The following are some of the professors of Agriculture & Life Science who have received patents. Topics covered include: plant-parasitic nematodes, keratinase technology, ultrapasterurization of liquid whole egg products, etc. To find additional information on any of the patents listed below, search the U.S. Patent Office Database by linking to: http://www.uspto.gov/patft/index.html
Method of Adjusting a Video Microscope System Incorporating Polarization or Interference Optics for Optimum Imaging Conditions
U.S. Patent Number 4,412,246
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Video microscopy is used to capture light microscopic images using video cameras. In 1978 most scientists felt that light microscopic images were best-captured on film and that video images were very inferior in resolution and contrast. That year Nina Strömgren Allen had a conversation with her father, the astrophysicist Bengt Strömgren, in which he pointed out that the recently developed video cameras used on telescopes might be equally effective when combined with microscopes. During a microscopy course taught by Robert and Nina Allen at the Marine Biological Laboratory (MBL) in Woods Hole, MA in 1979, a Hamamatsu analogue video camera was obtained and it became clear that with certain modifications to the microscope set-up and the camera, one could obtain images with better resolution, contrast, and magnification than those gotten by conventional means. Some additional and important advantages were that the rate of image recording was much faster than on 8-mm film and the costs were greatly reduced. The Allens and Shinya Inoué (who also was teaching in the microscopy course) described and illustrated the method at the same meeting of the American Society for Cell Biology and shortly after papers appeared detailing the method and demonstrating what it could do in terms of imaging dynamic events in living cells. In 1982 Nina and Bob Allen further extended the development of video microscopy for the first time by capturing the video images digitally and then using a frame memory to do image analysis and quantification. This development is described in the Journal of Microscopy paper from 1983. Robert and Nina Allen received U.S. Patent Number 4,412,246 for the development of AVEC (Allen Video Enhanced Contrast) light microscopic methods on October 25, 1983.
The use of Video Microscopy is now very widespread not just for the study of biology, but also for the detection of flaws in materials. Some of the first important discoveries using this method were the imaging in real time of bi-directional transport in nerve axons. It became possible to image microtubules moving on glass in real time and this ability eventually led to the discovery of the important motor molecule kinesin. The first observations of the endoplasmic reticulum in living cells were also made using this method. Almost every cell biology laboratory in the country now employs video microscopy routinely.
These images of Nina Strömgren Allen and Robert Day Allen in their laboratory at Dartmouth College were made from videotape produced by the National Science Foundation in 1981.That tape explained their recent development and testing of Video Microscopy. The microscope seen in the picture was a Zeiss Axiomat and Hamamatsu made the camera and frame memory. They worked with the Hamamatsu engineers to modify the video camera and frame memory for this new microscope use. Robert Allen died from cancer in 1986. Nina Allen moved to Wake Forest University in 1984 and Joined the Botany Department at North Carolina State University in 1995. She is Director of the Cellular and Molecular Imaging Facility housed in 4115 Gardner Hall. Video microscopy is used in that laboratory on a daily basis to image biological and material science objects.
Dr. Eric L. Davis
The research program of Dr. Eric L. Davis in the Department of Plant Pathology at NC State University investigates the interactions of plant-parasitic nematodes (microscopic worms) with their host plants. Although the nematodes are extremely small, their damage to plants is very large-more then $70 billion in damage to a wide variety of crops throughout the world every year (Figure 1).
Figure 1: Healthy soybean plants in the background compared with soybean plants that were severely damaged by nematodes (microscopic worms that infect plant roots) in the foreground.
Most plant-parasitic nematodes live in the soil and infect plant roots. The cyst nematodes (Heterodera and Globodera species) and root-knot nematodes (Meloidogyne species) are the most damaging groups of plant-parasitic nematodes, and they have evolved very complex interactions with their host plants. As with all nematodes, cyst and root-knot nematodes grow by a series of four molts to the adult stage. Worm-shaped juveniles at the second life stage (J2) penetrate plant roots completely. The nematodes migrate to the center of the roots, where they must transform selected plant root cells into an elaborate feeding site to support the further growth and reproduction of the nematode.
