BACKGROUND & AIMS: :Detergent-solubilized plasma membrane protein of either adult bovine or calf lens and high-performance liquid chromatography-purified major intrinsic protein (MIP) of the lens were reconstituted into unilamellar vesicles and planar lipid bilayers. Freeze-fracture studies showed that the density of intramembrane particles in the vesicles was proportional to the protein/lipid ratio. At high ratios, these particles crystallized into tetragonal arrays as does MIP in lens fibers. Channels induced by either purified MIP or detergent-solubilized protein had essentially identical properties. The conductance of multichannel membranes was maximal near 0 mV and decreased to 0.49 +/- 0.08 of the maximum value at voltages greater than 80 mV. The dependence of the conductance on voltage was well fit by a two-state Boltzmann distribution. Voltage steps greater than 30 mV elicited an ohmic current step followed by a slow (seconds) biexponential decrease. The amplitudes and time constants depended on the magnitude but not the sign of the voltage. Steps from 100 mV to voltages less than 30 mV caused the channels to open exponentially with a millisecond time constant. Analysis of latency to first closure after a voltage step gave nearly the same time constants as multichannel kinetics. Single-channel conductance is proportional to salt concentration from 0.1 to 1.0 M in KCl. In 0.1M KCl, the channel had two preferred conductance states with amplitudes of 380 and 160 pS, as well as three additional substates. Multi- and single-channel data suggest that the channel has two kinetically important open states. The channel is slightly anion selective. The properties of the channel do not vary appreciably from pH 7.4 to 5.8 or from pCa 7 to 2. We propose that a channel with these properties could contribute to maintenance of lens transparency and fluid balance.

背景与目标： ：成年牛或小牛晶状体的去污剂溶解质膜蛋白和高效液相色谱纯化的晶状体主要内在蛋白（MIP）重构为单层囊泡和平面脂质双层。冷冻断裂研究表明，囊泡中膜内颗粒的密度与蛋白质/脂质比成正比。在高比例下，这些颗粒与透镜纤维中的MIP一样结晶为四边形阵列。纯化的MIP或去污剂溶解的蛋白诱导的通道具有基本相同的特性。多通道膜的电导在0 mV附近最大，并且在大于80 mV的电压下降低到最大值的0.49 /-0.08。电导对电压的依赖性通过两态玻耳兹曼分布很好地拟合。大于30 mV的电压阶跃引起欧姆电流阶跃，然后缓慢（秒）双指数下降。幅度和时间常数取决于幅度，而不取决于电压的符号。从100 mV到低于30 mV的电压阶跃导致通道以毫秒的时间常数呈指数方式打开。电压阶跃后首次闭合的等待时间的分析给出了与多通道动力学几乎相同的时间常数。单通道电导与KCl中0.1至1.0 M的盐浓度成正比。在0.1M KCl中，通道具有两个优选的电导状态，其振幅分别为380和160 pS，以及三个其他子状态。多通道和单通道数据表明该通道具有两个动力学上重要的开放状态。该通道对阴离子具有轻微的选择性。通道的特性在pH 7.4至5.8或pCa 7至2范围内变化不大。我们建议具有这些特性的通道可有助于维持镜片的透明度和流体平衡。

BACKGROUND & AIMS: :The Slc5a6 gene expresses a plasma membrane protein involved in the transport of the water-soluble vitamin biotin; the transporter is commonly referred to as the sodium-dependent multivitamin transporter (SMVT) because it also transports pantothenic acid and lipoic acid. The relative contribution of the SMVT system toward carrier-mediated biotin uptake in the native intestine in vivo has not been established. We used a Cre/lox technology to generate an intestine-specific (conditional) SMVT knockout (KO) mouse model to address this issue. The KO mice exhibited absence of expression of SMVT in the intestine compared with sex-matched littermates as well as the expected normal SMVT expression in other tissues. About two-thirds of the KO mice died prematurely between the age of 6 and 10 wk. Growth retardation, decreased bone density, decreased bone length, and decreased biotin status were observed in the KO mice. Microscopic analysis showed histological abnormalities in the small bowel (shortened villi, dysplasia) and cecum (chronic active inflammation, dysplasia) of the KO mice. In vivo (and in vitro) transport studies showed complete inhibition in carrier-mediated biotin uptake in the intestine of the KO mice compared with their control littermates. These studies provide the first in vivo confirmation in native intestine that SMVT is solely responsible for intestinal biotin uptake. These studies also provide evidence for a casual association between SMVT function and normal intestinal health.

