SAMPLE ABSTRACT (2009 ABSTRACTS START ON NEXT PAGE!) :
Edible Vaccines: A study of the Norwalk virus capsid protein (NVCP) in potatoes and tobacco
Jane Doe, November 4, 200X
In an age where there is the medical knowledge to vaccinate children and ward of life-threatening diseases there are an estimated 900,000 children in the US and millions in developing nations that still remain unvaccinated today. In addition, about $17.5 billion are lost annually to livestock and poultry products as a result of disease even though we have the knowledge to vaccinate these animals as well. Although we have the knowledge to vaccinate all animals alike we do not have the capability. Most parenteral (injected vaccines) are costly to develop and ship, require refrigeration (cold-chain) as well as need trained personnel to inject it subcutaneously. These things together make it difficult for widespread vaccination efforts particularly in developing nations where most of these diseases remain endemic.
Edible vaccines might be a solution that will enable the positive effects of vaccines to be far reaching and to decrease some potential hazards associated with parenteral vaccines such as toxic compounds, allergic responses and risk of attenuated strains reverting to pathogenic strains. Edible vaccines offer a way to deliver a vaccine orally, without the need for the cold chain, decrease costs of production and shipping and may be ideal for facing bio-weapons and veterinary use among other benefits.
When choosing a plant to be used as a vaccine it is important that it is a hardy, palatable plant that is high in nutritive and protein content. The plant is also one that would best be indigenous to the country in which it is to be used and should be able to be transformed relatively easily. Tobacco plants have been used extensively in this research but new work is being done in potatoes, tomatoes, lettuce, corn and other crop plants. Little is known about the optimum dosage needed to confer immunity or how long this immunity lasts so there is still a lot of research to be done.
A study conducted by Mason et al. (1996) looked at the immunogenic effect of transgenic potato tubers and tobacco leaves carrying a Norwalk virus capsid protein (NVCP) in mice. Currently, there are no drugs are vaccines available to treat Norwalk infections yet this virus accounts for about 42% of the viral gastroenteritis seen in the US.
Mason et al. (1996) looked at three different plasmid constructions containing the NVCP gene that were transformed into plants using a binary T-DNA plasmid using Agrobacterium tumefaciens . They found varying expression of the antigenic protein due to the position effect and an increase in translational expression of the protein in plasmids that contained the TEV 5’-UTR. In addition, they obtained 0.23% and 0.37% TSP (total soluble protein) in tobacco leaves and potato tubers respectively, which are higher levels than previous work. Finally, both humoral and mucosal antibody responses were detected in the mice suggesting that edible vaccines might be a viable source for immunization. A follow up study in 2000 done by Tacket et al. looked at the human immune response to the NVCP expressed in potatoes. Overall, 95%, 19 out of 20 volunteers developed some kind of immune response, although the antibody increase in some cases was modest.
Sources used:
Arntzen, C. J. 2001. Keynote lecture: agricultural biotechnology. Journal of the Science of Food and Agriculture.
81: 805-809.
Carter, J. E. and W. H. R. Langridge. 2002. Plant-based vaccines for protection against infectious and autoimmune diseases. Critical Reviews in Plant Sciences. 21(2): 93-109.
Daniell, H., Streatfield, S. J., and Wycoff, K. 2001. Medical molecular farming: production of antibodies, biopharmaceuticals and edible vaccines in plants. Trends in Plant Science. 6(5): 219-226.
Mason, H. S., Ball, J. M., Shi, J. J., Jiang X., Estes, M. K. and Arntzen, C. J. 1996. Expression of Norwalk virus caspid protein in transgenic tobacco and potato and its oral immunogenicity in mice. Proc. Natl. Acad. Sci.
USA. 93: 5335-5340.
Sala, F. Rigano, M. M., Barbante, A. Basso, B. Walmsley, A. M., and Castiglione, S. 2003. Vaccine antigen production in transgenic plants: strategies, gene constructs and perspectives. Vaccine 22: 803-805.
Tacket, C. O., Mason, H. S., Losonsky, B., Estes, M. K., Levin M. M., and Arntzen C. J. 2000. Human immune responses to a novel Norwalk virus vaccine delivered in transgenic potatoes. Journal of Infectious Diseases
182: 302-305.
1

2009 Class Abstracts appear below with my editorial additions in red. –ams
Diabetes is the most costly disease that affects our nation today, and type 1 diabetes is at an all time high. Type 1 diabetes is an autoimmune disease in which the beta cells of the pancreas are attacked by the patient’s own immune system. These beta cells are no longer able to produce insulin which is responsible for converting sugars, starches and other foods into energy. This leads to an extreme level of glucose in the blood. There are many devastating side effects to the disease. The current treatments lead to side effects of their own, and the patient must constantly check their blood glucose levels.
A recent experiment by Rene Maehr et al. looks into the possibility of using induced pluripotent stem cells (iPS) to create insulin producing cells. These pluripotent cells will be generated from the patient’s fibroblasts obtained from a skin biopsy. These fibroblasts will be infected with retroviruses containing the necessary transcription factors. Immunostaining was used to determine if the cells had made antibodies for the transcription factors; the cells tested positive for these antibodies and for alkaline phosphatase enzyme activity. The induced pluripotent stem cells of made from a type 1 diabetes patient are called DiPS cells. These cells were then able to differentiate and express markers of all three cell types, which is as expected from a pluripotent cell. In order for these DiPS cells to differentiate into the insulin-producing cells they were aiming for, it must follow the endothermal/pancreatic lineage. There was a stepwise protocol to lead the cells down this pathway and to eventually obtain the beta-like cells. In order to find out if the cells are able to produce insulin in vitro, the cells were subjected to high and low concentrations of glucose. The DiPS cells released human
C-peptide after glucose stimulation. They concluded that the cells can be differentiated into insulin producing/glucose-responsive cells.
This research opens the door in many ways. This allows a model of the not-well understood type
1 diabetes, to be made. As of now this treatment is inefficient, and the protocol leads to a risk of potential integration into the genome. In the near future they hope to perfect the protocol to allow this to be a treatment.
