ELUCIDATING THE INFLUENCE OF CHARGE DENSITY AND … generation therapeutics require a fundamental understanding of structure-property-transfection relationships to improve non-viral

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 post-polymerization 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. DGE-0333378. 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


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


Phone: (540) 522-0762 Fax: (540) 231-3824


Kim Niewolny, PhD

Assistant Professor

Department of Agricultural & Extension Education


Phone: (540) 231-5784 Fax: (540) 231-3824


Susan Clark, RD, PhD

Associate Professor

Department of Human Nutrition, Foods and Exercise


Phone: (540) 231-8768 Fax: (540) 231-3916


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


Phone: 540-915-2734 Fax: 540-231-3652


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, 635-641.

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


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 anti-HIV therapeutics. We have synthesized and screened a 4,096 member library of branched peptides (Figure 1) to determine their capacity as ligands for HIV-1 TAR RNA. 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.

Figure 1. Branched peptide library bound to resin. At each position there are four possible amino acids, generating 4096 possible combinations.

Byker, Holmes, Shanks 1

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


Phone: 8152174608 Fax: 5402313916



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 pre-cursor 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.


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.

BIOENGINEERING OF CORNEAS FOR TRANSPLANTATION

Jin San Choi1, J. Koudy Williams1, Margaret Greven1, Keith Walter2, Patrick Laber2,

and Shay Soker1 1Wake Forest Institute for Regenerative Medicine, Wake Forest University Health Sciences and

2Department 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 X-

100/0.1% NH4OH for 3 days at 4C. 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:-


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


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 cavity

1. 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 “milky spots”

2. 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 cancer

1. 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]

1Wake 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. 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 CM-DiI (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).

Fig. 1:Fluorescently labeled myoblasts (green), HUVECs (red) and pericytes (blue) grown in co-culture and visualized by fluorescent (left) or confocal (right) microscopy.

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.

Desmin

MUTATIONAL ANALYSIS OF CWLJ1 FUNCTION IN BACILLUS ANTHRACIS SPORE GERMINATION

Cuffee, CW; Lambert, EA; Popham, DL

Junior

Department of Biology

Virginia Tech

Phone:7577736000


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 C-terminal 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

1Virginia Bioinformatics Institute, and 2Department of Plant Pathology, Physiology and Weed Science,

Virginia Polytechnic and State University, Blacksburg, Virginia

Phone: 276-783-3565


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

1Department 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


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.

0.0

2.0

4.0

6.0

8.0

10.0

12.0

14.0

Baked

Salmon

Broiled

Salmon

Poached

Salmon

Baked

Tilapia

Broiled

Tilapia

Pan-Fried

Tilapia

Product

Hard

ne

ss

(N

)

64 degrees C 74 degrees C

*

**

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


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.

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, including carcass weight, carcass length, weight of visceral fat, longissimus muscle area (LMA), and back fat at the 10th rib were evaluated. The LMA at the 10th rib of the control pigs was larger than HED pigs (P<0.05). The 10th rib back fat was thicker and there was an increased deposition of visceral fat in the HED carcasses, in addition to an observed increase in intramuscular fat.

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.

Time, min0 30 60 90 120 150 180 210 240

Blo

od g

luco

se, m

g/dL

40

50

60

70

80

90

100 Control High Fat

**

*** **

** **

* P < 0.05, ** P < 0.01

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


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 fluorescein-coated 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 UniversityBlacksburg, VA

Phone: 703-203-4437 Fax: 540-231-9293Email: [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 iron-treated 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 elicited higher incidences of the “angry”, “disgusted”, or “surprised” emotions compared to baseline or to neutral water responses for the majority of participants; there 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.

Figure 1. Example of changes in emotional response from baseline (no sample in mouth); with neutral (control) water in mouth and response to expectoration; and iron-treated water in mouth and the resulting aftertaste effect.

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).

mailto:[email protected]???????????

mailto:[email protected]???????????

http://www.cpe.vt.edu/accforum/abstract.html




http://www.noldus.com/human-behavior-research/products/facereader

http://www.noldus.com/human-behavior-research/products/facereader

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


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 DNA-binding 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

1Department of Chemical Engineering, 2Department of Chemistry

Virginia Tech, Blacksburg, VA 24061

Phone: (540)231-3378 Fax: (540)231-8517


Gene therapy is a very active field, primarily due to the potential treatment for a variety of genetic diseases.

