Non-proximate Sampling and Photoionization for Damage-Free Mass Spectrometric Analysis of Intact Native American Baskets.

Selected examples of Native American woven woodsplint basketry created between 1870 and 1983 are studied to recover traditional knowledge about their manufacture by identifying dyes or colorants. An ambient mass spectrometry system is designed to sample from intact objects with minimal invasiveness, neither cutting solids from the whole, exposing objects to liquid, nor leaving a mark on a surface. Baskets up to 60 cm wide in one dimension are placed on height-adjusted mounts. A timed jet of inert nitrogen from a finely positioned probe thermally desorbs neutral material from a mounted item, and a heated transport tube carries the analyte 2 m away at 4.9 L/min. Gas phase analyte is mixed with anisole dopant from an in-line permeation tube and photoionized in a reaction tee immediately before entering the mass spectrometer, identifying dye molecules in real time. Extensive optimization and exposure tests with flat and near-flat splints of dyed wood ensure that the analysis produces no discoloration on the curved and contoured basket splints.

Rescue of the first alphanucleorhabdovirus entirely from cloned complementary DNA: An efficient vector for systemic expression of foreign genes in maize and insect vectors.

Recent reverse genetics technologies have enabled genetic manipulation of plant negative-strand RNA virus (NSR) genomes. Here, we report construction of an infectious clone for the maize-infecting Alphanucleorhabdovirus maydis, the first efficient NSR vector for maize. The full-length infectious clone was established using agrobacterium-mediated delivery of full-length maize mosaic virus (MMV) antigenomic RNA and the viral core proteins (nucleoprotein N, phosphoprotein P, and RNA-directed RNA polymerase L) required for viral transcription and replication into Nicotiana benthamiana. Insertion of intron 2 ST-LS1 into the viral L gene increased stability of the infectious clone in Escherichia coli and Agrobacterium tumefaciens. To monitor virus infection in vivo, a green fluorescent protein (GFP) gene was inserted in between the N and P gene junctions to generate recombinant MMV-GFP. Complementary DNA (cDNA) clones of MMV-wild type (WT) and MMV-GFP replicated in single cells of agroinfiltrated N. benthamiana. Uniform systemic infection and high GFP expression were observed in maize inoculated with extracts of the infiltrated N. benthamiana leaves. Insect vectors supported virus infection when inoculated via feeding on infected maize or microinjection. Both MMV-WT and MMV-GFP were efficiently transmitted to maize by planthopper vectors. The GFP reporter gene was stable in the virus genome and expression remained high over three cycles of transmission in plants and insects. The MMV infectious clone will be a versatile tool for expression of proteins of interest in maize and cross-kingdom studies of virus replication in plant and insect hosts.

The physical interactome between Peregrinus maidis proteins and the maize mosaic virus glycoprotein provides insights into the cellular biology of a rhabdovirus in the insect vector.

Rhabdovirus glycoproteins (G) serve multifunctional roles in virus entry, assembly, and exit from animal cells. We hypothesize that maize mosaic virus (MMV) G is required for invasion, infection, and spread in Peregrinus maidis, the planthopper vector. Using a membrane-based yeast two-hybrid assay, we identified 107 P. maidis proteins that physically interacted with MMV G, of which approximately 53% matched proteins with known functions including endocytosis, vesicle-mediated transport, protein synthesis and turnover, nuclear export, metabolism and host defense. Physical interaction networks among conserved proteins indicated a possible cellular coordination of processes associated with MMV G translation, protein folding and trafficking. Non-annotated proteins contained predicted functional sites, including a diverse array of ligand binding sites. Cyclophilin A and apolipophorin III co-immunoprecipitated with MMV G, and each showed different patterns of localization with G in insect cells. This study describes the first protein interactome for a rhabdovirus spike protein and insect vector.

Paired inpatient & outpatient senior practicum clinical placement: Lessons learned.

The American Association of Ambulatory Care Nurses (AAACN) promotes addressing the increasing need for registered nurses with specialized knowledge and skills to be effective in the ambulatory care environment (2014). A large University Medical Center was interested in offering a senior practicum clinical experience in the ambulatory care environment, that might result in future hiring of new graduate RNs for positions in 17 ambulatory care centers, to address vacancies and in support of the AAACNs goals (2014). The same University's School of Nursing needed to address the need for additional senior practicum clinical placements for 200+ pre-licensure nursing students, each semester. Targeting both the needs of the Medical Center and the School of Nursing, an academic-practice partnership resulted in the creation of a combined inpatient and outpatient, ambulatory care senior practicum clinical experience. This project resulted in a clearer understanding of the utility of ambulatory care environments as a site for nursing student clinical placements. Additionally, the project informed participating students about the roles of the nurse in the ambulatory setting, but did not result in an increased number of placements nor the hiring of newly-graduated RNs into the ambulatory care environment, to date. Lessons learned and potential solutions are shared.

Diversity and epidemiology of plant rhabdoviruses.

