2015 Postdoc Research Symposium
Office of Postdoctoral Affairs
April 22, 2015
Emerson Alumni Hall
University of Florida
Poster and Oral
Presentations
Abstracts
Oral Presentation Abstracts
1. Giancarlo Cuadra, Oral Biology, Dentistry
Streptococcus intermedius Enhances Innate Immunity of Gingival Epithelial Cells
2. Viet D. Dang, Physiological Sciences, Veterinary Medicine
Bioaccumulation of Persistent Organic Pollutants in Fish via a Feeding Study
3. Lan Hoang-Minh, Neuroscience, Medicine
Targeting Pericentriolar Proteins to Decrease Glioblastoma Growth and Resistance to
Standard-of-care Therapies
4. Jing Hu, Soil and Water Science, Agricultural and Life Sciences
Greenhouse Gas Emissions from Peatlands
5. Priyanka Ingle-Jadhav, Pathology, Immunology and Laboratory Medicine, Medicine
Sharing for a cure!
6. Stephan C. Jahn, Medicinal Chemistry, Pharmacy
Factors Influencing the Anti-Cancer Success of Dichloroacetate Treatment
7. Nathan A. Jud, Vertebrate Paleontology, Florida Museum of Natural History
Fossil trees and climbers provide new insight to the history of tropical rainforests in Panama
8. Lisa Keyes, Molecular Genetics & Microbiology, Medicine
MHV68 lncRNAs regulate epigenetic machinery
9. Carla Nartuhi Mavian, Pathology, Immunology, and Laboratory Medicine, Medicine
Novel human immunodeficiency virus type 1 (HIV-1) protease inhibitors
10. Jacklyn Quinlan, Anthropology, Liberal Arts and Sciences
Blood Pressure Variation in African Americans Is Influenced by Genetics and Sociocultural
Factors
11. Nicholas M. Teets, Entomology and Nematology, IFAS
Improving the efficacy of Sterile Insect Technique with transgenic overexpression of antioxidant
defense systems
12. Rebecca Ann Wachs, Biomedical Engineering, Engineering
Development of a Proteoglycan Rich Matrix for Nucleus Pulposus Regeneration
13. Jonathan Whittamore, Pathology, Immunology and Laboratory Medicine, Medicine
Getting to the Guts of Calcium Oxalate
14. Ali Zarezadeh, Orthopaedics and Rehabilitation, Medicine
Proteasome inhibitors, a new window for Osteosarcoma patients.
1. Giancarlo Cuadra
Department: Oral Biology
College: Dentistry
Streptococcus intermedius Enhances Innate Immunity of Gingival
Epithelial Cells
Co-Authors: A. Michelotti, S. Lan, D. Culp
Abstract:
Objectives: Streptococcus intermedius is an oral commensal and member of the healthy
subgingival microbiota. In health, interactions between the host and commensal bacteria function
to maintain homeostasis. Conversely, perturbations of host-bacteria interactions likely lead to
dysbiosis and disease. Porphyromonas gingivalis is a hallmark pathogen that invades gingival
epithelial cells. We hypothesized that epithelial cell-S. intermedius interactions helps to protect
against adherence and/or invasion by P. gingivalis.
Methods: Monolayers of Telomerase-Immortalized Gingival Keratinocytes were challenged 3 h with
S. intermedius 0809. At 21 h post-challenge, TIGKs were incubated 30 min either with S.
intermedius 0809 or three different strains of P. gingivalis: 53977, 33277 or 381 (MOIs of 100).
TIGKs were washed and cell lysates plated on agar to determine CFU of adherent bacteria.
Alternatively, TIGKs were cultured 1 h in medium + gentamicin before lysing and plating to
determine invasion CFU.
Results: Challenge of TIGKs with S. intermedius 0809 significantly increases its own subsequent
adherence. In contrast, adherence and/or invasion of all P. gingivalis strains were decreased 40%
- 60%.
Conclusion: S. intermedius induces changes in the host surface that promote its own binding.
However, such changes also decrease subsequent adherence of three different strains of P.
gingivalis. S. intermedius challenge is thus protective. It is of interest to determine molecular
changes induced by S. intermedius, mechanisms for their induction as well as for their protective
effects.
2. Viet D. Dang
Department: Physiological Sciences
College: Veterinary Medicine
Bioaccumulation of Persistent Organic Pollutants in Fish via a
Feeding Study
Abstract:
Persistent organic pollutants (e.g., pesticides, insecticides, and pharmaceutical and personal care
products) in sediments are of concern because these contaminants are available for uptake by
benthic organisms and subsequently bioaccumulate at higher trophic levels like fish. Here, I
conducted a feeding study to determine uptake kinetics and concentration levels of contaminants
in fish feeding on contaminated worms. Contaminated worms were prepared by exposing worms
to only water spiked with two organochlorine pesticides p,p’-DDE and dieldrin, antimicrobials
triclosan and triclocarban, and a current-use insecticide fipronil for a week and then fed to fathead
minnow fish. Fish were collected at 2, 7, 14, and 21 days to determine their body burdens of
chemicals. Results showed that uptake of chemicals by fish reached steady state levels within a
week. In addition, concentrations of triclocarban and fipronil were close to limit of quantitation in
tissues suggesting that fish are able to biotransform chemicals. As fish provides a direct link to
human health risks via consumption, results from this study will be useful for both risk assessment
of contaminated sediments and decision making for site cleanup.
3. Lan Hoang-Minh
Department: Neuroscience
College: Medicine
Targeting Pericentriolar Proteins to Decrease Glioblastoma Growth
and Resistance to Standard-of-care Therapies
Co-Authors: Loic Deleyrolle, Susan Semple-Rowland, Brent Reynolds, Matthew
Sarkisian
Abstract:
A better understanding of the cellular and molecular mechanisms that ensure the successful
proliferation, migration and survival of glioblastoma multiforme (GBM) cells is urgently needed to
improve GBM treatment strategies and prolong patient survival. Cilia are microcellular antennae
that mediate signaling pathways regulating cell growth and survival. Our preliminary data show that
loss of cilia renders some GBM cells unresponsive to exposure to mitogens present in the adult
brain, such as sonic hedgehog, and delays the lethality of intracranial tumors. However, despite
cilia loss, cells continue to divide and presumably mobilize the cell division machinery to coordinate
mitosis. Centrioles and derived ciliary basal bodies are surrounded by satellite proteins, for
example PCM1, that regulate cell division, microtubule organization, and cell stress response.
Here, we find that PCM1 is expressed around centrioles and the base of cilia in GBM biopsies and
nearly every cell from patient-derived primary cell lines. Interestingly, PCM1 retains its
pericentriolar expression pattern despite cilia ablation, and stress-induced intracellular dispersion
of PCM1 also appears to be cilia-independent. Thus, the preservation of proteins like PCM1 may
support the continued proliferation of GBM cells despite the absence of cilia. To test whether
PCM1 is required for GBM cell proliferation/survival, we generated multiple PCM1-knockout cell
lines and found that the loss of PCM1 resulted in significantly decreased cell proliferation and a
concurrent increase in cell death in vitro. Further investigation of PCM1 or PCM1-interacting
proteins may lead to novel targets that could inhibit GBM growth or sensitize tumors to current
therapies.
