Protein Interactions and Fluctuations in a Proteomic Network using an Elastic Network Model

Abstract

A set of protein conformations are analyzed by normal mode analysis. An elastic network model is used to obtain fluctuation and cooperativity of residues with low amplitude fluctuations across different species. Slow modes that are associated with the function of proteins have common features among different protein structures. We show that the degree of flexibility of the protein is important for proteins to interact with other proteins and as the species gets more complex its proteins become more flexible. In the complex organism, higher cooperativity arises due to protein structure and connectivity.     

Selectivity is species-dependent: Characterization of standard agonists and antagonists at human,rat, and mouse adenosine receptors

Adenosine receptors (ARs) have emerged as new drug targets. The majority of data on affinity/potency and selectivity of AR ligands described in the literature has been obtained for the human species. However, preclinical studies are mostly performed in mouse or rat, and standard AR agonists and antagonists are frequently used for studies in rodents without knowing their selectivity in the investigated species. In the present study, we selected a set of frequently used standard AR ligands, 8 agonists and 16 antagonists, and investigated them in radioligand binding studies at all four AR subtypes, A₁, A_2A, A_2B, and A₃, of three species, human, rat, and mouse. Recommended, selective agonists include CCPA (for A₁AR of rat and mouse), CGS-21680 (for A_2A AR of rat), and Cl-IB-MECA (for A₃AR of all three species). The functionally selective partial A_2B agonist BAY60-6583 was found to additionally bind to A₁ and A₃AR and act as an antagonist at both receptor subtypes. The antagonists PSB-36 (A₁), preladenant (A_2A), and PSB-603 (A_2B) displayed high selectivity in all three investigated species. MRS-1523 acts as a selective A₃AR antagonist in human and rat, but is only moderately selective in mouse. The comprehensive data presented herein provide a solid basis for selecting suitable AR ligands for biological studies.

Electronic supplementary material

The online version of this article (doi:10.1007/s11302-015-9460-9) contains supplementary material, which is available to authorized users.     

First dating of a recombination event in mammalian tick-borne flaviviruses

The mammalian tick-borne flavivirus group (MTBFG) contains viruses associated with important human and animal diseases such as encephalitis and hemorrhagic fever. In contrast to mosquito-borne flaviviruses where recombination events are frequent, the evolutionary dynamic within the MTBFG was believed to be essentially clonal. This assumption was challenged with the recent report of several homologous recombinations within the Tick-borne encephalitis virus (TBEV). We performed a thorough analysis of publicly available genomes in this group and found no compelling evidence for the previously identified recombinations. However, our results show for the first time that demonstrable recombination (i.e., with large statistical support and strong phylogenetic evidences) has occurred in the MTBFG, more specifically within the Louping ill virus lineage. Putative parents, recombinant strains and breakpoints were further tested for statistical significance using phylogenetic methods. We investigated the time of divergence between the recombinant and parental strains in a Bayesian framework. The recombination was estimated to have occurred during a window of 282 to 76 years before the present. By unravelling the temporal setting of the event, we adduce hypotheses about the ecological conditions that could account for the observed recombination.     

Divergent responses of different endothelial cell types to infection with Candida albicans and Staphylococcus aureus

Endothelial cells are important in the pathogenesis of bloodstream infections caused by Candida albicans and Staphylococcus aureus. Numerous investigations have used human umbilical vein endothelial cells (HUVECs) to study microbial-endothelial cell interactions in vitro. However, the use of HUVECs requires a constant supply of umbilical cords, and there are significant donor-to-donor variations in these endothelial cells. The use of an immortalized endothelial cell line would obviate such difficulties. One candidate in this regard is HMEC-1, an immortalized human dermal microvascular endothelial cell line. To determine if HMEC-1 cells are suitable for studying the interactions of C. albicans and S. aureus with endothelial cells in vitro, we compared the interactions of these organisms with HMEC-1 cells and HUVECs. We found that wild-type C. albicans had significantly reduced adherence to and invasion of HMEC-1 cells as compared to HUVECs. Although wild-type S. aureus adhered to and invaded HMEC-1 cells similarly to HUVECs, an agr mutant strain had significantly reduced invasion of HMEC-1 cells, but not HUVECs. Furthermore, HMEC-1 cells were less susceptible to damage induced by C. albicans, but more susceptible to damage caused by S. aureus. In addition, HMEC-1 cells secreted very little IL-8 in response to infection with either organism, whereas infection of HUVECs induced substantial IL-8 secretion. This weak IL-8 response was likely due to the anatomic site from which HMEC-1 cells were obtained because infection of primary human dermal microvascular endothelial cells with C. albicans and S. aureus also induced little increase in IL-8 production above basal levels. Thus, C. albicans and S. aureus interact with HMEC-1 cells in a substantially different manner than with HUVECs, and data obtained with one type of endothelial cell cannot necessarily be extrapolated to other types.     

