Towards conserving natural diversity: A biotic inventory by observations, specimens, DNA barcoding and high-throughput sequencing methods.

The Kenai National Wildlife Refuge has been given a broad conservation mandate to conserve natural diversity. A prerequisite for fulfilling this purpose is to be able to identify the species and communities that make up that biodiversity. We tested a set of varied methods for inventory and monitoring of plants, birds and terrestrial invertebrates on a grid of 40 sites in a 938 ha study area in the Slikok Creek watershed, Kenai Peninsula, Alaska. We sampled plants and lichens through observation and specimen-based methods. We surveyed birds using bird call surveys on variable circular plots. We sampled terrestrial arthropods by sweep net sampling, processing samples with High Throughput Sequencing methods. We surveyed for earthworms, using the hot mustard extraction method and identified worm specimens by morphology and DNA barcoding. We examined community membership using clustering methods and Nonmetric Multidimensional Scaling. We documented a total of 4,764 occurrences of 984 species and molecular operational taxonomic units: 87 vascular plants, 51 mosses, 12 liverworts, 111 lichens, 43 vertebrates, 663 arthropods, 9 molluscs and 8 annelid worms. Amongst these records, 102 of the arthropod species appeared to be new records for Alaska. We found three non-native species: Deroceras agreste (Linnaeus, 1758) (Stylommatophora: Agriolimacidae), Dendrobaena octaedra (Savigny, 1826) (Crassiclitellata: Lumbricidae) and Heterarthrus nemoratus (Fallén, 1808) (Hymenoptera: Tenthredinidae). Both D. octaedra and H. nemoratus were found at sites distant from obvious human disturbance. The 40 sites were grouped into five community groups: upland mixed forest, black spruce forest, open deciduous forest, shrub-sedge bog and willow. We demonstrated that, at least for a subset of species that could be detected using these methods, we were able to document current species distributions and assemblages in a way that could be efficiently repeated for the purposes of biomonitoring. While our methods could be improved and additional methods and groups could be added, our combination of techniques yielded a substantial portion of the data necessary for fulfilling Kenai National Wildlife Refuge's broad conservation purposes.

Tracking a half century of media reporting on gray wolves.

Natural resource and wildlife managers must balance the disparate priorities of a diversity of stakeholders. To manage these priorities, a firm understanding of topics salient to the public is needed. The media often report on issues of importance to the public; therefore, these reports may be a useful measure of public interest. However, efficient methods for distinguishing diverse topics related to a wildlife management issue reported in the media and changes in the salience of those topics have been lacking. We used latent Dirichlet allocation, a Bayesian mixture model, to quantitatively assess the salience of topics surrounding the gray wolf (Canis lupus), which was reintroduced to Idaho (U.S.A.) in 1995. We analyzed articles published from 1960 to 2015 in an Idaho newspaper. We identified 6 distinct topics associated with gray wolves: policy, hunting, biological status, implementation of management, recovery, and human-wolf conflict. The salience of topics pre- and postreintroduction of wolves (1995) and pre- and postdelisting of wolves from the U.S. Endangered Species Act (2009) differed significantly, underscoring that these events were turning points in how issues were being publicly discussed and framed. Articles written by the local reporters were more likely to report on topics regarding conflict between humans and wolves, whereas articles sourced from a national outlet reported more on topics pertaining to wolf policy and biological status. In the context of managing a contentious, far-ranging, and long-lived wildlife species, our methods can help guide the location and timing of a suite of management strategies (e.g., media relation plans and stakeholder engagement) that promote human-wildlife coexistence across different landscapes.

