2. Current affiliation: Iowa State University, Iowa, USA 3. Research fields

271 Pages·2016·3.36 MB·English

1. Name: Liza Esther ALEXANDER （ID No.: SP16001）
2. Current affiliation: Iowa State University, Iowa, USA
3. Research fields and specialties:
Biological Sciences
4. Host institution: RIKEN- Center for Sustainable Resource, Kanagawa, Japan.
5. Host researcher: Dr. Kazuki SAITO (Group Director), Dr. Yozo OKAZAKI (Research
scientist)
6. Description of your current research
My PhD research is being conducted under the supervision of Dr. Basil J. Nikolau at Iowa
State University (ISU), in the interdepartmental program of Molecular, Cellular and
Developmental Biology. Plant epidermal cells express unique molecular machinery that
juxtaposes the assembly of intracellular lipid components and the unique extracellular lipids
that are unidirectionally secreted to the surface of the plant. The overarching objective of my
project is to functionally characterize the genetic and metabolic networks that interrelate the
intracellular and extracellular lipid metabolism. Physiologically this lipid-trafficking process
is genetically programmed, but can change in response to environmental pressures (e.g.,
drought, temperature, pathogens), making them important to agricultural crop productivity.
Additionally, these lipids are chemically most akin to petroleum hydrocarbons making this
research insightful towards the development of biorenewable fuels and chemicals.
My research builds on past research developments at ISU that has been and continues to
be supported by the National Science Foundation (NSF) and the Department Of Energy
(DOE). Specifically, ISU has developed maize systems for dissecting the genetic and
metabolic networks and technological platforms for analyzing and imaging the metabolic
intermediates of these processes. My research specifically focuses on the functional
characterization of one of these maize gene family in lipid biogenesis. The strategy I am
taking is two pronged: 1) expression of the protein encoded by the maize gene in a
heterologous host that lacks this function; three such hosts are being used, bacteria E.coli,
yeast Saccharomyces cerevisiae, and Arabidopsis mutant lines that carry mutations in a
homologous gene; and 2) characterization of maize plants carrying mutations at the gene. In
both strategies I have generated unique genetic stocks that are being analyzed by targeted and
non-targeted metabolomics analytical platforms. Since the genes I am characterizing are
known to affect extracellular cuticular lipid deposition I use GC-MS analysis to specifically
profile these lipids and ultra-high resolution Fourier transform ion cyclotron resonance mass
spectrometer (FT-ICR/MS) to spatially image metabolites to individual cells, using well
established procedures implemented by the Nikolau group at ISU.
The proteins encoded by these genes express its biochemical function within the cell
interior, specifically in the ER membranes possibly affecting fatty acid elongation. Therefore
more sophisticated analyses are required to comprehensively explore the lipid profiles that
can reveal the relationship between extracellular and intracellular lipid networks. This NSF
EAPSI award in collaboration with the JSPS has provided valuable insights and helped me
expand this area of my PhD thesis.
7. Research implementation and results under the program
Title of your research plan:

Evaluating intracellular and extracellular lipid metabolic networks at the level
of individual cells
Description of the research activities:

