Heterarchy of Transcription Factors Driving Basal and Luminal Cell Phenotypes in Human Urothelium

Cell differentiation is effected by complex networks of transcription factors that co-ordinate re-organisation of the chromatin landscape. The hierarchies of these relationships can be difficult to dissect. During in vitro differentiation of normal human uro-epithelial cells, formaldehyde-assisted isolation of regulatory elements (FAIRE-seq) and RNA-seq were used to identify alterations in chromatin accessibility and gene expression changes following activation of the nuclear receptor PPARG as a differentiation-initiating event. Regions of chromatin identified by FAIRE-seq as having altered accessibility during differentiation were found to be enriched with sequence-specific binding motifs for transcription factors predicted to be involved in driving basal and differentiated urothelial cell phenotypes, including FOXA1, P63, GRHL2, CTCF and GATA3. In addition, co-occurrence of GATA3 motifs was observed within sub-sets of differentiation-specific peaks containing P63 or FOXA1 after induction of differentiation. Changes in abundance of GRHL2, GATA3, and P63 were observed in immunoblots of chromatin-enriched extracts. Transient siRNA knockdown of P63 revealed that P63 favoured a basal-like phenotype by inhibiting differentiation and promoting expression of basal marker genes. GATA3 siRNA prevented differentiation-associated downregulation of P63 protein and transcript, and demonstrated positive feedback of GATA3 on PPARG transcript, but showed no effect on FOXA1 transcript or protein expression. This approach indicates that as a transcriptionally-regulated programme, urothelial differentiation operates as a heterarchy wherein GATA3 is able to co-operate with FOXA1 to drive expression of luminal marker genes, but that P63 has potential to transrepress expression of the same genes

In vivo PET imaging of the neuroinflammatory response in rat spinal cord injury using the TSPO tracer [F-18]GE-180 and effect of docosahexaenoic acid

Centre for Trauma Sciences, funded by the Barts & The London Charity, GE Healthcare Ltd, the Experimental Medicine Awards from the Blizard Institute and the Imaging Centre at the Barts Cancer Institute

Chromosomal Aberrations in Bladder Cancer: Fresh versus Formalin Fixed Paraffin Embedded Tissue and Targeted FISH versus Wide Microarray-Based CGH Analysis

Bladder carcinogenesis is believed to follow two alternative pathways driven by the loss of chromosome 9 and the gain of chromosome 7, albeit other nonrandom copy number alterations (CNAs) were identified. However, confirmation studies are needed since many aspects of this model remain unclear and considerable heterogeneity among cases has emerged. One of the purposes of this study was to evaluate the performance of a targeted test (UroVysion assay) widely used for the detection of Transitional Cell Carcinoma (TCC) of the bladder, in two different types of material derived from the same tumor. We compared the results of UroVysion test performed on Freshly Isolated interphasic Nuclei (FIN) and on Formalin Fixed Paraffin Embedded (FFPE) tissues from 22 TCCs and we didn't find substantial differences. A second goal was to assess the concordance between array-CGH profiles and the targeted chromosomal profiles of UroVysion assay on an additional set of 10 TCCs, in order to evaluate whether UroVysion is an adequately sensitive method for the identification of selected aneuploidies and nonrandom CNAs in TCCs. Our results confirmed the importance of global genomic screening methods, that is array based CGH, to comprehensively determine the genomic profiles of large series of TCCs tumors. However, this technique has yet some limitations, such as not being able to detect low level mosaicism, or not detecting any change in the number of copies for a kind of compensatory effect due to the presence of high cellular heterogeneity. Thus, it is still advisable to use complementary techniques such as array-CGH and FISH, as the former is able to detect alterations at the genome level not excluding any chromosome, but the latter is able to maintain the individual data at the level of single cells, even if it focuses on few genomic regions

Studies on the impact of mixing in brewing fermentation - Comparison of methods of effecting enhanced liquid circulation

