Determinants of a transcriptionally competent environment at the GM-CSF promoter

Granulocyte macrophage-colony stimulating factor (GM-CSF) is produced by T cells, but not B cells, in response to immune signals. GM-CSF gene activation in response to T-cell stimulation requires remodelling of chromatin associated with the gene promoter, and these changes do not occur in B cells. While the CpG methylation status of the murine GM-CSF promoter shows no correlation with the ability of the gene to respond to activation, we find that the basal chromatin environment of the gene promoter influences its ability to respond to immune signals. In unstimulated T cells but not B cells, the GM-CSF promoter is selectively marked by enrichment of histone acetylation, and association of the chromatin-remodelling protein BRG1. BRG1 is removed from the promoter upon activation concomitant with histone depletion and BRG1 is required for efficient chromatin remodelling and transcription. Increasing histone acetylation at the promoter in T cells is paralleled by increased BRG1 recruitment, resulting in more rapid chromatin remodelling, and an associated increase in GM-CSF mRNA levels. Furthermore, increasing histone acetylation in B cells removes the block in chromatin remodelling and transcriptional activation of the GM-CSF gene. These data are consistent with a model in which histone hyperacetylation and BRG1 enrichment at the GM-CSF promoter, generate a chromatin environment competent to respond to immune signals resulting in gene activation

Interplay between Transcription Factors and the Epigenome: Insight from the Role of RUNX1 in Leukemia

The genome has the ability to respond in a precise and co-ordinated manner to cellular signals. It achieves this through the concerted actions of transcription factors and the chromatin platform, which are targets of the signalling pathways. Our understanding of the molecular mechanisms through which transcription factors and the chromatin landscape each control gene activity has expanded dramatically over recent years, and attention has now turned to understanding the complex, multifaceted interplay between these regulatory layers in normal and disease states. It has become apparent that transcription factors as well as the components and modifiers of the epigenetic machinery are frequent targets of genomic alterations in cancer cells. Through the study of these factors, we can gain unique insight into the dynamic interplay between transcription factors and the epigenome, and how their dysregulation leads to aberrant gene expression programs in cancer. Here we will highlight how these factors normally co-operate to establish and maintain the transcriptional and epigenetic landscape of cells, and how this is reprogrammed in cancer, focusing on the RUNX1 transcription factor and oncogenic derivative RUNX1-ETO in leukemia as paradigms of transcriptional and epigenetic reprogramming

LINE Retrotransposon RNA Is an Essential Structural and Functional Epigenetic Component of a Core Neocentromeric Chromatin

We have previously identified and characterized the phenomenon of ectopic human centromeres, known as neocentromeres. Human neocentromeres form epigenetically at euchromatic chromosomal sites and are structurally and functionally similar to normal human centromeres. Recent studies have indicated that neocentromere formation provides a major mechanism for centromere repositioning, karyotype evolution, and speciation. Using a marker chromosome mardel(10) containing a neocentromere formed at the normal chromosomal 10q25 region, we have previously mapped a 330-kb CENP-A–binding domain and described an increased prevalence of L1 retrotransposons in the underlying DNA sequences of the CENP-A–binding clusters. Here, we investigated the potential role of the L1 retrotransposons in the regulation of neocentromere activity. Determination of the transcriptional activity of a panel of full-length L1s (FL-L1s) across a 6-Mb region spanning the 10q25 neocentromere chromatin identified one of the FL-L1 retrotransposons, designated FL-L1b and residing centrally within the CENP-A–binding clusters, to be transcriptionally active. We demonstrated the direct incorporation of the FL-L1b RNA transcripts into the CENP-A–associated chromatin. RNAi-mediated knockdown of the FL-L1b RNA transcripts led to a reduction in CENP-A binding and an impaired mitotic function of the 10q25 neocentromere. These results indicate that LINE retrotransposon RNA is a previously undescribed essential structural and functional component of the neocentromeric chromatin and that retrotransposable elements may serve as a critical epigenetic determinant in the chromatin remodelling events leading to neocentromere formation

GM-CSF promoter chromatin remodelling and gene transcription display distinct signal and transcription factor requirements

Granulocyte-macrophage colony stimulating factor (GM-CSF) plays a key role in myeloid cell function and is rapidly and transiently expressed in T cells in response to immune or inflammatory stimuli. Induction of GM-CSF gene expression is accompanied by changes in chromatin structure across the proximal promoter region of the gene. We show that the promoter remodelling and subsequent gene transcription occurs with distinct signal and transcription factor requirements. Activation of the protein kinase C (PKC) signalling pathway is sufficient to induce changes in chromatin structure across the promoter, but both the PKC and calcium signalling pathways are required for efficient gene transcription. Although NFAT transcription factors contribute to GM-CSF gene transcription, they are not required for promoter remodelling. However, the presence of the nuclear factor-κB transcription factor, c-Rel, in the nucleus is strongly correlated with and required for the events of chromatin remodelling

