、1级患者,差异有统计学意义(P0.05);经Perason相关性分析显示,p53、IL-1β、NF-κB表达量与胃癌患者的Hp感染呈正相关性(r分别为0.586、0.638、0.612,P0.05)。[结论]胃癌组织中的p53、IL-1β、NF-κB表达量与Hp感染呈正相关性。     

胃癌及癌前病变p53蛋白及Ki-67抗原检测的临床意义

应用免疫组织化学法检测胃癌和癌前病变p53蛋白、Ki-67抗原的表达.结果 正常胃黏膜无p53蛋白和Ki-67抗原表达.从萎缩性胃炎、肠上皮化生、胃黏膜异型增生至胃癌,p53蛋白和Ki-67抗原阳性表达率逐渐升高.胃癌及胃黏膜异型增生p53蛋白和Ki-67抗原阳性表达率显著高于萎缩性胃炎和肠上皮化生,差异有统计学意义（P＜0.05）;胃癌与胃黏膜异型增生、萎缩性胃炎与肠上皮化生比较p53蛋白和Ki-67抗原表达差异无统计学意义（P＞0.05）.认为p53蛋白和Ki-67抗原的检测有助于胃黏膜癌变的早期诊断.     

幽门螺杆菌感染与胃癌COX-2,Bcl-2表达关系的研究

目的　研究幽门螺杆菌 (Helicobacter pylori,Hp)感染与胃癌及癌前病变组织 COX- 2 ,Bcl- 2蛋白表达的关系 ,探讨 Hp感染与 COX-2 ,Bcl- 2表达的关系。方法　免疫组化方法检测 COX- 2 ,Bcl- 2蛋白表达 ,快速尿素酶方法和 HE染色检测 Hp感染情况。结果　 Bcl- 2 ,COX- 2在胃癌、异型增生的阳性表达率升高 ,与肠上皮化生、萎缩性胃炎、浅表性胃炎相比有显著性差异 (P<0 .0 5)。 COX- 2阳性与胃癌浸润深度、淋巴转移有关 ,且Bcl- 2 ,COX- 2与 Hp感染有关 (P<0 .0 5)。结论　 Hp感染可能作为促癌剂在胃癌的发生发展中起一定作用。     

幽门螺杆菌感染对老年人胃黏膜环氧合酶-2表达的影响及意义

目的:评估幽门螺杆菌(H pylori)感染对老年人胃黏膜COX-2表达的影响及意义.方法:取不同阶段的胃黏膜病变共200例,用速尿素酶试验结合组织学Giemsa染色或14C尿素呼气试验检测胃黏膜H pylori感染状况,应用免疫组织化学检测胃黏膜上皮细胞COX-2的表达.结果:不同组织类型H pylori检出率以胃癌最高,其次为不典型增生(AH)和肠上皮化生(IM).COX-2在慢性浅表性胃炎(CSG)、IM、AH和胃癌中的表达阳性率分别为8%、24%、46%和64%,呈递增趋势,其阳性率胃癌与非癌组织相比差异均有统计学意义(P<0.01).同一类型 H pylori 阳性组COX-2的表达高于H pylori 阴性组,2组比较有显著性差异(P＜0.05).结论:COX-2表达上调与H pylori感染的胃黏膜的癌变有关,可能在癌前病变早期阶段起作用.     

幽门螺杆菌检测在胃癌诊断中的临床意义

[目的]探讨幽门螺杆菌(Hp)检测在胃癌诊断中的价值。[方法]选取2015年5月~2016年5月在我院接受胃镜检查并作病理活检患者372例,其中慢性浅表性胃炎104例,慢性萎缩性胃炎110例,不典型增生71例,胃癌患者87例。采用苏木精-伊红染色观察各患者胃黏膜病变组织Hp感染,采用Hp特异性抗体试剂检测患者血液抗体情况。[结果]各患者Hp检测阳性比较差异有统计学意义(P0.05);随着病情的不断加重,苏木精-伊红染色和抗体检测阳性率增加,其中胃癌患者苏木精-伊红染色阳性和抗体检测阳性分别为80.46%和79.31%,明显高于其他患者(P0.05);早期胃癌苏木精-伊红染色阳性和抗体检测阳性分别为61.54%和65.38%,均明显低于进展期胃癌患者,差异比较有统计学意义(P0.05)。[结论]Hp感染在胃黏膜病变中起着重要的作用,及早诊断Hp感染并予以根除,对预防胃癌具有重要意义。     

