Genetic variation in Plethodon cinereus and Plethodon hubrichti from in and around a contact zone

Climate change poses several challenges to biological communities including changes in the frequency of encounters between closely related congeners as a result of range shifts. When climate change leads to increased hybridization, hybrid dysfunction or genetic swamping may increase extinction risk—particularly in range‐restricted species with low vagility. The Peaks of Otter Salamander, Plethodon hubrichti, is a fully terrestrial woodland salamander that is restricted to ~18 km of ridgeline in the mountains of southwestern Virginia, and its range is surrounded by the abundant and widespread Eastern Red‐backed Salamander, Plethodon cinereus. In order to determine whether these two species are hybridizing and how their range limits may be shifting, we assessed variation at eight microsatellite loci and a 1,008 bp region of Cytochrome B in both species at allopatric reference sites and within a contact zone. Our results show that hybridization between P. hubrichti and P. cinereus either does not occur or is very rare. However, we find that diversity and differentiation are substantially higher in the mountaintop endemic P. hubrichti than in the widespread P. cinereus, despite similar movement ability for the two species as assessed by a homing experiment. Furthermore, estimation of divergence times between reference and contact zone populations via approximate Bayesian computation is consistent with the idea that P. cinereus has expanded into the range of P. hubrichti. Given the apparent recent colonization of the contact zone by P. cinereus, future monitoring of P. cinereus range limits should be a priority for the management of P. hubrichti populations.     

Dynamics underlying hydroxylation selectivity of cytochrome P450cam

Structural heterogeneity and the dynamics of the complexes of enzymes with substrates can determine the selectivity of catalysis; however, fully characterizing how remains challenging as heterogeneity and dynamics can vary at the spatial level of an amino acid residue and involve rapid timescales. We demonstrate the nascent approach of site-specific two-dimensional infrared (IR) spectroscopy to investigate the archetypical cytochrome P450, P450cam, to better delineate the mechanism of the lower regioselectivity of hydroxylation of the substrate norcamphor in comparison to the native substrate camphor. Specific locations are targeted throughout the enzyme by selectively introducing cyano groups that have frequencies in a spectrally isolated region of the protein IR spectrum as local vibrational probes. Linear and two-dimensional IR spectroscopy were applied to measure the heterogeneity and dynamics at each probe and investigate how they differentiate camphor and norcamphor recognition. The IR data indicate that the norcamphor complex does not fully induce a large-scale conformational change to a closed state of the enzyme adopted in the camphor complex. Additionally, a probe directed at the bound substrate experiences rapidly interconverting states in the norcamphor complex that explain the hydroxylation product distribution. Altogether, the study reveals large- and small-scale structural heterogeneity and dynamics that could contribute to selectivity of a cytochrome P450 and illustrates the approach of site-selective IR spectroscopy to elucidate protein dynamics.     

Correlative Imaging of Motoneuronal Cell Elasticity by Pump and Probe Spectroscopy

Because of their role of information transmitter between the spinal cord and the muscle fibers, motor neurons are subject to physical stimulation and mechanical property modifications. We report on motoneuron elasticity investigated by time-resolved pump and probe spectroscopy. A dual picosecond geometry simultaneously probing the acoustic impedance mismatch at the cell-titanium transducer interface and acoustic wave propagation inside the motoneuron is presented. Such noncontact and nondestructive microscopy, correlated to standard atomic force microscopy or a fluorescent labels approach, has been carried out on a single cell to address some physical properties such as bulk modulus of elasticity, dynamical longitudinal viscosity, and adhesion.     

Characterization of binding kinetics of A2AR to Gαs protein by surface plasmon resonance

Because of their surface localization, G protein-coupled receptors (GPCRs) are often pharmaceutical targets as they respond to a variety of extracellular stimuli (e.g., light, hormones, small molecules) that may activate or inhibit a downstream signaling response. The adenosine A_2A receptor (A_2AR) is a well-characterized GPCR that is expressed widely throughout the human body, with over 10 crystal structures determined. Truncation of the A_2AR C-terminus is necessary for crystallization as this portion of the receptor is long and unstructured; however, previous work suggests shortening of the A_2AR C-terminus from 412 to 316 amino acids (A_2AΔ316R) ablates downstream signaling, as measured by cAMP production, to below that of constitutive full-length A_2AR levels. As cAMP production is downstream of the first activation event—coupling of G protein to its receptor—investigating that first step in activation is important in understanding how the truncation effects native GPCR function. Here, using purified receptor and Gα_s proteins, we characterize the association of A_2AR and A_2AΔ316R to Gα_s with and without GDP or GTPγs using surface plasmon resonance (SPR). Gα_s affinity for A_2AR was greatest for apo-Gα_s, moderately affected in the presence of GDP and nearly completely ablated by the addition of GTPγs. Truncation of the A_2AR C-terminus (A_2AΔ316R) decreased the affinity of the unliganded receptor for Gα_s by ～20%, suggesting small changes to binding can greatly impact downstream signaling.     

Modeling Cumulative Effects of Climate and Development on Moose,Wolf, and Caribou Populations

Wildlife models focused solely on a single strong influence (e.g., habitat components, wildlife harvest) are limited in their ability to detect key mechanisms influencing population change. Instead, we propose integrated modeling in the context of cumulative effects assessment using multispecies population dynamics models linked to landscape-climate simulation at large spatial and temporal scales. We developed an integrated landscape and population simulation model using ALCES Online as the model-building platform, and the model accounted for key ecological components and relationships among moose (Alces alces), grey wolves (Canis lupus nubilus), and woodland caribou (Rangifer tarandus caribou) in northern Ontario, Canada. We simulated multiple scenarios over 5 decades (beginning 2020) to explore sensitivity to climate change and land use and assessed effects at multiple scales. The magnitude of effect and the relative importance of key factors (climate change, roads, and habitat) differed depending on the scale of assessment. Across the full extent of the study area (654,311km² [ecozonal scale]), the caribou population declined by 26% largely because of climate change and associated predator-prey response, which led to caribou range recession in the southern part of the study area. At the caribou range scale (108,378 km²), which focused on 2 herds in the northern part of the study area, climate change led to a 10% decline in the population and development led to an additional 7% decline. At the project scale (8,331 km²), which was focused more narrowly on the landscape surrounding 4 proposed mines, the caribou population declined by 29% largely in response to simulated development. Given that observed caribou population dynamics were sensitive to the cumulative effects of climate change, land use, interspecific interactions, and scale, insights from the analysis might not emerge under a less complex model. Our integrated modeling framework provides valuable support for broader regional assessments, including estimation of risk to caribou and Indigenous food security, and for developing and evaluating potential caribou recovery strategies. © 2021 The Authors. The Journal of Wildlife Management published by Wiley Periodicals LLC on behalf of The Wildlife Society.