1 Synthetic, nonviral vectors are popular due to the tunable chemical composition and their

general lack of cytotoxicity.2, 3

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 the cell while also re-encoding defective genetic sequences.

4 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.

Acknowledgements:

This material is based upon work supported in part by the U.S. Army Research Office under grant number W911NF-07-1-0452 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. Heath, W. H.; Senyurt, A. F.; Layman, J.; Long, T. E., Macromolecular Chemistry and Physics

2007, 208 (12), 1243-1249. 2. Layman, J. M.; Ramirez, S. M.; Green, M. D.; Long, T. E., Biomacromolecules 2009, 10 (5),

1244-1252. 3. Ramirez, S. M.; Layman, J. M.; Long, T. E., Macromolecular Bioscience 2009, 9 (11), 1127-

1134. 4. Williams, S. R.; Lepene, B. S.; Thatcher, C. D.; Long, T. E., Biomacromolecules 2009, 10 (1),

155-161.

N

N

x

N

N

y

OO

S

Br

SG

N

N

x

N

N

y

R

Br

R = C2H5

C4H9

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


ABSTRACT:

The design and synthesis of selenium containing heterocycles has received considerable attention resulting from their ability to function as antioxidants, anti-virals, anti-inflammatories, 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.

RSe

R2

OO

N

SS

hv

fast

RSe

R2

OO

N

SS

fastR

SeR2

OO

SH8

ktrap

kcyclization

RSe +

RSe

HR2

+

+

S8

n=1,2

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


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 synthesis of imidazolium ionene segmented block copolymers

1 and hydrogen bonding incorporation through the synthesis of

9-vinylbenzyladenine and 9-vinylbenzylthymine containing triblock copolymers2. 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 functionalized triblock copolymer

3. 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-07-

1-0452 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), 6834-

6845. 3. Mather, B. D.; Baker, M. B.; Beyer, F. L.; Green, M. D.; Berg, M. A. G.; Long, T. E., Macromolecules 2007, 40 (13), 4396-

4398.

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


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

1Department of Biological Sciences Virginia Tech

2Department 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 seafood-associated 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


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 a-Chemistry, 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

1Department of Wood Science and Forest Products (0323)

2Department of Chemistry (0212)

Virginia Tech

Blacksburg, VA 24061

Phone: (540) 231-1421; Fax: (540) 231-8176


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

aDepartment of Chemistry,Virginia Tech, Blacksburg,Virginia 24061, USA. E-mail: [email protected]; Fax: +1 540 231 3255; Tel: +1 540 231 5041

bDepartment 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


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 nano-scaffolds 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


Florida State University

Phone: 850-645-9274 Fax: 850-645-8447


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, genome-wide 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

David L. Mack

1, Yu Zhou

2, Koudy Williams

1, Sayed-Hadi Mirmalek-Sani

1, Diego Lorenzetti

2,

Mark Furth1, Anthony Atala

1, Shay Soker

1

1Wake Forest Institute for Regenerative Medicine, Winston Salem, NC USA

2Plureon 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 stem cell source for β-cell differentiation

1. 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. Immuno-deficient (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. 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.

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 - - - - - - - - - -

Characterization of a novel transmembrane protein, Cr7TM, in the algal species Chlamydomonas reinhardtii.

Jennifer I. Middlebrooks and Laura R. Keller


Florida State University

Phone: 850-644-9814


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


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_25-26_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


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.

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.

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.

0.000.200.400.600.801.001.201.401.601.802.002.202.402.60

0 3 6 10 18 30

Time (weeks)

Delta TBARS/Total Protein (uM/g)

Delta TBARS‐HealthySubjectsDelta TBARS‐CancerPatients, HRDelat TBARS‐CancerPatients, LR

�16.95 16.91

USE OF ACELLULAR PANCREATIC MATRIX TO SUPPORT BETA CELL DIFFERENTIATION

Sayed-Hadi Mirmalek-Sani1, David L. Mack1, Giuseppe Orlando1,2, Yu Zhou3 & Shay Soker1

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]

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 pancreatic matrix showed prolonged viability and functionality1. 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 scaffold-seeded cultures, compared to no infection controls in induction media alone (Fig. 3).