Seguimiento de Medio Siglo de Reportes Mediáticos sobre Lobos Grises Resumen Los administradores de los recursos naturales y de la fauna deben balancear las prioridades discrepantes de una diversidad de accionistas. Para manejar estas prioridades se requiere de un entendimiento firme de los temas destacados para el público. Los medios frecuentemente reportan sobre temas de importancia para el público; por lo tanto, estos reportajes pueden ser una medida útil del interés del público. Sin embargo, hoy en día se carece de métodos eficientes para distinguir diferentes temas relacionados con un tema de manejo de fauna reportado en los medios y para reconocer los cambios en la prominencia de estos temas. Usamos la asignación latente Dirichlet, un modelo de mezcla bayesiana, para evaluar cuantitativamente la prominencia de los temas circundantes al lobo gris (Canis lupus), el cual fue reintroducido a Idaho (E.U.A.) en 1995. Analizamos artículos publicados en un periódico de Idaho desde 1960 y hasta 2015. Identificamos seis temas distintos asociados con el lobo gris: política, caza, estado biológico, implementación del manejo, recuperación, y conflicto humano-lobos. La prominencia de los temas previa y posterior a la introducción de los lobos (1995) y previa y posterior al retiro de los lobos del Acta de Especies Amenazadas de E.U.A (2009) varió significativamente, resaltando que estos eventos fueron puntos críticos para cómo se discutían y enmarcaban estos temas. Los artículos escritos por los reporteros locales tuvieron una mayor probabilidad de tratar temas sobre el conflicto entre humanos y lobos, mientras que los artículos surgidos en alguna fuente nacional reportaron más sobre temas correspondientes a la política y al estado biológico de los lobos. En el contexto de una especie silvestre contenciosa, de una extensión amplia y un ciclo de vida largo, nuestros métodos pueden ayudar a guiar la ubicación y el momento de un conjunto de estrategias de manejo (p. ej.: planes de relación de medios y compromiso de los accionistas) que promueva la coexistencia entre humanos y fauna a lo largo de diferentes paisajes.

A nanoporous gold membrane for sensing applications.

Design and fabrication of three-dimensionally structured, gold membranes containing hexagonally close-packed microcavities with nanopores in the base, are described. Our aim is to create a nanoporous structure with localized enhancement of the fluorescence or Raman scattering at, and in the nanopore when excited with light of approximately 600 nm, with a view to provide sensitive detection of biomolecules. A range of geometries of the nanopore integrated into hexagonally close-packed assemblies of gold micro-cavities was first evaluated theoretically. The optimal size and shape of the nanopore in a single microcavity were then considered to provide the highest localized plasmon enhancement (of fluorescence or Raman scattering) at the very center of the nanopore for a bioanalyte traversing through. The optimized design was established to be a 1200 nm diameter cavity of 600 nm depth with a 50 nm square nanopore with rounded corners in the base. A gold 3D-structured membrane containing these sized microcavities with the integrated nanopore was successfully fabricated and 'proof of concept' Raman scattering experiments are described.

Design and fabrication of a 3D-structured gold film with nanopores for local electric field enhancement in the pore.

Three-dimensionally structured gold membrane films with nanopores of defined, periodic geometries are designed and fabricated to provide the spatially localised enhancement of electric fields by manipulation of the plasmons inside nanopores. Square nanopores of different size and orientation relative to the pyramid are considered for films in aqueous and air environments, which allow for control of the position of electric fields within the structure. Designs suitable for use with 780 nm light were created. Here, periodic pyramidal cavities produced by potassium hydroxide etching to the {111} planes of (100) silicon substrates are used as templates for creating a periodic, pyramidal structured, free-standing thin gold film. Consistent with the findings from the theoretical studies, a nano-sized hole of 50 nm square was milled through the gold film at a specific location in the cavity to provide electric field control which can subsequently used for enhancement of fluorescence or Raman scattering of molecules in the nanopore.

Triplex-mediated analysis of cytosine methylation at CpA sites in DNA.

Modified triplex-forming oligonucleotides distinguish 5-methyl cytosine from unmethylated cytosine in DNA duplexes by differences in triplex melting temperatures. The discrimination is sequence-specific; dramatic differences in stabilisation are seen for CpA methylation, whereas CpG methylation is not detected. This direct detection of DNA methylation constitutes a new approach for epigenetic analysis.

Efficient self-assembly of DNA-functionalized fluorophores and gold nanoparticles with DNA functionalized silicon surfaces: the effect of oligomer spacers.

