海滨盐薄片菌——分离自海洋盐田的一个嗜盐古菌新种（英文）

【目的】研究分离自我国华南地区阳江盐田的一株嗜盐古菌菌株YJ-41~T,探究其分类学地位。【方法】运用多相分类学方法即通过表型和遗传型特征鉴定,研究菌株YJ-41T的分类学地位。【结果】菌株YJ-41~T的细胞为杆状、革兰氏染色阴性、菌落呈红色。菌株YJ-41T的生长温度范围20–50°C(最适为37°C)、Na Cl浓度范围2.1–4.8 mol/L(最适为3.1 mol/L)、MgCl_2浓度范围0–1.0 mol/L(最适为0.05 mol/L)、pH范围5.0–9.0(最适为pH 7.0)。细胞在蒸馏水中会裂解,维持细胞形态的最低Na Cl浓度为10%(质量体积比)。菌株YJ-41~T的极性脂为磷脂酸(Phosphatidic acid,PA)、磷脂酰甘油(Phosphatidyl glycerol,PG)、磷脂酰甘油磷酸甲基酯(Phosphatidyl glycerol phosphate methyl ester,PGP-Me)、磷脂酰甘油硫酸酯(Phosphatidyl glycerol sulphate,PGS)和8种糖脂;其中3种糖脂为硫酸甘露糖苷葡萄糖二醚(Sulfated mannosyl glucosyl diether,S-DGD-1)、半乳糖苷甘露糖苷葡萄糖二醚(Galactosyl mannosy glucosyl diether,TGD-1)和甘露糖苷葡萄糖二醚(Mannosyl glucosyl diether,DGD-1),其余为未知糖脂。菌株YJ-41~T的16S rRNA基因和rpo B′基因与盐薄片菌属(Halolamina)的成员相关基因相似性分别为97.5%–98.4%和93.1%–94.4%。菌株YJ-41T的G+C mol%为61.4 mol%。【结论】表型、化学分类和系统发育的特性表明,菌株YJ-41T(=CGMCC 1.12859T=JCM 30237T)代表Halolamina属的一个新种,建议命名为海滨盐薄片菌(Halolamina litorea)。     

来源于中国南海的微生物新种乙醇速生杆菌(Celeribacter ethanolicus)多相分类研究

【目的】研究来源于南海海水的一株速生杆菌属菌株NH195^T 的多相分类。【方法】采用表型、基因型和化学分类方法,并综合系统发育关系结果,分析菌株NH195^T 的分类学地位。【结果】菌株NH195^T 是一株革兰氏阴性、好氧、杆状、无运动性细菌;能积累Poly-β-hydroxybutyrate (PHB);能在0.5%-10.0% (质量体积比) NaCl 浓度,pH 5.0-9.0 和20-40 ℃ 条件下生长,最适NaCl 生长浓度为1.0%-3.0%;氧化酶、触酶和脲酶反应结果阳性。菌株NH195^T 主要呼吸醌为Q-10,主要脂肪酸为C_18:1ω7c、C_18:1ω6c 和11 methyl C_18:1ω7c,主要极性脂为磷脂酰胆碱、磷脂酰甘油、双磷脂酰甘油、一个未知的氨基脂和两个未知脂。基因组G+C 含量为61.3 mol%。基于16S rRNA 基因的系统发育结果显示,菌株NH195^T 隶属于速生杆菌属;其与速生杆菌属标准菌株的16S rRNA 基因相似性范围为94.4%-97.7%。菌株NH195^T 与速生杆菌属标准菌株C. halophilus ZXM137^T 和C. indicus P73^T 的平均核苷酸一致性(ANI)分别为78.6%和78.0%;基于基因组数据计算所得的DNA 杂交同源率分别为26.1%和23.0%。【结论】基于表型和基因型结果,菌株NH195^T 代表了速生杆菌属一个新物种,命名为Celeribacter ethanolicus,标准菌株为NH195^T (CGMCC 1.15406^T=JCM 31095^T)。     

