The Quest to Identify a New Virus Disease of Sunflower from Nebraska.

Between 2010 and 2018, sunflower plants exhibiting virus-like symptoms, including stunting, mottling, and chlorotic ringspots on leaves, were observed from commercial fields and research plots from four sites within three distinct counties of western Nebraska (Box Butte, Kimball, and Scotts Bluff). Near identical symptoms from field samples were reproduced on seedlings mechanically in the greenhouse on multiple occasions, confirming the presence of a sap-transmissible virus from each site. Symptomatic greenhouse-inoculated plants from the 2010 and 2011 Box Butte samples tested negative for sunflower mosaic virus (SuMV), sunflower chlorotic mottle virus (SuCMoV), and all potyviruses in general by ELISA and RT-PCR. Similar viral-like symptoms were later observed on plants in a commercial sunflower field in Kimball County in 2014, and again from volunteers in research plots in Scotts Bluff County in 2018. Samples from both of these years were again successfully reproduced on seedlings in the greenhouse as before following mechanical transmissions. Symptom expression for all years began 12 to 14 days after inoculation as mild yellow spots followed by the formation of chlorotic ringspots from the mottled pattern. The culture from 2014 tested negatively for three groups of nepoviruses via RT-PCR, ruling this group out. However, transmission electron microscopy assays of greenhouse-infected plants from both 2014 and 2018 revealed the presence of distinct, polyhedral virus particles. With the use of high throughput sequencing and RT-PCR, it was confirmed that the infections from both years were caused by a new virus in the tombusvirus genus and was proposed to be called Sunflower ring spot mottle virus (SuRSMV). Although the major objective of this project was to identify the causal agent of the disease, it became evident that the diagnostic journey itself, with all the barriers encountered on the 10-year trek, was actually more important and impactful than identification.

Response of Pemphigus betae (Hemiptera: Aphididae) and Beneficial Epigeal Arthropod Communities to Sugarbeet Plant Density and Seed-Applied Insecticide in Western Nebraska.

This study investigated the impact of a neonicotinoid seed-applied insecticide (Poncho Beta) and two plant densities (86,487 and 61,776 plants per hectare) on the sugarbeet root aphid (Pemphigus betae Doane), beneficial epigeal arthropods, and selected crop yield parameters in sugarbeet (Beta vulgaris L. var. vulgaris). Ground beetles and centipedes were the most commonly collected taxa during 2012 and 2013, respectively. Centipede, spider, and rove beetle activity densities were not affected by the seed-applied insecticide, whereas plant density had a marginal effect on centipede activity density during 2012. Ground beetle species richness, diversity, and evenness were also not impacted by the seed treatments. However, during 2013, ground beetle activity density was significantly higher in plots planted with untreated sugarbeet seeds due to the abundance of Bembidion quadrimaculatum oppositum Say. Sugarbeet root aphid populations were significantly higher in the untreated plots during both years. In 2012, sugarbeet tonnage and sugar yield were higher under the low plant density treatment, while higher sugar content was recorded from the seed-applied insecticide plots (2013). Seed-applied neonicotinoids and plant density had little impact on beneficial epigeal arthropod activity density. Seed treatment did result in decreased root aphid populations; however, these reductions were not sufficient to be considered as an adequate control. This limited aphid control likely contributed to inconsistent effects on yield parameters.

Characterization of management and environmental factors associated with regional variations in potato zebra chip occurrence.