背景与目标： ：Slc5a6基因表达参与水溶性维生素生物素运输的质膜蛋白；该转运蛋白通常也称为钠依赖性多种维生素转运蛋白（SMVT），因为它也可以转运泛酸和硫辛酸。 SMVT系统对体内天然肠道中载体介导的生物素摄取的相对贡献尚未确定。我们使用Cre / lox技术来生成特定于肠道的（条件性）SMVT敲除（KO）小鼠模型，以解决此问题。与性别匹配的同窝幼仔相比，KO小鼠在肠道中不存在SMVT表达，在其他组织中也表现出预期的正常SMVT表达。大约三分之二的KO小鼠在6至10周龄之间过早死亡。在KO小鼠中观察到生长迟缓，骨密度降低，骨长度降低和生物素状态降低。显微镜分析显示KO小鼠的小肠（绒毛缩短，发育不良）和盲肠（慢性活动性炎症，发育不良）的组织学异常。体内（和体外）转运研究表明，与对照组的同窝仔猪相比，KO小鼠肠道中的载体介导的生物素摄取被完全抑制。这些研究首次证实了天然肠中SMVT负责肠道生物素的摄取。这些研究还为SMVT功能与正常肠道健康之间的偶然关联提供了证据。

BACKGROUND & AIMS: OBJECTIVE:The intracellular signals that contribute to granulocyte colony-stimulating factor (G-CSF) receptor induced stem cell mobilization are poorly characterized. METHODS:We show enhanced G-CSF induced mobilization of stem cells in mice deficient in expression of Src family kinases (SFK-/-), which is associated with hypersensitivity of SFK-/- bone marrow cells to G-CSF as well as sustained activation of signal transducer and activator of transcription-3. RESULTS:A proteome map of the bone marrow fluid derived from wild-type and SFK-/- mice revealed a significant global reduction in the number of proteins in SFK-/- mice compared to controls, which was associated with elevated matrix metalloproteinase-9 levels, reduced stromal-derived factor-1 expression, and enhanced breakdown of vascular cell adhesion molecule-1. Transplantation of wild-type or SFK-/- stem cells into wild-type mice and treatment with G-CSF recapitulated the G-CSF-induced increase in stem cell mobilization noted in SFK-/- nontransplanted mice; however, the increase was significantly less. G-CSF treatment of SFK-/- mice engrafted with wild-type stem cells also demonstrated a modest increase in stem cell mobilization compared to controls, however, the observed increase was greatest in mice completely devoid of SFKs. CONCLUSIONS:These data suggest an involvement of both hematopoietic intrinsic and microenvironmental factors in Src kinase-mediated mobilization of stem cells and identify Src kinases as potential targets for modulating stem cell mobilization.

背景与目标： 目的：促成粒细胞集落刺激因子（G-CSF）受体诱导干细胞动员的细胞内信号的特征较差。
方法：我们显示了增强的G-CSF诱导缺乏Src家族激酶（SFK-/-）表达的小鼠干细胞动员，这与SFK-/-骨髓细胞对G-CSF的超敏性以及持续性有关激活信号转导子和转录激活子3。
结果：从野生型和SFK-/-小鼠衍生的骨髓液的蛋白质组图谱显示，与对照组相比，SFK-/-小鼠的蛋白质数量显着减少，这与基质金属蛋白酶9升高有关水平，减少基质衍生因子1表达，并增强血管细胞粘附分子1的分解。将野生型或SFK-/-干细胞移植到野生型小鼠中并用G-CSF进行治疗，概括了G-CSF诱导的SFK-/-非移植小鼠中干细胞动员的增加；但是，增加幅度明显较小。对植入野生型干细胞的SFK-/-小鼠进行G-CSF处理，与对照组相比，干细胞动员也有适度的增加，但是，观察到的这种增加在完全没有SFK的小鼠中最大。
结论：这些数据表明造血的内在因素和微环境因素都参与了Src激酶介导的干细胞动员，并确定Src激酶是调节干细胞动员的潜在靶标。

BACKGROUND & AIMS: :Breast cancer is a sex steroid hormone-dependent malignant neoplasia. The role of oestradiol in this malignancy has been well documented; however, the involvement of androgens has remained controversial. To determine the role of non-phenolic androgen metabolites in human breast cancer, we studied the metabolism of [(14)C] testosterone and [(14)C] androstenedione in oestrogen-dependent MCF-7 cells and non-oestrogen-dependent MDA-MB 231 cells, at different substrate concentrations (1-10 muM) and time periods (30 min-48 h). Cultured non-oestrogen-dependent HeLa and yeast cells served as controls. Metabolites were identified and quantified by reverse isotope dilution. A distinctive pattern of androgen metabolism was identified in MCF-7 cells, being the 5alpha-androstane-3alpha,17beta-diol (3alpha,5alpha-diol) and its 3beta epimer (3beta,5alpha-diol), the major conversion products of testosterone (48.3%), with 5alpha-dihydrotestosterone as intermediary. The formation of 3alpha,5alpha-diol and 3beta,5alpha-diol (diols) was substrate concentration- and time-dependent, and abolished by finasteride. In contrast, very little of any diol formation was observed in MDA-MB 231, HeLa and yeast cell incubations. Additional enzyme gene expression studies revealed an overexpression of 5alpha-steroid reductase type-1 in MCF-7 cells, as compared with MDA-MB 231 cells. The oestrogen-like activities of diols were assessed in HeLa cells co-transfected with expression vectors for alpha or beta subtypes of the human oestrogen receptor (hER) genes and for an oestrogen-responsive reporter gene. The results show that 3beta, 5alpha-diol and to a lesser extent 3alpha,5alpha-diol bind with high relative affinity to hERalpha and hERbeta. Both diols induced hER-mediated reporter gene transactivation in a dose-response manner, similar to that induced by oestradiol, though with lower potency, an effect that was abolished by ICI-182 780. Furthermore, 3beta,5alpha-diol and to lesser extent 3alpha,5alpha-diol induced MCF-7 cell proliferation. The overall results demonstrated that MCF-7 cells exhibit enhanced expression and activity of androgen-metabolising enzymes, leading to rapid and large diol formation, and provide evidence that these androgen metabolites exert a potent oestrogen-agonistic effect, at genomic level, in oestrogen-dependent breast cancer cells. The data suggest that diols may act as in situ intracrine factors in breast cancer and that its formation can be pharmacologically inhibited.

