Citation
Inhibition and Apoptotic Responses of Human Colorectal Carcinoma Cells by Anti-Survivin Small Interfering RNA

Material Information

Title:
Inhibition and Apoptotic Responses of Human Colorectal Carcinoma Cells by Anti-Survivin Small Interfering RNA
Series Title:
Journal of Undergraduate Research
Creator:
Lin, Philip W.
Liu, Chen ( Mentor )
Place of Publication:
Gainesville, Fla.
Publisher:
University of Florida
Publication Date:
Language:
English

Subjects

Genre:
serial ( sobekcm )

Notes

Abstract:
As evidenced by the 940,000 cases and 655,000 deaths worldwide per year, colon cancer is one of the most prominent diseases in the world. Within the United States alone, colon cancer has been linked to over 56,000 deaths and is considered the second leading cause of cancer death. Colon cancer, or colorectal cancer, develops from the growth of adenomatous polyps in the colon due to mutations in the DNA of the gastrointestinal epithelial cells. The advancement of the formation of polyps in the colon results in cancer that has the ability to metastasize to adjacent organs, such as the liver. Currently 3 different forms of treatment are used in combination. Surgical removal of the cancerous growth is the primary treatment for colon cancer. Through the colectomy procedure, the cancerous mass is removed and the colon resected, restoring the original purpose of the colon. Colostomy surgery also excises the cancerous growth, but the colon is instead attached to the anterior abdominal wall due to the unrestricted and increased manifestation of the cancerous cells. Despite the advancements in surgery, approximately 40-50% of patients will relapse and require additional therapies and treatments (2). Subsequent applications of chemotherapy or radiation are used to treat metastasized cancer and prevent such tumor recurrence after surgery. These treatments stimulate cancer cells to enter apoptotic pathways, causing cell death in these targeted cells. There have been well-documented cases of resistance to apoptotic stimuli. This resistance to chemotherapy and radiation could result from the over-expression of anti-apoptotic factors. These factors, such as survivin, are predominately expressed in cells of common human cancers. Cell division is coupled with checkpoints along the different stages of cell life. These checkpoints allow the prevention of the developments of abnormalities such as mutations, by inducing the abnormal cell to enter the apoptotic pathway. By this definition, apoptosis is a state of programmed cell death due to DNA damage or cellular aging. The balance between cell growth and checkpoints is an essential feature in preventing the development of any form of cancer. Failure of the induction of apoptosis in cells that contain abnormalities in the mitotic spindle, DNA structure, and mutations in oncogenes could result in increased resistance to chemotherapy and radiation. There are two major apoptotic pathways. The extrinsic pathway involves extracellular inducers such as toxins, hormones and growth factors that induce cellular signals for apoptosis. The intrinsic pathway results in the mitochondrial release of cytochrome c and a caspase cascade that mediates cellular apoptosis. Survivin is a protein belonging to the inhibitor of apoptosis protein (IAP) family and is an important regulator of the intrinsic apoptotic pathway. The survivin gene is located on chromosome 17, which encodes a 16.5 kD protein containing 142 amino acids and a Baculovirus IAP Repeat (BIR). The survivin protein regulates the intrinsic apoptotic pathway by interfering with caspase-3, caspase-7, and caspase-9 activity. Also, the association of survivin with the cell cycle and mitotic spindle is consistent with the 40-fold increase in expression levels during the G2/M phase in HeLa cells. Its expression is upregulated in most human cancer cells, but undetectable or found at very low levels in normal adult tissues. The increased activity of survivin in cancer cells in contrast to normal somatic cells provides an ideal target for cancer treatment. Current research provides the technology to synthetically produce a 21-23 RNA nucleotide molecule that would be able to effectively inhibit specific gene expression by RNA interference. This small interfering RNA (siRNA) disrupts the expression of a gene by down-regulating the desired target protein. The siRNA is designed by selecting target sequences from the RNA of a gene of interest. The sense and anti-sense templates are placed on both sides of a loop sequence in a plasmid DNA. The transcription of siRNA uses an RNA Polymerase III promoter (U6 or H1), which allows the transcription of short hairpin RNA. The single strand complementarily binds to the target mRNA and consequently down-regulates targeted gene expression through the RNA-induced silencing complex (RISC). The transfection of siRNA through plasmids is limited to in vitro use. In vivo delivery of siRNA may be optimized through the use of a viral vector such as an adeno-associated virus (AAV). The adenovirus (AV) and the adeno-associated virus (AAV) have been used successfully for delivery in gene therapy. Examples of this include the successful delivery of a survivin mutant to breast cancer cells by using adenoviruses, resulting in apoptosis in cancerous cells and inhibited growth of tumors (14). We hypothesize that by introducing siRNA that specifically inhibits survivin expression, cancer cells will be more susceptible to apoptotic stimuli and ultimately result in apoptosis. A vector will be constructed with four targets of the survivin gene. These 4 targets will be analyzed for their efficacy in in-vitro down-regulation of survivin protein in cancerous cell lines. The optimal siRNA target will then be cloned into an adeno-associated virus (AAV) for further in vivo studies. The inhibition of colorectal carcinoma cells by siRNA survivin could prove to be a potential therapy for colon cancer.

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University of Florida
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Inhibition and Apoptotic Responses of Human Colorectal
Carcinoma Cells by Anti-Survivin Small Interfering RNA


College of Medicine, University of Florida


INTRODUCTION

As evidenced by the 940,000 cases and 655,000 deaths
worldwide per year, colon cancer is one of the most
prominent diseases in the world (1). Within the United
States alone, colon cancer has been linked to over 56,000
deaths and is considered the second leading cause of cancer
death (1).
Colon cancer, or colorectal cancer, develops from the
growth of adenomatous polyps in the colon due to
mutations in the DNA of the gastrointestinal epithelial cells.
The advancement of the formation of polyps in the colon
results in cancer that has the ability to metastasize to
adjacent organs, such as the liver.
Currently 3 different forms of treatment are used in
combination. Surgical removal of the cancerous growth is
the primary treatment for colon cancer. Through the
colectomy procedure, the cancerous mass is removed and
the colon rejected, restoring the original purpose of the
colon. Colostomy surgery also excises the cancerous
growth, but the colon is instead attached to the anterior
abdominal wall due to the unrestricted and increased
manifestation of the cancerous cells. Despite the
advancements in surgery, approximately 40-50% of
patients will relapse and require additional therapies and
treatments (2). Subsequent applications of chemotherapy or
radiation are used to treat metastasized cancer and prevent
such tumor recurrence after surgery. These treatments
stimulate cancer cells to enter apoptotic pathways, causing
cell death in these targeted cells. There have been well-
documented cases of resistance to apoptotic stimuli. This
resistance to chemotherapy and radiation could result from
the over-expression of anti-apoptotic factors. These factors,
such as survivin, are predominately expressed in cells of
common human cancers.
Cell division is coupled with checkpoints along the
different stages of cell life. These checkpoints allow the
prevention of the developments of abnormalities such as
mutations, by inducing the abnormal cell to enter the
apoptotic pathway. By this definition, apoptosis is a state
of programmed cell death due to DNA damage or cellular
aging.

Rafal P. Witek, Xiaokui Zhang, Han Zongchao, Arun Srivastava,
Chen Liu


The balance between cell growth and checkpoints is an
essential feature in preventing the development of any form
of cancer (3). Failure of the induction of apoptosis in cells
that contain abnormalities in the mitotic spindle, DNA
structure, and mutations in oncogenes could result in
increased resistance to chemotherapy and radiation (4, 5).
There are two major apoptotic pathways. The extrinsic
pathway involves extracellular inducers such as toxins,
hormones and growth factors that induce cellular signals
for apoptosis. The intrinsic pathway results in the
mitochondrial release of cytochrome c and a caspase
cascade that mediates cellular apoptosis (6, 7).
Survivin is a protein belonging to the inhibitor of
apoptosis protein (IAP) family and is an important
regulator of the intrinsic apoptotic pathway (8). The
survivin gene is located on chromosome 17, which encodes
a 16.5 kD protein containing 142 amino acids and a
Baculovirus IAP Repeat (BIR) (9). The survivin protein
regulates the intrinsic apoptotic pathway by interfering
with caspase-3, caspase-7, and caspase-9 activity (10).
Also, the association of survivin with the cell cycle and
mitotic spindle is consistent with the 40-fold increase in
expression levels during the G2/M phase in HeLa cells (11).
Its expression is upregulated in most human cancer cells,
but undetectable or found at very low levels in normal
adult tissues (10).
The increased activity of survivin in cancer cells in
contrast to normal somatic cells provides an ideal target for
cancer treatment. Current research provides the technology
to synthetically produce a 21-23 RNA nucleotide molecule
that would be able to effectively inhibit specific gene
expression by RNA interference (12, 13). This small
interfering RNA (siRNA) disrupts the expression of a gene
by down-regulating the desired target protein. The siRNA
is designed by selecting target sequences from the RNA of
a gene of interest. The sense and anti-sense templates are
placed on both sides of a loop sequence in a plasmid DNA.
The transcription of siRNA uses an RNA Polymerase III
promoter (U6 or H1), which allows the transcription of
short hairpin RNA. The single strand complementarily
binds to the target mRNA and consequently down-
regulates targeted gene expression through the RNA-
induced silencing complex (RISC)
The transfection of siRNA through plasmids is limited
to in vitro use. In vivo delivery of siRNA may be


University of Florida I Journal of Undergraduate Research I Volume 11, Issue 1 I Fall 2009


Philip W. Lin*





PHILIP W. LIN


optimized through the use of a viral vector such as an
adeno-associated virus (AAV). The adenovirus (AV) and
the adeno-associated virus (AAV) have been used
successfully for delivery in gene therapy. Examples of this
include the successful delivery of a survivin mutant to
breast cancer cells by using adenoviruses, resulting in
apoptosis in cancerous cells and inhibited growth of tumors
(14).
We hypothesize that by introducing siRNA that
specifically inhibits survivin expression, cancer cells will
be more susceptible to apoptotic stimuli and ultimately
result in apoptosis. A vector will be constructed with four
targets of the survivin gene. These 4 targets will be
analyzed for their efficacy in in-vitro down-regulation of
survivin protein in cancerous cell lines. The optimal siRNA
target will then be cloned into an adeno-associated virus
(AAV) for further in vivo studies. The inhibition of
colorectal carcinoma cells by siRNA survivin could prove
to be a potential therapy for colon cancer.

MATERIALS AND METHODS

Materials

The siRNA expression vector, pSilencer 3.0-H1 (Fig. 1),
was ordered from Ambion, Inc (Austin, TX). The retroviral
vector containing the shRNA-TERT, pSHAG-Magic2-
shTERT, was ordered from Open Biosystems (Huntsville,
AL). All restriction endonucleases including Ecor I, Xho I,
Hind III, Xba I, and BamH I was purchased through
Promega Corp. (Madison, Wisconsin). Survivin
monoclonal antibody was purchased from Santa Cruz
Biotechnology Inc. (Santa Cruz, California). Lipofectin
Transfection Reagent was obtained from Invitrogen Corp.
(Carlsbad, California). Cell lines HT29, HCT 116, and
Huh7.5 were stored by our department.


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Figure 1: siRNA mammalian expression vector used for
Survivin siRNA ligation.


Design and Construction of Survivin siRNA Expression
Vector. The best possible target candidates for survivin
siRNA were chosen by processing human survivin mRNA
(NM001168) through the Ambion (Ambion, Inc; Austin,
TX) siRNA Target Finder. Target sequences were limited
to approximately 50% GC content. Four survivin DNA
templates corresponding to the gene survivin were
synthesized as following: survivin-67 sense, 5'-
GGACCACCGCATCTCTACA-3'; survivin-248 sense, 5'-
AGCATTCGTCCGGTTGCGC-3'; survivin-331 sense, 5'-
ACTGGACAGAGAAAGAGCC-3'; survivin - 401 sense,
5' - ACTGCGAAGAAAGTGCGCC-3'. Using the selected
target sequences, oligonucleotides will be designed using
the same Ambion software and prepared for cloning into
the siRNA expression vector pSilencer 3.0-H1.
The siRNA survivin sequences are inserted between the
restriction sites BamHI (496) and HindIII (557). Directly
upstream of the siRNA sequence is the H1 Promoter,
which is essential for the transcription process as provided
by RNA Polymerase III. The vector pSilencer 3.0-H1 also
has a region that confers ampicillin resistance, which will
be exploited for purification and isolation of the expression
vector.
Transformation of Survivin siRNA Expression Vector.
The siRNA expression was transformed into DH5a
bacterial cells. The transformation reactions were then
plated on LB plates containing the antibiotic ampicillin and
grown overnight at 370C.
Sequencing and Purification of Survivin Expression
Vector. Four colonies per target sequence were abraded,
isolated, and purified using QIAprep Spin Miniprep Kit.
Each of the colonies corresponding to the 4 survivin target
sequences was kindly sequenced by Han Zongchao from
Dr. Arun Srivastava's Lab. The following are the
sequencing primers as provided by Ambion: M13-forward
sequencing primer: 5'-GTTTTCCCAGTCACGAC-3'; 5'-
GAGTTAGCTCACTCATTAGGC-3'. After sequences
were confirmed and verified that the clones contain the
survivin target insert, the pSilencer plasmids were purified
through QIAprep Spin Midiprep Kit.
Western Blot. The HCT116 cell line was transfected
with survivin siRNA and protein was extracted 72 after
transfection. After purification of the protein, a Western
blot analysis was performed to determine the most efficient
siRNA survivin sequence that down-regulated the survivin
protein in cancerous cell lines. Western Blot Analysis was
kindly performed by Xiaokui Zhang.
Transfection of Survivin and hTERT at Different
Concentrations. Huh7.5 cell lines were grown to
approximately 50% confluency a day prior to transfection.
The cell lines were then transfected with the survivin
siRNA molecule that was most efficient in down-


University of Florida I Journal of Undergraduate Research I Volume 11, Issue 1 I Fall 2009
2





INHIBITION AND APOPTOTIC RESPONSES OF HUMAN COLORECTAL CARCINOMA CELLS


regulating survivin (survivin67). The concentrations at
which transfection occurred were at 1 ug, 2 ug and 3 ug. 72
hours after transfection, pictures were taken and cells were
counted.
The previously described protocol was applied to
hTERT at the same concentrations as described above.
Cells were counted after 72 hours from the start of the
transfection.
Co-Transfection of Survivin siRNA. Three cell lines
(Huh7.5, HCT116, and HT29) were cultured to
approximately 50% confluency the day prior to
transfection. The cell lines were transfected with a negative
plasmid, survivin-331 vector, hTERT vector, and both
vectors, respectively. Pictures were taken and cells were
counted 72 hours after transfection.
Adeno-Associated Virus (AAV) packaging. The
packaging of AAV requires approximately 800-1000 ug/ul
of the AAV vector and two helper vectors (pHelper,
pACG-2). Han Zhongchao kindly supplied the viral vectors
from Dr. Arun Srivastava's lab. The pSilencer3.0-H1
containing the sur67 siRNA sequence and the H1 promoter
were excised using the restriction enzymes EcorI and
HindIII. This segment was then inserted into the pdsAAV-
EGFP between the restriction site Acc65I (1334), which is
located directly downstream of the mutated ITR. The
successful ligation of the sur67 sequence into the AAV
vector was confirmed by restriction digest with BamH1,
BamH1 with XbaI, and BamH1 w/ HindIII, respectively.
The pHelper and pACG-2 vectors were also confirmed
by restriction digest with the restriction enzymes Clal and
XmnI. After restriction digest was performed on all 3
vectors, they were maxi-prepped and quantified for the
concentration. The vectors were then sent to the UF core
lab for further packaging of the virus for effective viral
delivery.




RESULTS

Western Blot Analysis

Western blot analysis was performed twice. In both
experiments, survivin-67 limited the greatest amount of
survivin protein expression in the HCT 116 cell line after
72 hours post-transfection. The results in the HCT116 cell
lines represent significant knockdown of the survivin
protein expression by siRNA treatment. These results
allowed us to discern that survivin-67 would be the best
survivin target to introduce as a siRNA for gene therapy.
The subsequent experiments after the Western blot was
completed with survivin-67 as the only target for the
siRNA.


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Figure 2: Western blot analysis of HCT116 cell lines transfected
for 72 hours with survivin-67, 259, 331, 401, respectively


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Figure 3: Western blot analysis of HCT 116 cell lines
transfected for 72 hours with survivin-331 (wells 1-3), survivin-
67 (wells 4-6), survivin-259 (wells 7-9), siu i ii\ i-4-1 (10-12)


Apoptotic Cell Death

The results of Huh7.5 cells transfected with hTERT
siRNA shows greater than a 50% decrease in cell viability
in a dose dependent manner (Fig. 4a). Increasing
concentrations of hTERT siRNA shows greater apoptotic
response. Transfection of Huh7.5 cells with survivin
siRNA shows a similar pattern in apoptotic response. 1 ug
of survivin siRNA decreased cell viability by
approximately 30%, while 2 ug and 4 ug of survivin


University of Florida I Journal of Undergraduate Research I Volume 11, Issue 1 I Fall 2009
3


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PHILIP W. LIN


siRNA decreased cell viability by greater than 50% (Fig.
3b).
Huh7.5 and HT29 cell lines all decreased in cell
viability in response to different treatments of siRNA (Fig.
6c). Cotransfection of survivin and hTERT results in
greater apoptotic response in HCT116 and HT29 cell lines.
HCT116 cells displayed virtually no increase in cell death
when treated with only survivin or hTERT, but decrease in
cell viability was observed when cotransfected with
survivin and hTERT.


A. Transfection of Huh7.5 cells with
hTERT -72 hours

120
o
S 100 0
S 80 -80
C 60
S40 -
ao 20
I 0
Control 1 ug 2 ug 3 ug
hTERT Plasmid Concentration



B. Transfection of Huh7.5 cells with
Survivin

5 120
S100
u 80-
0 60-
1 40
'O 20

Control lug 2ug 3ug
Survivin Plasmid Concentration



C. Transfection of Cell Lines with
Survivin and hTERT Treatments

120

e5 60 8 5*HCT1
4 HT29

Control 2 ug 2 ug 2 ug
SurImn hTERT Surymn
&
hTERT
siRNA Treatments


Figure 4: a) Transfection of Huh7.5 cell lines with different
concentrations of hTERT siRNA b) Transfection of Huh7.5 cell
lines with different concentrations of survivin siRNA c)
Transfection of Huh7.5, HCT116, and HT29 cell lines with
different treatments of survivin siRNA, hTERT siRNA, and
cotransfection of survivin and hTERT siRNA


Adeno-Associated Virus (AA V) packaging


Presented in Figure 5 are the vector maps for pdsAAV-
EGFP, pHelper, and pACG-2 paralleled with their
corresponding restriction digests that confirm their identity.
The confirmation of the viral vectors allows us to continue
with AAV packaging for further use for in vivo studying.


