44th Maine Biological and Medical Sciences Symposium
hosted by
MDI Biological Laboratory
With support from the
Maine IDeA Network of Biomedical Research Excellence (INBRE)
MDI Biological Laboratory Conference Center
Salisbury Cove, Maine
April 28-29, 2017
Poster Session A
Eden Parish Hall
Upstairs: Developmental and Cellular Biology/Regeneration
Downstairs: Neuroscience/Physiology
Listed alphabetically by presenting author
The regulatory role of a phosphorylated G protein: Implication for lipid microdomains
Alamer, SA1,2, Gundersen, RE1,2
1 Graduate School of Biomedical Sciences and Engineering, University of Maine, ME 04469
2 Department of Molecular and Biomedical Sciences, University of Maine, ME 04469
Heterotrimeric G-proteins play roles in many signaling pathways, where they act as molecular
switches in transducing a signal from G protein-coupled receptors to downstream effectors.
Post-translation modifications such as phosphorylation and palmitoylation can be important
factors in regulating G protein function, but the mechanism of their involvement remains
unknown. We used Dictyostelium discoideum as a cellular model that relies on chemotaxis
toward a secreted chemoattractant, cyclic adenosine monophosphate to examine the roles of G
protein palmitoylation and phosphorylation. The Gα2 of D. discoideum is required for the
chemotactic response. Our preliminary data demonstrate that the Gα2 is enriched in lowdensity lipid microdomains. This localization was palmitoyaltion-dependent. Activation
significantly shifts Gα2 out of these microdomains. Once activation occurs, Gα2 is known to be
phosphorylated on serine 113. Our goal was to determine if the phosphorylation of Gα2
regulates its function and membrane clustering. Exchange of serine residue 113 to alanine
allows starved cells to begin the aggregation phase several hours sooner when compared to
wild type, while exchanging this serine to aspartic acid showed a decrease in plasma membrane
localization. Cell fractionation experiment revealed that Gα2-S113A clusters in the low-density
fraction similar to the inactive Gα2. This clustering is decreased in case of the phosphorylation
mimic Gα2 ;Gα2-S113D. In addition, D.discoideum 14-3-3 protein has been found by
coimmunoprecipitation with activated Gα2.
Anecdata.org: Closing the citizen science “data loop”.
Duncan Bailey1 and Dr. Jane Disney1
1 Mount Desert Island Biological Laboratory, PO Box 35, Salisbury Cove, ME
1
Anecdata.org, developed at the MDI Biological Laboratory, is a free online platform for
environmental citizen science. Since launching in 2014, it has become home to over 400 citizen
scientists and 30 projects ranging from the Gulf of Maine to the Pacific Northwest. Anecdata
provides projects with a versatile data collection platform and makes it easy for citizen
scientists and members of the public to browse, analyze, and download project data.
Project coordinators tell us that showcasing data can be as important as data collection
because citizen scientists are encouraged and empowered by seeing their data in a broader
context. We believe continued development of tools like these will be greatly helpful in closing
the citizen science “data loop”, transforming data into knowledge and knowledge into action.
Motivation to self-administer cocaine as function of prenatal choline availability
Elizabeth Lucy Bainbridge
Previous research has demonstrated that prenatal supplementation of dietary choline in rats
protects against disease-induced anhedonia, or the inability to derive pleasure from that which
used to give it. These findings suggest that choline may be acting in the mesolimbic
dopaminergic pathway to alter motivation. Thus, the present study assessed rats’ motivation to
self-administer cocaine as a function of prenatal choline availability, and the neural bases of any
effects. Timed-pregnant Sprague-Dawley dams were fed either a choline-supplemented or
standard diet and the adult female offspring were given limited access (7 hr/day) to a twobottle choice of cocaine (0.1 mg/ml) or tap water for 16 days. Intake for each rat in mg/kg and
ratios of cocaine intake relative to total intake were calculated. On day 17, brains were
collected and planned histological analyses include quantification of dopamine transporter
activity in the nucleus accumbens and cell proliferation in the hippocampus.
Progression of cardiomyogenesis from embryonic stem cells in a three-dimensional gel matrix
Bomar, J.1, Sawyer, D.2, Collins, S.1,3, Smith, R.1,4
1GSBSE, University of Maine, Orono, ME; Myocardial Biology and Heart Failure Research Lab,
MMCRI, Scarborough, ME; 3Department of Chemistry, University of Maine, Orono, ME;
4
Electrical and Computer Engineering, University of Maine, Orono, ME
[email protected]
Each year, nearly one million Americans have a heart attack, all of whom suffer some loss of
cardiac function due to permanent tissue damage. Stem cell therapy is under investigation;
however, the efficacy is to date very low. In this project, murine embryonic stem cells
suspended in a three-dimensional gel matrix are differentiated in vitro into functional cardiac
tissue with a highly reproducible, complex spatial cooperation of multiple cardiac cell types.
Spontaneous contractions begin in an organized network of cells at approximately day 10.5,
shortly following upregulation of muscle protein expression. The spatial and temporal
progression of cardiomyogenesis was monitored using fluorescence imaging techniques. Shortterm goals are to use microfluidics and 3D printing technology in conjunction with our 3D
cultures to expose pluripotent cells to temporal and spatial chemical profiles in order to direct
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stem cell differentiation into functioning cardiac tissue and ultimately improve cardiomyogenic
yield for stem cell therapies.
Zebrafish as a Preclinical Model for Dystroglyclanopathies
Carter, E.1,2, Goody, M.2, Kilroy, E.1,2, Karunasiri, C.2, Belanger, J.2, Astumian, M.2, King, E.2,
Drinkert, D.3, Henry, C.1,2
1Graduate School of Biomedical Science and Engineering, University of Maine, Orono, ME
04469, 2School of Biology and Ecology, University of Maine, Orono, ME 04469, 3Orono High
School, Orono, ME 04473.
[email protected]
Muscular dystrophies are heritable diseases characterized by progressive muscle wasting and
weakness. Mutations in molecules that modify the extracellular matrix protein dystroglycan
cause a subset of muscular dystrophies known as secondary dystroglycanopathies. Because
these conditions present with diverse symptoms and severities, dystroglycanopathy research
lags behind. This project aims to establish zebrafish as a preclinical model for secondary
dystroglycanopathies. Our preliminary studies reveal that muscle structure, motility, and
innervation are all compromised in a zebrafish morphant model of secondary
dystroglycanopathy and that vitamin supplementation improves these defects. We found
similar defects in a novel secondary dystroglycanopathy engineered via modern genomic
technology, some of which are rescued with vitamins. Future work will involve comprehensive
analysis of muscle and nerve phenotypes in multiple mutant models and will address discrete
roles of muscle and nerve in these conditions with the goal of identifying potential therapeutic
targets.
Nanocellulose conduits for enhanced regeneration of peripheral nerve injury
Carter, N.1,2, Dewey, M.2, Millard, C.3, Castillo, R.3, Neivandt, D.1,2
1Graduate School of Biomedical Science and Engineering, University of Maine
2Department of Chemical and Biological Engineering, University of Maine
3Student Intern, Department of Chemical and Biological Engineering, University of Maine
[email protected]
Peripheral neuropathy is estimated to afflict 20 million people in the United States. Most cases
of neuropathy result from physical injuries and trauma arising from automobile accidents and
war. Peripheral nerves have the intrinsic ability to regenerate over time, bridging the injury gap
caused by the trauma. Current methods utilized to assist in the regeneration of peripheral
nerves include nerve autografts and implantation of conduits. Nerve autografts are regarded as
the most effective method, but require a second surgical site to access a donor nerve. Allografts
are similar to autografts except the donor nerve is from another individual and requires a
prolonged regimen of immunosuppressant medication. Conduits currently on the market have
equal or lower success rates relative to nerve autografts. Issues that arise with the use of the
current generation of conduits involve foreign body reaction and potential second surgeries to
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remove the conduit. It is proposed that a biocompatible material such as nanocellulose may
serve as a viable alternative conduit construction material. The current work determines means
by which cellulose nanofibril conduits may be produced, and evaluates their efficacy in
regeneration after a sciatic nerve injury in a murine model.
