Analysis of the relative frequencies of the multipartite BNYVV genomic RNAs in different plants and tissues.

Multipartite virus genomes are composed of two or more segments, each packaged into an independent viral particle. A potential advantage of multipartitism is the regulation of gene expression through changes in the segment copy number. Soil-borne beet necrotic yellow vein virus (BNYVV) is a typical example of multipartism, given its high number of genomic positive-sense RNAs (up to five). Here we analyse the relative frequencies of the four genomic RNAs of BNYVV type B during infection of different host plants (Chenopodium quinoa, Beta macrocarpa and Spinacia oleracea) and organs (leaves and roots). By successfully validating a two-step reverse-transcriptase digital droplet PCR protocol, we show that RNA1 and -2 genomic segments always replicate at low and comparable relative frequencies. In contrast, RNA3 and -4 accumulate with variable relative frequencies, resulting in distinct RNA1 : RNA2 : RNA3 : RNA4 ratios, depending on the infected host species and organ.

NRAM: a disruptive carbon-nanotube resistance-change memory.

Advanced memory technology based on carbon nanotubes (CNTs) (NRAM) possesses desired properties for implementation in a host of integrated systems due to demonstrated advantages of its operation including high speed (nanotubes can switch state in picoseconds), high endurance (over a trillion), and low power (with essential zero standby power). The applicable integrated systems for NRAM have markets that will see compound annual growth rates (CAGR) of over 62% between 2018 and 2023, with an embedded systems CAGR of 115% in 2018-2023 (http://bccresearch.com/pressroom/smc/bcc-research-predicts:-nram-(finally)-to-revolutionize-computer-memory). These opportunities are helping drive the realization of a shift from silicon-based to carbon-based (NRAM) memories. NRAM is a memory cell made up of an interlocking matrix of CNTs, either touching or slightly separated, leading to low or higher resistance states respectively. The small movement of atoms, as opposed to moving electrons for traditional silicon-based memories, renders NRAM with a more robust endurance and high temperature retention/operation which, along with high speed/low power, is expected to blossom in this memory technology to be a disruptive replacement for the current status quo of DRAM (dynamic RAM), SRAM (static RAM), and NAND flash memories.

[Etiology of the sugar beet rhizomania]. / Étiologie de la rhizomanie de la betterave sucrière.

Beet necrotic yellow vein virus is responsible for sugar beet rhizomania. Root proliferation is characteristic of the viral infection and lead to sugar losses. Pathogenicity is particularly linked to the expression of RNA-3-encoded p25. The extensive use of viral tolerant crops allows maintenance of sugar yields but also permits viruliferous vector to be maintained and therefore the appearance of resistance breaking isolates. The resistance breaking isolates present some amino acid variations within the p25 protein sequence, a key determinant in BNYVV pathogenicity. Here, we will review the molecular biology of BNYVV, of its vector and the antiviral strategies that may be used against rhizomania.

P42 movement protein of Beet necrotic yellow vein virus is targeted by the movement proteins P13 and P15 to punctate bodies associated with plasmodesmata.

Cell-to-cell movement of Beet necrotic yellow vein virus (BNYVV) is driven by a set of three movement proteins--P42, P13, and P15--organized into a triple gene block (TGB) on viral RNA 2. The first TGB protein, P42, has been fused to the green fluorescent protein (GFP) and fusion proteins between P42 and GFP were expressed from a BNYVV RNA 3-based replicon during virus infection. GFP-P42, in which the GFP was fused to the P42 N terminus, could drive viral cell-to-cell movement when the copy of the P42 gene on RNA 2 was disabled but the C-terminal fusion P42-GFP could not. Confocal microscopy of epidermal cells of Chenopodium quinoa near the leading edge of the infection revealed that GFP-P42 localized to punctate bodies apposed to the cell wall whereas free GFP, expressed from the replicon, was distributed uniformly throughout the cytoplasm. The punctate bodies sometimes appeared to traverse the cell wall or to form pairs of disconnected bodies on each side. The punctate bodies co-localized with callose, indicating that they are associated with plasmodesmata-rich regions such as pit fields. Point mutations in P42 that inhibited its ability to drive cell-to-cell movement also inhibited GFP-P42 punctate body formation. GFP-P42 punctate body formation was dependent on expression of P13 and P15 during the infection, indicating that these proteins act together or sequentially to localize P42 to the plasmodesmata.

