火电厂周边不同生物结皮细菌群落特征差异及其影响因素

为探究大气降尘重金属污染对矿区周边不同类型生物结皮细菌群落结构的影响,利用高通量测序技术分析位于宁东能源化工基地典型火电厂周边的3类生物结皮(藻结皮ZB、混生结皮HB、苔藓结皮TB)和对照(CK,裸土)的细菌丰度和群落结构,并探讨了影响细菌群落结构的环境因子。结果表明: 不同类型生物结皮的理化性质和重金属含量存在差异,且由于生物结皮对大气降尘重金属的富集作用造成各类结皮均达重度污染级别。在相对丰度排名前10的优势细菌门中,芽单胞菌门、蓝细菌门在不同类型生物结皮之间差异显著。细菌群落α多样性由高到低排序依次为CK>TB>HB>ZB。非度量多维排序(NMDS)结果显示,裸土细菌群落与其他3种生物结皮存在明显差异。相关性分析表明,生物结皮演替对细菌群落组成具有显著影响,细菌多样性和组成与pH、养分、重金属含量等密切相关。放线菌门、绿弯菌门相对丰度与pH值呈显著正相关关系,而与全氮(TN)、全磷(TP)、Pb、Zn、Cd均呈显著负相关关系;冗余分析结果表明,TN、pH、TP、有机碳(SOC)是影响3种生物结皮细菌群落α多样性以及一些优势菌群相对丰度的主要土壤环境因子,而重金属Pb、Zn、Cd是影响细菌群落结构的主要重金属元素,对细菌群落数量和多样性有抑制或刺激作用。说明pH、重金属和养分是影响结皮细菌群落组成的关键因子。总体而言,长期的重金属富集作用会对生物结皮的细菌多样性和群落组成产生影响。     

不同重金属污染水平下生物结皮及下层土壤细菌群落的差异性分析

为了探究生物结皮及下层土壤细菌群落多样性及结构在不同重金属污染水平下的差异及其影响因素,以宁夏东部工矿区和封育草地内广泛分布的两类生物结皮为研究对象,采用高通量测序技术,分析结皮层(A层)和结皮下层(B层)土壤细菌群落结构和多样性差异。采用相关分析、冗余分析探讨两个生境生物结皮各层次细菌群落与土壤环境因子的关系。结果表明：(1)相较于封育区,工矿区2类生物结皮对源自大气降尘的重金属元素具有更显著的表层富集作用,并表现出显著的种间差异：苔藓结皮>藻结皮。污染负荷指数评价结果显示：工矿区生物结皮各层次重金属污染程度更高,且均表现为A层污染程度高于B层。(2)两个生境的优势细菌门组成基本一致,均为放线菌门、变形菌门、酸杆菌门。受生物结皮类型、重金属污染程度的影响,细菌群落结构差异显著,主要表现为：工矿区藻结皮A、B层的α多样性指数均大于封育区,而苔藓结皮A、B层的细菌多样性指数高于封育区,丰富度指数低于封育区。主坐标分析结果显示,不同生境的生物结皮细菌群落结构差异明显,而同一生境的生物结皮细菌群落结构相似。重金属As、Pb、土壤黏粒含量和pH是影响生物结皮细菌群落结构的主要土壤环境因子...     