The Role of c-Fos and DCX in Encoding Novel Place Experience in Response to Choline
Supplementation
Chun, Y.1, Glenn, MJ.1
1Colby College
[email protected]
Choline is a vital dietary component for proper biological function, and supplementation
increases neurogenesis in the hippocampus, which is often used as a biomolecular marker of a
properly functioning hippocampus. How are the functions of new neurons altered with choline
supplementation? What role do these new neurons take on in response to a spatial
experience? Choline supplemented rats either received five days of the same spatial
experience, four days of the same maze, a novel maze on the fifth, or no spatial experiences.
Interestingly, there is robust DCX and c-Fos colabeling in the CA1 in control and treatment
groups, with marked physical differences when compared to expression in the DG. c-Fos
expressing cells in the CA1 are almost exclusively colabeled with DCX, although there is a
substantial amount of singly labeled DCX cells. What does this sort of expression tell us about
the function of these cells?
Evidence for mouse telomerase reverse transcriptase (mTERT) as a novel and unique marker
of adult neural stem cells.
Curtis, C.D.1, Jensen, G.2, Blaszkiewicz, M.2, Breault, D. 3, Townsend, K.L.1,2
1School of Biology and Ecology, University of Maine, Orono, ME, 2Graduate School of
Biomedical Sciences and Engineering, University of Maine, Orono, ME, 2Children’s Hospital
Boston and Harvard Medical School, Boston, MA.
[email protected]
Neurodegenerative diseases and brain injuries would likely benefit from novel treatments
targeting adult neural stem cells (ANSCs). However, there is currently not a single, specific
marker for ANSCs in the mouse brain, making the cells difficult to study. Mouse telomerase
reverse transcriptase (mTERT), an enzyme that lengthens telomeres after DNA replication, has
been shown to mark adult stem cells in the mouse intestine (Montgomery et al. PNAS 2011)
and in other tissues. By utilizing a specially-designed triple-transgenic mouse line capable of
lineage tracing mTERT+ cells, we have demonstrated that mTERT marks putative ANSCs in the
known neurogenic niches of the adult mouse brain. Cell sorting of mTERT+ cells, followed by
gene expression studies, have further shown that these cells express known ANSC markers,
including SOX2, Nestin and GFAP. Ongoing experiments are testing known neurogenic stimuli,
such as exercise, to determine how mTERT+ ANSCs behave in terms of proliferation, migration,
differentiation, and integration in the mature neural circuitry. We hypothesized that mTERT+
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ANSCs would line the lateral ventricle stem cell niche, and have for the first time have
demonstrated mTERT expression in the choroid plexus, which secretes cerebrospinal fluid and
may represent a novel adult stem cell niche in the mouse brain.
Development of the bumble bee as a model of near-infrared light therapy
Shain, K.1, Johnston, A.1., Barbosa, S.1, Dobrin, S.E.1
1Univesity of Maine at Presque Isle, Biology, Presque Isle ME
[email protected]
Near-infrared (NiR) light therapy has been investigated for its ability to promote wound healing,
stimulate muscle repair, and increase angiogenesis. Recently, NiR has been used clinically in
recovery following ischemic stroke and as a treatment for a variety of neurological disorders
and is reported to enhance cognition in humans. We aimed to establish the bumble bee
(Bombus impatiens) as an invertebrate model for human NiR therapy. The bumble bee has a
short lifespan, is amenable to molecular approaches, and displays complex social behaviors
which lend to studies of memory and cognition. We aimed to determine if 1) bumble bee
survival is affected by NiR irradiation, 2) the activity of cytochrome C oxidase, the purported
photoactive molecule, and ATP production are increased following irradiation, and 3) if changes
in cytochrome C oxidase activity is mediated via changes in gene expression. Here, we report
preliminary findings of aims 1 and 2.
Metabolic and Physiological Effects of Varying Dietary Fat Type and Amount
Blaszkiewicz, M.1, Dubois, A.1, Miller, J.L.2, Beaton, C.2, Kiebish, M. 3, Townsend, K.1,2
1Graduate School of Biomedical Sciences and Engineering, University of Maine, Orono, ME,
2School of Biology and Ecology, University of Maine, Orono, ME
3 Berg Bioscience, Framingham, MA
While the low-fat diet trends stemming from recommendations in the 1970s are now being
replaced by recommendations that we reduce sugar and increase consumption of healthy fats,
we still do not have a comprehensive understanding of how specific dietary fats affect our
physiology and metabolic health. We also do not know how dietary fat metabolites are
preferentially stored in some tissues versus others and how these affect cellular functions. For
this study, we designed custom mouse diets differing in the type and amount of dietary fats:
high or low saturated, high or low omega-6 polyunsaturated (PUFA), and high or low omega-3
PUFA. We began the study with the hypothesis that omega-3 diets would have greater health
benefits, as per the cardiovascular research in the field and findings from the Mediterranean
diet which is high in omega-3. However, we have uncovered important negative effects from
omega-3 supplements that are prone to peroxidation, and have discovered beneficial effects of
omega-6 diets on adipose tissue health. In addition, we have run lipidomics assessments on
brown adipose tissue, white adipose tissue and hypothalamus (the brain region that regulates
energy balance) and hypothesize that certain lipid metabolites may be affecting brain-adipose
communication.
ERK Regulates Transcription of JC polyomavirus
5
DuShane J.1, Maginnis M.1
1Department of Molecular and Biomedical Sciences, University of Maine, Orono, ME
[email protected]
JC polyomavirus (JCPyV) infects more than half of the population and presents as a persistent,
lifelong infection in the kidney. In immunosuppressed individuals, JCPyV infection can lead to
the onset of the fatal, demyelinating disease, progressive multifocal leukoencephalopathy.
Dynamic interactions between the virus and host-cell factors are responsible for driving the
infectious life cycle, yet the role of cellular signaling pathways in the infectious process remain
poorly understood. Previous work demonstrated that the activation of extracellular signalregulated kinase (ERK) occurs within minutes of viral challenge of host cells. Our research has
elucidated that both the presence and activation of ERK is necessary for JCPyV infection. While
ERK becomes activated immediately following infection, we found ERK activity is a critical
regulator of JCPyV transcription, as ERK inhibition demonstrated a decrease in viral promoter
activity and viral gene expression. These findings demonstrate how the ERK signaling cascade
impacts JCPyV infection and pathogenesis.
Advances on Translating Ribosome Affinity Purification (TRAP) to analyze mature white
adipocytes
Figueroa C.A.1,2, DeMambro V. E.1, Rosen C.J1.
1Maine Medical Center Research Institute (MMCRI), 81 Research Drive, Scarborough, ME,
04074. 2University of Maine, Orono, ME 04469.
[email protected]
Introduction, Our laboratory has shown that marrow adipose tissue (MAT) is a biologically
active adipose depot that reflects metabolic status and may influence skeletal remodeling2. For
further understanding of MAT function we aim to test the translating ribosome affinity
purification (TRAP) technology in visceral white adipose tissue from an EGFP-ribosomal tagged
mice3 as a tool to analyze actively translating mRNA profile from mature adipocytes.