The project focused on understanding the molecular genetics and metabolic
relationships between the intracellular and extracellular cuticular lipid networks. The
latter network is restricted to a single cell layer, the epidermis of plant aerial organs.
Genetic stocks of maize and Arabidopsis designed at ISU that revealed changes in the
extracellular lipid profile were used in this study, these include 1) transgenic Arabidopsis
stems carrying specific maize genes 2) maize seedling leaves and 3) maize silks from
inbred lines that showed differential expression in extracellular lipids. The
comprehensive intracellular lipid profiles were analyzed via liquid
chromatography/quadrupole time-of-flight mass spectrometry (LC/Q-TOF/MS) guided
by Dr. Yozo Okazaki, a Research Scientist in the Saito group. Integrating these new
intracellular lipid data with the already generated extracellular lipid data provided
invaluable insights on the interrelationship between the networks as disrupted by genetic
permutations.
The results from the transgenic Arabidopsis stems and maize seedling leaves showed
that the specific maize genes used in the study influenced the intracellular levels of fatty
acids of specific carbon chain lengths namely 30 and 32. The finding was able to support
already generated extracellular lipid data that had shown changes 30 and 32 carbon
length fatty acid derivatives. In addition to the changes in the fatty acid composition,
several other lipid species were also seen to be influenced such as sphingolipids,
glycolipids, acyl glycerols and polyprenols. Studies conducted at ISU by the Nikolau
and Yandeau-Nelson groups have shown that the extracellular lipids are also richly
expressed also in maize silks with a ~5-fold developmental increase in accumulation of
these lipids along the length of the emerging silk. The intracellular profile of these maize
silks also showed differences mainly between the different genotypes but with fewer
differences across the developmental gradient of the silk. Several uncharacterized
metabolites were also seen to be altered in the study and using tools developed at
RIKEN – namely MS-DIAL and MS-FINDER, some of these metabolites have fully
annotated and others partially identified. Although a lot more work needs to be done to
understand the gene functionality, this project has demonstrated the utility of integrating
molecular genetic strategies with advanced instrumentation for biochemical analyses
(i.e., PTV-GCMS, LC/Q-TOF/MS and MALDI-FTICR imaging), and thus generated
new knowledge concerning metabolic networks that occur uniquely within specialized
single cell tissues. A manuscript highlighting these findings is in progress.
8. Please add your comments, including any cultural experience during your stay in
Japan (if any):
Amazing experience at RIKEN and Japan as a whole. The work ethics, hospitality and
friendliness of those at RIKEN and around is something I would treasure forever. The
beauty of Japan lies in its terrains, food, and mostly its people. Festivities celebrated with
fireworks and dances, temple architectures, visits to Hakone and other surrounding areas
along with the exquisite variety of food at every spot have definitely made a mark!
9. Advisor’s remarks (if any):
I, Kazuki Saito hosting Liza Esther Alexander, must say that she made a magnificent job
together with my colleague, Yozo Okazaki, regarding the detailed lipid analysis of her
samples. She has been excellently integrated with our people in RIKEN for the last
several weeks. I am quite convinced she will be able to pave the way to success for her
scientific career in future.
RESEARCH REPORT
1. Name: Matthew Barcus （ID No.: SP16002 ）
2. Current affiliation:
Cornell University
3. Research fields and specialties:

Biological Sciences

4. Host institution: University of Tokyo
5. Host researcher: Prof. Shinya FUSHINOBU
6. Description of your current research
Chicken feathers are a large byproduct from the poultry industry, yet the high protein
content of the feathers (>85%) offer a unique opportunity to be used as an animal feed and
fertilizer, provided they are properly processed. Using enzymes to break down feathers has
the potential to reduce energy requirements during the rendering process and yield a better
end product. Wax esters are present on the surface of the feathers due to the birds
preening, which protects their plumage. By removing the waxy barrier created from
preening, we would expect the feather to be more easily rendered into coproducts. Soil
microorganisms associated with the ability to degrade feathers were screened for lipolytic
enzymes. These enzymes were further tested for the ability to degrade wax esters. The top
performing enzyme was selected to further study its catalytic mechanism. Due to low
sequence similarity with known protein structures, it was necessary to begin crystallization
work in order to gain insight on how the enzyme is able to hydrolyze the bulky,
hydrophobic substrates. Knowledge gained from the study may also provide insight on
how the enzyme can be optimized for chicken feather rendering and many other industrial
processes that have waxy substrates.
7. Research implementation and results under the program
Title of your research plan:

Structure determination and molecular modeling of the catalytic mechanism of a
wax-ester hydrolase isolated from a feather-degrading soil microorganism

Description of the research activities:

The main component to the research project was trying to crystalize the wax-ester
hydrolase. The purified enzyme was subjected to hundreds of different conditions to
find optimal parameters for crystals to grow. From over 400 conditions there were
two hits. Adjustments were made with the concentrations of salt, buffer, and
precipitant in an attempt to grow larger and better crystals. Some crystals were then
tested using an in-house X-ray to determine if the crystals were indeed protein and to
evaluate how well the crystals would diffract. A homology model of the wax-ester
hydrolase was also constructed to provide some insight on the enzyme and serve as
an alternative should the 3D structure not be determined. Furthermore, the enzyme
activities of the wax ester hydrolase were able to be evaluated against different
substrates using thin layer chromatography.

