CTCF mediates chromatin looping via N-terminal domain-dependent cohesin retention

The DNA-binding protein CCCTC-binding factor (CTCF) and the cohesin complex function together to shape chromatin architecture in mammalian cells, but the molecular details of this process remain unclear. Here, we demonstrate that a 79-aa region within the CTCF N terminus is essential for cohesin positioning at CTCF binding sites and chromatin loop formation. However, the N terminus of CTCF fused to artificial zinc fingers was not sufficient to redirect cohesin to non-CTCF binding sites, indicating a lack of an autonomously functioning domain in CTCF responsible for cohesin positioning. BORIS (CTCFL), a germline-specific paralog of CTCF, was unable to anchor cohesin to CTCF DNA binding sites. Furthermore, CTCF-BORIS chimeric constructs provided evidence that, besides the N terminus of CTCF, the first two CTCF zinc fingers, and likely the 3D geometry of CTCF-DNA complexes, are also involved in cohesin retention. Based on this knowledge, we were able to convert BORIS into CTCF with respect to cohesin positioning, thus providing additional molecular details of the ability of CTCF to retain cohesin. Taken together, our data provide insight into the process by which DNA-bound CTCF constrains cohesin movement to shape spatiotemporal genome organization

Pharmacokinetic characteristics and anticancer effects of 5-Fluorouracil loaded nanoparticles

<p>Abstract</p> <p>Background</p> <p>It is expected that prolonged circulation of anticancer drugs will increase their anticancer activity while decreasing their toxic side effects. The purpose of this study was to prepare 5-fluorouracil (5-FU) loaded block copolymers, with poly(γ-benzyl-L-glutamate) (PBLG) as the hydrophobic block and poly(ethylene glycol) (PEG) as the hydrophilic block, and then examine the 5-FU release characteristics, pharmacokinetics, and anticancer effects of this novel compound.</p> <p>Methods</p> <p>5-FU loaded PEG-PBLG (5-FU/PEG-PBLG) nanoparticles were prepared by dialysis and then scanning electron microscopy (SEM) and transmission electron microscopy (TEM) were used to observe the shape and size of the nanoparticles, and ultraviolet spectrophotometry was used to evaluate the 5-FU in vitro release characteristics. The pharmacokinetic parameters of 5-FU/PEG-PBLG nanoparticles in rabbit plasma were determined by measuring the 5-FUby high-performance liquid chromatography (HPLC). To study in vivo effects, LoVo cells (human colon cancer cell line) or Tca8113 cells (human oral squamous cell carcinoma cell line) were implanted in BALB/c nude mice that were subsequently treated with 5-FU or 5-FU/PEG-PBLG nanospheres.</p> <p>Results</p> <p>5-FU/PEG-PBLG nanoparticles had a core-shell spherical structure with a diameter of 200 nm and a shell thickness of 30 nm. The drug loading capacity was 27.1% and the drug encapsulation was 61.5%. Compared with 5-FU, 5-FU/PEG-PBLG nanoparticles had a longer elimination half-life (t_1/2, 33.3 h vs. 5 min), lower peak concentration (C, 4563.5 μg/L vs. 17047.3 μg/L), and greater distribution volume (V_D, 0.114 L vs. 0.069 L). Compared with a blank control, LoVo cell xenografts and Tca8113 cell xenografts treated with 5-FU or 5-FU/PEG-PBLG nanoparticles grew slower and had prolonged tumor doubling times. 5-FU/PEG-PBLG nanoparticles showed greater inhibition of tumor growth than 5-FU (p < 0.01). In the PEG-PBLG nanoparticle control group, there was no tumor inhibition (p > 0.05).</p> <p>Conclusion</p> <p>In our model system, 5-FU/PEG-PBLG nanoparticles changed the pharmacokinetic behavior of 5-FU, thus increasing its anticancer activity. 5-Fluorouracil loaded nanoparticles have potential as a novel anticancer drug that may have useful clinical applications.</p

Lipopolysaccharide modifies amiloride-sensitive Na+ transport processes across human airway cells: role of mitogen-activated protein kinases ERK 1/2 and 5

