E2F3 is responsible for frequent amplification of 6p22.3 in human bladder cancer

The 6p22 is generally regarded as one of the most important amplification sites in urinary bladder cancer. Investigations, encouraged by these findings, subsequently lead to the delineation of the amplicon. During this process the genomic region was narrowed down to 1.7 Mb at 6p22.3, including presumably 13 different genes. Some of these genes were withdrawn from additional investigations due to low-level or absent expression in 6p22.3- amplified bladder cancers. Two genes, however, showed unquestionable correlation between high-level amplification and subsequent overexpression. But the relevant target gene that drives the amplification remained unidentified, yet. This work was ultimately aimed in a comprehensive comparison of the two remaining candidate oncogenes. The major findings were: - By performing FISH on a large bladder cancer TMA we show that NM_017774 is amplified in 11.6% of 893 tested human bladder cancer samples. Thus, the gene reaches an amplification level that is comparable to E2F3. - Following case-by-case re-evaluation of a large-section FISH analysis, exhibiting 104 6p22.3-amplified bladder cancers, demonstrates that both genes are 100% coamplified. - Furthermore, we show that both candidate oncogenes are always co-overexpressed in 6p22.3-amplified bladder cancer cell lines, presumably as a consequence of the amplification. - Experimentally decreased expression levels of NM_017774 and/or E2F3 similarly lead to strongly inhibited cell proliferation (observed in normal bladder cancer cells CRL-7930; without 6p22.3-amplification). - This finding suggests that NM_017774 -the gene of hitherto unknown function –must be functionally connected to the cell cycle regulatory machinery. - Besides, decreased E2F3-expression results in proliferation-reduction, and thus confirms the previously predicted essential role of this transcriptionfactor in cell cycle progression. - Finally, functional analysis performed in the 6p22.3-amplified HTB-5 cell line, demonstrate that E2F3 –but not NM_017774 -captures a limiting role for enhanced cellular proliferation in 6p22.3-amplified bladder cancer cells. - Hence, our results suggest that NM_017774 is only accidentally co-amplified because of its spatial neighbourhood to E2F3 (like other genes in the area), but does not have a functional role in 6p22.3-amplification, whatsoever. Conclusively, the findings of this study consistently document that amplification of 6p22.3 leads to upregulated mRNA expression, and increased protein production of the transcriptionfactor E2F3. While also other genes localized in the amplified region may be coamplified and co-overexpressed as a by-product of the amplification, E2F3 represents the main target gene and is therefore responsible for the frequent amplification of 6p22.3 in urinary bladder cancer

Process Improvements by the Utilisation of an Annular Slit Reactor

For a strongly endothermic catalytic dehydrocyclisation a new design of an annular slit reactor was evaluated. The major advantage of the design is an optimised temperature profile. Consequently, both a higher selectivity and a higher conversion were achieved. Furthermore, the formation of a problematic side product was significantly reduced, leading to a simplification of the subsequent purification step and an increased productivity. From an economic point of view, yearly savings of up to CHF 1 million are anticipated compared to the present manufacturing costs

Experimental pulse technique for the study of microbial kinetics in continuous culture

A novel technique was developed for studying the growth kinetics of microorganisms in continuous culture. The method is based on following small perturbations of a chemostat culture by on-line measurement of the dynamic response in oxygen consumption rates. A mathematical model, incorporating microbial kinetics and mass transfer between gas and liquid phases, was applied to interpret the data. Facilitating the use of very small disturbances, the technique is non-disruptive as well as fast and accurate. The technique was used to study the growth kinetics of two cultures, Methylosinus trichosporium OB3b growing on methane, both in the presence and in the absence of copper, and Burkholderia (Pseudomonas) cepacia G4 growing on phenol. Using headspace flushes, gas blocks and liquid substrate pulse experiments, estimates for limiting substrate concentrations, maximum conversion rates Vmax and half saturation constants Ks could rapidly be obtained. For M. trichosporium OB3b it was found that it had a far higher affinity for methane when particulate methane monooxygenase (pMMO) was expressed than when the soluble form (sMMO) was expressed under copper limitation. While for B. cepacia G4 the oxygen consumption pattern during a phenol pulse in the chemostat indicated that phenol was transiently converted to an intermediate (4-hydroxy-2-oxovalerate), so that initially less oxygen was used per mole of phenol.