8. Please add your comments, including any cultural experience during your stay in
Japan (if any):
During my time in Japan, not only was I able to experience first-hand what lab culture is
was like in this part of the world, I was also able to visit many wonderful cultural spots in
and around Tokyo. I was able to visit Yamanashi during a lab outing where we enjoyed
onsen at the base of Mount Fuji, picked fresh peaches, and ate delicious houtou. A couple
other weekend trips I was able to see Odawara castle, travel through Hakone, and swim at
the beaches in Shimoda. My time in Japan was also filled with delicious food.
9. Advisor’s remarks (if any)
RESEARCH REPORT
1. Name: Jessica L. BERGMAN （ID No.: SP16003）
2. Current affiliation: California State University Northridge

3. Research fields and specialties:
Biological Sciences

4. Host institution: Tropical Biosphere Research Center, University of the Ryukyus
5. Host researcher: Dr. Saki Harii
6. Description of your current research
My current research focuses on the implications of intraspecific genetic variation of
phenotypic plasticity in a scleractinian coral under pCO conditions predicted to occur by
2
the end of the current century. Using Pocillopora damicornis as a study species, and
growth as a means to evaluate a plastic response in scleractinians, I have tested the
hypothesis that plasticity in growth differs between genotypes. Corals are well-known to
vary in plasticity among genotypes, however identifying those genotypes that differ in
plasticity is critical to testing the association of phenotypic plasticity with tolderance to
ocean acidification. Having identified differences in plasticity of growth among
genotypes, I am testing the hypothesis that under elevated pCO , genotypes with a high
2
phenotypic plasticity grow faster than those with a low plasticity. The result of these
experiments will be explored in a fitness context to evaluate the potential for natural
selection to act on plasticity as a means to respond in a beneficial way to ocean
acidification. This research will be done as part of fulfillment of my master’s thesis.

7. Research implementation and results under the program
Title of your research plan: The effects of climate change on coral larvae in relation

to coral reef connectivity
Description of the research activities: The purpose of my experiment was to focus on

the behavior and physiology of larvae from a common coral (Pocillopora
damicornis) and how these features will be affected by ocean acidification (OA), as
part of a larger objective to better understand how OA and climate change will alter
connectivity among coral reefs and mediate changes in species composition. My
approach tested for the effect of OA on the behavior of larvae in-situ at two different
depths: < 1 meter and 3 meters, over the course of the first 24 hours after larval
release. Additionally, I examined the effect of 24-hour exposure to OA on total lipid
content of the larvae, as a measure of larval physiology. Initial assessment of results
indicate that in-situ, at < 1 m of depth, larval position in the water column differs
significantly by time of day, with larvae tending to swim to the top of the water
column over a 24 hour period. However, at 3 meters, larval position in the water
column varies both with time of day and with treatment conditions. The larvae are
distributed more unevenly than at < 1 m, and can be found more often at the bottom
of the water column than at the top. These differences in behavior may be explained
by differing light conditions at different depths in the water column. Total lipid
content of larvae was not found to differ significantly after 24 hours of exposure to
OA conditions, although it decreased from initial measurements taken on the larvae.
The results of the research activities, and differences in larval behavior between
ambient and OA conditions, will provide insight to a critical knowledge gap of how
position of coral larvae in the water column will be affected by OA. On a larger
scale, they will add to existing research characterizing barriers to connectivity
between populations in future ocean conditions.
8. Please add your comments, including any cultural experience during your stay in
Japan (if any):
This was an incredible research experience – the constant collaboration and endless
academic resources at my fingertips, as well as experiencing a new and fascinating
culture, made the JSPS summer program an enriching and invaluable addition to my
master’s program.
RESEARCH REPORT
1. Name: Dena BLOCK （ID No.: SP16004）
2. Current affiliation: University of California, Berkeley

