tag:blogger.com,1999:blog-11952227270105373052024-03-13T22:26:31.073-05:00Aptamer Project Site - 10% APS for EveryoneThis space represents the ideas, views, opinions, projects and data of researchers within the Aptamer Stream of the Freshman Research Initiative, a program developed at the University of Texas at Austin. These are projects we currently have in the pipeline.<hr>
- We're not exclusive...we are <i>SELECTIVE</i>!Aptamer Staffhttp://www.blogger.com/profile/11584531632318072295noreply@blogger.comBlogger404125tag:blogger.com,1999:blog-1195222727010537305.post-84198520006533173342013-12-04T01:28:00.002-06:002013-12-04T01:28:30.947-06:00Lab Attendance Hours Fall 2013 (as of 12/1/13)<img 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" 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<div class="MsoNormal" style="text-align: center;">
<b style="mso-bidi-font-weight: normal;">Nucleic Acid Aptamer
Selection Against Amyloid Precursor Protein to Prevent Neurodegeneration in
Down’s Syndrome and Alzheimer’s Disease</b><br />
<br />
<b style="mso-bidi-font-weight: normal;">Charisma DeSai - Fall 2013 </b></div>
<div class="MsoNormal">
<br /></div>
<div class="MsoNormal">
In the United States, approximately 5.2 million people have
Alzheimer’s Disease and more than 400,000 individuals have Down’s Syndrome (Texas
Department of State Health Services) (National Down Syndrome Society). These
conditions are well-known, but as of yet, <a href="http://www.blogger.com/null" name="_GoBack"></a>no definitive
cause or cure has been found for either. Alzheimer’s disease is the most common
form of dementia in the human population. Amyloid plaques and neurofibrillary
tangles form in the brain, and the malfunction or loss of neurons causes
changes in memory and brain function. Many patients with Down’s syndrome also
develop Alzheimer’s later in life, but earlier than normal adults. Amyloid
deposition associated with Alzheimer’s disease is present in all Down’s
syndrome patients older than 30 (Egensperger 113). </div>
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<br /></div>
<div class="MsoNormal" style="mso-layout-grid-align: none; text-autospace: none;">
Amyloid
precursor protein (APP) is a transmembrane glycoprotein that is spliced into
multiple peptides. Evidence suggests that APP and its derivative, beta amyloid,
may contribute to Alzheimer’s disease. The beta amyloid protein makes up the
plaques that form in the brains of Alzheimer’s patients. The amyloid hypothesis
states that accumulation of beta-amyloid in the brain is the primary cause of
Alzheimer’s disease pathogenesis (Hermey 8). Down’s syndrome patients have
elevated APP levels since the gene for this protein is located on chromosome 21
(Arai 290). A small minority of Alzheimer’s disease patients (approximately
five percent) have an inherited form of the disease associated with an
autosomal dominant disorder that is linked to mutations in the genes APP (Van
Dijk 712). </div>
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<br /></div>
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The amyloid hypothesis suggests that reducing the amount of
beta-amyloid would mitigate the symptoms of Alzheimer’s disease (Hermey 8). Aptamers
have unique advantages for medical treatment since they are highly specific, efficiently produced, can be modified for added functions, and have very
low immunogenicity. An aptamer against APP would help reduce the amount of
plaques by reducing its functionality and preventing the creation of beta amyloid
protein.<span style="mso-spacerun: yes;"> </span>By inhibiting this pathway, the
formation of plaques from the beta amyloid protein could be reduced or perhaps
even prevented. This aptamer may even help prevent the development of dementia
in people with Down’s syndrome as they age or individuals with familial
Alzheimer’s disease.</div>
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<br /></div>
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" /></td></tr>
</tbody></table>
<div class="MsoNormal" style="margin-left: .5in;">
<br /></div>
<div class="MsoNormal" style="margin-left: .5in;">
Figure 1. The role of Amyloid
Precursor Protein, which is cleaved into beta amyloid protein. By inhibiting
APP, formation of amyloid plaques in the brain and neuronal death could be
prevented.</div>
<div class="MsoNormal">
<br /></div>
<div class="MsoNormal" style="margin-right: .5in; text-align: justify;">
<u>Specific Aim
1:</u> <span style="mso-fareast-font-family: SimSun; mso-fareast-language: ZH-CN;">Selection
of RNA aptamers against Amyloid Precursor Protein</span></div>
<div class="MsoNormal" style="margin-right: .5in; text-align: justify;">
The Amyloid
Precursor Protein (APP) differentiates into multiple proteins. One of these is
beta amyloid, which leads to the formation of plaques in the brain and
consecutive nerve cell death. Thus, the selection of RNA aptamers against APP
will inhibit the role of beta amyloid in neuronal death. </div>
<div class="MsoNormal" style="margin-bottom: .0001pt; margin-bottom: 0in; margin-left: .25in; margin-right: .5in; margin-top: 0in; text-align: justify;">
<br /></div>
<div class="MsoNormal" style="margin-right: .5in; text-align: justify;">
<b style="mso-bidi-font-weight: normal;">Ordering information:</b></div>
<div class="MsoNormal" style="margin-right: .5in; text-align: justify;">
<br /></div>
<div class="MsoNormal" style="margin-right: .5in; text-align: justify;">
Vendor: My Bio Source</div>
<div class="MsoNormal" style="margin-right: .5in; text-align: justify;">
Product Catalog
number: <a href="http://www.mybiosource.com/datasheet.php?products_id=204227" title="APP recombinant protein (Catalog #MBS204227)"><b>MBS204227</b></a> </div>
<div class="MsoNormal" style="margin-right: .5in; text-align: justify;">
Vendor Website:
<a href="http://www.mybiosource.com/datasheet.php?products_id=204227">http://www.mybiosource.com/datasheet.php?products_id=204227</a></div>
<div class="MsoNormal" style="margin-right: .5in; text-align: justify;">
Vendor
Telephone: 1-858-633-0165</div>
<div class="MsoNormal" style="margin-right: .5in; text-align: justify;">
Cost per Unit:
100 µg for $335</div>
<div class="MsoNormal" style="margin-right: .5in; text-align: justify;">
Cost per round:
$23.26</div>
<div class="MsoNormal" style="margin-bottom: .0001pt; margin-bottom: 0in; margin-left: .25in; margin-right: .5in; margin-top: 0in; text-align: justify;">
<br /></div>
<div class="MsoNormal">
<b>Sources:</b></div>
<div class="MsoNormal">
<br /></div>
<div class="MsoNormal">
"Alzheimer's Disease - Questions and Answers." <i>Alzheimer'
S Disease Questions and Answers</i>. Texas Department of State Health Services,
14 May 2013. Web. 20 Aug. 2013.</div>
<div class="MsoNormal">
<br /></div>
<div class="MsoNormal">
Arai, Y., Suzuki, A., Masashi, M., and Takashima, S. "Developmental and aging changes
in the expression of amyloid precursor protein in Down syndrome brains." <i>Brain
and Development</i> 19.4 (1997): 290-294.</div>
<div class="MsoNormal">
<br /></div>
<div class="MsoNormal">
"Down Syndrome Facts." <i>NDSS.org</i>. National
Down Syndrome Society, 2012. Web. 01 Sept. 2013.</div>
<div class="MsoNormal">
<br /></div>
<div class="MsoNormal">
Egensperger, R., Weggen, S. Multhaup, N., Schnabel, F., Beyreuther, K., and Bayer, T. "Reverse relationship
between β-amyloid precursor protein and β-amyloid peptide plaques in Down’s
syndrome versus sporadic/familial Alzheimer’s disease." <i>Acta
neuropathologica</i> 97.2 (1999): 113-118.</div>
<div class="MsoNormal">
<br /></div>
<div class="MsoNormal">
Hermey, Guido. "Targeting amyloid precursor
protein." <i>Annals of neurology</i> 69.1 (2011): 8-10.</div>
<div class="MsoNormal">
<br /></div>
<div class="MsoNormal">
Roizen, N. J., and Patterson, D. "Down's
syndrome." <i>The Lancet</i> 361.9365 (2003): 1281-1289.</div>
<div class="MsoNormal">
<br /></div>
<div class="MsoNormal">
Van Dijk, R., Fischer, D., Sluijs, J., Sonnemans, M., Hobo, B., Mercken, L., Mann, D., Hol, E., and van Leeuwen, F. "Frame shifted amyloid precursor
protein found in Alzheimer's disease and Down's syndrome increases levels of
secreted amyloid β40." <i>Journal of neurochemistry</i> 90.3 (2004):
712-723.</div>
<div class="MsoNormal">
<br />
Proposal: <a href="https://docs.google.com/file/d/0B9u9dHA7_FVXa3VpT0tjRGhFSjQ/edit?usp=sharing">Link to Proposal</a><br />
First Progress Report: <a href="https://docs.google.com/file/d/0B9u9dHA7_FVXMkNIMkpVUnYwZ2c/edit?usp=sharing"> Link to First Progress Report</a></div>
<div class="MsoNormal">
Second Progress Report: <a href="https://drive.google.com/file/d/0B9u9dHA7_FVXOFpSWUh1X0V1S0U/edit?usp=sharing">Link to Second Progress Report</a><br />
Final Progress Report: <a href="https://drive.google.com/file/d/0B9u9dHA7_FVXTlNvaTAwbENqcE0/edit?usp=sharing">Link to Final Progress Report</a> </div>
Charismahttp://www.blogger.com/profile/03034603870709812601noreply@blogger.com0tag:blogger.com,1999:blog-1195222727010537305.post-58097268884577656362013-09-17T18:14:00.002-05:002013-10-09T03:08:07.859-05:00Nucleic Acid Aptamer Selection for Myelin Basic Protein for the Prevention of Malignant Schwann Cells by Manjari Subramanian<div class="MsoNormal" style="line-height: 200%;">
<b style="mso-bidi-font-weight: normal;"><span style="font-family: Times; mso-bidi-font-family: "Times New Roman";">Abstract:<o:p></o:p></span></b></div>
<div class="MsoNormal" style="line-height: 200%; text-indent: .5in;">
<span style="font-family: Times; mso-bidi-font-family: "Times New Roman";">Myelin basic
protein (MBP) promotes growth of myelin sheath throughout the nervous system by
binding to the lipids on the myelin membrane (Boggs, 2006). However when a
malignant cell is formed, the abnormal cell inhibits the transmission of
electric signals across synapses (Kaur et al., 2008). MBP has the ability to
distinguish the difference between a normal and malignant Schwann cell due to
deletions within the MBP gene sequence (Kaur et al., 2008). <o:p></o:p></span></div>
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<div class="separator" style="clear: both; text-align: center;">
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<b style="mso-bidi-font-weight: normal;"><span style="font-family: Times; mso-bidi-font-family: "Times New Roman";"><br /></span></b></div>
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<b style="mso-bidi-font-weight: normal;"><span style="font-family: Times; mso-bidi-font-family: "Times New Roman";">Figure 1 Myelin Basic Protein</span></b><span style="font-family: Times; mso-bidi-font-family: "Times New Roman";">: Computer
model of the protein. Human myelin basic protein by binding it to a leukocyte
antigen (Smith et al., 1998).<o:p></o:p></span></div>
<div class="MsoNormal" style="line-height: 200%;">
<span style="font-family: Times; mso-bidi-font-family: "Times New Roman";">Aim 1: Select for the protein<span style="mso-spacerun: yes;"> </span><o:p></o:p></span></div>
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<span style="font-family: Times; mso-bidi-font-family: "Times New Roman";"><span style="mso-tab-count: 1;"> </span>A
multi-layered process will be involved in the selection for MBP. Aptamers, used
as RNA protein markers in this case, will be selected for myelin basic protein.
Aptamers bind to proteins with high specificity and affinity to the specific
protein target. This is because aptamers are about one-tenth the size of an
antibody (Boggs, 2006). This provides a set of tools that aid in area of
therapeutic diagnostics in medicine. MBP itself is not a large protein (only
18.5 kDa); thus using an aptamer, which is even smaller, will increase chances
of binding to the protein (Saxe et al., 1985). By selecting for MBP, the gene
sequence of the protein can be verified before further development of an
aptamer to distinguish between a normal and malignant cell. If successful,
further tests can be conducted to see if the aptamer can deliver a drug or
select against a mutate MBP. The first step is to find an aptamer that binds to
this specific protein. <o:p></o:p></span></div>
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<span style="font-family: Times; mso-bidi-font-family: "Times New Roman";">The target can
be ordered from US Biologicals at $327.00 per milligram and $2.40 per round
(400pmol).<o:p></o:p></span></div>
<div class="MsoNormal" style="line-height: 200%;">
<b style="mso-bidi-font-weight: normal;"><span style="font-family: Times; mso-bidi-font-family: Arial;">References:<o:p></o:p></span></b></div>
<div class="MsoNormal" style="line-height: 200%; margin-left: .5in; text-indent: -.5in;">
<span style="font-family: Times; mso-bidi-font-family: Arial;">Boggs JM (2006) Myelin
basic protein: a multifunctional protein. Cell Mol Life Sci. 63:
1945–1961.<span style="mso-spacerun: yes;"> </span><o:p></o:p></span></div>
<h1 style="background: white; line-height: 200%; margin-bottom: 4.5pt; margin-left: .5in; margin-right: 0in; margin-top: 4.5pt; text-indent: -.5in;">
<span style="color: windowtext; font-family: Times; font-size: 12.0pt; font-weight: normal; line-height: 200%; mso-bidi-font-family: Arial; mso-fareast-font-family: "MS 明朝"; mso-fareast-theme-font: minor-fareast;">Eylar EH, Brostoff S, Hashim G, Caccam J,
Burnett P (1971). Basic A1 protein of the myelin membrane. The complete amino
acid sequence. J. Biol. Chem. 246 (18): 5770–84. <o:p></o:p></span></h1>
<h1 style="background: white; line-height: 200%; margin-bottom: 4.5pt; margin-left: .5in; margin-right: 0in; margin-top: 4.5pt; text-indent: -.5in;">
<span style="color: windowtext; font-family: Times; font-size: 12.0pt; font-weight: normal; line-height: 200%; mso-bidi-font-family: Arial; mso-fareast-font-family: "MS 明朝"; mso-fareast-theme-font: minor-fareast;">Gragoudas ES, Adamis AP, Cunningham ET Jr,
Feinsod M, Guyer DR (2004). Pegaptanib for neovascular age-related macular
degeneration. N Engl J Med. 351(27):2805-16.<o:p></o:p></span></h1>
<h1 style="background: white; line-height: 200%; margin-bottom: 4.5pt; margin-left: .5in; margin-right: 0in; margin-top: 4.5pt; text-indent: -.5in;">
<a href="http://www.ncbi.nlm.nih.gov/pubmed?term=Kaur%20G%5BAuthor%5D&cauthor=true&cauthor_uid=18095918"><span style="color: windowtext; font-family: Times; font-size: 12.0pt; font-weight: normal; line-height: 200%; mso-bidi-font-family: Arial; mso-fareast-font-family: "MS 明朝"; mso-fareast-theme-font: minor-fareast; text-decoration: none; text-underline: none;">Kaur
G</span></a><span style="color: windowtext; font-family: Times; font-size: 12.0pt; font-weight: normal; line-height: 200%; mso-bidi-font-family: Arial; mso-fareast-font-family: "MS 明朝"; mso-fareast-theme-font: minor-fareast;">, </span><a href="http://www.ncbi.nlm.nih.gov/pubmed?term=Roy%20I%5BAuthor%5D&cauthor=true&cauthor_uid=18095918"><span style="color: windowtext; font-family: Times; font-size: 12.0pt; font-weight: normal; line-height: 200%; mso-bidi-font-family: Arial; mso-fareast-font-family: "MS 明朝"; mso-fareast-theme-font: minor-fareast; text-decoration: none; text-underline: none;">Roy
I</span></a><span style="color: windowtext; font-family: Times; font-size: 12.0pt; font-weight: normal; line-height: 200%; mso-bidi-font-family: Arial; mso-fareast-font-family: "MS 明朝"; mso-fareast-theme-font: minor-fareast;"> (2008).
Therapeutic applications of aptamers. National Institute of Pharmaceutical
Education and Research (NIPER). Expert Opin Investing Drugs. <o:p></o:p></span></h1>
<div class="MsoNormal" style="line-height: 200%; margin-left: .5in; text-indent: -.5in;">
<span style="font-family: Times; mso-bidi-font-family: Arial;">Meinl, Weber, Drexler,
Morelle, Ott, Saruhan-Direskeneli, Goebels, Ertl, Jechart, Giegerich (1993).
Myelin basic protein-specific T lymphocyte repertoire in multiple sclerosis.
Complexity of the response and dominance of nested epitopes due to recruitment
of multiple T cell clones. J Clin Invest. 92(6):2633–2643<o:p></o:p></span></div>
<div class="MsoNormal" style="line-height: 200%; margin-left: .5in; text-indent: -.5in;">
<span style="font-family: Times; mso-bidi-font-family: Arial;">Saxe DF, Takahashi N, Hood
L, Simon MI (1985). Localization of the human myelin basic protein gene (MBP)
to region 18q22----qter by in situ hybridization. Cytogenet. Cell Genet.
39 (4): 246–9.<o:p></o:p></span></div>
<div class="MsoNormal" style="line-height: 200%; margin-left: .5in; text-indent: -.5in;">
<span style="font-family: Times; mso-bidi-font-family: Arial;"> Smith, K.J., Pyrdol,
J., Gauthier, L., Wiley, D.C., Wucherpfennig, K (1998). Crystal structure of HLA-DR2 (DRA*0101, DRB1*1501) complexed with
a peptide from human myelin basic protein.
J.Exp.Med. 188: 1511-1520</span></div>
<div class="MsoNormal" style="line-height: 200%; margin-left: .5in; text-indent: -.5in;">
<span style="font-family: Times; mso-bidi-font-family: Arial;">Wang C, Neugebauer U,
Bürck J, Myllykoski M, Baumgärtel P, et al. (2011). Charge Isomers of Myelin
Basic Protein: Structure and Interactions with Membranes, Nucleotide Analogues,
and Calmodulin. PLoS ONE 6(5): e19915. <o:p></o:p></span></div>
<div class="MsoNormal" style="line-height: 200%; margin-left: .5in; text-indent: -.5in;">
<span style="font-family: Times; mso-bidi-font-family: Arial;">Warren, K.J., Catz, I.,
Steinman, L. (1995). Fine specificity of the antibody response to myelin basic
protein in the central nervous system in multiple sclerosis: The minimal B-cell
epitope and a model of its features. Proc. Natl. Acad. Sci. USA. 92:
11061-11065. <o:p></o:p></span></div>
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<span style="font-family: Times; mso-bidi-font-family: Arial;"><a href="http://www.ncbi.nlm.nih.gov/pubmed/?term=Zhang%20Y%5Bauth%5D"><span style="color: windowtext; text-decoration: none; text-underline: none;">Yin Zhang</span></a>,
<a href="http://www.ncbi.nlm.nih.gov/pubmed/?term=Hong%20H%5Bauth%5D"><span style="color: windowtext; text-decoration: none; text-underline: none;">Hao Hong</span></a>,
<a href="http://www.ncbi.nlm.nih.gov/pubmed/?term=Cai%20W%5Bauth%5D"><span style="color: windowtext; text-decoration: none; text-underline: none;">Weibo Ca</span></a>i
(2011). Tumor-Targeted Drug Delivery with Aptamers. Current Medical Chemistry 1<a href="http://www.ncbi.nlm.nih.gov/entrez/eutils/elink.fcgi?dbfrom=pubmed&retmode=ref&cmd=prlinks&id=21838687"><span style="color: windowtext; text-decoration: none; text-underline: none;">. 18(27):
4185–4194.</span></a><o:p></o:p></span></div>
<br />
Research Proposal:<br />
<br />
<a href="https://docs.google.com/document/d/1iiFltUcxxQ39oUpegZl-CNKEM1p1RdXQlP2_njopEnA/edit?usp=sharing">https://docs.google.com/document/d/1iiFltUcxxQ39oUpegZl-CNKEM1p1RdXQlP2_njopEnA/edit?usp=sharing</a><br />
<br />
1st Progress Report:<br />
<br />
<a href="https://docs.google.com/file/d/0By_LVGU6rig_aTc4b2I4aG5GWmc/edit?usp=sharing">https://docs.google.com/file/d/0By_LVGU6rig_aTc4b2I4aG5GWmc/edit?usp=sharing</a><br />
<br />
<br />Anonymoushttp://www.blogger.com/profile/10082346920107353729noreply@blogger.com0tag:blogger.com,1999:blog-1195222727010537305.post-43399914192271086852013-09-17T03:25:00.000-05:002013-12-03T20:00:12.928-06:00The Brain's New Hero: Nucleic Acid Aptamer Selection Against Beta-Glucuronidase<!--[if gte mso 9]><xml>
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<div class="MsoNormal" style="margin-bottom: 12.0pt; mso-layout-grid-align: none; mso-pagination: none; text-autospace: none;">
<br /></div>
<div class="MsoNormal">
<span style="font-family: Arial;">Nucleic Acid Aptamer
Selection Against Beta-Glucuronidase<o:p></o:p></span></div>
<div class="MsoNormal">
<span style="font-family: Arial;">Ellen Airhart<o:p></o:p></span></div>
<div class="MsoNormal">
<span style="font-family: Arial;">Nucleic Acid Pool: N71 RNA
Pool<o:p></o:p></span></div>
<div class="MsoNormal">
<span style="font-family: Arial;">Target: Beta-Glucuronidase<o:p></o:p></span></div>
<div class="MsoNormal">
<br /></div>
<div class="MsoNormal" style="margin-bottom: 12.0pt; mso-layout-grid-align: none; mso-pagination: none; text-autospace: none; text-indent: .5in;">
<span style="font-family: Calibri; mso-ascii-theme-font: major-latin; mso-bidi-font-family: Helvetica; mso-bidi-font-weight: bold; mso-hansi-theme-font: major-latin;">Microglia</span><span style="font-family: Calibri; mso-ascii-theme-font: major-latin; mso-bidi-font-family: Helvetica; mso-hansi-theme-font: major-latin;"> are a type of glial cell found in
the central nervous system. There, they act as the first line of defense of the
nervous system. </span><span style="font-family: Calibri; mso-ascii-theme-font: major-latin; mso-bidi-font-family: Arial; mso-hansi-theme-font: major-latin;"> Stimulation of microglia is a signal of
infectious, inflammatory, and degenerative illnesses of the CNS. This
inflammation could be indicative of many diseases, such as Alzheimer’s,
dementia, and other neurodegenerative conditions. Studies have shown that there
is a rise in the release of β- glucuronidase, or GUS, by stimulated microglia
into the extracellular space at an area of neuroinflammation. GUS is involved
in the hydrolysis of glycosaminoglycans on the cell surface and the
degeneration of the extracellular matrix. Therefore, GUS might be a biomarker
for continuing neurodegeneration induced by neuroinflammation (Antunes,
Doorduin, Haisma, Elsinga, Waarde, Willemson, Dierckx & Vries, 2012).<o:p></o:p></span></div>
<div class="MsoNormal" style="margin-bottom: 12.0pt; mso-layout-grid-align: none; mso-pagination: none; text-autospace: none;">
<span style="font-family: Calibri; mso-ascii-theme-font: major-latin; mso-bidi-font-family: Arial; mso-hansi-theme-font: major-latin;">An
aptamer, or an olglionucleic acid or peptide molecule that have been carefully
selected to bind to its target in a similar way to an antibody (Pai, Roberts
& Ellington, 2008), has not been discovered for Beta-Glucuronidase. If an
aptamer was developed to bind to GUS it could be used as a biomarker to assist
in diagnosing neurodegenerative diseases.</span><span style="font-family: Calibri; mso-ascii-theme-font: major-latin; mso-bidi-font-family: Times; mso-hansi-theme-font: major-latin;"><o:p></o:p></span></div>
<div class="MsoNormal">
<u><span style="font-family: Arial;">Primary Specific Aim: </span></u><span style="font-family: Arial;">Identifying
an aptamer that binds to GUS-His<o:p></o:p></span></div>
<div class="MsoNormal">
<span style="font-family: Arial;"><br /></span></div>
<div class="separator" style="clear: both; text-align: center;">
<a href="http://2.bp.blogspot.com/-eZKvUR2x2es/UiVn7dIvvZI/AAAAAAAAAAc/H8Nvv_mUlm8/s1600/Screen+Shot+2013-09-02+at+11.38.35+PM.png" imageanchor="1" style="margin-left: 1em; margin-right: 1em;"><img border="0" height="193" src="http://2.bp.blogspot.com/-eZKvUR2x2es/UiVn7dIvvZI/AAAAAAAAAAc/H8Nvv_mUlm8/s320/Screen+Shot+2013-09-02+at+11.38.35+PM.png" width="320" /></a></div>
<div class="MsoNormal">
<br /></div>
<div class="MsoNormal" style="margin-bottom: 12.0pt; mso-layout-grid-align: none; mso-pagination: none; text-autospace: none;">
<span style="font-family: Arial;">Figure 1 illustrates
the active sites Glu451, Tyr504, Glu540, and Asn450. This high number of active
sites increases the chance that there is an aptamer for this target. </span><o:p></o:p></div>
<div class="MsoNormal">
<u><span style="font-family: Arial;">Secondary Specific Aim: </span></u><span style="font-family: Arial;">Use
this aptamer as a biomarker for neurodegenerative inflammation<o:p></o:p></span></div>
<div class="MsoNormal" style="margin-bottom: 14.0pt; mso-layout-grid-align: none; mso-pagination: none; tab-stops: 11.0pt .5in; text-autospace: none; text-indent: -.5in;">
<br /></div>
<div class="MsoNormal">
<u><span style="font-family: Arial;">Target Order Information</span></u><span style="font-family: Arial;"><o:p></o:p></span></div>
<div class="MsoNormal">
<span style="font-family: Arial;">Vendor: Ellington Lab<o:p></o:p></span></div>
<div class="MsoNormal">
<span style="font-family: Arial;">Vendor Website:
https://www.ellingtonlab.org<o:p></o:p></span></div>
<div class="MsoNormal">
<span style="font-family: Arial;">Central Lab Telephone:
512-471-6445<o:p></o:p></span></div>
<div class="MsoNormal">
<span style="font-family: Arial;">Office Manager:
512-232-3426<o:p></o:p></span></div>
<div class="MsoNormal">
<span style="font-family: Arial;">Cost per Unit: $0<o:p></o:p></span></div>
<div class="MsoNormal" style="margin-bottom: 14.0pt; mso-layout-grid-align: none; mso-pagination: none; tab-stops: 11.0pt .5in; text-autospace: none; text-indent: -.5in;">
<span style="font-family: Arial;"> Cost
per Round: $0<o:p></o:p></span></div>
<div class="MsoNormal" style="margin-bottom: 14.0pt; margin-left: .25in; margin-right: 0in; margin-top: 0in; mso-layout-grid-align: none; mso-pagination: none; tab-stops: 11.0pt .5in; text-autospace: none; text-indent: -.5in;">
<span style="font-family: Arial;">Antunes,
I., Doorduin, J., Haisma, H., Elsinga, P., Waarde, A., Willemson, A., Dierckx,
R., & Vries, E. (2012). F-feanga for pet of β-glucuronidase activity in
neuroinflammation. <i>Journal of Nuclear Medicine, 53(3), </i>451-458. <o:p></o:p></span></div>
<div class="MsoNormal" style="margin-left: .25in; mso-layout-grid-align: none; mso-pagination: none; text-autospace: none; text-indent: -.5in;">
<span style="font-family: Helvetica; mso-bidi-font-family: Helvetica;">Lawson L J, Perry V H, Gordon S
(1992). "Turnover of resident microglia in the normal adult mouse
brain". <i>Neuroscience</i> <b>48</b>: 405–415.</span><span style="font-family: Arial;"><o:p></o:p></span></div>
<div class="MsoNormal" style="mso-layout-grid-align: none; mso-pagination: none; text-autospace: none; text-indent: -.5in;">
<br /></div>
<div class="MsoNormal" style="margin-bottom: 14.0pt; margin-left: .25in; margin-right: 0in; margin-top: 0in; mso-layout-grid-align: none; mso-pagination: none; tab-stops: 11.0pt .5in; text-autospace: none; text-indent: -.5in;">
<span style="font-family: Arial;">Pai,
S., Roberts , A., & Ellington , A. (2008). Aptamer amplification: divide
and signal. <i>Expert Opinion on Medical Diagnostics, 2(12), </i>1333-1346.
