Persistence of duplicated PAC<Subscript>1</Subscript> receptors in the teleost, <Emphasis Type="Italic">Sparus auratus</Emphasis>

Background:  

Duplicated genes are common in vertebrate genomes. Their persistence is assumed to be either a consequence of gain of novel function (neofunctionalisation) or partitioning of the function of the ancestral molecule (sub-functionalisation). Surprisingly few studies have evaluated the extent of such modifications despite the numerous duplicated receptor and ligand genes identified in vertebrate genomes to date. In order to study the importance of function in the maintenance of duplicated genes, sea bream (Sparus auratus) PAC₁ receptors, sequence homologues of the mammalian receptor specific for PACAP (Pituitary Adenylate Cyclase-Activating Polypeptide), were studied. These receptors belong to family 2 GPCRs and most of their members are duplicated in teleosts although the reason why both persist in the genome is unknown.     

Effects of grassland intensification on Whinchats Saxicola rubetra and implications for conservation in upland habitats

Modern, intensive grassland management has led to strong declines in ground‐nesting grassland birds, and is now increasingly threatening the last remaining strongholds of the Whinchat Saxicola rubetra in the Central European uplands. In this study, we assess key threats to Whinchat populations in these uplands in order to suggest appropriate conservation measures. We compared the direct threat of early mowing as well as the indirect threat resulting from a deteriorating arthropod food source in an inner‐alpine valley. Five of our seven study sites were mown too early with respect to the chicks' fledging date. Such early mowing was particularly evident on the more intensively farmed, earlier mown valley bottoms than on the valley slopes. Arthropod abundance and biomass did not differ between valley bottoms and slopes. However, valley bottoms had a greater amount of unprofitable prey items such as flies. Breeding bird density was mainly determined by the degree of overlap between the mowing schedule and breeding phenology. These findings suggest that in upland grasslands at an early stage of intensification, early mowing is of greater importance for populations than possible negative effects of a reduced food source. We suggest that mowing is delayed until a sufficient proportion of nestlings are safely fledged.     

In vitro expression of cytokeratin 18, 19 and tube formation of adipose‐derived stem cells induced by the breast epithelial cell line HBL‐100

Fat transplantation is increasingly used in breast augmentation; and recently, the issue of safety concerns from a cellular and molecular point of view has been raised. In this study, attentions were paid to the interaction between adipose‐derived stem cells (ADSC) and mammary epithelial cells: human breast cancer cell line ‐ 100 (HBL ‐ 100) cells were used to simulate the normal microenvironment in breast tissue, ADSCs were harvest from human and co‐cultured with HBL‐100 cells. It was found that ADSCs formed tube‐like structures in the co‐culture with HBL‐100 cells in contrast to the normal morphology of ADSCs in the control group. In addition, the immunofluorescence imaging showed that cytokeratin 18 and 19 (CK18 and 19) were significantly expressed in ADSCs after the co‐culture with HBL‐100 cells. The ultrastructure of those ADSCs also showed epithelial changes. In conclusion, ADSCs are not biological stable when co‐cultured with HBL‐100 cells. They differentiate into epithelial‐like cells with the expression of epithelial surface marks (CK 18, 19) and form tube‐like structures. This may offer an important evidence for the further study of clinical application of transplanting ADSCs rich adipose tissue into the breast in the future.     

Evidence for senescence in survival but not in reproduction in a short‐lived passerine

Senescence has been studied since a long time by theoreticians in ecology and evolution, but empirical support in natural population has only recently been accumulating. One of the current challenges is the investigation of senescence of multiple fitness components and the study of differences between sexes. Until now, studies have been more frequently conducted on females than on males and rather in long‐lived than in short‐lived species. To reach a more fundamental understanding of the evolution of senescence, it is critical to investigate age‐specific survival and reproduction performance in both sexes and in a large range of species with contrasting life histories. In this study, we present results on patterns of age‐specific and sex‐specific variation in survival and reproduction in the whinchat Saxicola rubetra, a short‐lived passerine. We compiled individual‐based long‐term datasets from seven populations that were jointly analyzed within a Bayesian modeling framework. We found evidence for senescence in survival with a continuous decline after the age of 1 year, but no evidence of reproductive senescence. Furthermore, we found no clear evidence for sex effects on these patterns. We discuss these results in light of previous studies documenting senescence in short‐lived birds. We note that most of them have been conducted in populations breeding in nest boxes, and we question the potential effect of the nest boxes on the shape of age‐reproductive trajectories.     

