p53-independent apoptosis in UV-irradiated mouse skin: possible inhibition by 50 Hz magnetic fields

Our recent results suggest that 50 Hz magnetic fields (MF) enhance ultraviolet (UV)-induced tumorigenesis in mouse skin. The aim of the present experiment was to study suppression of apoptosis as a possible mechanism for MF effects on skin tumorigenesis. Another aim was to test the importance of a UV and MF exposure schedule, particularly the role of MF exposure prior to UV irradiation. Female mice were exposed to a UV dose of 2 human MED and to 100 microT MF of 50 Hz, using the following exposure schedules: group 1 sham MF 24 h, UV 1 h, sham MF 24 h; group 2 sham MF 24 h, UV 1 h, MF 24 h; group 3 MF 24 h, UV 1 h, MF 24 h. Lamps emitting simulated solar radiation (SSR) were used for UV irradiation. Skin samples were analysed for apoptosis, expression of the p53 gene, activity of the enzyme ornithine decarboxylase (ODC) and polyamine concentrations. A significantly (p = 0.017) lower number of apoptotic cells was measured in group 2 compared to group 1. A similar but not statistically significant (p = 0.064) decrease was also detected in group 3. No p53 expression was detected in any sample. The levels of ODC and putrescine did not differ significantly between the UV-only and UV and MF-exposed groups. Spermidine and spermine levels were significantly (p = 0.014 and 0.014, respectively) lower in group 3 than in group 1, but no decrease was observed in group 2. Our findings suggest that SSR induces p53-independent apoptosis in mouse skin and that the apoptotic response may be inhibited by exposure to MF. The exposure schedule did not alter the MF effect. The results do not support a causal role for polyamines in MF effects on apoptosis.     

Resveratrol as a Pan-HDAC Inhibitor Alters the Acetylation Status of Jistone Proteins in Human-Derived Hepatoblastoma Cells

The polyphenolic alcohol resveratrol has demonstrated promising activities for the prevention and treatment of cancer. Different modes of action have been described for resveratrol including the activation of sirtuins, which represent the class III histone deacetylases (HDACs). However, little is known about the activity of resveratrol on the classical HDACs of class I, II and IV, although these classes are involved in cancer development or progression and inhibitors of HDACs (HDACi) are currently under investigation as promising novel anticancer drugs. We could show by in silico docking studies that resveratrol has the chemical structure to inhibit the activity of different human HDAC enzymes. In vitro analyses of overall HDAC inhibition and a detailed HDAC profiling showed that resveratrol inhibited all eleven human HDACs of class I, II and IV in a dose-dependent manner. Transferring this molecular mechanism into cancer therapy strategies, resveratrol treatment was analyzed on solid tumor cell lines. Despite the fact that hepatocellular carcinoma (HCC) is known to be particularly resistant against conventional chemotherapeutics, treatment of HCC with established HDACi already has shown promising results. Testing of resveratrol on hepatoma cell lines HepG2, Hep3B and HuH7 revealed a dose-dependent antiproliferative effect on all cell lines. Interestingly, only for HepG2 cells a specific inhibition of HDACs and in turn a histone hyperacetylation caused by resveratrol was detected. Additional testing of human blood samples demonstrated a HDACi activity by resveratrol ex vivo. Concluding toxicity studies showed that primary human hepatocytes tolerated resveratrol, whereas in vivo chicken embryotoxicity assays demonstrated severe toxicity at high concentrations. Taken together, this novel pan-HDACi activity opens up a new perspective of resveratrol for cancer therapy alone or in combination with other chemotherapeutics. Moreover, resveratrol may serve as a lead structure for chemical optimization of bioavailability, pharmacology or HDAC inhibition.     

MobiSeq: De novo SNP discovery in model and non‐model species through sequencing the flanking region of transposable elements

