Production and use of monoclonal antibodies for molecular characterization, affinity purification, and serological detection of economically significant plant viruses

Hybridomas secreting monoclonal antibodies (M-Abs) against three isolates of barley yellow dwarf virus (BYDV) were established. Two M-Abs were generated against the MAV isolate, one against RPV, and six against P-PAV. None of the M-Abs reacted with healthy host components. Reactions of M-Abs, or unlabelled polyclonal antisera, in an indirect enzyme-linked immunosorbent assay (ELISA) indicate the presence of a common epitope on all three virus isolates. BYDV dissociated when incubated in carbonate-bicarbonate coating buffer at pH 9.6; stabilization was achieved by predialysis with either 2% formaldehyde or 2% glutaraldehyde. In indirect ELISA, unlabelled polyclonal antisera bound to both stabilized and dissociated homologous and heterologous BYDV isolates. However, conjugated polyclonal antisera were incapable of binding to any dissociated isolates or to stabilized heterologous isolates;M-Abs, independently produced against each of the BYDV isolates, were able to efficiently detect all BYDV isolates in serologically specific electron microscopy (SSEM). SSEM would not, however, detect unrelated viruses. The procedure was highly sensitive, detecting 7.5 pg of virus, and specific; SSEM performed on a mixture of BYDV and soybean mosaic virus (SMV) could detect only BYDV particles;Two M-Abs, specific for different epitopes on SMV, were used in a double-antibody sandwich ELISA (M-Ab-ELISA). When compared witha polyclonal antibody solid phase radioimmunoassay (SPRIA), M-Ab-ELISA was more sensitive than SPRIA and detected less than 10 ng of SMV/ml as compared to 25 ng SMV/ml, respectively. When seeds from 33 field plots, in which 0%, 30%, or 50% of the soybean plants had been inoculated with SMV, were assayed by both systems, results of the two tests correlated for 31 of 33 (94%) seed samples;Immunoaffinity columns, prepared by covalently binding M-Abs to an agarose support matrix, were used to purify SMV. Affinity purified SMV was substantially identical in infectivity, u.v. absorbance profile, 280/260 nm absorbance ratio, sedimentation coefficient, electrophoretic pattern of coat protein, morphology, and antigenicity, to virus purified by standard procedures

New Zealand Guidelines for cyanobacteria in recreational fresh waters: Interim Guidelines

This document is divided into four main sections, plus 14 appendices. Section 1. Introduction provides an overview of the purpose and status of the document as well as advice on who should use it. Section 2. Framework provides a background to the overall guidelines approach, recommendations on agency roles and responsibilities, and information on the condition of use of this document. Section 3. Guidelines describes the recommended three-tier monitoring and action sequence for planktonic and benthic cyanobacteria. Section 4. Sampling provides advice on sampling planktonic and benthic cyanobacteria. The appendices give further background information and include templates for data collection and reporting, including: • background information on known cyanotoxins and their distribution in New Zealand • information on the derivation of guideline values • photographs of typical bloom events • a list of biovolumes for common New Zealand cyanobacteria • templates for field assessments • suggested media releases and warning sign templates. A glossary provides definitions for abbreviations and terms used in these guidelines

Effects of fire and treefalls on Japanese climbing fern and native species groundcover in a restored longleaf pine savanna

