Effect of high light on the efficiency of photochemical energy conversion in a variety of lichen species with green and blue-green phycobionts

Exposure to high light induced a quantitatively similar decrease in the rate of photosynthesis at limiting photon flux density (PFD) and of photosystem II (PSII) photochemical efficiency, F_V/F_M, in both green and blue-green algal lichens which were fully hydrated. Such depressions in the efficiency of photochemical energy conversion were generally reversible in green algal lichens but rather sustained in blue-green algal lichens. This greater susceptibility of blue-green algal lichens to sustained photoinhibition was not related to differences in the capacity to utilize light in photosynthesis, since the light-and CO₂-saturated rates of photosynthetic O₂ evolution were similar in the two groups. These reductions of PSII photochemical efficiency were, however, largely prevented in lichen thalli which were fully desiccated prior to exposure to high PFD. Thalli of green algal lichens which were allowed to desiccate during the exposure to high light exhibited similar recovery kinetics to those which were kept fully hydrated, whereas bluegreen algal lichens which became desiccated during a similar exposure exhibited greatly accelerated recovery compared to those which were kept fully hydrated. Thus, green algal lichens were able to recover from exposure to excessive PFDs when thalli were in either the hydrated or desiccated state during such an exposure, whereas in blue-green algal lichens the decrease in photochemical efficiency was reversible in thalli illuminated in the desiccated state but rather sustained subsequent to illumination of thalli in the hydrated state.Abbreviations and Symbols F_o yield of instantaneous fluorescence - F_M maximum yield of fluorescence induced by pulses of saturating light - F_V variable yield of fluorescence - PFD photon flux density (400–700 nm) - PSII photosystem II This work was supported by the Deutsche Forschungsgeneinschaft (Forscherguppe Ökophysiologic and Sonderforschungsbereich 251 of the University of Würzburg) and the Fonds der Chemischen Industrie. W.W.A. gratefully acknowledges the support of a fellowship from the Alexander von Humboldt Foundation. We thank Professor T.G.A. Green for identifying and supplying all of the New Zealand lichen material and Professor F.-C. Czygan for advice concerning the chlorophyll analyses which were performed by Johanna Leisner.     

Cryo‐Immunoelectron Microscopy of Adherent Cells Improved by the Use of Electrospun Cell Culture Substrates

Electrospun nanofibres are an excellent cell culture substrate, enabling the fast and non‐disruptive harvest and transfer of adherent cells for microscopical and biochemical analyses. Metabolic activity and cellular structures are maintained during the only half a minute‐long harvest and transfer process. We show here that such samples can be optimally processed by means of cryofixation combined either with freeze‐substitution, sample rehydration and cryosection‐immunolabelling or with freeze‐fracture replica‐immunolabelling. Moreover, electrospun fibre substrates are equally suitable for complementary approaches, such as biochemistry, fluorescence microscopy and cytochemistry.     

Emotion recognition in children and adolescents with attention-deficit/hyperactivity disorder (ADHD)

Children with attention-deficit/hyperactivity disorder (ADHD) are impaired in social adaptation and display deficits in social competence. Deficient emotion recognition has been discussed to underlie these social problems. However, comorbid conduct problems have not been considered in the majority of studies conducted so far, and the influence of medication on emotion recognition has rarely been studied. Here, emotion recognition performance was assessed in children with ADHD without medication compared with children with ADHD under stimulant medication and a matched control group. In order to rule out confounding by externalizing symptoms, children with comorbid conduct problems were excluded. Video clips with neutral faces developing a basic emotion (happiness, sadness, disgust, fear and anger) were presented in order to assess emotion recognition. Results indicated between-group differences neither concerning the number of correctly identified emotions nor concerning reaction times and their standard deviations. Thus, we suggest that ADHD per se is not associated with deficits in emotion recognition.     