Potato zebra chip (ZC), caused by the bacterial pathogen 'Candidatus Liberibacter solanacearum', which is vectored by the potato psyllid (Bactericera cockerelli), has caused widespread damage to U.S. potato production ever since its first discovery in south Texas in 2000. To determine the influence of environmental factors and management practices on ZC occurrence, data on management and meteorological variables, field locations, and psyllid counts were collected over a 3-year period (2010 to 2012) from six locations across the central United States (south Texas to Nebraska). At these locations, ZC-symptomatic plants were counted in 26 fields from systematically established 20 m × 30 m plots around the field edges and field interiors. Mean numbers of symptomatic plants per plot were classified into two intensity classes (ZC ≤ 3 or ZC > 3) and subjected to discriminant function and logistic regression analyses to determine which factors best distinguish between the two ZC intensity classes. Of all the variables, location, planting date, and maximum temperature were found to be the most important in distinguishing between ZC intensity classes. These variables correctly classified 88.5% of the fields into either of the two ZC-intensity classes. Logistic regression analysis of the individual variables showed that location accounted for 90% of the variations, followed by planting date (86%) and maximum temperature (70%). There was a low but significant (r = -0.44983, P = 0.0211) negative correlation between counts of psyllids testing positive for pathogen and latitudinal locations, indicating a south-to-north declining trend in counts of psyllids testing positive for the pathogen. A similar declining trend also was observed in ZC occurrence (r = -0.499, P = 0.0094).

Seasonal population dynamics of the potato psyllid (Hemiptera: Triozidae) and its associated pathogen "Candidatus Liberibacter solanacearum" in potatoes in the southern great plains of North America.

The potato psyllid, Bactericera cockerelli (Sulc) (Hemiptera: Triozidae), and its associated pathogen "Candidatus Liberibacter solanacearum" (Ca. L. solanacearum), the putative causal agent of zebra chip (ZC) disease in potatoes (Solanum tuberosum L.), were sampled in commercial potato fields and untreated control plots for 3 yr in multiple locations in Texas, Kansas, Nebraska, and Colorado. Populations of the potato psyllid varied across years and across potato growing regions. However, the percentage of potato psyllids infected with Ca. L. solanacearum although variable across years, was consistently highest in the Lower Rio Grande Valley of Texas (LRGV), the reported overwintering location for this pest. The numbers of Ca. L. solanacearum-infected psyllids collected on field traps and large nymphs counted on leaf samples were both positively correlated with the final percentage of ZC in tubers. In the LRGV, where vector and disease pressure is the highest, population levels of immature life stages of the psyllid and percentage of ZC differed greatly between commercial and untreated fields. These results show that the pest management program that was used can be effective at controlling development of the psyllid and ultimately reducing the incidence of ZC.

Relative feeding and development of armyworm on switchgrass and corn, and its potential effects on switchgrass grown for biomass.

To help assess the potential for damage by armyworms [Mythimna (Pseudaletia) unipuncta (Haworth) (Lepidoptera: Noctuidae)] to switchgrass (Panicum virgatum L.) and surrounding crops, survival and development were evaluated for larvae reared on leaves of switchgrass, corn (Zea mays L.), and miscanthus (Miscanthus x giganteus Greef and Deuter ex Hodkinson and Renvoize). Additional tests assessed the relationship between leaf position and the concentration of saponins (plant compounds which can provide protection from insect herbivores) and examined the effect of defoliation on switchgrass dry mass. Survival to adulthood was similar when larvae were reared on field-grown leaves of switchgrass and corn. However, lower larval mass (10 d) and delayed development of M. unipuncta (to pupation, adult emergence) suggest switchgrass is an inferior host relative to corn. When fed field-grown miscanthus, no larvae survived 10 d. Few differences were noted between switchgrass and corn grown under controlled (laboratory) conditions, but M. unipuncta survival seemed to decline rapidly when larvae were fed the fourth and fifth leaves of switchgrass. Switchgrass leaf samples collected from different leaf positions and stages of tiller maturity showed up to 10-fold differences in the concentration of the saponin protodioscin, with the greatest concentrations in the fourth and fifth leaves. However, other saponins showed an opposite pattern, indicating the role of protodioscin on insect development should be tested in isolation (e.g., by addition of the purified compound to an artificial diet). Defoliation trials indicated that extremely high M. unipuncta populations may be necessary to cause any significant reduction in switchgrass biomass. Collectively, results suggest M. unipuncta may not present a significant risk to biomass production in switchgrass, but that the spring emergence of switchgrass provides an alternate host for M. unipuncta before colonizing annual food and feed crops.

