(Pre)analytical imprecision, between-subject variability, and daily variations in serum and urine hepcidin: Implications for clinical studies

The utility of urine and serum hepcidin measurements in the clinic depends on their reproducibility. We sought to expand our previous work on the within-subject variability and between-subject variability of this novel iron parameter in the serum and urine of 24 healthy controls by time-of-flight mass spectrometry at four different time points during the day. A linear mixed model for repeated data was used to distinguish three components of the total variability in the measurements: within-day/within-subject variability, between-subject variability, and additional residual or (pre)analytical variability. Differences in diurnal hepcidin patterns were observed between urine and serum. Urine levels remained similar during the course of the morning and increased significantly during the afternoon, whereas serum levels increased significantly throughout both the morning and afternoon. Furthermore, in serum the (pre)analytical variability (28.6%) was smaller than the between-subject (48.1%) and within-day/within-subject variability (30.3%) compared with urine variability (97.2% vs. 67.7 and 77.3%, respectively). High serum ferritin levels were associated with higher serum hepcidin levels but not with urine levels. Transferrin saturation did not correlate with hepcidin levels. To minimize variability, we recommend (i) standardizing for sampling time and (ii) measuring serum hepcidin levels.     

Genetic analysis of ecologically relevant morphological variability in Plantago lanceolata L.

Summary Morphological variability was studied in two populations of Plantago lanceolata using diallel analysis. In each population, reciprocal crosses between all possible pairs of ten plants were made. In the greenhouse, six members of each family were grown and many characters were measured. Using the model of Cockerham and Weir, the contributions of the different genetic variance components were calculated. From earlier papers it was postulated in advance to what extent and by which effect the characters in both populations were genetically determined. The populations had been differentiated for life history and morphological characters, and varied also in the relative contribution of genetic components to variability. In the Merrevliet (Me) population, where strong biotic selection was assumed, low levels of additive-genetic variability were present and the relative dominance appeared to be high. The contrasting population, Westduinen (Wd), which is abiotically controlled and shows strong environmental variability, possessed higher levels of additive-genetic variability and lower levels of relative dominance. It is possible that differential natural selection has diminished additive-genetic variability to different extents in both populations: plasticity and environmental heterogeneity prevented the loss of additive-genetic variability in Wd, whereas in the stable population, Me, natural selection had the opportunity of not only changing the means of the characters but also of diminishing additive-genetic variability to a great extent.Grassland Species Research Group Publication No. 146     

Variability of methylation profiles of CpG sites in microRNA genes in leukocytes and vascular tissues of patients with atherosclerosis

In this study, we for the first time described the variability of methylation levels of 71 CpG sites in microRNA genes in leukocytes and blood vessels (coronary artery atherosclerotic plaques, intact internal thoracic arteries, and great saphenous veins) in patients with atherosclerosis using the Infinium HumanMethylation27 BeadChip microarray. Most of the analyzed CpG sites were characterized by the low variability, and most of these low-variable sites were hypomethylated in all tissue samples. CpG sites in coronary artery atherosclerotic plaques and leukocytes were similar in their methylation status. The highest variability of CpG methylation levels between different tissues was found for the CpG sites of the MIR10B gene; the methylation levels of these sites in leukocytes and atherosclerotic arteries were lower than in intact blood vessels. We also found that several cardiovascular disease risk factors, as well as medications, might affect methylation levels of CpG sites in microRNAs.     

The Influence of Translocation Strategy and Management Practices on the Genetic Variability of a Reestablished Elk (Cervus elaphus) Population

Reintroduction of terrestrial vertebrates with the goal of ecosystem restoration typically establishes small and isolated populations that may experience reduced genetic variability due to founder effects and genetic drift. Understanding the genetic structure of these populations and maintaining adequate genetic diversity is important for long‐term restoration success. We quantified genetic variability at six microsatellite loci for a reintroduced population of Cervus elaphus (elk) restored to the tallgrass prairie ecosystem of northeastern Kansas. Allelic richness, observed and expected heterozygosity were intermediate to levels reported in other North American elk populations. Current levels of genetic variability in restored North American elk populations were not well explained by founding population size, number of founding populations, or number of years since the last translocation. Simulation results suggest that the retention of genetic variability in isolated populations is strongly influenced by mating system while also being impacted by temporal variability in population size and population growth rate. Our results have implications for understanding how translocation strategies and post‐reintroduction management may influence genetic variability in restored populations.     

