ELUCIDATING THE INFLUENCE OF CHARGE DENSITY AND HYDROGEN
BONDING OF IMIDAZOLIUM COPOLYMERS FOR GENE DELIVERY
Michael H. Allen,1 Matthew D. Green,2 Hiwote K. Getaneh,1 and Timothy E. Long1
Macromolecules and Interfaces Institute
Departments of Chemistry1 and Chemical Engineering2 – (0212)
Virginia Tech
Blacksburg, VA 24061
Phone: (540) 231-3378; Fax: (540) 231-8517
Email: [email protected]
Next generation therapeutics require a fundamental understanding of structure-property-transfection
relationships to improve non-viral gene delivery for more effective gene therapy.1 Here, we explored the use of
novel, cationic imidazolium-based copolymers as efficient gene delivery therapeutics.2,3 We investigated the
influence of varying the substituent attached to the tertiary nitrogen of the imidazole ring and the impact of
charge density of these copolymers on cytotoxicity, DNA binding, and in vitro plasmid DNA delivery efficiency.
The novel copolymers were synthesized using conventional free radical polymerization and were modified postpolymerization resulting in a series of copolymers containing various quaternization percentages (Figure 1a).
Luciferase reporter gene expression decreased as both a function of increasing charge density and elimination
of a hydrogen bonding group (Figure 1b). In conclusion, our data suggested the difference in charge density of
the novel imidazolium copolymers affected transfection efficiency as well as varying the substituent on the
imidazole ring, providing a better understanding of structure-property-transfection relationships.
Figure 1. (a) Copolymer structures containing 25% quaternized comonomer. (b) Transfection efficiency of
copolymers as a function of increasing hydrogen bonding substituents.
Acknowledgements:
This material is based upon work supported in part by the Macromolecular Interfaces with Life Sciences (MILES) Integrative
Graduate Education and Research Traineeship (IGERT) of the National Science Foundation under Agreement No. DGE0333378.
References:
1. Layman, J. M.; Ramirez, S. M.; Green, M. D.; Long, T. E. Biomacromolecules 2009, 10, 1244-1252.
2. Green, M. D.; Long, T. E. Polymer Reviews 2009, 49, 291-314.
3. Anderson, E. B.; Long, T. E. Polymer 2010, 51, 2447-2454.
Using Steered Molecular Dynamics to Investigate the Mechanism of
Monoamine Oxidase B Inhibition
William J. Allen and David R. Bevan
Department of Biochemistry
Virginia Polytechnic Institute and State University
Phone: 540-231-9080 Fax: 540-231-9070
Email: [email protected]
ABSTRACT:
Monoamine oxidase B (MAO B) catalyzes the degradation of certain monoamine
neurotransmitters, including phenyethylamine, benzylamine, and dopamine. The
oxidative process results in decreased dopamine availability in neuronal cells as well as
increased production of hydrogen peroxide, a potent pro-oxidant species. For these
reasons, MAO B is widely considered a drug target for neurodegenerative diseases,
including Parkinson’s disease. In order to design more specific inhibitors for this
monotopic bilayer enzyme, it is important to determine what factors govern the inhibitor
binding process, including the role of the lipid bilayer, the active site loop, and several
key residues within the binding pocket. We have performed molecular dynamics
simulations of MAO B in two states - embedded in a lipid bilayer and free in solution.
We have discovered that the homodimeric enzyme and undergoes a slow periodic
rocking motion in the bilayer that facilitates the opening and closing of the active site
entrance depending on its height above or below the bilayer interface. Within the two
simulation conditions (membrane bound and unbound), we also simulated MAO B in
complex with seven different reversible inhibitors that represent a wide range of size,
functionalization, and Ki values. We used steered molecular dynamics to simulate the
release of these compounds from the active site, followed by binding to the active site of
each of the inhibitors in the presence and absence of the lipid bilayer. We have
identified several specific interactions that are important to inhibitor binding, and thus
should be exploited when designing new inhibitors.
USING THE LOGIC MODEL AS A PROGRAM DEVELOPMENT AND EVALUATION
TOOL TO STRATEGICALLY DEVELOP INTERDISCIPLINARY COMMUNITY FOOD
SYSTEM RESEARCH, TEACHING AND ENGAGEMENT ACTIVITIES
AUTHORS:
Matt Benson
Graduate Research Assistant
Department of Agricultural & Extension Education
Virginia Polytechnic Institute and State University
Phone: (540) 522-0762 Fax: (540) 231-3824
Email: [email protected]
Kim Niewolny, PhD
Assistant Professor
Department of Agricultural & Extension Education
Virginia Polytechnic Institute and State University
Phone: (540) 231-5784 Fax: (540) 231-3824
Email: [email protected]
Susan Clark, RD, PhD
Associate Professor
Department of Human Nutrition, Foods and Exercise
Virginia Polytechnic Institute and State University
Phone: (540) 231-8768 Fax: (540) 231-3916
Email: [email protected]
Abstract:
According to Lattuca (2001), interdisciplinary research and teaching agendas have
become prevalent in higher education to address a wide range of contemporary issues. Within
life sciences, improving agricultural sustainability through the development of local and regional
community-based food systems has become a priority issue that demands interdisciplinary
collaboration. For instance, the United States National Academy of Sciences (2009) states that
interdisciplinary curriculum is essential to transform agricultural and life science education in
higher education for better addressing our rapidly changing agricultural and food system needs.
Across Virginia, diverse institutions, agencies and organizations are beginning to
address this need for interdisciplinary collaboration around community food system issues. At
Virginia Tech, an interdisciplinary project team developed a new undergraduate minor in Civic
Agriculture and Food Systems (CAFS). Virginia Cooperative Extension has developed a
statewide educational program to address food system issues through community resource
development and capacity building. The Virginia Food System Council, a new statewide 501c3
nonprofit, has been established to promote awareness, educational programs and new policies
that will support stronger community food systems.
The purpose of this poster presentation is to illustrate how the logic model can be used
as an interdisciplinary program development and evaluation tool to strategically develop
collaborations around community food system research, teaching and engagement activities.
Specifically, this poster will focus on both the processes and outcomes these diverse groups are
coordinating to strengthen Virginia’s community food systems. Logic models describe logical
linkages among program resources, activities, outputs, audiences, and short-, intermediate-,
and long-term outcomes related to a specific problem or situation (McCawley, 2001). As part of
this poster, a framework and set of best practices for catalyzing interdisciplinary community food
system research, teaching and engagement opportunities will be described.
Literature Cited:
Lattuca, L.R. (2001). Creating Interdisciplinarity: Interdisciplinary Research and Teaching
among College and University Faculty. Nashville, TN: Vanderbilt University Press.
McCawley, P. F. (2001). The Logic Model for Program Planning and Evaluation. University of
Idaho Extension.
National Academy of Sciences. (2009). Transforming agricultural education for a changing
world. Washington, DC: The National Academies Press.
ASSOCIATION OF MATERNAL BELIEFS ABOUT EMOTIONS AND
SOCIAL STRESS WITH INTERNALIZING SYMPTOMS FOR CHILDREN
WITH OPPOSITIONAL DEFIANT DISORDER
Jordan A. Booker, Julie C. Dunsmore, Thomas H. Ollendick
Developmental and Biological Psychology
Department of Psychology
Virginia Polytechnic Institute and State University
Phone: 540-915-2734
Fax: 540-231-3652
Email: [email protected]
ABSTRACT:
Oppositional Defiant Disorder (ODD) is a disruptive behavior disorder involving antisocial
acts including defiance of authority and loss of temper (DSM-IV-TR, APA 2000). ODD is
believed to often be comorbid with internalizing disorders such as anxiety and depression
(Angold & Costello, 1993; Wolff & Ollendick, 2006). Children with ODD who also display
internalizing symptoms are more socially anxious, have lower self-worth, and show increased
sensitivity to hostile social cues and decreased sensitivity to positive social cues (Dodge, 1993;
Ginsburg et al., 1998).
Children diagnosed with ODD may also have difficulty communicating and managing
emotions (Burns et al., 2009) compared with non-diagnosed peers. These symptoms may make
peer relations difficult for children with ODD, increasing their experience of social stress.
Furthermore, children whose parents offer little social support or hold antagonistic beliefs toward
children’s emotions may be more likely to experience social stress (Pinderhughes et al., 2000).
Children whose mothers used a more authoritarian and empathic approach to guiding their
children’s emotions showed more positive social outcomes in regards to aggression and
withdrawal (Hastings & Rubin, 1999). In particular, parents’ beliefs about the value of emotions
and their role in guiding children’s emotions are associated with children’s peer relationships
(Dunsmore & Karn, 2004; Katz & Windecker-Nelson, 2004).
In this study we examined whether children’s experience of social stress and mothers’
beliefs about children’s emotions were related to internalizing symptoms for children with ODD.
One hundred three mothers with children (mean age = 9.58 years; range 7 – 14 years, 66 boys,
37 girls) diagnosed with ODD participated in a pre-treatment assessment. Mothers reported
beliefs about their role in guiding children’s emotions (Parents’ Beliefs about Children’s
Emotions, Halberstadt et al., 2007). Children reported social stress (Behavior Assessment
System for Children-2, Reynolds & Kamphaus, 1992) and internalizing symptoms for anxiety
and depression (Beck Youth Inventory, Beck et al., 2001).
Regression analyses predicting T-scores for children’s internalizing symptoms showed
that mothers’ belief about emotional guidance and children’s social stress both predicted
children’s anxiety. The effect of mothers’ belief about emotional guidance on children’s anxiety
was accounted for by social stress.
These results suggest that social stress and parents’ beliefs regarding children’s
emotions are associated with internalizing symptoms for children diagnosed with ODD.
Targeting parental perceptions about emotions and improvements in children’s social
experiences may therefore be beneficial in further intervention and prevention efforts for
children with ODD.
References:
1. American Psychiatric Association. (2000). Diagnostic and statistical manual of mental
disorders: DSM-IV-TR. Washington, DC: American Psychiatric Association.
2. Angold, A., & Costello, E. J. (1993). Depressive comorbidity in children and adolescents:
empirical, theoretical, and methodological issues. American Journal of
Psychiatry, 150, 1779-1791.
3. Beck et al., 2001 J. Beck, A. Beck and J. Jolly, Beck Youth Inventories of Emotional &
Social Impairment manual, Psychological Corporation, San Antonio, TX (2001).
4. Burns, G. L., Desmul, C., Walsh, J. A., Silpakit, C., & Ussahawanitchakit, P. (2009). A
multitrait (ADHD-IN, ADHD-HI, ODD toward adults, academic and social
competence) by multisource (mothers and fathers) evaluation of the invariance
and convergent/discriminant validity of the child and adolescent disruptive
behavior inventory with Thai adolescents. Psychological Assessment, 21, 635641.
5. Dodge, K. A. (1993). Social-cognitive mechanisms in the development of conduct
disorder and depression. Annual Review of Psychology, 44, 559-584.
6. Dunsmore, J. C., & Karn, M. A. (2004). The Influence of Peer Relationships and
Maternal Socialization on Kindergartners' Developing Emotion Knowledge. Early
Education & Development, 15, 39.
7. Ginsburg, G. S., La Greca, A. M., & Silverman, W. K. (1998). Social anxiety in children
with anxiety disorders: Relation with social and emotional functioning. Journal of
Abnormal Child Psychology, 26, 175-185.
8. Halberstadt, A.G., Dunsmore, J.C., Parker, A.E., Beale, K. S., Thompson, J.A, & Bryant,
A., Jr. (2007). Unpublished questionnaire.
9. Hastings, P. D., Rubin, K. H. (1999). Predicting mothers’ beliefs about preschool-aged
children’s social behavior: Evidence for maternal attitudes moderating child
effects. Child Development, 70, 722-741.
10. Katz, L. F., & Windecker-Nelson, B. (2004). Parental meta-emotion philosophy in
families with conduct-problem children: Links with peer relations. Journal of
Abnormal Child Psychology, 32, 385-398.
11. Pinderhughes, E. E., Dodge, K. A., Bates, J. E., Pettit, G. S., & Zelli, A. (2000).
Discipline responses: Influences of parents’ socioeconomic status, ethnicity,
beliefs about parenting, stress, and cognitive-emotional processes. Journal of
Family Psychology, 14, 380-400.
12. Reynolds, C., & Kamphaus, R. Behavior Assessment System for Children, (BASC-2).
13. Wolff, J. C., & Ollendick, T. H. (2006). The comorbidity of conduct problems and
depression in childhood and adolescence. Clinical Child and Faimly Psychology
Review, 9, 201-220.
BRANCHED PEPTIDES FOR BINDING HIV-1 TAR RNA
David I. Bryson, Wenyu Zhang, W. Keith Ray, Patrick M. McLendon, David Rekosh, Theresa Reineke,
Webster L. Santos
MILES IGERT Graduate Student
Department of Chemistry
Virginia Tech
Phone: 540-231-8233 Fax:None
Email: [email protected]
ABSTRACT:
Since the recognition that RNA is more than just an information carrier from
genetic code to fully functional protein, researchers have realized that RNA operates in
many more biological processes than previously realized.
Thus, RNA binding
molecules have potential as tools in molecular biology and medicine.1
Many of the biological functions of RNA result from specific proteins binding to
complex tertiary structures of RNA. This is the case for the HIV-1 transactivation
response element region (TAR) RNA, a conserved 59-nucleotide stem loop located at
the 5’ end of all nascent transcribed HIV-1 mRNA. The TAR structure is composed of a
conserved hexanucleotide loop and a tri-nucleotide bulge, which is flanked by two
double stranded stems.2 Binding of the transcriptional activator protein Tat and other
cofactors promotes efficient transcriptional elongation, allowing the HIV-1 virus to
replicate quickly.
Tat, an 86 amino acid
protein, contains an arginine
rich
motif
(ARM),
which
specifically interacts with the
TAR tri-nucleotide bulge (U23,
C24, and U25). By disrupting
the interaction between Tat and
TAR, a blockade of viral
replication
is
generated.
Therefore, this is a viable
strategy in developing new antiHIV therapeutics.
We have
synthesized and screened a
4,096
member
library
of
branched peptides (Figure 1) to
Figure 1. Branched peptide library bound to resin.
determine their capacity as
At each position there are four possible amino acids,
ligands for HIV-1 TAR RNA.
generating 4096 possible combinations.
The library was designed with
structurally diverse functional
groups in an effort to form multivalent interactions and enhanced selectivity with the
target RNA. Standard solid phase peptide synthesis and on-bead screening assays
using confocal microscopy generated 17 unique “hit” peptide sequences, displaying an
elevated affinity for the target sequence. Fluorescence polarization assays have
confirmed that several of the branched peptides were selective for the target sequence
over a mutant version of HIV-1 TAR, and that a linear version of one hit compound
suffered from reduced affinity to the target.3 Dot blot assays with 32P- labeled RNA
have been used to characterize the binding constants of the branched peptides.
Additionally, in vitro assays of our branched peptides in HeLa cells have displayed low
toxicity and good cell permeability. Additionally, our branched peptides display good to
fair stability in human serum. We have shown that the use of branched structures with
diverse functional groups and capacity to form multivalent interactions is important in
defining new molecular entities that can be selective for a target RNA.
References:
1. D. E. Draper, Ann. Rev. Biochem., 1995, 64, 593.
2. F. Aboul-ela, J. Karn and G. Varani, J. Mol. Biol., 1995, 253, 313; T. M. Rana and K. T. Jeang,
Arch. Biochem. Biophys., 1999, 365, 175.
3. D.I. Bryson, W. Zhang, W.K. Ray and W.L. Santos, Mol. BioSyst. 2009, 5, 1070. AN IMMERSION IN TEACHING AND LEARNING:
VIRGINIA TECH’S HEIFER INTERNATIONAL SPRING BREAK PROGRAM
Carmen Byker, PhD Student
Ashley Holmes, PhD Student
Justin Shanks, PhD Student
Dr. Susan Clark, Associate Professor
Department of Human Nutrition, Foods, and Exercise
Virginia Polytechnic Institute and State University
Phone: 8152174608 Fax: 5402313916
Email: [email protected]
Byker, Holmes, Shanks 1 ABSTRACT:
Aiming to teach individuals of all ages from across the country about the organization’s
mission to “work with communities to end hunger and poverty and to care for the earth”
Heifer International (HI) facilitates an Alternative Spring Break (ASB) in Perryville,
Arkansas. Virginia Tech partnered with HI to enhance their immersion curriculum and
educate college-students about methods for incorporating sustainable practices into
their daily lives and utilizing the organization’s sustainable community development
model to effect local and global changes in the areas of poverty and hunger. Employing
the experiential learning model, students visit the Learning Center at Heifer Ranch to
think globally and act locally by “experiencing some of the challenges of global hunger
and poverty—and come away with a re-energized determination to be a part of the
solution.” Sustainable food system education is appropriate and necessary for the
contemporary higher education environment as the tenants of sustainability—
environmental health, economic profitability, and social justice—with an addition of
health, are important issues for all students to consider and are themes that cross
curriculums.
Midway through the semester, students enrolled in Engaged Learning Environment I:
Agriculture Community Development Models traveled to the Heifer Ranch for an ASB
experience. In addition to its role in the course, the Heifer ASB also served as a precursor to and promotion tool for a new experiential based Civic Agriculture and Food
Systems (CAFS) minor within the College of Agriculture and Life Sciences (CALS). To
encourage interdisciplinary graduate education and incorporate HI’s sustainable
community development model, three Human Nutrition, Foods, and Exercise graduate
students were tasked with developing course content and facilitating class sessions with
the leadership of Susan Clark. Students completed pre-post surveys and wrote journal
entries about their perceptions, attitudes and knowledge regarding sustainable
community development, the course, and the ASB in order to measure the effectiveness
of Heifer’s ASB on student experiential learning engagement. Survey results will be
shared about the student’s knowledge and attitude change in their understanding of
hunger, poverty, and issues of sustainability.
Our contribution to the ACC Interdisciplinary Conference will involve a presentation
focused on the value of using graduate students as course instructors, provide
qualitative tools for experiential course evaluation, and provide undergraduate verbal
interviews about the Heifer ASB as well as the overall course experience.
References:
Aaker, J. (1996). The Heifer Model: Cornerstones Values-Based Development. Little
Rock, Arkansas: Heifer Project International.
Byker, Holmes, Shanks 2 Presentation bios:
Carmen Byker received a BS in Human Nutrition, Foods and Exercise—Dietetics and
is currently pursuing a PhD in the same department at Virginia Tech. With guidance
from Dr. Elena Serrano, she actively seeks to improve the quality and accessibility of
nutrition and foods in the local community through sustainable and civic-minded means.