4. Jing Hu
Department: Soil and Water Science
College: Agricultural and Life Sciences
Greenhouse Gas Emissions from Peatlands
Abstract:
The increasing atmospheric concentration of greenhouse gases, including carbon dioxide (CO2),
nitrous oxide (N2O) and methane (CH4), have contributed to global climate change and will lead to
potential dangerous consequences to human beings and the planet. Peatlands are potential
sources for the greenhouse gases, which are affected differently by the frequency and duration of
flooding and draining cycles of peatlands. The objective of this study was to compare greenhouse
gas emissions from peatlands under different flooding and draining cycles. A laboratory study was
performed using intact soil cores collected from peatlands of the Everglades Agricultural Area. Soil
cores were subjected to different hydrological treatments with continuous or alternating flooding
and draining for 6 months. Our results showed that CO2 was the dominant component of
greenhouse gas emissions, irrespective of different hydrological treatments, accounting for more
than 93% of the total greenhouse gas emissions, while CH4 and N2O accounted for less than 2%
and 2-6%, respectively. Overall, total greenhouse gas emissions were inversely proportional to the
total days of peatlands under flooded conditions. This study has implication for mitigating
greenhouse gas emissions from peatlands through water management. And the results can also
be applied at larger ecosystem scales for a more accurate estimation of greenhouse gas
emissions from peatlands.
5. Priyanka Ingle-Jadhav
Department: Pathology, Immunology and Laboratory Medicine
College: Medicine
Sharing for a cure!
Co-Authors: Dr. Mark Atkinson
Abstract:
Background:
The global incidence of type 1 diabetes is on rise targeting young and adolescents, destroying beta
cells of pancreas making them dependent on exogenous insulin for the rest of their life. There are
therapies under development with very little clinical success and the main reason being very
limited knowledge about the disease and pathophysiology. As the access and imaging of current
functional and pathological status of pancreas is difficult because of its retroperitoneal positioning
and proximity to vital organs making pancreatic biopsies difficult raising ethical and safety issues.
There is a longstanding need for a system allowing access to human pancreatic tissue and foster
the understanding of the disease.
Methods:
The Network for Pancreatic Organ donor for Diabetes (nPOD) was established, in 2007, to recover
and characterize pancreata and related organ from cadaveric donors with various risk levels to
type 1 diabetes or otherwise. These biospecimens are provided to the investigators worldwide, free
of cost, to look at the disease pathogenesis, progress and possibly cure in their own perspective.
Major findings:
As a result of this initiative, the investigators working on this valuable resource provided by the
families during the most tragic times, has grown from 7 in 2007 to 239 in 2015, resulting in over 90
scientific publications in the last years.
Conclusion:
These studies are making tremendous impact on the age-old dogma about the disease
progression and its pathogenesis supporting the research for the prevention and cure for the
betterment of type 1 diabetics.
6. Stephan C. Jahn
Department: Medicinal Chemistry
College: Pharmacy
Factors Influencing the Anti-Cancer Success of Dichloroacetate
Treatment
Co-Authors: Ryan J. Lorenzo, Peter W. Stacpoole, Margaret O. James
Abstract:
Background: Variability in response to a drug between patients is an issue that must be addressed
when developing any therapy. However, when the drug is treating a life-threatening illness,
predicting patient response is especially critical. Such is the case with dichloroacetate (DCA).
DCA is a small molecule commonly used to treat metabolic disorders and is under investigation as
an anti-cancer therapy due to its ability to normalize cancer cell metabolism and cause tumor
death. The drug has shown great promise in some patients, but has failed to provide much benefit
in others, so we aimed to elucidate the reason for these differences.
Methods: We analyzed human tissue samples with donor-matched normal tissue using Western
blot, an enzyme activity assay, and an HPLC-based method for determining chloride
concentration.
Results: Here, we present evidence that some tumors express glutathione transferase Z1
(GSTZ1) the enzyme responsible for the break-down of DCA in the body. When this enzyme,
normally expressed almost exclusively in the liver, is present in a tumor, it breaks down DCA as it
enters the tumor, protecting the cancer from the effects of the drug. Additionally, we found that
chloride is abnormally high in some tumors. Since chloride provides a stabilizing effect on GSTZ1,
high levels could exacerbate the resistance afforded by the GSTZ1 in these tumors.
Conclusions: Together, these data provide a way to predict which tumors will respond positively to
DCA treatment as well as opening the door to co-therapies that would eliminate resistance to DCA.
7. Nathan A. Jud
Department: Vertebrate Paleontology
College: Florida Museum of Natural History
Fossil trees and climbers provide new insight to the history of
tropical rainforests in Panama
Co-Authors: Chris W. Nelson
Abstract:
Neotropical rainforests are among the most diverse and productive ecosystems in the world, but a
poor fossil record limits our understanding of their history. New fossils collected as part of the
Panama Canal Project provide direct evidence of the plants and animals that inhabited Panama
during the Early Miocene. We examined thin sections of fossil woods to assess both plant diversity
and paleoclimate during the Early Miocene and the Late Miocene. Growth rings are not present in
any of the woods examined so far, suggesting that the trees did not experience a strong dry
season. Some of the wood specimens from the Early Miocene represent at least six different
species of climbing plants (lianas) rather than trees. Lianas provide a special insight into the
structure of the forest. Modern lianas are common in seasonal forests, but can be indicators of
disturbance in everwet forests. The absence of growth rings in the trees and the diversity of
climbers indicates the presence of an everwet tropical rainforest in Panama 19 Million years ago.
Co-occurring fossils and sedimentological evidence suggest that volcanism, fluvial processes, and
megafauna such as horses and rhinos caused natural disturbance that promoted the diversity of
lianas.
8. Lisa Keyes
Department: Molecular Genetics & Microbiology
College: Medicine
MHV68 lncRNAs regulate epigenetic machinery
Co-Authors: Mehmet Kara, Emily Feldman, Scott Tibbetts
Abstract:
Epigenetic modifications of histones plays a critical role in regulating the life cycle of
gammaherpesviruses. An emerging theme in noncoding RNA (ncRNA) biology is a central role for
these molecules in regulating epigenetic machinery. However, the role of ncRNAs in
gammaherpesvirus infections, and the relationship between viral ncRNAs and epigenetics in the
context of gammaherpesviruses, is poorly understood. Murine gammaherpesvirus 68 (MHV68) is
genetically and pathogenically related to the human gammaherpesviruses, and infection of mice
provides a unique system with which to study the interplay between epigenetics and viral ncRNAs.
Previous studies have demonstrated that epigenetic modifications to herpesvirus genomes plays a
central role in the regulation of lytic replication and the establishment/maintenance of EBV and
KSHV latency. Recent reports have demonstrated that ncRNAs have the capacity to actively
regulate epigenetic machinery. Such epi-ncRNAs are also likely to be crucial regulators of
epigenetic modifications of histones associated with the viral genome and thus overall viral gene
expression. Using MHV68-recombinant viruses lacking various ncRNAs, we have observed altered
regulation of key components of the chromatin remodeling machinery, strongly suggesting that
MHV68-encoded ncRNAs play an important role in regulation of epigenetically-regulated viral
processes. Further defining the nature of this relationship should elucidate the roles for these
ncRNAs in chronic gammaherpesvirus infection and pathogenesis.