Hepatitis C virus protease gene diversity in patients coinfected with human immunodeficiency virus

The clonal variability of the hepatitis C virus (HCV) protease gene in 24 individuals with HCV genotypes 1a, 1b, 2b, and 3a who were coinfected with the human immunodeficiency virus was evaluated. Within-genotype variability at the nucleotide and amino acid levels ranged from 6.5 to 8.6% and 2.2 to 3.8%, respectively. After adjustments were made for correlation of intrapatient clonal variation, mixed-model analysis indicated that nucleotide and amino acid variability among patients with different genotypes did not differ significantly. However, within individual patients, clonal variability differed by up to 5.3% and 5.8% at the nucleotide and amino acid levels, respectively, and genotype 1a had significantly greater nucleotide variability than other genotypes (P = 0.01). Significant variability exists within HCV protease gene variants at the patient level and could affect the effectiveness of HCV protease inhibitors.     

The role of the placenta in variability of fetal exposure to cocaine and cannabinoids: a twin study.

There is wide variability in the reported adverse fetal effects of cocaine and cannabinoids. The causes of this variability are largely unknown. We hypothesized that variability in placental handling of drugs affect fetal exposure. We used twin pregnancies as a paradigm to address the role of the placenta in this variability. We analyzed hair or meconium samples taken from dizygotic and monozygotic twins exposed in utero to illicit drugs. Out of 12 pairs, 5 had negative levels in both twins, and seven pairs of twins had chemical evidence of fetal exposure to cocaine (n = 5) or cannabinoids (n = 2). The one known monozygotic pair of twins had almost identical levels of cocaine. In contrast, the six dizygotic pairs had large disparities in either cocaine or cannabinoid concentrations. In three of these six dizygotic pairs, levels of cocaine (n = 2) or canabinoids (n = 1) were undetectable in one twin while positive in the other. Given that twins are theoretically exposed to similar maternal drug levels, our findings suggest that the placenta may have a major role in modulating the amounts of drug reaching the fetus.     

HGH20k species and variability of GH responses to long-duration exercise in male cyclists fed different food supplements.

Exercise studies dealing with hGH have always considered this hormone as a unique molecular entity. We postulated that the well-known variability in blood total hGH response could possibly be explained, at least in part, by concomitant changes in blood hGH20k levels, variant form possibly expressing some of the hGH anti-insulinic properties. Six male trained cyclists were imposed a 2-hr long ergocycle exercise. Food supplements were given prior to and/or during exertion to exacerbate a possible contribution from hGH20k to total hGH variability by modification of substrate availability. Both blood total hGH and hGH20k levels increased with exercise, the largest increases being observed in absence of supplementation. Large variability of responses were observed in both blood total hGH and hGH20k levels, the latter variant contributing minimally to total blood hGH response (4.3 +/- 0.8%), and being closely associated with the main species (r = 0.90; p less than 0.001). It was concluded that variations associated with hGH20k increases observed in response to prolonged exercise cannot explain the large intra-and inter-individual variability measured in blood total hGH response.     

