State-of-the art data normalization methods improve NMR-based metabolomic analysis

Extracting biomedical information from large metabolomic datasets by multivariate data analysis is of considerable complexity. Common challenges include among others screening for differentially produced metabolites, estimation of fold changes, and sample classification. Prior to these analysis steps, it is important to minimize contributions from unwanted biases and experimental variance. This is the goal of data preprocessing. In this work, different data normalization methods were compared systematically employing two different datasets generated by means of nuclear magnetic resonance (NMR) spectroscopy. To this end, two different types of normalization methods were used, one aiming to remove unwanted sample-to-sample variation while the other adjusts the variance of the different metabolites by variable scaling and variance stabilization methods. The impact of all methods tested on sample classification was evaluated on urinary NMR fingerprints obtained from healthy volunteers and patients suffering from autosomal polycystic kidney disease (ADPKD). Performance in terms of screening for differentially produced metabolites was investigated on a dataset following a Latin-square design, where varied amounts of 8 different metabolites were spiked into a human urine matrix while keeping the total spike-in amount constant. In addition, specific tests were conducted to systematically investigate the influence of the different preprocessing methods on the structure of the analyzed data. In conclusion, preprocessing methods originally developed for DNA microarray analysis, in particular, Quantile and Cubic-Spline Normalization, performed best in reducing bias, accurately detecting fold changes, and classifying samples.

     

Defective production of anti-herpes simplex virus antibody by neonatal mice. Reconstitution with Ia+ macrophages and T helper lymphocytes from nonimmune adult syngeneic mice

Both neonatal humans and mice are exquisitely susceptible to severe HSV infection. We have now documented a profound defect in the ability of neonatal C57BL/6 mice to produce anti-HSV ADCC antibody. This ability is acquired over the first 2 to 4 wk of life. Reconstitution of neonatal mice by i.p. injection of peritoneal cells from adult nonimmune syngeneic mice both affords dose-dependent protection against lethal HSV infection and reconstitutes the antibody-production defect. By cell-separation techniques (adherence, nylon wool column purification, B cell panning) and cell ablation techniques (silica treatment, irradiation, anti-T cell, anti-Ia, anti-Lyt-1.2 and anti-Lyt-2.2 monoclonal antibodies plus complement treatment) the subpopulations involved in the antibody production reconstitution of neonatal mice by adult cells were identified. These include both an Ia+, radioresistant, adherent, silica-sensitive macrophage population and a nylon wool column-purified, radiosensitive, anti-T, anti-Lyt-1.2-sensitive helper T cell population. The latter cell may be substituted for by concanavalin A-stimulated lymphokine-containing spleen cell supernatants or human recombinant IL 2. In addition to reconstitution of ADCC antibody production, the same cell populations, or cells plus lymphokine-containing supernatants or IL 2, protected the newborn mice from lethal HSV infection. Further characterization of this system and of soluble replacement factors has implications for therapy or immunoprophylaxis of human neonates with, or at risk of, HSV infection.     

Structural basis for altered soybean agglutinin lectin binding between a murine metastatic lymphoma and an adhesive low malignant variant

By selection for plastic adhesiveness we have previously established a variant tumor line (ESb-MP) from the metastatic murine lymphoma ESb. In contrast to the parental line, the adhesion variant is significantly decreased in malignancy and is altered in the capacity to bind soybean agglutinin (SBA) lectin. Here we show biochemically that the major SBA-binding cell-surface component of ESb-MP cells is the T200 glycoprotein. In ESb cells, T200 antigens bind SBA only after sialidase treatment. Enzymatic studies suggested that glycans detected by the lectin with or without sialidase treatment are different. Inhibition of N-glycosylation by tunicamycin and biosynthetic labeling revealed two T200 chains for ESb-MP cells that were larger in size than the single chain detected in ESb cells. Studies on the biosynthesis revealed that ESb-MP cells expressed two precursor chains for T200 whereas ESb cells displayed only one. There was no size difference detectable in the mature T200 molecules of ESb and ESb-MP cells. Our data suggest that the molecules differ in expression of O-linked glycans that can be recognized by SBA. Additional O-linked sugars on ESb-MP T200 molecules seem to be expressed in particular after trimming of the second T200 precursor chain.     

Plasmids in toxic and nontoxic strains of the cyanobacteriumMicrocystis aeruginosa

The plasmid content and toxicity of nine different strains ofMicrocystis aeruginosa have been analyzed. The two toxic strains of the HUB Culture Collection were found to carry each two plasmids, pMA1 and pMA2, of 2.9 kb and 8.5 kb, respectively. In strains PCC 7813 and PCC 7820, also toxic, two different plasmids of 2.6 kb and 16 kb were detected. Hybridization experiments showed that there exists no sequence homology between the pMA plasmids and the plasmids found in the PCC strains; but the pMA plasmids hybridized to chromosomal DNA of the toxic strains PCC 7820, PCC 7813, HUB 063, and the nontoxic strain HUB 5-3. In nontoxic strains no or at most one plasmid of unstable occurrence could be detected. Only one of the toxic strains investigated, SAG 14.85 (NRC-1), contained no plasmid.     

