芍药苷对APP/PS1小鼠的神经细胞保护作用及机制研究

目的:探讨芍药苷(paeoniflorin,PF)通过抑制细胞凋亡通路而产生神经细胞保护作用的机制。方法:分别选用15只5月龄雄性APP/PS1非显性小鼠作为正常对照组,15只5月龄雄性APP/PS1双转基因小鼠为模型组和15只5月龄雄性APP/PS1双转基因小鼠为给药组(5 mg/kg的PF腹腔注射)。采用水迷宫实验检测各组小鼠的学习和记忆能力。采用TUNEL荧光染色法检测脑内神经细胞凋亡情况。采用Western Blot检测脑内皮层及海马区PI3K、Akt、p-PI3K、p-Akt、caspase-3、caspase-9、Bcl-2和Bax的蛋白表达水平,并用免疫组化分析caspase-3和caspase-9的蛋白表达水平及分布情况。结果:(1)与正常对照组相比,APP/PS1模型组小鼠的学习和记忆能力明显下降;与APP/PS1模型组相比,PF明显改善小鼠的学习和记忆能力。(2)与正常对照组相比,APP/PS1模型组小鼠脑内神经细胞凋亡明显增多,分布区域较广,而PF给药组小鼠凋亡细胞明显减少。(3)与APP/PS1模型组相比,PF给药组能显著下调促凋亡因子caspase-3、caspase-9和Bax的表达水平(P0.05),同时上调抑凋亡因子pPI3K、p-Akt和Bcl-2的表达水平(P0.05)。结论:PF可能通过激活PI3K/Akt通路而上调Bcl-2,下调caspase-9、caspase-3和Bax的蛋白表达水平,从而抑制神经细胞凋亡和保护神经细胞,以治疗神经退行性疾病。     

金匮肾气丸对肾阳虚证雄性大鼠生精细胞中凋亡相关蛋白Bcl-2、Bax的影响

背景：肾虚可导致不育,金匮肾气丸是治疗肾阳虚证的经典药物,但其作用机制尚不明确。 目的：观察金匮肾气丸对凋亡相关蛋白Bcl-2、Bax在肾阳虚证雄性大鼠生精细胞中表达的影响。 方法：成年雄性SD大鼠100只随机分成5组,采用氢化可的松注射液制备肾阳虚模型。金匮肾气丸低、中、高剂量组在造模基础上每天灌胃含生药0.625,1.250,2.500 g/kg的肾气丸混悬液,连续30 d。正常组和模型组灌胃等量蒸馏水。检测各组大鼠左侧睾丸质量、血清睾丸酮水平、精子密度、精子活率改变情况。采用免疫组织化学方法检测Bcl-2、Bax蛋白在生精细胞中的表达。 结果与结论：模型组左侧睾丸质量、血清睾丸酮水平、精子密度、精子活率低于正常组、金匮肾气丸中、高剂量组(P < 0.05)。与模型组比较,正常组、金匮肾气丸低、中、高剂量组大鼠生精细胞中Bcl-2蛋白表达显著增高(P < 0.05),Bax蛋白表达显著降低(P < 0.05)。说明金匮肾气丸可使肾阳虚证雄性大鼠生精细胞中Bcl-2蛋白表达升高,Bax蛋白表达降低,从而抑制细胞凋亡,对肾阳虚证雄性大鼠生殖能力有较好的治疗作用。     

Doppel induces degeneration of cerebellar Purkinje cells independently of Bax

Doppel (Dpl) is a prion protein paralog that causes neurodegeneration when expressed ectopically in the brain. To investigate the cellular mechanism underlying this effect, we analyzed Dpl-expressing transgenic mice in which the gene for the proapoptotic protein Bax had been deleted. We found that Bax deletion does not alter either clinical symptoms or Purkinje cell degeneration in Dpl transgenic mice. In addition, we observed that degenerating Purkinje cells in these animals do not display DNA fragmentation or caspase-3 activation. Our results suggest that non-Bax-dependent pathways mediate the toxic effects of Dpl in Purkinje cells, highlighting a possible role for nonapoptotic mechanisms in the death of these neurons.     

