Loss of AQP3 protein expression is associated with worse progression-free and cancer-specific survival in patients with muscle-invasive bladder cancer

Purpose Urothelial carcinoma has recently been shown to express several aquaporins (AQP), with AQP3 being of particular interest as its expression is reduced or lost in tumours of higher grade and stage. Loss of AQP3 expression was associated with worse progression-free survival (PFS) in patients with pT1 bladder cancer. The objective of this study was to investigate the prognostic value of AQP3 expression in patients with muscle-invasive bladder carcinoma (MIBC). Methods Retrospective single-centre analysis of the oncological outcome of patients following radical cystectomy (Cx) due to MIBC. Immunohistochemistry was used to assess AQP3 protein expression in 100 Cx specimens. Expression levels of AQP3 were related to clinicopathological variables. The impact of biomarker expression on progression-free, cancer-specific and overall survival was determined by multivariate Cox regression analysis (MVA). Results High expression of AQP3 by the tumour was associated with a statistically significantly improved PFS (75 vs. 19 %, p = 0.043) and CSS (75 vs. 18 %, p = 0.030) and, alongside lymph node involvement, was an independent predictor of PFS (HR 2.871, CI 1.066–7.733, p = 0.037), CSS (HR 3.325, CI 1.204–8.774, p = 0.019) and OS (HR 2.001, CI 1.014–3.947) in MVA. Conclusions Although the results of the study would be strengthened by a larger, more appropriately powered, prospective, multi-institutional study, our findings strongly suggest that AQP3 expression status may represent an independent predictor of PFS and CSS in MIBC and may help select patients in need for (neo-)adjuvant chemotherapy

Disease proteomics

Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/62680/1/nature01514.pd

Real-time PCR probe optimization using design of experiments approach

AbstractPrimer and probe sequence designs are among the most critical input factors in real-time polymerase chain reaction (PCR) assay optimization. In this study, we present the use of statistical design of experiments (DOE) approach as a general guideline for probe optimization and more specifically focus on design optimization of label-free hydrolysis probes that are designated as mediator probes (MPs), which are used in reverse transcription MP PCR (RT-MP PCR). The effect of three input factors on assay performance was investigated: distance between primer and mediator probe cleavage site; dimer stability of MP and target sequence (influenza B virus); and dimer stability of the mediator and universal reporter (UR). The results indicated that the latter dimer stability had the greatest influence on assay performance, with RT-MP PCR efficiency increased by up to 10% with changes to this input factor. With an optimal design configuration, a detection limit of 3–14 target copies/10μl reaction could be achieved. This improved detection limit was confirmed for another UR design and for a second target sequence, human metapneumovirus, with 7–11 copies/10μl reaction detected in an optimum case. The DOE approach for improving oligonucleotide designs for real-time PCR not only produces excellent results but may also reduce the number of experiments that need to be performed, thus reducing costs and experimental times

Two distinct activities contribute to the oncogenic potential of the adenovirus type 5 E4orf6 protein

Previous studies have shown that the adenovirus type 5 (Ad5) E4orf6 gene product displays features of a viral oncoprotein. It initiates focal transformation of primary rat cells in cooperation with Ad5 E1 genes and confers multiple additional transformed properties on E1-expressing cells, including profound morphological alterations and dramatically accelerated tumor growth in nude mice. It has been reported that E4orf6 binds to p53 and, in the presence of the Ad5 E1B-55kDa protein, antagonizes p53 stability by targeting the tumor suppressor protein for active degradation. In the present study, we performed a comprehensive mutant analysis to assign transforming functions of E4orf6 to distinct regions within the viral polypeptide and to analyze a possible correlation between E4orf6-dependent p53 degradation and oncogenesis. Our results show that p53 destabilization maps to multiple regions within both amino- and carboxy-terminal parts of the viral protein and widely cosegregates with E4orf6-dependent acceleration of tumor growth, indicating that both effects are related. In contrast, promotion of focus formation and morphological transformation require only a carboxy-terminal segment of the E4 protein. Thus, these effects are completely independent of p53 stability, but may involve other interactions with the tumor suppressor. Our results demonstrate that at least two distinct activities contribute to the oncogenic potential of Ad5 E4orf6. Although genetically separable, both activities are largely mediated through a novel highly conserved, cysteine-rich motif and a recently described arginine-faced amphipathic alpha helix, which resides within a carboxy-terminal "oncodomain" of the viral protein

The adenovirus E4orf6 protein can promote E1A/E1B-induced focus formation by interfering with p53 tumor suppressor function

