BACKGROUND & AIMS: :Trace eyeblink conditioning is used as a translational model of declarative memory but restricted to the temporal domain. Potential spatial aspects have never been experimentally addressed. We employed a spatiotemporal trace eyeblink conditioning paradigm in which a spatial dimension (application side of the unconditioned stimulus) was differentially coded by tone frequency of the conditioned stimulus and recorded conditioned reactions from both eyes. We found more and stronger conditioned reactions at the side predicted by the conditioned stimulus but only in aware participants. Thus, spatial effects are present in trace eyeblink conditioning and may be differentially conditioned depending on the awareness about the spatial relation between conditioned and unconditioned stimulus.

背景与目标： ：Trace眨眼条件用作声明性记忆的翻译模型，但仅限于时域。潜在的空间方面从未实验性地解决过。我们采用了时空迹线眨眼条件范式，其中空间维度（未条件刺激的应用侧）通过条件刺激的音调频率进行差分编码，并记录了两只眼睛的条件反应。我们在条件刺激所预测的一侧发现了更多且更强的条件反应，但仅在有意识的参与者中。因此，空间效应存在于微量眨眼条件中，并且可以根据对条件刺激与非条件刺激之间的空间关系的了解而有区别地进行调节。

BACKGROUND & AIMS: :A nested PCR assay was employed to detect the presence of phytoplasmas in 127 blueberry plants exhibiting typical or a portion of blueberry stunt (BBS) syndrome collected in 2010 and 2011, from 11 commercial farms predominantly located in two counties in New Jersey, USA. Ninety plants exhibiting typical stunt syndrome tested positive for phytoplasma infection. Restriction fragment length polymorphism (RFLP) analysis indicated that two distinct phytoplasmas were associated with BBS-diseased plants. About 95% of phytoplasmas detected were very closely related to BBS phytoplasma strains BBS3-AR (subgroup 16SrI-E) and BBS1-MI (unidentified) identified previously, and 4.4% of phytoplasmas detected belonged to the pigeon pea witches'-broom phytoplasma group (16SrIX). Sequence and phylogenetic analysis of cloned 16S rDNA further indicated the subgroup 16SrI-E related phytoplasmas represented a variant of 16SrI-E reference strain BBS3-AR, while the 16SrIX related phytoplasmas were closely related to juniper witches'-broom (JunWB) phytoplasma (16SrIX-E), representing a 16SrIX-E variant. Ribosomal protein (rp) and secY gene-based phylogenies revealed that BBS3-AR and BBS-NJ 16SrI-E strains belonged to a closely related lineage, while BBS-NJ 16SrIX-E strains and JunWB strains represented two distinct lineages. Single nucleotide polymorphisms (SNPs) analyses of rp and secY gene sequences further revealed that no specific rp gene SNPs and only two specific secY gene SNPS were present between BBS-NJ 16SrI-E strains and BBS3-AR. In contrast, BBS-NJ 16SrIX-E strains/clones had 15 consensus rp SNPs and 28 consensus secY SNPs that separated them from JunWB strains/clones. For the first time, two distinct phytoplasmas that cause BBS-disease in the U.S. was revealed.

背景与目标： ：采用巢式PCR分析法检测了2010年和2011年收集的127株表现出典型或部分蓝莓特技（BBS）综合征的蓝莓植物中的植物浆原体，这些植物主要分布于美国新泽西州的两个县中的11个商业农场。表现出典型特技综合症的九十株植物检测出的植物浆体感染呈阳性。限制性片段长度多态性（RFLP）分析表明，两个不同的植物质原体与BBS病害的植物有关。之前鉴定出的约95％的植物原质与BBS植物原虫菌株BBS3-AR（亚组16SrI-E）和BBS1-MI（未鉴定）密切相关，而检出的植物原质中有4.4％属于木豆巫婆扫帚植物原质组。 （16SrIX）。克隆的16S rDNA的序列和系统发育分析进一步表明，亚组16SrI-E相关的植物质浆体代表16SrI-E参考菌株BBS3-AR的变体，而16SrIX相关的植物质体与杜松witch帚（JunWB）植物质体（16SrIX -E），代表16SrIX-E变体。核糖体蛋白（rp）和secY基因为基础的系统发育研究表明BBS3-AR和BBS-NJ 16SrI-E菌株属于密切相关的血统，而BBS-NJ 16SrIX-E菌株和JunWB菌株则代表两个不同的血统。 rp和secY基因序列的单核苷酸多态性（SNPs）分析进一步表明，在BBS-NJ 16SrI-E菌株和BBS3-AR之间不存在特定的rp基因SNP，而仅存在两个特定的secY基因SNPS。相反，BBS-NJ 16SrIX-E菌株/克隆具有15个共有rp SNP和28个共有secY SNP，将它们与JunWB菌株/克隆分开。首次揭示了在美国引起BBS疾病的两种不同的植物原质。

