Broad characterization of endogenous peptides in the tree shrew visual system

Endogenous neuropeptides, acting as neurotransmitters or hormones in the brain, carry out important functions including neural plasticity, metabolism and angiogenesis. Previous neuropeptide studies have focused on peptide-rich brain regions such as the striatum or hypothalamus. Here we present an investigation of peptides in the visual system, composed of brain regions that are generally less rich in peptides, with the aim of providing the first broad overview of peptides involved in mammalian visual functions. We target three important parts of the visual system: the primary visual cortex (V1), lateral geniculate nucleus (LGN) and superior colliculus (SC). Our study is performed in the tree shrew, a close relative of primates. Using a combination of data dependent acquisition and targeted LC-MS/MS based neuropeptidomics; we identified a total of 52 peptides from the tree shrew visual system. A total of 26 peptides, for example GAV and neuropeptide K were identified in the visual system for the first time. Out of the total 52 peptides, 27 peptides with high signal-to-noise-ratio (>10) in extracted ion chromatograms (EIC) were subjected to label-free quantitation. We observed generally lower abundance of peptides in the LGN compared to V1 and SC. Consistently, a number of individual peptides showed high abundance in V1 (such as neuropeptide Y or somatostatin 28) and in SC (such as somatostatin 28 AA1-12). This study provides the first in-depth characterization of peptides in the mammalian visual system. These findings now permit the investigation of neuropeptide-regulated mechanisms of visual perception.     

Decolonisation of MRSA, S. aureus and E. coli by cold-atmospheric plasma using a porcine skin model in vitro

In the last twenty years new antibacterial agents approved by the U.S. FDA decreased whereas in parallel the resistance situation of multi-resistant bacteria increased. Thus, community and nosocomial acquired infections of resistant bacteria led to a decrease in the efficacy of standard therapy, prolonging treatment time and increasing healthcare costs. Therefore, the aim of this work was to demonstrate the applicability of cold atmospheric plasma for decolonisation of Gram-positive (Methicillin-resistant Staphylococcus aureus (MRSA), Methicillin-sensitive Staphylococcus aureus) and Gram-negative bacteria (E. coli) using an ex vivo pig skin model. Freshly excised skin samples were taken from six month old female pigs (breed: Pietrain). After application of pure bacteria on the surface of the explants these were treated with cold atmospheric plasma for up to 15 min. Two different plasma devices were evaluated. A decolonisation efficacy of 3 log(10) steps was achieved already after 6 min of plasma treatment. Longer plasma treatment times achieved a killing rate of 5 log(10) steps independently from the applied bacteria strains. Histological evaluations of untreated and treated skin areas upon cold atmospheric plasma treatment within 24 h showed no morphological changes as well as no significant degree of necrosis or apoptosis determined by the TUNEL-assay indicating that the porcine skin is still vital. This study demonstrates for the first time that cold atmospheric plasma is able to very efficiently kill bacteria applied to an intact skin surface using an ex vivo porcine skin model. The results emphasize the potential of cold atmospheric plasma as a new possible treatment option for decolonisation of human skin from bacteria in patients in the future without harming the surrounding tissue.     

6-Arylpyrido[2,3-d]pyrimidines as Novel ATP-Competitive Inhibitors of Bacterial D-Alanine:D-Alanine Ligase

Background

ATP-dependent D-alanine:D-alanine ligase (Ddl) is a part of biochemical machinery involved in peptidoglycan biosynthesis, as it catalyzes the formation of the terminal D-ala-D-ala dipeptide of the peptidoglycan precursor UDPMurNAc-pentapeptide. Inhibition of Ddl prevents bacterial growth, which makes this enzyme an attractive and viable target in the urgent search of novel effective antimicrobial drugs. To address the problem of a relentless increase in resistance to known antimicrobial agents we focused our attention to discovery of novel ATP-competitive inhibitors of Ddl.

Methodology/Principal Findings

Encouraged by recent successful attempts to find selective ATP-competitive inhibitors of bacterial enzymes we designed, synthesized and evaluated a library of 6-arylpyrido[2,3-d]pyrimidine-based compounds as inhibitors of Escherichia coli DdlB. Inhibitor binding to the target enzyme was subsequently confirmed by surface plasmon resonance and studied with isothermal titration calorimetry. Since kinetic analysis indicated that 6-arylpyrido[2,3-d]pyrimidines compete with the enzyme substrate ATP, inhibitor binding to the ATP-binding site was additionally studied with docking. Some of these inhibitors were found to possess antibacterial activity against membrane-compromised and efflux pump-deficient strains of E. coli.