Methods, Western blot against EGFP were performed from different peripheral white adipose
tissue and bone marrow (BM). TRAP was performed in BM cells transfected with Adeno-Cre
virus by immunoprecipitation, followed by RT-qPCR.
Results, MAT and peripheral white adipose tissues from Adiponectin-CRE EGFP mice
differentially expresses EGFP. mRNA was isolated from TRAP technology after optimizing
immunoprecipitation and RNA integrity conditions using BM Adeno-Cre transfected cells.
Conclusion, TRAP technology successfully isolate actively translating mRNA, showing that it can
be used to study MAT gene expression profiles.
mir-21 directs the regenerative response in non-myocyte cells during cardiac regeneration in
the zebrafish
FitzSimons, M.1,3, Benjamin L. King2, Yin, V.P.1,3
1Graduate School of Biomedical Sciences and Engineering, University of Maine, Orono, ME
2 Department of Molecular and Biomedical Sciences, University of Maine, Orono, ME
6
3MDI
Biological Laboratory, Kathryn W. Davis Center for Regenerative Biology and Medicine,
Salisbury Cove, ME
[email protected]
Coronary heart disease (CHD) is the main cause of death worldwide. In humans, damaged
myocardium associated with CHD is replaced with non-functional scar tissue. In contrast,
zebrafish possess the remarkable ability to completely regenerate heart muscle without
residual scar. Using the zebrafish as a model organism, we hope to better understand the
genetic circuitry promoting cardiac regeneration.
Recent work has demonstrated that non-myocyte cells are essential to coordinating tissue
regeneration and repair; nevertheless, the mechanisms governing behavior of these cells are
incompletely understood. MicroRNAs are powerful post-transcriptional regulators of gene
expression. Our lab has identified miR-21 as one of the most highly upregulated miRNAs in
response to cardiac injury in the zebrafish, and found that miR-21 expression co-localizes
spatially and temporally with non-myocyte cells during regeneration. miR-21 depletion
compromises heart regeneration, underscoring the requirement for upregulation of this miRNA
during the regenerative process.
Detailed in vivo comparison of two Wnt1:Cre models revealing critical functions of primary
cilia of cardiac neural crest cells (cNCC) in heart development.
Fitzsimons, L. A.1,2, Budzsizewski, K.2, Pinz, I. M.1,3, Tucker, K. L.1,2
1Graduate School of Biomedical Science and Engineering, University of Maine, Orono, Maine,
U.S.A.; 2Department of Biomedical Sciences, College of Osteopathic Medicine, University of
New England, Biddeford, Maine, U.S.A.; 3Small Animal Imaging Core Facility, Maine Medical
Center Research Institute, Scarborough, Maine, U.S.A.
[email protected]
Eliminating primary cilia from cardiac neural crest cells (cNCC) using a Wnt1:Cre-driver line
(Danielian et al.,1998) to conditionally delete the Ift88 gene results in unique cardiac
developmental phenotypes combining a lethal outflow tract (OFT) defect with ventricular septal
defects (VSD) and non-compaction of the ventricular myocardium. Evidence demonstrating
ectopic expression of Wnt1 in this Wnt1:Cre driver prompted an additional investigation into
the Wnt1:Cre2 driver (Lewis et al.,2013). Comparing Wnt1:Cre-mediated phenotypes revealed
consistencies in the ventricular non-compaction and interventricular septum defects seen in the
original Wnt1:Cre-mediated mutants, but with an unexpected and intact OFT. The lack of
severe OFT defects in the Wnt1:Cre2-mediated mutant phenotype warrants further
investigation into the presence/nature of ectopic Wnt1 expression in cNCC lacking primary cilia
and the subsequent malformation of the developing OFT of the Wnt1:Cre-mediated mutant.
Our results support key modulatory roles for primary cilia of cNCC in both interventricular
septation and myocardial maturation.
Mutation in putative linker sequence prevents GLH-1 aggregation
Fuqua, H.¹˒², Updike, D.¹
7
¹The MDI Biological Laboratory, Kathryn W. Davis Center for Regenerative Biology and
Medicine, Salisbury Cove, ME,
²College of the Atlantic, Bar Harbor, ME
[email protected]
Germ granules, known as P granules in C. elegans, are specialized ribonucleoprotein (RNP)
aggregates that are necessary for proper germline development and the overall fertility of the
organism. GLH-1 is a P-granule protein that plays a role in maintaining this fertility and is
thought to be necessary for the formation of P granules themselves. Through a forward genetic
screen, we have identified a short amino acid sequence in a putative linker region of GLH-1 that
when mutated results in the diffusion of the protein throughout the cytoplasm of the germ cell.
This result may aid in understanding the process of P-granule assembly and could hold more
general significance for the study of RNP granule formation and protein aggregation.
Grant Support: NIGMS P20GM103423, NIGMS R01GM113933NTENT
Stress-induced developmental programming of the immune system
Ian Gans1,2, Elli Hartig1, Shusen Zhu1, James Coffman1,2
1MDI Biological Laboratory, Davis Center for Regenerative Biology and Medicine, Salsbury Cove,
ME 04672
2Graduate School of Biomedical Sciences and Engineering, University of Maine, Orono, ME
04469
[email protected]
Chronic stress during development is associated with an increased susceptibility to disease
throughout the lifetime, but the nature of this elevated risk is not fully understood. The stress
response in humans is mediated primarily by the hormone cortisol, a steroid hormone that
binds to the glucocorticoid receptor (GR), a transcription factor found in cells throughout the
body. Once bound by cortisol, the GR regulates transcription of a complex network of stressresponsive genes and the resulting physiological functions, including immunity and
inflammation. This mechanism is conserved in Zebrafish, a small vertebrate that has emerged
as a useful model organism for the study of immune system development. Our lab has shown
that zebrafish embryos directly exposed to elevated cortisol during early development mature
into adult fish with immune system dysregulation, and our research is now focused on
determining the specific cellular, molecular, and genetic mechanisms of this altered immune
function.
The role of CD40 in calcitonin gene related protein (CGRP) mediated antiviral effect in glial
cells
Grlickova-Duzevik, E. ,1,2 , Cao , L., 1,2
1Graduate School of Biomedical Sciences and Engineering, University of Maine, Orono, ME
04469 2Biomedical Sciences, University of New England, Biddeford, ME 04005
[email protected]
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LP-BM5 is a retrovirus that induces peripheral neuropathy and AIDS like immune deficiency in
B6 mice. Upon infection, primary sensory neurons in the spinal cord express elevated levels of
calcitonin gene related protein (CGRP). Previously, we showed that CGRP induced reduction of
LP-BM5 viral loads in primary mixed glial cells. The purpose of this current study was to
investigate possible CGRP downstream targets that may contribute to its antiretroviral
response. We hypothesized that CGRP decreases the viral load in glial cells by modifying the
CD40 expression; increasing in CD40 signaling may promote production of cytokines and
chemokines with antiretroviral properties. Using CD40 knockout mice we showed that CD40 is
required for CGRP mediated antiretroviral response in mixed glia. In microglial cell line, CD40
expression was elevated early after treatment with CGRP. While LP-BM5 increased microglial
CD40 expression gradually over time up to 7 days post-infection in primary mixed glia, CGRP
caused an early elevation (24 hr post-infection) of microglial CD40 expression in microglia and
stayed relatively steady levels 7 days after infection. Chemokine levels remained unchanged
upon infection and CGRP treatment. Future studies will be directed to specifically delineate
CD40 downstream target involved in antiretroviral response in glial cells.
Substrain differences in ethanol preference and running-wheel activity in C57BL/6J and
C57BL/6N mice.