Ashley Holmes received a B.S. in Human, Nutrition, Foods and Exercise- in
concentrations in both Science of Nutrition and Dietetics from Virginia Tech as well as a
German Language Minor primarily completed at University of Stuttgart in Germany.
Ashley also received her masters in Human, Nutrition, Foods, & Exercise from Virginia
Tech under the mentorship of Dr. Elena Serrano, examining the impact of a grocery
store intervention promoting the sales of healthy snacks to parents and their young
children. Ashley is currently pursuing her PhD, with Dr. Serrano, expanding on her work
promoting healthy foods to young children and their families using nutrition menu
labeling in the restaurant context.
Justin Shanks received a BA in Environmental Studies from Loyola University Chicago
(2003), earned a MA in English as well as a Masters of Urban and Regional Planning
(MURP) from Virginia Tech (both 2009), and is currently working on a PhD in Human
Nutrition, Foods, and Exercise at Virginia Tech (expected Spring 2012). Drawing upon
this humanistic and scientific background, Justin’s research is community-based,
socially-conscious, and strongly interdisciplinary. Under the tutelage of Dr. Elena
Serrano, Justin’s dissertation work explores the intersection of the built environment,
childhood obesity, food security, policy development, and the rhetoric of nutrition.
Byker, Holmes, Shanks 3 BIOENGINEERING OF CORNEAS FOR TRANSPLANTATION
Jin San Choi1, J. Koudy Williams1, Margaret Greven1, Keith Walter2, Patrick Laber2,
and Shay Soker1
1
Wake Forest Institute for Regenerative Medicine, Wake Forest University Health Sciences and
2
Department of Ophthalmology, Wake Forest University School of Medicine, Winston-Salem, North
Carolina
Phone: (336)713-7295 Fax: (336)713-7290
Email: [email protected]
ABSTRACT: The inner layer of the corneal is a single layer of neural-crest derived cells that form a barrier
between the cornea and the aqueous humor. These corneal endothelial cells (CEC) are essential for
transport of water from the corneal stroma. Damage or decompensation of the CEC pump results in
corneal edema and loss of vision. CEC loss is most well documented as a result of accidental damage
during cataract surgery or in an inherited condition known as Fuchs’ dystrophy. Bioengineered corneas,
created using an expandable population of human donor-derived corneal endothelial cells (HCEC), could
address this current shortage. The objectives of this study were to establish HCEC isolation and culture
protocols and to investigate the feasibility of bioengineering corneal tissue constructs by seeding the cells
on decellularized human corneal stroma (DHCS). HCEC were obtained from discarded of various aged
eye donors by digestion in collagenase II, expanded. HCEC were cultured on collagen IV-coated,
fibronectin-coated, and tissue culture plates to compare their adhesion, proliferation, and cellular
morphology. Corneal scaffolds were sliced using a microtome and decellularized with 2% Triton X100/0.1% NH4OH for 3 days at 4C. The mechanical properties and ECM components of native and
DHCS were examined. HCEC were seeded on the DHCS and investigated by scanning electron
microscopy (SEM), hematoxylin and eosin (H&E) staining, and Immunohistochemistry. Re-endothelialized
cornea was transplanted in a rabbit eye. Surgically treated eye was observed for 4 weeks after operation.
In the present study we explored clinically relevant sources of HCEC. HCEC doubling times from young
donors were less than those from older donors and grew best on fibronectin-coated plates.
Human DHCS were prepared by removing all cellular components chemically and leaving behind
the intact ECM proteins. SEM images of HCEC-seeded DHCS showed compact surface coverage with a
uniform monolayer of cells. Confluent cells expressed connexin-43, ZO-1, and Na+/K+ ATPase. The
photographs showed that engineered corneal endothelium maintained after transplantation and the
cornea was recovered its clarity. HCEC can be isolated from sclera rims remaining after the central
cornea has been removed for transplantation. These cells express markers typical of CEC. HCEC from
one donor can be expanded to re-endothelialize several engineered cornea constructs. Corneal scaffolds
can be obtained from tissue unfit for transplantation because of flow cell counts. Bioengineered cornea
tissue creates a new source of high quality corneal tissue for transplantation.
References: Joyce NC, Prog Retin Eye Res 2003 May;22(3):359-389.
PATHOGENICITY OF PSEUDOMONAS SYRINGAE ON TOMATO
BEYOND TYPE III EFFECTORS
Christopher Clarke, Haijie Liu, Rongman Cai, James Lewis, and Boris Vinatzer
Affiliation
Department of Plant Pathology, Physiology, and Weed Sciences
Virginia Tech
Phone: 540-231-4145 Fax:Email: [email protected]
ABSTRACT:
To begin assessing the diversity of Pseudomonas syringae pv. tomato (Pto) strains, we
sequenced five isolates of P. syringae collected from diseased tomato leaves and identified informative
single nucleotide polymorphisms (SNPs). We then performed a SNP analysis of over 100 Pto strains.
The majority of Pto strains were identified as members of the PtoT1-like clade based on their similarity to
the sequenced eponymous member PtoT1. Several of the identified SNPs help to elucidate the recent
success of the PtoT1-like strains. SNPs between PtoT1-like strains were disproportionately found in the
gene coding for flagellin, a known immune-triggering constituent, and genes associated with chemotaxis.
These identified SNPs and corroborating experimental evidence suggest that PtoT1-like strains owe their
relative success on tomato to being more adept at avoiding early detection by the plant and utilizing more
honed chemotactic pathways than more ancestral Pto strains. The current paradigm of P. syringae
pathogenicity posits that the virulence and avirulence activities of effector proteins are the primary
determinants of P. syringae pathogenicity. In contrast, these results inspire a more nuanced view of P.
syringae pathogenicity that considers multiple aspects of the infection process.
IMMUNE CELL MODULATION OF OMENTAL FAT BAND DURING PERITONEAL SEEDING AND
SPREAD OF OVARIAN CANCER.
Courtney A. Cohen*, C. Lynn Heffron*, Eva Schmelz** and P. Chris Roberts*
*Department of Biomedical and Veterinary Sciences, Virginia Tech
**Department of Human Nutrition, Food and Exercise, Virginia Tech
Phone: 231-5519
Email: [email protected]
ABSTRACT:
Ovarian cancer is the leading cause of death due to gynecological malignancy in the Western
world, the fourth most deadly cancer in women. Typically originating in the layer of epithelial cells
surrounding the ovary, cancer cells exfoliate to disseminate throughout the peritoneal cavity1. The
omental fat band (OFB) has been described as a site of preferential tumor cell adhesion and invasion
within the peritoneal cavity, composed of adipose tissue interspersed with immune cell aggregates, or
2
“milky spots” . The composition of these milky spots has been elucidated in naïve mice, but effects of
cancer cell adhesion on leukocyte populations have not yet been investigated. Our lab developed a
spontaneously transformed murine ovarian surface epithelial (MOSE) cell model that, following long-term
passaging, mimics the progressive stages of ovarian cancer1. Our interest lies in understanding the
immunomodulatory mechanisms that influence dissemination and propagation within the peritoneal
cavity. Specifically, we are interested in the mechanism by which cancer cells regulate monocyte
polarization towards a protective, inflammatory M1 phenotype, or an M2-like, tumor-associated
macrophage (TAM) or DC. EGFP-expressing MOSE-L (late) cells, which are highly aggressive, were
injected i.p. into 8 month-old C57BL/6 mice in order to verify immediate homing of cancer cells to the
omentum at 6 and 24 hours post-injection, and at 7 days. EGFP-positive colonies were detected in the
omentum at milky spots and vascular areas at 6 hrs (Fig 1). Flow cytometric analysis of immune
populations in response to early seeding and after long-term (4 wks) in vivo expansion of cancer cells are
underway. Preliminary results indicate that ovarian cancer cells preferentially invade the omentum early in
cancer metastasis, and that they are capable of escaping early innate immune surveillance mechanisms
to survive and establish peritoneal disease. Larger studies are underway to shed light on
immunomodulatory events associated with initial cancer cell seeding.
Figure 1a. Omentum, vascular milky spot
Figure 1b. EGFP-expressing MOSE-L
cells, invasion at 24 hours.
References:
1. Roberts, P.C., et. al. (2005). Sequential Molecular and Cellular Events during Neoplastic
Progression: A mouse syngeneic ovarian cancer model. Neoplasia, 7(10): 944-956.
2. Sorenson, E.W., et.al. (2009). Omental immune aggregates and tumor metástasis within the
peritoneal cavity. Immunol Res, 45(2-3): 185-194.
IMAGING VASCULOGENESIS IN REGENERATING SKELETAL MUSCLE
Tracy L. Criswell1, Benjamin T. Corona1, Zhan Wang1, Shay Soker1
Corresponding Author: [email protected]
1
Wake Forest Institute for Regenerative Medicine, Winston-Salem, North Carolina, USA
Introduction: A critical difficulty in tissue engineering is the ability to maintain large masses of living cells,
upon transfer from the in vitro culture conditions into the host, in vivo. To achieve the goals of engineering
large complex tissues, vascularization of the regenerating tissues has a high priority. Another significant
barrier for progress in tissue engineering is our inability to monitor in real time the dynamic process of
tissue regeneration, which makes optimization for clinical therapeutics extremely difficult. The objective of
this project is to develop a model culture system consisting of fluorescently labeled endothelial progenitor
cells, pericytes and myoblasts that will allow for the visualization of the neovascularization of regenerated
muscle tissue.
Fig. 1:Fluorescently labeled
myoblasts (green), HUVECs (red)
and pericytes (blue) grown in coculture and visualized by fluorescent
(left) or confocal (right) microscopy.
Materials and Methods: Human umbilical vein endothelial cells
(HUVECs) were used as the source of endothelial progenitor
cells. Murine 10T½ cells (ATCC) were used as pericytes and
myoblasts were derived from GFP+ mice (Jackson Labs).
Pericytes were labeled with Qtracker 705 Cell Labeling Kit
(Invitrogen, faux-blue) and the HUVECs were labeled with CMDiI (red). Muscle constructs were either collected for analysis 14
days after seeding on scaffold or implanted subcutaneously into
mice for 8 weeks.
Results: Using fluorescent microscopy, we are able to visualize
the self assembly of HUVECs into branching vessel-like
structures on the scaffold in the presence of
myoblasts and pericytes (Fig. 1). In vivo, 8 weeks
after implantation, the HUVECs had integrated into
the vasculature along with host endothelial cells. In
addition, the GFP-labeled myoblasts assembled into
mature skeletal muscle fibers, which is confirmed by
staining for GFP immunoreactivity and the presence
of desmin (Fig. 2).
Discussion and Conclusions: We have used our
model
to
visualize
cell
organization
and
differentiation on an acellular scaffold in vitro and in
vivo. These experiments demonstrate that the use of
fluorescently labeled cells is a viable method of
following cell fate during vasculogenesis and tissue
regeneration.
Acknowledgments:
The
authors
gratefully
acknowledge financial support for this work from the
Armed Forces Institute of Regenerative Medicine
(AFIRM).
Desmin Figure 2: (a) Gross image of scaffold in
subcutaneous space. (b-c) Fluorescently labeled
myoblasts and HUVECs were detected in
scaffolds 8 weeks after implantation (arrows).
(c) HUVECs have integrated into a blood vessel
along with host endothelial cells (arrow). Nuclei
labeled with DAPI (blue). (d) Mature skeletal
muscle indicated by desmin staining. MUTATIONAL ANALYSIS OF CWLJ1 FUNCTION IN BACILLUS
ANTHRACIS SPORE GERMINATION
Cuffee, CW; Lambert, EA; Popham, DL
Junior
Department of Biology
Virginia Tech
Phone:7577736000
Email: [email protected]
Bacillus anthracis spores are the cause of the infectious disease anthrax. Bacterial spores are
extremely resistant to environmental factors such as heat, extremes of pH, and desiccation and
are therefore extremely hard to kill. Spores are metabolically inactive and must undergo
germination in order to become metabolically active, toxin-producing vegetative cells. The cwlJ1
gene encodes a cell wall hydrolase enzyme that is required during spore germination. The
CwlJ1 protein is created during sporulation and becomes localized in an inactive form into the
developing spore. When CwlJ1 is activated during germination, it hydrolyzes the peptidoglycan
in the cortex of a spore, allowing for the spore core to become hydrated. Protein sequence
similarites suggest that CwlJ1 is a muramidase that cleaves the cortex glycan strands. The
purpose of this study is to analyze CwlJ1 protein functions by creating mutant forms of cwlJ1.
The gene was isolated using PCR of chromosomal DNA and was integrated into a replicative
plasmid that allowed complementation of a cwlJ1 mutation. The gene be modified with Six Cterminal histine codons to incorporated a 6His affinity-tag onto the protein. The plasmid will then
undergo site-directed mutagenesis transforming a predicted active-site glutamate into an
alanine, producing an inactive CwlJ1. Separately, mutations will be introduced to produce
several short C-terminal truncations of the protein. The wild type and mutagenized plasmids will
then be transformed into a B. anthracis strain that lacks cwlJ1. Transformed cells will be
allowed to form colonies and to complete sporulation. A tetrazolium reduction-based assay was
developed to screen for germination ability. The production, stability, and spore-localization of
the altered proteins will be analyzed by western blotting using antibody against the 6His tag.
Mutations will reveal the contributions of various protein regions to dormancy, activation, and
enzymatic activity of the protein. This research will contribute to possible artificial methods to
activate CwlJ1 and cause dormant spores to become vegetative, allowing for more efficient
eradication of bacteria in contaminated sites.
Fluorescence sensing of xenobiotic degradation and
development of metabolic biosensors
Michael Angelo-Anthony Daniele, Deepti Sharma, Iurii Bandera, Michael Sehorn, and
Stephen H. Foulger
Graduate Research Assistant
Department of Materials Science and Engineering
Clemson University
Phone: 864.656.1023 Email: [email protected]
ABSTRACT:
Xenobiotics, such as polycyclic aromatic hydrocarbons and heterocyclic aromatics (HCAs), play a
unique role in environmental science and current biotechnology. As common environmental pollutants
HCAs are prime candidates for bioremediation, and in pharmaceutics, they supply a unique substrate for
drug development [1-3]. Given HCAs' robust structures, conventional synthetic approaches have given
way to biological methods, and a series of microorganisms have the capacity to degrade HCAs into novel
substrates. For example, Pseudomonas spp. are able to survive with catabolic activity necessary to
degrade carbazole (CAR) at a sufficient rate, and P. resinovorans CA10 (CA10) has the capability to use
CAR as its sole source of energy[1,4]. This adaptation requires extraordinary physiological capabilities
and reflects the broad technological capacity of these organisms. Monitoring and probing the metabolism
of xenobiotic degradation is a critical step toward utilizing these biological capabilities for novel
technologies.
In this research, metabolite-functionalized colloids were assembled and introduced to CA10 to
probe CAR metabolism by monitoring the inherent CAR fluorescence. Propargyl-acrylate (PA) colloids
were synthesized by a standard emulsion polymerization. Surface-functionalization of PA particles with a
primary metabolite, 9-(3-azidopropyl)-9H-carbazole (AC), used the Huisgen 1,3-dipolar cyclo-addition of
terminal alkynes with azides, copper(I)-catalyzed production of 1,2,3-triazoles [5-7]. Both CAR and the
functionalized colloids (PAAC) were introduced to CA10 cell cultures and lysate. When isolate CA10 was
grown in nitrogen-free mineral medium with CAR or PAAC, optical density measurements showed
sustained CA10 growth after 4-day incubations. Reduced CAR and AC levels were monitored by
photoluminescence spectroscopy (PL) of extracted supernatant samples. Anthranilic acid (AN) was
positively identified, by PL, as intermediates of CAR degradation by isolate CA10 and lysate, but was not
present after the degradation of AC. The lack of expected metabolites suggests the degradation of PAAC
is through an alternate pathway or results in different metabolites.
References:
1. Pieper, D. H.; Reineke, W. Current Opinion in Biotechnology 2000, 11, 262-270.
2. Pieper, A. A.; et al. Cell 2010, 142, 39-51.
3. Widada, J.; et al. Chemosphere 2002, 49, 485-491.
4. Ouchiyama, N.; et al. Biosci. Biotechnol. Biochem. 1993, 57, 455-460.
5. Huisgen, R. Angewandte Chemie-International Edition 1968, 7, 321.
6. Evanoff, D. D.; et al. Advanced Materials 2007, 19, 3507.
7. Rungta, P.; et al. Soft Matter 2010, (in press).
USING INTERPOLATION TO ESTIMATE SYSTEM UNCERTAINTY IN
GENE EXPRESSION EXPERIMENTS
Lee J. Falin1, Brett M. Tyler1,2
1
Virginia Bioinformatics Institute, and 2Department of Plant Pathology, Physiology and Weed Science,
Virginia Polytechnic and State University, Blacksburg, Virginia
Phone: 276-783-3565
Email: [email protected]
ABSTRACT:
The widespread use of high-throughput experimental assays designed to
measure the entire complement of a cell’s genes or gene products in response to some
experimental condition has led to vast stores of data which are extremely plentiful in
terms of the number of items (e.g. genes) they can measure in a single sample, yet
necessarily sparse in the number of samples per experiment due to their high cost. This
often leads to datasets where the number of treatment levels or time points sampled is
limited, or where there are very small numbers of technical and/or biological replicates.
Here we introduce a novel algorithm to quantify the uncertainty in the
unmeasured intervals between biological measurements taken across a set of
quantitative treatments. The algorithm provides a probabilistic distribution of possible
gene expression values within unmeasured intervals, based on a plausible biological
constraint. We show how quantification of this uncertainty can be used to guide
researchers in further data collection by identifying which samples would likely add the
most information to the system under study.
CHANGES IN PHYSICAL ATTRIBUTES IN
COOKED ATLANTIC SALMON AND TILAPIA AS A FUNCTION OF ENDPOINT
TEMPERATURE
Renee J. Felice1, Susan E. Duncan1, Joseph E. Marcy1, Michael L. Jahncke2
and Kumar Mallikarjunan3
1
Department of Food Science and Technology; 2Virginia Seafood Agriculture Research and
Extension Center; 3Department of Biological Systems Engineering
Virginia Polytechnic Institute and State University
Phone: (540) 231 - 8697
Fax: (540) 231 - 9293
Email: [email protected]
Abstract:
Fish tissue is more fragile than beef or pork yet must be cooked enough to inactivate
pathogens while still maintaining optimal eating quality of the fish (NACMCF 2008). Overcooking
fish can lead to dryness, decreased flavor, toughness and excessive flakiness (Brown 2007).
