Seminal cell-free DNA levels measured by PicoGreen fluorochrome are associated with sperm fertility criteria.

Previous investigations suggested that elevated cell-free DNA (cfDNA) can indicate non-healthy states. However, the potential association between cfDNA seminal plasma levels and fertility sperm parameters has not yet been determined. Therefore, the present study evaluated the association between seminal cfDNA levels and sperm fertility criteria to determine the use of seminal cfDNA quantification. An in vivo protocol quantified cfDNA levels of semen samples obtained from 163 male patients using fluorescent PicoGreen dye staining. To confirm if semen cfDNA quantification is realistic, an in vitro complementary test was performed using three or four semen samples. The fresh sperm samples were exposed to paraquat that generates high levels of superoxide anion causing oxidative stress and cell mortality. The results showed significant association between dsDNA levels and several sperm fertility parameters, such as low viability and alterations of motility and morphology. The in vitro analysis confirmed the association between dsDNA levels and sperm viability. Together, these results suggest that dsDNA levels could be an important biomarker to test sperm fertility.

Adolescent growth: genes, hormones and the peer group. Proceedings of the 20th Aschauer Soiree, held at Glücksburg castle, Germany, 15th to 17th November 2013.

The association between poverty, malnutrition, illness and poor socioeconomic conditions on the one side, and poor growth and short adult stature on the other side, is well recognized. Yet, the simple assumption by implication that poor growth and short stature result from poor living conditions, should be questioned. Recent evidence on the impact of the social network on adolescent growth and adult height further challenges the traditional concept of growth being a mirror of health. Twenty-nine scientists met at Glücksburg castle, Northern Germany, November 15th - 17th 2013, to discuss genetic, endocrine, mathematical and psychological aspects and related issues, of child and adolescent growth and final height.

Final height, target height and the community.

Height varies with age, and it varies with historic time. Final height is determined by endocrine parameters and genetics, by nutrition and health, by environmental factors, by birth weight, early growth, BMI, and developmental tempo. European populations of the 19th century were short, but their shortness did not result from growth impairment at all ages. In those days, shortness was mainly due to a significantly blunted adolescent growth spurt. New modelling approaches suggest an independent regulation of adolescent growth and final height: the target for growth and final height appears to be set by the community. In order to test this hypothesis, we formed a geographic network of Switzerland consisting of 169 nodes (district capitals) and 335 connecting edges (roads), and investigated military conscript data obtained between 2004 and 2009. Average height of Swiss military conscripts was 178.2 cm (SD 6.5 cm). But conscripts from first order neighbouring districts were more similar in height than expected. Short stature districts have short, tall stature districts have tall neighbours. We found significant height correlations between 1st (r=0.58), 2nd (r=0.64), 3rd (r=0.45) and even 4th order neighbours (r=0.42). It appears that tall stature communities generate tall people, short stature communities generate short people, and migrants orientate towards the new height target of their host population (community effect on growth).

Harmonizing national growth references for multi-centre surveys, drug monitoring and international postmarketing surveillance.

AIM: National European growth references differ. We aimed to convert (harmonize) currently used charts into a single unified interchangeable LMS format for each European nation. METHODS: Nine currently used national European growth references from Belgium (2009), France (1979), Poland (2001), Sweden (2002), Switzerland (1989), the UK (1990), Italy (2006) and Germany (1979 and 1997) were harmonized and compared with the international WHO child growth standards and WHO growth reference data for 5-19 years. RESULTS: European growth charts can be harmonized. The approach appears useful as height, and body mass index (BMI) is inappropriately represented by WHO references. European height references exhibit warping when plotted against the WHO reference. The French appears too short, the other Europeans too tall. Also, the BMI is not appropriately represented by the WHO references. CONCLUSIONS: Harmonizing references is a novel, convenient and cost-effective approach for converting historic and/or incomplete local or national growth reference charts into a unified interchangeable LMS format. Harmonizing facilitates producing growth references 'on demand', for limited regional purposes, for ethnically, socio-economically or politically defined minorities, but also for matching geographically different groups of children and adolescents for international growth and registry studies.

Statistical agreement and cost-benefit: Comparison of methods for constructing growth reference charts.