Single cell variability of L. monocytogenes grown on liver pâté and cooked ham at 7 degrees C: comparing challenge test data to predictive simulations

AIMS: The variability in growth between individual Listeria monocytogenes cells was investigated on liver paté and cooked ham. These results were compared to Monte Carlo simulations based on data collected previously in broths (Francois et al., submitted for publication). METHODS AND RESULTS: Single cells were isolated by a dilution protocol and inoculated on 15 g samples of liver paté and cooked ham, pasteurized in the packaging. Of each product, 250 samples were inoculated, of which 50 samples were analysed for L. monocytogenes on each analysis day. Results were compared to simulations, based on distributions that describe the variability of the individual cell lag phases and generation times of L. monocytogenes cultivated in broths. Based on the same simulation techniques, the variability effect was investigated for different inoculum levels (10, 100, 10,00 and 10,000 cells). It was demonstrated that the expected variability of the outgrowth of L. monocytogenes in a challenge test is very high for low inoculum levels. CONCLUSIONS: The variability in growth characteristics observed between different single L. monocytogenes cells on foods is very large. The simulations based on the previously collected optical density data in broths, could be confirmed by foods inoculated with single L. monocytogenes cells. SIGNIFICANCE AND IMPACT OF THE STUDY: The large variability between different individual L. monocytogenes cells has serious consequences for the experimental design of a challenge test. One thousand cells per portion are necessary in order to reduce the variability to acceptable levels and quantify the behaviour of the pathogen consistently with a reasonable number of challenge tests.     

Effects of estradiol and progesterone on the variability of the micronucleus assay

To investigate chromosomal radiosensitivity of lymphocytes the micronucleus (MN) assay has been used for many years. The results of these studies suggest the use of the MN assay as a biomarker for cancer predisposition. However, the MN assay has still some limitations associated with the reproducibility and sensitivity. Especially a high intra-individual variability has been observed. An explanation for this high intra-individual variability is not yet available. In literature it is suggested that the high variability among females is attributable to hormonal status. In this study we investigated if the high intra-individual variability in micronucleus formation in lymphocytes of females after in vitro exposure to ionising radiation is caused by variations in hormone levels of estradiol (E2) and progesterone (PROG). For this, the MN assay was performed on blood samples of 18 healthy women during 7 consecutive weeks while the estradiol and progesterone levels were determined at the same time. The MN assay was also examined in cultures of isolated blood lymphocytes with estradiol or progesterone levels added in vitro. The results demonstrated that estradiol and progesterone levels have no influence on the variations in radiation-induced MN yields observed in blood samples of healthy women. These conclusions were confirmed by the "in vitro" experiments as no correlation between the MN yields and the concentrations of hormones (estradiol or progesterone) added in vitro to isolated lymphocytes cultures was observed.     

Effect of inversion polymorphism on the neutral nucleotide variability of linked chromosomal regions in Drosophila

Recombination is a main factor determining nucleotide variability in different regions of the genome. Chromosomal inversions, which are ubiquitous in the genus Drosophila, are known to reduce and redistribute recombination, and thus their specific effect on nucleotide variation may be of major importance as an explanatory factor for levels of DNA variation. Here, we use the coalescent approach to study this effect. First, we develop analytical expressions to predict nucleotide variability in old inversion polymorphisms that have reached mutation-drift-flux equilibrium. The effects on nucleotide variability of a new arrangement appearing in the population and reaching a stable polymorphism are then studied by computer simulation. We show that inversions modulate nucleotide variability in a complex way. The establishment of an inversion polymorphism involves a partial selective sweep that eliminates part of the variability in the population. This is followed by a slow convergence to the equilibrium values. During this convergence, regions close to the breakpoints exhibit much lower variability than central regions. However, at equilibrium, regions close to the breakpoints have higher levels of variability and differentiation between arrangements than regions in the middle of the inverted segment. The implications of these findings for overall variability levels during the evolution of Drosophila species are discussed.     

Investigation of genetic diversity in fragmented geometric tortoise (Psammobates geometricus) populations

Microsatellite DNA was used to investigatelevels of genetic variability in severelyfragmented populations of the geometrictortoise, Psammobates geometricus, themost endangered tortoise on mainland Africa.Eight microsatellite markers were used toassess genetic variability within and betweenthree naturally occurring populations of P. geometricus. These populations areseparated by the Cape Fold mountain ranges inthe western Cape region of South Africa. Levels of variability were quantified usingallelic diversity, genotypic frequencies andheterozygosity. Evidence for populationsub-structuring was examined using Fstatistics, R_st and ².High levels of variability were found in allthree populations. Low levels of populationdifferentiation were found suggestingsignificant gene flow between the populations.     