高产吲哚乙酸东乡野生稻内生微杆菌KlspL 18分离及鉴定

【目的】对分离自东乡野生稻叶组织的高产吲哚乙酸菌株KlspL18进行分类学鉴定。【方法】采用菌株形态、生理生化指标结合16S rRNA基因序列同源性比对以及全基因组测序等方法,对该菌株进行多相分类鉴定,并采用高效液相(HPLC)法测定其产吲哚乙酸的产量。【结果】菌株KlspL18为革兰氏阳性、不形成孢子的棒杆状,接触酶实验阳性,其温度、NaCl浓度和pH值生长范围分别为15–40 °C (最适为28 °C)、1%–10% (最适为1%)及6.0–11.0 (最适为7.0),能利用多种糖和有机酸作为碳源。菌株细胞壁氨基酸主要为鸟氨酸,细胞壁多糖为半乳糖和甘露糖,极性脂类为二磷脂酰甘油、磷脂酰甘油和2种未鉴定的糖脂,细胞脂肪酸主要为anteiso-C_15:0 (30.33%)、anteiso-C_17:0 (31.53%)和iso-C_16:0 (14.32%),萘醌类主要为MK-10和MK-11。16S rRNA基因序列分析表明,该菌株与Microbacterium proteolyticum RZ36^T相似度为97.64%;全基因组测序分析显示,其G+C含量70.2%,与近缘标准菌株核苷酸同源性(ANI)和数字DNA-DNA杂交值(dDDH)分别为83.35%和26.4%,均低于种间同源性的临界值。菌株KlspL18产IAA可达291.7 mg/L。【结论】菌株KlspL18是高产吲哚乙酸微杆菌属的一个新种,命名为Microbacterium dongxiang sp. nov.,模式菌株为KlspL18 (=CCTCC M2022446),具有应用于农业生产促进植物生长的潜能。     

4株高效产电菌的电化学活性及其生物学特征

采用循环伏安法对从湖南省吉首大田湾污水处理厂曝气池活性污泥中富集和筛选的37株产电菌的电化学活性进行考察。结果发现,4株菌株（F012、F015、F021、F026）的电化学活性较为显著,其中F026的电化学活性最好。对4株产电菌的系统发育分析表明,菌株F012属于Dyella属,与Dyella marensis CS5-B2^T系统发育关系最为密切（相似性为97.22%）;菌株F015属于Paludibacterium属,与该属的Paludibacterium yongneupense 5YN8-15^T系统发育关系最为密切（相似性为97.70%）;菌株F021和F026都属于Pseudomonas属,分别与该属的Pseudomonas simiae OLi^T和Pseudomonas otitidis MCC10330^T系统发育关系最为密切（相似性分别为99.60%和98.62%）。生物学特性研究表明,电化学活性最好的产电菌F026的生长温度范围为20~40 ℃,最适宜生长温度为30~35 ℃;生长pH范围为5~9,最适pH生长范围为8~9,适合作为微生物燃料电池的高效产电菌。     

N-十一烷酰基环戊酰胺对铜绿假单胞菌生物被膜及毒力的影响

【背景】铜绿假单胞菌(Pseudomonas aeruginosa)耐药性问题日趋严重的重要原因之一是细菌生物被膜的产生,群体感应(quorum sensing, QS)系统在其生物被膜形成过程中发挥了重要作用。QS抑制剂能够抑制生物被膜的形成和毒力因子的分泌,成为解决细菌耐药性问题的新策略。【目的】通过化学方法对las系统信号分子N-(3-氧十二烷基)-l-高丝氨酸内酯[N-3-(oxododecanoyl)-l-homoserine lactone, OdDHL]的母核和酰基侧链同时改变,合成N-十一烷酰基环戊酰胺,命名为Y0-C11-HSL,探讨其对P. aeruginosa生物被膜形成和毒力因子分泌的作用潜力和分子机制。【方法】采用结晶紫染色和扫描电子显微镜(scanning electron microscope, SEM)评价Y0-C11-HSL对生物被膜形成和结构的影响,通过测定毒力因子的产生和运动试验评析Y0-C11-HSL的抑制活性,通过傅里叶红外光谱(Fourier transform infrared spectrometer, FT-IR)研究Y0-C11-HSL对胞外聚合物(extracellular polymers, EPS)表面化学基团的影响,采用分子对接进一步解析Y0-C11-HSL的作用机制。【结果】与对照组相比,在10-200μmol/L浓度梯度下,Y0-C11-HSL能够减少P. aeruginosa生物被膜形成,且在200 μmol/L时减少率达24.1% (P＜0.01)。此外,在200 μmol/L处理下,Y0-C11-HSL能够显著抑制绿脓菌素、鼠李糖脂、胞外多糖和水解蛋白酶的分泌,抑制率分别为34.7% (P＜0.01)、33.1% (P＜0.01)、27.3% (P＜0.01)和37.3% (P＜0.01),抑制swarming和twitching运动,抑制率分别为45.6% (P＜0.01)和51.7% (P＜0.01),影响了EPS表面化学基团。分子对接结果表明,Y0-C11-HSL能与OdDHL结合的LasR受体蛋白竞争性结合。【结论】Y0-C11-HSL能与OdDHL结合的LasR受体蛋白竞争性结合,对转录蛋白产生影响,进而下调P. aeruginosa QS相关基因的表达。     

