Ohio University



Cloning and expression in Saccharomyces cerevisiae of the NAD(P)H-dependent xylose reductase- encoding gene (XYLI) from the xylose-assimilating yeast Pichia stifltisBianca Peixoto Correia, November 25, 2014Due to increasing population and industrial development, the market for machines as well as the automotive market is growing accordingly to meet current economic demands. Currently, the production of ethanol is predominantly based on the use of sugar cane and the conversion of starch derived from grain, especially corn. In recent decades because of the growing concern about global warming, sustainability and ethics, high investment has been applied in the development of alternative fuel sources, and the utilization of yeasts for the fermentation process of biomass and residual biomass has presented important role.Lignocellulosic biomass employed in the fermentation process is composed mainly of cellulose, hemicellulose and lignin, being xylose and cellulose the most abundant sugars in cellulose hydrolysis. Among the organisms used in industrial fermentation processes for producing ethanol, the yeast Saccharomyces cerevisiae is the better known and through an anaerobic process is capable of converting glucose to ethanol. Although this yeast presents the basic mechanism of xylose assimilation, this organism is not able to utilize xylose as a carbon source for the production of ethanol. Because of this inability of S. cerevisiae, efforts have been made in order to make this competent yeast for fermentation of xylose.Among the approaches used for the genetic modification of S. cerevisiae targeting the metabolic pathway of xylose, the main are based on modeling, flux analysis, and deletion and / or alteration in the expression of key genes. In this study conducted by Amore at al. The XYLI gene of the yeast Pichia stipitis encoding a key enzyme in the pathway for xylose was cloned and expressed in Saccharomyces cerevisiae in order to make this organism capable to conduct the fermentation of xylose to ethanol.This research represents a considerable industrial economic advantage, since it aims at the utilization of a major of part of the waste biomass that before could not be used in fermentation processes. Additionally, this technology opens scope for the use of alternative carbon sources, providing an environmentally sustainable technology.References:Amore Rene, Kotter Peter, Kiister Christina, Michael Ciriacy, Hoilenber P. Cornelis. 1991. Cloning and expression in Saccharomyces cerevisiae of the NAD(P)H-dependent xylose reductase- encoding gene (XYLI) from the xylose-assimilating yeast Pichia stifltis. Gene, 109 (1991) 89-97.Kim Soo rin, Park Yong-cheo, Jin Yong-su, Seo Jin-ho. 2013. Strain engineering of Saccharomyces cerevisiae for enhanced xylose metabolism. Biotechnology Advances, 31: 851-856Thomas W Jeffries. 2006. Engineering yeasts for xylose metabolism. Current Opinion in Biotechnology, 17:320-326.Watanabe Seiya, Saleh Ahmed Abu, Pack Pil Seung, Annaluru, Kodaki Tsutomu, Makino Keisuke. 2007. Ethanol production from xylose by recombinant Saccharomyces cerevisiae expressing protein engineered NADP+-dependent xylitol dehydrogenase. Journal of Biotechnology, 130: 316-319Abstract Received LateThe Flavr Savr TomatoLu Tian, November 25, 2014AbstractThe Flavr Savr tomato was introduced as the first genetically engineered whole food in 1994. The commercial event, resulting from transformation with an antisense expression cassette of polygalacturonase gene. Polygalacturonase is expressed in tomato only during the ripening stage of fruit development. PG becomes abundant during ripening and has a major role in cell wall degradation and fruit softening. Tomato plants were transformed to produce antisense RNA from a gene construct containing the cauliflower mosaic virus 35S promoter and a full-length PG cDNA in reverse orientation. The construct was integrated into the tomato genome by?Agrobacterium-mediated transformation. The constitutive synthesis of PG antisense RNA in transgenic plants resulted in a substantial reduction in the levels of PG mRNA and enzymatic activity in ripening fruit. However, after the gene was sequenced, people found that it contain two contiguous, linked, transfer DNA insertions. Also, they found polygalacturonase suppression correlates with accumulation of ’21-nt small interfering RNAs, the hallmark of an RNA interference-mediated suppression mechanism.Sources used:Sheehy RE, Kramer M, Hiatt WR. Reduction of polygalacturonase activity in tomato fruit by antisense RNA.?Proc Natl Acad Sci U S A.?1988 Dec;85(23):8805–8809.Krieger EK, Allen E, Gilbertson LA, Roberts JK.?The Flavr Savr tomato, an early example of RNAi technology.?HortScience?2008;?43: 962–964.RNAi can identify new components of the p53 pathway & Silence mutant p53 pathwayBy Chelsey MaagNovember 25, 2014Using experimental therapy to learn more about molecular carcinogenesis using the powerful tool of RNAi, RNA interference, to screen for loss-of-function genes and identifying components of cellular signaling pathways and its affect on tumourgenesis. I focus on identifying new components of the p53 suppression pathway. Additionally, I focus on using RNAi therapeutics on mutant p53 that accumulates typically in cancer cells. RNAi is a defense mechanism triggered by double-stranded RNAs, which processed into short interference RNAs, siRNAs. They target homologous RNAs for destruction. Searching for components of the p53 tumor-suppressor pathway. P53 tumor suppressor stops the formation