疏肝健脾针法对肝郁脾虚证腹泻型肠易激综合征患者肠道菌群和血清5-HT、NPY、CGRP的影响

目的:观察疏肝健脾针法治疗肝郁脾虚证腹泻型肠易激综合征(IBS-D)的临床疗效及对肠道菌群和血清5-羟色胺(5-HT)、降钙素基因相关肽(CGRP)、神经肽Y(NPY)的影响。方法:96例肝郁脾虚证IBS-D患者均来自天津市人民医院2018年5月~2021年2月期间收治的门诊或住院患者。根据双色球法将患者分为对照组和研究组,各为48例,对照组接受西医治疗,研究组在对照组基础上结合疏肝健脾针法治疗,对比两组疗效、中医证候积分、肠道菌群和血清5-HT、NPY、CGRP水平变化。结果:研究组的临床总有效率明显高于对照组(P<0.05)。疗程结束后,研究组大便稀溏、少腹胀痛、食后腹胀、食欲减退、精神疲乏、口苦口黏、四肢无力、烦躁易急、畏寒怕冷症状积分均低于对照组(P<0.05)。疗程结束后,研究组血清5-HT、NPY、CGRP水平均低于对照组(P<0.05)。疗程结束后,研究组双歧杆菌、双歧杆菌/大肠杆菌比值、乳酸杆菌数量均高于对照组,而大肠杆菌数量低于对照组(P<0.05)。结论:疏肝健脾针法治疗肝郁脾虚证IBS-D患者,可有效促进症状改善,调节肠道菌群,降低血清5-HT、NPY、CGRP水平,临床疗效显著。     

不同类型肠易激综合征患者肠道菌群与血清微小RNA的关系

目的 研究不同类型肠易激综合征（IBS）患者肠道菌群与血清微小RNA(miRNA)的关系。方法 选择2019年1月至2022年1月于我院接受治疗的93例IBS患者为研究对象,根据其IBS类型,分为腹泻型IBS组（44例）与便秘型IBS组（49例）,同时将我院30例行胃镜检查未患IBS者作为对照组。采集研究对象十二指肠液行肠道菌群计数与培养,分析不同类型IBS患者肠道菌群差异。采集研究对象外周血,检测血清miR-29a、miR-143-5p水平。分析肠道菌群与血清miRNAs之间的关系。结果 便秘型IBS患者回肠末端肠液中拟杆菌数量高于对照组,B/E比值低于对照组（均P<0.05）;腹泻型IBS患者肠道双歧杆菌、乳杆菌数量及B/E比值均低于对照组,肠杆菌数量高于对照组（均P<0.05）。便秘型IBS组和腹泻型IBS组患者血清miR-29a及miR-143-5p水平均高于对照组（均P<0.05）。相关性分析显示,便秘型IBS患者回肠内拟杆菌数量与其血清mi R-29a和miR-143-5p水平均呈正相关（r=0.326,P=0.046;r=0.343,P=0.041）;腹...     

肠道菌群与肠易激综合征分型及患者血清细胞间黏附分子 1表达的关系

目的分析肠道菌群与IBS分型及患者血清细胞间黏附分子 1(ICAM 1)表达的关系,为后续研究提供参考。方法选择2018年12月至2020年1月我院收治的152例符合标准的肠易激综合征(IBS)患者作为观察组,并依照罗马Ⅲ标准将观察组患者分为腹泻型组(62例)、便秘型组(56例)、混合型组(34例);选择同期我院健康体检者30例为对照组。检测受试者粪便中双歧杆菌、乳杆菌、肠杆菌和肠球菌数量,同时检测受试者血清ICAM 1水平。采用Pearson相关性分析患者肠道菌群与血清ICAM 1关系,并采用Logistics回归模型分析肠道菌群与IBS各亚型的关系。结果观察组患者血清ICAM 1、肠道双歧杆菌、乳杆菌水平显著低于对照组,肠道肠杆菌和肠球菌水平显著高于对照组,差异均有统计学意义(均P<0.05)。不同亚型IBS患者血清ICAM 1、肠道双歧杆菌、乳杆菌、肠杆菌和肠球菌水平存在明显差异,其中混合组患者血清ICAM 1、肠杆菌、肠球菌数量最高,双歧杆菌、乳杆菌数量最低;腹泻型组肠道肠杆菌、肠球菌数量最低,双歧杆菌、乳杆菌数量最高(均P<0.05)。肠道中双歧杆菌、乳杆菌、肠杆菌、肠球菌数量是IBS分型的独立影响因素(均P<0.05)。患者肠道双歧杆菌、乳杆菌数量与ICAM 1水平呈显著负相关,而肠道肠杆菌和肠球菌数量与ICAM 1水平呈显著正相关(均P<0.05)。结论肠道菌群可影响IBS患者分型,且肠道中部分菌群与ICAM 1水平存在显著相关关系。     

