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 Some of these genes were withdrawn from additional investigations due to low-level or absent expression in 6p22.3- amplified bladder cancers.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

Distinct domains of the β1-subunit cytosolic N-terminus control surface expression and functional properties of large conductance calcium-activated potassium (BK) channels

The properties and function of large conductance calcium- and voltage- activated potassium (BK) channels are modified by the tissue specific expression of regulatory β 1-subunits. Although the short cytosolic N-terminal domain of the β 1-subunit is important for controlling both BK channel trafficking and function, whether the same, or different, regions of the N-terminus control these distinct processes remain unknown. Here we demonstrate that the first six N-terminal residues, including K3, K4 and L5, are critical for controlling functional regulation, but not trafficking, of BK channels. This membrane distal region has features of an amphipathic helix that is predicted to control the orientation of the first transmembrane segment (TM1) of the β 1-subunit. In contrast, a membrane proximal leucine residue (L17) controls trafficking without affecting functional coupling, an effect that is in part dependent on controlling efficient ER exit of the pore-forming α-subunit. Thus cell surface trafficking and functional coupling with BK channels are controlled by distinct domains of the β 1-subunit N-terminus.</p

Role of KCNMA1 in breast cancer

KCNMA1 encodes the α-subunit of the large conductance, voltage and Ca(2+)-activated (BK) potassium channel and has been reported as a target gene of genomic amplification at 10q22 in prostate cancer. To investigate the prevalence of the amplification in other human cancers, the copy number of KCNMA1 was analyzed by fluorescence-in-situ-hybridization (FISH) in 2,445 tumors across 118 different tumor types. Amplification of KCNMA1 was restricted to a small but distinct fraction of breast, ovarian and endometrial cancer with the highest prevalence in invasive ductal breast cancers and serous carcinoma of ovary and endometrium (3-7%). We performed an extensive analysis on breast cancer tissue microarrays (TMA) of 1,200 tumors linked to prognosis. KCNMA1 amplification was significantly associated with high tumor stage, high grade, high tumor cell proliferation, and poor prognosis. Immunofluorescence revealed moderate or strong KCNMA1 protein expression in 8 out of 9 human breast cancers and in the breast cancer cell line MFM223. KCNMA1-function in breast cancer cell lines was confirmed by whole-cell patch clamp recordings and proliferation assays, using siRNA-knockdown, BK channel activators such as 17ß-estradiol and the BK-channel blocker paxilline. Our findings revealed that enhanced expression of KCNMA1 correlates with and contributes to high proliferation rate and malignancy of breast cancer

Understanding the impacts of the McKay creek wildfire on vegetation trajectories and St’at’imc cultural values to inform restoration of post-wildfire landscapes

Devastating mega-wildfires have become the new normal throughout the interior of British Columbia, Canada, in recent years. These are the result of decades of colonial land management practices that have resulted in increased fuel loads and simplification of forest communities, the impacts of which are compounded by a changing climate. In 2021, the day after the historic heat dome, the McKay Creek Wildfire ignited 11 km north of Lillooet and ultimately burned 46,000 ha of land within St’at’imc Nation territory. We investigated the impacts of the wildfire on vegetation recovery in collaboration with the St’at’imc Nation communities of: Ts'kw'aylaxw, T'ít'q'et, and Xwísten, as well as the Lillooet Tribal Council and St'át'imc Government Services. Together we explored factors driving post-wildfire vegetation trajectories including burn severity, previous presence of invasive plants, and topographical factors, and how St’at’imc knowledges and cultural values can inform post-wildfire restoration. Our objective was to contribute to data-driven post-wildfire restoration approaches that uphold and support the sovereignty rights of Indigenous communities. Vegetation trajectories were analyzed by surveys of plots stratified by burn severity and previous known presence of invasive plants then placed using a mixed preferential approach guided by St’at’imc co-researchers. Research methods and analyses brought together Indigenous and Western scientific knowledges guided by the ‘walking on two legs’ framework, wherein each knowledge system, the ‘legs’, are guided by an Indigenous worldview. Results showed that two years post-wildfire, elevation and aspect were more significant factors in vegetation recovery and susceptibility to invasive plant invasion than burn severity or prior presence of invasive species. Native plant cover increased and non-native plant cover decreased with increasing elevation. Further analyses of plants when grouped by lifecycle, status (native or non-native), and by cultural values gave insight into recovery, suggesting that conventional functional plant groupings may overestimate recovery and underestimate plant invasion risk. Our research brings to light the importance of Indigenized, data-driven post-wildfire restoration as it provides the nuance needed to prioritize interventions across vast landscapes, while upholding Indigenous sovereignty rights in recovery processes.Forestry, Faculty ofGraduat