New aspects of electron transfer revealed by the crystal structure of a truncated bovine adrenodoxin, Adx(4–108)

AbstractBackground: Adrenodoxin (Adx) is a [2Fe–2S] ferredoxin involved in steroid hormone biosynthesis in the adrenal gland mitochondrial matrix of mammals. Adx is a small soluble protein that transfers electrons from adrenodoxin reductase (AR) to different cytochrome P450 isoforms where they are consumed in hydroxylation reactions. A crystallographic study of Adx is expected to reveal the structural basis for an important electron transfer reaction mediated by a vertebrate [2Fe–2S] ferredoxin.Results: The crystal structure of a truncated bovine adrenodoxin, Adx(4–108), was determined at 1.85 å resolution and refined to a crystallographic R value of 0.195. The structure was determined using multiple wavelength anomalous dispersion phasing techniques, making use of the iron atoms in the [2Fe–2S] cluster of the protein. The protein displays the compact (α+β) fold typical for [2Fe–2S] ferredoxins. The polypeptide chain is organized into a large core domain and a smaller interaction domain which comprises 35 residues, including all those previously determined to be involved in binding to AR and cytochrome P450. A small interdomain motion is observed as a structural difference between the two independent molecules in the asymmetric unit of the crystal. Charged residues of Adx(4–108) are clustered to yield a strikingly asymmetric electric potential of the protein molecule.Conclusions: The crystal structure of Adx(4–108) provides the first detailed description of a vertebrate [2Fe–2S] ferredoxin and serves to explain a large body of biochemical studies in terms of a three-dimensional structure. The structure suggests how a change in the redox state of the [2Fe–2S] cluster may be coupled to a domain motion of the protein. It seems likely that the clearly asymmetric charge distribution on the surface of Adx(4–108) and the resulting strong molecular dipole are involved in electrostatic steering of the interactions with AR and cytochrome P450

Physiological and behavioral responses of dairy cattle to the introduction of robot scrapers

Autonomous mobile robot scrapers are increasingly used in order to clean the floors on dairy farms. Given the complexity of robot scraper operation, stress may occur in cows due to unpredictability and lack of control of the situation. Experiencing stress can impair animal welfare and in the long-term the health and milk production of the cows. Therefore, this study addressed potential stress responses of dairy cattle to the robot scraper after introducing the autonomous mobile machine. 36 cows in total were studied on three different farms to explore possible modifications in cardiac function, behavior, and adrenocortical activity. The research protocol on each farm consisted of four experimental periods including one baseline measurement without robot scraper operation followed by three test measurements, in which cows interacted with the robotic cleaning system. Interbeat intervals were recorded in order to calculate the heart rate variability parameter RMSSD, behavior was observed to determine time budgets, and fecal samples were collected for analysis of the cortisol metabolites concentration. A statistical analysis was carried out using linear mixed-effects models. Heart rate variability decline immediately after the introduction of the robot scraper and modified behavior in the subsequent experimental periods indicated a stress response. The cortisol metabolites concentration remained constant. It is hypothesized that after the initial phase of decrease, heart rate variability stabilized through the behavioral adjustments of the cows in the second part of the study. Persistent alterations in behavior gave rise to the assumption that the animals’ habituation process to the robot scraper was not yet completed. In summary, the present study illustrated that the cows showed minor signs of disturbance towards the robotic cleaning system. Thus, our findings suggest that dairy cattle can largely adjust their behavior to avoid aversive effects on animal welfare. Additional research can provide further insight into the development of the animal-machine interaction beyond the initial phase of robot scraper operation considered in this study

Impact of degradable magnesium implants on osteocytes in single and triple cultures

In vitro triple cultures of human primary osteoblasts, osteocytes and osteoclasts can potentially help to analyze the effect of drugs and degradation products of biomaterials as a model for native bone tissue. In the present study, degradation products of Magnesium (Mg), which has been successfully applied in the biomedical field, were studied with respect to their impact on bone cell morphology and differentiation both in osteocyte single cultures and in the triple culture model. Fluorescence microscopic and gene expression analysis, analysis of osteoclast- and osteoblast-specific enzyme activities as well as osteocalcin protein expression were performed separately for the three cell types after cultivation in triple culture in the presence of extracts, containing 5 and 10 mM Mg2+. All three cell species were viable in the presence of the extracts and did not show morphological changes compared to the Mg-free control. Osteoblasts and osteoclasts did not show significant changes in gene expression of ALPL, BSPII, osteocalcin, TRAP, CTSK and CA2. Likewise on protein level, no significant changes in ALP-, TRAP-, CTSK- and CAII activities were detected. Osteocytes showed a significant downregulation of MEPE, which codes for a protein playing an important role in regulation of phosphate homeostasis by osteocytes. This study is the first to analyze the effects of Mg degradation products on primary osteocytes in vitro, both in single and triple culture. Even if promoting effects on the three examined bone cell species were not found in the applied triple culture setup, it was shown, that Mg degradation products do not interfere with the activity of osteoblasts, osteoclasts and osteocytes in vitro

Hybridization capture of larch (Larix Mill) chloroplast genomes from sedimentary ancient DNA reveals past changes of Siberian forest

Siberian larch (Larix Mill.) forests dominate vast areas of northern Russia and contribute important ecosystem services to the world. It is important to understand the past dynamics of larches in order to predict their likely response to a changing climate in the future. Sedimentary ancient DNA extracted from lake sediment cores can serve as archives to study past vegetation. However, the traditional method of studying sedimentary ancient DNA - metabarcoding - focuses on small fragments which cannot resolve Larix to species level nor allow a detailed study of population dynamics. Here we use shotgun sequencing and hybridization capture with long-range PCR-generated baits covering the complete Larix chloroplast genome to study Larix populations from a sediment core reaching back to 6700 years from the Taymyr region in northern Siberia. In comparison to shotgun sequencing, hybridization capture results in an increase of taxonomically classified reads by several orders of magnitude and the recovery of complete chloroplast genomes of Larix. Variation in the chloroplast reads corroborate an invasion of Larix gmelinii into the range of Larix sibirica before 6700 years ago. Since then, both species have been present at the site, although larch populations have decreased with only a few trees remaining in what was once a forested area. This study demonstrates for the first time that hybridization capture applied directly to ancient DNA of plants extracted from lake sediments can provide genome-scale information and is a viable tool for studying past genomic changes in populations of single species, irrespective of a preservation as macrofossil.publishe