原发性肝癌患者肠道菌群变化与内毒素和炎性因子水平的关系

目的探讨原发性肝癌患者肠道菌群变化及其与内毒素、炎性因子水平的相关性。方法选取2018年2月至2019年10月我院收治的80例原发性肝癌患者为观察组,另选同时期80例健康体检者为对照组。观察两组对象肠道菌群情况,同时检测两组对象血清内毒素和白细胞介素6(IL-6)、白细胞介素1(IL-1)及肿瘤坏死因子α(TNF-α)水平。采用Pearson检验进行相关性分析。结果观察组患者肠道双歧杆菌、乳杆菌和拟杆菌数量低于对照组;而肠杆菌和酵母菌数量高于对照组(均P0.05)。观察组患者血清内毒素、IL-6、IL-1、TNF-α水平均高于对照组(均P0.05)。Pearson相关性分析显示,肠道双歧杆菌、乳杆菌和拟杆菌数量与血清内毒素和IL-6、IL-1、TNF-α水平呈负相关,肠道肠杆菌和酵母菌数量与内毒素和IL-6、IL-1、TNF-α水平呈正相关(均P0.05)。结论原发性肝癌患者肠道菌群紊乱与内毒素和炎症因子水平具有相关性,肠道菌群失衡可能是促进该病病情发展的原因之一。     

双歧杆菌四联活菌片对胆囊切除术后腹泻患者 肠道微生态及胃肠激素的影响

目的探讨双歧杆菌四联活菌片对胆囊切除术后腹泻(PCD)患者肠道微生态及胃肠激素的影响。方法选取72例PCD患者随机分为观察组与对照组。两组均给予饮食调整和蒙脱石散口服等治疗。观察组加用双歧杆菌四联活菌片1.5g/次,3次/d,温水送服,连用8周。评估两组治疗前后肠道菌群数量[乳酸杆菌、双歧杆菌、大肠杆菌和双歧杆菌(B)与大肠杆菌(E)的比值]及血清胃肠激素[血管活性肠肽(VIP)和5-羟色胺(5-HT)]的变化,并比较其临床效果。结果治疗8周后,两组双歧杆菌、乳酸杆菌数量及B/E比值与其治疗前数据比较上升,大肠杆菌数量与其治疗前数据比较下降(P0.05或P0.01),且治疗后观察组改善更显著(P0.05);两组血清VIP和5-HT与其治疗前数据比较下降(P0.05或P0.01),且治疗后观察组下降更显著(P0.05);同时观察组总有效率较对照组更佳(χ2=5.26,P0.05)。结论双歧杆菌四联活菌片治疗PCD的疗效确切,能调节肠道菌群紊乱,重建肠道微生态平衡;并能调节血清胃肠激素的分泌,降低血清VIP和5-HT水平,改善肠道功能。     

亚临床肝性脑病患者肠道菌群和血清炎症因子水平变化及益生菌的干预作用

目的探讨亚临床肝性脑病(SHE)患者肠道菌群及血清炎症因子水平的变化及益生菌对该病的干预作用。方法选取2015年1月至2018年10月在我院就诊的40例SHE患者为观察组。选择同期我院体检中心检查无明显肝胆胃肠道病变和急慢性炎症性疾病的30例健康者为对照组。观察组患者在低盐饮食和护肝降转氨酶基础上加用双歧杆菌三联活菌胶囊630 mg/次,2次/d,温开水送服,连用6周。观察两组对象肠道菌群数量及血清炎症因子水平的变化。结果观察组患者治疗前肠道双歧杆菌和乳杆菌的数量少于对照组,而大肠埃希菌数量多于对照组(均P0.05)。治疗6周后,观察组患者肠道双歧杆菌和乳杆菌的数量较治疗前明显上升,而大肠埃希菌数量较治疗前明显下降(均P0.05);同时观察组患者治疗前血清IL-6和TNF-α水平高于对照组,IL-10水平低于对照组(均P0.05)。治疗6周后,观察组患者血清IL-6和TNF-α水平较治疗前明显下降,而IL-10水平较治疗前明显上升(均P0.05)。结论双歧杆菌三联活菌胶囊能调节SHE患者肠道菌群紊乱,重建肠道菌群屏障,同时能调节患者血清炎症因子水平,控制局部炎症反应。     

