Role of Tachykinin 1 and 4 Gene-Derived Neuropeptides and the Neurokinin 1 Receptor in Adjuvant-Induced Chronic Arthritis of the Mouse

Objective

Substance P, encoded by the Tac1 gene, is involved in neurogenic inflammation and hyperalgesia via neurokinin 1 (NK1) receptor activation. Its non-neuronal counterpart, hemokinin-1, which is derived from the Tac4 gene, is also a potent NK1 agonist. Although hemokinin-1 has been described as a tachykinin of distinct origin and function compared to SP, its role in inflammatory and pain processes has not yet been elucidated in such detail. In this study, we analysed the involvement of tachykinins derived from the Tac1 and Tac4 genes, as well as the NK1 receptor in chronic arthritis of the mouse.

Methods

Complete Freund’s Adjuvant was injected intraplantarly and into the tail of Tac1^−/−, Tac4^−/−, Tacr1^−/− (NK1 receptor deficient) and Tac1^−/−/Tac4^−/− mice. Paw volume was measured by plethysmometry and mechanosensitivity using dynamic plantar aesthesiometry over a time period of 21 days. Semiquantitative histopathological scoring and ELISA measurement of IL-1β concentrations of the tibiotarsal joints were performed.

Results

Mechanical hyperalgesia was significantly reduced from day 11 in Tac4^−/− and Tacr1^−/− animals, while paw swelling was not altered in any strain. Inflammatory histopathological alterations (synovial swelling, leukocyte infiltration, cartilage destruction, bone damage) and IL-1β concentration in the joint homogenates were significantly smaller in Tac4^−/− and Tac1^−/−/Tac4^−/− mice.

Conclusions

Hemokinin-1, but not substance P increases inflammation and hyperalgesia in the late phase of adjuvant-induced arthritis. While NK1 receptors mediate its antihyperalgesic actions, the involvement of another receptor in histopathological changes and IL-1β production is suggested.     

Human Analogue Safe Haven Effect of the Owner: Behavioural and Heart Rate Response to Stressful Social Stimuli in Dogs

The secure base and safe haven effects of the attachment figure are central features of the human attachment theory. Recently, conclusive evidence for human analogue attachment behaviours in dogs has been provided, however, the owner’s security-providing role in danger has not been directly supported. We investigated the relationship between the behavioural and cardiac response in dogs (N = 30) while being approached by a threatening stranger in separation vs. in the presence of the owner, presented in a balanced order. Non-invasive telemetric measures of heart rate (HR) and heart rate variability (HRV) data during the threatening approaches was compared to periods before and after the encounters. Dogs that showed distress vocalisation during separation (N = 18) and that growled or barked at the stranger during the threatening approach (N = 17) were defined as behaviourally reactive in the given situation. While characteristic stress vocalisations were emitted during separations, the absence of the owner did not have an effect on dogs’ mean HR, but significantly increased the HRV. The threatening approach increased dogs’ mean HR, with a parallel decrease in the HRV, particularly in dogs that were behaviourally reactive to the encounter. Importantly, the HR increase was significantly less pronounced when dogs faced the stranger in the presence of the owner. Moreover, the test order, whether the dog encountered the stranger first with or without its owner, also proved important: HR increase associated with the encounter in separation seemed to be attenuated in dogs that faced the stranger first in the presence of their owner. We provided evidence for human analogue safe haven effect of the owner in a potentially dangerous situation. Similarly to parents of infants, owners can provide a buffer against stress in dogs, which can even reduce the effect of a subsequent encounter with the same threatening stimuli later when the owner is not present.     

Immobilized metal affinity chromatography optimized for the analysis of extracellular phosphorylation

Phosphorylation is the most widely studied posttranslational modification. Its role within the cell has been the focus of numerous large‐scale studies. Recently there is growing evidence on the biological significance of extracellular phosphorylation. The analysis of these phosphopeptides is complicated by the abundance of glycosylation in the extracellular space, since glycopeptides are also enriched by the methods used for phosphopeptide isolation. Thus, we optimized IMAC for phosphorylation analysis of secreted proteins, specifically in human serum. Selectivity and efficiency of different enrichment conditions used in earlier large‐scale phosphoproteomic studies were evaluated. We found that minimizing hydrophilic interactions in the enrichment allowed selective phosphopeptide isolation. Using a two‐step IMAC enrichment protocol under these conditions led to the identification of ～100 phosphorylation sites from the tryptic digest of as little as 40 μL human serum.     