罗氏沼虾(Macrobrachium rosenbergii)养殖池塘沉积物中细菌、硫酸盐还原菌和硫氧化菌的垂直分布特征

沉积物中微生物介导的硫循环在有机物分解和养分循环中发挥重要作用,但目前我们对水产养殖生态系统中的参与硫酸盐还原和硫氧化过程的微生物多样性及其调控机制仍知之甚少。【目的】探究硫酸盐还原菌(sulfate-reducing bacteria,SRB)和硫氧化菌(sulfur-oxidizing bacteria,SOB)的垂直分布特征及其主要的环境驱动因素。【方法】本研究利用高通量测序和荧光定量PCR (qPCR)分析了罗氏沼虾(Macrobrachium rosenbergii)养殖池塘沉积物表层(0–1 cm)、中层(10–11 cm)和底层(20–21 cm)中的细菌、SRB和SOB的丰度、多样性和群落组成。【结果】细菌(16S rRNA)、硫酸盐还原菌(dsrB)和硫氧化菌(soxB)的基因拷贝数呈现着从表层到中层急剧骤降的趋势(ANOVA,P<0.05),但中层和底层样品之间的差异却并不显著(P>0.05),以及α多样性分析显示3个群体的物种丰富度和均匀度都随深度而逐步降低,这都说明硫循环过程主要发生于沉积物的表层。γ-、δ-和β-变形菌分别是细菌、SRB和SOB的优势类群;其中SRB以Desulfobacca属和脱硫八叠球菌属(Desulfosarcina)为主,前者在表层有着最低的比重,而后者却与之相反;硫杆菌(Thiobacillus)作为细菌和SOB的优势属,更广泛地分布于中层沉积物中。RDA分析和Mantel检验揭示了影响细菌群落的主要环境因子是NO₃^–、SO₄^2–、TOC和TON,而SRB的群落变异主要是由As、TON、NO₃^–和Pb所驱动,以及SOB的群落变化则主要响应了TC、NO₂^–、NH₄⁺和TON浓度。【结论】养殖池塘底栖的细菌、SRB和SOB的丰度、多样性和群落结构的垂直分布特征可能受到多种环境因素共同的影响。     

wbnH2基因对北京欧文氏菌生物学特性及致病力的影响

【背景】北京欧文氏菌(Erwinia beijingensis)引起的刺芹侧耳细菌性软腐病(bacterial soft rot)给企业带来了严重的经济损失。wbnH2糖基转移酶基因在北京欧文氏菌中的生物学功能尚不明确。【目的】构建wbnH2基因的缺失株Δ-wbnH2和回补株C-wbnH2,探究wbnH2基因对北京欧文氏菌致病性的影响。【方法】采用同源重组方法构建北京欧文氏菌LMG 27579^TwbnH2基因缺失突变株Δ-wbnH2,并对基因缺失菌株的致病性、生长速度、运动能力、生物膜形成能力、黏附能力等生物学特性与野生型菌株进行比较分析;采用广宿主质粒pBBR1MCS2构建回补株C-wbnH2,排除了极性效应引起的突变株表型变化。【结果】与野生型菌株相比,基因缺失株Δ-wbnH2的生长速度无明显差别。但是wbnH2基因的缺失导致多糖分泌、生物膜形成能力、黏附能力、致病能力明显下降。【结论】wbnH2基因影响北京欧文氏菌多糖分泌、生物膜的形成能力、黏附能力及致病力,表明该基因在北京欧文氏菌致病过程中起重要作用,本研究为软腐病的防控提供了理论基础。     