References:
1. Maehr, Rene, Shuibing Chen, Melinda Snitow, Thomas Ludwig, Lisa Yagasaki, Robin Goland, Rudolph
Leibel, & Douglas Melton, 2009. “Generation of pluripotent stem cells from patients with type 1 diabetes.” PNAS, Vol. 106, No. 37, pg. 15768-15773
2. Shin-ichi Nishikawa, Robert A. Goldstein & Concepcion R. Nierras, 2008. “The promise of human induced pluripotent stem cells for research and therapy.” Nature Reviews: Molecular cell biology 9:
725-729
3. Xu, Yang, 2009. “Developing induced pluripotent stem cells into human therapeutics and disease models.” California Institute of Regenerative Medicine <http://www.cirm.ca.gov/node/2096>
2

Nick Cummings , November 5, 2009
To the world some of the most deadly biological include weaponized organisms such as Anthrax, smallpox and tuberculosis; among the most deadly toxins in the world include Ricin and botulism toxin.
The best defense to any biological terrorism act is the vast administration of vaccine to the general population. Among protein toxins such as ricin which is most effective in aerosol form, the best way to inactivate the toxin itself is by developing antibodies against it.
Ricin is a protein compound found in Castor beans. It’s composed of two chains one A-chain which is an N-glycoside hydrolase with three domains. The B-chain is the catalytically inactive portion that mediates entry into the cells. Upon entry into the cells the protein enters the ER where it’s believed to be cleaved releasing the catalytic site of the protein. The protein then is capable of cleaving rRNA where it then inactivates it essentially stopping protein synthesis.
In order to find an effective treatment toward ricin intoxication, Pelat et. all conducted an experiment in order to find an effective antibody fragment(scFv) that can neutralize the A-chain of ricin or ricin in general. To do this, they immunized a non-human primate with the A-chain of ricin. After a series of shots of the A-chain between different periods of time, they extracted bone marrow cells and lysed them freeing the mRNA encoding for ricin antibodies. After amplifying the DNA that was reversed transcribed from the mRNA, they inserted cDNA into a series of vectors that transformed bacteria. By using a phage display ed immune library they were able to isolate the most ideal antibody fragment that neutralized ricin at the most efficient rate. Among the most affective antibody fragments was the 43RCA clone which had a neutralizing efficiency of IC
50
= 23 ± 3 corresponding to a molar ratio of 4
[scFv]/[ricin]. Compared with the antibody obtained from hyperimmune mouse serum (anti-dgRCA) IgG the 43RCA cloned performed more efficiently. Another advantage of the 43RCA antibody is its similarity to human IgG germlines , meaning it can be further humanized and be safe for human use.
Sources:
1.
Pelat T, Et. All .
et al.
Isolation of a human like antibody fragment(scFv) that neutralizes ricin biological activity. Biomedcentral [Internet]. [cited 2009 Nov 1]; :1-14. Available from : http://www.biomedcentral.com/1472-6750/9/60 .
2.
. 2009 [cited 2009 Nov 1]. Antibody Structure and Isotopes. [Internet]. United States: abcam.
Available from: http://www.abcam.com/ps/CMS/Images/abstructure.JPG
.
3.
. 2009 [cited 2009 Nov 1]. Ricin and the Umbrella murder. [Internet]. CNN. Available from: http://www.cnn.com/2003/WORLD/europe/01/07/terror.poison.bulgarian/.
4.
. 2009 [cited 2009 Nov 1]. Plasmid map of pGEM-T vector. [Internet]. biovisualtech. Available from: http://www.biovisualtech.com/bvplasmid/pGEM-T_vector.jpg.
3

Nima Maria George, November 5, 2009
The remarkable brightly glowing green fluorescent protein, GFP, was first observed in the beautiful jellyfish, Aequorea victoria in 1962. Since then, this protein has become one of the most important tools used in contemporary bioscience. By using DNA technology, researchers can now connect GFP to other interesting, but otherwise invisible, proteins. With the aid of GFP, researchers have developed ways to watch processes that were previously invisible, such as the development of nerve cells in the brain or how cancer cells spread This glowing marker allows us to watch the movements, positions and interactions of the tagged proteins. Osamu Shimomura was the first person to isolate GFP and to find out which part of
GFP was responsible for its fluorescence. His meticulous research laid the solid foundations on which the
GFP revolution was built.
Heat shock proteins ( HSPs ) function as molecular chaperones and thus play a critical role in protein folding, intracellular trafficking of proteins, and coping with proteins denatured by heat and other stresses.
These activities are part of a cell's own repair system, called the "cellular stress response" or the "heatshock response". Accordingly, the study of stress proteins has undergone explosive growth. In recent years, the zebrafish has been increasingly used as a model for studying heat shock proteins. In this study a transgenic hsp27-gfp zebrafish line is established and the transgenic expression of GFP is compared with that of the endogenous hsp27.
Transgenic zebrafish were was responsive to heat shock with GFP induced in several tissues, with the highest expression in skeletal muscle and recapitulated the expression pattern of endogenous hsp27 RNAs. Also , the potential of transgenic zebrafish embryos for heavy metal induction is also tested and demonstrated the inducibility of GFP expression by heavy metal (i.e., arsenic).
References
Wu Y .L, Pan X, Mudumana S. P, Wang H, Kee P. W., and Gong Z. Development of a heat shock inducible gfp transgenic zebrafish line by using the zebrafish hsp27 promoter. Gene 408 (2007) 85-94
Chalfie M.GFP: Lighting up life. Proc Natl Acad Sci U S A . (2009) 106(25):10073-80.
Chalfie M, Tu Y, Euskirchen G, Ward W.W., and Prasher D.C. Green fluorescent protein as a marker for gene expression. Science (1994) 263(5148):802-5.
Gerdes H.H and Kaether C.Green fluorescent protein: applications in cell biology. FEBS Lett. (1996) 24;
389(1):44-7.
Rizzuto R, Brini M, Pizzo P, Murgia M., and Pozzan T.Chimeric green fluorescent protein as a tool for visualizing subcellular organelles in living cells. Curr Biol.
1995 5(6):635-42
4

Exercise in a Pill? It May Be Possible Thanks To The Marathon Mouse
By Grant Hutchinson
November 5, 2009
In today’s society, there is an ever increasing trend in towards obesity and obesity related problems. Researchers have been looking into ways to fix this growing problem. Recently, marathon mice have been used to examine whether genetic engineering could produce a solution. This transgenic mouse has the ability to run twice the distance of a normal mouse and is able to run an hour longer than the typical 90 minutes a normal mouse can run (Martindale 2004). This mouse also has the ability to be resistant to obesity.