0

5000

10000

15000

20000

25000

30000

35000

Single Co-culture Single Co-culture

Day 3 Day 7

Cel

l num

ber

0

500

1000

1500

2000

2500

No

infe

ctio

n co

ntro

l

Mon

olay

er

Sca

ffold

s

Mon

olay

er

Sca

ffold

s Day 3 Day 7

Rel

ativ

e IN

SU

LIN

Gen

e E

xpre

ssio

n N

orm

aliz

ed to

BE

TA-A

CTI

N

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).

Fig. 2: Seeding of co-cultures to acellular pancreatic scaffolds enhanced cell expansion compared with single cell types alone (n=3, mean±SD).

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,*

aDepartment of Chemistry, Virginia Tech, Blacksburg, VA 24061

bDepartment of Pharmacology, University of Virginia, Charlottesville, VA 22908

Phone:540-231-5742 Fax: 540-231-3255


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 two-dimensional 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. 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. 2. 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.

3. 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.

4. 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


Wake Forest University

Phone: 336-758-5096


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


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 glutamate-like 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 X-ray 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-phenyl-1,2,3,6-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.

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-methyllumiflavin-promoted 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.

N

O

O

N

N

NN

N

1 2

ClO4

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


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 life-stage 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 post-treatment 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


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 (di-tert-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


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.

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.

OOP

O

O

OH

P

O

O

OH

OH OH

HN

HN

S

O COOH

O

HO N

NH

O

O

n=1 analog 1n=5 analog 2

nO

OP

O

O

OH

P

O

O

OH

OH OH

HN

O COO

N

N

N

NH

O

O

O

analog 3

0

100

200

300

400

500

0.001 0.01 0.1 1 10 100 1000

mP

Un

its

AfUGM Concentration (uM)

Kd = 26 uM

0

100

200

300

400

500

0.001 0.01 0.1 1 10 100 1000

mP

Un

its

AfUGM Concentration (uM)

Kd = 2 uM

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

1Department of Mathematics, 4Department of Chemistry, Virginia Tech

2Department of Mathematical Sciences, New Jersey Institute of Technology

3Department of Physics, University of Western Australia

Phone: 540-320-1573 Fax:540-231-5960


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 non-conducting 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


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 pre-adolescent 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 post-survey 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. Pre- and 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.

*-denotes statistical significance

Ta-Paired T-Test Zb-Wilcoxon Signed Ranks Test

(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.

Quest for Health Survey Results

Variable Pre-Test

Mean

Pre-Test

STDEV

Post-Test Mean

Post-Test

STDEVTest Diff.

Ta or Z

b

Value P-

Value

Nutrition Behavior

40.69 (74.0%)

7.53 44.38

(80.7%) 5.83 -3.69 -1.969

b 0.05*

Physical Activity

Behavior

32.25 (80.6%)

6.51 34.31 (85.8%)

6.67 -2.06 -1.258b 0.208

Nutritional Knowledge

11.13 (69.6%)

1.71 12.69 (79.3%)

1.54 -1.56 -2.778a 0.014*

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. 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). 2. 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).

3. 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 PPG-based ionene having 36 wt% HS showed a monomodal peak using DMF-based SEC and the relative weight-average molecular weight was 21000 g/mol.

OO

O

33n

N CH2 N6

x

O

10N CH2 N

6 y

Br Br Br BrO

O

O

33n

N CH2 N6

x

O

10N CH2 N

6 y

Br Br Br Br

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

1Department of Civil and Environmental Engineering, Virginia Tech 2VT MEMS Lab, Department of Electrical and Computer Engineering, Virginia Tech

1Phone: (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

Tenax-TA SPME, and a μPC. All samples were analyzed 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

1

2

3

4

5 6

7

89

10

μPC PDMS-DVB SPMETenax SPME

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

1Vyas, D, 1Baptista, PM, 1Wang, Z, 1Atala, A, 1Soker S. 1Wake Forest Institute of Regenerative Medicine, Wake Forest University Health Sciences,

Winston-Salem, NC, USA Email: [email protected]

Introduction

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 re-cellularizing 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

also observed with Ki67 expression and modest apoptosis detected by TUNEL.

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


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 drug-loaded 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

Documents

ELUCIDATING THE INFLUENCE OF CHARGE DENSITY AND … generation therapeutics require a fundamental understanding of structure-property-transfection relationships to improve non-viral