Ba0Ill (1)
H1 Promoter siRNA-sur67
A oRI (j4699) \ | flII (64)
ApdA (24110) \ #


pSilencer-sur67
2797 bI6
Ap&] (1983)




Mutated ITR H1 Promoter
S sRNA-sur67

intron



pdsAAV-sur67-EGFP' EGF


- poly


ApUt (t17


Figure 5: a) vector maps of pSilencer3.0-H1 with survivin67
insert and ligation into pdsAAV-EGFP b) restriction digest of
pdsAAV-sur67-EGFP with BamH1, BamH1 + Xbal, BamH1 +
HindIII, respectively


University of Florida I Journal of Undergraduate Research I Volume 11, Issue 1 I Fall 2009






INHIBITION AND APOPTOTIC RESPONSES OF HUMAN COLORECTAL CARCINOMA CELLS


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Figure 6: a) Vector maps of pHelper and pACG-2 b) restriction
digest of pHelper and pACG-2 (last two wells, respectively) with
Clal and XmnI.

DISCUSSION

Inhibition of survivin through RNA interference has
provided the scientific community with new insight and
prospective therapies to cancer treatment. The importance
of targeting survivin stems from the identification that both
are expressed in cancerous cells, but are limited in their
expression in normal somatic cells.
Current Western blot results have shown that survivin-
67 is the most effective siRNA treatment in inhibiting
survivin protein expression. Experimental data provided
evidence that RNA interference through siRNA survivin
and hTERT was effective in inducing an apoptotic
response in Huh7.5 and HT29 cell lines. Cotransfection of
survivin and hTERT did produce an apoptotic response in
HCT116 and HT29 cells, but the difference in


cotransfection response was not distinguished enough from
the independent treatments of survivin and hTERT.
One problem that may present itself is the accuracy in
using the HCT116 alone in defining the most efficient
siRNA sequence for survivin. Despite the ability of
survivin-67 siRNA to down-regulate the survivin protein in
HCT116 cell lines, the transfection of survivin and/or
hTERT proved to induce a very small response in
apoptosis of the cells. It is suggested that the Huh7.5 and
HT29 cell lines also be used to determine the most efficient
siRNA sequence since the transfection of survivin and/or
hTERT produced a drastic response in apoptosis.
Future treatment could include such a therapy that
would combine the efficacy of chemotherapy and the use
of siRNA survivin to induce apoptosis in those cells that
were initially resistant to radiation. One still must consider
the in vivo results with the use of a mouse model and
determine the best mode of delivery of AAV into the
targeted region of the host. Even with the advancement of
biomedical technology, one can envision small-interfering
RNA and adeno-associated virus therapy to be novel
treatments in the fight against cancer. The survivin AAV
will hopefully open up new avenues for in vivo animal
studies in order to determine the usefulness of survivin
siRNA therapy against cancer.

REFERENCES

1. Jemal,A., Tiwari,R.C., Murray,T., Ghafoor,A., Samuels,A., Ward,E.,
Feuer,E.J., & Thun,M.J. Cancer statistics, 2004. CA Cancer J. Chn. 54, 8-29
(2004).

2. Obrand,D.I. & Gordon,P.H. Incidence and patterns of recurrence following
curative resection for colorectal carcinoma. Dzs. Colon Rectum 40, 15-24 (1997).

3. Hanahan,D. & Weinberg,R.A. The hallmarks of cancer. Cell 100, 57-70 (2000).

4. Reed,J.C. Dysregulation of apoptosis in cancer. J. Clin. Oncol. 17, 2941-2953
(1999).

5. Soengas,M.S., Capodieci,P., Polsky,D., Mora,J., Esteller,M., Opitz-Araya,X.,
McCombie,R., Herman,J.G., Gerald,W.L., Lazebnik,Y.A., Cordon-Cardo,C., &
Lowe,S.W. Inactivation of the apoptosis effector Apaf-1 in malignant melanoma.
Nature 409, 207-211 (2001).

6. Ravi,R. & Bedi,A. Potential methods to circumvent blocks in apoptosis in
lymphomas. Curr. Opin. Oncol. 14, 490-503 (2002).

7. Zaffaroni,N., Pennati,M., & Daidone,M.G. Survivin as a target for new
anticancer interventions. J. CellMolMed 9, 360-372 (2005).

8. Ambrosini,G., Adida,C., & Altieri,D.C. A novel anti-apoptosis gene, survivin,
expressed in cancer and lymphoma. Nat. Med3, 917-921 (1997).

9. LaCasse,E.C., Baird,S., Korneluk,R.G., & MacKenzie,A.E. The inhibitors of
apoptosis (IAPs) and their emerging role in cancer. Oncogene 17, 3247-3259
(1998).

10. Zaffaroni, N., Pennati, M., & Daidone, M.G. Survivin as a target for new
anticancer interventions. J. Cell Mol Med 9, 360-372 (2005)


University of Florida I Journal of Undergraduate Research I Volume 11, Issue 1 I Fall 2009
5






PHILIP W. LIN


11. Chen, J., Wu, W., Tahir, S.K., Kroeger, P.E., Rosenberg, S.H., Cowsert, L.M.,
Bennett, F., Karjewski, S., Karjewska, M., Welsh, K., Reed, J.C., % Ng, S.C.
Downregulation of surviving by antisense oligonucleotides increases apoptosis,
inhibits cytokinesis and anchorage-independent growth. Neoplasia. 2, 235-241
(2000).


13. Kappler,M., Bache,M., Bartel,F., Kotzsch,M., Panian,M., Wurl,P., Blumke,K.,
Schmidt,H., Meye,A., & Taubert,H. Knockdown of survivin expression by small
interfering RNA reduces the clonogenic survival of human sarcoma cell lines
independently of p53. Cancer Gene Ther 11, 186-193 (2004).


14. Mesri,M., Wall,N.R., Li,J., Kim,R.W., & Altieri,D.C. Cancer gene therapy
12. Chawla-Sarkar,M., Bae,S.I., Reu,F.J., Jacobs,B.S., Lindner,D.J., & using a surviving mutant adenovirus. J. Chn. Invest 108, 981-990 (2001).
Borden,E.C. Downregulation of Bcl-2, FLIP or IAPs (XIAP and survivin) by
siRNAs sensitizes resistant melanoma cells to Apo2L/TRAIL-induced apoptosis.
CellDec . 11, 915-923 (2004).



































































University of Florida I Journal of Undergraduate Research I Volume 11, Issue 1 I Fall 2009
6





JILLIAN CIMINO


Table 1: Demographic Information and Outcome Variables of Preterm Infants

Demographic Information Outcome Variables

NBRS
Age min min Weight Days to Weight Feed PO Feed Feed O Resp.
(gm) Score DC Gain days days Intol. Outcome

All Infants (Experimental and Control) N 67

Visit Status:
24.8 5.4 7.6 1145.4 2.3 9.8 44.4 14.9 28.4 51.0 3.6 7.5 57.0%
Low


High 25.4 5.4 7.0 1012.2 3.5 21.5 61.3 19.9 31.5 59.3 4.1 9.8 61.8%

Experimental Infants n 32

Visit Status:
26.8 5.6 7.9 1107.6 1.4 8.1 41.0 16.4 26.0 48.0 3.4 6.0 51.7%
Low


High 24.9 5.9 7.1 1069.7 3.3 27.1 61.8 21.0 27.4 56.7 2.8 9.6 58.4%


Control Infants n 35

Visit Status:
23.4 5.3 7.5 1170.5 2.8 10.9 46.6 13.8 30.5 52.3 3.4 8.4 60.7%
Low


High 25.9 4.9 6.9 949.1 3.9 15.5 60.8 18.2 36.5 61.8 5.3 10.0 65.4%



NBRS = Neurobehavioral Risk Score; DC = discharge; PO by mouth; Intol. = intolerance; NPO= nothing by mouth; Resp. =respiratory


Statistical Analyses. Data were analyzed using SAS (v8,
Cary, NC). Descriptive statistics were determined to
characterize the sample. A two-way ANOVA was used to
compare outcomes between groups. A level of significance
of P < 0.05 was used.


Results

Using a two-way ANOVA, variables related to
discharge timing were compared (Table 1). A near
significant finding was noted for episodes of feeding
intolerance. The number of episodes of feeding intolerance
was less for infants whose families visited more often, as
well as heard the maternal voice recordings. A near
significant difference was noted in between the high
visitation Experimental (or maternal voice group) and the
high visitation Control group (F = 3.16; p = .08;
Experimental mean = 2.8, Control mean = 5.5). Further, a
counterintuitive finding was noted. Infants in the both the
Experimental and Control groups whose family members
visited less were discharged earlier (F = 8.42; p<.01;


combined Experimental and Control low visitation mean =
44.4, combined Experimental and Control high visitation
mean = 61.3).

Discussion

Findings suggest that the combined effects of family
visitation and exposure to maternal voice recordings
(within a NICU without an organized program of
developmental care) does not significantly affect discharge
timing. A near significant difference (F = 3.16; p = .08),
however, was noted for the Experimental infants between
the Low versus High visitation groups in episodes of
feeding intolerance.
The finding related to episodes of feeding intolerance
may be explained by a variation in feeding type between
the Low versus High visitation status groups. The effect
may be due to the fact that the high visitation infants
experienced more days of breast milk feeds (F = 4.77;
p<.05; high visitation mean = 21.5 days, low visitation
mean =9.8 days). Breast milk could have influenced this
finding because it is known to decrease episodes of feeding


University of Florida I Journal of Undergraduate Research I Volume 11, Issue 1 I Fall 2009
4






INHIBITION AND APOPTOTIC RESPONSES OF HUMAN COLORECTAL CARCINOMA CELLS


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VP2


Figure 6: a) Vector maps of pHelper and pACG-2 b) restriction
digest of pHelper and pACG-2 (last two wells, respectively) with
Clal and XmnI.

DISCUSSION

Inhibition of survivin through RNA interference has
provided the scientific community with new insight and
prospective therapies to cancer treatment. The importance
of targeting survivin stems from the identification that both
are expressed in cancerous cells, but are limited in their
expression in normal somatic cells.
Current Western blot results have shown that survivin-
67 is the most effective siRNA treatment in inhibiting
survivin protein expression. Experimental data provided
evidence that RNA interference through siRNA survivin
and hTERT was effective in inducing an apoptotic
response in Huh7.5 and HT29 cell lines. Cotransfection of
survivin and hTERT did produce an apoptotic response in
HCT116 and HT29 cells, but the difference in


cotransfection response was not distinguished enough from
the independent treatments of survivin and hTERT.
One problem that may present itself is the accuracy in
using the HCT116 alone in defining the most efficient
siRNA sequence for survivin. Despite the ability of
survivin-67 siRNA to down-regulate the survivin protein in
HCT116 cell lines, the transfection of survivin and/or
hTERT proved to induce a very small response in
apoptosis of the cells. It is suggested that the Huh7.5 and
HT29 cell lines also be used to determine the most efficient
siRNA sequence since the transfection of survivin and/or
hTERT produced a drastic response in apoptosis.
Future treatment could include such a therapy that
would combine the efficacy of chemotherapy and the use
of siRNA survivin to induce apoptosis in those cells that
were initially resistant to radiation. One still must consider
the in vivo results with the use of a mouse model and
determine the best mode of delivery of AAV into the
targeted region of the host. Even with the advancement of
biomedical technology, one can envision small-interfering
RNA and adeno-associated virus therapy to be novel
treatments in the fight against cancer. The survivin AAV
will hopefully open up new avenues for in vivo animal
studies in order to determine the usefulness of survivin
siRNA therapy against cancer.

REFERENCES

1. Jemal,A., Tiwari,R.C., Murray,T., Ghafoor,A., Samuels,A., Ward,E.,
Feuer,E.J., & Thun,M.J. Cancer statistics, 2004. CA Cancer J. Chn. 54, 8-29
(2004).

2. Obrand,D.I. & Gordon,P.H. Incidence and patterns of recurrence following
curative resection for colorectal carcinoma. Dzs. Colon Rectum 40, 15-24 (1997).

3. Hanahan,D. & Weinberg,R.A. The hallmarks of cancer. Cell 100, 57-70 (2000).

4. Reed,J.C. Dysregulation of apoptosis in cancer. J. Clin. Oncol. 17, 2941-2953
(1999).

5. Soengas,M.S., Capodieci,P., Polsky,D., Mora,J., Esteller,M., Opitz-Araya,X.,
McCombie,R., Herman,J.G., Gerald,W.L., Lazebnik,Y.A., Cordon-Cardo,C., &
Lowe,S.W. Inactivation of the apoptosis effector Apaf-1 in malignant melanoma.
Nature 409, 207-211 (2001).

6. Ravi,R. & Bedi,A. Potential methods to circumvent blocks in apoptosis in
lymphomas. Curr. Opin. Oncol. 14, 490-503 (2002).

7. Zaffaroni,N., Pennati,M., & Daidone,M.G. Survivin as a target for new
anticancer interventions. J. CellMolMed 9, 360-372 (2005).

8. Ambrosini,G., Adida,C., & Altieri,D.C. A novel anti-apoptosis gene, survivin,
expressed in cancer and lymphoma. Nat. Med3, 917-921 (1997).

9. LaCasse,E.C., Baird,S., Korneluk,R.G., & MacKenzie,A.E. The inhibitors of
apoptosis (IAPs) and their emerging role in cancer. Oncogene 17, 3247-3259
(1998).

10. Zaffaroni, N., Pennati, M., & Daidone, M.G. Survivin as a target for new
anticancer interventions. J. Cell Mol Med 9, 360-372 (2005)


University of Florida I Journal of Undergraduate Research I Volume 11, Issue 1 I Fall 2009
5





CORY JAMES SMITH


promoter region that can be recognized by the same
sigma factor. Regulation of which sigma factors are
active serves as a master control switch in the coordinated
regulation of global gene expression.
Not only are sigma factors under strict transcriptional
control but they are also controlled post-translationally by
means of proprotein sequences and anti-sigma factors.
Proprotein sequences are segments of a nascent peptide
that must be proteolytically cleaved to activate the mature
functional sigma factor. Anti-sigma factors bind the
sigma factor preventing it from associating with RNAP
and transcribing its regulon. In response to specific
environmental signals the anti-sigma factor changes
conformation, releasing the sigma factor and allowing it
to transcribe its regulon.
The most common form of anti-sigma factor described
in the literature is the Extracellular function sigma factor.
ECF operons produce environmental response systems
that transduce stress signals to changes in global gene
regulation patterns (Missiakas, Helmann). The anti-sigma
factor is generally described as a transmembrane protein
that binds its cognate sigma factor sequestering it from
RNAP. This steric isolation prevents the transcription of
the regulon that is controlled by it's cognate sigma factor
(Helmann). In response to an environmental signal
specific to each anti-sigma factor, the integral protein
changes conformation, releasing the ECF sigma factor,
ans allowing it's re-association with the RNAP. The
active holoenzyme is now able to transcribe the regulon
of operons with the cognate promoter to the sigma factor
(Missiakas). The activated gene products respond to the
perceived environmental stress rendering the organism
viable for the new conditions.
The anti-sigma factor regulatory system shares many
aspects with the two-component regulatory system
characterized throughout eubacteria and in some
eukaryotes (Helmann, Stock). Two-component regulatory
systems typically consist of; a histidine kinase that binds
adenosine triphosphate and autophosphorylates at a
histidine residue, and a response regulator that receives
the phosphoryl group on an aspartate residue (Stock). The
histidine kinase is usually a membrane bound receptor
that autophosphorylates only in response to specific
environmental stimuli. Phosphoryl transfer from the
histidine kinase to an aspartate residue then activates the
response regulator. The phosphoryl group changes the
confirmation of the response regulator permitting it to
bind DNA at a specific sequence. The response regulator
alters transcription of genes in the local area.
Some virulence factors of P. gingivalis have been
shown to be controlled by two-component regulation.
FimA, a long fimbriae associated with invasion of host
cells, and a short fimbriae, mfal, shown to be involved in


interspecies communication Hayashi and, Wu are such
examples. These fimbrial genes are regulated by the
experimentally characterized two-component regulatory
system of FimR/FimS (Hasegawa N H). FimS is a
membrane bound sensor kinase and FimR is a response
regulator that is activated by FimS allowing it to bind
DNA, altering expression offimA, and mfa (Hasegawa N
H, Xie).
The regulator OxyR is directly involved in
transcriptional responses to oxidation in P. gingivalis
(Wu). OxyR changes conformation under oxidative
stress, allowing it to bind directly to the promoter offimA,
reducing transcription. OxyR also increases transcription
of sod, super-oxide dismutase, a critical enzyme involved
in the mild aerotolerance of P. gingivalis (Wu). Other
intracellular pathogens such as Mycobacterium
tuberculosis enter a dormant state upon oxidative stress in
an attempt to avoid the immune system (Manganelli).
Since the FimA protein is involved in TLR assisted
phagocytosis of P. gingivalis by macrophages, the
repression offimA by OxyR could be a form of antigenic
variation used to evade immunological response.


METHODS

Putative sigma factors were identified from the
complete annotated genome sequence of Porphyromonas
gingivalis strain W83 sequenced by The Institute for
Genomic Research (Frasier). The fully annotated
sequence can be obtained at the website for the National
Center for Biotechnology Information. The annotation
with features was viewed in the open source genome
viewer Artemis (Rutherford). Within Artemis a feature
search was conducted to identifying genes with "sigma"
in their putative descriptions. Manual reading of all terms
hitting sigma resulted in a list of putative sigma factor
genes.
The String database for known and predicted protein
interactions was then used to identify genes in operons
containing the putative sigma factors. The predictions are
made from genome context, high-throughput
experiments, conserved coexpression, and previous
knowledge. The predictions were viewed in Artemis to
ensure that the predicted genes are adjacent and in the
same transcriptional orientation as the putative sigma
factors.
The genes in predicted operons containing sigma
factors were further analyzed using several comparative
bioinformatics tools to detect the presence of hypothetical
transmembrane helices and proprotein cleavage sites. The
transmembrane domain prediction software TMHMM
was used to identify conserved membrane spanning helix


University of Florida I Journal of Undergraduate Research I Volume 11, Issue 1 I Fall 2009
2





CREATINE COMBINATION THERAPY


skeletal muscle fibers 24, 25. The multitude of degenerative
processes that influence the aging process manifest in the
total reduction of slow oxidative muscle fibers (Type I
muscle) and fast glycolytic fibers (Type IIB muscle) 8, 26. It
is hypothesized that the specific combination of CoQ10
and creatine may attenuate the deterioration of both Type
I/IIB fibers and provide a therapeutic effect 16 27. In aging
studies, oxidative stress is a major factor in diminishing
integrity of functional tissue by destroying nucleic acids
coding for appropriate proteins needed to build those
tissues. When considering the amount of antioxidants a
normal individual would assimilate from diet alone, this
amount appears to be deficient in producing changes in the
progression of tissue oxidation. Antioxidant supplements
help regulate oxidative damage by reducing the unbridled
activity of reactive oxygen species in the cell. However,
most over-the-counter products have poor bioavailability
when taken in a standard-quality, generic form, thus their
effects go unnoticed in the organism. Despite the benefits
associated with CoQ10 supplementation, its slow and
ineffective absorption into the body due to its lipophilic
nature tends to be the major obstacle in getting access to
the nutrient through ingestion 28. This very problem has
been addressed by biochemist in their development of the
innovative Eufortyn compound (Scharper Company,
Milan, Italy), which is mainly comprised of CoQ10,
creatine, and ginseng extract. Eufortyn's unique
terclatrated structural composition makes it a highly
soluble, multicomposite entity while maintaining the
integrity of the CoQ10 moiety, thereby greatly increasing
absorption into the mucosa.