The muscles of animals consist of slow twitch (type 1) and fast twitch (type 2) muscle fibers.
These fibers have distinct characteristics and different functions inside the body. The slow twitch muscles are rich in mitochondria and use oxidative metabolism to produce energy and the fast twitch fibers rely on glycolytic metabolism for energy. The slow twitch muscle fibers therefore are used for endurance and the fast twitch muscle fibers tire easily and are used for short powerful contractions.
It was recently found that altering the expression of peroxisome proliferator-activated receptor δ resulted in mice that contained skeletal muscle that had an increased number of slow twitch muscle fibers.
Under normal conditions PPARδ is activated by exercise. It may be activated by the co-activator PGC-1α or by an upstream signaling component (Goto et al. 2000). In the experiment a PPARδ cDNA was fused with a human skeleton actin promoter to create a high level of activation. This study which was conducted by Yong-Xu Wang et al. (2004) showed these mice had the ability to run significantly longer than their wild type counterparts. Along with the increased endurance the mice showed an ability to be obesity
“resistant” even under a high calorie and high fat diet. There was also an increase in oxidation enzymes, mitochondrial biogenesis, and type 1 fiber contractile protein. This lead to an overall discovery that the over expression of PPARδ allows for the effects of endurance training without actual training to occur.
This means that there could be weight loss in individuals who cannot exercise due to their excessive size.
There could be the potential for solving problems that are related to obesity such as heart disease and diabetes.
References:
Goto M, Terada S, Kato M, Katoh M, Yokozeki T, et al. 2000. “cDNA cloning and mRNA analysis of
PGC-1 in epitrochlearis muscle in swimming-exercised rats.” Biochem Biophys Res Commun 274: 350-
354.
Martindale D. 2004. "Muscle Twitch Switch." Scientific American 291 (6): 22-24.
Wang Y, Zhang C, Yu R, Cho H, Nelson M, et al. 2004. “Regulation of Muscle Fiber type and Running
Endurance by PPARδ.” PLoS Biology.
2:10. 1533-1539.
5

Anthrax: Therapies and Vaccinations through recombinant protective antigen (PA)
Christine Fisher, November 5, 2009
Anthrax is a disease caused by the bacteria Bacillus anthrasis.
Anthrasis comes from the greek word for coal which shares the color of the scabs from cutaneous anthrax. Anthrax comes in three different forms; skin, gastrointestinal, and inhalation. Skin anthrax is caused when spores from the bacteria enter a cut or abrasion in the skin and gastrointestinal anthrax is caused by the ingestion of anthrax spores usually from contaminated meat. The most deadly form is inhalation anthrax in which spores are inhaled into the lungs. The disease has flu-like symptoms which have led to the misdiagnosis of the disease by multiple doctors in the past. During the early days of infection, the macrophage cells ingest anthrax spores and transport them to the lymph nodes near the lungs where the spores turn into full bacteria and burst through the macrophages as they multiply. The bacteria then releases toxins into the blood stream which causes the swelling and bleeding of many tissues during the second stage. Shortly after this stage, the blood pressure drops, oxygen levels fall, organs fail, and death rapidly follows. The current vaccine for anthrax, anthrax vaccine adsorbed (AVA), is used for skin anthrax infections (not including the more dangerous inhalation anthrax infection) and requires multiple injections and booster shots. The lethal toxin (LeTx) is a major factor in anthrax pathogenesis that forms through the combination of the protective antigen (PA) and the lethal factor (LF). Phenylalanine-427 (F427) has been extensively studied as it is a vital contribution to the function of PA. Studies have been conducted in which mutants are produced with mutations at the F427 residue for the dominant-negative inhibitory
(DNI) phenotype, and are tested in the presence of LF. Individual amino acid replacements were shown to be responsible for different levels of DNI activity, for example F247D and F247N mutants had the highest
DNI activity. Both of these F247 mutants were able to inhibit LeTx based on the dose of the mutant. The current PA vaccine is dangerous when administered to patients that are in the first stages of anthrax infection because it increases the binding sites for the endema factor (EF) and LF. As an alternative, the
DNI phenotype occurs when PA mutants are used to coassemble with the wild-type protein (WPA) and block its ability to move parts of enzymes across membranes (a necessary step in the pathogenesis of anthrax). Other studies have been conducted in which recombinant PA was used in conjunction with a synthetic double stranded RNA for polyriboinosinic-polyribocytidylic acid (pI:C). This mutant PA and pI:C adjuvant have shown promising potential as a vaccine against inhalation anthrax based upon nasal immunization trials in mice.
References:
Brown K. 2001. A ‘Sure Killer’ Yields to Medicine.
Science . 294 : 1813-1814.
Heijne G.V. 2005. Translocation of Anthrax Toxin: Lord of the Rings. Science.
309(5735) : 709-710.
Sellman B.R., Mourez M., and Collier R.J. 2001. Dominant-Negative Mutants of a Toxin Subunit: An
Approach to Therapy of Anthrax. Science . 292(5517) : 695-697.
Sha C., Aizhen G., Ziduo L., Yadi T., Gaobing W., Chengcai Z., Yaxing Z., and Huanchun C. 2009.
Investigation of New Dominant-Negative Inhibitors of Anthrax Protective Antigen Mutants for
Use in Therapy and Vaccination. Infection and Immunity . 77(10) : 4679-4687.
Sloat B.R., Cui Z. 2006. Nasal Immunization with Anthrax Protective Antigen Protein Adjuvanted with
Polyriboinosinic–Polyribocytidylic Acid Induced Strong Mucosal and Systemic Immunities.
Pharmaceutical Research . 23(6) : 1217-1226.
6

Differentiation of Embryonic Stem Cells into Neural Cells to Provide Therapeutic Benefit
Julian Capito November 5 th , 2009
Embryonic stem cells are early stage developmental cells that have the ability to differentiate into any cell type, which is called pluripotency. These pluripotent cells are obtained by extracting cells from the embryoblast of the blastocyst embryonic stage. Once these cells are gathered, they can be cultured in various media to induce differentiation to various cellular phenotypes. In theory, these cells, once properly differentiated, can be transplanted into a patient to replace damaged cells that do not have the ability to regenerate, such as muscle and CNS cells.
