The present invention relates to isolated nucleic acid molecules encoding endogenous cotton chitinases and corresponding promoters, which are preferentially expressed in secondary walled cells during secondary wall deposition. The polypeptide encoded by the nucleic acid molecule, a DNA construct linking the isolated nucleic acid molecule with a promoter, the DNA construct incorporated in an expression system, a host cell, a plant, or a plant seed are also disclosed. The present invention also relates to a DNA construct linking the isolated promoters with a second DNA as well as expression systems, host cells, plants, or plant seeds containing the DNA construct. Methods of imparting resistance to insects and fungi, regulating the fiber cellulose content, and methods of expressing a gene preferentially in secondary walled cells during secondary wall deposition are also disclosed.

Fiber cells of cotton (Gossypium hirsutum L. and other Gossypium species including G. barbadense L., G. arboreum L., and G. herbaceous L.), a crop of enormous economic importance to world-wide agriculture, are differentiated epidermal cells of the seed coat. At maturity, the fiber cell, considered from inside to outside, consists of a cell lumen, secondary cell wall, primary cell wall, and thin waxy cuticle. The primary cell wall is made up of pectic compounds, hemicellulose components, cellulose, and protein. The secondary cell wall consists mainly (about 95%) of cellulose with small percentages of other components not yet conclusively identified.
Cotton fiber development is characterized by the stages of initiation, primary cell wall deposition, secondary cell wall deposition, and dessication. During the primary wall stage of fiber development, primary wall deposition occurs to facilitate fiber elongation. During the secondary wall stage of fiber development, secondary wall deposition occurs to accomplish fiber thickening. Primary and secondary wall deposition involve the synthesis of all the cell wall components characteristic of each stage and the assembly of the molecules into an organized cell wall outside the plasma membrane. Many hundred of genes are required for the differentiation and development of plant fiber. Work on in vitro translated fiber proteins (Delmer et al., "New Approaches to the Study of Cellulose Biosynthesis," J. Cell Sci. Suppl., 2:33–50 (1985)), protein isolated from fiber (Graves and Stewart, "Analysis of the Protein Constituency of Developing Cotton Fibers," J. Exp. Bot., 39:59–69 (1988)), and analysis of particular genes (Wilkins et al., "Molecular Genetics of Developing Cotton Fibers," in Basra, ed., Cotton Fibers: Developmental Biology, Quality Improvement, and Textile Processing, Haworth Press:New York, p. 231–270 (1999)) clearly suggests differential gene expression during various developmental stages of the cell. However, only a few of the genes involved in the biosynthesis of the large numbers of fiber-specific or fiber-enhanced structural proteins, enzymes, polysaccharides, or waxes have been identified (John et al., "Gene Expression in Cotton (Gossypium hirsutum L.) Fiber: Cloning of the mRNAs," Proc. Natl. Acad. Sci. USA, 89:5769–5773 (1992); John, "Characterization of a Cotton (Gossypium hirsutum L.) Fiber mRNA (Fb-B6)," Plant Physiol., 107:1477–1478 (1995)). Since these genes and their interactions with environment determine the quality of fiber, their identification and characterization is considered to be an important aspect of cotton crop improvement.
In particular, how secondary cell walls are synthesized, how they aid plant function, adaptation, and defense, and how their properties translate into industrial utility are important questions related to basic biological mechanisms, ecology, and plant improvement. Plant secondary cell walls are synthesized in some specialized cell types to facilitate particular functions, such as long-range conduction of water (tracheary elements), control of transpiration (guard cells), and dispersal of seeds (cotton fibers). These secondary cell walls have a higher content of high tensile strength cellulose, usually exceeding 40% by weight, and are much thicker than primary cell walls. Consequently, their hemicellulose, pectin, and protein content is reduced, with the most extreme reduction occurring in the case of cotton fiber secondary cell walls which are about 95% cellulose. On the other hand, during primary wall deposition, the cellulose content is typically 9–30% (w/w) (Meinert et al., "Changes in Biochemical Composition of the Cell Wall of the Cotton Fiber During Development," Plant Physiol., 59:1088–1097 (1977); Darvill et al., "The Primary Cell Walls of Flowering Plants," The Biochemistry of Plants, 1:91–162 (1980); Smook, Handbook for Pulp and Paper Technologists, Vancouver, Canada:Angus Wilde Publications, p. 15 (1992)). Because secondary cell walls are strong and represent a bulk source of chemical cellulose, they have been exploited as important renewable resources, for example in wood and cotton fibers.
Cotton fiber cells have two distinct developmental stages that include secondary wall deposition. Many studies of fiber length and weight increase, morphology, cell wall composition, cellulose synthesis rates, and gene expression have confirmed the summary of the stages of fiber development presented below, and recent reviews contain many primary references to confirm these facts (Delmer, "Cellulose Biosynthesis in Developing Cotton Fibers," in Basra, ed., Cotton Fibers: Developmental Biology, Quality Improvement, and Textile Processing," New York, N.Y.:Haworth Press, pp. 85–112 (1999); Ryser, "Cotton Fiber Initiation and Histodifferentiation," in Basra ed., Cotton Fibers: Developmental Biology, Quality Improvement, and Textile Processing," New York, N.Y.:Haworth Press, pp. 1–46 (1999); Wilkins et al., "Molecular Genetics of Developing Cotton Fibers, in Basra, ed., Cotton Fibers: Developmental Biology, Quality Improvement, and Textile Processing," New York, N.Y.:Haworth Press, pp. 231–270 (1999)). Following fiber initiation by bulging of an epidermal cell above the surface, fiber elongation begins. Primary wall deposition is required to facilitate fiber elongation, and primary wall deposition continues alone for at least 12 days after fiber initiation. This period represents exclusively the primary wall stage of fiber development. Then, secondary wall deposition begins while primary wall deposition continues, albeit usually at a slower rate. This stage of fiber development represents the transition between primary and secondary wall deposition, and it typically begins in G. hirsutum L. between 14–17 days post anthesis (DPA). Subsequently, fiber elongation and primary wall deposition cease, typically between 18–24 DPA, and secondary wall deposition persists exclusively until 34–50 DPA. Variation in the time of initiation and duration of each phase of fiber development depends on the cotton cultivar and the temperature conditions (DeLanghe, "Lint Development" in Mauney, eds., Cotton Physiology, pp. 325–349, The Cotton Foundation, Memphis, Tennessee (1986); Haigler et al., "Cultured Cotton Ovules as Models for Cotton Fiber Development Under Low Temperatures," Plant Physiol., 95:88–96 (1991); Thaker et al., "Genotypic Variation and Influence of Diurnal Temperature on Cotton Fiber Development," Field Crops Research, 22:129–141 (1989)). For example, in field-grown G. barbadense L., extensive secondary wall deposition did not occur until after 20 DPA, elongation continued until 39 DPA, and secondary wall deposition ceased at 48 DPA (Schubert et al., "Growth and Development of the Lint Fibers of Pima S-4 Cotton," Crop Sci., 16:539–543 (1976)).
The rates of cellulose synthesis change from low, to medium, to high, respectively, at the primary wall, transition, and secondary wall stages of fiber development (Meinert et al., "Changes in Biochemical Composition of the Cell Wall of the Cotton Fiber During Development," Plant Physiol., 59:1088–1097 (1977); Martin, "Cool-Temperature-Induced Changes in Metabolism Related to Cellulose Synthesis in Cotton Fibers, Ph.D. dissertation, Texas Tech University, Lubbock, Tex., U.S.A. (1999)). An example is found in fibers developing on cultured cotton ovules at an optimum temperature of constant 34° C., which maximizes the rate of progression through the stages of fiber development. Combining results from fibers on ovules of two cotton cultivars of G. hirsutum L. cultured under this condition, primary wall deposition along with a low rate of cellulose synthesis occurs until 12–14 DPA, the transition between primary and secondary wall deposition and an intermediate rate of cellulose synthesis begins at 14–16 DPA, and secondary wall deposition continues along with a high rate of cellulose synthesis beginning at 16–21 DPA (Martin, "Cool-Temperature-Induced Changes in Metabolism Related to Cellulose Synthesis in Cotton Fibers, Ph.D. dissertation, Texas Tech University, Lubbock, Tex. U.S.A. (1999)). Other biochemical features demonstrate that the initiation of secondary wall deposition via an intermediate rate of cellulose synthesis at the transition stage marks a distinct developmental event: the cellulose content of new wall material rises sharply so that the overall percentage of cellulose in the whole fiber wall doubles in one day (Meinert et al., "Changes in Biochemical Composition of the Cell Wall of the Cotton Fiber During Development," Plant Physiol., 59:1088–1097 (1977)), the respiration rate transiently declines, and the intercellular pools in the fiber of UDP-glucose and glucose-6-P begin to rise (Martin, "Cool-Temperature-Induced Changes in Metabolism Related to Cellulose Synthesis in Cotton Fibers, Ph.D. dissertation, Texas Tech University, Lubbock, Tex. U.S.A. (1999)). These are signs of an abrupt onset of secondary wall deposition (DeLanghe, "Lint Development" in Mauney, eds., Cotton Physiology, pp. 325–349, The Cotton Foundation, Memphis, Tenn. (1986)).
Primary and secondary wall deposition appear to be controlled by different genetic factors (Kohel et al., "Fiber Elongation and Dry Weight Changes in Mutant Lines of Cotton," Crop Sci., 14:471–474 (1974)), and both stages can likely be manipulated independently by genetic engineering to achieve the longer, stronger, finer (smaller diameter), and more mature (as related to the secondary wall thickness) fiber that the textile industry desires. However, this goal can only be achieved by knowing more about the genes that control and contribute to each stage of fiber development.
After the fibers mature, the protective boll opens and the dried fiber often hangs for several weeks on the plant until the whole crop matures (unless stopped by killing cold temperatures) and vegetative growth dies or is killed chemically to allow harvest. During this period, fibers are subject to degradation by enzymatic activity of fungi, which is often enhanced by wet fall weather (Simpson et al., "The Geographical Distribution of Certain Pre-Harvest Microbial Infections of Cotton Fiber in the U.S. Cotton Belt," Plant Disease Reporter, 55:714–718 (1971)). In some years, this field-waiting time causes substantial deterioration of the grade of the fiber so that the producer receives a discounted price and the production of quality yams and fabrics is jeopardized. Therefore, cotton production efficiency will be improved by more knowledge of how to bring the fibers undamaged from the field to textile plant. Relevant to achieving this goal is a better understanding of endogenous protections against fungal degradation that could be introduced or enhanced in the fiber.