茶多酚预防慢性酒精中毒对精子损伤的作用及机制研究

目的　探讨茶多酚预防慢性酒精中毒对精子损伤的作用及其可能机制。 方法　40只雄性小鼠随机分为4组：酒精损伤组（CA组）、茶多酚低剂量组（TP-L组）及茶多酚高剂量组（TP-H组）每天先喂服酒精,8 h后再分别喂服纯水、或浓度为1%或2%的茶多酚悬液。正常对照组（NC组）以同样方式和剂量每天2次喂服纯水。90 d后取材制备精子悬液用于精子密度、精子活动率及畸形率的检测。同时,取睾丸组织行免疫组织化学检测Bcl-2和Bax的表达。 结果　与NC组比较, CA组的精子密度和精子活动率明显下降,精子畸形率明显增加。相对于CA组,TP-L组及TP-H组精子密度和精子活动率增高, 精子畸形率降低,其中TP-H组的效果比TP-L组更显著。CA组睾丸组织中Bcl-2的阳性细胞比NC组显著减少,Bax的阳性率却明显升高。喂服茶多酚却能明显逆转由酒精导致的上述变化。 结论　茶多酚能预防酒精对精子的损伤,这可能与精子发生过程Bcl-2及Bax的表达变化有关。     

Bcl-2-caspase-9凋亡通路在哮喘小鼠睾丸组织中的相关表达

目的：通过建立小鼠哮喘模型,探讨哮喘对小鼠睾丸组织中细胞凋亡表达的影响。方法：将10只BALB/c小鼠分为两组：急性哮喘组和正常对照组。急性哮喘组采用连续7 d雾化吸入卵白蛋白(OVA)的方法致敏,而对照组则雾化吸入生理盐水,之后收集附睾和睾丸来确定精子计数和精子能动性。通过DNA Ladder、免疫组化和一系列相关的凋亡基因PCR扩增来判断睾丸细胞的凋亡。caspase-3和聚腺苷二磷酸 核糖聚合酶(PARP)的裂解是通过Western blot来测定,而caspase-9、caspase 3/7酶活性则是通过Caspase-Glo试剂盒来测定的。结果：与对照组相比,实验组小鼠的体重、睾丸重量、精子计数和精子活性显著降低(P<0.05)。在急性哮喘组中,DNA-Ladder和免疫组化结果均显示睾丸组织的凋亡指数显著增加（P<0.05）,而PCR结果显示Bcl-2的表达显著降低（P<0.01）,而Bax、BNIP3、caspase-9、AIF的表达显著增加。此外,在蛋白表达水平,AIF的表达显著增加,而Bcl-2的表达明显下调。而且在哮喘小鼠的睾丸组织中,caspase-9的表达明显高于正常对照组。结论：在哮喘小鼠的睾丸组织中Bcl-2-caspase-9凋亡通路的相关基因及蛋白表达显著增高。     

Degradation of the proapoptotic proteins Bik, Puma, and Bim with Bcl-2 domain 3 homology in Chlamydia trachomatis-infected cells

We have previously correlated Chlamydia trachomatis antiapoptotic activity with the blockade of mitochondrial cytochrome c release and the inhibition of Bax and Bak activation. We now report that C. trachomatis infection leads to degradation of Bik, Puma, and Bim, three upstream proapoptotic BH3-only proteins of the Bcl-2 family that can transmit death signals to mitochondria by inhibiting the Bcl-2 antiapoptotic proteins and/or activating the Bcl-2 proapoptotic members, such as Bax and Bak. This observation has provided new information on the chlamydial antiapoptosis mechanisms.     

EGFR ligands exert diverging effects on male reproductive organs

While the EGFR and most of its ligands are expressed in the male reproductive tract, their functions in male reproduction are poorly understood. Interestingly, male transgenic mice overexpressing EGF are sterile, and transgenic mice overexpressing TGFA, another EGFR ligand, show an enlarged coagulation gland (anterior prostate) due to severe hyperplasia with focal dysplasia. We studied the male reproductive tract of transgenic mice overexpressing betacellulin (BTC-tg) under the control of a promoter conferring widespread transgene expression. Despite strong overexpression of BTC in different parts of the male reproductive tract, the gross appearance and histology of the reproductive organs of BTC-tg males were normal and the same were true for sperm parameters and the in vitro fertilization rate. Collectively, our findings demonstrate that excess of BTC exerts no deleterious effects on the structure or function of the male reproductive tract in mice and indicates unique, non-overlapping functions of specific EGFR ligands in male reproduction.     