BACKGROUND & AIMS: :Satellite cells are skeletal muscle stem cells that provide myonuclei for postnatal muscle growth, maintenance, and repair/regeneration in adults. Normally, satellite cells are mitotically quiescent, but they are activated in response to muscle injury, in which case they proliferate extensively and exhibit up-regulated expression of the transcription factor MyoD, a master regulator of myogenesis. MyoD forms a heterodimer with E proteins through their basic helix-loop-helix domain, binds to E boxes in the genome and thereby activates transcription at muscle-specific promoters. The central role of MyoD in muscle differentiation has increased interest in finding potential MyoD regulators. Here we identified transducin-like enhancer of split (TLE3), one of the Groucho/TLE family members, as a regulator of MyoD function during myogenesis. TLE3 was expressed in activated and proliferative satellite cells in which increased TLE3 levels suppressed myogenic differentiation, and, conversely, reduced TLE3 levels promoted myogenesis with a concomitant increase in proliferation. We found that, via its glutamine- and serine/proline-rich domains, TLE3 interferes with MyoD function by disrupting the association between the basic helix-loop-helix domain of MyoD and E proteins. Our findings indicate that TLE3 participates in skeletal muscle homeostasis by dampening satellite cell differentiation via repression of MyoD transcriptional activity.

背景与目标： 卫星细胞是骨骼肌干细胞，可为成年后的肌肉生长，维持和修复/再生提供肌核。通常，卫星细胞是有丝分裂的静止状态，但是它们会响应肌肉损伤而被激活，在这种情况下，它们会大量增殖并表现出转录因子MyoD（肌生成的主要调控因子）的表达上调。 MyoD通过其基本的螺旋-环-螺旋结构域与E蛋白形成异二聚体，与基因组中的E box结合，从而激活肌肉特异性启动子处的转录。 MyoD在肌肉分化中的核心作用增加了寻找潜在的MyoD调节剂的兴趣。在这里，我们确定了Groucho / TLE家族成员之一的分裂（duc3）的转导蛋白样增强子，作为肌生成过程中MyoD功能的调节剂。 TLE3在活化的和增殖的卫星细胞中表达，其中升高的TLE3水平抑制了肌原性分化，相反，降低的TLE3水平促进了肌发生，同时伴随着增殖的增加。我们发现，TLE3通过其富含谷氨酰胺和丝氨酸/脯氨酸的结构域，通过破坏MyoD的基本螺旋-环-螺旋结构域与E蛋白之间的关联来干扰MyoD功能。我们的发现表明，TLE3通过抑制MyoD转录活性，通过抑制卫星细胞分化来参与骨骼肌稳态。

BACKGROUND & AIMS: :Several recent studies have shown that amphibian populations may exhibit high genetic subdivision in areas with recent fragmentation and urban development. Less is known about the potential for genetic differentiation in continuous habitats. We studied genetic differentiation of red-backed salamanders (Plethodon cinereus) across a 2-km transect through continuous forest in Virginia, USA. Mark-recapture studies suggest very little dispersal for this species, whereas homing experiments and post-Pleistocene range expansion both suggest greater dispersal abilities. We used six microsatellite loci to examine genetic population structure and differentiation between eight subpopulations of red-backed salamanders at distances from 200 m to 2 km. We also used several methods to extrapolate dispersal frequencies and test for sex-biased dispersal. We found small, but detectable differentiation among populations, even at distances as small as 200 m. Differentiation was closely correlated with distance and both Mantel tests and assignment tests were consistent with an isolation-by-distance model for the population. Extrapolations of intergenerational variance in spatial position (sigma(2)<15 m(2)) and pair-wise dispersal frequencies (4 Nm < 25 for plots separated by 300 m) both suggest limited gene flow. Additionally, tests for sex-biased dispersal imply that dispersal frequency is similarly low for both sexes. We suggest that these low levels of gene flow and the infrequent dispersal observed in mark-recapture studies may be reconciled with homing ability and range expansion if dispersing animals rarely succeed in breeding in saturated habitats, if dispersal is flexible depending on the availability of habitat, or if dispersal frequency varies across the geographic range of red-backed salamanders.

