Quantitative proteomic analysis by iTRAQ<Superscript>®</Superscript> for the identification of candidate biomarkers in ovarian cancer serum

Background

Ovarian cancer is the most lethal gynecologic malignancy, with the majority of cases diagnosed at an advanced stage when treatments are less successful. Novel serum protein markers are needed to detect ovarian cancer in its earliest stage; when detected early, survival rates are over 90%. The identification of new serum biomarkers is hindered by the presence of a small number of highly abundant proteins that comprise approximately 95% of serum total protein. In this study, we used pooled serum depleted of the most highly abundant proteins to reduce the dynamic range of proteins, and thereby enhance the identification of serum biomarkers using the quantitative proteomic method iTRAQ^®.

Results

Medium and low abundance proteins from 6 serum pools of 10 patients each from women with serous ovarian carcinoma, and 6 non-cancer control pools were labeled with isobaric tags using iTRAQ^® to determine the relative abundance of serum proteins identified by MS. A total of 220 unique proteins were identified and fourteen proteins were elevated in ovarian cancer compared to control serum pools, including several novel candidate ovarian cancer biomarkers: extracellular matrix protein-1, leucine-rich alpha-2 glycoprotein-1, lipopolysaccharide binding protein-1, and proteoglycan-4. Western immunoblotting validated the relative increases in serum protein levels for several of the proteins identified.

Conclusions

This study provides the first analysis of immunodepleted serum in combination with iTRAQ^® to measure relative protein expression in ovarian cancer patients for the pursuit of serum biomarkers. Several candidate biomarkers were identified which warrant further development.

     

Assessing combined methylation-sensitive high resolution melting and pyrosequencing for the analysis of heterogeneous DNA methylation

Heterogeneous DNA methylation leads to difficulties in accurate detection and quantification of methylation. Methylation-sensitive high resolution melting (MS-HRM) is unique among regularly used methods for DNA methylation analysis in that heterogeneous methylation can be readily identified, although not quantified, by inspection of the melting curves. Bisulfite pyrosequencing has been used to estimate the level of heterogeneous methylation by quantifying methylation levels present at individual CpG dinucleotides. Sequentially combining the two methodologies using MS-HRM to screen the amplification products prior to bisulfite pyrosequencing would be advantageous. This would not only replace the quality control step using agarose gel analysis prior to the pyrosequencing step but would also provide important qualitative information in its own right. We chose to analyze DAPK1 as it is an important tumor suppressor gene frequently heterogeneously methylated in a number of malignancies, including chronic lymphocytic leukemia (CLL). A region of the DAPK1 promoter was analyzed in ten CLL samples by MS-HRM. By using a biotinylated primer, bisulfite pyrosequencing could be used to directly analyze the samples. MS-HRM revealed the presence of various extents of heterogeneous DAPK1 methylation in all CLL samples. Further analysis of the biotinylated MS-HRM products by bisulfite pyrosequencing provided quantitative information for each CpG dinucleotide analyzed, and confirmed the presence of heterogeneous DNA methylation. Whereas each method could be used individually, MS-HRM and bisulfite pyrosequencing provided complementary information for the assessment of heterogeneous methylation.Key words: MS-HRM, pyrosequencing, digital PCR, heterogeneous DNA methylation, DAPK1, chronic lymphocytic leukemia     

A pilot study of rivastigmine in the treatment of delirium after stroke: A safe alternative

Background

Delirium is a common disorder in the early phase of stroke. Given the presumed cholinergic deficiency in delirium, we tested treatment with the acetylcholinesterase inhibitor rivastigmine.

Methods

This pilot study was performed within an epidemiological study. In 527 consecutive stroke patients presence of delirium was assessed during the first week with the confusion assessment method. Severity was scored with the delirium rating scale (DRS). Sixty-two patients developed a delirium in the acute phase of stroke. Only patients with a severe and persistent delirium (defined as a DRS of 12 or more for more than 24 hours) were enrolled in the present study. In total 26 fulfilled these criteria of whom 17 were treated with orally administered rivastigmine with a total dose between 3 and 12 mg a day. Eight patients could not be treated because of dysphagia and one because of early discharge.

Results

No major side effects were recorded. In 16 patients there was a considerable decrease in severity of delirium. The mean DRS declined from 14.8 on day one to 8.5 after therapy and 5.6 after tapering. The mean duration of delirium was 6.7 days (range; 2–17).