Particularly because of their defensive role in plants (Gooday, "Aggressive and Defensive Roles for Chitinases," in Jolles, eds., Chitin and Chitinases, Birkhäuser Verlag:Basel, pp. 157–170 (1999)), numerous chitinase genes and proteins have been characterized in diverse plant species. The chitinase gene and protein family has been the subject of many reviews (including Graham et al., "Cellular Coordination of Molecular Responses in Plant Defense," Molecular Plant-Microbe Interactions: MPMI, 4:415–422 (1991); Cutt et al., "Pathogenesis-Related Proteins," in Boller, eds., Genes Involved in Plant Defense, Springer Verlag/N.Y. pp. 209–243 (1992); Meins et al., "The Primary Structure of Plant Pathogenesis-Related Glucanohydrolases and Their Genes," in Boller, eds., Genes Involved in Plant Defense, Springer Verlag/N.Y., p. 245–282 (1992); Collinge et al., "Plant Chitinases," Plant J., 3:31–40 (1993); Sahai et al., "Chitinases of Fungi and Plants: Their Involvement in Morphogenesis and Host-Parasite Interaction," FEMS Microbiology Rev., 11:317–338 (1993); Meins et al., "Plant Chitinase Genes," Plant Molecular Biology Reporter, 12:522–528 (1994); Hamel et al., "Structural and Evolutionary Relationships Among Chitinases of Flowering Plants," Journal of Molecular Evolution, 44:614–624 (1997)) and of an edited book (Jolles, eds. Chitin and Chitinases, Birkäuser Verlag:Basel, 340 pp (1999)). These sources contain many primary references to the well known facts summarized below. Chitinases are among a group of genes that are inducible in plants by pathogen attack, corresponding to the frequent occurrence of chitin in fungal cell walls and insect exoskeletons. In their defensive role, chitinases catalyze the hydrolysis of chitin. Structural chitin occurs as crystalline microfibrils composed of a linear homopolymer of β-1,4-linked N-acetyl-D-glucosamine residues, (GlcNAc)n. Chitin hydrolysis defends the plant against predators or pathogens, particularly invading fungi, by weakening or dissolving their body structure. Especially in combination with β-1,3-glucanases that serve to uncoat the chitin microfibrils, chitinases can inhibit the growth of many fungi by causing hyphal tip lysis due to a weakened hyphal wall. This has been shown by inhibition of fungal growth in cultures as well as in transgenic plants that exhibit reduced pathogen damage in correlation with increased chitinase activity. Gene expression or activity of chitinases with a probable defensive function have previously been characterized in cotton leaves and roots (Liu et al., "Detection of Pathogenesis-Related Proteins in Cotton Plants," Physiological and Molecular Plant Pathology, 47:357–363 (1995); Hudspeth et al., "Characterization and Expression of Chitinase and 1,3-β-Glucanase Genes in Cotton," Plant Molecular Biology, 31:911–916 (1996); Dubery et al., "Induced Defence Responses in Cotton Leaf Disks by Elicitors From Verticillium dahliae," Phytochemistry, 44: 1429–1434 (1997)).
Some chitinases are induced upon fungal invasion, accumulating around invading fungal hyphae (Benhamou et al., "Subcellular Localization of Chitinase and Its Potential Substrate in Tomato Root Tissues Infected With Fusarium Oxysporium F.Sp. Radicislycopersici," Plant Physiology, 92:1108–1120 (1990); Wubben et al., "Subcellular Localization of Plant Chitinases and 1,3-β-Glucanases in Cladosporium Fulvum (Syn. Fulvia Fulva)-Infected Tomato Leaves," Physiological and Molecular Plant Pathology 41:23–32 (1992)). Other chitinases apparently occur constitutively in plant parts that are particularly susceptible to invasion such as epidermal cells, root cortical cells, stomates, flower parts, and vascular cells. These conclusions arise from both localization of chitinase mRNA by in situ hybridization and analysis of patterns of expression of the GUS reporter gene under control of chitinase promoters (Samac et al., "Developmental and Pathogen-Induced Activation of the Arabidopsis Acidic Chitinase Promoter," The Plant Cell, 3:1063–1072 (1991); Zhu et al., "Stress Induction and Developmental Regulation of a Rice Chitinase Promoter in Transgenic Tobacco," The Plant Journal, 3:203–212 (1993); Büchter et al., "Primary Structure and Expression of Acidic (Class II) Chitinase in Potato," Plant Molecular Biology, 35:749–761 (1997); Ancillo et al., "A Distinct Member of the Basic (Class I) Chitinase Gene Family in Potato is Specifically Expressed in Epidermal Cells,". Plant Molecular Biology, 39:1137–1151 (1999)). In another case of possible anticipation of fungal invasion in disrupted tissues, ethylene induces chitinase in bean abscission zones (del Campillo et al., "Identification and Kinetics of Accumulation of Proteins Induced by Ethylene in Bean Abscission Zones," Plant Physiology, 98:955–961 (1991)). None of these studies included analysis of cotton fibers or showed the presence of chitinase activity or chitinase-related proteins in cotton fibers.
Other studies show that some chitinases have a developmental role in plants, although authentic structural chitin is not a natural part of the plant body (Meins et al., "The Primary Structure of Plant Pathogenesis-Related Glucanohydrolases and Their Genes," In Boller, eds., Genes Involved in Plant Defense, Springer Verlag/N.Y., p. 245–282 (1992)). It has been shown that chitinase isoforms with developmental roles are at least sometimes distinct from those with roles in stress responses and defense (Mauch et al., "Antifungal Hydrolases in Pea Tissue. I. Purification and Characterization of Two Chitinases and Two β-1,3-Glucanases Differentially Regulated During Development and in Response to Fungal Infection," Plant Physiology, 87:325–333 (1988)). Defensive chitinases bind to short stretches (probably 3–6 residues) of a single N-acetyl-glucosamine chain prior to cleaving the inter-sugar bond (Robertus et al., "The Structure and Action of Chitinases," in Jolles, eds., Chitin and Chitinases, Birkäuser Verlag:Basel, pp. 125–136 (1999)). Therefore, enzymes in the chitinase family can also bind to oligomers of N-acetyl-glucosamine within other molecules such as glycoproteins or signalling molecules. Such molecules may have roles in signal transduction to regulate gene expression cascades required for developmental transitions or in the biosynthetic processes that implement the developmental program.
Previous research on the regulation of secondary wall deposition or function at the molecular level focused on a few genes involved in cellulose, hemicellulose, lignin, or protein biosynthesis. Among these pathways, lignin synthesis has been most fully explored and manipulated in transgenic plants (Merkle et al., "Forest Biotechnology," Current Opinion in Biotechnology, 11:298–302 (2000)). However, cotton fibers contain no lignin (Haigler, "The Functions and Biogenesis of Native Cellulose," in Zeronian, eds., Cellulose Chemistry and Its Applications, Ellis Horwood:Chichester, England, pp. 30–83 (1985)). Hemicellulose polysaccharides within some secondary walls include xylans and glucomannans, but cotton fiber secondary walls do not contain significant quantities of any similar molecule (Haigler, "The Functions and Biogenesis of Native Cellulose," in Zeronian, eds., Cellulose Chemistry and Its Applications, Ellis Horwood:Chichester, England, pp. 30–83 (1985)). Only two proteins with possible structural roles in the cotton fiber secondary wall have been identified. One of these, H6, is an arabinogalactan-type protein that accumulates to detectable levels during secondary wall deposition, although the expression of its gene begins during rapid elongation including primary wall deposition (John et al., "Characterization of mRNA for a Proline-Rich Protein of Cotton Fiber," Plant Physiology, 108:669–676 (1995)). The second, FbL2A, lacks homology to any known protein, but its highly repetitive sequence and high hydrophilicity suggest that it may have a structural role or protect cotton fibers during dessication. The expression of its gene begins weakly at the primary to secondary wall transition (15 DPA) and is stronger by 20 DPA (Rinehart et al., "Tissue-Specific and Developmental Regulation of Cotton Gene FbL2A," Plant Physiology, 112:1331–1341 (1996)).
Enzymes that increase in gene expression and/or activity during cotton fiber secondary wall deposition and that relate to the regulation of cellulose synthesis include cellulose synthase, sucrose synthase, sucrose phosphate synthase, and UDP-glucose pyrophosphorylase (Basra et al., "Sucrose Hydrolysis in Relation to Development of Cotton (Gossypium spp.) Fibres," Indian Journal of Experimental Botany, 28:985–988 (1990); Wäfler et al., "Enzyme Activities in Developing Cotton Fibres," Plant Physiology and Biochemistry, 32:697–702 (1994); Amor et al., "A Membrane-Associated Form of Sucrose Synthase and its Potential Role in Synthesis of Cellulose and Callose in Plants," Proc. Nat'l. Acad. Sci. U.S.A., 92:9353–9357 (1995); Pear et al., "Higher Plants Contain Homologs of the Bacterial celA Genes Encoding the Catalytic Subunit of Cellulose Synthase," Proc. Nat'l. Acad. Sci. U.S.A., 93:12637–12642 (1996); Tummala, "Response of Sucrose Phosphate Synthase Activity to Cool Temperatures in Cotton," M.S. thesis, Texas Tech University, Lubbock, Tex. (1996)). UDP-glucose pyrophosphorylase converts glucose-1-P to UDP-glucose or mediates the reverse reaction. In the context of cellulose synthesis, sucrose synthase is thought to degrade sucrose and supply UDP-glucose to cellulose synthase. Cellulose synthase transfers the glucose to the elongating β-1,4-linked cellulose polymer while free UDP is recycled to sucrose synthase. Sucrose phosphate synthase may use fructose-6-P (e.g. that derived from the fructose released by the degradative action of sucrose synthase) and UDP-glucose to synthesize additional sucrose to support cellulose synthesis (Delmer, "Cellulose Biosynthesis in Developing Cotton Fibers," in Basra, ed., Cotton Fibers: Developmental Biology, Quality Improvement, and Textile Processing, Haworth Press:New York, pp. 85–112 (1999)).
All of the enzymes just discussed operate within the pathways of sugar metabolism leading to the formation of the β-1,4-linked glucan polymer. Evidence from other systems and cotton fibers implicates other possible points of regulation of cellulose synthesis coincident with or after formation of the glucan polymer, although the relevant pathways and proteins are incompletely understood. Examples of other relevant proteins include β-1,4-glucanase that may act as a glucan chain editor or in some other role (Delmer, "Cellulose Biosynthesis: Exciting Times For a Difficult Field of Study," Ann. Rev. Plant Physiol. Mol. Biol., 50:245–276 (1999)) and glycoproteins that may act as primers for cellulose biosynthesis (Lukowitz et al., "Arabidopsis cyt1 Mutants are Deficient in a Mannose-1-Phosphate Guanyltransferase and Point to a Requirement of N-Linked Glycosylation for Cellulose Biosynthesis," Proc. Nat'l. Acad. Sci. U.S.A., 98:2262–2267 (2001)). Mutations that down-regulate β-1,4-glucanase or glycoprotein synthesis cause reduced cellulose content in other systems (Nicol et al., "Plant Cell Expansion: Scaling the Wall," Current Opinion in Cell Biology, 1:12–17 (1998); Lukowitz et al., "Arabidopsis cyt1 Mutants are Deficient in a Mannose-1-Phosphate Guanyltransferase and Point to a Requirement of N-Linked Glycosylation for Cellulose Biosynthesis," Proc. Nat'l. Acad. Sci. U.S.A., 98:2262–2267 (2001)). In Arabidopsis, mutation of xylem secondary wall specific cellulose synthase genes causes reduced cellulose content and weak xylem walls (Turner et al., "Collapsed Xylem Phenotype of Arabidopsis Identifies Mutants Deficient in Cellulose Deposition in the Secondary Cell Wall," Plant Cell, 9:689–701 (1997)). In cotton, increased expression of spinach sucrose phosphate synthase causes increased cellulose content in fiber walls of plants growing under a cool night cycle (Haigler et al., "Transgenic Cotton Over-Expressing Sucrose Phosphate Synthase Produces Higher Quality Fibers With Increased Cellulose Content and Has Enhanced Seed Cotton Yield," Abstract 477. In: Proceedings of Plant Biology 2000, July 15–19, San Diego, Calif., American Society of Plant Physiologists, Rockville, Md., (2000)). These findings indicate that it is possible to manipulate the quantity of cellulose in secondary walls, but presently there is insufficient identification of target genes that might be beneficially manipulated.