秦岭石生苔藓结皮的微生物群落组成和多样性特征北大核心CSCD

苔藓结皮是地表重要的活性覆盖物,具有改善土壤性状、固土持水、固碳固氮等多种生态功能,微生物群落是苔藓结皮发挥生态功能的主要成分。为了探究湿润区石生苔藓结皮的微生物群落组成及其多样性特征,揭示其发育形成机制以及功能机理,该研究以秦岭北麓的泥峪、车峪、骆峪5个样地的石生苔藓结皮为研究对象,利用高通量测序技术,分析了石生苔藓结皮层的微生物群落组成、丰度、多样性,并测定分析养分与微生物群落的关系,为裸露岩石创面的生态修复提供理论依据。结果表明:(1)秦岭石生苔藓结皮层的全氮和有机质含量在4.16~8.26 g/kg、89.44~131.05 g/kg之间,且样点间均差异显著;全磷和全钾含量较低,分别为0.88~1.21 g/kg和13.50~18.10 g/kg。(2)秦岭石生苔藓结皮层中细菌占微生物群落组成的80%以上,且细菌的多样性也远高于真菌;其中细菌优势菌群为变形菌门(Proteobateria)、拟杆菌门(Bateroidetes)、放线菌门(Actinobacteria)和酸杆菌门(Acidobacteria),真菌优势菌群为子囊菌门(Ascomycota)和被孢霉菌门(Mortierellomycota)。(3)冗余分析(RDA)显示,全钾和有机质对优势细菌群落结构影响显著,变形菌和拟杆菌适宜生活在湿度大、营养状况良好的基质中;全磷和有机质对优势真菌群落结构影响显著,被孢霉菌门在低磷基质中更具生长优势。研究发现,细菌是秦岭石生苔藓结皮层的优势菌群,与旱区土生状况相比,秦岭石生苔藓结皮层的放线菌和酸杆菌的含量有所下降,而拟杆菌含量显著增加;对于真菌而言,子囊菌门和担子菌门的相对丰度明显减少,被孢菌门丰度显著增加;有机质是影响结皮层微生物群落组成最主要的养分因子,丰富的养分使湿润区石生苔藓结皮层的微生物群落结构更加复杂多样、物种组成更加丰富。     

贵州省新路卡林型金矿区苔藓植物多样性及重金属污染监测

研究发现新路卡林型金矿区苔藓植物有10科17属28种,其中,丛藓科有9种,占总数的32.14%,真藓科有8种,占总数的28.57%。真藓科和丛藓科为优势科。分析该卡林型金矿区4个生境的β多样性,发现废石区—废渣区与相对污染区—清洁区的β多样性指数差异最大,苔藓群落结构差异性最大,相似性成分最低。测定该矿区苔藓植物及相应基质的重金属元素Pb、Zn、Cu、Cd、Hg、As含量,发现这些重金属元素含量呈现了一定的规律性,即：废渣区>废石区>相对污染区>清洁区,这可能与金矿的开采和冶炼有密切联系。因此,通过对苔藓植物的深入研究,可用以监测卡林型金矿区重金属污染情况。     

工矿区不同类型生物结皮对大气降尘重金属的富集规律及其影响因子

大气降尘是矿区土壤重金属的主要来源,而生物结皮对大气降尘重金属有显著的富集作用。为探究不同类型生物结皮对大气降尘重金属的富集规律及其影响因子,选取宁东典型火电厂周边生物结皮广布区作为试验样地,3类生物结皮作为研究对象,并以临近裸土作为对照,对比分析了生物结皮富集大气降尘过程中土壤理化性质、酶活性及重金属含量的变化,采用相关分析、冗余分析和方差分解方法探讨了不同类型生物结皮的结皮层(A层)和层下土壤(B层)重金属含量与其土壤物理、化学性质及酶活性之间的关系。结果表明：燃煤烟尘是矿区大气降尘重金属污染的主要来源,涉及重金属元素包括Cd、Cr、Hg、Pb、Zn、As。生物结皮对源自大气降尘的重金属元素均具有显著的富集作用,且在不同演替阶段生物结皮间的富集规律完全一致：藓结皮>混生结皮>藻结皮;重金属综合污染指数评价结果显示：生物结皮对重金属具有表层富集效应,表现为A层污染程度高于B层。不同类型生物结皮A、B层综合污染指数存在显著差异,排序为：藓结皮>混生结皮>藻结皮;和对照相比,三类生物结皮均能通过富集大气降尘增加其A、B层养分和细颗粒物含量并改善土壤质地。方差分解结...     

