Development of biomarkers based on diet-dependent metabolic serotypes: practical issues in development of expert system-based classification models in metabolomic studies

Dietary restriction (DR)-induced changes in the serum metabolome may be biomarkers for physiological status (e.g., relative risk of developing age-related diseases such as cancer). Megavariate analysis (unsupervised hierarchical cluster analysis [HCA]; principal components analysis [PCA]) of serum metabolites reproducibly distinguish DR from ad libitum fed rats. Component-based approaches (i.e., PCA) consistently perform as well as or better than distance-based metrics (i.e., HCA). We therefore tested the following: (A) Do identified subsets of serum metabolites contain sufficient information to construct mathematical models of class membership (i.e., expert systems)? (B) Do component-based metrics out-perform distance-based metrics? Testing was conducted using KNN (k-nearest neighbors, supervised HCA) and SIMCA (soft independent modeling of class analogy, supervised PCA). Models were built with single cohorts, combined cohorts or mixed samples from previously studied cohorts as training sets. Both algorithms over-fit models based on single cohort training sets. KNN models had >85% accuracy within training/test sets, but were unstable (i.e., values of k could not be accurately set in advance). SIMCA models had 100% accuracy within all training sets, 89 % accuracy in test sets, did not appear to over-fit mixed cohort training sets, and did not require post-hoc modeling adjustments. These data indicate that (i) previously defined metabolites are robust enough to construct classification models (expert systems) with SIMCA that can predict unknowns by dietary category; (ii) component-based analyses outperformed distance-based metrics; (iii) use of over-fitting controls is essential; and (iv) subtle inter-cohort variability may be a critical issue for high data density biomarker studies that lack state markers.     

Drought‐resistant fungi control soil organic matter decomposition and its response to temperature

Microbial‐mediated decomposition of soil organic matter (SOM) ultimately makes a considerable contribution to soil respiration, which is typically the main source of CO₂ arising from terrestrial ecosystems. Despite this central role in the decomposition of SOM, few studies have been conducted on how climate change may affect the soil microbial community and, furthermore, on how possible climate‐change induced alterations in the ecology of microbial communities may affect soil CO₂ emissions. Here we present the results of a seasonal study on soil microbial community structure, SOM decomposition and its temperature sensitivity in two representative Mediterranean ecosystems where precipitation/throughfall exclusion has taken place during the last 10 years. Bacterial and fungal diversity was estimated using the terminal restriction fragment length polymorphism technique. Our results show that fungal diversity was less sensitive to seasonal changes in moisture, temperature and plant activity than bacterial diversity. On the other hand, fungal communities showed the ability to dynamically adapt throughout the seasons. Fungi also coped better with the 10 years of precipitation/throughfall exclusion compared with bacteria. The high resistance of fungal diversity to changes with respect to bacteria may open the controversy as to whether future ‘drier conditions’ for Mediterranean regions might favor fungal dominated microbial communities. Finally, our results indicate that the fungal community exerted a strong influence over the temporal and spatial variability of SOM decomposition and its sensitivity to temperature. The results, therefore, highlight the important role of fungi in the decomposition of terrestrial SOM, especially under the harsh environmental conditions of Mediterranean ecosystems, for which models predict even drier conditions in the future.     

CCN2 promotes keratinocyte adhesion and migration via integrin α5β1

Background

CCN2, (a.k.a. connective tissue growth factor and CTGF) has emerged as a regulator of cell migration. While the importance of CCN2 for the fibrotic process in wound healing has been well studied, the effect of CCN2 on keratinocyte function is not well understood. In this study, we investigated the mechanism behind CCN2-driven keratinocyte adhesion and migration.Materials and methods: Adhesion assays were performed by coating wells with 10 μg/ml fibronectin (FN) or phosphate-buffered saline (PBS). Keratinocytes were seeded in the presence or absence of 200 ng/ml CCN2, 5 mmol/l ethylenediaminetetraacetic acid, 10 mmol/l cations, 500 μl arginine–glycine–aspartic acid (RGD), 500 μM arginine–glycine–glutamate–serine (RGES), and 10 μg/ml anti-integrin blocking antibodies. Migration studies were performed using a modified Boyden chamber assay. Quantitative PCR was used to study the effect of CCN2 on integrin subunit mRNA expression. To block intracellular pathways, keratinocytes were pretreated with 20 μM PD98059 (MEK-1 inhibitor) or 20 μM PF573228 (FAK inhibitor) for 60 min prior the addition of CCN2. Western blot was used to measure CCN2, p-ERK1/2, and ERK1/2.Results: CCN2 enhanced keratinocyte adhesion to fibronectin via integrin α5β1. The addition of anti-integrin α5β1 antibodies reduced CCN2-mediated keratinocyte migration. In addition, CCN2 regulated mRNA and protein expression of integrin subunits α5 and β1. CCN2 activated the FAK-MAPK signaling pathway, and pretreatment with MEK1-specific inhibitor PD98059 markedly reduced CCN2-induced keratinocyte migration.Conclusions: Our results demonstrate that CCN2 enhances keratinocyte adhesion and migration through integrin α5β1 and activation of the FAK-MAPK signaling cascade.     