Toxin depletion has no effect on antipredator responses in common toad (Bufo bufo) tadpoles

Antipredator responses often involve changes in several phenotypic traits and these changes interactively influence fitness. However, gaining insight into how the overall fitness effect of the overall response comes about is notoriously difficult. One promising avenue is to manipulate a single defensive trait and observe how that modifies fitness as well as the expression of other inducible responses. In chemically‐defended animals, toxins are likely to be costly to produce but it is still unknown how their depletion influences other characteristics. In the present study, we artificially depleted bufadienolide toxin stores in common toad (Bufo bufo) tadpoles, and assessed the effect of this with respect to the interaction with predator presence and limited food availability. We found that toxin depletion in tadpoles did not significantly affect any of the measured life‐history traits. Tadpoles in the predator treatment exhibited an elevated development rate, although this was only apparent when food availability was limited. Also, body mass at metamorphosis was lower in tadpoles exposed to chemical cues indicating a predation threat and when food availability was limited. These results provide evidence that, in larval common toads, the expression of inducible defences may incur fitness costs, whereas chemical defences are either expressed constitutively or, if inducible, elevated toxin production has negligible costs.     

Response of Organ Structure and Physiology to Autotetraploidization in Early Development of Energy Willow Salix viminalis

The biomass productivity of the energy willow Salix viminalis as a short-rotation woody crop depends on organ structure and functions that are under the control of genome size. Colchicine treatment of axillary buds resulted in a set of autotetraploid S. viminalis var. Energo genotypes (polyploid Energo [PP-E]; 2n = 4x = 76) with variation in the green pixel-based shoot surface area. In cases where increased shoot biomass was observed, it was primarily derived from larger leaf size and wider stem diameter. Autotetraploidy slowed primary growth and increased shoot diameter (a parameter of secondary growth). The duplicated genome size enlarged bark and wood layers in twigs sampled in the field. The PP-E plants developed wider leaves with thicker midrib and enlarged palisade parenchyma cells. Autotetraploid leaves contained significantly increased amounts of active gibberellins, cytokinins, salicylic acid, and jasmonate compared with diploid individuals. Greater net photosynthetic CO₂ uptake was detected in leaves of PP-E plants with increased chlorophyll and carotenoid contents. Improved photosynthetic functions in tetraploids were also shown by more efficient electron transport rates of photosystems I and II. Autotetraploidization increased the biomass of the root system of PP-E plants relative to diploids. Sections of tetraploid roots showed thickening with enlarged cortex cells. Elevated amounts of indole acetic acid, active cytokinins, active gibberellin, and salicylic acid were detected in the root tips of these plants. The presented variation in traits of tetraploid willow genotypes provides a basis to use autopolyploidization as a chromosome engineering technique to alter the organ development of energy plants in order to improve biomass productivity.Energy security and climate change as global problems urge increased efforts to use plants as renewable energy sources both for power generation and transportation fuel production. Selected wood species, such as willows (Salix spp.), can be cultivated as short-rotation coppice for the rapid accumulation of biomass and reduction of CO₂ emission. Coppicing reinvigorates shoot growth, resulting in a special woody plant life cycle that differs from natural tree development, which takes decades. In this cultivation system, small stem cuttings are planted at high densities (15,000–25,000 ha⁻¹). In the soil, these dormant wood cuttings first produce roots and shoots that emerge from reactivated buds. During the first year, the growing shoots mature to woody stems. In the winter, these stems are cut back, and in the following spring, the cut stumps develop multiple shoots. The short-rotation coppice plantations are characterized by a very short, 2- to 3-year rotation, and the most productive varieties can produce up to 15 tons of oven-dried wood per hectare per year (Cunniff and Cerasuolo, 2011). The high-density willow plantations can also be efficiently used for heavy metal or organic phytoremediation, as reviewed by Marmiroli et al. (2011).The biomass productivity of shrub willows is largely dependent on coppicing capability, early vigorous growth, shoot growth rate and final stem height, root system size, photosynthetic efficiency, formation and composition of woody stems, water and nutrient use, as well as abiotic and biotic stress tolerance. Genetic improvement of all these traits can be based on broad natural genetic resources represented by more than 400 species in the genus Salix. More than 200 species have hybrid origins, and ploidy levels vary from diploid up to dodecaploid (Suda and Argus, 1968; Newsholme, 1992). In addition to molecular marker-assisted clone selection, intraspecific and interspecific crosses have been shown to further extend genetic variability in breeding programs for biomass yield (Karp et al., 2011).During natural diversification and artificial crossings of Salix spp., the willow genomes frequently undergo polyploidization, resulting in triploid or tetraploid allopolyploids. In triploid hybrids, both heterosis and ploidy can contribute to the improved biomass yield (Serapiglia et al., 2014). While the alloploid triploids have attracted considerable attention in willow improvement, the potentials of autotetraploid willow genotypes have not been exploited so far. As shown for other short-rotation wood species (poplar [Populus spp.], black locust [Robinia pseudoacacia], Paulownia spp., and birch [Betula spp.]), doubling the chromosome set by colchicine treatment can cause significant changes in organ morphology or growth parameters (Tang et al., 2010; Cai and Kang, 2011; Harbard et al., 2012; Mu et al., 2012; Wang et al., 2013a, 2013b). In several polyploidization protocols, the in vitro cultured tissues are exposed to different doses of colchicine or other inhibitors of mitotic microtubule function, and plantlets are differentiated from polyploid somatic cells (Tang et al., 2010; Cai and Kang, 2011). Alternatively, seeds or apical meristems of germinating seedlings can be treated with a colchicine solution (Harbard et al., 2012). Allotetraploids of poplar were produced by zygotic chromosome doubling that was induced by colchicine and high-temperature treatment (Wang et al., 2013a).Since tetraploid willow plants with 2n = 4x = 76 chromosomes are expected to represent novel genetic variability, especially for organ development and physiological parameters, a polyploidization project was initiated that was based on a highly productive diploid energy willow (S. viminalis var. Energo). Colchicine treatment of reactivated axillary buds of the in vitro-grown energy willow plantlets resulted in autotetraploid shoots and, subsequently, plants. For comparison of diploid and tetraploid variants of willow plants, digital imaging of green organs and roots was used for phenotyping. Among the tetraploid lines, genotypes were identified with improved biomass production, better photosynthetic parameters, and altered organ structure and hormone composition. The new tetraploid willow variants produced can serve as a unique experimental material to uncover key factors in biomass production in this short-rotation energy plant. In the future, these plants can also serve as crossing partners of diploid lines for the production of novel triploid energy willow genotypes.     