of tumors the p53 protein binds to DNA that stimulates another gene to produce a protein called p21 that interacts with the next stage in cell division.Mutations in the?p53?gene, encoding the p53 tumor suppressor, are possibly the most common type of gene specific changes in human cancer. This emphasizes the essential role of p53 as a major support of the body’s built in anticancer defense mechanisms. P53 mutants possess the characteristics of oncogenes, suggesting that knockdown of mutant p53 may restrict or reverse the process of oncogenesis. Therapeutics based on RNA interference RNAi has become powerful and useful methods for the treatment of many diseases, including cancer; due to the high specificity, high efficacy and low toxicity of the RNAi trigger, small dsRNA. siRNA targeting mutant p53 could induce cell cycle arrest and apoptosis in human cancer cellsRNA interference (RNAi) is a powerful new tool with which to perform loss-of-function genetic screens in lower organisms and can greatly facilitate the identification of components of cellular signaling pathways and stoppage of mutant p53 constant cellular proliferation. A potentially useful application of short interfering siRNA screening is in the identification of man-made lethal interactions, which are a combination of two non-lethal mutations, that together result in cell death. The identification of such genetic interactions in mammalian cells may accelerate the development of new and more specific classes of anticancer drugs.References:Berns, Katrien, and Roderick L. Beijersbergen. "A Large-scale RNAi Screen in Human Cells Identifies New Components of the P53 Pathway."?. Nature Publishing Group, 25 Mar. 2004. Web. 16 Nov. 2014. < Zhu,, Hai-, and Kai Yang. "Silencing of Mutant P53 by SiRNA Induces Cell Cycle Arrest and Apoptosis in Human Bladder Cancer Cells."?WJSO. World Journal of Surgical Oncology, 28 Jan. 2013. Web. 16 Nov. 2014. Modified Cotton: To Bt or not to Bt?Bethany Zumwalde, November 25, 2014Cotton is one of the most important cash crops in the world accounting for over $25 billion of revenue annually in the United States (USDA 2014). Monsanto first genetically modified cotton in 1996 to express insecticidal genes derived from the soil bacterium Bacillus thuringiensis. This bacterium naturally produces toxic crystalline proteins (Cry-proteins) that target specific pests such as the bollworm. When the insect larvae ingest the plant, the Bt protein is then activated by the high pH of the gut and subsequently attaches to a specific receptor found only in lepidopteron species (Ostlie et al. 1997). The protein then dissolves the gut lining of the insect, ultimately resulting in death. Bt proteins have proven to be a powerful insecticidal tool for cotton due to its high specificity for certain insect species, non-toxicity to non-target species, biodegradability, increase in crop yield, and also the reduction of the use of other possibly harmful insecticides. However, the cost of the transgenic seeds can be relatively high and unaffordable for some farmers and contemporary studies have revealed that the first generation of transgenic Bt cotton has been proven to be susceptible to insect resistance (Tabashnik et al. 2008). A recent study in 2012 conducted by Ghazanfar et al. analyzes the inheritance of the insecticidal gene cry1Ab in genetically modified cotton. This study incorporates two transgenic lines (CEMB-3 and CEMB-11) and two non-transgenic lines (MNH-93 and CIM-482) to decrease recent field-evolved insect resistance to first generation Bt cotton. Results from PCR, southern blots, western blots, and lab biotoxicity assays were used to analyze and confirm high heritability of the cry1Ab gene in addition to other cry-genes for future breeding programs to combat insect resistance.ReferencesGhazanfar, M., Hhan, G. A., Bakhsh, A., Riazuddin, S., & Husnain, T. 2012. Inheritance of an insecticidal gene (cry1Ab) in genetically modified cotton. Russian Agricultural Sciences, 38(3), 210-217.Ostlie, K.R., W.D. Hutchison, and R.L. Hellmich. 1997. Bt corn and European corn borer. NCR Publication 602. University of Minnesota, St. Paul. Tabashnik, B. E., Gassmann, A. J., Crowder, D. W., & Carrière, Y. 2008. Insect resistance to Bt crops: evidence versus theory. Nature biotechnology. 26(2), 199-202.United States Department of Agriculture (USDA). 2014. Cotton and Wool. Retrieved from Ready corn: A case studyHarlan Svoboda, 25 November 2014Roundup Ready corn, a genetically modified (GM) strain of Zea mays designed to be resistant to the herbicide Roundup, has been in use in the United States since 1998 (Roundup Ready System, accessed 18 November 2014). The effectiveness of this system has been proven repeatedly (The Monsanto Company, 2002), but the toxicity of ingesting the GM food, although proven safe, has still been in question by the public and some sects of the scientific community.The first major criticism to the Roundup Ready corn system reported a myriad of health problems seen in lab rats (Séralini et al., 2012). Among many disturbing accusations, the 2-year-long study claimed that 1) all treated groups died 2-3 times more than controls, and more rapidly; 2) females produced large mammary tumors more often; 3) pituitary glands were the second most disabled organ; 4) sex hormonal balance was modified; 5) liver congestions and necrosis were 2.5-5.5 times higher; 6) kidney nephropathies were also generally 1.3-2.3 greater; and 7) 76% of the altered parameters were kidney related. Not only did the experimenters feed