Under-cooked fish may appear tender and moist, but can lead to food safety concerns such as
causing foodborne illness. The Food Code’s (2009) recommended cooking parameter for raw
fish and shellfish is 63°C (145°F) or above for 15 seconds. This was developed with Salmonella
as the target pathogen to eliminate, due to the large number of associated outbreaks. However,
limited research has been conducted on the thermal inactivation of Salmonella spp. in seafood
(NACMCF 2008).
The Food Code’s cooking guidelines are to help insure safety; however, it is uncertain
what eating quality is associated with fish and shellfish heated to the recommended internal
temperature. A higher thermal treatment recommendation would increase thermal inactivation of
Salmonella spp. and may be possible if it resulted in acceptable product quality to consumers.
This study explored physical attributes (cook loss, texture using Kramer Shear Cell (KSC) and
Texture Profile Analysis (TPA) and color) in farm-raised Atlantic salmon and Tilapia fillets. Each
of the two fish products were prepared by three different culinary methods. Atlantic salmon fillets
were baked, poached, and broiled; Tilapia fillets were baked, broiled and pan-fried. Products
were cooked to two endpoints (64°C and 74°C).
Significant statistical differences (p < 0.05) were found for cook loss between the two
endpoints (64°C and 74°C) for both salmon and Tilapia prepared by all respective culinary
methods. Significant statistical differences (p < 0.05) in max force (N) for the two endpoint
temperatures were determined for Tilapia prepared by all three culinary methods using KSC.
For salmon, baking was the only culinary method to result in statistical differences in TPA
parameters. Hardness values for salmon were much higher than for Tilapia, and the internal
temperature of 74°C tended to result in higher hardness values (Figure 1). For CIE Lab scores,
used as measure of color changes, Tilapia had less significant statistical differences among the
two internal temperatures and within culinary preparation methods than did salmon.
14.0
64 degrees C
74 degrees C
12.0
Hardness (N)
10.0
*
8.0
6.0
*
4.0
*
2.0
0.0
Baked
Salmon
Broiled
Salmon
Poached
Baked
Salmon
Tilapia
Product
Broiled
Tilapia
Pan-Fried
Tilapia
Figure 1: Hardness as Determined by Texture Profile Analysis for Atlantic Salmon and
Tilapia Prepared By Various Culinary Methods to Two Internal Temperature Endpoints.
(*) indicates significant statistical difference (p < 0.05) between internal temperature endpoints
References:
Brown, A. C. 2007. Understanding Food: Principles & Preparation. 3rd Ed. Belmont, CA:
Wadsworth Publishing. 696 p.
National Advisory Committee on Microbiological Criteria for Foods (NACMCF). 2008. Response
to the questions posed by the Food and Drug Administration and the National Marine Fisheries
Service regarding determination of cooking parameters for safe seafood for consumers.
NACMCF, Washington, DC. J Food Prot 71(6):1287-1308
United States Department of Health and Human Services, Public Health Service, Food and
Drug Administration. 2009. Food Code. United States Department of Health and Human
Services, College Park, MD. Internet. Accessed February 22, 2010.
<http://www.fda.gov/Food/FoodSafety/RetailFoodProtection/FoodCode/FoodCode2009/>
A MODEL FOR CHILDHOOD OBESITY: HIGH FAT, HIGH SUCROSE,
HIGH FRUCTOSE DIET IN YOUNG GROWING PIGS
K.D. Fisher, T.L. Scheffler, S.C. Kasten, B.M. Reinholt, G.R. van Eyk, J.E. Escobar, J.M. Scheffler, and
D.E. Gerrard
Department of Animal and Poultry Sciences
Virginia Tech University
Phone: 540-231-5861 Fax: 540-231-3713
Email: [email protected]
ABSTRACT:
The creation of a porcine model of obesity will enhance our understanding of the biological
mechanisms and consequences of childhood obesity. Porcine models of obesity provide similar biology
that will facilitate the application of results to humans easier than those from rodent models. Pigs and
humans have similar gastrointestinal physiology, metabolism, and cardiovascular systems, and
proportional organ sizes. Moreover, both species exhibit a taste preference for sucrose, lactose, and
fructose. The present study tested the hypothesis that a diet rich in fat and sugar will increase food intake
and fat deposition in young pigs, resulting in obesity and perturbed metabolism.
Pigs (age = 5 wk) were fed either a control or high energy diet (HED; 15% tallow, 20% sucrose,
15% fructose) for 16 weeks. Body weights and ultrasound fat and loin depth were measured weekly. At
week 15, pigs underwent an oral glucose tolerance test (OGTT). At the end of the feeding period, pigs
were sacrificed and carcass measurements were evaluated.
Blood glucose, mg/dL
HED pigs consumed less feed relative to body weight but consumed more kilocalories of
metabolizable energy per kilogram of body weight. Over the course of the study, HED pigs deposited
more back fat than control pigs (P<0.05). When pigs were subjected to the OGTT, blood glucose in
control pigs returned to baseline levels within two hours. In contrast, blood glucose of HED pigs did not
return to baseline, even after four hours (Figure 1). These data support that the HED led to impaired
glucose tolerance. Following slaughter, carcass traits,
Control
100
High Fat
including carcass weight, carcass length, weight of
**
90
visceral fat, longissimus muscle area (LMA), and back
**
th
th
fat at the 10 rib were evaluated. The LMA at the 10
*
80
**
**
rib of the control pigs was larger than HED pigs
th
(P<0.05). The 10 rib back fat was thicker and there
70
**
was an increased deposition of visceral fat in the HED
60
carcasses, in addition to an observed increase in
intramuscular fat.
50
Collectively, these data show that the HED
results in increased fat deposition and impaired
glucose tolerance and thus will be a beneficial model
for the study of juvenile obesity.
* P < 0.05, ** P < 0.01
40
0
30
60
90 120 150
Time, min
180
210
240
Figure 1: Blood glucose levels during a four
hour oral glucose tolerance test.
FÖRSTER RESONANCE ENERGY TRANSFER OF
FLOURESCEIN FUNCTIONALIZED COLLOIDS
Alexandra Foguth and Stephen Foulger
Graduate Research Assistant
Department of Materials Science and Engineering
Clemson University
Phone: 864-656-1023
Fax: 864-656-1099
Email: [email protected]
ABSTRACT:
Förster resonance energy transfer (FRET) between small molecule dyes can be utilized
to detect minor conformational changes.1 Nonetheless, small molecule dyes experience
problems including photobleaching, blinking characteristics, and fast clearance of bioimaging
markers.2-5 These problems were overcome through covalently linking a fluorescent dye to
colloidal particles via a 1,3-dipolar (3+2) cycloaddition, known as a Huisgen cycloaddition, of an
azide functionalized fluorescein and an alkyne functionalized collolid.6 The effect of covalently
linking dye to the colloid on FRET was studied with the resulting fluorescein-coated colloids and
rhodamine B in methanol. Utilizing an excitation of fluorescein at 443 nm and rhodamine
emission at 571 nm. Photoluminescence spectra show an energy transfer from the fluoresceincoated colloids to the rhodamine B small molecule through decreased fluorescein emission and
increased rhodamine B emission.
References:
1. Rudolf, R.; Mongillo, M.; Rizzuto, R.; Pozzan, T., Nature Reviews Molecular Cell Biology 2003, 4
(7), 579-586.
2. Dyba, M.; Hell, S. W., Applied Optics 2003, 42 (25), 5123-5129.
3. Galassi, L., Eur. J. Histochem. 2000, 44 (4), 419-432.
4. Wu, W. B.; Wang, M. L.; Sun, Y. M.; Huang, W.; Cui, Y. P.; Xu, C. X. Journal of Physics and
Chemistry of Solids 2008, 69 (1), 76-82.
5. Roberts, D. V.; Wittmershaus, B. P.; Zhang, Y. Z.; Swan, S.; Klinosky, M. P., Journal of
Luminescence 1998, 79 (4), 225-231.
6. Meldal, M.; Tornoe, C. W., Chemical Reviews 2008, 108 (8), 2952-3015.
USE OF FACIAL RECOGNITION TECHNOLOGY IN THE EVALUATION OF AFFECTIVE REACTIONS
TOWARDS METALLIC FLAVORED WATER
Sarah Fong1, Susan Duncan2, Ph.D, RD, Lisa Hightower3, Qin Li2
1Department
of Human Nutrition, Foods and Exercise; 2Department of Food Science and Technology;
3Agricultural Education and Extension
Virginia Polytechnic and State University
Blacksburg, VA
Phone: 703-203-4437 Fax: 540-231-9293
Email: [email protected]
ABSTRACT
Facial recognition software is capable of identifying a person by age, gender, ethnicity, and outward
emotions by analyzing muscle movements of the face and eyes. To evaluate effectiveness of this tool in
sensory evaluation, affective responses to untreated bottled water and mineral-treated bottled water were
evaluated. Some minerals in water, such as copper and iron, can contribute a lingering, unpleasant, metallic
aftertaste in the mouth.
Thirty-nine individuals from a Virginia Tech food
sensory class were videotape while evaluating water
samples using an electronic survey. Untreated and irontreated water samples were each swirled in the mouth for
10 seconds before expectorating into a cup.
Each
individual’s baseline emotions were compared with his or
her response to both water samples in order to see if and
how their emotions changed upon tasting the two
samples (Figure 1).
Only six participants had video footage that the
facial recognition software could accurately analyze; the
remaining participant videos were unable to be analyzed
due to problems with facial obstruction, lack of eye
contact with the camera, misidentification of panelist
gender and a number of other confounding variables to
which the software is sensitive. For the six videos that
produced usable footage, the iron-treated water sample Figure 1. Example of changes in emotional response from
elicited higher incidences of the “angry”, “disgusted”, or baseline (no sample in mouth); with neutral (control) water
“surprised” emotions compared to baseline or to neutral in mouth and response to expectoration; and iron-treated
water responses for the majority of participants; there water in mouth and the resulting aftertaste effect.
was great variance in baseline emotions. The higher
incidences of these emotions upon tasting the iron-treated water indicate that facial recognition software is
effective in measuring relative displeasure toward this stimulus.
Although the sensitivity of FaceReader to confounding variables reduces its current performance,
improvements in future versions of FaceReader pose exciting possibilities in the research of human reaction to
sensory stimuli. Other fields such as food product development, consumer research, and nutrition counseling
could apply the technologies of FaceReader for unique insight into food preferences and trends. The
applications of FaceReader in these fields of food and nutrition would further our understanding of people’s
reactions and emotions to various aspects of food and flavor.
References:
1. Atlantic Coast Conference Interdisciplinary Forum for Discovery in Life Sciences. Abstract Template. Internet: http://www.cpe.vt.edu/accforum/
abstract.html (accessed September 8 2010).
2. Noldus Information Technology. FaceReader. Internet: http://www.noldus.com/human-behavior-research/products/facereader (accessed September 8
2010).
Insights into the Mechanism of Ligand Responsiveness for
the Quorum-sensing Regulator EsaR from Pantoea stewartii
Daniel J. Schu, Jessica M. Scruggs, Jared S. Geissinger and Ann M. Stevens
Department of Biological Sciences
Virginia Tech
Phone: (540) 231-2342 Fax: (540) 231-4043
Email: [email protected]
ABSTRACT:
The mechanism whereby the LuxR family of proteins is controlled by acylated homoserine
lactone (AHL) ligands remains elusive. AHL is required for the purification of most LuxR family
members; this has limited the ability to study the ligand-free versus the ligand-associated form
of LuxR family members. This type of analysis is possible with a subset of LuxR proteins
represented by the homologue EsaR.
EsaR regulates production of exo/capsular polysaccharide (EPS) in the corn pathogen
Pantoea stewartii subsp. stewartii. At low cell densities, EPS production is repressed. However,
at high cell densities when high concentrations of its cognate AHL autoinducer signal are
present, EsaR is inactivated and derepression of EPS production occurs. Thus, EsaR responds
to AHL in a manner opposite to that of the majority of LuxR homologues. Further, EsaR has
two unique regions, (i) an extended linker region between the AHL-binding N-terminal domain
and the DNA binding C-terminal domain, and (ii) an extended C-terminus.
Biochemical and genetic methods have been used to examine the mechanistic differences
between EsaR and other LuxR homologues. Here, we describe the generation and analysis of
AHL-insensitive variants of EsaR. Six of these variants no longer bind AHL and have helped to
confirm the location of the AHL binding pocket. Interestingly, two EsaR variants still bind AHL,
but are not inactivated by it, providing further clues to the structural mechanism of AHL
responsiveness. Additional studies using fluorescence anisotropy to examine the relative DNAbinding affinities of these mutants are being performed.
The Effect of Polymer Structure on Nuclear Localization for DNA Delivery
Giovanna Grandinetti, Theresa M. Reineke
Department of Chemistry, Virginia Polytechnic Institute and State University,
Blacksburg, Virginia 24061-0001
Cationic polymers have been used for gene delivery, but their mechanism of
action remains largely unknown. The end goal for DNA delivery vehicles is to get the
therapeutic DNA into the nucleus. The Reineke group has developed gene delivery
polymers called poly(glycoamidoamine)s (PGAAs) that incorporate a sugar moiety as
well as an ethylamine moiety in their repeat unit; these polymers have proven to be
efficient and non-toxic compared to another well-known gene delivery polymer,
poly(ethylenimine) (PEI). Previous work done in our group has shown that the PGAAs
have different mechanisms of cellular uptake and intracellular trafficking patterns than
PEI. This research further investigates the role that polymer structure plays in nuclear
gene delivery and focuses on the mechanism of nuclear uptake.
Flow cytometry was used to determine nuclear membrane integrity and plasmid
DNA uptake during transfection, and confocal microscopy was used on isolated nuclei
to study the minimum requirements for plasmid DNA nuclear entry. It was found that
the type of carbohydrate influences nuclear trafficking as well as the polymers’ ability to
directly permeabilize the nuclear membrane.
DESIGN OF IMIDAZOLE-CONTAINING POLYMERS FOR
EMERGING LIFE SCIENCE APPLICATIONS
Matthew D. Green1, Michael H. Allen, Jr.2, and Timothy E. Long2
1
Department of Chemical Engineering, 2Department of Chemistry
Virginia Tech, Blacksburg, VA 24061
Phone: (540)231-3378
Fax: (540)231-8517
Email: [email protected]
Gene therapy is a very active field, primarily due to the potential treatment for a variety of genetic
1
diseases. Synthetic, nonviral vectors are popular due to the tunable chemical composition and their
2, 3
general lack of cytotoxicity.
The design of vectors that not only deliver therapeutic nucleic acids, but
also incorporate radical scavenging species would further restore the balance of oxidative species within
4
the cell while also re-encoding defective genetic sequences. Recent efforts have focused on formulating
systematic structure-property relationships that elucidate polymer parameters necessary for optimization
of nucleic acid delivery. Therefore, the synthesis of a series of functionalized poly(1-vinylimidazole)
polymers conjugated with glutathione and alkyl halides, left and right below, provided novel polymeric
vectors for analysis as efficient chaperones to bind and protect DNA using gel electrophoresis, dynamic
light scattering, and various in vitro assays. The glutathione-conjugated polymer also acts as an efficient
antioxidant delivery vehicle as glutathione scavenges radicals under imposed oxidative stress.
x
y
N
N
Br
N
O
O
N
y
x
N
N Br
R
N
N
R = C 2H 5
C 4H 9
S
SG
Acknowledgements:
This material is based upon work supported in part by the U.S. Army Research Office under grant number W911NF07-1-0452 Ionic Liquids in Electro-Active Devices (ILEAD) MURI. This material is also based upon work supported
in part by the Macromolecular Interfaces with Life Sciences (MILES) Integrative Graduate Education and Research
Traineeship (IGERT) of the National Science Foundation under Agreement No. DGE-0333378.
References:
1.
2.
3.
4.
Heath, W. H.; Senyurt, A. F.; Layman, J.; Long, T. E., Macromolecular Chemistry and Physics
2007, 208 (12), 1243-1249.
Layman, J. M.; Ramirez, S. M.; Green, M. D.; Long, T. E., Biomacromolecules 2009, 10 (5),
1244-1252.
Ramirez, S. M.; Layman, J. M.; Long, T. E., Macromolecular Bioscience 2009, 9 (11), 11271134.
Williams, S. R.; Lepene, B. S.; Thatcher, C. D.; Long, T. E., Biomacromolecules 2009, 10 (1),
155-161.
HOMOLYTIC SUBSTITUTION AT SELENIUM: FACTORS INFLUENCING
KINETICS OF CYCLIZATION
Amber N. Hancock*, Sofia Lobachevsky+ and Carl Schiesser+
+
University of Melbourne, Melbourne, Victoria 3053 AU
*
Virginia Polytechnic Institute and State University, Blacksburg, Virginia 24060 USA
Phone: 540-231-3254 Fax: 540-231-3255
Email: [email protected]
ABSTRACT:
The design and synthesis of selenium containing heterocycles has received considerable
attention resulting from their ability to function as antioxidants, anti-virals, antiinflammatories, and immunomodulators. Interested in establishing the synthetic feasibility
of intramolecular homolytic substitution at selenium for preparation of these compounds, we
set out to determine what factors influence the kinetics and energetics of these reactions.
We synthesized a series of photochemically labile Barton and Kim esters radical precursors
and determined rate constants and activation parameters for intramolecular homolytic
substitution of the corresponding radicals via competition experiments.
Results presented
will discuss the effect of leaving group stability as well as selenaheterocycle ring size on the
kinetics and activation parameters for these reactions. Our investigation of these reactions
revealed notable effect of both leaving radical stability and ring size.