Embryonic stem cells that have been differentiated into neural cells have many possible therapeutic benefits, such as repairing spinal cord damage, treatment of Parkinson’s disease and treatment of amyotrophic lateral sclerosis. However, there are many obstacles that need to be overcome for the practical implementation of therapeutic stem cells. There are issues over how to properly differentiate embryonic stem cells, deliverance of the differentiated cells to the proper site, avoiding possible tumorigenic cells once deliverance to the proper site has occurred, prevention of infection, etc. A significant amount of research still needs to be conducted to address these various concerns.
A study conducted by Kim et al .
(2009) addressed the issue of properly differentiating pluripotent cells into a specific neural phenotype. Previous to this study, embryonic stem cells were induced to become neural cells by the use of a medium containing retinoic acid and serum. Neural cells obtained by this method differentiate into various neural phenotypes, such as astrocytes or motor neurons. This study attempted to differentiate these pluripotent cells into neural cells by using a growth medium that contained no retinoids. Additionally, the medium was serum-free and the only exogenous proteins present were insulin, transferrin and bovine serum albumin. Basically, the goal of this experiment was to restrict the number of proteins exposed to the undifferentiated cells to help determine which factors contribute to the development of a specific neural phenotype. By establishing which factors contribute to the development of a certain neural phenotype, the therapeutic use of embryonic stem cells will soon become a reality.
References
Ehnert, Sabrina, Matthias Glanemann, Andreas Schmitt, Stephan Vogt, Naama Shanny, Natascha C.
Nussler, Ulrich Stockle, and Andreas Nussler. "The possible use of stem cells in regenerative medicine: dream or reality?" Langenbecks Archive of Surgery 394 (2009): 985-97.
Kim, Mijeong, Ayman Habiba, Jason M. Doherty, Jason C. Mills, Robert W. Mercer, and James E.
Huettner. "Regulation of mouse embryonic stem cell neural differentiation by retinoic acid."
Developmental Biology 328 (2009): 456-71.
Legge, M., and L. M. Jones. "Stem cell spinal cord regeneration: first do no harm." Journal of Medical
Ethics 34 (2008): 838-39.
Preynat-Seauve, Olivier, Pierre R. Burkhard, Jean Villard, Walter Zingg, Nathalie Ginovart, Anis Feki,
Michel Dubois-Dauphin, Samia A. Hurst, Alex Mauron, Marisa Jaconi, and Karl-Heinz Krause.
"Pluripotent stem cells as new drugs? The example of Parkinson's disease." International Journal of Pharmaceutics 381 (2009): 113-21.
Wang, Zi-kuan, Dong-Bo Ou, and Qiang-Sun Zheng. "Methods of maintaining human embryonic stem cells continues to be worth studying." Cell Biology International 33 (2009): 1123-124.
7

Ryan Scavinski, November 10, 2009
Type 1-diabetes is caused by the autoimmune destruction of insulin producing
β-cells. To stay healthy, diabetics must frequently monitor their glucose levels in the blood and inject themselves with insulin multiple times throughout the day.
The use of islet transplantation has been used in the past as a potential treatment.
However, problems such as the difficulty of obtaining sufficient numbers of islets or the need for immunosuppressive medication to control rejection have limited the widespread use of this treatment. For this reason, the use of embryonic stem (ES) cells has been studied for their potential to produce insulin producing cells (IPCs).
The study by S. Raikwar and N. Zavazava show two main strategies for the differentiation of embryonic stem cells into insulin producing cells. The first strategy involves the use of embryoid body (EB) formation. An EB is an arrangement of embryonic stem c ells destined to differentiate into the precursors of the endoderm, ectoderm and mesoderm. The treatment of EBs with multiple growth factors gives rise to Nestin cells, which eventually differentiate into IPCs.
The second strategy involves using Definitive Endoderm (DE). This involves bypassing the EB formation and selectively generating the endoderm. The ES cells which are treated with Activin A leads to the development of the DE. Retinoic acid
(RA) is also used to promote pancreatic differentiation and after maturation on serum free medium, differentiated cells expressed islet specific markers such as Cpeptide, insulin, glucagons and glut2. These cells were then transplanted under the renal capsules of diabetic mice of which 30% of the mice maintained normal blood glucose levels. With further research, the use of stem cells to cure diabetes will be a possibility in our near future.
Reference:
1.
Soria, B., Skoudy, A., and Martin, F. 2001. From stem cells to beta cells: new strategies in cell therapy of diabetes mellitus. Diabetologia 44 407-415
2.
Raikwar, S. and Zavazava, N. 2009. Insulin producing cells derived from embryonic stem cells: are we there yet?. Journal of Cellular Physiology, 218
256-263
3.
Jiang, W., Shi, Y., Zhao, D., Chen, S., Youg, J., Zhang, J., Qing, T., Sun, X.,
Zhang, P., Ding, M., Li, D., and Deng, H. 2007. In vitro derivation of functional insulin producing cells from human embryonic stem cells. Cell
Research. 17 333-344
8

Isolation of two storage protein promoters from Hordeum chilense and characterization of their expression patterns in transgenic wheat
Matthew Payne - November 10, 2009
Genetic modification of plants has been a cutting edge science, which has enabled scientists to provide farmers with plants which are disease resistant as well as plants which provide the consumer with increased nutritional content. Cereal grains, such as wheat, are a dietary staple of humans and domestic animals worldwide. Therefore, increasing the protein yield of such plants would greatly increase the quality of one’s diet, stately in developing countries, or provide resistance to plant disease, primarily of fungal origin, which affect many of these cultivated species.
In 2007 Piston, Leon, Lazzeri, and Barro published a paper describing research they had completed which increased the knowledge of the synthesis and deposition of storage protein in cereal grains. The researchers utilized available knowledge regarding the process of protein synthesis and deposition of said storage protein and incorporated it into an experimental design which permitted them to increase both the viability of plants with HMW protein production and increase their overall protein production.
The researchers grew the plants in a greenhouse to provide 12 hour light and dark cycles. Then, researchers isolated a 5’ UTR by genome walking (flanking genomic segments adjacent to a known sequence). The fragment containing gusA gene and the nopaline (nos) terminator excised from plasmid pAHC25 and inserted into pGerm3Z to create pGUS. PCR products were cloned into pGUS to create pHorB-GUS and pHorD-GUS. Bombardment of plasmids pHorB-GUS or pHorD-GUS in combination with pAHC20, which contains the selectable bar gene (confers tolerance to phosphinothricin) under the control of maize ubiquitin promoter, targeted the immature scutella of wheat. Surviving explants transferred to R regeneration medium supplemented with 2mg 1
-1
of PPT and cultured 2 additional rounds of 3 weeks each. The putative transgenic plants were then transferred to soil and grown to maturity in greenhouse.