乙型肝炎肝硬化患者肠道菌群变化与血清细胞因子水平的相关性

目的探讨乙型肝炎肝硬化患者肠道菌群变化与血清干扰素-α(IFN-α)、白细胞介素6(IL-6)、白细胞介素1β(IL-1β)水平的相关性。方法选择2017年12月至2019年12月我院收治的105例乙型肝炎肝硬化患者为A组,50例乙型肝炎患者为B组,同期于我院体检健康者50例为C组。比较3组患者粪便标本中菌群分布情况,同时比较3组患者血清IFN-α、IL-6、IL-1β水平,不同程度肠道菌群失调乙型肝炎肝硬化患者血清IFN-α、IL-6、IL-1β水平。采用Pearson相关分析乙型肝炎肝硬化患者肠道菌群与血清IFN-α、IL-6、IL-1β的相关性。结果 A组患者肠道乳杆菌、双歧杆菌数量显著低于B组,而肠杆菌、肠球菌及血清IFN-α、IL-6、IL-1β水平显著高于B组(均P0.05)。B组患者肠道乳杆菌、双歧杆菌数量显著低于C组,而肠杆菌、肠球菌及血清IFN-α、IL-6、IL-1β水平显著高于C组(均P0.05)。Ⅱ度菌群失调乙型肝炎肝硬化患者血清IFN-α、IL-6、IL-1β水平显著高于I度菌群失调者(均P0.05)。Ⅲ度菌群失调乙型肝炎肝硬化患者血清IFN-α、IL-6、IL-1β水平显著高于Ⅱ度菌群失调者(均P0.05)。乙型肝炎肝硬化患者肠道肠杆菌、肠球菌数量与血清IFN-α、IL-6、IL-1β水平呈正相关,而其肠道乳杆菌、双歧杆菌数量与血清IFN-α、IL-6、IL-1β水平呈负相关(均P0.05)。结论乙型肝炎肝硬化患者存在明显的肠道菌群失调,同时其外周血IFN-α、IL-6、IL-1β水平呈现上升趋势。肠道菌群与血清IFN-α、IL-6、IL-1β可能协同参与了乙型肝炎肝硬化的发生及发展。     

肠道菌群与呼吸道感染患者免疫球蛋白的关系及其肠内营养的疗效

目的分析肠道菌群紊乱对呼吸道感染患者免疫球蛋白水平的影响,探讨肠内营养对此类患者的疗效。方法选取2016年2月至2018年12月在我院接受治疗的116例呼吸道感染患者作为观察组,进一步将其分为非菌群失调组(50例)和菌群失调组(66例),选取同期60例健康体检者作为对照组,比较各组对象肠道菌群失调情况和血清免疫球蛋白水平,同时对菌群失调患者在常规治疗基础上联合应用肠内营养治疗,观察其治疗效果。结果 (1)观察组患者肠道乳杆菌、双歧杆菌数量及双歧杆菌/大肠埃希菌比值(B/E)显著低于对照组,而肠球菌、大肠埃希菌数量显著高于对照组(均P0.05);观察组患者血清IgM、IgA、IgG水平均显著低于对照组(均P0.05);菌群失调组患者血清IgM、IgA、IgG水平均显著低于非菌群失调组(均P0.05)。(2)观察组患者肠道乳杆菌、双歧杆菌数量及B/E值与血清IgM、IgA、IgG水平呈显著正相关(均P0.05),而大肠埃希菌和肠球菌数量与之呈显著负相关(均P0.05)。(3)菌群失调组患者血清ALB、PA水平随治疗时间的延长而逐渐升高,CRP、PCT水平逐渐降低,IgM、IgA、IgG水平逐渐升高,乳杆菌、双歧杆菌数量和B/E值逐渐增加,大肠埃希菌和肠球菌数量逐渐减少(均P0.05)。结论呼吸道感染患者存在明显的肠道菌群失调情况,其与患者免疫球蛋白水平的降低有关,这可能是引发呼吸道感染的主要危险因素。肠内营养支持疗法能有效改善患者肠道菌群失调,降低血清炎性因子水平,增强机体免疫力,提高患者营养状况及治疗效果。     

