In Vitro Selection of Short DNA Aptamers that Can Inhibit or Alleviate Cocaine and MK-801 Inhibition of Muscle-Type Nicotinic Acetylcholine Receptors.

Ligands of high specificity and selectivity have been selected for biological molecules of interest including nicotinic acetylcholine receptor (nAChR) using combinatorial libraries of nucleic acids. The nAChR belongs to a group of structurally related proteins that regulate signal transmission between ~ 1012 cells of the mammalian nervous system. It is inhibited by both therapeutic agents and abused drugs, including cocaine. A mechanism-based approach to alleviating noncompetitive inhibition of the mucle-type nAChR, including Torpedo, resulted in the selection of very short DNA aptamers only seven nucleotides long. By transient kinetic measurements, these DNA aptamers, which displaced cocaine from its binding site on the muscle-type nAChR, were classified into two groups based on their effects on the nAChR: Class I aptamers inhibit agonist-induced current in the muscle-type nAChR and Class II molecules alleviate inhibition by MK-801 [(+)-dizocilpine] without affecting the receptor function. The most potent Class I DNA aptamer, which inhibits the muscle-type nAChR, has an apparent dissociation constant (KIapt) of 5 µM, while the most efficient Class II DNA aptamer, which alleviates MK-801-induced inhibition, has an apparent dissociation constant (KApt) of 1.8 µM. An innovative aspect of the work is the identification of very short DNA aptamers with these properties that makes them attractive for therapeutic and diagnostic applications.

Metal contamination of river otters in North Carolina.

Aquatic apex predators are vulnerable to environmental contaminants due to biomagnification. North American river otter (Lontra canadensis) populations should be closely monitored across their range due to point and nonpoint pollution sources. Nonetheless, no information exists on environmental contaminants in the North Carolina otter population. Metals and metalloids occur naturally across the landscape, are essential for cellular function, and become toxic when concentrated unnaturally. We conducted our study across the three Furbearer Management Units (FMU) and 14 river basins of North Carolina. We determined the concentrations of arsenic, cadmium, calcium, cobalt, copper, iron, lead, magnesium, manganese, mercury, molybdenum, selenium, thallium, and zinc in liver and kidney samples from 317 otters harvested from 2009 to 2016. Arsenic, lead, and thallium samples were tested at levels below the limit of detection. With the exception of cadmium, we detected all other elements at higher levels in the liver compared with the kidney. Specifically, cadmium, cobalt, copper, iron, magnesium, manganese, mercury, molybdenum, and zinc levels differed by tissue type analyzed. Most element concentrations remained stable or increased with otter age. We detected higher levels of mercury and selenium in the Lower Pee Dee and Cape Fear river basins. River basins within the Mountain FMU were higher in cadmium, copper, iron, lead, and zinc, whereas the Coastal Plain FMU was lower in cobalt and manganese. None of the elements occurred at toxic levels. Our research establishes baseline concentration levels for North Carolina, which will benefit future monitoring efforts and provide insight into future changes in the otter population.

Beyond Impervious: Urban Land-Cover Pattern Variation and Implications for Watershed Management.

Impervious surfaces degrade urban water quality, but their over-coverage has not explained the persistent water quality variation observed among catchments with similar rates of imperviousness. Land-cover patterns likely explain much of this variation, although little is known about how they vary among watersheds. Our goal was to analyze a series of urban catchments within a range of impervious cover to evaluate how land-cover varies among them. We then highlight examples from the literature to explore the potential effects of land-cover pattern variability for urban watershed management. High-resolution (1 m(2)) land-cover data were used to quantify 23 land-cover pattern and stormwater infrastructure metrics within 32 catchments across the Triangle Region of North Carolina. These metrics were used to analyze variability in land-cover patterns among the study catchments. We used hierarchical clustering to organize the catchments into four groups, each with a distinct landscape pattern. Among these groups, the connectivity of combined land-cover patches accounted for 40 %, and the size and shape of lawns and buildings accounted for 20 %, of the overall variation in land-cover patterns among catchments. Storm water infrastructure metrics accounted for 8 % of the remaining variation. Our analysis demonstrates that land-cover patterns do vary among urban catchments, and that trees and grass (lawns) are divergent cover types in urban systems. The complex interactions among land-covers have several direct implications for the ongoing management of urban watersheds.