Although strategies for the immobilization of DNA oligonucleotides onto surfaces for bioanalytical and top-down bio-inspired nanobiofabrication approaches are well developed, the effect of introducing spacer molecules between the surface and the DNA oligonucleotide for the hybridization of nanoparticle-DNA conjugates has not been previously assessed in a quantitative manner. The hybridization efficiency of DNA oligonucleotides end-labelled with gold nanoparticles (1.4 or 10 nm diameter) with DNA sequences conjugated to silicon surfaces via hexaethylene glycol phosphate diester oligomer spacers (0, 1, 2, 6 oligomers) was found to be independent of spacer length. To quantify both the density of DNA strands attached to the surfaces and hybridization with the surface-attached DNA, new methodologies have been developed. Firstly, a simple approach based on fluorescence has been developed for determination of the immobilization density of DNA oligonucleotides. Secondly, an approach using mass spectrometry has been created to establish (i) the mean number of DNA oligonucleotides attached to the gold nanoparticles and (ii) the hybridization density of nanoparticle-oligonucleotide conjugates with the silicon surface-attached complementary sequence. These methods and results will be useful for application with nanosensors, the self-assembly of nanoelectronic devices and the attachment of nanoparticles to biomolecules for single-molecule biophysical studies.

The design of microfluidic affinity chromatography systems for the separation of bioanalytes.

The analytical (numerical) design of planar microfluidic affinity chromatography devices, which consist of multiple separation lanes and multiple, different surface-immobilised receptor patterns in each lane, is described. The model is based on the analytical solution of the transport-reaction equations in microfluidic systems of low Gratz number and for injection of small analyte plugs. The results reveal a simple approach for the design of microfluidic affinity chromatography devices tailored to the separation of bioanalytes, where receptors with high binding affinity are available. These devices have been designed for bioanalytical applications in mind, most notably for the proteomics field; the results are illustrated with an example using ß-Amyloid binding peptides.

Polyglutamine aggregate structure in vitro and in vivo; new avenues for coherent anti-Stokes Raman scattering microscopy.

Coherent anti-Stokes Raman scattering (CARS) microscopy is applied for the first time for the evaluation of the protein secondary structure of polyglutamine (polyQ) aggregates in vivo. Our approach demonstrates the potential for translating information about protein structure that has been obtained in vitro by X-ray diffraction into a microscopy technique that allows the same protein structure to be detected in vivo. For these studies, fibres of polyQ containing peptides (D(2)Q(15)K(2)) were assembled in vitro and examined by electron microscopy and X-ray diffraction methods; the fibril structure was shown to be cross ß-sheet. The same polyQ fibres were evaluated by Raman spectroscopy and this further confirmed the ß-sheet structure, but indicated that the structure is highly rigid, as indicated by the strong Amide I signal at 1659 cm(-1). CARS spectra were simulated using the Raman spectrum taking into account potential non-resonant contributions, providing evidence that the Amide I signal remains strong, but slightly shifted to lower wavenumbers. Combined CARS (1657 cm(-1)) and multi-photon fluorescence microscopy of chimeric fusions of yellow fluorescent protein (YFP) with polyQ (Q40) expressed in the body wall muscle cells of Caenorhabditis elegans nematodes (1 day old adult hermaphrodites) revealed diffuse and foci patterns of Q40-YFP that were both fluorescent and exhibited stronger CARS (1657 cm(-1)) signals than in surrounding tissues at the resonance for the cross ß-sheet polyQ in vitro.

The self-orientation of mammalian cells in optical tweezers--the importance of the nucleus.

Here we present the first evidence showing that eukaryotic cells can be stably trapped in a single focused Gaussian beam with an orientation that is defined by the nucleus. A mammalian eukaryotic cell (in suspension) is trapped and is re-oriented in the focus of a linearly polarized Gaussian beam with a waist of dimension smaller than the radius of the nucleus. The cell reaches a position relative to the focus that is dictated by the nucleus and nuclear components. Our studies illustrate that the force exerted by the optical tweezers at locations within the cell can be predicted theoretically; the data obtained in this way is consistent with the experimental observations.

Uniform aligned bioconjugation of biomolecule motifs for integration within microfabricated microfluidic devices.