Diurnal testosterone variability is differentially associated with parenting quality in mothers and fathers

Previous studies on the relation between testosterone (T) levels and parenting have found ample evidence for the challenge hypothesis, demonstrating that high T levels inhibit parental involvement and that becoming a parent is related to a decrease in T levels in both mothers and fathers. However, less is known about the relation between T levels and more qualitative aspects of parenting. In the current study we examined basal T levels and diurnal variability in T levels in relation to mothers' and fathers' parenting quality. Participants included 217 fathers and 124 mothers with two children (3 and 5 years of age). Evening and morning salivary T samples were analyzed with radio-immunoassays to determine circulating T levels. Parental sensitivity (i.e., child-centered responsiveness) and respect for children's autonomy were observed during free play in the family home. The results showed that higher evening T levels in mothers were associated with more sensitivity to the oldest and youngest child. Diurnal T variability was more consistently associated with parenting behavior towards their children than basal T levels. For fathers, more diurnal variability in T was associated with more sensitivity and more respect for autonomy with their youngest children. For mothers, more diurnal variability in T was associated with less sensitivity to both children and less respect for the youngest child's autonomy. These findings suggest that the T system might act differently in relation to parenting behavior in males and females.     

Integral evaluation of phenotypic and genotypic variability of morphologic and psychophysiologic human traits

The paper demonstrates the expedience of the developed populational-genetic approach to studying the genetic bases of a complex of quantitative characters pertaining to different levels of human organization: anthropometry, neurodynamics and psychodynamics. The studies were carried out in eight human populations of different degree of isolation and ethnical background. It is shown that correlation phenotypic variability is higher for these characters, as compared to genotypic variability, the number of general genes responsible for this variability being decreased, respectively.     

Exploring the contextual sensitivity of factors that determine cell-to-cell variability in receptor-mediated apoptosis

Stochastic fluctuations in gene expression give rise to cell-to-cell variability in protein levels which can potentially cause variability in cellular phenotype. For TRAIL (TNF-related apoptosis-inducing ligand) variability manifests itself as dramatic differences in the time between ligand exposure and the sudden activation of the effector caspases that kill cells. However, the contribution of individual proteins to phenotypic variability has not been explored in detail. In this paper we use feature-based sensitivity analysis as a means to estimate the impact of variation in key apoptosis regulators on variability in the dynamics of cell death. We use Monte Carlo sampling from measured protein concentration distributions in combination with a previously validated ordinary differential equation model of apoptosis to simulate the dynamics of receptor-mediated apoptosis. We find that variation in the concentrations of some proteins matters much more than variation in others and that precisely which proteins matter depends both on the concentrations of other proteins and on whether correlations in protein levels are taken into account. A prediction from simulation that we confirm experimentally is that variability in fate is sensitive to even small increases in the levels of Bcl-2. We also show that sensitivity to Bcl-2 levels is itself sensitive to the levels of interacting proteins. The contextual dependency is implicit in the mathematical formulation of sensitivity, but our data show that it is also important for biologically relevant parameter values. Our work provides a conceptual and practical means to study and understand the impact of cell-to-cell variability in protein expression levels on cell fate using deterministic models and sampling from parameter distributions.     

Transference of natural diversity from the apomictic germplasm of Paspalum notatum to a sexual synthetic population

Genetic improvement in apomictic forage species has been restricted because of the absence of genetic variability in sexual germplasm with the same ploidy level. Following a new breeding scheme, a sexual synthetic tetraploid population (SSTP) of Paspalum notatum has been generated. The objectives of this work were: (a) to evaluate the genetic variability in SSTP by means of molecular markers, morphologic and agronomic traits, and seed fertility and quality traits and (b) to assess the transference of genetic variability from the apomictic germplasm to the sexual one. Molecular markers revealed a twofold higher level of variability in the SSTP in comparison with the sexual germplasm utilised for its generation, and similar levels with the apomictic ones; moreover, markers showed that most of the variability was inherited from the apomictic germplasm. Morphologic and agronomic traits and seed fertility and quality traits showed high levels of variation in the three groups of genotypes indicating that the new breeding scheme was effective in transferring variability from the apomictic germplasm to the SSTP. This new population will be useful in breeding of P. notatum, and the breeding scheme used for its generation may be used in other apomictic species.     