A mouse model of familial oligoasthenoteratozoospermia

BACKGROUND: Hrb is an HIV-1 Rev-binding/interacting protein and is a cofactor for Rev export pathway. Hrb interacts with Eps15 homology (EH) domain-containing proteins and is a component of EH network and functions in vesicle sorting. Earlier, we reported that Hrb-deficient male mice are infertile and that they show oligozoospermia. Their sperm lack acrosomes and present globozoospermia. The aim of this study was: (i) to investigate the additional defects in spermatogenesis in Hrb-deficient mice; and (ii) to investigate the effect of acrosomelessness on spermatid differentiation in Hrb-deficient mice. METHODS: Hrb(-/-) testes, epididymides, spermatids and sperm were analyzed by histology and electron microscopy. Centrioles were analyzed in spermatids and sperm by indirect immunofluorescence technique. RESULTS: Hrb(-/-) male mice exhibited multiple anomalies during meiosis and spermiogenesis that produced developmentally impaired sperm with unshaped or deformed nuclei, loss in cell polarity, intracellular flagellar coiling, multinucleation, supernumerary centrioles and multiflagellation. A total of 13.0% Hrb(-/-) sperm showed macrocephaly. The Hrb(-/-) sperm exhibited variation in head size and shape, disarranged cellular organelles, nuclear and cytoplasmic vacuolization, mitochondrial loss or scattering and no forward motility. CONCLUSIONS: These aberrations, in Hrb(-/-) mouse spermatids and sperm, are reminiscent of human familial male infertility with oligoasthenoteratozoospermia syndrome. The Hrb-deficient mouse may be useful in understanding familial oligoasthenoteratozoospermia syndrome.     

Overexpression of Bcl-2 protects from ultraviolet B-induced apoptosis but promotes hair follicle regression and chemotherapy-induced alopecia

Hair follicle (HF) growth and regression is an exquisitely regulated process of cell proliferation followed by massive cell death and is accompanied by cyclical expression of the apoptosis regulatory gene pair, Bcl-2 and Bax. To further investigate the role of Bcl-2 expression in the control of hair growth and keratinocyte apoptosis, we have used transgenic mice that overexpress human Bcl-2 in basal epidermis and in the outer root sheath under the control of the human keratin-14 promoter (K14/Bcl-2). When irradiated with ultraviolet B (UVB) light, K14/Bcl-2 mice developed about 5-10-fold fewer sunburn cells (ie, apoptotic keratinocytes) in the basal layer of the epidermis, compared to wild-type mice, whereas cultures of primary keratinocytes from transgenic mice were completely resistant to UVB-induced histone formation, at doses that readily induced histone release from wild-type cells. K14/Bcl-2 mice show no alteration of neonatal hair follicle morphogenesis or of the onset of the first wave of HF regression (catagen). However, compared to wild-type controls, K14/Bcl-2 mice subsequently displayed a significant acceleration of spontaneous catagen progression. During chemotherapy-induced alopecia, follicular dystrophy was promoted in K14/Bcl-2 mice. Thus, although K14-driven overexpression of Bcl-2 protected murine epidermal keratinocytes from UVB-induced apoptosis, it surprisingly promoted catagen- and chemotherapy-associated keratinocyte apoptosis.     

Inactivation of prosurvival Bcl-2 proteins activates Bax/Bak through the outer mitochondrial membrane

The mechanism of Bax/Bak activation remains a central question in mitochondria-dependent apoptotic signaling. While it is established that all proapoptotic Bcl-2 homology 3 (BH3)-only proteins bind and neutralize the anti-apoptotic Bcl-2 family proteins, how this neutralization leads to Bax/Bak activation has been actively debated. Here, genome editing was used to generate cells deficient for all eight proapoptotic BH3-only proteins (OctaKO) and those that lack the entire Bcl-2 family (Bcl-2 allKO). Although the OctaKO cells were resistant to most apoptotic stimuli tested, they underwent Bax/Bak-dependent and p53/Rb-independent apoptosis efficiently when both Bcl-xL and Mcl-1, two anti-apoptotic Bcl-2 proteins, were inactivated or eliminated. Strikingly, when expressed in the Bcl-2 allKO cells, both Bax and Bak spontaneously associated with the outer mitochondrial membrane (OMM) through their respective helix 9, and this association triggered their homo-oligomerization/activation. Together, these results strongly suggest that the OMM, not BH3-only proteins or p53/Rb, is the long-sought-after direct activator of Bax/Bak following BH3-only-mediated neutralization of anti-apoptotic Bcl-2 proteins.     