C-myc、p16、p53基因与老年患者胃黏膜癌变幽门螺杆菌感染的关系研究

[目的]探讨老年患者幽门螺杆菌感染与胃黏膜癌变的相关性,并对可能存在的C-myc、p16、p53基因机制进行分析。[方法]对260例胃黏膜病变患者幽门螺杆菌感染状况进行检测,并对C-myc、p16、p53基因表达进行检测,比较不同胃黏膜病变患者检测结果。[结果]浅表性胃炎组除p16基因阳性表达率比胃癌组高外(P0.05),C-myc阳性率,p53阳性率,幽门螺杆菌感染率均低于胃癌组(P0.05);萎缩性胃炎组幽门螺杆菌感染率、C-myc阳性率,p53阳性率均低于胃癌组(P0.05);胃溃疡组患者p53表达阳性率低于胃癌组(P0.05);萎缩性胃炎组患者C-myc与p53基因表达阳性率与幽门螺杆菌感染率间关联具有统计学意义(P0.05),胃溃疡组患者C-myc与p16基因表达阳性率间关联具有统计学意义,胃癌组C-myc、p16、p53基因表达阳性率与幽门螺杆菌感染率间关联均具有统计学意义(P0.05)。[结论]老年胃癌患者幽门螺杆菌感染与C-myc、p16、p53基因表达存在一定的关联。     

瘦素和胃促生长素在胃黏膜癌变过程中表达及相关性的研究

目的检测瘦素(leptin)及胃促生长素(ghrelin)在胃癌及其癌前病变中的表达及相关性,探讨两者在胃黏膜癌变过程中的作用,为胃癌的早期诊断及治疗提供一定的理论依据。方法 139例患者分为慢性非萎缩性胃炎组30例、慢性萎缩性胃炎非肠上皮化生(简称肠化)组30例、慢性萎缩性胃炎伴肠化组30例、异型增生组19例、胃癌组30例,采用免疫组织化学法检测ghrelin和leptin在胃黏膜中的表达,分析两者在胃黏膜癌变过程中的表达变化及相关性。结果 ghrelin在非萎缩性胃炎组、萎缩非肠化组、萎缩伴肠化组、异型增生组、胃癌组中表达阳性率分别为80.00%、53.33%、43.33%、21.05%、16.67%,表达递减,萎缩非肠化组与萎缩伴肠化组差异无统计学意义(P0.05),萎缩伴肠化组与异型增生组差异无统计学意义(P0.05),异型增生组与胃癌组差异无统计学意义(P0.05),余各组间比较差异均有统计学意义(P0.05)。上述5组中leptin表达阳性率分别为10.00%、33.33%、53.33%、73.68%、80.00%,表达递增,非萎缩性胃炎组与萎缩非肠化组差异无统计学意义(P0.05),萎缩非肠化组与萎缩伴肠化组差异无统计学意义(P0.05),萎缩伴肠化组与异型增生组差异无统计学意义(P0.05),异型增生组与胃癌组差异无统计学意义(P0.05),余各组间比较差异均有统计学意义(P0.05)。在萎缩伴肠化组、异型增生组、胃癌组中leptin与ghrelin的表达呈显著负相关(P0.05)。结论 ghrelin在胃黏膜癌变过程中表达呈递减趋势,其在胃黏膜表达的特异性降低可作为胃癌诊断指标继续研究;leptin在胃黏膜癌变过程中表达呈递增趋势,提示leptin与胃癌发生相关,其在胃黏膜中高表达可被视为胃癌早期诊断的监测指标;胃黏膜癌变过程中,ghrelin与leptin可能相互作用,呈负相关,两者的异常表达参与了胃黏膜损伤及癌变,联合检测有助于胃癌的早期诊断。     