On-line estimation of biomass through pH control analysis in aerobic yeast fermentation systems

The amount of acid or base consumed in yeast cultures has been recently assigned to the pathway of nitrogen assimilation under respiratory conditions with no contribution by carbon metabolism (Castrillo et al., 1995). In this investigation, experiments under respirofermentative conditions have shown that production or consumption of ethanol does not contribute significantly to the specific rate of proton production (qH+), thus extending the previously obtained relationships for all aerobic conditions in which other major acid/base contributions are not involved. Tests in batch and chemostat culture confirm the validity of qH+ as a formal control parameter in aerobic fermentations.Junta Nacional de Investigação Científica e Tecnológica Gobierno Vasco (Departamento de Educación, Universidadese Investigación) Ministerio de Educación y Ciencia-Acciones Integrada

Ceramic foam plates: a new tool for processing fresh radical prostatectomy specimens

Procurement of fresh tissue of prostate cancer is critical for biobanking and generation of xenograft models as an important preclinical step towards new therapeutic strategies in advanced prostate cancer. However, handling of fresh radical prostatectomy specimens has been notoriously challenging given the distinctive physical properties of prostate tissue and the difficulty to identify cancer foci on gross examination. Here, we have developed a novel approach using ceramic foam plates for processing freshly cut whole mount sections from radical prostatectomy specimens without compromising further diagnostic assessment. Forty-nine radical prostatectomy specimens were processed and sectioned from the apex to the base in whole mount slices. Putative carcinoma foci were morphologically verified by frozen section analysis. The fresh whole mount slices were then laid between two ceramic foam plates and fixed overnight. To test tissue preservation after this procedure, formalin-fixed and paraffin-embedded whole mount sections were stained with hematoxylin and eosin (H&E) and analyzed by immunohistochemistry, fluorescence, and silver in situ hybridization (FISH and SISH, respectively). There were no morphological artifacts on H&E stained whole mount sections from slices that had been fixed between two plates of ceramic foam, and the histological architecture was fully retained. The quality of immunohistochemistry, FISH, and SISH was excellent. Fixing whole mount tissue slices between ceramic foam plates after frozen section examination is an excellent method for processing fresh radical prostatectomy specimens, allowing for a precise identification and collection of fresh tumor tissue without compromising further diagnostic analysis

Genome-Wide Analyses of Nkx2-1 Binding to Transcriptional Target Genes Uncover Novel Regulatory Patterns Conserved in Lung Development and Tumors

The homeodomain transcription factor Nkx2-1 is essential for normal lung development and homeostasis. In lung tumors, it is considered a lineage survival oncogene and prognostic factor depending on its expression levels. The target genes directly bound by Nkx2-1, that could be the primary effectors of its functions in the different cellular contexts where it is expressed, are mostly unknown. In embryonic day 11.5 (E11.5) mouse lung, epithelial cells expressing Nkx2-1 are predominantly expanding, and in E19.5 prenatal lungs, Nkx2-1-expressing cells are predominantly differentiating in preparation for birth. To evaluate Nkx2-1 regulated networks in these two cell contexts, we analyzed genome-wide binding of Nkx2-1 to DNA regulatory regions by chromatin immunoprecipitation followed by tiling array analysis, and intersected these data to expression data sets. We further determined expression patterns of Nkx2-1 developmental target genes in human lung tumors and correlated their expression levels to that of endogenous NKX2-1. In these studies we uncovered differential Nkx2-1 regulated networks in early and late lung development, and a direct function of Nkx2-1 in regulation of the cell cycle by controlling the expression of proliferation-related genes. New targets, validated in Nkx2-1 shRNA transduced cell lines, include E2f3, Cyclin B1, Cyclin B2, and c-Met. Expression levels of Nkx2-1 direct target genes identified in mouse development significantly correlate or anti-correlate to the levels of endogenous NKX2-1 in a dosage-dependent manner in multiple human lung tumor expression data sets, supporting alternative roles for Nkx2-1 as a transcriptional activator or repressor, and direct regulator of cell cycle progression in development and tumors

¿CUÁNTO PESA UNA CÉLULA?

Tan sólo una célula ¿Cuánto pesa una célula humana? En octubre 2018,investigadores en Suiza dieron a conocer esta pequeña balanza en voladizodiseñada para detectar las más mínimas fluctuaciones en la masa de una célula viva

On‐Line Exhaust Gas Analysis of Volatiles in Fermentation Using Mass Spectrometry

Liquid‐phase concentrations of volatile compounds were determined on‐line via gasphase analysis using mass spectrometry. It has been shown theoretically as well as experimentally that almost every volatile compound commonly produced by microorganisms can be measured by the method presented, if its partial pressure in the fermentor off‐gas exceeds 1 μbar. This has been shown in a baker's yeast as well as a Bacillus subtilis culture. The experimental results also indicated that these measurements are not controlled by mass‐transfer kinetics. A dimensionless parameter, E, allows us to check whether the analysis is determined by thermodynamics (Henry coefficient) or by mass‐transfer rates