doi: 10.1517/17530050802562016 <o:p></o:p></span></div>
<div class="MsoNormal" style="margin-bottom: 14.0pt; margin-left: .25in; margin-right: 0in; margin-top: 0in; mso-layout-grid-align: none; mso-pagination: none; tab-stops: 11.0pt .5in; text-autospace: none; text-indent: -.5in;">
<span style="font-family: Arial;">Roffler,
S., Wu, C., Schechinger, W., Chen, K., & Prijovic, Z. (2009). Human
beta-glucuronidase mutants with elevated enzymatic activity under physiological
conditions and methods for identifying as such. <o:p></o:p></span></div>
<!--EndFragment--><a href="https://docs.google.com/file/d/0B5AOFSinkMp4dU5FSk9veVU2c1U/edit?usp=sharing">First Target Proposal</a><br />
<a href="https://docs.google.com/file/d/0B5AOFSinkMp4TzNrX3QtTGZHc1k/edit?usp=drive_web">First Progress Report</a><br />
<a href="https://drive.google.com/file/d/0B5AOFSinkMp4ODE1TEdEM0JSLWs/edit?usp=sharing">Second Progress Report</a><br />
<!--EndFragment--><a href="https://drive.google.com/file/d/0B5AOFSinkMp4UEdNemZVb1AzWEU/edit?usp=sharing">Final Progress Report</a><br />
<!--EndFragment-->Ellen Airhart 6http://www.blogger.com/profile/14795146201253412670noreply@blogger.com0tag:blogger.com,1999:blog-1195222727010537305.post-56303184772229485532013-09-05T16:06:00.003-05:002013-12-03T21:07:22.291-06:00Modified Selection against EGFR Using Known Aptamer E07 to Optimize SELEX Efficiency<div class="MsoNormal">
<b>Modified Selection against EGFR Using Known Aptamer E07 to
Optimize SELEX Efficiency</b><o:p></o:p></div>
<div class="MsoNormal">
<br /></div>
<div class="MsoNormal">
Marc St. Cyr<o:p></o:p></div>
<div class="MsoNormal">
<br /></div>
<div class="MsoNormal">
Aptamer
research provides a wealth of possibilities for therapeutic, diagnostic, and
drug delivery purposes but the methods are constantly evolving and being
optimized. It is very difficult to stay updated with the most current and
innovative techniques and procedures. However, it is possible to, from time to
time, perform selection with positive controls and varying conditions in order
to determine the optimum conditions that should be used while performing a
Systematic Evolution of Ligands by Exponential Enrichment (SELEX). There are
many aspects of the SELEX procedure that can be optimized but the most crucial
for these purposes are the time-based and nucleic acid recovery components (Stoltenburg
2007). Reducing the amount of time spent on one round of selection allows for
faster results, which lead to expedited aptamer identification. The most
effective way to optimize experiments is by comparing the results provided by
reagents known to work with current conditions and conditions proposed to be
faster, more effective, or otherwise helpful. <o:p></o:p></div>
<div class="MsoNormal">
<br /></div>
<div class="MsoNormal" style="text-indent: .5in;">
The aptamer E07 was successfully
selected against epidermal growth factor receptor (EGFR) (Li 2011). Using this
aptamer in the SELEX procedure to replace the randomized RNA pool will allow
for the optimization of certain aspects of selection. The same reagents
provided in the published aptamer paper will be used while the conditions of
the selection itself will be varied and analyzed by assaying reactions,
incubations, etc. to provide data or comparisons (Avutu 2010).<o:p></o:p></div>
<div class="MsoNormal" style="text-indent: .5in;">
<br /></div>
<div class="MsoNormal" style="text-indent: .5in;">
<u>Specific Aim 1</u>: Provide the
FRI Aptamer Lab with optimized selection conditions with the goal of producing
more efficient results when using the SELEX method of aptamer selection. This
can include a wide and varying range of suggestions from shorter incubation
times, new procedures, or preferred versions of already established procedures.<o:p></o:p></div>
<br />
<div class="separator" style="clear: both; text-align: center;">
<a href="http://4.bp.blogspot.com/-AS7EZB5-nkA/UijyLJnb0fI/AAAAAAAAABA/Y9Yb4KT1Dr8/s1600/SELEX.png" imageanchor="1" style="margin-left: 1em; margin-right: 1em;"><img border="0" height="279" src="http://4.bp.blogspot.com/-AS7EZB5-nkA/UijyLJnb0fI/AAAAAAAAABA/Y9Yb4KT1Dr8/s320/SELEX.png" width="320" /></a></div>
<div class="MsoNormal" style="margin-left: 1.5in;">
<b>Figure 1.</b> Overall outline of the SELEX method of selection of LNA
against a target, including binding, washing, and amplification steps.<o:p></o:p></div>
<div class="MsoNormal" style="margin-left: 1.5in;">
<br /></div>
<div class="MsoNormal">
References:<o:p></o:p></div>
<div class="MsoListParagraphCxSpFirst" style="margin-left: .75in; mso-add-space: auto; mso-list: l0 level1 lfo1; text-indent: -.25in;">
<!--[if !supportLists]-->1)<span style="font-size: 7pt;">
</span><!--[endif]-->Stoltenburg R, Reinemann C, Strehlitz B (2007)
SELEX- A (R)evolutionary Method to Generate High-Affinity Nucleic Acid Ligands.
Biomedical Engineering 24(4): 381-403.<o:p></o:p></div>
<div class="MsoListParagraphCxSpMiddle" style="margin-left: .75in; mso-add-space: auto;">
<br /></div>
<div class="MsoListParagraphCxSpMiddle" style="margin-left: .75in; mso-add-space: auto; mso-list: l0 level1 lfo1; text-indent: -.25in;">
<!--[if !supportLists]-->2)<span style="font-size: 7pt;">
</span><!--[endif]-->Li N, Nguyen HH, Byrom M, Ellington AD (2011)
Inhibition of Cell Proliferation by an Anti-EGFR Aptamer. PLoS ONE 6(6):
e20299. doi:10.1371/journal.pone.0020299<o:p></o:p></div>
<div class="MsoListParagraphCxSpMiddle">
<br /></div>
<br />
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<!--[if !supportLists]-->3)<span style="font-size: 7pt;">
</span><!--[endif]-->Avutu V (2010) Avidity Effects of MinE07, an
anti-EGFR Aptamer, on Binding to A431 Cells <<a href="http://repositories.lib.utexas.edu/handle/2152/13407?show=full">http://repositories.lib.utexas.edu/handle/2152/13407?show=full</a>><o:p></o:p><br />
<br />
<br />
<div style="text-indent: 0px;">
To see full proposal click <a href="https://docs.google.com/file/d/0B-PM1BzK-aV1MkJrc3ZGdnVsdmM/edit?usp=sharing">here</a>.<br />
To see first progress report click <a href="https://docs.google.com/file/d/0B-PM1BzK-aV1amRDc2F4SFpfLU0/edit?usp=sharing">here</a>.<br />
To see second progress report click <a href="https://drive.google.com/file/d/0B-PM1BzK-aV1VHAzUXBnX01jS1U/edit?usp=sharing">here</a>.<br />
To see final report click <a href="https://drive.google.com/file/d/0B-PM1BzK-aV1a3NZRVFpbGpmR28/edit?usp=sharing">here</a>.</div>
</div>
Marc St. Cyrhttp://www.blogger.com/profile/08583337191431296132noreply@blogger.com0tag:blogger.com,1999:blog-1195222727010537305.post-36325842774372555642013-09-04T08:26:00.001-05:002013-12-03T23:47:21.656-06:00<style>
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<span style="font-family: Helvetica; mso-bidi-font-family: Helvetica;">Nucleic Acid Aptamer Selection Against
Progesterone for use in Preventing and Treating Osteoporosis</span></div>
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<span style="font-family: Helvetica; mso-bidi-font-family: Helvetica;"></span></div>
<div class="MsoNormal" style="mso-layout-grid-align: none; mso-pagination: none; tab-stops: 28.0pt 56.0pt 84.0pt 112.0pt 140.0pt 168.0pt 196.0pt 224.0pt 3.5in 280.0pt 308.0pt 336.0pt; text-autospace: none;">
<span style="font-family: Helvetica; mso-bidi-font-family: Helvetica;"></span></div>
<div class="MsoNormal" style="mso-layout-grid-align: none; mso-pagination: none; tab-stops: 28.0pt 56.0pt 84.0pt 112.0pt 140.0pt 168.0pt 196.0pt 224.0pt 3.5in 280.0pt 308.0pt 336.0pt; text-autospace: none;">
<span style="font-family: Helvetica; mso-bidi-font-family: Helvetica;">Kelsey Gardner</span></div>
<div class="MsoNormal" style="mso-layout-grid-align: none; mso-pagination: none; tab-stops: 28.0pt 56.0pt 84.0pt 112.0pt 140.0pt 168.0pt 196.0pt 224.0pt 3.5in 280.0pt 308.0pt 336.0pt; text-autospace: none;">
<span style="font-family: Helvetica; mso-bidi-font-family: Helvetica;">September 1, 3013<br style="mso-special-character: line-break;" />
<br style="mso-special-character: line-break;" />
</span></div>
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<span style="font-family: Times; mso-bidi-font-family: Times;"><span style="mso-tab-count: 1;"> </span></span><span style="font-family: Helvetica;">Nucleic Acid Aptamer Selection Against Progesterone
for use in Preventing and Treating Osteoporosis</span><br />
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<!--[if !supportLineBreakNewLine]--><br />
<!--[endif]--><o:p></o:p></span></div>
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<span style="font-family: "Times New Roman"; mso-bidi-font-family: "Times New Roman";">Osteoporosis
is a disease in which the body struggles to create enough new bone tissue to
maintain a bone density high enough to support a healthy skeletal system. Type
one osteoporosis is most commonly associated with menopausal women, and results
in bones become more brittle so that the incidence of fracture increases [1]. <br />
Estrogen and
progesterone are both hormones associated with maintaining women’s health, and
both drop to record lows during menopause, as depicted in Figure 1, and remain
low upon diagnosis of osteoporosis [1]. Hormone therapy along with calcium
supplements are considered to be somewhat successful in the prevention of
osteoporosis, and are sometimes used to help ease the symptoms of women
diagnosed with post-menopausal osteoporosis. Yet, these methods alone are not
directly targeted toward the exact progestins associated with early onset
osteoporosis. Several antibodies associated with estrogen and progesterone
hormone receptors do exist but are not successful because the therapeutic drug
does not specifically target the progestin receptors strictly associated with
bone growth [2]. <br />
Developing
an aptamer for progesterone specifically associated with receptors for bone growth
would increase the effectiveness of treating menopausal osteoporosis. Specifically targeting progesterone, so that it can function for drug delivery, would allow for progesterone molecules carrying a drug that increases bone growth to bind at various sites such as the TE85 osteosarcoma </span><span style="font-family: 'Times New Roman';">receptor</span><span style="font-family: 'Times New Roman';"> </span><span style="font-family: 'Times New Roman';">, a receptor that plays a large role in promoting regeneration a bone growth [4]. Progesterone is a very small steroid hormone that possesses
a high binding affinity inside of the cell membrane, and these characteristics
allow for a wide use of aptamers to treat varying degrees and stages of
osteoporosis uniquely [1]. Therefore, an anti-progesterone aptamer would be used as a means for highly specified drug delivery to progesterone receptors (via progesterone aptamers) associated with bone growth and result in a prevention of further bone density loss.</span><br />
<span style="font-family: "Times New Roman"; mso-bidi-font-family: "Times New Roman";">
Specific Aim: Develop an aptamer for progesterone hormones to prevent or treat
the symptoms of menopausal (stage one) osteoporosis.<o:p></o:p></span></div>
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<div class="separator" style="clear: both; text-align: center;">
<a href="http://1.bp.blogspot.com/-8Q6Igk6EHXs/Uic0rfZ2EFI/AAAAAAAAAAY/M-A9XWGkA20/s1600/abstract.png" imageanchor="1" style="margin-left: 1em; margin-right: 1em; text-align: center;"><img border="0" height="139" src="http://1.bp.blogspot.com/-8Q6Igk6EHXs/Uic0rfZ2EFI/AAAAAAAAAAY/M-A9XWGkA20/s200/abstract.png" width="200" /></a></div>
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<span style="color: windowtext;">Figure </span><span style="color: windowtext;">1</span><span style="color: windowtext;">- A decline in progesterone during menopause is a
leading </span></div>
<span style="color: windowtext;"></span><br />
<div style="text-align: center;">
<span style="color: windowtext;"><span style="color: windowtext;">factor in the onset of osteoporosis in women age 45-60[3].</span></span></div>
<span style="color: windowtext;">
</span></div>
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<br /></div>
<div class="MsoNormal" style="mso-layout-grid-align: none; mso-pagination: none; text-autospace: none;">
<span style="font-family: "Times New Roman"; mso-bidi-font-family: "Times New Roman";">Vendor: Cayman Chemical</span></div>
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<span style="font-family: "Times New Roman"; mso-bidi-font-family: "Times New Roman";">Product catalog number: 9000645</span></div>
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<span style="font-family: "Times New Roman"; mso-bidi-font-family: "Times New Roman";">Vendor website:
https://www.caymanchem.com/app/template/Product.vm/catalog/9000645</span></div>
<div class="MsoNormal" style="mso-layout-grid-align: none; mso-pagination: none; text-autospace: none;">
<span style="font-family: "Times New Roman"; mso-bidi-font-family: "Times New Roman";">Vendor telephone number: (800) 364-9897</span></div>
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<span style="font-family: "Times New Roman"; mso-bidi-font-family: "Times New Roman";">Cost per unit: $72 per 500ug</span></div>
<div class="MsoNormal">
<span style="font-family: "Times New Roman"; mso-bidi-font-family: "Times New Roman";">Cost per round: $0.02 per round</span></div>
<div class="MsoNormal">
<br /></div>
<div class="MsoNormal">
<span style="font-family: "Times New Roman"; mso-bidi-font-family: "Times New Roman";">References:</span></div>
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<br /></div>
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<span style="font-family: "Times New Roman"; mso-bidi-font-family: "Times New Roman";">1. Seifert-Klauss,
Vanadin, and Jerilynn C. Prior. (2010) "Progesterone and Bone."
National Center for Biotechnology Information. U.S. National Library of
Medicine.<o:p></o:p></span></div>
<div class="MsoNormal">
<br /></div>
<div class="MsoNormal">
<span style="font-family: "Times New Roman"; mso-bidi-font-family: "Times New Roman";">2. A.D.A.M.
Editorial. (2012) "Osteoporosis." Osteoporosis-Guideline. U.S.
National Library of Medicine.<o:p></o:p></span></div>
<div class="MsoNormal">
<br /></div>
<div class="MsoNormal">
<span style="font-family: "Times New Roman"; mso-bidi-font-family: "Times New Roman";">3. Lee, John R.
"Oestrogen Dominance." (2013) Wellsprings Natural Hormone Health.
WellSprings<o:p></o:p></span></div>
<!--EndFragment--></div>
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<span style="font-family: "Times New Roman"; mso-bidi-font-family: "Times New Roman";"><br /></span>
<span style="font-family: "Times New Roman"; mso-bidi-font-family: "Times New Roman";">4.</span>
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<!--StartFragment--><span style="font-family: Cambria; font-size: 12.0pt; mso-ansi-language: EN-US; mso-ascii-theme-font: minor-latin; mso-bidi-font-family: "Times New Roman"; mso-bidi-theme-font: minor-bidi; mso-fareast-font-family: Cambria; mso-fareast-language: EN-US; mso-fareast-theme-font: minor-latin; mso-hansi-theme-font: minor-latin;">Combs, Donald and Reese, Kimberly. Nonsteroidal Progesterone Receptor Ligands.(1995)
Selectivity for Bone Cell Progesterone Receptors, Med Chem, 38, 4880-4884</span><!--EndFragment--><br />
<span style="font-family: "Times New Roman"; mso-bidi-font-family: "Times New Roman";"><br /></span>
<span style="font-family: "Times New Roman"; mso-bidi-font-family: "Times New Roman";">View the full proposal <a href="https://docs.google.com/file/d/0Bys8WWuj29xgekVnVnItYXlJd0E/edit?usp=sharing" target="_blank">here</a>.</span><br />
<span style="font-family: "Times New Roman"; mso-bidi-font-family: "Times New Roman";">View the final progress report <a href="https://drive.google.com/file/d/0Bys8WWuj29xgUm02NUhUaXV4bUU/edit?usp=sharing">here.</a></span></div>
Anonymoushttp://www.blogger.com/profile/07814379283572171434noreply@blogger.com0tag:blogger.com,1999:blog-1195222727010537305.post-11783877143309099082013-09-04T04:07:00.001-05:002013-12-04T05:56:02.138-06:00Aptamer E07 Against EGFR for Use as an Anti-Tumor Therapeutic<div class="MsoNormal">
Zane Alami-Nassif</div>
<div class="MsoNormal">
September 3rd, 2013</div>
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<br /></div>
<div align="center" class="MsoNormal" style="text-align: center;">
<b>Aptamer E07 Against EGFR for Use as an Anti-Tumor Therapeutic<o:p></o:p></b></div>
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<br /></div>
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Epidermal
growth factor receptor (EGFR) leads to many cancers when over expressed due to
a mutation (Zhang et al., 2007). This is because EGFR is a tyrosine kinase receptor
that is responsible for binding to epidermal growth factor and initiating a
signal transduction cascade that will lead to DNA synthesis and cell
proliferation (Oda et al., 2005). If EGFR is constantly activated then uncontrolled
cell division will occur leading to malignant tumors. </div>
<div class="MsoNormal">
<br /></div>
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The
anti-EFGR aptamer, E07, will compete with epidermal growth factor for binding and
will thus inhibit the receptor (Li et al., 2011). In figure 1, the sequence of E07 can
be observed with the predicted motif that will bind to EFGR. Currently E07 is
not as efficient as alternative drugs such as Cetuximab and monoclonal
antibodies until a very high dose is used (Avutu 2010). Increasing the
efficiency of E07 is a goal that could be pursued in order to bring E07 into
viability for treatment against multiple cancers. The theory that avidity
effects could increase the efficiency of E07 was tested with limited results. It
was hypothesized that nucleic acid would behave in a similar manner to peptides, which obtain supradditve effects when monomers dimerize or multimerize. (Avutu
2010)</div>
<div class="MsoNormal">
<br /></div>
<div class="MsoNormal">
<u>Specific Aim 1</u>: The current goal is to better
understand the conditions of selection using E07 with two versions of the FT7
primer and positive controls. Whether or not a concentration of aptamer can be
maintained after undergoing selection is one focus. </div>
<div class="MsoNormal">
<br /></div>
<div class="MsoNormal">
<u>Specific Aim 2</u>: Another focus is to understand the
effects of using different buffers during the binding reaction. Buffers to be
tested are HBSS without calcium or magnesium, HBSS with calcium and magnesium, PBS and PBS with magnesium.</div>
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<br /></div>
<div class="separator" style="clear: both; text-align: center;">
<a href="http://2.bp.blogspot.com/-2RDci7Hib50/Uib4TsAsp_I/AAAAAAAAADE/oMbAAbT3IRE/s1600/E07.jpg" imageanchor="1" style="margin-left: 1em; margin-right: 1em;"><img border="0" height="226" src="http://2.bp.blogspot.com/-2RDci7Hib50/Uib4TsAsp_I/AAAAAAAAADE/oMbAAbT3IRE/s320/E07.jpg" width="320" /></a></div>
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<b><span style="font-size: 10.0pt;">Figure 1. </span></b><span style="font-size: 10.0pt;">Predicted secondary structure of Aptamer E01 and
Aptamer E07 <o:p></o:p></span><span style="font-size: 10pt; line-height: 200%;">(Li et al., 2011)</span><br />
<div align="center" class="MsoNormal" style="line-height: 200%;">
<span style="font-size: 10.0pt; line-height: 200%;"><o:p></o:p></span></div>
</div>
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<br />
<a href="https://docs.google.com/document/d/1ZUDPwJilKL1DPqakTlAr9HpS1eLUweqg-LuzJ_pVK6A/edit?usp=sharing">Link to Proposal</a></div>
<div class="MsoNormal">
<a href="https://docs.google.com/document/d/1tmvKzaW5I5kbaXyCEtJ42wnfI47qhkkw4rCwqB0k-k8/edit" target="_blank">Link to 1st Progress Report</a><br />
<a href="https://docs.google.com/document/d/1leGqcN4UfM6pkAO6vvcj4-EjrWxP3x6HH96LVf1MI2c/edit" target="_blank">Link to 2nd Progress Report</a><br />
<a href="https://docs.google.com/document/d/1GqtobWJtNIzAzcnRdACAvYHHWI7bN7lHI8O1kXnEOR8/edit?usp=sharing" target="_blank">Link to Final Report</a><br />
<br /></div>
<div class="MsoNormal">
<u>Ordering Information:</u><br />
<div class="MsoNormal" style="line-height: 200%;">
Target: Recombinant Human EGFR</div>
<div class="MsoNormal" style="line-height: 200%;">
Vendor: R&D Systems</div>
<div class="MsoNormal" style="line-height: 200%;">
Product Catalog Number: 344-ER</div>
<div class="MsoNormal" style="line-height: 200%;">
Vendor Website: <a href="http://www.rndsystems.com/Products/344-er/">http://www.rndsystems.com/Products/344-er/</a></div>
<div class="MsoNormal" style="line-height: 200%;">
Vendor Telephone: (612) 379-2956</div>
<div class="MsoNormal" style="line-height: 200%;">
Cost: EGFR will be provided by the
Altermune lab.</div>
</div>
<div class="MsoNormal">
<u>References:<o:p></o:p></u></div>
<div class="MsoNormal">
<br /></div>
<div class="MsoNormal">
1. Zhang, H., Berezov A., Wang Q., Zhang G., Drebin J.,
Murali R., Greene M. (2007). ErbB receptors: from oncogenes to
targeted cancer therapies. J Clin Invest. 117(8): 2051-2058. </div>
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<br /></div>
<div class="MsoNormal">
2. Oda K., Matsuoka Y., Funahashi A., Kitano H. (2005). A comprehensive pathway map of epidermal growth factor
signaling. Mol Syst Biol. 1: 2005.0010. </div>
<div class="MsoNormal">
<br /></div>
<div class="MsoNormal">
3. Li N., Nguyen HH., Byrom M., Ellington AD. (2011)
Inhibition of Cell Proliferation by an Anti-EGFR Aptamer. PLoS ONE 6(6):
e20299.</div>
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<br /></div>
<div class="MsoNormal">
4. Avutu, V., (2010) Avidity effects of MinE07, an anti-EFGR
aptamer, on binding to A431 cells. </div>
Zanehttp://www.blogger.com/profile/10447126158697465654noreply@blogger.com0tag:blogger.com,1999:blog-1195222727010537305.post-72835432710446921632013-09-03T21:48:00.000-05:002013-10-28T17:58:13.191-05:00Using Aptamers against HA33 to Supress the Reaction of Clostridium botulinum against Acetylcholine<div class="WordSection1">
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<div style="text-align: center;">
<b><u><span style="font-family: "Times New Roman","serif";">Using Aptamers against HA33 to Supress the Reaction of Clostridium botulinum against Acetylcholine</span></u></b></div>
<div style="text-align: center;">
<span style="font-family: Times New Roman, serif;"><b><br /></b></span></div>
<div style="text-align: center;">
<span style="font-family: Times New Roman, serif;"><b>Shashwath Nayak</b></span></div>
<b><u><span style="font-family: "Times New Roman","serif";"><br /></span></u></b>
<b><u><span style="font-family: "Times New Roman","serif";">Abstract</span></u></b><span style="font-family: "Times New Roman","serif";">:<o:p></o:p></span></div>
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<span style="font-family: "Times New Roman","serif";"><br /></span>
<span style="font-family: "Times New Roman","serif";">Clostridium
botulinum is a gram-positive bacterium which causes botulism, a very fatal
disease that prevents acetylcholine from binding to receptors therefore not
removing the muscle’s ability to contract, in people (Shukla 2005). The cells
of this bacterium secrete botulinum neurotoxin, (BoNT), which is considered the
deadliest toxin up to date and has been deliberated by biodefense researchers
as a possible agent for bioterrorism. BoNT, shown in Figure 1, works by binding
to the receptor in which acetylcholine is supposed to bind, blocking the
binding reaction from occurring. One very important protein in the process of
secreting BoNT is HA33. <o:p></o:p></span></div>
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<span style="font-family: "Times New Roman","serif";"><br /></span>
<span style="font-family: "Times New Roman","serif";">BoNT
forms a toxin complex (TC) that includes HA33 (Ito et all 2011). HA33 is a
protein from Clostridium botulinum that was shown to possibly protect and
activate BoNT. HA33 is a neurotoxin- associated protein (NAP) that protects
BoNT form acid denaturation in the stomach and gastric tract (Arndt et all
2005). These TCs are actually more toxic than pure BoNT. Without NAPs, like
HA33, BoNT are actually susceptible to the acid conditions in the gastric
tract. This allows the absorbance of BoNT throughout the body to increase due
to the more epithelium tissue being available for the BoNT (Ito et all 2011).