Every plant-parasitic nematode (see Figure 2) has a "stylet" (a hypodermic needle-like feeding spear in the nematode head) that they use to withdraw nutrients from plant cells and also to secrete substances into the plant. The substances secreted by the nematodes allow them to penetrate and migrate within the roots and to transform the plant cells into feeding sites. Dr. Davis' lab is beginning to understand this process at a molecular level-with the goal of interfering in successful nematode parasitism to prevent crop damage. Some of the discoveries have potential commercial application to prevent nematode damage of plants, and two of the patent disclosures from the Davis lab are described here.
Figure 2: Illustration of an infective juvenile of the soybean cyst nematode, Heterodera glycines, showing the (hypodermic needle-like) "stylet" in the nematode head that it uses to feed from plant cells. Substances produced in the (red and green) esophageal gland cells of the nematode are secreted out through the stylet into plant root tissue.
NCSU Patent Disclosure Number 99-28
Figure 3 shows a soybean cyst nematode (SCN) juvenile (Heterodera glycines) that had been induced to secrete substances (stained blue) from its stylet. Since the nematodes are extremely small, it is almost impossible to collect enough of the secretions for direct analysis. Instead, the Davis lab has collaborated with other scientists to clone the nematode genes that produce the secretions. Once the genes are cloned, the analysis becomes much easier. One of the SCN genes cloned encodes an enzyme called a cellulase (b-1,4-endoglucanase) that the nematode secretes from its stylet to degrade the cellulose walls around plant cells. This turned out to be the first cellulase gene ever cloned from any animal-cellulase genes had only previously been cloned from bacteria, fungi, and plants. This discovery was published in 1998 by Davis and his collaborators (Smant, et al.) in the Proceedings of the National Academy of Sciences. A year later, the Davis lab was able to show that the juveniles of SCN secrete the cellulase to allow the nematodes to penetrate and migrate in plant roots. This discovery was highlighted on the cover of Molecular Plant-Microbe Interactions (Figure 4), which shows a bright green fluorescence that indicates where the nematode has secreted its cellulase through its stylet into the plant root. Inhibition of the nematode cellulase activity could represent a commercial way to prevent cyst nematode parasitism of crop plants, and this is a primary reason why an application to protect the discovery of the novel nematode cellulases has been pursued by NC State University.
Endoglucanase Gene Promoter Specifically Upregulated by the Root-Knot Nematode
NCSU Patent Disclosure Number 00-74
Figure 3: Production of secretions (stained blue) out the stylet of an infective juvenile of the soybean cyst nematode, Heterodera glycines. The secreted substances allow the nematode to penetrate and feed from plant roots. Figure 4: A journal cover showing the secretion of an enzyme (bright green fluorescence) called cellulase (b-1,4-endoglucanase) by an infective soybean cyst nematode juvenile as it penetrates a plant root. The cellulase enzyme degrades plant cell walls and allows the nematode to invade the plant root. Inhibition of nematode cellulase activity could be a commercial application to reduce nematode damage to crop plants.
Figure 5: Juvenile stages of nematodes (stained red) that have entered a plant root and started to feed. The nematode on the left has begun to swell as it feeds from the same plant cells and progresses through several molts to the swollen and immobile adult (reproductive) life stage. Figure 7: The blue color demonstrates a plant cellulase gene that has been "turned-on" (upregulated) in the infection site of a root-knot nematode. The swollen portion of the root (called a gall) is a typical symptom of plant root infection by root-knot nematodes (as observed by many unfortunate home gardeners). The secretions of nematodes also induce many changes in the plant cells that the nematodes use for feeding. The root-knot nematodes (Meloidogyne species) are similar to cyst nematodes in that the worm-shaped juveniles enter plant roots completely and then begin to feed (Figure 5). The nematodes transform selected plant cells around their head into "giant-cells" that are much larger than normal plant cells and have multiple nuclei (Figure 6). The nematodes require the giant-cells to feed as they become swollen and immobile at the adult life stages. Many plant genes are "turned-on" (upregulated) in the giant-cells to make the feeding site for the nematode. A Ph.D. student (Melissa Goellner) in the Davis lab discovered that the root-knot nematodes upregulate a cellulase (endoglucanase) gene of plant origin specifically within the giant-cells. Goellner obtained plants from Oded Shoseyov at the Hebrew University of Jerusalem that were bioengineered to produce a blue color in any plant cells that expressed the (cel 1) plant cellulase gene. Goellner discovered that the cellulase gene (blue color) appeared to be specifically upregulated at the site of nematode infection (Figure 7). She observed under a microscope that the plant cellulase gene (blue color) was only expressed in the giant-cells that the root-knot nematode had induced for feeding within the plant root Figure 8). Because the plant cellulase gene is upregulated by nematode feeding, the portion of the plant cellulase gene that responds to the nematode (the "promoter") could be used to express "anti-nematode" genes when the nematode attempts its normal feeding. This discovery may have tremendous commercial applications for bioengineering crop plants with a novel form of resistance to root-knot nematodes, and this is one reason why an application to protect the discovery of this plant cellulase promoter response to nematode parasitism has been pursued by N.C. State University.