岩溶洞穴苔藓群落特征及其对重金属污染的指示意义: 以贵州织金洞为例

岩溶洞穴的开发与利用导致的重金属污染严重威胁洞穴环境及地下水, 因此对洞穴环境的监测具有重要的现实意义。本文通过对典型的暗河型溶洞贵州织金洞内的苔藓群落特征及其重金属污染状况进行研究, 拟揭示苔藓群落对洞穴重金属污染的指示意义。共发现苔藓群落类型34个, 苔藓植物种类20科33属49种, 含苔类8科8属12种, 藓类12科25属37种。优势科为丛藓科、凤尾藓科和提灯藓科, 物种占比率分别为16%、14%、14%。Hg污染超标倍数最高达59.45倍, 在距洞口不同深度均远高于Cr和Zn, 说明洞内Hg污染较为严重。苔藓群落指标随着重金属污染含量的上升总体呈下降趋势。所测定的重金属中基质Hg含量与苔藓群落指标的拟合程度最好, 与其对应的苔藓群落指标均呈显著负相关关系(P < 0.05), 说明苔藓群落特征能够很好地反映洞内Hg污染的变化趋势。RDA排序结果分析表明: 洞穴苔藓群落为适应低光度环境会表现出向光性运动、喜钙生基质、耐重金属等生理特征。蛇苔(Conocephalum conicum)植物体内的Hg含量与其基质Hg含量具有显著相关性(P < 0.05), 说明蛇苔受基质Hg污染影响较大。在今后的治理过程中, 可以考虑将蛇苔作为洞内环境的生物监测材料之一。     

放射性污染对铀矿区土壤微生物群落结构的影响

为探究放射性及其伴生污染对铀矿区附近土壤微生物群落功能多样性的影响，采集退役铀矿冶周围不同辐射值的土壤为研究对象，通过测定其土壤理化性质、土壤可培养微生物计数及Biolog-ECO微孔板培养，分析土壤放射性污染程度及理化性质对土壤微生物群落功能多样性和数量的影响。结果表明，矿区放射性污染土壤中重金属含量普遍较高，尤其是铅已超出管控标准。随着辐射值的增加，土壤中可培养微生物数量显著下降，特别是放线菌和真菌的数量；微生物群落功能多样性指数、物种丰富度指数、优势度、均一性指数显著下降；对碳源利用也逐渐下降，尤其是对酚酸类和胺类碳源，利用率分别下降25.8%、29.7%。RDA分析发现放射性核素是该区域驱动土壤微生物群落代谢发生变化的主要因素。铀矿区放射性污染及伴随的重金属污染明显改变土壤微生物群落结构，抑制了土壤微生物群落的代谢活性，造成土壤微生物群落功能多样性下降。     

毛乌素沙地不同类型生物结皮细菌群落差异及其驱动因子

生物结皮在干旱半干旱区具有极为重要的生态功能,细菌是生物结皮中的重要微生物类群,在生物结皮形成、养分循环与调控过程中发挥着关键作用。然而,对于毛乌素沙地生物结皮演替过程中细菌群落多样性和关键环境因子的认识尚不清楚。因此,本文通过q PCR和高通量测序方法调查了毛乌素沙地裸地、藻结皮、地衣结皮和藓结皮之间的细菌丰度和群落多样性,并探究了生物结皮中细菌群落及其与关键环境因子之间的关系。结果表明,随着生物结皮的演替,细菌16S rRNA基因丰度呈显著上升趋势,细菌Chao1、Shannon指数和系统发育多样性呈先降低后增加的模式。生物结皮不同阶段的细菌门、目和属的相对丰度均存在显著差异,在裸地阶段优势细菌类群是拟杆菌门的噬几丁质菌目、放线菌门的红色杆菌目和变形菌门的根瘤菌目;藻结皮和地衣结皮中均以蓝藻门的颤藻目占绝对优势;藓结皮的细菌以拟杆菌门的噬几丁质菌目和变形菌门的根瘤菌目占优势。全磷、全氮、pH和总有机碳是影响土壤细菌群落的关键环境因子。综上所述,生物结皮的演替通过改变土壤理化特性,为细菌群落提供了不同生态位,其中,土壤养分和pH等环境因子对毛乌素沙地生物结皮细菌物种筛选和群落塑造发挥...     