背景与目标： ：最近的几项研究表明，两栖动物种群在具有最近的支离破碎和城市发展的地区可能表现出较高的遗传细分。关于连续生境中遗传分化的潜力知之甚少。我们研究了红背sal（Plethodon cinereus）通过美国弗吉尼亚州连续森林跨越2公里的样带的遗传分化。标记捕获研究表明，该物种的扩散能力很小，而归巢实验和更新世后的范围扩展都表明具有较强的扩散能力。我们使用六个微卫星基因座来研究遗传种群的结构以及在200 m至2 km的距离上的红背sal的八个亚群之间的分化。我们还使用了几种方法来推断散布频率并测试性别偏向的散布。我们发现，即使在小至200 m的距离上，种群之间的差异也很小，但可检测到。分化与距离密切相关，Mantel检验和分配检验均与总体的按距离隔离模型一致。空间位置的代际方差的外推法（sigma（2）<15 m（2））和成对的散布频率（4 Nm <25对于由300 m分隔的地块）都表明基因流有限。此外，针对性别偏见的分散测试表明，男女的分散频率同样较低。我们建议，如果分散的动物很少能在饱和的栖息地中成功繁殖，那么这种低水平的基因流动和在标记捕获研究中观察到的不频繁的分散可能与归巢能力和范围扩展相一致；如果分散能够灵活地取决于栖息地的可用性，或分散频率在红背sal的地理范围内变化。

BACKGROUND & AIMS: :Class-switch recombination (CSR) is essential for humoral immunity. However, the regulation of CSR is not completely understood. Here we demonstrate that phosphatidylinositol 3-kinase (PI3K) actively suppressed the onset and frequency of CSR in primary B cells. Consistently, mice lacking the lipid phosphatase, PTEN, in B cells exhibited a hyper-IgM condition due to impaired CSR, which could be restored in vitro by specific inhibition of PI3Kdelta. Inhibition of CSR by PI3K was partially dependent on the transcription factor, BLIMP1, linking plasma cell commitment and cessation of CSR. PI3K-dependent activation of the serine-threonine kinase, Akt, suppressed CSR, in part, through the inactivation of the Forkhead Box family (Foxo) of transcription factors. Reduced PI3K signaling enhanced the expression of AID (activation-induced cytidine deaminase) and accelerated CSR. However, ectopic expression of AID could not fully overcome inhibition of CSR by PI3K, suggesting that PI3K regulates both the expression and function of AID.

背景与目标： ：Class-switch recombination（CSR）对于体液免疫至关重要。但是，关于企业社会责任的规定尚不完全清楚。在这里，我们证明磷脂酰肌醇3-激酶（PI3K）积极抑制原代B细胞中CSR的发生和频率。一致地，由于CSR受损，B细胞中缺乏脂质磷酸酶PTEN的小鼠表现出高IgM病状，可通过特异性抑制PI3Kdelta使其在体外恢复。 PI3K对CSR的抑制部分取决于转录因子BLIMP1，这与浆细胞的定型和CSR的终止有关。丝氨酸-苏氨酸激酶Akt的PI3K依赖性激活部分地通过使转录因子的Forkhead Box家族（Foxo）失活而抑制了CSR。减少的PI3K信号传导增强了AID（激活诱导的胞苷脱氨酶）的表达并加速了CSR。然而，AID的异位表达不能完全克服PI3K对CSR的抑制作用，这表明PI3K既可以调节AID的表达又可以调节AID的功能。

BACKGROUND & AIMS: :Control of self-renewal and differentiation of human ES cells (hESCs) remains a challenge. This is largely due to the use of culture systems that involve poorly defined animal products and do not mimic the normal developmental milieu. Routine protocols involve the propagation of hESCs on mouse fibroblast or human feeder layers, enzymatic cell removal, and spontaneous differentiation in cultures of embryoid bodies, and each of these steps involves significant variability of culture conditions. We report that a completely synthetic hydrogel matrix can support (i) long-term self-renewal of hESCs in the presence of conditioned medium from mouse embryonic fibroblast feeder layers, and (ii) direct cell differentiation. Hyaluronic acid (HA) hydrogels were selected because of the role of HA in early development and feeder layer cultures of hESCs and the controllability of hydrogel architecture, mechanics, and degradation. When encapsulated in 3D HA hydrogels (but not within other hydrogels or in monolayer cultures on HA), hESCs maintained their undifferentiated state, preserved their normal karyotype, and maintained their full differentiation capacity as indicated by embryoid body formation. Differentiation could be induced within the same hydrogel by simply altering soluble factors. We therefore propose that HA hydrogels, with their developmentally relevant composition and tunable physical properties, provide a unique microenvironment for the self-renewal and differentiation of hESCs.