Hartmann, M.1,2, McCulley III, W.2, Holbrook, S.3, Kumar, V.4, Rosenwasser, A.1,2,3
1
Graduate School of Biomedical Science and Engineering, 2Department of Psychology, 3School
of Biology and Ecology, University of Maine, Orono, ME, USA, 4The Jackson Laboratory, Bar
Harbor, ME, USA
While the C57BL/6 (B6) mouse originated at The Jackson Laboratory (JAX), separate breeding
colonies are now maintained at various sites, resulting in genetic drift that has led to both
genotypic and phenotypic differences among these colonies. Researchers currently recognize
two distinct substrains, C57BL/6J (B6J) and the C57BL/6N (B6N). B6 mice typically display high
levels drug self-administration, making them ideal model organisms for addiction research. B6J
mice typically display higher levels of ethanol preference and increased sensitivity to
psychostimulants relative to B6N. In the present study, we assessed ethanol preference
drinking and running-wheel activity in B6J and B6N mice from JAX. B6N mice displayed higher
levels of running-wheel activity, while B6J mice show greater ethanol preference. Subsequent
research, utilizing the CRISPR-Cas9 system, will examine whether these phenotypic differences
depend on similar or different genetic polymorphisms relative to those suggested to underlie
substrain differences seen in responses to other drugs of abuse.
Mechanistic distinctions between cancer-induced ongoing pain and movement-triggered
breakthrough pain
Joshua Havelin1,3, Jonathan Gentry1, Michael Dearborn1,2, Cosmin Iocaban2, Ian Imbert1,2,
Tamara King1,2,3
1Center for Excellence in the Neurosciences, University of New England, Biddeford, ME, 2College
of Osteopathic Medicine, University of New England, Biddeford, ME, 3Graduate School of
Biomedical Sciences and Engineering, University of Maine Orono, Orono, ME
[email protected]
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Cancer-induced bone pain is described as moderate to severe persistent pain that is often
controlled with opioids (e.g. morphine, fentanyl). While ongoing pain is managed with mu
opioid receptor (MOR) agonists, patients experience pain that "breaks through" (BT) their
medication, indicating that there may be distinct mechanisms mediating ongoing and BT pain.
We examined whether ongoing and BT pain are mediated by distinct populations of nociceptive
fibers in a rat model of cancer-induced bone pain. Ablation of TRPV1 expressing fibers failed to
block BT pain whereas ablation of IB4-binding fibers blocked BT pain. Systemic administration
of MOR agonists blocked ongoing pain but not BT pain. In contrast, movement-triggered BT
pain was blocked by administration of a DOR agonist. These observations indicate separate
populations of nociceptive fibers mediate ongoing and BT pain and indicate that DOR agonists
may block BT pain. This work was supported by NIH COBRE grant P20GM103643, Dr. Ian Meng.
Lithium Treatment and Impact on Health in Alzheimer’s Disease
Hayes. M1, Howard A.1
1Univeristy of Maine at Augusta, Augusta, ME
[email protected]
People are living longer than ever, but with increased lifespan also comes an increase in aging
associated diseases, particularly those associated with cognitive decline. Lithium is a common
cognitive drug used in the treatment of bipolar disease depression and schizophrenia. Recent
research has determined that lithium can increase the lifespan of model organisms, such as
Caenorhabditis elegans, but the impact on healthspan has not been determined. We assessed a
C. elegans strain modeling human Alzheimer’s disease during lithium treatment and monitored
overall health of the organism throughout its lifespan. The results thus far suggest that
although lithium increases lifespan it does not promote an increase in health for this disease
model.
Discovering the effects of TNFAIP8L1 on tumor growth in zebrafish.
Hayes, J.1, Mayhue, S.2, Sullivan, C.2
1
Department of Molecular and Biomedical Sciences, 2Graduate School of Biomedical Science
and Engineering
[email protected]
Lung cancer is the leading cause of cancer-related deaths in the United States. Recently, four
members of the human TNFAIP8 gene family (TNFAIP8, TNFAIP8L1, TNFAIP8L2, and TNFAIP8L3)
have been linked to multiple cancer types, including lung cancer. While little is known about the
TNFAIP8L1 gene, one report demonstrated a tumor suppressing role in hepatocellular
carcinoma. We are designing a zebrafish tumor xenotransplantation model with human NCIH1299 non-small cell lung cancer cells in an attempt to better understand the contribution
TNFAIP8L1 makes to tumorigenesis. We are developing flow cytometry-based in vivo
proliferation assays to test the effects of TNFAIP8L1 overexpression on tumorigenesis. We are
complementing these studies with in vitro proliferation assays intended to measure the effect
of overexpression on tumor cell growth and viability. A comprehensive understanding of
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TNFAIP8L1 function in tumor development may lead to the identification of alternative drug
targets that could reduce lung cancer mortality rates.
Extending aging in C. elegans
Henderson, C. Paluszek, M. Hannigan S. Sanborn, D. Laurence Montagne, M.
University of Maine at Fort Kent: Biological Sciences, Fort Kent, Maine
[email protected]
Aging is the birth and death of your cells; this is the cause of the eventual breakdown of an
organism's body. It used to be believed that that ageing was something that was incapable of
being stopped.In recent years this has been found that through C.elegans, two genes (daf-2 and
daf-16) have been found to influence the lifespan of these animals.These genes are
developmental regulators, and work as an insulin like system suggesting a programmed
component to ageing. Using this knowledge and research we were able to conduct two
experiments using DNA extraction with a PCR analysis, along with a heat stress worm lysis
analysis. We explored how the results of the alteration of these two genes caused the increase
in stress resistance and the increase of the aging lifespan of the C. elagen worms.
FRMD4B modifies retinal photoreceptor dysplasia by acting on cell-cell junctions
Kong, Y.1, 2, Charette, J.2, Hicks, W.2, Naggert, J.2, Zhao, L.2, Nishina, P.2
1 The Graduate School of Biomedical Science and Engineering, University of Maine, Orono, ME, 2
The Jackson Laboratory, 600 Main Street, Bar Harbor, ME
[email protected]
In this study, we aimed to identify and characterize genetic modifiers of Nr2e3rd7, which are capable
of modifying retinal dysplasia; and to unveil the targets of the modifiers that suppress the dysplastic
change.
B6.Cg-Nr2e3rd7/J mice, which lack functional Nr2e3, a transcription factor important for
photoreceptor development, were chemically mutagenized. Their G3 offspring were screened by
indirect ophthalmoscopy to detect mutants with reduced retinal fundus spotting. Quantitative trait
locus analysis combined with high-throughput sequencing was used to identify the mutant loci.
Subsequently, the modifier allele was transferred from the B6.Cg-Nr2e3rd7/J strain by crossing to
B6.Cg-Nrltm1Asw/J homozygous mice, which are deficient in Nrl, another transcription factor critical in
the developmental pathway of photoreceptor cells for the purpose of validating the modifying effect
of the identified allele. Finally, immunostaining of cell junction-associated molecules, including
zonula occludens (ZO)-1, β-catenin and cadherins, etc. was performed to reveal the underlying
anomaly associated with the retinal dysplasia in Nr2e3rd7 and Nrl-/- mice, and the molecular targets of
the identified modifier.
Firstly, Tvrm222 line, homozygous Nr2e3rd7 was established due to its heritable reduction of
retinal spots. It was determined that Tvrm222 mice carry a missense mutation in the Frmd4b gene on
Chromosome 6, leading to a serine to proline substitution. Morphologically, retinal folds associated
seen in Nr2e3rd7 retinopathy were suppressed in the Tvrm222 mice. Moreover, a pronounced
reduction in the number of retinal spots was also observed in Nrl-/- mice carrying Frmd4bTvrm222.