黄土高原北部水蚀风蚀交错区苔藓多样性及苔藓结皮发育的微生境特征

微生境因子对苔藓结皮的物种分布和发育有重要调控作用。为探明其作用途径和影响程度,以黄土高原水蚀风蚀交错区的苔藓结皮为研究对象,通过样线法调查了8种微生境中苔藓结皮的物种分布和发育特征,结合结构方程模型剖析了植物冠层覆盖、坡向和坡度对苔藓多样性和苔藓结皮发育的影响途径并量化了其影响系数。结果表明: 1)与无冠层相比,冠层覆盖使苔藓的Patrick、Shannon、Pielou和Simpson指数分别降低了63.4%、66.6%、91.0%和68.3%,但使苔藓结皮的厚度、生物量和叶绿素含量分别提高了0.5、0.2和1.3倍;2)北坡样地的4种苔藓物种多样性指数较南坡分别提高了0.6、0.9、5.6和0.9倍,而3种苔藓结皮发育指标分别提高了0.3、0.3和0.6倍;3)坡度从14°增加到34°,4种苔藓多样性指数分别降低了59.8%、84.1%、57.3%和68.0%,而3种苔藓结皮发育指标分别降低了15.2%、25.0%和16.5%;4)3种微生境因子对苔藓多样性和苔藓结皮发育的影响程度为冠层覆盖＞坡向＞坡度,其主要影响途径因微生境因子不同而存在差异。综上,植物冠层覆盖、坡度和坡向分异通过直接和间接途径显著影响了苔藓的物种分布和苔藓结皮发育,因此,在利用苔藓结皮进行荒漠化治理时应充分考虑微生境条件。     

石漠化程度对苔藓植物多样性及其结皮土壤化学性质的影响

以研究区域不同石漠化等级为代表,分析苔藓多样性特征及其结皮土壤化学性质变化规律,探索其对石漠化进程的响应机制。结果显示:(1)苔藓物种多样性:含苔藓14科29属84种,其中苔类2科2属2种;(2)多样性指数表现为:重度石漠化阶段中度石漠化轻度石漠化极重度石漠化无石漠化;其均匀度为:中度石漠化阶段轻度石漠化重度石漠化极重度石漠化无石漠化;(3)随着石漠化程度加剧,苔藓群落组成中混合群落比重下降而纯群落比重上升;群落生活型则呈现交织型递减而丛集型递增的规律。(4)结皮土壤中全氮、全磷和微生物量碳随石漠化加剧均都呈递减趋势。(5)通过Partial RDA分析发现密枝青藓(Brachythecium amnicolum)、褶叶藓(Palamocladium nilgheriense)和美灰藓(Eurohypnum leptothallum)等可用于石漠化生态修复。石漠化程度对苔藓多样性影响不显著,但对其群落特征和生活型影响显著。因此可以结合苔藓群落和生活型特征监测石漠化,同时也可使用苔藓作为先锋植物引进用于石漠化早期修复。     

Role for dopamine in malonate-induced damage in vivo in striatum and in vitro in mesencephalic cultures

Defects in mitochondrial energy metabolism have been implicated in the pathology of several neurodegenerative disorders. In addition, the reactive metabolites generated from the metabolism and oxidation of the neurotransmitter dopamine (DA) are thought to contribute to the damage to neurons of the basal ganglia. We have previously demonstrated that infusions of the metabolic inhibitor malonate into the striata of mice or rats produce degeneration of DA nerve terminals. In the present studies, we demonstrate that an intrastriatal infusion of malonate induces a substantial increase in DA efflux in awake, behaving mice as measured by in vivo microdialysis. Furthermore, pretreatment of mice with tetrabenazine (TBZ) or the TBZ analogue Ro 4-1284 (Ro-4), compounds that reversibly inhibit the vesicular storage of DA, attenuates the malonate-induced DA efflux as well as the damage to DA nerve terminals. Consistent with these findings, the damage to both DA and GABA neurons in mesencephalic cultures by malonate exposure was attenuated by pretreatment with TBZ or Ro-4. Treatment with these compounds did not affect the formation of free radicals or the inhibition of oxidative phosphorylation resulting from malonate exposure alone. Our data suggest that DA plays an important role in the neurotoxicity produced by malonate. These findings provide direct evidence that inhibition of succinate dehydrogenase causes an increase in extracellular DA levels and indicate that bioenergetic defects may contribute to the pathogenesis of chronic neurodegenerative diseases through a mechanism involving DA.     