Unique Properties of Eufortyn

Clatration (from the latin word, clatrum, i.e. cage) uses
mechanical energy to create a multicomposite substance;
that is, a combination of two or more chemical entities that
interact with each other without modifying their respective
physio-chemical properties, but end up with physical
characteristics that are unique to that multicomposite.
Clatration can be used when certain physical properties,
considered to be restrictive for pharmaceutical purposes,
need to be improved. According to Giorgio Bianchi, Ph.D,
a leading development expert of Qter, a terclatrate (ie,
three-in-a-cage) designates a multicomposite arising from
delivering mechanical energy to three moieties:
1. A biologically active substance whose
physical properties need to be modified.
2. A pharmacologically inactive polymer
acting as a passive matrix that traps single moieties
of the active ingredient inside each of its loops,
thus preventing the trapped moieties from getting
in contact and interacting with each other.


3. A small molecule that acts as a catalyst for
the formation of the clatrate, enabling clatration to
occur at room temperature thus, keeping chemical
reactions from occurring between the components
of the multicomposite.
Qter is a terclatrate multicomposite in which the active
moiety is coenzyme Qo1 (CoQo1), the inactive polymer is a
commonly used pharmaceutical excipient, and the catalyst
is a naturally occurring amino-acid. Native CoQo1 has poor
water solubility as a substance, forming a waxy and highly
electrostatic powder: impossible to be administered by
intravenous route, poorly absorbed by the GI mucosa when
orally administered, and troublesome in the industrial
pharmaceutical setting. Qter is aimed at overcoming these
limitations, by trapping CoQio in the passive matrix;
electrostatic interactions between moieties are prevented,
and a finely dispersible, water soluble powder is obtained,
showing a fairly improved bio-availability profile in
humans, when compared to native CoQ1o.

Materials and Methods

The Eufortyn pilot was conducted in accordance with
the National Institutes of Health guidelines for the care and
use of laboratory animals. In an effort to examine the
efficacy of Eufortyn on the physical, cognitive and
biochemical aspects of an organism, the combined therapy
(Eufortyn) was fed for 6 weeks to male Fischer 344 x
Brown Norway rats obtained from the National Institute of
Aging colony (Harlan Sprague Dawley, Indianapolis, IN)
at 6, 19, and 27 months of age. The F344xBN is an
excellent model often used for aging research. The stock
and strain of these hybrid models have been characterized
under well-defined environmental and genetic conditions.
All animals were singly housed in a temperature
(20+2.50C) and light-controlled (12:12h light-dark cycle)
environment. The animals were provided with water, food
(ad libitum NIH31 pellets #7017 and a daily Eufortyn
pellet dosed at either 938 mg/tab or 375 mg/tab depending
on rodent weight at time of feeding), and were given two
week acclimation time before the experiments began.
Animals with documented pathology were not included in
the final analyses. All three age groups were randomly
selected into either control or experimental groups (C6,
C19, E19, C27, or E27) followed by a further separation
into one of four cohorts (Table 1), which were then
staggered throughout the treatment process to allow for
sufficient time to perform all necessary physical and
cognitive analysis prior to sacrifice and tissue extraction.
Eufortyn treatment began in line with pilot schedule (Fig.
2). No treatments were given to these animals so as to limit
confounding variables in tissue biochemistry analysis.
Animals were euthanized by decapitation using a dedicated


University of Florida I Journal of Undergraduate Research I Volume 11, Issue 1 I Fall 2009
3






CREATINE COMBINATION THERAPY


Grip Strength Analysis


Grip strength results were expressed as total grip strength
force (kg of force) and total force divided by body weight
(kg of force/kg body weight) (Fig. 8). Control cohorts
expressed diminished grip strength capabilities with age
when the kg force/kg body weight were normalized with
body weight of the animal. The Eufortyn treated cohorts
did not exhibit significantly


Ag (Non-nonnazed


Odd Distance


E looo-
1000
o
5 760-
2 O
0
I-.


controls.


Day


Figure 9: Distance in maze measurement

Odd duration





30-
0
I-
20
Day

Figure 10: Duration in maze measurement


Figure 8: Grip Strength Analysis. Absolute grip strength force
did not change with age (Fig 8A upper Panel). In contrast, grip
strength expressed by body weight showed a decline in the 19-
month and 27-month old animals as compared to the 6-month old
controls (Fig 8A lower Panel). There was a marked increase
(12%) in total grip strength force for the 19-month old Eufortyn
group as compared to their age-matched controls after 4-weeks
of treatment with Eufortyn (Fig 8B upper panel). In addition,
forces normalized by body weight showed a similar (14%)
increase in the 19-month old Eufortyn treated group (Fig 8B
lower panel). The older 27-month old cohorts exhibited no
comparable difference due to Eufortyn treatment.


improved strength, however, there was a slight
improvement (12%) in total grip strength for the 19-month
old Eufortyn cohort compared to their control counterparts.
Eufortyn produced no distinguishable effects for the 27-
month old cohorts in terms of grip strength.


Morris Water Maze Spatial Discrimination

Initial (day 1) and terminal learning performance (day 3)
using the distance measure were determined. A significant
effect of both day (p < 0.001) and age (p = 0.02) was seen
in that all groups improved between initial and terminal
learning and this effect was larger in the younger versus
older animals (Fig. 9). Though, there was no significance
observed in either aging or treatment effect, there was a
promising trend observed for the initial (day 1) trial of the


Odd Velocity
27.S.
ao


o
17.a
15.0
12.5 .


o i 2
Day


0c1g
...E19
--0C27
* E27


3 4


Figure 11: Velocity in maze measurement

experiment; the Eufortyn groups achieved a shorter
distance to the platform. This indicated that the Eufortyn
groups experienced improved cognitive performance
relative to the
Over the course of the experiment, the learning curve of
the Eufortyn and control groups began to coincide and
finally no significant difference in duration was seen on the
final day of water maze. The similar pattern was observed
for the distance traveled by each cohort and group (Fig.
10). The Eufortyn groups started off with an advantage in
that they had to travel the shortest distance to reach the
hidden platform as compared to the control, however at the
final stage, all groups tended to travel the same small
distance resulting from the inevitable assimilation to the
water maze in general. The velocity at which the rats
traversed to the hidden platform was consistently greatly in
the Eufortyn groups than the control groups (Fig. 11). The


University of Florida I Journal of Undergraduate Research I Volume 11, Issue 1 I Fall 2009
7


N-1bn~d gaw -M
Nowumr-1.


AGO 01M-ftodl





CORY JAMES SMITH


Table 4: SignalP prediction of the presence of signal peptide cleavage loci.


Signal
Locus tag Max Ca Max Sa Mean Sa Max yb Dc peptide?d
PG 0016 0.018 0.784 0.229 0.077 0.153 No
PG 0148 0.192 0.707 0.122 0.143 0.133 No
PG 0162 0.250 0.037 0.016 0.018 0.017 No
PG 0214 0.047 0.057 0.033 0.034 0.033 No
PG 0594 0.049 0.073 0.035 0.026 0.030 No
PG 0747 0.216 0.542 0.096 0.190 0.143 No
PG 0985 0.282 0.116 0.039 0.061 0.050 No
PG 1105 0.022 0.091 0.031 0.024 0.028 No
PG 1318 0.145 0.151 0.047 0.038 0.042 No
PG 1660 0.494 0.396 0.050 0.044 0.047 No
PG 1827 0.199 0.666 0.262 0.184 0.223 No


a C-score and S-score are the two neural networks used.
b Y-max is a derivative of the C-score combined with the S-score.
c D is an average of S-mean and Y-max that provides the best view of whether the peptide contains a
cleavage site.
d Determination if the protein is a signal peptide is determined by a summation of these values


DISCUSSION

Regulation of gene expression in eubacterial species
occurs primarily at the level of transcription
(Haldenwang). The specificity of the holoenzyme is
determined by the interaction of the sigma subunit with
the promoter region on the DNA directly upstream of the
transcription initiation site (Haldenwang). Non sigma
factor DNA binding proteins such as repressors can alter
the efficiency of transcription but it is the sigma factor
that determines when and where transcription occurs
(Haldenwang). Genetic regulation in P. gingivalis, up to
this point, has only characterized non-sigma factor DNA
binding proteins. This paper suggests eleven operons are
potentially involved in sigma factor creation and
regulation.
A primary sigma factor such as sigma-70, or sigma-54
in E. coli is one that is essential and cannot be deleted
from the genome (Manganelli). They tend to be conserved
across bacterial species and are represented in P.
gingivalis by PG_0594 (sigma-70 like) and PG_1105
(sigma-54 like). A lethal gene knockout of these two
genes would confirm their role as primary essential sigma
factors.
All of the identified sigma factors in P. gingivalis are
merely predicted, to confirm their actual role in the cell
molecular biology tools must be used. A yeast two-hybrid
test is a molecular biology technique used to determine


protein-protein, and protein-DNA interactions. The test
uses activation of a reporter gene (usually lacZ) promoted
by a transcription factor (Gal4) binding to upstream
activating sequence (UAS). The transcription factor
consists of two domains: a DNA binding domain (BD)
and an activating domain (AD). The two domains are split
and added to the proteins of interest that might have
interactions. If the two proteins of interest interact, then
the activating domain is brought into close enough
proximity to recruit RNAP and to transcribe the reporter
gene (Finley). A yeast two-hybrid test could confirm that
the putative sigma factors both bind to RNAP as well as
identify promoter specificity.
Extracellular function (ECF) sigma factors are
environmental response systems that transduce a signal
from the environment to enact global changes in
transcription. ECF sigma factors are usually kept in an
inactive state until the environmental cue is received to
activate them (Helmann). Inactivation can be
accomplished through several diverse mechanisms such as
physical sequestration by a membrane protein, or initial
translation with a pro-protein inhibition sequence, or both
(Missiakas). Vital to the functioning of an ECF system is
the membrane bound receptor that recognizes
environmental stresses and begins the signal transduction
cascade. The sigma factors in the P. gingivalis genome are
generally associated with a predicted transmembrane
protein that could fulfill this role. Further studies could be


University of Florida I Journal of Undergraduate Research I Volume 11, Issue 1 I Fall 2009
6





SIGNAL TRANSDUCTION


domains that have been extensively characterized
throughout all domains of life.
SignalP, based on neural networks and hidden Markov
models, is used to predict the presence and location of
proprotein cleavage sites. Neural networks function in a
similar manor to the way biological neurons function in
processing information. Several inputs are taken in
(protein sequence) which are taken through several
calculations (statistical estimation, optimization, and
control theory) to output a single answer, in this case
whether or not the peptide contains a proprotein cleavage
site similar to other experimentally characterized systems
(Emanuelson).


RESULTS

The P. gingivalis W83 genome encodes 11 putative
sigma factors, four related to sigma-54 and seven related
to sigma-70. rpoD and rpoN are related to the classically
conserved primary sigma factors related to Escherichia
coli sigma-54 and sigma-70 respectively. Six of the sigma
factors are classified as ECF type related to
environmental response. Table 1 lists the 11 sigma factors
and their putative identification.
Ten of the 11 sigma factors are predicted to be
transcribed in an operon with an additional 1 to 7 genes
by the cotranscriptional prediction of the String database.


Although the majority of the genes in operons with sigma
factors are uncharacterized the few that are characterized
are generally involved in signal transduction. The results
of the string database prediction of functional
associations is available in Table 2. From the putative
description of genes in the operons of sigma factors
PG_0746, PG_0017, and PG_0151 are involved in signal
recognition of signal transduction. PG_0017 is a sensor
protein, PG_0746 is a sensor histidine kinase, and
PG_0151 is a signal recognition particle-docking protein.
However the majority of genes collocated in operons with
sigma factors are reported as uncharacterized hypothetical
proteins requiring further analysis using bioinformatics
tools newly developed since 2001 when the W83 genome
was annotated.
Of the 10 operons containing sigma factors, 7 of them
contain at least one predicted transmembrane protein as
determined by TMHMM. The data for TMHMM is
available in table 3. These integral proteins are likely
targets as anti-sigma factors because of the classically
conserved structure of the ECF operon.
The results of P. gingivalis sigma factors tested on the
signalP server for the presence of a signal peptide
cleavage site are listed in table 4. The results suggest that
none of the 11 sigma factors contain a cleavage site. For
each peptide two neural networks are used to predict
cleavage sites based on similarity to experimentally
characterized peptides.


Table 1: Putative sigma factors of P. gingivalis strain W83.


Locus tag Gene Putative identification
PG_0016 sigma-54 dependent DNA-binding response regulator
PG_0148 sigma-54-dependent transcriptional regulator
PG_0162 RNA polymerase sigma-70 factor, ECF subfamily
PG_0214 RNA polymerase sigma-70 factor, ECF subfamily
PG_0594 rpoD RNA polymerase sigma-70 factor
PG_0747 sigma-54 dependent DNA-binding response regulator
PG_0985 -RNA polymerase sigma-70 factor, ECF subfamily
PG_1105 rpoN RNA polymerase sigma-54 factor
PG_1318 -RNA polymerase sigma-70 factor, ECF subfamily
PG_1660 -RNA polymerase sigma-70 factor, ECF subfamily
PG_1827 -RNA polymerase sigma-70 factor, ECF subfamily


University of Florida I Journal of Undergraduate Research I Volume 11, Issue 1 I Fall 2009
3






DARYA VOROBYEVA


soleus, extensor digitorum longus (EDL), and quadriceps
muscle of the 19-month and 27-month old animals
progressively decreased in contrast with the 6-month old
rats (Fig. 6). No significant difference was seen in all
muscles weights when comparing age-matched controls







Gastrocnenius


0 19 27
Age
monthss)


EDL


with Eufortyn groups (Fig. 7). Heart weight, kidney weight
and liver weight of the 19-month and 27-month old rats
gradually increased with age as compared to the 6-month
old animals. In contrast, brain weight did not change with
age.


Plantaris


Soius


(mo 6) 19
Age Ag.
months. (month.)


27


Figure 6: Control Animal Muscle Tissue Weights. Aging effect on various muscle groups of control diet age-cohorts.


Gastrocnenlus mConol
Bjtfrtyn








(mnalnl)


Plantrts iCaord
lEuraftyn


o.oIl

""
~; O

-m,
C .M


Soleus icorm
iEul3ftn


Ag(
If tha)


EDL Canti
EIthtyn


IL
I, 27
Age
(morth.)


-I


0-0


Bs"


Quadriceps Control
mEulortyn


Age
month. )


Figure 7: Treatment Effect On Muscle Tissue. Eufortyn effect on various muscle groups consisting primarily from Type II skeletal
muscle tissue. Supplementation with Eufortyn did not affect muscle tissue weights in either cohort.






University of Florida I Journal of Undergraduate Research I Volume 11, Issue 1 I Fall 2009
6


Quadriceps
I"
b


0.-

6 19 27
Age
monthsl


* o


a,


Ss
-.a
- Cl C-
-|.. a





JILLIAN CIMINO

Zeskind, P. S., & Iacino, R. (1984). Effects of maternal visitation to preterm infants in the neonatal intensive care unit. ChildDev, 55(5), 1887-1893.

































































University of Florida I Journal of Undergraduate Research I Volume 11, Issue 1 I Fall 2009
6






CREATINE COMBINATION THERAPY


03
v,
E
0 0M
Z =


g.L-


Age
(Months)


Eufortyn

T


Control
=Eufortyn


19
Age
(Months)


Figure 13: Non-Heme Iron Aging and Eufortyn Effect. A substantial aging effect depicted by total non-heme iron content
measured in gastrocnemius muscle tissue of ad-libitum fed 6, 19, and 27-month old rats (left panel) normalized to micrograms of iron
per gram of wet tissue weight (p< 0.05 )by Tukey's Multiple Comparison Test, n= 7-8 per group. Total non-heme iron content
measured in gastrocnemius muscle tissue of 19 and 27-month old ad-libitum and Eufortyn fed rats (right panel) normalized to
micrograms of iron per gram of wet tissue weight. With respect to 19-month old control group, Eufotyn treatment showed no effect,
but in the 27-month old age Eufortyn treated animals show a remarkable suppression of non-heme iron (54%) as compared to control.
(p< 0.05), n =7-8 per group.


19 27
Age
;months


mContol
Eutartyn


Age
(months)


Figure 14: Effects of aging and eufortyn on DNA oxidation levels in control and eufortyn-treated gastrocnemius muscle. Levels
of oxidized DNA were assessed by examining levels of oxidative products 8-oxo-7.8-2'-deoxyguanosine using HPLC-ECD. One way
ANOVA exhibited no significant difference in DNA oxidative damage between control cohorts (Fig 14 left panel), but oxidative
damage in both 19 and 27 month old rats was diminished by 31% and 24% respectively in the Eufortyn groups as compared to the
control cohort matches (Fig 14 right Panel). Two-way ANOVA suggested a significant treatment effect (p< 0.10), indicating that
Eufortyn successfully lowered DNA oxidative damage in the gastrocnemius muscle.


of physical and cognitive improvements of the aging
organism. Our results demonstrated the treatment's role in
altering biomarkers specific to mitochondrial bioenergetics
and skeletal muscle aging. All tissue weights appeared
unaffected by Eufortyn treatment when compared with
age-matched controls indicating that the diet utilized has no
negative effects on organ weight and is safe for
consumption. Moreover, Eufortyn had no negative effects
on body weight, food intake and muscle weights in this


study. These findings establish the safety of Eufortyn as a
supplement. The distance measurements delineate that the
Eufortyn groups had a higher initial performance capacity
and were therefore able to exhibit outstanding performance
compared to the control groups. Resultantly, Eufortyn may
play a role in delaying cognitive deterioration with age by
extending its protective properties beyond the blood brain
barrier. The beginning trials of the water maze experiment
in which the Eufortyn groups display a distinct lead over


University of Florida I Journal of Undergraduate Research I Volume 11, Issue 1 I Fall 2009
9






CREATINE COMBINATION THERAPY


muscle: results from the Invecchiare in Chianti study. Am J Chn Nutr 2006
May;83(5):1142-8.

(25) Hiona A, Leeuwenburgh C. The role of mitochondrial DNA mutations
in aging and sarcopenia: implications for the mitochondrial vicious cycle theory
of aging. Exp Gerontol 2008 January;43(1):24-33.

(26) Phillips T, Leeuwenburgh C. Muscle fiber specific apoptosis and
TNF-alpha signaling in sarcopenia are attenuated by life-long calorie restriction.
FASEBJ 2005 April;19(6):668-70.

(27) Walsh B, Hooks RB, Homyak JE, Koch LG, Britton SL, Hogan MC.
Enhanced mitochondrial sensitivity to creatine in rats bred for high aerobic
capacity. JApplF ':. .. ..'2006 June; 100(6):1765-9.

(28) Schulz C, Obermuller-Jevic UC, Hasselwander O, Bernhardt J,
Biesalski HK. Comparison of the relative bioavailability of different coenzyme
Q10 formulations with a novel solubilizate (Solu Q10). IntJFoodSci Nutr 2006
November;57(7-8):546-55.

(29) Pandolfo M. Iron and Friedreich ataxia. JNeural Transm Suppl
2006;(70):143-6.

