Protein interaction verification and functional annotation by integrated analysis of genome-scale data

Assays capable of determining the properties of thousands of genes in parallel present challenges with regard to accurate data processing and functional annotation. Collections of microarray expression data are applied here to assess the quality of different high-throughput protein interaction data sets. Significant differences are found. Confidence in 973 out of 5342 putative two-hybrid interactions from S. cerevisiae is increased. Besides verification, integration of expression and interaction data is employed to provide functional annotation for over 300 previously uncharacterized genes. The robustness of these approaches is demonstrated by experiments that test the in silico predictions made. This study shows how integration improves the utility of different types of functional genomic data and how well this contributes to functional annotation.     

Proliferation assays for estrogenicity testing with high predictive value for the in vivo uterotrophic effect

Proliferation assays based on human cell lines are the most used in vitro tests to determine estrogenic properties of compounds. Our objective was to characterise to what extent these in vitro tests provide alternatives for the in vivo Allen and Doisy test, a uterotrophic assay in immature or ovariectomised rodents with uterus weight as a crucial read-out parameter. In the present study four different human cell lines derived from three different female estrogen-sensitive tissues, i.e. breast (MCF-7/BOS and T47D), endometrial (ECC-1) and ovarian (BG-1) cells, were characterised by investigating their relative ERα and ERβ amounts, as the ERα/ERβ ratio is a dominant factor determining their estrogen-dependent proliferative responses. All four cell lines clearly expressed the ERα type and a very low but detectable amount of ERβ on both the mRNA and protein level, with the T47D cell line expressing the highest level of the ERβ type. Subsequently, a set of reference compounds representing different modes of estrogen action and estrogenic potency were used to investigate the proliferative response in the four cell lines, to determine which cell line most accurately predicts the effect observed in vivo. All four cell lines revealed a reasonable to good correlation with the in vivo uterotrophic effect, with the correlation being highest for the MCF-7/BOS cell line (R2=0.85). The main differences between the in vivo uterotrophic assay and the in vitro proliferation assays were observed for tamoxifen and testosterone. The proliferative response of the MCF-7/BOS cells to testosterone was partially caused by its conversion to estradiol by aromatase or via androstenedione to estrone. It is concluded that of the four cell lines tested, the best assay to include in an integrated testing strategy for replacement of the in vivo uterotrophic assay is the human MCF-7/BOS breast cancer cell line.     

High-sensitivity cardiac troponin T: risk stratification tool in patients with symptoms of chest discomfort

Background

Recent studies have demonstrated the association between increased concentrations of high-sensitivity cardiac troponin T (hs-cTnT) and the incidence of myocardial infarction, heart failure, and mortality. However, most prognostic studies to date focus on the value of hs-cTnT in the elderly or general population. The value of hs-cTnT in symptomatic patients visiting the outpatient department remains unclear. The aim of this study was to investigate the prognostic value of hs-cTnT as a biomarker in patients with symptoms of chest discomfort suspected for coronary artery disease and to assess its additional value in combination with other risk stratification tools in predicting cardiac events.

Methods

We studied 1,088 patients (follow-up 2.2±0.8 years) with chest discomfort who underwent coronary calcium scoring and coronary CT-angiography. Traditional cardiovascular risk factors and concentrations of hs-cTnT, N-terminal pro-brain-type natriuretic peptide (NT-proBNP) and high-sensitivity C-reactive protein (hsCRP) were assessed. Study endpoint was the occurrence of late coronary revascularization (>90 days), acute coronary syndrome, and cardiac mortality.

Results

Hs-cTnT was a significant predictor for the composite endpoint (highest quartile [Q4]>6.7 ng/L, HR 3.55; 95%CI 1.88–6.70; P<0.001). Survival analysis showed that hs-cTnT had significant predictive value on top of current risk stratification tools (Chi-square change P<0.01). In patients with hs-cTnT in Q4 versus P<0.01). This was not the case for hsCRP and NT-proBNP.

Conclusions

Hs-cTnT is a useful prognostic biomarker in patients with chest discomfort suspected for coronary artery disease. In addition, hs-cTnT was an independent predictor for cardiac events when corrected for cardiovascular risk profiling, calcium score and CT-angiography results.     