Studies that identify genes that are under tissue-specific and developmental regulation are important in understanding the roles of proteins in fiber development and cell-wall architecture (John, "Structural Characterization of Genes Corresponding to Cotton Fiber mRNA, E6: Reduced E6 Protein in Transgenic Plants by Antisense Gene," Plant Mol. Biol., 30:297–306 (1996)). In addition, such genes and their regulatory elements are important tools for fiber modification through genetic engineering (John, "Prospects for Modification of Fibers Through Genetic Engineering of Cotton," in Gebelein, eds., Industrial Biotechnological Polymers, Lancaster, Pa.:Technomic, pp. 69–79 (1995); John, "Genetic Engineering Strategies for Cotton Fiber Modification. In: A. S. Basra (ed.), Cotton Fibers: Developmental Biology, Quality Improvement, and Textile Processing, The Haworth Press, New York, pp. 271–289 (1999)).
In many instances, it would be desirable for a transgene to be developmentally regulated to have exclusive or preferential expression in fiber cells at a proper developmental stage. This regulation can be most expeditiously accomplished by a promoter capable of preferential promotion.
Promoters are DNA elements that direct the transcription of RNA in cells. Together with other regulatory elements that specify tissue and temporal specificity of gene expression, promoters control the development of organisms. Thus, there has been a concerted effort in identifying and isolating promoters from a wide variety of plants and animals.
Many promoters function properly in heterologous systems. For example, promoters taken from plant genes such as rbcS, Cab, chalcone synthase, and protease inhibitor from tobacco and Arabidopsis are functional in heterologous transgenic plants. (Benfey et al., "Regulated Genes in Transgenic Plants," Science, 244:174–181, (1989)). Specific examples of transgenic plants include tissue-specific and developmentally regulated expression of soybean 7s seed storage protein gene in transgenic tobacco plants (Chen et al., "A DNA Sequence Element That Confers Seed-Specific Enhancement to a Constitutive Promoter," EMBO J., 7:297–302, (1988)) and light-dependent organ-specific expression of Arabidopsis thaliana chlorophyll a/b binding protein gene promoter in transgenic tobacco (Ha et al., "Identification of Upstream Regulatory Elements Involved in the Developmental Expression of the Arabidopsis thaliana Cab-1 Gene," Proc. Natl. Acad. Sci. USA, 85:8017–8021, (1988)). Similarly, anaerobically inducible maize sucrose synthase-1 promoter activity was demonstrated in transgenic tobacco (Yang et al., "Maize Sucrose Synthase-1 Promoter Directs Phloem Cell-Specific Expression of Gus Gene in Transgenic Tobacco Plants," Proc. Natl. Acad. Sci. USA, 87: 4144–4148, (1990)). Tomato pollen promoters were found to direct tissue-specific and developmentally regulated gene expression in transgenic Arabidopsis and tobacco (Twell et al., "Pollen-Specific Gene Expression in Transgenic Plants Coordinate Regulation of Two Different Tomato Gene Promoters During Microsporogenesis," Development, 109:705–714, (1990)). Thus, some plant promoters can be utilized to express foreign proteins in plant tissues in a developmentally regulated fashion.
Tissue-specific and developmentally regulated expression of genes has also been shown in fiber cells. Several of these, such as E6, vacuolar ATPase, and lipid transfer-type proteins, have strong expression in cotton fibers during primary wall deposition (Wilkins et al., "Molecular Genetics of Developing Cotton Fibers," in Basra, ed., Cotton Fibers: Developmental Biology, Quality Improvement, and Textile Processing, Haworth Press:New York, p. 231–270 (1999); Orford et al., "Expression of a Lipid Transfer Protein Gene Family During Cotton Fibre Development," Biochimica and Biophysica Acta, 1483:275–284 (2000)). Other genes show transient expression during fiber development. For example, Rae is transiently expressed at the primary to secondary wall stage transition (Delmer et al., "Genes Encoding Small GTP-Binding Proteins Analogous to Mammalian Rae are Preferentially Expressed in Developing Cotton Fibers," Mol. Gen. Genet., 248:43–51 (1995)) and another lipid transfer-type protein, FS18A (Orford et al., "Characterization of a Cotton Gene Expressed Late in Fibre Cell Elongation," Theoretical and Applied Genetics, 98:757–764 (1999)), is transiently expressed at 24 DPA during secondary wall deposition. Another gene, H6, is expressed between 10–24 DPA, which includes both primary and early secondary wall deposition, but, after the promoter of this gene has commenced activity during primary wall deposition, there is post-transcriptional control of gene expression so that H6 protein accumulates only during secondary wall deposition at 15–40 DPA (John et al., "Characterization of mRNA for a Proline-Rich Protein of Cotton Fiber," Plant Physiology, 108:669–676 (1995)). At the level of gene expression, only certain cellulose synthase genes have been previously shown to have preferential and prolonged expression in cotton fibers during secondary wall deposition, although there were lower levels of expression during primary wall deposition and in other parts of the plant. In addition, cellulose synthase did not show strong expression until 20 DPA, with only weak expression observed at 17 DPA (Pear et al., "Higher Plants Contain Homologs of the Bacterial celA Genes Encoding the Catalytic Subunit of Cellulose Synthase," Proc. Nat'l. Acad. Sci. U.S.A., 93:12637–12642 (1996)). Another gene, FbL2A, is up-regulated at the primary to secondary wall transition (weakly at 15 DPA and strongly at 20 DPA) (Rinehart et al., "Tissue-Specific and Developmental Regulation of Cotton Gene FbL2A," Plant Physiology, 112:1331–1341 (1996)).
Any of the genes expressed in cotton fibers are candidates for containing useful promoters, with a particular class of useful promoter being those that are preferentially expressed in fibers and/or secondary walled cells compared to other cell types and/or developmental stages. Promoters expressing preferentially during the primary wall stage of fiber development will also have particular uses. It may also be advantageous to have promoters that express in cotton fibers and certain restricted classes of cells (for example, secondary walled cells) in other parts of the cotton plant. Promoters with different strength are also valuable because different genetic engineering goals may be best accomplished with different amounts of the foreign protein being present in the cell or tissue. The biotechnology industry dealing with any particular species will ultimately desire and need a "toolbox" of promoters so that the most appropriate one for any particular use may be chosen. Each promoter will have a combination of unique characteristics in terms of cell, tissue, or developmental specificity of driving gene expression, strength of gene expression, degree of susceptibility to positional effects of gene insertion on level of gene expression, susceptibility to gene silencing, and any number of other similar phenomena that affect transcription.
A few promoters of genes expressed in cotton fibers have been isolated and tested in stably transformed cotton and with relationship to two or more time points in the time course of fiber development, where the testing involved promoter fusions with "reporter" genes or genes that are part of putatively useful genetic engineering strategies. Three major patterns were observed, one typified by the Gh10 promoter (for an acyl carrier protein), which drives foreign gene expression throughout fiber development (Song et al., "Expression of a Promoter from a Fiber-Specific Acyl Carrier Protein Gene in Transgenic Cotton Plants," Proc. Beltwide Cotton Conf., 1:486–488 (1998)). A second pattern was typified by the E6 promoter, which drives gene expression preferentially at the primary wall stage of cotton fiber development (U.S. Pat. No. 5,521,078 to John). A third pattern was shown by the promoter of the FbL2A gene. This promoter was tested by fusion to two reporter genes (polyhydroxyalkanoic acid synthase or PHA synthase, an enzyme that was detected with a specific antibody, and acetoacetyl-CoA reductase, an enzyme with activity that was monitored by enzyme assay) and transformation of cotton (Rinehart et al., "Tissue-Specific and Developmental Regulation of Cotton Gene FbL2A," Plant Physiology, 112:1331–1341 (1996)). However, by use of the two promoter/reporter gene constructs, somewhat contradictory data were provided on the utility of the promoter of FbL2A. When the acetoacetyl-CoA reductase gene was under control of the FbL2A promoter, acetoacetyl-CoA reductase activity was detected in cotton fibers during primary wall deposition at 5–10 DPA, and there was consistent, substantial activity by 20 DPA during secondary wall deposition, peak activity at 35 DPA, and continued activity until 45 DPA among a family of independently transformed plants (Rinehart et al., "Tissue-Specific and Developmental Regulation of Cotton Gene FbL2A," Plant Physiology, 112:1331–1341 (1996)). However, the enzyme activity after 20 DPA could be due to long-lived messenger RNA or protein synthesized at 15–20 DPA, which is the period when the data directly show FbL2A gene expression under control of its own promoter. In contrast, when the PHA synthase gene was under the control of the FbL2A promoter, Western blotting to detect PHA synthase immunologically in a transformed plant showed only very weak signal during secondary wall deposition at 20 DPA, a trace signal at 25 DPA, and no signal at 30 or 35 DPA (Rinehart et al., "Tissue-Specific and Developmental Regulation of Cotton Gene FbL2A," Plant Physiology, 112:1331–1341 (1996)). Furthermore, the PHA synthase gene under the control of the putatively consitutive 35S promoter from CaMV virus correlated with detection of PHA synthase protein strongly during primary wall deposition at 10 DPA and weakly continuing through 35 DPA of secondary wall deposition. The comparative patterns during secondary wall deposition are consistent with transient expression of the FbL2A promoter around 20 DPA of secondary wall deposition. The data also show that the FbL2A promoter will drive weak foreign gene expression in cotton fibers during primary wall deposition (Rinehart et al., "Tissue-Specific and Developmental Regulation of Cotton Gene FbL2A," Plant Physiology, 112:1331–1341 (1996)). Further, graphical data in U.S. Pat. No. 6,211,430 to John showed that the FbL2A promoter activity was not substantial until 20 DPA (about 5 days past the onset of secondary wall deposition) and that 50% of maximal foreign acetoacetyl-CoA reductase activity was not detectable until 31 DPA (about 16 days past onset of secondary wall deposition).
Thus, it would be useful to have a promoter that would drive gene expression preferentially and strongly in secondary walled cells—fibers, in particular—throughout secondary wall deposition, i.e., strongly and continuously (e.g. at ≧50% of its maximal activity) from the initiation of secondary wall deposition to its termination. The initiation of secondary wall deposition is defined as the time when the dry weight/unit length of a cotton fiber begins to increase or when the dry weight/unit surface area of any cell begins to increase via synthesis of new wall material containing more than 30% (w/w) of cellulose. In the case of cotton fiber of G. hirsutum L., this is expected to occur between 14–17 DPA when cotton plants are grown under typical conditions in the greenhouse or the field (day temperature of 26–34° C., night temperature of 20–26° C., light intensity greater than or equal to 1000 μeinsteins/m2/s, with adequate water and mineral nutrition). Furthermore, it would be useful to have a promoter that would drive gene expression only or preferentially in secondary walled cells such as fibers while excluding or minimizing expression in other cell types.
The present invention is directed to achieving these objectives.