Tritium-labeled agonists as tools for studying adenosine A<Subscript>2B</Subscript> receptors

A selective agonist radioligand for A_2B adenosine receptors (A_2BARs) is currently not available. Such a tool would be useful for labeling the active conformation of the receptors. Therefore, we prepared BAY 60-6583, a potent and functionally selective A_2BAR (partial) agonist, in a tritium-labeled form. Despite extensive efforts, however, we have not been able to establish a radioligand binding assay using [³H]BAY 60-6583. This is probably due to its high non-specific binding and its moderate affinity, which had previously been overestimated based on functional data. As an alternative, we evaluated the non-selective A_2BAR agonist [³H]NECA for its potential to label A_2BARs. [³H]NECA showed specific, saturable, and reversible binding to membrane preparations of Chinese hamster ovary (CHO) or human embryonic kidney (HEK) cells stably expressing human, rat, or mouse A_2BARs. In competition binding experiments, the AR agonists 2-chloroadenosine (CADO) and NECA displayed significantly higher affinity when tested versus [³H]NECA than versus the A_2B-antagonist radioligand [³H]PSB-603 while structurally diverse AR antagonists showed the opposite effects. Although BAY 60-6583 is an A_2BAR agonist, it displayed higher affinity versus [³H]PSB-603 than versus [³H]NECA. These results indicate that nucleoside and non-nucleoside agonists are binding to very different conformations of the A_2BAR. In conclusion, [³H]NECA is currently the only useful radioligand for determining the affinity of ligands for an active A_2BAR conformation.     

Can spelt wheat be used as heterotic group for hybrid wheat breeding?

Key message

Spelt wheat is a distinct genetic group to elite bread wheat, but heterosis for yield and protein quality is too low for spelt to be recommended as heterotic group for hybrid breeding in wheat.

Abstract

The feasibility to switch from line to hybrid breeding is currently a hot topic in the wheat community. One limitation seems to be the lack of divergent heterotic groups within wheat adapted to a certain region. Spelt wheat is a hexaploid wheat that can easily be crossed with bread wheat and that forms a divergent genetic group when compared to elite bread wheat. The aim of this study was to investigate the potential of Central European spelt as a heterotic group for Central European bread wheat. We performed two large experimental field studies comprising in total 43 spelt lines, 14 wheat lines, and 273 wheat–spelt hybrids, and determined yield, heading time, plant height, resistance against yellow rust, leaf rust, and powdery mildew, as well as protein content and sedimentation volume. Heterosis of yield was found to be lower than that of hybrids made between elite wheat lines. Moreover, heterosis of the quality trait sedimentation volume was negative. Consequently, spelt wheat does not appear suited to be used as heterotic group in hybrid wheat breeding. Nevertheless, high combining abilities of a few spelt lines with elite bread wheat lines make them interesting resources for pre-breeding in bread wheat. Thereby, the low correlation between line per se performance and combining ability of these spelt lines shows the potential to unravel the breeding value of genetic resources by crossing them to an elite tester.