背景与目标： ：乳腺癌是性激素依赖的恶性肿瘤。雌二醇在这种恶性肿瘤中的作用已被充分证明。然而，雄激素的参与仍存在争议。为了确定非酚类雄激素代谢物在人类乳腺癌中的作用，我们研究了雌激素依赖性MCF-7细胞和非雌激素依赖性MDA中[（14）C]睾丸激素和[（14）C]雄烯二酮的代谢-MB 231细胞，处于不同的底物浓度（1-10μM）和时间段（30分钟-48小时）。培养的非雌激素依赖性HeLa和酵母细胞用作对照。通过反向同位素稀释对代谢物进行鉴定和定量。在MCF-7细胞中发现了一种独特的雄激素代谢模式，即5alpha-androstane-3alpha，17beta-diol（3alpha，5alpha-diol）及其3beta epimer（3beta，5alpha-diol），这是睾丸激素的主要转化产物。 （48.3％），其中以5alpha-dihydrotestosterone为中介。 3α，5α－二醇和3β，5α－二醇（二醇）的形成与底物浓度和时间有关，并被非那雄胺废除。相反，在MDA-MB 231，HeLa和酵母细胞培养中几乎没有观察到任何二醇的形成。额外的酶基因表达研究表明，与MDA-MB 231细胞相比，MCF-7细胞中5α-类固醇还原酶1型过度表达。在共转染了人类雌激素受体（hER）基因的α或β亚型的表达载体的HeLa细胞中，以及针对雌激素反应性报告基因的HeLa细胞中，评估了二醇的类雌激素活性。结果表明3β，5α-二醇和较小程度的3α，5α-二醇以高相对亲和力与hERα和hERβ结合。两种二醇均以剂量响应方式诱导hER介导的报告基因反式激活，与雌二醇诱导的类似，尽管效力较低，但被ICI-182 780所废除。此外，3beta，5alpha-二醇程度较低3alpha，5alpha-diol诱导MCF-7细胞增殖。总体结果表明，MCF-7细胞显示出增强的雄激素代谢酶表达和活性，导致快速大量的二醇形成，并提供了证据表明这些雄激素代谢物在基因组水平上在雌激素中发挥了强大的雌激素激动作用。依赖性乳腺癌细胞。数据表明，二醇可能在乳腺癌中充当原位内分泌因子，并且其形成可以在药理学上得到抑制。

BACKGROUND & AIMS: :Glucocorticoid eye drops are one of the most widely used medications in ophthalmology. However, little is known about the effects of glucocorticoids on corneal epithelial cells that are directly exposed to topically-administered glucocorticoids. Here we investigated the effects of prednisolone, a synthetic glucocorticoid analogue frequently used in the clinic, on corneal epithelial cells. Results showed that prednisolone decreased survival of corneal epithelial cells by inhibiting proliferation and inducing apoptosis in a dose-dependent manner. The levels of mitochondrial reactive oxygen species (mtROS), cleaved caspase-3, and -9 were increased by prednisolone. The effects of prednisolone on apoptosis and mtROS were blocked 1) by the glucocorticoid receptor (GR) antagonist RU-38486, 2) in cells with GR siRNA knockdown, and 3) by treatment with N-acetylcysteine. Transcript levels of pro-inflammatory cytokines were increased in corneal epithelial cells upon hyperosmolar stress, but repressed by prednisolone. In NOD.B10.H2b mice, topical administration of 1% prednisolone increased apoptotic cells in the corneal epithelium. Together, data indicate that prednisolone induces apoptosis in corneal epithelial cells through GR and the intrinsic pathway involving mtROS, caspase-9, and -3. The pro-apoptotic effects of glucocorticoids along with their anti-inflammatory effects should be considered when glucocorticoid eye drops are used in patients with ocular surface disease.

