Optimization of a Chemiluminescent Dot-Blot Immunoassay for Detection of Potato Viruses

Vegetative propagation of potato leads to virus accumulation, resulting in significant yield losses and reduced quality. Virus identification is critical for developing disease management strategies and measuring seed lot health. The most widely used method of virus diagnosis in seed potatoes is a post-harvest test, for which the enzyme-linked immunosorbent assay (ELISA) is often used. ELISA was previously modified by substituting microtiter plates with membranes to develop a more flexible and inexpensive assay. We optimized a dot-blot immunoassay with viral proteins bound to a polyvinylidene fluoride (PVDF) membrane and detection of the proteins with alkaline phosphatase-labeled antibodies and a chemiluminescence reagent. The assay was tested for detection of viruses of seven genera. We have also altered the assay by spotting an antibody array onto a PVDF membrane and tested it for its potential uses as a diagnostic tool for Potato virus Y, Hosta virus X, and Potato leafroll virus.     

Reactive histiocytosis of the orbit and posterior segment in a dog

We present a case of reactive histiocytic disease involving the orbit, optic nerve, retina, and choroid in a Border Collie dog initially presenting for vision loss. Long-term partial return of vision has been achieved with systemic immunosuppression. Anterior segment and ocular surface manifestations of reactive histiocytic disease in dogs are relatively common. Posterior segment and orbital involvement, however, are minimally documented in the existing literature. To the authors' knowledge, this is the first report of disease confined to the orbit and posterior segment as well as the first report of vision loss as a presenting complaint for reactive histiocytic disease. Clinical, magnetic resonance imaging, cytologic, and histopathologic findings are reviewed.     

Fire and Nitrogen Effects on Purple Threeawn (Aristida purpurea) Abundance in Northern Mixed-Grass Prairie Old Fields

Purple threeawn (Aristida purpurea Nutt. varieties) is a native grass capable of increasing on rangelands, forming near monocultures, and creating a stable state. Productive rangelands throughout the Great Plains and Intermountain West have experienced increases in purple threeawn abundance, reducing overall forage quality. Our objectives were to 1) reveal the effects of prescribed fire and nitrogen amendments on purple threeawn abundance and 2) assess nontarget plant response posttreatment. Season of fire (no fire, summer fire, fall fire) and nitrogen addition (0 kg N · ha^?1, 46 kg N · ha^?1, and 80 kg N · ha^?1) were factorially arranged in a completely randomized design and applied to two similar sites in southeastern Montana. We evaluated fire and nitrogen effects on purple threeawn basal cover, relative composition, and current-year biomass one growing season postfire at two sites treated during different years. Spring weather following fire treatments was very different between years and subsequently impacted community response. Initial purple threeawn biomass at both sites was 1 214 ± 46 kg · ha^?1 SEc. When postfire growing conditions were wet, current-year biomass of purple threeawn was reduced 90% and 73% with summer and fall fire, respectively. Under dry postfire growing conditions, purple threeawn current-year biomass was reduced 73% and 58% with summer and fall fire, respectively. Nitrogen additions had no effect on purple threeawn current-year biomass at either site. Current-year biomass of C₃ perennial grass doubled with nitrogen additions and was not impacted by fire during a wet spring. Nitrogen additions and fire had no effect on C₃ perennial grass current-year biomass following a dry spring. Prescribed fire appears to be a highly effective tool for reducing purple threeawn abundance on semiarid rangelands, with limited detrimental impacts to nontarget species.     

Production and Environmental Implications of Equine Grazing

Horses' physical and digestive well-being is often enhanced when allowed to graze on pastures. Furthermore, a well-managed pasture can contribute to economic viability. Grazing can however have deleterious effects on the environment if not properly managed. Although equine grazing, defecating, and ground trampling behavior is unique from that of other livestock species, pasture management practices are often based on those derived from cattle grazing. This review summarizes the current knowledge of impacts of equine grazing on pasture quality and environment and identifies gaps where further information is needed to formulate and recommend sustainable grazing methods specific to equine.     