Development and feeding of fall armyworm on Miscanthus x giganteus and switchgrass.

Observations of fall armyworm, Spodoptera frugiperda (J.E. Smith) (Lepidoptera: Noctuidae), larvae infesting plots of Miscanthus x giganteus Greef and Deuter ex Hodkinson and Renvoize prompted laboratory-based tests of survival, development, and feeding preferences on leaf tissue from M. x giganteus and switchgrass, Panicum virgatum L. Survival from hatch to pupation was >70 and 50% for fall armyworms reared on switchgrass and M. x giganteus, respectively, although survival of the S. frugiperda rice strain was significantly greater than the corn strain on both crops. Developmental times from hatch to pupation or adult emergence showed effects of crop and S. frugiperda host strain, but analysis of an interaction revealed developmental times for the rice strain were similar on both crops, whereas corn strain larvae showed delayed development on M. x giganteus relative to switchgrass. Analysis of larval (10 d) and pupal masses showed a similar pattern, with effects of crop and an interaction (at 10 d), but only the mass of corn strain larvae feeding on M. x giganteus was reduced relative to the other crop and strain combinations. In choice tests, neonates of both corn and rice strains showed a strong preference for feeding on young tissues rather than mature leaves of M. x giganteus or switchgrass, but they also clearly favored corn, Zea mays L., leaves over either of the perennial grasses. Results indicate both plants are potential hosts for S. frugiperda, but additional information is needed to understand under which scenarios and to what degree fall armyworms may damage perennial grasses grown for biofuel production.

Macaque blood-derived antigen-presenting cells elicit SIV-specific immune responses.

Natural blood-borne antigen-presenting cells (APCs) were tested for their ability to augment antigen presentation for SIV vaccines. Fibrocytes and monocyte-derived dendritic cells (DCs) were isolated from multiple Macaca fascicularis. Macaque fibrocytes displayed the characteristic cellular morphology and stained positive for CD34 and collagen, as observed in human and murine fibrocytes. Macaque DCs were generated from monocytes by culturing in granulocyte-macrophage colony stimulating factor and interleukin-4 (IL-4). Two days after maturation, cells were enriched for the DC marker CD83. Fibrocytes and DCs were each transfected with green fluorescence protein expression plasmids or DNA expression vectors encoding all of the SIVmne structural and regulatory genes. Autologous DCs were re-infused into macaques subcutaneously (sc) following transfection; mixing with recombinant SIV antigens or inactivated whole SIV in vitro; or mock-treatment. Autologous monocyte-derived DCs pulsed with whole inactivated SIV were re-infused and elicited cellular and/or humoral responses in vivo in eight of ten vaccinated macaques.

Interaction of the cytoplasmic tail of CTLA-4 (CD152) with a clathrin-associated protein is negatively regulated by tyrosine phosphorylation.

CTLA-4 (CD152), high-avidity receptor for CD80 and CD86, is a powerful regulator of T cell activation. While CTLA-4 functions at the cell surface, it is primarily localized in intracellular vesicles and cycles to the cell surface. The CTLA-4 cytoplasmic domain contains sequences that direct its intracellular localization and regulate its signaling. Here we demonstrate that effector molecules involved in receptor trafficking and signaling interact with distinct, but overlapping, sequences in the CTLA-4 cytoplasmic domain. Using the yeast two-hybrid method, we demonstrate association of the mu2 subunit of AP-2, the clathrin-associated complex found in plasma membrane-associated coated pits, with the cytoplasmic tail of CTLA-4, but not CD28. The mu1 subunit of AP-1, found in Golgi-associated coated pits, associated with neither CTLA-4 nor CD28. Sequences required for interaction of mu2 and CTLA-4 were localized to residues, 161TTGVY in CTLA-4; this sequence is N-terminal to, but overlaps with, a previously identified SH2 binding motif, 165YVKM, involved in CTLA-4 signaling. Mu2 interacted preferentially with CTLA-4 when residue 165Y was nonphosphorylated, whereas a PI3 kinase SH2 domain interacted preferentially when 165Y was phosphorylated. In co-transfection experiments, both tyrosine residues in the cytoplasmic tail of CTLA-4 (165Y and 182Y) were phosphorylated by the T lymphocyte-associated tyrosine kinase, p56lck. Thus, phosphorylation of CTLA-4 residue 165Y may reciprocally regulate signaling and trafficking of CTLA-4 by determining which effector molecules bind to its cytoplasmic tail.