9. Carla Nartuhi Mavian
Department: Pathology, Immunology and Laboratory Medicine
College: Medicine
Novel human immunodeficiency virus type 1 (HIV-1) protease
inhibitors
Co-Authors: Roxana M Coman, Steve Pomeroy, David Ostrov, Ben M Dunn,
Maureen M Goodenow
Abstract:
Background: Introduction of HIV-1 protease (PR) inhibitors (PIs) to anti-viral therapy has
significantly improved the life expectancy of HIV-infected individuals. However, current PIs that
bind PR in the active site present disadvantages as poor pharmacokinetics and high toxicity.
Moreover, due to therapeutic pressure, HIV-1 PR accumulates mutations leading to drug
resistance and therapy failure. Identification of molecules that inhibit the PR exploiting non-active
site mechanisms of inhibition is essential to overcome resistance to current PIs.
Methods: Molecular docking of 140,000 compounds (NCI/NIH) to the conserved residues of the
elbow of the PR flaps was performed in silico. Inhibitory activity of best docking compounds was
tested in an in vitro PR inhibition biochemical assay. Antiviral activity of non-toxic compounds was
tested against PI-sensitive HIV-1 in primary peripheral blood mononuclear cells (PBMCs) and
Tzmb-l cells. Ritonavir (RTV) was used as reference for anti-viral activity.
Results: Twelve of the best predicted docking compounds induced partial to complete inhibition of
HIV-1 PR activity in vitro and less than 20% toxicity in PBMCs or Tzmb-l cells (10-200 μM range).
Compounds suppressed HIV-1 replication by 60-90% and, in combination with RTV, increased
anti-viral activity compared to any compound or RTV alone.
Conclusions: These novel PIs with multi-cell type effectiveness can be delivered with classic drugs
for a better inhibition of the virus and are a promising alternative to attenuate or even prevent
therapy resistance. Further chemical engineering of these PIs would improve concentrations more
suitable for pharmaceutical use.
10. Jacklyn Quinlan
Department: Anthropology
College: Liberal Arts and Sciences
Blood Pressure Variation in African Americans Is Influenced by
Genetics and Sociocultural Factors
Co-Authors: Laurel Pearson, Chris Clukay, Clarence C. Gravlee, Connie J.
Mulligan
Abstract:
Background: African Americans are 40% more likely to have high blood pressure and 50% more
likely to die from heart disease than whites. Blood pressure is affected by many factors, including
genetic and environmental factors. The purpose of this study is to examine how genetics and
exposure to sociocultural risk factors, such as unfair treatment, influences blood pressure in
African Americans.
Methods: Using a community-based study, African Americans living in Tallahassee were
interviewed to obtain extensive demographic, socio-cultural, and medical history data. Saliva
samples for DNA were collected to assay variants in candidate genes for hypertension and related
phenotypes and blood pressure as measured as our outcome variable. Novel measures of unfair
treatment were developed that asked, for the first time, for experiences of unfair treatment by
individuals close to the study participant.
Results: We identified both genetic and socio-cultural variables that explain variation in blood
pressure in African Americans. Unfair treatment was only significantly associated with blood
pressure when genetic data were included. In addition to unfair treatment to themselves, 22% of
participants reported that someone close to them had experienced unfair treatment. Depending on
the type of unfair treatment experienced, different genetic factors were significantly enriched in
certain biological pathways and associated with blood pressure, suggesting that different types of
unfair treatment may act through different biological pathways.
Conclusion: Our results indicate the importance of including both genetic and sociocultural data in
future studies on complex diseases, particularly to identify interacting factors that contribute to
racial health disparities.
11. Nicholas M. Teets
Department: Entomology and Nematology
College: IFAS
Improving the efficacy of Sterile Insect Technique with transgenic
overexpression of antioxidant defense systems
Abstract:
The Sterile Insect Technique (SIT) is a pest management strategy in which large numbers of
sterilized insects are released into the environment to suppress pest populations. SIT is an
environmentally friendly, species-specific control strategy that has effectively suppressed several
agricultural pests. However, cost remains a significant burden for expanding the scope of SIT; the
US spends >$30 million annually on SIT for a single species of fruit fly, with Florida alone spending
>$6 million. One of the greatest economic barriers of SIT is the poor mating performance of
released males, as ionizing radiation used for sterilization causes substantial oxidative stress to
flies. Thus, I am using transgenic techniques to overexpress antioxidant enzymes, with the aim of
reducing irradiation-induced oxidative damage and improving the health and performance of
males. My model for this study is the Caribbean fruit fly, Anastrepha suspensa, an invasive pest in
Florida with a history of SIT research and well-developed tools for genetic transformation. Using
piggyBac-mediated germline transformation, I have generated seven lines of A. suspensa that
overexpress mitochondrial superoxide dismutase, which is the cell’s primary defense against
damaging reactive oxygen species produced by mitochondria. These lines overexpress the
enzyme between 40-130%, depending on genotype, and we are testing whether overexpression
reduces cellular oxidative damage and enhances male mating performance following sterilizing
irradiation. Improving the quality of sterile males would reduce the number of males that need to be
released, thereby enhancing the efficiency of ongoing SIT programs and lowering economic
hurdles for establishing new programs.
12. Rebecca Ann Wachs
Department: Biomedical Engineering
College: Engineering
Development of a Proteoglycan Rich Matrix for Nucleus Pulposus
Regeneration
Co-Authors: H Huda, RC Cornelison, S Xin, CE Schmidt
Abstract:
Disc degeneration is leading cause of low back pain is and is characterized by a loss in
intervertebral disc height from breakdown of the nucleus pulposus (NP) matrix. The NP is rich in
collagen type II and chondroitin sulfate proteoglycans with sulfated glycosominoglycans (sGAG)
side chains. Fragmentation of chondroitin sulfate proteoglycans and removal of sGAGs has been
implicated in disease progression. Tissue specific decellularized extracellular matrix holds promise
for regeneration of the NP. This research seeks to develop a gentle decellularization method for
use on the NP to remove cells, but maintain intact proteoglycans.
Intact spines were dissected from female porcine spines and the NP was isolated. Samples were
processed in a two day decellularization series including washing in water, followed by washes
detergents and enzymes. NP samples were all macroscopically intact after processing.
Decellularized samples were tested for removal of DNA and maintenance of sGAGs using
standard techniques. Results revealed >97% removal of DNA and maintenance of >58% of sGAG
in the decellularized NP versus unprocessed controls. Additionally, samples were labeled for nuclei
and CSPGs. Confocal imaging revealed nearly complete removal of nuclei and cellular debris, and
substantial maintenance of CSPGs in decellularized samples versus controls.
These results indicate that we have successfully removed cellular components from the NP while
maintaining an intact extracellular matrix with CSPGs thereby creating a hydrogel with potential for
use in NP regeneration. Future experiments will include in vitro and in vivo experiments to explore
the efficacy of this tissue analog for regeneration.