(30) Rotig A, de LP, Chretien D, Foury F, Koenig M, Sidi D, Munnich A,
Rustin P. Aconitase and mitochondrial iron-sulphur protein deficiency in
Friedreich ataxia. Nat Genet 1997 October;17(2):215-7.

(31) Xu J, Knutson MD, Carter CS, Leeuwenburgh C. Iron accumulation
with age, oxidative stress and functional decline. PLoS ONE 2008;3(8):e2865.

(32) Di LF, Bernardi P. Mitochondrial function and myocardial aging. A
critical analysis of the role of permeability transition. Cardiovasc Res 2005 May
1;66(2):222-32.

(33) Seo AY, Hofer T, Sung B, Judge S, Chung HY, Leeuwenburgh C.
Hepatic oxidative stress during aging: effects of 8% long-term calorie restriction
and lifelong exercise. AntioxidRedox Signal 2006 March;8(3-4):529-38.

(34) Hofer T, Seo AY, Prudencio M, Leeuwenburgh C. A method to
determine RNA and DNA oxidation simultaneously by HPLC-ECD: greater
RNA than DNA oxidation in rat liver after doxorubicin administration. Biol
Chem 2006 January;387(1):103-11.































University of Florida I Journal of Undergraduate Research I Volume 11, Issue 1 I Fall 2009
11





RUSSELL J. OWEN


Matthews, C.K., K.E. v. Holde, and K.G. Ahem. 2000. Biochemistry, p. 650.
Benjamin-Cummings Publishing Company, San Francisco, CA.


Nelson, A. L. 2001. High-Fiber Ingredients Handbook. Eagan Press, St. Paul,
Minnesota


I kt I..i B.V., and L. Prosky. 2001. Advanced Dietary Fibre Technology, p. Pfeifer, H.H., and Thiele, E. 2005. Low-glycemic-index treatment: A liberalized
157. Blackwell Science, United Kingdom. ketogenic diet for treatment of intractable epilepsy. Neurology. 65:1810-1812.


Appendix A


Broccoli Bites Recipe


Yield: 16 6.5 oz Broccoli Bites
Serving Size 4 ea
Servings 4

Ingredients

32.5 g Frozen Chopped Broccoli (thawed, rung dry, and chopped)
24.9 g Sargento Cheddar Jack Cheese Blend (pre-shredded)
17.5 g Canola Oil
7.5 g Crisco (Trans-Fat Free)
31.1 g Philadelphia Original Cream Cheese-Room Temp
10.0 g Fresh Garlic (minced)
0.8 g -Kosher Salt
Oil for frying (amount will depend) on method used)


Utensils for Preparation

3 Med Size Mixing Bowls
Cheesecloth or clean dry towel
Cutting Board
Chefs Knife
3 Sheet pans
Wax paper
Digital food scale
Freezer Bags
Paper Towels


Utensils for Cooking

2 Qt Pot or Deep Fryer
Fry Skimmer


Preparation Directions

1) Allow broccoli to thaw overnight in refrigerator. If not completely thawed, run water over broccoli while still in package.
2) Slice cream cheese into smaller portions (2in x 2in), and allow to come to room temperature (about 1 hour).
3) Remove broccoli from package and rind dry using a clean dry towel or cheesecloth.
4) Roughly chop broccoli so that no large chunks are visible.
5) Place chopped broccoli in mixing bowl.
6) Mix cream cheese with broccoli.
7) Add remaining ingredients and mix thoroughly.
8) Refrigerate for 1 hour, or until chilled.
9) In small batches, weight out 6.5g portions of broccoli mix. Roll portions into ball shape, place on sheet pan covered with wax
paper, and freeze. Repeat process until all mix has been weighed, shaped, and froze (Figure A).











University of Florida I Journal of Undergraduate Research I Volume 11, Issue 1 I Fall 2009
6







Formulation and Sensory Analysis of a Ketogenic Snack to
Improve Compliance with Ketogenic Therapy


Russell J. Owen*


College of Agricultural and Life Sciences, University of Florida


Limited dietary choices in the ketogenic diet may compromise compliance and reduce overall quality of life, and the low provision of
fiber may further diminish quality of life. The purpose of this study was to develop highly acceptable high fiber, ketogenic snacks.
Broccoli bites and crab rangoon were developed approximately at a 3.5 to 1 ketogenic ratio. The snacks were formulated using fiber
isolates, pea hull fiber, hydroxypropyl methylcellulose and inulin as an alternative breading for frying. Sensory evaluation was carried
out by students and staff at the University of Florida to determine the acceptability of overall taste, mouthfeel, and appearance of the
snacks. Using a hedonic scaling method, panelists (n=67) determined acceptability, with 1 indicating extreme dislike, and 9 indicating
extreme liking. For the broccoli bites, the mean hedonic rankings for overall taste, mouthfeel, and appearance were 6.54 1.78 (mean
SD), 6.27 1.71, and 5.85 1.73, respectively. For the crab rangoon, the mean hedonic rankings for overall taste, mouthfeel, and
appearance were 5.60 1.86, 4.93 2.00, and 5.79 1.78, respectively. In addition, hedonic rankings for the overall taste, mouthfeel,
and appearance for the crab rangoon were rated as 6 (like slightly) or higher by 58.2%, 47.8%, and 67.2% of panelists, respectively.
Hedonic rankings for the overall taste, mouthfeel, and appearance for the broccoli bites were rated as 6 (like slightly) or higher by
76.1%, 73.1%, and 62.7% of panelists, respectively.


Introduction

The ketogenic diet, first developed in the 1920s, is
widely used to treat intractable epilepsy. Originally,
bromides and phenobarbital were used for the treatment
of epilepsy. The heavy sedating effects of these anti-
epileptic drugs contributed to the acceptance of
ketogenic therapy in treatment of intractable epilepsy.
Hugh Conklin, an osteopathic physician, believed that
epilepsy was caused by intoxication of the brain from
substances originating in the intestines (Freeman and
Kossoff, 2007). Conklin hypothesized that putting the
intestines at rest would prevent intoxication and prevent
seizures. Conklin used "water therapy" to treat epilepsy,
giving nothing but water for as long as 25 days. Conklin
reported prolonged reduction in seizures activity, and
news of his findings spread rapidly (Freeman and
Kossoff, 2007).
The discovery that diets high in fat and low in
carbohydrates could mimic starvation gave rise to
ketogenic therapy. Ketogenic therapy consists of a high
fat (up to 90% of total intake), low carbohydrate, and
adequate protein diet. The mechanism behind the neural
protection of ketogenic therapy remains a mystery, but in
many cases is very effective in treating intractable
epilepsy. Ketogenic therapy has been shown to reduce
seizures by >50 % in 60-75% of children who maintain

* with Wendy J. Dahl and Charles A. Sims


ketogenic therapy (Freeman and Vining, 1998). The dietary
restrictions of ketogenic therapy are accompanied by
psychosocial issues: patients feel isolated from peers because
they eat completely distinct foods (Pfeifer and Thiele, 2005).
GI disturbances are frequently reported among KD
patients with nausea/vomiting, diarrhea, and constipation
being most common (Kang and Chung, 2004). The restriction
of carbohydrate in ketogenic therapy limits intake of fiber,
and may be a factor contributing GI disturbances. Dietary
fiber is derived from the cell wall of edible plants or
analogous carbohydrates that are resistant to digestion and
absorption in the human small intestine with partial or
complete fermentation in the large intestine (Nelson, 2001).
Carbohydrates that are indigestible in the small intestine are
fermented in the large bowel to produce short chain fatty
acids (SCFA's). Increased fiber intake reduces the risk of
prevalent Western diseases ((McCleary and Prosky, 2001),
and demonstrates the importance of maintaining a healthy
gut. Insufficient intake of soluble and insoluble fiber in
ketogenic therapy may impart additional stress, and
potentially increase the frequency and severity of seizures.
Ketogenic therapy alters the metabolism of the brain by
limiting carbohydrates and forcing the brain and body to
utilize alternative sources of fuel. Utilizing fatty acids as
energy substrates in times of food depravation is crucial for
survival. The brains ability to adapt to these metabolic
changes in times of starvation is thought to be the foundation
of ketogenic therapy. Ketogenic therapy mimics starvation by
limiting glucose and providing high amounts of fatty acids,


University of Florida I Journal of Undergraduate Research I Volume 11, Issue 1 I Fall 2009





KETOGENIC SNACKS


time and consistency. Following a series of steps to
achieve the desired products (see Appendix A & B), the
ketogenic snacks were fried and stored for sensory
evaluation to be performed at a later time.
The fiber mix consisted of Inulin (Fruitafit-TEX!,
Sensus America Inc., Monmouth Junction, NJ, USA) .
Guar gum (3500F-D, Tic Gums, Belcamp, MD, USA).
Hydropropyl Methyl Cellulose (Methocel A4M, Dow
Chemicals, Midland, MI, USA). Gluten (Arise 8000,
MGP Ingredients, Atchison, KS, USA). Pea Fibre (Best
Pea Fibre, Best Cooking Pulses, Portaga la Prairie,
Manitoba, Canada). A dry mixture consisting of 33.5 %
inulin, 33.5 % pea fiber, 23.5 % gluten, 7.0% cellulose,
and 2.5% guar was prepared. The dry ingredients were
combined in a mixing bowl and mixed until a
homogenous color and consistency was reached.
The ketogenic snacks products were prepared under
commercial-like conditions in the pilot plant of the Food
Science and Human Nutrition Department. The snacks
were prepared at approximately a 3.5:1 ratio (3.5 parts
fat to 1 part carbohydrate plus protein). The ratio of the
ketogenic snacks were determined using an excel
spreadsheet that yields accurate percentages of protein,
carbohydrate, and lipid based on gram measurements of
food items used to prepare the ketogenic snacks.

2.2 Sensory Evaluation

Sensory evaluation was performed on both the
broccoli bite (Appendix A) and crab rangoon (Appendix
B) in the sensory lab at the University of Florida.
Panelists signed informed consent (standing protocol #
2003-U-0491).


Panelists were asked to respond to a series of demographic
questions to determine age, gender, and frequency of fried
food consumption. Panelists were given a small cup of water
and crackers to cleanse their palate between samples.
Panelists were presented with two foods samples, broccoli
bite and crab rangoon, and responded to a series of computer
generated questions (Appendix C) regarding the overall
appearance, taste, and mouthfeel of the product being
sampled. Panelists were also instructed to comment on
appearance, taste, and mouthfeel.

Results

The resulting ketogenic snacks included broccoli bites
(Appendix A) and crab rangoon (Appendix B) formulated at a
ketogenic ratio of 3.47 to 1 and 3.42 to 1, respectively.
Overall, 67 people including students and staff at the
University of Florida participated in sensory evaluation.
Hedonic scaling results for the overall appearance, taste, and
mouthfeel of the broccoli bite and crab rangoon were 5.85
1.72 and 5.79 1.78, 6.54 1.78 and 5.60 1.86, and 6.27
1.71 and 4.93 2.00, respectively (Figure 1). In addition,
hedonic rankings for the overall taste, mouthfeel, and
appearance for the crab rangoon were rated as 6 (like slightly)
or higher by 58.2%, 47.8%, and 67.2%, respectively. Hedonic
rankings for the overall taste, mouthfeel, and appearance for
the broccoli bites were rated as 6 (like slightly) or higher by
76.1%, 73.1%, and 62.7%, respectively. Figure 1 depicts the
sensory evaluation hedonic results for overall appearance,
taste, and mouthfeel of the crab rangoon, while figure 2
depicts the sensory evaluation hedonic results for overall
appearance, taste, and mouthfeel of the broccoli bites.


Hedonic Results for Crab Rangoon


20

30
JL


Hed~onkr Ranki..


Figure 1: Sensory Results for Crab Rangoon






University of Florida I Journal of Undergraduate Research I Volume 11, Issue 1 I Fall 2009
3


-oucrall all~~nrance
-Ov-rall+rrfl-
~C)vCrlll M~lflrfCCI





CORY JAMES SMITH


Table 2: P. gingivalis predicted operons containing sigma factors


Sigma factors in W83 Operon
Locus tag Locus tag Description
PG 0016 PG 0017 Sensor protein (EC 2.7.13.3)


PG 0018


Putative uncharacterized protein


PG_0148 PG_0146 Putative uncharacterized protein
PG_0147 Putative uncharacterized protein
PG_0149 conserved domain protein
PG_0150 conserved hypothetical protein
PG_0151 Signal recognition particle-docking protein
PG_0152 Carboxynorspermidine decarboxylase
PG_0153 Aspartyl-tRNA synthetase (EC 6.1.1.12)

PG_0162 PG_0161 Putative uncharacterized protein
PG_0163 phosphofructokinase

PG_0214 PG_0215 Putative uncharacterized protein
PG_0216 Putative uncharacterized protein
PG_0217 Putative uncharacterized protein
PG_0218 Putative uncharacterized protein

PG_0594 PG_0543 htrA protein

PG_0747 PG_0745 Lactoylglutathione lyase, putative
PG 0746 Sensor histidine kinase

PG_0985 PG_0984 Putative uncharacterized protein
PG_0986 Putative uncharacterized protein
PG_0987 Putative uncharacterized protein

PG_1105 PG_1106 Putative uncharacterized protein

Peptidyl-prolyl cis-trans isomerase SlyD, FKBP-
PG_1318 PG_1315 type
PG_1316 Putative uncharacterized protein
PG_1317 Putative uncharacterized protein

PG_1660 PG_1659 Putative uncharacterized protein
PG_1661 Putative uncharacterized protein
PG_1662 Putative uncharacterized protein

PG 1827








University of Florida I Journal of Undergraduate Research I Volume 11, Issue 1 I Fall 2009







Inhibition and Apoptotic Responses of Human Colorectal
Carcinoma Cells by Anti-Survivin Small Interfering RNA


College of Medicine, University of Florida


INTRODUCTION

As evidenced by the 940,000 cases and 655,000 deaths
worldwide per year, colon cancer is one of the most
prominent diseases in the world (1). Within the United
States alone, colon cancer has been linked to over 56,000
deaths and is considered the second leading cause of cancer
death (1).
Colon cancer, or colorectal cancer, develops from the
growth of adenomatous polyps in the colon due to
mutations in the DNA of the gastrointestinal epithelial cells.
The advancement of the formation of polyps in the colon
results in cancer that has the ability to metastasize to
adjacent organs, such as the liver.
Currently 3 different forms of treatment are used in
combination. Surgical removal of the cancerous growth is
the primary treatment for colon cancer. Through the
colectomy procedure, the cancerous mass is removed and
the colon rejected, restoring the original purpose of the
colon. Colostomy surgery also excises the cancerous
growth, but the colon is instead attached to the anterior
abdominal wall due to the unrestricted and increased
manifestation of the cancerous cells. Despite the
advancements in surgery, approximately 40-50% of
patients will relapse and require additional therapies and
treatments (2). Subsequent applications of chemotherapy or
radiation are used to treat metastasized cancer and prevent
such tumor recurrence after surgery. These treatments
stimulate cancer cells to enter apoptotic pathways, causing
cell death in these targeted cells. There have been well-
documented cases of resistance to apoptotic stimuli. This
resistance to chemotherapy and radiation could result from
the over-expression of anti-apoptotic factors. These factors,
such as survivin, are predominately expressed in cells of
common human cancers.
Cell division is coupled with checkpoints along the
different stages of cell life. These checkpoints allow the
prevention of the developments of abnormalities such as
mutations, by inducing the abnormal cell to enter the
apoptotic pathway. By this definition, apoptosis is a state
of programmed cell death due to DNA damage or cellular
aging.

Rafal P. Witek, Xiaokui Zhang, Han Zongchao, Arun Srivastava,
Chen Liu


The balance between cell growth and checkpoints is an
essential feature in preventing the development of any form
of cancer (3). Failure of the induction of apoptosis in cells
that contain abnormalities in the mitotic spindle, DNA
structure, and mutations in oncogenes could result in
increased resistance to chemotherapy and radiation (4, 5).
There are two major apoptotic pathways. The extrinsic
pathway involves extracellular inducers such as toxins,
hormones and growth factors that induce cellular signals
for apoptosis. The intrinsic pathway results in the
mitochondrial release of cytochrome c and a caspase
cascade that mediates cellular apoptosis (6, 7).
Survivin is a protein belonging to the inhibitor of
apoptosis protein (IAP) family and is an important
regulator of the intrinsic apoptotic pathway (8). The
survivin gene is located on chromosome 17, which encodes
a 16.5 kD protein containing 142 amino acids and a
Baculovirus IAP Repeat (BIR) (9). The survivin protein
regulates the intrinsic apoptotic pathway by interfering
with caspase-3, caspase-7, and caspase-9 activity (10).
Also, the association of survivin with the cell cycle and
mitotic spindle is consistent with the 40-fold increase in
expression levels during the G2/M phase in HeLa cells (11).
Its expression is upregulated in most human cancer cells,
but undetectable or found at very low levels in normal
adult tissues (10).
The increased activity of survivin in cancer cells in
contrast to normal somatic cells provides an ideal target for
cancer treatment. Current research provides the technology
to synthetically produce a 21-23 RNA nucleotide molecule
that would be able to effectively inhibit specific gene
expression by RNA interference (12, 13). This small
interfering RNA (siRNA) disrupts the expression of a gene
by down-regulating the desired target protein. The siRNA
is designed by selecting target sequences from the RNA of
a gene of interest. The sense and anti-sense templates are
placed on both sides of a loop sequence in a plasmid DNA.
The transcription of siRNA uses an RNA Polymerase III
promoter (U6 or H1), which allows the transcription of
short hairpin RNA. The single strand complementarily
binds to the target mRNA and consequently down-
regulates targeted gene expression through the RNA-
induced silencing complex (RISC)
The transfection of siRNA through plasmids is limited
to in vitro use. In vivo delivery of siRNA may be


University of Florida I Journal of Undergraduate Research I Volume 11, Issue 1 I Fall 2009


Philip W. Lin*





CREATINE COMBINATION THERAPY


180
160 lcium Injection :
140 I
120
100
80 -
60
40 PTP Ope-
20
0
0:00:00 0:02:53 0:05:46 0:08:38 0:11:31 0:14:24

Figure 4: Example of mitochondrial permeability transition
pore. Mitochondria were energized with succinate and a known
amount of Ca2+ (10 uM Ca2+; Calcium Green 5N) was added
stepwise after each minute, reflective of fluorescence pulses. The
increase of fluorescence is directly linked to an increase of extra-
mitochondrial Ca2+ and the reduction in fluorescence pulses
shows the uptake of calcium by the mitochondria. The total
content of calcium intake prior to PTP is used as data for PTP.
The control data points (blue) reach maximum capacity
prematurely as indicated by the expulsion of mitochondrial
contents whereas the Eufortyn treated mitochondria (red)
indicate imperviousness to additional Ca2+ at the same point,
opening substantially later in the injection process.


samples were infused with Calcium Green 5N and the
uptakes were measured using micro plate reader with
automatic injectors (Fig. 4).