Chitinase encoding DNA molecules from cotton expressed preferentially in secondary walled cells during secondary wall deposition and a corresponding promoter


An extendable stirrup has a main stirrup and a retractable step parallel to and slideably attached to the main stirrup. The retractable step has an upper lift portion and a lower step portion. A latch holds the retractable step in the retracted position for riding. For mounting, the user releases the latch thereby allowing the retractable step to automatically descend to an extended position. The user places a foot on the lower step portion of the retractable step to assist in mounting a saddled animal. After mounting the saddled animal, the user engages the upper lift portion with his boot and raises the retractable step to a retracted position whereby the latch holds the retractable step in the retracted position adjacent to the main stirrup.

1. Field of the Invention
This invention relates primarily to the area of equestrian activities and the general area of safe horsemanship. More specifically, it is directed toward a device for enhancing a horseman's ability to more easily mount a four legged animal such as a horse.
2. Prior Art
The purpose of the traditional saddle stirrup is to aid the horseman in mounting a horse and getting into the saddle. It functions by loosely, but securely, hanging vertically lower than and in line with the position of the horseman's legs when in the saddle. This provides both a means for the horseman to step up into the saddle and a means for the horseman to support and balance their self when in the saddle. Additionally, once the horseman is in the saddle, the stirrup provides the horseman with a means of balance and stability while riding the horse.
For various reasons, a person often has difficulty mounting a horse. The primary difficulty is that a person must step very high in order to place their foot into the stirrup. Shorter people usually have the greatest difficulty because they must step proportionately higher than taller people and this disparity is even greater when the horse is a taller horse.
Often riders must find some sort of aide such as standing on a stool, a rock, a fence, or anything that they can stand on to shorten the step they have to take in order for them to get their foot into the stirrup. This can be both problematic and a safety hazard. Most often there are no aiding devices available at the time and place that the rider needs them. On a trail ride, for example, one cannot always find something of the right height to stand on. It is a safety hazard because random aiding devises are not typically very solid and may tend to move when mounting the horse. Additionally, a horse is often nervous and fidgety when being asked to stand still next to an inanimate object while being mounted. This is sometimes a hazardous situation.
Horse saddle stirrups are historically designed to be attached to a saddle and held in place by a leather strap. This strap is typically adjustable to accommodate different leg lengths or preferences of riders. Typically the desired length of the stirrup strap while riding in the saddle is too high for the rider to comfortably step up to mount the horse. An adjustment to the length of the stirrup strap to minimize the step up requires the rider to be dismounted. Unfortunately, once adjusted the height is fixed and now the rider must ride with a stirrup well below his foot.
Prior art includes a somewhat retractable device disclosed in U.S. Pat. No. 6,688,088 issued to Aweeka. Aweeka discloses a device that assists a rider by providing a lowered step to insert the right foot into and then the left foot is inserted into the riding stirrup in a manner similar to the steps of a ladder. Although this device can be of assistance to the rider in mounting a horse, it is cumbersome and leaves part of the device hanging below the stirrup where it is exposed to other objects such as brush or low lying limbs of a tree that may snag it or, in some cases, it could be something the horse or the rider could possibly become entangled in. This is an unacceptable risk level for the trail rider or any riding that takes place outside of the safe confines of the clinical setting of a professional arena.
Another device disclosed in U.S. Pat. No. 6,026,633 issued to Burke is a stirrup device that "includes a crossbar, a first U-shaped member, a second U-shaped member slidably connected to the first U-shaped member and a retraction mechanism" for facilitating the mounting of a riding animal. This device has a very complicated retraction and retention mechanism that requires a spring latching feature that would easily become non-functional with a minimal amount of dirt and wear. The complication of this device would make it extremely expensive to manufacture.
Another device disclosed in U.S. Pat. No. 5,809,754 issued to Schulte provides an auxiliary mounting step which is pivotal and is generally under and adjacent to the tread of the stirrup. The rider is assisted in mounting the saddle by unfolding and rotating the auxiliary step so it is in a position to provide a step below the stirrup thereby assisting the rider by providing a lower step. This device employs a spring which is very vulnerable to wear and its reliability under heavy use is questionable. Additionally, it is awkward to fold up and even in the retracted or folded position the device cannot be completely out of the way and therefore is exposed to being snagged.
Another mounting device disclosed in U.S. Pat. No. 5,661,957 issued to Daly employs a ratchet assembly mechanism that utilizes a gear rack and spring assembly approach to lower the stirrup for easier mounting. This device does provide the rider with an adjustable height. However, this method leaves an awkward and cumbersome piece of equipment that must be tolerated by both the horse and the rider.
Prior art has, in one fashion or another, attempted to address the issue of providing a device to make it easier for a rider to mount an animal under a saddle. The result, in most cases, has been a device that either compromises the utility, dependability, appearance or level of safety of the traditional western stirrup.
There is a need for a device for assisting in the mounting of a saddled animal, such as a horse, that is simplistic in function for ease of manufacture, dependable, and safe for the rider as well as the animal.


An aerial ladder roller assembly to improve the function of the ladder during extension and retraction of sections of a multiple-section aerial ladder. One embodiment has a plurality of aligned apertures for rotatably mounting and a roller that is rotatably mounted in a housing. The housing has a roller pad and an aperture through which a roller extends. The housing is adapted for pivotal mounting at a point approximately central to the length of the axis. A roller is rotatably mounted in the housing on each side of the pivotal mounting point. A pair of rollers is rotatably mounted in the housing on each side of the pivotal mounting point. Also provided is a multiple-section ladder, having at least two nested sections with a housing having at least one roller is rotatably and pivotally mounted extending through an aperture. The slide plate of the second section juxtaposes the roller assembly.

1. Field of the Invention
The present invention generally relates to multiple-section aerial ladders and particularly to an aerial ladder roller assembly to improve the function of the ladder during extension and retraction of ladder sections.
2. Discussion of Related Art
A common problem in the extension of telescoping ladders, especially very large ladders, such as aerial ladders found on fire trucks, is the slipping, sticking, and surging that may occur during the extension and retraction of the ladder sections. Ladder sections typically follow tracks or slides that require a high degree of maintenance through regular greasing to reduce the defects cited above. Otherwise, the surging motion reduces the life of cables used to extend and retract ladder sections due to the dynamic loads encountered. Since dependability in fire truck aerial ladders is essential, there has been a resistance in the art to cure these problems.
One possible solution to the problems is the use of roller assemblies at contact points of the ladder sections. No successful such roller assemblies are known in the art most likely because such application would need to be highly dependable yet easily maintained. Further, single metal rollers have not been successful due to line contacts between the ladder rail and the roller causing high stresses and distortion of the base rails of the ladder.
Rollers attached to ladders are known in the art, but not for the solution of problems cited above. For example, U.S. Pat. No. 4,754,843 to Anderson discloses a roller assembly attached to the top of a ladder to assist in ladder increasing and decreasing inclination. A similar device is also found in U.S. Pat. No. 5,123,503 to Clarke. U.S. Pat. No. 5,226,703 to Norman discloses the use of rollers to facilitate movement of "endless tracks" in large construction equipment, but only for support rollers. U.S. Pat. No. 5,172,952 to Lasnetski also discloses the use of rollers on ladders but only to assist in storing the ladder. Here a roller is placed at the end of the ladder to roll on a track suspended from the ceiling of a truck. The roller is not integral to a slide mechanism and is not designed to support a load on the ladder while in use.
Thus, there is a desire and need in the art to provide a dependable and easily maintainable roller assembly integral to the slide tracks of a multi-section ladder.


There is disclosed a thermal bonding pattern for nonwoven fabric comprising a pattern having an element aspect ratio between about 2 and about 20 and an unbonded fiber aspect ratio of between about 3 and about 10. It has been unexpectedly found that such a fabric has a higher abrasion resistance and strength than a similar fabric bonded with different bond patterns of similar bond areas. This combination of strength and abrasion resistance has long been sought after.