2018年中国植物科学若干领域重要研究进展

2018年中国植物科学继续呈现快速发展的态势, 我国科学家在国际植物科学主流学术刊物发表论文数量大幅增加, 取得了多项具有重要影响的成果。调控植物生长-代谢平衡实现可持续农业发展入选2018年度中国科学十大进展; 中国被子植物区系进化历史研究入选2018年度中国生命科学十大进展。以水稻为代表的农作物和果蔬等经济作物研究在国际上已呈现出明显的优势, 若干领域已从“追赶”状态跨越到“领跑”地位。该文对2018年中国科学家在植物科学若干领域取得的重要研究成果进行了概括性评述, 旨在全面追踪和报道当前中国植物科学领域的发展前沿和热点, 展示中国科学家所取得的杰出成就。     

2017年中国植物科学若干领域重要研究进展

2017年中国植物科学继续保持高速发展态势, 重大成果频出, 具体表现在中国植物学家在国际顶级学术期刊发表的文章数量平稳上升。中国植物科学领域的研究工作者成果精彩纷呈, 如新型广谱抗病机制的发现、水稻广谱抗病遗传基础及机制和疫霉菌诱发病害成灾机制研究等。2017年中国生命科学领域十大进展评选中, 有两项植物科学领域的研究成果入选。水稻生物学、进化与基因组学和激素生物学等领域学科发展突出。另外, 值得一提的是, 长期从事高等植物与代谢途径调控分子网络研究和水稻品种设计育种的李家洋院士的研究成果“水稻高产优质性状形成的分子机理及品种设计”荣获2017年国家自然科学一等奖。这一具有重大国际影响的开创性贡献标志着中国植物科学在该领域的国际科学前沿居于引领和卓越地位。该文对2017年中国本土科学家在植物科学若干领域取得的重要研究成果进行了系统梳理, 旨在全面追踪和报道当前中国植物科学领域发展的最新前沿动态, 与广大读者共同分享我国科学家所取得的辉煌成就。     

我国葫芦科植物离体培养研究进展

葫芦科植物包括多种瓜类蔬菜,对其进行离体培养研究具有重要的理论和实践意义。综述了国内在葫芦科植物器官培养、体细胞胚胎发生、花药培养、原生质体培养和体细胞杂交及离体遗传转化等方面取得的研究进展,并对葫芦科植物离体培养、遗传转化与育种的前景作了展望。     

SSTR5 ablation in islet results in alterations in glucose homeostasis in mice

Somatostatin (SST) peptide is a potent inhibitor of insulin secretion and its effect is mediated via somatostatin receptor 5 (SSTR5) in the endocrine pancreas. To investigate the consequences of gene ablation of SSTR5 in the mouse pancreas, we have generated a mouse model in which the SSTR5 gene was specifically knocked down in the pancreatic beta cells (betaSSTR5Kd) using the Cre-lox system. Immunohistochemistry analysis showed that SSTR5 gene expression was absent in beta cells at three months of age. At the time of gene ablation, betaSSTR5Kd mice demonstrated glucose intolerance with lack of insulin response and significantly reduced serum insulin levels. Insulin tolerance test demonstrated a significant increase of insulin clearance in vivo at the same age. In vitro studies demonstrated an absence of response to SST-28 stimulation in the betaSSTR5Kd mouse islet, which was associated with a significantly reduced SST expression level in betaSSTR5Kd mice pancreata. In addition, betaSSTR5Kd mice had significantly reduced serum glucose levels and increased serum insulin levels at 12 months of age. Glucose tolerance test at an older age also indicated a persistently higher insulin level in betaSSTR5Kd mice. Further studies of betaSSTR5Kd mice had revealed elevated serum C-peptide levels at both 3 and 12 months of age, suggesting that these mice are capable of producing and releasing insulin to the periphery. These results support the hypothesis that SSTR5 plays a pivotal role in the regulation of insulin secretion in the mouse pancreas.     

2018年中国植物科学若干领域重要研究进展

2018年中国植物科学继续呈现快速发展的态势, 我国科学家在国际植物科学主流学术刊物发表论文数量大幅增加, 取得了多项具有重要影响的成果。调控植物生长-代谢平衡实现可持续农业发展入选2018年度中国科学十大进展; 中国被子植物区系进化历史研究入选2018年度中国生命科学十大进展。以水稻为代表的农作物和果蔬等经济作物研究在国际上已呈现出明显的优势, 若干领域已从“追赶”状态跨越到“领跑”地位。该文对2018年中国科学家在植物科学若干领域取得的重要研究成果进行了概括性评述, 旨在全面追踪和报道当前中国植物科学领域的发展前沿和热点, 展示中国科学家所取得的杰出成就。     

我国葫芦科植物离体培养研究进展

葫芦科植物包括多种瓜类蔬菜,对其进行离体培养研究具有重要的理论和实践意义.综述了国内在葫芦科植物器官培养、体细胞胚胎发生、花药培养、原生质体培养和体细胞杂交及离体遗传转化等方面取得的研究进展,并对葫芦科植物离体培养、遗传转化与育种的前景作了展望.     