Roundup Ready corn to the rats, comprising 11% of the rats’ diet, but also added in concentrated Roundup herbicide into their water. The paper concluded that both Roundup herbicide and Roundup Ready corn were toxic to rats and implied the same for human consumption.Almost immediately, the Séralini et al. (2012) study came under scrutiny from the scientific community. Butler (2012) articulated these concerns in a Nature article, reporting that “the design, reporting, and analysis of the study are inadequate,” and that the paper is “of insufficient scientific quality to be considered as valid for risk assessment.” Nearly a year after the initial study, Arjó et al. (2013) summarized the discourse within the scientific community regarding the poor science behind Séralini’s article, asserting that the study was designed poorly, unsubstantiated statements were made, control data was not presented, and that the rats were treated inhumanely. Ultimately, Arjó et al. (2013) and others insisted that the original Séralini et al. (2012) article be retracted by the publishing journal, which it later was.References2014. Roundup Ready System. The Monsanto Company (accessed online at ).Arjó, G., M. Portero, C. Pi?ol, J. Vi?as, X. Matias-Guiu, T. Capell, A. Bartholomaeus, W. Parrott, & P. Christou. 2013. Plurality of opinion, scientific discourse and pseudoscience: an in depth analysis of the Séralini et al. study claiming that RoundupTM Ready corn or the herbicide RoundupTM cause cancer in rats. Transgenic Research 22: 255–267.Butler, D. 2012. Hyped GM maize study faces growing scrutiny. Nature 490: 158.The Monsanto Company. 2002. Safety Assessment of Roundup Ready Corn Event GA21 (retrieved from ).Séralini, G.-E., E. Clair, R. Mesnage, S. Gress, N. Defarge, M. Malatesta, D. Hennequin, J. Spiroux de Vend?mois. 2012. Long term toxicity of a Roundup herbicide and a Roundup-tolerant genetically modified maize. Food and Chemical Toxicology 50: 4221–4231.Abstract Received LatePhotobiological Hydrogen Production UsingBioengineered Algae and CyanobacteriaCurtis Moore, November 25, 2014No matter ones stance on climate change it is obvious that emissions from ICEs (internal combustion engines) are no conducive to a healthy environment, any naysayer can feel free to huff a tailpipe. On the other hand, if one were to huff a tailpipe of a fuel-cell vehicle powered by clean hydrogen, they would be taking in only heat and water.As of yet there are few feasible methods to produce clean hydrogen without the addition of non-renewable energies to purify the gas. One method of production being considered by scientists is the use of Algae and cyanobacteria to produce this gas for us. Hydrogenase enzymes are used as a catalyst by these organisms to fix Co2 into carbohydrates using ATP and NADPH. In the absence of CO2 and under anaerobic conditions, reduced ferrodoxin, the main electron donor to NADPH, reduces protons to yield hydrogen gas.Though promising, there are still challenges to using this technology to produce hydrogen on a commercial scale. Specifically, for algae light conversion efficiency o H2 is theoretically about 10%, and the Fe-Fe hydrogenases are inhibited by oxygen produced in one of their photosystems. These limitations are overcome by truncating the chlorophyll antenna size of PS-II using RNAi and Sulfur deprivation.1 Another main limitation of this technology is competition between hydrogenases and the NADPH-dependent carbon dioxide fixation.2 Recent experiments have shown that bioengineering a ferredoxin-hydrognease fusion enzyme can switch the bias of electron transfer from FNR, to hydrogenase and results in an increased rate of hydrogen photoproduction.2 In response to the experiments described above, it was concluded that a certain level of FNR activity is necessary for stable photosynthetic growth. In response another group of researchers genetically modified residues of PETF (photosynthetic electron transport ferredoxin) which are essential for differential recognition of FNR and HYDA1.3Sources used:Singh, Shailendra Kumar.; Dixit, Kritika.; Sundaram, Shanthy. Bioengineering of Biochemical Pathways for Enhanced Photobiological Hydrogen Production in Algae and Cyanobacteria. Int. J. Biotech. Bioen. Re. [Online] 2013 511-518 Yacobya, Iftach.; Pochekailova, Sergii.; Toporikb, Hila.; Ghirardic, Maria L.; Kingc, Paul W.; Zhang, Shuguang. Photosynthetic electron partitioning between [FeFe]-hydrogenase and ferredoxin:NADP+-oxidoreductase (FNR) enzymes in vitro J Biol Chem 2013 288 (49) 35192-35209 Rumpel, Sigrun.; Siebel, Judith F.;? Farès,?Christophe.;? Duan, Jifu.;?Reijerse, Edward.; Happe,?Thomas.; Lubitz, Wolfgang.; Winkler, Martin. Enhancing hydrogen production of microalgae by redirecting electrons from photosystem I to hydrogenase R. Soc. Chem. 2014 3296-3301Genetically engineering bean golden mosaic virus resistance in the common beanMichael Xie—December 2, 2014Bean golden mosaic virus (BGMV) is a plant virus that infects the common bean plant (Phaseolus vulgaris L.). The whitefly (Bemisia tabaci) is virus’ insect vector. Those infected will demonstrate the following symptoms; stunted growth, yellow-green mosaic pattern of leaves, and distorted pods. BGMV is currently a large contributor to the annual yield loss of bean production in Latin America. It has been estimated that the annual yield losses are 90,000-280,000 tons in Brazil due to BGMV. Currently no bean plant has developed or exhibited a natural immunity to this virus. In order to combat BGMV, a transgenic bean line has been engineered