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.

     

Specified species in gingival crevicular fluid predict bacterial diversity

Background

Analysis of gingival crevicular fluid (GCF) samples may give information of unattached (planktonic) subgingival bacteria. Our study represents the first one targeting the identity of bacteria in GCF.

Methodology/Principal Findings

We determined bacterial species diversity in GCF samples of a group of periodontitis patients and delineated contributing bacterial and host-associated factors. Subgingival paper point (PP) samples from the same sites were taken for comparison. After DNA extraction, 16S rRNA genes were PCR amplified and DNA-DNA hybridization was performed using a microarray for over 300 bacterial species or groups. Altogether 133 species from 41 genera and 8 phyla were detected with 9 to 62 and 18 to 64 species in GCF and PP samples, respectively, per patient. Projection to latent structures by means of partial least squares (PLS) was applied to the multivariate data analysis. PLS regression analysis showed that species of genera including Campylobacter, Selenomonas, Porphyromonas, Catonella, Tannerella, Dialister, Peptostreptococcus, Streptococcus and Eubacterium had significant positive correlations and the number of teeth with low-grade attachment loss a significant negative correlation to species diversity in GCF samples. OPLS/O2PLS discriminant analysis revealed significant positive correlations to GCF sample group membership for species of genera Campylobacter, Leptotrichia, Prevotella, Dialister, Tannerella, Haemophilus, Fusobacterium, Eubacterium, and Actinomyces.

Conclusions/Significance

Among a variety of detected species those traditionally classified as Gram-negative anaerobes growing in mature subgingival biofilms were the main predictors for species diversity in GCF samples as well as responsible for distinguishing GCF samples from PP samples. GCF bacteria may provide new prospects for studying dynamic properties of subgingival biofilms.     

The Impact of Helicobacter pylori Infection on the Gastric Microbiota of the Rhesus Macaque

Helicobacter pylori colonization is highly prevalent among humans and causes significant gastric disease in a subset of those infected. When present, this bacterium dominates the gastric microbiota of humans and induces antimicrobial responses in the host. Since the microbial context of H. pylori colonization influences the disease outcome in a mouse model, we sought to assess the impact of H. pylori challenge upon the pre-existing gastric microbial community members in the rhesus macaque model. Deep sequencing of the bacterial 16S rRNA gene identified a community profile of 221 phylotypes that was distinct from that of the rhesus macaque distal gut and mouth, although there were taxa in common. High proportions of both H. pylori and H. suis were observed in the post-challenge libraries, but at a given time, only one Helicobacter species was dominant. However, the relative abundance of non-Helicobacter taxa was not significantly different before and after challenge with H. pylori. These results suggest that while different gastric species may show competitive exclusion in the gastric niche, the rhesus gastric microbial community is largely stable despite immune and physiological changes due to H. pylori infection.     

Comparison of the gut microbiota from soldier and worker castes of the termite Reticulitermes grassei

The bacterial microbiota from the whole gut of soldier and worker castes of the termite Reticulitermes grassei was isolated and studied. In addition, the 16S rDNA bacterial genes from gut DNA were PCR-amplified using Bacteria-selective primers, and the 16S rDNA amplicons subsequently cloned into Escherichia coli. Sequences of the cloned inserts were then used to determine closest relatives by comparison with published sequences and with sequences from our previous work. The clones were found to be affiliated with the phyla Spirochaetes, Proteobacteria, Firmicutes, Bacteroidetes, Actinobacteria, Synergistetes, Verrucomicrobia, and candidate phyla Termite Group 1 (TG1) and Termite Group 2 (TG2). No significant differences were observed with respect to the relative bacterial abundances between soldier and worker phylotypes. The phylotypes obtained in this study were compared with reported sequences from other termites, especially those of phylotypes related to Spirochaetes, Wolbachia (an Alphaproteobacteria), Actinobacteria, and TG1. Many of the clone phylotypes detected in soldiers grouped with those of workers. Moreover, clones CRgS91 (soldiers) and CRgW68 (workers), both affiliated with 'Endomicrobia', were the same phylotype. Soldiers and workers also seemed to have similar relative protist abundances. Heterotrophic, poly-β-hydroxyalkanoate-accumulating bacteria were isolated from the gut of soldiers and shown to be affiliated with Actinobacteria and Gammaproteobacteria. We noted that Wolbachia was detected in soldiers but not in workers. Overall, the maintenance by soldiers and workers of comparable axial and radial redox gradients in the gut is consistent with the similarities in the prokaryotes and protists comprising their microbiota.     

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 characterization of epibiotic bacteria in the accessory nidamental gland and egg capsules of the squid Loligo pealei (Cephalopoda:Loliginidae)

Sexually mature female squid Loligo pealei harbour dense bacterial communities in their accessory nidamental glands (ANGs) and in their egg capsules (ECs). This study describes a molecular approach using the 16S rRNA gene (rDNA) to identify bacterial populations within the ANG and the ECs of the North Atlantic squid species L. pealei. Fluorescent in situ hybridization (FISH) and 16S rDNA analysis showed that predominantly alpha- and, to a lesser extent, gamma-proteobacteria were the predominant components of the ANG and EC bacterial communities. Sequencing results showed the presence of alpha-proteobacterial populations affiliated with the Roseobacter group and additional deep-branching alpha-proteobacterial lineages. In contrast, isolates from the ANG and ECs contained only a few alpha-proteobacteria of the Roseobacter group compared with several gamma-proteobacterial isolates, mostly Shewanella and Pseudoalteromonas species. Most of the ANG-associated bacterial populations were also found within the ECs of L. pealei. The molecular approach allowed the visualization of alpha-proteobacteria as major constituents of a bacterial symbiosis within the reproductive system of the Loliginidae.     

The stabilities of mammalian apomyoglobins vary over a 600-fold range and can be enhanced by comparative mutagenesis

Apomyoglobins from 13 different mammals were examined for resistance to denaturation by guanidinium chloride. Unfolding was followed by circular dichroism and tryptophan fluorescence and analyzed globally using the two-step, three-state mechanism first described by Barrick and Baldwin (Barrick, D., and Baldwin, R. L. (1993) Biochemistry 32, 3790-3796). With one exception, the rise and fall of Trp fluorescence intensity correlates quantitatively with the native to intermediate to unfolded steps seen in the CD curves. Although the O(2) binding properties of the holoproteins are nearly identical, the unfolding transitions of the apomyoglobins show 600-fold differences in resistance to guanidinium chloride denaturation. Apomyoglobins from diving mammals, particularly from sperm whales, are the most stable, whereas the apoproteins from pig, horse, and sheep are the least stable, indicating selective pressure for resistance to denaturation in the whale proteins. Sequence comparisons suggest that the key stabilizing residues in whale globins are Ala(5), His(12), Ile(28), Thr(51), Ala(53), Ala(74), Lys(87), Lys(140), and Ile(142). Combinations of these residues were substituted into pig myoglobin. The resultant multiple mutants showed stabilities approaching that of recombinant sperm whale apomyoglobin. Thus, comparative mutagenesis can be used to increase heme protein stability and improve expression yields in bacteria without compromising function.