The sequence of B hordein promoter reported showed structure of the prolamin promoters and contain characteristic element ‘Prolamin box’ formed by the E motif and the N motif, whereas, the sequence of the H. chilense D hordein promoter showed the ‘HMW-enhancer’characteristic of the HMW gene promoters, and the G-box like and the RY-element. The fragments of the B and D hordein promoters fused with the gus A gene to characterize their activities in bread wheat; 5 and 9 independent transgenic lines (respectively) were obtained with plasmids pHorB-GUS and pHorD-GUS. The expression of the gus A gene was only detected in the seeds of the transgenic lines. GUS staining increased in both transgenic lines with the development of the endosperm, reaching max at 28 DPA when the accumulation of the β-glucuronidase protein was maximal; staining (protein accumulation) appeared to not be uniform in all of the endosperm. The use of QRT-PCR assisted in determining the expression profiles of both promoters. Expression of the gus A gene first detected at 13 days post anthesis (DPA) in both groups of transgenic plants; gus A transcripts driven by the B hordein promoter increased in level to a peak at 25 DPA and subsequently declined, whereas gus A transcripts driven by the D hordein promoter maintained the same transcript level from 23 to 25 DPA, and then increased expression from 25 to 28
DPA.
References needed!
9

Golden Rice: Introduce Provitamin A in Rice Endosperm
Yingying Wu-- November 10, 2009
Vitamin A deficiency can result in permanent blindness and increase the incidence and severity of infectious diseases.
It is estimated that a quarter of a million children go blind each year because of nutritional deficiency in southern Asia. Success may be got by using recombinant technologies to produce provitamin A in a major staple food, rice to overcome this worldwide problem. No rice cultivars produce provitamin A in endosperm, which is the edible part of the grain. ‘Golden Rice’ is engineered to produce beta-carotene (provitamin A). The carotenoids produced in the endosperm of the gains give rise to a characteristic yellow color.
The synthesis of beta-carotene from geranylgeranyl diphosphate that is synthesized in immature rice endosperm requires plant enzymes: phytoene synthase, phytoene desaturase, zeta-carotene desaturase and lycopene cyclase that is encoded by lcy gene. The original golden rice was produced in 2000, which used
Agrobacterium-mediated transformation to introduce the entire beta-carotene biosynthetic pathway into rice endosperm with three vectors. In the single transformation, the vector pB19hpc combined the sequences for a plant phytoene synthase (psy) originating from daffodil with the sequence coding for a bacterial phytoene desaturase (crtI) originating from Erwinia uredovora. The psy and crt1 genes were placed under the control of an endosperm-specific glutelin (Gt1), so that they are only expressed in the endosperm. The end product of the engineered pathway is lycopene, but if the plant accumulates lycopene the rice would be red. Co-transformation was using vectors pZPsC and pZLcyH. Vector pZPsC carries psy and crtI and vector pZLcyH provides lycopene

cyclase from Narcissus pseudonarcissus. Lycopene

-cyclase carried a functional transit peptide sequence attached so that it is targeted to the plastid, where geranylgeranyl diphosphate formation occurs. The pB19hpc single transformants, engineered to synthesize only lycopene (red), were similar in color to the pZPsC/pZLcyH cotransformants engineered for beta-carotene (yellow) synthesis. In fact, further research showed that lcy gene is already being produced in wild-type rice endosperm.
Under greenhouse conditions, the original golden rice could produce 1.6 ug/g carotenoids. The Golden Rice 2, which came out in 2005, reported has up to 37ug/g carotenoid of which 31 ug/g is beta-carotene. Golden Rice 2 produces 23 times more carotenoid than the original golden rice. Phytoene synthase is the limiting step for carotenoid biosynthesis and is a major regulatory step. The Golden Rice 2 combines the phytoene synthase gene from maize with crt1 from the original golden rice.
References:
Ye et al. 2000. Engineering the provitamin A (beta-carotene) biosynthetic pathway into (carotenoid-free) rice endosperm. Science 287 (5451): 303-305 PMID 10634784
Paine et al. 2005. Improving the nutritional value of Golden Rice through increased pro-vitamin A content. Nature Biotechnology doi:10.1038/nbt1082
10

Abbie Hunt-- November 10, 2009
All different types of cancer plague our medical community, but perhaps one of the most dreaded forms of cancer is brain cancer. Glioblastoma multiforme (GBM) is one of the most dismal forms of brain cancer, with only a 5% survival rate in a 5-y time period. In order to try and increase these odds, Curtin et al. (2009) are studying a way to increase the brain's ability to produce an immune response to tumor invasion which when combined with cytotoxic methods, result in tumor regression.
The immune system is often the human body's greatest weapon against disease, and exploring ways in which to enhance this complex system to fight challenging diseases typically treated with therapies with low therapeutic indices is an important alternative to explore.
Immune response to brain tumors is an important treatment option to consider, due to the limited radiotherapy that can be performed on this sensitive organ and the destructive nature of the therapy when used for cancers in other locations throughout the body. In order to study and enhance the immune system's response to tumors, administration of adenoviral vectors, Fms-like tyrosine kinase ligands, and thymidine kinase to mutant mice suffering from induced brain tumors was performed. This therapy shows the specific T cells necessary for tumor regression and can actually increase the amount of cells instrumental in the initiation of immune response, which are usually sparse in the brain and are dependent upon signaling molecules released from dying tumor cells. These tumor cells are often dying as a result of the established thymidine kinase and glacyclovir treatment. These signaling molecules as well as the development of immunological memory, enable the regression of brain tumors to occur more effectively.
A better understanding of this pathway is extremely beneficial to the fight against brain cancer for those patients whose prognosis is grim due to a mutation in the genes that code for these signaling molecules or their receptors. If the molecules can be administered and effective in mice, those with aberrant receptors or molecules may benefit the most of all.
References:
1. Curtin J.F., Liu N., Candolfi M., et al. 2009. HMB1 Mediate Endogenous TLR2 Activation and Brain Tumor
Regression. PLoS Medicine. 6(1): 84-104.