Nonomuraea candida HMC10T中新结构套索肽noncaromin生物合成基因簇的克隆及异源表达

【目的】本研究旨在通过定向克隆菌株Nonomuraea candida HMC10^T中一个新的Ⅱ型套索肽类生物合成基因簇,通过在放线菌底盘宿主中的异源表达,获得新结构套索肽noncaromin,并完成其抑菌活性分析。【方法】通过antiSMASH软件分析菌株N.candida HMC10^T全基因组序列,确定新的Ⅱ型套索肽noncaromin的生物合成基因簇(biosynthetic gene cluster of noncaromin,nonc-BGC)。然后,利用ExoCET重组技术(exonuclease combined with RecET recombination)获得完整的nonc-BGC,得到重组质粒pJQK652,并通过λ-Red重组技术改造得到整合型质粒pJQK653。采用接合转移方法,将该质粒分别导入白色链霉菌、2株变铅青链霉菌、2株天蓝色链霉菌和红色糖多孢菌宿主中进行异源表达,再通过发酵和分离纯化获得目标套索肽noncaromin。最后,利用QTOF-ESI-MS²完成套索肽noncaromin的结构鉴定,并通过抗菌活性检测确定该化合物的生物活性。【结果】本研究利用ExoCET技术成功获得了完整的nonc-BGC,在6种放线菌宿主中成功异源表达,完成了noncaromin的结构鉴定,确定了其具有微弱的抗枯草芽孢杆菌活性。【结论】本研究在克隆得到新结构套索肽nonc-BGC的基础上,实现了该基因簇在6个放线菌底盘宿主中的成功表达,获得了1个具有微弱抑制枯草芽孢杆菌活性的新结构Ⅱ型套索肽noncaromin。本研究结果为发掘菌株N.candida HMC10^T及其他放线菌中的新结构化合物提供了借鉴。     

一株耐碱变形假单胞菌ZY-3的鉴定及其脱氮特性

【背景】目前缺少具有高效脱氮能力、较高生物安全性、能处理高碱含氮污水的好氧反硝化菌株,难以使用生物方法处理高碱性的工业、养殖废水。【目的】对前期于佛山市一水产养殖池塘底泥中分离得到的耐碱高效好氧反硝化细菌ZY-3进行研究,期望获得一株能用于不同酸碱环境脱氮的高效、安全的好氧反硝化细菌。【方法】通过形态学、生理生化试验及16S rRNA基因序列分析方法对菌株种属进行鉴定,采用抗生素试验及斑马鱼攻毒试验进行菌株的环境生物安全性评估,利用3种含不同氮素的含氮模拟废水进行脱氮能力的测定。【结果】确定ZY-3为假单胞菌属变形假单胞菌（Pseudomonas plecoglossicida）,其对多种临床常用抗生素敏感,对水生生物的毒性低,该菌株在高浓度含氮模拟废水中以28℃、180 r/min振荡培养时,其对数期出现在4—12 h,在12 h时NH₄⁺-N、NO₃^--N和NO₂^--N的去除率分别达到94.87%、81.44%和98.02%,其pH耐受范围为6.0—10.0。【结论】得到一株安全、高效、具有广泛pH适应范围的耐碱好氧反硝化细菌P. plecoglossicida ZY-3,其在有氧条件下对3种氮素（NH₄⁺-N、NO₃^–-N、NO₂^–-N）具有快速去除能力。     