13. Jonathan Whittamore
Department: Pathology, Immunology and Laboratory Medicine
College: Medicine
Getting to the Guts of Calcium Oxalate
Abstract:
A primary constituent of kidney stones is calcium oxalate, and elevated levels of urinary oxalate
(hyperoxaluria) are a major risk factor for stone formation. Oxalate is a non-functional, waste
metabolite of the liver, and also part of the diet, with the kidney responsible for its excretion.
Primary Hyperoxaluria type 1 (PH1) is a rare, inherited disease caused by a liver enzyme
deficiency resulting in the vast over-production of oxalate. This leads to kidney stone formation
together with oxalate crystal deposition around the body, and ultimately results in early death due
to kidney failure. With no effective drug treatments and the only available cure a combined liverkidney transplant, there is clearly the need to find an effective form of treatment for this devastating
disease. In the Hatch lab we are focused on the important contribution made by the intestine,
particularly when colonized by the non-pathogenic, commensal bacterium Oxalobacter formigenes.
What makes Oxalobacter unique is not only does it survive exclusively on oxalate, but it draws on
the endogenous burden by stimulating the intestine to secrete oxalate from the blood into the gut.
Previous studies from our lab using a mouse model of PH1 have shown that colonizing the
intestine with Oxalobacter normalizes oxalate levels in the blood and urine relative to healthy
controls. Our goal is to understand this relationship between Oxalobacter and the gut to support
the development of future probiotic-based treatments for the benefit of PH1 patients as well as the
much larger population of kidney stone formers.
14. Ali Zarezadeh
Department: Orthopaedics and Rehabilitation
College: Medicine
Proteasome inhibitors, a new window for Osteosarcoma patients.
Co-Authors: Elham Nasri, Pardraic P. Levings, Maria V. Guijjaro, Steven C.
Ghivizzani, C. Parker Gibbs.
Abstract:
Osteosarcoma (OS) is the most common primary bone tumor in childhood and adolescence.
Despite the utilization of modern chemotherapy in combination with surgery, the 5-year survival for
non-metastatic high grade OS patients has not been exceeded more than 60-70% over the last 30
years. In fact the 5-year survival rate for the OS patients with pulmonary metastasis is still about
18-33%. This failure may be explained by the fact that the mechanism of tumorigenicity in
osteosarcoma is still not clear. Additionally OS tumors are widely heterogenic, not only intertumoral but also intra-tumoral; an individual tumor composed of subpopulations with varying
phenotypes. This adds another level of complexity to treatment of OS. With a new approach to
intratumoral heterogeneity, we could successfully identify and fractionate at least two different cell
populations in OS tumors, of which one population is 100 times more tumorigenic and the other
population is at least 3 times more resistant to current chemotherapeutic regimen. Comparing their
distinct global gene expression pattern, we identified the pathways that contribute to tumor
initiation and drug resistance in our tumors. Based on this data, we chose Bortezomib, an FDA
approved proteasome inhibitor, to investigate its effects in OS. We showed that it induces a range
of favorable dose dependent anti-cancer effects in OS tumor initiating cells including differentiation,
cell cycle arrest and apoptosis. Last but not the least; the resistant population is at least 5 times
more sensitive to Bortezomib than current treatment.
Poster Presentations Abstracts
1. Carolina Abrahan Neuroscience, Medicine
Reprogramming human cells to study new therapies for nerve diseases.
2. Nima Afshar-Mohajer Environmental Engineering Sciences, Engineering
Estimation of Particulate Matters and Total VOC Emissions from Binder Jetting 3D Printers
3. Kalina Rosenova Atanasova Periodontology, Dentistry
Porphyromonas gingivalis’ nucleoside diphosphate kinase is secreted from primary gingival
epithelial cells through pannexin-1 hemichannels
4. Manas Biswal Molecular Genetics and Microbiology, Medicine
Gene based antioxidant therapy delays retinal degeneration in a mouse model of progressive
retinal degeneration
5. Sasanka Chukkapalli Periodontology, Dentistry
Periodontal microbiome orchestrates a distinct inflammatory response
6. Giancarlo Cuadra Oral Biology, Dentistry
Effects of Oral Commensals on Host Epithelial Cells in Adherence and Invasion
7. Amy L. Donate Pediatrics, Medicine
Urine Derived Epithelial Cells as a Model for Neuromuscular Disease
8. Mehrnaz Hazrati Electrical and Computer Engineering, Engineering
Quantifying Functional Connectivity in Spatio-Temporal Brain Networks
9. Jing Hu Soil and Water Science, Agricultural and Life Sciences
Greenhouse Gas Fluxes under Different Flooding and Draining Regimes of Cultivated
Peatlands
10. Rachel Alexandra Jones Chemistry, Liberal Arts and Sciences
Synthetic Strategies for the Bioconjugation of NSAIDs with Glucosamine
11. Amber L. LaCrosse Psychology, Liberal Arts and Sciences
Ceftriaxone requires both xCT and GLT-1 up-regulation in the nucleus accumbens to attenuate
the reinstatement of cocaine-seeking
12. Jungnam Lee Periodontology, Dentistry
Porphyromonas gingivalis-Nucleoside Diphosphate Kinase Suppresses Epithelial Cell
Apoptosis by Interacting with Human Heat-Shock Protein 27
13. Stefanie Marquez Hematology and Oncology, Medicine
BRG1 is Silenced in Human Tumors by Non-mutational Mechanisms
14. Biswapriya Biswavas Misra Biology, Liberal Arts and Sciences
Time-Resolved Cell-type Specific Metabolomes Reveal Differential Metabolomic Responses to
Elevated [CO2] in Guard Cells and Mesophyll Cells
15. Manjunatha K Nanjappa Physiological Sciences, Veterinary Medicine
Rat stem Leydig cells can transdifferentiate into uterine and prostatic epithelium, but not
epidermis or adipocytes
16. Abolfazl Ravanshad Civil and Coastal Engineering, Engineering
Application of Scanning Electron Microscopy to Evaluate Composition and Polishing Behavior
of Limestone and Granitic Aggregates
17. Michael D. Williams Pathology, Immunology, and Laboratory Medicine, Medicine
An Optimized Method for Extraction of Red Blood Cell Metabolites
1. Carolina Abrahan
Department: Neuroscience
College: Medicine
Reprogramming human cells to study new therapies for nerve
diseases.
Co-authors: Aaron Shepherd, Sooyeon Lee, Nao Terada, Guangbin Xia, Katherine
Santostefan and Lucia Notterpek
Abstract:
There have been many studies to find new therapies for diseases that affect the insulating material
for nerves that connect the brain and spinal cord with the body. Although some of these studies
have been successful in rodents, they have been found to be ineffective in humans. This indicates
that there are some important differences between rodent nerves and human nerves and that we
need to develop other models. One such model uses human cells. By using methods for genetic
engineering, collaborators of the lab can “reprogram” cells extracted from human skin and
generate cells that are able to become different kinds of cells. Our objective is to find the right type
of environment that these cells need, to create the insulating material for nerves. In this way we
may be able to create a human cell-based model to study nerves diseases, and that with a
personalized medicine approach.