In recent years, the availability of reduced representation library (RRL) methods has catalysed an expansion of genome‐scale studies to characterize both model and non‐model organisms. Most of these methods rely on the use of restriction enzymes to obtain DNA sequences at a genome‐wide level. These approaches have been widely used to sequence thousands of markers across individuals for many organisms at a reasonable cost, revolutionizing the field of population genomics. However, there are still some limitations associated with these methods, in particular the high molecular weight DNA required as starting material, the reduced number of common loci among investigated samples, and the short length of the sequenced site‐associated DNA. Here, we present MobiSeq, a RRL protocol exploiting simple laboratory techniques, that generates genomic data based on PCR targeted enrichment of transposable elements and the sequencing of the associated flanking region. We validate its performance across 103 DNA extracts derived from three mammalian species: grey wolf (Canis lupus), red deer complex (Cervus sp.) and brown rat (Rattus norvegicus). MobiSeq enables the sequencing of hundreds of thousands loci across the genome and performs SNP discovery with relatively low rates of clonality. Given the ease and flexibility of MobiSeq protocol, the method has the potential to be implemented for marker discovery and population genomics across a wide range of organisms—enabling the exploration of diverse evolutionary and conservation questions.     

Role of CLASP2 in microtubule stabilization and the regulation of persistent motility

In motile fibroblasts, stable microtubules (MTs) are oriented toward the leading edge of cells. How these polarized MT arrays are established and maintained, and the cellular processes they control, have been the subject of many investigations. Several MT "plus-end-tracking proteins," or +TIPs, have been proposed to regulate selective MT stabilization, including the CLASPs, a complex of CLIP-170, IQGAP1, activated Cdc42 or Rac1, a complex of APC, EB1, and mDia1, and the actin-MT crosslinking factor ACF7. By using mouse embryonic fibroblasts (MEFs) in a wound-healing assay, we show here that CLASP2 is required for the formation of a stable, polarized MT array but that CLIP-170 and an APC-EB1 interaction are not essential. Persistent motility is also hampered in CLASP2-deficient MEFs. We find that ACF7 regulates cortical CLASP localization in HeLa cells, indicating it acts upstream of CLASP2. Fluorescence-based approaches show that GFP-CLASP2 is immobilized in a bimodal manner in regions near cell edges. Our results suggest that the regional immobilization of CLASP2 allows MT stabilization and promotes directionally persistent motility in fibroblasts.     

Phenotypic plasticity in growth and fecundity induced by strong population fluctuations affects reproductive traits of female fish

Fish are known for their high phenotypic plasticity in life‐history traits in relation to environmental variability, and this is particularly pronounced among salmonids in the Northern Hemisphere. Resource limitation leads to trade‐offs in phenotypic plasticity between life‐history traits related to the reproduction, growth, and survival of individual fish, which have consequences for the age and size distributions of populations, as well as their dynamics and productivity. We studied the effect of plasticity in growth and fecundity of vendace females on their reproductive traits using a series of long‐term incubation experiments. The wild parental fish originated from four separate populations with markedly different densities, and hence naturally induced differences in their growth and fecundity. The energy allocation to somatic tissues and eggs prior to spawning served as a proxy for total resource availability to individual females, and its effects on offspring survival and growth were analyzed. Vendace females allocated a rather constant proportion of available energy to eggs (per body mass) despite different growth patterns depending on the total resources in the different lakes; investment into eggs thus dictated the share remaining for growth. The energy allocation to eggs per mass was higher in young than in old spawners and the egg size and the relative fecundity differed between them: Young females produced more and smaller eggs and larvae than old spawners. In contrast to earlier observations of salmonids, a shortage of maternal food resources did not increase offspring size and survival. Vendace females in sparse populations with ample resources and high growth produced larger eggs and larvae. Vendace accommodate strong population fluctuations by their high plasticity in growth and fecundity, which affect their offspring size and consequently their recruitment and productivity, and account for their persistence and resilience in the face of high fishing mortality.     

Does Serum 25-Hydroxyvitamin D Influence Muscle Development during Puberty in Girls? - A 7-Year Longitudinal Study