一株中度嗜盐芽孢杆菌(Bacillus sp. BZ-SZ-XJ39)的微生物学特性

【目的】了解一株来自新疆盐碱湖的中度嗜盐菌(Bacillus sp.BZ-SZ-XJ39)的微生物学特性,为进一步阐明该菌独特的盐适应机制奠定基础。【方法】通过16S rRNA基因序列分析、G+C mol%含量测定、细胞形态和菌落观察、培养条件确定、营养与生理生化指标测定以及细胞化学组分分析等描述该菌的生物特性。【结果】基于16S rRNA基因序列的同源性和系统发育树分析,菌株BZ-SZ-XJ39与Bacillus saliphilus 6AGT(序列相似性为97.5%),Bacillus daliensis DLS13T(96.5%),Bacillus luteus JC167T(96.2%),Bacillus chagannorensis CG-15T(95.5%)和Bacillus agaradhaerens DSM8721T(95.3%)有密切的亲缘关系。基因组DNA G+C含量为44.4 mol%±1.2 mol%(Tm)。菌株BZ-SZ-XJ39为好氧、短杆状、革兰氏阳性菌。菌落呈黄色、圆形凸起、表面光滑且边缘整齐。菌株生长盐度范围为0.5%–28%(最适8%),pH范围为5.5–9.5(最适pH 8.0),温度范围为4–41oC(最适33oC)。菌株BZ-SZ-XJ39合成的主要脂肪酸包括anteiso-C15:0(50.2%)和anteiso-C17:0(16.3%)。合成极性脂为磷脂(PL)、磷脂酰乙醇胺(PE)、磷脂酰甘油(PG)、二磷脂酰甘油(DPG)和磺酸基异鼠李糖基二脂酰基甘油(SQDG)。呼吸醌类型为甲基萘醌(MK-7)。菌株BZ-SZ-XJ39已经在中国普通微生物菌种保藏管理中心(CGMCC1.12936)和日本微生物菌种保藏中心(JCM30194)冻干保藏,在Gen Bank中的序列注册号为KP456019。【结论】基于BZ-SZ-XJ39菌株的遗传型、表型、生理生化以及化学组成特征,初步确定该菌为Bacillus属中的一个新成员。研究将为探索生命在高盐环境中存在的本质提供新素材,也可为生物技术的潜在应用提供新视角。     

Westerdykella globosa,a proposal for a new combination

The surface ornamentation of ascospores ofPreussia globosa was compared in an isolate from paddy soil in Japan and a culture derived from the holotype. The ascospores of two cultures were characterized by the surface ornamentation of a single, semicircular spiral ridge. This new finding strongly suggested that the fungus should be transferred to the genusWesterdykella. Therefore, the morphological and cultural characters of the fungus were re-examined, and the new combinationWesterdykella globosa is herein proposed.     

Inbreeding in a french peasant population

Genealogical pedigrees were collected from 976 farming couples who lived in a canton of central France in the XIXth and XXth century. Several methods (genealogical, isonymic and probability of origin of genes) were used to appraise the evolution of the biological structure of this population, which has gone through two phases. The first phase from 1800 to the World War I, shows gene flows between the farming population of the canton and those of neighbouring rural districts which are linked to an increase in consanguineous marriages. The second phase, from 1920 onwards, shows important gene flow from distant areas. This trend implies a decrease in consanguineous marriages.     

Cyanobacteria dynamics in a restored Finnish lake: a long term simulation study

Several explanations have been presented for the formation of lacustrine cyanobacteria blooms. These include elevated water temperature, nutrient enrichment, low N/P ratios, low light energy requirements, high pH and/or low carbon dioxide concentration, selective zooplankton grazing, excretion of compounds which suppress the growth of competing algae, and migration. The impact of the first four were studied in Lake Tuusulanjärvi, a eutrophic Finnish lake, using deterministic simulation. The study, which covered 15 years, included a sequence of cases on the recovery of the lake after the implementation of two restoration measures: aeration of the hypolimnion, and termination of municipal waste discharge, both of which were done during the study period. The focus was on Microcystis and Oscillatoria, and on the N-fixing cyanobacteria, Anabaena and Aphanizomenon. Eukaryotic phytoplankton were also included in the study. Monitoring data were used. The roles of the hypotheses were discussed, from the point of view of the utility of the information in water resources management.     

<Emphasis Type="Italic">hedgehog</Emphasis> is a segment polarity gene in a crustacean and a chelicerate

The evolution of arthropod segmentation has been studied by comparing expression patterns of pair-rule and segment polarity genes in various species. In Drosophila, the formation and maintenance of the parasegmental boundaries depend on the interactions between the wingless (wg), engrailed (en) and hedgehog (hh) genes. Until now, the expression pattern of hh has not been analysed to such a great extent as en or wg. We report the cloning and expression analysis of hh genes from Euscorpius flavicaudis, a chelicerate, and Artemia franciscana, a branchiopod crustacean. Our data provide evidence that hh, being expressed in the posterior part of every segment, is a segment polarity gene in both organisms. Additional hh expression sites were observed in the rostrum and appendages of Euscorpius and in the gut of Artemia. From the available data on hh expression in various bilaterians, we review the various hypotheses on the evolution of hh function and we suggest an ancestral role of hh in proctodeum specification and gut formation.Edited by D. Tautz     

