磷酸三邻甲苯酯对母鸡神经组织线粒体超微结构及功能的影响

目的观察磷酸三邻甲苯酯(tri-ortho-cresyl phosphate,TOCP)对母鸡神经组织线粒体超微结构及功能的影响,探讨有机磷化合物诱导的迟发性神经毒性(organophosphorus easter-induced delayed neurotoxicity,OPIDN)的机制。方法成年罗曼母鸡16只,随机分为750mg/kg TOCP染毒组(n=12)和对照组(n=4)。TOCP按0.65ml/kg灌胃量一次性染毒,对照组给予等体积玉米油。分别于染毒后第1、5、15、21天观察线粒体结构的改变。另取成年罗曼母鸡96只,随机分为低、中、高剂量染毒组和1个对照组(n=24)。染毒组分别按185、375、750mg/kg经灌胃一次性染毒TOCP,对照组给予等体积玉米油。分别于染毒后第1、5、15、21天采用MTT比色法检测大脑、脊髓和周围神经(坐骨神经和胫神经)中琥珀酸脱氢酶活力的改变来间接代表线粒体呼吸链酶活力的改变。结果 TOCP组母鸡中枢神经系统大脑和脊髓中线粒体结构从染毒第5天开始出现病理改变,并随着OPIDN症状的发展逐渐加重;胫神经在第1天即可观察到线粒体及髓鞘的病理改变,且逐渐加重。染毒组与对照组相比,大脑中线粒体呼吸链酶活力的改变不明显,高剂量组在第15天和第21天出现明显降低,差异有统计学意义(P0.05);脊髓与周围神经中线粒体呼吸链酶活力的变化趋势大致相同,中、高剂量组均从第5天开始明显下降,差异有统计学意义(P0.05),低剂量组的最低峰值较中、高剂量组出现稍晚。各剂量组间及各时间点之间没有显著差异。结论 TOCP能使母鸡神经组织线粒体结构发生改变,并影响线粒体呼吸链关键酶的活力。     

三邻甲苯基磷酸酯诱发的迟发性神经病动物模型的病理和微管相关蛋白的改变

目的 建立母鸡迟发性神经病(OPIDN)模型,观察其病理改变及微管相关蛋白2(MAP2)的改变.方法 选取12月龄的产卵母鸡48只,随机分为对照组和3个三邻甲苯基磷酸酯(TOCP)染毒组,每组12只.TOCP染毒组的动物经口一次性灌胃染毒,剂量分别为250、500、750 mg/kg,观察中毒症状,染毒后第21天处死动物,其中6只动物取脑、脊髓和坐骨神经,进行HE染色和髓鞘染色;另6只动物的脑组织匀浆后用免疫印迹(Western blot)法测定MAP2的蛋白表达.结果 500和750mg/kg剂量组动物出现不同程度共济失调症状,脊髓、坐骨神经出现神经胶质增生、髓鞘脱失及轴突变性.500和750 mg/kg剂量组MAP2蛋白表达明显升高,分别较对照组升高了25%和23%,差异有统计学意义(P<0.05或P<0.01).结论 TOCP引起各剂量组动物出现不同程度的OPIDN病理改变;TOCP可引起鸡神经组织中的MAP2含量发生改变,这种改变可能与TOCP引起的迟发性神经毒性有关.     

磷酸三邻甲苯酯对母鸡中枢神经组织ATP含量的影响

[目的]研究母鸡中枢神经组织ATP含量在磷酸三邻甲苯酯（TOCP）诱导迟发性神经毒性（OPIDN）过程的变化,进一步探讨TOCP诱发OPIDN的分子机制。[方法]成年罗曼母鸡40只,随机分为d0（正常对照组）、d5、d10、d15和d21组（每组8只）,除d0组外,其余各组动物一次性经口灌胃TOCP750mg／kg。每天观察母鸡OPIDN症状并按8级评分标准进行评价,利用高效液相色谱法（HPLC）测定母鸡大脑、小脑和脊髓组织中ATP含量。[结果]母鸡在染毒后第5天即出现间或的双腿运动轻微不协调,第21天完全瘫痪。大脑组织中ATP含量在染毒后第5天升高了20．62％（P〈0．01）,第15天和第21天分别降低了15．5％和17．96％（均为P〈0．01）;小脑组织中ATP含量在染毒后第5天和第10天分别升高了76．95％（P〈0．01）和20.36％（P〈0．05）,第21天降低了20．49％（P〈0．05）;脊髓组织中ATP含量在染毒后第5天、第10天和第15天分别升高了32．45％、42．89％和87．1％（均为P〈0．01）。[结论]在母鸡表现出明显的OPIDN症状之前中枢神经组织ATP含量即出现明显的升高,提示能量的变化可能在OPIDN的发生过程中起重要作用。     