背景与目标： 糖皮质激素滴眼液是眼科最广泛使用的药物之一。然而，关于糖皮质激素对直接暴露于局部施用的糖皮质激素的角膜上皮细胞的影响知之甚少。在这里，我们研究了强的松龙（一种在临床上经常使用的合成糖皮质激素类似物）对角膜上皮细胞的作用。结果表明泼尼松龙通过抑制增殖并诱导细胞凋亡以剂量依赖的方式降低了角膜上皮细胞的存活率。泼尼松龙可增加线粒体活性氧（mtROS），裂解的caspase-3和-9的水平。泼尼松龙对细胞凋亡和mtROS的作用被1）糖皮质激素受体（GR）拮抗剂RU-38486阻断； 2）GR siRNA敲除的细胞被阻断； 3）N-乙酰半胱氨酸处理。高渗压后角膜上皮细胞中促炎细胞因子的转录水平增加，但被泼尼松龙抑制。在NOD.B10.H2b小鼠中，局部给药1％泼尼松龙可增加角膜上皮中的凋亡细胞。总之，数据表明泼尼松龙通过GR和涉及mtROS，caspase-9和-3的内在途径通过GR诱导角膜上皮细胞凋亡。当将糖皮质激素滴眼剂用于眼表疾病患者时，应考虑糖皮质激素的促凋亡作用及其抗炎作用。

BACKGROUND & AIMS: :Mutations in the gene encoding hepatocyte nuclear factor (HNF)1beta result in maturity-onset diabetes of the young-(MODY)5, by impairing insulin secretory responses and, possibly, by reducing beta-cell mass. The functional role of HNF1beta in normal beta-cells is poorly understood; therefore, in the present study, wild-type (WT) HNF1beta, or one of two naturally occurring MODY5 mutations (an activating mutation, P328L329del, or a dominant-negative form, A263insGG) were conditionally expressed in the pancreatic beta-cell line, insulin-1 (INS-1), and the functional consequences examined. Surprisingly, overexpression of the dominant-negative mutant did not modify any of the functional properties of the cells studied (including insulin secretion, cell growth and viability). By contrast, expression of WT HNF1beta was associated with a time- and dose-dependent inhibition of INS-1 cell proliferation and a marked increase in apoptosis. Induction of WT HNF1beta also inhibited the insulin secretory response to nutrient stimuli, membrane depolarisation or activation of protein kinases A and C and this correlated with a significant decrease in pancrease-duodenum homeobox-1 protein levels. The attenuation of insulin secretion was, however, dissociated from the inhibition of proliferation and loss of viability, since expression of the P328L329del mutant led to a reduced rate of cell proliferation, but failed to induce apoptosis or to alter insulin secretion. Taken together, the present results suggest that mature rodent beta-cells are sensitive to increased expression of WT HNF1beta and they imply that the levels of this protein are tightly regulated to maintain secretory competence and cell viability.

背景与目标： ：编码肝细胞核因子（HNF）1beta的基因突变会削弱胰岛素分泌反应，并可能减少β细胞的数量，从而导致年轻的（MODY）5发病。 HNF1beta在正常的β细胞中的功能作用了解甚少。因此，在本研究中，野生型（WT）HNF1beta或两个自然发生的MODY5突变之一（激活突变P328L329del或显性负性形式A263insGG）在胰腺β细胞系中有条件表达，胰岛素-1（INS-1），并检查其功能后果。出人意料的是，显性负突变体的过表达并未改变所研究细胞的任何功能特性（包括胰岛素分泌，细胞生长和生存力）。相比之下，WT HNF1beta的表达与INS-1细胞增殖的时间和剂量依赖性抑制以及凋亡的显着增加有关。 WT HNF1beta的诱导还抑制了胰岛素对营养刺激，膜去极化或蛋白激酶A和C活化的分泌反应，这与胰十二指肠同源盒1蛋白水平的显着降低有关。然而，由于P328L329del突变体的表达导致细胞增殖率降低，但是胰岛素分泌的减弱与增殖抑制和生存力丧失分离，但不能诱导细胞凋亡或改变胰岛素分泌。综上所述，本发明结果表明成熟的啮齿动物β细胞对WTHNF1β表达的增加敏感，并且暗示该蛋白的水平被严格调节以维持分泌能力和细胞生存力。

BACKGROUND & AIMS: :Loss of function mouse models comprise knock-out mice, where a gene is deleted in the germline, and conditional knock-out mice with somatic deletion of a floxed allele in defined tissues. Both types of mice are used for comprehensive studies of gene functions in vivo. Here, we describe a simple method for simultaneous generation of mice with conditional or knock-out alleles for the transcription factor fra-2 (Fos-related antigen 2) using a single embryonic stem (ES) cell clone. ES cells with a floxed fra-2 allele were transiently transfected with a Cre-recombinase expression plasmid and plated at low density. Most of the resulting ES cell colonies consisted of a mixture of cells that have either retained or lost the conditional allele. We demonstrate that these mixed ES cell clones can be directly used for generation of chimeras that give rise to offspring with conditional or knock-out alleles simultaneously. This strategy shortens the time and reduces the number of germline transmission events to generate genetically modified mice.