Conclusions/Significance

We discovered new ATP-competitive inhibitors of DdlB, which may serve as a starting point for development of more potent inhibitors of DdlB that could include both, an ATP-competitive and D-Ala competitive moiety.     

Are host-parasite interactions influenced by adaptation to predators? A test with guppies and Gyrodactylus in experimental stream channels

Natural populations often face multiple mortality sources. Adaptive responses to one mortality source might also be beneficial with respect to other sources of mortality, resulting in "reinforcing adaptations"; or they might be detrimental with respect to other sources of mortality, resulting in "conflicting adaptations". We explored these possibilities by testing experimentally if the responses of guppies (Poecilia reticulata) to the monogenean ectoparasitic worm Gyrodactylus differed between populations adapted to different predation regimes. In experimental stream channels designed to replicate the natural environment, we exposed eight guppy populations (high-predation and low-predation populations from each of four separate rivers) either to their local Gyrodactylus parasites (infection treatment) or to the absence of those parasites (control). We found that infection dynamics varied dramatically among populations in a repeatable fashion, but that this variation was not related to the predation regime of origin. Consistent with previous work, high-predation guppy females gained more mass, had lower reproductive investment, and had more but smaller embryos than did low-predation females. Relative to control (no parasite) channels, guppies from treatment (infected) channels gained less mass but produced similar numbers and sizes of embryos-and thus had a higher reproductive effort. However, no interaction was evident between infection treatment and predation regime. We conclude that parasitism by Gyrodactylus and predation are both likely selective forces for guppies, but that adaptation to predation does not have an obvious deterministic effect on host-parasite dynamics or on life-history traits of female guppies.     

The PI3-kinase/mTOR-targeting drug NVP-BEZ235 inhibits growth and IgE-dependent activation of human mast cells and basophils

The phosphoinositide 3-kinase (PI3-kinase) and the mammalian target of rapamycin (mTOR) are two major signaling molecules involved in growth and activation of mast cells (MC) and basophils (BA). We examined the effects of the dual PI3-kinase/mTOR blocker NVP-BEZ235 on growth of normal and neoplastic BA and MC as well as immunoglobulin E (IgE)-dependent cell activation. Growth of MC and BA were determined by measuring (3)H-thymidine uptake and apoptosis. Cell activation was determined in histamine release experiments and by measuring upregulation of CD63 and CD203c after challenging with IgE plus anti-IgE or allergen. We found that NVP-BEZ235 exerts profound inhibitory effects on growth of primary and cloned neoplastic MC. In the MC leukemia cell line HMC-1, NVP-BEZ235 showed similar IC(50) values in the HMC-1.1 subclone lacking KIT D816V (0.025 μM) and the HMC-1.2 subclone expressing KIT D816V (0.005 μM). Moreover, NVP-BEZ235 was found to exert strong growth-inhibitory effects on neoplastic MC in a xenotransplant-mouse model employing NMR1-Foxn1(nu) mice. NVP-BEZ235 also exerted inhibitory effects on cytokine-dependent differentiation of normal BA and MC, but did not induce growth inhibition or apoptosis in mature MC or normal bone marrow cells. Finally, NVP-BEZ235 was found to inhibit IgE-dependent histamine release in BA and MC (IC(50) 0.5-1 μM) as well as anti-IgE-induced upregulation of CD203c in BA and IgE-dependent upregulation of CD63 in MC. In summary, NVP-BEZ235 produces growth-inhibitory effects in immature neoplastic MC and inhibits IgE-dependent activation of mature BA and MC. Whether these potentially beneficial drug effects have clinical implications is currently under investigation.     

Large SNP arrays for genotyping in crop plants

Genotyping with large numbers of molecular markers is now an indispensable tool within plant genetics and breeding. Especially through the identification of large numbers of single nucleotide polymorphism (SNP) markers using the novel high-throughput sequencing technologies, it is now possible to reliably identify many thousands of SNPs at many different loci in a given plant genome. For a number of important crop plants, SNP markers are now being used to design genotyping arrays containing thousands of markers spread over the entire genome and to analyse large numbers of samples. In this article, we discuss aspects that should be considered during the design of such large genotyping arrays and the analysis of individuals. The fact that crop plants are also often autopolyploid or allopolyploid is given due consideration. Furthermore, we outline some potential applications of large genotyping arrays including high-density genetic mapping, characterization (fingerprinting) of genetic material and breeding-related aspects such as association studies and genomic selection.     