Figure 8: Section of a plant root observed under a microscope that shows plant cellulase gene expression (blue color) exclusively within the giant-cells surrounding a swollen (clear) root-knot nematode (Meloidogyne species). This plant cellulase gene may be modified to produce an "anti-nematode" product when the nematode feeds, which could have commercial applications to prevent crop damage by root-knot nematodes.
Figure 6: Section of a plant root observed under a microscope that shows six "giant-cells" surrounding a swollen root-knot nematode (Meloidogyne species). The nematode transformed these plant cells into these giant cells for feeding. The expression of plant genes within the many nuclei (red dots) of the giant
Dr. Jean Beagle Ristaino
PCR Assays for Phytophthora Species
U.S. Patent Number 5,780,271
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Dr. Jean Beagle Ristaino is a professor in the Department of Plant Pathology at NC State University. Her patent, "PCR [Polymerase Chain Reaction] assays for phytophthora species," was issued under U.S. Patent Number 5,780,271 in 1998. This invention is a method of screening for the presence of pathogens in potatoes, tomatoes and other plant species using oligonucleotide primers. Her research is being used to determine the source of the potato late-blight pathogen, Phytophthora infestans that infected the Irish potato and caused the great Irish potato famine of the 1840s. The research is also being used to shed light on the origin of the modern form of the late-blight pathogen.
Windows to the Past:
Tracking 19th-century Irish potato famine epidemics using herbarium specimens
Contributed by Dr. Jean Beagle Ristaino, Professor, Department of Plant Pathology, NC State University, Raleigh, N.C.
More than 150 years ago, the late-blight pathogen Phytophthora infestans struck the Irish potato crop, leading to famine. Epidemics first began in North America in 1843 and in late-summer of 1845 and swept across Europe to Ireland, destroying potato crops in its wake. In its aftermath, more than one million people died and another two million emigrated from Ireland. As depicted in this painting by Daniel MacDonald, "The Discovery of Potato Blight," c. 1852, the Irish people were dependent on potatoes as a sole food source, and when the crop failed millions suffered the consequences.
Late-Blight in Sampson County, North Carolina, 1998
Late-blight has become a reemerging disease on both potato and tomato crops in the United States and many other areas of the developed and developing word because of widespread occurrence of new genotypes of the pathogen that are highly resistant to the commonly applied fungicide metalaxyl. Most isolates of the pathogen found worldwide are not resistant to this fungicide. This potato field in Sampson County, North Carolina was destroyed by the pathogen in a matter of weeks.
Sporangia of the Late-Blight Pathogen
(Slide courtesy of W. Fry, Cornell University) Late-Blight Infected Potato Tuber Phytophthora infestans
The late-blight pathogen produces asexual spores known as sporangia. The microscopic, lemon-shaped sporangia are borne on tiny branched stalks called sporangiosphores. Sporangia are easily dislodged from the sporangiophores and spread by wind and rain.
Some of the fungicide resistant isolates of Phytophthora infestans are not only surviving but also reproducing sexually and forming overwintering, thick-walled oospores in the presence of a number of commonly used fungicides on potatoes. This clever pathogen has adapted well to the pesticides that have been developed to manage it. Clearly, new and innovative research on the biology of the pathogen is needed to manage effectively late-blight epidemics.