by the company Embrapa to be resistant by using RNA interference. The gene most important for viral replication is the AC1 gene in DNA-A, which encodes for the complex, multifunctional transcription factor needed for replication. The linear vector, pBGMVRNAiAHAS, was engineered to encode RNA sequence specifically corresponding to AC1 in the sense and anti-sense orientation in order to elicit RNA interference. When AC1 is present in the plant cell, a double stranded RNA complex will form and be cleaved by a Dicer (RNase III), into siRNA that will bind to target mRNA that will be degraded by the RISC protein complex. The transgene was introduced into the common bean genome through micro-projectile bombardment. Several lines of transgenic beans were created and screened by virus inoculation and one was found to have the highest expression of resistance rest (5.1). There was no viral DNA found in 5.1 when screened through PCR with primers flanking BGMV. When 5.1 were allowed to set seed as the founder line, Southern blot analysis revealed that the transgene were not independently segregating. Utilizing RNAi in plants is a novel method that has been approved by Brazil’s Nation Technical Commission for Biosecurity. Thus, it has been approved to be substantially equivalent and safe to its nontransgenic counterpart. Controversy surrounds safety, affecting biodiversity, and creating an unfair advantage over smaller scale farmers.References: Arag?o, F., Nogueira, E., Tinoco, M., & Faria, J. (2013). Molecular characterization of the first commercial transgenic common bean immune to the Bean golden mosaic virus. Journal of Biotechnology, 166, 42-50.Bonfim, K., Faria, J., Nogueira, E., Aragao, F., & Mendes, E. (2007). RNAi-Mediated Resistance to Bean golden mosaic virus in Genetically Engineered Common Bean. The American Phytopathological Society, 6(10.1094), 717-726. Retrieved November 26, 2014, from . (2010, January 1). Technical Opinion No. 3024/2011 - Commercial Release of genetically modified bean resistant to Bean Golden Mosaic Virus (Bean golden mosaic virus - BGMV), event Embrapa 5.1 - Case No. 01200.005161/2010-86. Retrieved November 26, 2014, from Abstract ReceivedOwen Riemer, December 2, 2014AbstractNot receivedNo Abstract Received and No Topic ChosenColin Hinton, December 2, 2014AbstractNot receivedAbstract Received LateTargeting and killing of malignant gliomas with specific stem cells expressing the suicide gene, thymidine kinase, from the herpes simplex virusEthan Whipp, December 2, 2014In today’s society, despite continual advances in neurosurgery and advanced radio- and chemotherapy, a poor prognosis is often given when diagnosed with malignant gliomas. A glioma is a tumor that arises from glial cells in the brain or spinal cord, and is responsible for 80% of malignant brain tumors (Goodenberger, Jenkins 2012). The survival rates are low, a reported 30% of diagnosed patients live one year and only 14% of patients make it to two years (Rachet et. al. 2008). The low survival rates are attributed to the ability of a single tumor cell, from a glioma, to migrate to a distant area and affect healthy brain tissue, forming new tumors. Adult stem cells can be used to target these tumors, and have the ability to target mobile glioma cells that metastasize to healthy CNS tissues. Cultivating these adult stem cells on a large scale is limited by low population doublings, and by the inability to subculture these stem cells effectively. Clinical use of adult stem cells would therefore be impractical at this time, due to these limitations. In this study, a promising strategy was developed to treat gliomas with tumor infiltrative adult stem cells that express therapeutic genes. The adult stem cells were selected to be highly proliferative, able to migrate to and infiltrate tumor cells, and able to be subcultured in order to maintain cell lines. The stem cells used in this experiment were a subcategory of mesenchymal stem cells named bone marrow-derived tumor-infiltrating cells (BM-TICs). The BM-TICs were transduced by the retro-viral vector expressing the thymidine kinase gene from the herpes simplex virus as well as the green fluorescent protein gene variant (BM-TIC-tk-GFP). BM-TIC-tk-GFPs were injected into or nearby malignant gliomas in a rat model. Gene expression was monitored via positron emission tomography using a specific tracer to confirm expression of thymidine kinase. Fluorescence microscopy of histological sections confirmed GFP containing cells location around the gliomas as well as infiltrating into the tumors. Rats injected with BM-TIC-tk-GFPs were treated with the prodrug ganciclovir. Ganciclovir is an antiviral prodrug that is phosphorylated by the viral thymidine kinase to produce a highly toxic triphosphate. The triphosphate is then incorporated into DNA, halting replication in the S phase and in turn signaling apoptosis. Gap junctions formed between therapeutic cells and glioma cells allow viral thymidine kinase diffusion into the tumor, thus making them susceptible to ganciclovir. The rats with BM-TIC-tk-GFPs treated ganciclovir lived significantly longer than control groups. The survival analysis showed a 65% long term survival and was statistically significant (P<0.001). Therapeutic genes incorporated into tumor infiltrating cells were shown to be highly effective in the treatment of brain tumors in rats, and shows promise for the treatment of humans with malignant gliomas in the future.References: HYPERLINK "" \t "_blank" Rachet B, Mitry E, Quinn MJ, et al; Survival from brain tumours in England and Wales up to 2001. Br J Cancer. 