Solution and displacement in monolayer and multilayer binary films of SF6 and CF4 on graphite.

Infrared reflection absorption spectroscopy is used to study the evolution of binary physisorbed films on graphite. A predeposited monolayer of SF6 is exposed to slowly increasing pressure of CF4 at constant temperature between 80 and 113 K. Shifts in the frequencies of the dominant vibrational mode of each species due to resonant dipole-dipole coupling serve as proxies for the areal density of each species in the monolayer. If the initial SF6 film is far below saturation (coexistence with bulk solid), the SF6 can be largely displaced by continuous solution of CF4. However, if the initial SF6 layer is at or near saturation, a layer of CF4 condenses on top at a well defined CF4 pressure after only 2%-3% dilution of the SF6 layer. Simultaneously, most of the dissolved CF4 is withdrawn from the SF6 layer. With further increase in CF4 pressure, the CF4 layer is compressed and additional layers condense, while the SF6 layer is again diluted. Still, the SF6 layer retains about 90% concentration until the CF4 pressure is very close to saturation, at which point the SF6 is rapidly displaced, apparently going into dilute solution in the rapidly growing CF4 multilayer. Monte Carlo simulations are used to quantitatively relate measured frequency shifts to concentrations in the binary monolayer.

Phase diagram of the CF4 monolayer and bilayer on graphite.

We report an experimental study of physisorbed monolayers and bilayers of CF4 on graphite using infrared reflection absorption spectroscopy supplemented by ellipsometry. The symmetric C-F stretch mode ν3 near 1283 cm(-1) in the gas is strongly blue shifted in the film by dynamic dipole coupling. This blue shift provides a very sensitive measure of the inter-molecular spacing in the monolayer and, less directly, in the bilayer. We find that important corrections are necessary to the volumetric coverage scales used in previous heat capacity and x-ray diffraction studies of this system. This requires quantitative and some qualitative changes to the previously proposed phase diagram. We find evidence for a new phase transition in the middle of the hexagonal incommensurate region and construct new phase diagrams in both the variables coverage-temperature and chemical potential-temperature. We determine the compressibility and thermal expansion in the low-pressure hexagonal incommensurate phase and values for the entropy change in several phase transitions. Below about 55 K there is evidence of solution of up to 7% of an impurity, most likely CO, in our monolayer but not the bilayer film.

Land trust evaluation of progress toward conservation goals.

In the United States local land trusts preserve and conserve land to protect water quality and supply, farmlands, habitat for native plants and animals, areas of cultural or historical significance, and scenic views. We surveyed the 24 active, local land trusts in North Carolina (U.S.A.) to determine how they report progress toward attaining such conservation goals. Twenty-two land trusts responded to our survey. Of these, eight reported developing specific conservation goals for all of the properties they protect, five identified conservation targets on all properties, and two reported monitoring biological indicators on all of their protected properties. On the basis of these results, we believe most of the land trusts surveyed could not determine whether they were meeting conservation goals because most did not identify explicit, measurable goals and monitor progress toward them. Instead they reported success in terms of the amount of land protected and money raised. We think this is a lost opportunity for land trusts to build sound approaches to environmental management, engage the public, entice new donors, and attain funding for additional conservation activities. We propose conservation professionals help local land trusts adopt the Open Standards for the Practice of Conservation, a framework developed by a consortium of international conservation organizations, to develop conservation goals and measure whether the goals are achieved.

SNAPSHOT USA 2020: A second coordinated national camera trap survey of the United States during the COVID-19 pandemic.