Worldwide distribution and theoretical spreading of Trichoferus campestris (Coleoptera: Cerambycidae) depending on the main climatic elements

The velvet longhorned beetle, Trichoferus campestris (Coleoptera: Cerambycidae) is a serious wood‐boring pest that is a major threat to the phytosanitary condition of forests and orchards. Its worldwide expansion is a major concern for plant health. We have collected all bibliographical references, phytosanitary reports and authentic photographic evidence from entomological websites to determine the worldwide distribution of T. campestris. The theoretical directions of the spreading and actually occupied area of this arthropod pest were determined over the whole Holarctic range. Furthermore, the potential distribution area was calculated using cumulated temperature in the growing season averaged over 15 years both in the Palearctic and Nearctic regions. Holarctic expansion of the species, including the main parts of North America and Eurasia, is clearly indicated. Its populations occur in 29 countries to date, supported by documentation from 64 publications and 30 online forums. Its spread is continuously westward in the Palearctic; in the Nearctic, the spread was first southward from the Great Lakes region then eastward from Utah. The species has excellently adapted to circumstances of freight by ship and plane, as wood is often used, ensuring optimal conditions for the pest. In addition, the active spreading achieved by flying is an important factor contributing to its expansion. The primary criterion for controlling the species would be the introduction of a monitoring system in affected and exposed areas.     

Trait‐based approach confirms the importance of propagule limitation and assembly rules in old‐field restoration