2008 Sep 23;99 Suppl 1:S98-101Goodenberger ML, Jenkins RB (2012). “Genetics of adult glioma”. Cancer genet. doi:10.1016/j.cancergen.2012.10.009Miletic, Hrvoie, et. al. “Bystander Killing Of Malignant Glioma By Bone Marrow-Derived Tumor-Infiltrating Progenitor Cells Expressing A Suicide Gene.” Molecular Therapy: The Journal Of The American Society Of Gene Therapy 15.7 (2007): 1373-1381. Abstract Received LateTransgenetic Cell Base Therapy to ALSTianyi Cai, December 2, 2014 In this experiment, researchers utilized encapsulated genomic modified human mesenchymal stromal cells (hMSC) which can produce Glucagon-like peptide 1 (GLP1) to reduce the onset of ALS because the neuroprotective potential of GLP1 in the SOD1(G93A) transgenetic mice model that has familial ALS. Superoxide dismutase 1(SOD1) is an enzyme that is encoded by SOD1 gene which located on human chromosome 21; and Glycine 93 changed to alanine (G93A) represents a mutation which will influence the produce level of SOD1, this is one of the mutations causes familial ALS. GLP1 will be utilized in the experiment is a candidate for treatment of neurodegenerative disorder. According to previous study, GLP shows anti-oxidant capacity and neuroprotective against excitotoxicity in vitro and vivo. To achieve the constant drug delivery, the researcher used microcapsules contain cells; the transgenetic cells in the capsules can produce GLP constantly like an osmotic pump. However, the cells contained capsule do not require maintains and partials replacement. Genomic modified MSCs were immortalized by transduction with the human Telomerase Reverse Transcriptase gene. The cells were sealed in the spherical shape alginate matrix. And then the microcapsules were injected to the brains of mice. According to the visual comparison between the control group and treated group, mice in the treated group had longer lives and slower disease onset. On the other hand, the histological analysis of brain tissue of mice shows that the mice in treated group has lower brain injury which expressed as high microtubule associated protein 2 percentage when stain. Therefore, the experiment proved that the GLP1-hMSC therapy exhibited to control ALS onset and increase the life time of disease. References:Knippenberg S, Thau N, Dengler R, Brinker T, Petris; Intracerebroventricular injection of encapsulated human mesenchymal cells producing glucagon-like peptide 1 prolong survival in a mouse model of ALS. PLoS One, 2012. Lewis C, Suzuki M; Therapeutic applications of mesenchymal stem cells for amyotrophic lateral sclerosis. Stem Cell Research & Therapy, 2014, 5. Keifer O, O’Connor D, Boulis N; Gene and protein therapies utilizing VEGF for ALS. Pharmacology & Therapeutics, 2014, 141.Novel Molecular Beacon Probe-Based Real Time RT-PCR Assay for Diagnosis of Crimean-Congo Hemorrhagic Fever Encountered in IndiaSawyer Ellis- December 2, 2014One of the most specific chemical reactions to take place in nature is the pairing of a nucleic acid to its complement (Sanjay 1). Using this concept, molecular beacons were developed to show when this specific binding occurs. Molecular beacons are single stranded nucleic acids that contain two main regions. These regions are the stem and loop. The loop section contains a DNA target sequence, and the stem is composed of two arm segments. One arm segment contains a fluorophore, and the other arm possesses a quencher molecule. The model fluorophore and quencher Sanjay uses are EDANS and DABCYL, respectively. Normally the fluorophore absorbs light and emits it back off, but fluorescence resonance energy transfer occurs and the DABCYL absorbs the emitted energy and gives it back off as heat, thus no light is produced. When the compliment strand of DNA binds to the DNA sequence of the loop, a conformational change occurs that causes the quencher molecule to no longer absorb the light energy emitted by the fluorophore and an observable change in light may be observed. Kamboj et. al. has developed a method using this premise, coined MB Real Time RT-PCR in order to detect Crimean-Congo Hemorrhagic Fever (CCHF) which is an emerging zoonotic disease. This method has been proven to be sensitive, specific, and reproducible using these molecular beacons. The TaqMan Assay was the previously used method, but in this study, the MB real-time RT-PCR method proves to be more sensitive, contain less error, and has a higher efficiency. References:Garrison, A. R., Alakbarova, S., Kulesh, D. A., Shezmukhamedova, D., Khodjaev, S., Endy, T. P., & Paragas, J. (2007). Development of a TaqMan?–Minor Groove Binding Protein Assay for the Detection and Quantification of Crimean-Congo Hemorrhagic Fever Virus. The American journal of tropical medicine and hygiene, 77(3), 514-520.Kamboj, A., Pateriya, A. K., Mishra, A., Ranaware, P., Kulkarni, D. D., & Raut, A. A. (2014). Novel Molecular Beacon Probe-Based Real-Time RT-PCR Assay for Diagnosis of Crimean-Congo Hemorrhagic Fever Encountered in India. BioMed research international, 2014.Tyagi, S., & Kramer, F. R. (1996). Molecular beacons: probes that fluoresce upon hybridization. Nature biotechnology, 14(3), 303-308.Golden Rice 1 and Golden Rice 2 – Eliminating vitamin-A deficiency in third world countriesKameron Starr, December 4, 2014In many third world countries rice is the main source of food. Rice has many health benefits but lacks any precursors to vitamin-A.As a result, many people in these countries suffer from vitamin-A deficiencies. Golden