Natural15N abundance as a method of estimating the contribution of biologically fixed nitrogen to N2-fixing systems: Potential for non-legumes

The¹⁵N abundance of plants usually closely reflects the¹⁵N abundance of their major immediate N source(s); plant-available soil N in the case of non-N₂-fixing plants and atmospheric N₂ in the case of N₂ fixing plants. The¹⁵N abundance values of these sources are usually sufficiently different from each other that a significant and systematic difference in the¹⁵N abundance between the two kinds of plants can be detected. This difference provides the basis for the natural¹⁵N abundance method of estimating the relative contribution of atmospheric N₂ to N₂-fixing plants growing in natural and agricultural settings. The natural¹⁵N abundance method has certain advantages over more conventional methods, particularly in natural ecosystems, since disturbance of the system is not required and the measurements may be made on samples dried in the field. This method has been tested mainly with legumes in agricultural settings. The tests have demonstrated the validity of this method of arriving at semi-quantitative estimates of biological N₂-fixation in these settings. More limited tests and applications have been made for legumes in natural ecosystems. An understanding of the limits and utility of this method in these systems is beginning to emerge. Examples of systematic measurements of differences in¹⁵N abundance between non-legume N₂-fixing systems and neighbouring non-fixing systems are more unusual. In principle, application of the method to estimate N₂-fixation by nodulated non-legumes, using the natural¹⁵N abundance method, is as feasible as estimating N₂-fixation by legumes. Most of the studies involving N₂-fixing non-legumes are with this type of system (e.g., Ceanothus, Chamabatia, Eleagnus, Alnus, Myrica, and so forth). Resuls of these studies are described. Applicability for associative N₂-fixation is an empirical question, the answer to which probably depends upon the degree to which fixed N goes predominantly to the plant rather than to the soil N pool. The natural¹⁵N abundance method is probably not well suited to assessing the contribution of N₂-fixation by free-living microorganisms in their natural habitat, particularly soil microorganisms.This work was supported in part by subcontracts under grants from the US National Science Foundation (DEB79-21971 and BSR821618)     

Leukocyte LFA-1, OKM1, p150,95 deficiency syndrome: functional and biosynthetic studies of three kindreds

Three patients (2 female, 1 male) with recurrent infection, granulocytosis, impaired pus formation, and/or delayed umbilical cord separation were identified. Assessments of polymorphonuclear leukocytes (PMN)/monocyte function in each patient revealed profound abnormalities of adherence and adherence-dependent functions. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) of their PMN lysates demonstrated a deficient or absent protein(s) of 138 kilodaltons (gp 138). Na3HB4 labeling demonstrated the absence of a major cell surface glycoprotein complex in each patient. Among parental and sibling PMN suspensions, functional assessments revealed no consistent abnormalities, although variably diminished gp138 was identified by SDS-PAGE and Na3HB4 labeling. Analysis by fluorescence-activated cell sorting and monoclonal antibodies (MAb) to LFA-1 alpha, OKM1 alpha, and their common beta subunit demonstrated a severe or total deficiency of PMN/monocyte surface expression of each protein among all patients; intermediate values were observed for parental and affected sibling suspensions, findings consistent with an autosomal recessive mode of inheritance for this disorder. Cell surface labeling (125I) and immunoprecipitation with the same MAb demonstrated the absence of these glycoproteins in addition to a 150-kilodalton protein (p150,95). Identical abnormalities of surface expression of patient lymphocytes blast-transformed with phytohemagglutinin (PHA) or Epstein-Barr virus were demonstrated. Further, significantly diminished natural killer cell cytotoxicity was observed for each patient tested. PHA blast-transformed patient lymphocytes labeled with [35S]methionine demonstrated a total absence of the beta molecule but indicated the presence of an LFA-1 alpha precursor. These findings indicate that LFA-1 alpha synthesis and surface expression require beta association. It is concluded that impaired inflammatory function in this disorder is casually related to a heritable deficiency of critical "adhesive" leukocyte glycoproteins.     

Synthesis of 3 beta-hydroxy-5 alpha-cholest-8-en-7-one and 3 beta-hydroxy-5 alpha-cholest-8-en-11-one: evaluation as potential hypocholesterolemic agents

An efficient procedure for the chemical synthesis of 3 beta-hydroxy-5 alpha-cholest-8-en-7-one and 3 beta-hydroxy-5 alpha-cholest-8-en-11-one is described. These ketosterols have been shown to have possible significant hypocholesterolemic effects when fed to normal rats at a level of 0.15% in a laboratory chow diet. The diets containing the steroids caused significant decreases in food consumption which were associated with decreases in the rate of gain in body weight.