Mixing during beer production by natural CO2 evolution has been enhanced at the bench scale by headspace gas recirculation to the base of a 3.51 cylindroconical fermenter or by mechanical agitation in a 41 fermenter. Standardized lager fermentations were used to compare the two methods at a range of mean specific energy dissipation rates (ε̄T, W kg-1), including ∼5 × 10-2 W kg-1. The latter corresponds approximately to the maximum ε̄T found at the production scale (∼400 m3) due to natural CO2 evolution. The work has shown that gas recirculation is a viable technique that avoids the loss of volatiles found if mixing is enhanced by sparging a separate gas such as nitrogen. For both types of mixing, it was found that ε̄T resulted in increased yeast growth and fermentation rates and an alteration in the balance of key volatile compounds, with, in general, an enhanced formation of higher alcohols and a suppression of the formation of esters. However, with gas recirculation this enhanced performance was limited to an ε̄T of ∼5 × 10-2 W kg-1 because of the tendency for excessive foaming at higher recirculation rates, whilst values up to ∼2.5 × 10-2 W kg-1 proved effective under agitated conditions. On comparison, at an ε̄T of ∼5 × 10-2 W kg-1, the two modes of operation gave similar results, although gas recirculation produced a slightly increased fermentation rate. The possible reasons for this are briefly discussed. Both methods seem to have the potential for reducing fermentation time and enhancing reproducibility, especially at the small scale

Studies on the impact of mixing in brewing fermentation - Comparison of methods of effecting enhanced liquid circulation

Mixing during beer production by natural CO2 evolution has been enhanced at the bench scale by headspace gas recirculation to the base of a 3.51 cylindroconical fermenter or by mechanical agitation in a 41 fermenter. Standardized lager fermentations were used to compare the two methods at a range of mean specific energy dissipation rates (ε̄T, W kg-1), including ∼5 × 10-2 W kg-1. The latter corresponds approximately to the maximum ε̄T found at the production scale (∼400 m3) due to natural CO2 evolution. The work has shown that gas recirculation is a viable technique that avoids the loss of volatiles found if mixing is enhanced by sparging a separate gas such as nitrogen. For both types of mixing, it was found that ε̄T resulted in increased yeast growth and fermentation rates and an alteration in the balance of key volatile compounds, with, in general, an enhanced formation of higher alcohols and a suppression of the formation of esters. However, with gas recirculation this enhanced performance was limited to an ε̄T of ∼5 × 10-2 W kg-1 because of the tendency for excessive foaming at higher recirculation rates, whilst values up to ∼2.5 × 10-2 W kg-1 proved effective under agitated conditions. On comparison, at an ε̄T of ∼5 × 10-2 W kg-1, the two modes of operation gave similar results, although gas recirculation produced a slightly increased fermentation rate. The possible reasons for this are briefly discussed. Both methods seem to have the potential for reducing fermentation time and enhancing reproducibility, especially at the small scale

Comparison of tensile strength of different carbon fabric reinforced epoxy composites

Carbon fabric/epoxy composites are materials used in aeronautical industry to manufacture several components as flaps, aileron, landing-gear doors and others. To evaluate these materials become important to know their mechanical properties, for example, the tensile strength. Tensile tests are usually performed in aeronautical industry to determinate tensile property data for material specifications, quality assurance and structural analysis. For this work, it was manufactured four different laminate families (F155/PW, F155/HS, F584/PW and F584/HS) using pre-impregnated materials (prepregs) based on F155TM and F584TM epoxy resins reinforced with carbon fiber fabric styles Plain Weave (PW) and Eight Harness Satin (8HS). The matrix F155TM code is an epoxy resin type DGEBA (diglycidil ether of bisphenol A) that contains a curing agent and the F584TM code is a modified epoxy resin type. The laminates were obtained by handing lay-up process following an appropriate curing cycle in autoclave. The samples were evaluated by tensile tests according to the ASTM D3039. The F584/PW laminates presented the highest values of tensile strength. However, the highest modulus results were determined for the 8HS composite laminates. The correlation of these results emphasizes the importance of the adequate combination of the polymeric matrix and the reinforcement arrangement in the structural composite manufacture. The microscopic analyses of the tested specimens show valid failure modes for composites used in aeronautical industry