Non-heme iron assay

In homeostatic biological organisms, free-radical formation
is a natural, continuous phenomenon and is typically kept
in check by endogenously occurring antioxidants localized
in specific areas of the skeletal muscle 9. Despite the
unremitting scavenging of free-radicals by antioxidants,
oxidant production in aging may surpass the capability of
antioxidants to fully shield oxidants from incurring
extensive oxidative damage on the muscle cell 2. Iron is
widely recognized as a powerful pro-oxidant that catalyzes
the formation of free radicals within cells. Despite a natural
tendency to form free-radicals, iron may inflict further
oxidation creating a summation effect of oxidation 31
Conducting the non-heme assay provided insight into non-
heme iron accumulation in muscle tissue with normal aging
as well as non-heme iron status in Eufortyn-treated muscle
tissue. Gastrocnemius muscle non-heme iron content was
measured after performing an iron assay described by Xu
et al.


Food Intake


26-
20-
16-

5,1.


Fo
25-
20


10-


I9
19


Age
(months)
od Intake Control
SEufortyn


Age
(months)


i2
27


Figure 5: Food Intake Trends. Aging effect on food intake of
control diet age-cohort animals (left panel). Eufortyn-effect on
food intake of age-match groups.

RNA and DNA oxidation measurement using HPLC-
ECD

Previously, studies have observed a strong correlation
between state of oxidation and mitochondrial levels of iron.
It is hypothesized that the antioxidant properties of
Eufortyn may have mediated some of the effects of non-
heme mitochondrial iron on vulnerable nucleic acids
resulting in greater longevity of the organelle in an aging
organism. Investigation of the oxidation status of muscle
tissue established the intracellular role of Eufortyn on pro-
oxidant regulation. Total RNA and DNA oxidation levels
and RNA/DNA ratios of plantaris muscles were analyzed
using a novel HPLC-ECD method 34

Results

Eufortyn supplementation during the 6-weeks showed a
significant increased in body weight between 6 and 19
months of age, but did not show a further increase at 27
months of age. No adverse changes in body weight were
experienced by any of the groups. Food intake increased in
relation to age-cohorts; both 19-month and 27-month old
animals consumed greater amounts of food as compared to
6-month old controls. There was no significant difference
in food intake between Eufortyn groups and age-matched
control groups in the animals at 19 and 27 months of age
(Fig. 5). Muscle weight in the gastrocnemius, plantaris,


University of Florida I Journal of Undergraduate Research I Volume 11, Issue 1 I Fall 2009
5


B





DARYA VOROBYEVA


Extracellular

Intracellular Fe2++ 0
H202
Fenton rac"on FeZ+ O-
,OH-+ *OH Fe3+

I- Calorie Restriction

RNA/DNA damage
Protein modification (Aiu eta 2oT
Lipid peroxidation (Cook anYd vi19



Sarcopenia

Figure 1: Intracellular Free-Radical Generation Cycle. The
release of iron from heme-protein or ferritin protein via H202
and superoxide ion results in free iron capable of reacting with
diatomic oxygen to generate the highly-reactive hydroxyl
radical via Fenton chemistry. This free-radical, in turn, has the
capacity to alter the physiological structure of nucleic acids,
proteins, and lipids, thus compromising the integrity of the cell.
This ultimately leads to cellular apoptosis a widely
recognized contributor of sarcopenia.

then at liberty to modify the delicate structure of nucleic
acids, lipids, and proteins 14 15. Cell death, the theorized
culprit behind degenerative disease, is facilitated by the
compromised integrity of the cell.

Coenzyme Q10

Naturally present within the inner mitochondrial
membranes is Coenzyme Q10 (CoQ10), a critical
biomolecule crucial to ATP synthesis. Functioning as a
ubiquinone and an important Complex II electron carrier in
the mitochondrial electron transport chain (ETC), CoQ10 is
a powerful antioxidant that scavenges free-radicals and
thus, maintains tissue health, potentiates cell growth, and
enhances vigor 16. Comparative studies on various
mammalian species have observed an inverse relationship
between the generation of super oxide anion radical and
sub mitochondrial CoQ10 content, implying the effective
antioxidant properties of CoQ10 16. Another study
investigated age-related changes in lipid peroxidation and
functionality of liver and skeletal-muscle mitochondria in
rats fed a diet rich in polyunsaturated fatty acids that was
either supplemented or not with CoQ10. Results for the


supplemented groups showed a decrease in peroxidizability
index, an increase in catalase activity in skeletal muscle,
and modulation of the age-related changes in the
mitochondrial ETC components. The shifts in these
biomarkers from CoQ10 supplementation suggest key
underlying mechanisms associated with the age-delaying
properties of CoQ10 17

Creatine Monohydrate

Creatine monohydrate (C) is a high energy compound
that anaerobically recycles ATP during intense muscular
exertion and is therefore concentrated in fast-twitch (Type
IIB fiber) muscle. Synthesized in the liver, pancreas, and
kidneys, creatine is transported in the bloodstream to
muscle cells where 95% of all creatine is found.
Supplementation can improve stores of phosphocreatines
and thus optimize muscular output during periods of high-
intensity exercise as well as lessen recovery time thereafter
18 Recent studies suggested that created aids patients
suffering from muscular dystrophy as well as attenuate
sarcopenia by rehabilitating disuse atrophy 19. Resistance
training for the elderly has proved beneficial, yet some
muscular loss is still problematic in the elderly; a lack of a
nutritional component may be the culprit. Creatine
supplementation has the potential to override muscle
atrophy during resistance training, although the mechanism
for its ergogenic effect is unclear 20. Additionally, creatine
descreases cytoplasm Ca2+ levels and increases
phosphocreatine stores intramuscularly which allows for
possible musculoskeletal effects, including cellular
hydration, increases in myogenic transciption factors, and
up-regulation of myosin heavy chains possibly involved in
muscle hypertrophy 19

Ginseng Extract

Ginseng extract is regarded widely as an effective
adaptogen a natural herb product used to increase an
organism's resistance to stress, trauma, anxiety, and fatigue
21. Prolonged administration of standardized ginseng to rats
reduced oxidative stress in certain tissues by modifying
specific antioxidant enzyme activities that are required to
eliminate free radicals, thus mitigating tissue peroxidation
end-products 22, 23. Some earlier studies reported greater
oxygen uptake and transport in elderly subjects as well as
enhanced energy levels in athletes.

Recognizing the Need for Bioavailable Combination
Therapy

With the progression of age, the susceptibility to
reactive oxygen species expedites the deterioration of


University of Florida I Journal of Undergraduate Research I Volume 11, Issue 1 I Fall 2009
2





JILLIAN CIMINO


numerous benefits for infants and parents resulting from
this program of care, the use of developmental care has
become more prevalent. Family visitation is one important
component of developmental care programs.
Family Visitation. While currently there is research
establishing the effect of maternal visitations on a preterm
infant's days to discharge, there is no information linking
whether or not visitations by other family members would
also improve discharging timing.
A study by Zeskind and lacino (1984) looked at
quantities of maternal visitation with a set of 2 groups (1
intervention, 1 control) composed of 32 mothers and their
infants ranging from 30-36 weeks of age. Both groups
experienced routine care offered by the NICU staff. The
intervention group, however, had the addition of help from
a project interventionist who provided advocacy, explained
the many actions of the NICU staff so the mothers might
better understand the processes, and made an appointment
each week for the mother to visit with her infant.
Comparisons of the 2 groups revealed that the intervention
group mothers independently (not including those set up
appointments to visit infant) visited their infants more than
twice as often as the mothers in the control group. Length
of hospitalization was looked at as well, and the
intervention infants stayed an average of 8 days fewer than
the control group. This established maternal visitation (in
conjunction with a program directed towards educating the
mother about her infant's health care) as a factor in
decreasing a preterm infant's days to discharge.
Family visitations, in addition to maternal visitations,
may be even more beneficial to the infant's discharge
timing than just solely by the one member. Perhaps any
increased exposure to family members is more advan-
tageous compared to the absence of any exposure.
Including other family members in family visitation may
assist in providing a nurturing environment for these
vulnerable babies by exposing them to a positive source of
sound. Preterm infants in the NICU are constantly exposed
to negative sources of sound through monitor alarms,
health care professional talk, and nearby infants crying.
Family visitation by multiple family members, especially
the mother, would provide more opportunities of positive
auditory stimulation for the preterm infant. While there is
general theoretical support indicating the significance of
exposure to maternal voice in the fetus and preterm infant
(Lickliter, 2000), research evaluating exposure to maternal
voice in preterm infants is equivocal.
Maternal Voice. In a study done by Chapman (1978)
and Malloy (1979), infants 26-33 weeks post-menstrual age
were split into 3 groups (Group 1, Group 2, and control),


with all receiving standard NICU care. Group 1 was
exposed daily to a recording of maternal voice while Group
2 was exposed daily to an orchestra playing a lullaby.
Chapman reported that the infants exposed to their
mothers' voice demonstrated the gross motor pattern of
laterality (preference for use of one side) more often than
those infants listening to the lullaby and those in the
control group. In Malloy's following study with these same
infants, the weight gain and developmental outcomes were
evaluated at 1 day following discharge and at 9 months of
age using components of Rosenblith's Behavioral
Examination of the Neonate and Bayley Scales of Infant
Development (Bayley, 1969; Malloy, 1979: Rosenblith,
n.d.). There were no statistical between-group differences
noted; however, infants exposed to maternal voice gained
more weight.
More recently, Krueger (in press) reviewed all studies
addressing exposure to maternal voice in preterm infants
and found that all used unsafe sound levels. All studies
used decibel levels ranging between 75-80 decibels, which
is much more than what is recommended. Sound levels are
important in preterm infant exposure, because depending
on the gestational age, the infant is undergoing neuro-
behavioral advances that can be negatively affected. Out of
the 7 studies viewed, the true significance of the findings
are difficult to evaluate because of these high sound levels.
Taken together, the above studies suggest that both the
frequency of maternal visitation and exposure to maternal
voice positively impact the number of days to discharge.
We therefore sought to describe whether this combination
(using safe sound levels) could potentially be an effective
and efficient way of decreasing length of hospitalization
for preterm infants.


Study Design and Methods

A retrospective comparative design was used with a
convenience sample of 67 preterm infants. Thirty-two
infants within this group took part in an experimental
component; 35 infants were retrospectively selected to
create a control group comparison. All infants were cared
for within the same time period within a level 3 NICU at an
academic teaching institution in the southeastern United
States. During this time period there was no ongoing
program of developmental or family-centered care
occurring within the NICU.
Following Institutional Review Board approval, criteria
selected for inclusion were: 1) birth between 27 and 28
weeks post-menstrual age, and 2) English as a native


University of Florida I Journal of Undergraduate Research I Volume 11, Issue 1 I Fall 2009
2





KETOGENIC SNACKS


Age Demographics


m Female
18-20 21-24 25-29 30-34 35-39 40-44 45-49 50-54 55-59
Age Range


Figure 3: Age and gender demographics for sensory panelists of ketogenic snacks


Included in the fiber breading mix were gluten
(wheat protein), guar (gum), hydropropyl methyl
cellulose, inulin, and pea fibre. Gluten provided structure
to the fiber mix, creating a protein matrix that stabilized
the fiber mix when moistened. Hydropropyl methyl
cellulose and guar form thermal gels at high
temperatures, preventing the leakage and absorption of
oils while cooking. Inulin and pea fiber were used to
fortify the fiber mixture, and replace the digestible forms
of carbohydrate commonly found in flour. The
application of the fiber mixture is similar to the
application of wheat based flours.
Fiber intake, and the effects of fiber on those who
suffer from intractable epilepsy, is a topic which needs
greater exploration to determine the importance of fiber
in the diet of ketogenic patients. The importance of fiber
is well documented in the general population, but it is
uncertain the effects that fiber supplementation will have
on ketogenic patients. However, our current knowledge
of fiber, and the role it plays in maintaining the integrity
of the large intestine, indicates that the effects of fiber
fortification in the ketogenic population may be
beneficial.
The ethics of ketogenic therapy are called into
question when considering the high fat, and often times,
caloric restriction of ketogenic therapy. However, not
implementing ketogenic therapy leaves few alternatives
for those who suffer from intractable epilepsy.
Individuals who are unresponsive to antiepileptic
therapy in the form of pharmaceuticals are limited in
options for the management of intractable epilepsy.
Ketogenic therapy may be the most effective means of
treating intractable epilepsy, as scientific studies support
the theory that ketogenic therapy is more effective in
treating intractable epilepsy than traditional
pharmacological approaches. Justification for utilizing
ketogenic therapy is evident when examining the
beneficial effects of ketogenic therapy in managing


intractable epilepsy, however, ketogenic therapy could be
vastly improved by understanding the effects that dietary
components such as fiber have on ketogenic patients.
To maximize the efficacy of ketogenic therapy, research is
needed to understand the role of fiber in ketogenic therapy,
and how it may influence patients on ketogenic therapy.
Understanding the intestinal environment before and after
supplementation with fiber may help to determine how much
fiber is needed to maximize the efficacy of ketogenic therapy.
High fiber ketogenic snacks may provide an adequate vehicle
for the delivery of fiber, while possibly improving
compliance with ketogenic diet. Further research is needed to
determine how ketogenic snacks may impact ketogenic
therapy, and if the ketogenic snacks are acceptable to those on
ketogenic therapy.

Acknowledgements

Thanks to Dr. Wendy Dahl and Charles Sims and his staff
for assistance with sensory analysis and evaluation and to
Sensus America Inc, Tic Gums, Dow Chemicals, MGP
Ingredients, and Best Cooking Pulses for their generous
donation of products.

Literature Cited

Amari, A., L. Dahlquist, E.H. Kossoff, E.P. Vining, W.H. Trescher, and K.J. Slifer.
2007. Children with seizures exhibit preferences for foods compatible with the
ketogenic diet. Epilepsy Behav. 11:98-104.

Freeman, J. M., E. H. Kossoff, and A. L. Hartman. 2007. The ketogenic diet: one
decade later. Pediatrics. 119:535-543.United States

John M. Freeman, Eileen P. G. Vining, Diana J. Pillas, Paula L. Pyzik, Jane C.
Casey, and LCSW, and and Millicent T. Kelly. 1998. The Efficacy of the Ketogenic
Diet: A Prospective Evaluation of Intervention in 150 Children. PEDIATRICS.
102:1358-1363.

Kang, H. C., D. E. Chung, D. W. Kim, and H. D. Kim. 2004. Early- and late-onset
complications of the ketogenic diet for intractable epilepsy. International League
Against Epilepsy. Epilepsia. 45:1116-1123.


University of Florida I Journal of Undergraduate Research I Volume 11, Issue 1 I Fall 2009
5





PHILIP W. LIN


optimized through the use of a viral vector such as an
adeno-associated virus (AAV). The adenovirus (AV) and
the adeno-associated virus (AAV) have been used
successfully for delivery in gene therapy. Examples of this
include the successful delivery of a survivin mutant to
breast cancer cells by using adenoviruses, resulting in
apoptosis in cancerous cells and inhibited growth of tumors
(14).
We hypothesize that by introducing siRNA that
specifically inhibits survivin expression, cancer cells will
be more susceptible to apoptotic stimuli and ultimately
result in apoptosis. A vector will be constructed with four
targets of the survivin gene. These 4 targets will be
analyzed for their efficacy in in-vitro down-regulation of
survivin protein in cancerous cell lines. The optimal siRNA
target will then be cloned into an adeno-associated virus
(AAV) for further in vivo studies. The inhibition of
colorectal carcinoma cells by siRNA survivin could prove
to be a potential therapy for colon cancer.

MATERIALS AND METHODS

Materials

The siRNA expression vector, pSilencer 3.0-H1 (Fig. 1),
was ordered from Ambion, Inc (Austin, TX). The retroviral
vector containing the shRNA-TERT, pSHAG-Magic2-
shTERT, was ordered from Open Biosystems (Huntsville,
AL). All restriction endonucleases including Ecor I, Xho I,
Hind III, Xba I, and BamH I was purchased through
Promega Corp. (Madison, Wisconsin). Survivin
monoclonal antibody was purchased from Santa Cruz
Biotechnology Inc. (Santa Cruz, California). Lipofectin
Transfection Reagent was obtained from Invitrogen Corp.
(Carlsbad, California). Cell lines HT29, HCT 116, and
Huh7.5 were stored by our department.


Ampicliln -


HI Promrnow 397496
CoiEloriin 790 If17


SColE1 origin


Figure 1: siRNA mammalian expression vector used for
Survivin siRNA ligation.


Design and Construction of Survivin siRNA Expression
Vector. The best possible target candidates for survivin
siRNA were chosen by processing human survivin mRNA
(NM001168) through the Ambion (Ambion, Inc; Austin,
TX) siRNA Target Finder. Target sequences were limited
to approximately 50% GC content. Four survivin DNA
templates corresponding to the gene survivin were
synthesized as following: survivin-67 sense, 5'-
GGACCACCGCATCTCTACA-3'; survivin-248 sense, 5'-
AGCATTCGTCCGGTTGCGC-3'; survivin-331 sense, 5'-
ACTGGACAGAGAAAGAGCC-3'; survivin 401 sense,
5' ACTGCGAAGAAAGTGCGCC-3'. Using the selected
target sequences, oligonucleotides will be designed using
the same Ambion software and prepared for cloning into
the siRNA expression vector pSilencer 3.0-H1.
The siRNA survivin sequences are inserted between the
restriction sites BamHI (496) and HindIII (557). Directly
upstream of the siRNA sequence is the H1 Promoter,
which is essential for the transcription process as provided
by RNA Polymerase III. The vector pSilencer 3.0-H1 also
has a region that confers ampicillin resistance, which will
be exploited for purification and isolation of the expression
vector.
Transformation of Survivin siRNA Expression Vector.
The siRNA expression was transformed into DH5a
bacterial cells. The transformation reactions were then
plated on LB plates containing the antibiotic ampicillin and
grown overnight at 370C.
Sequencing and Purification of Survivin Expression
Vector. Four colonies per target sequence were abraded,
isolated, and purified using QIAprep Spin Miniprep Kit.
Each of the colonies corresponding to the 4 survivin target
sequences was kindly sequenced by Han Zongchao from
Dr. Arun Srivastava's Lab. The following are the
sequencing primers as provided by Ambion: M13-forward
sequencing primer: 5'-GTTTTCCCAGTCACGAC-3'; 5'-
GAGTTAGCTCACTCATTAGGC-3'. After sequences
were confirmed and verified that the clones contain the
survivin target insert, the pSilencer plasmids were purified
through QIAprep Spin Midiprep Kit.
Western Blot. The HCT116 cell line was transfected
with survivin siRNA and protein was extracted 72 after
transfection. After purification of the protein, a Western
blot analysis was performed to determine the most efficient
siRNA survivin sequence that down-regulated the survivin
protein in cancerous cell lines. Western Blot Analysis was
kindly performed by Xiaokui Zhang.
Transfection of Survivin and hTERT at Different
Concentrations. Huh7.5 cell lines were grown to
approximately 50% confluency a day prior to transfection.
The cell lines were then transfected with the survivin
siRNA molecule that was most efficient in down-


University of Florida I Journal of Undergraduate Research I Volume 11, Issue 1 I Fall 2009
2






PHILIP W. LIN


11. Chen, J., Wu, W., Tahir, S.K., Kroeger, P.E., Rosenberg, S.H., Cowsert, L.M.,
Bennett, F., Karjewski, S., Karjewska, M., Welsh, K., Reed, J.C., % Ng, S.C.
Downregulation of surviving by antisense oligonucleotides increases apoptosis,
inhibits cytokinesis and anchorage-independent growth. Neoplasia. 2, 235-241
(2000).