Abnormal gastric myoelectrical activity and delayed gastric emptying in patients with symptoms suggestive of gastroparesis

Gastric myoelectrical activity modulates gastric motor activity. Abnormalities in gastric myoelectrical activity may be associated with gastric motility disorders. The aim of this study was to investigate the correlation of gastric myoelectrical activity with gastric emptying in symptomatic patients with and without gastroparesis. Ninety-seven patients with symptoms suggestive of gastroparesis participated in the study. Gastric myoelectrical activity was recorded using surface electrogastrography. The electrogastrogram (EGG) was recorded for 30 min in the fasting state and for 120 min after a solid test meal. Gastric emptying of the solid meal was simultaneously monitored for 120 min. Patients with delayed gastric emptying showed a significantly lower percentage of normal gastric slow waves (P<0.03) and a significantly reduced increase of the dominant power in the postprandial EGG (P<0.02). Postprandial EGG parameters were found to be able to predict delayed emptying of the stomach. Postprandial gastric dysrhythmia predicts delayed gastric emptying with an accuracy of 78%, while the abnormality in postprandial EGG power predicts delayed gastric emptying with an accuracy of 75%. All patients with abnormalities in both the rhythmicity and the power had delayed gastric emptying. Patients with delayed gastric emptying have a lower percentage of normal gastric slow waves in the EGG and a lower postprandial increase in the dominant power. Abnormalities in the postprandial EGG seem to be able to predict delayed emptying of the stomach. However, a normal EGG does not seem to guarantee normal emptying of the stomach.     

胃溃疡大鼠胃粘膜血流量与泌酸变化的研究

用手术将十二指肠内容物持续胃内反流制成大鼠胃溃疡及经转流后的溃疡愈合模型进行研究。结果表明,溃疡组于胃窦小弯侧可见8.84±3.08(m~2)~(-3)的慢性溃疡形成,并显示胃粘膜血流量降低,G细胞密度、壁细胞数增加。溃疡愈合组经转流后大部分溃疡已愈合,G细胞密度、壁细胞数降低,粘膜血流量增加。本实验提示,泌酸细胞增多,泌酸量增加和胃粘膜缺血可能是溃疡形成的重要因素,增加胃粘膜的血液供应,降低胃酸分泌可促进溃疡愈合。     

胃微生态与胃癌的研究进展

胃微生态平衡是人体健康的重要前提,幽门螺杆菌(Helicobacter pylori,Hp)是目前已发现的与胃癌相关的关键病原体之一,普遍存在于人胃黏膜上皮。Hp感染可引起胃内其他菌群的改变,还可引起长期慢性的胃黏膜损伤,导致一系列胃黏膜上皮恶性进展和胃癌的发生。本文就胃微生态与Hp感染的关系、Hp感染在胃癌发生中的作用、胃内其他菌群在胃癌发生中的作用及微生态制剂在胃癌治疗的作用进行综述。进一步揭示Hp感染对胃微生态平衡的影响,胃微生态平衡和Hp感染在胃癌发生发展中的作用及微生态制剂在胃癌治疗中的意义。     

Relationship between gastric mucosal hemodynamics and gastric motility

Continuous measurement of gastric mucosal hemodynamics (the index of mucosal hemoglobin concentration, the index of oxygen saturation and blood flow) in rats showed oscillatory changes. The mechanism of the oscillations was investigated using a probe specially designed for simultaneous measurement of hemodynamics and intragastric pressure. A hemodynamics-measuring probe for either reflectance spectrophotometry or laser-Doppler flowmetry was tied to a pressure microtransducer, inserted through an incision in the forestomach, and brought into gentle contact with the corpus mucosa. Synchronous oscillatory changes (4-6 cycles/min) in hemodynamics and motility were observed in the resting state (mean blood pressure: 120 mmHg). During moderate hemorrhagic hypotension (mean: 81 mmHg), oscillations in the hemodynamics increased in both amplitude and frequency, while motility remained constant. Oscillations in the hemodynamics were also affected by fluctuations in blood pressure and by topical application of norepinephrine to the corpus serosa. In water-immersion restraint rats, changes in the oscillations in the hemodynamics and motility were virtually synchronous; frequency decreased and amplitude increased. These findings suggest that oscillatory changes in gastric mucosal hemodynamics are regulated not only by gastric motility but also by arteriolar vasomotion of the gastric wall.     