2. Nima Afshar-Mohajer
Department: Environmental Engineering Sciences
College: Engineering
Estimation of Particulate Matters and Total VOC Emissions from
Binder Jetting 3D Printers
Co-authors: Chang-Yu Wu, Thomas Ladun, Didier Rajon, and Yong Huang
Abstract:
Binder jetting 3D printing is a popular type of additive manufacturing and a powerful tool for
creating parts as well as prototypes especially in medical research labs and architectural
workshops. Due to continuous movement of dry powders inside printer chambers and injection of
resin-like binder fluid during printing, binder jetting 3D printers are a potential emission source of
fine particulate matters (PM) from the powder and volatile organic compounds (VOCs) from the
binder solution. In this study, real-time measurements of total volatile organic compounds (TVOCs)
and a wide size range of aerosol (10 nm to 10 µm) during a 2-hr continuous operation of a binder
jetting printer incorporated into a time-varying mass balance equation at a space under
investigation to obtain the emission rates. The maximum amount of the TVOC was slightly below
the permissible exposure level (PEL) recommended by OSHA for formaldehyde, 750 ppb, but the
PM2.5 mass concentration exceeded the USEPA standard of 25 µg/m3. Comparison of the results
to other indoor air quality results measured in previous studies from indoor environments with
similar sources of particulate emitters such as blackboard-chalk classrooms revealed that
particulate emission rates of a binder jetting 3D printer is several order of magnitude greater than
that of writing or erasing a blackboard but exposure level of the TVOC was comparable. Caution
should be taken for printing jobs with runtimes longer than 8 hr.
3. Kalina Rosenova Atanasova
Department: Periodontology
College: Dentistry
Porphyromonas gingivalis’ nucleoside diphosphate kinase is
secreted from primary gingival epithelial cells through pannexin-1
hemichannels.
Co-authors: Jungnam Lee, JoAnn Roberts and Ozlem Yilmaz
Abstract:
Background: Nucleoside diphosphate kinases (NDKs) are evolutionarily conserved small
multifunctional enzymes. Their secretion is important for latency of opportunistic pathogens, such
as P. gingivalis (Pg). The NDK-homolog of Pg (Pg-NDK) contributes to bacterial persistence in
primary gingival epithelial cells (GECs), and its secretion from GECs modulates danger-signal
“extracellular ATP” (eATP)-mediated reactive-oxygen-species production by interfering with P2X7receptor activation via hydrolysis of eATP. Although bacterial NDK can be secreted during infection
and human homologs were found to be secreted by cancer cells, mechanism(s) of NDK
extracellular translocation are still unrecognized. Based on our previous studies where depletion of
the P2X7-coupled pannexin-1-hemichannel (PNX1) inhibited NDK-associated bacterial effects, we
hypothesized that NDK may utilize PNX1 pathway. Here we examined the role of PNX1, along with
classical secretion pathways (e.g.ER-Golgi, lipid rafts) on Pg-NDK-secretion. Methods: GECs
infected with Pg or transfected with green fluorescent protein-NDK construct were examined for
NDK localization using TEM and fluorescence confocal microscopies. Inhibitors of ER-Golgi
(Brefeldin A), lipid rafts (methyl-β-cyclodextrin), PNX1 (Probenecid), and RNAi approach for PNX1,
were utilized. Secreted Pg-NDK was detected via ATPase assay. Results: Microscopically Pg-NDK
localized both in cytoplasm and Pg-containing autophagy vacuoles. eATP stimulation induced
significant relocation of NDK to host-cell membrane. All used inhibitors and RNAi-depletion of
PNX1 resulted in significant reduction of Pg-NDK secretion. Conclusions: This is the first study
showing NDK secretion outside host cells via multiple pathways working jointly. Our novel findings
may aid multiple fields in human health and identify novel targets to control Pg- and other chronic
bacterial infections.
4. Manas Biswal
Department: Molecular Genetics and Microbiology
College: Medicine
Gene based antioxidant therapy delays retinal degeneration in a
mouse model of progressive retinal degeneration
Co-authors: Christhian Ildefonso, Haoyu Mao, Hong Li, and Alfred S Lewin.
Abstract:
Purpose: We established a model of RPE (retinal pigment epithelium) oxidative stress by Cre-lox
mediated deletion of the Sod2 gene, that codes for the protective enzyme manganese superoxide
dismutase (MnSOD), leading to features of geographic atrophy. We asked whether delivery of
Sod2 using adeno-associated virus (AAV) could prevent retinal degeneration once it has begun
and whether there is an optimal window of opportunity for such intervention.
Methods: Deletion of Sod2 was induced in mice with a “floxed” allele of Sod2 and an RPE-specific
tet-transactivator controlling expression of cre. Retinal degeneration was monitored by
electroretinography (ERG) and spectral domain optical coherence tomography (OCT) over a period
of 9 months. Experimental mice at different ages received subretinal injection of a selfcomplementary AAV1 vector expressing mouse Sod2 cDNA in their right eyes and ScAAV1-GFP
in left eyes.
Results: Following doxycycline induction of Cre, mice demonstrated signs of oxidative stress in
RPE, a gradual decline in the ERG response and thinning of the outer nuclear layer which were
statistically significant by 6 months. When injected early, ScAAV1-Sod2 delayed the progressive
retinal degeneration in RPE specific Sod2 knockout mice.
Conclusions: Deletion of Sod2 in the RPE leads to some of the salient features of dry AMD.
ScAAV1 delivery of Sod2 led to expression in RPE. Delivery of Sod2 vector can be used as a tool
to reverse oxidative stress in this mouse model of dry AMD.
5. Sasanka Chukkapalli
Department: Periodontology
College: Dentistry
Periodontal microbiome orchestrates a distinct inflammatory
response
Co-authors: Irina Velsko, Merceds Rivera-Kweh, Dong Hang, Alexandra Lucas,
Kesavalu Lakshmyya
Abstract:
OBJECTIVES: Periodontal disease (PD) develops from a synergy of complex subgingival oral
microbiome and is linked to systemic inflammatory atherosclerotic vascular disease (ASVD).
However, the complex molecular mechanisms establishing such a relationship are not understood
completely. This study investigated how a polybacterial infection accelerates atherosclerotic
plaque progression in vivo.
METHODS: ApoEnull mice were periodontally infected for 12 and 24 weeks with a polybacterial
consortium of 4 well-characterized pathogens Porphyromonas gingivalis, Treponema denticola,
Tannerella forsythia and Fusobacterium nucleatum. We assessed PD characteristics, bacterial
dissemination, peripheral T cell response, serum inflammatory cytokines, atherosclerosis risk
factors, atherosclerotic plaque development, and alteration of aortic gene expression after 12 and
24 weeks of infection.