We have recently shown that the adenovirus type 5 E4orf6 protein interacts with the cellular tumor suppressor protein p53 and blocks p53 transcriptional functions. Here we report that the E4orf6 protein can promote focus formation of primary rodent epithelial cells in cooperation with adenovirus E1A and E1A plus E1B proteins. The E4orf6 protein can also inhibit p53-mediated suppression of E1A plus E1B-19kDa-induced focus formation. Mutant analysis of the E4orf6 protein demonstrates that these activities correlate with the ability of the adenovirus protein to relieve transcriptional repression mediated by the carboxyl-terminal region of p53 in transient transfection assays. We further demonstrate that expression of wild-type E4orf6 correlates with a dramatic reduction of p53 steady-state levels in transformed rat cells. Our data demonstrate that adenovirus type 5 encodes two different proteins, E1B-55kDa and E4orf6, that bind to p53 and contribute to transformation by modulating p53 transcriptional functions

Structural analysis of the adenovirus type 5 E1B 55-kilodalton-E4orf6 protein complex.

The adenovirus type 5 (Ad5) early 1B (E1B) 55-kDa (E1B-55kDa)-E4orf6 protein complex has been implicated in the selective modulation of nucleocytoplasmic mRNA transport at late times after infection. Using a combined immunoprecipitation-immunoblotting assay, we mapped the domains in E1B-55kDa required for the interaction with the E4orf6 protein in lytically infected A549 cells. Several domains in the 496-residue 55-kDa polypeptide contributed to a stable association with the E4orf6 protein in E1B mutant virus-infected cells. Linker insertion mutations at amino acids 180 and 224 caused reduced binding of the E4orf6 protein, whereas linker insertion mutations at amino acid 143 and in the central domain of E1B-55kDa eliminated the binding of the E4orf6 protein. Earlier work showing that the central domain of E1B-55kDa is required for binding to p53 and the recent observation that the E4orf6 protein also interacts with the tumor suppressor protein led us to suspect that p53 might play a role in the E1B-E4 protein interaction. However, coimmunoprecipitation assays with extracts prepared from infected p53-negative H1299 cells established that p53 is not needed for the E1B-E4 protein interaction in adenovirus-infected cells. Using two different protein-protein interaction assays, we also mapped the region in the E4orf6 protein required for E1B-55kDa interaction to the amino-terminal 55 amino acid residues. Interestingly, both binding assays established that the same region in the E4orf6/7 protein can potentially interact with E1B-55kDa. Our results demonstrate that two distinct segments in the 55-kDa protein encoding the transformation and late lytic functions independently interact with p53 and the E4orf6 protein in vivo and provide further insight by which the multifunctional 55-kDa EIB protein can exert its multiple activities in lytically infected cells and in adenovirus transformation

Structural analysis of the adenovirus type 5 E1B 55-kilodalton-E4orf6 protein complex

The adenovirus type 5 (Ad5) early 1B (E1B) 55-kDa (E1B-55kDa)-E4orf6 protein complex has been implicated in the selective modulation of nucleocytoplasmic mRNA transport at late times after infection. Using a combined immunoprecipitation-immunoblotting assay, we mapped the domains in E1B-55kDa required for the interaction with the E4orf6 protein in lytically infected A549 cells. Several domains in the 496-residue 55-kDa polypeptide contributed to a stable association with the E4orf6 protein in E1B mutant virus-infected cells. Linker insertion mutations at amino acids 180 and 224 caused reduced binding of the E4orf6 protein, whereas linker insertion mutations at amino acid 143 and in the central domain of E1B-55kDa eliminated the binding of the E4orf6 protein. Earlier work showing that the central domain of E1B-55kDa is required for binding to p53 and the recent observation that the E4orf6 protein also interacts with the tumor suppressor protein led us to suspect that p53 might play a role in the E1B-E4 protein interaction. However, coimmunoprecipitation assays with extracts prepared from infected p53-negative H1299 cells established that p53 is not needed for the E1B-E4 protein interaction in adenovirus-infected cells. Using two different protein-protein interaction assays, we also mapped the region in the E4orf6 protein required for E1B-55kDa interaction to the amino-terminal 55 amino acid residues. Interestingly, both binding assays established that the same region in the E4orf6/7 protein can potentially interact with E1B-55kDa. Our results demonstrate that two distinct segments in the 55-kDa protein encoding the transformation and late lytic functions independently interact with p53 and the E4orf6 protein in vivo and provide further insight by which the multifunctional 55-kDa EIB protein can exert its multiple activities in lytically infected cells and in adenovirus transformation