Leaf Lesion on Potato
Caused by Phytophthora infestansSporangia infect leaves of potatoes or tomatoes and cause grayish black lesions. The pathogen then produces millions of additional sporangia in white tufts on the leaves, stems, tubers, or fruit that can be easily spread to the next susceptible plant.
Herbarium materials are being used in the Laboratory of Dr. Jean Ristaino (shown on right) at North Carolina State University with the help of Dr. Carol Trout Groves (shown on left), USDA, Orono, Maine, and research specialist Greg Parra to help clarify present-day questions about the biology of the late-blight disease. The tools of molecular biology coupled with herbarium specimens infected with Phytophthora infestans offer a unique approach to addressing questions concerning the nature and source of populations of old epidemics and the migration of the pathogen worldwide. "We are most interested in identifying the actual genotype of the pathogen that caused the 19th-century Irish, European and United States epidemics.
It has been proposed that a particular genotype of Phytophthora infestans referred to as the US-1 genotype, was the genotype that caused the original epidemics during the famine in Ireland. The previous studies were based on present-day collections of the pathogen. No one has ever tested actual specimens from the 19th-century epidemics to confirm or refute our current hypotheses. We plan to track historical migration patterns of the devastating potato pathogen using 19th-century dried potato leaves and identify the genotype(s) responsible in our work funded in part by a grant from the Committee for Research and Exploration of the National Geographic Society."
Infected Dried Modern Leaf and Herbarium Sheet
What are the unsolved mysteries of potato late-blight? There are many questions that remain to be answered concerning the origins of the late-blight pathogen, Phytophthora infestans. Some scientists ascribe to the Mexican theory believing that the pathogen originated in Mexico, which is clearly a present-day center of diversity of the pathogen. Other scientists ascribe to the Peruvian theory that the pathogen evolved in South America, the ancestral home of the potato. In fact, the first potato cultivatars to succumb to the disease in Europe in the 1840s included South American types such as "Lima," "Cordilleres," and "Peruviennes." Bat guano was used as fertilizer on potatoes and imported from Peru during the 1840s. The development of steamships also led to greater export of potato tubers from Peru to European countries in the 1840s. A third group of scientists has suggested that the pathogen originated in Mexico but inoculum for the 19th-century epidemics came from Peru. Development of a clear understanding of the origins of the pathogen will be useful as present-day potato and tomato breeders search for sources of host plant resistance to the disease.
Dr. John Lindley collected this specimen of potato in 1846 in the Royal Botanic Gardens in Dublin, Ireland. It is one of several of the oldest known specimens of potato that still exists from potato famine epidemics. The pathogen was then called Botrytis infestans by British mycologist Miles Berkeley, and the specimen was part of his herbarium collection.
DNA Gel Showing Bands of rDNA for 19th-Century Specimens
North Carolina State University scientists used molecular methods and a PCR primer called HERB1 in combination with PINF, a Phytophthora infestans specific primer, to amplify successfully a 100 pair fragment of the ribosomal DNA for 19th-century dried potato leaves from herbarium samples. Researchers in the Ristaino lab have successfully amplified DNA from more than 30 percent of the sampled tested to date which include Irish, British, and French samples collected in 1845, 1846, and 1847. Molecular studies of herbarium specimens have the potential to open a new window to study epidemics of the past.
Dr. Jason Shih
Two different technologies were invented from Dr. Jason Shih's laboratory. One derived from the discovery of a feather-degrading bacterium, which can break down chicken feathers. This discovery is significant, because feathers, like hair, are made of keratin protein that is resistant to common digestive enzymes. Because keratin is difficult to digest, feathers and hair have never been used as dietary protein. During a series of studies, the enzyme keratinase, which catalyzes the hydrolysis of feathers, was isolated. The gene that encodes keratinase was also isolated and sequenced. Cloning of this gene for hyper-production of this enzyme is now possible. More importantly, it was demonstrated that feathers after treatment with this enzyme were indeed hydrolyzed to peptides and amino acids. Feathers processed with the keratinase, therefore, are convertible to dietary protein used in animal feeds. The poultry industry in the U.S. produces eight billion chickens each year. From processing these chickens, one million tons of feathers are generated each year as a major by-product, mostly wasted or underutilized. If the keratinase technology can be adapted to process feathers, it can create a $400 million market based on the value of digestible protein. The development of the keratinase technology is a good case study in converting the waste into a value-added product.
