An atlas of altered expression of deubiquitinating enzymes in human cancer

Background

Deubiquitinating enzymes (DUBs) are proteases that process ubiquitin (Ub) or ubiquitin-like gene products, remodel polyubiquitin(-like) chains on target proteins, and counteract protein ubiquitination exerted by E3 ubiquitin-ligases. A wealth of studies has established the relevance of DUBs to the control of physiological processes whose subversion is known to cause cellular transformation, including cell cycle progression, DNA repair, endocytosis and signal transduction. Altered expression of DUBs might, therefore, subvert both the proteolytic and signaling functions of the Ub system.

Methodology/Principal Findings

In this study, we report the first comprehensive screening of DUB dysregulation in human cancers by in situ hybridization on tissue microarrays (ISH-TMA). ISH-TMA has proven to be a reliable methodology to conduct this kind of study, particularly because it allows the precise identification of the cellular origin of the signals. Thus, signals associated with the tumor component can be distinguished from those associated with the tumor microenvironment. Specimens derived from various normal and malignant tumor tissues were analyzed, and the “normal” samples were derived, whenever possible, from the same patients from whom tumors were obtained. Of the ∼90 DUBs encoded by the human genome, 33 were found to be expressed in at least one of the analyzed tissues, of which 22 were altered in cancers. Selected DUBs were subjected to further validation, by analyzing their expression in large cohorts of tumor samples. This analysis unveiled significant correlations between DUB expression and relevant clinical and pathological parameters, which were in some cases indicative of aggressive disease.

Conclusions/Significance

The results presented here demonstrate that DUB dysregulation is a frequent event in cancer, and have implications for therapeutic approaches based on DUB inhibition.     

Publication delay of randomized trials on 2009 influenza A (H1N1) vaccination

Background

Randomized evidence for vaccine immunogenicity and safety is urgently needed in the setting of pandemics with new emerging infectious agents. We carried out an observational survey to evaluate how many randomized controlled trials testing 2009 H1N1 vaccines were published among those registered, and what was the time lag from their start to publication and from their completion to publication.

Methods

PubMed, EMBASE and 9 clinical trial registries were searched for eligible randomized controlled trials. The units of the analysis were single randomized trials on any individual receiving influenza vaccines in any setting.

Results

73 eligible trials were identified that had been registered in 2009–2010. By June 30, 2011 only 21 (29%) of these trials had been published, representing 38% of the randomized sample size (19905 of 52765). Trials starting later were published less rapidly (hazard ratio 0.42 per month; 95% Confidence Interval: 0.27 to 0.64; p<0.001). Similarly, trials completed later were published less rapidly (hazard ratio 0.43 per month; 95% CI: 0.27 to 0.67; p<0.001). Randomized controlled trials were completed promptly (median, 5 months from start to completion), but only a minority were subsequently published.

Conclusions

Most registered randomized trials on vaccines for the H1N1 pandemic are not published in the peer-reviewed literature.     

Fetal DNA detection in maternal plasma throughout gestation

The presence of fetal DNA in maternal plasma may represent a source of genetic material which can be obtained noninvasively. We wanted to assess whether fetal DNA is detectable in all pregnant women, to define the range and distribution of fetal DNA concentration at different gestational ages, to identify the optimal period to obtain a maternal blood sample yielding an adequate amount of fetal DNA for prenatal diagnosis, and to evaluate accuracy and predictive values of this approach. This information is crucial to develop safe and reliable non-invasive genetic testing in early pregnancy and monitoring of pregnancy complications in late gestation. Fetal DNA quantification in maternal plasma was carried out by real-time PCR on the SRY gene in male-bearing pregnancies to distinguish between maternal and fetal DNA. A cohort of 1,837 pregnant women was investigated. Fetal DNA could be detected from the sixth week and could be retrieved at any gestational week. No false-positive results were obtained in 163 women with previous embryo loss or previous male babies. Fetal DNA analysis performed blindly on a subset of 464 women displayed 99.4, 97.8 and 100% accuracy in fetal gender determination during the first, second, and third trimester of pregnancy, respectively. No SRY amplification was obtained in seven out of the 246 (2.8%) male-bearing pregnancies. Fetal DNA from maternal plasma seems to be an adequate and reliable source of genetic material for a noninvasive prenatal diagnostic approach.     