背景与目标： 功能丧失的小鼠模型包括敲除小鼠（其中种系中的基因缺失）和条件性敲除小鼠，其在定义的组织中体细胞性等位基因缺失。两种类型的小鼠都用于体内基因功能的综合研究。在这里，我们描述了使用单个胚胎干（ES）细胞克隆同时生成具有转录因子fra-2（Fos相关抗原2）的条件或敲除等位基因的小鼠的简单方法。用Cre-重组酶表达质粒瞬时转染带有fra-2等位基因的ES细胞，并以低密度铺板。产生的大多数ES细胞集落由保留或丢失条件等位基因的细胞混合物组成。我们证明，这些混合的ES细胞克隆可直接用于产生嵌合体，这些嵌合体同时产生条件等位基因或敲除等位基因。该策略缩短了时间并减少了产生遗传修饰小鼠的种系传播事件的数量。

BACKGROUND & AIMS: :Denervation-induced plastic changes impair locomotor recovery after spinal cord injury (SCI). Spinal motoneurons become hyperexcitable after SCI, but the plastic responses of locomotor network interneurons (INs) after SCI have not been studied. Using an adult mouse SCI model, we analyzed the effects of complete spinal cord lesions on the intrinsic electrophysiological properties, excitability, and neuromodulatory responses to serotonin (5-HT) in mouse lumbar V2a spinal INs, which help regulate left-right alternation during locomotion. Four weeks after SCI, V2a INs showed almost no changes in baseline excitability or action potential properties; the only parameter that changed was a reduced input resistance. However, V2a INs became 100- to 1000-fold more sensitive to 5-HT. Immunocytochemical analysis showed that SCI caused a coordinated loss of serotonergic fibers and the 5-HT transporter (SERT). Blocking the SERT with citalopram in intact mice did not increase 5-HT sensitivity to the level seen after SCI. SCI also evoked an increase in 5-HT(2C) receptor cluster number and intensity, suggesting that several plastic changes cooperate in increasing 5-HT sensitivity. Our results suggest that different components of the spinal neuronal network responsible for coordinating locomotion are differentially affected by SCI, and highlight the importance of understanding these changes when considering therapies targeted at functional recovery.

背景与目标： ：神经变性引起的塑料变化会损害脊髓损伤（SCI）后的运动能力恢复。脊髓运动神经元在SCI后变得过度兴奋，但尚未研究SCI后运动网络内部神经元（IN）的塑性响应。使用成年小鼠SCI模型，我们分析了完整的脊髓损伤对小鼠腰V2a脊髓IN中固有的电生理特性，兴奋性和对血清素（5-HT）的神经调节反应的影响，这有助于调节运动中的左右交替。 SCI后四周，V2a INs几乎没有显示出基线兴奋性或动作电位特性的变化。唯一改变的参数是减小的输入电阻。但是，V2a IN对5-HT的敏感性提高了100到1000倍。免疫细胞化学分析表明，SCI导致了血清素能纤维和5-HT转运蛋白（SERT）的协同损失。在完整小鼠中用西酞普兰阻断SERT不会使5-HT敏感性增加到SCI后的水平。 SCI还引起了5-HT（2C）受体簇数量和强度的增加，这表明一些塑性变化可协同提高5-HT敏感性。我们的结果表明，负责协调运动的脊髓神经网络的不同组成部分受SCI的影响不同，并且在考虑针对功能恢复的疗法时强调了理解这些变化的重要性。

BACKGROUND & AIMS: OBJECTIVE:To determine the validity of intrinsic value and therapeutic utility. DESIGN:The medicines prescribed in a random sample of 50 cases of common cold from each health centre evaluated were classified according to their intrinsic value (ordinal classification using five categories) and therapeutic use (dichotomized classification based on their intrinsic value). Patients with immunodeficiency or underlying pathology (COPD, cardiopathy, diabetes) were excluded. Each of the generic and non-specific indicators were studied to determine sensitivity and specificity for detecting problems in the rational prescription of medicines. SETTING:A representative sample from 8 primary care centres in the Murcia region. MEASUREMENTS AND MAIN RESULTS:The operative replies curve for intrinsic value revealed that this indicator was of little use in evaluating the quality of prescription. CONCLUSIONS:The low validity of the generic prescription indicators suggests that systems to monitor quality based on a specific focus (linking the prescription to the disease treated) should be designed.

