SiDCoN: a tool to aid scoring of DNA copy number changes in SNP chip data

The recent application of genome-wide, single nucleotide polymorphism (SNP) microarrays to investigate DNA copy number aberrations in cancer has provided unparalleled sensitivity for identifying genomic changes. In some instances the complexity of these changes makes them difficult to interpret, particularly when tumour samples are contaminated with normal (stromal) tissue. Current automated scoring algorithms require considerable manual data checking and correction, especially when assessing uncultured tumour specimens. To address these limitations we have developed a visual tool to aid in the analysis of DNA copy number data. Simulated DNA Copy Number (SiDCoN) is a spreadsheet-based application designed to simulate the appearance of B-allele and logR plots for all known types of tumour DNA copy number changes, in the presence or absence of stromal contamination. The system allows the user to determine the level of stromal contamination, as well as specify up to 3 different DNA copy number aberrations for up to 5000 data points (representing individual SNPs). This allows users great flexibility to assess simple or complex DNA copy number combinations. We demonstrate how this utility can be used to estimate the level of stromal contamination within tumour samples and its application in deciphering the complex heterogeneous copy number changes we have observed in a series of tumours. We believe this tool will prove useful to others working in the area, both as a training tool, and to aid in the interpretation of complex copy number changes.     

Evidence for the formation of a novel nitrosothiol from the gaseous mediators nitric oxide and hydrogen sulphide

The gaseous mediators hydrogen sulphide (H2S) and nitric oxide (*NO) are synthesised in the body from L-cysteine and L-arginine, respectively. In the cardiovascular system, *NO is an important regulator of vascular tone and its over- or under-production has been linked to a variety of diseases. The physiological significance of H2S is not yet clear but, like *NO, it exhibits vasodilator activity and may play a part in septic and haemorrhagic shock, hypertension, regulation of cardiac contractility, and in inflammation. To date, there have been no reports of a chemical interaction between H2S and *NO. Here we show that incubation of the H2S donor, sodium hydrosulphide, with a range of *NO donors and *NO gas in vitro leads to the formation of a nitrosothiol molecule as determined by a combination of techniques; electron paramagnetic resonance, amperometry, and measurement of nitrite. We further show that this nitrosothiol did not induce cGMP accumulation in cultured RAW264.7 cells unless *NO was released with Cu2+. Finally, using liver homogenates from LPS treated rats we present evidence for the endogenous formation of this nitrosothiol. These findings provide the first evidence for the formation of a novel nitrosothiol generated by reaction between H2S and *NO. We propose that generation of this nitrosothiol in the body may regulate the physiological effects of both *NO and H2S.     

Cardioprotective effects of nitroparacetamol and paracetamol in acute phase of myocardial infarction in experimental rats

We aimed to determine whether nitroparacetamol (NO-paracetamol) and paracetamol exhibit cardioprotective effects. Myocardial infarction (MI) was induced in rats, and drug treatment was started 1 wk before surgery. Mortality rate and infarct size at 2 days after MI were compared. Treatment groups included vehicle (saline), paracetamol (5 mg x kg(-1) x day(-1)) and NO-paracetamol (15 mg x kg(-1) x day(-1)). Mortality rates for vehicle (n = 80), paracetamol (n = 79), and NO-paracetamol (n = 76) groups were 37.5%, 21.5%, and 26.3%, respectively. Infarct size for the vehicle group was 44.8% (+/-6.1%) of the left ventricle (LV). For the paracetamol and NO-paracetamol groups, infarct size was 31.3% (+/-5.6%) and 30.7% (+/-8.1%) of the LV, respectively. Both paracetamol- and NO-paracetamol-treated groups showed increased activities of catalase and SOD compared with the vehicle group. They could attenuate endothelial, inducible, and neuronal nitric oxide synthase and cyclooxygenase-1 and -2 gene expression after MI. The observation indicates the potential clinical significance of the cardioprotective effects of these drugs.     

Eight polymorphic microsatellite markers isolated from the widespread avian louse Colpocephalum turbinatum (Phthiraptera: Amblycera: Menoponidae)

We report eight novel microsatellite loci for Colpocephalum turbinatum, a parasitic louse of the endangered Galápagos hawk (Buteo galapagoensis). Two island populations of C. turbinatum (N = 30) were genotyped for each locus. We found between two and 12 alleles per locus, polymorphic information content from 0.268 to 0.798, observed heterozygosity from 0.067 to 0.667 and no linkage disequilibrium was detected between loci. These markers will be useful in understanding contemporary gene flow of C. turbinatum among islands in the Galápagos and in understanding transmission dynamics between B. galapagoensis hosts, within and between social groups. Because this louse is unusually widespread among avian host taxa, parasitizing at least 53 bird species in the Falconiformes, Strigiformes and Columbiformes, these markers are likely to be useful outside the context of the Galápagos Islands.     