BACKGROUND: Growth reference charts are important tools for adequate paediatric decisions. AIM: In view of the workload required to construct empirical growth reference charts we debate practicable and less demanding alternatives and took the recent national 2000-2002 Lithuanian growth charts as an example. Two options appeared reasonable: (1) applying international WHO child growth standards and WHO growth reference data for 5-19 years that are recommended for global use; or (2) replacing the costly empirical method of deriving national growth references by more convenient low-cost statistics, e.g. the method of generating synthetic references for the Lithuanian population. METHODS: We analysed the degree of agreement between the 2000-2002 Lithuanian growth charts, and the international WHO child growth standards and WHO growth reference data for 5-19 years and synthetic references for the Lithuanian population using the Bland-Altman method. RESULTS: Synthetically generated references for the Lithuanian population slightly surpassed the national Lithuanian reference for body height (males +0.3 (SD 0.9) cm; females +0.2 (SD 0.6) cm) particularly at young age, which may be regarded clinically irrelevant. WHO international child growth standards and the WHO growth reference data for 5-19 years, however, failed to match the Lithuanian references as they underestimated mean height in boys by -2.8 (SD 1.4) cm and in girls by -2.9 (SD 1.1) cm, with extremely discrepant estimates of more than -6 cm occurring in several adolescent cohorts. CONCLUSIONS: The analysis revitalizes the debate on clinically relevant and at the same time practicable but less demanding alternatives for constructing growth reference charts, and for economic reasons, strongly suggests replacing the traditional empirical methods by synthetic growth references.

Multiple imputation as one tool to provide longitudinal databases for modelling human height and weight development.

BACKGROUND/OBJECTIVES: Besides large efforts regarding field work, provision of valid databases requires statistical and informational infrastructure to enable long-term access to longitudinal data sets on height, weight and related issues. SUBJECTS/METHODS: To foster use of longitudinal data sets within the scientific community, provision of valid databases has to address data-protection regulations. It is, therefore, of major importance to hinder identifiability of individuals from publicly available databases. To reach this goal, one possible strategy is to provide a synthetic database to the public allowing for pretesting strategies for data analysis. The synthetic databases can be established using multiple imputation tools. Given the approval of the strategy, verification is based on the original data. RESULTS: Multiple imputation by chained equations is illustrated to facilitate provision of synthetic databases as it allows for capturing a wide range of statistical interdependencies. Also missing values, typically occurring within longitudinal databases for reasons of item non-response, can be addressed via multiple imputation when providing databases. CONCLUSIONS: The provision of synthetic databases using multiple imputation techniques is one possible strategy to ensure data protection, increase visibility of longitudinal databases and enhance the analytical potential.

Modelling human height and weight: a Bayesian approach towards model comparison.

BACKGROUND/OBJECTIVES: Given the availability of large longitudinal data sets on human height and weight, different modelling approaches are at hand to access quantities of interest relating to important diagnostic aims. SUBJECTS/METHODS: Statistical modelling frameworks for longitudinal data on human height and weight have to consider the issues of individual heterogeneity and time dependence to provide an accurate statistical characterisation. Further, missing values inevitably occurring within longitudinal data sets have to be addressed adequately to allow for valid inference. The Bayesian framework is illustrated to facilitate stringent comparison of available non-nested model frameworks addressing these issues using simulated and empirical data sets. RESULTS: Comparing random-effects and fixed-effects modelling approaches with the Preece-Baines (PB) model reveals that, for simulated data, the Bayesian approach towards model comparison is effective in discriminating between different model specifications. With regard to analysis of 14 longitudinal data sets, the implicit trade-off between model fit, that is, description of the data, and a parsimonious parameterisation favouring prediction is often best addressed via the PB model. CONCLUSIONS: The Bayesian approach is illustrated to allow for effective comparison in case model specifications for longitudinal data are not linked directly via parametric restrictions.

Monte Carlo simulation of body height in a spatial network.

BACKGROUND/OBJECTIVES: Recent evidence suggests clustering of human body height. We want to assess the consequences of connectedness in a spatial network on height clustering in an artificial society. SUBJECTS/METHODS: We used an agent-based computer modelling technique (Monte Carlo simulation) and compared simulated height in a spatial network with characteristics of the observed geographic height distribution of three historic cohorts of Swiss military conscripts (conscripted in 1884-1891; 1908-1910; and 2004-2009). RESULTS: Conscript height shows several characteristic features: (1) height distributions are overdispersed. (2) Conscripts from districts with direct inter-district road connections tend to be similar in height. (3) Clusters of tall and clusters of short stature districts vary over time. Autocorrelations in height between late 19th and early 21st century districts are low. (4) Mean district height depends on the number of connecting roads and on the number of conscripts per district. Using Monte Carlo simulation, we were able to generate these natural characteristics in an artificial society. Already 5% height information from directly connected districts is sufficient to simulate the characteristics of natural height distribution. Very similar observations in regular rectangular networks indicate that the characteristics of Swiss conscript height distributions do not so much result from the particular Swiss geography but rather appear to be general features of spatial networks. CONCLUSIONS: Spatial connectedness can affect height clustering in an artificial society, similar to that seen in natural cohorts of military conscripts, and strengthen the concept of connectedness being involved in the regulation of human height.