Relationship of peroxyacetyl nitrate to active and total odd nitrogen at northern high latitudes: influence of reservoir species on NOx and O3.

Measurements of peroxyacetyl nitrate (PAN), NO, NO2, HNO3, NOy (total odd nitrogen), and O3 were made in the high-latitude troposphere over North America and Greenland (35 degrees to 82 degrees N) during the Arctic Boundary Layer Expedition (ABLE 3A) (July-August 1988) throughout 0-to 6-km altitudes. These data are analyzed to quantitatively describe the relationships between various odd nitrogen species and assess their significance to global tropospheric chemistry. In the free troposphere, PAN was as much as 25 times more abundant than NOx. PAN to NOx ratio increased with increasing altitude and latitude. PAN was found to be the single most abundant reactive nitrogen species in the free troposphere and constituted a major fraction of NOy, PAN to NOy ratios were about 0.1 in the boundary layer and increased to 0.4 in the free troposphere. A 2-D global photochemical model with C1-C3 hydrocarbon chemistry is used to compare model predictions with measured results. A sizable portion (approximately 50%) of the gaseous reactive nitrogen budget is unaccounted for, and unknown organic nitrates and pernitrates are expected to be present. Model calculations (August 1, 70 degrees N) show that a major fraction of the observed NOx (50 to 70% of median) may find its source in the available PAN reservoir. PAN and the unknown reservoir species may have the potential to control virtually the entire NOx availability of the high latitude troposphere. It is predicted that the summer NOx and O3 mixing ratios in the Arctic/sub-Arctic troposphere would be considerably lower in the absence of the ubiquitous PAN reservoir. Conversely, this PAN reservoir may be responsible for the observed temporal increase in tropospheric O3 at high latitudes.

Regulation of plasminogen activator inhibitor mRNA levels in lipopolysaccharide-stimulated human monocytes. Correlation with production of the protein.

Peripheral blood monocytes are essential participants in processes that require pericellular plasminogen activation, a regulated proteolytic pathway that is greatly influenced by the relative concentrations of urokinase-type plasminogen activator (profibrinolytic) and plasminogen activator inhibitor type 2 (PAI-2) (anti-fibrinolytic). Monocyte synthesis of these molecules is inducible by bacterial lipopolysaccharide (LPS) although PAI-2 production is regulated over a much wider concentration range than is urokinase-type PA. The PAI-2 response of LPS-stimulated monocytes was investigated and found to be biphasic, with a peak of mRNA at 4-6 h after stimulation, a decrement in mRNA levels at 8-10 h, and a secondary increase at 16 h. The primary (early phase) response was studied in detail wherein PAI-2 protein production was found to depend on the levels of PAI-2 mRNA. The profiles of steady-state PAI-2 mRNA levels and PAI-2 protein production were parallel with respect to LPS concentration, time of exposure to LPS, and persistence of the response. PAI-2 mRNA accumulation was inducible by cycloheximide but prevented by actinomycin D. The increase in steady-state PAI-2 mRNA was mediated both by an increase in gene transcription and by stabilization of the mRNA once formed. Therefore, the initial phase of PAI-2 production by LPS-stimulated monocytes is determined by the amount of PAI-2 mRNA in these cells; levels of PAI-2 mRNA are controlled by several mechanisms, allowing for rapid variations in production of this molecule.

