...Month 2012 Research Communication Fluorescent Protein Engineering by In Vivo Site-directed Mutagenesis Melvys Valledor1,2, Qinghua Hu3, Paul Schiller1,2,4, and Richard S. Myers1 1 Department of Biochemistry and Molecular Biology, University of Miami Miller School of Medicine, Miami, FL, USA Geriatric Research, Education, and Clinical Center, and Research Service, Veteran’s Affairs Medical Center, Miami, FL, USA 3 Department of Medicine, University of Miami Miller School of Medicine, Miami, FL, USA 4 Department of Orthopedics, University of Miami Miller School of Medicine, Miami, FL, USA 2 Summary In vivo site-directed mutagenesis by single-stranded deoxyribonucleic acid recombineering is a facile method to change the color of fluorescent proteins (FPs) without cloning. Two different starting alleles of GFP were targeted for mutagenesis: gfpmut3* residing in the Escherichia coli genome and egfp carried by a bacterial/mammalian dual expression lentiviral plasmid vector. Fluorescent protein spectra were shifted by subtle modification of the chromophore region and residues interacting with the chromophore of the FP. Eight different FPs (Violeta, Azure, Aqua, Mar, Celeste, Amarillo, Mostaza, and Bronze) were isolated and shown to be useful in multicolor imaging and flow cytometry of bacteria and transgenic human stem cells. To make in vivo sitedirected mutagenesis more efficient, the recombineering method was optimized using the fluorescence change as a sensitive...
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...Nucleic Acid Hybridization Nucleic acid hybridization is a general method in determining the sequence of homologous DNA, it is also used to differentiate order of genes in a haploid set of chromosomes of a particular organism and the size of limitation fragments that contain such sequence. Meaning, it is possible to study the genetic differences between different organisms and individual here on Earth. A nucleic Acid Hybridization analysis uses five laboratory skills the Restriction fragment preparation: where a restriction enzyme is added to a DNA sample, Electrophoresis: separate the restriction fragment from DNA sample, Blotting: single strands are transferred onto special paper or nylon membranes through capillary action, Radioactive probe: it is added to DNA bands, probe is a single-stranded DNA that is complementary to it, Autoradiography: after the probe hybridizes and excess probe is rinsed off, a sheet of photographic film is laid over the gel, radioactivity in the bound probe exposes the film to form an image corresponding to specific DNA bands-the bands containing DNA that base pairs with the probe. The key on detecting specific nucleic sequence in the mixture of different DNA molecules is in the base pairing between complementary strands. Cytosine and guanine is paired by three hydrogen bonds, thymine and adenine is paired by two hydrogen bonds when DNA is heated the DNA attain heat that drive them to vigorous jiggling as the temperature extend to 95° that may...
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...Be it resolved that the labeling of GMOs in food and drinks should be mandatory. Just as the US House of Assembly did in July of this year, this side says resoundingly; No No, No way. While our opponents did a credibly job of defining the key terms in this debate, let me take a moment to highlight a few things which are critical to our objection of this moot. This series of second round debates all fall under the theme of health and safety. Thus, it is important when defining GMOs, that the issue of health and safety is aptly captured by the definition. Thus, this side adopts the following definition offered by The World Health Organisation 2015. A GMO is an organism in which the genetic material has been modified in a way that does not occur naturally through fertilisation and/or natural recombination and food and drinks which contain them undergo rigorous safety requirements before they are authorized. As first speaker, I will present uncontestable proof that food and drinks containing GMOs do not require mandatory labelling because they do not pose any health concerns and furthermore, I will expose the minority of persons who are calling for their mandatory labelling as nothing but a bunch of hypocrites. Our second speaker will look at some economic concerns as well as issues relating to consumer choices. Now, let us get one thing straight. Calling for mandatory labels on GM food and drinks imply that consumers have a right to know what they are consuming and that this...
