EUROCarbDB: An open-access platform for glycoinformatics

The EUROCarbDB project is a design study for a technical framework, which provides sophisticated, freely accessible, open-source informatics tools and databases to support glycobiology and glycomic research. EUROCarbDB is a relational database containing glycan structures, their biological context and, when available, primary and interpreted analytical data from high-performance liquid chromatography, mass spectrometry and nuclear magnetic resonance experiments. Database content can be accessed via a web-based user interface. The database is complemented by a suite of glycoinformatics tools, specifically designed to assist the elucidation and submission of glycan structure and experimental data when used in conjunction with contemporary carbohydrate research workflows. All software tools and source code are licensed under the terms of the Lesser General Public License, and publicly contributed structures and data are freely accessible. The public test version of the web interface to the EUROCarbDB can be found at http://www.ebi.ac.uk/eurocarb.     

Synthesis and characterisation of a novel tubulin-directed DO3A-colchicine conjugate with potential theranostic features

Colchicine is a known tubulin binding agent enabling necrosis in tumors. A novel tubulin-directed DO3A-colchicine conjugate and its Gd(III) complex were prepared from N-deacetylcolchicine, coupling alkaloid and polyaza-alicyclic functions via a peptide coupling methodology. The longitudinal proton relaxivity of the Gd(III) complex in water at 4.7 T is 2.86 mM⁻¹ s⁻¹ and a similar efficacy as colchicine towards ovarian carcinoma cells in vitro.     

Imaging the subcellular structure of human coronary atherosclerosis using micro-optical coherence tomography

Progress in understanding, diagnosis, and treatment of coronary artery disease (CAD) has been hindered by our inability to observe cells and extracellular components associated with human coronary atherosclerosis in situ. The current standards for microstructural investigation, histology and electron microscopy are destructive and prone to artifacts. The highest-resolution intracoronary imaging modality, optical coherence tomography (OCT), has a resolution of ~10 μm, which is too coarse for visualizing most cells. Here we report a new form of OCT, termed micro-optical coherence tomography (μOCT), whose resolution is improved by an order of magnitude. We show that μOCT images of cadaver coronary arteries provide clear pictures of cellular and subcellular features associated with atherogenesis, thrombosis and responses to interventional therapy. These results suggest that μOCT can complement existing diagnostic techniques for investigating atherosclerotic specimens, and that μOCT may eventually become a useful tool for cellular and subcellular characterization of the human coronary wall in vivo.     

Chemical defense by the native winter ant (Prenolepis imparis) against the invasive Argentine ant (Linepithema humile)

The invasive Argentine ant (Linepithema humile) is established worldwide and displaces native ant species. In northern California, however, the native winter ant (Prenolepis imparis) persists in invaded areas. We found that in aggressive interactions between the two species, P. imparis employs a potent defensive secretion. Field observations were conducted at P. imparis nest sites both in the presence and absence of L. humile. These observations suggested and laboratory assays confirmed that P. imparis workers are more likely to secrete when outnumbered by L. humile. Workers of P. imparis were also more likely to secrete near their nest entrances than when foraging on trees. One-on-one laboratory trials showed that the P. imparis secretion is highly lethal to L. humile, causing 79% mortality. The nonpolar fraction of the secretion was chemically analyzed with gas chromatography/mass spectrometry, and found to be composed of long-chain and cyclic hydrocarbons. Chemical analysis of dissected P. imparis workers showed that the nonpolar fraction is derived from the Dufour's gland. Based on these conclusions, we hypothesize that this chemical defense may help P. imparis to resist displacement by L. humile.     

A novel peptide ELISA for universal detection of antibodies to human H5N1 influenza viruses

Background

Active serologic surveillance of H5N1 highly pathogenic avian influenza (HPAI) virus in humans and poultry is critical to control this disease. However, the need for a robust, sensitive and specific serologic test for the rapid detection of antibodies to H5N1 viruses has not been met.

Methodology/Principal Findings

Previously, we reported a universal epitope (CNTKCQTP) in H5 hemagglutinin (HA) that is 100% conserved in H5N1 human isolates and 96.9% in avian isolates. Here, we describe a peptide ELISA to detect antibodies to H5N1 virus by using synthetic peptide that comprises the amino acid sequence of this highly conserved and antigenic epitope as the capture antigen. The sensitivity and specificity of the peptide ELISA were evaluated using experimental chicken antisera to H5N1 viruses from divergent clades and other subtype influenza viruses, as well as human serum samples from patients infected with H5N1 or seasonal influenza viruses. The peptide ELISA results were compared with hemagglutinin inhibition (HI), and immunofluorescence assay and immunodot blot that utilize recombinant HA1 as the capture antigen. The peptide ELISA detected antibodies to H5N1 in immunized animals or convalescent human sera whereas some degree of cross-reactivity was observed in HI, immunofluorescence assay and immunodot blot. Antibodies to other influenza subtypes tested negative in the peptide-ELISA.