Porcine Bladder Urothelial, Myofibroblast, and Detrusor Muscle Cells: Characterization and ATP Release

ATP is released from the bladder mucosa in response to stretch, but the cell types responsible are unclear. Our aim was to isolate and characterize individual populations of urothelial, myofibroblast, and detrusor muscle cells in culture, and to examine agonist-stimulated ATP release. Using female pig bladders, urothelial cells were isolated from bladder mucosa following trypsin-digestion of the luminal surface. The underlying myofibroblast layer was dissected, minced, digested, and cultured until confluent (10–14 days). A similar protocol was used for muscle cells. Cultures were used for immunocytochemical staining and/or ATP release investigations. In urothelial cultures, immunoreactivity was present for the cytokeratin marker AE1/AE3 but not the contractile protein α-smooth muscle actin (α-SMA) or the cytoskeletal filament vimentin. Neither myofibroblast nor muscle cell cultures stained for AE1/AE3. Myofibroblast cultures partially stained for α-SMA, whereas muscle cultures were 100% stained. Both myofibroblast and muscle stained for vimentin, however, they were morphologically distinct. Ultrastructural studies verified that the suburothelial layer of pig bladder contained abundant myofibroblasts, characterized by high densities of rough endoplasmic reticulum. Baseline ATP release was higher in urothelial and myofibroblast cultures, compared with muscle. ATP release was significantly stimulated by stretch in all three cell populations. Only urothelial cells released ATP in response to acid, and only muscle cells were stimulated by capsaicin. Tachykinins had no effect on ATP release. In conclusion, we have established a method for culture of three cell populations from porcine bladder, a well-known human bladder model, and shown that these are distinct morphologically, immunologically, and pharmacologically

Andromeda's Parachute: A Bright Quadruply Lensed Quasar at z=2.377

We present Keck Cosmic Web Imager spectroscopy of the four putative images of the lensed quasar candidate J014709+463037 recently discovered by Berghea et al. (2017). The data verify the source as a quadruply lensed, broad absorption-line quasar having z_S = 2.377 +/- 0.007. We detect intervening absorption in the FeII 2586, 2600, MgII 2796, 2803, and/or CIV 1548, 1550 transitions in eight foreground systems, three of which have redshifts consistent with the photometric-redshift estimate reported for the lensing galaxy (z_L ~ 0.57). By virtue of their positions on the sky, the source images probe these absorbers over transverse physical scales of ~0.3-21 kpc, permitting assessment of the variation in metal-line equivalent width W_r as a function of sight-line separation. We measure differences in W_r,2796 of <40% across all sight-line pairs subtending 7-21 kpc, suggestive of a high degree of spatial coherence for MgII-absorbing material. W_r,2600 is observed to vary by >50% over the same scales across the majority of sight-line pairs, while CIV absorption exhibits a wide range in W_r,1548 differences of ~5-80% within transverse distances less than ~3 kpc. J014709+463037 is one of only a handful of z > 2 quadruply lensed systems for which all four source images are very bright (r = 15.4-17.7 mag) and are easily separated in ground-based seeing conditions. As such, it is an ideal candidate for higher-resolution spectroscopy probing the spatial variation in the kinematic structure and physical state of intervening absorbers.Comment: Submitted to ApJL. 9 pages, 3 figures. Uses aastex61 forma

CCN Data Interpretation Under Dynamic Operation Conditions

We have developed a new numerical model for the non-steadystate operation of the Droplet Measurement Technologies (DMT) Cloud Condensation Nuclei (CCN) counter. The model simulates the Scanning Flow CCN Analysis (SFCA) instrument mode, where a wide supersaturation range is continuously scanned by cycling the flowrate over 20–120 s. Model accuracy is verified using a broad set of data which include ammonium sulfate calibration data (under conditions of low CCN concentration) and airborne measurements where either the instrument pressure was not controlled or where exceptionally high CCN loadings were observed. It is shown here for the first time that small pressure and flow fluctuations can have a disproportionately large effect on the instrument supersaturation due to localized compressive/expansive heating and cooling. The model shows that, for fast scan times, these effects can explain the observed shape of the SFCA supersaturation-flow calibration curve and transients in the outlet droplet sizes. The extent of supersaturation depletion from the presence of CCN during SFCA operation is also examined; we found that depletion effects can be neglected below 4000 cm−3 for CCN number