Improvement of a FRET-based indicator for cAMP by linker design and stabilization of donor-acceptor interaction

F?rster resonance energy transfer (FRET) technology has been used to develop genetically encoded fluorescent indicators for a variety of intracellular molecular events. Often, however, the poor dynamic range of such reporters prevents detection of subtle but physiologically relevant signals. Here we present a strategy for improving FRET efficiency between donor and acceptor fluorophores in a green fluorescent protein (GFP)-based protein indicator for cAMP. Such indicator is based on protein kinase A (PKA) and was generated by fusion of CFP and YFP to the regulatory and catalytic subunits of PKA, respectively. Our approach to improve FRET efficiency was to perform molecular dynamic simulations and modelling studies of the linker peptide (L11) joining the CFP moiety and the regulatory subunit in order to define its structure and use this information to design an improved linker. We found that L11 contains the X-Y-P-Y-D motif, which adopts a turn-like conformation that is stiffly conserved along the simulation time. Based on this finding, we designed a new linker, L22 in which the YPY motif was doubled in order to generate a stiffer peptide and reduce the mobility of the chromophore within the protein complex, thus favouring CFP/YFP dipole-dipole interaction and improving FRET efficiency. Molecular dynamic simulations of L22 showed, unexpectedly, that the conformational behaviour of L22 was very loose. Based on the analysis of the three principal conformational states visited by L22 during the simulation time, we modified its sequence in order to increase its rigidity. The resulting linker L20 displayed lower flexibility and higher helical content than L22. When inserted in the cAMP indicator, L20 yielded a probe showing almost doubled FRET efficiency and a substantially improved dynamic range.     

Conformational changes of murine polyomavirus capsid proteins induced by sialic acid binding

Murine polyomavirus (Py) infection initiates by the recognition of cell membrane molecules containing terminal sialic acid (SA) residues through specific binding pockets formed at the major capsid protein VP1 surface. VP1 Pockets 1, 2, and 3 bind terminal SA, Gal, and second branched SA, respectively. The consequence of recognition on viral cell entry remains elusive. In this work, we show that preincubation of Py with soluble compounds within Pocket 1 (N-acetyl or N-glycolyl neuraminic acids) increases Py cell binding and infectivity in murine 3T6 fibroblasts. In contrast, Gal does not significantly alter Py binding nor infectivity, whereas sialyllactose, in Pockets 1 and 2, decreases cell binding and infectivity. Binding experiments with Py virus-like particles confirmed the direct involvement of VP1 in this effect. To determine whether such results could reflect VP1 conformational changes induced by SA binding, protease digestion assays were performed after pretreatment of Py or virus-like particles with soluble receptor fragments. Binding of SA with the VP1 Pocket 1, but not of compounds interacting with Pocket 2, was associated with a transition of this protein from a protease-sensitive to a protease-resistant state. This effect was transmitted to the minor capsid proteins VP2 and VP3 in virus particles. Attachment of Py to cell monolayers similarly led to a VP1 trypsin-resistant pattern. Taken together, these data present evidence that initial binding of Py to terminal SA induces conformational changes in the viral capsid, which may influence subsequent virus cell entry steps.     

New N-(phenoxydecyl)phthalimide derivatives displaying potent inhibition activity towards α-glucosidase

Several members of a new family of non-sugar-type α-glucosidase inhibitors, bearing a phthalimide moiety connected to a variously substituted phenoxy ring by an alkyl chain, were synthesized and their activities were investigated. The efficacy of the inhibition activity appeared to be governed by the chain length of the substrate. Substrates possessing 10 carbons afforded the highest levels of activity, which were one to two orders of magnitude more potent than the known inhibitor 1-deoxynojirimycin (dNM). Furthermore, structure–activity relationship studies indicated a critical role of electron-withdrawing substituents at the phenoxy group for the activity. Derivatives bearing a chlorine atom along with a strong electron-withdrawing group, such as a nitro group, were the most potent of the series.     

Substrate preference of Bifidobacterium adolescentis MB 239: compared growth on single and mixed carbohydrates

The utilization of mono-, di-, and oligosaccharides by Bifidobacterium adolescentis MB 239 was investigated. Raffinose, fructooligosaccharides (FOS), lactose, and the monomeric moieties glucose and fructose were used. To establish a hierarchy of sugars preference, the kinetics of growth and sugar consumption were determined on individual and mixed carbohydrates. On single carbon sources, higher specific growth rates and cell yields were attained on di- and oligosaccharides compared to monosaccharides. Analysis of the carbohydrates in steady-state chemostat cultures, growing at the same dilution rate on FOS, lactose, or raffinose, showed that monomeric units and hydrolysis products were present. In chemostat cultures on individual carbohydrates, B. adolescentis MB 239 simultaneously displayed α-galactosidase, β-galactosidase, and β-fructofuranosidase activities on all the sugars, including monosaccharides. Glycosyl hydrolytic activities were found in cytosol, cell surface, and growth medium. Batch experiments on mixtures of carbohydrates showed that they were co-metabolized by B. adolescentis MB 239, even if different disappearance kinetics were registered. When mono-, di-, and oligosaccharides were simultaneously present in the medium, no precedence for monosaccharides utilization was observed, and di- and oligosaccharides were consumed before their constitutive moieties.