Bayesian independent component analysis recovers pathway signatures from blood metabolomics data

Interpreting the complex interplay of metabolites in heterogeneous biosamples still poses a challenging task. In this study, we propose independent component analysis (ICA) as a multivariate analysis tool for the interpretation of large-scale metabolomics data. In particular, we employ a Bayesian ICA method based on a mean-field approach, which allows us to statistically infer the number of independent components to be reconstructed. The advantage of ICA over correlation-based methods like principal component analysis (PCA) is the utilization of higher order statistical dependencies, which not only yield additional information but also allow a more meaningful representation of the data with fewer components. We performed the described ICA approach on a large-scale metabolomics data set of human serum samples, comprising a total of 1764 study probands with 218 measured metabolites. Inspecting the source matrix of statistically independent metabolite profiles using a weighted enrichment algorithm, we observe strong enrichment of specific metabolic pathways in all components. This includes signatures from amino acid metabolism, energy-related processes, carbohydrate metabolism, and lipid metabolism. Our results imply that the human blood metabolome is composed of a distinct set of overlaying, statistically independent signals. ICA furthermore produces a mixing matrix, describing the strength of each independent component for each of the study probands. Correlating these values with plasma high-density lipoprotein (HDL) levels, we establish a novel association between HDL plasma levels and the branched-chain amino acid pathway. We conclude that the Bayesian ICA methodology has the power and flexibility to replace many of the nowadays common PCA and clustering-based analyses common in the research field.     

Metabolic footprint of diabetes: a multiplatform metabolomics study in an epidemiological setting

Background

Metabolomics is the rapidly evolving field of the comprehensive measurement of ideally all endogenous metabolites in a biological fluid. However, no single analytic technique covers the entire spectrum of the human metabolome. Here we present results from a multiplatform study, in which we investigate what kind of results can presently be obtained in the field of diabetes research when combining metabolomics data collected on a complementary set of analytical platforms in the framework of an epidemiological study.

Methodology/Principal Findings

40 individuals with self-reported diabetes and 60 controls (male, over 54 years) were randomly selected from the participants of the population-based KORA (Cooperative Health Research in the Region of Augsburg) study, representing an extensively phenotyped sample of the general German population. Concentrations of over 420 unique small molecules were determined in overnight-fasting blood using three different techniques, covering nuclear magnetic resonance and tandem mass spectrometry. Known biomarkers of diabetes could be replicated by this multiple metabolomic platform approach, including sugar metabolites (1,5-anhydroglucoitol), ketone bodies (3-hydroxybutyrate), and branched chain amino acids. In some cases, diabetes-related medication can be detected (pioglitazone, salicylic acid).

Conclusions/Significance

Our study depicts the promising potential of metabolomics in diabetes research by identification of a series of known and also novel, deregulated metabolites that associate with diabetes. Key observations include perturbations of metabolic pathways linked to kidney dysfunction (3-indoxyl sulfate), lipid metabolism (glycerophospholipids, free fatty acids), and interaction with the gut microflora (bile acids). Our study suggests that metabolic markers hold the potential to detect diabetes-related complications already under sub-clinical conditions in the general population.     

Synthesis of thiophene-2-carboxamidines containing 2-aminothiazoles and their biological evaluation as urokinase inhibitors

The serine protease urokinase (uPa) has been implicated in the progression of both breast and prostate cancer. Utilizing structure based design, the synthesis of a series of substituted 4-[2-amino-1,3-thiazolyl]-thiophene-2-carboxamidines is described. Further optimization of this series by substitution of the terminal amine yielded urokinase inhibitors with excellent activities.     

Amidinohydrazones as guanidine bioisosteres: application to a new class of potent, selective and orally bioavailable, non-amide-based small-molecule thrombin inhibitors

We describe a new class of potent, non-amide-based small molecule thrombin inhibitors in which an amidinohydrazone is used as a guanidine bioisostere on a non-peptide scaffold. Compound 4 exhibits nM inhibition of thrombin, is selective for thrombin, and shows 60 and 23% bioavailability in rabbits and dogs, respectively. Crystallographic analysis of 4 bound to thrombin confirmed the amindinohydrazone binding mode.     

Serum-free culture of fractionated bovine bronchial epithelial cells

Summary Procedures for the serum-free culture of a density fractionated population of bovine bronchial epithelial cells have been established. Epithelial cells dispersed by protease digestion were fractionated by density equilibrium centrifugation, followed by plating of the small basal-like population on type I collagen-coated culture dishes. Two or three passages of 1:4 split enriched for a population of actively dividing cells, which could be stored in liquid nitrogen for subsequent use. Clonal growth assays revealed optimum proliferation using a 1:1 mixture of medium RPMI 1640 and LHC-9, a medium employed for human bronchial epithelial cells. Cellular growth rate, which was 0.6 to 1.3 doublings per day depending on the cell preparation, was conveniently decreased by supplementing LHC-9 medium with less than 50% RPMI. In contrast to airway epithelial cell cultures from other species, serum stimulated the growth of bovine bronchial epithelial cells in this system. Transforming growth factorβ1, however, inhibited growth and induced differentiation into a squamous phenotype. Also in contrast with other systems, the bovine cells were resistant to growth inhibition by 100 nM tetradecanoyl phorbol acetate or 1μM calcium ionophore A23187. Combination of phorbol ester with ionophore decreased mitotic activity, although induction of squamous morphology was not observed. Therefore, growth inhibition and squamous differentiation were not tightly coupled in this system. Finally, biologically synthesized matrix deposited by these cells stimulated growth rate. This culture system will therefore be useful in assessing the activities of both soluble and matrix-associated factors in the absence of serum.     