2. Southgate T., Kroeger K.M., Liu C., Lowenstein P.R. (2008). Gene transfer into meural cells in vivo using adenoviral vectors. Published online at Wiley Interscience. Available online at http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2659706/ Accessed 11/4/09.
3. Sandmair A., Vapalahti M., Yla-Herttuala S. (2000). Adenovirus-Mediated Herpes Simplex Thymidine Kinase
Gene Therapy for Brain Tumors. Cancer Gene Therapy: Past Achievements and Future Challenges. Plenum
Publishers, New York. 163-170.
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Harika Nandigam- Nov 10 th 2009
Somatic cell nuclear transfer is a technique for creating an ovum with a donor nucleus. This somatic cell nuclear transfer allows trans-acting factors present in the mammalian oocyte to reprogram somatic cell nuclei to an undifferentiated state.
Junying Yu et al showed that four factors are enough to make human somatic cells into the pluripotent cells with all the essential characteristics of embryonic stem cells and also the potential to develop the germ layers. Cloning of Dolly led to a search for factors that could mediate similar reprogramming without the somatic nuclear transfer. By testing all the genes they found that OCT4, SOX2, NANOG and
LIN28 are capable of reprogramming human somatic cells with a mesenchymal phenotype. OCT4 and
SOX2 will help in the appearance of reprogrammed clones where as NANOG and LIN28 helps to increase the number of reprogrammed clones.
To test whether these four are sufficient to reprogram primary, genetically unmodified, diploid human fibroblasts they took IMR90 fetal fibroblasts and the human newborn foreskin fibroblasts. The induced pluripotent human cells have the characteristics similar to that of the embryonic stem cells. Those cells have normal karyotype, express telomerase activity, express cell surface markers and the genes that characterize the human embryonic stem cells and also maintain the developmental potential to differentiate into advanced derivatives of all the three germ layers.
Induced pluripotent stem cell lines can be used in different ways. This helps us to study different human tissues, for discovering and testing new drugs. Patient specific iPS cells could be generated to treat various diseases.
References:
Junying Yu, Maxim A. Vodyanik, Kim Smuga-Otto, Jessica Antosiewicz-Bourget, Jennifer L. Frane,
Shulan Tian, Jeff Nie, Gudrun A. Jonsdottir, Victor Ruotti, Ron Stewart, Igor I. Slukvin, James A.
Thomson. Induced Pluripotent Stem Cell Lines Derived from Human Somatic cells. Science, Vol
318(2007)
Liu shuang and Duan Enkui. Advances in the study on induced pluripotent stem cells. Chinese science bulletin. Vol 53(2008). 709-717
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2+
Heart failure (HF) is one of the leading causes of death in developed countries. Traditional treatments for heart failure concentrate on increasing the presence of calcium in cardiac muscle to improve cardiac output. This positive inotropic treatment does indeed increase cardiac contractility. However, the long term use of calcium regulating drugs has been linked to increased mortality by causing fatal arrhythmias. Therefore, it would be beneficial to explore positive inotropic therapies that do not rely on the availability of calcium within cardiac muscle.
Myosin is a motor protein which is responsible for actin based motility. Myosin and actin are the main structural proteins of cardiac muscle. It has been shown that α-myosin comprises ~10% of the total ventricular myosin in normal human hearts, while β-myosin comprises the remaining 90%. The α-myosin isoform has a higher energy demand and is the heart’s fast molecular motor. The β-myosin isoform is the slower motor.
A recombinant adenovirus (AdMYH6) was produced to deliver the human fast α-myosin motor gene (MYH6) to rabbit and human β-myosin-dominant myocytes
. By increasing the α-myosin isoform expression, there was an increase in contractile amplitude of failing human and failing rabbit myocytes without changing intracellular calcium transient amplitude.
"Cardiac action potential."
. Web.
<http://en.wikipedia.org/wiki/Cardiac_action_potential>.
Freeman, Scott.
. 2. Upper Saddle River: Pearson Prentice Hall, 2005.
1018-1021.
Herron, Todd. "Ca2+ - independent positive molecular inotropy for failing rabbit and human cardiac muscle by a-muosin motor gene transfer."
. 24.
(2009): 1-10.
"Heart failure."
. Web. <http://en.wikipedia.org/wiki/Heart_failure>.
"Inotrope."
. Web. <http://en.wikipedia.org/wiki/Inotropy>.
"Ischemia."
. Web. <http://en.wikipedia.org/wiki/Ischemia>.
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Craig Schenck, November 12, 2009
Eludidating the biochemical pathways of plant nutrients in order to engineer plants for increased nutritional value has been an area of intense research. One pathway of particular interest is the vitamin E biosynthetic pathway. Vitamin E collectively represents a class of lipid-soluble molecules α-, β-, γ- and
δ-tocopherols and tocotrienols. α-tocopherol is considered to be the most important form of vitamin E to humans because it has the highest activity due to its ability to be retained in humans longer than other forms. Vitamin E has strong antioxidant properties and deficiencies cause loss of vision, muscle weakness and loss of muscle mass. Thus , engineering a way to increase its activity (abundance?) could greatly benefit human health.
A precursor to the active form of vitamin E, γ-tocopherol, is the most prevalent form of vitamin E found in the seeds of soybeans, 60-65% (Tavva et al. 2007). However the activity of γ-tocopherol is about one-tenth the activity of α-tocopherol. It is thus advantageous to be able to convert γ-tocopherol to
α-tocopherol in a more efficient way to increase the amount of α-tocopherol in the seeds of soybeans.
Overexpression of the final enzyme in the tocopherol biosynthetic pathway, γ-tocopherol methyltransferase (γ-TMT), was shown to increase α-tocopherol content 80-fold in Arabidopsis (Shintani and DellaPenna 1998).
α-tocopherol content was increased in two different crops soybeans and perilla. In both cases γ-
TMT was amplified from cDNA of the seeds of Perilla frutescens. The gene was expressed under the control of a seed specific promoter, vicilin, and a poly A coding sequence from the octopine synthase gene, was added. In soybeans, the gene construct was inserted via particle bombardment with a gene gun.
In perilla, the gene was inserted using Agrobacterium -mediated transformation. Multiple transgenic plants were generated and through northern blot analysis the expression pattern of γ-TMT was only seen in immature cotyledons. High-performance liquid chromatography (HPLC) was used to quantify αtocopherol content in seeds. HPLC analysis of soybeans revealed an increase of α-tocopherol content from 8.4% in wild-type to 85-89% in the transgenic lines. In perilla seeds, α-tocopherol content increased from .9-11.4 mg/100 g in wild-type to 153.2-200.4 mg/100g in transgenic lines.