Ice-COLD-PCR enables rapid amplification and robust enrichment for low-abundance unknown DNA mutations

Identifying low-abundance mutations within wild-type DNA is important in several fields of medicine, including cancer, prenatal diagnosis and infectious diseases. However, utilizing the clinical and diagnostic potential of rare mutations is limited by sensitivity of the molecular techniques employed, especially when the type and position of mutations are unknown. We have developed a novel platform that incorporates a synthetic reference sequence within a polymerase chain reaction (PCR) reaction, designed to enhance amplification of unknown mutant sequences during COLD-PCR (CO-amplification at Lower Denaturation temperature). This new platform enables an Improved and Complete Enrichment (ice-COLD-PCR) for all mutation types and eliminates shortcomings of previous formats of COLD-PCR. We evaluated ice-COLD-PCR enrichment in regions of TP53 in serially diluted mutant and wild-type DNA mixtures. Conventional-PCR, COLD-PCR and ice-COLD-PCR amplicons were run in parallel and sequenced to determine final mutation abundance for a range of mutations representing all possible single base changes. Amplification by ice-COLD-PCR enriched all mutation types and allowed identification of mutation abundances down to 1%, and 0.1% by Sanger sequencing or pyrosequencing, respectively, surpassing the capabilities of other forms of PCR. Ice-COLD-PCR will help elucidate the clinical significance of low-abundance mutations and our understanding of cancer origin, evolution, recurrence-risk and treatment diagnostics.     

Functional changes in human leukemic cell line HL-60. A model for myeloid differentiation

Polar solvents induce terminal differentiation in the human promyelocytic leukemia cell line HL-60. The present studies describe the functional changes that accompany the morphologic progression from promyelocytes to bands and poly-morphonuclear leukocytes (PMN) over 9 d of culture in 1.3 percent dimethylsulfoxide (DMSO). As the HL-60 cells mature, the rate of O(2-) production increase 18-fold, with a progressive shortening of the lag time required for activation. Hexosemonophosphate shunt activity rises concomitantly. Ingestin of paraffin oil droplets opsonized with complement or Ig increases 10-fold over 9 d in DMSO. Latex ingestion per cell by each morphologic type does not change significantly, but total latex ingestion by groups of cells increases with the rise in the proportion of mature cells with greater ingestion capacities. Degranulation, as measured by release of β-glucuronidase, lysozyme, and peroxidase, reaches maximum after 3-6 d in DMSO, then declines. HL-60 cells contain no detectable lactoferrin, suggesting that their secondary granules are absent or defective. However, they kill staphylococci by day 6 in DMSO. Morphologically immature cells (days 1-3 in DMSO) are capable of O(2-) generation, hexosemonophosphate shunt activity, ingestion, degranulation, and bacterial killing. Maximal performance of each function by cells incubated in DMSO for longer periods of time is 50-100 percent that of normal PMN. DMSO- induced differentiation of HL-60 cells is a promising model for myeloid development.     

Morphology agrees with molecular data: phylogenetic affinities of Euptychiina butterflies (Nymphalidae: Satyrinae)

Euptychiina is the most species‐rich subtribe of Neotropical Satyrinae, with over 450 known species in 47 genera (14 monotypic). Here, we use morphological characters to examine the phylogenetic relationships within Euptychiina. Taxonomic sampling included 105 species representing the majority of the genera, as well as five outgroups. A total of 103 characters were obtained: 45 from wing pattern, 48 from genitalia and 10 from wing venation. The data matrix was analysed using maximum parsimony under both equal and extended implied weights. Euptychiina was recovered as monophyletic with ten monophyletic genera, contrasting previous DNA sequence‐based phylogenies that did not recover the monophyly of the group. In agreement with sequence‐based hypotheses, however, three main clades were recognized: the ‘Megisto clade’ with six monophyletic and three polyphyletic genera, the ‘Taygetis clade’ with nine genera of which three were monophyletic, and the ‘Pareuptyhia clade’ with four monophyletic and two polyphyletic genera. This is the first morphology‐based phylogenetic hypothesis for Euptychiina and the results will be used to complement molecular data in a combined analysis and to provide critical synapomorphies for clades and genera in this taxonomically confused group.     