Plant rhabdoviruses are recognized by their large bacilliform particles and for being able to replicate in both their plant hosts and arthropod vectors. This review highlights selected, better studied examples of plant rhabdoviruses, their genetic diversity, epidemiology and interactions with plant hosts and arthropod vectors: Alfalfa dwarf virus is classified as a cytorhabdovirus, but its multifunctional phosphoprotein is localized to the plant cell nucleus. Lettuce necrotic yellows virus subtypes may differentially interact with their aphid vectors leading to changes in virus population diversity. Interactions of rhabdoviruses that infect rice, maize and other grains are tightly associated with their specific leafhopper and planthopper vectors. Future outbreaks of vector-borne nucleorhabdoviruses may be predicted based on a world distribution map of the insect vectors. The epidemiology of coffee ringspot virus and its Brevipalpus mite vector is illustrated highlighting the symptomatology and biology of a dichorhavirus and potential impacts of climate change on its epidemiology.

Discovery of Novel Thrips Vector Proteins That Bind to the Viral Attachment Protein of the Plant Bunyavirus Tomato Spotted Wilt Virus.

The plant-pathogenic virus tomato spotted wilt virus (TSWV) encodes a structural glycoprotein (GN) that, like with other bunyavirus/vector interactions, serves a role in viral attachment and possibly in entry into arthropod vector host cells. It is well documented that Frankliniella occidentalis is one of nine competent thrips vectors of TSWV transmission to plant hosts. However, the insect molecules that interact with viral proteins, such as GN, during infection and dissemination in thrips vector tissues are unknown. The goals of this project were to identify TSWV-interacting proteins (TIPs) that interact directly with TSWV GN and to localize the expression of these proteins in relation to virus in thrips tissues of principal importance along the route of dissemination. We report here the identification of six TIPs from first-instar larvae (L1), the most acquisition-efficient developmental stage of the thrips vector. Sequence analyses of these TIPs revealed homology to proteins associated with the infection cycle of other vector-borne viruses. Immunolocalization of the TIPs in L1 revealed robust expression in the midgut and salivary glands of F. occidentalis, the tissues most important during virus infection, replication, and plant inoculation. The TIPs and GN interactions were validated using protein-protein interaction assays. Two of the thrips proteins, endocuticle structural glycoprotein and cyclophilin, were found to be consistent interactors with GN These newly discovered thrips protein-GN interactions are important for a better understanding of the transmission mechanism of persistent propagative plant viruses by their vectors, as well as for developing new strategies of insect pest management and virus resistance in plants.IMPORTANCE Thrips-transmitted viruses cause devastating losses to numerous food crops worldwide. For negative-sense RNA viruses that infect plants, the arthropod serves as a host as well by supporting virus replication in specific tissues and organs of the vector. The goal of this work was to identify thrips proteins that bind directly to the viral attachment protein and thus may play a role in the infection cycle in the insect. Using the model plant bunyavirus tomato spotted wilt virus (TSWV), and the most efficient thrips vector, we identified and validated six TSWV-interacting proteins from Frankliniella occidentalis first-instar larvae. Two proteins, an endocuticle structural glycoprotein and cyclophilin, were able to interact directly with the TSWV attachment protein, GN, in insect cells. The TSWV GN-interacting proteins provide new targets for disrupting the viral disease cycle in the arthropod vector and could be putative determinants of vector competence.

Complete Genome Sequence of Maize Mosaic Nucleorhabdovirus.

The complete genome sequence of maize mosaic virus (MMV) was obtained using next-generation sequencing from infected Peregrinus maidis and rapid amplification of cDNA ends from infected Zea mays The genome of MMV is 12,170 bases, and this project completed the 5' and 3' ends and amended the polymerase sequence.

Plant rhabdoviruses-their origins and vector interactions.

Classical plant rhabdoviruses infect monocot and dicot plants, have unsegmented negative-sense RNA genomes and have been taxonomically classified in the genera Cytorhabdovirus and Nucleorhabdovirus. These viruses replicate in their hemipteran vectors and are transmitted in a circulative-propagative mode and virus infection persists for the life of the insect. Based on the discovery of numerous novel rhabdoviruses in arthropods during metagenomic studies and extensive phylogenetic analyses of the family Rhabdoviridae, it is hypothesized that plant-infecting rhabdoviruses are derived from insect viruses. Analyses of viral gene function in plants and insects is beginning to reveal conserved and unique biology for these plant viruses in the two diverse hosts. New tools for insect molecular biology and infectious clones for plant rhabdoviruses are increasing our understanding of the lifestyles of these viruses.

Cellular localization and interactions of nucleorhabdovirus proteins are conserved between insect and plant cells.