背景与目标： 目的：确定内在价值和治疗实用性的有效性。
设计：按照评估的内在价值（按五类常规分类）和治疗用途（根据内在价值二等分类）对来自每个评估中心的50例普通感冒的随机样本中指定的药物进行分类。具有免疫缺陷或基础病理（COPD，心脏病，糖尿病）的患者被排除在外。对每种通用和非特异性指标进行了研究，以确定敏感性和特异性，以检测药物合理处方中的问题。
地点：穆尔西亚地区8个初级保健中心的代表性样本。
测量和主要结果：内在价值的手术回复曲线表明，该指标在评估处方质量方面几乎没有用。
结论：通用处方指标的有效性较低，表明应设计基于特定重点（将处方与治疗的疾病联系起来）的质量监控系统。

BACKGROUND & AIMS: :Diffuse intrinsic pontine gliomas arise almost exclusively in children, and despite advances in treatment, the majority of patients die within 2 years after initial diagnosis. Because of their infiltrative nature and anatomic location in an eloquent area of the brain, most pontine gliomas are treated without a surgical biopsy. The corresponding lack of tissue samples has resulted in a limited understanding of the underlying genetic and molecular biologic abnormalities associated with pontine gliomas, and is a substantial obstacle for the preclinical testing of targeted therapeutic agents for these tumors. We have established a human glioma cell line that originated from surgical biopsy performed on a patient with a pontine glioma. To insure sustainable in vitro propagation, tumor cells were modified with hTERT (human telomerase ribonucleoprotein reverse transcriptase), and with a luciferase reporter to enable non-invasive bioluminescence imaging. The hTERT modified cells are tumorigenic in athymic rodents, and produce brainstem tumors that recapitulate the infiltrative growth of brainstem gliomas in patients.

背景与目标： ：弥漫性桥脑神经胶质瘤几乎仅在儿童中发生，尽管治疗有所进步，但大多数患者在初诊后2年内死亡。由于其浸润性和大脑雄辩区域的解剖位置，大多数桥脑神经胶质瘤无需手术活检即可治疗。组织样品的相应缺乏导致对与桥脑神经胶质瘤相关的潜在遗传和分子生​​物学异常的了解有限，并且是针对这些肿瘤的靶向治疗剂进行临床前测试的主要障碍。我们已经建立了人类脑胶质瘤细胞系，该细胞系源自对桥脑胶质瘤患者进行的手术活检。为了确保可持续的体外繁殖，使用hTERT（人端粒酶核糖核蛋白逆转录酶）和荧光素酶报道分子修饰肿瘤细胞，以实现非侵入性生物发光成像。 hTERT修饰的细胞在无胸腺啮齿动物中具有致瘤性，并产生脑干肿瘤，可概括患者脑干神经胶质瘤的浸润性生长。

BACKGROUND & AIMS: :Proteins are often classified in a binary fashion as either structured or disordered. However this approach has several deficits. Firstly, protein folding is always conditional on the physiochemical environment. A protein which is structured in some circumstances will be disordered in others. Secondly, it hides a fundamental asymmetry in behavior. While all structured proteins can be unfolded through a change in environment, not all disordered proteins have the capacity for folding. Failure to accommodate these complexities confuses the definition of both protein structural domains and intrinsically disordered regions. We illustrate these points with an experimental study of a family of small binding domains, drawn from the RNA polymerase of mumps virus and its closest relatives. Assessed at face value the domains fall on a structural continuum, with folded, partially folded, and near unstructured members. Yet the disorder present in the family is conditional, and these closely related polypeptides can access the same folded state under appropriate conditions. Any heuristic definition of the protein domain emphasizing conformational stability divides this domain family in two, in a way that makes no biological sense. Structural domains would be better defined by their ability to adopt a specific tertiary structure: a structure that may or may not be realized, dependent on the circumstances. This explicitly allows for the conditional nature of protein folding, and more clearly demarcates structural domains from intrinsically disordered regions that may function without folding.

背景与目标： ：Protein通常以二进制方式分类为结构化或无序。但是，这种方法存在一些缺陷。首先，蛋白质折叠总是以物理化学环境为条件。在某些情况下结构化的蛋白质在其他情况下会变得无序。其次，它掩盖了行为的基本不对称性。虽然所有结构化蛋白质都可以通过环境变化来展开，但并非所有无序蛋白质都具有折叠能力。无法适应这些复杂性会混淆蛋白质结构域和内在无序区域的定义。我们通过对腮腺炎病毒及其近亲的RNA聚合酶绘制的一系列小结合域的实验研究说明了这些观点。按面值评估，这些域位于结构连续体上，具有折叠，部分折叠和接近非结构化的成员。然而，该家族中存在的病症是有条件的，并且这些紧密相关的多肽可以在适当的条件下进入相同的折叠状态。强调构象稳定性的蛋白质结构域的任何启发式定义都将该结构域家族分为两部分，这是没有生物学意义的。结构域可以采用特定的三级结构的能力来更好地定义：根据情况，该结构可能会实现，也可能不会实现。这明确地允许蛋白质折叠的条件性质，并且更清楚地从可能无折叠而起作用的内在无序的区域划定结构域。