     

Tick-Borne Encephalitis Virus Sequenced Directly from Questing and Blood-Feeding Ticks Reveals Quasispecies Variance

The increased distribution of the tick-borne encephalitis virus (TBEV) in Scandinavia highlights the importance of characterizing novel sequences within the natural foci. In this study, two TBEV strains: the Norwegian Mandal 2009 (questing nymphs pool) and the Swedish Saringe 2009 (blood-fed nymph) were sequenced and phylogenetically characterized. Interestingly, the sequence of Mandal 2009 revealed the shorter form of the TBEV genome, similar to the highly virulent Hypr strain, within the 3′ non-coding region (3′NCR). A different genomic structure was found in the 3′NCR of Saringe 2009, as in-depth analysis demonstrated TBEV variants with different lengths within the poly(A) tract. This shows that TBEV quasispecies exists in nature and indicates a putative shift in the quasispecies pool when the virus switches between invertebrate and vertebrate environments. This prompted us to further sequence and analyze the 3′NCRs of additional Scandinavian TBEV strains and control strains, Hypr and Neudoerfl. Toro 2003 and Habo 2011 contained mainly a short (A)3C(A)6 poly(A) tract. A similar pattern was observed for the human TBEV isolates 1993/783 and 1991/4944; however, one clone of 1991/4944 contained an (A)3C(A)11 poly(A) sequence, demonstrating that quasispecies with longer poly(A) could be present in human isolates. Neudoerfl has previously been reported to contain a poly(A) region, but to our surprise the re-sequenced genome contained two major quasispecies variants, both lacking the poly(A) tract. We speculate that the observed differences are important factors for the understanding of virulence, spread, and control of the TBEV.     

Effects of Surface Asymmetry on Neuronal Growth

Detailed knowledge of how the surface physical properties, such as mechanics, topography and texture influence axonal outgrowth and guidance is essential for understanding the processes that control neuron development, the formation of functional neuronal connections and nerve regeneration. Here we synthesize asymmetric surfaces with well-controlled topography and texture and perform a systematic experimental and theoretical investigation of axonal outgrowth on these substrates. We demonstrate unidirectional axonal bias imparted by the surface ratchet-based topography and quantify the topographical guidance cues that control neuronal growth. We describe the growth cone dynamics using a general stochastic model (Fokker-Planck formalism) and use this model to extract two key dynamical parameters: diffusion (cell motility) coefficient and asymmetric drift coefficient. The drift coefficient is identified with the torque caused by the asymmetric ratchet topography. We relate the observed directional bias in axonal outgrowth to cellular contact guidance behavior, which results in an increase in the cell-surface coupling with increased surface anisotropy. We also demonstrate that the disruption of cytoskeletal dynamics through application of Taxol (stabilizer of microtubules) and Blebbistatin (inhibitor of myosin II activity) greatly reduces the directional bias imparted by these asymmetric surfaces. These results provide new insight into the role played by topographical cues in neuronal growth and could lead to new methods for stimulating neuronal regeneration and the engineering of artificial neuronal tissue.     

Experimental endocarditis model of methicillin resistant Staphylococcus aureus (MRSA) in rat

Endovascular infections, including endocarditis, are life-threatening infectious syndromes. Staphylococcus aureus is the most common world-wide cause of such syndromes with unacceptably high morbidity and mortality even with appropriate antimicrobial agent treatments. The increase in infections due to methicillin-resistant S. aureus (MRSA), the high rates of vancomycin clinical treatment failures and growing problems of linezolid and daptomycin resistance have all further complicated the management of patients with such infections, and led to high healthcare costs. In addition, it should be emphasized that most recent studies with antibiotic treatment outcomes have been based in clinical settings, and thus might well be influenced by host factors varying from patient-to-patient. Therefore, a relevant animal model of endovascular infection in which host factors are similar from animal-to-animal is more crucial to investigate microbial pathogenesis, as well as the efficacy of novel antimicrobial agents. Endocarditis in rat is a well-established experimental animal model that closely approximates human native valve endocarditis. This model has been used to examine the role of particular staphylococcal virulence factors and the efficacy of antibiotic treatment regimens for staphylococcal endocarditis. In this report, we describe the experimental endocarditis model due to MRSA that could be used to investigate bacterial pathogenesis and response to antibiotic treatment.