S
S
8 SH
8 S
+
S
hv
+
R2
Se
R
O
HR2
Se
R
O f ast
O
N
f ast
O
R2
Se
R
R2
Se
R
ktrap
kcyclization
Se
+
S
N
O
O
n=1,2
R
DNA INSPIRED HIERARCHICAL POLYMER DESIGN: HYDROGEN
BONDING AND ELECTROSTATICS IN CONCERT
Sean T. Hemp and Timothy E. Long
Macromolecules and Interfaces Institute
Department of Chemistry – (0212)
Virginia Tech
Blacksburg, VA 24061
Phone: (540) 231-6587; Fax: (540) 231-8517
Email: [email protected]
Biomacromolecules, for example DNA and proteins, utilize electrostatics and hydrogen bonding to generate
complex secondary and tertiary structures which subsequently produce unique properties. Electrostatic or hydrogen
bonding interactions significantly influence the properties of synthetic polymers; ionic groups enhance conductivity and
improve mechanical properties while hydrogen bonding interactions provide stimuli responsive properties and enable
supramolecular assembly. Our research group investigates both areas of polymer research: ionic incorporation through the
1
synthesis of imidazolium ionene segmented block copolymers and hydrogen bonding incorporation through the synthesis of
2
9-vinylbenzyladenine and 9-vinylbenzylthymine containing triblock copolymers . Very few reports study the effect of
electrostatic and hydrogen bonding interactions together in concert in the same polymer structure. Recently, our group
incorporated the two interactions together through the addition of a uracil-containing phosphonium salt to an adenine
3
functionalized triblock copolymer . To further our advances in the integration of electrostatic and hydrogen bonding forces
together, we synthesized random copolymers of tert-butyl acrylate and 9-vinylbenzyladenine as shown in Figure 1. Upon
hydrolysis of the tert-butyl esters and neutralization, the resulting copolymers contained both nucleobases for hydrogen
bonding and carboxylate anions for electrostatic interactions. To generate a cationic analog, we also performed the random
copolymerization of 2-(dimethylamino)ethyl methacrylate(DMAEMA) and 9-vinylbenzyladenine as shown in Figure 1 and the
resulting random copolymer was protonated with HCl. Solution and melt rheology revealed the complementary nature of the
forces and the polymers were fully characterized utilizing NMR, DSC, TGA, SEC, and in situ IR.
Figure 1. Synthesis of poly(tert-butyl acrylate-co-9-vinylbenzyladenine) and poly(DMAEMA-co-9-vinylbenzyladenine) and
their subsequent deprotection/neutralization and protonation, respectively.
Acknowledgements: U.S. Army Research Laboratory and the U.S Army Research Office under grant number W911NF-071-0452 Ionic Liquids in Electroactive Devices (ILEAD) MURI and in addition, this material is based upon work supported in
part by the U.S. Army Research Laboratory and the U.S. Army Research Office under the Army Materials Center of
Excellence Program, contract W911NF-06-2-0014.
References:
1. Williams, S. R.; Salas-de la Cruz, D.; Winey, K. I.; Long, T. E., Polymer 2010, 51 (6), 1252-1257.
2. Mather, B. D.; Baker, M. B.; Beyer, F. L.; Berg, M. A. G.; Green, M. D.; Long, T. E., Macromolecules 2007, 40 (19), 68346845.
3. Mather, B. D.; Baker, M. B.; Beyer, F. L.; Green, M. D.; Berg, M. A. G.; Long, T. E., Macromolecules 2007, 40 (13), 43964398.
A GROCERY STORE INTERVENTION DESIGNED TO INCREASE FRUIT,
VEGETABLE, AND HEALTHY SNACK PURCHASES AMONG PARENTS OF YOUNG
CHILDREN
For Oral Presentation
Ashley Holmes, M.S.; PhD Student
Elena Serrano, PhD; Associate Professor
Department of Human Nutrition, Foods, & Exercise
Virginia Polytechnic Institute & State University
Blacksburg, VA
Phone: 804-928-6062 Fax: 540-231-3916
Email: [email protected]
ABSTRACT:
Grocery stores have the ability to manipulate the availability, access, pricing, promotion and
information of food products sold. Encouraging grocery stores to be a vehicle for utilizing its influence
towards more healthful food purchase has been strongly suggested by researchers. Grocery stores have
the potential for broad reach, as evidenced by family households averaging two visits to the grocery store
per week, spending an average of $93 a week, based on data from 2006. Further, a survey by the Food
Marketing Institute found that the majority of shoppers in their study were concerned about the nutritional
quality of their food, whereby contributing to a greater consumer demand for more point-of-purchase
nutrition information. As a result, grocery stores represent a unique opportunity to promote the purchase
of healthy foods to their consumers, while still making a profit.
The aim of this study was to develop and evaluate a multi-faceted, child-focused grocery store
intervention. The intervention included a point-of-purchase kiosk featuring 32 fresh fruits, vegetables,
and processed goods chosen using the MyPyramid guidelines for children, as well as a sampling pod,
comprised of food items from the kiosk. Items featured in the sampling pod were creatively combined
into tasty, but healthy snacks to not only give parents ideas for integrating these items into their child’s
diet, but also to give children an opportunity to try foods without having to purchase it.
An observational uninterrupted time-series design was implemented in one grocery store for twelve
weeks. The study measures consisted of three components: examination of changes in sales data for
featured products, provided by the grocery chain; candid, unobtrusive, blind observations of customers
near and around the intervention; and brief questionnaires of customers, who engaged at some level with
the kiosk and sampling pod. The results yielded an overall increase in the proportion of the sales of the
featured items to total store sales during the intervention period. Individual items that increased sales
during the intervention period, included whole-wheat mini bagels, bananas, radishes, honey, sunflower,
baked tortilla chips, and almond butter (p<.05). Almost two-thirds (61.7%) of the patrons interviewed
noticed the healthy kids kiosk, with about one-quarter (28.7%) indicating that they purchased at least one
item. 58% of patrons surveyed reported that the kiosk encouraged them to buy healthier foods.
Promoting healthy foods at point-of purchase locations can result in increased purchases of these foods
among parents with young children. These findings have provided insight into the effectiveness of
grocery store interventions on purchasing patterns and behaviors of families.
References:
1. Glanz, K. Mullis, R. 1998. Environmental interventions to promote healthy eating: a
review of models, programs, and evidence. Health Education Quarterly 15(4): 395-415.
2. Food Marketing Institute. 2006. Consumer attitudes and the supermarket. Washington, DC: Food
Marketing Inst.
3. Tuttle, M. 1995. Trends and Changes in Consumer Attitudes about Nutrition and Food Shopping.
Available at: http://www.nutrientdataconf.org/PastConf/NDBC20/3-1_Tuttle.pdf Accessed on
September 1, 2009.
4. Levy, A., Mathews, O., Stephenson, M., et al. The impact of a nutrition information program on food
purchases. Prev. Med. 39(2): 108-136.
5. Ernst, N., Wu, M., Frommer, P., et al. 1986. Nutrition education at the point of purchase: The Foods for
Health Project evaluated. Prev. Med. 15:60-73.
6. Pennington, J., Wisniowski, L., Logan, G. 1988. In-store nutrition information programs. J Nutr Educ.
20(1):5-10.
Presentor bio:
Ashley Holmes received a B.S. in Human, Nutrition, Foods and Exercise- in concentrations in
both Science of Nutrition and Dietetics from Virginia Tech as well as a German Language Minor
primarily completed at University of Stuttgart in Germany. Ashley also received her masters in
Human, Nutrition, Foods, & Exercise from Virginia Tech under the mentorship of Dr. Elena
Serrano, examining the impact of a grocery store intervention promoting the sales of healthy
snacks to parents and their young children. Ashley is currently pursuing her PhD, with Dr.
Serrano, expanding on her work promoting healthy foods to young children and their families
using nutrition menu labeling in the restaurant context.
GENOME-SCALE COMPARATIVE ANALYSIS OF TWO VIBRIO
PARAHAEMOLYTICUS STRAINS
Saylem M. Ingalls1, Thero Modise1, Tian Hong1, Cimarron Smith1, Linda M. McCarter2,
Roderick V. Jensen1, Ann M. Stevens1
1
Department of Biological Sciences
Virginia Tech
2
Department of Microbiology
University of Iowa
Phone: (540) 231-2342
Fax: (540) 231-4043
Email: [email protected]
Abstract:
The marine bacterium, Vibrio parahaemolyticus, is a leading cause of seafoodassociated gastroenteritis in the United States and an emergent food-borne pathogen
worldwide. Its virulence factors are distinct from those of the better-studied Vibrio
cholerae system. The genome of a pandemic strain (RIMD 2210633), first isolated in
1996 in Japan, has previously been sequenced. The genome of another V.
parahaemolyticus strain, BB22OP, has now been sequenced. The Roche/454 GS FLX
Titanium sequencing system provided greater than 200x coverage of the BB22OP
genome, allowing for a comparative analysis with the RIMD strain. Strain BB22OP, an
environmental isolate from Bangladesh, was isolated before 1980 and is one of the best
genetically characterized V. parahaemolyticus strains. BB22OP has an opaque colony
morphology and is capable of undergoing a phase variable switch to a translucent
colony phenotype (BB22TR). This switch alters many cell surface characteristics of the
cell, including capsular polysaccharide production. Strain BB22OP is not pathogenic;
whereas, genetic alterations in either opaR or luxO cause the translucent strain to be
pathogenic. opaR encodes the major quorum-sensing output regulator, and luxO
encodes a transcriptional regulator upstream of opaR in the quorum-sensing network. It
is hypothesized that a genome-level sequence analysis of BB22OP compared to the
RIMD strain will reveal novel genetic components in the BB22OP strain. A de novo
genome sequence assembly using the Roche/454 Newbler software resulted in the
alignment of 98% of the reads into 63 large contigs covering 5.04 Mbp. These contigs
have been assembled into two chromosomes. Preliminary sequence alignment to the
RIMD sequence confirms gaps in the BB22OP genome originally identified by DNA
microarray data from the McCarter lab. Alignment to the RIMD sequence has revealed
the presence of approximately 300 genes (240,000 bp) novel to BB22OP. Further
comparative analysis will provide insight into the conserved colonization and virulence
factors essential to the infectious process utilized by V. parahaemolyticus, as well as the
distinctive genetic determinants in the two strains under investigation.
Enhanced emission for near-infrared fluorescence of nanoparticles
surface modified with indocyanine green and protein activation for
application in photodynamic therapy
Parul Rungta, Ragini Jetty, Yuriy P. Bandera, Ryan D. Roeder,
and Stephen H. Foulger
Center for Optical Materials Science and Engineering Technologies
School of Materials Science and Engineering
Clemson University, Clemson, SC 29634-0971, USA
Phone: 864-656-1023 Fax: 864-656-1023
Email: [email protected]
ABSTRACT:
In the current effort, studies on enhancing the emission of surface modified
nanoparticles with indocyanine green (ICG) is carried out to allow ICG to operate as a
photosensitizer in Photodynamic therapy (PDT). The low fluorescence quantum yield
and non-specific quenching of ICG is overcome by adding a terminal azide to ICG and
poly(ethylene glycol) (PEG), and then attaching them to the poly(propargyl acrylate)
(PA) colloids via a click transformation, specifically the copper-catalyzed azide/alkyne
cycloaddition. Attaching the PEG chains did not change the emission characteristics.
However, the PEG attached particles showed an immediate increase in emission when
Bovine albumin solution (BSA) was mixed. For PA particles attached with azide
modified ICG and PEG, it has been observed that they attain 63% of their total one day
emitting increase within the first 15 minutes. From the studies of cancer cells, it can be
noted that the surface modified PA particles do not alter the growth of cancer cells
significantly.
References:
1. T. J. Dougherty, C. J. Gomer, B. W. Henderson, G. Jori, D. Kessel, M. Korbelik, J. Moan, and Q.
Peng. Photodynamic therapy. Journal of the National Cancer Institute, 90(12):889–905, 1998.
2. S. Reindl, A. Penzkofer, S. H. Gong, M. Landthaler, R. M. Szeimies, C. Abels, and W. Baumler.
Quantum yield of triplet formation for indocyanine green. Journal of Photochemistry and
Photobiology a-Chemistry,
105(1):65–68, 1997.
3. W. Baumler, C. Abels, S. Karrer, T. Weiss, H. Messmann, M. Landthaler, and R. M. Szeimies.
Photo-oxidative killing of human colonic cancer cells using indocyanine green and infrared light.
British Journal of Cancer, 80(3-4):360–363, 1999.
4. R. Philip, A. Penzkofer, W. Baumler, R. M. Szeimies, and C. Abels. Absorption and fluorescence
spectroscopic investigation of indocyanine green. Journal of Photochemistry and Photobiology aChemistry, 96(1-3):137–148, 1996.
EFFECTS OF THE NON-IONIC SURFACTANT TWEEN 80 ON THE ENZYMATIC
HYDROLYSIS OF MODEL CELLULOSE SURFACES
Feng Jiang,1 Alan Esker,2 and Maren Roman*,1
1
Department of Wood Science and Forest Products (0323)
2
Department of Chemistry (0212)
Virginia Tech
Blacksburg, VA 24061
Phone: (540) 231-1421; Fax: (540) 231-8176
Email: [email protected]
ABSTRACT:
Non-ionic surfactants, such as Tween 80, have been found to enhance the enzymatic
conversion of lignocellulosic biomass to bioethanol. The mechanism for this enhancement,
however, is incompletely understood. To gain further insight into the mechanism, we studied the
effects of Tween 80 on the enzymatic hydrolysis of model cellulose surfaces. The interactions
between Tween 80 and cellulase enzymes in solution were analyzed by tensiometry and
fluorescence spectroscopy. The surfactant’s critical micelle concentration (CMC) was found to
be higher in the presence of cellulase enzymes than in their absence, indicating the formation of
surfactant-enzyme complexes. The adsorption of Tween 80 onto model cellulose surfaces was
studied with a quartz crystal microbalance with dissipation monitoring (QCM-D) and by surface
plasmon resonance spectroscopy (SPR). The adsorption data was well described by Langmuir
adsorption isotherms. The effect of Tween 80 on the enzymatic hydrolysis of the model surfaces
was analyzed by two sets of QCM-D experiments. In both sets of experiments, Tween 80 had
little or no effect on the enzymatic hydrolysis of CNC model surfaces. Our results indicate that
the enhancement of lignocellulose conversion by Tween 80 is not due to an enhancement of the
cellulose-enzyme interactions but may be related to lignin-enzyme interactions.
“CLICK” CHEMISTRY AS A ROUTE TO PHOTOACTIVE POLYESTERS
FOR TAILORABLE INTERFACES
Stephen M. June, Philippe Bissel, Takeo Suga, and Timothy E. Long
Department of Chemistry
Virginia Tech
Blacksburg, VA 24061
Phone: 5402313378 Fax: 5402318517
Email: [email protected]
ABSTRACT:
With the increasing level of miniaturization and advancement of microelectronic devices, the need
for fabrication techniques that address the size and fragility of these devices has arisen.1 Due to the
thinness of microelectronic devices, current manufacturing processes require that the device substrate be
adhered to a support layer for grinding, fabrication, and cutting. In order to subsequently remove the
device from the support substrate, the adhesives utilized must exhibit reversibility.1 Currently,
thermoplastic hot melt adhesives are primarily utilized. After manufacturing the device, the device and
support are heated beyond the melt temperature of the adhesive, which allows for debonding. However,
the repeated heating and cooling cycles that the fabrication and subsequent debonding require often
cause thermal stress, leading to warping and fracture of the device. Therefore, adhesives containing
photo-reversible properties are hereby proposed.1-3
Figure 1 – Photo-cleaveage of o-nitro benzyl esters and subsequent adhesive debonding
In order to achieve this goal, a series of polymers containing o-nitro benzyl (ONB) esters are
proposed. 2-Nitro-p-xylene glycol (NXG) was utilized to incorporate ONB functionality. NXG was
functionalized into a propiolate with propiolic acid, to afford a bisalkyne for “click” chemistry. “Click”
chemistry allows for the synthesis of polyesters in the absence of precious metal catalysts and at
significantly lower temperatures. Furthermore, polyesters synthesized by “click” chemistry exhibit
significantly higher glass transitions than analogous polyesters synthesized by standard methods. 1H
NMR will confirm photocleavage upon UV exposure.
Acknowledgements
This material is based upon work supported in part by the U.S. Army Research Laboratory and the
U.S. Army Research Office under the Army Materials Center of Excellence 15 Program, contract
W911NF-06-2-0014; and by the Macromolecular Interfaces with Life Sciences (MILES) Integrative
Graduate Education and Research Traineeship (IGERT) of the National Science Foundation grant under
agreement No. DGE-0333378.
References:
1) June, S. M.; Suga, T.; Heath, W. H.; Lin, Q.; Puligadda, R.; Long, T. E. J. Adhes. 2010, In Press.
2) Trenor, S. R.; Long, T. E.; Love, B. J. J. Adhes. 2005, 81, pp 213-229.
3) Trenor, S. R.; Long, T. E.; Love, B. J. Macromol. Chem. Phys. 2004, 205, pp 715-723.
N-Terminal peptidic boronic acids selectively inhibit human
ClpXP
Kenneth Knott,a Jennifer Fishovitz,b Steven B. Thorpe,a Irene Leeb and Webster L. Santos*a
a
Department of Chemistry,Virginia Tech, Blacksburg,Virginia 24061, USA.
E-mail: [email protected]; Fax: +1 540 231 3255; Tel: +1 540 231 5041
b
Department of Chemistry, Case Western Reserve University, Cleveland,
Ohio 44106, USA
Phone: 540-267-6502 Email: [email protected]
ABSTRACT:
The synthesis and development of N-terminal peptidic boronic acids as protease inhibitors is reported. N-Terminal
peptidic boronic acids interrogate the S’ sites of the target protein for selectivity and provide a new strategy that
complements the currently known peptidic α-amino boronic acids (C-terminal boronic acids). After screening a series
of N-terminal peptidic boronic acids, the first selective inhibitor of human ClpXP, an ATP-dependent serine protease
present in the mitochondrial matrix, was discovered. This should facilitate the understanding of the physiological
function of this protease
References:
1. Org. Biomol. Chem., 2010, 8, 3451–3456
BIOCOMPATIBLE DETACHABLE POLYELECTROLYTE MULTILAYER
FILMS FOR APPLICATIONS IN TISSUE ENGINEERING
Adam L. Larkin, Richey M. Davis and Padma Rajagopalan
Department of Chemical Engineering, Virginia Polytechnic Institute and State University
Phone: 7174050849
Email: [email protected]
ABSTRACT:
We report the design and assembly of novel, detachable, free-standing polyelectrolyte multilayer
(PEM) films. PEMs have been used to design anti-bacterial coatings, non-cytotoxic surfaces, and to
assemble three dimensional cellular architectures [1-3]. The majority of reports in the literature are studies
on PEMs deposited and adherent on a solid substrate [4, 5].
We have assembled PEMs on inert hydrophobic substrates such as polypropylene or
polytetrafluoroethylene substrates with water contact angles of 102.31 ± 2.81° and 109.64 ± 1.59°,
respectively. A typical PEM consists of 30-50 bilayers of HA and chitosan. To achieve detachability, PE
deposition time and concentration were varied. After PEM assembly, the films were detached from the
solid substrate (Figure 1) and subsequently cross-linked to enhance stability. Crosslinked films placed in
a phosphate buffered solution at 37°C, exhibited 6-8% weight loss over a 7-day period.
Figure 1. A typical 50 bilayer free-standing, HA and chitosan PEM.