Recurrent disturbances play an important role in maintaining longleaf pine savannas. Windstorms and fires contribute to the heterogeneity of pine savanna understory through generation of canopy gaps and fuel accumulation, distribution and consumption. The combination of windstorms and fires promotes native herbaceous species, but also promotes invasion of Japanese climbing fern (Lygodium japonicum). Lygodium japonicum is an invasive fern with subterranean rhizomes and copious spore production invading pine savannas along the southeastern Gulf Coast Region. This dissertation examines how fire, previous windstorm disturbances, and animal disruptions influence L. japonicum and native species abundance in a restored longleaf pine savanna. Studies were conducted at Girl Scout Camp Whispering Pines, an upland mesic site containing longleaf pine in Tangipahoa Parish, Louisiana. I experimentally examined L. japonicum response to various fire severities during prescribed fire by manipulating fine fuels in the understory. Three fine-fuel treatments (increased, reduction, unaltered) were applied to plots containing fern genets. The effect of fire severity through duration of heating was transient on frond emergence, suggesting that established fern genets survive subsequent fires. Native groundcover species abundance and composition was also transiently influenced by fire severity, as examined in a post hoc fuel manipulation study. Abundance and composition of native species within localized areas differed based on heterogeneity of fire severity, indicating sensitivity to prolonged heating. Heat released, a product of fuel accumulation, may stunt recovery of native groundcover in localized areas and contribute to understory patches susceptible to invasion by non-native species. Animal biopedturbations were strongly associated with stump locations, which provided refuge and foraging opportunities in the understory. Biopedturbations did not, however, promote or suppress L. japonicum frequency or density. Windstorms and fire disturbances contributed to heterogeneity of groundcover and, thus influenced L. japonicum spread. Particular growth characteristics of L. japonicum enabled persistence in certain locations. Areas with open canopy, where fire severity was low, were at higher risk of invasion than areas under pine trees. Fire is essential for restoration, yet promotes invasion by L. japonicum. Careful consideration of disturbance regime and characteristics of invasive species are required for successful maintenance of longleaf pine savannas

The Black Death and the Future of the Plague

This essay summarizes what we know about the spread of Yersinia pestis today, assesses the potential risks of tomorrow, and suggests avenues for future collaboration among scientists and humanists. Plague is both a re-emerging infectious disease and a developed biological weapon, and it can be found in enzootic foci on every inhabited continent except Australia. Studies of the Black Death and successive epidemics can help us to prepare for and mitigate future outbreaks (and other pandemics) because analysis of medieval plagues provides a crucial context for modern scientific discoveries and theories. These studies prevent us from stopping at easy answers, and they force us to acknowledge that there is still much that we do not understand

Systems Biology Of Gene Regulation Across Scales: From Single Molecules To Cellular Identities

Gene regulation takes many forms and is responsible for phenotypes at the scale of individual molecules up through the scale of complex tissue functions. At the smallest level, single-base modifications of individual mRNA molecules transcribed from the same gene can lead to functionally different protein products. In the first chapter of this thesis, I develop a new method, inoFISH, and associated analytical tools to visualize and quantify RNA editing with single molecule resolution in single mammalian cells. Using this new method in conjunction with mathematical modeling I show that the heterogeneity of single-cell mRNA editing rates across a population depends on the gene of interest. Further, I characterize subcellular localization patterns of edited and unedited mRNAs. At the other end of the spectrum, the regulation of transcriptome-wide patterns of gene expression can underpin cellular identities. In the second chapter of this thesis I develop a new experimental design and analytical framework for prioritizing lists of transcription factors that can be used for directed changes of cellular identity. With Perturbation Panel Profiling (P3), I show that cardiomyocyte lineage-driving transcription factors are more frequently up-regulated, or “perturbable”, than other highly expressed transcription factor genes. I subsequently demonstrate that a known cocktail of cardiomyocyte-perturbable transcription factors enables cardiac transdifferentiation of several types of human fibroblasts. Lastly I extend perturbability-based selection of transcription factors to another biological context, i.e., fibroblast reprogramming to pluripotency. I show that fibroblast-perturbable factor knockdown often enables more efficient fibroblast reprogramming. Together, my thesis makes critical steps toward understanding and engineering gene regulation through the development of a diverse array of methods, experimental designs, and analytical frameworks

An Assessment of the Evolutionary Stability of Distyly in Hedyotis Caerulea (Rubiaceae)