背景与目标： ：控制人类ES细胞（hESCs）的自我更新和分化仍然是一个挑战。这主要是由于使用了涉及定义不明确的动物产品且不模仿正常发育环境的培养系统。常规方案涉及hESC在小鼠成纤维细胞或人饲养层上的繁殖，酶细胞的去除以及胚状体培养物中的自发分化，并且这些步骤中的每一个都涉及培养条件的显着可变性。我们报告完全合成的水凝胶基质可以支持（i）从小鼠胚胎成纤维细胞饲养层的条件培养基存在下hESCs的长期自我更新，和（ii）直接细胞分化。选择透明质酸（HA）水凝胶是因为HA在hESC的早期发育和饲养层培养中的作用以及水凝胶结构，力学和降解的可控性。当封装在3D HA水凝胶中（但不在其他水凝胶中或在HA的单层培养物中）时，hESC保持其未分化状态，保留其正常核型，并保持其完整的分化能力（如胚状体形成所示）。只需改变可溶性因子，即可在同一水凝胶中诱导分化。因此，我们建议HA水凝胶具有与发育相关的组成和可调节的物理特性，可为hESCs的自我更新和分化提供独特的微环境。

BACKGROUND & AIMS: :Unc119 is an adaptor protein that is involved in the development of the vertebrate nervous system. We have shown that Unc119 stimulates the induction of alpha-smooth muscle actin (alpha-SMA) and myofibroblast differentiation by TGF-beta in human lung fibroblasts. Unc119 increases the kinase activity of Fyn and associates with it in coprecipitation and colocalization studies. Phosphorylation and activation of Fyn in response to TGF-beta and platelet-derived growth factor is delayed in Unc119-deficient cells. This delay translates into suppressed cell proliferation. In Src family kinase-deficient (SYF) cells, Unc119 knockdown does not affect cell proliferation. The result suggests that Unc119 interacts with Fyn in the early stages of signal generation and its presence is essential for conducive signal transduction. Unc119 overexpression does not stimulate alpha-SMA in SYF cells and this defect is restored upon reconstitution with Fyn indicating that Unc119 stimulation of alpha-SMA requires at least Fyn. Unc119 overexpression stimulated p38, but not JNK, phosphorylation. Blocking p38 MAPK resulted in reduced alpha-SMA expression by Unc119 suggesting that the p38 pathway regulates Unc119-induced myofibroblast differentiation. Unc119 stimulates the production of TGF-beta and IL-6, known inducers of myofibroblast differentiation. Thus, Unc119 regulates receptor-mediated signal transduction and myofibroblast differentiation by activating Fyn and the p38 MAPK pathway. Using primary lung fibroblasts from patients with fibrotic lung diseases and control subjects, we show that the expression of alpha-smooth muscle actin is highly correlated with that of Unc119. Taken together, our results suggest that Unc119 plays an important role in fibrotic processes through myofibroblast differentiation.

背景与目标： ：Unc119是与脊椎动物神经系统发育有关的衔接蛋白。我们已经显示Unc119刺激人肺成纤维细胞中TGF-β诱导α平滑肌肌动蛋白（α-SMA）和成肌纤维细胞分化。 Unc119增加Fyn的激酶活性，并在共沉淀和共定位研究中与其关联。 Unc119缺陷细胞中，响应于TGF-β和血小板衍生的生长因子的Fyn的磷酸化和激活被延迟。这种延迟转化为抑制的细胞增殖。在Src家族激酶缺陷（SYF）细胞中，Unc119敲低不影响细胞增殖。结果表明，Unc119在信号生成的早期阶段与Fyn相互作用，并且它的存在对于有益的信号转导至关重要。 Unc119的过表达不会刺激SYF细胞中的α-SMA，并且用Fyn重建后该缺陷得以恢复，这表明Unc119刺激α-SMA至少需要Fyn。 Unc119过表达刺激p38磷酸化，但不刺激JNK磷酸化。阻断p38 MAPK导致Unc119降低α-SMA表达，表明p38途径调节Unc119诱导的成肌纤维细胞分化。 Unc119刺激成纤维细胞分化的已知诱导物TGF-β和IL-6的产生。因此，Unc119通过激活Fyn和p38 MAPK通路来调节受体介导的信号转导和成纤维细胞分化。使用来自纤维化性肺病患者和对照对象的原发性肺成纤维细胞，我们显示α-平滑肌肌动蛋白的表达与Unc119的表达高度相关。综上所述，我们的结果表明Unc119在通过成肌纤维细胞分化的纤维化过程中起着重要作用。

BACKGROUND & AIMS: :Recent studies have suggested that wild-type p53 blocks cell cycle progression near the G1-S boundary and is involved in both differentiation and apoptosis in many types of cells including cancer cells. p53 expression is enhanced upon DNA-damaging apoptotic stimuli while Fas/Apo-1, a member of the tumor necrosis factor receptor family expressed on cell surface, transduces a signal for apoptosis upon specific ligand or antibody engagement. We demonstrated that stable transfection of the wild-type p53 gene under the control of CMV promoter induced differentiation and apoptosis under restricted conditions in cancer cells, and often caused sensitization of p53-transfected cells to Fas/Apo-1 signal. To investigate the interaction between two major apoptotic pathways involving p53 and Fas/Apo-1 we have established a system that allows to induce wild-type p53 overexpression and apoptosis in cancer cells upon treatment with anti-Fas antibody. The system also allows to investigate other factors interacting with p53 and Fas/Apo-1, and should provide a clue to understanding the biological and biochemical aspects of apoptosis.