This means that HA33 is a problem because it allows BoNT to be even more of a
threat than it already is just by allowing it to further penetrate the body. A
way that has been discovered to possibly stop this protein from helping BoNT is
by the use of aptamers.<o:p></o:p></span></div>
<div class="MsoNormal" style="text-indent: .5in;">
<span style="font-family: "Times New Roman","serif";">Aptamers
are oglionucleiotide that can specifically bind to a specific target (Nobuko,
Ellington & Stanton, 2001). Aptamers use an in vitro selection process, a
process that can be done outside of an organism.<o:p></o:p></span></div>
<div class="MsoNormal" style="text-indent: .5in;">
<span style="font-family: "Times New Roman","serif";"><br /></span>
<span style="font-family: "Times New Roman","serif";">Specific
Aim 1: Use aptamers that can bind to HA33 and change its shape in order to
prevent the neurotoxin from causing problems in the future. <o:p></o:p></span></div>
<div class="MsoNormal" style="text-indent: .5in;">
<br />
<span style="font-family: "Times New Roman","serif";">The aptamer would look for the specific
sequence in HA33 by going through a binding selection. The binding selection
would take place in a series of rounds in order to narrow a pool of RNA to a
specific sequence that will bind to the HA33 protein.<o:p></o:p></span></div>
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<div class="MsoCaption">
<div class="separator" style="clear: both; text-align: center;">
<a href="http://2.bp.blogspot.com/-pmFha0RnOcc/UiadiEMCB5I/AAAAAAAAAEE/XeTMOkguvqg/s1600/Figure.png" imageanchor="1" style="margin-left: 1em; margin-right: 1em;"><img border="0" height="260" src="http://2.bp.blogspot.com/-pmFha0RnOcc/UiadiEMCB5I/AAAAAAAAAEE/XeTMOkguvqg/s320/Figure.png" width="320" /></a></div>
<b><span style="color: windowtext;"><span style="color: #2e75b6;"><span style="font-size: x-small;"><br /></span></span></span></b><b><span style="color: windowtext;"><span style="color: #2e75b6;"><span style="font-size: x-small;">Figure 1: A crystal structure of BoNT in Clostridium botuinum.
The arrow in the picture is pointing towards the HA33 present in the toxin
com</span></span><span style="color: #3d85c6; font-size: x-small;">plex <o:p></o:p></span></span></b><span style="font-family: 'Times New Roman', serif;"><b><span style="color: #3d85c6; font-size: x-small;">(Arndt et all 2005)</span></b></span></div>
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<span style="font-family: Times New Roman, serif;"><b>Target Order Information:</b></span><br />
<span style="font-family: Times New Roman, serif;"><b>Vendor: Ellington Lab</b></span><br />
<span style="font-family: Times New Roman, serif;"><b>Vendor Website: https://www.ellingtonlab.org</b></span><br />
<span style="font-family: Times New Roman, serif;"><b>Central Lab Telephone: 512-471-6445</b></span><br />
<span style="font-family: Times New Roman, serif;"><b>Office Manager: 512-232-3426</b></span><br />
<span style="font-family: Times New Roman, serif;"><b>Cost per Unit: $0</b></span><br />
<span style="font-family: Times New Roman, serif;"><b>Cost per Round: $0</b></span></div>
<div class="MsoNormal" style="text-indent: .5in;">
<br /></div>
<div class="MsoNormal" style="margin: 0in 0in 0.0001pt 5pt;">
<span style="font-family: Times New Roman, serif;"><b>References:</b></span><br />
<span style="font-family: Times New Roman, serif;"><b><br /></b></span>
<span style="font-family: Times New Roman, serif;"><b>1. Shukla, H. (2005). Clostridium botulinum: a bug with beauty and weapon. Retrieved from http://www.ncbi.nlm.nih.gov/pubmed/15839401</b></span><br />
<span style="font-family: Times New Roman, serif;"><b><br /></b></span>
<span style="font-family: Times New Roman, serif;"><b><br /></b></span>
<span style="font-family: Times New Roman, serif;"><b>2. Arndt, J. W., Gu, J., Jaroszewski, L., Schwarzenbacher, R., Hanson, M. A., Lebeda, F. J., & Stevens, R. C. (2005, 01 01). The structure of the neurotoxin-associated protein ha33/a from clostridium botulinum suggests a reoccurring β-trefoil fold in the progenitor toxin complex. Retrieved from http://www.sciencedirect.com/science/article/pii/S0022283604016092</b></span><br />
<span style="font-family: Times New Roman, serif;"><b><br /></b></span>
<span style="font-family: Times New Roman, serif;"><b><br /></b></span>
<span style="font-family: Times New Roman, serif;"><b>3. Ito, H., Sagane, Y., Miyata, K., Inui, K., Matsuo, T., Horiuchi, R., Ikeda, T., & Suzuki, T. (2011, 02</b></span><br />
<span style="font-family: Times New Roman, serif;"><b>01). Ha-33 facilitates transport of the serotype d botulinum toxin across a rat intestinal epithelial cell monolayer. Retrieved from http://onlinelibrary.wiley.com/doi/10.1111/j.1574-695X.2011.00779.x/full</b></span><br />
<span style="font-family: Times New Roman, serif;"><b><br /></b></span>
<span style="font-family: Times New Roman, serif;"><b>4. Nobuko, H., Ellington, A., & Stanton, M. (2001, June 15).Aptamer beacons for the direct detection of proteins. Retrieved from http://www.sciencedirect.com/science/article/pii/S0003269701951693</b></span><br />
<span style="font-family: Times New Roman, serif;"><b><br /></b></span>
<span style="font-family: Times New Roman, serif;"><b>5. Yoshimasa , S., Ken, I., Shin-Ichiro, M., Keita, M., Tomonori, S., Koichi, N., & Toshihiro, W. (2012, August 22). Botulinum toxin complex: A delivery vehicle of botulinum neurotoxin traveling digestive tract. Retrieved from http://www.intechopen.com/books/structure-and-function-of-food- engineering/botulinum-toxin-complex-a-delivery-vehicle-of-botulinum-neurotoxin-traveling-digestive-</b></span><br />
<span style="font-family: Times New Roman, serif;"><b>tract</b></span><br />
<span style="font-family: Times New Roman, serif;"><b><br /></b></span>
<span style="font-family: Times New Roman, serif;"><b><br /></b></span>
<span style="font-family: Times New Roman, serif;"><b>6. Sugiyama, H. (1980). Clostridium botulinum neurotoxin.Microbiological Reviews, 44(3), 419-448. Retrieved from http://www.ncbi.nlm.nih.gov/pmc/articles/PMC373187/</b></span><br />
<span style="font-family: Times New Roman, serif;"><b><br /></b></span>
<span style="font-family: Times New Roman, serif;"><b><br /></b></span>
<span style="font-family: Times New Roman, serif;"><b><br /></b></span>
<span style="font-family: Times New Roman, serif;"><b><a href="https://docs.google.com/document/d/12C46JHV3v1byvZAH-INnjHFcU6YAJqXwuHh8MSYxsI0/edit?usp=sharing">First Proposal</a></b></span><br />
<span style="font-family: Times New Roman, serif;"><a href="https://drive.google.com/file/d/0Bz7_eX4Ye8cFUTJPSDc0Wnh5bVU/edit?usp=sharing">First Progress Report</a></span></div>
</div>
shash.nayak93http://www.blogger.com/profile/08218183537775725773noreply@blogger.com0tag:blogger.com,1999:blog-1195222727010537305.post-82625383598950363982013-09-03T20:01:00.003-05:002013-12-04T00:33:07.710-06:00Identification of Aptamer for Human Telomeric Repeat Binding Factor (TRF1) for the Control of Telomerase Activity<div style="text-align: center;">
</div>
<span style="background-color: black;"><span style="color: white; font-family: Times, "Times New Roman", serif;"></span><br /></span>
<br />
<div class="MsoCaption" style="margin: 0in 0in 10pt; text-align: center;">
<span style="background-color: black;"><span style="color: white; font-family: Times, "Times New Roman", serif;"></span><br /></span>
<span style="background-color: black;"><span style="color: white; font-family: Times, "Times New Roman", serif;"></span><br /></span>
<br />
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<span style="font-family: Times, "Times New Roman", serif;"><span style="background-color: black; color: white;">Identification of Aptamer for Human Telomeric Repeat Binding
Factor (TRF1) for the Control of Telomerase Activity<o:p></o:p></span></span></div>
<span style="background-color: black; color: white; font-family: Times, "Times New Roman", serif;">
</span><br />
<span style="background-color: black;"><span style="color: white; font-family: Times, "Times New Roman", serif;"></span><br /></span></div>
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<span style="background-color: black; color: white; font-family: Times, "Times New Roman", serif;">
</span></div>
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<span style="background-color: black; color: white; font-family: Times, "Times New Roman", serif;">Helen Jung</span></div>
<span style="background-color: black;"><span style="color: white; font-family: Times, "Times New Roman", serif;">
</span>
</span><br />
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<span style="background-color: black; color: white; font-family: Times, "Times New Roman", serif;">3 September 2013</span></div>
<span style="font-family: Times, "Times New Roman", serif;"><span style="color: windowtext; font-family: "Times New Roman","serif"; font-size: 11pt;"><span style="font-family: Times, "Times New Roman", serif;"><span style="background-color: black; color: white; font-size: small;">
</span></span></span></span><br />
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<span style="background-color: black;"><span style="font-family: Times, "Times New Roman", serif;"><span style="color: windowtext; font-family: "Times New Roman","serif"; font-size: 11pt;"><span style="color: white; font-family: Times, "Times New Roman", serif; font-size: small;"></span><br /></span></span>
<span style="font-family: Times, "Times New Roman", serif;"><span style="color: windowtext; font-family: "Times New Roman","serif"; font-size: 11pt;"><span style="color: white; font-family: Times, "Times New Roman", serif; font-size: small;">
</span></span></span></span><br />
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<span style="background-color: black;"><span style="font-family: Times, "Times New Roman", serif;"><span style="color: windowtext; font-family: "Times New Roman","serif"; font-size: 11pt;"><span style="color: white; font-family: Times, "Times New Roman", serif; font-size: small;"></span><br /></span></span>
<span style="font-family: Times, "Times New Roman", serif;"><span style="color: windowtext; font-family: "Times New Roman","serif"; font-size: 11pt;"><span style="line-height: 200%;"><span style="color: white; font-family: Times, "Times New Roman", serif; font-size: small;"></span><br />
<span style="color: white; font-family: Times, "Times New Roman", serif; font-size: small;">
</span><br />
</span></span></span></span><br />
<div class="MsoNormal" style="line-height: 200%; margin: 0in 0in 10pt; text-indent: 0.5in;">
<span style="font-family: Times, "Times New Roman", serif;"><span style="color: windowtext; font-family: "Times New Roman","serif"; font-size: 11pt;"><span style="line-height: 200%;"><span style="font-family: "Times New Roman","serif"; line-height: 200%;"><span style="font-family: Times, "Times New Roman", serif;"><span style="font-size: small;"><span style="background-color: black; color: white;">With
the rise of nuclear and linear DNA, eukaryotes had to overcome the imperfect
DNA replicating mechanism that arises from DNA polymerases’ inability to start
their own sequence. The advance of telomeres, non-coding DNA sequences at the
end of linear DNA, allows the preservation of genetic information in coding
sequences of DNA. These telomeres shorten in length as the specific DNA
molecule repeats replication, but excessive shortening of telomere can have
devastating consequences, such as HHS to be further described below. In
response, yeast and mammals maintain their telomere within a certain size range
through telomerase activity (de Lange, 2005). The access to each telomeric end
is strictly controlled by a telomere nucleoprotein complex, and they guard
telomeres against indiscriminate elongation by telomerase or inappropriate
modification by DNA damage repair system. Especially, telomeric repeat binding
factor (TRF1), a protein within the telomere nucleoprotein complex, negatively
regulates telomere length; its over-expression causes telomere shortening while
its under-expression results telomere elongation. (Kishi, <i style="mso-bidi-font-style: normal;">etal</i>, 2001).<o:p></o:p></span></span></span></span></span></span></span></div>
<span style="font-family: Times, "Times New Roman", serif;"><span style="font-family: "Times New Roman","serif"; font-size: 11pt;"><span style="background-color: black; line-height: 200%;">
<span style="color: white; font-family: Times, "Times New Roman", serif; font-size: small;">
</span><br />
</span></span></span><br />
<div class="MsoNormal" style="color: windowtext; line-height: 200%; margin: 0in 0in 10pt;">
<span style="font-family: Times, "Times New Roman", serif;"><span style="font-family: "Times New Roman","serif"; font-size: 11pt;"><span style="line-height: 200%;"><span style="font-family: "Times New Roman","serif"; line-height: 200%;"><span style="font-family: Times, "Times New Roman", serif;"><span style="background-color: black; font-size: small;"><span style="color: white;">oyeraal-Hreidarsson
Syndrome (HHS) is an X-linked genetic disorder that shows abnormalities in
telomere length and its maintenance, including defects in RTEL1 (Le Guen, <i style="mso-bidi-font-style: normal;">etal</i>, 2013). This condition impedes
development and shows symptoms of bone marrow failure and immunodeficiency.
Currently, there is no effective treatment for this condition. The objective of
this study is to select an aptamer—an oligonucleotide of three-dimensional
orientation that is selected to bind to and inhibit a specific target
protein—using repetitive talon bead-based selection method. In a previous
study, the overexpression of the protein TRF1 has shown strong telomere
shortening and damage, whereas under-expression of the protein has shown
telomere elongation (Kishi, <i style="mso-bidi-font-style: normal;">etal,</i>
2001). Thus, development of an aptamer against TRF1 can provide an outlook for
forced TRF1 inactivation. TRF1 inhibition results promoted telomeric repair
activities and </span><span style="color: white;">offers potential aptamer therapeutics to alleviate the symptoms
of HHS.<o:p></o:p></span></span></span></span></span></span></span></div>
<span style="font-family: Times, "Times New Roman", serif;"><span style="font-family: "Times New Roman","serif"; font-size: 11pt;"><span style="background-color: black; line-height: 200%;">
<span style="color: white; font-family: Times, "Times New Roman", serif; font-size: small;">
</span><br />
</span></span></span><br />
<div class="MsoNormal" style="color: windowtext; line-height: 200%; margin: 0in 0in 10pt; text-indent: 0.5in;">
<span style="font-family: Times, "Times New Roman", serif;"><span style="font-family: "Times New Roman","serif"; font-size: 11pt;"><span style="line-height: 200%;"><span style="font-family: "Times New Roman","serif"; line-height: 200%;"><span style="font-family: Times, "Times New Roman", serif;"><span style="font-size: small;"><span style="background-color: black; color: white;">This
study specifically aims to identify aptamer against TRF1 protein target and to
develop realistic potential therapeutic purposes of this aptamer against HHS.
Currently, there is no effective way of identification or remedy for the
symptoms. Potential medical applications for HHS will further be investigated.<o:p></o:p></span></span></span></span></span></span></span></div>
<span style="font-family: Times, "Times New Roman", serif;"><span style="background-color: black; font-family: "Times New Roman","serif"; font-size: 11pt;"><span style="line-height: 200%;">
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</span><span style="color: white;"><br />
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<span style="font-family: Times, "Times New Roman", serif;"><span style="background-color: black; font-family: "Times New Roman","serif"; font-size: 11pt;"><span style="line-height: 200%;"><span style="font-family: "Times New Roman","serif"; line-height: 200%;"><span style="font-family: Times, "Times New Roman", serif;"><span style="font-size: small;"><span style="color: white;">His-tagged
TRF1 protein (Catalog number: 13044-H07B) can
be purchased from Sino Biological Inc. (sinobiological.com; Phone: 86-400-890-9989). Fifty micrograms, or about 990.01
picomoles, of TRF1 protein cost $290. The molecular weight of the target is
50.5Da. Each round of selection will be performed with about 100 picomoles of
TRF1 protein and is expected to cost about $30. The selection is performed
using talon beads, which are known to and are used in different laboratory (Stovall,
2013) to select against polyhistidine-tagged targets. These beads are already
available in the lab with other necessary equipment without any additional
cost. <o:p></o:p></span></span></span></span></span></span></span></div>
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<span style="font-family: Times, "Times New Roman", serif;"><span style="background-color: black; color: white; font-family: Times, "Times New Roman", serif; font-size: small;">References:</span></span></div>
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<span style="font-family: Times, "Times New Roman", serif;"><a href="https://drive.google.com/file/d/0BwhvTPTt0GX-QnJsa0tJM2xkY2c/edit?usp=sharing"><span style="background-color: black; color: white; font-family: Times, "Times New Roman", serif; font-size: small;">https://drive.google.com/file/d/0BwhvTPTt0GX-QnJsa0tJM2xkY2c/edit?usp=sharing</span></a></span></div>
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<span style="background-color: black;"><span style="font-family: Times, "Times New Roman", serif;"><span style="color: white; font-family: Times, "Times New Roman", serif; font-size: small;">Target Proposal: </span></span></span></div>
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<span style="color: white; font-family: Times, "Times New Roman", serif; font-size: small;"> </span><a href="https://docs.google.com/file/d/0BwhvTPTt0GX-ZDVLYnQ4SkRKUG8/edit?usp=sharing"><span style="color: white; font-family: Times, "Times New Roman", serif; font-size: small;">https://docs.google.com/file/d/0BwhvTPTt0GX-ZDVLYnQ4SkRKUG8/edit?usp=sharing</span></a></div>
<div style="color: windowtext; font-family: "Times New Roman", serif; font-size: 11pt;">
<span style="color: white; font-family: Times, "Times New Roman", serif; font-size: small;"></span><br /></div>
<div style="color: windowtext; font-family: "Times New Roman", serif; font-size: 11pt;">
<span style="color: white; font-family: Times, "Times New Roman", serif; font-size: small;">Progress Report:</span></div>
<span style="color: white;"><u>https://drive.google.com/file/d/0BwhvTPTt0GX-ZDVLYnQ4SkRKUG8/edit?usp=sharing</u></span><br />
<div style="color: windowtext; font-family: "Times New Roman", serif; font-size: 11pt;">
<span style="color: white; font-family: Times, "Times New Roman", serif; font-size: small;"></span><br /></div>
<div style="color: windowtext; font-family: "Times New Roman", serif; font-size: 11pt;">
<span style="color: white; font-family: Times, "Times New Roman", serif; font-size: small;">Second Progress Report</span></div>
<div style="color: windowtext; font-family: "Times New Roman", serif; font-size: 11pt;">
<a href="https://drive.google.com/file/d/0BwhvTPTt0GX-QVNVVXk3SGNMUzQ/edit?usp=sharing"><span style="color: white; font-family: Times, "Times New Roman", serif; font-size: small;">https://drive.google.com/file/d/0BwhvTPTt0GX-QVNVVXk3SGNMUzQ/edit?usp=sharing</span></a><br />
<span style="color: white;">Final Manuscript</span><br />
<a href="https://drive.google.com/file/d/0BwhvTPTt0GX-d2IzdjJLS3NBaEE/edit?usp=sharing"><span style="color: white;">https://drive.google.com/file/d/0BwhvTPTt0GX-d2IzdjJLS3NBaEE/edit?usp=sharing</span></a></div>
</div>
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<a href="http://3.bp.blogspot.com/-3-hhc_xW-xs/UiaHauymz1I/AAAAAAAAACQ/Kj0kTjfgXgg/s1600/Capture.JPG" imageanchor="1" style="margin-left: 1em; margin-right: 1em;"><img border="0" src="http://3.bp.blogspot.com/-3-hhc_xW-xs/UiaHauymz1I/AAAAAAAAACQ/Kj0kTjfgXgg/s1600/Capture.JPG" /></a></div>
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</span>Figure 1. The role of TRF1 in telomerase activity regulation and facilitation during S phase of the cell cycle.</span><br />
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Anonymoushttp://www.blogger.com/profile/11778132916806559306noreply@blogger.com0tag:blogger.com,1999:blog-1195222727010537305.post-77117841840882461382013-09-03T17:31:00.002-05:002013-12-04T01:38:46.834-06:00<div class="MsoNormalCxSpFirst">
<span style="font-family: Times New Roman, serif;"><span style="line-height: 18px;">Arianne Alcantara</span></span></div>
<div class="MsoNormalCxSpFirst">
<span style="font-family: Times New Roman, serif;"><span style="line-height: 18px;">September 3, 2013</span></span></div>
<div class="MsoNormalCxSpFirst">
<b><span style="font-family: "Times New Roman","serif"; font-size: 12.0pt; line-height: 115%;"><br /></span></b></div>
<div class="MsoNormalCxSpFirst">
<b><span style="font-family: "Times New Roman","serif"; font-size: 12.0pt; line-height: 115%;">Inhibition
of FGF8b Protein Oncogenic Characteristics through Nucleic Acid Aptamer
Selection<o:p></o:p></span></b></div>
<div class="MsoNormalCxSpMiddle" style="text-indent: .5in;">
<br /></div>
<div class="MsoNormalCxSpMiddle" style="text-indent: .5in;">
<span style="font-family: "Times New Roman","serif"; font-size: 12.0pt; line-height: 115%;">Prevalent
throughout the world and always seemingly one step ahead of society’s most
brilliant physicians, cancer has continued to ravage the human population for
centuries. As a result, oncology methods to combat the disease seem to shift
every year, with experts believing surgery to be the solution in the eighteenth
century, to radiation, chemotherapy, and targeted therapy as alternatives in
the twenty-first ("Evolution of Cancer Treatments," 2012). The protein present in humans, Fibroblast Growth
Factor 8 (FGF8), can be attributed directly to the third of the aforementioned
techniques, targeted therapy, due to its significant role in cell processes
such as mitogenesis and angiogenesis. Its isoform FGF8b is medically known as
an androgen-induced on dominant in tumorigenesis and hormonal carcinogenesis,
with one of the more popular diseases being prostate cancer (Henderson and Feigelson, 1999).<o:p></o:p></span></div>
<div class="MsoNormalCxSpMiddle" style="text-indent: .5in;">
<span style="font-family: "Times New Roman","serif"; font-size: 12.0pt; line-height: 115%;">Although FGF8b
expression is linked to hormone-related cancers, speculation still remains as
to whether it <i>is</i> the cause;
furthermore, like all proteins, FGF8 performs various other functions such as
those pertaining to embryo development, tissue repair, and cell survival (White and MacArthur, 1995).