Pedunculated gastric carcinoma demonstrating a gastric foveolar phenotype

We report a case of gastric cancer complicated with very well differentiated adenocarcinoma containing signet ring cells. An endoscopic examination revealed a pedunculated polyp in the fornix of the stomach. A surgical operation was performed and the pathological findings showed very well differentiated adenocarcinoma mimicking gastric foveolae with a poorly differentiated component containing signet ring cells. This is the first case of pedunculated gastric cancer complicated with very well differentiated adenocarcinoma containing signet ring cells and also demonstrating a gastric foveolar phenotype.     

胃癌及良性胃病患者血和胃粘膜细胞内维生素的变化

目的 进一步评价维生素在胃癌发生中的变化,为维生素防治胃癌提供理论依据。 方法 以微量生化法检测胃癌、萎缩性胃炎、胃溃疡外周静脉血,供癌或溃疡灶动静脉血,手术切除胃癌标本各部位粘膜细胞内,内镜下活检粘膜组织细胞内维生素A,C,E及β-胡萝卜素的含量。 结果 胃癌与胃溃疡外周血清4种维生素含量均低于常人(P<0.01),供癌灶动静脉血中β-胡萝卜素含量之差明显大于供良性溃疡灶者(P<0.01),胃癌者静脉血中维生素C含量高于动脉血。而良性溃疡则相反;胃癌手术标本粘膜细胞内4种维生素的含量基本上按癌区→癌旁→外周正常区顺序递增,萎缩性胃炎活检胃粘膜细胞内4种维生素含量高于大体标本中类似病理改变的粘膜细胞。 结论 胃癌的发生发展中4种抗氧化维生素,尤其是β-胡萝卜素有代谢异常。     

Relationship between gastric xanthoma,gastric precancerous lesions,and gastric cancer: A retrospective study

Objective

To evaluate the relationship between gastric cancer and its precancerous lesions and gastric xanthoma.

Methods

Medical records of 47 736 patients who underwent gastroscopy in our center from January 2020 to December 2021 were reviewed. Patients’ age, sex, endoscopic and histopathological findings, and the presence, number and location of gastric xanthoma were recorded. To investigate the detection rate of gastric xanthoma at different stages of gastric lesions, the participants were further divided into the chronic gastritis group (n = 42 758), the precancerous lesion group (n = 3672), and the gastric cancer group (n = 1306), respectively.

Results

The overall detection rate of gastric xanthoma was 2.85%, and it was most commonly observed in the gastric antrum (52.50%). In addition, gastric xanthoma was more common in men and more likely to be single lesion. It was most detected in the precancerous lesion group (8.39%), followed by the gastric cancer group (5.44%), and least in the chronic gastritis group (2.29%). Multivariate analysis showed that gastric xanthoma was closely related to precancerous lesions (odds ratio [OR] 3.197, 95% confidence interval [CI] 2.791–3.662, P < 0.001) and gastric cancer (OR 1.794, 95% CI 1.394–2.309, P < 0.001).

Conclusion

Gastric xanthoma is closely related to gastric precancerous lesions and gastric cancer.     