三邻甲苯磷酸酯诱导母鸡神经组织中自噬相关蛋白含量的变化

目的 研究三邻甲苯磷酸酯(TOCP)诱导的迟发性神经病(OPIDN)中自噬相关蛋白Atg1、Atg5和Beclin1的变化,探讨OPIDN发生的分子机制.方法 取成年罗曼母鸡30只,随机分为对照组、1d、5d、10d和21d组(每组6只),对照组给予同体积的玉米油,各染毒组动物均按750 mg/kg经一次性灌胃染毒TOCP,分别在相应时间点处死动物取胫神经和脊髓,免疫印迹法检测胫神经和脊髓中Atg1、Atg5和Beclin1的相对含量.结果 与对照组相比,TOCP染毒后胫神经中Atg1含量1、5、10、21d分别下降了 29.8％、64.4％、43.5％和19.8％(P＜0.05);TOCP染毒后Atg 5含量1、5、10、21d分别下降了36.8％、49.6％ 、51.2％和31.5％(P＜0.05);TOCP染毒后beclin1含量1、5、10天分别下降了 68.5％、66.3％和32.2％(P＜0.05).与对照组相比,TOCP染毒后脊髓组织中Atg1含量1、5、10d分别下降了23.5％、48.7％和20％(P＜0.05);Atg5含量在TOCP染毒后1、5、10、21d分别下降了32.7％ 、51.5％、47.3％和39.6％(P＜0.05);beclin1含量在TOCP染毒后1、5、10、21d分别下降了28.9％、50.2％、43.2％和28.3％(P＜0.05).结论 TOCP染毒干扰了母鸡神经组织中自噬相关蛋白的表达,可能与OPIDN的发病机制有关.     

三邻甲苯磷酸酯染毒母鸡神经组织中自噬相关蛋白ATG4表达与磷酸化的变化

目的 研究三邻甲苯磷酸酯(TOCP)染毒母鸡以及提前应用苯甲基磺酰氟(PMSF)后神经组织中自噬相关蛋白ATG4A与p-ATG4A表达的变化,探讨迟发性神经病(OPIDN)的发病机制.方法 选取成年罗曼母鸡60只,其中30只动物用于TOCP染毒实验,即动物随机分为对照组,1、5、10、21 dTOCP染毒组,剂量为600 mg/kg,一次性灌胃染毒.另外30只动物随机分为对照组,1、5、10、21 d PMSF干预组,提前24 h皮下注射90 mg/kg PMSF,然后再染毒600 mg/kg TOCP,用免疫印迹法检测胫神经ATG4A与p-ATG4A的相对含量.结果 与对照组比较,TOCP染毒后第1、5、10天胫神经ATG4A含量分别下降36％、43.7％,41％,染毒后第1、5、10天胫神经p-ATG4A含量分别下降22.5％、25％,21％差异有统计学意义(P＜0.05).PMSF干预组动物胫神经中ATG4A 、p-ATG4A与对照组相比没有明显改变.结论 TOCP染毒后干扰了自噬相关蛋白ATG4A、p-ATG4A表达的变化,可能与OPIDN中神经元自噬活性变化有关.     

