Development of an in vitro method for activation of X-succinate synthases for fumarate hydroalkylation.

Anaerobic microbial degradation of hydrocarbons is often initiated through addition of the hydrocarbon to fumarate by enzymes known as X-succinate synthases (XSSs). XSSs use a glycyl radical cofactor, which is installed by an activating enzyme (XSS-AE), to catalyze this carbon-carbon coupling reaction. The activation step, although crucial for catalysis, has not previously been possible in vitro because of insolubility of XSS-AEs. Here, we take a genome mining approach to find an XSS-AE, a 4-isopropylbenzylsuccinate synthase (IBSS)-AE (IbsAE) that can be solubly expressed in Escherichia coli. This soluble XSS-AE can activate both IBSS and the well-studied benzylsuccinate synthase (BSS) in vitro, allowing us to explore XSSs biochemically. To start, we examine the role of BSS subunits and find that the beta subunit accelerates the rate of hydrocarbon addition. Looking forward, the methodology and insight gathered here can be used more broadly to understand and engineer XSSs as synthetically useful biocatalysts.

Fear of the human 'super predator' reduces feeding time in large carnivores.

Large carnivores' fear of the human 'super predator' has the potential to alter their feeding behaviour and result in human-induced trophic cascades. However, it has yet to be experimentally tested if large carnivores perceive humans as predators and react strongly enough to have cascading effects on their prey. We conducted a predator playback experiment exposing pumas to predator (human) and non-predator control (frog) sounds at puma feeding sites to measure immediate fear responses to humans and the subsequent impacts on feeding. We found that pumas fled more frequently, took longer to return, and reduced their overall feeding time by more than half in response to hearing the human 'super predator'. Combined with our previous work showing higher kill rates of deer in more urbanized landscapes, this study reveals that fear is the mechanism driving an ecological cascade from humans to increased puma predation on deer. By demonstrating that the fear of humans can cause a strong reduction in feeding by pumas, our results support that non-consumptive forms of human disturbance may alter the ecological role of large carnivores.

Fear of large carnivores causes a trophic cascade.

The fear large carnivores inspire, independent of their direct killing of prey, may itself cause cascading effects down food webs potentially critical for conserving ecosystem function, particularly by affecting large herbivores and mesocarnivores. However, the evidence of this has been repeatedly challenged because it remains experimentally untested. Here we show that experimentally manipulating fear itself in free-living mesocarnivore (raccoon) populations using month-long playbacks of large carnivore vocalizations caused just such cascading effects, reducing mesocarnivore foraging to the benefit of the mesocarnivore's prey, which in turn affected a competitor and prey of the mesocarnivore's prey. We further report that by experimentally restoring the fear of large carnivores in our study system, where most large carnivores have been extirpated, we succeeded in reversing this mesocarnivore's impacts. We suggest that our results reinforce the need to conserve large carnivores given the significant "ecosystem service" the fear of them provides.

Accessory muscle activation during the superimposed burst technique.

Quadriceps muscle activation is assessed using the superimposed burst technique. This technique involves percutaneous muscle stimulation superimposed during maximal isometric volitional knee extension. It is unknown whether accessory muscle activation during maximal knee extension influences estimates of quadriceps muscle activation. Our aim was to compare accessory muscle activation while performing the superimposed burst technique using investigator delivered verbal instruction to constrain the system (CS) and a participant preferred (PP) technique. Twenty five healthy, active individuals (13M/12F, age=23.8 ± 3.35, height=72.73 ± 14.51 cm, and weight=175.29 ± 9.59 kg) were recruited for this study. All participants performed superimposed burst testing with (CS) and without (PP) verbal instruction to encourage isolated quadriceps activation during maximal isometric knee extension. The main outcome variables measured were knee extension torque, quadriceps central activation ratio and mean EMG of vastus lateralis, biceps femoris, and lumbar paraspinal muscles. There were significant differences in knee extension torque (CS=2.87 ± 0.93 Nm/kg, PP=3.40 ± 1.12 Nm/kg, p<0.001), superimposed burst torque (CS=3.40 ±0.98 Nm/kg, PP=3.75 ± 1.11 Nm/kg, p=0.002) and quadriceps CAR (CS=84.1 ± 12.0%, PP=90.2 ± 9.9%, p<0.001) between the techniques. There was also a significant difference in lumbar paraspinal EMG (CS=6.40 ± 8.52%, PP=11.86 ± 14.89%, p=0.043) between the techniques however vastus lateralis EMG was not significantly different. Patient instruction via verbal instruction to constrain proximal structures may help patient minimize confounders to knee extension torque generation while maximizing quadriceps activation.