Mitogen-Activated Protein Kinase-Activated Protein Kinase 2 Deficiency Reduces Insulin Sensitivity in High-Fat Diet-Fed Mice

Adipose tissue inflammation is considered an important contributor to insulin resistance. Mitogen-activated protein kinase-activated protein kinase 2 (MK2) is a major downstream target of p38 MAPK and enhances inflammatory processes. In line with the role of MK2 as contributor to inflammation, MK2^−/− mice are protected against inflammation in different disease models. Therefore, MK2 is considered an attractive therapeutic target for the treatment of chronic inflammatory diseases. This study tested the impact of MK2-deficiency on high-fat diet (HFD)-induced adipose tissue inflammation and insulin resistance. After feeding MK2^−/− and WT control mice a HFD (60% energy from fat) for 24 weeks, body weight was not different between groups. Also, liver weight and the amount of abdominal fat remained unchanged. However, in MK2^−/− mice plasma cholesterol levels were significantly increased. Surprisingly, macrophage infiltration in adipose tissue was not altered. However, adipose tissue macrophages were more skewed to the inflammatory M1 phenotype in MK2^−/− mice. This differerence in macrophage polarization did however not translate in significantly altered expression levels of Mcp-1, Tnfα and Il6. Glucose and insulin tolerance tests demonstrated that MK2^−/− mice had a significantly reduced glucose tolerance and increased insulin resistance. Noteworthy, the expression of the insulin-responsive glucose transporter type 4 (GLUT4) in adipose tissue of MK2^−/− mice was reduced by 55% (p<0.05) and 33% (p<0.05) on the mRNA and protein level, respectively, compared to WT mice. In conclusion, HFD-fed MK2^−/− display decreased glucose tolerance and increased insulin resistance compared to WT controls. Decreased adipose tissue expression of GLUT4 might contribute to this phenotype. The data obtained in this study indicate that clinical use of MK2 inhibitors has to be evaluated with caution, taking potential metabolic adverse effects into account.     

Construction and Rescue of a Molecular Clone of Deformed Wing Virus (DWV)

European honey bees are highly important in crop pollination, increasing the value of global agricultural production by billions of dollars. Current knowledge about virulence and pathogenicity of Deformed wing virus (DWV), a major factor in honey bee colony mortality, is limited. With this study, we close the gap between field research and laboratory investigations by establishing a complete in vitro model for DWV pathogenesis. Infectious DWV was rescued from a molecular clone of a DWV-A genome that induces DWV symptoms such as crippled wings and discoloration. The expression of DWV proteins, production of infectious virus progeny, and DWV host cell tropism could be confirmed using newly generated anti-DWV monoclonal antibodies. The recombinant RNA fulfills Koch’s postulates circumventing the need of virus isolation and propagation of pure virus cultures. In conclusion, we describe the development and application of a reverse genetics system for the study of DWV pathogenesis.     

BiPPred: Combined sequence‐ and structure‐based prediction of peptide binding to the Hsp70 chaperone BiP

Substrate binding to Hsp70 chaperones is involved in many biological processes, and the identification of potential substrates is important for a comprehensive understanding of these events. We present a multi‐scale pipeline for an accurate, yet efficient prediction of peptides binding to the Hsp70 chaperone BiP by combining sequence‐based prediction with molecular docking and MMPBSA calculations. First, we measured the binding of 15mer peptides from known substrate proteins of BiP by peptide array (PA) experiments and performed an accuracy assessment of the PA data by fluorescence anisotropy studies. Several sequence‐based prediction models were fitted using this and other peptide binding data. A structure‐based position‐specific scoring matrix (SB‐PSSM) derived solely from structural modeling data forms the core of all models. The matrix elements are based on a combination of binding energy estimations, molecular dynamics simulations, and analysis of the BiP binding site, which led to new insights into the peptide binding specificities of the chaperone. Using this SB‐PSSM, peptide binders could be predicted with high selectivity even without training of the model on experimental data. Additional training further increased the prediction accuracies. Subsequent molecular docking (DynaDock) and MMGBSA/MMPBSA‐based binding affinity estimations for predicted binders allowed the identification of the correct binding mode of the peptides as well as the calculation of nearly quantitative binding affinities. The general concept behind the developed multi‐scale pipeline can readily be applied to other protein‐peptide complexes with linearly bound peptides, for which sufficient experimental binding data for the training of classical sequence‐based prediction models is not available. Proteins 2016; 84:1390–1407. © 2016 Wiley Periodicals, Inc.     