Managing wildlife populations in the face of global change requires regular data on the abundance and distribution of wild animals, but acquiring these over appropriate spatial scales in a sustainable way has proven challenging. Here we present the data from Snapshot USA 2020, a second annual national mammal survey of the USA. This project involved 152 scientists setting camera traps in a standardized protocol at 1485 locations across 103 arrays in 43 states for a total of 52,710 trap-nights of survey effort. Most (58) of these arrays were also sampled during the same months (September and October) in 2019, providing a direct comparison of animal populations in 2 years that includes data from both during and before the COVID-19 pandemic. All data were managed by the eMammal system, with all species identifications checked by at least two reviewers. In total, we recorded 117,415 detections of 78 species of wild mammals, 9236 detections of at least 43 species of birds, 15,851 detections of six domestic animals and 23,825 detections of humans or their vehicles. Spatial differences across arrays explained more variation in the relative abundance than temporal variation across years for all 38 species modeled, although there are examples of significant site-level differences among years for many species. Temporal results show how species allocate their time and can be used to study species interactions, including between humans and wildlife. These data provide a snapshot of the mammal community of the USA for 2020 and will be useful for exploring the drivers of spatial and temporal changes in relative abundance and distribution, and the impacts of species interactions on daily activity patterns. There are no copyright restrictions, and please cite this paper when using these data, or a subset of these data, for publication.

Adsorption of CF4 on graphite preplated with a monolayer of CF3Cl.

We report a study of the adsorption of CF(4) on graphite preplated with a monolayer of CF(3)Cl, using infrared reflection absorption spectroscopy combined with ellipsometry. The saturated vapor pressure of CF(3)Cl is nearly 3 orders of magnitude smaller than that of CF(4) at the same temperature, so the main control variables are the temperature and the pressure (or chemical potential) of CF(4), together with the initial coverage of CF(3)Cl. The temperature range covered is 60-105 K. We find that, if the initial monolayer of CF(3)Cl is liquid, CF(4) continuously displaces CF(3)Cl by substitution in the monolayer. If the initial monolayer of CF(3)Cl is solid, due to either lower temperature or compression, CF(4) condenses as a second layer on the top of the CF(3)Cl layer, with only slight mixing with the original layer. This behavior persists to multiple layers of CF(4).

Carolina critters: a collection of camera-trap data from wildlife surveys across North Carolina.

Camera trap surveys are useful to understand animal species population trends, distribution, habitat preference, behavior, community dynamics, periods of activity, and species associations with environmental conditions. This information is ecologically important, because many species play important roles in local ecosystems as predators, herbivores, seed dispersers, and disease vectors. Additionally, many of the larger wildlife species detected by camera traps are economically important through hunting, trapping, or ecotourism. Here we present a data set of camera-trap surveys from 6,043 locations across all 100 counties of North Carolina, USA from 2009 to 2019. These data come from 26 survey initiatives and contain 215,108 records of 36 mammal species and three species of terrestrial birds. This large data set increases the geographical distribution data for these 39 mammal and bird species by >500% over what is available for North Carolina in the Global Biodiversity Information Facility (GBIF). These data can be used to conduct inquiries about species, populations, communities, or ecosystems, and to produce useful information on wildlife behavior, distribution, and interactions. There are no copyright restrictions. Please cite this paper when using the data for publication.

SNAPSHOT USA 2019: a coordinated national camera trap survey of the United States.