BACKGROUND & AIMS: :Cooperation declines in repeated public good games because individuals behave as conditional cooperators. This is because individuals imitate the social behaviour of successful individuals when their payoff information is available. However, in human societies, individuals cooperate in many situations involving social dilemmas. We hypothesize that humans are sensitive to both success (payoffs) and how that success was obtained, by cheating (not socially sanctioned) or good behaviour (socially sanctioned and adds to prestige or reputation), when information is available about payoffs and prestige. We propose and model a repeated public good game with heterogeneous conditional cooperators where an agent's donation in a public goods game depends on comparing the number of donations in the population in the previous round and with the agent's arbitrary chosen conditional cooperative criterion. Such individuals imitate the social behaviour of role models based on their payoffs and prestige. The dependence is modelled by two population-level parameters: affinity towards payoff and affinity towards prestige. These affinities influence the degree to which agents value the payoff and prestige of role models. Agents update their conditional strategies by considering both parameters. The simulations in this study show that high levels of cooperation are established in a population consisting of heterogeneous conditional cooperators for a certain range of affinity parameters in repeated public good games. The results show that social value (prestige) is important in establishing cooperation.

背景与目标： ：在反复的公益活动中，合作减少了，因为个人表现为有条件的合作者。这是因为，当可获得他们的回报信息时，个人会模仿成功人士的社交行为。但是，在人类社会中，个人在许多涉及社会困境的情况下进行合作。我们假设人们对成功（收益）以及成功与否（无论是通过作弊（未受到社会制裁）还是良好行为（受到社会认可并会增加声望或声誉））都非常敏感，只要他们可以获得有关收益和声望的信息。我们提出了一个具有异类条件合作者的重复公共物品博弈并对其进行建模，其中代理在公共物品博弈中的捐赠取决于将前一轮中人口捐赠的数量与代理的任意选择的条件合作标准进行比较。这些人根据他们的回报和声望来模仿榜样的社会行为。依赖关系由两个总体级别的参数建模：对收益的亲和力和对声望的亲和力。这些亲和力会影响代理人重视榜样的回报和声望的程度。代理通过考虑两个参数来更新其条件策略。本研究中的模拟显示，在重复的公益游戏中，对于特定范围的亲和力参数，在由异质条件合作者组成的群体中建立了高水平的合作。结果表明，社会价值（信誉）在建立合作中很重要。

BACKGROUND & AIMS: OBJECTIVES:The aim of this study was to derive longitudinal reference values of fetal growth (estimated fetal weight [EFW] and abdominal circumference [AC]) during the third trimester and to develop coefficients for conditional growth assessment. PATIENTS AND METHODS:A prospective cohort study was conducted involving consecutive singleton pregnancies in a low-risk population for a routine third-trimester scan at 30+0-34+6 weeks and follow-up at 37+0-38+6 weeks for an additional ultrasound. Statistical analysis was based on multilevel modeling using MLwiN software. Unconditional centiles were calculated from z-values at each gestational age, and conditional centiles were calculated from z-values at a given measurement (30-34 weeks) and the expected measurement (37-38 weeks). RESULTS:At 30-34 weeks, 8 and 9.3% of the fetuses had an unconditional EFW below the 10th and above the 90th centile, respectively. At 37-38 weeks, these figures were 10.3 and 9.3%, respectively. Regarding the unconditional AC, at the first scan, 8.9 and 9.6% had values below the 10th and above the 90th centile, while at the second scan 10.5 and 10.5% had values below the 10th and above the 90th centile, respectively. The proportion with a conditional EFW below the 10th and above the 90th centile was 10.2 and 9.4% at the second scan, respectively. For conditional AC, these figures were 10.7 and 10.3%, respectively. CONCLUSION:We have produced reference centiles for EFW and AC growth during the third trimester as a useful tool for quantifying growth.

背景与目标： 目的：本研究的目的是得出妊娠中期胎儿生长的纵向参考值（估计的胎儿体重[EFW]和腹围[AC]），并建立条件生长评估的系数。
患者和方法：进行了一项前瞻性队列研究，涉及低危人群中连续单胎妊娠，在30 0-34 6周进行常规的孕中期扫描，并在37 0-38 6周进行随访，以进行额外的超声检查。统计分析基于使用MLwiN软件的多级建模。根据每个胎龄的z值计算无条件的百分位数，根据给定的测量值（30-34周）和预期的测量值（37-38周）的z值计算条件的百分位。
结果：在30-34周时，分别有8％和9.3％的胎儿在第10个百分位以下和第90个百分位以上有无条件的EFW。在第37-38周时，这些数字分别为10.3和9.3％。对于无条件AC，在第一次扫描时，8.9和9.6％的值低于第10个百分位，在第90个百分点之上，而在第二次扫描中，10.5和10.5％的值分别在第10个百分位数和第90个百分率以上。在第二次扫描时，条件EFW低于第10个百分位和高于第90个百分位的比例分别为10.2％和9.4％。对于有条件的AC，这些数字分别为10.7％和10.3％。
结论：我们为妊娠晚期的EFW和AC增长绘制了参考百分位，作为量化增长的有用工具。