The association of body height, height variability and inequality.

Body height is associated with environmental conditions. It has been suggested that under poor conditions when inequality within a population increases, also the variability in height tends to increase. We studied the association of body height, within-country variability in height and geographic and historic origin in 767 growth studies carried out in 80 countries, published between 1794 and 2013, with data on N = 78,184 infants age 2 years, and N = 2,130,729 juveniles age 7 years. The studies represent almost the whole spectrum of economic diversity in human societies since the end-18(th) century. 207 studies contained data for both infants and juveniles with 50,819 subjects (age 2), and 123,078 subjects (age 7). Multiple linear regressions showed significant interactions between height, sex, historic year of the study, geographic origin, and within-study standard deviation for height with multiple R-squared = 0.527, p < 0.001, at age 2, and multiple R-squared = 0.436, p < 0.001, at age 7. Yet, the two age groups differed in respect to within-study standard deviation for height. We found a significant association between body height and within-study standard deviation for height only at age 2: tall infant populations are less variable in height (r = -0.27, p < 0.01). There was no such association in children aged 7 years. Tall children from affluent and short children from less affluent countries do not differ in the variability of body height. The data suggest that the 'environmental adversity' hypothesis for variation in growth: small mean values for height go along with large standard deviations for height, does not apply for children at age 7.

Detection of vancomycin resistances in enterococci within 3 ½ hours.

Vancomycin resistant enterococci (VRE) constitute a challenging problem in health care institutions worldwide. Novel methods to rapidly identify resistances are highly required to ensure an early start of tailored therapy and to prevent further spread of the bacteria. Here, a spectroscopy-based rapid test is presented that reveals resistances of enterococci towards vancomycin within 3.5 hours. Without any specific knowledge on the strain, VRE can be recognized with high accuracy in two different enterococci species. By means of dielectrophoresis, bacteria are directly captured from dilute suspensions, making sample preparation very easy. Raman spectroscopic analysis of the trapped bacteria over a time span of two hours in absence and presence of antibiotics reveals characteristic differences in the molecular response of sensitive as well as resistant Enterococcus faecalis and Enterococcus faecium. Furthermore, the spectroscopic fingerprints provide an indication on the mechanisms of induced resistance in VRE.

Cytoprotective and genoprotective effects of ß-glucans against aflatoxin B1-induced DNA damage in broiler chicken lymphocytes.

The polysaccharide ß-glucan presents beneficial effects on the immune system, although the mechanisms of the immunomodulatory effect remain poorly understood. The potential cytoprotective and genoprotective effects of ß-glucans were evaluated in broiler chicken lymphocytes exposed to increasing concentrations of aflatoxin B1 (AFB1) and/or ß-glucans. AFB1 significantly decreased cell viability at the concentrations of 10 and 20 µg/ml at 72 h of incubation (p<0.01 and p<0.001, respectively). Moreover, the AFB1 concentrations of 1, 10 and 20 µg/ml increased DNA fragmentation levels at 24 h (p<0.001). Conversely, lymphocyte death was prevented by ß-glucans at the concentrations of 1% and 10%, indicating a cytoprotective effect. Reactive oxygen species levels were increased in the cells treated with 20 µg/ml AFB1 at 24 h (p<0.05) and 10% ß-glucans with or without AFB1 at 24, 48 and 72 h of incubation (p<0.001). DNA damage increased by more than 100% in AFB1-treated lymphocytes when compared to control group. ß-glucans at 1% was able to fully revert the AFB1-induced lymphocyte DNA damage, indicating a genoprotective effect and maintaining DNA integrity. In conclusion, ß-glucans showed in vitro dose-dependent cytoprotective and genoprotective effects in broiler chicken lymphocytes exposed to AFB1.

Retroviral immortalization of phagocytic and dendritic cell clones as a tool to investigate functional heterogeneity.

We have developed a method to generate immortalized phagocytic and dendritic cell clones from various mouse tissues such as spleen, thymus, brain and bone marrow. The clones were phenotypically characterized and shown to retain the ability to respond to immune or inflammatory signals, e.g., IFN-gamma. Functional cytokine activity and nitric oxide production were maintained in activated macrophages, microglial and dendritic cell clones. Immune functions, such as antigen presentation was exhibited by all clones whereas tissue-specific properties such as the ability to respond to corticotropin-releasing hormone and produce beta-endorphin was shown in microglial cell clones but not in macrophage cell clones, indicating that heterogeneity of cells of the mononuclear-phagocytic lineage can be maintained in vitro after the immortalization procedure. Moreover, the continuous proliferation of the clones could be inhibited by various stimuli and further differentiation of the cells could be achieved in vitro.