Individualised pelvic floor muscle training in women with pelvic organ prolapse: a multicentre randomised controlled trial

&lt;br&gt;Background: Pelvic organ prolapse is common and is strongly associated with childbirth and increasing age. Women with prolapsed are often advised to do pelvic floor muscle exercises, but supporting evidence is limited. Our aim was to establish if one-to-one individualised pelvic floor muscle training (PFMT) is effective in reducing prolapse symptoms.&lt;/br&gt; &lt;br&gt;Methods: A parallel‐group multicentre randomised controlled trial (ISRCTN35911035) in female outpatients with newly-diagnosed, symptomatic stage I, II or III prolapse, comparing five PFMT appointments over 16 weeks (n=225) versus a lifestyle advice leaflet (n=222). Treatment allocation was by remote computer allocation using minimisation. Our primary endpoint was participants’ self-report of prolapsed symptoms at 12 months. Group assignment was masked from outcome assessors. We compared outcomes between trial groups in an intention-to-treat analysis. The cost of PFMT and savings on subsequent treatments were calculated to estimate cost-effectiveness.&lt;/br&gt; &lt;br&gt;Findings: Compared to the control group, the intervention group reported fewer prolapse symptoms at 12 months (mean difference between groups in change score 1.52, 95% CI [0.46, 2.59], p=0.0053); reported their prolapse to be “better” more often (57.2% versus 44.7%, difference 12.6%, 95% CI [1.1%, 24.1%], p=0.0336); and had an increased but non-significant odds of having less severe stage of prolapse at their 6-month clinical examination, (OR 1.47, 95% CI [0.97, 2.27], p=0.07). The control group had a greater uptake of other prolapse treatment (49.6% versus 24.1%, difference 25.5%, 95% CI [14.5%, 36.0%], p &#60;0.0001). Findings were robust to missing data. The net cost of the 25 intervention was £131.61 per woman and the cost per one-point reduction in the symptom score was £86.59, 95% CI [£50.81, £286.11]. &lt;/br&gt

Therapeutic targeting of integrin αvβ6 in breast cancer

BACKGROUND: Integrin ?v?6 promotes migration, invasion, and survival of cancer cells; however, the relevance and role of ?v?6 has yet to be elucidated in breast cancer.METHODS: Protein expression of integrin subunit beta6 (?6) was measured in breast cancers by immunohistochemistry (n &gt; 2000) and ITGB6 mRNA expression measured in the Molecular Taxonomy of Breast Cancer International Consortium dataset. Overall survival was assessed using Kaplan Meier curves, and bioinformatics statistical analyses were performed (Cox proportional hazards model, Wald test, and Chi-square test of association). Using antibody (264RAD) blockade and siRNA knockdown of ?6 in breast cell lines, the role of ?v?6 in Human Epidermal Growth Factor Receptor 2 (HER2) biology (expression, proliferation, invasion, growth in vivo) was assessed by flow cytometry, MTT, Transwell invasion, proximity ligation assay, and xenografts (n ? 3), respectively. A student's t-test was used for two variables; three-plus variables used one-way analysis of variance with Bonferroni's Multiple Comparison Test. Xenograft growth was analyzed using linear mixed model analysis, followed by Wald testing and survival, analyzed using the Log-Rank test. All statistical tests were two sided.RESULTS: High expression of either the mRNA or protein for the integrin subunit ?6 was associated with very poor survival (HR = 1.60, 95% CI = 1.19 to 2.15, P = .002) and increased metastases to distant sites. Co-expression of ?6 and HER2 was associated with worse prognosis (HR = 1.97, 95% CI = 1.16 to 3.35, P = .01). Monotherapy with 264RAD or trastuzumab slowed growth of MCF-7/HER2-18 and BT-474 xenografts similarly (P &lt; .001), but combining 264RAD with trastuzumab effectively stopped tumor growth, even in trastuzumab-resistant MCF-7/HER2-18 xenografts.CONCLUSIONS: Targeting ?v?6 with 264RAD alone or in combination with trastuzumab may provide a novel therapy for treating high-risk and trastuzumab-resistant breast cancer patients.<br/

Synthesis and characterisation of lamellar ZnS nanosheets containing intercalated diamines

A solvothermal method has been used to preparehybrid inorganic-organic composites with a lamellar structure in which layers of wurtzite ZnS are separated by intercalated diamine molecules. A hybrid composite prepared with diethylenetriamine has been isolated and characterisedand its structure and properties compared with those of the composite prepared using ethylenediamine. Comparative structural and morphological studies of the two lamellar hybrid composites are described on the basis of powder XRD, electron and scanning probe microscopies and thermal analysis of the materials