Nonwoven fabrics produced by the meltblowing and spunbonding process have found great utility in many diverse applications from car and boat covers to incontinence products. Different attributes or properties of the fabric are required depending on the application. A car cover, for example, must have great tensile strength and resistance to ultraviolet radiation, while a feminine hygiene product must exhibit great absorbency and softness. Developing just the right combination of properties for the application is a complex task requiring the focused attention of many highly qualified individuals.
The bonding pattern used in either bonding the fibers of the nonwoven fabric to itself or in bonding the nonwoven fabric to other material layers can cause great changes in the fabric properties. Bonding patterns with large bond areas, for example, tend to make a strongly bonded but rough feeling fabric. Those with small bond areas tend to make soft feeling but very weak fabric.
Various attempts have been made at overcoming the disadvantage seemingly inherent in higher bond areas, i.e. decreased softness. One such attempt is taught in U.S. Pat. No. 5,620,779 to Levy and Mcormack and is a nonwoven fabric with a bond pattern having a certain required spacing ratio which is then stretched to produce ribs.
A number of treatments have also been developed to soften nonwoven fabrics such as multiple washings and chemical treatments.
There remains a need, however, for an unribbed fabric without chemical treatments having good bonding strength (i.e. tensile strength and abrasion resistance) yet also having good fabric softness without excessive bonding area.
Accordingly, it is an object of this invention to provide a nonwoven fabric with a bonding area comparable to fabrics bonded with known patterns yet having greater softness and comparable or better tensile strength and abrasion resistance.

Nonwoven bonding patterns producing fabrics with improved strength and abrasion resistance


A method of operating a robot includes receiving image data from an image capture device of the robot. The image data is representative of a glyph viewed by the image capture device on the display of a computing device within a field of view of the image capture device. The method further includes determining, at a controller, a command message based on the glyph represented in the image data and issuing a command to at least one resource or component of the robot based on the command message.

Mobile robots are used in many different fields. For example, mobile robots are used in home settings, office settings, manufacturing settings, and military settings. Many of these robots have limited user interfaces due to limited surface area along the respective housings of the robots. In many instances, the only user interfaces are an on/off switch and/or a small LED screen. Thus, it is very difficult to provide complex commands to the mobile robot. As such, users may find configurations and control of these robots to be difficult.


A vehicle includes at least one autonomous driving sensor configured to detect a traffic flow pattern relative to an intersection. An autonomous mode controller is configured to determine the state of the traffic control device. The autonomous mode controller may estimate when the state of the traffic control device is likely to change based on the traffic flow pattern.

Drivers use visual clues to estimate when a traffic light will change color. Some drivers will speed up the vehicle when the traffic light is green to clear the intersection before the light changes to yellow or red. Seeing a traffic light turn yellow prior to reach the intersection will likely cause the driver to slow the vehicle in anticipation of the traffic light turning red. Drivers can use other visual clues from the traffic light, such as a left-turn arrow, to guess when the traffic light will change.


Disclosed is a process for the upgrading and demetallizing of heavy oils and bitumens. A crude heavy oil and/or bitumen feed is supplied to a solvent extraction process 104 wherein DAO and asphaltenes are separated. The DAO is supplied to an FCC unit 106 having a low conversion activity catalyst for the removal of metals contained therein. The demetallized distillate fraction is supplied to a hydrotreater 110 for upgrading and collected as a synthetic crude product stream. The asphaltene fraction can be supplied to a gasifier 108 for the recovery of power, steam and hydrogen, which can be supplied to the hydrotreater 110 or otherwise within the process or exported. An optional coker 234 can be used to convert excess asphaltenes and/or decant oil to naphtha, distillate and gas oil, which can be supplied to the hydrotreater 220.

The present invention generally relates to the upgrading of heavy oils and bitumens. More particularly, the present invention relates to a process for the upgrading of heavy oils and bitumens including one or more of the steps of production, fractionation, solvent extraction, fluid catalytic cracking and hydrotreating to produce synthetic crude and/or naphtha, distillate and gas oil streams having reduced metal and/or sulfur content.
As world reserves of light, sweet crudes diminish and worldwide consumption of oil increases, refiners seek methods for extracting useful oils from heavier crude resources. The heavier crudes, which can include bitumens, heavy oils and tar sands, pose processing problems due to significantly higher concentration of metals, most notably nickel and vanadium. In addition, the heavier crudes typically have higher sulfur and asphaltene content, posing additional problems in the upgrading of crudes. Finally, tar sands, bitumens and heavy oils are extremely viscous, resulting in problems in transporting the raw materials by traditional means. Heavy oils and bitumens often must be maintained at elevated temperatures to remain flowable, and/or mixed with a lighter hydrocarbon diluent for pipeline transportation. The diluent can be expensive and additional cost can be incurred in transporting it to the location where production is occurring.
As the prices of light oil and natural gas continue to increase, the price of heavy oils and bitumens remains relatively low due to the difficulty in the recovery and upgrading to useable oils. The recovery of bitumens and other heavy crudes is expensive due to substantial energy requirements in the production.
Extensive reserves in the form of "heavy crudes" exist in a number of countries, including Western Canada, Venezuela, Russia, the United States, and elsewhere. These deposits of heavy crudes often exist in areas that are inaccessible by normal means. Generally, the term "heavy crude" refers to a hydrocarbon material having an API gravity of less than 20. Typical heavy crude oils are not fluid at ambient temperatures, and contain a high fraction of materials boiling above 343° C. (650° F.) and a significant portion with a boiling point greater than 566° C. (1050° F.). The high proportion of high boiling point hydrocarbons materials typical in heavy oils make fractionation difficult without resorting to vacuum fractionation.
High metals content in the hydrocarbon feed presents similar processing difficulties. Metals and asphaltenes in the heavy hydrocarbon materials are undesirable in the separated oil fractions as the metals tend to poison catalysts conventionally used in upgrading the oil fractions to other useful products. Asphaltenes will tend to foul/plug downstream equipment. Because of such difficulties during processing by conventional methods, the highest boiling portions are often thermally upgraded by coking or visbreaking processes. The heaviest fractions of heavy oil and bitumen containing the bulk of the metal and asphaltene can be separated by fractionation to recover lighter oils, which can be upgraded catalytically. However, the heavier fraction is still left with some usable oils that can not be extracted using fractionation techniques.
Metals present in heavy oils can include, for example, vanadium and nickel. Vanadium is typically present in excess of 100 wt ppm, often greater than 200 wt ppm. Nickel is typically present in excess of 50 wt ppm, with 75 wt ppm and greater also common.
Solvent extraction of the residuum oil has been known since the 1930's, as previously described in U.S. Pat. No. 2,940,920, to Garwin. With the introduction of the commercially available ROSE(r) process technology, solvent deasphalting processes have become more efficient and cost effective. Solvent deasphalting technology is commonly used today as one method of bottom-of-the-barrel upgrading in a deep conversion refinery and can also be used to produce fluid catalytic cracker (FCC) feeds, lube bright stocks, deasphalted gas oil feeds for hydrotreating and hydrocracking units, specialty resins, and heavy fuel and asphalt blending components from heavy oil feedstocks. Improved techniques in solvent extraction have been disclosed in U.S. Pat. No. 5,843,303 to Ganeshan.
Prior studies have focused on methods of increasing the transportability of heavy crudes by decreasing their viscosities. U.S. Pat. No. 5,192,421 to Audeh et al., discloses an improved method of demetallization during the deasphalting process, including the steps of deasphalting heavy asphalt-rich crudes, followed by thermal treatment, to produce deasphalted crude having a reduced metal content.
In U.S. Pat. No. 4,875,998, Rendall discloses the extraction of bitumen oils from tar-sands with hot water. Specifically, bitumen oils are conditioned in hot water and then extracted with a water immiscible hydrocarbon solvent to form a mixture which settles into several phases. Each phase can be processed to produce product bitumen oils and recycled process components. Other water or solvent extraction processes are disclosed in U.S. Pat. No. 4,160,718 to Rendall; U.S. Pat. No. 4,347,118 to Funk, et al.; U.S. Pat. No. 3,925,189 to Wicks, III; and U.S. Pat. No. 4,424,112 to Rendall. All patents and publications referenced to herein are hereby incorporated by reference in their entireties.


The present invention relates to methods of monitoring the menstrual cycle and/or the first half of pregnancy in a female by assessing the adhesion of lymphocytes from the female to uterine or lymphoid histological tissues from a pregnant animal or an animal that has been treated with gestational hormones. The method can be used to determine the ability of the lymphocyte donor's immune system to recognize and respond to an environment suitable for sustaining a pregnancy.