Atmospheric ammonia measurement using a VUV/photo-fragmentation laser-induced fluorescence technique.

Vacuum ultraviolet/photofragmentation laser-induced fluorescence has been demonstrated to be a highly specific and sensitive method for the quantitative measurement of atmospheric ammonia (NH(3)). The fluorescence detected in this approach results from the two 193-nm photon photofragmentation step NH(3)?NH(2)? NH(b(1)Sigma(+)) followed by the excitation of the NH(b(1)Sigma(+)) NH(c(1)Pi) transition via a 450-nm photon with final emission being observed from the NH(c(1) Pi) NH(a(1)Delta) transition at 325 nm. Limits of detection for the instrumentpresented here are < 10 pptv and < 4 pptv for 1- and 5-min integration periods, respectively, in ambient sampling conditions. The technique is free from interferences and system performance does not significantly degrade in adverse sampling conditions (i.e., rain, fog, clouds, haze, etc.). Spectroscopic selectivity in the NH(b(1)Sigma(+))?NH(c(1)Pi) transition is sufficient to resolve (15)NH(3) and (14)NH(3) contributions for use in atmospheric tracer studies. Average ammonia measurements at Stone Mountain, GA, ranged from approximately 110 pptv for air temperatures <5 degrees C to approximately 240 pptv for air temperatures >/=<5 degrees C over the period from Dec. 1987 to the end of Apr. 1988.

Differential regulation of tissue factor and plasminogen activator inhibitor by human mononuclear cells.

Fibrin is a hallmark of immune-mediated tissue lesions. The presence of fibrin in such lesions implies both the formation of fibrin via coagulation and the accompanying restriction of fibrinolysis, allowing fibrin to persist. Previous work has shown that human monocytes exposed to an inflammatory stimulus such as lipopolysaccharide (LPS) produce both tissue factor (TF) and plasminogen activator inhibitor--type 2 (PAI-2). These two proteins favor fibrin deposition, and evidence implies that cellular production of these two molecules may be linked. Another proinflammatory process pertinent to immune-mediated tissue damage and fibrin deposition is the response to alloantigen. Peripheral-blood mononuclear cells (PBM), consisting of lymphocytes and monocytes together, responded to alloantigen stimulation with differential expression of TF and PAI-2. PBM exposed to alloantigen developed high levels of TF activity, with no concomitant increase in PAI-2 activity or antigen. Alloantigen-stimulated PBM did not accumulate intracellular PAI-2, nor did they degrade PAI-2 added to cultures. This lack of PAI-2 production was not due to inadequate stimulation, as tritiated thymidine uptake and TF production demonstrated recognition of, and a vigorous reaction to, alloantigen. The divergent TF and PAI-2 responses of PBM exposed to alloantigen was maintained over 5 days and was reflected by mRNA profiles. These results imply that under specific physiologically relevant conditions, the procoagulant and antifibrinolytic effectors of inflammatory mononuclear cells can be independently regulated. This would imply more flexibility to monocyte mechanisms that favor fibrin deposition than previously thought.

Endotoxin-induced production of plasminogen activator inhibitor by human monocytes is autonomous and can be inhibited by lipid X.