Rice may solve this problem. Golden rice 1 was first produces by means of introducing the biosynthesis pathway of provitamin A (β-carotene). Agrobacterium-mediated transformation of plasmid pB19hpc makes this possible. Plasmid pB19hpc includes the sequences for a plant phytoene synthase (psy) and the sequence coding for bacterial phytoene desaturase (crtI) from a daffodil and erwinia uredovora, respectively. This plasmid produces lycopene from naturally produced GGPP. To the researcher’s surprise, the endosperm of the rice contained the machinery necessary to convert this lycopene into β-carotene without the need for a second plasmid encoding the enzyme lycopene β-cyclase. The β-carotene is what gives the rice its golden color. Although promising, to meet the daily recommended value of vitamin-A, a two year old would have to eat 7 pounds of rice per day, and an adult would have to eat 20 pounds a day. This was solved by Golden Rice 2. Researchers found that the limiting factor in the production of β-carotene is the psy gene (from a daffodil in Golden Rice 1). Genes encoding psy from various organisms were tested to see which would have the greatest yield of β-carotene. The psy gene from maize produced the greatest level of β-carotene (23 times that of Golden Rice 1).Much controversy is involved in this topic and will be described in my talk. References:Beyer, Peter, Salim Al-Babili, Xudong Ye, Paola Lucca, Patrick Schaub, Ralf Welsch, and Ingo Potrykus. "Golden Rice: Introducing the Beta-Carotene Biosynthesis Pathway into Rice Endosperm by Genetic Engineering to Defeat Vitamin A Deficiency." THE JOURNAL OF NUTRITION (2002)Jacqueline A Paine1, Catherine A Shipton1, Sunandha Chaggar1, Rhian M Howells1, Mike J Kennedy1, Gareth Vernon1, Susan Y Wright1, Edward Hinchliffe2, Jessica L Adams3, Aron L Silverstone3 & Rachel Drake1, “Improving the nutritional value of Golden Rice through increased pro-vitamin A content” Nature (2005)Smith, Jeffrey M. "Golden Rice Is the Wrong Way to Supplement Vitamin A."Genetic Roulette: The Documented Health Risks of Genetically Engineered Foods. Fairfield, IA: Yes!, 2007. 243-44. Print. Unlikely Remediator: Engineering D. radiodurans for toxic metal cleanupMorgan Etheridge, December 4, 2014Deinococcus radiodurans is a soil bacterium capable of withstanding large amounts of ionizing and ultraviolet radioactivity. The bacteria is 1.5 – 3.5 micrometers in diameter, spherical, and pigmented. The natural habitat of this particular species of bacteria is unknown, though members of the genus have been found all over the world. D. radiodurans was discovered when an attempt was made to completely sterilize canned food using radioactivity; the bacteria was the only organism remaining after the attempt. Bacteria within the entire genus Deinococcus have the ability to withstand lethal amounts of radioactivity; the bacteria are the most DNA damage-tolerant organisms every identified. It is believed that the DNA repair system in D. radiodurans is responsible for the ability to withstand high levels of IR. Scientists engineered Deinococcus radiodurans to remediate toxic heavy metal waste. Heavy metal waste was targeted due to the high levels present as a result of global nuclear weapons production between 1945 and 1986. About one third of the reported 3,000 waste sites containing production materials are still radioactive. The wastes include uranium, mercury, and toluene, among others. Metal resistance genes were cloned into D. radiodurans, conferring resistance to the most common toxic metal wastes and granting the ability to transform those metals into less toxic and less soluble chemicals. Scientists took merA locus and inserted in into D. radiodurans. merA encodes mercuric ion reductase, which reduces highly toxic, thiol-reactive mercuric ion Hg (II) to Hg (0), a much less toxic and nearly inert ion. The cloned mer encoded six proteins that confer mercury resistance functions. Four different strains were created. After insertion into D. radiodurans strains, the strains were inoculated into liquid medium containing Merbromin or HgCl2. The most functional strain in Merbromin and HgCl2 was the recombinative transformation strain. The researchers also showed that it is possible to introduce several remediating functions into a single radiodurans host by combining the TDO functions of strain MD560 into MD737. D. radiodurans has also been a study organism for information storage in a potential catastrophe. References:Brim, H., McFarlan, S.C., Fredrickson, JK., Minton, K.W., Zhai, M., Wackett, L.P., Daly, M.J. 1999. Engineering Deinococcus radiodurans for metal remediation in radioactive mixed waste environments. Nature Biotechnology: 18. 85 – 90. Battista, J.R. 1997. Against all odds: The survival strategies of Deinococcus radiodurans.Annual Review of Microbiology: 51. 203 – 224. Wong, P.C., Wong, K., Foot, H. 2003. Organic data memory: Using the DNA approach. Communications of the ACM: 46. 