纳米硒对实验性糖尿病小鼠心肌的保护作用及其可能的机制

目的： 观察纳米硒对实验性糖尿病小鼠心肌的保护作用。方法: 选取60只健康雄性昆明种小鼠，随机抽取10只小鼠作为正常对照组，其余50只小鼠禁食24 h后，左下腹腔注射链脲佐菌素(streptozotocin，STZ)50 mg/kgBW连续5 d，7 d后尾静脉取血测血糖，随机选择血糖值>16.65 mmol/L的40只小鼠分为4组，分别是阳性对照组、低剂量(25 μg/kg)纳米硒组、中剂量(50 μg/kg)纳米硒组、高剂量(100 μg/kg)纳米硒组。各组小鼠分别每天以0.2 mL溶液灌胃补充生理盐水及相应剂量的纳米硒。每周称体重1次，各组剂量根据体重做相应调整。8周后摘除眼球放血处死小鼠，迅速取出心脏，取左心室部分心肌，制成10%心肌匀浆，测定SOD、GSH-Px活力及MDA含量；剩下心肌采用原位缺口末端标记法(TUNEL)检测心肌细胞凋亡，细胞免疫组织化学检测Bcl-2、Bax蛋白的表达。结果: 与正常对照组比较，阳性对照组心肌SOD、GSH-Px活性降低，MDA水平升高，细胞凋亡率明显增加, Bcl-2表达下降,Bax蛋白表达增强；与阳性对照组比较，低、中剂量纳米硒组心肌SOD、GSH-Px活性升高，MDA水平降低，细胞凋亡率下降, Bcl-2蛋白表达增强,Bax蛋白表达下降；而高剂量纳米硒组心肌SOD、GSH-Px活性较中剂量钠米硒组有明显的下降，相应地MDA明显上升，细胞凋亡率明显增加, Bcl-2蛋白表达下降,Bax蛋白表达增强,与阳性对照组比较SOD、GSH-Px活性、MDA含量及心肌细胞凋亡率无明显差异。结论: 给糖尿病小鼠补充一定剂量的纳米硒可以保护心肌，其机制可能与抗氧化、调节血糖及增强Bcl-2表达有关。     

2-5A synthetase expression in mice infected with Trypanosoma cruzi.

This study describes the production and action of interferon in mice infected with Colombian and Y strain T. cruzi. The production of interferon was monitored by an in vitro assay of plasma and extract of spleen, lung and heart for interferon activity. The action of interferon in mice was assessed by measuring an interferon-mediated enzyme activity, 2-5A synthetase. Infected mice (strain Balb/c) were sacrificed at different time intervals, and the level of this enzyme was measured in extracts of spleen, lung and heart. Colombian strain infection induced higher levels of interferon than Y strain under the same conditions; consequently, a greater increase in 2-5A synthetase induction was observed in the former of the two strains. These results suggest that interferon produced by T. cruzi infected mice is active, since a variety of organs respond to its presence by producing elevated levels of 2-5A synthetase.     

Differential expression of cell death regulators in response to thapsigargin and adriamycin in Bcl-2 transfected DU145 prostatic cancer cells