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Likewise, the retinal pseudo-rosettes apparent in Nrl-/- mouse retinas were markedly suppressed.
Finally, immunostaining revealed a discontinuous external limiting membrane (ELM), in both Nr2e3rd7
and Nrl-/- mice, whereas the integrity of the ELM was restored in each by the presence of the
Frmd4bTvrm222 allele.
In conclusion, the photoreceptor dysplasia due to genetic flaws in both Nr2e3rd7 and Nrl-/mice is associated with fragmented ELM. Frmd4bTvrm222 restores the ELM integrity and suppresses
dysplasia by acting on the cell-junction molecules. This suggests that fragmentation of the ELM
underlies the retinal dysplasia observed in both of these models.
Caffeine’s effect on aging and progression of Huntington’s disease
Masha LaChance1 and Amber Howard1
1University of Maine Augusta
Advanced age increases the incidence of neurological impairment and disease. Although there
are many drugs designed to improve cognitive function, the health impact of prolonged usage is
unclear. In model organisms, such as C. elegans, low doses of the neurological stimulant
caffeine extends lifespan whereas higher doses creates stress. We chronically exposed worms to
a low dose of caffeine and monitored them for endoplasmic reticulum (ER) stress and health
throughout their lifespan. We also examined the effect of caffeine on a worm strain modeling
Huntington’s disease, marked by a fluorescent poly-glutamate aggregation, and monitored
health and aggregate fluorescence. We determined that 5mM caffeine exposure from the
beginning of life slowed down development but reduced ER stress. We also found that chronic
caffeine exposure decreased Huntington’s disease progression.
Characterization of LincRNA dysregulation upon Pseudomonas aeruginosa infection in Danio
rerio
Longfellow, J.1, Sullivan, C.1, King, B.1, Kim, C.1, 2
1Department of Molecular and Biomedical Sciences, University of Maine, Orono, ME 2Graduate
School of Biomedical Sciences and Engineering, University of Maine, Orono, ME
[email protected]
LincRNAs are involved in many important biological processes. The Kim lab is particularly
interested in the role of lincRNAs in response to Pseudomonas aeruginosa infection, which is
very common in hospital-bound patients with weakened immune systems and can lead to
sepsis and death. In order to better understand lincRNAs in this role, it is critical to study their
mechanisms in vivo. Danio rerio is an excellent animal model, because it can be genetically
manipulated, it is optically clear, and it relies solely on its innate immune system during early
development.
The Kim lab recently completed RNA sequencing to measure the dysregulation of lincRNAs in
response to P. aeruginosa infection in D. rerio. Following the validation of candidate lincRNAs,
experiments will be performed to better understand the role of lincRNAs in infection. These
finding could further our understanding of innate immunity and potentially lead to better
therapies for P. aeruginosa infection.
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Innate immune response to group B Streptococcus infection in zebrafish larvae
MacIsaac, M.1, Neely, M.1
1University of Maine Department of Molecular and Biomedical Sciences, Orono, Maine
[email protected]
Streptococcus agalactiae is a Group B Streptococcal bacterium that can both colonize the
vaginal and rectal mucosa as a commensal organism as well as cause systemic invasive
infections in older adults and newborns. Since the polysaccharide capsule of a bacterial cell is
important for bacterial virulence, we are investigating how mutations in the CpsA protein of the
capsule of S. agalactiae affect the innate immune response in zebrafish larvae. Characterization
of the innate immune response will verify that the polysaccharide capsule is a valid candidate
for use as a target for antimicrobial therapies and vaccine development. We are injecting
zebrafish larvae with wild-type S. agalactiae and with two strains that carry significantly less
polysaccharide capsule on the outside of the cell. Mortality over a 3-day period is being
recorded, and time-lapse microscopy will be performed to document immune cell recruitment
in response to infection with the different bacterial strains.
A potential correlation between p53 expression and the TNFAIP8 gene family in
tumorigenesis
Mayhue, S.1, Sullivan, C.1
1Department of Molecular and Biomedical Sciences, University of Maine, Orono, ME
[email protected]
Lung cancer diagnoses largely result in a poor prognosis due to less than adequate therapeutic
interventions. Widely used lung cancer therapies, including chemotherapy, radiation, and
invasive surgery, are often too aggressive and frequently result in cancer relapse. Genetargeted therapies lack long-term efficacy due to poorly understood lung cancer-associated
gene mechanisms in tumorigenesis. It has been recently demonstrated that the TNFAIP8 (tumor
necrosis factor alpha induced protein-8) gene family plays a role in tumor development and
metastasis, although the mechanisms by which they participate in tumorigenesis have not been
well established. The TP53 gene is commonly known for its role in tumor suppression. Our lab is
investigating the functional interaction between TP53 (and common mutants found in lung
cancer) and the TNFAIP8 gene family. Newly acquired data will offer a better insight to
improved cancer therapies that will allow for less invasiveness and extend life expectancy.
Research reported in this project was supported by an Institutional Development Award (IDeA)
from the National Institute of General Medical Sciences of the National Institutes of Health
under grant number P20GM103423.
Crosstalk between TGFβ and WNT in CAKUT
McCarthy, S.S.1,2, Oxburgh, L.1,2
1Graduate School of Biomedical Sciences and Engineering, University of Maine, Orono, ME
2Maine Medical Center Research Institute, Scarborough, ME
[email protected]
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Congenital anomalies of the kidney and urinary tract (CAKUT) are a heterogeneous
group of renal malformations resulting from defects in embryonic kidney development. CAKUT
are the primary cause of juvenile chronic kidney disease (CKD) and can necessitate dialysis or
transplantation. Despite its clinical significance, the pathological mechanism responsible for the
majority of CAKUT cases remains unknown.
TGFβ plays a pivotal role in kidney fibrosis through activation of the Smad signaling
cascade. However, the function of TGFβ in the developing renal interstitium is not known. Our
in vivo studies reveal inactivation of Smad4 in kidney interstitial cell progenitors results in
unrestricted proliferation, expansion of the medullary interstitium, and displacement of the
collecting duct epithelium. Previous studies have shown that paracrine WNT signaling from the
collecting duct promotes interstitial cell proliferation. Furthermore, the expanded interstitium
observed in Smad4 mutants displays elevated levels of the WNT pathway components LEF1 and
CyclinD1. These data suggest opposing roles for TGFβ and WNT in regulating interstitial cell
proliferation. To investigate this in vitro, we have developed a novel method to isolate primary
renal interstitial cells. Identifying the mechanism of crosstalk between the TGFβ and WNT
pathways will promote the identification of CAKUT candidate genes and therapeutic targets for
CKD.
Aberrant morphological digit tip regeneration in agr2 KO mice
Miller, J.L.1, Thomas S. Lisse2, Rieger, S.1
1 MDI Biological Laboratory, Kathryn W. Davis Center for Regenerative Biology and Medicine,
Salisbury Cove, ME, 2 The Jackson Laboratory, 600 Main Street, Bar Harbor, ME 04609
[email protected]
Salamanders, such as newts and axolotl, frog tadpoles and zebrafish are the only vertebrates
known to fully regenerate their appendages following amputation. While mammals lack the
inherent ability to regrow their limbs, limited regeneration occurs in embryos and juveniles
upon removal of the distal third of a digit. Limb regeneration involves the formation of a
blastema that harbors dedifferentiated cells, which subsequently redifferentiate to give rise to
a new limb. Blastema formation and proliferation critically depend on nerve reinnervation of
the limb. A protein that has been shown in newts to regulate blastema proliferation in a nervedependent manner is the Anterior gradient protein 2 (Agr2). Here we asked whether Agr2
plays a role in wound healing and digit regeneration in mice. We show that Agr2 appears to
control wound repair but its function in digit regeneration remains to be established. Future
studies are planned to further assess this question.