BACKGROUND & AIMS: :Age-related changes in primary lymphoid organs are well described. Less is known about age-related changes affecting peripheral lymphoid organs, although defects in old peripheral lymph nodes (pLNs) were recently described in both steady state and during viral infection. To address whether such pLN defects were intrinsic to old T cells or extrinsic (due to aging microenvironment), we employed heterochronic parabiosis. We found no age-related intrinsic or extrinsic barriers to T cell circulation and seeding of pLN, spleen, and bone marrow. However, heterochronic parabiosis failed to improve cellularity of old pLN, suggesting an environment-based limit on pLN cellularity. Furthermore, upon parabiosis, pLN of the adult partner exhibited reduced, old-like stromal and T cell cellularity, which was restored following separation of parabionts. Decay measurement of adult and old T cell subsets following separation of heterochronic parabionts delineated both T cell-intrinsic and environmental changes in T cell maintenance. Moreover, parabiotic separation revealed differences between CD4 and CD8 T cell subset maintenance with aging, the basis of which will require further investigation. Reasons for this asymmetric and subset-specific pattern of differential maintenance are discussed in light of possible age-related changes in lymph nodes as the key sites for peripheral T cell maintenance.

背景与目标： ：与淋巴管原发性器官的年龄相关的变化已被很好地描述。关于年龄相关变化影响外周淋巴器官的知之甚少，尽管最近已在稳定状态和病毒感染期间描述了旧的外周淋巴结（pLNs）的缺陷。为了解决此类pLN缺陷是旧T细胞固有的还是外部T细胞固有的（由于老化的微环境），我们采用了异时共生。我们没有发现年龄相关的内在或外在障碍对T细胞循环和pLN，脾脏和骨髓的播种。但是，异时生物共生不能改善旧pLN的细胞性，这表明基于环境的pLN细胞性受到限制。此外，在发生共生关系时，成年伴侣的pLN表现出减少的，老样的基质和T细胞细胞性，这在分离代谢物后得以恢复。异时生物体分离后，成年和老T细胞亚群的衰变测量描述了T细胞内在性和T细胞维持环境的变化。此外，共生生物分离揭示了CD4和CD8 T细胞亚群维持与衰老之间的差异，其基础将需要进一步研究。鉴于可能与年龄相关的淋巴结变化是外周T细胞维持的关键部位，讨论了这种差异维护的非对称和子集特定模式的原因。

BACKGROUND & AIMS: OBJECTIVE:To investigate the functions of Fibroblast Growth Factor Receptor-2 (FGFR2) at different stages of cell differentiation. The engineered murine embryonic stem (ES) cells with conditional knockout of FGFR2 were developed depending on Cre-loxP. METHODS:Cre-loxP system was used in a conditional targeting vector. The competent AM-1 bacteria, which expressed Cre-recombinase, was used to confirm the Cre-mediated deletion of the floxed exons 7 and 8 of FGFR2. The targeting vector was electroporated into the ES cells, and the transfected ES cells were screened with G418 and Ganciclovir. Finally, the ES clones with correct targeting events were identified by Southern Blot and PCR. RESULTS:The targeting vector with conditional knockout of murine FGFR2 was successfully constructed and confirmed by PCR and digestion analysis in bacteria. 86 ES clones were collected by selective culture with G418 and Ganciclovir. Four of the 86 ES clones were found containing the targeting gene sequence in genomic DNA proved by Southern Blot with a 5'-end flank probe. Two of the four ES clones had the correct targeting events that included the insertion of the targeting gene sequence in genomic DNA and were checked by Southern Blot with a 3'-end flanking probe. Finally, the insertion of loxP (loxP3) between exons 8 and 9 in genomic DNA was identified in one of the two ES clones by Southern Blot and PCR. CONCLUSION:FGFR2 conditional knockout depending on Cre-loxP can be successfully used in ES cells.

背景与目标： 目的：探讨成纤维细胞生长因子受体2（FGFR2）在不同分化阶段的功能。根据Cre-loxP，开发了具有条件敲除FGFR2的工程鼠胚胎干（ES）细胞。
方法：将Cre-loxP系统用于条件靶向载体。表达Cre重组酶的感受态AM-1细菌用于确认Cre介导的FGFR2外显子7和8的缺失。将靶向载体电穿孔到ES细胞中，并用G418和更昔洛韦筛选转染的ES细胞。最后，通过Southern印迹和PCR鉴定具有正确靶向事件的ES克隆。
结果：成功构建了有条件敲除鼠FGFR2的靶向载体，并通过PCR和酶切分析鉴定。通过用G418和更昔洛韦选择性培养收集了86个ES克隆。发现86个ES克隆中有四个在基因组DNA中包含靶向基因序列，该DNA由Southern Blot用5'端侧翼探针证实。四个ES克隆中的两个具有正确的靶向事件，包括在基因组DNA中插入了靶向基因序列，并使用3'端侧翼探针通过Southern Blot检测。最后，通过Southern印迹和PCR在两个ES克隆之一中鉴定出loxP（loxP3）在基因组DNA的外显子8和9之间的插入。
结论：依赖于Cre-loxP的FGFR2条件敲除可成功用于ES细胞。