Peripheral blood mononuclear cells (PBMs) produce both tissue factor and plasminogen activator inhibitor type 2 (PAI-2) in response to gram-negative bacterial lipopolysaccharide (LPS). The cellular roles in the tissue factor response have been previously elucidated, and we now report those roles in PAI-2 production. Monocytes are the only cells among LPS-stimulated PBMs that produce PAI-2 as assessed by measurement of PAI-2 activity and antigen. Concomitant immunohistochemistry demonstrated that monocytes contain PAI-2, with a greater number staining positively and more intensely after exposure to LPS. LPS-stimulated monocytes produced increased amounts of PAI-2 with or without addition of lymphocytes. Lymphocytes prestimulated with LPS and then washed did not induce PAI-2 production in monocytes to which they were added. Lipid X, a precursor in the biosynthetic pathway of lipid A and LPS, was able to inhibit LPS induction of monocyte PAI-2 in a dose-dependent manner. This inhibition was not due to cellular toxicity, the phospholipidlike nature of lipid X, interference with the PAI-2 assay, or monocyte production of a substance interfering with PAI-2. Lipid X was an effective inhibitor of PAI-2 production even when added up to 30 minutes after LPS.

Primary intracerebral lymphoma: a clinicopathological analysis of 14 patients presenting over a 10-year period in Sheffield.

Intracerebral disease was diagnosed in 14 out of 450 patients who presented with non-Hodgkin's lymphoma between January 1976 and January 1987. Twelve of the 14 presented after June 1980. Age ranged from 31 to 73 years and eight patients were male. Two patients had other tumours, and three had relevant associated immunosuppressive disorders. Radiological assessment of one further patient showed a cavitating bronchial carcinoma. Five patients were untreated, and one died before radiotherapy was complete. Eight patients completed courses of whole-brain irradiation; four of these received higher doses. All entered remission. Three patients are alive, between eight months and seven years after treatment. Of the remaining five, one never recovered intellectual function and died of bronchopneumonia; three died between eight and 30 months after treatment and autopsy showed severe radionecrosis of the brain with no residual tumour. All three had received higher doses of radiation and had undergone burrhole aspiration before treatment. Autopsy was refused on one patient who also appeared to have died from radionecrosis of the brain. Immunohistological examination in eight cases confirms that cerebral non-Hodgkin's lymphoma is a B-cell tumour. As other groups have found, its incidence appears to be rising. Survival rate is poor, and at least some deaths are related to both radiation necrosis and the bulk of residual tumour after diagnostic surgery.

Sequential two-photon laser-induced fluorescence detection of mercury.

We present initial experimental results on the two-photon laser-induced-fluorescence (TP-LIF) detection of mercury. This technique uses the sequential absorption of two photons, involving real atomic levels, to produce atomic fluorescence at a wavelength shorter than either pump wavelength. In this system, the pumping occurs at 254 and 408 nm, and optical sampling occurs at 185 nm. Based on preliminary findings using a first-generation system, together with modest evaluations of future improvements, we have estimated that the sensitivity of the TP-LIF mercury-detection system, under atmospheric sampling conditions, could approach ~10(3) atoms/cm(3). Under ideal sampling conditions, e.g., using He or Ar diluent gases, the detection limit could be as low as a few atoms per cubic centimeter. Attempts to realize both of these goals will soon be undertaken.

Single photon laser-induced fluorescence detection of NO and SO(2) for atmospheric conditions of composition and pressure.

Reported here are laboratory results from a laser-induced fluorescence (LIF) study of the molecules NO and SO(2) in which both the selectivity and sensitivity of the LIF method are examined. The laser excitation of these molecules occurred at 226 and 222 nm, respectively. The laser system employed consisted of a Nd: YAG-driven Quanta-Ray PDL dye laser, the fundamental of which was frequency doubled, and this output, in turn, was then frequency mixed with the Nd:YAG fundamental at 1064 nm. Two different dyes were required for generating the 226- and 222-nm wavelengths. To make these results as relevant as possible to the ultimate development of an atmospheric airborne field sampling system all experiments were carried out in atmospheric conditions of pressure and composition. In addition to the experimental data provided there has also been presented a theoretical assessment of the signal strength for both the NO and SO(2) LIF systems, and these results have been compared with the experimentally measured values. Current state-of-the-art technology would suggest that both NO and SO(2) can be measured by the LIF technique in atmospheric conditions at concentration levels of a few pptv.