A Personalized Gene Therapy Approach for Charcot-Marie-Tooth Disease Type 2D
Morelli, K.1 2, Pyne, N.3, Fowler, A.3, Harper, S.3, Burgess, R. 1 2
1 The Jackson Laboratory, Bar Harbor, ME, 04609, USA
2 Graduate School of Biomedical Science & Engineering, The University of Maine, Orono, ME,
04469, USA
3 Center for Gene Therapy, The Research Institute at Nationwide Children's Hospital, Columbus,
Ohio, USA
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[email protected]
Dominant mutations in GARS (glycyl-tRNA synthetase) cause Charcot-Marie-Tooth Disease Type
2D (CMT2D), a dominantly inherited axonal neuropathy. Although the mechanisms through
which mutant forms of GARS cause axon degeneration remain controversial, preliminary data
from CMT2D patients and mouse models of the disease suggest that the expression of mutant
GARS may cause toxic gain-of-function effects in peripheral nerves. As such, the selective
silencing of mutant GARS expression should benefit patients with this disorder. In response, we
have developed a gene therapy strategy that reduces the expression of mutant Gars transcripts
through allele-specific RNAi. Importantly, in vivo we found that, when injected at birth, our
mutant-allele targeting gene therapy significantly improves gross motor function and prevents
axon atrophy in an established mouse model of CMT2D. These data confirm that our gene
therapy approach can prevent the onset of CMT2D.
Hp1bp3 mediates neuronal excitability and hippocampus-dependent memory
Neuner, S.M.1,2, Ding, S.2, O’Connell, K.M.S2, Kaczorowski, C.C2
1 University of Tennessee Health Science Center, Memphis, TN, 2 The Jackson Laboratory, Bar
Harbor, ME 04609
[email protected]
Recently, our lab functionally validated heterochromatin protein 1 binding protein 3 (Hp1bp3)
as a novel regulator of cognitive aging using a combination of forward genetics and a global
knock-out mouse model. To test the hypothesis that this effect was mediated via loss of
hippocampal rather than peripheral Hp1bp3, we performed a targeted knockdown of Hp1bp3.
Adult mice receiving bilateral hippocampal injections of Hp1bp3 shRNA exhibited impairments
in both working and long-term memory. These mice also exhibited decreased hippocampal
neuronal excitability as evidenced by an increase in the slow after-hyperpolarization (sAHP).
Changes in hippocampal neuronal sAHP plasticity have been posited to influence information
storage, providing a candidate mechanism for Hp1bp3-mediated changes in cognitive function.
In addition, hippocampal sAHP plasticity is disrupted in mouse models of Alzheimer’s disease,
suggesting Hp1bp3 may play a role in AD-related cognitive deficits. Ongoing work in the lab is
investigating the role of Hp1bp3 in AD.
The Hedgehog and Retinoic Acid cell signaling pathways interact to influence early Danio
rerio odontogenesis
Park, C.1, Jackman, W.R.1
1Department of Biology, Bowdoin College, Brunswick, ME
[email protected]
Hedgehog and Retinoic Acid signaling are required to direct proper vertebrate odontogenesis.
However, few studies of early tooth development have investigated how conserved signaling
pathways interact to influence normal odontogenesis and have instead primarily characterized
gene expression following manipulation of individual pathways. We here report that
simultaneous overactivation of Hedgehog and Retinoic Acid signaling during early zebrafish
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(Danio rerio) development produces a relatively normal dentition, as opposed to the
supernumerary tooth phenotype induced by overactive Retinoic Acid signaling alone. We also
find that overactive Retinoic Acid signaling upregulates Ptch2 expression, a marker of Hedgehog
activity, suggesting that Retinoic Acid regulates Hedgehog signaling during normal tooth
development. Furthermore, overactive Retinoic Acid signaling appears to permit cells of dental
tissue to alter phenotype in response to increased Hedgehog signaling, which by itself does not
appear to affect normal tooth development. These results implicate cell signaling interactions
as essential to normal organogenesis.
Epidermal damage as underlying cause of paclitaxel-induced peripheral neuropathy
Pellegrini, A.1, Bolduc, J.1, Dominy J.1, Rieger, S.1
¹MDI Biological Laboratory, Kathryn W. Davis Center of Regenerative Biology and Medicine, 159
Old Bar Harbor Road, Maine 04672, [email protected]
Paclitaxel is a microtubule (MT)-stabilizing chemotherapeutic agent that, despite its
effectiveness, also damages the axons of peripheral sensory neurons. Mammalian studies
suggest that neuron-intrinsic damage may be a cause of axon degeneration, but no direct
evidence has yet been established. We assessed MT stabilization in live zebrafish using the MT
cap-end protein EB3:GFP to visualize MT dynamics and with an antibody detecting
detyrosinated MTs. This revealed that paclitaxel preferentially stabilizes MTs in keratinocytes,
consistent with our previous findings that paclitaxel preferentially damages the epidermis. We
previously also showed that epidermal damage is mediated by upregulation of MMP-13, a
collagenase degrading primarily basement membrane (BM) proteins. Consistently, we now find
that paclitaxel treatment causes defects in the epidermal BM, which is traversed by
unmyelinated sensory axons. MMP-13 upregulation appears to be caused by oxidative stress
formation in the skin. Future studies will assess the molecular mechanisms underlying
paclitaxel-dependent MMP-13 regulation.
sPQN-75 is secreted from the pharyngeal gland cells of C. elegans and is dispensable for
germline development
Jesse Rochester, Paige Tanner, Kevin Strange, & Dustin Updike
The Mount Desert Island Biological Laboratory
Presenting Author: jrochest.mdibl.org
Corresponding Author: [email protected]
C. elegans P granules are dynamic RNPs that regulate translation in the germline. Disordered
phenylalanine-glycine (FG) repeats within P granules create a phase that extends the size
exclusion properties of the nuclear pore complex into the cytoplasm. A mis-sense allele of pqn75, which encodes a novel protein with both polyQ and FG peptide repeats, was isolated in a
mutagenesis screen for factors that affect P-granule assembly; however, the P-granule
phenotype was later attributed to a non-sense mutation in a linked gene. Using CRISPR, we
tagged endogenous pqn-75 with GFP and found no evidence of germline expression. PQN-75
expression was found to be in all G1 and G2 pharyngeal gland cells and associated excretion
ducts, thought to aid in digestion, lubrication of bacteria or pharyngeal lumen, cuticle
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formation, and disassociation during larval stage transition periods. Here we report our
progress to understand the consequence of unchecked development in pqn-75 mutants.
Beat the Heat: Worming Your Way to Longer Life
Tilley, L.1, Barbosa, S1., Carpenter, S. 1, Kimball, A. 1, Sewell, J1, Dube, E.1, Postell, M.1 Hannigan,
S.2, Henderson, C. 2 Montagne, M. L. 2, Paluszek, M. 2, Sanborn, D. 2, Theriault, J. 2, Rogers, A.3,
Roe, J.L. 1
1University of Maine at Presque Isle, Presque Isle, ME, 2University of Maine at Ft. Kent, Ft. Kent,
Maine, 3MDI Biological Laboratory, Salisbury Cove, ME.
[email protected]
The roundworm Caenorhabditis elegans is used as a model system to study the biology of
human health because their genes and biochemical pathways are similar to those found in
humans. A worm’s tolerance to stress is reduced when given excessive nutrients and increased
when caloric intake is restricted. By modifying genes used in nutrient-sensing pathways, the
animal’s lifespan can be affected, and observing these changes may allow further
understanding of the genetics of human aging and the role of nutrition in longevity. RNA
interference targeting the daf-2/insulin/IGF-1 pathway was employed by feeding worms with
gene expression-altering bacteria. Worms were then exposed to heat stress and their survival
was measured. The prolonged survival of daf-2 inhibition may be attributed to an increased
expression of stress-resistance genes promoted by DAF-16.