Stomatal conductance in a tropical xerophilous shrubland at a lava substratum

Diurnal variation in leaf stomatal conductance (g _s) of three xerophilous species (Buddleia cordata, Senecio praecox and Dodonaea viscosa) was measured over a 10-month period during the dry and wet seasons in a shrubland that is developing in a lava substratum in Mexico. Averaged stomatal conductances were 147 and 60.2 (B. cordata), 145 and 24.8 (D. viscosa) and 142.8 and 14.1 mmol m^–2 s^–1 (S. praecox) during the wet and dry season respectively. Leaf water potential () varied in a range of –0.6 to –1.2 (S. praecox), –0.6 to –1.8 (B. cordata) and –0.9 to –3.4 MPa (D. viscosa) during the same measurement periods. Stomata were more sensitive to changes in irradiance, air temperature and leaf–air vapour pressure difference in the rainy season than the dry season. Although stomatal responses to were difficult to distinguish in any season (dry or rainy), data for the entire period of measurement showed a positive correlation, stomata tending to open as increased, but there is strong evidence of isohydric behaviour in S. praecox and B. cordata. A multiplicative model relating g _s to environmental variables and to accounted for 79%–83% of the variation of g _s in three sites (pooled data); however, the performance of the model was poorer (60%–76%) for individual species from other sites not included in the pooled data.     

Site selection by a social wasp in a nesting association (Hymenoptera: Vespidae)

The tropical social waspMischocyttarus immarginatus is a nesting associate ofPolybia occidentalis in northwestern Costa Rica. We show that an activeP. occidentalis colony is a necessary component of aM. immarginatus nesting site in this part of the latter’s range.M. immarginatus foundresses actively seekP. occidentalis colonies to initiate nests at a modal distance of 8 cm from them. There is an 18-mm lower limit to how closeM. immarginatus can nest in relation toP. occidentalis. At distances of less than this,P. occidentalis workers can reach theM. immarginatus nest while standing on their own nest and robM. immarginatus of its brood when the adults are absent.     

Utilisation of a tropical bay as a nursery area by sharks of the families Carcharhinidae and Sphyrnidae

Synopsis At least eight species of sharks of the families Carcharhinidae and Sphyrnidae use Cleveland Bay in northern Australia as a communal nursery area.Carcharhinus dussumieri, C. fitzroyensis, C. limbatus andC. tilstoni use the bay as a seasonal primary nursery, with juveniles occurring in it for only a few months each year immediately after birth. Alternatively,Carcharhinus sorrah, Rhizoprionodon acutus andR. taylori use the bay as a year-round primary and secondary nursery, with juveniles remaining in it up to the size at maturity. AdultR. taylori also persist in the bay, a behavioural pattern possibly explained by their small maximum size. While present immediately after birth the type of utilisation pattern displayed bySphyrna lewini could not be clarified in this study. Although diets of these species in the bay are similar, there is probably little direct competition for food due to the highly productive habitats in the bay supporting an abundance of food resources. The highest numbers of juveniles occur when prey species are the most abundant, and when temporal separation of some seasonally-occurring species of sharks in effect.     

Aspergillus myositis in a patient with a myelodysplastic syndrome

We present the case of an elderly man who, while being treated with corticosteroids for a myelodysplastic syndrome, developed myositis of the calf due to Aspergillus fumigatus. Despite therapy with amphotericin B the myositis failed to resolve and he died. At autopsy, a localized necrotizing myositis of the right calf was found with no evidence of disseminated Aspergillus infection. Myositis in the setting of disseminated candidiasis or cryptococcosis has been previously reported. This case is unique in that it is the first reported case of localized fungal myositis and of myositis caused by Aspergillus.     

The Lipomycetaceae,a model family for phylogenetic studies

The Lipomycetaceae (Endomycetales) are known from the generaDipodascopsis, Lipomyces andZygozyma with budding anamorphic states inMyxozyma. The family is easily recognized culturally and physiologically but is phenotypically and ecologically extremely diverse. This natural taxon is phylogenetically distinct from the Saccharomycetaceae, but probably related to the Dipodascaceae. The possible evolution of the lipomycetaceous anamorphs is discussed.