背景与目标： ：最近的研究表明，野生型p53阻断了G1-S边界附近的细胞周期进程，并参与了包括癌细胞在内的许多类型细胞的分化和凋亡。 DNA损伤凋亡刺激后p53的表达增强，而Fas / Apo-1（在细胞表面表达的肿瘤坏死因子受体家族的成员）在特异性配体或抗体参与时转导凋亡信号。我们证明了在CMV启动子的控制下，野生型p53基因的稳定转染在癌细胞的限制性条件下诱导了分化和凋亡，并经常引起p53转染的细胞对Fas / Apo-1信号的敏感性。为了研究涉及p53和Fas / Apo-1的两个主要凋亡途径之间的相互作用，我们建立了一个系统，该系统可在用抗Fas抗体治疗后诱导癌细胞中野生型p53过表达和凋亡。该系统还允许研究与p53和Fas / Apo-1相互作用的其他因素，并应为了解凋亡的生物学和生化方面提供线索。

BACKGROUND & AIMS: :Deregulated epigenetic mechanisms are likely involved in the pathogenesis of myelodysplastic syndromes (MDSs). Which genes are silenced by aberrant promotor methylation during MDS hematopoiesis has not been equivalently investigated. Using an in vitro differentiation model of human hematopoiesis, we generated defined differentiation stages (day 0, day 4, day 7, day 11) of erythro-, thrombo- and granulopoiesis from 13 MDS patients and seven healthy donors. Promotor methylation analysis of key regulatory genes involved in cell cycle control (p14, p15, p16, CHK2), DNA repair (hMLH1), apoptosis (p73, survivin, DAPK), and differentiation (RARb, WT1) was performed by methylation-specific polymerase chain reaction. Corresponding gene expression was analyzed by microarray (Affymetrix, HG-U133A). We provide evidence that p16, survivin, CHK2, and WT1 are affected by promotor hypermethylation in MDSs displaying a selective International Prognostic Scoring System risk association. A methylation-associated mRNA downregulation for specific hematopoietic lineages and differentiation stages is demonstrated for survivin, CHK2, and WT1. We identified a suppressed survivin mRNA expression in methylated samples during erythropoiesis, whereas WT1 and CHK2 methylation-related reduction of mRNA expression was found during granulopoiesis in all MDS risk types. Our data suggest that lineage-specific methylation-associated gene silencing of survivin, CHK2, and WT1 in MDS hematopoietic precursor cells may contribute to the MDS-specific phenotype

背景与目标： ：表观遗传机制失调可能与骨髓增生异常综合症（MDSs）的发病机制有关。尚未对MDS造血过程中异常启动子甲基化沉默哪些基因进行了研究。使用人类造血作用的体外分化模型，我们从13名MDS患者和7位健康供体中生成了定义的分化阶段（第0天，第4天，第7天，第11天）。通过甲基化特异性的方法对参与细胞周期控制（p14，p15，p16，CHK2），DNA修复（hMLH1），凋亡（p73，survivin，DAPK）和分化（RARb，WT1）的关键调控基因的启动子甲基化分析聚合酶链反应。通过微阵列（Affymetrix，HG-U133A）分析相应的基因表达。我们提供的证据表明p16，survivin，CHK2和WT1受MDS中启动子过甲基化的影响，显示出选择性的国际预后评分系统风险关联。针对survivin，CHK2和WT1证实了特定造血谱系和分化阶段的甲基化相关mRNA下调。我们在红细胞生成过程中发现甲基化样本中survivin mRNA表达受到抑制，而在所有MDS风险类型的粒细胞生成过程中，发现了WT1和CHK2甲基化相关的mRNA表达下降。我们的数据表明，MDS造血前体细胞中survivin，CHK2和WT1的谱系特异性甲基化相关基因沉默可能有助于MDS特异性表型

BACKGROUND & AIMS: :We reported previously that onset of oligodendrocyte precursor cell (OPC) differentiation is accompanied by an increase in intracellular pH (pH(i)). We show that OPC differentiation is dependent primarily on a permissive pH(i) value. The highest differentiation levels were observed for pH(i) values around 7.15 and inhibition of differentiation was observed at slightly more acidic or alkaline values. Clamping the pH(i) of OPCs at 7.15 caused a transient activation of ERK1/2 that was not observed at more acidic or alkaline values. Furthermore, inhibition of ERK activation with the UO126 compound totally prevented OPC differentiation in response to pH(i) shift. These results indicate that pH(i), acting through the ERK1/2 pathway, is a key determinant for oligodendrocyte differentiation. We also show that this pH(i) pathway is involved in the process of retinoic acid-induced OPC differentiation.

