Rotation gene set testing for longitudinal expression data

Gene set analysis methods are popular tools for identifying differentially expressed gene sets in microarray data. Most existing methods use a permutation test to assess significance for each gene set. The permutation test's assumption of exchangeable samples is often not satisfied for time‐series data and complex experimental designs, and in addition it requires a certain number of samples to compute p‐values accurately. The method presented here uses a rotation test rather than a permutation test to assess significance. The rotation test can compute accurate p‐values also for very small sample sizes. The method can handle complex designs and is particularly suited for longitudinal microarray data where the samples may have complex correlation structures. Dependencies between genes, modeled with the use of gene networks, are incorporated in the estimation of correlations between samples. In addition, the method can test for both gene sets that are differentially expressed and gene sets that show strong time trends. We show on simulated longitudinal data that the ability to identify important gene sets may be improved by taking the correlation structure between samples into account. Applied to real data, the method identifies both gene sets with constant expression and gene sets with strong time trends.     

Feasibility of MR Metabolomics for Immediate Analysis of Resection Margins during Breast Cancer Surgery

In this study, the feasibility of high resolution magic angle spinning (HR MAS) magnetic resonance spectroscopy (MRS) of small tissue biopsies to distinguish between tumor and non-involved adjacent tissue was investigated. With the current methods, delineation of the tumor borders during breast cancer surgery is a challenging task for the surgeon, and a significant number of re-surgeries occur. We analyzed 328 tissue samples from 228 breast cancer patients using HR MAS MRS. Partial least squares discriminant analysis (PLS-DA) was applied to discriminate between tumor and non-involved adjacent tissue. Using proper double cross validation, high sensitivity and specificity of 91% and 93%, respectively was achieved. Analysis of the loading profiles from both principal component analysis (PCA) and PLS-DA showed the choline-containing metabolites as main biomarkers for tumor content, with phosphocholine being especially high in tumor tissue. Other indicative metabolites include glycine, taurine and glucose. We conclude that metabolic profiling by HR MAS MRS may be a potential method for on-line analysis of resection margins during breast cancer surgery to reduce the number of re-surgeries and risk of local recurrence.     

Dramatic increase of nitrite levels in hearts of anoxia-exposed crucian carp supporting a role in cardioprotection

Nitrite (NO(2)(-)) functions as an important nitric oxide (NO) donor under hypoxic conditions. Both nitrite and NO have been found to protect the mammalian heart and other tissues against ischemia (anoxia)-reoxygenation injury by interacting with mitochondrial electron transport complexes and limiting the generation of reactive oxygen species upon reoxygenation. The crucian carp naturally survives extended periods without oxygen in an active state, which has made it a model for studying how evolution has solved the problems of anoxic survival. We investigated the role of nitrite and NO in the anoxia tolerance of this fish by measuring NO metabolites in normoxic, anoxic, and reoxygenated crucian carp. We also cloned and sequenced crucian carp NO synthase variants and quantified their mRNA levels in several tissues in normoxia and anoxia. Despite falling levels of blood plasma nitrite, the crucian carp showed massive increases in nitrite, S-nitrosothiols (SNO), and iron-nitrosyl (FeNO) compounds in anoxic heart tissue. NO(2)(-) levels were maintained in anoxic brain, liver, and gill tissues, whereas SNO and FeNO increased in a tissue-specific manner. Reoxygenation reestablished normoxic values. We conclude that NO(2)(-) is shifted into the tissues where it acts as NO donor during anoxia, inducing cytoprotection under anoxia/reoxygenation. This can be especially important in the crucian carp heart, which maintains output in anoxia. NO(2)(-) is currently tested as a therapeutic drug against reperfusion damage of ischemic hearts, and the present study provides evolutionary precedent for such an approach.     