RESULTS: Polybacterial infections have enhanced gingival colonization in hyperlipidemic ApoEnull
mice. Bacterial infections were more invasive with hematogenous dissemination into
cardiovascular tissues such as the heart and aorta. Infection-induced significantly higher levels of
serum risk factors (oxidized LDL, nitric oxide), altered lipid profiles (cholesterol, triglycerides,
chylomicrons, VLDL) as well as accelerated aortic plaque formation. Polybacterial infection is
associated with significant decreases in Apoa1, Apob, Birc3, Fga, FgB genes associated with
atherosclerosis. Periodontal infection after 12 weeks showed decrease in inflammatory molecules
Fas ligand, IL-13, SDF-1 and increase in chemokine RANTES levels. In contrast, 24 weeks of
infection reduced inflammatory molecules KC, MCSF and enhanced the expression of GM-CSF,
IFNγ, IL-1β, IL-13, IL-4, IL-13, lymphotactin, and RANTES.
CONCLUSIONS: Periodontal pathogens synergistically colonized gingiva, enhanced periodontal
disease outcome measures, augmented risk factors, altered inflammatory markers, and
accelerated atherosclerosis progression.
6. Giancarlo Cuadra
Department: Oral Biology
College: Dentistry
Effects of Oral Commensals on Host Epithelial Cells in Adherence
and Invasion
Co-authors: A. Michelotti, S. Lan, D. Culp
Abstract:
Objectives: Streptococcus intermedius is an oral commensal and member of the healthy
subgingival microbiota. In health, interactions between the host and commensal bacteria function
to maintain homeostasis. Conversely, perturbations of host-bacteria interactions likely lead to
dysbiosis and disease. Porphyromonas gingivalis is a hallmark pathogen that invades gingival
epithelial cells. We hypothesized that epithelial cell-S. intermedius interactions helps to protect
against adherence and/or invasion by P. gingivalis.
Methods: Monolayers of Telomerase-Immortalized Gingival Keratinocytes were challenged 3 h with
S. intermedius 0809. At 21 h post-challenge, TIGKs were incubated 30 min either with S.
intermedius 0809 or three different strains of P. gingivalis: 53977, 33277 or 381 (MOIs of 100).
TIGKs were washed and cell lysates plated on agar to determine CFU of adherent bacteria.
Alternatively, TIGKs were cultured 1 h in medium + gentamicin before lysing and plating to
determine invasion CFU.
Results: Challenge of TIGKs with S. intermedius 0809 significantly increases its own subsequent
adherence. In contrast, adherence and/or invasion of all P. gingivalis strains were decreased 40%
- 60%.
Conclusion: S. intermedius induces changes in the host surface that promote its own binding.
However, such changes also decrease subsequent adherence of three different strains of P.
gingivalis. S. intermedius challenge is thus protective. It is of interest to determine molecular
changes induced by S. intermedius, mechanisms for their induction as well as for their protective
effects.
7. Amy L. Donate
Department: Pediatrics
College: Medicine
Urine Derived Epithelial Cells as a Model for Neuromuscular Disease
Co-authors: Ramanathan, M Rizzo SA, Mccall A, Love-Leonor T, Cade T, Byrne
BJ, Pacak CA
Abstract:
Renal epithelial cells (RECs) enter the urine following detachment from kidney tubules. Their
abundance in urine samples and the straightforward techniques necessary for isolation and culture
make RECs a useful in vitro model of disease. The goal of this study is to determine if RECs can
be directly differentiated into skeletal myoblasts and thus, serve as a model for neuromuscular
disease. RECs derived from patients with Barth Syndrome, Myotubular Myopathy, and Friedreich
Ataxia were differentiated using adenovirus – MyoD (Ad-MyoD). After 48 hours, media was
switched to DMEM + 2% horse serum to further encourage differentiation and subsequently, media
was replaced every 3-5 days for 1-2 weeks. Increasing multiplicities of infection (MOIs 1-100***)
were added to RECs to establish the minimum effective dose. Differentiated cells were analyzed
by qPCR and immunofluorescence to confirm expression of the myocyte markers, DES and
MYOG. Results show that RECs efficiently differentiate into skeletal myotubes by day 7. Further
studies will include functional evaluations relevant to each disease-type. In conclusion, directly
differentiated RECs display standard myotube morphology and gene expression profiles making
these highly advantageous in vitro models of neuromuscular disease.
8. Mehrnaz Hazrati
Department: Electrical and Computer Engineering
College: Engineering
Quantifying Functional Connectivity in Spatio-Temporal Brain
Networks
Co-authors: Andreas Keil, Jose C. Principe
Abstract:
Cognitive processes are grounded in dynamic communication between brain regions. Therefore,
the quantification of functional connectivity at high temporal resolution is greatly desirable for
understanding brain function. In this study, we proposed a data-driven algorithm that is sufficiently
sensitive to detect relatively rapid (on the order of hundred milliseconds) changes in the
establishment of functional links between distinct cortical regions. Our method is a modified version
of a recently developed algorithm called the generalized measure of association (GMA). Healthy
human subjects participated in a cognitive experiment and instructed either to avoid (active) or to
accept (passive) an aversive loud noise by means of a key press. For active compared to passive
trials, heightened connectivity between visual and central areas was observed in time segments
preceding and surrounding the avoidance cue. Local neural coupling within extended visual
regions was sustained throughout major parts of the viewing epoch.
9. Jing Hu
Department: Soil and Water Science
College: Agricultural and Life Sciences
Greenhouse Gas Fluxes under Different Flooding and Draining
Regimes of Cultivated Peatlands
Co-authors: Christine M. VanZomeren, Kanika S. Inglett, Alan L Wright, Mark W.
Clark, and K. Ramesh Reddy
Abstract:
Globally, approximately 10–20 % of peatlands have been drained for agricultural purposes.
Drainage of peatlands increases soil organic matter oxidation, resulting in soil subsidence and net
CO2 emission to the atmosphere. A strategy to protect peatlands and mitigate CO2 emissions,
while continuing agricultural production is intermittent flooding and drainage, accompanied by
growth of crops which are tolerant of periodic flooding. A potential drawback of this strategy of
alternating flooding-drainage cycles could be increases in CH4 and N2O emissions. The objective
of this study was to compare greenhouse gas emissions (CO2, CH4, and N2O) from peatlands
under different flooding-drainage cycles. A laboratory study was performed using intact soil cores
(40 cm in depth) of peatlands from the Everglades Agricultural Area (EAA) in south Florida. Soil
cores were subjected to different durations of flooding and drainage for 6 months. Average daily
emissions of CO2 and N2O were significantly higher under drained than under flooded conditions
(P < 0.001). Emissions of CO2 and N2O averaged 667 mg CO2-C m-2 d-1 and 135 μg N2O-N m-2
d-1 under drained conditions, respectively, but only 86 mg CO2-C m-2 d-1 and 48 μg N2O-N m-2
d-1 under flooded conditions. Methane emissions were not influenced by drained/flooded
conditions, with an average emission of 116 μg CH4-C m-2 d-1. Peaks of CH4 and N2O emissions
were observed after flooding events and lasted no longer than 24 h. The highest emissions were
observed 0-1 h after flooding, which were approximately 8 and 19 times higher than the mean CH4
and N2O emissions, respectively. Carbon dioxide was the dominant component of greenhouse gas
emissions, irrespective of different hydrological treatments, accounting for more than 92% of
overall global warming potential (GWP) of greenhouse gas emissions. Global warming potential
was inversely proportional to the flooding period of peatlands, indicating that prolong the flooding
period of peatlands would help mitigate soil oxidation and greenhouse gas emissions, and
enhance sustainability of these agricultural peatlands.
