Alterations of biochemical marker levels and myonuclear numbers in rat skeletal muscle after ischemia–reperfusion

Prolonged ischemia–reperfusion results in various damages in skeletal muscle. Following reperfusion, although the damaged muscles undergo regeneration, the precise process and mechanism of regeneration have not yet been fully understood. Here, we show the altered levels of plasma biochemical markers of muscle damage, and the change in myonuclear numbers in adult rat skeletal muscle by ischemia–reperfusion. Male Wistar rats were subjected to unilateral hindlimb ischemia by clamping the anterior tibial artery for 2 h before reperfusion. Both plasma creatine kinase activity and C-reactive protein levels in plasma were increased significantly at 0.5 h of reperfusion and returned to the control level at 24 h. The transverse sectional area of muscle belly of the anterior tibial muscles in ischemic side was significantly decreased by 20 % compared with those in sham-ischemic (control) side at 2 days, and returned to the control level at 5 days of reperfusion. Moreover, the number of interstitial nuclei in the ischemic side were significantly increased at 5–14 days and returned to the control level at 21 days of reperfusion. Central nuclei that are specifically observed in regenerating muscle, appeared at 5 days, reached a peak at 14 days, and disappeared at 28 days of reperfusion. Furthermore, MyoD, a regulatory factor for myogenesis, showed a transient expression at 5 days of reperfusion. These results indicate that, although the size of muscle seems to be recovered by 5 days of reperfusion, the most active muscle regeneration occurs much later, as shown by the increase in central nuclei.     

Retention of dissolved silica within the fluvial system of the conterminous USA

Dissolved silica (DSi) is an important nutrient in aquatic ecosystems. Increased DSi retention within the fluvial system due to damming and eutrophication has led to a decrease in DSi exports to coastal waters, which can have severe consequences for coastal areas where ecosystem functioning depends on fluvial DSi inputs. The analysis of fluvial DSi fluxes and DSi retention at regional to global scales is thus an important research topic. This study explores the possibility to empirically assess regional DSi retention based on a spatially explicit estimation of DSi mobilization and fluvial DSi fluxes calculated from hydrochemical monitoring data. The uncertainty of DSi retention rates (r_DSi) estimated for particular rivers is high. Nevertheless, for the St. Lawrence River (r_DSi = 91 %) and the Mississippi River (r_DSi = 13 %) the estimated DSi retention rates are reasonable and are supported by literature values. The variety of sources of the uncertainty in the DSi retention assessment is discussed.     

Flow cytometric analysis of the cadmium-exposed green alga Chlamydomonas reinhardtii (Chlorophyceae)

A flow-cytometric method was developed and evaluated as a rapid ecotoxicological tool using cultures of the microalga Chlamydomonas reinhardtii (Chlorophyceae) under cadmium exposure. Three staining protocols were developed to assess the toxicological impact of this trace metal on algal physiology. Algal cells were exposed to total nominal cadmium concentrations of 5 and 100 µM. After 48 and 72 h exposure the fluorescent probes, fluorescein diacetate (FDA), dihydrorhodamine 123 (DHR123) and tetramethylrhodamine methyl ester (TMRM), were used to assess esterase activity, presence of reactive oxygen species and membrane potential, respectively. Results indicated that cell size, cell granularity and internal complexity were influenced by cadmium, confirming earlier findings on ultrastructural changes in microalgae exposed to trace metals. An increase was observed in the percentage of DHR123 positive cells as well as in their mean fluorescence intensity, on increasing cadmium concentration, confirming that this metal exerts its toxicity through the generation of reactive oxygen species. Furthermore, cadmium exposure resulted in an increase in esterase activity, as reflected in fluorescein fluorescence. We suggest this observation was linked to possible detoxification activity and defence mechanisms. Measurements of control samples during protocol optimization for TMRM proved not to be reproducible, leading us to defer any judgment on results of exposed samples and to conclude that TMRM does not seem suitable for flow cytometric use in algae. Our results demonstrate that although very rarely used in ecotoxicology, flow cytometry is a quick and convenient technique to assess toxic effects that can generate mechanistic information on the mode of action of contaminants.     

Evaluation of the INS-1 832/13 Cell Line as a Beta-Cell Based Screening System to Assess Pollutant Effects on Beta-Cell Function

Environmental pollutants have recently emerged as potential risk factors for metabolic diseases, urging systematic investigation of pollutant effects on metabolic disease processes. To enable risk assessment of these so-called metabolic disruptors the use of stable, robust and well-defined cell based screening systems has recently been encouraged. Since beta-cell (dys)functionality is central in diabetes pathophysiology, the need to develop beta-cell based pollutant screening systems is evident. In this context, the present research evaluated the strengths and weaknesses of the INS-1 832/13 pancreatic beta-cell line as diabetogenic pollutant screening system with a focus on beta-cell function. After optimization of exposure conditions, positive (exendin-4, glibenclamide) and negative (diazoxide) control compounds for acute insulin secretion responses were tested and those with the most profound effects were selected to allow potency estimations and ranking of pollutants. This was followed by a first explorative screening of acute bisphenol A and bis(2-ethylhexyl)phthalate effects. The same approach was applied for chronic exposures, focusing primarily on evaluation of acknowledged chronic stimulators (diazoxide, T0901317, exendin-4) or inhibitors (glibenclamide) of insulin secretion responses to select the most responsive ones for use as control compounds in a chronic pollutant testing framework. Our results showed that INS-1 832/13 cells responded conform previous observations regarding acute effects of control compounds on insulin secretion, while bisphenol A and bis(2-ethylhexyl)phthalate had limited acute effects. Furthermore, chronic exposure to known beta-cell reactive compounds resulted in deviating insulin secretion and insulin content profiles compared to previous reports. In conclusion, this INS-1 subclone appears to lack certain characteristics needed to respond appropriately to acute pollutant exposure or long term exposure to known beta-cell reactive compounds and thus seems to be, in our setting, inadequate as a diabetogenic pollutant screening system.     