Conclusion

Rivastigmine is safe in stroke patients with delirium even after rapid titration. In the majority of patients the delirium improved after treatment. A randomized controlled trial is needed to establish the usefulness of rivastigmine in delirium after stroke.

Trial registration

Nederlands Trial Register NTR1395

     

Apolipoprotein C3 SstI polymorphism and triglyceride levels in Asian Indians

Background  

A close association between Sst I polymorphism in the 3' untranslated region of the apolipoproteinC3 (APOC3 ) gene and levels of plasma triglycerides (TG) had been reported by different investigators. Hypertriglyceridemia(HTG) is a known risk factor for coronary artery disease (CAD) in the context of Asian Indians. We conducted a study on the relationship between APOC3 SstI polymorphism (S1S1, S1S2 and S2S2 genotypes) and plasma TG levels in a group of 139 male healthy volunteers from Northern India.     

Phylogenetic analysis of the outer-membrane-protein genes of Chlamydiae, and its implication for vaccine development

Examination of 18 complete and 6 partial sequences of the major outer- membrane protein from 24 chlamydiae isolates was used to reconstruct their evolutionary relationships. From this analysis, assuming that the clades with 100% bootstrap support are correct, come the following conclusions: (1) The tree of these sequences is not congruent with the phylogeny of the hosts, and thus host switching would seem to have occurred, thereby limiting the extent to which there has been coevolution of parasite and host. (2) The tree is also noncongruent with clustering by type of cell infected, thereby limiting the extent to which there has been coevolution of parasite and the cell type that it infects. (3) The tree is also noncongruent with clustering by the organ infected (eyes or genitalia), thereby limiting the extent to which there has been coevolution of parasite and the organ that it infects. (4) The tree is also noncongruent with genital strains arising from lymphogranuloma venereum strains. (5) The tree is also noncongruent with the geographic site at which the isolates were obtained, thereby limiting the extent of divergence explained by geographic separation. (6) There are estimated to be 185 amino acid positions that are invariable (as opposed to unvaried) in the major outer-membrane protein. There are 10 unvaried positions in the variable domains, of which 9 appear to be invariable, giving some reason to hope that development of a vaccine might be possible. (7) The rate of change of this protein is too small to see increased divergence over the time span of isolation of these genes, giving hope to any vaccine having longevity. Bootstrapping supports those portions of the tree on which the first five conclusions above depend. The picture that these results provide is more one of pathogen versatility than one of coevolutionary constraints. In addition, we examined 10 60-KDa, outer-membrane protein- 2 genes, all but one of which were from these same strains. The tree was not, among the trachomatis strains, congruent with the major-outer- membrane protein tree, suggesting that gene exchange could be occurring among strains. Moreover, there is an apparent slowdown in divergence in this gene, among the trachomatis strains.      

Western Palaearctic phylogeography of an inquiline oak gall wasp,Synergus umbraculus

Insect‐induced galls on plants comprise species‐rich but self‐contained communities of herbivores and natural enemies. In the present study, we focus on galls induced by cynipid gall wasps on oaks, and on the least‐known trophic level that these galls contain: inquilines. These insects, also cynipids, feed on gall tissue and are an abundant but taxonomically poorly understood part of an otherwise well‐studied system. We used DNA sequence data to examine spatial patterns in the genetic diversity of Synergus umbraculus Olivier 1791 (Hymenoptera: Cynipidae: Synergini), a widespread species attacking many host galls across the Western Palaearctic. Analysis of 239 cytochrome b sequences revealed eight haplogroups showing significant phylogeographic pattern across the Western Palaearctic, corresponding to putative glacial refugia in Iberia, Central Europe, Turkey, and Iran. There were significant genetic discontinuities across the Pyrenees and the Anatolian diagonal but no impact of the Alps, suggesting that significant discontinuities have biotic rather than physical causes. Detailed analysis of sites in the Carpathian Basin reveal a high diversity and low spatial structure, and identify Central Europe as the source of colonists for Quaternary colonization of Germany, France, and Britain. We found no evidence for host‐associated differentiation of S. umbraculus lineages associated with the most common cynipid host galls, suggesting frequent shifts within the host gall assemblage by inquiline lineages. © 2011 The Linnean Society of London, Biological Journal of the Linnean Society, 2011, 102 , 750–764.