10. Rachael Alexandra Jones
Department: Chemistry
College: Liberal Arts and Sciences
Synthetic Strategies for the Bioconjugation of NSAIDs with
Glucosamine
Co-authors: Yann Thillier, Christoper S. Sebastiano Jr., C. Dennis Hall
Abstract:
Non-steroidal anti-inflammatory drugs (NSAIDs), an over the counter medication, are used for the
management of pain associated with conditions including osteo- and rheumatoid arthritis. The
continued use of NSAIDs, including naproxen, indomethacin and diclofenac can result in damage
to the gastrointestinal tract, in addition to diverse risks such as nephrotoxicity, hepatotoxicity and
an increased risk of heart disease.
A number of methods have been proposed to reduce the side effects of NSAIDs, including
prescribing the lowest effective dose for a short period of time to control symptoms and the
concomitant use of gastro-protective drugs. The design and synthesis of novel NSAIDs has met
with limited success.
Glucosamine is ubiquitous in human tissue; in particular it is highly concentrated in connective
tissue, especially in cartilage. This has led to the widespread use of glucosamine supplements as
an alternative therapy for the treatment of arthritis.
Drug hybrids have emerged over the past decade in which two or more compounds, known to be
pharmaceutically active, are linked via a covalent bond to form a drug that exhibits greater activity
through a synergistic effect. We envisaged that glucosamine-NSAID drug hybrids might behave as
novel pro-drugs that display reduced adverse side-effects associated with NSAIDs, and would
therefore be a useful addition to current pain management therapies. We describe hereon
synthetic strategies that we have used to prepare glucosamine-NSAID bioconjugates.
11. Amber L. LaCrosse
Department: Psychology
College: Liberal Arts and Sciences
Ceftriaxone requires both xCT and GLT-1 up-regulation in the
nucleus accumbens to attenuate the reinstatement of cocaineseeking
Co-authors: Lori A Knackstedt
Abstract:
Two key transporters for regulating glutamate homeostasis in the nucleus accumbens are: 1) the
major glial glutamate transporter GLT-1 which performs glutamate reuptake in a sodium-dependent
manner and 2) system xc-, which exports glutamate into the extrasynaptic space in exchange for
cystine in a sodium-independent manner. Following cocaine self-administration and 2-3 weeks of
withdrawal, the expression and function of GLT-1 and xCT, the catalytic subunit of system xc-, are
significantly decreased relative to drug-naive controls. Ceftriaxone increases GLT-1 and xCT
expression and attenuates both cue- and cocaine-induced reinstatement. It is not known if
ceftriaxone exerts its therapeutic actions through upregulation of GLT-1 or xCT, or both. Here, we
reduced GLT-1 or xCT expression using in vivo morpholino antisense technology and examined
the role of specific GLT-1 or xCT upregulation in the ameliorative effects of ceftriaxone on
reinstatement and the expression of transporters and AMPA receptor subunits. Both GLT-1 and
xCT knockdown prevented ceftriaxone from attenuating reinstatement. xCT knockdown
significantly increased GluA1 surface expression and GLT-1 knockdown showed a trend for doing
so. This phenomenon has been associated with the formation of GluA2-lacking calcium-permeable
AMPA receptors and elevated cocaine-seeking, and this mechanism may contribute to the ability
of xCT and GLT-1 knockdown to prevent ceftriaxone from attenuating reinstatement. GLT-1
expression was reduced by both the GLT-1 and the xCT morpholino, and so the behavioral effects
of the xCT morpholino are not specific to xCT knockdown. This is evidence for co-regulation of the
transporters.
12. Jungnam Lee
Department: Periodontology
College: Dentistry
Porphyromonas gingivalis-Nucleoside Diphosphate Kinase
Suppresses Epithelial Cell Apoptosis by Interacting with Human
Heat-Shock Protein 27
Co-authors: Kalina R Atanasova, JoAnn Roberts, and Özlem Yilmaz
Abstract:
Background: Porphyromonas gingivalis is a Gram-negative anaerobe that has been implicated in
the pathogenesis and progression of periodontal diseases in humans. P. gingivalis produces a
plethora of virulence factors that allow it to invade and replicate intracellularly in gingival
epithelium. Recent studies have shown the ability of P. gingivalis to modulate host mitochondrial
apoptotic pathways and to render gingival epithelial cells (GEC) resistant to apoptosis induced by
host danger signals and pro-apoptotic reagents. One of the recently identified virulence factors
involved in the anti-apoptotic features of P. gingivalis is nucleoside diphosphate kinase (NDK). In
this study, we tried to identify possible target cellular molecules for the NDK modulation of host
apoptotic signaling and to construct candidate host cell signaling pathways which could be
modulated by anti-apoptotic characteristics of NDK. Methods: Through pull-down assay and
MALDI-TOF approach, we identified heat-shock protein 27 (HSP27) as a binding target to NDK
and confirmed the binding of the two proteins using ELISA assay. In addition, we demonstrated
direct phosphorylation of human HSP27 by NDK. Results: Increased HSP27 phoshporylation was
observed in GECs infected with P. gingivalis ATCC 33277, compared to the infected cells with P.
gingivalis NDK-deficient mutant. We treated P. gingivalis-infected GECs with staurosporine and the
result showed that P. gingivalis NDK successfully suppresses staurosporine –induced cell
apoptosis by inhibiting cytochrome c release and caspase 9 activation. Conclusions: Taken
together, this study demonstrates the role of P. gingivalis NDK in the anti-apoptosis and unveils
HSP27 as a crucial target molecule in the process.
13. Stefanie Marquez
Department: Hematology and Oncology
College: Medicine
BRG1 is Silenced in Human Tumors by Non-mutational Mechanisms
Co-authors: Bhaskar Kahali, Kenneth Thompson, Li Lu, David Reisman
Abstract:
The SWI/SNF chromatin remodeling complex is a major regulator of gene expression where its
role is to facilitate the shifting and exposure of certain segments of the DNA to transcription factors
and other key proteins. SWI/SNF functions in a plethora of processes including DNA repair,
differentiation, development, cell adhesion and growth control. Given its participation in these
cellular processes, SWI/SNF is often targeted during cancer initiation and progression. Brahmarelated gene 1 (BRG1), is one of two catalytic ATPase subunits and not surprisingly, BRG1 is
frequently lost in a variety of human tumor types, including renal clear cell carcinoma, lung, liver,
breast and colon cancers, among others. The loss of both BRG1 and its homolog Brahma (BRM)
renders this complex inactive. Both BRG1 and BRM are known to bind to Rb in order to facilitate
growth inhibition. Similarly, these two proteins have been linked to p53 function. Without BRG1
and BRM, Rb- and p53-mediated growth inhibition are severely impaired. In studies of cancerderived cell lines, BRG1 has been reported to be mutated, and consequently, the dogma has been
that BRG1 is also mutated in primary tumors. However, the rate of reported loss by
immunohistochemistry (IHC) far exceeds the reported abrogating mutation rates of this subunit.