X-Chromosomal Maternal and Fetal SNPs and the Risk of Spontaneous Preterm Delivery in a Danish/Norwegian Genome-Wide Association Study

Background

Recent epidemiological studies suggest that the maternal genome is an important contributor to spontaneous preterm delivery (PTD). There is also a significant excess of males among preterm born infants, which may imply an X-linked mode of inheritance for a subset of cases. To explore this, we examined the effect of maternal and fetal X-chromosomal single nucleotide polymorphisms (SNPs) on the risk of PTD in two independent genome-wide association studies and one replication study.

Methods

Participants were recruited from the Danish National Birth Cohort and the Norwegian Mother and Child cohort studies. Data from these two populations were first analyzed independently, and then combined in a meta-analysis. Overall, we evaluated 12,211 SNPs in 1,535 case-mother dyads and 1,487 control-mother dyads. Analyses were done using a hybrid design that combines case-mother dyads and control-mother dyads, as implemented in the Haplin statistical software package. A sex-stratified analysis was performed for the fetal SNPs. In the replication study, 10 maternal and 16 fetal SNPs were analyzed using case-parent triads from independent studies of PTD in the United States, Argentina and Denmark.

Results

In the meta-analysis, the G allele at the maternal SNP rs2747022 in the FERM domain containing 7 gene (FRMD7) increased the risk of spontaneous PTD by 1.2 (95% confidence interval (CI): 1.1, 1.4). Although an association with this SNP was confirmed in the replication study, it was no longer statistically significant after a Bonferroni correction for multiple testing.

Conclusion

We did not find strong evidence in our data to implicate X-chromosomal SNPs in the etiology of spontaneous PTD. Although non-significant after correction for multiple testing, the mother’s G allele at rs2747022 in FRMD7 increased the risk of spontaneous PTD across all populations in this study, thus warranting further investigation in other populations.     

Cartilage tissue engineering and bioreactor systems for the cultivation and stimulation of chondrocytes

Damage to and degeneration of articular cartilage is a major health issue in industrialized nations. Articular cartilage has a particularly limited capacity for auto regeneration. At present, there is no established therapy for a sufficiently reliable and durable replacement of damaged articular cartilage. In this, as well as in other areas of regenerative medicine, tissue engineering methods are considered to be a promising therapeutic component. Nevertheless, there remain obstacles to the establishment of tissue-engineered cartilage as a part of the routine therapy for cartilage defects. One necessary aspect of potential tissue engineering-based therapies for cartilage damage that requires both elucidation and progress toward practical solutions is the reliable, cost effective cultivation of suitable tissue. Bioreactors and associated methods and equipment are the tools with which it is hoped that such a supply of tissue-engineered cartilage can be provided. The fact that in vivo adaptive physical stimulation influences chondrocyte function by affecting mechanotransduction leads to the development of specifically designed bioreactor devices that transmit forces like shear, hydrostatic pressure, compression, and combinations thereof to articular and artificial cartilage in vitro. This review summarizes the basic knowledge of chondrocyte biology and cartilage dynamics together with the exploration of the various biophysical principles of cause and effect that have been integrated into bioreactor systems for the cultivation and stimulation of chondrocytes. Dedicated to Prof. K. Arnold on the occasion of his 65th birthday.     

Ungulate impact on vegetation in a two-level trophic system

A central challenge in ecology is to predict long-term consequences of high herbivore densities on their resources. Here we describe vegetation changes during 26 years following the reintroduction of a wild Svalbard reindeer (Rangifer tarandus platyrhynchus) population that was not subject to predation. The population irrupted, crashed and subsequently fluctuated around lower densities throughout the study period. The irruption generated strong trophic top-down effects on the vegetation including a decline in the cover of mosses, vascular plants and lichens. Previously dominant fruticose lichens, which are preferred forage during winter, were almost depleted and showed no response to the relief in grazing pressure. This in turn indirectly affected rates of recovery in other plant groups subsequent to the crash. Mosses recovered completely and even exceeded pre-reindeer levels 6 years after the population peak. Recovery of vascular plants was more delayed and only partial due to a long-term suppression of common species that are important as reindeer forage. Thus, a suppression of major forage species and a sustained reduction in herbivore densities suggest that overgrazing occurred during the population irruption, possibly inducing a long-term decline in reindeer carrying capacity K. This supports the “exploitation ecosystem hypothesis” predicting top-down control of vegetation by herbivores when predators are absent.