三邻甲苯磷酸酯暴露鸡脊髓神经中Stathmin的差异表达

目的 从三邻甲苯磷酸酯(TOCP)暴露鸡的脊髓组织中筛选可能与调控微管解聚作用相关的差异表达蛋白,为探讨有机磷化合物诱发的迟发性神经毒性( OPIDN)作用机制提供靶蛋白依据.方法 42只罗曼鹤母鸡随机分成1000 mg/kg TOCP组、给予40 mg/kg苯甲基磺酰氟(PMSF)后再给予1000 mg/kg TOCP的干预组和生理盐水对照组,每组14只.染毒第5和20天,每组分别处死4只动物,低温环境下分离腰髓,提取总蛋白.用双向电泳和质谱分析技术,筛选和鉴定可能与调控微管解聚作用相关的差异表达蛋白.结果 染毒第5天,TOCP暴露组部分鸡出现OPIDN症状,并随时间的推移其症状逐渐加重.双向电泳和质谱分析结果表明,在染毒早.期与对照组和PMSF前干预组比较,TOCP组鸡腰髓组织中微管解聚蛋白Stathmin表达分别下调3.4和2.8倍,而PMSF前干预组与对照组之间,Stathmin的表达无明显差异.结论 TOCP暴露诱导鸡脊髓神经组织Stathmin表达明显下调,而且该蛋白表达下调与微管的大量聚集及其OPIDN发生的机制可能有关.     

苯甲基磺酰氟预处理磷酸三邻甲苯酯染毒母鸡对其脊髓中神经丝的影响

目的 研究用苯甲基磺酰氟(PMSF)预处理对有机磷化合物诱导的迟发性神经病(OPIDN)母鸡脊髓中神经丝(NFs)动态变化的影响,探索OPIDN发生的可能机制,方法成年母鸡随机分为正常对照组、磷酸三邻甲苯酯(TOCP)组和PMSF+TOCP组,每组32只,PMSF+TOCP组的动物提前给予皮下注射60ms/ksPMSF,24 h后给予TOCP组和PMSF+TOCP组动物一次性经口灌胃750mykg TOCP,对照组给予等体积生理盐水.分别在TOCP染毒后1、5、10、21 d处死动物,取脊髓组织匀浆分离出上清和沉淀2种组分,利用十二烷基磺酸钠-聚丙烯酰胺凝胶电泳(SDS.PAGE)和蛋白免疫印迹法(Westem-blotting)检测上清和沉淀中高相对分子质量神经丝蛋白(NF-H)、中相对分子质量神经丝蛋白(NF-M)、低相对分子质量神经丝蛋白(NF-L)的相对含量.结果 TOCP染毒组母鸡在21 d内均出现瘫痪,PMSF+TOCP组动物未出现明显的OPIDN表现,与正常对照相比,脊髓沉淀中3种NF亚单位的含量均表现为先降低后恢复,均在10 d达到最低,差异有统计学意义(P<0.01).上清中3种NF亚单位的变化趋势与沉淀中基本一致,均在染毒后10d时明显降低,到21 d时恢复到正常水平.其中,10 d时NF-L、NF-M和NF-H的含量降低幅度分别达到51%、86%和38%,与对照组比较,差异有统计学意义(P<0.05).PMSF预处理再染毒TOCP组母鸡在21 d内未出现瘫痪,且脊髓中NFs紊乱也明显减轻.与正常对照相比,脊髓样品匀浆离心后的沉淀中只有NF-M在21 d时增加了21%,差异有统计学意义(P<0.05);上清中只有NF-H和NF-M在21 d时增加了19%和35%,差异有统计学意义(P<0.05).结论 TOCP染毒母鸡出现OPIDN,脊髓组织中NFs含量发生紊乱.用PMSF预处理后能阻断OPIDN的发生,并且可明显地抑制脊髓中NFs含量的紊乱.     

莎稗磷对鸡迟发性神经毒性研究

选取8~12月龄的成年莱亨母鸡60只,随机分为溶剂对照组、阳性对照组和莎稗磷低、中、高(1 000、2 500、5 000 mg/kg)剂量染毒组。溶剂对照组给予玉米油,阳性对照组给予500 mg/kg三邻甲苯磷酸酯(TOCP)。莎稗磷高剂量染毒组动物出现活动减少、呈卧位等改变,并有动物死亡;全血及血清胆碱酯酶活力分别与溶剂对照组比较明显降低(P0.01或P0.05);各莎稗磷染毒组均未出现迟发性神经毒性症状,脊髓病理组织学检查也未发现神经毒性病理改变。因此,在本实验条件下,未见莎稗磷产生迟发性神经毒性效应。     