Full details and a step-by-step guide suitable for printing proteins aligned to micron-sized sensors and subsequent integration and alignment of microfluidic structures are presented. The precise alignment and grafting of micron-sized biomolecule patterns with an underlying substrate at predefined locations is achieved using a novel semi-automated microcontact printer. Through integration of optical alignment methods in the x, y, and z directions, uniform contact of micron-sized stamps is achieved. Feature compression of the stamp is avoided by fine control of the stamp during contact. This printing method has been developed in combination with robust, compatible bioconjugate chemistry for patterning of a dextran-functionalized silicon oxide substrate with a NeutrAvidin-"inked" stamp and subsequent incubation with a biotin-functionalized protein. The bioconjugate chemistry is such that uniform coverage of the protein (without denaturation) over the printed motif is obtained and reproduction of the initial mask shape and dimensions is achieved. Later integration with a microfluidic structure aligned with the printed motif on the substrate is also described.

Flexible acoustic particle manipulation device with integrated optical waveguide for enhanced microbead assays.

Realisation of a device intended for the manipulation and detection of bead-tagged DNA and other bio-molecules is presented. Acoustic radiation forces are used to manipulate polystyrene micro-beads into an optical evanescent field generated by a laser pumped ion-exchanged waveguide. The evanescent field only excites fluorophores brought within approximately 100 nm of the waveguide, allowing the system to differentiate between targets bound to the beads and those unbound and still held in suspension. The radiation forces are generated in a standing-wave chamber that supports multiple acoustic modes, permitting particles to be both attracted to the waveguide surface and also repelled. To provide further control over particle position, a novel method of switching rapidly between different acoustic modes is demonstrated, through which particles are manipulated into an arbitrary position within the chamber. A novel type of assay is presented: a mixture of streptavidin coated and control beads are driven towards a biotin functionalised surface, then a repulsive force is applied, making it possible to determine which beads became bound to the surface. It is shown that the quarter-wave mode can enhance bead to surface interaction, overcoming potential barriers caused by surface charges. It is demonstrated that by measuring the time of flight of a microsphere across the device the bead size can be determined, providing a means of multiplexing the detection, potentially detecting a range of different target molecules, or varying bead mass.

Fluorescent properties of oligonucleotide-conjugated thiazole orange probes.

The fluorescence properties of thiazole orange, linked via a (1) hydrophobic alkyl or a (2) hydrophilic ethylene glycol chain to the central internucleotidic phosphate group of a pentadeca-2'-deoxyriboadenylate (dA15), are evaluated. Linkage at the phosphate group yields two stereoisomers, S-isomer of the phosphorus chiral center (Sp) and R-isomer of the phosphorus chiral center (Rp); these are studied separately. The character of the linkage chain and the chirality of the internucleotidic phosphate linkage site influence the fluorescent properties of these thiazole orange-oligonucleotide conjugates (TO-probes). Quantum yields of fluorescence (phifl) of between 0.04 and 0.07 were determined for the single-stranded conjugates. The fluorescence yield increased by up to five times upon hybridization with the complementary sequence (d5'[CACT15CAC3']); (phifl values of between 0.06-0.35 were determined for the double-stranded conjugates. The phifl value (0.17) of thiazole orange, 1-(N,N'-trimethylaminopropyl)-4-[3-methyl-2,3-dihydro-(benzo-1,3-thiazole)-2-methylidene]-quinolinium iodide (TO-Pro 1) in the presence of the oligonucleotide duplex (TO-Pro 1: dA15.d5'[CACT15CAC3'] (1:1)) is much less than that for some of the hybrids of the conjugates. Our studies, using steady-state and time-resolved fluorescence experiments, show that a number of discrete fluorescent association species between the thiazole orange and the helix are formed. Time-resolved studies on the four double-stranded TO-probes revealed that the fluorescent oligonucleotide-thiazole orange complexes are common, only the distribution of the species varies with the character of the chain and the chirality at the internucleotidic phosphate site. Those TO-probes in which the isomeric structure of the phosphate-chain linkage is Rp, and therefore such that the fluorophore is directed toward the minor groove, have higher phifl values than the Sp isomer. Of the systems studied, thiazole orange linked by an alkyl chain to the internucleotidic phosphate (Rp isomer) has the highest phifl and the greatest fraction of the longest-lived fluorescent thiazole orange species (in the hybrid form).