Features of early gastric cancer and gastric adenoma by enhanced-magnification endoscopy

Background Changes to the mucosal surface of early gastric carcinomas and gastric adenomas as viewed by enhanced-magnification endoscopy with acetic acid have not been investigated thoroughly. Using this technology, we investigated the appearance of the gastric surface patterns of neoplastic and surrounding nonneoplastic mucosa. Methods Forty-seven consecutive patients with early gastric carcinomas or gastric adenomas underwent enhanced-magnification endoscopy following 1.5% acetic acid instillation. All biopsy specimens were taken from the area at which the enhanced-magnified endoscopic image was obtained. Results Surface patterns of gastric tumors and the surrounding mucosa were classified into five types: type I, small round pits of uniform size and shape; type II, slit-like pits; type III, gyrus and villous patterns; type IV, irregular arrangements and sizes of pattern types I, II and III; type V, destructive patterns of types I, II and III. The predominant pattern of the surrounding mucosa was type III, and most type III mucosa had characteristics of intestinal metaplasia. Although all elevated adenomas showed type II or type III surface patterns, both depressed adenomas showed type IV. Elevated carcinomas showed type III (42.9%) or type IV (57.1%) surface patterns, while depressed carcinomas showed type IV (70%) or type V (30%). Although differentiated tubular adenocarcinomas showed type III (10.3%), type IV (86.2%), or type V (3.5%) surface patterns, all of the signet-ring cell carcinomas and poorly differentiated tubular adenocarcinomas showed type V. Conclusions Enhanced-magnification endoscopy may be useful for identifying gastric tumors and determining the extent of horizontal spread, especially in tumors of the depressed type.     

金葵胃药抗胃黏膜损伤作用的实验研究

[目的]观察金葵胃药（JK）对胃黏膜损伤的保护作用，并探讨其机制。[方法]采用盐酸一乙醇灌胃致大鼠胃黏膜损伤模型，观察JK用药组和对照组的胃黏膜损伤指数，并测定血清和胃组织超氧化物歧化酶（SOD）活性和丙二醛（MDA）水平。[结果]JK（90、180、360mg／kg）能明显降低大鼠胃黏膜损伤指数，并明显提高血清和胃组织中SOD活性，降低其MDA水平。[结论]JK对大鼠胃黏膜损伤有明显的保护作用，其作用机制可能与抗氧化作用有关。     

Shift of the gastric T-cell response in gastric carcinoma

BACKGROUND AND AIMS: The etiology and pathophysiology of stomach carcinoma is complex, and the mechanism whereby H. pylori directly or indirectly induces carcinoma remains unclear. In this study, interleukin (IL)-8, IL-4 and interferon (IFN)-gamma were measured in the tissue culture supernatant of gastric organ cultures from subjects with chronic gastritis with or without H. pylori infection, and with or without gastric cancer and gastric dysplasia. RESULTS: Interleukin-8 levels were higher in cancer- and H. pylori-infected gastritis subjects than in H. pylori-negative subjects (12.95 +/- 3.16, 10.48 +/- 1.55 and 4.49 +/- 1.28 ng/mL, respectively). Elevated levels of IFN-gamma were detected in both H. pylori-infected and non-infected subjects with uncomplicated gastritis (72.23 +/- 19.0 and 34.61 +/- 5.30 pg/mL) and in non-infected dysplasia subjects (88 +/- 20.5 pg/mL). Background levels of IL-4 (< or = 9.4 pg/mL) in uncomplicated gastritis subjects and relatively high levels of IL-4 in dysplasia subjects (25.8 +/- 7.3 pg/mL) were detected. In contrast, trace amounts of IFN-gamma (16.01 +/- 0.35 pg/mL) and high levels of IL-4 (42.81 +/- 8.49 pg/mL) in gastric biopsy culture supernatants were found in cancer subjects. Mucosal IL-4 levels (but not IL-8 levels) correlated with infection and mucosal anti-H. pylori immunoglobulin G antibody. CONCLUSIONS: The significant differences between gastritis with and without cancer and dysplasia indicated a shift from a Th1 to a Th2 helper cell pattern of cytokine secretion. This study has identified a local mucosal defect in gastric cancer. The near absence of IFN-gamma production from the mucosa at the margins of the tumor may be a critical factor in promoting growth of neoplastic cells.