三邻甲苯磷酸酯诱发迟发性神经毒性动物模型的机制初探

选取12月龄的来亨母鸡24只,随机分为4组,分别作为赋性剂对照组和3个三邻甲苯磷酸酯(TOCP)染毒组。TCOP染毒组的动物经口一次性灌胃染毒,剂量分别为250,500,750 mg/kg,观察中毒症状,染毒后第21天处死动物,取脑组织,匀浆后Western blot测定大脑组织蛋白激酶A(PKA)、磷酸化环腺苷酸反应元件结合蛋白(CREB)、微管相关蛋白2(MAP-2)和Tau-2蛋白的含量。结果500和750mg/kg剂量组动物出现不同程度共济失调症状。低、中、高剂量组PKA含量显著升高,分别较对照组升高了23%、46%、70%(P<0.01);中、高剂量组CREB显著升高,分别较对照组升高了23%(P<0.05)、37%(P<0.01);中、高剂量组MAP2显著升高,分别较对照组升高了25%(P<0.01)、23%(P<0.05);Tau-2均未见显著性改变。 更多还原     

OPIDN致鸡大脑脑型肌酸激酶差异表达

目的 探讨有机磷化合物致迟发性神经毒性（OPIDN）鸡脑组织中脑型肌酸激酶同工酶（CK-BB）表达变化。方法 罗曼鹤母鸡24只,随机分成三磷甲苯磷酸酯（TOCP）染毒组（1 000 mg/kg）、苯甲基磺酰氟（PMSF）干预组（在染毒之前给予40 mg/kg PMSF）和对照组（生理盐水）,每组8只。染毒第5和第21天,每组分别处死8只动物,分离大脑,提取总蛋白;用双向电泳和质谱分析技术,筛选相关差异表达蛋白。结果 蛋白双向电泳结果显示,在染毒第5天,TOCP组与对照组、PMSF干预组与对照组的CK-BB蛋白点灰度比值分别为0.858 3和0.913 3,各组间差异无统计学意义（P>0.05）;染毒第21天,TOCP组和PMSF干预组与对照组差异蛋白点CK-BB灰度比值分别为0.327 5与0.675 7,前者表达下调达3.0倍;对该蛋白点的质谱分析结果在Swiss-por数据库中进行比对,其与鸡种属、蛋白序列号为gi|45384340的creatine kinase B-type（CK-BB）蛋白的肽段匹配数为26,序列覆盖率为75%,评分值为1 603（可信度高）,显示该蛋白点即为CK-BB蛋白。结论 OPIDN可致鸡大脑组织CK-BB蛋白表达显著下调,其变化可能与OPIDN模型动物神经组织损伤有关。     

PMSF对TOCP暴露鸡腰髓组织蛋白表达谱影响

目的 从三邻甲苯磷酸酯(TOCP)暴露鸡的腰髓组织中筛选迟发性神经毒性(OPIDN)相关差异表达蛋白点,为进一步质谱分析提供依据。方法 成年罗曼鹤母鸡24只,随机分成1 000 mg/kg TOCP组,先给40 mg/kg苯甲基磺酰氟(PMSF)后24 h再投1 000 mg/kg TOCP组(PMSF干预组)和给予生理盐水的对照组,每组8只。染毒后5 d天处死动物,分离腰髓后提取总蛋白;通过双向凝胶电泳获得完整的全蛋白质图谱,运用图像分析软件(ImageMaster2D)对银离子染色的电泳图谱进行分析。结果 根据PMSF的作用特点,在TOCP组中选择与对照组比较差异有统计学意义,与PMSF干预组差异无统计学意义的蛋白匹配点,共获得上调点23个,下调点113个,其中>4倍的上调点9个,>4倍的下调点11个。结论 TOCP暴露鸡腰髓组织中9个上调点和11个下调点的差异表达蛋白与其迟发性神经毒性可能密切相关。     

