Minimizing proteome redundancy in the UniProt Knowledgebase.

Advances in high-throughput sequencing have led to an unprecedented growth in genome sequences being submitted to biological databases. In particular, the sequencing of large numbers of nearly identical bacterial genomes during infection outbreaks and for other large-scale studies has resulted in a high level of redundancy in nucleotide databases and consequently in the UniProt Knowledgebase (UniProtKB). Redundancy negatively impacts on database searches by causing slower searches, an increase in statistical bias and cumbersome result analysis. The redundancy combined with the large data volume increases the computational costs for most reuses of UniProtKB data. All of this poses challenges for effective discovery in this wealth of data. With the continuing development of sequencing technologies, it is clear that finding ways to minimize redundancy is crucial to maintaining UniProt's essential contribution to data interpretation by our users. We have developed a methodology to identify and remove highly redundant proteomes from UniProtKB. The procedure identifies redundant proteomes by performing pairwise alignments of sets of sequences for pairs of proteomes and subsequently, applies graph theory to find dominating sets that provide a set of non-redundant proteomes with a minimal loss of information. This method was implemented for bacteria in mid-2015, resulting in a removal of 50 million proteins in UniProtKB. With every new release, this procedure is used to filter new incoming proteomes, resulting in a more scalable and scientifically valuable growth of UniProtKB.Database URL: http://www.uniprot.org/proteomes/.

GPSy: a cross-species gene prioritization system for conserved biological processes--application in male gamete development.

We present gene prioritization system (GPSy), a cross-species gene prioritization system that facilitates the arduous but critical task of prioritizing genes for follow-up functional analyses. GPSy's modular design with regard to species, data sets and scoring strategies enables users to formulate queries in a highly flexible manner. Currently, the system encompasses 20 topics related to conserved biological processes including male gamete development discussed in this article. The web server-based tool is freely available at http://gpsy.genouest.org.

Cytotoxicity of half sandwich ruthenium(II) complexes with strong hydrogen bond acceptor ligands and their mechanism of action.

Neutral and cationic organometallic ruthenium(II) piano stool complexes of the type [(eta(6)-cymene)RuCl(X)(Y)] (complexes R1-R8) has been synthesized and characterized. In cationic complexes, X, Y is either a eta(2) phosphorus ligand such as 1,1-bis(diphenylphosphino)methane (DPPM) and 1,2-bis(diphenylphosphino)ethane (DPPE) or partially oxidized ligands such as 1,2-bis(diphenylphosphino)methane monooxide (DPPMO) and 1,2-bis(diphenylphosphino)ethane monooxide (DPPEO) which are strong hydrogen bond acceptors. In neutral complexes, X is chloride and Y is a monodentate phosphorous donor. Complexes with DPPM and DPPMO ligands ([(eta(6)-cymene)Ru(eta(2)-DPPM)Cl]PF(6) (R2), [(eta(6)-cymene)Ru(eta(2)-DPPMO)Cl]PF(6) (R3), [(eta(6)-cymene)Ru(eta(1)-DPPM)Cl(2)] (R5) and [(eta(6)-cymene)Ru(eta(1)-DPPMO)Cl(2)] (R6) show good cytotoxicity. Growth inhibition study of several human cancer cell lines by these complexes has been carried out. Mechanistic studies for R5 and R6 show that inhibition of cancer cell growth involves both cell cycle arrest and apoptosis induction. Using an apoptosis PCR array, we identified the sets of anti-apoptotic genes that were down regulated and pro-apoptotic genes that were up regulated. These complexes were also found to be potent metastasis inhibitors as they prevented cell invasion through matrigel. The complexes were shown to bind DNA in a non intercalative fashion and cause unwinding of plasmid DNA in cell-free medium by competitive ethidium bromide binding, viscosity measurements, thermal denaturation and gel mobility shift assays.

Novel glioblastoma markers with diagnostic and prognostic value identified through transcriptome analysis.

PURPOSE: Current methods of classification of astrocytoma based on histopathologic methods are often subjective and less accurate. Although patients with glioblastoma have grave prognosis, significant variability in patient outcome is observed. Therefore, the aim of this study was to identify glioblastoma diagnostic and prognostic markers through microarray analysis. EXPERIMENTAL DESIGN: We carried out transcriptome analysis of 25 diffusely infiltrating astrocytoma samples [WHO grade II--diffuse astrocytoma, grade III--anaplastic astrocytoma, and grade IV--glioblastoma (GBM)] using cDNA microarrays containing 18,981 genes. Several of the markers identified were also validated by real-time reverse transcription quantitative PCR and immunohistochemical analysis on an independent set of tumor samples (n = 100). Survival analysis was carried out for two markers on another independent set of retrospective cases (n = 51). RESULTS: We identified several differentially regulated grade-specific genes. Independent validation by real-time reverse transcription quantitative PCR analysis found growth arrest and DNA-damage-inducible alpha (GADD45alpha) and follistatin-like 1 (FSTL1) to be up-regulated in most GBMs (both primary and secondary), whereas superoxide dismutase 2 and adipocyte enhancer binding protein 1 were up-regulated in the majority of primary GBM. Further, identification of the grade-specific expression of GADD45alpha and FSTL1 by immunohistochemical staining reinforced our findings. Analysis of retrospective GBM cases with known survival data revealed that cytoplasmic overexpression of GADD45alpha conferred better survival while the coexpression of FSTL1 with p53 was associated with poor survival. CONCLUSIONS: Our study reveals that GADD45alpha and FSTLI are GBM-specific whereas superoxide dismutase 2 and adipocyte enhancer binding protein 1 are primary GBM-specific diagnostic markers. Whereas GADD45alpha overexpression confers a favorable prognosis, FSTL1 overexpression is a hallmark of poor prognosis in GBM patients.