Consequently, recognizing its role in human development forces the necessary
realization that completely eradicating the protein will produce more harmful
side effects than good ones. Employing more selective techniques designed to
inhibit FGF8 after a certain point instead of abolishing it are sought in this
study. <o:p></o:p></span></div>
<div class="MsoNormalCxSpMiddle" style="text-indent: .5in;">
<span style="font-family: "Times New Roman","serif"; font-size: 12.0pt; line-height: 115%;"><br /></span></div>
<div class="separator" style="clear: both; text-align: center;">
<a href="http://1.bp.blogspot.com/-W3C_H0M6ZzQ/UiZh6fl8LEI/AAAAAAAAAPY/qNQPnmjJrpI/s1600/FGF8+Abstract+Fig+1.jpg" imageanchor="1" style="margin-left: 1em; margin-right: 1em;"><img border="0" height="285" src="http://1.bp.blogspot.com/-W3C_H0M6ZzQ/UiZh6fl8LEI/AAAAAAAAAPY/qNQPnmjJrpI/s320/FGF8+Abstract+Fig+1.jpg" width="320" /></a></div>
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<br /></div>
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<span style="font-family: "Times New Roman","serif"; font-size: 12.0pt; line-height: 115%;">Figure 1 illustrates the affect of an
RTK inhibitor if a growth factor like FGF8, binds to a cell. The discovery of
an FGF8b aptamer would lead to the prevention of a possibly cancerous cell to
divide, and as such remain benign ("Cancer Growth Blockers," 2013).<o:p></o:p></span></div>
<div class="MsoNormalCxSpMiddle">
<br /></div>
<div class="MsoNormalCxSpMiddle">
<u><span style="font-family: "Times New Roman","serif"; font-size: 12.0pt; line-height: 115%;">Specific Aim 1:</span></u><span style="font-family: "Times New Roman","serif"; font-size: 12.0pt; line-height: 115%;">
Employing therapeutic strategies, to determine an aptamer, substance with the
capability to bind to a target molecule, through bead-based selection for FGF8 that
once bound together, possesses the ability to trigger inhibition or at least
antagonization and alter the protein’s affects on an organism. <o:p></o:p></span></div>
<div class="MsoNormalCxSpMiddle">
<u><span style="font-family: "Times New Roman","serif"; font-size: 12.0pt; line-height: 115%;"><br /></span></u></div>
<div class="MsoNormalCxSpMiddle">
<u><span style="font-family: "Times New Roman","serif"; font-size: 12.0pt; line-height: 115%;">Specific Aim 2:</span></u><span style="font-family: "Times New Roman","serif"; font-size: 12.0pt; line-height: 115%;">
For cases like prostate cancer, once an aptamer is determined, it can be
utilized in blocking the interaction between tumor cells and stromal cells by
binding to and preventing the activation of cell surface receptor tyrosine kinases
(RTKs) as shown in Figure 1 (Henderson and Feigelson, 1999). This would in turn alter the expected
biological response that would typically occur, hopefully leading to decreased
oncogenic symptoms, and thus later applied to other types of cancer besides
prostate. <o:p></o:p></span></div>
<div class="MsoNormalCxSpMiddle" style="text-indent: .5in;">
<span style="font-family: "Times New Roman","serif"; font-size: 12.0pt; line-height: 115%;">The FGF8b
protein is made available by Shawn Piasecki through the Keatinge-Clay lab, with
other stock sold through mybiosource.com for 0.005mg at $140.00. <o:p></o:p></span></div>
<div class="MsoNormalCxSpMiddle">
<br /></div>
<div class="MsoNormalCxSpLast">
<b><span style="font-family: "Times New Roman","serif"; font-size: 12.0pt; line-height: 115%;">References<o:p></o:p></span></b></div>
<div class="MsoNormalCxSpLast">
</div>
<ol>
<li><span style="font-family: Times New Roman, serif;"><span style="line-height: 18px;"><b> </b>"Evolution of Cancer Treatments: Targeted Therapy." Evolution of Cancer Treatments: Targeted Therapy. American Cancer Society, 8 June 2012. Web. 02 Sept. 2013. <http://www.cancer.org/cancer/cancerbasics/thehistoryofcancer/the-history-of-cancer-cancer-treatment-targeted-therapy>.</span></span></li>
<li><span style="font-family: 'Times New Roman', serif; line-height: 18px;">Henderson, Brian E., and Heather S. Feigelson. "Hormonal Carcinogenesis." Oxford Journals 21.3 (1999): 427-33. Oxford Journals. Oxford University Press, 1999. Web. 2 Sept. 2013. <3. http://carcin.oxfordjournals.org/content/21/3/427.full>.</span></li>
<li><span style="font-family: Times New Roman, serif;"><span style="line-height: 18px;">White, Robert, and Craig MacArthur. "Assignment OfFGF8to Human Chromosome 10q25–q26: Mutations InFGF8May Be Responsible for Some Types of Acrocephalosyndactyly Linked to This Region." Genomics 30.1 (1995): 109-11. Print.</span></span></li>
<li><span style="font-family: Times New Roman, serif;"><span style="line-height: 18px;">"Cancer Growth Blockers." : Cancer Research UK : CancerHelp UK. Cancer Research UK, 15 Apr. 2013. Web. 02 Sept. 2013. <http://www.cancerresearchuk.org/cancer-help/about-cancer/treatment/biological/types/cancer-growth-blockers>.</span></span></li>
</ol>
<a href="https://docs.google.com/file/d/0B9pbGUM07jvNTXFRUUlNSWdBbTg/edit?usp=sharing" target="_blank">Click here for full proposal</a><br />
<a href="https://drive.google.com/file/d/0B6Vrfx3pY1UxLU84REhzVXRxMTQ/edit?usp=sharing" target="_blank">Click here for Progress Report 1</a><br />
<a href="https://drive.google.com/file/d/0B6Vrfx3pY1UxRllRRTBQVzZRWm8/edit?usp=sharing" target="_blank">Click here for Progress Report 2</a><br />
<a href="https://drive.google.com/file/d/0B6Vrfx3pY1UxS200Z3VTLTJEb1U/edit?usp=sharing" target="_blank">Click here for Final Progress Report</a><br />
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Ariannehttp://www.blogger.com/profile/08432625874379259785noreply@blogger.com0tag:blogger.com,1999:blog-1195222727010537305.post-2586744814727850902013-09-03T15:40:00.001-05:002013-12-04T02:50:41.513-06:00<div align="center" class="MsoNormal" style="text-align: center;">
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<b style="mso-bidi-font-weight: normal;"><span lang="EN-US" style="font-family: "Times New Roman"; font-size: 22.0pt; mso-bidi-font-size: 12.0pt;">Selection of an Aptamer Against FGF8b for Breast
Cancer and prostate Cancer Treatment<o:p></o:p></span></b></div>
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<b style="mso-bidi-font-weight: normal;"><span lang="EN-US">Shuhui Wan</span></b></div>
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<!--EndFragment--><span lang="EN-US" style="font-family: "Times New Roman";"><span style="font-size: 12pt; line-height: 150%; text-indent: 24pt;">FGF8, the fibroblast growth factor (1), a protein that is inducible by
androgen in breast cancer cells, is involved in many hormone related cancers,
such as breast cancer and prostate cancer (2,3).</span> More than 67% of breast
cancer patients and over 93% of prostate cancer patients showed expression of FGF8
(3). And the breast cancer tissues were show to express much higher level of
FGF8 mRNA than the non-malignant tissues (4). One specific isoform, FGF8b, was
proved to induce progression of prostate cancer (5). <o:p></o:p></span></div>
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<span lang="EN-US" style="font-family: "Times New Roman";">Because
of FGF8b’s significant involvement with tumor growth, it was thought that by
inhibiting the activities of this growth protein the tumor growth could also be
greatly suppressed. One promising way of doing this would be finding an
aptamer, a RNA ligand that binds to specific targets, for FGF8b. The apatamer
for FGF8b may bind to it and inhibiting the protein from functioning in promoting
tumor growth. In other words, the aptamer could be used for therapeutic
purposes to treat breast cancer and prostate cancer in future. <o:p></o:p></span></div>
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<span lang="EN-US" style="font-family: "Times New Roman";">Specific
aim #1: find an aptamer that binds specifically to FGF8b. <o:p></o:p></span></div>
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<span lang="EN-US" style="font-family: "Times New Roman";">Specific
aim #2: modify the aptamer to be used for therapeutic purposes. <o:p></o:p></span></div>
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<span lang="EN-US">FGF-8b is prepared by Shawn Piasecki and available in lab.<o:p></o:p></span><br />
<span lang="EN-US"><br /></span>
<span lang="EN-US"><br /></span>
<span lang="EN-US">...so the references are not showing up, here's the PDF version file:</span><br />
<span lang="EN-US">https://docs.google.com/file/d/0B38waIP26ToBdDRBX1lLMXItQU0/edit?usp=sharing</span><br />
<span lang="EN-US"><br /></span>
<span lang="EN-US">progress report 1</span><br />
<span lang="EN-US">https://docs.google.com/file/d/0B38waIP26ToBZUpkX3N1a1pvdzg/edit?usp=sharing</span><br />
<span lang="EN-US"><br /></span>
<span lang="EN-US">progress report 2</span><br />
<span lang="EN-US">https://drive.google.com/file/d/0B38waIP26ToBQUZWSjFCRU83NUE/edit?usp=sharing</span><br />
<span lang="EN-US"><br /></span>
<span lang="EN-US"><a href="https://drive.google.com/file/d/0B38waIP26ToBajNOSlltX0RiZzQ/edit?usp=sharing">Final report</a></span></div>
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<span lang="EN-US"><span style="font-family: Cambria;">(1)</span> </span><span lang="EN-US" style="font-family: "Times New Roman"; mso-bidi-font-family: "Times New Roman"; mso-bidi-theme-font: minor-bidi;">Goetz
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<span lang="EN-US"><span style="font-family: Cambria;">(7)</span> </span><span lang="EN-US" style="font-family: "Times New Roman"; mso-bidi-font-family: "Times New Roman"; mso-bidi-theme-font: minor-bidi;">Gwen
Stovall, PowerPoint - “Bead Based RNA Selection, Lecture 09”, slide 21, 2013<o:p></o:p></span></div>
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Anonymoushttp://www.blogger.com/profile/17269343692061979533noreply@blogger.com0tag:blogger.com,1999:blog-1195222727010537305.post-79886744610581869622013-09-03T15:39:00.000-05:002013-09-06T11:52:58.138-05:00Manjari Subramanian: Nucleic Acid Aptamer Selection for Myelin Basic Protein for the Prevention of Malignant Schwann Cells <div>
Nucleic Acid Aptamer Selection for Myelin Basic Protein for the Prevention of Malignant Schwann Cells</div>
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Manjari Subramanian</div>
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<a href="http://www.blogger.com/blogger.g?blogID=1195222727010537305" imageanchor="1" style="clear: right; float: right; margin-bottom: 1em; margin-left: 1em;"></a><a href="http://www.blogger.com/blogger.g?blogID=1195222727010537305" imageanchor="1" style="clear: right; float: right; margin-bottom: 1em; margin-left: 1em;"></a><a href="http://www.blogger.com/blogger.g?blogID=1195222727010537305" imageanchor="1" style="clear: right; float: right; margin-bottom: 1em; margin-left: 1em;"></a><a href="http://www.blogger.com/blogger.g?blogID=1195222727010537305" imageanchor="1" style="clear: right; float: right; margin-bottom: 1em; margin-left: 1em;"></a><a href="http://www.blogger.com/blogger.g?blogID=1195222727010537305" imageanchor="1" style="clear: right; float: right; margin-bottom: 1em; margin-left: 1em;"></a> <span class="Apple-tab-span" style="white-space: pre;"> </span>Myelin basic protein (MBP) promotes growth of myelin sheath throughout the nervous system. This is done by the protein binding to the lipids on the myelin membrane (Boggs, 2006). However occasionally a malignant myelin sheath cell forms in the process. When a malignant cell is formed, this inhibits the transmission of electric signals across synapses within the body (Kaur et al., 2008). MBP has the ability to distinguish the difference between a normal and malignant Schwann cell due to minute differences in the gene sequence (Kaur et al., 2008).</div>
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Figure 1 Myelin Basic Protein: Computer model of the protein (Meinl et al., 1993).</div>
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Aim 1: Select for the protein </div>
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<span class="Apple-tab-span" style="white-space: pre;"> </span>A multi-layered process will be involved in the selection for MBP. Aptamers, RNA protein markers, will be selected for myelin basic protein. Aptamers bind to proteins with complex specificity and high affinity to the specific protein target. This is because of the size advantage that aptamers have. Most aptamers are around one-tenth the size of an antibody (Boggs, 2006). This provides a set of tools that aid in area of therapeutic diagnostics in medicine. By selecting for MBP, the gene sequence of the protein can be verified before further development of an aptamer to distinguish between a normal and malignant cell.</div>
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Myelin basic protein has the newfound ability to help differentiate between a normal and a malignant Schwann cell. Researchers at the Max-Planck-Institute of Psychiatry in Germany is to find an aptamer for MBP in T cells (1993). The study is still ongoing but in later stages and is now trying to pinpoint the exact location of where the aptamer binds to the target T cell (Meinl et al., 1993). Similarly, the first step is to find an aptamer that binds to this specific protein. Once found, further research as to how this aptamer can be utilized in cell differentiation will be determined.</div>
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Target Information:</div>
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Myelin Basic Protein</div>
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US Biologicals</div>
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(800-520-3011)</div>
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Catalog # M9758-10</div>
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Target Cost: $327.00</div>
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References:</div>
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Boggs JM (2006) Myelin basic protein: a multifunctional protein. Cell Mol Life Sci 63: 1945–1961. </div>
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<div>
Kaur G, Roy I. Therapeutic applications of aptamers. National Institute of Pharmaceutical Education and Research (NIPER). Expert Opin Investing Drugs 17 January 2008. <http://www.ncbi.nlm.nih.gov/pubmed/18095918></div>
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Meinl, Weber, Drexler, Morelle, Ott, Saruhan-Direskeneli, Goebels, Ertl, Jechart, Giegerich. Myelin basic protein-specific T lymphocyte repertoire in multiple sclerosis. Complexity of the response and dominance of nested epitopes due to recruitment of multiple T cell clones. Published in Volume 92, Issue 6</div>
<div>
J Clin Invest. 1993; 92(6):2633–2643 doi:10.1172/JCI116879</div>
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<br /></div>
Anonymoushttp://www.blogger.com/profile/10082346920107353729noreply@blogger.com0tag:blogger.com,1999:blog-1195222727010537305.post-81749016566094410662013-09-03T11:35:00.002-05:002013-12-03T23:23:18.306-06:00Selection of Aptamers Against FGF8b Oncoprotein for Diagnostic Application in Detecting Cancerous Cells by Finding Overexpression of Oncoprote<div class="MsoNormal" style="text-indent: .5in;">
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Taher Jamali<br />
Target: mFGF8b<br />
Pool: N71<br />
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<span style="font-family: "Times New Roman";">Breast
and prostate cancer(Jemal et al. 2009). Nasopharyngeal carcinoma. Structure of
the midbrain and cerebellum. What do these three seemingly unrelated phenomena
of the body have in common? Mammalian fibroblast growth factor 8b (mFGF8b).
This unique protein has been found to be involved in the cell signaling pathway
that can lead to uncontrolled growth in hormonal cancers, such as breast and
prostate(Tanaka et al. 1998; Mattila and Harkonen, 2007), as well as
nasopharyngeal cancer. mFGF8b is also interestingly involved with other
isoforms and receptors to control the structure of midbrain and cerebellum
during development(Olsen et al. 2006).<o:p></o:p></span></div>
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<a href="file://localhost/Users/tj_2152/Library/Caches/TemporaryItems/msoclip/0/clip_image002.png" imageanchor="1" style="clear: right; float: right; margin-bottom: 1em; margin-left: 1em;"></a><a href="file://localhost/Users/tj_2152/Library/Caches/TemporaryItems/msoclip/0/clip_image002.png" imageanchor="1" style="clear: right; float: right; margin-bottom: 1em; margin-left: 1em;"></a><span style="font-family: "Times New Roman";"> An intricate approach to dealing
with many of these possibly fatal cancerous diseases involves detection, and
conceivably, inhibition of the mFGF8b protein using aptamers. Aptamers are
small nucleic acid strands made up of DNA or RNA that can be selected against a
protein of interest for utilization in a specific purpose. In this case, the
aptamer selected against mFGF8b can attach to the protein of interest, and-
with the proper detection signal such as radioactivity or fluorescence- help to
identify overexpression of the oncoprotein. After finding overexpressed proteins
in specific cells in the body, further action can be taken to remove the cells
before the cancer can progress. The aptamer may also be utilized
therapeutically if allowed to attach to mFGF8b in its cellular pathways, and
hopefully, block any unnecessary effects of the protein in cells showing
uncontrolled growth by inhibiting the entire protein. A representation of this
therapeutic inhibition can be seen in <i>Figure
1</i>.<o:p></o:p></span></div>
<div class="separator" style="clear: both; text-align: center;">
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<i><span style="font-family: "Times New Roman"; font-size: 8.0pt;">Figure 1: </span></i><span style="font-family: "Times New Roman"; font-size: 8.0pt;">By preventing mFGF8b from
binding to FGFR with the use of aptamers in cells such as prostate cells, we
can prevent the oncoprotein’s effect in cellular pathways affecting
uncontrolled cancerous growth.<o:p></o:p></span></div>
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<span style="font-family: 'Times New Roman';"><br /></span>
<span style="font-family: 'Times New Roman';">Specific Aim 1:
Selection of RNA aptamers against mFGF8b</span></div>
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<span style="font-family: 'Times New Roman';">The first major goal is to complete rounds of
selection to find a specific aptamer that can bind to the mFGF8b protein.</span></div>
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<br /></div>
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<span style="font-family: "Times New Roman";">Specific Aim 2:
Perform Binding Assay to Test Binding Affinity of mFGF8b aptamers<o:p></o:p></span></div>
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<span style="font-family: 'Times New Roman';">The second major
goal is to test the binding affinity of the selected aptamer to the mFGF8b
protein so that it can be possible to see how effectively the aptamer will bind
and inhibit the protein in its cellular pathways in the future.</span><br />
<u style="text-indent: 0.5in;"><span style="font-family: "Times New Roman";"><br /></span></u>
<u style="text-indent: 0.5in;"><span style="font-family: "Times New Roman";">Target Order
Information:</span></u><br />
<span style="font-family: 'Times New Roman'; text-indent: 0.5in;">Keatinge-Clay
Lab</span><br />
<span style="font-family: 'Times New Roman'; text-indent: 0.5in;">mFGF8b is
currently available in the lab (Purified by Shawn Piasecki)</span><br />
<span style="font-family: 'Times New Roman'; text-indent: 0.5in;">Lab Info is as
follows-</span><br />
<span style="font-family: 'Times New Roman'; text-indent: 0.5in;">Office: WEL 4.230B</span><br />
<span style="font-family: 'Times New Roman'; text-indent: 0.5in;">Office Phone: (512) 471-2977</span><br />
<span style="font-family: 'Times New Roman'; text-indent: 0.5in;">E-mail: adriankc@utexas.edu</span><br />
<span style="font-family: 'Times New Roman'; text-indent: 0.5in;">Lab: WEL 4.234</span><br />
<span style="font-family: 'Times New Roman'; text-indent: 0.5in;">Website: http://keatinge-clay.cm.utexas.edu/research/index.html</span><br />
<span style="font-family: 'Times New Roman'; text-indent: 0.5in;">Target Cost: $0</span><br />
<span style="font-family: 'Times New Roman'; text-indent: 0.5in;">Cost Per Round: $0</span></div>
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<div class="MsoNormal" style="line-height: 150%; text-align: justify; text-justify: inter-ideograph;">
<b><span style="font-family: "Times New Roman";">References:<o:p></o:p></span></b></div>
<div class="MsoListParagraphCxSpFirst" style="line-height: 150%; mso-list: l0 level1 lfo1; text-align: justify; text-indent: -.25in; text-justify: inter-ideograph;">
<!--[if !supportLists]--><span style="font-family: "Times New Roman"; mso-fareast-font-family: "Times New Roman";">1.<span style="font-size: 7pt; line-height: normal;">
</span></span><!--[endif]--><span style="font-family: "Times New Roman";">"His-Tagged
Protein Isolation & Pulldown Using Dynabeads®." <i>His-Tagged Protein
Isolation & Pulldown Using Dynabeads</i>. Life Technologies Corportation,
2013. Web. 21 Apr. 2013.<o:p></o:p></span></div>
<div class="MsoListParagraphCxSpMiddle" style="line-height: 150%; mso-list: l0 level1 lfo1; text-align: justify; text-indent: -.25in; text-justify: inter-ideograph;">
<!--[if !supportLists]--><span style="font-family: "Times New Roman"; mso-fareast-font-family: "Times New Roman";">2.<span style="font-size: 7pt; line-height: normal;">
</span></span><!--[endif]--><span style="font-family: "Times New Roman";">Jemal
A, Siegel R, Ward E, Yongping H, Xu Jiaquan, Thun, Michael. (2009). Cancer
Statistics, 2009. <i>A Cancer Journal for
Clinicians. <b>59, 4. pp 215- 275</b></i><o:p></o:p></span></div>
<div class="MsoListParagraphCxSpMiddle" style="line-height: 150%; mso-list: l0 level1 lfo1; text-align: justify; text-indent: -.25in; text-justify: inter-ideograph;">
<!--[if !supportLists]--><span style="font-family: "Times New Roman"; mso-fareast-font-family: "Times New Roman";">3.<span style="font-size: 7pt; line-height: normal;">
</span></span><!--[endif]--><span style="font-family: "Times New Roman";">Mattila
MM, Harkonen PL. (2007). Role of fibroblast growth factor 8 in growth and
progression of hormonal cancer. <i>Cytokine Growth Factor Rev</i> 18: 257–266.<o:p></o:p></span></div>
<div class="MsoListParagraphCxSpMiddle" style="line-height: 150%; mso-list: l0 level1 lfo1; text-align: justify; text-indent: -.25in; text-justify: inter-ideograph;">
<!--[if !supportLists]--><span style="font-family: "Times New Roman"; mso-fareast-font-family: "Times New Roman";">4.<span style="font-size: 7pt; line-height: normal;">
</span></span><!--[endif]--><span style="font-family: "Times New Roman";">Olsen
S, Li J, Bromleigh C, et al. (2006) Structural basis by which alternative
splicing modulates the organizer activity of FGF8 in the brain. <i> Genes
Dev. </i>20: 185-198.<o:p></o:p></span></div>
<div class="MsoListParagraphCxSpMiddle" style="line-height: 150%; mso-list: l0 level1 lfo1; text-align: justify; text-indent: -.25in; text-justify: inter-ideograph;">
<!--[if !supportLists]--><span style="font-family: "Times New Roman"; mso-fareast-font-family: "Times New Roman";">5.<span style="font-size: 7pt; line-height: normal;">
</span></span><!--[endif]--><span style="font-family: "Times New Roman";">Tanaka
A, Furuya A, Yamasaki M, Hanai N, Kuriki K, Kamiakito T <i>et al</i>. (1998).
High frequency of fibroblast growth factor (FGF) 8 expression in clinical
prostate cancers and breast tissues, immunohistochemically demonstrated by a
newly established neutralizing monoclonal antibody against FGF 8. <i>Cancer Res</i>
<b>58</b>: 2053–2056.<o:p></o:p></span></div>
<!--EndFragment--><br />
<div class="MsoListParagraphCxSpLast" style="text-align: justify; text-indent: -0.25in;">
<div style="line-height: 150%;">
<!--[if !supportLists]--><span style="font-family: "Times New Roman"; mso-fareast-font-family: "Times New Roman";"><span style="mso-list: Ignore;">6.<span style="font: 7.0pt "Times New Roman";">
</span></span></span><!--[endif]--><span style="font-family: "Times New Roman";">V W
Y Lui, D M-S Yau, C S-F Cheung, S C C Wong, A K-C Chan, et al. (2010). FGF8b
oncogene mediates proliferation and invasion of Espstein-Barr virus-associated
nasopharyngeal carcinoma cells: implication for viral-mediated FGF8b
upregulation. <i style="mso-bidi-font-style: normal;">Nature Publishing Group. </i>Oncogene (2011) <b>30</b>, pp 1518-1530<o:p></o:p></span></div>
<div style="line-height: 150%;">
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Proposal:</div>
<span style="line-height: 24px;"><a href="https://docs.google.com/file/d/0B8agoD1PeGsSNHlCMWN6VUhWbG8/edit?usp=sharing">https://docs.google.com/file/d/0B8agoD1PeGsSNHlCMWN6VUhWbG8/edit?usp=sharing</a></span><br />
First Progress Report:<br />
<a href="https://docs.google.com/file/d/0B8agoD1PeGsSY2pOUlpRYkNtZVU/edit?usp=sharing">https://docs.google.com/file/d/0B8agoD1PeGsSY2pOUlpRYkNtZVU/edit?usp=sharing</a><br />
Second Progress Report:<br />
<a href="https://drive.google.com/file/d/0B8agoD1PeGsScmxSUnlLOFJiZUE/edit?usp=sharing">https://drive.google.com/file/d/0B8agoD1PeGsScmxSUnlLOFJiZUE/edit?usp=sharing</a><br />
Final Report:<br />
<a href="https://drive.google.com/file/d/0B8agoD1PeGsSMWxsa3Budk55djQ/edit?usp=sharing">https://drive.google.com/file/d/0B8agoD1PeGsSMWxsa3Budk55djQ/edit?usp=sharing</a><br />
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Anonymoushttp://www.blogger.com/profile/08502568041036023185noreply@blogger.com0tag:blogger.com,1999:blog-1195222727010537305.post-8184772317641816442013-09-03T11:08:00.000-05:002013-12-03T23:51:51.835-06:00RNA Aptamer selection against HA33 for Neurotoxin sequestion<div class="separator" style="clear: both; text-align: center;">
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Wesley Yu<o:p></o:p></div>
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<br /></div>
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RNA Aptamer Selection against HA33 for Neurotoxin sequestion<o:p></o:p></div>
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HA33 is a portion of three botulism
neurotoxins secreted in a progenitor form from <i>Clostridium botulinum. </i>The HA33 protein can bind to glycoproteins
that facilitate internal transportation through the bloodstream, and as such,
can cause artery blockages leading to many circulatory problems associated with
botulism, a unique but potentially fatal paralytic illness (Arndt et al. 2005).
By binding to these glycolipids and glycoproteins, HA33 can cause flaccid
muscle paralysis amongst other symptoms. <o:p></o:p></div>
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The HA33 protein occupies up to 30%
of the progenitor complex, and acts as a dimer linking the entire progenitor
complex with the other protein subunits HA17 and HA70. Although the HA33
protein complex’s specific structural role in the progenitor complex has not
been properly defined yet, nucleic acids bind in general to the crystalline
B-trefoil regions of HA33 (Arndt et al. 2005). Most of the other applications
developed for this target revolve around its role in the progenitor structure,
as well as its own complex crystalline structure. Some of these applications
involve data collection about HA33’s relation to the other protein subunits in
the progenitor complex, as well as HA33’s binding affiliation to carbohydrates.
<o:p></o:p></div>
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Aptamers are oligonucleic acid or
peptide molecules created from random sequence pools for the purpose of binding
to specific targets. An aptamer developed for this protein would be useful in
the medical field for its ability to prevent or relieve muscle paralysis by
potentially changing the HA33 protein configuration. Consequently, new potential
uses for the HA33 protein include applications in pharmaceutical drugs used for
muscle paralysis and blood clotting. The aptamer would bind and induce a
conformational change in HA33, preventing the binding of glycoproteins to the
blood stream, allowing blood flow throughout the muscular and circulatory
systems to be less restricted. Furthermore, the glycoproteins could be
enchanced by the apatmer bounded HA33 to actually promote blood flow throughout
the human circulatory system, if they bind to cells typically responsible for
blood clots.<o:p></o:p><br />
<br />
<u style="text-indent: 0px;">Specific Aim 1</u><span style="text-indent: 0px;">: Select an RNA aptamer against the HA33 protein that can be used to induce a conformational change in the target’s protein structure.</span></div>
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<span style="font-family: "Times New Roman","serif"; font-size: 10.0pt; line-height: 150%;">Figure 1: Progenitor
toxin complex of <i>C. botulinum </i>comprised
of the HA33 protein complex (Dark Blue), HA17 complex (Sky Blue) and HA70
complex (Yellow). The BoNT neurotoxin protein is indicated by the red segment,
while the green segment indicates the NTNHA non-toxic, non-hemagglutinin
protein. Adapted from Arndt (2005).<o:p></o:p></span></div>
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<span style="font-family: "Times New Roman","serif"; font-size: 10.0pt; line-height: 150%;"><b><br /></b></span></div>
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<u>Target Order Information: <o:p></o:p></u></div>
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<u><br /></u></div>
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Vendor: Ellington Lab<o:p></o:p></div>
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Vendor Website: <a href="https://www.ellingtonlab.org/">https://www.ellingtonlab.org</a><o:p></o:p></div>
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Central Lab Telephone: 512-471-6445<o:p></o:p></div>
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Office Manager: 512-232-3426<o:p></o:p></div>
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Cost per Unit: $0<o:p></o:p></div>
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Cost per Round: $0<o:p></o:p></div>
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<u>References</u>:<o:p></o:p></div>
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<div class="MsoListParagraphCxSpFirst" style="line-height: 150%; mso-list: l0 level1 lfo1; text-indent: -.25in;">
<!--[if !supportLists]-->1.<span style="font-size: 7pt; line-height: normal;"> </span>Arndt
JW, Gu J, Jaroszewski L, Schwarzenbacher R, Hanson MA, Lebeda FJ, Stevens RC.