Bacterial lipopolysaccharides (LPS) are potent inducers of proinflammatory signaling pathways via the activation of nuclear factor-kappa B (NF-κB) and mitogen-activated protein kinase (MAPK), causing changes in the processes that control lung fluid homeostasis and contributing to the pathogenesis of lung disease. In human H441 airway epithelial cells, incubation of cells with 15 µg ml−1 LPS caused a significant reduction in amiloride-sensitive Isc from 15 ± 2 to 8 ± 2 µA cm−2 (p = 0.01, n = 13) and a shift in IC50 amiloride of currents from 6.8 × 10−7 to 6.4 × 10−6 M. This effect was associated with a decrease in the activity of 5 pS, highly Na+ selective, amiloride-sensitive <1 µM channels (HSC) and an increase in the activity of ∼18 pS, nonselective, amiloride-sensitive >10 µM cation channels (NSC) in the apical membrane. LPS decreased αENaC mRNA and protein abundance, inferring that LPS inhibited αENaC gene expression. This correlated with the decrease in HSC activity, indicating that these channels, but not NSCs, were comprised of at least αENaC protein. LPS increased NF-κB DNA binding activity and phosphorylation of extracellular signal-related kinase (ERK)1/2, but decreased phosphorylation of ERK5 in H441 cells. Pretreatment of monolayers with PD98059 (20 µM) inhibited ERK1/2 phosphorylation, promoted phosphorylation of ERK5, increased αENaC protein abundance, and reversed the effect of LPS on Isc and the shift in amiloride sensitivity. Inhibitors of NF-κB activation were without effect. Taken together, our data indicate that LPS acts via ERK signaling pathways to decrease αENaC transcription, reducing HSC/ENaC channel abundance, activity, and transepithelial Na+ transport in H441 airway epithelial cells

IL-10 transcription is negatively regulated by BAF180, a component of the SWI/SNF chromatin remodeling enzyme

<p>Abstract</p> <p>Background</p> <p>SWI/SNF chromatin remodeling enzymes play a critical role in the development of T helper lymphocytes, including Th2 cells, and directly program chromatin structure at Th2 cytokine genes. Different versions of SWI/SNF complexes, including BAF and PBAF, have been described based on unique subunit composition. However, the relative role of BAF and PBAF in Th cell function and cytokine expression has not been reported.</p> <p>Results</p> <p>Here we examine the role of the PBAF SWI/SNF complex in Th cell development and gene expression using mice deficient for a PBAF-specific component, BAF180. We find that T cell development in the thymus and lymphoid periphery is largely normal when the BAF180 gene is deleted late in thymic development. However, BAF180-deficient Th2 cells express high levels of the immunoregulatory cytokine IL-10. BAF180 binds directly to regulatory elements in the Il-10 locus but is replaced by BAF250 BAF complexes in the absence of BAF180, resulting in increased histone acetylation and CBP recruitment to the IL-10 locus.</p> <p>Conclusions</p> <p>These results demonstrate that BAF180 is a repressor of IL-10 transcription in Th2 cells and suggest that the differential recruitment of different SWI/SNF subtypes can have direct consequences on chromatin structure and gene transcription.</p

The Musicality of Non-Musicians: An Index for Assessing Musical Sophistication in the General Population

Musical skills and expertise vary greatly in Western societies. Individuals can differ in their repertoire of musical behaviours as well as in the level of skill they display for any single musical behaviour. The types of musical behaviours we refer to here are broad, ranging from performance on an instrument and listening expertise, to the ability to employ music in functional settings or to communicate about music. In this paper, we first describe the concept of ‘musical sophistication’ which can be used to describe the multi-faceted nature of musical expertise. Next, we develop a novel measurement instrument, the Goldsmiths Musical Sophistication Index (Gold-MSI) to assess self-reported musical skills and behaviours on multiple dimensions in the general population using a large Internet sample (n = 147,636). Thirdly, we report results from several lab studies, demonstrating that the Gold-MSI possesses good psychometric properties, and that self-reported musical sophistication is associated with performance on two listening tasks. Finally, we identify occupation, occupational status, age, gender, and wealth as the main socio-demographic factors associated with musical sophistication. Results are discussed in terms of theoretical accounts of implicit and statistical music learning and with regard to social conditions of sophisticated musical engagement

Uncovering Enhancer Functions Using the α-Globin Locus

Over the last three decades, studies of the α- and β-globin genes clusters have led to elucidation of the general principles of mammalian gene regulation, such as RNA stability, termination of transcription, and, more importantly, the identification of remote regulatory elements. More recently, detailed studies of α-globin regulation, using both mouse and human loci, allowed the dissection of the sequential order in which transcription factors are recruited to the locus during lineage specification. These studies demonstrated the importance of the remote regulatory elements in the recruitment of RNA polymerase II (PolII) together with their role in the generation of intrachromosomal loops within the locus and the removal of polycomb complexes during differentiation. The multiple roles attributed to remote regulatory elements that have emerged from these studies will be discussed