13. Kappler,M., Bache,M., Bartel,F., Kotzsch,M., Panian,M., Wurl,P., Blumke,K.,
Schmidt,H., Meye,A., & Taubert,H. Knockdown of survivin expression by small
interfering RNA reduces the clonogenic survival of human sarcoma cell lines
independently of p53. Cancer Gene Ther 11, 186-193 (2004).


14. Mesri,M., Wall,N.R., Li,J., Kim,R.W., & Altieri,D.C. Cancer gene therapy
12. Chawla-Sarkar,M., Bae,S.I., Reu,F.J., Jacobs,B.S., Lindner,D.J., & using a surviving mutant adenovirus. J. Chn. Invest 108, 981-990 (2001).
Borden,E.C. Downregulation of Bcl-2, FLIP or IAPs (XIAP and survivin) by
siRNAs sensitizes resistant melanoma cells to Apo2L/TRAIL-induced apoptosis.
CellDec 11, 915-923 (2004).



































































University of Florida I Journal of Undergraduate Research I Volume 11, Issue 1 I Fall 2009
6





KETOGENIC SNACKS


Fig. 1: rolled, shaped, and frozen


Fig. 2: freeze in fiber mix


Fig. 3: breaded product


10) Once completely frozen, remove broccoli bites from freezer and allow to sit at room temperature for five minutes. Using a spray
bottle, lightly mist bites with water, and roll in hands to form a moist round ball.
11) Dredge bites in dry mix, and return to freezer while still in dry mix. Bites should be covered with dry mix (Figure B).
12) Allow outer coating to freeze (about 45 minutes), and remove from dry mix. Lightly mist with water, and toss lightly in dry mix
(Figure 3).
13) Place coated bites on sheet pan covered with wax paper. Freeze bites until outer coating is completely frozen.

Cooking Instructions

1) Preheat deep fryer or fry oil to 3500F
2) Remove bites from freezer and very lightly mist with water
3) In small batches, fry bites for 15-25 seconds (If you will be eating immediately fry for
35-45 seconds).
4) Remove bites using skimmer, and lay on sheet pan covered with paper towels to
absorb excess oil.
5) Once cool, place in freezer. For long-term storage, place bites in freezer bags once
completely frozen.
6) To reheat, bake in over for approximately 15-20 minutes at 200F, serve immediately.



Figure 4: Finished Product




Appendix B Crab Rangoon Snacks Recipe

Yield: 11- 6.5 oz Crab Rangoon Snacks
Serving Size 4 ea
Servings 2.5

Ingredients

12.3 g -White Crab Meat
1.7 g Fresh Ginger
8.2 g Bok Choy
36.1 g- Philadelphia Original Cream Cheese
0.8 g Green Onion
2.6 g Worcestershire
7.0 g Canola Oil
7.0 g Crisco (Trans-Fat Free)
0.2 g Guar Gum




University of Florida I Journal of Undergraduate Research I Volume 11, Issue 1 I Fall 2009
7






DISCHARGE TIMING IN PRETERM INFANTS


intolerance (Boyd, 2007). This suggests that further studies
are needed to confirm whether our combination of family
visitation and exposure to maternal voice is effective. The
differences in number of days breastfed however could not
have been known beforehand.
Additional limitations to this study are related to the
accuracy and reliability of medically charted variables. A
retrospective review of medical records allowed for a bias
of accurate interpretations because no precise protocol was
followed by the nurses for documentation of the visitations.
There was a lack of identifying which family member
visited, the duration of visit, and what type of interaction
(whether the infant was held, spoken to, etc.) occurred.
Due to this limitation, we chose to opt for the most
accurate option and simply measure the absence or
presence of a family member at the bedside each day.
Further, infant feeding treatment and decisions on when to
progress feeding are largely subjective and vary between
clinicians, thus reducing the reliability of the findings.
Future studies using a prospective design are needed to
increase confidence in the findings. Further, the use of
quota sampling is recommended in order to balance risk
status and feed type between groups in this study. In order
to overcome limitations related to the reliability of taking
the frequency of family visitations from the medical
records, family members could be asked to record their
visitations on a log. The use of a log would also allow
future researchers to obtain the length of time families
stayed by the bedside.
If it is true that episodes of feeding intolerance are
affected by a combination of these simple interventions
(exposure to maternal voice and frequent visitation by
family members), additional research is needed to
investigate other areas that may be impacted by exposure
to maternal voice. For example, studies investigating
whether mother-infant/family-infant interactions are
affected by providing a combination of exposure to
maternal voice and frequent visits by the family. Future
investigations such as these could show immense reasoning
for the importance of maternal voice and high frequencies
of family visitation in the health and wellbeing of preterm
infants.

References

Als, H., Gilkerson, L., Duffy, F. H., McAnulty, G. B., Buehler, D. M.,
Vandenberg, K. et al., (2003). A three-center, randomized, controlled trial of
individualized developmental care for very low birth weight preterm infants:
medical, neurodevelopmental, parenting, and caregiving effects. Journal of
developmental and behavioral pediatrics, 24(6), 399-408.

Bayley N. (1969). 5,i..i scales of infant development: Birth to two years. New
York: The Psychological Corporation.

Becker, P. T., Grunwald, P. C., Moorman, J., & Stuhr, S. (1991). Outcomes of
developmentally supportive nursing care for very low birth weight infants.
Nursing Research, 40, 150-155.


Boyd CA, Quigley MA, Brocklehurst P. (2007). Donor breast milk versus infant
formula for preterm infants: a systematic review and meta-analysis. Arch Dzs
Child Fetal Neonatal. 92 (3):F169 F175

Brown, L. D., & Heermann, J. A. (1997). The effect of developmental care on
preterm infant outcome. ApphedNursing Research, 10(4), 190-197.

Chapman, J.S. (1978). The relationship between auditory stimulation and gross
motor activity of short-gestation infants. Researchin Nursing and Health, 1(1),
29-36.

Clark, R., Powers, R., White, R., Bloom, B., Sanchez, P., & Benjamin, D.K.
(2004). Nosocomial infection in the NICU: A medical complication or
unavoidable problem? Journal of . 24, 382-388.

Committee on Hospital Care. American Academy of Pediatrics. (2003). Family-
centered care and the pediatrician's role policy statement. Pediatrics, 112(3),
691-696.

Cooper, L.G., Gooding, J.S., Gallagher, J., Sternesky, L., Ledsky, R., Berns,
S.D. (2007). Impact of a family-centered care initiative on NICU care, staff and
families. Journalof 27, S32-S37

Diehl-Jones, W. L., & Askin, D. F. (2004). Nutritional modulation of neonatal
outcomes. AACN Clinicallssues, 15(1), 83-96.

Fleisher, B. E., VandenBerg, K., Constantinou, J., Heller, C., Benitz, W. E.,
Johnson, A. et al. (1995). Individualized developmental care for very-low-birth-
weight premature infants. Chn Pediatr (Phila), 34(10), 523-529.

Gerhardt, K. (1989) Characteristics of the fetal sheep sound environment.
Seminars n ... 13(5):362-70

Graven, S.N. (2000). Sound and the developing infant in the NICU: conclusions
and recommendations for care. Journal of . 20(8), 88-93.

Krueger, C. (2008). Variation in Care Practice And Discharge Timing in Preterm
Infants. Poster presented at the meeting of the International Society for
Developmental Psychobiology, Washington, D.C.

Lau, C., Smith, E. O., & Schanler, R. J. (2003). Coordination of suck-swallow
and swallow respiration in preterm infants. Acta Paediatr, 92(6), 721-727.

Lickliter R. (2000). The role of sensory stimulation perinatal development:
Insights from comparative research for care of the high-risk infant. Journal of
Developmental and Behavioral Pediatrics, 21(6), 437-467.

Malloy, G.B. (1979). The relationship between maternal and auditory stimulation
and the developmental behavior of premature infants. Birth Defects: Original
Article Series 15(7), 181-89.

Melnyk, B. M., Feinstein, N. F., Alpert-Gillis, L., Fairbanks, E., Crean, H. F.,
Sinkin, R. A. et al., (2006). Reducing premature infants' length of stay and
improving parents' mental health outcomes with the Creating Opportunities for
Parent Empowerment (COPE) neonatal intensive care unit program: a
randomized, controlled trial. Pediatrics, 118(5), e1414-1427.

Mihatsch, W. A., von Schoenaich, P., Fahnenstich, H., Dehne, N., Ebbecke, H.,
Plath, C. et al., (2002). The significance of gastric residuals in the early enteral
feeding advancement of extremely low birth weight infants. Pediatrics, 109(3),
457-459.

Petrou, S. (2003). Economic consequences of preterm birth and low birthweight.
BJOG: An International Journal of Obstetrics & Gynaecology, L, '. '21 17 2

Simon & Schuster. (1985). Opposites: Nursery rhyme concept books. New York,
New York: Little Simon Publishing.
Westrup, B., Kleberg, A., von Eichwald, K., Stjemqvist, K., & Lagercrantz, H.
(2000). A randomized, controlled trial to evaluate the effects of the newborn
individualized developmental care and assessment program in a Swedish setting.
Pediatrics, 105(1 Pt 1), 66-72.


University of Florida I Journal of Undergraduate Research I Volume 11, Issue 1 I Fall 2009
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RUSSELL J. OWEN


Hedonic Results for Broccoli Bites


11 ----

3 2
25
JL 0


-cdonjc Ratkitng


8


Figure 2: Sensory Results for Broccoli Bites


Panelists were also asked to comment on the
appearance, taste, and mouthfeel of the broccoli bites
and crab rangoon. Comments for the broccoli bites
included "looks crunchy, nice golden color", "It's very
good. It's a nice mix of natural, fried, and cheesy flavor.
I would recommend this item because of the
taste...seems like a perfect snack", and "not very
broccoli taste, sort of tastes like dirt with a gritty crust".
Comments for the crab rangoon included "batter looks
crispy with some flakiness, looks good", "no real shape,
could be darker in color", "I really like the fried taste
and the gooeyness of the sample...YUMMY! It wasn't
too fishy either", and "it tastes like old bad tuna"
Figure 3 depicts the demographic results for age and
gender of the 67 panelists that participated in sensory
evaluation are as follows. The majority of panelists fell
between the ages of 18 and 24, with 30% of panelists
reporting to be between 18 and 20 years of age, and 46%
of panelists reporting to be between 21 and 24 years of
age. The highest reported age was between 55 and 59
years of age.

Discussion

The purpose of the research project was to develop
high fiber ketogenic snacks, and to determine the
sensory acceptability of the developed food products. It
has been shown that the developed high-fiber ketogenic
snacks are acceptable sensory participants in this study,
but acceptability amongst patients on ketogenic therapy
is undetermined. Underlying factors that contribute to
acceptability of the ketogenic snacks amongst the public
are likely to influence acceptability amongst ketogenic
patients. For instance, preferences for certain types of
foods will have a major influence on acceptability, as
was evident in sensory evaluation of the ketogenic
snacks. Hedonic scaling results varied from a score of 1


(disliked extremely) to a score of 9 (liked extremely) for
overall appearance, taste, and mouthfeel. The discrepancy in
acceptability may be attributed to preference for certain types
of food and methods of preparation. Interestingly, sensory
panelists who consumed fried foods on an average times per year reported a lower acceptability for both
ketogenic snacks in terms of overall appearance, taste, and
mouthfeel with the exception of the overall appearance of the
crab rangoon. It is expected that children who suffer from
intractable epilepsy will have a greater preference for high-fat
foods, however, it is not clear if ketogenic patients will prefer
the types of ketogenic snacks evaluated by the students and
staff at the Univesity of Florida. Amari et al. (2008) has
shown that children who have seizures show a significant
higher preference for high-fat foods. The children who
participated in this study had never been initiated on
ketogenic therapy, but showed a significant preference for
high fat foods when compared to a control group of children
who did not have seizures. One child in the study with
seizures stated that "this one makes me feel better" after
consuming samples of butter, cream, cheese, and mayonnaise.
Development of the ketogenic snacks offered many
challenges, but yielded a novel method of preparing
ketogenic foods. Frying foods would be the ideal method of
delivering high fat food items to ketogenic patients, but the
usage of flours to bread foods prior to being fried presents
many challenges. Using a fiber mix to replace the
carbohydrates in flour creates an opportunity to provide fried
foods to ketogenic patients. Fiber is similar in structure to
carbohydrate, except it is indigestible by the humans.
Interestingly, soluble fibers may be fermented by bacteria in
the large intestine to yield short-chain fatty acids, as opposed
to being broken down into mono and disaccharides for
absorption. The effects of increasing the fiber intake of those
on ketogenic therapy is uncertain, however, determining how
fiber intake influences ketogenic therapy may present
opportunities to improve the efficacy of ketogenic therapy.


University of Florida I Journal of Undergraduate Research I Volume 11, Issue 1 I Fall 2009


ApprlllpcrlS





INHIBITION AND APOPTOTIC RESPONSES OF HUMAN COLORECTAL CARCINOMA CELLS


regulating survivin (survivin67). The concentrations at
which transfection occurred were at 1 ug, 2 ug and 3 ug. 72
hours after transfection, pictures were taken and cells were
counted.
The previously described protocol was applied to
hTERT at the same concentrations as described above.
Cells were counted after 72 hours from the start of the
transfection.
Co-Transfection of Survivin siRNA. Three cell lines
(Huh7.5, HCT116, and HT29) were cultured to
approximately 50% confluency the day prior to
transfection. The cell lines were transfected with a negative
plasmid, survivin-331 vector, hTERT vector, and both
vectors, respectively. Pictures were taken and cells were
counted 72 hours after transfection.
Adeno-Associated Virus (AAV) packaging. The
packaging of AAV requires approximately 800-1000 ug/ul
of the AAV vector and two helper vectors (pHelper,
pACG-2). Han Zhongchao kindly supplied the viral vectors
from Dr. Arun Srivastava's lab. The pSilencer3.0-H1
containing the sur67 siRNA sequence and the H1 promoter
were excised using the restriction enzymes EcorI and
HindIII. This segment was then inserted into the pdsAAV-
EGFP between the restriction site Acc65I (1334), which is
located directly downstream of the mutated ITR. The
successful ligation of the sur67 sequence into the AAV
vector was confirmed by restriction digest with BamH1,
BamH1 with XbaI, and BamH1 w/ HindIII, respectively.
The pHelper and pACG-2 vectors were also confirmed
by restriction digest with the restriction enzymes Clal and
XmnI. After restriction digest was performed on all 3
vectors, they were maxi-prepped and quantified for the
concentration. The vectors were then sent to the UF core
lab for further packaging of the virus for effective viral
delivery.




RESULTS

Western Blot Analysis

Western blot analysis was performed twice. In both
experiments, survivin-67 limited the greatest amount of
survivin protein expression in the HCT 116 cell line after
72 hours post-transfection. The results in the HCT116 cell
lines represent significant knockdown of the survivin
protein expression by siRNA treatment. These results
allowed us to discern that survivin-67 would be the best
survivin target to introduce as a siRNA for gene therapy.
The subsequent experiments after the Western blot was
completed with survivin-67 as the only target for the
siRNA.


- -U..


a.I
- -c


S.. .w
*" *.

Figure 2: Western blot analysis of HCT116 cell lines transfected
for 72 hours with survivin-67, 259, 331, 401, respectively


AI A '4r


~ 19


'5 -


U .i9 l


* 1! 3 4


;. s *


-, ,f.
-r li.


4Z '


Figure 3: Western blot analysis of HCT 116 cell lines
transfected for 72 hours with survivin-331 (wells 1-3), survivin-
67 (wells 4-6), survivin-259 (wells 7-9), siu i ii\ i-4-1 (10-12)


Apoptotic Cell Death

The results of Huh7.5 cells transfected with hTERT
siRNA shows greater than a 50% decrease in cell viability
in a dose dependent manner (Fig. 4a). Increasing
concentrations of hTERT siRNA shows greater apoptotic
response. Transfection of Huh7.5 cells with survivin
siRNA shows a similar pattern in apoptotic response. 1 ug
of survivin siRNA decreased cell viability by
approximately 30%, while 2 ug and 4 ug of survivin


University of Florida I Journal of Undergraduate Research I Volume 11, Issue 1 I Fall 2009
3


--,,,
"f~






PHILIP W. LIN


siRNA decreased cell viability by greater than 50% (Fig.
3b).
Huh7.5 and HT29 cell lines all decreased in cell
viability in response to different treatments of siRNA (Fig.
6c). Cotransfection of survivin and hTERT results in
greater apoptotic response in HCT116 and HT29 cell lines.
HCT116 cells displayed virtually no increase in cell death
when treated with only survivin or hTERT, but decrease in
cell viability was observed when cotransfected with
survivin and hTERT.


A. Transfection of Huh7.5 cells with
hTERT -72 hours

120
o
S 100 0
S 80 -80
C 60
S40 -
ao 20
I 0
Control 1 ug 2 ug 3 ug
hTERT Plasmid Concentration



B. Transfection of Huh7.5 cells with
Survivin

5 120
S100
u 80-
0 60-
1 40
'O 20

Control lug 2ug 3ug
Survivin Plasmid Concentration



C. Transfection of Cell Lines with
Survivin and hTERT Treatments

120

e5 60 8 5*HCT1
4 HT29

Control 2 ug 2 ug 2 ug
SurImn hTERT Surymn
&
hTERT
siRNA Treatments


Figure 4: a) Transfection of Huh7.5 cell lines with different
concentrations of hTERT siRNA b) Transfection of Huh7.5 cell
lines with different concentrations of survivin siRNA c)
Transfection of Huh7.5, HCT116, and HT29 cell lines with
different treatments of survivin siRNA, hTERT siRNA, and
cotransfection of survivin and hTERT siRNA


Adeno-Associated Virus (AA V) packaging


Presented in Figure 5 are the vector maps for pdsAAV-
EGFP, pHelper, and pACG-2 paralleled with their
corresponding restriction digests that confirm their identity.
The confirmation of the viral vectors allows us to continue
with AAV packaging for further use for in vivo studying.


Ba0Ill (1)
H1 Promoter siRNA-sur67
A oRI (j4699) \ | flII (64)
ApdA (24110) \ #


pSilencer-sur67
2797 bI6
Ap&] (1983)




Mutated ITR H1 Promoter
S sRNA-sur67

intron



pdsAAV-sur67-EGFP' EGF


poly


ApUt (t17


Figure 5: a) vector maps of pSilencer3.0-H1 with survivin67
insert and ligation into pdsAAV-EGFP b) restriction digest of
pdsAAV-sur67-EGFP with BamH1, BamH1 + Xbal, BamH1 +
HindIII, respectively


University of Florida I Journal of Undergraduate Research I Volume 11, Issue 1 I Fall 2009






DARYA VOROBYEVA


their control counterparts support the notion that the
Eufortyn treatment boosts mental acuity. The velocity
(cm/s) measure confirms the two previous measures of
distance and duration in corroborating the positive effect of
the treatment on cognition and therefore, facilitation of
learning, habituation, and application of problem solving
strategy in the 19 and 27-month old animal models.
In the PTP analysis of the Eufortyn treated groups, the
calcium retention capacity increased by 66% in the 19-
month old rats and 19% in the 27-month old rats
establishing sufficient evidence that Eufortyn prevented
mitochondrial-mediated apoptosis and reduced inclinations
toward cell death in vivo; this is a remarkable result
pointing out the treatment's preventative features in
delaying mitochondrial-mediated apoptosis and reducing
the susceptibility to cell death in vivo.
The non-heme iron assay results provided a positively
correlating trend between the aging rats and non-heme iron
concentration found in gastrocnemius muscle tissue. The
typical increase of iron seen in aging skeletal muscle has
been substantially suppressed by supplementation of
Eufortyn just over a six week period providing strong
evidence that Eufortyn helped diminish non-heme iron
levels in aging muscle.
Nucleic acid oxidation results propose that the treatment
is clearly more promising in the middle-aged animals than
in the 27-month old rats; 27-month olds failed to benefit as
much as 19-month olds because of irreversible damage.
The intervention with Eufortyn needs to be initiated at an
earlier age than 27-month old to see optimal effects.
Considering all the trends, it is imperative for future
clinical trials to take place. Further, in-depth studies will
work to validate Eufortyn as an effective therapy in
delaying aging effects while improving the strength,
fatigue resistance, and independence of the elderly
population.