Myogenic differentiation of mesenchymal stem cells co‐cultured with primary myoblasts

TE (tissue engineering) of skeletal muscle is a promising method to reconstruct loss of muscle tissue. This study evaluates MSCs (mesenchymal stem cells) as new cell source for this application. As a new approach to differentiate the MSCs towards the myogenic lineage, co‐cultivation with primary myoblasts has been developed and the myogenic potential of GFP (green fluorescent protein)‐transduced rat MSC co‐cultured with primary rat myoblasts was assessed by ICC (immunocytochemistry). Myogenic potential of MSC was analysed by ICC, FACS and qPCR (quantitative PCR). MSC—myoblast fusion phenomena leading to hybrid myotubes were evaluated using a novel method to evaluate myotube fusion ratios based on phase contrast and fluorescence microscopy. Furthermore, MSC constitutively expressed the myogenic markers MEF2 (myogenic enhancer factor 2) and α‐sarcomeric actin, and MEF2 expression was up‐regulated upon co‐cultivation with primary myoblasts and the addition of myogenic medium supplements. Significantly higher numbers of MSC nuclei were involved in myotube formations when bFGF (basic fibroblast growth factor) and dexamethasone were added to co‐cultures. In summary, we have determined optimal co‐culture conditions for MSC myogenic differentiation up to myotube formations as a promising step towards applicability of MSC as a cell source for skeletal muscle TE as well as other muscle cell‐based therapies.     

Role of guanylate binding protein‐1 in vascular defects associated with chronic inflammatory diseases

Rheumatic autoimmune disorders are characterized by a sustained pro‐inflammatory microenvironment associated with impaired function of endothelial progenitor cells (EPC) and concomitant vascular defects. Guanylate binding protein‐1 (GBP‐1) is a marker and intracellular regulator of the inhibition of proliferation, migration and invasion of endothelial cells induced by several pro‐inflammatory cytokines. In addition, GBP‐1 is actively secreted by endothelial cells. In this study, significantly increased levels of GBP‐1 were detected in the sera of patients with chronic inflammatory disorders. Accordingly we investigated the function of GBP‐1 in EPC. Interestingly, stable expression of GBP‐1 in T17b EPC induced premature differentiation of these cells, as indicated by a robust up‐regulation of both Flk‐1 and von Willebrand factor expression. In addition, GBP‐1 inhibited the proliferation and migration of EPC in vitro. We confirmed that GBP‐1 inhibited vessel‐directed migration of EPC at the tissue level using the rat arterio‐venous loop model as a novel quantitative in vivo migration assay. Overall, our findings indicate that GBP‐1 contributes to vascular dysfunction in chronic inflammatory diseases by inhibiting EPC angiogenic activity via the induction of premature EPC differentiation.     

Osteoinduction and survival of osteoblasts and bone-marrow stromal cells in 3D biphasic calcium phosphate scaffolds under static and dynamic culture conditions

In many tissue engineering approaches, the basic difference between in vitro and in vivo conditions for cells within three‐dimensional (3D) constructs is the nutrition flow dynamics. To achieve comparable results in vitro, bioreactors are advised for improved cell survival, as they are able to provide a controlled flow through the scaffold. We hypothesize that a bioreactor would enhance long‐term differentiation conditions of osteogenic cells in 3D scaffolds. To achieve this either primary rat osteoblasts or bone marrow stromal cells (BMSC) were implanted on uniform‐sized biphasic calcium phosphate (BCP) scaffolds produced by a 3D printing method. Three types of culture conditions were applied: static culture without osteoinduction (Group A); static culture with osteoinduction (Group B); dynamic culture with osteoinduction (Group C). After 3 and 6 weeks, the scaffolds were analysed by alkaline phosphatase (ALP), dsDNA amount, SEM, fluorescent labelled live‐dead assay, and real‐time RT‐PCR in addition to weekly alamarBlue assays. With osteoinduction, increased ALP values and calcium deposition are observed; however, under static conditions, a significant decrease in the cell number on the biomaterial is observed. Interestingly, the bioreactor system not only reversed the decreased cell numbers but also increased their differentiation potential. We conclude from this study that a continuous flow bioreactor not only preserves the number of osteogenic cells but also keeps their differentiation ability in balance providing a suitable cell‐seeded scaffold product for applications in regenerative medicine.