Maize mosaic virus (MMV), similar to other nucleorhabdoviruses, replicates in divergent hosts: plants and insects. To compare MMV protein localization and interactions, we visualized autofluorescent protein fusions in both cell types. Nucleoprotein (N) and glycoprotein (G) localized to the nucleus and cytoplasm, phosphoprotein (P) was only found in the nucleus, and 3 (movement) and matrix (M) were present in the cytoplasm. This localization pattern is consistent with the model of nucleorhabdoviral replication of N, P, L and viral RNA forming a complex in the nucleus and the subvirion associating with M and then G during budding into perinuclear space. The comparable localization patterns in both organisms indicates a similar replication cycle. Changes in localization when proteins were co-expressed suggested viral proteins interact thus altering organelle targeting. We documented a limited number of direct protein interactions indicating host factors play a role in the virus protein interactions during the infection cycle.

Transcriptomic response of the insect vector, Peregrinus maidis, to Maize mosaic rhabdovirus and identification of conserved responses to propagative viruses in hopper vectors.

Maize mosaic virus (MMV) is a plant-pathogenic rhabdovirus that is transmitted by the corn planthopper, Peregrinus maidis, in a propagative manner. P. maidis supports long-term MMV infections with no negative effects on insect performance. To elucidate whole-body transcriptome responses to virus infection, RNA-Seq was used to examine differential gene expression of virus-infected adult insects, and libraries were prepared from replicated groups of virus-exposed insects and non-exposed insects. From the 68,003 de novo-assembled transcripts, 144 were differentially-expressed (DE) during viral infection with comparable numbers up- and down-regulated. DE transcripts with similarity to genes associated with transposable elements (i.e., RNA-directed DNA polymerases) were enriched and may represent a mechanisim for modulating virus infection. Comparison of the P. maidis DE transcripts to published propagative virus-responsive transcript databases for two other hopper vectors revealed that 16% of the DE transcripts were shared across the three systems and may represent conserved responses to propagative viruses.

In planta localization and interactions of impatiens necrotic spot tospovirus proteins.

Impatiens necrotic spot tospovirus (INSV) is a significant pathogen of ornamentals. The tripartite negative- and ambi-sense RNA genome encodes six proteins that are involved in cytoplasmic replication, movement, assembly, insect transmission and defence. To gain insight into the associations of these viral proteins, we determined their intracellular localization and interactions in living plant cells. Nucleotide sequences encoding the nucleoprotein N, non-structural proteins NSs and NSm, and glycoproteins Gn and Gc of a Kentucky isolate of INSV were amplified by RT-PCR, cloned, sequenced and transiently expressed as fusions with autofluorescent proteins in leaf epidermal cells of Nicotiana benthamiana. All proteins accumulated at the cell periphery and co-localized with an endoplasmic reticulum marker. The Gc protein fusion also localized to the nucleus. N and NSm protein self-interactions and an NSm-N interaction were observed by using bimolecular fluorescence complementation. A tospovirus NSm homotypic interaction had not been reported previously.

Lettuce necrotic yellows cytorhabdovirus protein localization and interaction map, and comparison with nucleorhabdoviruses.

Lettuce necrotic yellows virus (LNYV), Sonchus yellow net virus (SYNV) and Potato yellow dwarf virus (PYDV) are members of the family Rhabdoviridae that infect plants. LNYV is a cytorhabdovirus that replicates in the cytoplasm, while SYNV and PYDV are nucleorhabdoviruses that replicate in the nuclei of infected cells. LNYV and SYNV share a similar genome organization with a gene order of nucleoprotein (N), phosphoprotein (P), putative movement protein (Mv), matrix protein (M), glycoprotein (G) and polymerase (L). PYDV contains an additional predicted gene of unknown function located between N and P. In order to gain insight into the associations of viral structural and non-structural proteins and the mechanisms by which they may function, we constructed protein localization and interaction maps. Subcellular localization was determined by transiently expressing the viral proteins fused to green or red fluorescent protein in leaf epidermal cells of Nicotiana benthamiana. Protein interactions were tested in planta by using bimolecular fluorescence complementation. All three viruses showed Mv to be localized at the cell periphery and the G protein to be membrane associated. Comparing the interaction maps revealed that only the N-P and M-M interactions are common to all three viruses. Associations unique to only one virus include P-M for LNYV, G-Mv for SYNV and M-Mv, M-G and N-M for PYDV. The cognate N-P proteins of all three viruses interacted and exhibited characteristic changes in localization when co-expressed.

Tomorrow's challenges - today's realities making the leap, 2002 Ryley-Jeffs Memorial Lecture.

The premise of the presentation is a challenge to health care providers to examine the quality of services currently provided in health care facilities across the country. While the Canadian health care system is under scrutiny with numerous reviews and commissions, the underlying question is: are the structural changes making a difference? We need to consider the recommendations in the latest report from the Institute of Medicine, Crossing the Quality Chasm. The report calls for a sweeping redesign and suggests a set often new rules to guide patient/clinician relationships. Dietitians must take the lead on implementation of systematic changes, model the way and get involved in the necessary changes. As the report suggests, the gap between where we are and where we need to go in providing quality health care services is not just a crack, it is in fact a chasm.