Mapping the promoter for subgenomic RNA synthesis on beet necrotic yellow vein virus RNA 3.

During infection of Tetragonia expansa leaves, RNA 3 of the quadripartite genome of beet necrotic yellow vein virus directs synthesis of a subgenomic RNA (RNA 3sub) which corresponds to the 3'-terminal 600 residues of the RNA 3 molecule. Biologically active run-off transcripts have been prepared from full-length cDNA of RNA 3 cloned behind a bacteriophage T7-RNA polymerase promoter. RNA 3 transcripts carrying deletions in the vicinity of the RNA 3sub initiation site were produced by site-directed mutagenesis at the cDNA level and then tested for their capacity to direct RNA 3sub synthesis in infected leaves. The cis-acting domain essential for normal levels of RNA 3sub production in planta (the 'core' promoter) did not extend in the 5'-direction beyond position -16 relative to the RNA 3sub transcription initiation site. The 3'-boundary of the core promoter domain was located somewhere between positions +100 and +208. Displacement of the promoter domain to an upstream site in RNA 3 produced a new subgenomic RNA starting at or near the predicted upstream site.

Multiple memory states in resistive switching devices through controlled size and orientation of the conductive filament.

Multilevel operation in resistive switching memory (RRAM) based on HfOx is demonstrated through variable sizes and orientations of the conductive filament. Memory states with the same resistance, but opposite orientation of defects, display a different response to an applied read voltage, therefore allowing an improvement of the information stored in each physical cell. The multilevel scheme allows a 50% increase (from 2 to 3 bits) of the stored information.

Subcellular localization of the Triple Gene Block movement proteins of Beet necrotic yellow vein virus by electron microscopy.

The Triple Gene Block proteins TGBp1, TGBp2, and TGBp3 of Beet necrotic yellow vein virus (BNYVV) are required for efficient cell-to-cell spread of the infection. The TGB proteins can drive cell-to-cell movement of BNYVV in trans when expressed from a co-inoculated BNYVV RNA 3-based 'replicon'. TGBp2 and TGBp3 expressed from the replicon were nonfunctional in this assay if they were fused to the green fluorescent protein (GFP), but addition of a hemagglutinin (HA) tag to their C-termini did not incapacitate movement. Immunogold labeling of ultrathin sections treated with HA-specific antibodies localized TGBp2-HA and TGBp3-HA to what are probably structurally modified plasmodesmata (Pd) in infected cells. A similar subcellular localization was observed for TGBp1. Large gold-decorated membrane-rich bodies containing what appear to be short fragments of endoplasmic reticulum were observed near the cell periphery. The modified gold-decorated Pd and the membrane-rich bodies were not observed when the TGB proteins were produced individually in infections using the Tobacco mosaic virus P30 protein to drive cell-to-cell movement, indicating that these modifications are specific for TGB-mediated movement.

Visualisation of transgene expression at the single protoplast level.

Protoplasts are currently used to study the expression of genes following transformation. Expression is followed on a population of protoplasts after total protein extraction by conventional western blotting or measure of the enzymatic activity of the transgenic protein. We describe here a new method, called protoplast printing, allowing easy detection of the fraction of cells expressing a certain protein within a population of protoplasts. It consists of immobilization of the protoplast proteins on a nitrocellulose filter, so as to retain the outlines of the cell, followed by immunological detection of the protein of interest. The only special requirement is an antibody specific for the protein. We have studied the expression of the BNYVV coat protein after electroporation of Chenopodium quinoa protoplasts with viral RNAs, and the expression of the NPT II gene in protoplasts isolated from transgenic tobacco plants as well as after direct transfer of plasmid DNA into tobacco protoplasts. In both cases - infection with viral RNAs and transformation with plasmid DNA - expressing and non-expressing cells can be distinguished as early as 12h after transfer of the transgenes.

Effects of structural modifications upon the accumulation in planta of replicons derived from beet necrotic yellow vein virus RNA 3.