Functional overexpression of Bcl-2 has been reported to confer an anti-apoptotic potential in a variety of cell types. The role of Bcl-2 in epithelial cell-cycle control and in interactions with other cell-cycle regulators is not clearly understood. Its expression has been correlated with the hormono- and chemo-resistant phenotype in advanced prostate cancer. The aim of this study was to investigate the mechanisms through which Bcl-2 mediates increased cytotoxic chemoresistance by assessing alterations in the expression of cell death regulatory molecules. The DU145 human prostatic adenocarcinoma cell line was stably transfected with a Bcl-2 encoding expression plasmid. Two Bcl-2 transfectants, DKC9 and DKC11, were expanded for further study. The effects of Bcl-2 expression on cellular proliferation, cell death (+/- adriamycin or thapsigargin), and expression of cell-cycle/death regulators (p53, PCNA, Bax, Bak, Bcl-X(L)) were evaluated. Compared with controls, Bcl-2 transfectants showed no difference in the rate of proliferation, a decrease in p53 (approximately two-fold), an increase in Bax (approximately two-fold) and PCNA (approximately three-fold), and no change in the levels of Bcl-X(L) and Bak proteins. DKC9 and DKC11 also exhibited a significantly increased chemoresistance to adriamycin (0.0025-5 microM) and thapsigargin (0.0025-5 microM) compared with controls. In the presence of thapsigargin or adriamycin, levels of Bcl-2 and its heterodimeric partner Bax were elevated approximately two-fold with no change in Bak in Bcl-2 transfectants in contrast to controls, where Bak was increased (two-fold). This is the first study to demonstrate that Bcl-2 transfection modulates the expression of mutant p53, Bax, and PCNA in prostate cancer cells. Moreover, Bcl-2 overexpression conferred a significant cytotoxic chemoresistance and altered the balance of expression of death promoters (from Bak, a dominant death promoter in controls, to Bax) in response to thapsigargin and adriamycin.     

Deletion of the fcgamma receptor IIb in thymic stromal lymphopoietin transgenic mice aggravates membranoproliferative glomerulonephritis

Engagement of immunoglobulin-binding receptors (FcgammaR) on leukocytes and other cell types is one means by which immunoglobulins and immune complexes activate effector cells. One of these FcgammaRs, FcgammaRIIb, is thought to contribute to protection from autoimmune disease by down-regulation of B-cell responsiveness and myeloid cell activation. We assessed the role of FcgammaRIIb in a mouse model of cryoglobulin-associated membranoproliferative glomerulonephritis induced by overexpression of thymic stromal lymphopoietin (TSLP). TSLP transgenic mice were crossbred with animals deficient for FcgammaRIIb on the same genetic background (C57BL/6). Renal pathology was assessed in female and male animals (wild-type, FcgammaRIIb-/-, TSLP transgenic, and combined TSLP transgenic/FcgammaRIIb-/- mice) after 50 and 120 days, respectively. FcgammaRIIb-/- mice had no significant renal pathology, whereas overexpression of TSLP induced a membranoproliferative glomerulonephritis, as previously established. TSLP transgenic FcgammaRIIb-/- mice appeared sick with increased mortality. Kidney function was significantly impaired in male mice corresponding to aggravated glomerular pathology with increases in glomerular matrix and cellularity. This resulted from both a large influx of infiltrating macrophages and increased cellular proliferation. These results emphasize the important role of FcgammaRIIb in regulating immune responses and suggest that modulation of Fcgamma receptor activation or expression may be a useful therapeutic approach for treating glomerular diseases.     

Specific arrest of spermatogenesis caused by apoptotic cell death in transgenic mice

Background: The c-myc protooncogene has been implicated in the control of cell proliferation, differentiation and/or apoptosis in various cellular systems. However, the role of c-myc in germ cell lineage is largely unknown.
Results: We have produced transgenic mouse lines carrying the rat c-myc protooncogene under the control of human metallothionein promoter (hMT-c-myc). It was found that the male transgenic mice were sterile. In contrast, all of the female transgenic mice were completely fertile and transmitted the transgene to the next generation. However, male transgenic mice from the female transgenic founders were also found to be sterile. This sterility was due to a defect in spermatogenic cell differentiation, since virtually no sperm were seen within the seminiferous tubules or the cauda epididymis. Histological examination revealed that germ cell death occurred approximately 7 days after birth and, consequently, spermatogenesis was arrested at an early stage in meiotic division in the transgenic mice. Moreover, this germ cell death was found to be caused by apoptosis.
Conclusion: We conclude that an excess level of c-myc expression in differentiating spermatogenic cells is responsible for the apoptotic death of germ cell, and that a decrease in c-myc level would be an obligatory step for the completion of normal spermatogenesis.     