Effect of vitamin E and eccentric exercise on selected biomarkers of oxidative stress in young and elderly men

Muscle damage resulting from eccentric exercise provides a useful model of oxyradical-induced injury and can be used to examine age-related responses to oxidative stress. Sixteen young (26.4 ± 3.3 years) and 16 older (71.1 ± 4.0 years) healthy men were randomly assigned to 1000 IU/d vitamin E or placebo for 12 weeks and ran downhill for 45 min at 75% VO₂max, once before and following supplementation. Blood samples were obtained before (baseline) and immediately postexercise (0 h), and at 6, 24, and 72 h postexercise to determine antioxidant status, muscle damage, lipid peroxidation, and DNA damage. Following exercise, young and older men experienced similar increases in serum creatine kinase (CK), F₂-isoprostanes (iPF₂; p < .001) and malondialdehyde (MDA; p < .01), although iPF₂ peaked at 72 h postexercise and MDA peaked at 0 h. Oxygen Radical Absorbance Capacity (ORAC) decreased at 72 h (p < .01) and correlated with the rise in iPF₂, MDA, and CK in the young men (p < .05). Leukocyte 8-hydroxy-2′-deoxyguanosine (8-OHdG) was unaffected by exercise. Vitamin E decreased peak CK in young men, while in older men it decreased resting levels of iPF₂ and suppressed the 24 h postexercise increases in iPF₂ (p < .05). Thus, vitamin E supplementation induced modest changes eccentric exercise-induced oxidative stress, although differentially between the young and older subjects, while age had no direct influence on these responses among this group of physically fit subjects.     

Carbon and nitrogen content based estimation of the fat content of animal carcasses in various species

The objective of this study was to explore whether the C and N content can be used to estimate the fat content of animal carcasses. Considering the mean C and N contents of body fat and body protein, the fat content (EE) [%] can be predicted from C and N values [%] according to the generally valid equation EE=1.3038·C – 4.237·N. The application of this equation to estimate the total fat content of all animal carcasses results in significant differences in fat content between predicted and measured values. Therefore, we derived specific equations for rats, pigs, cattle, sheep, broilers and mice to predict the fat content by dual linear regression analysis (y=EE [% DM], x₁=C [% DM], x₂=N [% DM]) based on measured fat, C, and N contents of animal body samples. The specific equations for different animals showed residual standard deviations of 1.55, 1.63, 1.12, 1.35, 1.85 and 0.92% fat for rats, pigs, cattle, sheep, broilers and mice, respectively.     

Diversity in Mediterranean farm ponds: trade‐offs and synergies between irrigation modernisation and biodiversity conservation

1. Agricultural intensification has caused dramatic biodiversity loss in many agricultural landscapes over the last century. Here, we investigated whether new types of farm ponds (made of artificial substrata) in intensive systems and natural‐substratum ponds in traditional farming systems differ in their value for aquatic biodiversity conservation. 2. We analysed the main patterns of environmental variation, compared α‐, β‐ and γ‐diversity of macroinvertebrates between ponds types and evaluated the role of submerged aquatic vegetation (SAV). Generalised additive models (GAM) were used to analyse the relationships of α‐ and β‐diversity with environmental predictors, and variation partitioning to separate the effect of environmental and spatial characteristics on the variation in macroinvertebrate assemblages. Moran’s eigenvector maps (MEMs) were used to define spatial variables. 3. A principal coordinate analysis (PCoA) detected a primary environmental gradient that separated nutrient‐rich ponds from those dominated by SAV; a secondary morphometric gradient distinguished natural‐substratum ponds, with large surface area and structural complexity, from artificial‐substratum ponds with steeper slopes. Natural‐substratum ponds had almost twice the α‐ and γ‐diversity of artificial‐substratum ponds, and diversity significantly increased when SAV was present, particularly in artificial‐substratum ponds. Total phosphorus (TP) strongly contributed to explain the patterns in diversity, while SAV was a significant predictor of assemblage composition and diversity. GAMs revealed optima of both α‐diversity at intermediate SAV covers and β‐diversity at intermediate–high TP concentrations. 4. These findings have important implications for conservation planning. Adaptation of artificial‐substratum ponds by adding natural substratum and smoothing the gradient of pond margins would improve their conservation value. Development of SAV with occasional harvests and certain cautionary measures to control nutrient levels may also improve both the agronomical and environmental function of ponds.