Bibliography


(1) Marzetti E, Leeuwenburgh C. Skeletal muscle apoptosis, sarcopenia
and frailty at old age. Exp Gerontol 2006 October 17;41(12):1234-8.

(2) Judge S, Jang YM, Smith A, Hagen T, Leeuwenburgh C. Age-
associated increases in oxidative stress and antioxidant enzyme activities in
cardiac interfibrillar mitochondria: implications for the mitochondrial theory of
aging. FASEB J 2005 March; 19(3):419-21.

(3) Judge S, Leeuwenburgh C. Cardiac mitochondrial bioenergetics,
oxidative stress, and aging. Am JP-: ... Cell : *... .,' 2007 June;292(6):C1983-
C1992.

(4) Steller H. Mechanisms and genes of cellular suicide. Science 1995
March 10;267(5203):1445-9.

(5) Duke RC, Ojcius DM, Young JD. Cell suicide in health and disease.
Scz Am 1996 December;275(6):80-7.


(6) Warner HR. Apoptosis: a two-edged sword in aging. Ann N YAcad
Sci 1999;887:1-11.

(7) Cortopassi GA, Wong A. Mitochondria in organismal aging and
degeneration. Bzochim 5,,. ..- th 1999 February 9;1410(2):183-93.

(8) Dirks AJ, Leeuwenburgh C. The role of apoptosis in age-related
skeletal muscle atrophy. Sports Med 2005;35(6):473-83.

(9) Leeuwenburgh C, Heinecke JW. Oxidative stress and antioxidants in
exercise. Curr Med Chem 2001 June;8(7):829-38.

(10) Sohal RS, Weindruch R. Oxidative stress, caloric restriction, and
aging. Science 1996 July 5;273(5271):59-63.

(11) Sohal RS. Oxidative stress hypothesis of aging. Free Radzc BiolMed
2002 September 1;33(5):573-4.

(12) Leeuwenburgh C, Fiebig R, Chandwaney R, Ji LL. Aging and
exercise training in skeletal muscle: responses of glutathione and antioxidant
enzyme systems. Am JP:: '... ,' 1994 August;267(2 Pt 2):R439-R445.

(13) Powers SK, Kavazis AN, Deruisseau KC. Mechanisms of disuse
muscle atrophy: role of oxidative stress. Am J F-' ... ..' Regul ntegr Compi U r., .
2005 February;288(2):R337-R344.

(14) Altun M, Edstrom E, Spooner E, Flores-Moralez A, Bergman E,
Tollet-Egnell P, Norstedt G, Kessler BM, Ulfhake B. Iron load and redox stress
in skeletal muscle of aged rats. Muscle Nerve 2007 August;36(2):223-33.

(15) Cook CI, Yu BP. Iron accumulation in aging: modulation by dietary
restriction. Mech Ageing Dev 1998 May 1;102(1):1-13.

(16) Mizuno K, Tanaka M, Nozaki S, Mizuma H, Ataka S, Tahara T,
Sugino T, Shirai T, Kajimoto Y, Kuratsune H, Kajimoto O, Watanabe Y.
Antifatigue effects of coenzyme Q10 during physical fatigue. Nutrition 2008
April;24(4):293-9.

(17) Ochoa JJ, Quiles JL, Lopez-Frias M, Huertas JR, Mataix J. Effect of
lifelong coenzyme Q10 supplementation on age-related oxidative stress and
mitochondrial function in liver and skeletal muscle of rats fed on a
polyunsaturated fatty acid (PUFA)-rich diet. J GerontolA Biol Sci MedSci 2007
November;62(11):1211-8.

(18) Jager R, Metzger J, Lautmann K, Shushakov V, Purpura M, Geiss KR,
Maassen N. The effects of creatine pyruvate and creatine citrate on performance
during high intensity exercise. JIntSoc Sports Nutr 2008;5:4.

(19) Pearlman JP, Fielding RA. Creatine monohydrate as a therapeutic aid
in muscular dystrophy. Nutr Rev 2006 February;64(2 Pt 1):80-8.

(20) Candow DG, Chilibeck PD. Effect of creatine supplementation during
resistance training on muscle accretion in the elderly. JNutr HealthAging 2007
March;11(2):185-8.

(21) Chen TS, Liou SY, Chang YL. Antioxidant evaluation of three
adaptogen extracts. Am J Chin Med 2008;36(6):1209-17.

(22) Kitts D, Hu C. Efficacy and safety of ginseng. Publc Health Nutr
2000 December;3(4A):473-85.

(23) Nitta H, Matsumoto K, Shimizu M, Ni XH, Watanabe H. Panax
ginseng extract improves the scopolamine-induced disruption of 8-arm radial
maze performance in rats. BiolPharm Bull 1995 October; 18(10):1439-42.

(24) Cesari M, Leeuwenburgh C, Lauretani F, Onder G, Bandinelli S,
Maraldi C, Guralnik JM, Pahor M, Ferrucci L. Frailty syndrome and skeletal


University of Florida I Journal of Undergraduate Research I Volume 11, Issue 1 I Fall 2009
10







Assessing the Efficacy of Eufortyn A Terclatrated CoQ10 and

Creatine Combination Therapy on the Aging Rat Model


Darya Vorobyeva*


College of Medicine, University of Florida


With the progression of age, the organism enters a highly oxidized state resulting from impaired mitochondrial function. As the
susceptibility to reactive oxygen species heightens, a decline of both size and number of skeletal muscle fibers manifests in chronic
fatigue syndrome known as sarcopenia in the elderly. Consumption of antioxidant as part of a diet may help regulate oxidative damage
by reducing the rampant activity of reactive oxygen species in the cell, yet the poor bioavailability of most commercial supplements
limit the capacity of the antioxidant to quench free-radicals. Eufortyn is a terclatrated formulation of Coenzyme Q10, creatine, and
ginseng extract that retains the integrity of the antioxidant moiety, while allowing maximal absorption into the intestinal mucosa. A
Eufortyn pellet was fed orally to 19 and 27-month old Fischer 344 x BNF1 rats for six weeks. Grip strength improved (12%) in 19-
mos, but not in 27-mos old rats. Water maze analysis exhibited sharpened cognitive performance in Eufortyn-treated old-age rats
compared to their age-match peers. Significant advancement in mitochondrial calcium retention capacity (66% in 19-mos and 19% in
27-mos), diminished non-heme iron levels (54% in 27-mos), and suppressed nucleic acid oxidation (31% in 19-mos and 24% in 27-
mos) were observed in skeletal muscle in Eufortyn-treated cohorts. These findings indicate that treatment is more effective in the
middle-aged animals in comparison to the 27-month old rats suggesting that intervention with Eufortyn needs to be initiated at an
earlier age than 27-month old to see optimal effects.


Introduction

Aging is a natural, degenerative process occurring
throughout the life cycle of all organisms. In the elderly,
chronic fatigue syndrome is an expected, yet degenerative
symptom of aging evidenced by the decline of muscle
fibers, diminished cognitive function resulting in the
departure from a self-sufficient lifestyle '. The impairment
of mitochondrial bioenergetics is theorized to be the central
mechanism behind tissue dysfunction, fatigue and aging 2,
. In normal aging, cellular matter undergoes a highly
regulated form of cell death characterized by
morphological, biochemical, and molecular events referred
to as apoptosis 4-7. When apoptosis afflicts skeletal muscle,
the decline in both the size and number of both kinds of
muscle fibers, particularly the Type II (fast-twitch fiber)
contributes to the condition known as sarcopenia.
Sarcopenia is a debilitating condition commonly associated
with diminished muscle mass, strength and increased
disability and dependence 8. Apoptosis, in healthy cells,
allows for turnover and removal of defective cellular
matter as a way to promote tissue homeostasis.
Regrettably, once apoptosis strikes post-mitotic cells
(skeletal muscle fibers), regeneration is not possible. Thus,

*with Jinze Xu, Ph.D, Christiaan Leeuwenburgh, Ph.D.


progression of apoptosis is linked to the increasing frailty
experienced in aging. Being that apoptosis is strictly
regulated by controlled signaling platlh a ~ ', it is apparent
that any oxidative damage afflicting internal cellular
homeostasis will inevitably lead the cell to programmed
death. The central theory of aging states that mitochondrial
free-radical formation destabilizes the internal cellular
balance because endogenously occurring antioxidants fail
to manage the rampant pro-oxidant activity 9

Free-Radical Chemistry

Senescence is tied to the accrual of tissue damage by
free-radicals that results from a marked shift in pro-oxidant
activity 10 11. Normally, there is a balance between pro-
oxidant generation and anti-oxidant defense. However, in
aging, the ratio of pro-oxidants increases relative to the
endogenously occurring anti-oxidant activity 12. The free-
radical formation pathway begins in the mitochondria of
the skeletal muscle where superoxide anion (02-) and
hydrogen peroxide (H202) diffuse in and out of skeletal
muscle cells. When ferrous (Fe2+) iron, a pro-oxidant,
reacts with diatomic oxygen, the produced H202 is
converted to the highly unstable hydroxyl radical (.OH) via
Fenton chemistry (Fig. 1). Additionally, ferric (Fe3+) iron
can in turn react with hydroxyl radical and hydroxide ion to
further contribute to overall superoxide ion concentration
via Haber-Weiss reactions 3. The resulting oxidation is


University of Florida I Journal of Undergraduate Research I Volume 11, Issue 1 I Fall 2009





RUSSELL J. OWEN


Utensils for Preparation

3 Med Size Mixing Bowls
Cutting Board
Chefs Knife
3 Sheet pans
Wax paper
Digital food scale
Freezer Bags
Paper Towels


Utensils for Cooking

2Qt Pot or Deep Fryer
Oil for frying (amount will depend on method used)
Fry Skimmer


Preparation Directions

1) Drain excess water from crab meat.
2) Slice cream cheese into smaller portions (2in x 2in), and allow to come to room temperature (about 1 hour).
3) Cut tops and bottoms (1") off of bok choy, and finely chop the remainder (use half greens and half stalk for mixture).
4) Place crab and bok choy into mixing bowl.
6) Mix cream cheese with crab and bok choy.
7) Add remaining ingredients and mix thoroughly.
8) Refrigerate for 1 hour, or until chilled (it may be necessary to cool in freezer to achieve desired shape as described below).
9) In small batches, weight out 6.5g portions of crab mix. Roll portions into ball shape, place on sheet pan covered with wax paper,
and freeze.
Repeat process until all mix has been weighed, shaped, and froze.
10) Once completely frozen, remove crab rangoon from freezer and allow to sit at room temperature for five minutes. Using a spray
bottle, lightly mist bites with water, and roll in hands to form a moist round ball.
11) Dredge crab rangoon in dry mix, and return to freezer while still in dry mix. Bites should be covered with dry mix (Figure 1).













Fig. 1: freeze in fiber mix Fig. 2: breaded product

12) Allow outer coating to freeze (about 45 minutes), and remove from dry mix. Lightly mist with water, and toss lightly in dry mix
(Figure 2).
13) Place coated bites on sheet pan covered with wax paper. Freeze bites until outer coating is completely frozen.

Cooking Instructions

1) Preheat deep fryer or fry oil to 3500F
2) Remove crab rangoon from freezer and very lightly mist with water
3) In small batches, fry crab rangoon for 15-25 seconds (If you will be eating
immediately fry for 35-45 seconds).
4) Remove using skimmer, and lay on sheet pan covered with paper towels to
absorb excess oil.
5) Once cool, place in freezer. For long-term storage, place bites in freezer bags
once completely frozen.

6) To reheat, bake in oven at 2000F for 15-20 minutes; serve immediately.
Fig. 3: Finished Product


University of Florida I Journal of Undergraduate Research I Volume 11, Issue 1 I Fall 2009
8








Signal Transduction: Environmental Stimulus to Changes in

Global Transcription in Porphyromonas gingivalis


Cory James Smith


College of Dentistry, University of Florida


Porphyromonas gingivalis is an anaerobic oral pathogen that has been associated with atherosclerosis and coronary heart disease.
Genetic regulation in eubacteria occurs primarily at the level of transcription. The specificity of RNA polymerase for promoters is
determined by the sigma subunit. Regulation of sigma factors is generally achieved by an attached proprotein sequence or an anti-
sigma factor capable of responding to environmental cues. The string database was used to determine the genes in the operons
containing putative sigma factors in the P. gingivalis genome. The putative sigma factors were tested for proprotein sequences using
signalP peptide cleavage site prediction software. This test concluded that none of the putative sigma factors contained a cleavage site
similar to any previously characterized system. The genes in cotranscriptional units with sigma factors were tested for transmembrane
helicies using TMHMM, revealing that seven of the ten operons containing a sigma factor had an integral protein. These results
provide a set of possible environmentally reactive transcriptional regulators that can be confirmed with a yeast two-hybrid test to
confirm protein-protein and protein-DNA interactions.


Within our bodies resides a dynamic population of
microbial cells that are estimated to outnumber human
cells ten to one (Backhed). This consortium of microbiota
and their fluctuating collective genomes encode
metabolic and physiological functions that are not
encoded by the human genome (Backhed). This new view
of the human body as a consortium consisting of
symbiotic eukarya, archaea, and eubacteria is forcing
scientists to redefine what is considered self. We are more
than just the familiar eukaryote with 23 diploid
chromosomes that we know as human but also all of our
microbial inhabitants living together symbiotically.
(Nicholson).
It has been estimated that 700 different species of
microorganisms can colonize the oral cavity (Aas).
Colonization of dental plaque occurs in distinct waves of
microbial species over time (Hasegawa N). The primary
colonization of the oral cavity occurs by streptococci and
actinomyces species (Aas, Janeway). The initial col-
onization alters the microenvironment of the mouth
permitting the establishment of gram positive rods and
gram negative bacteria such as Fusobacterium nucleatum.
The environment is again altered by its new colonizers
creating new a new niche that gram-negative anaerobes
such as Porphyromonas gingivalis can exploit (Hasegawa
N).
P. gingivalis is strongly implicated as an etiological
agent of periodontal disease, a chronic inflammatory
infection of the tissues that surround and support the teeth
(Dor). Historically periodontal disease has been an
associated risk factor with heart disease and theories


about the causes of coronary heart disease are changing to
include pathogenic factors (Dor). Recently, viable P.
gingivalis was isolated and proven invasive from
atherosclerotic plaque (Kozarov). Oral bacteria have an
entry route into the circulatory system in patients afflicted
with periodontitis simply when patients floss, brush or
chew (Sconyers).
Atherosclerosis is a chronic inflammatory disease
caused by prolonged damage from the accumulation of
immune cells along the arterial wall. (Libby) During the
course of atherosclerosis normal endothelial functions are
altered to express adhesion cytokines such as integrin and
selection that induce leukocyte recruitment and
inflammation (Libby).
Eubacterial RNA polymerase (RNAP) typically
consists of a four subunit core (3p'a2) and a sigma
subunit that reversibly binds with RNAP, forming an
active holoenzyme (pp'a20). The active holoenzyme is
capable of forming an open promoter complex and
initiating transcription. The sigma subunit is responsible
for direct contact and recognition with the -10 and -35
boxes of the promoter and determines promoter
specificity (Klimple). The number of sigma factors
encoded in studied eubacterial genomes is highly
variable, ranging from 1 in Mycoplasma sp. to as many as
65 in Streptomyces coelicolor (Manganelli). Each sigma
factor has unique specificity for a promoter sequence.
Often genes of related function are grouped into operons
with a single promoter that are transcribed in one
polycistronic mRNA. Regulons are sets of operons that
are expressed simultaneously because they share a similar


University of Florida I Journal of Undergraduate Research I Volume 11, Issue 1 I Fall 2009





DARYA VOROBYEVA


Groups Control Control Eufortyn Control Eufortyn

Age 6 19 19 27 27
(months)
Rats 7 8 8 7 7

Table 1: Eufortyn Pilot Animal Distribution. Randomized
selection of animals into experimental and control groups
followed by placement into cohorts C6, C19, E19, C27 or
E27.


Figure 2: Eufortyn Pilot Study Design Plan. Cohorts were
staggered so as to allow for sufficient assimilation,
treatment, physical study, and sacrifice time.

guillotine to avoid interference of anesthesia on
mitochondrial functions. All discomfort, distress, pain, or
injury was accounted for in this pilot study. Select muscle
gastrocnemiuss, plantaris, soleus, EDL, and quadriceps)
and organ tissue (heart, kidney, liver, and brain) were
extracted fresh, weighed, and flash-frozen in liquid
nitrogen followed by storage in -800C until analysis. These
methods were all consistent with the recommendation of
the Panel of Euthanasia of the American Veterinary
Medical Association .

Grip strength-behavioral/functional testing

Five weeks into treatment, functionality analyses of the
forelimb grip were assessed to determine treatment effect
on retention of musculoskeletal performance of the three
age groups. An automated grip strength meter was used as
a standard measuring device for all animals (Fig. 3). The
mean force in grams was determined with a computerized
electronic pull strain gauge fitted directly to the grasping
ring. To normalize, the resulting measurement would then


Figure 3: Grip Strength Analysis. The grip strength apparatus
measured forelimb strength and is predictive of future physical
disability. Grip strength results were expressed as total grip
strength force (kg of force) and total force divided by body
weight (kg of force/kg body weight).


be divided by body mass. After 3 successful trials were
conducted, an average was taken to determine the final
outcome.

Permeability Transition Pore

In aging, progressive deregulation of the mitochondria
organelle leads to decreased life-sustaining functions such
as ATP production, intracellular Ca2+ buffering, and
regulation of cellular redox balance and apoptosis 29, 30
Additionally, increases in non-heme iron positively
correlate with age-related mRNA oxidative damage,
diminishing mitochondrial capacity to handle influxes of
Ca2 in skeletal muscle. This theory is central to the idea
that the pro-oxidant effects of non-heme iron influences
mitochondrial impermanence thus, triggering cellular
apoptosis and the overall dilemma of neuromuscular
degeneration 31. In a highly oxidized state, such as in
senescence, mitochondrial integrity is compromised by its
inability to tolerate membrane impermeable calcium
uptakes before opening and releasing cytotoxic factors.
This mitochondrial suicide is catalyzed by the permeability
transition pore (mPTP), a voltage-dependent, high-
conductance, non-specific passive pore that infiltrates the
mitochondrial matrix and the outer and inner mitochondrial
membranes. The opening of mPTP compromises
mitochondrial membrane potential and leads to a transition
in membrane permeability 32. The negative relationship
between Ca2+-retention capacity and iron contents implies
that higher iron content renders the mitochondria more
susceptible to Ca2+- induced mPTP opening and thus,
apoptosis 3. The mPTP of both control and Eufortyn


University of Florida I Journal of Undergraduate Research I Volume 11, Issue 1 I Fall 2009
4





DARYA VOROBYEVA


SSM


to-
Gno-
50.
d'~
40.