Community assembly theory is suggested as a guiding principle for ecological restoration to help understand the mechanisms that structure biological communities and identify where restoration interventions are needed. We studied three hypotheses related to propagule limitation, stress‐dominance, and limiting similarity concepts in community assembly in a restoration field experiment with a trait‐based null model approach. The experiment aimed to assist the recovery of sand grassland on former arable land in the Kiskunság, Pannonian biogeographic region, Europe. Treatments included initial seeding of five grassland species, carbon amendment, low‐intensity mowing, and combinations in 1 m by 1 m plots in three old fields from 2003 to 2008. The distribution of 10 individual plant traits was compared to the null model and the effect of time and treatments were tested with linear mixed effect models. Initial seeding had the most visible impact on species and trait composition confirming propagule limitation in grassland recovery. Reducing nutrient availability through carbon amendment strengthened trait convergence for length of flowering as expected based on the stress‐dominance hypothesis. Mowing changed trait divergence to convergence for plant height with a strengthening impact with time, supporting our hypothesis of increasing dominance of limiting similarity with time. Our results support the idea that community assembly is simultaneously influenced by propagule limitation and multiple trait‐based processes that act through different traits. The limited impact of manipulating environmental filtering and limiting similarity compared to seeding, however, supports the view that only targeting the dispersal and environmental filters in parallel would improve restoration outcome.     

Synthesis of non-glycosidic 4,6'-thioether-linked disaccharides as hydrolytically stable glycomimetics

Michael addition of 1,2:3,4-di-O-isopropylidene-6-thio-alpha-D-galactose (2) to 2-propyl 6-O-acetyl-3,4-dideoxy-alpha-D-glycero-hex-3-enopyranosid-2-ulose (1) afforded, as the major diastereoisomer, 2-propyl 6-O-acetyl-3-deoxy-4-S-(6-deoxy-1,2:3,4-di-O-isopropylidene-alpha-D-galactopyranos-6-yl)-4-thio-alpha-D-threo-hexopyranosid-2-ulose (3, 91% yield). Reduction of the carbonyl group of 3, followed by O-deacetylation gave the two epimers 7 (alpha-D-lyxo) and 8 (alpha-D-xylo) in a 1:2 ratio. On removal of the protecting groups of 8 by acid hydrolysis, formation of an 1,6-anhydro bridge was observed in the 3-deoxy-4-thiohexopyranose unit (10). The free non-glycosidic thioether-linked disaccharide 3-deoxy-4-S-(6-deoxy-alpha,beta-D-galactopyranos-6-yl)-4-thio-alpha,beta-D-xylo-hexopyranose (11) was obtained by acetolysis of 10 followed by O-deacetylation. A similar sequence starting from the enone 1 and methyl 2,3,4-tri-O-benzoyl-6-thio-alpha-D-glucopyranoside (12) led successfully to 2-propyl 3-deoxy-4-S-(methyl 6-deoxy-alpha-D-glucopyranos-6-yl)-4-thio-alpha-D-lyxo-hexopyranoside (17) and its alpha-D-xylo analog (19, major product). In this synthetic route, orthogonal sets of protecting groups were employed to preserve the configuration of both reducing ends and to avoid the formation of the 1,6-anhydro ring.     

Active site closure facilitates juxtaposition of reactant atoms for initiation of catalysis by human dUTPase

Human dUTPase, essential for DNA integrity, is an important survival factor for cancer cells. We determined the crystal structure of the enzyme:alpha,beta-imino-dUTP:Mg complex and performed equilibrium binding experiments in solution. Ordering of the C-terminus upon the active site induces close juxtaposition of the incoming nucleophile attacker water oxygen and the alpha-phosphorus of the substrate, decreasing their distance below the van der Waals limit. Complex interactions of the C-terminus with both substrate and product were observed via a specifically designed tryptophan sensor, suitable for further detailed kinetic and ligand binding studies. Results explain the key functional role of the C-terminus.     

When to measure plumage reflectance: a lesson from Collared Flycatchers Ficedula albicollis

Sexually selected colour traits of bird plumage are widely studied. Although the plumage is replaced only at one or two yearly moults, plumage colour has long been shown to change between moults. Nevertheless, most studies measure colour weeks to months after the courtship period, typically at nestling rearing, and it is unclear whether these measurements yield relevant data concerning the primary process of sexual selection. Here we analyse repeated spectrometric data taken from male Collared Flycatchers during social courtship and nestling rearing. We show that some spectral traits are not correlated between the two measurements and that within‐individual correlation declines significantly with the likely exposure of the plumage area to damage and soiling. There is an overall decline in spectral trait exaggeration during breeding, but trait decline is not closely related to measurement latency, especially not in the damage‐exposed areas. Finally, sexual selection estimates differ depending on whether they are derived from spectra measured during courtship or during nestling rearing. These results suggest that, contrary to current practice, measurements of plumage reflectance should be made during the primary period of sexual signalling. Spectral trait decline during breeding could also be studied as a possible signal for mates and neighbours.