老年2型糖尿病合并慢性肾脏病患者肠道菌群变化

目的探讨合并慢性肾脏病的老年2型糖尿病患者肠道菌群变化情况,为后续研究提供参考。方法选择2018年11月至2019年7月在我院内科确诊的老年2型糖尿病及并发慢性肾脏病患者作为糖尿病组(20例)及糖尿病肾病组(20例),选择本院健康老年体检者(30例)为对照组。测定各组对象血清甲状旁腺激素(PTH)、超敏C反应蛋白(hs-CRP)、血肌酐(Scr)、血磷(P)、血钙(Ca~(2+))、碱性磷酸酶(ALP)、糖化血红蛋白(HbA1c)水平,同时分析各组对象肠道大肠埃希菌、双歧杆菌、乳杆菌、酵母菌、真杆菌的差异。结果糖尿病肾病组患者肠道大肠埃希菌数量高于糖尿病组和对照组,糖尿病组患者肠道大肠埃希菌数量高于对照组(均P0.05)。糖尿病肾病组患者肠道双歧杆菌、真杆菌数量低于糖尿病组和对照组,同时糖尿病组患者肠道双歧杆菌、真杆菌数量低于对照组(均P0.05)。糖尿病肾病组患者血清Scr、P、Ca~(2+)水平高于糖尿病组和对照组,糖尿病组患者血清Scr、P、Ca~(2+)水平高于对照组(均P0.05)。糖尿病肾病组患者血清HbA1c、ALP、hs-CRP水平高于糖尿病组和对照组,同时糖尿病组患者血清HbA1c、ALP、hs-CRP水平高于对照组(均P0.05)。大肠埃希菌数量与Scr、P、Ca~(2+)、HbA1c、ALP、hs-CRP水平呈正相关,双歧杆菌、真杆菌数量与Scr、P、Ca~(2+)、HbA1c、ALP、hs-CRP水平呈负相关(均P0.05)。大肠埃希菌数量升高(OR=4.26,95%CI:1.87～9.71),双歧杆菌(OR=5.02,95%CI:2.36～10.68)和真杆菌(OR=17.89,95%CI:12.67～25.89)数量降低均为糖尿病并发慢性肾病的危险因素。结论糖尿病肾病患者肠道菌群失调,其特征为大肠埃希菌数量增多,双歧杆菌、真杆菌数量减少。大肠埃希菌数量升高,双歧杆菌和真杆菌数量降低均为糖尿病并发慢性肾脏病的危险因素。     

双歧杆菌三联活菌胶囊联合思密达对儿童腹泻血浆VIP、NPY及肠黏膜5-HT的影响

目的探讨双歧杆菌三联活菌胶囊联合思密达对儿童腹泻血浆血管活性肽(VIP)、神经肽Y(NPY)及肠黏膜5-羟色胺(5-HT)的影响。方法 2014年1月至2014年12月选取延安大学附属医院儿科收治的124例腹泻患儿为研究对象,根据随机数字表将患儿分为观察组及对照组各62例,对照组给予思密达治疗,观察组在对照组基础上应用双歧杆菌三联活菌胶囊治疗。观察两组治疗效果及治疗前后血浆VIP、5-HT和NPY的变化。结果观察组总有效率(96.77%)高于对照组(83.87%),差异有统计学意义(P0.05)。观察组大便性状变稠、腹泻次数正常、腹泻停止、腹痛停止、大便镜检正常时间、大便细菌培养恢复正常时间显著短于对照组(P0.05)。两组治疗后血浆VIP、5-HT显著低于治疗前(P0.05),而NPY显著高于治疗前(P0.05)。观察组治疗后血浆VIP、肠黏膜5-HT显著低于对照组(P0.05),而血浆NPY水平显著高于对照组(P0.05)。结论双歧杆菌三联活菌胶能显著降低VIP及5-HT水平,促进NPY生成,从而有效改善腹泻患儿临床症状,提高治疗效果。     