Vitamin k-antagonists accelerate atherosclerotic calcification and induce a vulnerable plaque phenotype

Background

Vitamin K-antagonists (VKA) are treatment of choice and standard care for patients with venous thrombosis and thromboembolic risk. In experimental animal models as well as humans, VKA have been shown to promote medial elastocalcinosis. As vascular calcification is considered an independent risk factor for plaque instability, we here investigated the effect of VKA on coronary calcification in patients and on calcification of atherosclerotic plaques in the ApoE^−/− model of atherosclerosis.

Methodology/Principal Findings

A total of 266 patients (133 VKA users and 133 gender and Framingham Risk Score matched non-VKA users) underwent 64-slice MDCT to assess the degree of coronary artery disease (CAD). VKA-users developed significantly more calcified coronary plaques as compared to non-VKA users. ApoE^−/− mice (10 weeks) received a Western type diet (WTD) for 12 weeks, after which mice were fed a WTD supplemented with vitamin K₁ (VK₁, 1.5 mg/g) or vitamin K₁ and warfarin (VK₁&W; 1.5 mg/g & 3.0 mg/g) for 1 or 4 weeks, after which mice were sacrificed. Warfarin significantly increased frequency and extent of vascular calcification. Also, plaque calcification comprised microcalcification of the intimal layer. Furthermore, warfarin treatment decreased plaque expression of calcification regulatory protein carboxylated matrix Gla-protein, increased apoptosis and, surprisingly outward plaque remodeling, without affecting overall plaque burden.

Conclusions/Significance

VKA use is associated with coronary artery plaque calcification in patients with suspected CAD and causes changes in plaque morphology with features of plaque vulnerability in ApoE^−/− mice. Our findings underscore the need for alternative anticoagulants that do not interfere with the vitamin K cycle.     

Bifidobacterium microbiota and parameters of immune function in elderly subjects

Faecal and serum samples were collected over a period of 6 months from 55 institutionalized elderly subjects, who were enrolled in a double-blind placebo-controlled study. Participants were randomized in one of the three treatment groups: intervention (two probiotic Bifidobacterium longum strains: 2C and 46), placebo and commercial control (Bifidobacterium lactis Bb-12). The faecal Bifidobacterium microbiota was characterized by genus and species-specific PCR. Serum levels of the cytokines IL-10, tumor necrosis factor (TNF)-alpha and transforming growth factor (TGF)-beta1 were determined by enzyme-linked immunosorbent assay. Each participant harboured on average approximately three different bifidobacterial species. The most frequently detected species were B. longum, Bifidobacterium adolescentis and Bifidobacterium bifidum. Depending on the treatment, the intervention resulted in specific changes in the levels of certain Bifidobacterium species, and positive correlations were found between the different species. Negative correlations were observed between the levels of Bifidobacterium species and the pro-inflammatory cytokine TNF-alpha and the regulatory cytokine IL-10. The presence of faecal B. longum and Bifidobacterium animalis correlated with reduced serum IL-10. The anti-inflammatory TGF-beta1 levels were increased over time in all three groups, and the presence of Bifidobacterium breve correlated with higher serum TGF-beta1 levels. This indicates that modulation of the faecal Bifidobacterium microbiota may provide a means of influencing inflammatory responses.     

Single-molecule observations of topotecan-mediated TopIB activity at a unique DNA sequence

The rate of DNA supercoil removal by human topoisomerase IB (TopIB) is slowed down by the presence of the camptothecin class of antitumor drugs. By preventing religation, these drugs also prolong the lifetime of the covalent TopIB-DNA complex. Here, we use magnetic tweezers to measure the rate of supercoil removal by drug-bound TopIB at a single DNA sequence in real time. This is accomplished by covalently linking camptothecins to a triple helix-forming oligonucleotide that binds at one location on the DNA molecule monitored. Surprisingly, we find that the DNA dynamics with the TopIB-drug interaction restricted to a single DNA sequence are indistinguishable from the dynamics observed when the TopIB-drug interaction takes place at multiple sites. Specifically, the DNA sequence does not affect the instantaneous supercoil removal rate or the degree to which camptothecins increase the lifetime of the covalent complex. Our data suggest that sequence-dependent dynamics need not to be taken into account in efforts to develop novel camptothecins.     