The role of the polo kinase Cdc5 in controlling Cdc14 localization

In budding yeast, the protein phosphatase Cdc14 controls exit from mitosis. Its activity is regulated by a competitive inhibitor Cfi1/Net1, which binds to and sequesters Cdc14 in the nucleolus. During anaphase, Cdc14 is released from its inhibitor by the action of two regulatory networks. The Cdc Fourteen Early Anaphase Release (FEAR) network initiates Cdc14 release from Cfi1/Net1 during early anaphase, and the Mitotic Exit Network (MEN) promotes Cdc14 release during late anaphase. Here, we investigate the relationship among FEAR network components and propose an order in which they function to promote Cdc14 release from the nucleolus. Furthermore, we examine the role of the protein kinase Cdc5, which is a component of both the FEAR network and the MEN, in Cdc14 release from the nucleolus. We find that overexpression of CDC5 led to Cdc14 release from the nucleolus in S phase-arrested cells, which correlated with the appearance of phosphorylated forms of Cdc14 and Cfi1/Net1. Cdc5 promotes Cdc14 phosphorylation and, by stimulating the MEN, Cfi1/Net1 phosphorylation. Furthermore, we suggest that Cdc14 release from the nucleolus only occurs when Cdc14 and Cfi1/Net1 are both phosphorylated.     

Variable expression of lineage regulators in differentiated stromal cells indicates distinct mechanisms of differentiation towards common cell fate

Mesenchymal stem cells (MSCs) possess a multi-lineage differentiation capacity that makes them important players in the field of regenerative medicine. MSC populations derived from different tissues or donors have been shown to exhibit variable gene expression patterns. Further, it is widely acknowledged that MSC isolates are heterogeneous mixtures of cells at different developmental stages. However, the heterogeneity of expression of lineage regulators has not been linked to differentiation potential of different MSC populations towards mesenchymal lineages. Here, we analyzed variation of expression of differentiation markers across whole population and between single differentiating cells of multipotent stromal cell populations derived from adipose tissue (AdMSCs) and skin (FBs) of seven donors. The results of the analyses show that all cell populations exhibit similar differentiation potential towards adipocyte, osteoblast and chondrocyte lineages despite tissue type- and donor-specific variations of expression of differentiation-associated genes. Further, we detected variable expression of lineage regulators in individual differentiating cells. Together, our data indicate that single cells of stromal cell populations could use distinct molecular mechanisms to reach a common cell fate.     

Conservation and evolutionary modifications of neuroblast expression patterns in insects

One of the major questions in evolutionary developmental neurobiology is how neuronal networks have been adapted to different morphologies and behaviour during evolution. Analyses of neurogenesis in representatives of all arthropod species have revealed evolutionary modifications of various developmental mechanisms. Among others, variations can be seen in mechanisms that are associated with changes in neural progenitor identity, which in turn determines the neuronal subtype of their progeny. Comparative analyses of the molecular processes that underlie the generation of neuronal identity might therefore uncover the steps of evolutionary changes that eventually resulted in modifications in neuronal networks. Here we address this question in the flour beetle Tribolium castaneum by analyzing and comparing the development and expression profile of neural stem cells (neuroblasts) to the published neuroblast map of the fruit fly Drosophila melanogaster. We show that substantial changes in the identity of neuroblasts have occurred during insect evolution. In almost all neuroblasts the relative positions in the ventral hemi-neuromeres are conserved; however, in over half of the neuroblasts the time of formation as well as the gene expression profile has changed. The neuroblast map presented here can be used for future comparative studies on individual neuroblast lineages in D. melanogaster and T. castaneum and additional markers and information on lineages can be added. Our data suggest that evolutionary changes in the expression profile of individual neuroblasts might have contributed to the evolution of neural diversity and subsequently to changes in neuronal networks in arthropod.