Hydrated films transmitted 90 to 97% of light, making these films good candidates for applications
requiring optical microscopy. Surface topography was investigated using atomic force microscopy (AFM)
and our results indicate that the films are smooth and exhibit surface features ranging from 10-30nm. The
Young’s modulus of the PEMs was measured for crosslinked and unmodified films using a
nanoindentation method. BALB/c 3T3 fibroblasts were cultured on PEMs. The interactions of fibroblasts
with PEMs were determined by measuring cell viability, cell proliferation and cytoskeletal organization.
Our current studies are focused upon incorporating PEMs as tissue scaffolds in 3D liver mimics.
References:
1.
Wong SY, Li Q, Veselinovic J, Kim BS, Klibanov AM, Hammond PT. Bactericidal and virucidal
ultrathin films assembled layer by layer from polycationic N-alkylated polyethylenimines and polyanions.
Biomaterials 2010;31(14):4079-87.
2.
Berg MC, Yang SY, Hammond PT, Rubner MF. Controlling mammalian cell interactions on
patterned polyelectrolyte multilayer surfaces. Langmuir 2004;20(4):1362-8.
3.
Rajagopalan P, Shen CJ, Berthiaume F, Tilles AW, Toner M, Yarmush ML. Polyelectrolyte nanoscaffolds for the design of layered cellular Architectures. Tissue Engineering 2006;12(6):1553-63.
4.
Decher G. Fuzzy nanoassemblies: Toward layered polymeric multicomposites. Science
1997;277(5330):1232-7.
5.
Yang SY, Berg MC, Hammond PT, Rubner MF. Primary hepatocyte and mammalian cell
response to polyelectrolyte multilayers containing polyacrylamide polymers. Abstracts of Papers of the
American Chemical Society 2003;226:U466-U.
ATOMISTIC SIMULATIONS OF THE INTERACTIONS BETWEEN THE
ALZHEIMER’S AMYLOID -PEPTIDE AND LIPID RAFTS
Justin A. Lemkul and David R. Bevan
Department of Biochemistry
Virginia Polytechnic Institute & State University
Phone: (540) 231-9080 Fax: (540) 231-9070
Email: [email protected]
ABSTRACT:
The “amyloid hypothesis” of Alzheimer’s disease holds that the aggregation and deposition of
the amyloid -peptide (A) in neural tissue is a key pathological event in the etiology of the
disease. Toxicity of A is specifically linked to its interactions with cellular membranes, which
result in ion leakage and ultimately cell death. A is produced within the cell membrane, but the
mechanism by which it exits this environment to begin the aggregation cascade is unclear. We
have conducted molecular dynamics (MD) simulations in different models of lipid rafts, which
are the specific microdomains in which A is produced. We find that the presence of a specific
lipid type, ganglioside GM1, promotes the exit of A from the membrane, an effect that is not
observed in rafts lacking this lipid. GM1 induces tilting of A and provides a hydrogen-bonding
scaffold for the peptide to adopt aggregation-prone -strand configurations. Experimental
evidence has long suggested that GM1 plays a role in the aggregation cascade, but until now,
its specific function has remained a mystery. The knowledge of specific A-GM1 interactions
gives new insight into the early stages of the amyloid cascade and Alzheimer’s disease, a time
when therapeutic intervention is most effective.
Global and local chromatin structural changes upon HIV reactivation
Sarah Lueking, Justin Fincher, and Jonathan Dennis
Department of Biological Sciences
Florida State University
Phone: 850-645-9274 Fax: 850-645-8447
Email: [email protected]
ABSTRACT:
A major unanswered question in HIV biology involves the transition from latent HIV infection to
AIDS. While several methods of treatment have proved effective, reservoirs of latent viral DNA
maintained in memory T-cells inevitably impede a complete purge of the virus from host cells. Thus,
efforts to characterize the virus-host cell interactions during viral latency and subsequent reactivation aim
to elucidate mechanisms controlling viral gene expression in these cells.
As a retrovirus, HIV infection requires genomic RNA to be reverse transcribed into a DNA
provirus and integrated into host DNA; as such, HIV must function in the context of chromatin. Chromatin
is the nucleoprotein complex into which DNA is organized in the nucleus; the fundamental subunit of
chromatin is the nucleosome, which consists of 150 bp of DNA wrapped around a histone octomer.
Several lines of evidence suggest that HIV proteins interact with chromatin regulatory machinery to assist
in reactivation from latency. Further evidence indicates that this transcriptional reactivation alters host
gene expression programs. We assume that changes in gene expression are linked with changes in
chromatin structure; however, this assumption is largely untested, as there are no systematic, genomewide studies of changes in chromatin in response to viral reactivation.
Our lab has developed innovative technology using microarrays to study the functional
relationship reactivated HIV has with host chromatin under the modulatory influence of the HIV
reactivating agent, phorbol 12-myristate 13-acetate (PMA). These microarrays allow us to visualize
changes in chromatin structure on two levels of resolution: individual nucleosome position and global
nuclease accessibility. Cells from the latently HIV infected cell line J1.1 and its parental line, Jurkat, were
treated with PMA to reactivate HIV. From a single growth, both chromatin, for nuclease accessibility and
nucleosome position studies, and RNA, for correlating gene expression studies, were harvested. PMA
treatment of J1.1 and Jurkat cells showed differences in nuclease accessibility and nucleosome position
measurements, indicating an HIV reactivation-driven global effect on host cell chromatin. Correlations of
these chromatin structural data with expressed RNA from the above cell lines will lead to the first
description of the relationship between HIV reactivation-induced changes in gene expression and
chromatin structure.
DIFFERENTIATION OF PRIMATE AMNIOTIC FLUID-DERIVED STEM CELLS INTO BETA CELLS
IN VITRO AND IN VIVO
1
2
1
1
2
David L. Mack , Yu Zhou , Koudy Williams , Sayed-Hadi Mirmalek-Sani , Diego Lorenzetti ,
1
1
1
Mark Furth , Anthony Atala , Shay Soker
1
Wake Forest Institute for Regenerative Medicine, Winston Salem, NC USA
2
Plureon Corporation, Winston Salem, NC USA
phone: 336 713-1193 Fax: 336 713-7290 email: [email protected]
Insulin therapy for Type 1 diabetes (T1D) does not prevent serious long-term complications including
vascular disease, neuropathy, retinopathy and renal failure. Stem cells, including amniotic fluid-derived stem (AFS)
cells--highly expansive, multipotent, and non-tumorigenic cells described in our lab--could serve as an appropriate
1
stem cell source for β-cell differentiation . In the current study we tested whether nonhuman primate (NHP) AFS
cells ectopically expressing key pancreatic transcription factors were capable of differentiating into a beta-like cell
phenotype in vitro and whether these cells would engraft in the pancreas of diabetic mice and restore normal
glucose metabolism.
NHP-AFS cells were obtained from Cynomolgus monkey amniotic fluid by immunomagnetic selection for a
CD117 (c-kit) positive population. For the in vitro studies, RT-PCR for endodermal and pancreatic lineage-specific
markers was performed on AFS cells after adenovirally transduced expression of Pdx1, Ngn3 and MafA. Immunodeficient (NOD/scid) streptozotocin (STZ)-induced diabetic mice were used for the in vivo studies. Mice were
implanted with slow-release insulin pellets to maintain euglycemia. Two days later, GFP-labeled NHP-AFS cells
were injected into the arterial vasculature and blood glucose concentrations measured periodically. Insulin pellets
were removed after 4 weeks and plasma glucose concentrations monitored for an additional 12 days.
Forced expression of MafA was sufficient to
induce insulin mRNA expression in NHP-AFS cells
and a higher induction was obtained in combination
with Pdx1 and Ngn3. In addition to insulin, other
important endocrine cell genes such as NEUROD1,
NKX2.2 and ISL1 were coordinately expressed in
response to the combination of all 3 transcription
factors (table at right). Lineage color coding: red =
definitive endoderm; green = pancreatic progenitor;
purple = endocrine progenitors and more mature
endocrine cells.
NHP AFS 7231
SOX17 FOXA2 SOX9 PDX1 NEUROD1 NKX2.2 NKX6.1 PAX6 ISL1 INS
Pdx1+NGN3+MafA
++
-
+
+
+++
++
+
+
+
+++
Pdx1+NGN3
-
-
-
-
+
-
-
-
-
-
Pdx1+MafA
++
+
+
+
-
-
-
+
-
+
NGN3+MafA
++
-
-
+
++
+
+
+
++
++
MafA
++
-
-
++
-
-
-
-
-
++
GFP
-
-
-
-
-
-
-
-
-
-
In diabetic mice whose plasma glucose
concentration was between 300-350 mg/dL prior to
insulin administration, delivery of NHP-AFS cells
resulted in euglycemia after withdrawal of insulin. In
contrast, mice whose initial plasma glucose
concentration exceeded 450 mg/dL, nhpAFS cell
delivery did not restore euglycemia after withdrawal
of insulin (figure at right).
NHP-AFS cells differentiated into a β-cell
like phenotype in vitro as evidenced by the
coordinated expression of pancreatic-specific
markers.
Undifferentiated NHP-AFS cells also
engrafted in the pancreas of a transplanted host,
responded to the inductive signals of the native
pancreatic microenvironment and acquired certain
differentiated properties of β-cells. The most notable
changes were the expression of specific pancreatic transcription factors and the production of insulin, leading to the
regulation of plasma glucose concentrations. Therefore, NHP-AFS cells could be a cell source for β-cell
differentiation and NHP-AFS cells could be used to develop a nonhuman primate model for evaluating cell therapy
in the treatment of diabetes.
1. De Coppi, P. at al. Nat Biotechnol. 2007; 25:100.
Characterization of a novel transmembrane protein, Cr7TM, in the
algal species Chlamydomonas reinhardtii.
Jennifer I. Middlebrooks and Laura R. Keller
Department of Biological Sciences
Florida State University
Phone: 850-644-9814
Email: [email protected]
ABSTRACT:
Primary cilia, once dismissed as evolutionary remnants, are now recognized as
the sensory antennae of the cell. Their importance is demonstrated by the diversity and
severity of conditions caused by the genetic mutations in ciliary genes (Reveiwed by
Marshall and Nonaka, 2006). Historically most of knowledge about cilia has come from
studies of flagella in unicellular organisms, such as the algal species Chlamydomonas
reinhardtii. Chlamydomonas is of particular interest due to its ability to excise and
regenerate its anterior pair of flagella (aka cilia). Even though there is a large amount of
biochemical and pharmacological data pertaining to the response pathway leading to
flagellar excision, the identification of the exact players in the pathway is yet to be
determined. To further investigate the flagellar excision response and the genes and
gene products associated with it the Keller lab did a microarray looking at the change in
gene expression over time after flagellar excision (Chamberlain et. al., 2008). This
study focused on genes whose expression changed 2-fold or more after excision. One
of the genes identified in the study was a seven-pass transmembrane protein of
unknown function, Cr7TM. Extensive literature searching revealed that there has been
no functional data published about Cr7TM or any of its homologues. A comprehensive
bioinformatic search also revealed that Cr7TM and its protein homologues do not show
significant homology to any other class of proteins, making them unique in composition.
Furthermore, the use of multiple sequence alignments showed that, while Cr7TM
homologues are found in all animals, they remain highly conserved throughout
evolutionary time, implying a critical role in eukaryotic cells. To elucidate the function of
Cr7TM we have created genetic knock down lines in Chlamydomonas using an
inducible, plasmid-based system. Preliminary phenotypes of the knockdown lines
include diminished cell size and accelerated division rates. These results imply a role in
cell cycle regulation. A critical cellular role like cell cycle regulation is consistent with
Cr7TM’s highly regulated evolution. Further characterization of Cr7TM and its
interaction partners may reveal novel signaling pathways. Such a discovery could
prove to be highly beneficial in the medical and research science communities.
References
Chamberlain KL, Miller SH, Keller LR. Gene expression profiling of flagellar disassembly in
Chlamydomonas reinhardtii. Genetics 2008;179(1):7-19.
Marshall WF, Nonaka S. Cilia: Tuning in to the cell's antenna. Current Biology 2006;16(15):R604-R614.
Discovery of Technology: Tools for Research Management and
Interdisciplinary Collaboration
Rebecca Miller and Allison Scripa
University Libraries
Virginia Polytechnic Institute and State University
Phone: 540-231-9669 Fax: 540-231-9263
Email: [email protected] and [email protected]
ABSTRACT:
Meaningful scientific exchange in the 21st century relies heavily on the ability of the
researchers involved to successfully collaborate and communicate with each other. In an
environment of information overload and fiscal restraint, researchers must utilize available tools
in order to organize and manage their own research while collaborating effectively with peers.
Many Web 2.0 tools foster this sort of information management and distribution, and recent
literature is full of examples promoting appropriate and innovative uses of these tools.
The June 2009 NIH-sponsored conference Future of Telehealth: Essential Tools and
Technologies for Clinical Research and Care focused on exploring ways to "leverage evolving
information and communication technologies to advance the field," ultimately highlighting the
ways that various communication technologies assist in broadening participation in research
and improving outcomes (National Center for Research Resources, 2010, p. 2). Similarly, in a
directly relevant example of use of Web 2.0 technologies, librarians at the Research Medical
Library at the University of Texas MD Anderson Medical Center describe the power of
purposefully implementing appropriate technologies to support clinicians and ensure that they
stay up to date with current literature and research (Damani and Fulton, 2010). In this article,
the librarians of the Research Medical Library assert that emerging technologies add a distinct
value to clinical practice and dissemination of research, and that information professionals are
poised to offer guidance and instruction in this particular area (Damani and Fulton, 2010).
Librarians at large research universities like Virginia Tech, Wake Forest, and other ACC
universities are prepared to offer guidance in selecting and using appropriate Web 2.0
technologies. During this oral session, presenters will demonstrate a variety of Web 2.0 tools
that can assist researchers in managing information and collaborating effectively. These tools
will include Endnote (and EndNote Web), Zotero, Evernote, XMind, and RSS. Ultimately, the
presenters aspire to introduce attendees to free or easily accessible tools that support scientific
exchange between ACC graduate students, undergraduate students, and faculty.
References:
Damani, S., & Fulton, S. (2010). Collaborating and Delivering Literature Search Results to
Clinical Teams Using Web 2.0 Tools. Medical Reference Services Quarterly, 29(3), 207-217.
doi:10.1080/02763869.2010.494476.
National Center for Research Resources, National Institutes of Health. NIH conference on the
future of telehealth: essential tools and technologies for clinical research and care: final
workshop report. (2010). [Web]. Retrieved from
http://www.ncrr.nih.gov/publications/clinical/Future_of_Telehealth_Final_Report_June_2526_2009.pdf.
METALLIC FLAVOR PERCEPTION AND THE EFFECTS OF CANCER
THERAPIES
Susan Mirlohi1, M.S
Andrea M. Dietrich1, PhD
Susan E. Duncan1, PhD
Glenn J. Lesser2, MD
Affiliation
Department of Civil and Environmental Engineering
Department of Food Science and Technology
Virginia Tech1
Wake Forest University School of Medicine2
Phone: (540) 231-4595 Fax: (540) 231-7916
Email: [email protected]
September 10, 2010
ABSTRACT
Consumer complaints of metallic flavor in foods and beverages are usually caused by dissolved
iron or copper. In drinking water, these metals derive from metal distribution materials or groundwater.
Cancer patients commonly report metallic and related off-flavors that have detrimental impacts on their
quality of life and nutritional intake1. This study investigated effects of cancer therapy on iron-induced oral
lipid oxidation. Lipid oxidation in the oral cavity (OLO), a measure of oxidative stress, has been used as
an indicator for metallic flavor production associated with ingestion of iron containing drinking water2.
Delta TBARS/Total Protein (uM/g)
Twenty-two brain cancer patients who were diagnosed with primary malignant glioma were
monitored over a 30-week period; they underwent a 6-week combined chemo/radiation therapy, followed
by administration of the chemotherapy drug Temodar. Their salivary OLO and sensory responses were
determined prior to the cancer treatment and at several time points during their treatment. The study
indicated that drinking ferrous containing water at 10-12 mg/L typically caused an increase, and
occasional decrease, in OLO as measured by thiobarbituric reactive substances (TBARS). The OLO
responses were grouped by “low” (< 5 µM TBARS/g Protein) and “high” responders (> 5 µM TBARS/g
protein). In “high-responding” cancer patients, OLO increased dramatically at 10- and 18- weeks time
points (9.2 - 41.0 µM TBARS/g protein); the total salivary protein levels in this group showed a declining
trend over the course of their treatment. Patient-reported taste alterations showed fluctuations over the
course of the cancer treatment. In a corresponding group of healthy subjects, iron-induced oral lipid
oxidation was considerably lower than those of cancer patients (1.1 ± 1.01 µM/g protein), and the total
salivary protein levels showed little change over time. Knowledge of the flavor impacts of metals in
drinking water can aid consumers in making choices that maintain their quality of life. Study of salivary
constituents and oxidative reactions can provide clues and insights in resolving metallic taste disorders in
cancer patients.
16.95
16.91
2.60
2.40
2.20
2.00
1.80
1.60
1.40
1.20
1.00
0.80
0.60
0.40
0.20
0.00
Delta TBARS‐Healthy
Subjects
Delta TBARS‐Cancer
Patients, HR
Delat TBARS‐Cancer
Patients, LR
0
3
6
10
18
30
Time (weeks)
Figure 1: The bars graphs represent change (delta) in oral lipid oxidation (OLO) as measured by thiobarbituric acid reactive
substances (TBARS) for the three groups: healthy subjects, “high-responding” (HR) cancer patients, and “low-responding” (LR)
cancer patients. Delta TBARS is the difference between OLO response resulting from drinking the control (purified water) water
sample and the metallic (ferrous spiked) water sample.
References:
1. Hong, JH, P. Ömür-Özbek, B.T. Stanek, A.M., Dietrich, S.E. Duncan, YW Lee, G. Lesser; Taste
and smell abnormalities in cancer patients, Journal of Supportive Oncology, 7:2:58-65, 2009.
2.
Ömür-Özbek, P. and A.M. Dietrich. Retronasal perception and flavor thresholds of iron and
copper in drinking water; accepted to Journal of Water and Health, 2010.