Figure 1: Dr. Jason Shih and his graduate student (now Dr. Scott Carter) are inspecting the culture of bacteria growing on feathers. These bacteria produce and secrete the keratinase enzyme that hydrolyzes feather keratin.
Pertinent Patents to Keratinase Technology
U.S. Patent Number Title Receiver of Patent and Year Issued #4,908,220 Feather-Lysate, A Hydrolyzed Feather Feed Ingredient and Animal Feeds Containing the Same Jason C. H. Shih and C. Michael Williams (1990) #4,959,311 Method of Degrading Keratinaceous Material and Bacteria Useful Therefor Jason C. H. Shih and C. Michael Williams (1990) #5,063,161 Method of Degrading Keratinaceous Material and Bacteria Useful Therefor, A Divisional Patent Jason C. H. Shih and C. Michael Williams (1991) #5,171,682 Purified Bacillus licheniformis PWD-1 Enzyme Jason C. H. Shih and C. Michael Williams (1992) #5,186,961 Method and Composition for Maintaining Animals on a Keratin Containing Diet Jason C. H. Shih and Chun-Ginn Lee (1993) #5,712,147 DNA Encoding Bacillus licheniformis PWD-1 Keratinase Jason C. H. Shih, Xiang Lin, and Eric S. Miller (1998)
In addition to the U.S., these patents have been issued or pending in many foreign countries, including Canada, Mexico, European Union, South Africa, Australia, New Zealand, Brazil, China, Taiwan, South Korea, and Japan.
Thermophilic Anaerobic Digestion
U.S. Patent Number 5,525,229
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Another technology invented in Dr. Shih's laboratory is called thermophilic anaerobic digestion (TAnD). It is a microbiological process by which organic matter can be degraded and converted to methane and carbon dioxide in the absence of air or oxygen. The same process can be used to manage animal waste that is generated in large amount on a poultry or livestock farm. It was in Dr. Shih's laboratory that the process was found to be very efficient when operated at higher (thermophilic) temperatures, first in the laboratory and then on the university farm. After a successful test with the prototype at NCSU, the full-scale system was tested successfully in Taiwan and China. In North Carolina, a full-scale TAnD and associated Integrated Farming system has been proposed as an alternative method for waste management on farms. The Integrated Farming system is designed to fully use all resources or by-products generated by TAnD.
Figure 2: Thermophilic anaerobic digestion (TAnD) and integrated farming is a new agricultural ecosystem. TAnD converts animal waste into useful resources including biogas (65% methane and 35% carbon dioxide) energy, nutrients for aquaculture and bio-fertilizer for horticultural produce.
Dr. Harold E. Swaisgood
Process of Removing the Cooked Flavor from Milk
U.S. Patent Number 4,053,644
This patent was issued in 1977 to Dr. Harold E. Swaisgood of the food science department at NC State University. Dr. Swaisgood's invention is a process for removing the "cooked" flavor from milk by contacting the heat-treated fluid milk with an immobilized sulfhydryl oxidase enzyme. This method eliminates the unpleasant taste and smell resembling boiled cabbage that resulted from heating milk in excess of 155º Fahrenheit.
Purification and Immobilization of Sulfhydryl Oxidase
U.S. Patent Number 4,087,328
This patent was issued in 1978 to Dr. Harold E. Swaisgood of the food science department at NC State University. This invention is a process for purification and immobilization of sulfhydryl oxidase enzyme.
Detection of Antibiotics in Milk
U.S. Patent Number 4,347,312
This patent was issued in 1982 to Rodney J. Brown of Logan, Utah, and Harold E. Swaisgood of NC State University's food science department. This invention is a process for detecting the presence of antibiotics in milk.