Conclusion/Significance

The peptide-ELISA based on the highly conserved and antigenic H5 epitope (CNTKCQTP) provides sensitive and highly specific detection of antibodies to H5N1 influenza viruses. This study highlighted the use of synthetic peptide as a capture antigen in rapid detection of antibodies to H5N1 in human and animal sera that is robust, simple and cost effective and is particularly beneficial for developing countries and rural areas.     

Qualitative and quantitative multiplexed proteomic analysis of complex yeast protein fractions that modulate the assembly of the yeast prion Sup35p

Background

The aggregation of the baker''s yeast prion Sup35p is at the origin of the transmissible [PSI⁺] trait. We and others have shown that molecular chaperones modulate Sup35p aggregation. However, other protein classes might be involved in [PSI⁺] formation.

Results

We designed a functional proteomic study that combines two techniques to identify modulators of Sup35p aggregation and describe the changes associated to [PSI⁺] formation. The first allows measuring the effect of fractionated Saccharomyces cerevisiae cytosolic extracts from [PSI⁺] and [psi⁻] yeast cells on Sup35p assembly. The second is a multiplex qualitative and quantitative comparison of protein composition of active and inactive fractions using a gel-free and label-free LC-MS approach. We identify changes in proteins involved in translation, folding, degradation, oxido-reduction and metabolic processes.

Conclusion

Our functional proteomic study provides the first inventory list of over 300 proteins that directly or indirectly affect Sup35p aggregation and [PSI⁺] formation. Our results highlight the complexity of the cellular changes accompanying [PSI⁺] formation and pave the way for in vitro studies aimed to document the effect of individual and/or combinations of proteins identified here, susceptible of affecting Sup35p assembly.     

Accumulation of mitochondrial DNA deletions is age, tissue and folate-dependent in rats

Folate is essential for the synthesis, repair and methylation of DNA. Folate depletion causes nuclear genetic and epigenetic aberrations in cell culture, rodents and humans. We hypothesized that folate depletion may also damage mitochondrial (Mt) DNA and induce large-scale deletions due to DNA breakage. MtDNA deletions and mutations accumulate during aging and tumorogenesis and may play causative roles in these processes. Weanling and adult (12 months) Sprague Dawley rats consumed folate deplete, replete and supplemented diets (0, 2 and 8 mg/kg folate, respectively) for 20 weeks. The presence of random and common (4.8 kb) MtDNA deletions was measured in colonic mucosa and liver. Six Mt genomes (<16 kb) harboring random deletions were detected in the liver (3.5-7.0 kb) and three in the colon (3.8-8 kb). Older rats had significantly more random hepatic MtDNA deletions than young rats (64 and 3.2% of samples, respectively, P < 0.0001), while age had no effect on these deletions in the colon (3.1 and 7.7% in young and old, respectively). Folate intake had no effect on the frequency of random deletions in either tissue. There was no discrete effect of aging on the common 4.8 kb deletion in the liver or colon. However, in the liver of old rats, increasing amounts of dietary folate reduced the deletion frequency, with replete and supplemented rats having 2.2- and 3.2-fold less deletions than the depleted rats. Our results confirm that random MtDNA deletions accumulate with age in a tissue-specific fashion. Furthermore, in contrast to previous work, we report that the common 4.8 kb deletion was not modulated by age, but is reduced by folate supplementation in the liver of rats.     

Expression of heat shock proteins and heat shock protein messenger ribonucleic acid in human prostate carcinoma in vitro and in tumors in vivo

Heat shock proteins (HSPs) are thought to play a role in the development of cancer and to modulate tumor response to cytotoxic therapy. In this study, we have examined the expression of hsf and HSP genes in normal human prostate epithelial cells and a range of prostate carcinoma cell lines derived from human tumors. We have observed elevated expressions of HSF1, HSP60, and HSP70 in the aggressively malignant cell lines PC-3, DU-145, and CA-HPV-10. Elevated HSP expression in cancer cell lines appeared to be regulated at the post-messenger ribonucleic acid (mRNA) levels, as indicated by gene chip microarray studies, which indicated little difference in heat shock factor (HSF) or HSP mRNA expression between the normal and malignant prostate cell lines. When we compared the expression patterns of constitutive HSP genes between PC-3 prostate carcinoma cells growing as monolayers in vitro and as tumor xenografts growing in nude mice in vivo, we found a marked reduction in expression of a wide spectrum of the HSPs in PC-3 tumors. This decreased HSP expression pattern in tumors may underlie the increased sensitivity to heat shock of PC-3 tumors. However, the induction by heat shock of HSP genes was not markedly altered by growth in the tumor microenvironment, and HSP40, HSP70, and HSP110 were expressed abundantly after stress in each growth condition. Our experiments indicate therefore that HSF and HSP levels are elevated in the more highly malignant prostate carcinoma cells and also show the dominant nature of the heat shock-induced gene expression, leading to abundant HSP induction in vitro or in vivo.