USE OF ACELLULAR PANCREATIC MATRIX TO SUPPORT BETA CELL
DIFFERENTIATION
1
1
1,2
3
Sayed-Hadi Mirmalek-Sani , David L. Mack , Giuseppe Orlando , Yu Zhou & Shay Soker
1. Wake Forest Institute for Regenerative Medicine, Winston-Salem, NC 27157, U.S.A.
2. The University of Oxford, Nuffield Dept. of Surgery, Oxford, OX3 9DU, U.K.
3. Plureon Corporation, Winston-Salem, NC 27157, U.S.A.
Tel: (336) 713-7295, fax: (336) 713-7290, email: [email protected]
1
Introduction: Diabetes is an increasing healthcare burden, affecting more than ten percent of the U.S.
population and with significant financial cost. Islet transplantation offers not only blood glucose control, but also
amelioration of the sequela of diabetes. Recent literature has shown that pancreatic islets seeded onto acellular
1
pancreatic matrix showed prolonged viability and functionality . The goal of this study was to determine if
decellularized porcine pancreas could be used to support stem cell differentiation towards pancreatic beta cells.
Materials and Methods: Porcine pancreata were obtained following euthanasia then cannulated.
Following perfusion of 1% heparinized phosphate buffered solution (PBS) to remove residual blood, organs were
decellularized by the perfusion of 1% TRITON X-100 and 0.1% ammonium hydroxide in PBS for 24hrs, followed
by 5d rinsing in PBS. Acellular tissue was frozen prior to the creation of 6mm scaffolds via a biopsy punch (Fig.
1). Cell seeding experiments employed amniotic fluid-derived stem (AFS) cells, endothelial cells or bone marrow
stromal cells, or a co-culture of all three seeded onto scaffolds. For beta cell differentiation, AFS cells were
infected with adenoviral vectors encoding for Pdx1, NGN3 or MafA, or a combination of all three factors.
Pancreatic gene expression was assayed via quantitative PCR.
Results: Cannulation of both pancreatic duct and superior mesenteric vein resulted in complete
decellularization of the porcine pancreas. Neither scaffold-conditioned media nor culture in the direct presence of
scaffolds had any deleterious effects on cell growth (Fig. 1). Seeding of AFS, bone marrow stromal cell and
endothelial cell co-cultures enhanced seeding number and expansion on acellular scaffolds over 7 days in
comparison to individual cell types alone (Fig. 2). Infection with all three adenoviral factors Pdx1, NGN3 and MafA
resulted in robust increase of INSULIN and NEUROD1 gene expression for both monolayer cultures and scaffoldseeded cultures, compared to no infection controls in induction media alone (Fig. 3).
10000
5000
0
Single
Co-culture Single
Day 3
Fig. 1: (A) Native porcine pancreas (B)
Decellularized
pancreas
following
detergent perfusion (C) 6mm acellular
pancreas scaffolds (D) Day 7 scaffolds
with AFS cells (H&E, scale = 200µm).
Co-culture
Day 7
Fig. 2: Seeding of co-cultures to
acellular pancreatic scaffolds
enhanced
cell
expansion
compared with single cell types
alone (n=3, mean±SD).
500
0
Day 3
Scaffolds
15000
1000
Monolayer
20000
1500
Scaffolds
Cell number
25000
2000
Monolayer
30000
2500
No infection control
Relative INSULIN Gene Expression
Normalized to BETA-ACTIN
35000
Day 7
Fig. 3: INSULIN gene expression of
adenovirally-infected AFS cells
matched or exceeded monolayer
cultures (no infection control set as
1, n=2, mean±SD).
Discussion and Conclusions: This study demonstrated the production of an acellular pancreatic matrix
from porcine pancreata that can support AFS cell growth. The acellular pancreas matrix may be used for
bioengineering of pancreatic tissue, using genetically modified AFS cells, in order to achieve glucose-responsive,
insulin-secreting beta cells in vivo.
Acknowledgments and Disclosures: The authors wish to acknowledge the support of the JDRF and
NIBIB. The authors have nothing to disclose.
References
1. De Carlo E et al. Pancreatic acellular matrix supports islet survival and function in a synthetic tubular device: in
vitro and in vivo studies. Int J Mol Med. 2010 Feb;25(2):195-202.
STRUCTURE-ACTIVITY RELATIONSHIP STUDIES OF SPHINGOSINE KINASE
INHIBITORS
Mithun R. Rajea, Yugesh Kharelb, Kevin Lynchb and Webster L. Santosa,*
a
Department of Chemistry, Virginia Tech, Blacksburg, VA 24061
b
Department of Pharmacology, University of Virginia, Charlottesville, VA 22908
Phone:540-231-5742 Fax: 540-231-3255
Email: [email protected]
ABSTRACT:
Sphingosine kinase (SphK), an enzyme that rapidly phosphorylates sphingosine (Sph) to form
sphingosine-1-phosphate (S1P), is overexpressed in a variety of tumor types. Sph and its
precursor, ceramide promote apoptosis while S1P promotes survival and cell proliferation. The
dynamic balance between S1P and Sph/ceramide is regulated by SphK which makes it an
attractive target for anticancer drugs. However, the lack of selective inhibitors of SphK and the
scarcity of isoenzyme-selective inhibitors leaves a lot to be done in this research area. We have
synthesized a library of SphK inhibitors and discovered subtype selective inhibitors with Ki of 9
µM. We are currently investigating the structure-activity relationship studies of these inhibitors
by exploring different regions of the pharmacophore.
STRUCTURAL BASIS OF LIGAND RECOGNITION BY THE TOLLIP C2 AND CUE DOMAINS
Sharmistha Mitra§, Gayatri Ankem§, Iriscilla Ayala§, Camille Moreno§, Hugo Azurmendi*, Carla V.
Finkielstein⊥, Liwu Li and Daniel G.S. Capelluto§
§
Protein Signaling Domains Laboratory, ⊥Integrated Cellular Responses Laboratory, and Laboratory of
Innate Immunity and Inflammation, Department of Biological Sciences, and *Department of Chemistry,
Virginia Polytechnic Institute and State University. E-mail: [email protected]; Phone: (540) 231-8386;
Fax: (540) 231-3414.
Toll-like receptors (TLRs) provide a mechanism of host defense responses by activating the
innate and adaptative immune responses (1). Although the mechanism that triggers TLR signaling
activation is unknown, subsequent events result in the recruitment of one or more adaptor proteins, a
process mediated by the cytosolic tail of TLRs. Downstream events promote the activation of kinases
including the interleukin-1 receptor-associated kinases (IRAKs) 1, 2, M, and 4 that act upon their
transcription factor targets to influence the expression of genes involved in the innate immune
response. The Toll-interacting protein (Tollip) controls IRAK function in the TLR signaling pathway (2).
Tollip is modular in nature with an N-terminal Tom1-binding domain (TBD), a central conserved 2 (C2)
domain, and a C-terminal coupling of ubiquitin to endoplasmic reticulum degradation (CUE) domain.
We have biophysically and structurally investigated the molecular interactions of the Tollip C2 domain.
We found that the Tollip C2 domain preferentially interacts with phosphoinositides including
phosphatidylinositol 3-phosphate (PI3P) and phosphatidylinositol 4,5-bisphosphate (PI(4,5)P2) in a
calcium-independent manner. Nuclear magnetic resonance (NMR) and lipid-protein overlay analyses
suggest that PI3P and PI(4,5)P2 share the same binding site in the protein and that phosphorylation
must be present in the head group of the lipid for recognition. Kinetic analysis reveals that the Tollip C2
domain reversibly binds PI3P and PI(4,5)P2 with affinities in the low micromolar range. Mutational
analysis identifies key phosphoinositide-binding basic residues in the Tollip C2 domain located in a
flexible region nearby the beta-groove. We have also structurally characterized the Tollip CUE domain.
We have recently reported the backbone resonance assignments, secondary structure, and oligomeric
state of the Tollip CUE domain (3). The CUE domain binds ubiquitin (4), although the biological
consequences of the association as well the molecular basis of the interaction are unknown. Using twodimensional NMR spectroscopy, we have identified the Tollip CUE domain residues that recognize
ubiquitin as well as the ubiquitin residues that bind to the Tollip CUE domain. Structural and kinetical
analyses suggest that a dimeric Tollip CUE domain forms a complex with ubiquitin in conserved binding
pockets with nanomolar affinity. Overall, our findings will provide the basis to understand how Tollip is
intracellularly partitioned in a ligand-dependent manner and how these molecular interactions modulate
TLR signaling.
1.
2.
3.
4.
Kawai, T., and Akira, S. (2010) The role of pattern-recognition receptors in innate immunity:
update on Toll-like receptors, Nat Immunol 11, 373-384.
Burns, K., Clatworthy, J., Martin, L., Martinon, F., Plumpton, C., Maschera, B., Lewis, A., Ray,
K., Tschopp, J., and Volpe, F. (2000) Tollip, a new component of the IL-1RI pathway, links IRAK
to the IL-1 receptor, Nat Cell Biol 2, 346-351.
Azurmendi, H., Mitra, S., Ayala, I., Li, L., Finkielstein, C. V., and Capelluto, D. G. S. (2010) 1H,
15N, and 13C resonance assignments and secondary structure of the Tollip CUE domain., Mol
Cells 30 (6), in press.
Kang, R. S., Daniels, C. M., Francis, S. A., Shih, S. C., Salerno, W. J., Hicke, L., and
Radhakrishnan, I. (2003) Solution structure of a CUE-ubiquitin complex reveals a conserved
mode of ubiquitin binding, Cell 113, 621-630.
How Solvent Modulates Hydroxyl Radical Reactivity in Hydrogen Atom
Abstractions.
Susan Mitroka, Stephanie Zimmeck, Diego Troya, James Tanko
Abstract:
The hydroxyl radical (OH) is an extremely reactive oxidant, important in
chemistry, biology, medicine, materials, and the environment. The rate at which OH
reacts with organic compounds in solution is generally thought to be near diffusion
controlled. In this research, the notion that water enhances the rate of reaction of the
hydroxyl radical with organic substrates in solution was tested—by conducting the
reaction in a dipolar, aprotic solvent (acetonitrile). Due to the popularity of pulse
radiolysis as a means of generating OH, relatively little is known about the reactivity of
OH in solvents other than water. We have determined the first absolute rate constants
for reaction of OH with various organic substrates measured in acetonitrile, and
compare them to the same reactions in an aqueous solution.1
1.
Mitroka, S. Z., S.; Troya, D.; Tanko, J. M., , How Solvent Modulates Hydroxyl
Radical Reactivity in Hydrogen Atom Abstractions. . Journal of the American Chemical
Society 2010, 132, (9), 2907-2913.
REACTIONS OF HNO WITH BIOLOGICAL TARGETS: THIOL MODIFICATION IN CYSTEINE
CONTAINING PROTEINS
Susan Mitroka, Mai Shoman, Bruce King
Department of Chemistry
Wake Forest University
Phone: 336-758-5096
Email: [email protected]
ABSTRACT:
As nitric oxide is an important biological mediator, the notion that other nitrogen
oxides may have possible biological importance has received much attention. The
structurally similar one-electron reduced and protonated compound, nitroxyl (HNO) has
been the focus of much interest over the past 20 years. Having a chemistry distinct from
nitric oxide, HNO shows promise of therapeutic utilityfor several diseases including
alcoholism, congestive heart failure and cancer. While an established, biologically
active compound, the chemistry driving HNO’s biology has yet to be sufficiently studied.
The post-translational modification of thiols to form sulfinamides or disulfide bonds is
thought to be the main mode of action of HNO.. In this work, we examine the reactions
of HNO with the free thiol groups of cysteine containing proteins, focusing on thiol
modification as well as the inhibition of activity. Initial studies have focused on the
treatment of glyceraldehyde 3-phosphate dehydrogenase (GAPDH) and human serum
albumin (HSA). Treatment of these pure proteins clearly results in structural and activity
changes that are currently being evaluated and will be discussed.
FUNCTIONAL ANALYSIS OF PLANT GLUTAMATE RECEPTOR HOMOLOGS
(GLRs)
Shelley Moore*, Altaf Ahmad, and Sakiko Okumoto
Virginia Tech
Department of Plant Pathology, Physiology and Weed Science
Phone: 304-952-5792 Fax: 540-231-7477
Email: [email protected]
Abstract:
Amino acids have long been suggested in N signaling, but the mechanisms for
amino acid perception are far from being understood. Searches for nitrogen sensors
resulted in the identification of 20 genes in Arabidopsis with sequence and structural
homology to mammalian ionotropic glutamate receptors (iGluRs), known as glutamatelike receptors (GLRs)(1). Mammalian ionotropic GluRs are known to sense extracellular
glutamate and regulate membrane potential of synapses through conduction of
cations(2). While evidence is emerging that plant GLRs also function as non-selective
cation channels, the function of plant glutamate receptors in vivo is not well understood,
particularly with regard to ligand specificity(3).
Plant GLRs are homologous to NMDA receptors that require the formation of
tetramers or pentamers for channel functionality(4). This research aims to investigate
whether these proteins also require the formation of oligomers to form a functional
channel. Interaction between different GLR subunits have been examined using a
modified yeast-2-hybrid technique known as mating-based split ubiquitin system.
Additionally, Förster Resonance Energy Transfer (FRET) will be utilized to examine
intra- and inter- molecular interactions of GLR genes. Preliminary results suggest that
AtGLR3.3 and 3.4, mutations in which were shown to attenuate glutamate responses
(5), may interact with each other. These constructs, along with other interacting GLRs,
will be used to analyze conformational changes induced by potential ligands of these
proteins.
References:
1) Lam, H. M., J. Chiu, et al. (1998). Nature 396(6707): 125-126.
2) Dingledine, R., K. Borges, et al. (1999). Pharmacol Rev 51(1): 7-61.
3) Davenport, R. (2002). Ann Bot 90(5): 549-557.
4) Rosenmund, C., Y. Stern-Bach, et al. (1998). Science 280(5369): 1596-1599.
5) Stephens, N. R., Z. Qi, et al. (2008). Plant Physiol 146(2): 529-538.
Chemical Modification of Multi-Wall Carbon Nanotubes for use in
Biomedical Applications
Erin B. Murphy*, David Inglefield, Sean M. Ramirez, and Timothy E. Long
Macromolecules and Interfaces Institute, Department of Chemistry, Virginia Tech,
Blacksburg, VA 24061
*[email protected]
Carbon nanotubes (CNTs) have attracted significant research interest due to
their unique properties, including high tensile strength and electrical conductivity. They
show promise as an excellent substitute for traditionally inorganic-based components for
any number of engineering and biomedical applications; for instance, CNTs have been
investigated for use as biosensors, scaffolds for tissue engineering, drug delivery
carriers, and bioimaging agents. While the carbon lattice structure of CNTs offers many
desirable properties, the high surface area and strong van der Waals interactions leads
to strong aggregation, thus limiting their effectiveness in aqueous media and composite
materials. Herein we discuss the chemical modification of multi-wall carbon nanotubes
(MWCNT) with a range of supramolecular functionality to aid in the dispersion of the
tubes in aqueous media and in polymer matrices containing complementary functional
groups. The addition of moities capable of hydrogen bonding (urethane, urea, amide),
ionic interaction (cationic, anionic, zwitterionic), as well as metal-ligand coordination
(gold and ferrite nanoparticles) allow for these functionalized MWCNT to be stabilized in
aqueous solutions and form complex interactions within a polymer matrix. The synthesis
of these modified MWCNT will be discussed, as well as their characterization through Xray photoelectric spectroscopy (XPS), thermogravimetric analysis (TGA), field emission
scanning electron microscopy (FE-SEM), transmission electron microscopy (TEM), and
Raman spectroscopy. In addition, the fabrication of MWCNT/polyurethane composites
will be discussed, as well as the mechanical and thermomechanical properties observed
through tensile testing and dynamic mechanical analysis (DMA).
1. Liang, F.; Chen, B. Current Medicinal Chemistry 2010, 17, 10-24.
2. Li, X.; Fan, Y.; Watari, F. Biomedical Materials 2010, 5, 022001.
3. Wu, H-C.; Change, X.; Liu, L.; Zhao, F.; Zhao, Y. J. Mater. Chem. 2010, 20,
1036-1052.
MECHANISM OF THE MONOAMINE OXIDASE-CATALYZED OXIDATION OF
TERTIARY-ALLYLAMINS USING MODEL CHEMISTRY
Akiko Nakamura, Neal Castagnoli Jr., and James M. Tanko
Affiliation
Department of Chemistry
Virginia Polytechnic Institute and State University
Phone: 540-231-5391 Fax: 540-231-3255
Email: [email protected]
ABSTRACT:
The neurogenerative properties of the parkinsonian-inducing agent 1-methyl-4-phenyl1,2,3,6-tetrahydropyridine (MPTP) are dependent on its brain monoamine oxidase B (MAO-B)
catalyzed bioactivation to the corresponding pyridiniumyl metabolite. Pretreatment with the
mechanism-based MAO-B inactivator (/R/)-deprenyl protects experimental animals from MPTP's
neurotoxicity. Furthermore, MAO-B inhibitors such as (/R/)-deprenyl and (/R/)-rasagiline have
beneficial effects in patients with idiopathic Parkinsons's disease. Studies on the mechanism of
MAO catalysis have not led so far to an unambiguous description of its turnover pathway.
We present the results of our model studies on the ring-alpha-carbon oxidation of a
pyrrolyl analog (1) of MPTP by the 5-ethyl-3-methyllumiflavinium species (2), an FAD analog
that has been shown to oxidize primary and secondary, but not tertiary, amines. The present
studies have documented that 1 is converted to the corresponding pyridiniumyl product in the
presence of 2.
N
N
N
N
N
N
1
ClO4
O
O
2
References:
1. Birkmayer, W.; Knoll, J.; Riederer, P.; Youdim, M. B.; Hars, V.; Marton, J., Increased life
expectancy resulting from addition of L-deprenyl to Madopar treatment in Parkinson's disease: a
longterm study. J Neural Transm 1985, 64 (2), 113-27.
2. Kim, J. M.; Bogdan, M. A.; Mariano, P. S., Mechanistic analysis of the 3-methyllumiflavinpromoted oxidative deamination of benzylamine. A potential model for monoamine oxidase
catalysis. Journal of the American Chemical Society 1993, 115 (23), 10591-5
3. Inoue, H.; Castagnoli, K.; Van Der Schyf, C.; Mabic, S.; Igarashi, K.; Castagnoli, N., Jr.,
Species-dependent differences in monoamine oxidase A and B-catalyzed oxidation of various
C4 substituted 1-methyl-4-phenyl-1,2,3, 6-tetrahydropyridinyl derivatives. The Journal of
pharmacology and experimental therapeutics 1999, 291 (2), 856-64.
PROBIOTIC COMPARED TO DRUG THERAPY FOR TREATMENT OF
ACUTE STRESS COLITIS IN AN ADULT CANINE SHELTER
POPULATION
J.Oliver, K.Saker, C.B.Lanier, B.Stevens, K.Ferris
College of Veterinary Medicine
Department of Clinical Nutrition
North Carolina State University
Phone: 919-513-6871 Fax:919-513-6465
Email: [email protected]
Diarrhea resulting from stress colitis is a common occurrence among shelter-housed canines.