Serum Levels of Choline-Containing Compounds Are Associated with Aerobic Fitness Level: The HUNT-Study

Background

Cardiovascular disease (CVD) is a leading cause of death worldwide, and the number of people at risk is continuously growing. New methods for early risk prediction are therefore needed to actuate prevention strategies before the individuals are diagnosed with CVD. Several studies report that aerobic fitness level, measured as maximal oxygen uptake (VO_2max), is the single best predictor of future CVD mortality in healthy people. Based on this, we wanted to study differences between healthy individuals with a large difference in VO_2max-level to identify new biomarkers of low aerobic fitness that may also have potential as early biomarkers of CVD risk.

Methodology/Principal Findings

Serum samples from 218 healthy individuals with a low VO_2max (n = 108, 63 women) or high VO_2max (n = 110, 64 women) were analysed with MR metabolomics. In addition, standard clinical-chemical analyses for glucose, lipids, liver enzymes, micro-CRP, and colorimetric analysis on circulating choline were performed. Individuals in the low VO_2max-group had increased serum levels of free choline, decreased phosphatidylcholine, increased glucosę and decreased unsaturated fatty acids compared to the individuals in the high VO_2max–group.

Conclusions/Significance

Aerobic fitness dependent differences in serum levels of free choline and phosphatidylcholine are observed. They should be further studied as potential early markers of CVD risk.     

A Gene Panel,Including LRP12, Is Frequently Hypermethylated in Major Types of B-Cell Lymphoma

Epigenetic modifications and DNA methylation in particular, have been recognized as important mechanisms to alter gene expression in malignant cells. Here, we identified candidate genes which were upregulated after an epigenetic treatment of B-cell lymphoma cell lines (Burkitt''s lymphoma, BL; Follicular lymphoma, FL; Diffuse large B-cell lymphoma, DLBCL activated B-cell like, ABC; and germinal center like, GCB) and simultaneously expressed at low levels in samples from lymphoma patients. Qualitative methylation analysis of 24 candidate genes in cell lines revealed five methylated genes (BMP7, BMPER, CDH1, DUSP4 and LRP12), which were further subjected to quantitative methylation analysis in clinical samples from 59 lymphoma patients (BL, FL, DLBCL ABC and GCB; and primary mediastinal B-cell lymphoma, PMBL). The genes LRP12 and CDH1 showed the highest methylation frequencies (94% and 92%, respectively). BMPER (58%), DUSP4 (32%) and BMP7 (22%), were also frequently methylated in patient samples. Importantly, all gene promoters were unmethylated in various control samples (CD19+ peripheral blood B cells, peripheral blood mononuclear cells and tonsils) as well as in follicular hyperplasia samples, underscoring a high specificity. The combination of LRP12 and CDH1 methylation could successfully discriminate between the vast majority of the lymphoma and control samples, emphasized by receiver operating characteristic analysis with a c-statistic of 0.999. These two genes represent promising epigenetic markers which may be suitable for monitoring of B-cell lymphoma.     

Norwegian PUQE (Pregnancy-Unique Quantification of Emesis and Nausea) Identifies Patients with Hyperemesis Gravidarum and Poor Nutritional Intake: A Prospective Cohort Validation Study

Objective

The English questionnaire Pregnancy-Unique Quantification of Emesis and nausea (PUQE) identifies women with severe Hyperemesis Gravidarum. Our aim was to investigate whether scores from the translated Norwegian version; SUKK (SvangerskapsUtløst Kvalme Kvantifisering) was associated with severity of hyperemesis and nutritional intake.

Design

A prospective cohort validation study.

Setting

Hospital cohort of Hyperemesis Gravidarum (HG) patients from western Norway and healthy pregnant women from Bergen, Norway.

Sample

38 women hospitalized due to HG and 31 healthy pregnant controls attending routine antenatal check-up at health centers.

Methods

Data were collected May 2013-January 2014. The study participants answered the Norwegian PUQE-questionnaire (scores ranging from 3 to15) and registered prospectively 24-hours nutritional intake by a food list form.

Main outcome measures

Differences of PUQE-scores, QOL-score and nutritional intake between hyperemesis patients and controls.