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...2. OBJECTIVES 2.1 PRIMARY OBJECTIVE : To compare the differences in the glycosylation pattern of Human Papilloma virus 16 L1 protein expressed in mammalian cells and Plant cells and reduce the binding capacity of the plant glycosylation enzyme. 2.2 SECONDARY OBJECTIVE : To predict the differences in the antigenicity of HPV 16 L1 gene expressed in recombinant mammalian cells and Plant cells. 3. LITERATURE REVIEW Markéta Šmídková et al.,(1998) mentioned in “Plant Production of Vaccine Against HPV: A New Perspectives”, the considerations of post translational modifications in therapeutic vaccine production including differences in glycosylation between plant and mammalian systems which gave us a basis for our work. Michael Karbiener et al,.(2004) “Humanization of the N-glycosylation pathway in plants and plant production systems” states the need to prevent addition of immunogenic N-glycans as this could induce IgE binding and rapid immune clearance of plant glycosylated therapeutics when injected into a mammal. Hence we chose 3 specific glycosylating enzymes to modify by mutation. Chandrasekaran et al in their work et al,. (2008) “Isolation and properties of alpha-D-mannose: beta-1,2-N-acetylglucosaminyltransferase from trachea mucosa” stated in detail the properties of the N acetyl glucosaminyl transferase enzyme including its specificity to terminal-branched mannosyl residues of glycoproteins. We...
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...The objectives of the experiments performed in the past three weeks were to induce mutagenesis in the E.coli K-12 strain W3110 through short ultra violet light (UV). This particular strain of E.coli lacked photolyase (phr) and the uvr system, which are non-mutagenic repair mechanisms. A survival curve was constructed based off various irradiation times and number of viable cells obtained. Mutant strains were then isolated and purified on MacConkey (MAC) plates to rule out false positives. During week 7, the generation time of the potential Lac+ and lac- strands of E.coli were also determined. The lac operon was also induced via IPTG to determine if β-galactosidase induction is affected by mutations. During week 8, we determined if β-galactosidase...
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...adapted with structural features that give them the ability to function up to 10 times higher catalytic efficiency than their mesophilic homologues. The overall goal of the proposed research is to use a biochemical and genetics approach to study the stability, structure, and function of psychrophilic proteins involved or believed to be involved in the nitrogen metabolism of Methanococcoides burtonii. The specific objectives are: 1. Investigate the structural and functional properties of M. burtonii’s nifH and nifD gene products using sequence analysis, UV-VIS spectroscopy, and two-hybrid studies; 2. Investigate the structure, stability, and kinetics of M. burtonii’s glutamate dehydrogenase through sequence analysis, site-directed mutagenesis, protein modeling, and kinetic assays. Cold-enzyme study can increase knowledge in the field of protein folding and catalysis and the broader impacts of this project include (1) potential biotechnological applications of cold-enzymes (2) the involvement of high school students and science teacher(s) in hands-on research; and (3) the presentation of research results to a non-scientific audience in understandable terms. Project Description Research Objectives and Significance About 75% of our biosphere’s temperature is permanently cold (≤ 5°C) and many microorganisms have adapted to this type (psychrophilic) of environment (1). Despite...
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...domain (NTD) comprising seven α-helices and a β-hairpin, a carboxy-terminal domain (CTD) comprising four α-helices, and a flexible linker with a 310-helix connecting the two structural domains. Structures of the capsid-protein assembly units have been determined by X-ray crystallography; however, structural information regarding the assembled capsid and the contacts between the assembly units is incomplete. Here we report the cryo-electron microscopy structure of a tubular HIV-1 capsid-protein assembly at 8 Å resolution and the three-dimensional structure of a native HIV-1 core by cryo-electron tomography. The structure of the tubular assembly shows, at the three-fold interface, a three-helix bundle with critical hydrophobic interactions. Mutagenesis studies confirm that hydrophobic residues in the centre of the three-helix bundle are crucial for capsid assembly and stability, and for viral infectivity. The cryo-electron-microscopy structures enable modelling by large-scale molecular dynamics simulation, resulting in all-atom models for the hexamer-of-hexamer and pentamer-of-hexamer elements as well as for the entire capsid. Incorporation of pentamers results in closer trimer contacts and induces acute surface curvature. The complete atomic HIV-1 capsid model provides a platform for further studies of capsid function and for targeted pharmacological...
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...Cystic fibrosis, also known as mucoviscidosis, is an autosomal recessive disorder.1,2,22 It is the most common inherited disease in the Caucasian population affecting 1 in 3000 children in Western Europe.3 It is a multiorgan disorder caused by mutations in the cystic fibrosis transmembrane conductance regulator (CFTR) protein, which is located on the long arm of chromosome 7 and encodes for a special chloride ion channel.4,5 The vast majority of mutations involve three or fewer nucleotides and result in primarily amino acid substitutions, frameshifts, splice site, or nonsense mutations.11 Of more than 800 identified CFTR mutations, the 3 base pair deletion of phenylalanine at position 508 is found worldwide in 70% of cystic fibrosis sufferers, therefore making F508 CFTR the most common deadly mutant in the Caucasian populations.6 Since cystic fibrosis has a genetic origin the opportunity to treat by replacing the defective gene with a normal healthy gene (gene therapy) offers a ‘novel therapeutic approach’ for sufferers.7 The estimated survival age of cystic fibrosis sufferers is 33.4 years (Fig 1). In this essay we will discuss the aetiology and symptoms of cystic fibrosis and the current available treatments, with particular emphasis on gene therapy and furanones, which prevent the build up of bacterial biofilms and thus reduce lung infection. Mutations in the CF gene can disrupt CFTR function within epithelial cells in different ways, ranging from complete loss...