背景与目标： ：我们之前报道过，少突胶质前体细胞（OPC）分化的开始伴随着细胞内pH值（pH（i））的增加。我们表明，OPC的差异主要取决于允许的pH（i）值。对于pH（i）值在7.15附近观察到最高的分化水平，在酸性或碱性稍高的情况下观察到分化的抑制作用。将OPC的pH（i）固定在7.15会导致ERK1 / 2的瞬时活化，而在更多的酸性或碱性条件下则无法观察到。此外，用UO126化合物抑制ERK活化完全阻止了响应pH（i）移位的OPC分化。这些结果表明，通过ERK1 / 2途径起作用的pH（i）是少突胶质细胞分化的关键决定因素。我们还表明，此pH（i）通路参与视黄酸诱导的OPC分化过程中。

BACKGROUND & AIMS: In the present study, we examine rod photoreceptor development in dissociated-cell cultures of neonatal mouse retina. We show that, although very few rhodopsin+ rods develop in the presence of 10% foetal calf serum (FCS), large numbers develop in the absence of serum, but only if the cell density in the cultures is high. The rods all develop from nondividing rhodopsin- cells, and new rods continue to develop from rhodopsin- cells for at least 6-8 days, indicating that there can be a long delay between when a precursor cell withdraws from the cell cycle and when it becomes a rhodopsin+ rod. We show that FCS arrests rod development in these cultures at a postmitotic, rhodopsin-, pre-rod stage. We present evidence that FCS acts indirectly by stimulating the proliferation of Müller cells, which arrest rod differentiation by releasing leukaemia inhibitory factor (LIF). These findings identify an inhibitory cell-cell interaction, which may help to explain the long delay that can occur both in vitro and in vivo between cell-cycle withdrawal and rhodopsin expression during rod development.

背景与目标： 在本研究中，我们检查了新生小鼠视网膜的游离细胞培养中杆感光细胞的发育。我们显示，尽管在10％的胎牛血清（FCS）存在的情况下，视紫红质棒很少，但在无血清的情况下会形成大量视紫红质棒，但前提是培养物中的细胞密度很高。视杆全部从不分裂的视紫红质细胞发育而来，新视杆继续从视紫红质细胞发育至少6-8天，这表明从前体细胞退出细胞周期到其退出细胞周期之间可能会有很长的延迟。视紫红质棒。我们显示，FCS在有丝分裂后，视紫红质，杆前阶段逮捕这些文化中的杆发育。我们提供的证据表明，FCS通过刺激Müller细胞的增殖而间接起作用，Müller细胞通过释放白血病抑制因子（LIF）阻止杆分化。这些发现确定了抑制性的细胞间相互作用，这可能有助于解释杆发育过程中细胞周期停药和视紫红质表达之间可能在体外和体内发生的长时间延迟。

BACKGROUND & AIMS: :The transcription factor Evi1 has an outstanding role in the formation and transformation of hematopoietic cells. Its activation by chromosomal rearrangement induces a myelodysplastic syndrome with progression to acute myeloid leukemia of poor prognosis. Similarly, retroviral insertion-mediated upregulation confers a competitive advantage to transplanted hematopoietic cells, triggering clonal dominance or even leukemia. To study the molecular and functional response of primary murine hematopoietic progenitor cells to the activation of Evi1, we established an inducible lentiviral expression system. EVI1 had a biphasic effect with initial growth inhibition and retarded myeloid differentiation linked to enhanced survival of myeloblasts in long-term cultures. Gene expression microarray analysis revealed that within 24 h EVI1 upregulated 'stemness' genes characteristic for long-term hematopoietic stem cells (Aldh1a1, Abca1, Cdkn1b, Cdkn1c, Epcam, among others) but downregulated genes involved in DNA replication (Cyclins and their kinases, among others) and DNA repair (including Brca1, Brca2, Rad51). Cell cycle analysis demonstrated EVI1's anti-proliferative effect to be strictly dose-dependent with accumulation of cells in G0/G1, but preservation of a small fraction of long-term proliferating cells. Although confined to cultured cells, our study contributes to new hypotheses addressing the mechanisms and molecular targets involved in preleukemic clonal dominance or leukemic transformation by Evi1.