Shift in AP-2alpha localization characterizes astrocytoma progression.

Activator protein 2alpha (AP-2alpha) has been shown to be lost in the advanced stages of many cancers, including gliomas. In this study, we wanted to analyze the expression of AP-2alpha in astrocytoma samples of different grades both at the RNA level, by real-time qPCR and at the protein level, by immunohistochemistry, and to examine its correlation, if any, with patient outcome. Five Grade I, 14 Grade II, 18 Grade III, 72 Grade IV samples and 13 normal brain controls were included. We did not find any clear pattern of regulation at the RNA level with tumor grade. The RNA expression levels however, correlated to a large extent with the nuclear AP-2alpha staining in these samples (72.09%; 31/43). Further, we did not find a complete loss of nuclear AP-2alpha expression in the higher grades, in contrast to previous reports. Interestingly, we found cytoplasmic AP-2alpha expression in a majority of higher grade astrocytomas (Grade IV-85%; 33/39 and Grade III-74%; 14/19) in comparison to lower grades (Grade I-0%; 0/5 and Grade II-37.5%; 3/8) suggesting that the translocation of this protein from the nucleus to the cytoplasm may be responsible for the increased malignancy. The nuclear expression in these grades was found to be concomitantly reduced. Within GBMs, we found that decreased nuclear expression was indicative of a better prognosis. The striking observation was the shift in localization of this protein from the nucleus to the cytoplasm with increasing tumor grade, pointing to a crucial role for this transcription factor in the progression of astrocytomas.

Apoptosis induction by activator protein 2alpha involves transcriptional repression of Bcl-2.

Activator protein 2alpha (AP-2alpha) induces cytotoxicity by inducing cell cycle arrest and apoptosis. In this study we investigated the mechanism of apoptosis induction by AP-2alpha. We found that AP-2alpha induced apoptosis efficiently in cells treated with benzyloxycar-bonyl-IETD-fluoromethyl ketone or FADD-silenced cells but failed to do so in benzyloxycarbonyl-LEHD-fluoromethyl ketone-treated or apoptosis protease activation factor-1 (Apaf1)-silenced cells, suggesting the central role of mitochondria in AP-2alpha-induced apoptosis. In good correlation, cells overexpressing AP-2alpha showed a reduction in mitochondrial membrane potential (Deltapsi(m)), cytochrome c and Smac/DIABLO release into cytosol, and Bax translocation into mitochondria. We found that the pro-apoptotic protein Bax is important for AP-2alpha-induced apoptosis as adenovirus AP2 failed to induce apoptosis in HCT116 Bax(-/-) cells. However, we found the IAP (inhibitor of apoptosis) inhibitor Smac/DIABLO may have a limited role in AP-2alpha-induced apoptosis as we found the IAP member Survivin down-regulated by AP-2alpha. Although the total Bax level remains unaltered, we found a time-dependent increase in the activated form of Bax in adenovirus AP2-infected cells. In addition, we show that AP-2alpha transcriptionally represses Bcl-2 by binding to its promoter both in vitro and in vivo and that this is essential for AP-2alpha-induced apoptosis as ectopic expression of Bcl-2 efficiently inhibited apoptosis induced by AP-2alpha. Furthermore, we show that chemotherapy-induced endogenous AP-2alpha down-regulates Bcl-2 and induces apoptosis in an AP-2alpha-dependent manner. Moreover, we demonstrate that inhibition of okadaic acid or staurosporine-sensitive pathways in AP-2alpha overexpressing breast cancer cells resulted in AP-2alpha-dependent apoptosis induction. These results suggest that AP-2alpha induces apoptosis by down-regulating Bcl-2 and utilizing a bax/cytochrome c/Apaf1/caspase 9-dependent mitochondrial pathway.

Upregulation of ASCL1 and inhibition of Notch signaling pathway characterize progressive astrocytoma.

Astrocytoma is the most common type of brain cancer constituting more than half of all brain tumors. With an aim to identify markers describing astrocytoma progression, we have carried out microarray analysis of astrocytoma samples of different grades using cDNA microarray containing 1152 cancer-specific genes. Data analysis identified several differentially regulated genes between normal brain tissue and astrocytoma as well as between grades II/III astrocytoma and glioblastoma multiforme (GBM; grade IV). We found several genes known to be involved in malignancy including Achaete-scute complex-like 1 (Drosophila) (ASCL1; Hash 1). As ASCL has been implicated in neuroendocrine, medullary thyroid and small-cell lung cancers, we chose to examine the role of ASCL1 in the astrocytoma development. Our data revealed that ASCL1 is overexpressed in progressive astrocytoma as evidenced by increased levels of ASCL1 transcripts in 85.71% (6/7) of grade II diffuse astrocytoma (DA), 90% (9/10) of grade III anaplastic astrocytoma (AA) and 87.5% (7/8) of secondary GBMs, while the majority of primary de novo GBMs expressed similar to or less than normal brain levels (66.67%; 8/12). ASCL1 upregulation in progressive astrocytoma is accompanied by inhibition of Notch signaling as seen by uninduced levels of HES1, a transcriptional target of Notch1, increased levels of HES6, a dominant-negative inhibitor of HES1-mediated repression of ASCL1, and increased levels of Notch ligand Delta1, which is capable of inhibiting Notch signaling by forming intracellular Notch ligand autonomous complexes. Our results imply that inhibition of Notch signaling may be an important early event in the development of grade II DA and subsequent progression to grade III AA and secondary GBM. Furthermore, ASCL1 appears to be a putative marker to distinguish primary GBM from secondary GBM.