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...DEVELOPMENTAL BIOLOGY The right slant Cell 121, 633–644 (2005) One of the biggest questions in developmental biology is how embryos that begin as uniform balls of cells end up asymmetric. Experiments in mice provided a clue when researchers discovered that hair-like cilia protruding from embryonic cells in mice rotate, somehow setting up a flow in the surrounding fluid that defines the left–right axis. A team led by Nobutaka Hirokawa of the University of Tokyo supplies another piece of the puzzle by showing exactly how the cilia’s movement sets up the directional flow. The researchers found that the cilia rotate around an axis tilted 40 backwards. The same rotation was observed in rabbit and medakafish embryos, suggesting that the mechanism, previously studied only in mice, defines asymmetry in other vertebrates. Paraphrasing of Research Highlight: When studying developmental biology researchers found that embryos start as a uniform ball of cells, however, they were questioning why do these embryos end up asymmetric. Research experiments were done on mice, researchers found that the cilia on the embryos on mice rotate and this rotation controls the flow of the surrounding fluid explaining the left-right axis. Further research was done and the experiments showed that the rotation of the cilia on the embryo is rotated 40 degrees backward on an axis. Experiments were also made in rabbits and medakafish embryos and the rotation in the cilia was observed. These results explain...
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...In addition to lac operator O1, “pseudo-operators” O2 and O3 might play a role in the repression of the lac operon. Therefore, the purpose of the experiment was to address how each of these operators contribute to lac operon repression. It was hypothesized that DNA loop formation had the potential to mediate repression of O1, O2, and O3. The mutant lacI gene (i^adi), which is a stable and active, shortened Lac repressor protein, was used to investigate the stated hypothesis. Eight constructs, each with the lacZ gene under the native lac promoter and activated or inactivated O1, O2, and O3 in all possible combinations, were cloned in phage λIPI to yield phages λEwt123 and λEwt000. All three lac operators were mutated by site directed mutagenesis and were infected into strain CHS 9 rec A (i^+,z^-) cells. Results of β-galactosidase assay under repressed and induced conditions showed an almost total loss of repression when O1 is destroyed. When either O2 or O3 is inactivated there was slight decrease of repression, but a 70-fold decrease of repression was seen when both pseudo-operators combined were lost. Repression value was obtained by dividing β-galactosidase activity in the presence of 1 mM IPTG by β-galactosidase activity in the absence of inducer. To examine how O2 and O3 contributed to repression, the i^adi was expressed to make loop formation impossible when binding of operators was not weakened. Gel filtration showed that molecular weight of mutant i^adi (75 kd) was similar...
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...ordinary human exposure type that occurs due to some pharmaceuticals or environmental agents. • Chronic toxicity. This relates to exposure of toxic substances for longer duration and the diverse effects are low. For instance, chronic bronchitis for tobacco addict and cirrhosis for alcohol addict • Carcinogenicity. This group refers to a complex abnormal process in cells which leads to cancer. • Developmental toxicity. This group refers to toxic responses to the developing embryo. This occurs due to the exposure of toxic substances to your partners prior to the act of conception. This leads to the death of cells of the fetus. • Genetic toxicity. The major cause of this kind of toxicity is a severe damage of DNA. This process is known as mutagenesis. It leads to cancer, or death of the cell. • Organ toxicity. This group is associated to toxic responses to different organs. It involves blood and cardiovascular toxicity, eye toxicity, dermal toxicity, reproductive toxicity, respiratory toxicity and various forms. (Hodgson, 2004, p.275) References Hodgson, E. (2004) A Textbook of Modern Toxicology. Wiley-Interscience; 3-rd edition. Question 2 List and provide a brief explanation of the types of Phase I reactions of metabolism of foreign substances. The types of Phase 1 reactions are: Oxidation- This is the additional of oxygen or removal of hydrogen to a substrate will make the compound more...