背景与目标： ：转录因子Evi1在造血细胞的形成和转化中具有杰出的作用。其通过染色体重排的激活诱导骨髓增生异常综合症，并发展为预后较差的急性髓细胞性白血病。同样，逆转录病毒插入介导的上调赋予移植的造血细胞竞争优势，引发克隆优势甚至白血病。为了研究原代小鼠造血祖细胞对Evi1激活的分子和功能反应，我们建立了可诱导的慢病毒表达系统。 EVI1具有双相作用，具有初始生长抑制作用，并且延缓了骨髓分化，与长期培养中成纤维细胞的存活率提高有关。基因表达芯片分析表明，EVI1在24h内上调了长期造血干细胞（Aldh1a1，Abca1，Cdkn1b，Cdkn1c，Epcam等）特有的“茎”基因，但下调了与DNA复制有关的基因（Cyclins及其激酶，以及DNA修复（包括Brca1，Brca2，Rad51）。细胞周期分析表明，EVI1的抗增殖作用与G0 / G1中的细胞积累严格地呈剂量依赖性，但保留了一小部分长期增殖的细胞。尽管仅限于培养的细胞，但我们的研究为解决Evi1在白血病前克隆优势或白血病转化中涉及的机制和分子靶点的新假设做出了贡献。

BACKGROUND & AIMS: :Canine distemper virus (CDV) is the cause of a severe and highly contagious disease in dogs. Practical diagnosis of canine distemper based on clinical signs and laboratory tests are required to confirm CDV infection. The present study aimed to develop a molecular assay to detect and differentiate field and vaccine CDV strains. Reverse transcription followed by nested real time polymerase chain reaction (RT-nqPCR) was developed, which exhibited analytical specificity (all the samples from healthy dogs and other canine infectious agents were not incorrectly detected) and sensitivity (all replicates of a vaccine strain were positive up to the 3125-fold dilution - 10(0.7) TCID50). RT-nqPCR was validated for CDV detection on different clinical samples (blood, urine, rectal and conjunctival swabs) of 103 animals suspected to have distemper. A total of 53 animals were found to be positive based on RT-nqPCR in at least one clinical sample. Blood resulted in more positive samples (50 out of 53, 94.3%), followed by urine (44/53, 83.0%), rectal (38/53, 71%) and conjunctival (27/53, 50.9%) swabs. A commercial immunochromatography (IC) assay had detected CDV in only 30 conjunctival samples of these positive dogs. Nucleoprotein (NC) gene sequencing of 25 samples demonstrated that 23 of them were closer to other Brazilian field strains and the remaining two to vaccine strains. A single nucleotide sequences difference, which creates an Msp I restriction enzyme digestion, was used to differentiate between field and vaccine CDV strains by restriction fragment length polymorphism (RFLP) analysis. The complete assay was more sensitive than was IC for the detection of CDV. Blood was the more frequently positive specimen and the addition of a restriction enzyme step allowed the differentiation of vaccine and Brazilian field strains.

背景与目标： ：犬瘟热病毒（CDV）是导致犬种严重和高度传染性疾病的原因。需要根据临床体征和实验室检查对犬瘟热进行实际诊断，以确认CDV感染。本研究旨在开发一种分子检测方法，以检测和区分野外和疫苗的CDV毒株。开发了逆转录，随后进行了巢式实时聚合酶链反应（RT-nqPCR），该方法具有分析特异性（未正确检测出健康犬和其他犬类传染病菌的所有样品）和灵敏度（疫苗株的所有重复均为阳性）稀释至3125倍-10（0.7）TCID50）。验证RT-nqPCR可在103例怀疑患有脾热的动物的不同临床样品（血液，尿液，直肠和结膜拭子）上进行CDV检测。基于RT-nqPCR，在至少一个临床样品中发现总共53只动物是阳性的。血液产生更多阳性样本（53个中有50个，占94.3％），其次是尿液（44 / 53，83.0％），直肠（38 / 53，71％）和结膜（27 / 53，50.9％）拭子。商业免疫色谱法（IC）分析仅在这些阳性犬的30个结膜样本中检测到CDV。对25个样品进行的核蛋白（NC）基因测序表明，其中23个更接近其他巴西田间毒株，其余两个更接近疫苗株。通过限制性片段长度多态性（RFLP）分析，使用产生Msp I限制性酶消化的单核苷酸序列差异来区分田间和疫苗CDV菌株。完整的检测方法比IC检测CDV更为灵敏。血液是更常见的阳性标本，加上限制性内切酶步骤可以区分疫苗和巴西田间菌株。