Definitions,measurements, and classifications of stimuli,situations, and environments

A review is presented of issues relevant to the definition, measurement, and classification of stimuli, situations, and environments. Problems such as the lack of adequate definitions of concepts, error and bias in measurement procedures, confusion between measurement of a concept and measurement of its behavioral effects, and the lack of agreement among alternative measures are emphasized. It is suggested that concepts be defined in terms of objective characteristics while allowing for the study of the transactional relationship between organism and environment. The work of the ethologists in defining stimuli while studying their relationship to different organismic states and situational contexts is emphasized in this regard. Following Brunswik, it is also suggested that wherever possible there be a representative sampling of variables in natural settings. Note from the editors: From time to time, Human Ecology will publish a review article. Our first in this series is a review by a psychologist of basic definitional and conceptual problems in environmental studies.This paper was prepared while the author was a Visiting Research Fellow at the Educational Testing Service. The support of ETS and my colleagues in the Division of Psychological Studies is gratefully acknowledged. The review was also supported in part by a grant from the Rutgers University Research Council.     

Synthesis,tautomerism, and antimicrobial,anti-HCV,anti-SSPE,antioxidant, and antitumor activities of arylazobenzosuberones

2-Dimethylaminomethylene-1-benzosuberone 1 was coupled with diazotized aniline derivatives to afford a series of the hitherto unreported 2-arylazo-1-benzosuberones 3a–i. The tautomeric structure and the effect of substituents on the tautomeric form (s) of the products 3a–i were discussed. Similar coupling of the enaminone 1 with diazonium salts of heterocyclic amines gave the respective fused azolotriazino-benzosuberones. Some of the newly synthesized compounds showed potent antimicrobial, anti-HCV, antioxidant, antitumor (as topoisomerase I inhibitors), and antimicrobial activities.     

The effect of malathion,diazinon, and various concentrations of zinc,copper, nickel,lead, iron,and mercury on fish

Acute and chronic toxicity tests for malathion, diazinon, copper (Cu), mercury (Hg), lead (Pb), zinc (Zn), nickel (Ni), and iron (Fe) were conducted. Mortalities ofBarilius vagra andCyprinus carpio (common carp) were variable but LC50-96 hr were similar for pesticides. AdultB. vagra seem to be more sensitive to malathion than juvenile carp. Both juvenile carp and adultB. vagra were extremely sensitive to diazinon. Long-term exposure to pesticides modified morphology and behavior. The LC50-96 values for Cu, Hg, and Pb were 0.3, 0.16, and 0.44, respectively, for smaller fish and 1.0, 0.77, and 1.33, respectively, for larger fish. Replicate LC50 values for Zn, Ni, and Fe were somewhat variable, and for these metals, the size of the fish seemed to affect response because LC50 values increased as fish size increased. Cooper, Pb, Zn, and Fe residues following exposure to sublethal concentrations of these metals for 15 d were significantly greater in whole juvenile common carp than in controls.     

Low-frequency dynamics and Raman scattering of crystals, of B-, A-, and Z-DNA, and fibers of C-DNA

Normal modes of vibration of DNA in the low-frequency region (10-300 cm-1 interval) have been identified from Raman spectra of crystals of B-DNA [d(CGCAAATTTGCG)], A-DNA [r(GCG)d(CGC) and d(CCCCGGGG)], and Z-DNA [d(CGCGCG) and d(CGCGTG)]. The lowest vibrational frequencies detected in the canonical DNA structures--at 18 +/- 2 cm-1 in the B-DNA crystal, near 24 +/- 2 cm-1 in A-DNA crystals, and near 30 +/- 2 cm-1 in Z-DNA crystals--are shown to correlate well with the degree of DNA hydration in the crystal structures, as well as with the level of hydration in calf thymus DNA fibers. These findings support the assignment [H. Urabe et al. (1985) J. Chem. Phys. 82, 531-535; C. Demarco et al. (1985) Biopolymers 24, 2035-2040] of the lowest frequency Raman band of each DNA to a helix mode, which is dependent primarily upon the degree of helix hydration, rather than upon the intrahelical conformation. The present results show also that B-, A-, C-, and Z-DNA structures can be distinguished from one another on the basis of their characteristic Raman intensity profiles in the region of 40-140 cm-1, even though all structures display two rather similar and complex bands centered within the intervals of 66-72 and 90-120 cm-1. The similarity of Raman frequencies for B-, A-, C-, and Z-DNA suggests that these modes originate from concerted motions of the bases (librations), which are not strongly dependent upon helix backbone geometry or handedness. Correlation of the Raman frequencies and intensities with the DNA base compositions suggests that the complex band near 90-120 cm-1 in all double-helix structures is due to in-plane librational motions of the bases, which involve stretching of the purine-pyrimidine hydrogen bonds. This would explain the centering of the band at higher frequencies in structures containing G.C pairs (greater than 100 cm-1) than in structures containing A.T pairs (less than 100 cm-1), consistent with the strengths of G.C and A.T hydrogen bonding.     