CRISPR-Based, Germline Specific Protein Overexpression and Visualization in C. elegans
Sharp, C.1, Updike D.1
Mount Desert Island Biological Laboratory, Salisbury Cove, ME1
[email protected]
Currently, systems of protein overexpression do not exist for the C. elegans germline.
Traditionally, transgenic expression in the germline utilizes extrachromosomal arrays that
become silenced. Our goal is to provide a more targeted method that consolidates effective
overexpression and fluorescence of desired proteins in the germline of C. elegans. GLH-1, which
is expressed abundantly in the germline, is linked via bicistronic spacer to achieve co-expression
with a target protein and GFP in our base plasmid PDU92, allowing for germline exclusive
protein overexpression and fluorescence. Red and blue plasmid variants are currently in
production to allow for greater versatility in imaging and expressing multiple proteins
simultaneously. All alternate color plasmids produced from the PDU92 base retain the same
restriction sites surrounding the target protein for overexpression. Culmination of the project
will result in a toolkit of three vibrantly colored, easily customizable plasmids that over-express
a target protein exclusive to the germline.
Discovery and genome analysis of L2 cluster mycobacteriophage Miley16
Shevenell, B.1, Michaud, E.1, Doty, J..2, Breton, T.2
1University of Maine at Farmington, Farmington, Maine, 2 MDI Biological Laboratory, Kathryn
W. Davis Center for Regenerative Biology and Medicine, Salisbury Cove, ME
17
[email protected]
Mycobacteriophage research helps to develop a better understanding of evolution and could
lead to medical advancements, yet these viruses are amongst the least genetically understood
organisms. This study aims to increase that limited knowledge by investigating the novel phage,
Miley16. Miley16 was isolated from an environmental sample, and its DNA was sequenced and
annotated to further understand the genome. Miley16 is an L2 cluster siphoviridae phage
consisting of 76,653 bp, 133 genes, and 12 potential tRNAs. The genome has been analyzed and
was found to contain one ribosomal frameshift mutation associated with a tail assembly
chaperone sequence. Completed research will provide information regarding
mycobacteriophage genetic diversity and further our understanding of genome evolution.
In vivo translational profiling of motor neurons in two mouse models of Charcot-Marie-Tooth
disease Type 2D suggests impaired translation and mitochondrial dysfunction.
Spaulding, E.1,2, Burgess, R.1,2.
1The Jackson Laboratory, Bar Harbor, USA; 2Graduate School of Biomedical Sciences and
Engineering, The University of Maine, Orono, USA.
[email protected]
Charcot-Marie-Tooth disease (CMT) is a collection of debilitating peripheral neuropathies
caused by mutations in over 80 genes. Disease heterogeneity and the technical challenge of
studying the mammalian peripheral neuron in vivo contribute to the lack of a cure. Dominant
mutations in glycyl tRNA synthetase (GARS), an enzyme crucial for translation, cause CMT Type
2D (CMT2D). We are using two in vivo, cell type and compartment-specific approaches to
profile translation in motor neuron cell bodies and axons of two mouse models of CMT2D: (1)
non-canonical amino acid-tagging (NCAT) provides the location, identity, and quantity of newly
translated proteins, and (2) ribosome-tagging catalogs ribosome-associated RNA. Our NCAT
studies suggest that CMT2D motor axons show a regenerative phenotype, therefore, we are
profiling regenerating wild-type motor neurons after sciatic nerve crush for comparison against
mutant Gars samples. Our data have confirmed impaired translation in CMT2D motor neuron
cell bodies, and further suggest mitochondrial dysfunction.
Using CRISPR/Cas9 technology in a classroom context to modify the P-Granule gene (PGL-2) in
Caenorhabditis elegans
Stinson, J1, mealey, H.1, Adkins, E.2, Breton, T.1, Updike, D.2
1University of Maine at Farmington, ME, 2Biological Laboratory, Kathryn W. Davis Center for
Regenerative Biology and Medicine, Salisbury Cove, ME
[email protected]
Mount Desert Island Biological Laboratory (MDIBL) conducts the IDeA Network of Biomedical
Research Excellence short courses, offering students opportunities to conduct research in a
professional setting. University of Maine system undergraduates worked with Updike
Laboratory at MDIBL to evaluate the success of a CRISPR/Cas9 experiment aimed at inserting a
degron sequence into the Caenorhabditis elegans germ granule (P-granule)-associated gene,
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PGL-2. Research methods carried out, including isolating dumpy worms that had been injected
using CRISPR/Cas9 technology, extracting DNA from individual worms, performing PCR using
primers that flank the putative gene insert, and visualizing product sizes by running agarose gel
electrophoresis, found that CRISPR/Cas9 did modify the PGL-2 gene. The PCR product was 791
base pairs long. Sequencing by the Updike Lab confirmed the degron sequence was present.
Using this gene, research will be done to address questions on P-granules and their relationship
with totipotency and germline properties.
Claw force measurements of four genetically different populations in the invasive European
green crab, Carcinus maenas
Stumper, J.1, Frederich, M.1
1 Marine Science Center, University of New England, Biddeford, ME
[email protected]
Multiple invasions of Carcinus maenas, have led to genetically different populations on the east
coast of North America and within its native range. Previous work has shown that green crabs
from Maine, Nova Scotia, Newfoundland, and Iceland have different stress tolerance and
behavior. Crabs from Nova Scotia are more aggressive than any other crabs. However, the more
docile Maine crabs caused more damage to eelgrass plants in an artificial mesocosm.
To correlate aggressiveness and eelgrass destruction to claw force we measured claw pinch
force of green crabs from 4 populations using a Tekscan Flexiforce sensor. Peak claw force was
positively correlated to claw volume in all 4 populations, but no difference between the
populations was found. Average peak force of all crabs did not differ among populations.
Elucidating the population specific differences in this invasive species can help predict damage
of future invasions or range expansions of green crab populations.
Assessment of methods for fluorescent retrograde labeling of corneal neurons
Bushey,W.1, Sullivan,C.2, Mecum, N.3, Meng,I.1,2
1 College of Osteopathic Medicine, University of New England, Biddeford, Maine
2
Graduate School of Biomedical Sciences and Engineering, University of Maine, Orono, Maine
3 Department of Molecular & Biomedical Sciences, University of Maine, Orono, Maine
[email protected]
The overall goal of this study is to specifically label corneal sensory neurons within an adult
mouse trigeminal ganglion. Correct identification of corneal cell bodies is critical for
downstream experiments that require accurate isolation of this cell population. In this study
we evaluated the efficacy of retrograde delivery methods for FM-143 and Fluoro-Gold when
applied to the cornea. Two methods were evaluated for each tracer and fluorescent labelling
specificity was compared. One method used iontophoresis to deliver electrically charged tracer
molecules to the cornea and drive their transport across the epithelium. The other used direct
application of Fluoro-Gold to the cornea after removing the epithelium with n-heptanol or
direct application of FM-143 followed by thermal and mechanical stimulation of the
cornea. Each of these methods offers advantages and disadvantages.
