Differential expression of equine muscle biopsy proteins during normal training and intensified training in young standardbred horses using proteomics technology

The major aim of the present study was to investigate the proteome of standardbred horses at different stages of training and intensified training. We searched for biomarkers using small skeletal muscle biopsies of live animals. 2D gel electrophoresis and mass spectrometry were successfully applied to investigate training-induced differential expression of equine muscle biopsy proteins. Despite the poor resolution of the equine genome and proteome, we were able to identify the proteins of 20 differential spots representing 16 different proteins. Evaluation of those proteins complies with adaptation of the skeletal muscle after normal training involving structural changes towards a higher oxidative capacity, an increased capacity to take up long-chain fatty acids, and to store energy in the form of glycogen. Intensified training leads to additional changed spots. Alpha-1-antitrypsin was found increased after intensified training but not after normal training. This protein may thus be considered as a marker for overtraining in horses and also linked to overtraining in human athletes.     

Causes and consequences of oscillations in the cerebellar cortex

Cerebellar high-frequency oscillations have been observed for many decades, but their underlying mechanisms have remained enigmatic. In this issue of Neuron, two papers indicate that specific intrinsic mechanisms in the cerebellar cortex contribute to the generation of these oscillations. Middleton et al. show that GABA(A) receptor activation and nonchemical transmission are required for nicotine-dependent oscillations at 30-80 Hz and 80-160 Hz, respectively, while de Solages et al. provide evidence that recurrent inhibition by Purkinje cells is essential for oscillations around 200 Hz.     

Carbonisation and morphological changes in modern dehusked and husked Triticum dicoccum and Triticum aestivum grains

Modern Triticum dicoccum and Triticum aestivum grains, with and without glumes, were subjected to experimental carbonisation under anoxic conditions. Experimental variables were the presence or absence of glumes, temperature, exposure time and heating rate. The maximum temperature was 600°C, the time of exposure was 60 min and the heating rate between 1 and 100°C/min. Length, width, area, mass loss and reflectance of uncarbonised and carbonised grains were measured as a function of the variables. The main effects of charring are an increase in width, decrease in length and formation of protrusions. Reflectance measurements allow for the determination of the temperature at which carbonisation occurred. The occurrence of protrusions on the pericarp, longitudinal imprints in the pericarp and concave flanks are observed and discussed. The calculated shape factor 100L/W is a useful tool for distinguishing between T. dicoccum and T. aestivum grains in samples that contain at least thirty specimens, but for single grains this method is problematic.     

Effects of changing climate on species diversification in tropical forest butterflies of the genus Cymothoe (Lepidoptera: Nymphalidae)

Extant clades may differ greatly in their species richness, suggesting differential rates of species diversification. Based on phylogenetic trees, it is possible to identify potential correlates of such differences. Here, we examine species diversification in a clade of 82 tropical African forest butterfly species (Cymothoe), together with its monotypic sister genus Harma. Our aim was to test whether the diversification of the Harma–Cymothoe clade correlates with end‐Miocene global cooling and desiccation, or with Pleistocene habitat range oscillations, both postulated to have led to habitat fragmentation. We first generated a species‐level phylogenetic tree for Harma and Cymothoe, calibrated within an absolute time scale, and then identified temporal and phylogenetic shifts in species diversification. Finally, we assessed correlations between species diversification and reconstructed global temperatures. Results show that, after the divergence of Harma and Cymothoe in the Miocene (15 Mya), net species diversification was low during the first 7 Myr. Coinciding with the onset of diversification of Cymothoe around 7.5 Mya, there was a sharp and significant increase in diversification rate, suggesting a rapid radiation, and correlating with a reconstructed period of global cooling and desiccation in the late Miocene, rather than with Pleistocene oscillations. Our estimated age of 4 Myr for a clade of montane species corresponds well with the uplift of the Eastern Arc Mountains where they occur. We conclude that forest fragmentation caused by changing climate in the late Miocene as well as the Eastern Arc Mountain uplift are both likely to have promoted species diversification in the Harma–Cymothoe clade. Cymothoe colonized Madagascar much later than most other insect lineages and, consequently, had less time available for diversification on the island. We consider the diversification of Cymothoe to be a special case compared with other butterfly clades studied so far, both in terms of its abrupt diversification rate increase and its recent occurrence (7 Myr). It is clear that larval host plant shift(s) cannot explain the difference in diversification between Cymothoe and Harma; however, such a shift(s) may have triggered differential diversification rates within Cymothoe. © 2013 The Linnean Society of London, Biological Journal of the Linnean Society, 2013, ●● , ●●–●●.     