Dr. Kenneth R. Swartzel
Pasteurization and Aseptic-Packaging for Extended Shelf-Life Liquid Egg
Liquid egg has become extremely important to commercial users of egg because of its convenience and safety. All liquid egg is pasteurized to control salmonella, listeria, and other food borne pathogens, as required by regulatory officials. But egg pasteurization by conventional methods results in a short shelf-life. The NCSU research team (Hershell R. Ball, Jr., Mohammad-Hossein Hamid-Samimi, and Kenneth R. Swartzel developed and patented in 1989, 1990, and 1991 the process technology that allowed liquid egg products to be marketed free of pathogens (Listeria as well as Salmonella) and possessing an extended refrigerated shelf-life. The patents were based upon the equivalent point concept and a basic study of the rheological properties of egg as a function of temperature. Until this investigation was completed, pasteurization temperatures had to be low to avoid egg coagulation. The research team demonstrated that egg could tolerate higher temperatures than previously believed, yielding greater destruction of both spoilage and pathogenic-microorganisms. In follow-up investigations, the processing technology was perfected and combined with aseptic-packaging to pave the way for commercialization. The concept used to provide extended shelf-life egg is now being applied to in-shell pasteurization. The research efforts of the research team have revolutionized the liquid egg industry. Morning Glory Egg Company of North Carolina was quick to recognize the potential of the patents and introduced Easy Eggs in December 1988. In order to capitalize on the national market, Michael Foods, Inc., of Minnesota, acquired Morning Glory and now processes and packages more than 200 million pounds of egg annually under these patents. In an article about Michael Foods published in March 1992, Newsweek stated, "Its biggest success is Easy Eggs, a salmonella (pathogen)-proof liquid egg with a 10-week shelf-life; sales have risen to $80 million annually since its 1989 introduction." The convenience, safety, and portion-control features of eggs produced with this process are causing previous shell-egg users to move to extended shelf-life refrigerated liquid eggs. The acceptance of the new egg product by major fast food firms (e.g. Burger King) and institutional users is resulting in a fundamental change in the structure of how eggs are marketed. The exponential growth of this product has resulted in a production of nearly 1.5 billion pounds since Easy Eggs were introduced. Egg, without the shell, offers opportunity for further treatments such as cholesterol removal. Today, the egg industry has a bright future with increased international trade possibilities and the potential for providing consumers with a low-cost, nutritious, stable product that promotes good health.
Cover of the September 1994 journal, Food Technology, shows Hershell R. Ball, Jr. (left) and Kenneth R. Swartzel (right) measuring the temperature of an ultrapasteurized liquid whole egg product coming from an aseptic-packaging machine.
The article details the 1994 IFT Food Technology Industrial Achievement Award given to Kenneth R. Swartzel, Hershell R. Ball, Jr., and Mohammad-Hossein Hamid-Samimi of NC State University. To view in pdf format click: Food Technology Article
Pertinent Patents to Liquid Egg Products
U.S. Patent Number Title Receiver of Patent and Year Issued #4,808,425 Method for the Ultrapasteurization of Liquid Whole Egg Products Kenneth R. Swartzel, Hershell R. Ball, Jr., and Mohammad-Hossein Hamid-Samimi (1989) #4,957,759 Method for the Ultrapasterurization of Liquid Whole Egg Products Kenneth R. Swartzel, Hershell R. Ball, Jr., and Mohammad-Hossein Hamid-Samimi (1990) #4,957,760 Ultrapasteurization of Liquid Whole Egg Products with Direct Heat Kenneth R. Swartzel, Hershell R. Ball, Jr., and Jeffrey W. Liebrecht (1990) #4,994,291 Method for the Ultrapasterurization of Liquid Whole Egg Kenneth R. Swartzel, Hershell R. Ball, Jr., and Mohammad-Hossein Hamid-Samimi (1991) #5,019,407 Method for Pasteurizing Liquid Whole Egg Products
Kenneth R. Swartzel and Hershell R. Ball, Jr. (1991)
#5,019,408 Method for the Ultrapasteurization of Liquid Whole Egg Products Kenneth R. Swartzel, Hershell R. Ball, Jr., and Mohammad-Hossein Hamid-Samimi (1991)