Accuracy of mean arterial pressure and blood pressure measurements in predicting pre-eclampsia: systematic review and meta-analysis

Objective To determine the accuracy of using systolic and diastolic blood pressure, mean arterial pressure, and increase of blood pressure to predict pre-eclampsia.Design Systematic review with meta-analysis of data on test accuracy.Data sources Medline, Embase, Cochrane Library, Medion, checking reference lists of included articles and reviews, contact with authors.Review methods Without language restrictions, two reviewers independently selected the articles in which the accuracy of blood pressure measurement during pregnancy was evaluated to predict pre-eclampsia. Data were extracted on study characteristics, quality, and results to construct 2×2 tables. Summary receiver operating characteristic curves and likelihood ratios were generated for the various levels and their thresholds.Results 34 studies, testing 60 599 women (3341 cases of pre-eclampsia), were included. In women at low risk for pre-eclampsia, the areas under the summary receiver operating characteristic curves for blood pressure measurement in the second trimester were 0.68 (95% confidence interval 0.64 to 0.72) for systolic blood pressure, 0.66 (0.59 to 0.72) for diastolic blood pressure, and 0.76 (0.70 to 0.82) for mean arterial pressure. Findings for the first trimester showed a similar pattern. Second trimester mean arterial pressure of 90 mm Hg or more showed a positive likelihood ratio of 3.5 (95% confidence interval 2.0 to 5.0) and a negative likelihood ratio of 0.46 (0.16 to 0.75). In women deemed to be at high risk, a diastolic blood pressure of 75 mm Hg or more at 13 to 20 weeks’ gestation best predicted pre-eclampsia: positive likelihood ratio 2.8 (1.8 to 3.6), negative likelihood ratio 0.39 (0.18 to 0.71). Additional subgroup analyses did not show improved predictive accuracy.Conclusion When blood pressure is measured in the first or second trimester of pregnancy, the mean arterial pressure is a better predictor for pre-eclampsia than systolic blood pressure, diastolic blood pressure, or an increase of blood pressure.     

Molecular and Clinical Characteristics of MSH6 Variants: An Analysis of 25 Index Carriers of a Germline Variant

The MSH6 gene is one of the mismatch-repair genes involved in hereditary nonpolyposis colorectal cancer (HNPCC). Three hundred sixteen individuals who were known or suspected to have HNPCC were analyzed for MSH6 germline mutations. For 25 index patients and 8 relatives with MSH6 variants, molecular and clinical features are described. For analysis of microsatellite instability (MSI), the five consensus markers were used. Immunohistochemical analysis of the MLH1, MSH2, and MSH6 proteins was performed. Five truncating MSH6 mutations, of which one was detected seven times, were found in 12 index patients, and 10 MSH6 variants with unknown pathogenicity were found in 13 index patients. Fourteen (54%) of 26 colorectal cancers (CRCs) and endometrial cancers showed no, or only weak, MSI. Twelve of 18 tumors of truncating-mutation carriers and 3 of 17 tumors of missense-mutation carriers showed loss of MSH6 staining. Six of the families that we studied fulfilled the original Amsterdam criteria; most families with MSH6, however, were only suspected to have HNPCC. In families that did not fulfill the revised Amsterdam criteria, the prevalence of MSH6 variants is about the same as the prevalence of those in MLH1/MSH2. Endometrial cancer and/or atypical hyperplasia were diagnosed in 8 of 12 female carriers of MSH6 truncating mutations. Most CRCs were localized distally in the colon. Although, molecularly, missense variants are labeled as doubtfully pathogenic, clinical data disclose a great resemblance between missense-variant carriers and truncating-mutation carriers. We conclude that, in all patients suspected to have HNPCC, MSH6-mutation analysis should be considered. Neither MSI nor immunohistochemistry should be a definitive selection criterion for MSH6-mutation analysis.