95 – 98. Genetic Screening for Alzheimer’s DiseaseErin Thorstensen, December 4, 2014Alzheimer’s disease (AD) is currently the most common form of dementia in the elderly, affecting more than five million people in just the United States. The cause of cell death and tissue loss in the brain due to AD is not entirely known, however scientists believe amyloid plaques and neurofibrillary tangles are most likely responsible. Four genes, APP, PS1, PS2, and APOE, have been determined as causative elements of AD. This presentation will focus on the APOE or Apolipoprotein E gene, which is proposed to play a role in amyloid β aggregation and clearance as well as intracellular signaling through low-density lipoprotein receptor-related protein (LRP). As we have learned, single-nucleotide polymorphisms (SNPs) are often used as biomarkers. The APOE gene has several SNPs, which are used to explore the genetic basis of AD. The research article being reviewed in this presentation concentrates on identifying deleterious nonsynonymous SNPs, which, upon mutation, result in amino acid substitution likely to cause a change in structure and therefore function of a protein. These SNPs, associated with APOE, could be the cause of the amyloid plaques or neurofibrillary tangles characteristic of AD.AD has a complex genetic background as well as non-genetic factors, which makes research for a cure difficult. However, research in the association between SNPs and disease risk is making progress in our understanding of diseases such as AD to where we could be able to predict and even prevent amyloid plaque or neurofibrillary tangle formation. ReferencesAlzheimer's Association. (2011). Alzheimer's Disease and the Brain. Retrieved?November?25, 2014, from , T. A., Al Shammari, S. A., Al-Muammar, M. N., & Alhamdan, A. A. (2012). Screening and Evaluation of Deleterious SNPs in APOE Gene of Alzheimer's Disease. Neurology Research International, 1-8. doi:10.1155/2012/480609Abstract Received LateExpression of human growth hormone in the milk of transgenic rabbits with transgene mapped to the telomere region of chromosome 7qAna Carolina Amaral Coutinho, December 4, 2014Production of highly purified proteins at large scale has been a challenge in the biotechnology field, and to accomplish a larger production of those pharmaceuticals the transgenic technology has been used. Growth hormone, a factor controlling growth in the vertebrates and metabolism of mammals, is an example of pharmaceutical in which researchers have tried to implement the transgenic technology to obtain a bigger production. The absence of human growth hormone promotes a series of deficiencies that has been treated with Growth Hormone Replacement Therapy, an effective treatment that has stimulated a profitable market once this hormone is required on a strong demand, and also presents big production costs. In this article, Lipinski et al discusses the mammary gland as an efficient bioreactor from which it is possible to obtain a relatively large amount of purified proteins once the protein production might occur at high levels in the milk of lactating animals. In this experiment, the recombinant human growth hormone (rhGH) was expressed in transgenic rabbits that carry in their genome an hGH genomic sequence which is controlled by the rat WAP promoter. This 969 bp promoter sequence contains an essential sequence that directs the expression of rhGH only in the mammary gland. The transgene was mapped and identified as a stable transgene that can be transmitted for the next generations maintaining its paternal position. The presence of recombinant human growth hormone was also verified in the milk of the transgenic rabbits female in a range up to the level of 10 μl/ml.Reference:Daniel Lipinski & Joanna Zeyland & Marlena Szalata & Andrzej Plawski & Malgorzata Jarmuz & Jacek Jura & Aleksandra Korcz & Zdzislaw Smorag &?Marek Pienkowski & Ryszard Slomski. 2012. Expression of human growth hormone in the milk of transgenic rabbits with transgene mapped?to the telomere region of chromosome 7q. J Appl Genetics 53 (2012) 435–442.Abstract Received LateProduction of the antimalarial drug precursor artemisinic acid in engineered yeastAna Virginia Guimaraes, December 4, 2014AbstractMalaria is a global health problem. Globally, an estimated 3.4 billion people are at risk of malaria. WHO estimated that 207 million cases of malaria occurred globally in 2012 (uncertainty range 135–287 million) and 627 000 deaths (uncertainty range 473 000–789 000). Disease control is hampered by the occurrence of multi-drug-resistant strains of the malaria parasite Plasmodium falciparum. Artemisinin, a sesquiterpene lactone endoperoxide extracted from Artemisia annua (family Asteraceae; commonly known as sweet wormwood), is highly effective against multidrug- resistant Plasmodium spp., but is in short supply and unaffordable to most malaria sufferers. Although total synthesis of artemisinin is difficult and costly, the semi-synthesis of artemisinin from microbially sourced artemisinic acid, its immediate precursor, could be a cost-effective, environmentally friendly, high-quality and reliable source of artemisinin. The engineering of Saccharomyces cerevisiae to produce artemisinic acid was done by using an engineered mevalonate pathway, amorphadiene synthase, and a novel cytochrome P450 monooxygenase (CYP71AV1) from A. annua that performs a three-step oxidation of amorpha-4,11-diene to artemisinic acid. The synthesized artemisinic acid is transported out and retained on the outside of the engineered yeast, meaning that a simple and inexpensive purification process can be used to obtain the desired product. Although the engineered yeast is already capable of producing artemisinic acid at a significantly higher specific productivity than A. annua, yield optimization and industrial scale-up will be required to raise artemisinic acid production to a level high enough to reduce artemisinin combination therapies to significantly below their current prices.Abstract (modified) retrieved from: Ro, D. K. et al. Production of the antimalarial drug precursor artemisinic acid in engineered yeast. Nature 440, 940-943 (2006) doi:10.1038/nature04640.ReferencesCDC and Malaria (April, 2014). Centers for Disease Control and Prevention, Center for Global Health, Division of Parasitic Diseases and Malaria. Retrieved from: malaria.Paddon, C. & Keasling, J. Semi-synthetic artemisinin: a model for use of synthetic biology in pharmaceutical development. Nature Reviews. 