Reevaluating the role of LTD in cerebellar motor learning

Long-term depression at parallel fiber-Purkinje cell synapses (PF-PC LTD) has been proposed to be required for cerebellar motor learning. To date, tests of this hypothesis have sought to interfere with receptors (mGluR1) and enzymes (PKC, PKG, or αCamKII) necessary for induction of PF-PC LTD and thereby determine if cerebellar motor learning is impaired. Here, we tested three mutant mice that target the expression of PF-PC LTD by blocking internalization of AMPA receptors. Using three different cerebellar coordination tasks (adaptation of the vestibulo-ocular reflex, eyeblink conditioning, and locomotion learning on the Erasmus Ladder), we show that there is no motor learning impairment in these mutant mice that lack PF-PC LTD. These findings demonstrate that PF-PC LTD is not essential for cerebellar motor learning.     

Anti-inflammatory actions of acupuncture

Acupuncture has a beneficial effect when treating many diseases and painful conditions, and therefore is thought to be useful as a complementary therapy or to replace generally accepted pharmacological intervention. The attributive effect of acupuncture has been investigated in inflammatory diseases, including asthma, rhinitis, inflammatory bowel disease, rheumatoid arthritis, epicondylitis, complex regional pain syndrome type 1 and vasculitis. Large randomised trials demonstrating the immediate and sustained effect of acupuncture are missing. Mechanisms underlying the ascribed immunosuppressive actions of acupuncture are reviewed in this communication. The acupuncture-controlled release of neuropeptides from nerve endings and subsequent vasodilative and anti-inflammatory effects through calcitonine gene-related peptide is hypothesised. The complex interactions with substance P, the analgesic contribution of beta-endorphin and the balance between cell-specific pro-inflammatory and anti-inflammatory cytokines tumour necrosis factor-alpha and interleukin-10 are discussed.     

GPS Based Daily Activity Patterns in European Red Deer and North American Elk (Cervus elaphus): Indication for a Weak Circadian Clock in Ungulates

Long-term tracking using global positioning systems (GPS) is widely used to study vertebrate movement ecology, including fine-scale habitat selection as well as large-scale migrations. These data have the potential to provide much more information about the behavior and ecology of wild vertebrates: here we explore the potential of using GPS datasets to assess timing of activity in a chronobiological context. We compared two different populations of deer (Cervus elaphus), one in the Netherlands (red deer), the other in Canada (elk). GPS tracking data were used to calculate the speed of the animals as a measure for activity to deduce unbiased daily activity rhythms over prolonged periods of time. Speed proved a valid measure for activity, this being validated by comparing GPS based activity data with head movements recorded by activity sensors, and the use of GPS locations was effective for generating long term chronobiological data. Deer showed crepuscular activity rhythms with activity peaks at sunrise (the Netherlands) or after sunrise (Canada) and at the end of civil twilight at dusk. The deer in Canada were mostly diurnal while the deer in the Netherlands were mostly nocturnal. On an annual scale, Canadian deer were more active during the summer months while deer in the Netherlands were more active during winter. We suggest that these differences were mainly driven by human disturbance (on a daily scale) and local weather (on an annual scale). In both populations, the crepuscular activity peaks in the morning and evening showed a stable timing relative to dawn and dusk twilight throughout the year, but marked periods of daily a-rhythmicity occurred in the individual records. We suggest that this might indicate that (changes in) light levels around twilight elicit a direct behavioral response while the contribution of an internal circadian timing mechanism might be weak or even absent.     