Atypical Development in Plant and Soil Nematodes

Observations of atypical developmental and anatomical characteristics have been recorded for many taxa of soil nematodes. They include the unusual occurrence of extra feeding structures, aberrant configuration of features of both male and female reproductive systems, and the occurrence of intersexes assumed to be functionally female, functionally male, or non-functional. In many cases, hypotheses have been advanced regarding the genetic or developmental mechanisms and environmental stimuli that control, regulate, or facilitate abnormalities, but many are quite speculative and lack experimental verification. Further, the fitness costs or advantages, and the heritability of aberrant characters are largely unknown, except where they clearly preclude reproduction, either apomictic or amphimictic. Underlying mechanisms and ecological consequences may be difficult to study in organisms that are not readily cultured under axenic or sterile laboratory conditions, however information on developmental processes in Caenorhabditis elegans represents an important resource in which to seek homologies.     

Conservation of threatened relict trees through living <Emphasis Type="Italic">ex situ</Emphasis> collections: lessons from the global survey of the genus <Emphasis Type="Italic">Zelkova</Emphasis> (Ulmaceae)

Maintaining living ex situ collections is one of the key conservation methods in botanic gardens worldwide. Despite of the existence of many other conservation approaches used nowadays, it offers for many endangered plants an important insurance policy for the future, especially for rare and threatened relict trees. The aim of this research was to investigate the global extent of living ex situ collections, to assess and discuss their viability and inform the development of conservation approaches that respond to latest global conservation challenges. We used as a model taxon the tree genus Zelkova (Ulmaceae). The genus includes six prominent Tertiary relict trees which survived the last glaciation in disjunct and isolated refugial regions. Our comprehensive worldwide survey shows that the majority of botanic institutions with Zelkova collections are in countries with a strong horticultural tradition and not in locations of their origin. More importantly, the acutely threatened Zelkova species are not the most represented in collections, and thus safeguarded through ex situ conservation. Less than 20% of the ex situ collections surveyed contain plant material of known wild provenance while the majority (90%) of collections are generally very small (1–10 trees). Botanic gardens and arboreta particularly in regions where iconic relict trees naturally occur should play a vital role in the conservation of these species. The coordination of conservation efforts between gardens has to be enhanced to prioritise action for the most threatened relict trees. Large scale genetic studies should be undertaken, ideally at genus level, in order to verify or clarify the provenance of ex situ collections of relict trees in cultivation. For the most threatened relict tree genera, well-coordinated specialist groups should be created.     

Pleistocene survival on central Alpine nunataks: genetic evidence from the jumping bristletail Machilis pallida

Mechanisms of survival during the Pleistocene glaciation periods have been studied for more than a century. Until now, molecular studies that confirmed animal survival on Alpine nunataks, that is, ice‐free summits surrounded by glaciers, were restricted to peripheral areas. Here, we search for molecular signatures of inner‐Alpine survival of the narrow endemic and putatively parthenogenetic Alpine jumping bristletail Machilis pallida combining mitochondrial and AFLP data from its three known populations. The mitochondrial data indicate survival on both peripheral and central nunataks, the latter suggesting that refugia in the centre of the Alpine main ridge were more widespread than previously recognized. Incongruences between mitochondrial and AFLP patterns suggest a complex evolutionary history of the species and may be explained via parallel fixation of parthenogenesis of different origins during the last glacial maximum. We suggest that the inferred parthenogenesis may have been essential for central nunatak survival, but may pose a serious threat for M. pallida in consideration of the present climatic changes.     

The C-terminal rod 2 fragment of filamin A forms a compact structure that can be extended

Filamins are large proteins that cross-link actin filaments and connect to other cellular components. The C-terminal rod 2 region of FLNa (filamin A) mediates dimerization and interacts with several transmembrane receptors and intracellular signalling adaptors. SAXS (small-angle X-ray scattering) experiments were used to make a model of a six immunoglobulin-like domain fragment of the FLNa rod 2 (domains 16-21). This fragment had a surprising three-branched structural arrangement, where each branch was made of a tightly packed two-domain pair. Peptides derived from transmembrane receptors and intracellular signalling proteins induced a more open structure of the six domain fragment. Mutagenesis studies suggested that these changes are caused by peptides binding to the CD faces on domains 19 and 21 which displace the preceding domain A-strands (18 and 20 respectively), thus opening the individual domain pairs. A single particle cryo-EM map of a nine domain rod 2 fragment (domains 16-24), showed a relatively compact dimeric particle and confirmed the three-branched arrangement as well as the peptide-induced conformation changes. These findings reveal features of filamin structure that are important for its interactions and mechanical properties.