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...and TK. The enzymatic process induced by GCV leads to the final death of the cell. The HS and TK enzyme which is present in mammalian cells has extremely low affinity for the GCV, systemic toxicity closely to this mechanism is not observed in any type of manner. This type of vivo gene transfer has several features. First, these retroviral-vectors will integrate and express their genes in cells which are actively synthesizing the DNA. Therefore, surrounding the non-proliferating normal brain tissue should not acquire any of the HS-tk gene and will keep it remaining insensitive to the GCV. Secondly all of the transduced tumor cells will be killed by the host immune response and the GCV treatment eliminating all concern about insertional mutagenesis giving the rise to the malignant cells. This is the first ever clinical attempt to treat malignant tumors in human beings by in-vivo genetic manipulation of the tumor's genome which is explained as the use of gene therapy for the malignant brain tumors....
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...The text Television is an important form of communication and has been for many years. Television brings important historical events, both positive and negative, directly into our homes. People don’t just read about current events, they experience them in real time. Television can also be a fine teaching tool – there are many television shows on the air that are informative and educational. The right television shows can teach our children about morals and values as well as right from wrong. Educational television is not only for children however; Public television as well as cable television channels such as National Geographic and The History Channel serve as learning tools for adults. In fact many colleges and universities use local channels to provide distance learners the opportunity to attend lectures given on television. Along with the advantages listed above there are also disadvantages to television. First the same proliferation of new channels that provides additional educational opportunities also provides opportunities for children to be exposed to more inappropriate content than ever before. It is not uncommon for children to have access to movies and programs intended to be viewed only by mature audiences. While technology has been put into place to help conscientious parents censor the channels and shows their children can access, these methods are far from fool proof and they are only as effective as the parents who choose whether or not to utilize them. 02...
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...methotrexate for this purpose has not been evaluated. Studies determining the safety of Rasuvo® for the intended purpose of managing pJIA in patients 2 to 16 years old showed incidence of nausea/vomiting and a smaller incidence of stomatitis, diarrhea, leukopenia (low white blood cell count), and dizziness. 4. Geriatric Use – The difference in safety and response to Rasuvo® between elderly and younger patients could not be determined as clinical studies did not include enough patients over 65 years old. Elderly patients though should be monitored for toxic reactions as they more commonly have decreased kidney function, have decreased folate levels, or are concurrently taking multiple drug therapies. Nonclinical Toxicology: 1. Carcinogenesis, Mutagenesis, Impairment of Fertility – There is no human data evaluating the risk of abnormal growth with administration of methotrexate. Animal studies evaluating the potential of methotrexate to cause cancer were inconclusive. Chromosomal damage to animal somatic cells and human bone marrow cells has been found but the clinical significance of these findings cannot be established. Low doses of methotrexate may cause tumors and Non-Hodgkin’s lymphoma but have regressed after withdrawal of the drug. Methotrexate causes birth defects and fetal death in humans along with infertility, low sperm count, and abnormal menstrual cycles. ...
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...A. (1996). Factories of Death: Japanese Biological Warfare, 1932-1945, and the American Cover-Up. The Hastings Center Report, (5), 37 Huffington, A. (2011). Huffington post. Retrieved 25, October, from http://www.huffingtonpost.com/2011/02/04/world-cancer-day-a-declar_n_818472.html Hutchison, C. A., Peterson, S. N., Gill, S. R., Cline, R. T., White, O., Fraser, C. M., & ... Venter, J. C. (1999). Global Transposon Mutagenesis and a Minimal Mycoplasma Genome.Science, (5447). 2165. Jackson, R., Ramsay, A., Christensen, C., Beaton, S., Hall, D., & Ramshaw, I. (2001). Expression of mouse interleukin-4 by a recombinant ectromelia virus suppresses cytolytic lymphocyte responses and overcomes genetic resistance to mousepox. Journal Of Virology, 75(3), 1205-1210 Jaton, K., & Greub, G. (2014). Clinical microbiologists facing an anthrax alert. Clinical Microbiology & Infection,20(6), 503-506. doi:10.1111/1469-0691.12682 Johnson, D., Puzanov, I., & Kelley, M. (2015). Talimogene laherparepvec (T-VEC) for the treatment of advanced melanoma. Immunotherapy, 7(6), 611-619. Kido, N., Ohta, M., Iida, K., Hasegawa, T., Ito, H., Arakawa, Y., … Kato, N. (1989). Partial deletion...
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