With the accelerating pace of global change, it is imperative that we obtain rapid inventories of the status and distribution of wildlife for ecological inferences and conservation planning. To address this challenge, we launched the SNAPSHOT USA project, a collaborative survey of terrestrial wildlife populations using camera traps across the United States. For our first annual survey, we compiled data across all 50 states during a 14-week period (17 August-24 November of 2019). We sampled wildlife at 1,509 camera trap sites from 110 camera trap arrays covering 12 different ecoregions across four development zones. This effort resulted in 166,036 unique detections of 83 species of mammals and 17 species of birds. All images were processed through the Smithsonian's eMammal camera trap data repository and included an expert review phase to ensure taxonomic accuracy of data, resulting in each picture being reviewed at least twice. The results represent a timely and standardized camera trap survey of the United States. All of the 2019 survey data are made available herein. We are currently repeating surveys in fall 2020, opening up the opportunity to other institutions and cooperators to expand coverage of all the urban-wild gradients and ecophysiographic regions of the country. Future data will be available as the database is updated at eMammal.si.edu/snapshot-usa, as will future data paper submissions. These data will be useful for local and macroecological research including the examination of community assembly, effects of environmental and anthropogenic landscape variables, effects of fragmentation and extinction debt dynamics, as well as species-specific population dynamics and conservation action plans. There are no copyright restrictions; please cite this paper when using the data for publication.

Mechanism of potentiation of a dysfunctional epilepsy-linked mutated GABA(A) receptor by a neurosteroid (3alpha, 21-dihydroxy-5alpha-pregnan-20-one): transient kinetic investigations.

The malfunction of a mutated GABA(A) receptor (alpha1beta2gamma2L(K289M)) in an inheritable form of epilepsy (GEFS+, generalized epilepsy with febrile seizures plus) in humans [Baulac, S., Huberfeld, G., Gourfinkel-An, I., Mitropoulou, G., Beranger, A., Prud'homme, J. F., Baulac, M., Brice, A., Bruzzone, R., and LeGuern, E. (2001) Nat. Genet. 28, 46-48] has been accounted for by a 5-fold decrease in the channel-opening equilibrium of the mutated receptor compared to the wild type [Ramakrishnan, L., and Hess, G. P. (2004) Biochemistry 43, 7534-7540]. Here we describe the mechanism by which the neurosteroid 3alpha, 21-dihydroxy-5alpha-pregnan-20-one (5alpha-THDOC) alleviates this malfunction of the mutated receptor transiently expressed in HEK293 cells. Two rapid reaction techniques, the cell-flow and the laser-pulse photolysis methods, were used in combination with whole-cell current recordings. 150-muM 5alpha-THDOC does not affect the rate constant for channel opening (k(op)) of approximately 250 s(-1) but does decrease the rate constant for channel closing (k(cl)) from 121 +/- 11 s(-1) to 56 +/- 21 s(-1). This results in an increase in the channel-opening equilibrium constant ((Phi(-1) = k(op)/k(cl)) by a factor of about 2, leading to about 50% alleviation of the malfunction of the inheritable mutated (alpha1beta2gamma2L(K289M)) GABA(A) receptor linked to GEFS+.

Dihydropyrimidinone positive modulation of delta-subunit-containing gamma-aminobutyric acid type A receptors, including an epilepsy-linked mutant variant.

Gamma-aminobutyric acid type A receptors (GABA(A) receptors) are ligand-gated chloride channels that play a central role in signal transmission within the mammalian central nervous system. Compounds that modulate specific GABA(A) receptor subtypes containing the delta-subunit are scarce but would be valuable research tools and starting points for potential therapeutic agents. Here we report a class of dihydropyrimidinone (DHPM) heterocycles that preferentially potentiate peak currents of recombinant GABA(A) receptor subtypes containing the delta-subunit expressed in HEK293T cells. Using the three-component Biginelli reaction, 13 DHPMs with structural features similar to those of the barbiturate phenobarbital were synthesized; one DHPM used (monastrol) is commercially available. An up to approximately 3-fold increase in the current from recombinant alpha1beta2delta receptors was observed with the DHPM compound JM-II-43A or monastrol when co-applied with saturating GABA concentrations, similar to the current potentiation observed with the nonselective potentiating compounds phenobarbital and tracazolate. No agonist activity was observed for the DHPMs at the concentrations tested. A kinetic model was used in conjunction with dose-dependent measurements to calculate apparent dissociation constant values for JM-II-43A (400 muM) and monastrol (200 microM) at saturating GABA concentrations. We examined recombinant receptors composed of combinations of subunits alpha1, alpha4, alpha5, alpha6, beta2, beta3, gamma2L, and delta with JM-II-43A to demonstrate the preference for potentiation of delta-subunit-containing receptors. Lastly, reduced currents from receptors containing the mutated delta(E177A) subunit, described by Dibbens et al. [(2004) Hum. Mol. Genet. 13, 1315-1319] as a heritable susceptibility allele for generalized epilepsy with febrile seizures plus, are also potentiated by these DHPMs.