Beet necrotic yellow vein virus (BNYVV) RNA 3 from which all but the 3' and 5' 'core' replication origins (promoters) have been deleted replicates when coinoculated to Chenopodium quinoa with viral RNAs 1 and 2. The resulting 'replicon' can be used to express inserted heterologous sequences in planta. The effects of alterations of replicon structure on its efficiency of accumulation in planta were examined. Inclusion of up to approximately 240 nucleotides of sequence from the region immediately upstream of the core 3'-promoter sequence increased replicon accumulation, suggesting that this region contains specific replication enhancer elements. Insertion of non-viral 'spacer' sequences between the core promoters also increased replicon accumulation, provided that no strong secondary structure was present. The highly homologous 3'-terminal core promoters of BNYVV RNAs 1, 2 and 4 could substitute for the RNA 3 core promoter but were generally somewhat less effective. Co-inoculation of full-length RNA 3 but not RNA 4 interfered with accumulation of the RNA 3-based replicons.

Adolescents with special health care needs in transition: three life histories.

PURPOSE: This study was part of a large multi-method inquiry designed to examine the service and support needs of adolescents with special health care needs who are transitioning to adulthood. METHODS: A multiple case study methodology relying on life history was used to ascertain perspectives of the parents on the longitudinal events and factors in the lives of adolescents with special health care needs that shaped or currently influence adolescent transition to adulthood. Three informants were purposively selected to depict a range of health and socio-economic conditions. Long interviews were conducted and audiotaped and transcribed data were thematically and taxonomically analyzed. RESULTS: Six major themes emerged in the analysis: (1) Begetting a service system, (2) Pathology or not pathology, (3) Educational stability vs. interruptions, (4) Role blurring of parents and providers, (5) Private life made public, and (6) Independence vs. burden. CONCLUSIONS: The study revealed that, consistent with the literature, adolescents with special health care needs do not follow typical developmental sequences, although their needs and desires are no different than those of typical adolescents. The lives of the adolescents and their families are significantly influenced by the timing and nature of the diagnosis, the family's articulation with service providers, and the degree to which school systems are responsive to atypical adolescents. Implications for health providers and future inquiry are advanced.

cis-active sequences near the 5'-termini of beet necrotic yellow vein virus RNAs 3 and 4.

RNAs 3 and 4 of the multicomponent genome of beet necrotic yellow vein virus are dispensable for infection of Chenopodium quinoa leaves. We have used mutagenesis of biologically active RNA 3 transcripts to identify 5'-proximal sequences essential in cis for RNA 3 amplification. One such element, Box I, (nucleotides 283-292) was complementary to the first 10 residues (Box I') following the 5'-terminal cap. A second cis-active element (Box II) was identified between nucleotides 237-244 and was complementary to nucleotides 16-23 (Box II'). Other cis-active sequences exist between Box II' and II but have not been mapped to fine scale. Most sequence substitutions in Boxes I and II or in the 5'-proximal complementary sequences were lethal but compensatory mutations designed to restore Box I/I' or Box II/II' base pairing restored viability, suggesting that secondary structure involving these elements rather than their exact sequence is the critical feature. Transcripts bearing short deletions near residue 200 were replicated but did not assemble into virions, indicating that this region contains or contributes to a cis-active encapsidation signal. Similar experiments with RNA 4 transcript have shown that 5'-proximal cis-essential elements are limited to the first 400 residues of this RNA. Essential subdomains within this region have not been mapped but there are no structures obviously homologous to Boxes I/I' and II/II' of RNA 3.

Efficient inoculation with CaMV 35 S promoter-driven DNA clones of the tobravirus PEBV.

Clones have been constructed containing full-length cDNA copies of PEBV RNA1 and RNA2, flanked by the CaMV 35 S RNA promoter and the nopaline synthase terminator. The clones are infectious when inoculated onto Nicotiana benthamiana plants. Both the viral RNAs and the virus particles were identified in infected plants.

Organochlorine and mercury residues in Swainson's and ferruginous hawk eggs collected in North and South Dakota, 1974-79.

Residues of organochlorine pesticides, polychlorinated biphenyls (PCBs), and mercury were measured in eggs of Swainson's hawks (Buteo swainsoni) and ferruginous hawks (B. regalis) collected in North and South Dakota during 1974-79. DDE was the most common compound detected in the eggs, but residues were below levels known to have adverse effects on reproduction. Other organochlorine compounds and mercury were found at low levels. Eggs of ferruginous hawks tended to contain more compounds with higher residues than eggs of Swainson's hawks.