Distylous species of flowers possess two distinct floral morphs that are generally found in equal numbers in naturally occurring populations. The flower form that has a relatively long style is called a pin the form with a short style is a thrum. Within the distylous mating system, selfing and intramorph mating are prevented due to the spatial separation of stigmas and anthers and by a self-incompatibility system that is inherited along with the dimorphic floral structure and ancillary polymorphisms. However, a breakdown of distyly has been documented in several families, either through the development of completely separate sexes (dioecy), or, more frequently, through the development of monomorphy and self-fertile homostyles. Here several populations of Hedyotis. caerulea, a perennial spring-flowering herb native to eastern North America, were surveyed in the Cleveland Metroparks and sampled to investigate the degree of distyly present and to detect any movement of the mating system towards selfing or dioecy. Morphometric analysis of the variation within and across populations of stigma height and anther height indicate that this species exhibits reciprocal herkogamy. Nearly all populations surveyed were isoplethic (i.e., contained a morph ratio of pins to thrums that was not different from 1:1), which is found when the species\u27 mating system promotes intermorph pollen transfer (dissortative mating) with nearly 100 outcrossing. There was no indication that the self-incompatibility system has broken down with no movement towards homostyly. Variation was present, however, in these primary traits as well as in stigma length, corolla tube width, pollen diameter, pollen count, dry weight, and seed set of the flowers collected among populations. Several ancillary floral traits, including corolla tube shape, pollen diameter, and length of stigmatic papilla, likewise varied. Across populations, the two morphs invested a similar amount of biomass in the production of pollen and seed, suggesting that each morph ha

An Assessment of the Evolutionary Stability of Distyly in Hedyotis Caerulea (Rubiaceae)

Distylous species of flowers possess two distinct floral morphs that are generally found in equal numbers in naturally occurring populations. The flower form that has a relatively long style is called a pin the form with a short style is a thrum. Within the distylous mating system, selfing and intramorph mating are prevented due to the spatial separation of stigmas and anthers and by a self-incompatibility system that is inherited along with the dimorphic floral structure and ancillary polymorphisms. However, a breakdown of distyly has been documented in several families, either through the development of completely separate sexes (dioecy), or, more frequently, through the development of monomorphy and self-fertile homostyles. Here several populations of Hedyotis. caerulea, a perennial spring-flowering herb native to eastern North America, were surveyed in the Cleveland Metroparks and sampled to investigate the degree of distyly present and to detect any movement of the mating system towards selfing or dioecy. Morphometric analysis of the variation within and across populations of stigma height and anther height indicate that this species exhibits reciprocal herkogamy. Nearly all populations surveyed were isoplethic (i.e., contained a morph ratio of pins to thrums that was not different from 1:1), which is found when the species\u27 mating system promotes intermorph pollen transfer (dissortative mating) with nearly 100 outcrossing. There was no indication that the self-incompatibility system has broken down with no movement towards homostyly. Variation was present, however, in these primary traits as well as in stigma length, corolla tube width, pollen diameter, pollen count, dry weight, and seed set of the flowers collected among populations. Several ancillary floral traits, including corolla tube shape, pollen diameter, and length of stigmatic papilla, likewise varied. Across populations, the two morphs invested a similar amount of biomass in the production of pollen and seed, suggesting that each morph ha

The development of serodiagnostic procedures for the detection and quantitation of soybean mosaic virus in seeds

A direct sandwich solid-phase immunoassay, using antibody-coated polystyrene beads, was developed for the detection and quantitation of soybean mosaic virus (SMV) in seeds. Virus-specific IgG was passively adsorbed to the solid phase, and virus antigen bound by the immobilized antibody was demonstrated by subsequent binding of anti-SMV IgG labelled with either (\u273)H (for SPRIA) or alkaline phosphatase (for ELISA). Optimal conditions for coating the solid phase, specificity of the immune reaction, and other kinetics and sensitivities of the assay were also defined for the purpose of standardizing the assay. A sensitivity level of 5 to 25 ng of purified SMV/ml was obtained, depending on the nature of the label used. The addition of healthy seed extracts did not interfere with the assay\u27s ability to detect purified virus, indicating its potential for seed analysis. The assay easily distinguished infected from healthy seed extracts, as well as identifying various levels of infection;Technical simplicity, reproducibility and versatility of the solid-phase procedure make it diagnostically useful for detecting SMV in individual and bulked seeds

Biology and control of foot rot of French beans

Imperial Users onl