Transient granulated lymphocytes are described in the pregnant uteri of >20 species1. In women and mice, these cells are Natural Killer (NK) cells and their activation/maturation depends upon uterine decidualization rather than presence of conceptuses.2,3 The life history and functions of uterine NK (uNK) cells are more fully known in rodents than women. In women, uNK cells are most frequent in first trimester, representing over 70% of the nucleated bone marrow-derived cells in decidual cell suspensions. Human data also suggest that uNK cells are distinctive, tissue based cells. Most circulating human NK cells are CD16+ CD56dim; uNK cells are CD16− CD56bright 2,4. The minor circulating CD56bright subset preferentially expresses (95%) very high levels of L-selectin5, a molecule central to initiation of extravasation. Fewer CD56dim circulating cells (24%) express L-selection and at a much lower surface density5. The two major NK cells functions, target cell lysis and cytokine production, may be displayed separately or dually by single cells6. Human uNK cells display both functions in vitro7-9 but their in vivo functions are undefined. Many current studies of human uNK function address interactions with trophoblast10-12. Other recent reports indicate human uNK cells express angiogenic factors including Ang-213, an antagonist to endothelial cell TIE-2 and thus, a vessel destabilizing molecule and NKG5, a potent endothelial cell mitogen14.
In vivo studies of murine uNK cell functions advanced rapidly after availability of strains genetically deficient in NK cells15, 16. Histological studies established that NK cell deficient mice do not differentiate uNK cells17, 18. In NK-deficient strains with unrelated genetic changes, a common uterine phenotype was developed by 48 hr after implantation. The anomalies were endothelial cell hypertrophy and damage in mesometrially-positioned uterine vessels, lack of uterine arteriole wall and lumen changes indicative of pregnancy and hypocellularity of decidua. Absence of lytic NK cell function does not explain these results, thus, a cytokine deficiency hypothesis was pursued. Interferon-gamma (IFN-γ) is the prototypic cytokine product of NK cells. IFN-γ is an induced molecule known to regulate expression of >1000 genes, many of which are expressed by vascular and decidual tissues19, 20. IFN-γ is expressed in human and murine uteri during normal gestation21-23 but many authors regard IFN-γ as detrimental to pregnancy24, 25. In an experimental series, it was found (i) IFN-γ peaks in mouse mesometrial uterus at gestation day (gd) 10 at 10 IU/implantation site and (ii) only 10% of this comes from non uNK cells26. Transplantation showed that higher levels of uNK-cell derived IFN-γ are essential for pregnancy-induced modification of the spiral arteries and integrity of decidua while the lower level, non lymphocyte-derived IFN-γ is adequate for maturing uNK cells and limiting their numbers. Daily recombinant mulFN-γ (100–1000 IU/6 days) in alymphoid mice promoted full uterine artery modification and normalized deciduas27. Tumor necrosis factor-alpha, another NK and uNK cell product, lacked these effects26, 27.
Mechanisms that transform endometrium to decidua are endocrine-related. In humans, decidualization begins 7–14 days after the surge in luteinizing hormone (LH) (LH+7–14) and continues if pregnancy occurs28-30. Early decidual development appears important for implantation and most human pregnancy wastage occurs in this peri-implantation interval. Human uNK cells begin to increase in number about LH+3, encircling arteries and uterine glands. Stromal cell changes that cuff the spiral arteries (Streeter's columns) are seen at LH+8. By LH+11 to +13, very large numbers of uNK cells are found throughout the stroma accounting of 30–40% of all cells4. Gap junction-like contacts are found between some human uNK cells and early decidua, that appear essential to the continued differentiation of both cell types31, suggesting that normal uNK cell numbers and levels of function contribute to human implantation success.
Reproductive cycles of mice differ to human by virtual absence of a luteal phase. Decidualization and uNK cell activation are initiated by implantation, thus, uNK cell deficits do not influence mouse embryo implantation17, 22. UNK cells proliferate rapidly within decidua2, 3 but recently it was established that self-renewing uNK progenitor cells do not reside there. Uterine segments from normal mice were grafted by end-to-end anastomosis into uNK cell deficient or normal recipients who were then mated. uNK cells were generated only when hosts had NK cell progenitors33,34. In pregnant mammals, thymus and marrow involute during pregnancy35, 36 while spleen and lymph nodes (LN) become hypercellular37, 38. By transplanting to NK cell deficient mice with established pregnancies, it was found that gestation induces acute uNK cell recruitment from spleen but not from marrow or thymus. uNK cells appear to be recruited to decidua basalis and then move into the mesometrial triangle, the entry portal for nerves and vessels supplying the uterus and developing feto-placenta units3,39,40. In decidua basalis of normal mice at mid gestation, 7% of uNK cells are within vessels homologous to spiral arteries, another 30% are in walls of these vessels and the remaining cells, as resolved in paraffin-embedded sections, are associated with other tissues41.
In normal mice, uNK cells are a major source of inducible nitric oxide synthase22, an IFN-γ regulated enzyme producing the powerful vasodilator nitric oxide (NO). In uNK cell deficient mice, expression of this enzyme is induced in trophoblast but at very low levels22 that cannot dilate the spiral arteries (Kiso and Croy, unpublished vascular casting data). Ineffective dilation of spiral arteries is a hallmark of the human gestational complication hypertension/pre-eclampsia42, 43. Despite extensive study of this syndrome, its frequency remains constant and there is no consensus on underlying causes44, 45. Systemic endothelial cell damage underlies clinical symptoms and may be mediated by dysregulated blood cytokine balance46, 47. Some authors suggest immunological contributions48-50 but assessment of changes in frequency or functional activities of uNK cells is just beginning51-53. Women achieving pregnancy by assisted reproductive technology (ART) are reported at higher risks for pre-eclampsia than women carrying naturally conceived conceptuses54-60. Thus, inappropriate uterine recruitment of human NK cells may contribute to two health-related problems: implantation failure through lack of decidual maintenance and predisposition in pregnant women to preeclampsia. Therefore, it is critically important to define the molecules contributing to the movement of human uNK cells and their progenitors into the uterus and to the specification of their intrauterine locations. Subnormal uNK cell frequencies are reported in women with recurrent spontaneous abortion61, suggesting an additional obstetrical group that may benefit from the proposed studies.
Movement of leukocytes from vessels into tissue has been extensively characterized in non-reproductive organs and many techniques have been validated for such work62-65. Lymphocytes constitutively express the tethering molecule L-selectin, which interacts with Peripheral LN Addressin (PNAd) and Mucosal Vascular Addressin-1 (MAdCAM-1) expressed by the microvillous surface of endothelium in LN and Peyers Patches (PP). Avidity of these interactions is modulated by physiological responses including cytokines, inflammation and fever which trigger rolling for egress of non activated lymphocytes from vessels5, 66-68. Firmer adhesion and trans-endothelial migration involve integrins, particularly α4β7, which uses MAdCAM-1. Recruitment of activated cells requires only the latter mechanism and down regulation of L-selectin is paired with upregulation of α4β7 as naive cells begin to roll and dock. In the presence of cytokines, Vascular Cell Adhesion Molecule 1 (VCAM-1) is induced on endothelium and utilized by lymphocytes69. The β2 integrin, Leukocyte Function Associated Antigen-1, (LFA-1) interacting with its ligands Intercellular Adhesion Molecules (ICAM)-1 and -2 also mediates firm adhesion but is not modified by fever ranges similar to those seen at human ovulation68.
In view of the foregoing, there is a need in the art to define the molecules contributing to the movement of human uterine NK cells and their progenitors into the uterus in order to determine if the uterine environment is amenable to sustaining a pregnancy.

Method of monitoring the menstrual cycle and/or pregnancy in a female

Health Research, Inc., Roswell Park Division


Cage systems that improve upon the prior art in various ways. In the preferred embodiments, devices are radiolucent, with markers, thereby allowing visualization of placement without excessive obscuration. Devices according to the invention eliminate multiple steps, instruments and trays, while being capable of a custom fit. The devices according to the invention permit easier and greater access to end plate surface area, and can be used with autografts, allografts, and biologics.

There are many intervertebral implants to assist with stabilization and fixation, including pre-assembled mesh rings of varying size; cylindrical threaded cages; carbon fiber boxes; and bone dowels, rings, and wedges. However, all existing devices have certain drawbacks, including the requirement of multiple steps and tools to prepare and mold the intervertebral plates for acceptance of the devices.
Existing implants also exhibit the need to remove/move a distraction device such as a spreader or plugs around a decorticate. Such plugs/distraction devices often get in the way of cage placement. Mesh cages in bone materials can deform or break with attempts to force them into the inner space. Typically, only a small surface of the end plate is exposed to bone graft, this being dictated by the size and position of the cage. Existing devices also require large trays with many instruments and many cages, and it is difficult to see the bone fused in mass inside metal cages, which are radiopaque.


The present invention is related to a image compression and transmission method, and particularly to a method of relocating a wavelet packet coefficient for a zerotree coding in order to optimize an encoder to be suitable for transmitting image off-line. To realizing the present invention, for the wavelet packet coefficients, located on frequency bands which are divided in several subbands according to frequency of an image data thereon, the present invention collects coefficients in same location on each of the subbands, and relocates the collected coefficients in a same location in an integrated frequency band, formed by integrating the subbands.

