Unsupervised Analysis of Classical Biomedical Markers: Robustness and Medical Relevance of Patient Clustering Using Bioinformatics Tools

Motivation: It has been proposed that clustering clinical markers, such as blood test results, can be used to stratify patients. However, the robustness of clusters formed with this approach to data pre-processing and clustering algorithm choices has not been evaluated, nor has clustering reproducibility. Here, we made use of the NHANES survey to compare clusters generated with various combinations of pre-processing and clustering algorithms, and tested their reproducibility in two separate samples. Method: Values of 44 biomarkers and 19 health/life style traits were extracted from the National Health and Nutrition Examination Survey (NHANES). The 1999–2002 survey was used for training, while data from the 2003–2006 survey was tested as a validation set. Twelve combinations of pre-processing and clustering algorithms were applied to the training set. The quality of the resulting clusters was evaluated both by considering their properties and by comparative enrichment analysis. Cluster assignments were projected to the validation set (using an artificial neural network) and enrichment in health/life style traits in the resulting clusters was compared to the clusters generated from the original training set. Results: The clusters obtained with different pre-processing and clustering combinations differed both in terms of cluster quality measures and in terms of reproducibility of enrichment with health/life style properties. Z-score normalization, for example, dramatically improved cluster quality and enrichments, as compared to unprocessed data, regardless of the clustering algorithm used. Clustering diabetes patients revealed a group of patients enriched with retinopathies. This coul

Non-homologous DNA end joining in normal and cancer cells and its dependence on break structures

DNA double-strand breaks (DSBs) are a serious threat to the cell, for if not or miss-repaired, they can lead to chromosomal aberration, mutation and cancer. DSBs in human cells are repaired via non-homologous DNA end joining (NHEJ) and homologous recombination repair pathways. In the former process, the structure of DNA termini plays an important role, as does the genetic constitution of the cells, through being different in normal and pathological cells. In order to investigate the dependence of NHEJ on DSB structure in normal and cancer cells, we used linearized plasmids with various, complementary or non-complementary, single-stranded or blunt DNA termini, as well as whole-cell extract isolated from normal human lymphocytes, chronic myeloid leukemia K562 cells and lung cancer A549 cells. We observed a pronounced variability in the efficacy of NHEJ reaction depending on the type of ends. Plasmids with complementary and blunt termini were more efficiently repaired than the substrate with 3' protruding single-strand ends. The hierarchy of the effectiveness of NHEJ was on average, from the most effective to the least, A549/ normal lymphocytes/ K562. Our results suggest that the genetic constitution of the cells together with the substrate terminal structure may contribute to the efficacy of the NHEJ reaction. This should be taken into account on considering its applicability in cancer chemo- or radiotherapy by pharmacologically modulating NHEJ cellular responses

Non-random peroxidation of different classes of membrane phospholipids in live cells detected by metabolically integrated cis-parinaric acid

Quantitative assays of lipid peroxidation in intact, living cells are essential for evaluating oxidative damage from various sources and for testing the efficacy of antioxidant interventions. We report a novel method based on the use of cis-parinaric acid (PnA) as a reporter molecule for membrane lipid peroxidation in intact mammalian cells. Using four different cell lines (human leukemia HL-60, K562 and K/VP.5 cells, and Chinese hamster ovary (CHO) fibroblasts), we developed a technique to metabolically integrate PnA into all major classes of membrane phospholipids, i.e., phosphatidylcholine, phosphatidylethanolamine, phosphatidylserine, phosphatidylinositol and cardiolipin, that can be quantified by HPLC with fluorescence detection. Integrated PnA constituted less than 1% of lipid fatty acid residues, suggesting that membrane structure and characteristics were not significantly altered. Low concentrations (20-40 mu M) of tert-butyl hydroperoxide (t-BuOOH) caused selective oxidation of PnA residues in phosphatidylserine and phosphatidylethanolamine of K562 cells and K/VP.5 cells while cell viability was unaffected. At higher t-BuOOH concentrations (exceeding 100 mu M), however, a progressive, random oxidation of all major phospholipid classes occurred and was accompanied by significant cell death. In HL-60 cells, phosphatidylethanolamine, phosphatidylserine and cardiolipin were sensitive to low concentrations of t-BuOOH, while phosphatidylcholine and phosphatidylinositol were not affected. Phosphatidylinositol was the only phospholipid that responded to the low concentrations of t-BuOOH in CHO cells, At high t-BuOOH concentrations, again, all phospholipid classes underwent extensive oxidation, All phospholipids were nearly equally affected by peroxidation induced by a initiator of peroxyl radicals, 2,2'-azobis-(2,4-dimethylvaleronitrile) (AMVN), in K562 cells. In gamma-irradiated (4-128 Gy) CHO cells, phosphatidylserine was the most affected phospholipid class (34% peroxidation) followed by phosphatidylinositol (24% peroxidation) while the other three phospholipid classes were apparently unaffected, Since loss of PnA fluorescence is a direct result of irreparable oxidative loss of its conjugated double bond system, the method described allows for selective and sensitive monitoring of oxidative stress in live cells without interference from cell repair mechanisms