Results

Hyperemesis patients had shorter gestational age compared to controls (median 9.7 weeks; 95% CI 8.6-10.6 versus 11.9; 95% CI 10.1-12.9, p=0.004), and larger weight-change from pre-pregnant weight (loss of median 3 kg; 95% CI 3-4 versus gain of 2 kg; 95% CI 0.5-2, p<0.001) otherwise groups were similar regarding pre-pregnant BMI, age, gravidity, and inclusion weight. Compared to controls, hyperemesis patients had significant higher PUQE-score (median 13; 95% CI 11-14 vs. 7; 95% CI 4-8), lower QOL (median score 3; 95% CI 2-4 vs. 6; 95% CI 4.5-8) and lower nutritional intake (energy intake median 990 kcal/24 hours; 95% CI 709-1233 vs. 1652; 95% CI 1558-1880 all p<0.001). PUQE-score was inversely correlated to nutritional intake (-0.5, p<0.001). At discharge PUQE-score had fallen to median 6 (95% CI 5-8) and QOL score risen to 7 (95% CI 6-8) in the HG group, (both p<0.001 compared to admission values).

Conclusion

PUQE-scoring has been validated as a robust indicator of severe hyperemesis gravidarum and insufficient nutritional intake in a Norwegian setting.     

Stem cell mobilization and harvesting by leukapheresis alters systemic cytokine levels in patients with multiple myeloma

Background aimsStem cell mobilization and harvesting by peripheral blood leukapheresis in patients with myeloma can alter plasma levels of certain cytokines. In the present study, we investigated the effects of these interventions on a larger group of cytokines.MethodsPlasma cytokine levels were determined in 15 patients with myeloma who were undergoing peripheral blood stem cell harvesting, and we compared the patients with healthy donors who were undergoing platelet apheresis.ResultsSeveral cytokines showed altered levels in patients with myeloma when examined after chemotherapy plus granulocyte colony-stimulating factor–induced stem cell mobilization. The most striking effect was increased levels of several CCL (CCL2/3/4) and CXCL (CXCL5/8/10/11) chemokines as well as increased thrombopoietin, interleukin 1 receptor antagonist, interleukin-4, granulocyte colony-stimulating factor and hepatocyte growth factor. Stem cell harvesting by apheresis altered the plasma levels of several mediators (CD40 ligand, interleukin 1 receptor antagonist, CCL5 and CXCL5/8/10/11). Apheresis in patients with myeloma had divergent effects on these chemokine levels, although they were all still significantly higher than for healthy individuals. Thrombapheresis in healthy individuals had only minor effects on plasma cytokine levels. Stem cell graft supernatants showed high levels of several cytokines, especially CCL and CXCL chemokines. Analyses of chemokine profiles in pre-apheresis plasma and graft supernatants suggested that such profiling can be used to detect prognostically relevant differences between patients.ConclusionsOur results demonstrate that patients with myeloma have an altered cytokine network during stem cell mobilization, and the network is further altered during stem cell harvesting by leukapheresis. These treatment- or procedure-induced alterations involve several mediators known to affect myeloma cell proliferation, migration and survival.     

Histone-like proteins from Atlantic cod milt: stimulatory effect on Atlantic salmon leucocytes in vivo and in vitro

This study was carried out to reveal some characteristics of cationic proteins from Atlantic cod (Gadus morhua) milt chromatin and to investigate their ability to activate Atlantic salmon (Salmo salar) macrophages. Cationic proteins extracted from cod milt chromatin were fractionated on a cation exchange chromatography column. SDS-PAGE and amino acid analyses of the resulting fractions indicated that these proteins are similar to calf thymus histones. Two cationic protein fractions were used to stimulate leucocytes from Atlantic salmon in vitro and in vivo. Increased production of superoxide, measured as reduction of nitroblue tetrazolium (NBT), was used as indication of macrophage activation. Both fractions induced elevated superoxide anion production in the macrophages after 3 and 6 days of in vitro stimulation. Intraperitoneal injection of the cationic protein fractions in Atlantic salmon (100 mg kg(-1)) four days prior to slaughtering stimulated superoxide production when assayed after one and two days of cell cultivation. In macrophages from fish slaughtered two days after injection, activation could first be seen after two days of cell cultivation.