BACKGROUND & AIMS: :Skeletal muscle injury and regeneration are closely associated with an inflammatory reaction that is usually characterized by sequential recruitment of neutrophils and monocytes or macrophages. Selective macrophage depletion models have shown that macrophages are essential for complete regeneration of muscle fibers after freeze injuries, toxin injuries, ischemia-reperfusion, and hindlimb unloading and reloading. Although there is growing evidence that macrophages possess major myogenic capacities, it is not known whether the positive effects of macrophages can be optimized to stimulate muscle regrowth. We used in vivo and in vitro mouse models of atrophy to investigate the effects of stimulating macrophages with macrophage colony-stimulating factor (M-CSF) on muscle regrowth. When atrophied soleus muscles were injected intramuscularly with M-CSF, we observed a 1.6-fold increase in macrophage density and a faster recovery in muscle force (20%), combined with an increase in muscle fiber diameter (10%), after 7 days of reloading, compared with PBS-injected soleus muscles. Furthermore, coculture of atrophied myotubes with or without bone marrow-derived macrophages (BMDM) and/or M-CSF revealed that the combination of BMDMs and M-CSF was required to promote myotube growth (15%). More specifically, M-CSF promoted the anti-inflammatory macrophage phenotype, which in turn decreased protein degradation and MuRF-1 expression by 25% in growing myotubes. These results indicate that specific macrophage subsets can be stimulated to promote muscle cell regrowth after atrophy.

背景与目标： ：骨骼肌的损伤和再生与炎症反应密切相关，炎症反应通常以嗜中性粒细胞和单核细胞或巨噬细胞的顺序募集为特征。选择性巨噬细胞耗竭模型显示，巨噬细胞对于冷冻损伤，毒素损伤，局部缺血-再灌注以及后肢卸载和再加载后肌肉纤维的完全再生至关重要。尽管越来越多的证据表明巨噬细胞具有主要的生肌能力，但尚不清楚是否可以优化巨噬细胞的积极作用来刺激肌肉再生。我们在体内和体外萎缩小鼠模型中研究了巨噬细胞集落刺激因子（M-CSF）刺激巨噬细胞对肌肉再生的影响。当肌肉萎缩的比目鱼肌肌肉内注射M-CSF时，我们观察到7天后巨噬细胞密度增加了1.6倍，肌肉力量恢复更快（20％），同时肌肉纤维直径增加了（10％）与注射PBS的比目鱼肌相比，此外，有或没有骨髓源性巨噬细胞（BMDM）和/或M-CSF的萎缩肌管的共培养显示，BMDM和M-CSF的组合需要促进肌管生长（15％）。更具体地说，M-CSF促进了抗炎巨噬细胞的表型，从而使生长中的肌管中的蛋白质降解和MuRF-1表达降低了25％。这些结果表明，萎缩后可以刺激特定的巨噬细胞亚群以促进肌肉细胞的再生。

BACKGROUND & AIMS: :Lymphedema is a dreaded complication of cancer treatment. However, despite the fact that >5 million Americans are affected by this disorder, the development of effective treatments is limited by the fact that the pathology of lymphedema remains unknown. The purpose of these studies was to determine the role of inflammatory responses in lymphedema pathology. Using mouse models of lymphedema, as well as clinical lymphedema specimens, we show that lymphatic stasis results in a CD4 T-cell inflammation and T-helper 2 (Th2) differentiation. Using mice deficient in T cells or CD4 cells, we show that this inflammatory response is necessary for the pathological changes of lymphedema, including fibrosis, adipose deposition, and lymphatic dysfunction. Further, we show that inhibition of Th2 differentiation using interleukin-4 (IL-4) or IL-13 blockade prevents initiation and progression of lymphedema by decreasing tissue fibrosis and significantly improving lymphatic function, independent of lymphangiogenic growth factors. We show that CD4 inflammation is a critical regulator of tissue fibrosis and lymphatic dysfunction in lymphedema and that inhibition of Th2 differentiation markedly improves lymphatic function independent of lymphangiogenic cytokine expression. Notably, preventing and/or reversing the development of pathological tissue changes that occur in lymphedema may be a viable treatment strategy for this disorder.

背景与目标： ：淋巴水肿是一种可怕的癌症治疗并发症。然而，尽管事实上有超过500万美国人受到这种疾病的影响，但淋巴水肿的病理学仍然未知，这限制了有效疗法的发展。这些研究的目的是确定炎症反应在淋巴水肿病理中的作用。使用小鼠模型的淋巴水肿，以及临床淋巴水肿标本，我们显示淋巴淤积导致CD4 T细胞炎症和T辅助2（Th2）分化。使用缺乏T细胞或CD4细胞的小鼠，我们表明这种炎症反应对于淋巴水肿的病理变化（包括纤维化，脂肪沉积和淋巴功能障碍）是必需的。此外，我们显示，使用白介素4（IL-4）或IL-13阻滞抑制Th2分化可通过减少组织纤维化并显着改善淋巴功能来防止淋巴水肿的发生和进展，而与淋巴管生成生长因子无关。我们显示，CD4炎症是淋巴水肿中组织纤维化和淋巴功能障碍的关键调节器，抑制Th2分化显着提高了淋巴功能，而与淋巴管生成细胞因子的表达无关。值得注意的是，预防和/或逆转在淋巴水肿中发生的病理组织改变的发展可能是该疾病的可行治疗策略。