三磷酸甲酯中毒致母鸡脊髓神经元凋亡的研究

目的　研究细胞凋亡机制在有机磷中毒后的动物迟发性神经病 (OPIDN)中所起的作用及其动态病理改变。方法　采用给母鸡一次性肌内注射三磷酸甲酯染毒的方法建立OPIDN动物模型 ,以染毒后 3、5、7、10、14、18d为时间点分别取材 ,以HE染色、尼氏法和原位末端标记法 (TUNEL)观察其第三腰髓 (L3 )的病理改变、神经元数量及细胞凋亡的变化。结果　母鸡于肌内注射三磷酸甲酯后第 9天前后出现进行性共济失调和肌无力等OPIDN的典型症状。HE染色示染毒后 5d后母鸡脊髓前角大神经元出现细胞核红色深染 ,至 18d消失 ;尼氏法染色示母鸡脊髓前角神经元数量呈进行性减少 [从 (82±4)个 /mm2 到 (66± 6)个 /mm2 ] ;TUNEL法示母鸡腰段脊髓TUNEL阳性细胞于染毒后 5d左右开始出现[(2 2± 2 )个 /mm2 ] ,以 7d[(2 7± 3 )个 /mm2 ]数量最多 ,18d消失。结论　在母鸡OPIDN发病过程中出现腰髓前角神经元细胞凋亡现象。提示细胞凋亡可能在OPIDN发病机制中起重要作用     

Assessment of the delayed neurotoxicity of tributyl phosphate, tributoxyethyl phosphate, and dibutylphenyl phosphate

There industrial organophosphorus compounds were tested for their ability to cause organophosphorus compound-induced delayed neurotoxicity (OPIDN) in the adult hen. The compounds tested were tributyl phosphate (TBP), tributoxyethyl phosphate (TBEP), and dibutylphenyl phosphate (DBPP). The acute oral LD50 of TBP and DBPP were estimated to be 1,863 and 1,500 mg/kg, respectively, and the dose equal to the LD50 was used as a test dose. The acute oral LD50 of TBEP was greater than 5,000 mg/kg and 5,000 mg/kg was used as a test dose. An oral dose of 750 mg tri-o-cresyl phosphate (TOCP) was used as a positive control. For the acute delayed neurotoxicity test, hens were given two test doses of the test materials 21 days apart and killed 21 days after the second dose. None of the hens given TBP, TBEP, or DBPP exhibited nerve damage or clinical signs which distinguished them from untreated control animals. A single dose of TOCP resulted in paralysis and a histopathological profile typical of a distal neuropathy. For the assay of the inhibition of esterases, hens were killed 24 hours after a single dose equal to the greater of either the LD50 or 5000 mg/kg. TOCP administration resulted in over 90% inhibition of brain neurotoxic esterase (NTE), but none of the other three compounds inhibited NTE to an extent (greater than 70%) which would be expected to result in OPIDN. Administration of TOCP, TBEP, or DBPP resulted in approximately a 70% decrease in plasma butyrylcholinesterase (BuChE) activity. TBP caused a 2-3 fold increase in BuChE activity. TBEP administration resulted in about 45% inhibition of acetycholinesterase (AChE) in brain. These results indicate that TBP, TBEP, and DBPP are all unlikely to cause OPIDN with any single sublethal dose.     

Effects of prednisolone and complex of vitamin B1, B2, B6 and B12 on organophosphorus compound-induced delayed neurotoxicity

Protective effects of prednisolone as a synthetic adrenal cortical hormone and complex of vitamin B(1), B(2), B(6) and B (12) on organophosphorus compound-induced delayed neurotoxicity (OPIDN) caused by leptophos and tri-o-cresyl phosphate (TOCP) as organophosphates (OPs) were examined. Nine groups of hens (six for each) were used. Eight groups received intravenous injection of 30 mg/kg of leptophos or 40 mg/kg of TOCP (four groups in each). Among them, three groups which received leptophos were given (p.o.) predonisolone (2 mg/body), vitamin B complex (25 mg/body) or both 3 h after OPs injection and then every day for 15 d (one group for each); the same treatment was performed on three groups which received TOCP. The remaining one group served as controls. It was observed that delayed neuropathy induced by OPs could not be resisted completely by the treatment with prednisolone or vitamin B complex, but clinical signs of OPIDN and pathological changes in hens that received these two protective agents after OPs were less severe than those in hens that received only OPs. Of these groups, the improvement in clinical signs was best shown in hens that received the both two protective agents. In addition, improvement in clinical signs among the hens that did not deteriorate to paralysis was observed. In particular, those which developed mild ataxia recovered well. It is indicated that combining administration of prednisolone and vitamin B complex early before clinical signs are manifest is effective in alleviating neuropathy. It is also suggested that recovery or good prognosis will be expected, as long as progression of the clinical signs is prevented before paralysis develops in delayed neuropathy.     