Synergistic effect of genetic polymorphisms in TLR6 and TLR10 genes on the risk of pulmonary tuberculosis in a Moldavian population

Polymorphisms in genes that control immune function and regulation may influence susceptibility to pulmonary tuberculosis (TB). In this study, 14 polymorphisms in 12 key genes involved in the immune response (VDR, MR1, TLR1, TLR2, TLR10, SLC11A1, IL1B, IL10, IFNG, TNF, IRAK1, and FOXP3) were tested for their association with pulmonary TB in 271 patients with TB and 251 community-matched controls from the Republic of Moldova. In addition, gene–gene interactions involved in TB susceptibility were analyzed for a total of 43 genetic loci. Single nucleotide polymorphism (SNP) analysis revealed a nominal association between TNF rs1800629 and pulmonary TB (Fisher exact test P = 0.01843). In the pairwise interaction analysis, the combination of the genotypes TLR6 rs5743810 GA and TLR10 rs11096957 GT was significantly associated with an increased genetic risk of pulmonary TB (OR = 2.48, 95% CI = 1.62–3.85; Fisher exact test P value = 1.5 × 10⁻⁵, significant after Bonferroni correction). In conclusion, the TLR6 rs5743810 and TLR10 rs11096957 two-locus interaction confers a significantly higher risk for pulmonary TB; due to its high frequency in the population, this SNP combination may serve as a novel biomarker for predicting TB susceptibility.     

Discovery of novel FMS kinase inhibitors as anti-inflammatory agents

The optimization of the arylamide lead 2 resulted in identification of a highly potent series of 2,4-disubstituted arylamides. Compound 8 (FMS kinase IC(50)=0.0008 microM) served as a proof-of-concept candidate in a collagen-induced model of arthritis in mice.     

Free-tissue transfer in patients with peripheral vascular disease: a 10-year experience

Advances in free-tissue transfer have allowed for lower limb salvage in patients with significant peripheral vascular disease and limb-threatening soft-tissue wounds. The authors retrospectively reviewed their 10-year experience with free flaps for limb salvage in patients with peripheral vascular disease to assess postoperative complication rates and long-term functional outcome. They identified all patients undergoing free-tissue transfer with significant peripheral vascular disease and otherwise unreconstructible soft-tissue defects. Charts were reviewed for perioperative and long-term outcome. Parameters studied included perioperative morbidity and mortality, flap success, bypass graft patency, ambulatory results, and long-term limb and patient survival. Survival data were analyzed using life-table analysis, Kaplan-Meier survival analysis, and Cox testing. A total of 79 flaps were examined in 75 patients with peripheral vascular disease from July of 1990 to November of 1999. All patients would have required a major amputation had free-tissue transfer not been performed. Mean age was 60 years, average hospital stay was 32 days, and perioperative mortality was 5 percent. Within the first 30 days after operation, there were four cases of primary flap loss, and another two were lost as the result of bypass graft failure (8 percent); five of these cases resulted in amputation. There were no primary flap failures after 30 days. Follow-up ranged to 91 months (mean, 24 months). During this time, another 14 limbs were lost, most commonly because of progressive gangrene and/or infection in sites remote from the still-viable free flap. Using Kaplan-Meier survival analysis, 5-year flap survival was 77 percent, limb salvage 63 percent, and patient survival 67 percent. Sixty-six percent of patients were able to ambulate independently with the use of their reconstructed limb at least 1 year after hospital discharge, although some of these later went on to amputation. Free-tissue transfer for lower extremity reconstruction can be performed with acceptable morbidity and mortality in patients with peripheral vascular disease. Flap loss is low, and limb salvage, ambulation, and long-term survival rates in these patients are excellent.     

Linkage disequilibrium patterns and tagSNP transferability among European populations

The pattern of linkage disequilibrium (LD) is critical for association studies, in which disease-causing variants are identified by allelic association with adjacent markers. The aim of this study is to compare the LD patterns in several distinct European populations. We analyzed four genomic regions (in total, 749 kb) containing candidate genes for complex traits. Individuals were genotyped for markers that are evenly distributed at an average spacing of approximately 2-4 kb in eight population-based samples from ongoing epidemiological studies across Europe. The Centre d'Etude du Polymorphisme Humain (CEPH) trios of the HapMap project were included and were used as a reference population. In general, we observed a conservation of the LD patterns across European samples. Nevertheless, shifts in the positions of the boundaries of high-LD regions can be demonstrated between populations, when assessed by a novel procedure based on bootstrapping. Transferability of LD information among populations was also tested. In two of the analyzed gene regions, sets of tagging single-nucleotide polymorphisms (tagSNPs) selected from the HapMap CEPH trios performed surprisingly well in all local European samples. However, significant variation in the other two gene regions predicts a restricted applicability of CEPH-derived tagging markers. Simulations based on our data set show the extent to which further gain in tagSNP efficiency and transferability can be achieved by increased SNP density.