Minimal RNA aptamer sequences that can inhibit or alleviate noncompetitive inhibition of the muscle-type nicotinic acetylcholine receptor.

Combinatorially synthesized nucleotide polymers have been used during the last decade to find ligands that bind to specific sites on biological molecules, including membrane-bound proteins such as the nicotinic acetylcholine receptors (nAChRs). The neurotransmitter receptors belong to a group of four structurally related proteins that regulate signal transmission between ~10(11) neurons of the mammalian nervous system. The nAChRs are inhibited by compounds such as the anticonvulsant MK-801 [(+)-dizocilpine] and abused drugs such as cocaine. Based on predictions arising from the mechanism of receptor inhibition by MK-801 and cocaine, we developed two classes of RNA aptamers: class I members, which inhibit the nAChR, and class II members, which alleviate inhibition of the receptor by MK-801 and cocaine. The systematic evolution of ligands by the exponential enrichment (SELEX) method was used to obtain these compounds. Here, we report that we have truncated RNA aptamers in each class to determine the minimal nucleic acid sequence that retains the characteristic function for which the aptamer was originally selected. We demonstrate that a truncated class I aptamer containing a sequence of seven nucleotides inhibits the nAChR and that a truncated class II aptamer containing a sequence of only four nucleotides can alleviate MK-801 inhibition.

Comparative proteomics profiling of a phospholamban mutant mouse model of dilated cardiomyopathy reveals progressive intracellular stress responses.

Defective mobilization of Ca2+ by cardiomyocytes can lead to cardiac insufficiency, but the causative mechanisms leading to congestive heart failure (HF) remain unclear. In the present study we performed exhaustive global proteomics surveys of cardiac ventricle isolated from a mouse model of cardiomyopathy overexpressing a phospholamban mutant, R9C (PLN-R9C), and exhibiting impaired Ca2+ handling and death at 24 weeks and compared them with normal control littermates. The relative expression patterns of 6190 high confidence proteins were monitored by shotgun tandem mass spectrometry at 8, 16, and 24 weeks of disease progression. Significant differential abundance of 593 proteins was detected. These proteins mapped to select biological pathways such as endoplasmic reticulum stress response, cytoskeletal remodeling, and apoptosis and included known biomarkers of HF (e.g. brain natriuretic peptide/atrial natriuretic factor and angiotensin-converting enzyme) and other indicators of presymptomatic functional impairment. These altered proteomic profiles were concordant with cognate mRNA patterns recorded in parallel using high density mRNA microarrays, and top candidates were validated by RT-PCR and Western blotting. Mapping of our highest ranked proteins against a human diseased explant and to available data sets indicated that many of these proteins could serve as markers of disease. Indeed we showed that several of these proteins are detectable in mouse and human plasma and display differential abundance in the plasma of diseased mice and affected patients. These results offer a systems-wide perspective of the dynamic maladaptions associated with impaired Ca2+ homeostasis that perturb myocyte function and ultimately converge to cause HF.

LEPTOSPIRA, PARVOVIRUS, AND TOXOPLASMA IN THE NORTH AMERICAN RIVER OTTER (LONTRA CANADENSIS) IN NORTH CAROLINA, USA.