1. Technical field
The present invention relates to an image compression and transmission method, and particularly to a method of relocating a wavelet packet coefficient for a zerotree coding in order to optimize an encoder to be suitable for transmitting image off-line.
2. Description of the Prior Art
This invention is incorporated by the reference, xe2x80x9cImage coding using wavelet transformxe2x80x9d, IEEE Trans. Image Processing, vol.1, pp.205-220, April 1992, by M.Antonini, M.Barlaoud, P.Mathieu, and I.Daubechies; xe2x80x9cEmbedded image coding using zerotree of wavelets coefficientsxe2x80x9d, IEEE Trans. Signal Processing, vol.41, pp.3445-3462, December 1993, by J. M. Sharpiro; xe2x80x9cA new fast and efficient image coding based on set partitioning in hierarchical treesxe2x80x9d, IEEE Trans. Circuits Syst. Video Technol. vol.6, pp.243-250, June 1996, A.Said and W. A. Pearlman; xe2x80x9cBest wavelet packet bases in a rate-distortion sensexe2x80x9d, IEEE Trans. Image Processing, vol.2, pp.160-175, April 1993, by K.Ramchandran and M.Vetterli.
FIG. 1 shows a conventional image compression and transmission method. As shown in the figure, the conventional image compression and transmission method executes a wavelet transform process 10 for dividing image data into several frequency bands, so as to allow most energy to be focused on a few coefficients on the band. Then, the image data is compressed by executing a zerotree coding process 20 of coding the wavelet coefficients by forming a tree structure with the wavelet coefficients.
The image transformed by the wavelet transform process 10 is divided into several frequency bands, as shown in FIG. 2. In the figure, it is indicated that a left and upper portion of the frequency band corresponds to a low frequency band and a right and lower portion also corresponds to a higher frequency band. Therefore, a smallest band, positioned leftmost and uppermost in the frequency band, shows a lowest frequency. Also a largest band, positioned rightmost and lowermost, shows a highest frequency. At this time, because most of the image data energy flock to the low frequency bands, most of the image data can be represented by using only the small frequency bands (low frequency bands). That is, because the energy gathers in a few coefficients, the image data can be compressed by coding only the coefficients.
As shown in FIG. 3, in the zerotree coding process 20, the wavelet transformed coefficients are assembled in a tree structure. In the tree structure, a coefficient in a top position is called xe2x80x9cpatentxe2x80x9d, and coefficients just under the top coefficient, or the parent, are named as xe2x80x9cchildxe2x80x9d. And all coefficients, including the child, under the parent can be commonly designated as xe2x80x9cdescendantxe2x80x9d.
Now, an operation of the above prior art is explained below.
In the wavelet transform process 10, at first, the image is transformed in order to express with a few coefficients in the frequency band. Then in the zerotree coding process 20, the wavelet coefficients are assembled into the tree structure, and a zerotree coding is executed, based on a specific threshold, so as to compress image.
For accomplishing the wavelet transform process 10, a filtering and subsampling is used. The image data can be divided into a low frequency band and a high frequency band by a low frequency filtering and subsampling and a high frequency filtering and subsampling. At this time, because the low frequency band has room to be compressed, the filtering and subsampling is repeated to the low frequency band. As a result, the wavelet transformed image having several frequency bands as shown in FIG. 2 can be obtained.
Then, the zerotree coding is carried out about the wavelet transformed image. In the zerotree coding process, when a magnitude of a coefficient is smaller than a threshold T, the coefficient is defined as xe2x80x9czeroxe2x80x9d or xe2x80x9cinsignificant coefficientxe2x80x9d, while, when a magnitude of a coefficient is bigger than the threshold T, the coefficient is defined as xe2x80x9cnon-zeroxe2x80x9d, or xe2x80x9csignificant coefficientxe2x80x9d. In case of the non-zero, the coefficient value is transmitted after executing quantization about the non-zero coefficient. However, in case of the coefficient defined in zero, the coefficient is defined as a xe2x80x9cZerotree Rootxe2x80x9d symbol when all of the children is zero. In the zero coefficient case, however, when there is non-zero child, the coefficient is defined as a xe2x80x9cIsolated Zeroxe2x80x9d symbol. Then, the wavelet coefficients in the tree structure are scanned and transmitted from parent to child and from left to right in FIG. 3. The wavelet coefficient of image data commonly has strong correlation with a position on a picture. So, when the parent coefficient is zero, the descendant coefficients are much probable to be zero. Therefore, defining that the all zero descendant coefficients as well as the zero parent coefficient are one symbol, that is, a zerotree root, it is permissible not to transmit coefficient values of the descendants in the zerotree root symbol, which gives a high compression effect.
Whole operation of the zerotree coding is accomplished as below. At first, after quantizing all the coefficients on Zerotree on the basis of the initial threshold T, the encoder transmits quantized values of the non-zero coefficients, the isolated zero symbols and the zerotree root symbols. At second, after quantizing all the coefficients on Zerotree on the basis of the second threshold T/2, the encoder transmits secondly calculated quantized values of the non-zero coefficients, the isolated zero symbols and the zerotree root symbols. Then, after repeating the above process successively, when it reaches a required bit rate, the coding is completed.
A decoder performs a reverse operation to the encoder for acquiring a reconstructed image.
It may be understood that the above zerotree coding is designed to be suitable for the wavelet transformed coefficient. As shown in FIG. 2, it is difficult that the tree structure for the zerotree coding is assembled in other form except the wavelet transform.
However, the zerotree coding can not be applied to a wavelet packet transform, which is generalized from the wavelet transform, and provides better performance than the wavelet transform in many kinds of image data. Therefore, there is need for an image compression and transmission technique for applying the zerotree coding to the wavelet packet coefficients.
Therefore, the present invention is designed to overcome the above problems. An object of the invention is to provide a method of relocating wavelet packet coefficients for a zerotree coding. The zerotree coding of the wave packet coefficients can be effectively accomplished by a method which is as follows; collecting coefficients at the same position of different frequency subbands and relocating them at the same position of integrating frequency band.
In order to accomplish the above object, the present invention provides a method for relocating wavelet packet coefficients from frequency subbands into the frequency band integrating the frequency subbands, comprising the steps of collecting coefficients in same locations of the subbands, and relocating the collected coefficients in a corresponding location in an integrating frequency band, formed by integrating the subbands.
Additionally, in another embodiment, the present invention provides a method for relocating wavelet packet coefficients comprising a wavelet packet transform process for executing a filtering and subsampling of image data and transforming the image data into wavelet packet coefficients having various frequency bands; and a coefficient relocating process for collecting coefficients corresponding to same locations on frequency subbands among the wavelet packet transformed coefficients, and relocating the coefficients in a corresponding location on an integrated frequency band so as to assemble a tree structure for a zerotree coding.

Method of relocating wavelet packet coefficient


A system and method of providing a telephone calling card service including the steps of providing a user with an access number to a telephone network and a personal identification code, prompting the user to input a personal identification code to initiate automatic dialing of at least one pre-selected telephone number associated with the personal identification code, and determining whether a call has been successfully completed to a called party via the pre-selected telephone number. The system may continue dialing pre-selected telephone numbers until a call has been successfully connected. In the alternative, the system may automatically and simultaneously dial all of the pre-selected phone numbers. An arrangement for determining whether an answering machine has answered a call is also provided. To implement the method, a telephone communications apparatus is used including a structure for recording a series of telephone numbers associated with a personal identification code, and a structure for retrieving the telephone numbers associated with the code and automatically dialing the telephone numbers until a call has been successfully completed to an individual.

Advances in technology have made individuals increasingly mobile. Although telephone technology has advanced so as to enhance communication ability, this increase in mobility often makes it particularly difficult to locate an individual with whom communication is desired, as it may be necessary to dial several phone numbers (home, office, etc.) to locate the individual.
Mobility has also led to widespread use of various telephone services, such as telephone calling cards. Of these calling cards, the use of pre-paid telephone cards has also become particularly widespread. For example, U.S. Pat. Nos. 5,353,335 and 5,511,114 illustrate two different forms of pre-paid telephone calling cards.
The implementation and use of such telephone calling cards and such advanced telephone technology, while convenient in many ways, has failed to address the needs of users who are often incapable of effectively utilizing telephone technology to locate and communicate with an individual or individuals, who may be at any one of several different telephone numbers. In particular, users such as children, the disabled and/or the elderly may find it particularly difficult and cumbersome to locate and communicate with an individual who may be at any one of several phone numbers. This difficulty can be compounded if communication with several individuals is desired. These problems can in fact become particularly acute in urgent situations where such communication can become vital.


The present invention provides methods for rapid detection of bioagents for environmental and product testing. The methods can be used for testing air, water, soil, surfaces of buildings, containers, towers and the like, as well as testing of foodstuff and cosmetics.