Positive selection in phytotoxic protein-encoding genes of Botrytis species

Evolutionary patterns of sequence divergence were analyzed in genes from the fungal genus Botrytis (Ascomycota), encoding phytotoxic proteins homologous to a necrosis and ethylene-inducing protein from Fusarium oxysporum. Fragments of two paralogous genes (designated NEP1 and NEP2) were amplified from all known Botrytis species and sequenced. NEP1 sequences of two Botrytis species contain premature stop codons, indicating that they may be non-functional. Both paralogs of all species encode proteins with a remarkably similar predicted secondary structure, however, they contain different types of post-translational modification motifs, which are conserved across the genus. While both NEP genes are, overall, under purifying selection, we identified a number of amino acids under positive selection based on inference using maximum likelihood models. Positively selected amino acids in NEP1 were not under selection in corresponding positions in NEP2. The biological significance of positively selected residues and the role of NEP proteins in pathogenesis remain to be resolved.     

Matrix-M Adjuvated Seasonal Virosomal Influenza Vaccine Induces Partial Protection in Mice and Ferrets against Avian H5 and H7 Challenge

There is a constant threat of zoonotic influenza viruses causing a pandemic outbreak in humans. It is virtually impossible to predict which virus strain will cause the next pandemic and it takes a considerable amount of time before a safe and effective vaccine will be available once a pandemic occurs. In addition, development of pandemic vaccines is hampered by the generally poor immunogenicity of avian influenza viruses in humans. An effective pre-pandemic vaccine is therefore required as a first line of defense. Broadening of the protective efficacy of current seasonal vaccines by adding an adjuvant may be a way to provide such first line of defense. Here we evaluate whether a seasonal trivalent virosomal vaccine (TVV) adjuvated with the saponin-based adjuvant Matrix-M (MM) can confer protection against avian influenza H5 and H7 virus strains in mice and ferrets. We demonstrate that mice were protected from death against challenges with H5N1 and H7N7, but that the protection was not complete as evidenced by severe clinical signs. In ferrets, protection against H7N9 was not observed. In contrast, reduced upper and lower respiratory tract viral loads and reduced lung pathology, was achieved in H5N1 challenged ferrets. Together these results suggest that, at least to some extent, Matrix-M adjuvated seasonal virosomal influenza vaccine can serve as an interim measure to decrease morbidity and mortality associated with a pandemic outbreak.     

A natural substrate-based fluorescence assay for inhibitor screening on diacylglycerol lipase α

The endocannabinoid 2-arachidonoylglycerol (2-AG) is predominantly biosynthesized by sn-1-diacylglycerol lipase α (DAGL-α) in the CNS. Selective inhibitors of DAGL-α will provide valuable insights in the role of 2-AG in endocannabinoid signaling processes and are potential therapeutics for the treatment of obesity and neurodegenerative diseases. Here, we describe the development of a natural substrate-based fluorescence assay for DAGL-α, using a coupled enzyme approach. The continuous setup of our assay allows monitoring of DAGL-α activity in real-time and in a 96-well plate format. This constitutes a major improvement to the currently available radiometric and LC/MS-based methods, which can be executed only in low-throughput formats. In addition, our assay circumvents the use of radioactive material. We demonstrate that our assay can be used to screen inhibitors of DAGL-α activity, using 1-stearoyl-2-arachidonoyl-sn-glycerol as the physiologically relevant natural substrate of DAGL-α. Furthermore, our method can be employed to measure DAGL activity and inhibition in the mouse brain membrane proteome. Consequently, our assay should serve as a valuable tool for rapid hit validation and lead optimization of DAGL-α inhibitors.