The Structure of the neurotoxin-associated protein HA33/A from Clostridium botulinum
suggests a reoccurring beta-trefoil fold in the progenitor toxin complex,
Molecular Biology 2005; 346 (4): 1083-1093.<o:p></o:p></div>
<div class="MsoListParagraphCxSpFirst" style="line-height: 150%; mso-list: l0 level1 lfo1; text-indent: -.25in;">
<br /></div>
<div class="MsoListParagraphCxSpMiddle" style="line-height: 150%; mso-list: l0 level1 lfo1; text-indent: -.25in;">
<!--[if !supportLists]-->2.<span style="font-size: 7pt; line-height: normal;"> </span><!--[endif]-->Nakamura
T, Tonozuka T, Ito S, Takeda Y, Sato R, Matsuo I, Ito Y, Oguma K, Nishikawa A. Molecular
diversity of the two sugar-binding sites of the Beta-trefoil lectin HA33/C
(HA1) from Clostridium botulinum type C neurotoxin, Biochemistry and Biophysics
2011; 1,512 (1): 69-77.<o:p></o:p></div>
<div class="MsoListParagraphCxSpMiddle" style="line-height: 150%; mso-list: l0 level1 lfo1; text-indent: -.25in;">
<br /></div>
<div class="MsoListParagraphCxSpMiddle" style="line-height: 150%; mso-list: l0 level1 lfo1; text-indent: -.25in;">
<!--[if !supportLists]-->3.<span style="font-size: 7pt; line-height: normal;"> </span><!--[endif]-->Van
Damme EJ, Nakamura-Tsuruta S, Hirabayashi J, Rouge P, Peumans WJ. The
Sclerotinia sclerotiorum agglutinin represents a novel family of fungal lectins
remotely related to the Clostridium botulinum non-toxin haemagglutinin HA33/A,
Molecular Biotechnology 2007; 24 (2-3): 143-156.<o:p></o:p></div>
<div class="MsoListParagraphCxSpMiddle" style="line-height: 150%; mso-list: l0 level1 lfo1; text-indent: -.25in;">
<br /></div>
<div class="MsoListParagraphCxSpMiddle" style="line-height: 150%; mso-list: l0 level1 lfo1; text-indent: -.25in;">
<!--[if !supportLists]-->4.<span style="font-size: 7pt; line-height: normal;"> </span><!--[endif]-->Yamashita
S, Yoshida H, Uchiyama N, Nakakita Y, Nakakita S, Tonozuka T, Oguma K,
Nishikawa A, Kamitori S. Carbohydrate recognition mechanism of HA70 from
Clostridium botulinum deduced from X-ray structures in complexes with
sialylated oligosaccharides, Science Research and Medicine 2012; 586 (16):
2404-2410.<o:p></o:p></div>
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<div class="MsoListParagraphCxSpLast" style="line-height: 150%; mso-list: l0 level1 lfo1; text-indent: -.25in;">
<!--[if !supportLists]-->5.<span style="font-size: 7pt; line-height: normal;"> </span><!--[endif]-->Umeda
K, Seto Y, Kohda T, Mukamoto M, Kozaki S. A novel multiplex PCR method for
Clostridium botulinum neurotoxin type A gene cluster typing, Microbiology 2010;
54 (5): 308-312.<br />
<div class="MsoListParagraphCxSpLast" style="line-height: 150%; mso-list: l0 level1 lfo1; text-indent: -.25in;">
<br /></div>
View Full Proposal: <a href="https://docs.google.com/file/d/0B7B7lE6h5mELNTN2WXBxTGtqNVU/edit?usp=sharing">https://docs.google.com/file/d/0B7B7lE6h5mELNTN2WXBxTGtqNVU/edit?usp=sharing</a><br />
View First Progress Report: <a href="https://docs.google.com/file/d/0B7B7lE6h5mELa2lKZkp0eFVsb00/edit?usp=sharing">https://docs.google.com/file/d/0B7B7lE6h5mELa2lKZkp0eFVsb00/edit?usp=sharing</a><br />
View Second Progress Report: <a href="https://drive.google.com/file/d/0B7B7lE6h5mELdERSRlJfdENkbG8/edit?usp=sharing">https://drive.google.com/file/d/0B7B7lE6h5mELdERSRlJfdENkbG8/edit?usp=sharing</a><br />
View Final Progress Report: <a href="https://drive.google.com/file/d/0B7B7lE6h5mELcFVydWw0N2ZDRjA/edit?usp=sharing">https://drive.google.com/file/d/0B7B7lE6h5mELcFVydWw0N2ZDRjA/edit?usp=sharing</a><br />
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Wesley Yuhttp://www.blogger.com/profile/10793145043162216622noreply@blogger.com0tag:blogger.com,1999:blog-1195222727010537305.post-41741291511292295102013-09-03T01:48:00.000-05:002013-12-03T23:32:09.280-06:00Aptamer Screening of HA33 for the Diagnose of Botulism Neurotoxin<div align="center" class="MsoNoSpacing" style="text-align: center;">
<b><span style="font-family: "Times New Roman","serif"; font-size: 12.0pt;">Aptamer Screening of HA33 for the Diagnose of
Botulism Neurotoxin<o:p></o:p></span></b></div>
<div align="center" class="MsoNoSpacing" style="text-align: center;">
<br /></div>
<div align="center" class="MsoNoSpacing" style="text-align: center;">
<span style="font-family: "Times New Roman","serif"; font-size: 12.0pt;">Norma Arroyo –
Fall 2013<o:p></o:p></span></div>
<div class="MsoNoSpacing">
<div class="MsoNoSpacing">
<b><span style="font-family: "Times New Roman","serif"; font-size: 12.0pt;">Abstract<o:p></o:p></span></b></div>
<div class="MsoNoSpacing" style="line-height: 200%; text-indent: .5in;">
<span style="font-family: "Times New Roman","serif"; font-size: 12.0pt; line-height: 200%;">Botulism
is an illness that leads to flaccid paralysis of the muscles and even causes
death from respiratory failure. Botulism is caused by a neurotoxin produced by
the Bacterium <i>Clostridium botulinum</i>. HA33 is a nontoxic neurotoxin-associated
protein (NAPs). HA33 helps protect the <i>Clostridium Botulinum</i> neurotoxin (BoNTs)
from acid denaturation in the stomach and from attacks from proteolytic enzymes
found in the gastro intestinal tract. HA33 have been discovered to potentially
serve as internalization or an activator for BoNTs [1].<o:p></o:p></span></div>
<div class="MsoNormal" style="line-height: 200%; margin-bottom: .0001pt; margin-bottom: 0in; text-indent: .5in;">
<span style="font-family: "Times New Roman","serif"; font-size: 12.0pt; line-height: 200%;">Botulism’s symptoms can be very similar
to other illnesses and disease of muscle paralysis, therefore many tests may be
performed to eliminate other possible illnesses. Botulism is diagnosed through
a mouse inoculation test. This and other test may take up to days or weeks to
finalize a concrete diagnosis of botulism[2].</span><span style="font-family: "Times New Roman","serif"; font-size: 12.0pt; line-height: 200%; mso-fareast-font-family: "Times New Roman";"> </span><span style="font-family: "Times New Roman","serif"; font-size: 12.0pt; line-height: 200%;">An alternative diagnostic method for
diagnosing botulism could be through utilizing aptamers.<o:p></o:p></span></div>
<br />
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<span style="font-family: "Times New Roman","serif"; font-size: 12.0pt; line-height: 200%;">Aptamers have a high specifically
affinity to their target, HA33 in this case. Modifying an aptamer to carry a
fluorescein will allow HA33 to be label and track in the small intestine. Similar
to the CFP/YEP-based sensors used to detect the activity of BoNTs in cells as
shown in <b>Figure 1. </b>[3]. Screening for the botulism neurotoxin can possibly help to estimate
the amount of Heptavalent Botulism Antitoxin necessary for the treatment. This
can also prevent the waste of Heptavalent Botulism Antitoxin. </span></div>
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<a href="http://1.bp.blogspot.com/-EnWWC-Ygh2A/UjkQfH8rkqI/AAAAAAAAAA0/2MZyQ_rAuLE/s1600/Abstract+picture.jpg" imageanchor="1" style="clear: left; float: left; margin-bottom: 1em; margin-right: 1em;"><img border="0" height="150" src="http://1.bp.blogspot.com/-EnWWC-Ygh2A/UjkQfH8rkqI/AAAAAAAAAA0/2MZyQ_rAuLE/s320/Abstract+picture.jpg" width="320" /></a></div>
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<b><span style="font-size: 9.0pt; line-height: 200%; mso-bidi-font-size: 11.0pt;">Figure 1. CFP/YFP-based sensors for detecting BoNT
protease activity<i> in vitro</i>. [3]</span></b></div>
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<span style="font-size: 9.0pt; line-height: 200%; mso-bidi-font-size: 11.0pt;"> </span><u><span style="font-family: "Times New Roman","serif"; font-size: 12.0pt; line-height: 200%;">Specific Aim 1: </span></u><span style="font-family: "Times New Roman","serif"; font-size: 12.0pt; line-height: 200%;"> Identify an aptamer against HA33 that
can be modified to carry a fluorescein and be allowed to be tracked within the
small intestine.</span> <span style="font-family: "Times New Roman","serif"; font-size: 12.0pt; line-height: 200%;">Identifying the binding locations of
these BoNTs can not only help for a sceening to diagnose botulism but possibly
to deliver a pay load to the specific region.</span><br />
<span style="font-family: 'Times New Roman', serif; line-height: 200%; text-indent: 0.5in;">Link to proposal: <a href="https://docs.google.com/file/d/0BwaR2WWmhjiOVnJTejJUc2pmMzQ/edit?usp=sharing">https://docs.google.com/file/d/0BwaR2WWmhjiOVnJTejJUc2pmMzQ/edit?usp=sharing</a></span><br />
<span style="line-height: 200%; text-indent: 0.5in;">Link to first progress report: </span><a href="https://docs.google.com/file/d/0BwaR2WWmhjiON1Z5NWQ3TThfdVE/edit?usp=sharing" style="line-height: 200%; text-indent: 0.5in;">https://docs.google.com/file/d/0BwaR2WWmhjiON1Z5NWQ3TThfdVE/edit?usp=sharing</a><br />
Link to second progress report:<br />
<span style="line-height: 200%; text-indent: 0.5in;"> </span><a href="https://drive.google.com/file/d/0BwaR2WWmhjiOUlZScVRWTjZuSlk/edit?usp=sharing" style="line-height: 200%; text-indent: 0.5in;">https://drive.google.com/file/d/0BwaR2WWmhjiOUlZScVRWTjZuSlk/edit?usp=sharing</a><br />
Link to final progress report:<br />
<a href="https://drive.google.com/file/d/0BwaR2WWmhjiOZDA0ZFp1b3A2akk/edit?usp=sharing">https://drive.google.com/file/d/0BwaR2WWmhjiOZDA0ZFp1b3A2akk/edit?usp=sharing</a></div>
</div>
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<span style="font-family: "Times New Roman","serif"; font-size: 12pt;"><b>Target
Order Information:</b><u><o:p></o:p></u></span></div>
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<span style="font-family: "Times New Roman","serif"; font-size: 12.0pt;">Vendor:
Ellington Lab<o:p></o:p></span></div>
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<span style="font-family: "Times New Roman","serif"; font-size: 12.0pt;">Vendor
Website: https://www.ellingtonlab.org<o:p></o:p></span></div>
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<span style="font-family: "Times New Roman","serif"; font-size: 12.0pt;">Central
Lab Telephone: 512-471-6445<o:p></o:p></span></div>
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<span style="font-family: Times New Roman, serif;">Cost per Round: $0.00 (Available in Lab)</span><br />
<span style="font-family: Times New Roman, serif;">Cost of Target: $0.00</span></div>
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<b><span style="font-family: "Times New Roman","serif"; font-size: 12.0pt;">References</span></b></div>
<div class="MsoListParagraphCxSpFirst" style="text-indent: -0.25in;">
<!--[if !supportLists]--><span style="font-family: "Times New Roman","serif"; font-size: 12.0pt; mso-fareast-font-family: "Times New Roman";">1.<span style="font-family: 'Times New Roman'; font-size: 7pt;">
</span></span><!--[endif]--><span style="font-family: "Times New Roman","serif"; font-size: 12.0pt;">Arndt,
J.W., Gu, J., Jaroszewski, L., Schwarzenbacher, R., Hanson, H.A., Lebeda, F.J.,
and Stevens, R.C. (2005). The Structure of the Neurotoxin-associated Protein
HA33/A from Clostridium botulinum Suggests a Reoccurring β-Trefoil Fold in the
Progenitor Toxin Complex. Jornal of Molecular Biology. 365(4): 1083-1093.<o:p></o:p></span></div>
<div class="MsoListParagraphCxSpFirst" style="text-indent: -0.25in;">
<span style="font-family: "Times New Roman","serif"; font-size: 12.0pt;"><br /></span></div>
<div class="MsoListParagraphCxSpMiddle" style="margin-bottom: 0.0001pt; text-indent: -0.25in;">
<!--[if !supportLists]-->2.<span style="font-size: 7pt;">
</span><!--[endif]--><span style="font-family: "Times New Roman","serif"; font-size: 12.0pt; mso-fareast-font-family: "Times New Roman";">Centers for Disease Control and
Prevention [CDC], National Center for Emerging and Zoonotic Infectious Diseases
[NCEZID], Division of Foodborne, Waterborne, and Environmental Diseases [DFWED]
(2011) Botulism.</span><o:p></o:p></div>
<div class="MsoListParagraphCxSpMiddle" style="margin-bottom: 0.0001pt; text-indent: -0.25in;">
<span style="font-family: "Times New Roman","serif"; font-size: 12.0pt; mso-fareast-font-family: "Times New Roman";"><br /></span></div>
<div class="MsoListParagraphCxSpMiddle" style="margin-bottom: 0.0001pt; text-indent: -0.25in;">
<!--[if !supportLists]--><span style="font-family: "Times New Roman","serif"; font-size: 12.0pt; mso-fareast-font-family: "Times New Roman";">3.<span style="font-family: 'Times New Roman'; font-size: 7pt;">
</span></span><!--[endif]--><span style="font-family: "Times New Roman","serif"; font-size: 12.0pt;">Dong,
M., Tepp, W.H., Johnson, E.A., and Chapman, E.R. (2004). Using fluorescent
sensors to detect botulinum neurotoxin activity <i>in vitro</i> and in living cells. Proc Natl Acad Sci USA. 10(41):
14701-14706<o:p></o:p></span></div>
<div class="MsoListParagraphCxSpMiddle" style="margin-bottom: 0.0001pt; text-indent: -0.25in;">
<span style="font-family: "Times New Roman","serif"; font-size: 12.0pt;"><br /></span></div>
<div class="MsoListParagraphCxSpLast" style="margin-bottom: 0.0001pt; text-indent: -0.25in;">
<!--[if !supportLists]-->4.<span style="font-size: 7pt;"> </span><span style="font-size: 7pt; text-indent: -0.25in;"> </span><span style="font-family: 'Times New Roman', serif; font-size: 12pt; line-height: 200%; text-indent: -0.25in;">Davis, R.G., (2004). The ABCs of
bioterrorism for veterinarians focusing on Category A Agents. J Am Vet Med
Assoc 2004; 224:1084-1095</span><br />
<div class="MsoListParagraph" style="line-height: 200%; margin-bottom: .0001pt; margin-bottom: 0in; mso-add-space: auto; mso-list: l0 level1 lfo1; text-indent: -.25in;">
<o:p></o:p></div>
</div>
<div class="MsoListParagraphCxSpLast" style="margin-bottom: 0.0001pt; text-indent: -0.25in;">
<span style="font-family: "Times New Roman","serif"; font-size: 12.0pt;"><br /></span></div>
<div class="MsoListParagraphCxSpLast" style="margin-bottom: 0.0001pt; text-indent: -0.25in;">
<i style="text-indent: -0.25in;"><span style="font-family: "Times New Roman","serif"; font-size: 12.0pt; mso-fareast-font-family: "Times New Roman";">5.<span style="font-family: 'Times New Roman'; font-size: 7pt; font-style: normal;">
</span></span></i><span style="font-family: 'Times New Roman', serif; font-size: 12pt; text-indent: -0.25in;">Davis, Charles P., Stöppler, Melissa C. (2012) Botulism.
WebMD</span></div>
<div class="MsoListParagraphCxSpLast" style="margin-bottom: 0.0001pt; text-indent: -0.25in;">
<span style="font-family: 'Times New Roman', serif; font-size: 12pt; text-indent: -0.25in;"><br /></span></div>
<div class="MsoListParagraphCxSpLast" style="margin-bottom: 0.0001pt; text-indent: -0.25in;">
<span style="font-family: 'Times New Roman', serif; font-size: 12pt; text-indent: -0.25in;">6.<span style="font-family: 'Times New Roman'; font-size: 7pt;">
</span></span><span style="font-family: 'Times New Roman', serif; font-size: 12pt; text-indent: -0.25in;">Hutson
R.A., Zhou Y., Collins M.D., Johnson E.A., Hatheway C.L., and Sugiyama H. J.,
(1996). Genetic characterization of Clostridium botulinum type A containing
silent type B neurotoxin gene sequences. Biol. Chem. 271:10786-10792</span></div>
<div class="MsoListParagraphCxSpLast" style="margin-bottom: 0.0001pt; text-indent: -0.25in;">
<span style="font-family: 'Times New Roman', serif; font-size: 12pt; text-indent: -0.25in;"><br /></span></div>
<div class="MsoListParagraphCxSpLast" style="margin-bottom: 0.0001pt; text-indent: -0.25in;">
<span style="text-indent: -0.25in;">7.</span><span style="font-size: 7pt; text-indent: -0.25in;">
</span><span style="font-family: 'Times New Roman', serif; font-size: 12pt; text-indent: -0.25in;">Pestronk,
Alan ( 2012). Botulism. Neuromuscular Disease Center. Washington University,
St. Louis, MO USA</span><br />
<span style="font-family: 'Times New Roman', serif; font-size: 12pt; text-indent: -0.25in;"><br /></span>
<span style="font-family: 'Times New Roman', serif; font-size: 12pt; text-indent: -0.25in;">8.</span><span style="font-size: 7pt; text-indent: -0.25in;"> </span><span style="font-family: 'Times New Roman', serif; font-size: 12pt; line-height: 200%; text-indent: -0.25in;">Hoon
S., Zhou B.., Janda KD., Brenner S., Scolnick J., (2011). Aptamer selection by
high-throughput sequencing and informatics analysis. BioTechniques. 51(6):413-6</span><br />
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Anonymoushttp://www.blogger.com/profile/05709636568406433013noreply@blogger.com0tag:blogger.com,1999:blog-1195222727010537305.post-63647774297082159862013-09-03T01:08:00.001-05:002013-10-10T16:16:20.616-05:00 Nucleic Acid Aptamer Selection against HA-33 of C. botulinum for an Early Diagnosis and a Treatment for Botulism<div dir="ltr" style="text-align: left;" trbidi="on">
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Nucleic Acid Aptamer Selection against HA-33 of C. botulinum for an Early Diagnosis and a Treatment for Botulism<br />
<br />
Sharon Joy</div>
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Botulism, an uncommon yet fatal illness characteristically manifests as an acute paralysis that begins with the cranial nerves and moves toward the lower extremities is caused by the rod-shaped and spore-forming bacterium Clostridium botulinum. The neurotoxins produced by this certain bacterium are among some of the most toxic bacterial toxins ever known. This protein neurotoxin interrupts or inhibits the neuromuscular transmission that leads to paralysis. The bacterium produces seven neurotoxins in which four of them affect humans. There are different kinds of botulism which include: food-borne botulism, wound botulism, infant botulism, adult infectious botulism and inadvertent botulism (World Health Organization). Treatment of this illness includes botulinus antitoxins which inhibits the function of the toxins in the blood stream. However, early diagnosis and treatment of this illness is key, since delayed treatment can lead to an advance in paralysis in patients including respiratory muscle paralysis which can lead to a patient requiring ventilatory care, and ICU care; not diagnosing early enough could also lead to death in patients (MedlinePlus Medical Encyclopedia).</div>
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<span class="Apple-tab-span"></span> A botulinum toxin complex consists of a neurotoxic, a nontoxic nonhaemagglutinin and a haemagglutinin or the HA complex. The HA complex consists of arms which include two HA-33 arms. A study was conducted on the absorption of the botulinum toxin in rat intestinal epithelial cell line, which showed that cell binding and monolayer transport of serotype D toxins significantly increased with an increase of HA-33/HA-17 arms. This led to the conclusion that the HA-33/HA-17 arm was not required yet it was a vital component in facilitating the transport of the toxins (Federation of European Microbiological Societies). HA-33 is also crucial for toxin movement, activation and protection (Journal of Molecular Biology). <span class="s1">A selected aptamer against HA33 can be used for an early diagnosis of this fatal illness and can be modified for a therapeutic application. This aptamer can be tagged with a molecule that can result in a change of shape for HA33, which results in an inhibition of the function of the protein. By changing the shape of the protein, it can obstruct the protein from entering through the microvilli to the small intestine membrane. By inhibiting the protein’s function, which is responsible for botulism, it can in turn help in preventing the fatal disease that leads to paralysis.</span><br />
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<a href="http://3.bp.blogspot.com/-AiZ7vw_TQt0/Uie6uTbQWnI/AAAAAAAAAV8/t0Qx6GpGPOE/s1600/Botulism.png" imageanchor="1" style="clear: left; float: left; margin-bottom: 1em; margin-right: 1em;"><img border="0" height="320" src="http://3.bp.blogspot.com/-AiZ7vw_TQt0/Uie6uTbQWnI/AAAAAAAAAV8/t0Qx6GpGPOE/s320/Botulism.png" width="270" /></a></div>
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Figure 1: This figure depicts the large botulinum complex which consists of </div>
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a neurotoxic (BoNT), a nontoxic nonhaemagglutinin (NTHA) and a haemagglutinin (HA) complex with three components: HA-33, HA-17 and HA-70. The HA-33/HA-17 arms greatly facilitate the transportation of the toxins. By selecting an aptamer against HA-33, which has an unique organization and a carbohydrate recognition site, it can be modified for a therapeutic application which can inhibit the toxin from entering the intestinal membrane (InTech).<br />
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Specific Aim 1: The identification and amplification of a RNA aptamer selected against HA-33. </div>
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Specific Aim 2: Modification of the aptamer for a therapeutic application. By tagging the aptamer with a molecule that changes the shape of the protein target when it binds to it, it can inhibit the function of the paralysis responsible for the fatal illness. </div>
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<span class="s2">Target Order Information</span></div>
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Vendor: Ellington Lab</div>
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<span class="s3">Vendor Website: <a href="https://www.ellingtonlab.org/"><span class="s2">https://www.ellingtonlab.org</span></a></span></div>
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Central Lab Telephone: 512-471-6445</div>
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Office Manager: 512-232-3426</div>
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Cost per Unit: $0</div>
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Cost per Round: $0</div>
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<span class="s4">Arndt, J. W., J. Gu, R. Schwarzenbacher, M. </span>A. Hanson, F. J. Lebeda, and R. C. Stevens. "The Structure of the Neurotoxin-associated Protein HA33/A from Clostridium Botulinum Suggests a Reoccurring β-Trefoil Fold in the Progenitor Toxin Complex." <span class="s4"><i>Journal of Molecular Biology</i> 4th ser. 346.4 (2005): 1083-093. </span></div>
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"Botulism: Treatment." <i>MedlinePlus Medical Encyclopedia</i>. U.S. National Library of Medicine, 2012. Web. 03 Sept. 2013.</div>
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"Clostridium Botulinum." <i>International Programme on Chemical Safety Poisons Information Monograph858 Bacteria</i>. World Health Organization, n.d. Web. 02 Sept. 2013. <http://www.who.int/csr/delibepidemics/clostridiumbotulism.pdf>.</div>
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Ito, H., Y. Sagane, K. Miyata, K. Inui, T. Matsuo, R. Horiuchi, and T. Ikeda. "HA-33 Facilitates Transport of the Serotype D Botulinum Toxin across a Rat Intestinal Epithelial Cell Monolayer." <i>Federation of European Microbiological Societies </i>(2011): n. pag. </div>
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Sagane, Y., K. Inui, S. Miyashita, K. Miyata, K. Niwa, T. Watanabe, and T. Suzuk. "Botulinum Toxin Complex: A Delivery Vehicle of Botulinum Neurotoxin Traveling Digestive Tract." <i>InTech</i>. N.p., 22 Aug. 2012. Web.<br />
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Proposal Link: <a href="https://docs.google.com/file/d/0B8u9l0gKo2jddHZkbHFSdWFmTW8/edit?usp=sharing">https://docs.google.com/file/d/0B8u9l0gKo2jddHZkbHFSdWFmTW8/edit?usp=sharing</a><br />
Link to first report: <a href="https://docs.google.com/file/d/0B8u9l0gKo2jdcmZFUUJ5NjlNTVk/edit?usp=sharing">https://docs.google.com/file/d/0B8u9l0gKo2jdcmZFUUJ5NjlNTVk/edit?usp=sharing</a> </div>
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Anonymoushttp://www.blogger.com/profile/02695795088353206608noreply@blogger.com0tag:blogger.com,1999:blog-1195222727010537305.post-81684204081412271872013-09-03T01:01:00.000-05:002013-12-04T02:22:27.392-06:00RNA Aptamer Selection Against Progesterone 3-biotin to Prevent Breastfeeding Complications Abstract<div class="MsoNormal" style="line-height: 150%; text-align: left;">
<span style="font-family: Times New Roman, serif;">Areeb Syed</span></div>
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<span style="font-family: Times New Roman, serif;">September 1, 2013</span></div>
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<b><span style="font-family: "Times New Roman","serif"; font-size: 12.0pt; line-height: 150%;">RNA Aptamer Selection Against
Progesterone 3-biotin to Prevent Breastfeeding Complications</span></b><br />
<b><span style="font-family: "Times New Roman","serif"; font-size: 12.0pt; line-height: 150%;"><br /></span></b></div>
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<span style="font-family: "Times New Roman","serif"; font-size: 12.0pt; line-height: 115%;"> Progesterone
is a key hormone produced by human animal alike to allow for reproduction and
in turn ensure the survivability and viability of the species (Harris, 2011). The molecule is synthesized from cholesterol
and serves as a precursor to several other steroid hormones. In humans,
progesterone is produced in the ovaries of the female and is produced in the
adrenal glands in both males and females. Progesterone also serves a vital role
in breastfeeding. During a normal pregnancy cycle, after delivery, the
progesterone levels in the female body decreases significantly while the
oxytocin level increases which allows for the mother to breastfeed, which not
only provides benefits to the newborn child, such as allowing the child to be
more resistant to disease and infection early in their life, but also provides
benefits for the mother such as delaying and even preventing osteoporosis, and
reducing the risk of several types of cancers that predominately affect women
(NRDC, 2005). For some women however, progesterone levels might not decrease
after giving birth, thereby preventing the mother from breastfeeding, since
progesterone is a major inhibitor of lactation (Fuller, 2008). <o:p></o:p></span></div>
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<span style="font-family: "Times New Roman","serif"; font-size: 12.0pt; line-height: 115%;"> Aptamers are nucleic acid species that tightly
bind to and inhibit a specific target. An aptamer selected against progesterone
could not only prevent breastfeeding complications in the form of
pharmaceuticals, which can provide numerous benefits to both the mother and
child, but can also serve as a form of birth control. Progesterone is a
required hormone to carry a child, and if there aren’t sufficient levels of the
hormone, a miscarriage could occur (Lydon, 1995). That is why progesterone
levels spike during the pregnancy term. By selecting for an aptamer against
progesterone could not only serve as a way to correct a lactation disorder, but
also serves as a potential form of birth control. <o:p></o:p></span></div>
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<span style="font-family: "Times New Roman","serif"; font-size: 12.0pt; line-height: 115%;"> The
mother’s ability to lactate after delivery and her potential to carry a child
in the womb without complications all revolve around the hormone progesterone.