This can be associated with or compounded by parasitism, infectious disease, and the abrupt change in
diet that all shelter dogs experience on admission. The clinical and social ramifications of diarrhea
impact individual and group animal health, facility hygiene, and the adoptability of the symptomatic dogs.
Standard treatment modalities, such as anthelmentics and/or antimicrobial drugs impart a physiological
stress on the gastrointestinal (GI) tract, which can compromise full recovery. Probiotics are live
microorganisms, which when administered in adequate amounts have reported mechanisms of action to
reduce bacteria-associated diarrhea, promote healthy gut microflora, and enhance resistance to
penetration of pathogens. Despite the fact that probiotic products are becoming increasingly used in
small animal medicine, there are few published studies regarding their clinical efficacy and none to date
that look at the shelter population. Probiotics may be a viable alternative for the treatment of diarrhea in
shelter dogs. The goal of our study was to utilize a fecal scoring system, select blood parameters and
fecal cultures to determine the benefit of probiotic as compared to antibiotic treatment for acute diarrhea
caused by stress colitis and associated factors in shelter dogs.
Dogs presenting to the Wake County Animal Center, Raleigh, NC, with a severe diarrhea as
determined by fecal score, were identified for this study. A physical examination, body weight, body
condition score (BCS), complete blood count, chemistry profile, urinalysis, and fecal flotation were
performed on all potential study dogs. Blood samples were also obtained for serum immunoglobulin
analysis. Dogs were randomly assigned to probiotic (P, Prostora MAX®), or antibiotic (M, Metronidazole)
treatment groups, both of which were administered according to label dosing directions. Animals in the M
group with a fecal score of 4 or greater at the end of treatment, (considered unresponsive), were
subsequently placed on P treatment (M-P group). All dogs were fed an adult maintenance or growth lifestage diet once daily, based on calculated daily energy requirements. Fecal scores and food intake were
recorded daily throughout the treatment period.
Complete data was collected on 50 shelter dogs (25 P, 25 M), with 11 M-P dogs. Body weight
and condition score did not change significantly during the treatment period for any study dogs. Fecal
scores improved in the P, M and M-P groups by a factor of approximately 2-fold, with ending fecal scores
of 3.52, 3.95, and 3.50, respectively. Parasite-infected (Ancylostoma caninum, Toxocara canis, or
Trichuris vulpis) dogs (n=8,P; n=9, M) exhibited an approximate 2-fold improvement in fecal scores posttreatment as well. Based on fecal score data alone, this probiotic is an equally effective treatment to the
traditional antibiotic regime for the treatment of acute diarrhea in shelter dogs. Antibiotic-treated dogs with
limited improvement appeared to benefit significantly from subsequent probiotic treatment. Determination
of the GI microbe population before and after treatment and evaluation of select blood measures will
further elucidate the clinical value of probiotics for shelter-housed dogs.
References:
1. Strompfova, V,Laukova, A,Ouwehand, AC. Selection of enterococci for potential canine probiotic
additives. Veterinary Microbiology, V. 100, 2004
2. Kelley, R.L,Minikhiem, D,Kiely, B. Clinical benefits of probiotic canine-derived Bifidobocterium
animalis strain AHC7 in dogs with acute idiopathic diarrhea. Alternative Medicine Review, V. 15,
2010
3.
O'Mahony, D; Murphy, K Barry; MacSharry, J; Boileau, T; Sunvold, G; Reinhart,G;
Kiely, B; Shanahan, F; O'Mahony, L. Portrait of a canine probiotic Bifidobacterium-from gut to gut. Veterinary microbiology Vol: 139 Issue: 1-2 ISSN: 1873-2542 Date:
10/2009 Start Page: 106.
4.
Herstad, H.K; Nesheim, B.B; L'Abee-lund, T; Larsen, S; Skancke, E.
Effects of a probiotic intervention in acute canine gastroenteritis - a controlled
clinical trial. Journal of Small Animal Practice Vol: 51 Issue: 1 ISSN: 0022-4510
Date: 01/2010 Start Page: 34
5.
Manninen, Titta J K; Rinkinen, Minna L; Beasley, Shea S; Saris, Per E J. Alteration
of the canine small-intestinal lactic acid bacterium microbiota by feeding of potential
probiotics. Applied and environmental microbiology Vol: 72 Issue: 10 ISSN:
0099-2240 Date: 10/2006 Start Page: 6539
6.
Ogue-Bon, Eva; Khoo, Christina; McCartney, Anne L; Gibson, Glenn R; Rastall,
Robert A. In vitro effects of synbiotic fermentation on the canine faecal microbiota.
FEMS Microbiology Ecology Vol: 73 Issue: 3 ISSN: 0168-6496 Date: 09/2010 Start
Page: 587
7.
Rastall RA. Bacteria in the gut: friends and foes and how to alter the balance
Journal of Nutrition [NLM - MEDLINE] Vol: 134 Issue: 8 ISSN: 0022-3166 Date:
08/2004 Start Page: 2022S
8.
Sokolow, Susanne H; Rand, Courtney; Marks, Stanley L;Drazenovich, Niki L;
Kather, Elizabeth J; Foley, Janet E. Epidemiologic evaluation of diarrhea in dogs in
an animal shelter.
“RADICALLY” GREEN APPROACHES TO HYDROCARBON AND
ETHER FUNCTIONALIZATION VIA ALLYL TRANSFER REACTION
J M Tanko and Shraddha Patil
Department of Chemistry
Virginia Tech, Blacksburg, VA, 24061
Phone: 231-3254 Email: [email protected], [email protected]
ABSTRACT:
Hydrocarbon functionalization via an allyl transfer reaction (Scheme 1, X = Br) using
different allyl bromide substrates, has been studied in our group. Replacement of Br• by
phthalimido-N-oxyl (PINO•) was successful, and has helped make this chemistry
environmentally benign. Reactions of allyl-phthalimido-N-oxyl (PINO) compounds for
hydrocarbon functionalization have shown excellent results using high temperature initiators (ditert-butylperoxide) and reactions are under investigation with respect to low temperature
initiators (triethylborane, di-tert-bytylhyponitrite). Similarly, functionalization of ethers
(tetrahydrofuran, 2-methyltetrahydrofuran, dioxane, diethyl ether etc.) has shown interesting
results and is being studied further.
Scheme 1
Low temperature initiators and/or Lewis acid catalysts are being examined to allow these
reactions to be performed at lower temperatures. This will allow the examination of the
stereoselectivity of the process. We are currently exploring better initiators for this reaction as
well as improved methods for the synthesis of the allyl-PINO substrates. We plan to study
kinetics of radical addition step involved in the allyl transfer reactions and compare the rates of
catalyzed and uncatalyzed reactions.
References:
1. Tanko, J. M.; Sadeghipour, M., Angew. Chemie .1999, 111 , 219-222
2. Struss, J. A.; Sadeghipour, M.; Tanko, J. M., Tetrahedron Let. 2009, 50 , 2119-2120
3. Koshino, N.; Cai, Y.; Espenson, J. H., J Phys Chem .A 2003, 107 , 4262-4267
4. Russell, G. A.; Kochi, J. K., In free Radicals. Wiley & Sons, New York 1973, 1 (1A)
5. Sibi, M. P.; Ji, J. J Org Chem. 1997, 62, 3800-3801
HIGH THROUGHPUT ASSAY TO IDENTIFY INHIBITORS
AGAINST UDP-GALACTOPYRANOSE MUTASE FROM
EUKARYOTIC PATHOGENS
Jun Qi, and Pablo Sobrado
Department of Biochemistry, Virginia Tech, Blacksburg, VA 24061
Phone: (540)239-7445
Email: [email protected]
The flavoenzyme UDP-galactopyranose mutase (UGM) specifically catalyzes the
transformation of UDP-galactopyranose (UDP-Galp) to UDP-galactofuranose (UDP-Galf), which
is an important precursor in the biosynthesis of galactofuranose (Galf). Galf residues are
essential components in the cell wall of many pathogenic prokaryotes and lower eukaryotes such
as bacterium M. tuberculosis (Mt), fungus A. fumigatus (Af), and parasite T. cruzi (Tc), and play
vital roles in their growth. Importantly, Galf is not present in mammals, therefore inhibitors of UGM
that block the biosynthesis of Galf could lead to novel therapeutics. Up to date, only a few
inhibitors of prokaryotic UGM have been identified by fluorescence polarization (FP) assay,1 but
no inhibitors of eukaryotic UGM have been reported. Here we present the synthesis of three
fluorescently labeled UDP analogs that can be used to develop a high throughput fluorescence
polarization assay to identify specific eukaryotic UGM inhibitors. The preliminary FP assay
indicates that the fluorescein analog 2 with a longer alkyl linker shows better binding to
prokaryotic MtUGM (Kd = 0.1 μM) than shorter-linker fluorescein analog 1 (Kd > 20 μM) and
rhodamine analog 3 (Kd = 0.7 μM). A specific binding to eukaryotic AfUGM was obtained using
the rhodamine analog 3 with a Kd value of 2 μM, compared to analog 2 (Kd = 26 μM). UDP and
two known prokaryotic UGM inhibitors were tested in FP competition assays. All these
compounds bind to MtUGM, but only UDP binds to AfUGM specifically. Binding of these three
chromophores to eukaryotic TcUGM is not very specific. Our results indicate that analog 3
exhibits the potential to be a good candidate as a binding ligand for developing a high throughput
assay to identify specific inhibitors of AfUGM. The experimental details will be presented.
O
O
NH
H
N
HO
H
N
S
O
O
n O P O P O
OH OH
N
O
N
O
O
O
n=1
n=5
analog 3
N
Kd = 2 uM
Kd = 26 uM
500
400
400
mP Units
mP Units
O
O
OH OH
analog 1
analog 2
500
300
200
300
200
100
100
0
0.001
N
COO
OH OH
COOH
O
NH
O
O
O P O P O
OH OH
HN
O
0.01
0.1
1
10
100
AfUGM Concentration (uM)
1000
0
0.001
0.01
0.1
1
10
100
1000
AfUGM Concentration (uM)
Reference
1. Soltero-Higgin, M.; Carlson, E. E.; Phillips, J. H.; Kiessling, L. L. Identification of inhibitors for
UDP-galactopyranose mutase, J. Am. Chem. Soc. 2004, 126, 10532-10533.
IDENTIFICATION OF THE QUORUM SENSING REGULON IN THE
CORN PATHOGEN PANTOEA STEWARTII
Revathy Ramachandran, Daniel J. Schu, Ann M. Stevens
Department of Biological Sciences,
Virginia Tech
Phone: (540) 231-2342, fax: (540) 231-4043
Email: [email protected]
Pantoea stewartii is a Gram-negative bacterium which causes Stewart’s wilt disease in
maize. This results in substantial yield losses in susceptible hybrids and is a major concern for
seed trade. The disease is transmitted to the seedlings via an insect vector, the corn flee beetle.
The success of disease pathogenesis lies in the timing of specific bacterial virulence factors
through the different stages of the infection. The cell density-dependent regulatory system of
quorum sensing (QS) is responsible for regulating the progress of virulence by timing capsule
production, one of the key virulence factors in P. stewartii infections.
The expression of capsular genes is controlled through two key proteins, EsaR and EsaI.
The DNA binding regulator, EsaR represses transcription from promoters of genes that control
capsule production at low cell densities. This repression is relieved at high cell density when
EsaI synthesizes sufficient levels of a signaling molecule, an acylated homoserine lactone
(AHL) ligand, which inactivates EsaR. Interestingly, EsaR also functions as a transcriptional
activator at low cell densities, indicating that there may be two sets of genes under control of QS
that in turn modulate the timing of virulence.
In order to identify additional components of the EsaR regulon, two-dimensional gel
electrophoresis was performed on P. stewartii strains proficient and deficient in EsaR and AHL
production. Differentially expressed proteins were excised and identified by mass spectrometry.
Promoters of genes encoding these regulon proteins were analyzed for possible EsaR binding
sites via electrophoretic mobility shift assays. This would help us in distinguishing the direct from
indirect targets and thus generate a more inclusive picture of the quorum-sensing regulon.
COMBINED EXPERIMENTAL AND NUMERICAL STUDY OF A
BIOCOMPATIBLE SUPERPARAMAGNETIC HYDROPHOBIC
FERROFLUID DROP IN A UNIFORM MAGNETIC FIELD
Y. Renardy1, S. Afkhami2, A. J. Tyler3, T. G. St Pierre3, R. C. Woodward3, J. S. Riffle4
1
Department of Mathematics, 4Department of Chemistry, Virginia Tech
2
Department of Mathematical Sciences, New Jersey Institute of Technology
3
Department of Physics, University of Western Australia
Phone: 540-320-1573 Fax:540-231-5960
Email: [email protected]
ABSTRACT:
We focus on a hydrophobic ferrofluid for the potential treatment of retinal detachment. The
material consists of magnetite nano-particles coated with biocompatible polydimethylsiloxane oligomers.
It is synthesized by J. S. Riffle’s group without the addition of a carrier fluid, thus minimizing phase
separation in strong magnetic fields. The effect of applied magnetic fields on the ferrofluid drop
suspended in a fluid model for the vitreous humor is investigated with direct numerical simulations and
compared with experimental data of T. G. St Pierre’s group (U. Western Australia). Our numerical
algorithm for the time-dependent simulation of the magnetohydrodynamics of two immiscible nonconducting fluids is formulated as a semi-implicit volume-of-fluid scheme for fully deformable interfaces.
At low magnetic fields, asymptotic small deformation theory is a benchmark against which both
experimental and numerical results are checked. At any given magnetic field, we can deduce the value of
interfacial tension, which is not given a priori, from a comparison of an optimal fit of a numerically
simulated shape with the experimentally obtained drop shape. At high magnetic fields, experimental
measurements deviate from numerical results if the constant interfacial tension deduced at low fields is
implemented. The difference can be represented as a dependence of apparent interfacial tension on the
magnetic field. This idea is investigated computationally by varying the interfacial tension as a function of
the applied magnetic field, and by comparing the drop shapes with experimental data until a perfect
match is found. This estimation method provides a consistent correlation for the variation in interfacial
tension at high magnetic fields. With the advent of focused superconducting magnets which operate at
high fields, the study of the dependence of the physical properties of ferrofluidic droplets under operating
conditions is necessary in order to control and use them.
References: S. Afkhami, A. J. Tyler, Y. Renardy, M. Renardy, T. G. St. Pierre, R. C. Woodward, J. S.
Riffle. Deformation of a hydrophobic ferrofluid droplet suspended in a viscous medium under uniform
magnetic fields. J. Fluid Mech.in press,2010.
SEPARATION AND CHARACTERIZATION OF PROCYANIDINS IN
PEANUT SKINS BY LC-MSN
Sarnoski, P.1, Tanko, J.2, Johnson, J.3, and O’Keefe, S.1
Department of Food Science and Technology1
Virginia Polytechnic Institute and State University
Department of Chemistry2
Virginia Polytechnic Institute and State University
Department of Chemistry3
University of Florida
Procyanidins are a type of flavanol that are commonly found in many plant
species. Procyanidins can provide potential health benefits by acting as
antioxidants and vasodilators. In this instance procyanidins were extracted from
peanut skins, an agricultural waste product. Procyanidin composition of single
solvent and multistep extraction procedures of peanut skins were compared by
UV-vis absorbance.
The multistep extraction procedure yielded more
procyanidins on a per weight basis. Thus this extraction procedure was chosen
for subsequent fractionation. Fractionation was performed by size exclusion
(Toyopearl HW-40S) and normal phase (porus silica) high performance liquid
chromatography (HPLC).
For size exclusion chromatography (SEC) the
Toyopearl stationary phase was manually packed into a HPLC column.
Procyanidin separation on the normal phase column was better compared to
SEC, and thus was chosen for characterization by mass spectrometry (MS).
Samples were analyzed on triple quadrupole and ion trap LC-MS instruments.
Background noise was lower on the triple quadrupole instrument, but the ion trap
provided more clear structural information due to its ability to trap and fragment
ions better than the triple quadrupole. Peanut skin procyanidins were found to
separate in order of increasing molecular weight on the normal phase column.
Molecular ion and fragmentation pattern data were used to identify specific
procyanidins.
COMPARING THE EFFECTIVENESS AND ACCEPTABILITY OF THE JUMP INTO
FOODS AND FITNESS (JIFF) AND QUEST FOR HEALTH NUTRITION
INTERVENTIONS WITHIN AN AFRICAN-AMERICAN POPULATION
Jermaine J. Shaw, M.S.
Department of Food Science and Human Nutrition
Clemson University
Phone: (803) 316-8412 Email: [email protected]
ABSTRACT:
The severity of the obesity epidemic has increased the necessity for nutrition
education programs. These programs must be culturally and age appropriate in order
to be more effective in bringing about positive behavior changes in the target
populations.
This study examined the effectiveness and acceptability of the Jump into Foods
and Fitness (JIFF) and Quest for Health nutrition education programs within a preadolescent African-American population. Two classes from the Boys and Girls Club of
Sumter, S.C. were randomly assigned to one of the nutrition education programs. Class
one was assigned to the JIFF curriculum, and included 23 participants. Class two was
assigned to Quest for Health, and included 16 participants. Each class received three
lessons from its assigned curriculum. The three lessons discussed 1) My Pyramid, 2)
Importance of Physical Activity, and 3) Nutrition Facts Labels. Before and after the
three lessons, the participants completed the evaluation survey tools for their
curriculum. At the conclusion of the post-surveys, each class participated in its own
focus group session. SPSS statistical software was used to compare pre- and postsurvey results for both curricula, and the focus group responses were transcribed and
grouped by themes.
The results indicate that both programs led to positive changes in the physical
activity behavior, nutritional knowledge, and nutrition behavior of the participants. Preand post-survey responses showed increases in scores for all three variables for both
curricula. Neither program held a statistically significant advantage over the other
program. Focus group responses indicated that participants from both groups believed
their curriculum was age and culturally appropriate for their population. The games and
physical activities were found to be great enhancements in both programs. Nutrition
education programs such as JIFF and Quest for Health can be an integral component in
efforts to reduce the prevalence of obesity.