Beet necrotic yellow vein virus 42 kDa triple gene block protein binds nucleic acid in vitro.

The triple gene block (TGB) of beet necrotic yellow vein virus RNA 2 is required for cell-to-cell movement of the virus RNA. The protein P42 encoded by the 5'-proximal gene of the TGB has consensus sequence motifs characteristic of an ATP/GTP-dependent helicase. P42 was over-expressed in Escherichia coli and shown to bind both single- and double-stranded RNA and DNA by Northwestern blotting. Site-directed mutagenesis located the nucleic acid-binding domain to the N-terminal 24 amino acids of the protein and a point mutation or deletions in the region of P42 containing the helicase consensus sequences did not affect nucleic acid-binding activity of the immobilized protein. Electrophoretic mobility-shift assays revealed that P42 also binds nucleic acids in solution and that deletion of the N-terminal region inhibits this binding. Mutations in both the N-terminal nucleic acid-binding domain and the helicase domain blocked infection of leaves, indicating that both regions of P42 are important for its activity in vivo.

Conformation of the 3'-end of beet necrotic yellow vein benevirus RNA 3 analysed by chemical and enzymatic probing and mutagenesis.

Secondary structure-sensitive chemical and enzymatic probes have been used to produce a model for the folding of the last 68 residues of the 3'-non-coding region of beet necrotic yellow vein benevirus RNA 3. The structure consists of two stem-loops separated by a single-stranded region. RNA 3-derived transcripts were produced containing mutations which either disrupted base pairing in the helices or maintained the helices but with alterations in the base pairing scheme. Other mutants contained substitutions in single-stranded regions (loops or bulged sequences). With a few exceptions all three types of mutation abolished RNA 3 replication in vivo, suggesting that both secondary structure and specific sequences are required for efficient recognition of the 3'-terminal region of RNA 3 by viral RNA-dependent RNA polymerase.

The secondary structure of the 5'-noncoding region of beet necrotic yellow vein virus RNA 3: evidence for a role in viral RNA replication.

Secondary structure-sensitive chemical and enzymatic probes have been used to produce a model for the folding of the first 312 residues of the long 5'-noncoding region of beet necrotic yellow vein virus RNA 3. The structure consists of two major domains, one of which includes long distance base-pairing interactions between two short sequence elements (Box I and Box II) situated between positions 237 and 292 and complementary elements (Box I' and II') near the 5'-terminus. Previous studies have shown that base pairing between these sequence elements (in either the plus-strand or minus-strand RNA) is important for RNA 3 accumulation during infection. RNA 3 transcripts were produced containing mutations which preferentially disrupted Box II-II' base pairing in either the plus- or minus-strand. In infection experiments, transcripts with mutations which disrupted the Box II-II' interaction in the plus-strand structure replicated less efficiently than mutants in which the Box II-II' interaction was disrupted in the minus-strand. These findings indicate that the complex 5'-proximal plus-strand structure to which the Box II-II' interaction contributes comprises at least part of the promoter for plus-strand RNA synthesis.

Efficient cell-to-cell movement of beet necrotic yellow vein virus requires 3' proximal genes located on RNA 2.

RNA 2 of beet necrotic yellow vein virus (BNYVV) carries six open reading frames. The four 3' proximal frames encode the proteins P42, P13, P15, and P14. The first three species present homologies to proteins encoded by three overlapping open reading frames (the triple gene block) in potexviruses, carlaviruses, and barley stripe mosaic virus. P14 does not display homology with other known plant viral proteins. The functions of P42, P13, P15, and P14 were investigated by site-directed mutagenesis. Full-length transcripts of wild-type BNYVV RNAs 1 and 2 were infectious when coinoculated to protoplasts or leaves of Chenopodium quinoa. RNA 2 transcripts in which P42, P13, and P15 were prematurely terminated by frameshift mutations replicated in protoplasts (when inoculated with wild-type RNA 1) but were not infectious to leaves, indicating that the triple gene block proteins of BNYVV are essential for viral cell-to-cell spread. Mutations in P14 were not lethal in leaf infections but smaller local lesions and lesser amounts of viral RNA were produced. RNA 2-related subgenomic RNA species of 2.6, 1.4, and 0.7 kb were detected; they presumably direct synthesis of P42, P13, and P14. No species of the length predicted for a P15-specific subgenomic RNA was detected.