miR-150-5p靶向SIRT1提高肝癌细胞系HepG2放疗敏感性

目的探究miR-150-5p靶向SIRT1提高肝癌细胞放射敏感性的作用机制。方法用分次放疗放射递增法诱导建立放射抵抗型细胞株(RR-HepG2);RT-qPCR检测HepG2和RR-HepG2在不同放射剂量下miR-150-5p的表达水平;细胞克隆实验检测相同放射剂量下两种细胞的放疗敏感性;流式细胞计量术和Western blot检测过表达miR-150-5p对HepG2凋亡的影响;双荧光素酶报告基因法检测miR-150-5p与SIRT1的关联;细胞克隆实验和Western blot检测过表达SIRT1后,细胞的放疗敏感性和凋亡蛋白的变化。结果与亲代HepG2比较,相同放射剂量下,RRHepG2组miR-150-5p的表达量均显著降低(P<0. 05);放射处理后,与control、agomiR-NC组比较,agomiR-150-5p组的细胞存活分数显著降低,敏感性高(P<0. 05),Bax、caspase-9蛋白表达水平显著升高,Bcl-2蛋白表达水平显著降低,细胞凋亡率显著升高;双荧光素酶报告基因法验证miR-150-5p靶向调控SIRT1。与agomiR-NC组比较,野生型(WT) agomiR-150-5p荧光素酶活性显著降低,SIRT1蛋白水平显著降低(P<0. 05);与anta-agomiR-NC比较,野生型(WT) anta-agomiR-150-5p荧光素酶活性显著升高,SIRT1蛋白水平显著升高(P<0. 05);放射处理后,与agomiR-NC组比较,agomiR-150-5p组的细胞存活分数显著降低,Bax、caspase-9蛋白表达水平显著升高,Bcl-2蛋白表达水平显著降低(P<0. 05);与agomiR-150-5p+vector组比较,agomiR-150-5p+SIRT1组的细胞存活分数显著升高,Bax、caspase-9蛋白表达水平显著降低,Bcl-2蛋白表达水平显著升高(P<0. 05)。结论 miR-150-5p靶向SIRT1,下调其表达,提高肝癌细胞放射敏感性,可为临床肝癌放射治疗增敏提供靶点。     

Redox mechanisms of cytoprotection by Bcl-2

Bcl-2 is a multifunctional protein that protects against cell death induced by a wide variety of stimuli. The best characterized antiapoptotic Bcl-2 mechanism of action involves direct binding to proapoptotic proteins, e.g., Bax, inhibiting their ability to oligomerize and form pores in the mitochondrial outer membrane, through which soluble mitochondrial proapoptotic proteins, e.g., cytochrome c, are released into the cytosol. Bcl-2 also exerts antiapoptotic and antinecrotic effects that are mediated by its influence on cellular redox state and apparently independent of its interaction with proapoptotic proteins. Bcl-2 expression increases cell resistance to oxidants, augments the expression of intracellular defenses against reactive oxygen species, and may affect mitochondrial generation of superoxide radicals and hydrogen peroxide. This review focuses on the protective effects of Bcl-2 related to changes in mitochondrial redox capacity.     

Assessment of carcinogenicity of di(2-ethylhexyl)phthalate in a short-term assay using Xpa-/- and Xpa-/-/p53+/- mice

The potential of Xpa-/- and Xpa-/-/p53+/- mice for short-term carcinogenicity assays was evaluated with di(2-ethylhexyl)phthalate (DEHP). Groups of 15 male and female Xpa-/- mice, received diets containing 0, 1, 500, 3,000, or 6,000 ppm DEHP, and wild-type (WT) and Xpa-/-p53+/-mice 0 or 6,000 ppm DEHP for 39 weeks. Xpa-/-, Xpa-/-/p53+/-, and WT males, fed 2,500 ppm p-cresidine, served as a positive control. In all models, the survival was not altered by DEHP. Increased incidences of nonneoplastic lesions were recorded in testes and kidneys with no apparent difference between the models. The only liver tumors in all models were adenomas in males with no statistically significantly increased incidence. For p-cresidine. the survival was decreased (p < 0.05) only in transgenic models. Statistically significantly increased incidences of nonneoplastic lesions were recorded in the liver, urinary bladder, and nasal cavity in all models, and in kidneys in transgenic models. The only tumors with statistically significantly increased incidence were liver adenomas in transgenic models (XPA: I vs 7: 'XPA/p53': 0 vs 12; WT: 0 vs 5, p = 0.053) and urinary bladder carcinomas in XPA/p53 model (0 vs 7). The negative carcinogenic response to DEHP and the positive response to p-cresidine support the expected sensitivity to genotoxic carcinogens in these transgenic models.