During a typical pregnancy cycle, progesterone levels spike in order to care
for the developing child, and after delivery the levels drop significantly to
allow for lactation. By developing an aptamer against progesterone, one could
develop a cost effective pharmaceutical that not only resolves breastfeeding
complications that may arise after delivery, but also serves as a “morning-after”
pill to prevent unwanted pregnancies. <o:p></o:p></span></div>
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<u><span style="font-family: "Times New Roman","serif"; font-size: 12.0pt; line-height: 115%;">Specific Aim 1:</span></u><span style="font-family: "Times New Roman","serif"; font-size: 12.0pt; line-height: 115%;">
Select for an aptamer against progesterone that inhibits the molecule to
resolve lactations complications.</span></div>
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<span style="font-family: "Times New Roman","serif"; font-size: 12.0pt; line-height: 115%;"><o:p></o:p></span><u><span style="font-family: 'Times New Roman', serif; font-size: 12pt; line-height: 18px;">Specific Aim 2:</span></u><span style="font-family: 'Times New Roman', serif; font-size: 12pt; line-height: 18px;"> Select for an aptamer against progesterone that serves as a cost effective birth control pill. </span></div>
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<a href="http://1.bp.blogspot.com/-R7S7G47YDBE/UiV6O3QSLUI/AAAAAAAAArc/Ggg6HG2iifw/s1600/Doc1-page-001+(1).jpg" imageanchor="1" style="margin-left: 1em; margin-right: 1em;"><img border="0" height="640" src="http://1.bp.blogspot.com/-R7S7G47YDBE/UiV6O3QSLUI/AAAAAAAAArc/Ggg6HG2iifw/s640/Doc1-page-001+(1).jpg" width="492" /></a></div>
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<u><span style="font-family: "Times New Roman","serif"; font-size: 12.0pt; line-height: 115%;">Ordering Information:<o:p></o:p></span></u></div>
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<span style="font-family: "Times New Roman","serif"; font-size: 12.0pt; line-height: 115%;">Cayman Chemical<o:p></o:p></span></div>
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<span style="font-family: "Times New Roman","serif"; font-size: 12.0pt; line-height: 115%;">Product Item Number: 9000644<o:p></o:p></span></div>
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<span style="font-family: "Times New Roman","serif"; font-size: 12.0pt; line-height: 115%;">Website: </span><a href="http://www.caymanchem.com/"><span style="font-family: "Times New Roman","serif"; font-size: 12.0pt; line-height: 115%;">www.caymanchem.com</span></a><span style="font-family: "Times New Roman","serif"; font-size: 12.0pt; line-height: 115%;"><o:p></o:p></span></div>
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<span style="font-family: "Times New Roman","serif"; font-size: 12.0pt; line-height: 115%;">Telephone: 800-364-9897<o:p></o:p></span></div>
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<span style="font-family: "Times New Roman","serif"; font-size: 12.0pt; line-height: 115%;">Cost/unit: $56.00 per 500 ug<o:p></o:p></span></div>
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<span style="font-family: "Times New Roman","serif"; font-size: 12.0pt; line-height: 115%;">Cost/round: $0 (Available in lab)<o:p></o:p></span></div>
<div class="MsoNormal">
<br /></div>
<div class="MsoNormal">
<u><span style="font-family: "Times New Roman","serif"; font-size: 12.0pt; line-height: 115%;">References:</span></u><span style="font-family: "Times New Roman","serif"; font-size: 12.0pt; line-height: 115%;"><o:p></o:p></span></div>
<div class="MsoNormal" style="line-height: 150%;">
<span style="font-family: "Times New Roman","serif"; font-size: 12.0pt; line-height: 150%;">Fuller, Zoe (2008)
“Breastfeeding Limitations: Are some women incapable of breastfeeding? Are
formula mothers neglectful?”. Bryn Mawr College<o:p></o:p></span></div>
<div class="MsoNormal" style="line-height: 150%;">
<span style="font-family: "Times New Roman","serif"; font-size: 12.0pt; line-height: 150%;">Harris, Gardiner (2011)
“Hormone is Said to Cut Risk of Premature Birth”. New York Times<o:p></o:p></span></div>
<div class="MsoNormal" style="line-height: 150%;">
<span style="font-family: "Times New Roman","serif"; font-size: 12.0pt; line-height: 150%;">Lydon, John (1995)
“Mice lacking progesterone receptor exhibit pleotropic reproductive
abnormalities”. <o:p></o:p></span></div>
<div class="separator" style="clear: both; text-align: center;">
</div>
<div class="MsoNormal" style="line-height: 150%;">
<span style="font-family: "Times New Roman","serif"; font-size: 12.0pt; line-height: 150%;">NRDC (2005) “Benefits
of Breastfeeding”.<o:p></o:p></span><br />
<span style="font-family: "Times New Roman","serif"; font-size: 12.0pt; line-height: 150%;"><br /></span>
<span style="font-family: "Times New Roman","serif"; font-size: 12.0pt; line-height: 150%;"><a href="https://www.dropbox.com/s/0wpjjmv4xoeunae/RNA%20Aptamer%20Selection%20against%20Progesterone%203.pdf" target="_blank">Link to Proposal</a></span><br />
<a href="https://docs.google.com/file/d/0BzGgK0LqIUopSk5MRkktN2JmOWs/edit?usp=sharing" target="_blank">Link to Progress Report 10.8</a><br />
<a href="https://drive.google.com/file/d/0BzGgK0LqIUopYm1YZlBhUDNfZU0/edit?usp=sharing" target="_blank">Link to Progress Report 11.5</a><br />
<a href="https://drive.google.com/file/d/0BzGgK0LqIUopbnZHVk1uemp4VHM/edit?usp=sharing" target="_blank">Link to Final Manuscript 12.3</a></div>
<div class="separator" style="clear: both; text-align: center;">
<br /></div>
Anonymoushttp://www.blogger.com/profile/14246872868634958045noreply@blogger.com0tag:blogger.com,1999:blog-1195222727010537305.post-69100777834695969952013-09-03T00:35:00.001-05:002013-12-02T20:33:19.250-06:00<div class="MsoNormal">
<h3 style="text-align: center;">
<b>Selecting an Aptamer Against the RCAS1 Biomarker for a Novel Tool in the Diagnosis and Treatment of Cancer</b></h3>
</div>
<div style="text-align: center;">
Daniel Liaou</div>
<div class="MsoNormal" style="-webkit-text-stroke-width: 0px; color: black; font-family: 'Times New Roman'; font-size: medium; font-style: normal; font-variant: normal; font-weight: normal; letter-spacing: normal; line-height: normal; orphans: auto; text-align: start; text-indent: 0px; text-transform: none; white-space: normal; widows: auto; word-spacing: 0px;">
</div>
<br />
<span style="font-family: inherit;"> RCAS1
(receptor-binding cancer antigen expressed on SiSo cells) is a protein expressed
by the EBAG9 (estrogen receptor-binding fragment-associated gene
9) gene<sup>1</sup>.<b> </b>Overexpression
of this biomarker has been identified to be present in nearly all gastric
cancers<sup>2</sup> and the degree of overexpression has been shown to be
indicative of the prognosis in 14 different types of cancer. In addition to
this, levels of RCAS1 have been linked to "clinicopathological parameters
including the histological type of cancer, its differentiation, tumor size,
clinical stage, [and] the depth of
invasion<b>..</b>."<sup>3 </sup>RCAS1
is also a biologically active molecule. For example, in gastric cancers, it has
been implicated to facilitate tumor cell evasion of the immune system via
growth-inhibition and apoptosis-induction in lymphocytes expressing the
receptor for the antigen<sup>3</sup>. Another role that has been associated
with RCAS1 is remodeling of connective tissue<sup>4</sup>. The capability of
remodeling connective tissue is an essential factor contributing to tumor malignancy
and eventual metastasis - the ultimate cause of death in over 90% of cancers<sup>5</sup>.</span><br />
<br />
<div class="MsoNormal">
Aptamers,
short sequences of RNA or DNA, provide a double-faceted approach to utilizing
the RCAS1 protein. First, because of the high affi<span style="font-family: inherit;">nity and specificity of
aptamers as well as their relative ease of production, an aptamer selected
against RCAS1 could provide a high-yield method of detecting RCAS1 levels and a
novel test for diagnosing cancers associated</span> with its overexpression. If a
cancer is found to be present, additional qualitative information could be
revealed relating to the disease progression. Second, because of the innate
specificity which is required for antigens to trigger a response, a selected
aptamer that can bind uniquely to and alter the surface of RCAS1 holds the
potential to suppress key mechanisms in cancer development by targeting key
steps in the disease pathway dependent on the biological role RCAS1. With its
close association and implicated roles in a number of different cancers, an
aptamer against RCAS1 would provide medically-relevant tools in a number of
clinical contexts.</div>
<div class="MsoNormal">
<br /></div>
<div class="MsoNormal">
<u>Specific Aim 1:</u> Select an RNA aptamer against the
RCAS1 protein using Systematic Evolution of Ligands by Exponential Enrichment
(SELEX).</div>
<div class="MsoNormal">
<br /></div>
<div class="MsoNormal">
<a href="http://2.bp.blogspot.com/-0FED0ZvyYew/Uieohu8eY1I/AAAAAAAAABk/QOkJLJ4wTXc/s1600/Selective+Binding+Aptamer.png" imageanchor="1" style="clear: right; float: right; margin-bottom: 1em; margin-left: 1em;"><img border="0" height="165" src="http://2.bp.blogspot.com/-0FED0ZvyYew/Uieohu8eY1I/AAAAAAAAABk/QOkJLJ4wTXc/s320/Selective+Binding+Aptamer.png" width="320" /></a><u>Specific Aim 2:</u> Modify the aptamer for use in
diagnostic tests for RCAS1-associated cancers. Once an aptamer is isolated that
can discriminately identify RCAS1 in a complex fluid, it can be used to detect
RCAS1 levels that would implicate the presence of cancer (Figure 1).</div>
<div class="MsoNormal">
<br /></div>
<div class="MsoNormal">
<u></u><br />
<div class="separator" style="clear: both; text-align: center;">
<a href="http://1.bp.blogspot.com/-cIqwMQeMk0s/Uieod1YWsPI/AAAAAAAAABU/ul0Wo4oBbmI/s1600/Selective+Binding+Aptamer.jpg" imageanchor="1" style="clear: right; float: right; margin-bottom: 1em; margin-left: 1em;"><img border="0" height="292" src="http://1.bp.blogspot.com/-cIqwMQeMk0s/Uieod1YWsPI/AAAAAAAAABU/ul0Wo4oBbmI/s320/Selective+Binding+Aptamer.jpg" width="320" /></a></div>
<u>Specific Aim 3:</u> Modify the aptamer to be an inhibitor
of RCAS1. If RCAS1 plays a key biological role in the pathogenesis of the
cancer, aptamer-inhibition could be used therapeutically to impede the
progression of the disease (Figure 2).<br />
<br /></div>
<div class="separator" style="clear: both; text-align: center;">
</div>
<div class="MsoNormal">
<span style="line-height: 115%;"> The RCAS1 protein will be ordered from Creative Biomart at a cost of $265.00
per 100 ug, which corresponds to a cost of $12.40 per round. The catalog number
is EBAG9-26510TH at the<span class="apple-converted-space"> </span></span>http://www.creativebiomart.net/<span class="apple-converted-space"> website.
The contact phone number is 1-631-559-9269.</span><br />
<br />
<br />
<br />
<u>References</u><br />
<br />
1. Nakashima, Manabu, Kenzo Sonoda, and Takeshi Watanabe. "Inhibition of cell growth and induction of apoptotic cell death by the human tumor-associated antigen RCAS1." <i>Nature Medicine</i>. 5.8 (1999): 940. Web. 29 Aug. 2013.<br />
<br />
2. Bohunicky, Brian, and Shaker Mousa. "Biosensors: the new wave in cancer diagnosis." <i>Nanotechnology, Science and Applications</i>. 2011.4 (2010): 4. Web. 29 Aug. 2013<br />
<br />
3. Sonoda, Kenzo, Shingo Miyamoto, Manabu Nakashima, Norio Wake. "The Biological Role of the Unique Molecule RCAS1: A Bioactive Marker that Induces Connective Tissue Remodeling and Lymphocyte Apoptosis." <i>Frontiers in Bioscience.</i> 13 (2008): 1106-16. Web. 29 Aug. 2013.<br />
<br />
4. Nakamura, Yuichi, Koichi Yamazaki, Satoshi Oizumi, Manabu Nakashima,Takeshi Watanabe, Hirotoshi Dosaka-Akita, and Masaharu Nishimura. "Expression of RCAS1 in human gastric carcinoma: A potential mechanism of immune escape." <i>Cancer Science</i>. 95.3 (2004): 260–265. Web. 29 Aug. 2013.<br />
<br />
5. Cox, Thomas, and Janine Erler. "Remodeling and homeostasis of the extracellular matrix: implications for fibrotic diseases and cancer." <i>Disease Models and Mechanisms</i>. 4.2 (2011): 165-178. Web. 29 Aug. 2013.<span class="apple-converted-space"><br /></span><br />
<br />
Click <a href="https://www.dropbox.com/s/u6jtmnib8tgthmv/Liaou_Daniel%20-%20Target%20Proposal.pdf">here </a>to view full target proposal.<br />
<a href="https://www.dropbox.com/s/u6jtmnib8tgthmv/Liaou_Daniel%20-%20Target%20Proposal.pdf">https://www.dropbox.com/s/u6jtmnib8tgthmv/Liaou_Daniel%20-%20Target%20Proposal.pdf</a><br />
Click <a href="https://www.dropbox.com/s/gugg5733mjf1ikb/Liaou_Daniel%20-%20Progress%20Report%201.pdf">here </a>to view Progress Report 1.<br />
<a href="https://www.dropbox.com/s/gugg5733mjf1ikb/Liaou_Daniel%20-%20Progress%20Report%201.pdf">https://www.dropbox.com/s/gugg5733mjf1ikb/Liaou_Daniel%20-%20Progress%20Report%201.pdf</a><br />
Click <a href="https://www.dropbox.com/s/m8n0ydk3kjfq9no/Liaou_Daniel%20-%20Progress%20Report%202.pdf">here </a>to view Progress Report 2.<br />
<a href="https://www.dropbox.com/s/m8n0ydk3kjfq9no/Liaou_Daniel%20-%20Progress%20Report%202.pdf">https://www.dropbox.com/s/m8n0ydk3kjfq9no/Liaou_Daniel%20-%20Progress%20Report%202.pdf</a><br />
Click <a href="https://www.dropbox.com/s/8suxn9jssocc4gu/Liaou_Daniel%20-%20Final%20Report.pdf">here </a>to view Final Report.<br />
<a href="https://www.dropbox.com/s/8suxn9jssocc4gu/Liaou_Daniel%20-%20Final%20Report.pdf">https://www.dropbox.com/s/8suxn9jssocc4gu/Liaou_Daniel%20-%20Final%20Report.pdf</a><br />
<br /></div>
Anonymoushttp://www.blogger.com/profile/10253590404889720890noreply@blogger.com0tag:blogger.com,1999:blog-1195222727010537305.post-82535051697810884432013-09-02T23:58:00.001-05:002013-12-03T22:51:30.600-06:00BRAINSTORM: NUCLEIC ACID APTAMER SELECTION AGAINST S100B<div class="MsoNormal">
By: Theresa Deike<br />
<br />
<a href="https://docs.google.com/file/d/0B63bDUZAJTrHdzFrQ2I4U0c3T2s/edit?usp=sharing">Full Proposal </a><br />
<a href="https://docs.google.com/file/d/0B63bDUZAJTrHT3pOZF9mbEliNm8/edit?usp=sharing">Progress Report 1</a><br />
<a href="https://www.dropbox.com/s/7dxauwpyol0jjj5/progressreport2_deike.pdf">Progress Report 2</a><br />
<a href="https://www.dropbox.com/s/f4cdf46i3z0pf4p/finalreport_deike.pdf">Final Report</a><br />
<b><u><i>Abstract</i></u></b><br />
<div class="MsoNormal" style="margin-right: -1.5in;">
S100B is a Calcium binding
peptide commonly associated with disorders and injuries sustained to the CNS.
Its presence has been associated with medical conditions such as traumatic
brain injury, Alzheimer’s, Down Syndrome, dementia, and other neurological
disorders. Currently the levels of S100B mRNA found intra- and extra- cellular
are used to determine levels of astrocyte cell activity and or death in the brain
and spinal cord after injury (<b>Figure 1).</b>
When the blood brain barrier ,BBB, is
functioning normally, increases in the concentration of the protein in the
brain correlate with an increased immune response due to the creation of auto
antibodies against it, a trait commonly seen in disorders such as Alzheimer’s
and dementia. <o:p></o:p></div>
<div class="MsoNormal" style="margin-right: -1.5in;">
An aptamer for S100B could not
only serve as a diagnostic tool for these CNS disorders but also serve as a way
to get in therapeutic drugs to help counteract the disruption of the blood
brain barrier once it has occurred. In
cases such as Alzheimer’s, the binding of an aptamer to the protein could help
to inhibit its interaction with glial cells by changing the shape so that it
can no longer bind to calcium ,receptors, or its own antibody.</div>
<div class="MsoNormal" style="margin-right: -1.5in;">
<br /></div>
<div class="MsoNormal" style="margin-right: -1.5in;">
Specific Aim 1: Selection of RNA
aptamers against S100B.
</div>
<div class="MsoNormal" style="margin-right: -1.5in;">
A
high affinity RNA aptamer could serve the purpose of inhibiting or detecting
S100B. By doing so, once levels of S100B reach certain levels the treatment,
detection, and possible prevention of CNS disorders associated with antibodies
against S100B could become more effective.<o:p></o:p></div>
<div class="MsoNormal" style="margin-right: -1.5in; page-break-after: avoid;">
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<br />
<div class="separator" style="clear: both; text-align: center;">
<a href="http://3.bp.blogspot.com/-KJMuQ1Uj4JA/UlTfms3X3kI/AAAAAAAAABc/pheMSp1An28/s1600/blood+brain+barrier.gif" imageanchor="1" style="margin-left: 1em; margin-right: 1em;"><img border="0" height="316" src="http://3.bp.blogspot.com/-KJMuQ1Uj4JA/UlTfms3X3kI/AAAAAAAAABc/pheMSp1An28/s320/blood+brain+barrier.gif" width="320" /></a></div>
<div class="MsoCaption" style="margin-right: -1.5in;">
<span style="font-size: x-small;"><br /></span></div>
<div class="MsoCaption" style="margin-right: -1.5in;">
<span style="font-size: x-small;"><b>Figure <!--[if supportFields]><span
style='mso-element:field-begin'></span><span
style='mso-spacerun:yes'> </span>SEQ Figure \* ARABIC <span style='mso-element:
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(Frances et al. 2003) </b> </span>
<span style="font-size: x-small;">Normally,
S100B is used in glial activation and for the CNS system on the brain side of
BBB in cells, such as the astrocyte cells pictured. Once the blood brain
barrier has been penetrated, excess amounts of S100B are secreted into the
blood allowing S100B to be used to test for the functionality of BBB. Levels on
the brain side of the barrier can also be used to test for neurological disorders
associated with high levels of S100B when the barrier is undisturbed.</span></div>
<div class="MsoCaption" style="margin-right: -1.5in;">
<o:p></o:p></div>
</div>
<div class="MsoNormal">
<br />
<div class="MsoNormal" style="margin-right: -1.5in;">
S100B is
currently in the lab so there are no additional costs associated with its
acquisition. It was provided by RayBiotech Inc. and is listed as catalog number 230-00002, Recombinant Human S100 Calcium
binding protein B. It can be purchased purchased for $326/100 ug with the
estimated cost per round totaling $7.44. RayBiotech Inc.’s contact information is <a href="http://www.raybiotech.com/">http://www.raybiotech.com/</a> and<span style="background: white; color: #444444; font-family: "Arial","sans-serif";"> (</span>770)
729-2992. <o:p></o:p></div>
<br /></div>
<div class="MsoNormal">
<br /></div>
<div class="MsoNormal">
<b><i><u>Works Cited </u></i></b> <br />
<div class="Reference" style="margin-right: -1.5in;">
<br /></div>
<div class="Reference" style="margin-right: -1.5in;">
"Blood Brain Barrier Working Group." <i>Blood
Brain Barrier</i>. John Hopkins University.<o:p></o:p></div>
<div class="Reference" style="margin-right: -1.5in;">
<br /></div>
<div class="Reference" style="margin-right: -1.5in;">
Verma, Neeraj, Madhumita Karmakar, Krishna P. Singh,
and Suchi Smita. "Structural and Dynamic Insights into S100B Protein
Activity Inhibition by Melittin for the Treatment of Epilepsy." <i>International
Journal of Computer Applications</i>. International Journal of Computer
Applications, 2013. <o:p></o:p></div>
<div class="Reference" style="margin-right: -1.5in;">
<br /></div>
<div class="Reference" style="margin-right: -1.5in;">
Wang, Doris D., and Angélique Bordey. "The
Astrocyte Odyssey." <i>PMC</i>. U.S. National Institutes of Health's
National Library of Medicine, 1 Dec. 2009. <o:p></o:p></div>
<div class="Reference" style="margin-right: -1.5in;">
<br /></div>
<div class="Reference" style="margin-right: -1.5in;">
Yardan, Turker, Ali Kemal Erenler, Ahmet Baydin,
Cengiz Cokluk, and Keramettin Aydin. "Usefulness of S100B Protein in
Neurological Disorders." Journal of Pakistan Medical Association, Mar.
2011. <o:p></o:p></div>
<div class="Reference" style="margin-right: -1.5in;">
<br /></div>
<div class="Reference" style="margin-right: -1.5in;">
Frances, Karen, Johan Van Beek, Cecile Canova, Jim W.
Neal, and Philippe Gasque. "The Bloodöbrain Barrier (BBB)." <i>Expert
Reviews in Molecular Medicine</i>. Cambridge University Press, 23 May 2003. <o:p></o:p></div>
<div class="Reference" style="margin-right: -1.5in;">
<br /></div>
<div class="Reference" style="margin-right: -1.5in;">
Marchi N, Bazarian JJ, Puvenna
V, Janigro M, Ghosh C, et al. “Consequences
of Repeated Blood-Brain Barrier Disruption in Football Players” <i>Plos One.</i>
Plos.org, Mar. 2013. <o:p></o:p></div>
<div class="Reference" style="margin-right: -1.5in;">
<br /></div>
<div class="Reference" style="margin-right: -1.5in;">
W, Pan, Xin P, Patrick S, Dean
S, Keating C, and Clawson G. "Primer-free Aptamer Selection Using a Random
DNA Library." <i>PubMed</i>. N.p., 26 July 2010. <o:p></o:p></div>
<div class="Reference" style="margin-right: -1.5in;">
<br /></div>
<div class="Reference" style="margin-right: -1.5in;">
Donato, Rosario, and Claus W.
Heizmann. "S100B Protein in the Nervous System and Cardiovascular
Apparatus in Normal and Pathological Conditions: Figure 2." <i>Hinawi</i>.
Cardiovascular Psychiatry and Neurology, 2010. <o:p></o:p></div>
<div class="Reference" style="margin-right: -1.5in;">
<br /></div>
<div class="Reference" style="margin-right: -1.5in;">
Smith, S. P., and G. S. Shaw.
"RCSB Protein Data Bank - RCSB PDB - 1UWO Structure Summary." <i>RCSB
Protein Data Bank - RCSB PDB - 1UWO Structure Summary</i>. RCSB, 1998. <o:p></o:p></div>
<div class="Reference" style="margin-right: -1.5in;">
<br /></div>
<div class="Reference" style="margin-right: -1.5in;">
"S100B [Homo
Sapiens]." <i>National Center for Biotechnology Information</i>. U.S.