These two papers exhibit an innovative way of overexpressing an enzyme in a biosynthetic pathway which can lead to increased nutritional value in agriculturally important crops.
References:
1) Tavva VK, Kim YH, Kagan IA, Dinkins RD, Kim KH, Collins GB (2007) Increased α-tocopherol content in soybean seed overexpressing the Perilla frutescens γ-tocopherol methyltransferase gene. Plant
Cell Rep 26 :61–70.
2) Lee BK, Kim SL, Kim KH, Yu SH, Lee SC, Zhang Z, Kim MS, Park HM, Lee JY (2008) Seed specific expression of perilla γ-tocopherol methyltransferase gene increases α-tocopherol content in transgenic perilla (Perilla frutescens). Plant Cell Tissue and Organ Culture 92 :47-54.
3) Maeda H, DellaPenna D (2007) Tocopherol functions in photosynthetic organisms. Current Opinion in
Plant biology 10 :260-265.
4) Department of Human Nutrition, "Vitamin E." November 2004.http://ohioline.osu.edu/hygfact/5000/5554.html (accessed 11/8/09).
5) Office of Dietary Supplements, "Vitamin E Fact Sheet." October 4,
2007.http://ods.od.nih.gov/factsheets/vitamine.asp#en4 (accessed 11/8/09).
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Alex Weber, November 12, 2009
Genetically modified organisms are nothing new to biotechnology. From the first genetically modified bacteria to the FLAVR SAVR Tomato and Golden Rice, genetic engineering and modification to organisms has become an invaluable and profitable technique for biomedicine and biotechnology.
Genetic modification to eukaryotes, specifically mammals, has the potential to be more beneficial than any other type of genetic modification. The application of genetically modified mammals includes models for human disease, bioreactors for the production of commercial products such as pharmaceuticals and the genetic improvement of livestock to name a few. One challenge that has yet to be completely solved is an inexpensive and effective method for introducing foreign DNA into mammals. All of the current methods used to genetically modify mammals rely on the integration of DNA into the mammal’s genome. This will sometimes lead to insertional mutagenesis and variable transgene expression as well as undergo silencing. Though these methods are capable of producing genetically modified organisms that are of the desired type, their yields are low and there is still work to be done.
When attempting to produce a new method for introducing foreign DNA into mammals, there are three guidelines that should be followed. The genetically modified animals should have the foreign DNA stably retained, both structurally and mitotically. The foreign DNA should not contain viral DNA sequences or encode for viral proteins that bear the risk of cellular transformation. Also, there should be long term expression of the introduced DNA. Prokaryotes provide a good example of such a vector know n as a plasmid. The only problem with this is that naturally occurring mammalian plasmid vectors have never been observed. In a recent study done by Stefano Manzini et al. one such vector that follows these guidelines is a nonviral episomal vector known as pEPI-EGFP. This vector contains what is known as a human scaffold/matrix attachment region (S/MAR). This is a segment that allows for the vector to be retained in the cell cytoplasm as an episome. An episome is genetic material that can replicate independently of the host chromosome without being integrated. The S/MAR sequence is chromatin regions that bind the nuclear matrix, and with this sequence present, it ensures episomality of the plasmid in cultured cells, as well as preventing integration of the plasmid into the genome of the host. The study done by Stefano Manzini et al. was done by adding a 2-Kbp S/MAR sequence to pGFP-C1 which is a commercial ly available plamid s . The newly created plasmid with the S/MAR sequence present was then introduced into pigs via the sperm mediated gene transfer method. The results showed that pEPI-EGFP was present in 12 of 18 fetuses (67%). This study shows that there is a possible method for introducing foreign DNA into mammalian cells with high efficiency and retention as well as low levels of undesired products. This vector system may also provide the basis for future development of germ line gene therapy.
References:
1.
Stefano Manzini , Alessia Vargiolu , Isa M Stehle , Maria Laura Bacci , Maria Grazia Cerrito , Roberto Giovannoni , Augusta
Zannoni , Maria Rosaria Bianco , Monica Forni , Pierluigi Donini , Michele Papa , Hans J Lipps , and Marialuisa Lavitrano ,
2006. “Genetically modified pigs produced with a nonviral episomal vector.” PNAS, Vol. 103, no. 47, pg. 17672-17677
2.
Joost H. A. Martens, Matty Verlaan, Eric Kalkhoven, Josephine C. Dorsman, and Alt Zantema, 2002. “ Scaffold/Matrix
Attachment Region Elements Interact with a p300-Scaffold Attachment Factor A Complex and Are Bound by Acetylated
Nucleosomes.” Molecular and Cell Biology, Vol. 22, No. 8, pg. 2598-2606
3.
Marialuisa Lavitrano, Marco Busnelli, Maria Grazia Cerrito, Roberto Giovannoni, Stefano Manzini, and Alessia Vargiolu,
2006. “Sperm Mediated Gene Transfer.” Reproduction, Fertility and Development, Vol. 18, no. 1 and 2, pg. 19-23
4.
“Episomes.” http://science.jrank.org/pages/2548/Episomes.html
. Web. 8 November, 2009.
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th
The demand of the pharmaceutical industry for large quantities of mammalian proteins has led to the development of heterologous expression systems for the production of proteins and peptides.
Plant-based production is now used to synthesize foreign proteins on a commercial scale. As with all protein production, regardless of plant or mammal, three factors must be considered. These factors include transcription, translation, and post-translation conditions. Also, since we are dealing with recombinant protein production in plants, environmental factors need to be considered.
In a recent study conducted by Ko et al., (2005) monoclonal antibodies (mAb) were developed to bind a recombinant antigen associated with colorectal carcinoma cells. The study was promising because the plant-derived mAb (mAb P ) differed considerably from the mammalian-derived mAb (mAb M ) glycosylation pattern yet both were shown to be biologically similar. Cyanovirin-N is also a mAB candidate and is used to inactivate a wide range of HIV strains that bind to gp120 but to have a global impact proteins must be developed on a large scale and at low costs, according to Sexton et al. (2006).
This study successfully demonstrated that plants would be able to develop antibodies at a concentration high enough to affectively bind the antigen of choice.