200]
E 0



ii



1~1 1001
Z


SSM


t-I
40]

L
10


iControt
Eufortyn


-r-


Age
(months)


10
Age
(months)


Figure 12: SSM and IFM Calcium Retention. The calcium retention capacity of the subsarcolemmal mitochondria
(SSM) decreased significantly with age in the 19- and 27-month old animals compared to the 6-month old control
(Fig 12A lower panel). One-way ANOVA revealed a significant age effect for SSM (Fig 12A lower panel; p< 0.05).
Eufortyn treated mitochondrial demonstrated a marked increase in the calcium retention capacity of the SSM
isolated from the 19-month old rats (66%) and a modest increase in the 27-month old rats (19%) (Fig 12B upper
panel). IFM mitochondria did not exhibit any significant trend.


19-month Eufortyn treated rats appeared to be fastest
initially, but eventually leveled off in speed with the 27-
month Eufortyn treated rats. Nevertheless, the control
groups consistently lagged behind their age-matched
Eufortyn groups throughout the course of the experiment.

Permeability transition pore (PTP) opening

The maximal calcium loading capacity of isolated
mitochondria was determined by using membrane
impermeable fluorescent probe, Calcium Green-5N.The
calcium uptakes of subsarcolemmal (SSM) and
intermyofibrillar (IFM) mitochondria were measured by
using the microplate reader with automatic injectors. In
control 19 and 27-month old rats as compared to the 6-
month olds, the SSM endured more calcium additions
before pore opening and expulsion of cell-death markers
(Fig. 12). In other words, with age the mitochondria uptake
less calcium additions (in nmol of calcium/ mg of
mitochondrial protein) for the opening of the permeability
transition pore and release of cytotoxic factors such as the
pro-apoptotic proteins cytochrome c and apoptosis-
inducing factor.

Eufortyn and non-heme iron

The total non-heme iron content of gastrocnemius


muscle in control cohorts showed significant iron
accumulation as part of a normal aging effect (Fig. 13).
Eufortyn treatment of age-matched cohorts showed no
distinction at the 6 and 19-months, but a significant (54%,
p< 0.05) mitigation in iron content was observed in the 27-
month old senescent rats.

Eufortyn and oxidative stress

Oxidized DNA was assessed by the levels of oxidative
products 8-oxo-7,8-2'-deoxyguanosine from control and
Eufortyn rats at 6, 19, and 27 months of age. One-way
ANOVA showed no significant age effect for the DNA
oxidative damage (Fig. 14 left panel). However, DNA
oxidative damage in both 19- and 27-month old rats was
attenuated by 31% and 24% individually in the Eufortyn
groups as compared to age-matched controls (Fig. 14 right
panel). Two-way ANOVA indicated a significant treatment
effect (p < 0.10), suggesting that Eufortyn effectively
mitigates the DNA oxidative damage in the gastrocnemius
muscle.

Conclusion

As a whole, these results have advanced our
understanding of Eufortyn's effectiveness on intracellular
energy regulation in skeletal muscle mitochondria by way


University of Florida I Journal of Undergraduate Research I Volume 11, Issue 1 I Fall 2009
8





RUSSELL J. OWEN


ultimately shifting the metabolism of the brain to utilize
ketone bodies as a major fuel (Matthews and Van Holde,
2000).
Challenges of administration of ketogenic therapy
can be contributed to numerous factors. An obvious
obstacle is the lack of commercial foods that are
consistent with the nutritional demands of ketogenic
diet. A major hurdle in developing widely available
ketogenic foods is creating foods that are acceptable
amongst large populations. In order to have widely
available foods compatible with ketogenic therapy a
large enough market must exist to produce affordable
products. Producing foods that are acceptable amongst
the general public creates the possibility of marketing
ketogenic foods within grocery stores, and making
ketogenic foods widely available to patients utilizing
ketogenic therapy. Meal components of ketogenic
therapy must be viewed as typical for the average diet,
and not therapeutic in nature to appeal to the average
consumer. Creating foods that appeal to the average
consumer, while therapeutic in composition, may be a
promising approach to improving compliance with
ketogenic therapy. Sensory evaluation could determine
the acceptability of these foods amongst the normal
population.
Affective tests measure subjective attitudes towards a
product based on sensory properties. Affective testing
methods may include paired comparison, hedonic scale,
and ranking. Hedonic scaling tests are effective in
measuring a degree of liking for a particular food
product. Hedonic scaling is commonly performed with a
nine point scale, ranging from "like extremely" to
"dislike extremely." A neutral response exists in the
central location, with the response corresponding to
"neither like or dislike." Hedonic scaling is a useful tool
in determining if a given food product would be
successful in the market place.
The effects fiber intake of the efficacy of ketogenic
diet are unknown, however, with isolated fiber (devoid
of protein and with very low levels of available
carbohydrate), we may be able to provide adequate
levels of fiber in the ketogenic diet. Increasing fiber
intake in the diet of those on ketogenic therapy has the
potential to enhance quality of life while easing the
burden of implementing ketogenic therapy. Through
product development and sensory testing it may be
possible to create highly acceptable foods that are
compliant with ketogenic therapy.

Materials and Methods

Fiber isolates were utilized to supplement fiber into
the ketogenic snacks, and sensory analysis was used to


determine the acceptability amongst students and staff at the
University of Florida.

2.1 Product Development

Product development initially focused on designing a
cracker type snack that may be consumed with, or between
meals. After numerous attempts to formulate snack-like
crackers, cookies, and breads, it became apparent that
minimization of carbohydrate and protein content would limit
the type of snacks that could be developed. Through trial and
error, it was discovered that the fiber isolates, in combination
with gluten, could function as a breading for fried foods. To
formulate a snack that would provide maximum clinical
effectiveness, it was necessary to minimize protein and
carbohydrate content of the fiber breading, while maximizing
fiber content.
The fiber mixture contained the protein gluten to provide
stability to the fiber breading. The amount of protein used in
the fiber mix was determined by reducing the gluten content
until the fiber mix was unstable when fried. Gluten content
was minimized as alternative sources of protein need to be
included in the ketogenic diet. Guar was utilized to provide
additional stability to the fiber breading when moistened with
water. It was speculated that the absorptive capacity of guar
was very effective in stabilizing the snack in the uncooked
state. Also, guar seemed to form a protective barrier to oil
when used in frying applications, as thermal gelation occurs
when guar is exposed to high temperatures (Sahin and
Sumnu, 2005). The ketogenic snacks contained a high fat
content, mostly in the form unsaturated oils. The goal was to
develop high fiber snacks compliant with ketogenic therapy
(high fat, low available carbohydrate and protein) that were
stable throughout the cooking process. The stability and
consistency of the ketogenic snack was dependent on
preventing the leaking of internal oils into the cooking
medium. Hydropropyl methyl cellulose behaved similarly to
guar when exposed to temperature extremes, forming a
protective gel and stabilizing the ketogenic snack while
cooking.
Initially, the fiber mix was combined with water and
rolled thin in a dough-like consistency. The fiber mix was cut
into 2in x 2in squares, stuffed with fillings, and folded into a
triangle. The products were then fried, and sampled. A
variety of fillings were sampled, including spicy chicken
pizza, cheese sticks, vanilla ice cream, and cheese cake.
Overall, this method of preparation proved to be very time-
consuming. The products chosen for sensory analysis
included crab rangoon, and broccoli bites. Instead of
preparing in the manner previously mentioned, the broccoli
bites and crab rangoon were dredged in a dry fiber mix, and
then moistened with water. Moistening the fiber mix after
coating the food products was more efficient in preparation


University of Florida I Journal of Undergraduate Research I Volume 11, Issue 1 I Fall 2009
2





SIGNAL TRANSDUCTION


Table 3: TMHMM prediction of transmembrane helicies of sigma factor operon genes


Gene locus
PG 0017
PG 0018
PG 0146
PG 0147
PG 0149
PG 0150
PG 0151
PG 0152
PG 0153
PG 0161
PG 0163
PG 0215
PG 0216
PG 0217
PG 0218
PG 0543
PG 0745
PG 0746
PG 0984
PG 0986
PG 0987
PG 1106
PG 1315
PG 1316
PG 1317
PG 1659
PG 1661
PG 1662


Number of
predicted
TMH
2
0
1
1
1
0
0
0
0
0
0
1
1
0
0
0
0
2
0
1
0
0
0
0
1
2
0
2


a A protein is considered a transmembrane protein if it contains 18 or more AA in transmembrane
helicies
b probability that the active domain is on the cytoplasmic side of the membrane
















University of Florida I Journal of Undergraduate Research I Volume 11, Issue 1 I Fall 2009
5


# of AA in
TMH
47
25
17
23
21
0
0
0
0
0
0
20
20
10
3
0
5
50
0
21
2
0
0
14
23
43
0
45


n-in
probability
0.98899
0.65987
0.14824
0.71989
0.84813
0.04123
0.00296
0.02918
0.00252
0.38057
0.00235
0.97667
0.98486
0.44654
0.15684
0.54632
0.53876
0.99960
0.61118
0.94097
0.17779
0.02673
0.12121
0.67223
0.15081
1.00000
0.30846
0.98909


Transmembrane
protein?a
Yes
Yes
No
Yes
Yes
No
No
No
No
No
No
Yes
Yes
No
No
No
No
Yes
No
Yes
No
No
No
No
Yes
Yes
No
yes






SIGNAL TRANSDUCTION


conducted by genetic inactivation of the predicted
membrane proteins and testing the mutant's ability to
respond to various environmental stimuli.
From the analysis of signalP results, it is predicted that
none of the 11 sigma factors are secreted in an inactive
form requiring proteolytic activation. This result simply
means that the sigma factors in the P. gingivalis genome
do not have peptide cleavage sites similar in homology to
experimentally characterized cleavage sites. A novel
cleavage site signal in P. gingivalis would not be
recognized by the neural networks of signalP because it
has not been trained to recognize this signal. In most
organisms that have had their sigma factors extensively
studied such as E. coli, M tuberculosis, and B. subtilis,
they have shown signal recognition sites that must be
cleaved to be activated as a further regulation opportunity
to fine tune genetic regulation (Manganelli, Haldenwang).
The use of either a proprotein sequence or cognate
anti-sigma factor allows for an intricate regulatory system
that readily adapts to a constantly changing environment.
The genetic regulatory system of P. gingivalis remains
highly uncharacterized but through the advent of high
throughput sequencing technology and comparative
bioinformatics many insights can be revealed by its
similarity to characterized systems in other species. These
predictions will point researchers in the right directions to
make discoveries with experimental tools.



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14(2):99-107.


University of Florida I Journal of Undergraduate Research I Volume 11, Issue 1 I Fall 2009
7








Preterm Infants and Maternal Voice, Family Visitation, and

Discharge Timing


Jillian Cimino*


College of Nursing, University of Florida


The purpose of this pilot study was to describe the combined effect of exposure to a recording of maternal voice and family visitation
on discharge timing in preterm infants cared for within a neonatal intensive care unit (NICU) without an ongoing program of
developmental care. Using a retrospective comparative design, a convenience sample of 67 preterm infants participated. Experimental
infants participating in a larger ongoing study listened twice a day to a recording of their mother's voice. Control infants received
routine NICU care. The median number of family visits (3 per week) was used to differentiate between high e 3) versus low (< 3)
levels of visitation. A near significant reduction in the number of episodes of feeding intolerance (F = 3.16; p = .08) was noted
between infants who heard the maternal voice recordings and whose families visited more often (experimental mean = 2.8; control
mean = 5.5). A significant, yet counterintuitive finding, was noted in the number of days to discharge (i.e., the length of stay),
independent of exposure to the maternal voice recordings (F = 8.42; p<.01). The number of days to discharge were fewer if family
members visited less frequently (mean = 44.4 days) compared to those infants whose family visited more (mean = 61.3 days).
Findings suggest that the combination of maternal voice and family visitation may have a positive effect on decreasing the number of
days to discharge. Future research is needed to verify this combination's effect on discharge outcomes in preterm infants.


Introduction

Measurement of discharge timing (i.e., length of stay)
for preterm infants from neonatal intensive care units
(NICU) includes several milestones. These milestones
include respiratory stability and the ability to oral feed,
which may be improved by the inclusion of developmental
care, family visitation, and exposure to maternal voice.
Health milestones maintain importance because the longer
an infant remains hospitalized, the greater the risk of
contracting a nosocomial infection (Clark et al., 2004), the
more cost incurred (Clark et al., 2004; Committee on
Hospital Care, 2003), less time for interaction with the
family, and increased family stress (Cooper et al., 2007;
Petrou, 2003). The purpose of this pilot study was to
discern the effect of maternal voice and family visitation
on discharge timing in preterm infants.

Methods to Improve Discharge Timing

Methods to improve discharge timing include programs
that are centered on developmental care, which improves
ability to oral feed, and maternal visitation and voice,
which both decrease days to discharge.


*with Charlene Krueger, PhD, ARNP, and Leslie Parker, MSN,
APNP


Developmental Care. Programs of developmental care
which began in 1986 by Heidi Als and colleagues,
individualized care based on the maturity and health status
of each individual infant and focused on oral feeding and
other health milestones. Such emphasis is placed on the
ability to oral feed because most newborn preterm infants
are incapable of doing basic functions such as breathing,
sucking, and swallowing during feeding (Lau, Smith, &
Schanler, 2003) and to digest nutrients properly (Diehl-
Jones & Askin, 2004). This inability forces the infant to
stay within the hospital until standard function
achievement is complete, confirming the importance of this
health milestone. The program later was altered to
incorporate an emphasis on parent-infant interactions to
form a bonding relationship between the infant and mother
early on.
Infants participating in developmental care programs
have been shown to require significantly fewer days of
mechanical ventilation and supplemental oxygen support
(Als et al., 2003; Becker et al., 1991; Fleisher et al., 1995).
Research shows that the use of developmental care
programs has also been associated with decreased length of
hospital stay for preterm infants (Als et al., 2003; Becker et
al., 1991; Brown & Heermann 1997; Melnyk et al., 2006;
Zeskind & Iacino, 1984), diminished parental stress
(Melnyk et al., 2006), and quicker weight gain (Westrup et
al., 2000). With the findings from multiple studies showing


University of Florida I Journal of Undergraduate Research I Volume 11, Issue 1 I Fall 2009





DISCHARGE TIMING IN PRETERM INFANTS


language. Exclusion criteria consisted of: 1) prenatally
transmitted viral/bacterial infections, 2) abnormal head
ultrasound, 3) sensorineural hearing loss, 4) cardiac
abnormalities, or 5) abdominal disorders. Ethnicity was
obtained for 66 infants (one subject's chart was not
available to obtain demographic information). Among
these infants, 60.6% were Caucasian 36.4% were African
American, and 3% were Hispanic.
Additional demographic information obtained was the
infant's gestational age at birth, Apgar score, and
neurobehavioral risk score (NBRS). An Apgar score is
calculated at both 1 minute and 5 minutes post birth. The
infant is rated on a scale of 0-4 (0 being bad and 4 being
well) on 5 categories related to the infant's health. The
higher the score, the better the infant's health. The average
is between 7 and 10. The NBRS score is the status of the
preterm infant while hospitalized to differentiate between
high risk and low risk infants.
Participants in the experimental component of this study
were part of a larger quasi-experimental study entitled,
Heart Rate Variability and Learning in the 28-34 Week
Old Preterm (NIH/NINR P20 NR07791; NIH/NCRR M01
RR00082; Southern Nursing Research Society) in which
32 experimental infants listened to a CD recording of a
nursery rhyme recited by their mothers twice a day for 2-6
weeks. These infants were cared for within the NICU
during the same time period as the experimental infants but
received standard NICU care and no exposure to maternal
voice recordings.


Variables

Nursery Rhyme. A CD recording of the mother reciting
a nursery rhyme was used in the experimental component
of the study. The untitled rhyme (Simon & Schuster, 1985)
was 9 lines long, took approximately 15 seconds to recite,
and was not a common verse (making it unlikely that
infants would be unexpectedly exposed to it). Recordings
lasted approximately 45 seconds and were played twice a
day over a 12.5-cm speaker positioned 20 cm from the
infant's ear. Sound levels were measured using an A-scale
of a Bruel-Kjaer (220SLM) sound level meter. Overall
stimulus intensity was 50-55 dB (M=53.9; SD=2.35), with
background NICU sound levels just prior to initiation of
recordings ranging between 48.6 to 69.2 dB (M=57.90;
SD=4.01). Fifty to 55 decibels was chosen in order to
maintain the decibel level just below the normal level of
human speech (58-60 dB) (Gerhardt, 1989) and to remain
within recommended sound levels for the preterm infant
(Graven, 2000).


Family visitation was defined as the average number of
days in a week (0-7 days) for a total of 6 weeks that a
family member visited the infant's bedside. Multiple
family visits per day were counted as 1 visit (due to
inability to quantify the length of time spent at the infant's
bedside). Family members included mothers, fathers,
grandparents and/or guardians. The median # of visits (3
per week) was used to differentiate between hig) (
versus low (<3) levels of visitation.
Number of days to discharge was defined by the
number of days from birth to the infant's discharge home
or transfer to another facility.
Average daily weight gain was obtained by dividing the
infant's total weight gained in grams (from birth) by the
number of days cared for within the NICU.
Days to full enteral feedings was defined as the number
of days from birth to the day the infant tolerated
120ml/kg/day of either breast milk or formula feedings.
Days to full oral feeding was defined as the number of
days from birth to the day the infant ingested all feedings
via breast or bottle for 24 hours.
Number episodes of feeding intolerance was defined as
the number of times the infant had gastric residuals equal
to or greater than 3 ml/kg or was placed in NPO (receiving
nothing by mouth) status due to events other than routine
preparation for a procedure or intervention. Gastric
residuals of less than 3 ml/kg have been shown to be safe
in previous research concerning VLBW infants (Mihatsch
et al., 2002).
Percent days on respiratory support was defined as the
percentage of hospital days on respiratory support (nasal,
CPAP, ventilator) provided.
Days of NPO status was defined as the number of days
the infant was placed on NPO status for over 50% of a 24-
hour period.

Procedure

A retrospective chart review for infants participating in
both the experimental and control component was
conducted in order to determine the quantity of family
visitation and nutritional and respiratory outcomes. Data
retrieval was initiated at >95% inter-rater reliability and
maintained at the same by evaluating 10% of the charts
once data retrieval was completed. The frequency of family
visitation was extracted by 2 research assistants whose
inter-rater reliability was maintained at >95% agreement.
All variables related to achievement of oral feeding and
respiratory support were similarly extracted by one
Advanced Practice registered nurse and maintained at
>95% inter-rater agreement.


University of Florida I Journal of Undergraduate Research I Volume 11, Issue 1 I Fall 2009
3