Synthesis and properties of N-, O-, and S-phospho derivatives of amino acids, peptides, and proteins

The literature on chemical (i.e., nonenzymic) phosphorylation of amino acids, peptides, and proteins is reviewed through 1982. The review covers synthetic methods, chemical reactions, and physical properties, with emphasis on the techniques used for separation and characterization of the products. Synthetic methods are classified by reagent rather than product, and are illustrated by experimental procedures for the most important methods. Chemical reactions are classified into four groups depending on whether the reaction site is the phospho group, the amino group, the carboxyl group, or in the case of serine the hydroxyl group. Physical data are given for all of the known N-, O-, and S-phospho derivatives of the amino acids, peptides, and proteins, within certain limitations, and are discussed in detail in the section on physical properties. Emphasis is given to the techniques used for separation of the products, such as chromatography and electrophoresis, and for characterization of the products, particularly spectroscopy. Medical and other uses of the products are mentioned.     

Cultural, Biochemical, and Immunological Properties of Mima, Herellea, and Flavobacterium Species

From study of cultural and biochemical characteristics of 40 strains of Herellea, Mima, or Flavobacterium species, a proposed schema for identification was developed. The reactions observed by agglutination, gel diffusion, and immunofluorescence suggest antigenic heterogeneity of this group of organisms.     

Pet Loss and Representations of Death,Attachment, Depression,and Euthanasia

Studies that have examined pet loss hypothesize that attachment, representations of death, and the belief in an afterlife for animals may influence owners’ bereavement and depressive outcomes. The following instruments were administered to 159 Italian participants recruited through snowball sampling: the Lexington Attachment to Pets Scale (LAPS), the Pet Bereavement Questionnaire (PBQ), the Testoni Death Representation Scale (TDRS), and Beck’s Depression Inventory II (BDI-II). Questions concerning pet euthanasia-related issues and the relationship between owners and veterinarians were also submitted to the participants. A path model was conducted, showing that the representation of death and the attachment to a pet had a direct effect on pet grief, which in turn had a direct effect on depression. The results show a positive correlation between the LAPS and PBQ factors, particularly with the PBQ factor Grief. The LAPS factors positively correlated with the TDRS representation of Death as a Passage and negatively correlated with the TDRS representation of Death as Annihilation. The LAPS People Substituting factor positively correlated with the total score and the Cognitive-Affective factor of the BDI-II. The PBQ factors positively correlated with the BDI-II, whereas only the TDRS Death as Annihilation factor positively correlated with the BDI-II. Belief in a transcendent dimension was associated with higher scores on the PBQ Guilt factor and the TDRS factors of Death as a Passage and Death as Change, whereas these beliefs were associated with lower scores on the TDRS factor Death as Annihilation.

The results indicated that the sensitivity of the veterinarian and a veterinarian who helps owners make conscious and informed decisions for their pet and choose the right time to perform euthanasia are important variables in the management of pet loss. However, these factors are not sufficient and psychological support should be improved to help owners better cope with grief.     

Count data, detection probabilities, and the demography, dynamics, distribution, and decline of amphibians

The evidence for amphibian population declines is based on count data that were not adjusted for detection probabilities. Such data are not reliable even when collected using standard methods. The formula C = Np (where C is a count, N the true parameter value, and p is a detection probability) relates count data to demography, population size, or distributions. With unadjusted count data, one assumes a linear relationship between C and N and that p is constant. These assumptions are unlikely to be met in studies of amphibian populations. Amphibian population data should be based on methods that account for detection probabilities.