Genetic similarity, breeding distribution range and sexual selection

Large populations with extensive breeding distributions may sustain greater genetic variability, thus producing a positive relationship between genetic variation and population size. Levels of genetic variability may also be affected by sexual selection, which could either reduce levels because a small fraction of males contribute to the following generation, or augment them by generating genetic variability through elevated rates of mutations. We investigated to what extent genetic variability, as estimated from band sharing coefficients for minisatellite markers, could be predicted by breeding distribution range, population size and intensity of sexual selection (as reflected by degree of polygyny and extra-pair paternity). Across a sample of 62 species of birds in the Western Palearctic, we found extensive interspecific variation in band sharing coefficients. High band sharing coefficients (implying low local genetic variability among individuals) were associated with restricted breeding distributions, a conclusion confirmed by analysis of statistically independent linear contrasts. Independently, species with large population sizes had small band sharing coefficients. Furthermore, bird species with a high richness of subspecies for their breeding distribution range had higher band sharing coefficients. Finally, bird species with high levels of polygyny and extra-pair paternity had small band sharing coefficients. These results suggest that breeding distribution range, population size and intensity of sexual selection are important predictors of levels of genetic variability in extant populations.     

Variation in the large-scale organization of gene expression levels in the hippocampus relates to stable epigenetic variability in behavior

Background

Despite sharing the same genes, identical twins demonstrate substantial variability in behavioral traits and in their risk for disease. Epigenetic factors–DNA and chromatin modifications that affect levels of gene expression without affecting the DNA sequence–are thought to be important in establishing this variability. Epigenetically-mediated differences in the levels of gene expression that are associated with individual variability traditionally are thought to occur only in a gene-specific manner. We challenge this idea by exploring the large-scale organizational patterns of gene expression in an epigenetic model of behavioral variability.

Methodology/Findings

To study the effects of epigenetic influences on behavioral variability, we examine gene expression in genetically identical mice. Using a novel approach to microarray analysis, we show that variability in the large-scale organization of gene expression levels, rather than differences in the expression levels of specific genes, is associated with individual differences in behavior. Specifically, increased activity in the open field is associated with increased variance of log-transformed measures of gene expression in the hippocampus, a brain region involved in open field activity. Early life experience that increases adult activity in the open field also similarly modifies the variance of gene expression levels. The same association of the variance of gene expression levels with behavioral variability is found with levels of gene expression in the hippocampus of genetically heterogeneous outbred populations of mice, suggesting that variation in the large-scale organization of gene expression levels may also be relevant to phenotypic differences in outbred populations such as humans. We find that the increased variance in gene expression levels is attributable to an increasing separation of several large, log-normally distributed families of gene expression levels. We also show that the presence of these multiple log-normal distributions of gene expression levels is a universal characteristic of gene expression in eurkaryotes. We use data from the MicroArray Quality Control Project (MAQC) to demonstrate that our method is robust and that it reliably detects biological differences in the large-scale organization of gene expression levels.

Conclusions

Our results contrast with the traditional belief that epigenetic effects on gene expression occur only at the level of specific genes and suggest instead that the large-scale organization of gene expression levels provides important insights into the relationship of gene expression with behavioral variability. Understanding the epigenetic, genetic, and environmental factors that regulate the large-scale organization of gene expression levels, and how changes in this large-scale organization influences brain development and behavior will be a major future challenge in the field of behavioral genomics.     