These two studies were both performed by transforming the plant cells of Nicotiana tabacum with plasmid vectors. pGEM-T vector was used for colorectal carcinoma cells and pMON530 was used for the HIV strains. Various assays ranging from western blots to ELISA were used to determine the presence of protein within the transformed plants.
Molecular farming has many attractive features for the production of therapeutic proteins but at the moment, is not currently satisfactory in a wide commercial scale. The plant species used and the type of vector employed are crucial in the development of this field.
References:
1. Jamal, Kinarm Ko, Kim, Choo, Joung, Kisung Ko, 2009. “Role of genetic factors and environmental conditions in recombinant protein production for molecular farming.” Biotechnology Advances, Vol.
27, No. 6, pg. 914-923
2. Ko, Steplewski, Glogowska, Koprowski. “Inhibition of tumor growth by plant-derived mAb.” PNAS,
Vol. 102, No. 19, pg. 7026-7030
3. Sexton, Drake, Mahmood, Harman, Shattock, Ma. “Transgenic plant production of Cyanovirin-N, an
HIV microbicide.” The FASEB Journal, Vol. 20, pg. 356-358
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Antibodies Used to Discover More about Parkinson’s Disease
Christina Ufholz, November 12, 2009
Parkinson’s disease (PD) is a neurodegenerative disease that is caused by the buildup of protein inclusions containing α-synuclein (αS) and ubiquitin. Some rare inherited forms are caused by autosomal dominant mutations in α-synuclein or by autosomal recessive (AR) mutations in parkin. Some motor symptoms are bradykinesia, rigidity, resting tremor, and postural instability. Some other symptoms are hyposmia, cognitive decline, mood disorders, pain, autonomic dysfunction and sleep disorders. About one million Americans suffer from this disease with around sixty thousand new case s every year.
The experiment by Hideki Shimura et al. (2001) showed the functions and locations of two proteins,
α-synuclein and parkin. This study created the antibodies HP2A, HP6A, HP7A, HP8A, LB509, syn-1, and
UbcH7. Frozen brain tissue and regular brain tissue are tested and Western blotted and/or immunoblotted.
The results were a direct functional relationship in the human brain between parkin and αS that are associated with inherited, monogenic forms of PD. Also, a multimeric ubiquitination complex was identified. It was confirmed that neural parkin is a n E3 Ub ligase and that WT parkin binds to αSp22 and
UbcH7. In vitro assay s were preformed to discover the molecular effects of naturally occurring parkin missense mutations. Future work will be needed to determine whether O-glycosylated αSp22 mediates specific physiological functions and/or has neurotoxic effects in PD.
More recent research by van der Vegt et al. showed that the presence of a single mutant Parkin allele does affect nigrostriatal dopaminergic function which triggers adaptive changes in motor cortical areas.
They also found that multimodal assessment of brain structure and function in non-manifesting carriers yielded a single mutant Parkin allele and different imaging modalities provided important pieces to resolve the puzzle of preclinical reorganization of PD.
References:
1.
Berg, Daniela, Antje Bornemann, Heike Fischer, Thomas Gasser, Takafumi Hasegawa, et al. 2009.
Parkin protects mitochondrial genome integrityand supports mitochondrial DNA repair. Human
Molecular Genetics. 18(20): 3832–3850
2.
Bloem, B.R., C. Klein, H.R. Siebner, J.P.M. van der Vegt , and B.F.L
.
van Nuenen.
2009. Imaging the impact of genes on Parkinson's disease. Neuroscience. 164(1): 191-204
3.
Farrer, Matthew James. 2006. Genetics of Parkinson disease: paradigm shifts and future prospects.
Nature Reviews Genetics. 7: 306-318
4.
Frosch, Matthew P., Nobutaka Hattori, Kenneth S. Kosik, Yoshikuni Mizuno, Michael G.
Schlossmacher, et al. 2001. Ubiquitination of a New Form of alpha -Synuclein by Parkin from Human
Brain: Implications for Parkinson's Disease. Science. 293(5528): 263 – 269
5.
The National Parkinson Foundation. 2007. cited: November 8, 2009 http://www.parkinson.org/Page.aspx?pid=225
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Genetic and Genomic approaches to Improving Biofuel: Sweet Sorghum
Donny Sparrow November 12, 2009
Petroleum supplies the largest fraction of the world’s energy, coming to a total of 37 %. Petroleum is a non-renewable resource and it is imperative that we replace our dependency on it for economic and environmental reasons. Ethanol is an excellent fuel for internal combustion and has a high sustainability rate. It has been proven as a good competitor for petroleum in the United States and especially in Brazil. It has a high heat of vaporization, is a more efficient fuel, is less volatile than gasoline, and has less photochemical activity making the emissions less potent. It also has another incentive to gasoline. Ethanol is renewable as it is made from the fermentation of sugars harvested from plant material. Sweet Sorghum is a relative of sugarcane and also accumulates sugar. It has a potential economic advantage, as it is possible to ferment the hydrolized sugar and sorghum bargasse to produce ethanol and then burn the lignin residuals as heat and electricity. 7682 L of ethanol can be produced per hectare can be produced by sweet sorghum under good conditions. Sweet sorghum belongs to the grass family and has high photosynthetic efficiency. It is also considered to be one of the most drought resistant plants. It can be a perennial or annual depending on the strain. Because of its high potential as a ethanol producing crop research has been done to maximize the efficiency of the plants sugar and cellulose production capabilities through genetic mapping and manipulation.
References:
Dauriat, A., & Wyman, C. (2005). Refining sweet sorghum to ethanol and sugar: economic trade-offs in the context of North China. BIORESOURCE TECHNOLOGY , 96 (9), 985-1002.
Seth C. Murray, William L. Rooney, Martha T. Hamblin, Sharon E. Mitchell, and Stephen
Kresovich (2009).
Plant Gen. March 2009 2:48-62; doi:10.3835/plantgenome2008.10.0011
Jordan, D., Chapman, S., Godwin, I., Mace, E., & McIntyre, C. (2008). Identification of QTL for sugarrelated traits in a sweet x grain sorghum (Sorghum bicolor L. Moench) recombinant inbred population. MOLECULAR BREEDING , 22 (3), 367-384.
Dolat, A., Steinberger, Y., Wang, X., Osman, A., & Xie, G. (2009). Biomass yield and changes in chemical composition of sweet sorghum cultivars grown for biofuel. Field Crops Research , 111 (1-
2), 55-64.
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