National Library of Medicine<o:p></o:p></div>
<div class="Reference" style="margin-right: -1.5in;">
<br /></div>
<div class="Reference" style="margin-right: -1.5in;">
Putnam, Chris. "Protein
Calculator." <i>Protein Calculator</i>. <b><span style="font-size: 10.0pt;"><o:p></o:p></span></b></div>
</div>
Anonymoushttp://www.blogger.com/profile/02579568452190295666noreply@blogger.com0tag:blogger.com,1999:blog-1195222727010537305.post-70096578420245295542013-09-02T23:09:00.000-05:002013-12-03T20:36:40.138-06:00Nucleic Acid Aptamer Selection against Fibroblast Growth Factor 8b for Development of Diagnostics and Therapeutics for Cancer<!--[if gte mso 9]><xml>
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<b style="mso-bidi-font-weight: normal;"><u><span class="Apple-style-span" style="font-family: Arial, Helvetica, sans-serif;">Nucleic Acid Aptamer Selection against
Fibroblast Growth Factor 8b for Development of Diagnostics and Therapeutics for
Cancer</span><span class="Apple-style-span" style="font-family: 'Times New Roman'; font-size: medium;"><o:p></o:p></span></u></b></div>
<div align="center" class="MsoNormal" style="tab-stops: 89.3pt; text-align: center;">
<br /></div>
<div class="MsoNormal" style="text-align: left;">
<span class="Apple-style-span" style="font-family: Arial, Helvetica, sans-serif;">Katherine Klein</span></div>
<div class="MsoNormal" style="text-align: left;">
<span class="Apple-style-span" style="font-family: Arial, Helvetica, sans-serif;"><br /></span></div>
<div class="MsoNormal" style="line-height: 150%; tab-stops: .5in;">
<span class="Apple-style-span" style="font-family: 'Times New Roman';"> </span><span class="Apple-style-span" style="font-family: Arial, Helvetica, sans-serif;"> Cancer
is a disease that affects millions of people yet is still not curable and often
difficult to diagnose early. Cancer arises when a certain type of gene, called
oncogenes, undergoes a mutation and leads to uncontrollable cell growth. One of
these oncogenes is the fibroblast growth factor 8b, or fgf8b. Mutation or
overexpression of the fgf8b gene can often be found in cancer, in particular
hormone-related cancers such as prostate cancer. An aptamer for fgf8b could
have many possible benefits as a diagnostic tool and potentially even a
therapeutic drug. <o:p></o:p></span></div>
<div class="MsoNormal" style="line-height: 150%; tab-stops: .5in;">
<span class="Apple-style-span" style="font-family: Arial, Helvetica, sans-serif;"><span style="mso-tab-count: 1;"> </span>The
fibroblast growth factor 8b (FGF8b) is an oncogene that is involved in
embryogenesis and later in life can be related to uncontrolled cell growth
ultimately leading to the development of cancer. (Lui, 2011) Specifically,
FGF8b is found to be overexpressed in hormone-related cancers. Such hormone
related cancers that can be affected by FGF8b include breast, prostate,
endometrial, ovarian, testis, and thyroid cancers.<span style="mso-spacerun: yes;"> </span>In embryogenesis, FGF8b has found to mediate
embryonic epithetial-mesenchymal transition<b style="mso-bidi-font-weight: normal;"><span style="line-height: 150%;">
</span></b>in addition to playing
an important role in gastrulation and early organization of the blastula. (Sato,
2004)<b style="mso-bidi-font-weight: normal;"><span style="line-height: 150%;"><o:p></o:p></span></b></span></div>
<div class="MsoNormal" style="line-height: 150%; tab-stops: .5in;">
<span class="Apple-style-span" style="font-family: Arial, Helvetica, sans-serif;"><b style="mso-bidi-font-weight: normal;"><span style="line-height: 150%;"><span style="mso-tab-count: 1;"> </span></span></b>Discovery of an aptamer for FGF8b could be very beneficial,
especially in the realm of diagnostics. Overexpression of FGF8b in a person
could suggest that that person has developed cancer or is at a high risk for
cancer. (Matilla, 2007) There have been several studies involving expression of
FGF8b and prostate cancer. Usually, high levels of FGF8b are associated with a
poor prognosis in prostate cancer. (Zhong, 2006). Therefore finding an aptamer
for FGF8b could be very beneficial as it can be used as a diagnostic tool to
determine levels of FGF8b expression. Risk for certain cancers, such as
prostate cancer, could be determined early on, increasing the change of
survival from these cancers. In addition, an aptamer against FGF8b has the
potential for a therapeutic drug if the aptamer is able to inhibit or “turn
off” FGF8b expression, therefore reducing the overgrowth of cancerous
cells.<span style="mso-spacerun: yes;"> </span></span><b style="mso-bidi-font-weight: normal;"><span style="line-height: 150%;"><span class="Apple-style-span" style="font-family: Arial, Helvetica, sans-serif;"> </span><span class="Apple-style-span" style="font-family: 'Times New Roman'; font-size: medium;"><o:p></o:p></span></span></b></div>
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<b style="mso-bidi-font-weight: normal;"><span style="line-height: 150%;"><span class="Apple-style-span" style="font-family: Arial, Helvetica, sans-serif;"><br /></span></span></b></div>
<div class="separator" style="clear: both; text-align: center;">
<a href="http://www.nanotemper-technologies.com/uploads/pics/Aptamer_Thrombin_01.JPG" imageanchor="1" style="margin-left: 1em; margin-right: 1em;"><img border="0" height="103" src="http://www.nanotemper-technologies.com/uploads/pics/Aptamer_Thrombin_01.JPG" width="320" /></a></div>
<div class="separator" style="clear: both; text-align: center;">
<span class="Apple-style-span" style="font-family: Arial, Helvetica, sans-serif; font-size: x-small;"><b>Figure 1. </b>The basic mechanism of an aptamer. An aptamer containing a marker or drug binds specifically to the target of interest (in this case the target is FGF8b). Figure adapted from NanoTemper Technologies. </span></div>
<div class="MsoNormal" style="line-height: 150%; tab-stops: .5in;">
<b style="mso-bidi-font-weight: normal;"><span style="line-height: 150%;"><span class="Apple-style-span" style="font-family: Arial, Helvetica, sans-serif;"><br /></span></span></b></div>
<div class="MsoNormal">
<span class="Apple-style-span" style="font-family: 'Times New Roman';"><span class="Apple-style-span" style="font-size: 19px; line-height: 28px;"><u>Budget</u></span></span><br />
<span class="Apple-style-span" style="font-family: 'Times New Roman';"><span class="Apple-style-span" style="line-height: 28px;"><span class="Apple-style-span" style="font-size: x-small;">
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<span class="Apple-style-span" style="font-family: 'Times New Roman';"><span style="font-family: Arial;"><span class="Apple-style-span" style="font-size: x-small;">FGF8b is currently available in the lab (purified by
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</span><br />
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<span class="Apple-style-span" style="font-family: 'Times New Roman';"><span style="font-family: Arial;"><span class="Apple-style-span" style="font-size: x-small;">Provided by: Keatinge-Clay Lab<o:p></o:p></span></span></span></div>
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<span class="Apple-style-span" style="font-family: 'Times New Roman';"><span style="font-family: Arial;"><span class="Apple-style-span" style="font-size: x-small;"><span style="mso-tab-count: 2;"> </span>Office:
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</span>
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<span class="Apple-style-span" style="font-size: x-small;"><span style="font-family: Arial;"><span style="mso-tab-count: 2;"> </span>Email:
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<span style="font-family: Arial;"><span class="Apple-style-span" style="font-size: x-small;"><span style="mso-tab-count: 2;"> </span>Lab:
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<span class="Apple-style-span" style="font-size: x-small;"><span style="font-family: Arial;"><span style="mso-tab-count: 2;"> </span>Website:
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<span style="font-family: Arial;"><span class="Apple-style-span" style="font-size: x-small;">Target Cost: $0 <o:p></o:p></span></span></div>
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<span class="Apple-style-span" style="font-family: Arial, Helvetica, sans-serif;"><u>References</u><o:p></o:p></span></div>
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<br /></div>
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<span class="Apple-style-span" style="font-family: Arial, Helvetica, sans-serif;">Lui, VW, and DM Yau. "FGF8b Oncogene
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<span class="Apple-style-span" style="font-family: Arial, Helvetica, sans-serif;"><span style="mso-tab-count: 1;"> </span>from
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<span class="Apple-style-span" style="font-family: Arial, Helvetica, sans-serif;"><span style="mso-tab-count: 1;"> </span><i>Development,
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<span class="Apple-style-span" style="font-family: Arial, Helvetica, sans-serif;">Zhong, C. "Cooperation between FGF8b
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<span class="Apple-style-span" style="font-family: Arial, Helvetica, sans-serif;"><a href="https://docs.google.com/file/d/0B4s5qozntOSCMkdFc1Q4S1JMQlk/edit?usp=sharing">Target Proposal</a></span><br />
<span class="Apple-style-span" style="font-family: Arial, Helvetica, sans-serif;"><a href="https://drive.google.com/file/d/0B4s5qozntOSCWm5NamRCM1AzSzA/edit?usp=sharing">1st Progress Report</a></span><br />
<span style="font-family: Arial, Helvetica, sans-serif;"><a href="https://drive.google.com/file/d/0B4s5qozntOSCWDZLd2RWTk5PdTg/edit?usp=sharing">2nd Progress Report</a></span><br />
<a href="https://drive.google.com/file/d/0B4s5qozntOSCaGtQSHEzT0JwNmM/edit?usp=sharing"><span style="font-family: Arial, Helvetica, sans-serif;">Final Report</span></a></div>
<!--EndFragment--><br />
<!--EndFragment-->Anonymoushttp://www.blogger.com/profile/15550464499720706406noreply@blogger.com0tag:blogger.com,1999:blog-1195222727010537305.post-90068787768203870422013-09-02T22:08:00.001-05:002013-12-03T23:24:07.954-06:00Nucleic Acid Aptamer Selection Using Beta-glucuronidase for Monitoring Gene Expression<div class="separator" style="clear: both; text-align: center;">
</div>
<br />
<h2 style="text-align: center;">
<b><span style="font-family: "Times New Roman","serif"; font-size: 12.0pt;">Nucleic Acid Aptamer Selection Using
Beta-glucuronidase for Monitoring Gene Expression</span></b><span style="font-family: 'Times New Roman', serif; font-size: 12pt; text-align: center;"> </span></h2>
<div style="text-align: center;">
<span style="font-family: "Times New Roman","serif"; font-size: 12.0pt;">Sarah
Kaddour-Djebbar, Fall 2013</span></div>
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<div class="MsoNormal" style="line-height: 150%; mso-layout-grid-align: none; tab-stops: 2.5in; text-autospace: none;">
<u>Abstract<o:p></o:p></u></div>
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Aptamers are oligonucleic acids
or peptide molecules that bind to a specific target molecule and are utilized
as biomolecular recognition units. Aptamers are usually created by selecting
them from a large random sequence pool. Nucleic acid aptamers are single
stranded ssDNA or RNA. Selection is carried out through many oligonucleotides
that have a central region of variable size and random sequence and two
flanking regions of known sequence to allow for PCR amplification. Aptamers
have been engineered through repeated rounds of in vitro selection to specifically
bind to various targets, such as proteins, small molecules, nucleic acids, and
even cells and tissues. They are capable of identifying a target molecule with
high affinity and specificity, which allows for potential as diagnostic tools. Application
of aptamers is also used for both basic research and clinical purposes as
macromolecular drugs. Beta-glucuronidases, shown in Figure 1, are members of
the glycosidase family of enzymes that catalyze the breakdown of complex
carbohydrates. Beta-glucuronidase has a molecular weight of 57,000 g/mol. It functions as a negatively charged tetrameric protein
with an isoelectric charge of 4.8. Human beta-glucuronidase is located in the
lysosome. In the gut, beta-glucuronidase converts conjugated bilirubin to the
unconjugated for reabsorption. Human beta-glucuronidase is similar to the E.
coli enzyme beta-galactosidase. <o:p></o:p></div>
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Beta-glucuronidase can have
effects on a patient’s health as well as other medically related factors. Deficiencies
in beta-glucuronidase result in the non-recessive inherited metabolic disease
known as Sly syndrome. This results in the build-up of non-hydrolyzed mucopolysaccharides
in a patient. This disease can be extremely debilitating for the patient or can result in hydrops fetalis, which is a condition in the fetus characterized by an accumulation of fluid prior to birth. In addition, mental retardation, short stature, coarse facial features, spinal abnormalities, and enlargement of liver and spleen are observed in surviving patients. A certain amount of beta-glucuronidase
activity appears to be important for normal enterohepatic recirculation of
endogenous compounds such as vitamin D, thyroid hormone, and estrogen. Elevated
beta-glucuronidase can deconjugate potential toxins, increasing the formation
of carcinogens in the bowel and promoting the enterohepatic recirculation of
toxins, hormones, and various drugs in the body. Research correlates elevated
levels of beta-glucuronidase with increased colon cancer risk. In fact,
excessive beta-glucuronidase activity may be a primary factor in the etiology
of colon cancer. Beta-glucuronidase can break down epithelium cells and can
cause the spread of cancer, specifically colon cancer. There is an aptamer that
can bind and inhibit beta-glucuronidase which can detect cancer in the
epithelium cells. <o:p></o:p></div>
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Successfully
isolating an aptamer that can locate beta-glucuronidase could help doctors
detect colon cancer by observing levels of beta-glucuronidase in the body.<o:p></o:p></div>
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<u>Specific Aim 1<o:p></o:p></u></div>
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The primary specific aim of this
project focuses on generating a specific aptamer the can bind to GUS-His.<o:p></o:p></div>
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<u>Specific Aim 2<o:p></o:p></u></div>
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The next specific aim is using beta-glucuronidase
to monitor gene expression for colon cancer.<o:p></o:p><br />
<br /></div>
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<a href="http://upload.wikimedia.org/wikipedia/commons/c/cc/Beta-Glucuronidase_Homotetramer.jpg" imageanchor="1" style="margin-left: 1em; margin-right: 1em;"><img border="0" height="285" src="http://upload.wikimedia.org/wikipedia/commons/c/cc/Beta-Glucuronidase_Homotetramer.jpg" width="400" /></a></div>
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<span style="line-height: normal; text-align: center;">Figure 1: Beta-glucuronidase binding with the target proteinBeta-glucuronidases</span><span class="apple-converted-space" style="line-height: normal; text-align: center;"> </span><span style="line-height: normal; text-align: center;">are members of the</span><span class="apple-converted-space" style="line-height: normal; text-align: center;"> </span><span style="line-height: normal; text-align: center;">glycosidase</span><span class="apple-converted-space" style="line-height: normal; text-align: center;"> </span><span style="line-height: normal; text-align: center;">family of</span><span class="apple-converted-space" style="line-height: normal; text-align: center;"> </span><span style="line-height: normal; text-align: center;">enzymes</span><span class="apple-converted-space" style="line-height: normal; text-align: center;"> </span><span style="line-height: normal; text-align: center;">that</span><span class="apple-converted-space" style="line-height: normal; text-align: center;"> </span><span style="line-height: normal; text-align: center;">catalyze</span><span class="apple-converted-space" style="line-height: normal; text-align: center;"> </span><span style="line-height: normal; text-align: center;">breakdown of complex</span><span class="apple-converted-space" style="line-height: normal; text-align: center;"> </span><span style="line-height: normal; text-align: center;">carbohydrates</span><span style="line-height: normal; text-align: center;">. Human β-glucuronidase is synthesized as an 80 </span><span style="line-height: normal; text-align: center;">kDa</span><span style="line-height: normal; text-align: center;"> </span><span style="line-height: normal; text-align: center;">monomer</span><span style="line-height: normal; text-align: center;"> (653 </span><span style="line-height: normal; text-align: center;">amino acids</span><span style="line-height: normal; text-align: center;">) before </span><span style="line-height: normal; text-align: center;">proteolysis</span><span style="line-height: normal; text-align: center;"> removes 18 amino acids from the </span><span style="line-height: normal; text-align: center;">C-terminal</span><span style="line-height: normal; text-align: center;"> end to form a 78 kDa monomer. Beta-glucuronidase exists as a 332 kDa </span><span style="line-height: normal; text-align: center;">homotetramer</span><span style="line-height: normal; text-align: center;">. Beta-glucuronidase contains several notable structural formations, including a type of </span><span style="line-height: normal; text-align: center;">beta barrel</span><span style="line-height: normal; text-align: center;"> known as a jelly roll barrel and a </span><span style="line-height: normal; text-align: center;">TIM barrel</span><span style="line-height: normal; text-align: center;">.</span><br />
<br />
View the full proposal <a href="https://www.dropbox.com/s/f9kb04rrstc5h4n/Target%20Proposal.pdf" target="_blank">here</a>.<br />
View the first progress report <a href="https://www.dropbox.com/s/207wbz2c0qdf8or/Progress%20Report%201.pdf" target="_blank">here</a>.<br />
View the second progress report <a href="https://www.dropbox.com/s/pbt4rfp1l0fiqq5/Progress%20Report%202.pdf" target="_blank">here</a>.<br />
View the final report <a href="https://www.dropbox.com/s/h26jk6u4zjh32hn/Final%20Report.pdf" target="_blank">here</a>.<br />
<div>
<br /></div>
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<u>Target Order Information<o:p></o:p></u></div>
<div class="MsoNormal" style="line-height: 150%;">
Vendor: Ellington Lab<o:p></o:p></div>
<div class="MsoNormal" style="line-height: 150%;">
Vendor Website: https://www.ellingtonlab.org<o:p></o:p></div>
<div class="MsoNormal" style="line-height: 150%;">
Vendor Telephone Number:<o:p></o:p></div>
<div class="MsoNormal" style="line-height: 150%;">
Central Lab Telephone 512-471-6445<o:p></o:p></div>
<div class="MsoNormal" style="line-height: 150%;">
Office Manager 512-232-3426<o:p></o:p></div>
<div class="MsoNormal" style="line-height: 150%;">
Cost per Unit: $0<o:p></o:p></div>
<div class="MsoNormal" style="line-height: 150%;">
Cost per Round: $0<o:p></o:p></div>
<div class="MsoNormal" style="line-height: 150%;">
<br /></div>
<div class="MsoNormal" style="line-height: 150%;">
<u>References<o:p></o:p></u><br />
Sinnott, Michael, ed. (1998).<i> Comprehensive Biological Catalysis</i>. Manchester, UK: Academic Press. pp. 119-138.<br />
Nyhan, William L.; Bruce Barshop, Pinar Ozand (2005). Atlas of Metabolic Diseases (2 ed). London, UK: Hodder Arnold. pp. 501-503, 546-550.<br />
Henrissat B, Bairoch A (1993). "New families in the classification of glycosylhydrolases". Biochem J 293: 781-788.<br />
Henrissat B (1991). "A classification of glycosyl hydrolases based on amino-acid sequence similarities". Biochem J 280: 309-316.</div>
<div class="MsoNormal" style="line-height: 150%;">
<span style="line-height: 150%;">Adlercreutz H, Martin F, Pulkkinen
M, et al. Intestinal metabolism of estrogens. J Clin Endocrin Metab
1976;43(3):497-505.</span></div>
<div class="MsoNormal" style="line-height: 150%;">
<o:p></o:p></div>
<div class="MsoNormal" style="line-height: 150%;">
Kuhn JG. Pharmacology of
irinotecan. Oncology 1998;12(8 Suppl 6):39-42.<o:p></o:p></div>
<span style="line-height: 150%;">Reddy BS, Wynder EL. Large-bowel
carcinogenesis: Fecal constituents of populations with diverse incidence rates
of colon cancer. J Natl Cancer Insti 1973;50(6):1437-42.</span><br />
<div class="MsoNormal" style="line-height: 150%;">
<o:p></o:p></div>
Sarah Kaddourhttp://www.blogger.com/profile/16759704664633043091noreply@blogger.com0tag:blogger.com,1999:blog-1195222727010537305.post-4590287407925817232013-09-02T21:18:00.000-05:002013-12-03T19:04:09.454-06:00Nucleic Acid Aptamer Selection against Superoxide Dismutase 1 for the Treatment of Amyotrophic Lateral Sclerosis<h4>
<span style="font-family: inherit;">Elizabeth Adams 9/2/13</span></h4>
<h2 class="MsoNormal" style="margin: 0in 0in 10pt;">
<span style="font-family: inherit;">Nucleic Acid Aptamer Selection against Superoxide Dismutase
1 for the Treatment of Amyotrophic Lateral Sclerosis</span></h2>
<div class="MsoNormal" style="margin: 0in 0in 10pt;">
<span style="font-family: inherit;"><br />
<br />Superoxide Dismutase 1 (SOD1) is a soluble protein, found primarily in the cytoplasm of cells, that converts harmful superoxide radicals to oxygen and hydrogen peroxide [1]. Recent research advances have found that the mutant form of SOD1 plays an active role in the pathogenesis of Amyotrophic Lateral Sclerosis (ALS), or Lou Gehrig’s disease [1]. ALS is a neurodegenerative disorder that causes the death of motor neurons in the brain and spinal cord, eventually leading to fatal paralysis in 2 to 5 years (see Figure 1) [2,3,4]. More specifically, it is the first known causative gene responsible for the familial form of ALS [3]. Some evidence even shows that mutations within the exons of the gene that codes for SOD1 contribute to the onset of sporadic ALS, which makes up 90% of ALS cases [1,2]. <br />
<br />An aptamer, an RNA strand that binds with high affinity, can be found for SOD1 to develop treatments for both familial and sporadic ALS cases. It is also believed that the accumulation of SOD1 in sites directly affected by the disease, like the spinal cord and brain stem, further progresses the degeneration of nerve cells [1,5]. An aptamer could be used to detect this elevated level, and also, by some mechanism, inhibit the harmful accumulation of SOD1. <br />
<br />The mutant form of SOD1 only directly causes 2% of ALS cases worldwide, so a more important goal would be to develop a treatment based off this knowledge that could be effective in treating all the other cases of ALS [1]. Researchers are continually learning more about ALS, but much is left unknown. An aptamer effective in treating ALS cases associated with the mutation of SOD1 could be the breakthrough to creating a world without ALS. </span><br />
<span style="font-family: inherit;">
</span>
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<div class="MsoNormal" style="margin: 0in 0in 10pt;">
<u><span style="font-family: inherit;">Specific Aims: </span></u></div>
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<div class="MsoNormal" style="margin: 0in 0in 10pt;">
<span style="font-family: inherit;">The selection of RNA aptamers against SOD1 could be used to
develop treatments to halt the devastating effects of Amyotrophic Lateral
Sclerosis. Also, by inhibiting the accumulation of SOD1 that has known to exacerbate the disease, treatments can be developed. Treatment developed based on an aptamer for SOD1, to treat ALS cases caused by the mutation of SOD1 could be used and modified to develop treatments, later on, for sporadic ALS cases in which the specific cause is still unknown. </span></div>
<span style="font-family: inherit;">
</span><br />
<div class="separator" style="clear: both; text-align: center;">
<a href="http://1.bp.blogspot.com/-EcM-0xk1Eqo/UiVDQw6F-fI/AAAAAAAAAAY/2d_gF5w8gcU/s1600/ALS.jpg" imageanchor="1" style="margin-left: 1em; margin-right: 1em;"><span style="font-family: inherit;"><img border="0" height="301" src="http://1.bp.blogspot.com/-EcM-0xk1Eqo/UiVDQw6F-fI/AAAAAAAAAAY/2d_gF5w8gcU/s320/ALS.jpg" width="320" /></span></a></div>
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<span style="font-family: inherit;">
<span style="font-size: 9pt; line-height: 115%; mso-bidi-font-size: 11.0pt;"><span style="font-size: x-small;">Figure 1: Figure 1, taken from the Yale Medical Group
website, depicts a normal nerve cell compared to a nerve cell affected by
amyotrophic lateral sclerosis. The nerve cell that has been affected by ALS is
weak, shriveled, and ultimately prevents the muscle from being stimulated
properly, which causes the muscle to degenerate over time [4]. An aptamer which
halts the destruction of the nerve cell would limit the damage to the muscle.</span> </span></span><span style="font-family: inherit;"><br />
</span><br />
<div class="MsoNormal" style="margin: 0in 0in 10pt;">
<div class="MsoNormal" style="margin: 0in 0in 10pt;">
<span style="font-family: inherit;"><u>Ordering Information:</u><span style="mso-spacerun: yes;">
</span></span><br />
<br />
Superoxide Dismutase Protein (His Tag) (MW=16.8 kDa) can be ordered from Sino Biological, Inc.<br />
Catalog Number: 11727-H07E<br />
Cost for 100ug = $200<br />
Cost per Round = $6.72<br />
<u><span style="font-family: inherit;"></span></u><br />
<u><span style="font-family: inherit;">References:</span></u></div>
</div>
<div class="MsoNormal" style="margin: 0in 0in 10pt;">
<span style="font-family: inherit;">[1] <span lang="EN" style="mso-ansi-language: EN;">Milani P,
Gagliardi S, Cova E, Cereda C. “SOD1 transcriptional and posttranscriptional
regulation and its potential implications in ALS.” <em><span style="font-family: "Calibri","sans-serif"; mso-ascii-theme-font: minor-latin; mso-bidi-font-family: "Times New Roman"; mso-bidi-theme-font: minor-bidi; mso-hansi-theme-font: minor-latin;">Neurology
Research International</span></em><span class="ref-journal">, 2011. </span>Web.
30 Aug. 2013.</span></span></div>
<div class="MsoNormal" style="margin: 0in 0in 10pt;">
<span style="font-family: inherit;">[2] <span lang="EN" style="mso-ansi-language: EN;">"Genetics
of ALS." <i>Alsa.org</i>. The ALS Association, n.d. Web. 30 Aug. 2013.</span></span></div>
<div class="MsoNormal" style="margin: 0in 0in 10pt;">
<span style="font-family: inherit;">[3] <span lang="EN" style="mso-ansi-language: EN;">"Recombinant
Human SOD1 / Superoxide." <i>Sinobiological.org</i>. Sino Biological, Inc,
n.d. Web. 30 Aug. 2013.</span></span></div>
<div class="MsoNormal" style="margin: 0in 0in 10pt;">
<span style="font-family: inherit;">[4] <span lang="EN" style="mso-ansi-language: EN;">"Amyotrophic
Lateral Sclerosis (ALS)." <i>Yalemedicalgroup.org</i>. Yale School of
Medicine, 2013. Web. 30 Aug. 2013.</span></span></div>
<div class="MsoNormal" style="margin: 0in 0in 10pt;">
<span style="font-family: inherit;"><span lang="EN" style="mso-ansi-language: EN;">[5] "Laboratory
Models of ALS." <i>Alsa.org</i>. The ALS Association, n.d. Web. 30 Aug.