12, 355-367 (2014).Ro, D. K. et al. Production of the antimalarial drug precursor artemisinic acid in engineered yeast. Nature 440, 940-943 (2006).Schmid, G. & Hofheinz, W. Total synthesis of Qinghaosu. J. Am. Chem. Soc. 105, 624–-625 (1983).World Health Organization. World Malaria Report 2013. (WHO, 2013)Cloning of Extinct and Endangered AnimalsKellie RableyDecember 4, 2014Some of the hottest topics in science today relate to the cloning of plants and animals. There are several different methods used to accomplish this task in animals, two of which are DNA microinjection and embryonic stem cells. This presentation will focus on creating transgenic animals by using embryonic stem cells, which has been successful in several different cases. As the interest and success of cloning of animals has grown, so has the debate of the ethics surrounding it. To give a broad overview of this topic, I chose to cover two cases that demonstrate cloning used as a conservation tool and using it a method of de-extinction. The first of two cases that I will explore covers the genetic rescue of the endangered wild sheep, the Mouflon. Scientists used cumulus-oocyte complexes gathered from two deceased Mouflon found in a field and enucleated oocytes from another sheep species, Ovis aries. The embryo was then implanted in a domestic female sheep, where it eventually developed into clone of one of the female Mouflon. Scientists performed a PCR amplification of nine microsatellites to confirm that the clone was genetically identical to the Mouflon donor nuclei. The second case study I review is one where an extinct goat subspecies, the Bucardo, was successfully cloned. Scientists used fibroblasts derived from a cryopreserved specimen to create the embryos and enucleated oocytes from domestic goats to create the embryo. In this case two experiments were run to determine what stage is embryonic development was best for implantation. There was one successful pregnancy that resulted from implanting an embryo at the 3- to 6-cell stage of embryo development. Even though the clone died minutes within birth, they were still able to confirm that it genetically identical to the donor cells using microsatellite markers. There are definite pros and cons of cloning, most of which are related to ethics. Some benefits to cloning extinct or endangered animals include species conservation and indefinite research opportunities. On the other hand, potential consequences of cloning revolve around the welfare of the clone animal and the cost. Overall it can be said that were is an exciting outlook for the cloning of extinct; however it will be met with both scientific and ethical challenges before it becomes a staple in today’s society. Resources:Folch, J., M.j. Cocero, P. Chesné, J.l. Alabart, V. Domínguez, Y. Cognié, A. Roche, A. Fernández-?rias, J.i. Martí, P. Sánchez, E. Echegoyen, J.f. Beckers, A. Sánchez Bonastre, and X. Vignon. "First Birth of an Animal from an Extinct Subspecies (Capra Pyrenaica Pyrenaica) by Cloning."?Theriogenology?71.6 (2009): 1026-034. Web. 23 Nov. 2014.Holt, W. V. "Wildlife Conservation and Reproductive Cloning."?Reproduction?127.3 (2004): 317-24. Web. 23 Nov. 2014.Loi, Pasqualino, et al. "Genetic Rescue Of An Endangered Mammal By Cross-Species Nuclear Transfer Using Post-Mortem Somatic Cells." Nature Biotechnology 19.10 (2001): 962. Food Science Source. Web. 1 Dec. 2014.Abstract Received LateEngineering of E.coli for the production of P3HPTallyne Vasconcelos Souza Gomes, December 4, 2014ABSTRACTP3HP is a kind of thermoplastic that is one of the alternatives for the petrochemical derived plastic. Its biosynthesis needs expensive compounds to happen so, in order to make the process cheaper, scientists decided to create a recombinant strain in E.coli to produce P3HP.Previous studies with the plasmids pwQ02 and pwQ04 generated 10.1 g/L of P3HP. To optimize this, the scientist created 5 strategies:Use only the plasmids pwQ02, pwQ04 like what was done before;Use a plasmid addition system with phenylalanine and tyrosine to work in a way that only cells able to be cloned would survive;Insert the genetic information necessary for the production of P3HP directly into the chromosome;Insert part of the information directly and part using plasmids;A way to improve the fourth strategy, using the plasmid addition system with phenylalanine and tyrosine.To determine the plasmid stability, the samples were removed from the flask and put into plates with and without ampicillin, to see if this antibiotic has any impact on the experiment.The results showed that ampicillin helped to generate more stable plasmids, resulting in a better yield of P3HP. However, since plasmids carry redundant DNA, which leads to metabolic burden and plasmid loss, the scientists decided to come up with two strategies to overcome this: the first one used an amino acid plasmid addition system and the second one used chromosomal integration. They found out that those strategies worked better together, and mixed them.The best final result was the production of 25.7 g/L of P3HP in aerobic fermentation. ................
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