Quest for Health Survey Results
PreTest
Variable
Pre-Test
Mean
Nutrition
Behavior
40.69
(74.0%)
7.53
Physical
Activity
Behavior
32.25
(80.6%)
6.51
Nutritional
Knowledge
11.13
(69.6%)
1.71
STDEV
PostTest
Mean
44.38
(80.7%)
34.31
(85.8%)
12.69
(79.3%)
PostTest
STDEV
Test
Diff.
a
b
T or Z
Value
5.83
-3.69
-1.969
6.67
-2.06
-1.258
1.54
-1.56
-2.778
b
b
a
PValue
0.05*
0.208
0.014*
*
-denotes statistical significance
Ta-Paired T-Test
Z -Wilcoxon Signed Ranks Test
b
(Shaw 45)
References:
1. Shaw, Jermaine. “Comparing the effectiveness and acceptability of the Jump Into
Foods and Fitness (JIFF) and Quest for Health nutrition interventions within an
African-American population”. MS Thesis. Clemson University, 2010.
CHARACTERIZATION OF THE CELLULAR SIGNALING NETWORKS OF
PROSTATE CANCER THROUGH PHOSPHOPROTEOME ANALYSIS
Paul A. Stewart, Xu Wang, Alan G. Marshall, and Qing-Xiang A. Sang
Phone: 850-366-8668 Email: [email protected]
Affiliation: Department of Chemistry and Biochemistry, Florida State University,
95 Chieftain Way, Tallahassee, FL 32306
Abstract
Prostate cancer is responsible for 25% of all cancer diagnosis of men in
the United States (1). Understanding the molecules involved in signal
transduction pathways and networks in a cancerous cell and the changes that
occur as a result of these signals is vital for the identification of therapeutic
targets. We analyzed the phosphoproteome of two types of human prostate
cancer cell lines to gain a better understanding of the active signal transduction
pathways and their roles in the cancerous transformation of a prostate cell. Wang
et al. used a novel sequential CPP and TiO2 enrichment strategy with ultrahigh
mass accuracy and stringent filtering to identify high confidence phosphoproteins
in ARCaP (human androgen repressed cancer of the prostate). We present some
of this unpublished data for the first time. Chen et al. used in-gel IEF LC-MS/MS
to identify phosphoproteins in LNCaP (cancer of human prostate that has
metastasized to lymph nodes) (2). The phosphoproteins from ARCaP and
LNCaP were then analyzed by use of the NCI-Nature Pathway Database and
Reactome (3, 4). We identified approximately 700 phosphoproteins over 61
pathways in ARCaP. Chen’s analysis uncovered 296 phosphoproteins over 42
active pathways in LNCaP. We found 145 phosphoproteins (about 49% of the
LNCaP sample) in 22 pathways in common between the two samples. Out of the
pathways that were characterized, several can be directly related to tumorogenic
activity including p53 and mTOR pathways. (Work supported by grants from
DOD W81XWH-07-1-0225 and DAMD17-02-1-0238, the Elsa U. Pardee
Foundation, Florida State University, NSF DMR-06-54118, and the State of
Florida.)
1.
2.
3.
A. Jemal, R. Siegel, E. Ward, Y. Hao, J. Xu, T. Murray, and M.J. Thun,
Cancer statistics, 2008. CA Cancer J Clin. 58, 71-96 (2008).
L. Chen, F. Giorgianni, and S. Beranova-Giorgianni, Characterization of
the phosphoproteome in LNCaP prostate cancer cells by in-gel isoelectric
focusing and tandem mass spectrometry. J Proteome Res. 9, 174-178
(2010).
C.F. Schaefer, K. Anthony, S. Krupa, J. Buchoff, M. Day, T. Hannay, and
K.H. Buetow, PID: the Pathway Interaction Database. Nucleic Acids Res.
37, D674-679 (2009).
4.
G. Joshi-Tope, M. Gillespie, I. Vastrik, P. D'Eustachio, E. Schmidt, B. de
Bono, B. Jassal, G.R. Gopinath, G.R. Wu, L. Matthews, S. Lewis, E.
Birney, and L. Stein, Reactome: a knowledgebase of biological pathways.
Nucleic Acids Res. 33, D428-432 (2005).
SYNTHESIS AND CHARACTERIZATION OF POLY(PROPYLENE GLYCOL)-BASED
AMMONIUM IONENES
Mana Tamami and Timothy E. Long
Macromolecules and Interfaces Institute
Department of Chemistry – (0212)
Virginia Tech
Blacksburg, VA 24061
Phone: (540) 231-3378; Fax: (540) 231-8517
Email: [email protected], [email protected]
Ionenes are ion-containing polymers that have quaternized nitrogen atoms
throughout their main chain. The ionene is named based on the number of methylene
spacers, which correspond to the diamine (x) and dihalide (y) monomers, respectively
(i.e. x,y-ionene). Two general types of ionenes are: segmented and non-segmented,
which are either linear, crosslinked, or branched.1 Ionenes have potential biomedical
applications as antimicrobial agents, flocculants for waste water treatment, gene
delivery models and medical uses. Herein, we describe the synthesis and
characterization of series of segmented poly(propylene glycol) (PPG)-based ammonium
ionenes having various hard segment content (HS). The HS content is comprised of
N,N,N′,N′-tetramethyl-1,6-hexanediamine and 1,12-dibromododecane and the soft
segment content includes PPG. The 4K PPG-based ionenes having 33 to 74 wt% HS
were synthesized and differential scanning calorimetry (DSC) showed microphase
separation; the first Tg corresponded to the soft segment and the second Tg
corresponded to the hard segment which increased from 55 to 94 °C with the increase
in HS content. In addition atomic force microscopy was consistent with microphase
separation (Figure 1). The dynamic mechanical analysis (DMA) showed that the rubbery
plateau modulus increased from 36 wt% HS to 74 wt% HS accordingly. The 4K PPGbased ionene having 36 wt% HS showed a monomodal peak using DMF-based SEC
and the relative weight-average molecular weight was 21000 g/mol.
O
3
O
Br
O
n
O
3
N
CH2
6
Br
Br
N
x
10
N
Br
CH2 N
6
y
Figure 1. AFM of 4K PPG-based ionene having 74 wt% HS
References
1. Williams, K. A.; Dreyer, D. R.; Bielawski, C. W., MRS Bull. 2008, 33 (8), 759-765.
2. Williams, S. R.; Salas-de la Cruz, D.; Winey, K. I.; Long, T. E., Polymer 2010, 51 (6),
1252-1257.
3. Williams, S. R.; Borgerding, E. M.; Layman, J. M.; Wang, W.; Winey, K. I.; Long, T.
E., Macromolecules 2008, 41 (14), 5216-5222.
Acknowledgements
The authors would like to acknowledge the financial support of Kimberly Clark.
Engineering a selfish genetic element for the common good?
Zhijian Jake Tu, Department of Biochemistry, Virginia Tech, VA 24061
Maternal-effect dominant embryonic arrest (Medea) is a selfish genetic element
that can spread quickly in natural populations. Although the precise molecular
mechanism of Medea is unknown, a synthetic Medea has been constructed in
the model insect Drosophila melanogaster. I will describe our initial effort to
identify components needed in a synthetic Medea in mosquitoes. I will discuss
the opportunities and challenges associated with using Medea to spread
disease-resistant genes into mosquito populations in order to control infectious
diseases such as malaria and dengue. I will also discuss some unexpected
results relating to mosquito embryonic development that came from our efforts to
identify Medea components in mosquitoes.
MONITORING LIPID OXIDATION PRODUCTS IN BREATH USING MEMS-BASED
PRECONCENTRATORS
Heather Vereb1*, Bassam Alfeeli2*, Andrea Dietrich1, and Masoud Agah2
1
Department of Civil and Environmental Engineering, Virginia Tech
2
VT MEMS Lab, Department of Electrical and Computer Engineering, Virginia Tech
1
Phone: (540) 231-6131 Fax: (540) 231-7916 Email: [email protected]
ABSTRACT:
Breath analysis is increasingly used as a clinical tool to diagnose medical conditions, such as heart
disease, measure oxidative stress, and monitor environmental and occupational exposure to chemicals. Such
analysis is complicated by the large number of chemicals found in breath, the relatively low pg-ug/Lair
concentrations of target analytes, and high humidity levels. When reactive metals such as iron and copper in
drinking water or foods interact with lipids in the human oral cavity, a series of odorous volatiles are formed which
produce a metallic flavor. Expected odorous volatiles are numerous but could include aldehydes and ketones, such
as 1-octen-3-one which has mushroom-metallic like odor with an odor threshold near 50 pg/Lair and hexanal which
has a green grassy odor with an odor threshold concentration near 0.140 ug/Lair.
Solid phase microextraction (SPME) is commonly used in extraction and concentration of analytes in
breath. However, recently developed MEMS-based silicon-on-glass preconcentrators (μPC) have demonstrated
chemical amplification of analytes suitable for analysis of the expected pg-ug quantities in breaths. In this work we
compare the performance of SPME and μPC technologies to demonstrate the superiority of the latter in extracting
and concentrating volatile organic compounds (VOCs), including lipid oxidation products, in human breath.
The subject provided approximately 50 mL oral-cavity breath samples after gargling a 80 mg/L ferrous
metal solution which was previously shown to induce lipid oxidation and metallic flavor. Samples were collected in
commercial breath-collection bags and then analyzed
using a commercial PDMS-DVB SPME, a lab-produced
μPC
4
PDMS-DVB SPME
1
Tenax-TA SPME, and a μPC. All samples were analyzed
Tenax SPME
using gas chromatography at an initial temperature of
35°C, held for four minutes, followed by a ramp rate of
10°C/min to 175°C on a 30 m DB-17MS column.
Fig. 1 shows an overlay of μPC, PDMS-DVB
SPME, and Tenax-TA SPME chromatograms. The peaks
were identified based on library-matching using mass
spectrometry analysis. This method detected known lipid
oxidation products as minor compounds, including octane,
methyl heptanol, dimethyl pentanol, and methyl nonene in
nanogram per liter amounts. It’s clear that the μPC was
successful in concentrating the VOCs which would
otherwise not be concentrated by the SPME fibers.
References:
1. M. Phillips, R.N. Cataneo, J. Greenberg, M.I. Munawar, S. Nachnani,
and S. Samtani, “Pilot study of a breath test for volatile organic
compounds associated with oral malodor: evidence for the role of
oxidative stress,” Oral Dis., vol. 11 (Suppl. 1), pp. 32-34, 2005.
2. B. Alfeeli, and M. Agah, “MEMS-Based Selective Preconcentration of
Trace Level Breath Analytes,” IEEE Sensors J., vol. 9, pp. 1068–1075,
2009.
*
Equally contributed to this work 7
10
5 6 8 9
3
2
Fig. 1. Overlay of μPC, PDMS-DVB SPME, and Tenax-TA SPME
chromatograms; major compounds detected include: (1) toluene, (2)
hexanal, (3) ethylbenzene and p-xylene, (4) styrene, (5) ethylmethylfulvene, (6) ethylmethylbenzene, (7) ethyldimethylbenzene,
(8), thienyl benzoate, (9) isopropylcyclohexylamine, (10) dimethylethoxy
benzene.
THE USE OF WHOLE ORGAN DECELLULARIZATION FOR THE BIOENGINEERING OF A HUMAN
VASCULARIZED LIVER
1
Vyas, D, 1Baptista, PM, 1Wang, Z, 1Atala, A, 1Soker S.
1
Wake Forest Institute of Regenerative Medicine, Wake Forest University Health Sciences,
Winston-Salem, NC, USA
Email: [email protected]
Introduction
also observed with Ki67 expression and
modest apoptosis detected by TUNEL.
Our laboratory recently developed a
decellularization method able to generate an
entire organ scaffold from a whole liver,
preserving its vascular network. Preliminary
studies showed the possibility to efficiently
re-cellularize the bioscaffold using perfusion
cell seeding in a bioreactor.The purpose of
this study was to investigate the feasibilty of
generating a bioengineered liver by recellularizing the liver bioscaffold with primary
human fetal liver cells (hFLCs) and
endothelial cells (hECs).
Materials and Methods
Human endothelial cells (hECs) and
freshly isolated human fetal liver progenitor
cells (hFLPCs) were seeded through the
portal vein of the bioscaffold. The seeded
bioscaffolds remained in a bioreactor with
constant culture medium perfusion up to one
week. Microscopy was used to determine
cell density and seeding efficiency.
Immunohistochemistry was used to identify
the engrafted cells and to detect hepatic
tissue associated functions. Urea, albumin
and prostacyclin secretion were quantified
as paramaters of cell function. Cell
proliferation and apoptosis was also
determined.
Results
The perfused hECs attached and
formed a monolayer on the luminal side of
the vascular channels. The primary hFLPCs
showed heterogeneous seeding and high
cell density in numerous large clusters
distributed throughout the bioscaffold.
Immunohistochemistry showed progressive
tissue formation with expression of albumin,
alpha fetoprotein, cytochrome P450 3A,
Hep-1 and EpCAM by the hFLPCs and von
Willebrand Factor, VE-cadherin and eNOS
by the hECs. Urea and albumin secretion
was higher in the hepatoblasts seeded on
the bioscaffold. Similarly, prostacyclin
secretion was was higher in hECs seeded in
the bioscaffold than hECs seeded in petri
dishes. Widespread cell proliferation was
Fig. 1. Bioengineered Liver – (A)
Decellularized liver bioscaffold; (B) Liver
bioscaffold seeded with hFLPCs and hECs
after 7 days in a continous perfusion
bioreactor; (C) Human fetal liver cells
expressing cytochrome P450 3A; (D)
Human endothelial cells coating a vascular
structure and expressing vWF.
Discussion and Conclusions
Our results demonstrate the efficient
generation of a bioengineered human liver
organoids with hFLPCs and hECs in a liver
bioscaffold. Hepatic and endothelial tissue
functions were detected along with
progressive liver tissue formation in vitro.
This technology may provide a new
approach for liver bioengineering, critical for
drug discovery and treatment of terminal
liver diseases.
PREPARATION, CHARACTERIZATION, AND IN VITRO DRUG RELEASE
PROPERTIES OF CHITOSAN-CELLULOSE NANOCRYSTAL IONIC COMPLEXES
Hezhong Wang and Maren Roman
Department of Wood Science and Forest Products (0323)
Virginia Tech
Blacksburg, VA 24061
Phone: (540) 231-1421; Fax: (540) 231-8176
Email: [email protected]
Ionic complexes between chitosan, a cationic polysaccharide, and cellulose
nanocrystals, rod-like, anionic cellulose nanoparticles, have potential applications in oral
drug delivery. The purpose of this research was to prepare and characterize chitosan–
cellulose nanocrystal ionic complexes and to determine their in vitro drug release
properties, using caffeine and ibuprofen as model drugs. Cellulose nanocrystals,
prepared by sulfuric acid hydrolysis of bleached wood pulp, were complexed with
medium-molecular-weight chitosan. The properties of the ionic complexes, such as size
and shape, depended strongly on the mixing ratio of the two components and the pH of
the reaction medium. The complexes were loaded with caffeine and ibuprofen and
characterized by FTIR spectroscopy and scanning electron microscopy. The drugloaded complexes were spherical in shape and had diameters of a few hundred
nanometers to a few micrometers. Release of caffeine from the complexes was rapid
and uncontrolled. Ibuprofen-loaded complexes showed no release in simulated gastric
fluid and rapid release in simulated intestinal fluid. Future evaluation studies will focus
on the expected mucoadhesive and permeability-enhancing properties of chitosan–
cellulose nanocrystal ionic complexes.
Hypoxic conditions have differential effects on colony
formation, proliferation and in vitro angiogenesis of endothelial
progenitors.
Zhan Wang1, Jill Mendelson2, Shay Soker1+
1. Wake Forest Institute for Regenerative Medicine,Wake Forest University Health Sciences,
Winston-Salem, NC 27101, USA. 2. Rensselaer Polytechnic Institute, Troy, NY 12180, USA.
+ Corresponding author [email protected]
Introduction. Bioengineered tissue requires a vascular network in order to access oxygen and
nutrients. Cord blood derived endothelial progenitor cells (CEPC) and umbilical vein endothelial
cells (HUVEC) are widely used to facilitate neo-vascularization (1-2); however, CEPCs or HUVECs
delivered in vivo are facing hypoxic conditions. It is not completely understood how and to which
degree hypoxic conditions affect angiogenesis results. In this current study, we examined the effect
of hypoxic conditions on CEPCs and HUVECs viability, clonogenic ability, proliferation and
angiogenesis potentials. Materials and Methods. Colony forming assay: HUVECs and CEPCs
cells were sorted by BD FACSAria II and dispersed as single cells into single well of 96-well-plate,
filled with 200µL of EGM10 media. The cells were cultured in normoxic and hypoxic conditions
(0.1%, 3%, or 20% O2) for seven days and then stained with Crystal Violet. Colonies were then
counted, as well the number of cells for each colony. Angiogenesis potential (Dynamic Tube
Forming Assay): HUVECs and CEPCs were seeded on Matrigel (BD Biosciences) with a seeding
density of 5000 cells per well of 96-well-plate. They were then cultured in normoxic and hypoxic
conditions for eight days. Images were taken each day at 40X with a Zeiss Axiovert microscope and
analyzed using Image J plug-in NeuronJ to quantify tube length. MTS cell proliferation assay
Vascular endothelial growth factor (VEGF) or fibroblast growth factor (FGF) was also added to some
samples. The MTS assay was performed on HUVECs and CEPCs at daily time points in normoxic and
hypoxic conditions to determine cellular proliferation. Results. Colony forming assay showed that
about 50% of the wells had colonies and there was no difference between the different oxygen
conditions. However, at high oxygen here was an increase in the number of larger colonies for both
cell types. Dynamic tube forming assay demonstrated that under low oxygen (0.1% and 3%),
HUVEC and CEPC were able to form and maintain complex tube networks for 7 days, while in high
oxygen (20%) the tube networks disintegrated. Proliferation assay have shown that the
proliferation of both cell types slowed down in hypoxic condition. However, compared to the
baseline culture condition (EBM2 culture medium), 10ng/mL bFGF or 50ng/mL of VEGF were able
to significantly rescue the declines of cell number under hypoxic conditions. Discussion and
Conclusion. In the present study we observed that hypoxic conditions affect the proliferative
ability and angiogenic potential of CEPCs and HUVEC, but colony forming ability was not altered.
These findings demonstrate the separated effects of hypoxic conditions on colony forming,
proliferation and tube forming and thus, they may represent three independent biological
responses to hypoxic stress. Overall, this study indicates that low oxygen concentrations, existing
at the center of implanted bioengineered tissues, have a significant effect on EPCs activities and
thus, their ability to contribute to tissue neovascularization.
References
1.
Au P, Daheron LM, Duda DG, Cohen KS, Tyrrell JA, et al. 2008. Blood 111: 1302-5
2.
Melero-Martin JM, De Obaldia ME, Kang SY, Khan ZA, Yuan L, et al. 2008. Circ Res 103:
194-202