Changes in aquatic vegetation in Rhine floodplain streams in Alsace in relation to disturbance

Abstract. Changes are described in aquatic vegetation in oligotrophic, groundwater-fed Rhine floodplain streams in Alsace (eastern France), resulting from disturbance. Disturbance factors include changes in nutrients, either permanent ones - effluent from a waste water treatment plant or trout hatcheries - or periodic ones: flooding. Regular inputs of high levels of phosphate and ammonia modified the macrophyte vegetation in these streams. The floristic composition, which was characteristic of oligotrophic waters upstream of the eutrophicated sector, changed to that of a eutrophic situation as originally found downstream. Periodic disturbance by floods which normally occur once a year, irregularly eutrophicates the small streams, causing the development of a mixture of eutrophic and oligotrophic species. Six macrophyte communities are distinguished, indicating different trophic levels. The aquatic vegetation is adapted to the variations of phosphate and ammonia levels. Hence, aquatic macrophytes can be used as bio-indicators of fluctuations in water nutrient levels in relation to the type of disturbance.     

Apoptosis in experimental myocardial infarction in situ and in the perfused heart in vitro

We report the appearance of apoptotic cells in experimental myocardial infarction (rabbit heart) in in situ and in vitro preparations. Apoptosis was recognized by intravital staining with Hoechst 33342 (Ho342), by nick-end labeling (TUNEL) and by DNA laddering. A steady rise in the relative number of apoptotic cardiomyocytes (apoptotic index) was noted in in situ preparations. Apoptosis was first noted 6 h after the onset of ischemia with its highest value occurring after 72 h. Apoptotic nuclei were absent in remote areas of the left and right ventricles. Apoptotic nuclei within the infarcted area showed diminished intensity of Ho342 fluorescence. Three days after ischemia, a border zone adjacent to the infarcted area consisting of apoptotic macrophages was recognized. A novel finding was the appearance of apoptotic cardiomyocytes in the isolated perfused ischemic heart. Occurring as early as 50 min after the onset of ischemia, a high apoptotic index was present adjacent to the ligature placed around the coronary artery. This observation provides the opportunity to selectively examine factors leading to apoptosis in the ischemic heart under controlled experimental conditions.     

Morphine-potentiated platelet aggregation in in vitro and platelet plug formation in in vivo experiments

The detailed mechanisms underlying morphine-signaling pathways in platelets remain obscure. Therefore, we systematically examined the influence of morphine on washed human platelets. In this study, washed human platelet suspensions were used for in vitro studies. Furthermore, platelet thrombus formation induced by irradiation of mesenteric venules with filtered light in mice pretreated with fluorescein sodium was used for an in vivo thrombotic study. Morphine concentration dependently (0.6, 1, and 5 microM) potentiated platelet aggregation and the ATP release reaction stimulated by agonists (i.e., collagen and U46619) in washed human platelets. Yohimbine (0.1 microM), a specific alpha(2)-adrenoceptor antagonist, markedly abolished the potentiation of morphine in platelet aggregation stimulated by agonists. Morphine also potentiated phosphoinositide breakdown and intracellular Ca(2+) mobilization in human platelets stimulated by collagen (1 microg/ml). Moreover, morphine (0.6-5 microM) markedly inhibited prostaglandin E(1) (10 microM)-induced cyclic AMP formation in human platelets, while yohimbine (0.1 microM) significantly reversed the inhibition of cyclic AMP by morphine (0.6 and 1 microM) in this study. The thrombin-evoked increase in pH(i) was markedly potentiated in the presence of morphine (1 and 5 microM). Morphine (2 and 5 mg/g) significantly shortened the time require to induce platelet plug formation in mesenteric venules. We concluded that morphine may exert its potentiation in platelet aggregation by binding to alpha(2)-adrenoceptors in human platelets, with a resulting inhibition of adenylate cyclase, thereby reducing intracellular cyclic AMP formation followed by increased activation of phospholipase C and the Na(+)/H(+) exchanger. This leads to increased intracellular Ca(2+) mobilization, and finally potentiation of platelet aggregation and of the ATP release reaction.