The Marine Natural Product Manzamine A Targets Vacuolar ATPases and Inhibits Autophagy in Pancreatic Cancer Cells

Manzamine A, a member of the manzamine alkaloids, was originally isolated from marine sponges of the genus Haliclona. It was recently shown to have activity against pancreatic cancer cells, but the precise mechanism of action remained unclear. To further our understanding of the mechanism of action of manzamine A, chemogenomic profiling in the yeast S. cerevisiae was performed, suggesting that manzamine A is an uncoupler of vacuolar ATPases. Fluorescence microscopy confirmed this effect on yeast vacuoles, where manzamine A produced a phenotype very similar to that of the established v-ATPase inhibitor bafilomycin A1. In pancreatic cancer cells, 10 µM manzamine A affected vacuolar ATPase activity and significantly increased the level of autophagosome marker LC3-II and p62/SQSTM1 as observed by western blot analysis. Treatment with manzamine A in combination with bafilomycin A1 (inhibitor of autophagosome-lysosome fusion) did not change the levels of LC3-II when compared to cells treated with bafilomycin A1 alone, suggesting that manzamine A is a potential inhibitor of autophagy by preventing autophagosome turnover. As autophagy is essential for pancreatic tumor growth, blocking this pathway with manzamine A suggests a promising strategy for the treatment of pancreatic cancer.     

Trypanocidal Activity of Marine Natural Products

Marine natural products are a diverse, unique collection of compounds with immense therapeutic potential. This has resulted in these molecules being evaluated for a number of different disease indications including the neglected protozoan diseases, human African trypanosomiasis and Chagas disease, for which very few drugs are currently available. This article will review the marine natural products for which activity against the kinetoplastid parasites; Trypanosoma brucei brucei, T.b. rhodesiense and T. cruzi has been reported. As it is important to know the selectivity of a compound when evaluating its trypanocidal activity, this article will only cover molecules which have simultaneously been tested for cytotoxicity against a mammalian cell line. Compounds have been grouped according to their chemical structure and representative examples from each class were selected for detailed discussion.     

Are hotspots of evolutionary potential adequately protected in southern California?

Reserves are often designed to protect rare habitats, or “typical” exemplars of ecoregions and geomorphic provinces. This approach focuses on current patterns of organismal and ecosystem-level biodiversity, but typically ignores the evolutionary processes that control the gain and loss of biodiversity at these and other levels (e.g., genetic, ecological). In order to include evolutionary processes in conservation planning efforts, their spatial components must first be identified and mapped. We describe a GIS-based approach for explicitly mapping patterns of genetic divergence and diversity for multiple species (a “multi-species genetic landscape”). Using this approach, we analyzed mitochondrial DNA datasets from 21 vertebrate and invertebrate species in southern California to identify areas with common phylogeographic breaks and high intrapopulation diversity. The result is an evolutionary framework for southern California within which patterns of genetic diversity can be analyzed in the context of historical processes, future evolutionary potential and current reserve design. Our multi-species genetic landscapes pinpoint six hotspots where interpopulation genetic divergence is consistently high, five evolutionary hotspots within which genetic connectivity is high, and three hotspots where intrapopulation genetic diversity is high. These 14 hotspots can be grouped into eight geographic areas, of which five largely are unprotected at this time. The multi-species genetic landscape approach may provide an avenue to readily incorporate measures of evolutionary process into GIS-based systematic conservation assessment and land-use planning.     

Evaluation of Commercial‐scale Approaches for Cryopreservation of White Crappie,Pomoxis annularis,Sperm

Crappie, Pomoxis spp., are popular game fish throughout North America and are produced by public and private hatcheries. However, production is limited by a lack of information on tank culture and induced spawning methods. Development of techniques for storage of sperm and in vitro fertilization would increase flexibility in spawning. Therefore, techniques for sperm cryopreservation were examined in white crappie, Pomoxis annularis. Sperm from adult wild white crappie were used to evaluate sperm extender, cryoprotectant agent and concentration, and cooling technique based on post‐thaw sperm motility. Percent egg fertilization was also compared between sperm stored in the two best cryopreservation protocols and two different osmotic activator solutions. Sperm were cryopreserved using treatment combinations of two extenders (350 mOsmol/kg Hanks' balanced salt solution [HBSS] and 350 mOsmol/kg Ca²⁺free HBSS) and two cryoprotectants (dimethyl sulfoxide [DMSO] and methanol) at concentrations of 5, 10, and 15% that were cooled at four different rates: 5, 10, 20, and 40 C/min. Post‐thaw sperm motility and fertilization rates indicated white crappie sperm can be cryopreserved using either extender, cryoprotectants of either 5% DMSO or 10% methanol, and cooling at 40 C/min. A follow‐up experiment demonstrated sperm in suspensions on ice retained viability after overnight transport.