Vitamin D is well known for its regulatory role in calcium and phosphate homeostasis, but its role in muscle mass and strength during growth remains inconclusive. We explored the association of serum 25-hydroxyvitamin D (25(OH)D) with muscle development in girls from 11 to 18-years old. Whole body lean tissue mass (LM_WB), appendicular lean mass (aLM), muscle cross-sectional area at the lower leg (mCSA), maximal voluntary contraction of elbow flexors (MVC_elbow) and knee extensors (MVC_knee) were assessed in 217 girls aged 10–13 years (at baseline), 215 in 2-year and 226 in 7.5-year follow-up. Serum concentration of 25(OH)D and intact parathyroid hormone (PTH) were analyzed retrospectively and girls were categorized according to their 25(OH)D levels (consistently insufficient 25(OH)D G_LL <50 nmol/l and consistently sufficient G_HH >50 nmol/l from baseline to 7-year follow-up). We found that 25(OH)D level declined until menarche (p<0.05) while LM_WB, aLM, mCSA, MVC_elbow and MVC_knee continued to increase (p<0.001 for all) post menarche. At pre-menarche, the G_LL (n = 34) had higher LM_WB and aLM than the G_HH (n = 21, p<0.05), while post-menarche the G_HH (n = 15) had a greater catch-up gain in LM_WB (p = 0.004), aLM (p = 0.001) and mCSA (p = 0.027) compared to the G_LL (n = 65) over the first 2-year period. At the age of 18, no differences in muscle mass/strength between the low (n = 151) and high (n = 77) levels of 25(OH)D groups were found. This finding was independent of vitamin D receptor genotype and other confounders. In conclusion, our results showed that levels of 25(OH)D have no significant negative influence on the development of muscle mass and strength during pubertal growth both with longitudinal and cross-sectional comparison. On the contrary, our results suggest that the temporary negative association between 25(OH)D and muscle mass arises as a consequence of fast growth prior to menarche, and this negative association is diminished through catch-up growth after menarche.     

Phosphorus Flux Through a Littoral Ecosystem: The Importance of Cladoceran Zooplankton and Young Fish

Foodchain relationships in water within a helophytic vegetation stand were experimentally investigated using the radioactive isotope ³³P as a tracer. The results proved the importance of cladoceran zooplankton (mainly Bosmina longispina LEYDIG) in the control of phytoplankton and the importance of young fish in the control of Bosmina abundance in the littoral water.     

Does Forest Continuity Enhance the Resilience of Trees to Environmental Change?

There is ample evidence that continuously existing forests and afforestations on previously agricultural land differ with regard to ecosystem functions and services such as carbon sequestration, nutrient cycling and biodiversity. However, no studies have so far been conducted on possible long-term (>100 years) impacts on tree growth caused by differences in the ecological continuity of forest stands. In the present study we analysed the variation in tree-ring width of sessile oak (Quercus petraea (Matt.) Liebl.) trees (mean age 115–136 years) due to different land-use histories (continuously existing forests, afforestations both on arable land and on heathland). We also analysed the relation of growth patterns to soil nutrient stores and to climatic parameters (temperature, precipitation). Tree rings formed between 1896 and 2005 were widest in trees afforested on arable land. This can be attributed to higher nitrogen and phosphorous availability and indicates that former fertilisation may continue to affect the nutritional status of forest soils for more than one century after those activities have ceased. Moreover, these trees responded more strongly to environmental changes – as shown by a higher mean sensitivity of the tree-ring widths – than trees of continuously existing forests. However, the impact of climatic parameters on the variability in tree-ring width was generally small, but trees on former arable land showed the highest susceptibility to annually changing climatic conditions. We assume that incompletely developed humus horizons as well as differences in the edaphon are responsible for the more sensitive response of oak trees of recent forests (former arable land and former heathland) to variation in environmental conditions. We conclude that forests characterised by a long ecological continuity may be better adapted to global change than recent forest ecosystems.     

An automated PCR platform with homogeneous time-resolved fluorescence detection and dry chemistry assay kits

We have developed a novel instrument platform, GenomEra, for small-scale analysis of nucleic acids. The platform combines a rapid thermal cycler, an integrated time-resolved fluorescence measurement unit, and user-friendly software for the analysis of results. Disposable low-cost plastic reaction vessels are designed specifically for the instrument and contain all of the assay-specific reagents in dry form. The appropriate assay protocol is specified on barcodes printed under the vessels and is automatically initiated by the software. Detection is based on the use of sequence-specific probes labeled with intrinsically fluorescent europium or terbium chelates and complementary quencher probes, which enable sensitive, homogeneous closed-tube assays without the risk of carryover contamination. The detection limit of the instrument (background + 3 SD) is approximately 20 pmol/L for both chelates with a dynamic range of nearly four orders of magnitude. The functionality of the platform is demonstrated with a dual-label homogeneous polymerase chain reaction (PCR) assay for the detection of Salmonella using a Magda CA Salmonella assay kit. An internal amplification control is included in each reaction to eliminate false negative results caused by PCR inhibition. Qualitative assay results are automatically interpreted by the software and are available 45 min after sample addition.