Testing for potentially harmful microbes in both natural and human-modified environments represents a significant public health challenge. Microbial contamination of waters can lead to numerous serious issues. Bacterial contamination can cause adverse economic impacts for the affected areas by closing popular recreational areas for extended periods of time. This is particularly relevant in the heavily visited recreational waters and the swimming beaches in urban centers. In addition, bacterial contamination can cause severe illnesses such as gastrointestinal disorders. Microbial contaminants may not be limited to surface or recreational waters. In fact, a far more costly migration of these microbial contaminants is their movement to groundwater supplies which are used for drinking water.
The cysts of Cryptosporidium are of increasing importance because of their presence in water supplies. When in the gut, four spindle-shaped motile sporozooites burst from the cyst to infect gut epithelial cells and continue their life cycle. Entamoeba histolytica, another water-borne pathogen, can cause diarrhea or a more serious invasive liver abscess. When in contact with human cells, these amebae are cytotoxic. Giardia is found in contaminated rivers and lakes and is also be contracted via contaminated foods. There is some evidence that a heavy infection of attached Giardia physically blocks the important transport of nutrients across the epithelium (see, for example, www.cellsalive.com/parasit.htm).
Detection of airborne pathogens has also garnered much attention in light of the recent anthrax attack where anthrax spores were disseminated from mail packages.
A recent news report has stated that the anthrax mailed to a Senate office last fall was able to become airborne again even after it settled in the office. The fact that ordinary movement in the office was enough to send anthrax back into the air provides evidence that the spores were altered to make them more dangerous. Scientists thought that once the anthrax-laden envelope was opened, the spores would settle in the office and would be unlikely to become airborne again. But simulation of normal workplace activities such as paper handling, walking around the office and mail sorting several weeks after the envelope arrived, caused the spores to become airborne. Measurements of anthrax in the air of the office were substantially higher after researchers simulated everyday office activity than when the office was more still. And more than 80% of the airborne anthrax spores were of a size that could easily be breathed into the lungs (see, for example, www.ph.ucla.edu/epi/bioter/officeactivityanthrax.html).
According to a study published in 1999 by the Centers for Disease Control and Prevention (Atlanta), there were almost 14 million cases of foodborne illness caused by known pathogenic microbes (bacteria, parasites, and viruses) in the United States in 1997, and these illnesses caused more than 60,000 hospitalizations and almost 2000 deaths (1). Although ˜70% of the reported illnesses were caused by food contaminated with Norwalk-like viruses, almost half of the deaths were the result of infection with Listeria monocytogenes and Salmonella species bacteria. Foodborne microbial contamination comes in a variety of guises and from myriad sources. Perhaps the best-known foodborne illnesses come from the various subspecies of E. coli. Largely associated with raw or undercooked ground beef, E coli. infection leads to gastrointestinal disorders. Infection is usually self-limited and can last for about eight days. In some cases, however, infection can lead to hemolytic uremic syndrome, which causes renal failure and anemia, and it can be deadly to children, the elderly, and those with weakened immune systems. Certain subtypes of E. coli can cause severe, even lethal illness. For example, in the spring of 1992–93, a multi-state outbreak of over 500 cases of E. coli O157:H7 infections was associated with a restaurant chain (www.dhss.state.mo.us/MoEpi/moepi161.pdf).
One of the least understood but increasingly prevalent illnesses is caused by Listeria monocytogenes. Associated with raw milk, soft cheeses, and raw meats, L. monocytogenes infection can lead to meningitis, encephalitis, and intrauterine or cervical infections in pregnant women that can cause spontaneous abortion or stillbirth. Listeria is able to grow under a wide variety of conditions, including refrigeration.
Another common infection is caused by Salmonella species. Associated with raw meats, poultry, and eggs, Salmonella infection can lead to nausea, vomiting, cramps, and diarrhea. Typically, acute symptoms subside after a couple of days. In severe cases of S. typhi or paratyphi, however, infection can cause a typhoid-like fever and possible septicemia.
As with many other bacteria, Campylobacter jejuni infections also lead to gastrointestinal disorders. Infection is usually self-limiting and lasts 7–10 days. Associated largely with poultry, Campylobacter is also found in unchlorinated water but can be eliminated by boiling.
The family of Norwalk-like viruses (NLVs) is spread through feces-contaminated water, and are thus largely associated with shellfish. Although most people have been exposed to NLVs at some point in their lives, they rarely exhibit any symptoms. Disease associated with infection is usually mild, with symptoms of vomiting, nausea, and abdominal pain, but infection is usually self-limiting and symptoms subside within 2 days.
Corporations and government health officials have put much effort into food testing to prevent human suffering and avoid lawsuits. The two big challenges to large-scale testing of samples, both during food processing and from a clinical perspective, are time and sensitivity. Traditional microbiological methods require that the microbes be cultured and characterized for a variety of metabolic and physical markers. This process can take days to weeks, depending on the organism. For example, in a test to detect Salmonella, one bacterial pre-enrichment step takes 16–20 h, a Salmonella-specific enrichment takes another 24 h, and a final identification step in which cultures are streaked out onto selective media can take 24–48 h. If the results are positive, they must then be confirmed by sub-cultivation and serological testing. Thus, this assay can take anywhere from 3 to 6 days. During this time, the food products might decay beyond the selling point. Consumers might contract food poisoning from the products, putting them at risk for sickness or death. It is therefore preferable to have an assay that can locate and identify the offending microbes in a timeframe that is measured in hours, not days.
Similarly, the assay must also be very sensitive because the tested samples might have no more (and possibly less) than one cell per milliliter or gram of starting material. Listeria infections can start from as few as 10 cells. As with most traditional microbiological tests, this requires some form of microbe culture enrichment using a growth medium, but it is critical that this step not take too much time (see, for example, pubs.acs.org/subscribe/journals/tcaw/12/i03/pdf/303willis.pdf).
Bacteria and their enzymes, along with some fungi and critical nutrient additives are cost effective agents for in-situ remediation (otherwise known as bioremediation) of hazardous wastes and subsurface pollution in soils, sediments and wastewaters. The ability of each bacterial strain to degrade toxic waste depends on the nature of each contaminant. Since most sites are typically comprised of multiple pollutant types, the most effective approach to bioremediation is to use a mixture of bacterial species/strains, each specific to the degradation of one or more types of contaminants. It is critical to monitor the composition of the indigenous and added bacterial consortium in order to evaluate the activity level of the bacteria, and to permit modifications of the nutrients and other conditions for optimizing the bioremediation process. Additionally, it is desirable to return a bioremediation site to its natural state following the bioremediation process. Thus, monitoring of levels of bioremediating bacteria becomes necessary in assessment of the return of the site to the natural state. Fungal bioremediation is also possible. This technology utilizes white-rot fungi to clean up a wide spectrum of soil pollutants, such as wood preservatives, polycyclic aromatic hydrocarbons, organochlorines, polychlorinated biphyenyls, dyes, pesticides, fungicides, herbicides, and others. Rapid throughput methods for detection and identification of the members of the indigenous and added bacterial and/or fungal consortium would greatly facilitate characterization of such bioremediative processes.
Household mold, is a growing concern for homeowners. Molds not only pose serious threats to a home's construction and survival, they pose serious threats to one's health. More than 100,000 types of molds have been discovered yet little is known about the life of these molds and their allergens, and how they become airborne.
Stachybotrys is a species of mold which has earned the title "toxic black mold," as it is one of the most lethal, yet common forms. Stachybotrys can become airborne and cause serious respiratory difficulties, memory and hearing loss, hemorrhaging, dizziness and sometimes death. Prolonged exposure to this strain can impair memory.
Cladosporium, Penicillium and Alternaria are more frequently detected in household mold problems. While not as likely to pose a fatal threat, these molds are known for causing asthma-related symptoms. Studies suggest that such molds are culpable for, or at least connected to, the tripled asthma rate in the past 20 years (see, for example, www.paloaltoonline.com/paw/paonline/news_features/real_estate/spring2002/2002—03—13.m old.shtml).
Identification of species of molds would also benefit from a rapid method for detection and identification.
Mass spectrometry provides detailed information about the molecules being analyzed, including high mass accuracy. It is also a process that can be easily automated. Low-resolution MS may be unreliable when used to detect some known agents, if their spectral lines are sufficiently weak or sufficiently close to those from other living organisms in the sample. DNA chips with specific probes can only determine the presence or absence of specifically anticipated organisms. Because there are hundreds of thousands of species of benign bacteria, some very similar in sequence to threat organisms, even arrays with 10,000 probes lack the breadth needed to detect a particular organism.
Antibodies face more severe diversity limitations than arrays. If antibodies are designed against highly conserved targets to increase diversity, the false alarm problem will dominate, again because threat organisms are very similar to benign ones. Antibodies are only capable of detecting known agents in relatively uncluttered environments.
Several groups have described detection of PCR products using high resolution electrospray ionization-Fourier transform-ion cyclotron resonance mass spectrometry (ESI-FT-ICR MS). Accurate measurement of exact mass combined with knowledge of the number of at least one nucleotide allowed calculation of the total base composition for PCR duplex products of approximately 100 base pairs. (Aaserud et al., J. Am. Soc. Mass Spec., 1996, 7, 1266–1269; Muddiman et al., Anal. Chem., 1997, 69, 1543–1549; Wunschel et al., Anal. Chem., 1998, 70, 1203–1207; Muddiman et al., Rev. Anal. Chem., 1998, 17, 1–68). Electrospray ionization-Fourier transform-ion cyclotron resistance (ESI-FT-ICR) MS may be used to determine the mass of double-stranded, 500 base-pair PCR products via the average molecular mass (Hurst et al., Rapid Commun. Mass Spec. 1996, 10, 377–382). The use of matrix-assisted laser desorption ionization-time of flight (MALDI-TOF) mass spectrometry for characterization of PCR products has been described. (Muddiman et al., Rapid Commun. Mass Spec., 1999, 13, 1201–1204). However, the degradation of DNAs over about 75 nucleotides observed with MALDI limited the utility of this method.
U.S. Pat. No. 5,849,492 describes a method for retrieval of phylogenetically informative DNA sequences which comprise searching for a highly divergent segment of genomic DNA surrounded by two highly conserved segments, designing the universal primers for PCR amplification of the highly divergent region, amplifying the genomic DNA by PCR technique using universal primers, and then sequencing the gene to determine the identity of the organism.
U.S. Pat. No. 5,965,363 discloses methods for screening nucleic acids for polymorphisms by analyzing amplified target nucleic acids using mass spectrometric techniques and to procedures for improving mass resolution and mass accuracy of these methods.
WO 99/14375 describes methods, PCR primers and kits for use in analyzing preselected DNA tandem nucleotide repeat alleles by mass spectrometry.
WO 98/12355 discloses methods of determining the mass of a target nucleic acid by mass spectrometric analysis, by cleaving the target nucleic acid to reduce its length, making the target single-stranded and using MS to determine the mass of the single-stranded shortened target. Also disclosed are methods of preparing a double-stranded target nucleic acid for MS analysis comprising amplification of the target nucleic acid, binding one of the strands to a solid support, releasing the second strand and then releasing the first strand which is then analyzed by MS. Kits for target nucleic acid preparation are also provided.
PCT W097/33000 discloses methods for detecting mutations in a target nucleic acid by nonrandomly fragmenting the target into a set of single-stranded nonrandom length fragments and determining their masses by MS.
U.S. Pat. No. 5,605,798 describes a fast and highly accurate mass spectrometer-based process for detecting the presence of a particular nucleic acid in a biological sample for diagnostic purposes.
WO 98/21066 describes processes for determining the sequence of a particular target nucleic acid by mass spectrometry. Processes for detecting a target nucleic acid present in a biological sample by PCR amplification and mass spectrometry detection are disclosed, as are methods for detecting a target nucleic acid in a sample by amplifying the target with primers that contain restriction sites and tags, extending and cleaving the amplified nucleic acid, and detecting the presence of extended product, wherein the presence of a DNA fragment of a mass different from wild-type is indicative of a mutation. Methods of sequencing a nucleic acid via mass spectrometry methods are also described.
WO 97/37041, WO 99/31278 and U.S. Pat. No. 5,547,835 describe methods of sequencing nucleic acids using mass spectrometry. U.S. Pat. Nos. 5,622,824, 5,872,003 and 5,691,141 describe methods, systems and kits for exonuclease-mediated mass spectrometric sequencing.
Thus, there is a need for a method for bioagent detection and identification which is both specific and rapid, and in which no nucleic acid sequencing is required. The present invention addresses this need.

This patent is subject to a terminal disclaimer.

Methods for rapid detection and identification of bioagents for environmental and product testing


A retractable banner display includes an easily replaceable banner core. Housing for the banner core includes an end cap with a reentrant opening on its top edge that receives and retains an end peg of the banner core. The end peg has a height greater than its width so that its movement is restricted within the reentrant opening. A retention member, such as a notch, may be included to hold the core within the reentrant opening when the banner is pulled to its extended position. A plurality of replacement banner cores may be included with a display assembly. The banner core may be configured to lock in a first orientation and unlock in a second orientation to assist in loading the core into the housing and assist in maintaining tension in the core. An adapter may be used to adjust the display angle of the banner.

1. Technical Field
Aspects of this document relate generally to signs used in advertising and other communication.
2. Background Art
The use of signs is found in almost every commercial or business setting. Choice of sign materials and structure is often determined by whether a sign will be permanent or temporary and to what surface it will be mounted. For temporary signs, a wide variety of sign designs attempt to balance factors such as size, cost, ease of installation, image quality, and others to meet specific needs. Among these designs are retractable banner displays. Conventional signs utilize a flexible banner displaying graphics and other information that is capable of retracting into a housing.


A connector for connecting to helically corrugated conduit comprises a bolt member having a passageway therethrough and a connector body having a hollow end portion that receives the bolt member. The connector includes a collet clamp within the passageway that has a grooved gripping surface adapted to be received within the helical corrugations of the conduit. The collet clamp includes a plurality of fingers each having a radially inward directing gripping surface and a wedge-shaped outer surface. A bushing having a frustoconically shaped bore is advanced over the fingers of the collet clamp when the bolt member is attached to the connector body in order to force the fingers radially inward into gripping contact with the corrugated conduit.

This invention relates generally to a connector for helically corrugated conduit, and in particular, to a connector for helically corrugated coaxially conducting conduit.
Several different types of connectors for helically corrugated conduit are known. For instance, U.S. Pat. No. 4,995,832 to Thommen et al. describes a connector that axially compresses a helical spring within a connector body using a bolt member. The conduit is held in place within the connector body because the helical spring is received within the corrugations of the conduit. Another connector described in U.S. Pat. No. 4,273,404 to Law employs a grounding ring having a plurality of arms that are deformed to project into the valleys of the corrugated conduit when the connector is threaded together. The arms of the grounding ring thus prevent the corrugated conduit from being withdrawn from the connector. Other connector structures of interest with respect to the present invention include U.S. Pat. Nos. 4,824,401 and 4,687,272 to Spinner, U.S. Pat. No. 3,291,895 to Van Dyke and U.S. Pat. No. 3,184,706 to Atkins.

Connector for helically corrugated conduit