The North American river otter (Lontra canadensis) is the largest mustelid in North Carolina, US, and was once extirpated from the central and western portions of the state. Over time and after a successful reintroduction project, otters are now abundant and occur throughout North Carolina. However, there is a concern that diseases may have an impact on the otter population, as well as on other aquatic mammals, either through exposure to emerging diseases, contact with domestic animals such as domestic cats (Felis catus), or less robust condition of individuals through declines in water quality. We tested brain and kidney tissue from harvested otters for the pathogens that cause leptospirosis, parvovirus, and toxoplasmosis. Leptospirosis and toxoplasmosis are priority zoonoses and are maintained by domestic and wild mammals. Although parvovirus is not zoonotic, it does affect pets, causing mild to fatal symptoms. Across the 2014-15 and 2015-16 trapping seasons, we tested 220 otters (76 females, 144 males) using real-time PCR for Leptospira interrogans, parvovirus, and Toxoplasma gondii. Of the otters tested, 1% (3/220) were positive for L. interrogans, 19% (41/220) were positive for parvovirus, and 24% (53/220) were positive for T. gondii. Although the pathogens for parvovirus and toxoplasmosis are relatively common in North Carolina otters, the otter harvest has remained steady and the population appears to be abundant and self-sustaining. Therefore, parvovirus and toxoplasmosis do not currently appear to be negatively impacting the population. However, subsequent research should examine transmission parameters between domestic and wild species and the sublethal effects of infection.

A new synthesis of caged GABA compounds for studying GABAA receptors.

A short and convergent synthetic approach to new photoactivatable precursors of gamma-aminobutyric acid (GABA) is described. When photolyzed, the 'caged' GABA precursor efficiently releases GABA, as judged by depolarization measurements on the mammalian GABA(A) receptor.

Biodiversity conservation in local planning.

Local land-use policy is increasingly being recognized as fundamental to biodiversity conservation in the United States. Many planners and conservation scientists have called for broader use of planning and regulatory tools to support the conservation of biodiversity at local scales. Yet little is known about the pervasiveness of these practices. We conducted an on-line survey of county, municipal, and tribal planning directors (n =116) in 3 geographic regions of the United States: metropolitan Seattle, Washington; metropolitan Des Moines, Iowa; and the Research Triangle, North Carolina. Our objectives were to gauge the extent to which local planning departments address biodiversity conservation and to identify factors that facilitate or hinder conservation actions in local planning. We found that biodiversity conservation was seldom a major consideration in these departments. Staff time was mainly devoted to development mandates and little time was spent on biodiversity conservation. Regulations requiring conservation actions that might benefit biodiversity were uncommon, with the exception of rules governing water quality in all 3 regions and the protection of threatened and endangered species in the Seattle region. Planning tools that could enhance habitat conservation were used infrequently. Collaboration across jurisdictions was widespread, but rarely focused on conservation. Departments with a conservation specialist on staff tended to be associated with higher levels of conservation actions. Jurisdictions in the Seattle region also reported higher levels of conservation action, largely driven by state and federal mandates. Increased funding was most frequently cited as a factor that would facilitate greater consideration of biodiversity in local planning. There are numerous opportunities for conservation biologists to play a role in improving conservation planning at local scales.

Study of SF6 adsorption on graphite using infrared spectroscopy.

We report an experimental study of adsorbed monolayers of SF(6) on graphite using infrared reflection absorption spectroscopy supplemented by ellipsometry. The asymmetric S-F stretch mode nu(3) near 948 cm(-1) in the gas is strongly blueshifted in the film by dynamic dipole coupling. This blueshift is very sensitive to the intermolecular spacing in the SF(6) layer. We convert the measured frequency nu(3) to a lattice spacing a, using a self-consistent field calculation, calibrated by the frequency in the commensurate phase. The resolution in lattice spacing is 0.002 A, although there is a larger systematic uncertainty associated with nondynamic-dipole contributions to the frequency shift. We map the commensurate-incommensurate transition, a transition between two incommensurate phases, and the melting transition. These results are compared to previous x-ray data. We provide a new determination of the layer critical point (156 K), the layer condensation line down to 110 K, and the spreading pressure at saturation in this temperature range.