Genetic analyses of the federally endangered Echinacea laevigata using amplified fragment length polymorphisms (AFLP)—Inferences in population genetic structure and mating system

Echinacea laevigata (Boynton and Beadle) Blake is a federally endangered flowering plant species restricted to four states in the southeastern United States. To determine the population structure and outcrossing rate across the range of the species, we conducted AFLP analysis using four primer combinations for 22 populations. The genetic diversity of this species was high based on the level of polymorphic loci (200 of 210 loci; 95.24%) and Nei’s gene diversity (ranging from 0.1398 to 0.2606; overall 0.2611). There was significant population genetic differentiation (G_ST = 0.294; Ө^II = 0.218 from the Bayesian f = 0 model). Results from the AMOVA analysis suggest that a majority of the genetic variance is attributed to variation within populations (70.26%), which is also evident from the PCoA. However, 82% of individuals were assigned back to the original population based on the results of the assignment test. An isolation by distance analysis indicated that genetic differentiation among populations was a function of geographic distance, although long-distance gene dispersal between some populations was suggested from an analysis of relatedness between populations using the neighbor-joining method. An estimate of the outcrossing rate based on genotypes of progenies from six of the 22 populations using the multilocus method from the program MLTR ranged from 0.780 to 0.912, suggesting that the species is predominantly outcrossing. These results are encouraging for conservation, signifying that populations may persist due to continued genetic exchange sustained by the outcrossing mating system of the species.     

Differences in straggling rates between two genera of dove lice (Insecta: Phthiraptera) reinforce population genetic and cophylogenetic patterns

Differences in dispersal abilities have been implicated for causing disparate evolutionary patterns between Columbicola and Physconelloides lice (Insecta: Phthiraptera). However, no study has documented straggling (when lice are found on atypical hosts) rates within these lineages. We used the fact that the Galapagos Hawk, Buteo galapagoensis (Gould) (Falconiformes) feeds on the Galapagos Dove Zenaida galapagoensis Gould (Columbiformes) within an ecologically simplified setting. The Galapagos Dove is the only typical host of Columbicola macrourae (Wilson) and Physconelloides galapagensis (Kellogg and Huwana) in Galapagos. We quantitatively sampled and found these lice on both bird species. A DNA barcoding approach confirmed that stragglers were derived from Galapagos doves. We also collected a Bovicola sp. louse, likely originating from a goat (Capra hircus). On hawks, C. macrourae was significantly more prevalent than P. galapagensis. On doves, the two lice were equally prevalent and abundant. Differences in prevalence on hawks was a function of differences in straggling rate between lice, and not a reflection of their relative representation within the dove population. This provides further evidence that differences in dispersal abilities may drive differences in the degree of cospeciation in Columbicola and Phyconelloides lice, which have become model systems in evolutionary biology.     

Characterization of antioxidant and antiglycation properties and isolation of active ingredients from traditional chinese medicines

There is considerable interest in the isolation of more potent antioxidant compounds to treat diseases involving oxidative stress. Thirty-three traditional Chinese medicine (TCM) extracts were examined for their antioxidant activity using the 2,2′-azinobis[3-ethylbenzothiazoline-6-sulfonate] (ABTS) assay. Five extracts with high activity (Cratoxylum cochinchinense, Cortex magnoliae officinalis, Psoralea corylifolia L, Curculigo orchioides Gaertn, and Glycyrrhiza uralensis Fisch.) were selected for further characterization. C. cochinchinense outperformed other extracts in most of the assays tested except phospholipid peroxidation inhibition, where P. corylifolia L showed higher activity. C. cochinchinense was particularly potent in inhibiting the formation of advanced glycation end products on proteins and strongly inhibited hypochlorous acid-induced DNA damage. We attempted to isolate the active ingredients from C. cochinchinense and obtained an extract (YCT) containing at least 90% mangiferin as identified by HPLC and mass spectrometry. However, YCT showed significantly higher activity in assays of phospholipid peroxidation, inhibition of protein glycation, and superoxide (O₂^√−) and peroxynitrite (ONOO⁻) scavenging, as compared with mangiferin, suggesting that the nonmangiferin constituents of YCT contribute to its additional antioxidant activities.     

Fucosebeta-1-P-Ser is a new type of glycosylation: using antibodies to identify a novel structure in Dictyostelium discoideum and study multiple types of fucosylation during growth and development

Three antibodies that recognize distinct fucose epitopes were used to study fucosylation during growth and development of Dictyostelium discoideum. mAb83.5 is known to recognize an undefined "fucose epitope" on several proteins with serine-rich domains, while mAb CAB4, and a component of anti-horse-radish peroxidase, specifically recognize Fucalpha1,6GlcNAc and Fucalpha1,3GlcNAc residues respectively in the core of N-linked oligosaccharides. We show that mAb 83.5 defines a new type of O-glycosylation. Serine-containing peptides incubated with GDPbeta[3H]Fuc and microsomes formed two fucosylated products. A neutral product accounting for 30% of the label did not react with the antibody, while the rest of the label was incorporated into a charged product which contained all the mAb83.5 reactive material. beta- Elimination of the labeled peptide or endogenous products produced [3H]Fuc-1-P, indicating phosphodiester linkage to serine. Fucbeta-1-P and GDP-betaFuc at 100 microM blocked mAb83.5 binding to endogenous and peptide products, but their alpha-linked anomers did not. Electrospray ionization mass spectra of the neutral and anionic labeled products showed major peaks of mass units corresponding to O-Fuc-Ser peptide and O-Fuc-phospho-Ser peptide, respectively. The activity of Fuc- phosphotransferase exactly paralleled the accumulation of reactive glycans during growth and development. The expressions of N-glycan core Fucalpha1,6GlcNAc and Fucalpha1,3GlcNAc and their respective fucosyl transferase activities were also synchronous, but their developmental regulation differed from one another. Fucalpha1, 6GlcNAc was expressed maximally during growth but declined during development. In contrast core Fucalpha1,3GlcNAc epitopes were expressed almost exclusively during development. These findings provide direct evidence for a novel type of O-phosphofucosylation, demonstrate the existence of an O- fucosyl transferase, and identify two different types of core fucosylation in the N-glycans of Dictyostelium.