We hypothesized that mechanisms other than mutations must contribute to BRG1 silencing and
that BRG1 might be re-expressed with compounds previously shown to induce its homolog, BRM.
To do this, we first determined the rate of BRG1 loss in different tumor types by IHC. We then
performed IHC to detect BRG1-negative tumors, obtained the matched frozen specimens and
sequenced the DNA to determine any mutations; we also investigated the possibility of splice
variants and gene deletion. As with BRM, the ability to re-express BRG1 has potential clinical
applications.
14. Biswapriya Biswavas Misra
Department: Biology
College: Liberal Arts and Sciences
Time-Resolved Cell-type Specific Metabolomes Reveal Differential
Metabolomic Responses to Elevated [CO2] in Guard Cells and
Mesophyll Cells
Co-authors: Evaldo D. Armas, Sisi Geng, Ning Zhu, Zhaohui Tong, Sixue Chen
Abstract:
Background
Anthropogenic [CO2] levels presently at 400 ppm is expected to reach 550 ppm in 2050, an
increment that is expected to affect plant growth and productivity, thus challenging global food
security. We aimed at deciphering this change in CO2-responsive metabolism at the levels of
single cells to predict the future of plant adaptation and survival.
Methods
We used two different cell types, i.e., stomatal guard cells (GC) and mesophyll cells (MC) from
canola (Brassica napus) to profile metabolomic changes upon increased [CO2] through
supplementation with bicarbonate [HCO3]-, the functional form of absorbed CO2. Gaschromatography (GC) and liquid chromatography (LC)-mass spectrometry (MS)-based platforms
enabled quantification of 285 metabolites in a time-course study providing short-term responses of
plant cellular metabolism. Metabolomic data sets were processed through uni- and multi-variate
statistical approaches to obtain physiological insights.
Results
The CO2-responsive plant metabolomes of the cell types differed, i.e., with increased carbon (C) in
the form of [HCO3]-, the levels of nitrogenous (N) metabolites showed directly proportional
response in MC and inverse relationship in GC, showing differential C:N ratio homeostasis. In
addition, the MCs showed incorporation of the CO2 into primary carbon metabolites and defenseresponsive pathways (i.e., phenolics and flavonoids), whereas the GCs displayed enriched Nmetabolites (i.e., amino acids).
Conclusions
Functional dimorphism of the cell-types to environmental perturbation such as CO2 is evident. MCs
are the bulk cell-types in green plants and their photobiosynthetic potentials regulate global food
production, these results provide insights into future crop productivity upon elevated CO2
conditions.
15. Manjunatha K. Nanjappa
Department: Physiological Sciences
College: Veterinary Medicine
Rat stem Leydig cells can transdifferentiate into uterine and
prostatic epithelium, but not epidermis or adipocytes
Co-authors: Theresa I. Medrano, Barry R. Zirkin, Haolin Chen, Paul S. Cooke
Abstract:
Stem Leydig cells (SLCs) are precursors of Leydig cells, which secrete testosterone, the androgen
required to maintain testicular spermatogenesis. The SLCs have therapeutic potential for treating
infertility in chemotherapy patients and also as a source of cells for regenerative medicine. The
objective of this study was to investigate the ability of rat SLCs to transdifferentiate into cell types
from all three germ layers: prostatic epithelium (endoderm), uterine epithelium (mesoderm) and
epidermis (ectoderm). The SLCs isolated from adult rat testis were combined with fetal or neonatal
mouse mesenchyme from these tissues, grafted under the renal capsule of nude mouse hosts and
grown in vivo. To differentiate cell lineages, mesenchyme from green fluorescent protein (GFP)
expressing mice was used. After 5 weeks, tissue recombinants of urogenital sinus mesenchyme
(a potent prostate inducer) and SLCs had formed fluid filled ductal structures which histologically
resembled prostate. Epithelium was of SLC origin as determined by absence of GFP. Similarly,
uterine mesenchyme/SLC recombinants grew into fluid filled cysts which resembled uterus
histologically, with the lumen lined by columnar epithelium of SLC origin. In contrast, skin
mesenchyme/SLC recombinants did not form epidermis and consisted only of dermis, suggesting
SLCs did not form skin epithelium. Furthermore, culturing SLCs in adipogenic media did not result
in adipocyte differentiation. Thus, SLCs have the capacity to differentiate into uterine and prostatic
epithelium but not epidermis or adipocytes. These results suggest that SLCs may have limited
therapeutic potential in regenerative medicine.
16. Abolfazl Ravanshad
Department: Civil and Coastal Engineering
College: Engineering
Application of Scanning Electron Microscopy to Evaluate
Composition and Polishing Behavior of Limestone and Granitic
Aggregates
Co-authors: Paul Carpinone, Reynaldo Roque
Abstract:
Aggregates in the asphalt mixture have varying ability to withstand the polishing action of traffic
and maintain microtexture based upon their mineralogy; therefore, different aggregates have
different polishing behavior. Limestone is the most susceptible to polishing, and granite has
intermediate resistance against polishing. In a previous study, authors used aggregate Image
Measurement System (AIMS) to capture surface roughness of aggregates. The results showed a
significant difference between limestone and granitic aggregates. This study was defined in order
to visually evaluate the surface characteristics of these aggregates using SEM and evaluate AIMS
performance.
17. Michael D. Williams
Department: Pathology, Immunology, and Laboratory Medicine
College: Medicine
An Optimized Method for Extraction of Red Blood Cell Metabolites
Co-authors: Ann Guggisberg; Christopher Beecher; Audrey Odom; Timothy Garrett
Abstract:
In the field of metabolomics, the need for standardized and effective methods for sample
processing, sample analysis and data mining are increasingly important. Careful study is needed
In order to establish methods that optimize metabolite extraction, sample throughput, and dynamic
range of detection. In a direct method comparison, we extracted metabolites from RBCs using
three methods. The first method combined use of ice-cold water, methanol (MeOH), chloroform,
and acetonitrile, added at key points in a multistep process, to extract metabolites. In the second
method, ice-cold acetonitrile (0.1% FA) was used to extract the metabolites from the RBCs. RBC
metabolites were extracted with warm (40°C) MeOH in the third method. In all methods, extraction
was followed by centrifugation, collection of supernatant, drying under N2 and reconstitution in
0.1% formic acid (FA). Extractions were analyzed via ultra-high pressure liquid chromatography –
high resolution mass spectrometry (UHPLC-HRMS). After initial data analysis, we determined the
most effective method and then tried several variations to optimize extraction results.
We found that using acetonitrile/water (A/W, 1:1) instead of methanol alone, following the
procedure of the third method gave dramatically better extraction of numerous metabolites. This
underlines importance that the extraction method has in the process of metabolomic analysis. We
demonstrated that using appropriate extraction methods for the sample type under investigation
can greatly enhance the number and type of features that are observed in the analysis. Further
analysis, in other vectors, is also needed to find the most effective methods of metabolite
extraction for specific sample types.