三邻甲苯磷酸酯染毒鸡大脑组织中迟发性神经毒性相关蛋白的筛选

目的 从三邻甲苯磷酸酯(TOCP)染毒鸡大脑组织中筛选迟发性神经毒性(OPIDN)相关差异表达蛋白,为进一步探讨OPIDN机制提供靶标蛋白.方法 成年罗曼鹤母鸡32只随机分成1000mg/kg TOCP组(染毒组)、先给40mg/kg PMSF后24 h再给予1000 mg/kg TOCP组(PMSF干预组)、给予40 mg/kg PMSF组(PMSF组)和给予生理盐水的对照组,每组8只.一次性染毒后第5天处死动物,低温环境下分离大脑,匀浆,高速离心提取总蛋白.通过双向凝胶电泳获得完整的全蛋白质图谱,运用图像分析软件(Image Master 2D)对银离子染色的电泳图谱进行分析,并对所选取的蛋白点进行质谱鉴定.结果 染毒组、PMSF干预组和PMSF组鸡大脑组织总蛋白中,分别检测到1185、1294和1063个蛋白点,对照组检测到1332个蛋白点.与对照组比较,染毒组、PMSF干预组和PMSF组匹配率分别为78.32%、79.56%、80.93%.与对照组比较,染毒组差异表达蛋白点有235个,其中,上调点有158个,下调点有77个.根据PMSF的作用特点,在TOCP组中选择与对照组比较差异明显且与PMSF干预组无明显差异的蛋白匹配点,共获得有102个.其中差异表达4倍以上且与PMSF组二次匹配差异无统计学意义的蛋白点有13个.对这些蛋白点做进一步质谱分析和鉴定,获得7个蛋白:homer-1b、Destrin蛋白、热休克蛋白70、真核翻译起始因子、蛋白酶体α1亚基、乳酸脱氢酶B、代谢性谷氨酰胺合成酶.结论 TOCP染毒鸡大脑组织中有112个差异表达蛋白点,可能与OPIDN诱发有关,其中13个差异表达蛋白点可能与OPIDN诱发有密切的关联性.质谱鉴定出7个可能与OPIDN机制关联的蛋白.

Abstract：

Objective To screen the proteins with differential expression levels in the cerebral tissue of hens exposed to tri-ortho-cresyl phosphate (TOCP),and to provide target proteins for studying the mechanism of organophosphoms ester-induced delayed neurotoxicity (OPIDN). Methods Thirty two adult Roman hens were randomly divided into four groups: TOCP group was exposed to 1000 mg/kg TOCP, PMSF group was exposed to 40 mg/kg PMSF, PMSF plus TOCP group was exposed to 40 mg/kg PMSF and after 24 h exposed to 1000 mg/kg TOCP, control group was exposed to normal saline. All hens exposed to chemicals by gastro-intestine for 5 days were sacrificed, and the cerebral tissue were dissected and homogenized in ice bath. Total proteins extracted from the cerebral tissue were separated by isoelectric focusing as the first dimension and SDS-PAGE as the second dimension. The 2-DE maps were visualized after silver staining and analyzed by Image Master 2D software. At last ,the expressed protein spots were identified by Mass spectrometry. Results From total proteins in TOCP group, the PMSF plus TOCP group and PMSF group, 1185, 1294 and 1063 spots were detected, respectively. One thousand three hundred thirty two spots from total proteins in control group were detected. The match rates of protein spots in TOCP group, the PMSF plus TOCP group and PMSF group were 78.32 %, 79.56 % and 80.93%, respectively. There were 235 protein spots with differential expression levels between TOCP group and control group, which included 158 up regulation spots and 77 down regulation spots. According to the PMSF features, there were 102 spots with differential expression levels between TOCP group and control group and without differential expression levels between TOCP group and PMSF plus TOCP group, among them there were 13 spots with 4 fold differential expression levels between TOCP group and control group and without differential expression levels between TOCP group and PMSF group. Seven protein spots (homer-1b, Destrin, heat shock protein 70, eukaryotic translation initiation factors, proteasome α1 subunit, lactate dehydrogenase B, glutamine synthetase) were detected by Mass spectrometry. Conclusion There are 112 protein spots with differential expression levels of the cerebral tissue in TOCP group,which may be related to OPIDN,among them 13 protein spots with differential expression levels are associated closely with OPIDN.Seven protein spots detected by Mass spectrometry may be related to the mechanism induced by OPIDN.