Phosphoproteome Profiling of the Receptor Tyrosine Kinase MuSK Identifies Tyrosine Phosphorylation of Rab GTPases

Muscle-specific receptor tyrosine kinase (MuSK) agonist antibodies were developed 2 decades ago to explore the benefits of receptor activation at the neuromuscular junction. Unlike agrin, the endogenous agonist of MuSK, agonist antibodies function independently of its coreceptor low-density lipoprotein receptor–related protein 4 to delay the onset of muscle denervation in mouse models of ALS. Here, we performed dose–response and time-course experiments on myotubes to systematically compare site-specific phosphorylation downstream of each agonist. Remarkably, both agonists elicited similar intracellular responses at known and newly identified MuSK signaling components. Among these was inducible tyrosine phosphorylation of multiple Rab GTPases that was blocked by MuSK inhibition. Importantly, mutation of this site in Rab10 disrupts association with its effector proteins, molecule interacting with CasL 1/3. Together, these data provide in-depth characterization of MuSK signaling, describe two novel MuSK inhibitors, and expose phosphorylation of Rab GTPases downstream of receptor tyrosine kinase activation in myotubes.     

Improved Stability of Methanolic Wright's Stain with Additive Reagents

Additive reagents have been investigated to improve the stability of methanolic Wright's stain. The addition of ammonium halides, monoalkyiamine hydrochlorides, dialkylamine hydrochlorides or trialkylamine hydrochlorides to methanolic Wright's stain was found to enhance the stability of stain components in methanol. No change in performance is observed with these additives present. Random precipitation in the stain solution was still observed with the addition of ammonium halides and monoalkyiamine hydrochlorides. No precipitation was found in stain solutions containing hydrochlorides of most dialkylamines and trialkylamines. Of the compounds evaluated, 0.6% diethylamine hydrochloride added to methanolic stain solutions produced the most desirable overall results. Mechanisms of stabilization and precipitation in these stain solutions are proposed, Essentially, separation of the thiazine-eosinate ion pair through interaction with an appropriate additive increases stain stability. The solubilities of thiazine-eosinate or additive cation-eosinate ion pairs in methanol determine the formation of precipitate in such stain solutions.     

An order-specific monoclonal antibody to Diptera reveals the impact of alternative prey on spider feeding behavior in a complex food web

Generalist predators have the capacity to restrict pest population growth, especially early in the season before densities increase. However, their polyphagous feeding habits sometimes translate into reduced pest consumption when they target alternative prey. An order-specific monoclonal antibody was developed to examine the strength of trophic connections between Diptera, a major category of non-pest prey, and linyphiid spiders in alfalfa. We report the development and characterization of a monoclonal antibody with order-level specificity to Diptera. This antibody elicited strong absorbance to 22 Diptera from 13 families, no false-positive reactivity to non-dipteran invertebrates, and antigen detection periods following prey consumption that were comparable between spiders. Over 900 field-collected females of the linyphiid spiders Erigone autumnalis and Bathyphantes pallidus were screened for Diptera antigen. Significantly more B. pallidus screened positive for Diptera (40%) compared to E. autumnalis (16%), indicating differential reliance on these prey. In parallel with the collection of spiders for gut-content analysis, prey availability was estimated at web sites. The two spiders exhibited different feeding responses to prey availability. Consumption of Diptera by B. pallidus was strongly correlated with Diptera abundance whilst the availability of other potential prey did not influence predation rates. Conversely, E. autumnalis did not prey upon Diptera in proportion to availability, but increased Collembola activity-density reduced dipteran consumption. Integration of molecular gut-content analysis with precise sampling of prey demonstrated how two closely related linyphiid spiders exhibit different feeding responses to the availability of prey under natural field conditions. Elucidating the feeding preferences of natural enemies is critical to effective incorporation of biological control by generalist predators in the management of agricultural pests.     

Strength and physical fitness predict the perception of looming sounds

Listeners consistently perceive approaching sounds to be closer than they actually are and perceptually underestimate the time to arrival of looming sound sources. In a natural environment, this underestimation results in more time than expected to evade or engage the source and affords a “margin of safety” that may provide a selective advantage. However, a key component in the proposed evolutionary origins of the perceptual bias is the appropriate timing of anticipatory motor behaviors. Here we show that listeners with poorer physical fitness respond sooner to looming sounds and with a larger margin of safety than listeners with better physical fitness. The anticipatory perceptual bias for looming sounds is negatively correlated with physical strength and positively correlated with recovery heart rate (a measure of aerobic fitness). The results suggest that the auditory perception of looming sounds may be modulated by the response capacity of the motor system.     

Differetial sensitivity of spontaneously hypertensive rats to the hypothermic effect of dopaminergic drugs

The dopaminergic agonist apomorphine produces dose-related hypothermia in naive rats as does L-DOPA in carbidopa-pretreated rats. The hypothermic responses to these two dopaminergic drugs were significantly more pronounced and prolonged in the spontaneously hypertensive rat than in normotensive Wistar control rats. The greater sensitivity of the SHR to these drugs was reflected as a leftward shift of the dose-response curves for apomorphine- and L-DOPA-induced hypothermias.     

Seed germination,seedling inoculation and establishment ofAlnus spp. in containers in greenhouse trials

Methods for production of containerized seedlings ofAlnus species were developed which permit nitrogen-fixing nodules to form on the root systems prior to outplanting, in order to provide an early nitrogen input during seedling establishment. The methods are based on procedures for inoculating root systems with suspensions ofFrankia (Actinomycetales), applied either directly in the container cell as a soil drench at the time of seeding, or as a root dip for seedlings transplanted into the containers. Germination of dried, stored seed was enhanced by light and by presoaking for 16 h in water. Pretreatments to overcome seed dormancy or to eliminate fungal pathogens did not further enhance germination. Some loss of seedlings was recorded in the early stages of growth shortly after germination, which is a factor in calculating projected seedling yield. Nodulation and seedling growth were evaluated in terms of growth media characteristics. Seedlings performed well in peat-vermiculite, at soil pH between 5.5 and 8.0.     

Changes in the Gut Microbiome of the Sea Lamprey during Metamorphosis

Vertebrate metamorphosis is often marked by dramatic morphological and physiological changes of the alimentary tract, along with major shifts in diet following development from larva to adult. Little is known about how these developmental changes impact the gut microbiome of the host organism. The metamorphosis of the sea lamprey (Petromyzon marinus) from a sedentary filter-feeding larva to a free-swimming sanguivorous parasite is characterized by major physiological and morphological changes to all organ systems. The transformation of the alimentary canal includes closure of the larval esophagus and the physical isolation of the pharynx from the remainder of the gut, which results in a nonfeeding period that can last up to 8 months. To determine how the gut microbiome is affected by metamorphosis, the microbial communities of feeding and nonfeeding larval and parasitic sea lamprey were surveyed using both culture-dependent and -independent methods. Our results show that the gut of the filter-feeding larva contains a greater diversity of bacteria than that of the blood-feeding parasite, with the parasite gut being dominated by Aeromonas and, to a lesser extent, Citrobacter and Shewanella. Phylogenetic analysis of the culturable Aeromonas from both the larval and parasitic gut revealed that at least five distinct species were represented. Phenotypic characterization of these isolates revealed that over half were capable of sheep red blood cell hemolysis, but all were capable of trout red blood cell hemolysis. This suggests that the enrichment of Aeromonas that accompanies metamorphosis is likely related to the sanguivorous lifestyle of the parasitic sea lamprey.