2013.</span></span><br />
<span style="font-family: inherit;"></span><br />
<span style="font-family: inherit;">View Full Target Proposal: <o:p></o:p></span><a href="https://docs.google.com/file/d/0B9sjx3wzW_h4Q2xyTDZSZENtYnM/edit?usp=sharing">https://docs.google.com/file/d/0B9sjx3wzW_h4Q2xyTDZSZENtYnM/edit?usp=sharing</a><br />
<br />
View Progress Report 1: <a href="https://docs.google.com/file/d/0B9sjx3wzW_h4SjNZY3l4S3VlUWc/edit?usp=sharing">https://docs.google.com/file/d/0B9sjx3wzW_h4SjNZY3l4S3VlUWc/edit?usp=sharing</a><br />
<br />
View Progress Report 2: <a href="https://drive.google.com/file/d/0B9sjx3wzW_h4cGtLNTRmZzY1TVk/edit?usp=sharing">https://drive.google.com/file/d/0B9sjx3wzW_h4cGtLNTRmZzY1TVk/edit?usp=sharing</a><br />
<br />
View Final Report: <a href="https://drive.google.com/file/d/0B9sjx3wzW_h4R0JSMkJodTlfUXc/edit?usp=sharing">https://drive.google.com/file/d/0B9sjx3wzW_h4R0JSMkJodTlfUXc/edit?usp=sharing</a></div>
<span style="font-family: inherit;">
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Anonymoushttp://www.blogger.com/profile/10043023535671654423noreply@blogger.com0tag:blogger.com,1999:blog-1195222727010537305.post-46006691097955556752013-09-02T19:45:00.003-05:002013-12-03T23:01:17.488-06:00Nucleic acid selection against PDL1 as a means of inhibiting immunosuppression by cancerous cells<!--[if !mso]>
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<span style="font-family: "Arial Narrow";">Protein A bead
based selection against PD-L1<o:p></o:p></span></div>
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<span style="font-family: "Arial Narrow";">Aptamer Stream</span></div>
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<b><span style="font-family: "Arial Narrow"; font-size: 22.0pt; line-height: 200%;">Aptamer selection against PD-L1 as a means of
inhibiting immunosuppressive properties of cancerous cells<o:p></o:p></span></b></div>
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Abstract<o:p></o:p><br />
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<span style="font-family: 'Arial Narrow'; line-height: 200%; text-indent: 0.5in;">Recent studies in the field of cancer
immunotherapy have shown that the Programmed death 1 receptor/Programed death
ligand1 (PD-1/PD-L1) pathway contributes to the ability of cancer cells to
evade the immune system. Programmed death ligand 1 (PD-L1) interacts with the
surface receptor site of cytotoxic T lymphocytes PD1, causing T cell inhibition
and apoptosis (Haile, S. et al. 2013).</span><br />
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<span style="font-family: "Arial Narrow";">Studies using flow cytometry show increased
up regulation of PD-L1 in the tissues of patients with lymphoma, suggesting
PDL1 can help promote carcinogenesis (Li, Y. et al. 2013). Iwai Y et al. (2004)
demonstrated that blocking the PD-1/PD-L1 pathway—by genetic manipulations or
antibody treatment— inhibited the metastasis of melanoma cells to the liver and
the metastasis of colon cancer cells to the lung by enhancing the recruitment
of T cells.<o:p></o:p></span></div>
<div class="MsoNormal" style="line-height: 200%; text-indent: .5in;">
<span style="font-family: "Arial Narrow";">The use of aptamers as a means of blocking
the PD-1/PD-L1 pathway is introduced as a way of promoting inhibition of
immunosuppressive properties characterized by cancerous cells. Aptamers are
short oligonucleotide sequences that have the ability to bind to a wide range
of target molecules, including drugs, proteins, and other organic or inorganic
molecules with high affinity and specificity (Ellington, A. D., 1990).<o:p></o:p></span></div>
<div class="MsoNormal" style="line-height: 200%; text-indent: .5in;">
<span style="font-family: "Arial Narrow";">Specific Aim – Find an aptamer that will
bind to PDL1. Binding an aptamer to PD-L1 has the potential to inhibit the
immunosuppression of cytotoxic T cells and promote the recruitment of cytotoxic
T cells for the destruction of cancer cells.<o:p></o:p></span></div>
<!--EndFragment--></div>
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<br /></div>
<div class="Body1">
<br /></div>
<div class="separator" style="clear: both; text-align: center;">
<a href="http://4.bp.blogspot.com/-idd4xOnCTo4/UiUwvYv6VGI/AAAAAAAAABE/Oion0bl2-V4/s1600/chart.jpg" imageanchor="1" style="margin-left: 1em; margin-right: 1em;"><img border="0" height="152" src="http://4.bp.blogspot.com/-idd4xOnCTo4/UiUwvYv6VGI/AAAAAAAAABE/Oion0bl2-V4/s320/chart.jpg" width="320" /></a></div>
<br />
<br />
<div align="center" class="Body1" style="text-align: center;">
<br /></div>
<div align="center" class="Body1" style="text-align: center;">
<b><span style="font-size: 10.0pt; mso-hansi-font-family: "Arial Unicode MS";">Figure
1.- </span></b><span style="font-size: 10.0pt; mso-hansi-font-family: "Arial Unicode MS";">An
aptamer against PDL1 can be used to block the PD1/PDL1 pathway inhibiting
immunosuppression of T cells and promoting tumor-immunity. <o:p></o:p></span></div>
<div class="Body1">
<span style="font-size: 10.0pt; mso-hansi-font-family: "Arial Unicode MS";"><br /></span>
<br />
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<span style="font-size: 10.0pt; mso-hansi-font-family: "Arial Unicode MS";">Budget</span><br />
<span style="font-size: 10.0pt; mso-hansi-font-family: "Arial Unicode MS";"><br /></span>
<span style="font-size: 10.0pt; mso-hansi-font-family: "Arial Unicode MS";">
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<span style="font-size: 10.0pt; mso-hansi-font-family: "Arial Unicode MS";"><span style="font-family: "Arial Narrow";">PD-L1 was obtained from R&D systems
(www.rndsystems.com) and is listed under the catalog number 1019-B7-100. The
protein was order as 100ug lyophilized for $329, and was reconstituted in
twenty microliters of 1X PBS plus 5mM MgCl<sub>2</sub> for a final
concentration of 100</span><span style="font-family: "Times New Roman";">μ</span><span style="font-family: "Arial Narrow";">M. The stock solution was then made into
200pmol aliquots. The cost of per round was calculated to be $32.9 for a total
of 10 rounds and was received functionalized with F/C chimera.<o:p></o:p></span></span></div>
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<span style="font-size: 10.0pt; mso-hansi-font-family: "Arial Unicode MS";"><span style="font-family: "Arial Narrow";"><br /></span></span></div>
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<!--EndFragment--></span><br />
<span style="font-size: 10.0pt; mso-hansi-font-family: "Arial Unicode MS";"><br /></span><span style="font-size: 10.0pt; mso-hansi-font-family: "Arial Unicode MS";"></span>
<span style="font-size: 10.0pt; mso-hansi-font-family: "Arial Unicode MS";"><br /></span>
<span style="font-size: 10.0pt; mso-hansi-font-family: "Arial Unicode MS";"><br /></span>
<span style="font-size: 10.0pt; mso-hansi-font-family: "Arial Unicode MS";"><br /></span>
<span style="font-size: 10.0pt; mso-hansi-font-family: "Arial Unicode MS";"><br /></span>
<span style="font-size: 10.0pt; mso-hansi-font-family: "Arial Unicode MS";">For a link to the proposal <a href="https://docs.google.com/file/d/0BxVthRhGoT08S2JaMnh4S2tFRUE/edit?usp=sharing" target="_blank">click here</a></span><span style="font-size: 10.0pt; mso-hansi-font-family: "Arial Unicode MS";"><br /></span>
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<span style="font-size: 10.0pt; mso-hansi-font-family: "Arial Unicode MS";"></span>
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<span style="font-size: 10.0pt; mso-hansi-font-family: "Arial Unicode MS";"></span>
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<span style="font-size: 10.0pt; mso-hansi-font-family: "Arial Unicode MS";"></span>
<span style="font-size: 10.0pt; mso-hansi-font-family: "Arial Unicode MS";"></span><span style="font-size: 10.0pt; mso-hansi-font-family: "Arial Unicode MS";"></span>
<span style="font-size: 10.0pt; mso-hansi-font-family: "Arial Unicode MS";"></span>
<span style="font-size: 10.0pt; mso-hansi-font-family: "Arial Unicode MS";"></span>
<span style="font-size: 10.0pt; mso-hansi-font-family: "Arial Unicode MS";"></span>
<span style="font-size: 10.0pt; mso-hansi-font-family: "Arial Unicode MS";"></span>
<span style="font-size: 10.0pt; mso-hansi-font-family: "Arial Unicode MS";"></span>
<span style="font-size: 10.0pt; mso-hansi-font-family: "Arial Unicode MS";"></span><span style="font-size: 10.0pt; mso-hansi-font-family: "Arial Unicode MS";"></span>
<span style="font-size: 10.0pt; mso-hansi-font-family: "Arial Unicode MS";"></span>
<span style="font-size: 10.0pt; mso-hansi-font-family: "Arial Unicode MS";"></span>
<span style="font-size: 10.0pt; mso-hansi-font-family: "Arial Unicode MS";"></span>
<span style="font-size: 10.0pt; mso-hansi-font-family: "Arial Unicode MS";"></span>
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<span style="font-size: 10.0pt; mso-hansi-font-family: "Arial Unicode MS";"></span>
<span style="font-size: 10.0pt; mso-hansi-font-family: "Arial Unicode MS";"></span>
<span style="font-size: 10.0pt; mso-hansi-font-family: "Arial Unicode MS";"></span>
<span style="font-size: 10.0pt; mso-hansi-font-family: "Arial Unicode MS";"></span><span style="font-size: 10.0pt; mso-hansi-font-family: "Arial Unicode MS";"></span>
<span style="font-size: 10.0pt; mso-hansi-font-family: "Arial Unicode MS";"></span>
<span style="font-size: 10.0pt; mso-hansi-font-family: "Arial Unicode MS";"></span>
<span style="font-size: 10.0pt; mso-hansi-font-family: "Arial Unicode MS";"></span><span style="font-size: 10.0pt; mso-hansi-font-family: "Arial Unicode MS";"></span>
<span style="font-size: 10.0pt; mso-hansi-font-family: "Arial Unicode MS";"></span>
<span style="font-size: 10.0pt; mso-hansi-font-family: "Arial Unicode MS";"></span><span style="font-size: 10.0pt; mso-hansi-font-family: "Arial Unicode MS";"></span>
<span style="font-size: 10.0pt; mso-hansi-font-family: "Arial Unicode MS";"><br /></span><br />
<span style="font-size: 10.0pt; mso-hansi-font-family: "Arial Unicode MS";">For a link to the first progress report <a href="https://docs.google.com/file/d/0BxVthRhGoT08TEFSSEFIdFQ0Q0U/edit?usp=sharing" target="_blank">click here</a></span>
<br />
<br />
<span style="font-size: x-small;">For a link to the second progress report <a href="https://drive.google.com/file/d/0BxVthRhGoT08YWJGWVRIVmtBaEk/edit?usp=sharing" target="_blank">click here</a></span><br />
<br />
For a link to the final report <a href="https://drive.google.com/file/d/0BxVthRhGoT08U0tkSEVBdF9wQWM/edit?usp=sharing" target="_blank">click here</a><br />
<br />
<br /></div>
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<span style="font-size: 10.0pt; mso-hansi-font-family: "Arial Unicode MS";"><br /></span>
<span style="font-size: 10.0pt; mso-hansi-font-family: "Arial Unicode MS";"><br /></span>
<span style="font-size: 10.0pt; mso-hansi-font-family: "Arial Unicode MS";"><br /></span>
<span style="font-size: 10.0pt; mso-hansi-font-family: "Arial Unicode MS";"><br /></span>
<span style="font-size: 10.0pt; mso-hansi-font-family: "Arial Unicode MS";"><br /></span>
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<span style="font-size: 10.0pt; mso-hansi-font-family: "Arial Unicode MS";">References</span></div>
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Specific Ligands. Nature, 346(6287), 818-822.<o:p></o:p></span></div>
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<!--EndFragment-->Adrian Larahttp://www.blogger.com/profile/06103088424455902879noreply@blogger.com0tag:blogger.com,1999:blog-1195222727010537305.post-18489296717546328532013-09-02T19:34:00.005-05:002013-12-03T21:10:27.497-06:00Nucleic Acid Aptamer Selection Against Burkholderia Pseudomallei for the diagnostic of Meliodosis<!--[if !mso]>
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Nucleic Acid Aptamer Selection Against Burkholderia Pseudomallei for the diagnostic of Meliodosis<br />
by Fidel Fernandez<br />
<br /></div>
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Burkholderia Pseudomallei
is the bacterium responsible for causing the disease Meliodosis. Burkholderia Pseudomallei
infects primarily Southeast Asia and Northern Australia and is common among
rice field workers. With four different types of infections caused by
Burkholderia Pseudomallei, and a Category B bioterrorism agent listing by the
Center of Disease Control (CDC), the need for a better diagnostic test is
needed. The current diagnostic test is the simple yet effective blood culture.
The problem however, with the blood culture, is that this test may take
anywhere from 24-48 hours to cultivate and the time spent diagnosing the
infection could be better spent on treating the infection. <o:p></o:p></div>
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The four types
of the infections include the localized, pulmonary, septicemia, and chronic
forms. The localized form causes swollen lymph nodes along with the development
of ulcers. Pulmonary abscesses and Pneumonia are both common symptoms
associated with the pulmonary form of Meliodosis. In the septicemia form, 90% of patients die
within the first 24-48 hours due to splenic, cutaneous, and hepatic
difficulties. Multiple abscesses form on the liver, spleen, skin, and muscle
tissues, which become more reactive with passing years. <o:p></o:p></div>
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An aptamer
against the flagella of Burkholderia Pseudomallei would be beneficial because
of the time it takes to diagnose the disease. As listed above, the septicemia
form of the disease can kill the infected within the first 24-48 hours, which
is the same amount of time that it takes to diagnose the disease with the
current diagnostic tool, the blood culture. If an aptamer against the flagella
of Burkholderia Pseudomallei was found, then the time needed to diagnose the
infection could be reduced to only a few minutes. <o:p></o:p></div>
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<a href="http://1.bp.blogspot.com/-9SjbzCWDxL0/UiUs5AGgEmI/AAAAAAAAAAY/4YuMUhrlChs/s1600/629px-Flagellum_base_diagram_en.svg.png" imageanchor="1" style="clear: left; float: left; margin-bottom: 1em; margin-right: 1em;"><img border="0" height="285" src="http://1.bp.blogspot.com/-9SjbzCWDxL0/UiUs5AGgEmI/AAAAAAAAAAY/4YuMUhrlChs/s320/629px-Flagellum_base_diagram_en.svg.png" width="320" /></a> In Figure 1, located
to the left, a flagella is looked at more closely <o:p></o:p></div>
and located in the filament
of the flagella, proteins are found that are the binding site for the potential
aptamer. An aptamer would bind to those proteins on the filament of the
flagella.<br />
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<br /></div>
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Specific Aim:<o:p></o:p></div>
<div class="MsoNormal" style="margin-right: .5in;">
To isolate an aptamer against the
Burkholderia Pseudomallei Flagella for the purpose to diagnostics.<o:p></o:p></div>
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<br />
<div class="MsoNormal">
<b>Aptamer Selection
Target<o:p></o:p></b></div>
<div class="MsoNormal">
Target Name (full name): BPSL3319 Flagellin (native)<o:p></o:p></div>
<div class="MsoNormal">
Abbreviated Name(s): FLA <o:p></o:p></div>
<div class="MsoNormal">
Date Obtained: June 2013<o:p></o:p></div>
<div class="MsoNormal">
Acquired Target from: Dr. Annie Gnanam, Dr. Katy Brown’s lab<o:p></o:p></div>
<div class="MsoNormal">
Functionalized/tagged: No<o:p></o:p></div>
<div class="MsoNormal">
Molecular Weight: 39,300 g/mol </div>
<br />
Full Proposal:<br />
<a href="https://docs.google.com/file/d/0B87nMSoAVDzpRzhzQWNuY2dxZU0/edit?usp=sharing">https://docs.google.com/file/d/0B87nMSoAVDzpRzhzQWNuY2dxZU0/edit?usp=sharing</a><br />
<br />
First Progress Report:<br />
<a href="https://docs.google.com/file/d/0B87nMSoAVDzpekVjaFlHRmt0Yk0/edit?usp=sharing">https://docs.google.com/file/d/0B87nMSoAVDzpekVjaFlHRmt0Yk0/edit?usp=sharing</a><br />
<br />
Second Progress Report:<br />
<a href="https://drive.google.com/file/d/0B87nMSoAVDzpX1Z1OWl2UmZPb2s/edit?usp=sharing">https://drive.google.com/file/d/0B87nMSoAVDzpX1Z1OWl2UmZPb2s/edit?usp=sharing</a><br />
<br />
Final Report:<br />
<a href="https://drive.google.com/file/d/0B87nMSoAVDzpQjZqeVBzNlJDWWs/edit?usp=sharing">https://drive.google.com/file/d/0B87nMSoAVDzpQjZqeVBzNlJDWWs/edit?usp=sharing</a><br />
<br />
<br /></div>
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References:</div>
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<br /></div>
<div class="MsoNormal" style="margin-right: .5in;">
</div>
<div style="font-family: 'Times New Roman'; font-size: 12px; margin-left: 36px; text-indent: -36px;">
"Burkholderia Pseudomallei." <i>Burkholderia Pseudomallei</i>. Pathema, 18 May 2006. Web. 02 Sept. 2013. </div>
<div style="font-family: 'Times New Roman'; font-size: 12px; margin-left: 36px; text-indent: -36px;">
"Meliodosis." <i>Centers for Disease Control and Prevention</i>. Centers for Disease Control and Prevention, 26 Jan. 2012. Web. 02 Sept. 2013. </div>
<div style="font-family: 'Times New Roman'; font-size: 12px; margin-left: 36px; text-indent: -36px;">
MLST. "Burkholderia Pseudomallei - Organismal Information." <i>Burkholderia Pseudomallei - Organismal Information</i>. MLST, n.d. Web. 02 Sept. 2013. </div>
<br />
<!--EndFragment-->Anonymoushttp://www.blogger.com/profile/06944679715893930628noreply@blogger.com0tag:blogger.com,1999:blog-1195222727010537305.post-7938515279769559162013-09-02T17:48:00.001-05:002013-12-03T19:29:16.926-06:00Progesterone 3-Biotin Aptamer for Breast Cancer Drug Delivery<div class="MsoNormalCxSpFirst" style="text-align: justify; text-indent: 0.5in;">
<div style="text-align: justify;">
<span style="line-height: 24px;">Progesterone 3-Biotin Aptamer for Breast Cancer Drug Delivery</span></div>
<div style="text-align: justify;">
<span style="font-size: x-small;"><span style="line-height: 24px;">Jasmin Sheth</span></span><br />
<div style="text-align: left;">
<span style="line-height: 150%; text-indent: 0.5in;"><br /></span><span style="line-height: 150%; text-indent: 0.5in;">Final Progress Report (due 12/2):</span><br />
<span style="line-height: 24px; text-indent: 0.5in;"><a href="https://drive.google.com/file/d/0B6dZaNXiPWYHRlk3T2JzMmMteXc/edit?usp=sharing">https://drive.google.com/file/d/0B6dZaNXiPWYHRlk3T2JzMmMteXc/edit?usp=sharing</a></span><br />
<span style="line-height: 150%; text-indent: 0.5in;"><br /></span>
<span style="line-height: 150%; text-indent: 0.5in;">Progress (due 11/4) Report: </span><br />
<span style="line-height: 150%; text-indent: 0.5in;"><a href="http://share.pdfonline.com/98ca5223ecce4b4792ff972d569faf2b/Sheth_Jasmin-Second%20Progress%20Report-ProgesteroneN40B.htm">http://share.pdfonline.com/98ca5223ecce4b4792ff972d569faf2b/Sheth_Jasmin-Second Progress Report-ProgesteroneN40B.htm</a></span><br />
<br />
Second Progress Report: <a href="http://share.pdfonline.com/2a1066c2d2984488bdf5d198016d428b/Sheth_Jasmin-Progress%20Report-ProgesteroneN40B-10.08.13.htm" style="text-indent: 0.5in;">http://share.pdfonline.com/2a1066c2d2984488bdf5d198016d428b/Sheth_Jasmin-Progress%20Report-ProgesteroneN40B-10.08.13.htm</a></div>
</div>
<div>
<div style="line-height: 150%;">
<br /></div>
<div style="line-height: 150%;">
Progesterone
(<b>Figure 1</b>) is a versatile steroid
hormone that belongs to the group of hormones, progestogens. In humans,
progesterone is involved with pregnancy, menstrual cycle, and embryogenesis.
Progesterone is produced in the ovaries during ovulation, the adrenal glands,
and during pregnancy, in the placenta. It is also synthesized by the central
nervous system and can act as a neurosteroid. Many steroid hormones are derived
from progesterone.<o:p></o:p></div>
</div>
</div>
<div class="MsoNormalCxSpMiddle" style="line-height: 150%; text-align: justify; text-indent: 0.5in;">
Progesterone
is sometimes called the “hormone of pregnancy”.<sup>2</sup> But besides its
role in pregnancy, progesterone also helps with water and sodium retention in
the kidneys, decreases cancer risks, increases libido, regulates apoptotic
genes<sup>3</sup>, protects against atherosclerosis<sup>4</sup>, and acts as an
anti-inflammatory, antidepressant, and anti-anxiety agent among many other
things.<sup>5</sup> Progesterone reaches every part of the body and the cell.
It can directly impact transcription in the nucleus or signal human sperm
non-genomically.<sup>6</sup> Progesterone is a small hormone and can easily
pass through the cell membrane due to its hydrophobic characteristic. Such
diversity in its actions allows for a variety of aptamers for or against
progesterone to carry out many different functions. The ease with which
progesterone travels around the body and enters the cell would allow for a more
direct and efficient way of drug delivery with the help of an aptamer. <o:p></o:p></div>
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<b style='mso-bidi-font-weight:
normal'><span style='font-size:10.0pt;mso-bidi-font-size:11.0pt'>Figure 1.
Progesterone molecule. </span></b><span style='font-size:10.0pt;mso-bidi-font-size:
11.0pt'>A steroid hormone that is produced by the ovaries and adrenal
glands and plays an imperative role in pregnancy.<sup>1<o:p></o:p></sup></span></p>
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<o:p> </o:p></p>
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o:title="Benefits of Natural Progesterone Cream"/>
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</v:shape><![endif]--><!--[if !vml]--><!--[endif]-->Human
breast cancer tumors are often hormone-dependent. A hormone, progesterone and
estrogen in the case of breast cancer, binds to its receptor and promotes tumor
growth. The epidermal growth factor receptor is important in normal cell growth
as well as tumor growth. Current research shows that a cancer drug, Iressa, can
effectively block this receptor and inhibit the growth of tumor cells along
with normal cells. An aptamer for progesterone can be found to deliver
specialized cancer drugs specific to certain breast cancer cells and further
inhibit metastasis and tumor growth.<sup>7<o:p></o:p></sup></div>
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<sup><br /></sup></div>
<div class="MsoNormalCxSpLast" style="line-height: 150%; text-align: justify;">
<u>Specific aim:</u> Select
for an aptamer for progesterone that delivers specific breast cancer drugs
straight to the nucleus of the cell.<o:p></o:p></div>
<div class="MsoNormalCxSpLast" style="line-height: 150%;">
<br /></div>
<div class="separator" style="clear: both; text-align: center;">
<a href="http://3.bp.blogspot.com/-GiBFaaXkebc/UiUKW2_qK_I/AAAAAAAAABQ/slJeCqzbxG0/s1600/fotolia_2018396_XS.jpg" imageanchor="1" style="margin-left: 1em; margin-right: 1em;"><img border="0" height="212" src="http://3.bp.blogspot.com/-GiBFaaXkebc/UiUKW2_qK_I/AAAAAAAAABQ/slJeCqzbxG0/s320/fotolia_2018396_XS.jpg" width="320" /></a></div>
<div class="MsoNormalCxSpFirst">
<b><span style="font-size: 10.0pt; mso-bidi-font-size: 11.0pt;">Figure 1.
Progesterone molecule. </span></b><span style="font-size: 10.0pt; mso-bidi-font-size: 11.0pt;">A steroid hormone that is produced by the ovaries and adrenal glands
and plays an imperative role in pregnancy.<sup>1<o:p></o:p></sup></span></div>
<div class="MsoNormalCxSpLast" style="line-height: 150%;">
<br /></div>
<div class="MsoListParagraphCxSpFirst" style="line-height: 150%; tab-stops: 1.0in;">
1. Jackson-Michel, Shavon. "Benefits of Natural Progesterone Cream." Livestrong.com.<br />
Demand Media, Inc., 03 Jun 2010. Web. 8 Apr 2013.<br />
<http://www.livestrong.com/article/138465-benefits-natural-progesterone-cream/>.<br />
2. Bowen, R. "Placental Hormones." Pathophysiology of the Reproductive System.<br />
Colorado <span style="line-height: 150%;">State University, 06 Aug 2000. Web. 7 Apr 2013</span><br />
3. Maruo T, Matsuo H, Samoto T, Shimomura Y, Kurachi O, Gao Z, Wang Y, Spitz<br />
IM, <span style="line-height: 150%;">Johansson E. (2000) “Effects of progesterone on uterine leiomyoma </span><br />
<span style="line-height: 150%;"> growth and apoptosis.”</span><span style="line-height: 150%;"> Steroids. 65(10-11):585-92.</span><br />
4. Lee WS, Harder JA, Yoshizumi M, Lee ME, Haber E. (1997) “Progesterone inhibits<br />
arterial smooth muscle cell proliferation.” Nat Med. 3(9):1005-8.<br />
5. Taylor, Eldred B., and Ava Bell-Taylor, M.D. Are Your Hormones Making You<br />
Sick?. Maximum Health Education, 2000. Print.<br />
6. Correia JN, Conner SJ, Kirkman-Brown JC. (2007) ‘Non-genomic steroid actions in<br />
human spermatozoa. "Persistent tickling from a laden environment.”’<br />
Semin Reprod Med. <span style="line-height: 150%;">25(3):208-19.</span><br />
7. "Researchers unlock how progesterone increases breast cancer risk." Phys.org.<br />
Michigan<span style="line-height: 150%;"> State University, 18 Jan 2011. Web. <http://phys.org/news/</span><br />
<span style="line-height: 150%;"> 2011-01-progesterone-breast-c</span><span style="line-height: 150%;">ancer.html>.</span><br />
<span style="line-height: 150%;"><br /></span>
Vendor: Cayman Chemicals</div>
<div class="MsoListParagraphCxSpFirst">
<span style="line-height: 24px;">Product Item Number: 9000644</span></div>
<div class="MsoListParagraphCxSpFirst">
<span style="line-height: 24px;">Website: www.caymanchem.com</span></div>
<div class="MsoListParagraphCxSpFirst">
<span style="line-height: 24px;">Telephone Number: (800) 364-9897</span></div>
<div class="MsoListParagraphCxSpFirst">
<span style="line-height: 24px;">Cost per unit: $56.00 per 500 ug</span></div>
<div class="MsoListParagraphCxSpFirst">
<span style="background-color: white; font-family: 'Segoe UI', 'Lucida Sans Unicode', 'Lucida Grande', 'Arial Unicode MS', 'DejaVu Sans', serif; font-size: 14px; line-height: 14px;"><br /></span></div>
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<o:p></o:p></div>
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