THE ASSOCIATION BETWEEN FLUCTUATING ASYMMETRY,TRAIT VARIABILITY,TRAIT HERITABILITY,AND STRESS: A MULTIPLY REPLICATED EXPERIMENT ON COMBINED STRESSES IN DROSOPHILA MELANOGASTER

A number of hypotheses have been proposed about the association between developmental stability phenotypic variability, heritability, and environmental stress. Stress is often considered to increase both the asymmetry and phenotypic variability of bilateral traits, although this may depend on trait heritability. Empirical studies of such associations often yield inconsistent results. This may reflect the diversity of traits and conditions used or a low repeatability of any associations. To test for repeatable associations between these variables, multiply replicated experiments were undertaken on Drosophila melanogaster using a combination stress at the egg, larval and adult stages of reduced protein, ethanol in the medium, and a cold shock. Both metric and meristic traits were measured and levels of heritable variation for each trait estimated by maximum likelihood and parent-offspring regression over three generations. Trait means were reduced by stress, whereas among-individual variation increased Fluctuating asymmetry (FA) was increased by stress in some cases, but few comparisons were significant. Only one trait orbital bristle, showed consistent increases in FA. Changes in trait means, trait phenotypic variability, and developmental stability as a result of stress were not correlated. Extreme phenotypes tended to have higher levels of FA but only the results for orbital bristles were significant. All traits had low to intermediate heritabilities except orbital bristle, which showed no heritable variation. Only traits with low heritability and high levels of phenotypic variability may show consistent increases in FA under stress. Overall, the independence of phenotypic variability, plasticity, and the developmental stability of traits extend to changes in these measures under stressful conditions.     

A study of spatial variability of domoic acid in razor clams: recommendations for resource management on the Washington coast

Domoic acid (DA) levels in Washington State razor clams (Siliqua patula) have been extremely variable and unpredictable, resulting in emergency closures of harvest areas in 1991, 1998, and 1999. Information concerning toxin variability relative to sampling location is important in developing a more reliable plan for managing DA outbreaks. In November 1998, record levels of DA in razor clams (up to 295 ppm) were reported at Kalaloch beach, Washington. A razor clam resource survey conducted there during the summer of 1999, along with the long retention time of DA in the clams, permitted a study of DA levels as a function of tidal elevation and north-south beach location. During the summer low tides (28–31 July 1999) razor clams were collected from six east-west transects, approximately 1.6 km apart. Each clam was individually analyzed for DA to determine the distribution of toxin between (interspecific variability) and within (intraspecific variability) transects. While average DA levels were similar between transects, toxin levels varied substantially among individual clams. The coefficient of variation among all samples (n=445) was 122%, indicating that harvest closures based upon composite analyses of only six clams could be in error. Here we recommend a sampling strategy of razor clams for regulatory purposes that will result in DA measurements more representative of total population toxin values.     

Group size structure affects patterns of aggression in larval salamanders

The potential importance of intrapopulation phenotypic variabilityto population-level ecology has been demonstrated in both theoreticaland field studies. One way to connect individuals to the dynamicsof populations they compose is to study behavioral response(an individual-level characteristic) to variability in conspecificphenotypes (a population-level characteristic). We examinedeffects of variation in size of individuals on patterns of aggressionin larval tiger salamanders (Ambystoma tigrinum nebulosum) byobserving aggressive behavior in groups of three larvae in alaboratory experment. We assessed effects of variability insize of conspecifics independently of mean larval size and larvaldensity Overall levels of aggression were generally higher ingroups in which all individuals were similprly sized than ingroups of variably sized individuals. Medium-sized individualsexhibited significantly higher levels of aggression and wereattacked significantly more often when in groups consistingonly of similarly sized larvae as compared to groups composedof larvae representing a wider range of body sizes. Activitylevels of larvae were also generally lower when all individualswere the same size, resulting in a negative correlation betweenactivity and levels of iggressititi. These results suggest thatgioups of similarly sized individuals experience a more aggressivesocial environment than groups of variably sized individuals,and they suggest a promising avenue of research for connectingindividual behavioral and physiological responses to size structure(phenotypic variability) with population dynamics.