Diversity in auxology: between theory and practice. Proceedings of the 18th Aschauer Soiree, 13th November 2010.

Auxology has developed from mere describing child and adolescent growth into a vivid and interdisciplinary research area encompassing human biologists, physicians, social scientists, economists and biostatisticians. The meeting illustrated the diversity in auxology, with the various social, medical, biological and biostatistical aspects in studies on child growth and development.

Growth variation, final height and secular trend. Proceedings of the 17th Aschauer Soiree, 7th November 2009.

Growth and body height have always been topics interesting to the public. In particular, the stupendous increase of some 15-19cm in final adult height during the last 150 years in most European countries (the "secular trend"), the concomitant changes in body and head proportions, the tendency towards early onset of sexual maturation, the changes in the age when final height is being reached, and the very recent trend in body mass index, have generated much scientific literature. The marked plasticity of growth in height and weight over time causes problems. Child growth references differ between nations, they tend to quickly become out of date, and raise a number of questions regarding fitting methods, effects caused by selective drop-out, etc. New findings contradict common beliefs about the primary importance of nutritional and health related factors for secular changes in growth. There appears to be a broad age span from mid-childhood to early adolescence that is characterised by a peculiar insusceptibility. Environmental factors that are known to influence growth during this age span appear to have only little or no impact on final height. Major re-arrangements in height occur at an age when puberty has almost been completed and final height has almost been reached, implying that factors, which drive the secular trend in height, are limited to early childhood and late adolescence.

Determination of the redox properties of the Rieske [2Fe-2S] cluster of bovine heart bc1 complex by direct electrochemistry of a water-soluble fragment.

The redox potential of the Rieske [2Fe-2S] cluster of the bc1 complex from bovine heart mitochondria was determined by cyclic voltammetry of a water-soluble fragment of the iron/sulfur protein. At the nitric-acid-treated bare glassy-carbon electrode, the fragment gave an immediate and stable quasireversible response. The midpoint potential at pH 7.2, 25 degrees C and I of 0.01 M was Em = +312 +/- 3 mV. This value corresponds within 20 mV to results of an EPR-monitored dye-mediated redox titration. With increasing ionic strength, the midpoint potential decreased linearly with square root of I up to I = 2.5 M. From the cathodic-to-anodic peak separation, the heterogeneous rate constant, k degrees, was calculated to be approximately 2 x 10(-3) cm/s at low ionic strength; the rate constant increased with increasing ionic strength. From the temperature dependence of the midpoint potential, the standard reaction entropy was calculated as delta S degrees = -155 J.K-1.mol-1. The pH dependence of the midpoint potential was followed over pH 5.5-10. Above pH 7, redox-state-dependent pK changes were observed. The slope of the curve, -120 mV/pH above pH9, indicated two deprotonations of the oxidized protein. The pKa values of the oxidized protein, obtained by curve fitting, were 7.6 and 9.2, respectively. A group with a pKa,ox of approximately 7.5 could also be observed in the optical spectrum of the oxidized protein. Redox-dependent pK values of the iron/sulfur protein are considered to be essential for semiquinone oxidation at the Qo center of the bc1 complex.

Isolation, characterisation and crystallisation of a water-soluble fragment of the Rieske iron-sulfur protein of bovine heart mitochondrial bc1 complex.

A water-soluble fragment of the bc1 complex from bovine heart mitochondria was isolated containing the intact Rieske [2Fe-2S] cluster. The fragment consists of the last 129 amino acid residues of the Rieske iron-sulfur protein and has a molecular mass of 14592 Da including two iron atoms. The absorption, visible CD, and EPR spectra of the fragment are indistinguishable from those of the membrane-bound iron-sulfur protein. The redox potential as determined by EPR-monitored redox titration was + 306 mV. The far-ultraviolet CD spectrum is indicative of a protein with little regular secondary structure, while significant alpha-helix content was detected in the membrane anchor of the complete iron-sulfur protein. The fragment could be crystallized using poly(ethylene glycol) 6000 as precipitant. Needle-shaped single crystals have been grown by the hanging-drop vapor diffusion technique. These crystals belong to the space group P21 and diffract well beyond 0.2 nm resolution. Phase determination using the multiple-wavelength anomalous-scattering technique is underway.