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Determining Role of LytR Protein in Pathogenicity of Streptococcus agalactiae
Thompson R.1, Neely M.1
1University of Maine, Department of Molecular and Biomedical sciences, Orono, Maine
[email protected]
Streptococcus agalactiae is a bacterial pathogen that can exist in our normal microbiome, only
causing disease when it gains access to certain tissues. To find ways to combat these infections,
it is important to determine the bacterial defenses which allow it to evade our immune
responses. Because of the importance of the capsule in the establishment of infection, we are
researching the function of two proteins, LytR and CpsA, which are involved in cell envelope
regulation and stability. We will be investigating how the presence or absence of the proteins
affects the recruitment of immune cells in systemic and localized infections in zebra fish larvae.
Results from this study will provide new information on how the S. agalactiae cell envelope
protects the bacteria from immune responses.
Assessing Gabapentin’s Influence on Health in Aging and Huntington’s Disease
Thor, Nallie1 and Amber Howard
1University of Maine, Augusta
Huntington’s disease is a progressive genetic disorder causing abnormal protein aggregation of
polyglutamine in the basal ganglia, the part of the brain responsible for voluntary motor
control, cognition, learning, and emotion. A decrease in the levels of inhibitory
neurotransmitter GABA, commonly found at neuromuscular junctions, is also involved in the
disease process. Gabapentin is an anticonvulsant medication that may act as a neuroprotective
agent and is structurally similar to GABA. We hypothesized that gabapentin would promote
health and reduce Huntingon’s disease progression. Mobility assessments were performed
using C. elegans normal and Huntington’s disease strains after 24 hours of gabapentin exposure
beginning at young adulthood and reassessed after a week of drug exposure. Results thus far
indicate that gabapentin does not improve motor control during the normal aging process or
for Huntingon’s disease.
The Effect of Kombucha and Mating Status to Modulate Ethanol-Induced Sedation in
Drosophila
Venditti, L., Lukic, T., Ahmad, S.T.
Department of Biology, Colby College, Waterville, ME.
[email protected], [email protected]
Alcohol addiction is characterized by loss of functionality in everyday life. In Drosophila,
ethanol consumption can cause disruption in physiological and behavioral processes. Using
sedation and tolerance assays we can measure the effectiveness of both pharmacological and
behavioral regimens on ethanol-induced sedation. Using a Drosophila model system we are
investigating if a Kombucha-rich diet modulates ethanol-induced sedation. Kombucha is a
beverage that is fermented in sugared black tea with acetic acid bacteria and yeast. This
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beverage has many claimed health benefits due to its antioxidant properties. This study will
help in characterizing the beneficial effects of including antioxidants in diet. In addition, we are
investigating the possibility that mating status affects ethanol sensitivity. We hypothesize that
virgin flies will exhibit decreased ethanol sensitivity in comparison to mated flies. Preliminary
data shows that both virgin male and female flies expressed decreased ethanol sensitivity
compared to mated females and males. Moreover, this link between mating status and ethanol
sensitivity in Drosophila depends on sex. Female flies sedate faster than male flies and this
pattern is seen in the case of both virgin and mated flies. This study will improve our
understanding of how ethanol sensitivity changes and which factors influence this change.
Study of functional conservation between the zebrafish and human orthologs of the
TNFAIP8L3 gene
White, B. and Sullivan, C.
Department of Molecular and Biomedical Sciences, University of Maine, Orono
[email protected]
The TNFAIP8L3 protein, a member of the tumor necrosis factor-alpha-induced protein 8
(TNFAIP8) family, is known to affect tumorigenesis by increasing phosphoinositide signaling in
cancer cells. Through recent analysis of conserved synteny, we identified a zebrafish ortholog to
the human TNFAIP8L3 gene. We are determining if the zebrafish ortholog functions in a similar
manner to the human protein so that we can model the role the zebrafish tnfaip8l3 gene plays
in tumorigenesis. To investigate the similarities between the zebrafish and human orthologs,
stable transgenic H1299 lung cancer cells designed to overexpress zebrafish Tnfaip8l3 protein
were transplanted into the yolk sacs of 48 h post-fertilization zebrafish embryos. In vivo
proliferation assays are being developed to observe the effects of zebrafish Tnfaip8l3
overexpression on tumorigenesis. If functional conservation with human TNFAIP8L3 is
observed, it will be possible to develop alternative cancer models to study tumor angiogenesis
along with tumor cell migration and metastasis.
Research reported in this project was supported by an Institutional Development Award (IDeA)
from the National Institute of General Medical Sciences of the National Institutes of Health
under grant number P20GM103423.
Elucidating the viral mechanisms of JC polyomavirus infection and disease progression in
astrocytes
Wilczek, M.1 and Maginnis, M.1
1University of Maine, the Department of Molecular and Biomedical Sciences, Orono, Maine
[email protected]
JC polyomavirus (JCPyV) infects 50% of the population and causes a persistent infection in the
kidney. In immunocompromised individuals, the virus can spread to the central nervous system,
causing a disease known as progressive multifocal leukoencephalopy (PML). There is no
treatment for PML and the disease is fatal within 1-2 years. It is thought that glial cells,
astrocytes and oligodendrocytes, are the predominant cell types infected in the brain. The
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mechanisms by which astrocytes become infected and lead to PML pathogenesis are poorly
understood. The objective of this project is to illuminate the mechanisms of JCPyV infection
utilizing an in vitro approach to characterize infection in primary human astrocytes (PHAs).
Interestingly, we have found differential expression of viral early and late genes in PHAs as
measured by immunofluorescence and qPCR. This research will provide a better understanding
of the role of astrocytes in JCPyV infection and PML pathogenesis.
NAD+ supply critically modulates mitochondrial vulnerability and neurodegeneration in aged
mice
Pete A Williams*, Jeffrey M Harder, Nicole E Foxworth, Kelly E Cochran, Vivek M Phillip, Simon
W M John
[email protected]
Affiliations: The Jackson Laboratory, Bar Harbor, ME, 04609, USA.
Glaucomas are common neurodegenerative diseases that cause vision loss, especially in the
elderly. The mechanisms initiating glaucoma and which drive neuronal vulnerability during
normal aging are unknown. Using RNA sequencing and other molecular methods to study
glaucoma-prone mice, we show that mitochondrial abnormalities are an early driver of
neuronal dysfunction, occurring prior to detectable degeneration. Importantly, retinal levels of
nicotinamide adenine dinucleotide (NAD+; a key molecule in energy and redox metabolism)
decrease with age. These decreases render aging neurons vulnerable to disease-related insults.
Supporting this, oral administration of the NAD + precursor nicotinamide (vitamin B3), and/or
gene therapy (driving expression of Nmnat1, a key NAD+ producing enzyme), was profoundly
protective (93% of eyes having no glaucomatous neurodegeneration). My findings encourage
the therapeutic use of targeted dietary supplements to prevent glaucoma and other agedependent neurodegenerations.
Defensive Mechanism of Epidermal Growth Factor Receptor in Host Response to Infection
Zwirner, Christian1
1The University of Maine
This project involves an analysis of the defensive roles of the epidermal growth factor receptor
(EGFR) in response to infection by Candida albicans, a commensal fungus. The ultimate goal is
to answer questions about how EGFR protects against mucosal candidiasis (C. albicans
infection) using a zebrafish model developed in the Wheeler lab at the University of Maine. It is
speculated that EGFR regulates epithelial barrier homeostasis, upregulates mucus production,
and recruits neutrophils; however, very few studies have been conducted to support these
claims. Preliminary experiments performed in zebrafish show that C. albicans causes greater
mortality following the inhibition of EGFR and cell-to-cell linkages may be affected. The
continuation of this research will involve progressing data involving EGFR’s role in epithelial
barrier strengthening as well as the investigation of mucus production and neutrophil
recruitment. Understanding the defensive mechanisms of EGFR could improve treatments
against C. albicans infections and fungal infections in general.
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