The adenovirus E4orf6 protein can promote E1A/E1B-induced focus formation by interfering with p53 tumor suppressor function

We have recently shown that the adenovirus type 5 E4orf6 protein interacts with the cellular tumor suppressor protein p53 and blocks p53 transcriptional functions. Here we report that the E4orf6 protein can promote focus formation of primary rodent epithelial cells in cooperation with adenovirus E1A and E1A plus E1B proteins. The E4orf6 protein can also inhibit p53-mediated suppression of E1A plus E1B-19kDa-induced focus formation. Mutant analysis of the E4orf6 protein demonstrates that these activities correlate with the ability of the adenovirus protein to relieve transcriptional repression mediated by the carboxyl-terminal region of p53 in transient transfection assays. We further demonstrate that expression of wild-type E4orf6 correlates with a dramatic reduction of p53 steady-state levels in transformed rat cells. Our data demonstrate that adenovirus type 5 encodes two different proteins, E1B-55kDa and E4orf6, that bind to p53 and contribute to transformation by modulating p53 transcriptional functions

Structural analysis of the adenovirus type 5 E1B 55-kilodalton-E4orf6 protein complex

The adenovirus type 5 (Ad5) early 1B (E1B) 55-kDa (E1B-55kDa)-E4orf6 protein complex has been implicated in the selective modulation of nucleocytoplasmic mRNA transport at late times after infection. Using a combined immunoprecipitation-immunoblotting assay, we mapped the domains in E1B-55kDa required for the interaction with the E4orf6 protein in lytically infected A549 cells. Several domains in the 496-residue 55-kDa polypeptide contributed to a stable association with the E4orf6 protein in E1B mutant virus-infected cells. Linker insertion mutations at amino acids 180 and 224 caused reduced binding of the E4orf6 protein, whereas linker insertion mutations at amino acid 143 and in the central domain of E1B-55kDa eliminated the binding of the E4orf6 protein. Earlier work showing that the central domain of E1B-55kDa is required for binding to p53 and the recent observation that the E4orf6 protein also interacts with the tumor suppressor protein led us to suspect that p53 might play a role in the E1B-E4 protein interaction. However, coimmunoprecipitation assays with extracts prepared from infected p53-negative H1299 cells established that p53 is not needed for the E1B-E4 protein interaction in adenovirus-infected cells. Using two different protein-protein interaction assays, we also mapped the region in the E4orf6 protein required for E1B-55kDa interaction to the amino-terminal 55 amino acid residues. Interestingly, both binding assays established that the same region in the E4orf6/7 protein can potentially interact with E1B-55kDa. Our results demonstrate that two distinct segments in the 55-kDa protein encoding the transformation and late lytic functions independently interact with p53 and the E4orf6 protein in vivo and provide further insight by which the multifunctional 55-kDa EIB protein can exert its multiple activities in lytically infected cells and in adenovirus transformation.</jats:p

A highly efficient buckypaper-based electrode material for mediatorless laccase-catalyzed dioxygen reduction

a b s t r a c t The redox enzyme laccase from Trametes versicolor efficiently catalyzes the oxygen reduction reaction (ORR) in mediatorless biofuel cell cathodes when adsorbed onto multi-walled carbon nanotubes (MWCNTs). In this work we demonstrate that the fabrication of MWCNTs in form of buckypaper (BP) results in an excellent electrode material for laccase-catalyzed cathodes. BPs are mechanically stable, self-entangling mats with high dispersion of MWCNTs resulting in easy to handle homogeneous layers with highly mesoporous structures and excellent electrical conductivities. All biocathodes have been electrochemically investigated in oxygen-saturated buffer at pH 5 by galvanostatic polarization and potentiodynamic linear sweep voltammetry. Both methods confirm an efficient direct interaction of laccase with BP with a high open circuit potential of 0.882 V vs. normal hydrogen electrode (NHE). The high oxygen reduction performance leads to high current densities of 422 ± 71 A cm −2 at a typical cathode potential of 0.744 V vs. NHE. When the current density is normalized to the mass of the electrode material (mass activity), the BPbased film electrodes exhibit a 68-fold higher current density at 0.744 V vs. NHE than electrodes fabricated from the same MWCNTs in a non-dispersed agglomerated form as packed electrodes. This clearly shows that MWCNTs can act more efficiently as cathode when prepared in form of BP. This can be attributed to reduced diffusional mass transfer limitations and enhanced electrical conductivity. BP is thus a very promising material for the construction of mediatorless laccase cathodes for ORR in biofuel cells. In addition we demonstrated that these electrodes exhibit a high tolerance towards glucose, the most common bioanode fuel