3. Research fields and specialties:
Biological Sciences
4. Host institution: Nagoya University
5. Host researcher: Professor Kunihiro MATSUMOTO
6. Description of your current research
Trade-offs between reproduction and maintenance of the soma are a general feature of
organismal physiology reflecting choices in energy allocation and affecting stress
resistance and lifespan. How organisms coordinate the complex interplay between
environmental inputs, metabolism, stress response, and reproduction remains unclear.
Members of the transthyretin-like (ttr) gene family of the nematode C. elegans have been
shown to respond to stress and affect lifespan, but the actual roles of these proteins in
worm physiology remain elusive. While little is known about ttrs in C. elegans, the single
vertebrate transthyretin is a carrier of lipophilic vitamins and hormones. I am currently
focusing on the function of one ttr, TTR-1, by evaluating which types of lipids TTR-1 can
bind in vitro. The project will shed light on a gene family of unknown function and its
potential role in integration of stress response and lifespan by hormonal signaling.

7. Research implementation and results under the program
Title of your research plan:

Investigating the role of a putative lipid-binding protein in hormonal signaling and
control of lifespan in the worm C. elegans
Description of the research activities:

In order to asses the lipid binding capabilities of TTR-1, I proposed to express
tagged TTR-1 in HEK293 mammalian cells and purify the protein for use in a
protein lipid overlay assay. In this assay, a lipid-spotted nitrocellulose membrane is
incubated with tagged protein, and protein bound to spotted lipids is detected by
tag-specific antibodies. This assay has already been used to demonstrate binding of
another member of the ttr family, TTR-52, to phosphatidylserine.
Most of the summer was spent optimizing conditions for expression of tagged TTR-1
in HEK293 mammalian cells. I found that I needed to use a specific transfection
reagent, and that TTR-1 is degraded by the proteasome. While treatment with a
proteasome inhibitor allowed expression of TTR-1, immunoprecipitation of the
protein was complicated by poor efficiency of elution from the beads. More work
will need to be done to optimize the expression and immunoprecipitation efficiency
of TTR-1 protein.
I was also able to do some other work characterizing TTR-1, such as examining its
upstream regulation using a TTR-1::GFP translational reporter, and beginning to
construct a transcriptional reporter by fusing GFP to the promoter region of TTR-1.

8. Please add your comments, including any cultural experience during your stay in
Japan (if any):
My experience during this program was invaluable. I am extremely grateful to the
Matsumoto lab for teaching me new techniques in the lab and for making me feel
welcome. I enjoyed many experiences with my labmates, such as going to a summer
festival, going to the Higashiyama Zoo, and eating ramen, mochi, and the Nagoya
specialty Hitsumabushi. I was also able to visit my host family again and go sightseeing
with other JSPS fellows.
9. Advisor’s remarks (if any):
RESEARCH REPORT
1. Name: Jared Broddrick （ID No.: SP16005 ）
2. Current affiliation:
University of California at San Diego
3. Research fields and specialties:

Biological Sciences
4. Host institution: Kwansai Gyakuin University, Sanda, Hyogo
5. Host researcher: MATSUDA Yusuke
6. Description of your current research
Exploring and developing new sustainable resources to counter increasing consumption has
become a focus of research efforts in the academic and private sector. Considerable hope in
meeting these challenges has been placed on leveraging photosynthetic metabolism;
organisms that are able to fix carbon dioxide and subsequently produce energy-dense
products. Modeling photosynthetic metabolic capabilities mandates a systems biology
framework for hypothesis generation, data analysis and simulation of cellular fitness. The
systems biology approach of genome-scale constraint based modeling coupled with flux
balance analysis (FBA) has a proven record of contextualizing organism specific
information and enabling the characterization of cellular. A genome-scale reconstruction,
the first step in genome-scale modeling, is a repository of cellular metabolic functions based
on the genome annotation of the modeled organism. It represents the connectivity of the
metabolic network resulting in a mathematically solvable representation of metabolic
capability. This provides a framework for hypothesis generation through predictive in silico
biology. A genome-scale reconstruction of the model diatom Phaeodactylum tricornutum,
an important photosynthetic marine organism, has recently been completed by the
researcher enabling evaluation of metabolic changes to cellular fitness. During the
development of this metabolic reconstruction, unique aspects of diatom metabolism were
revealed. These aspects were investigated in conjunction with Dr. Yusuke Matsuda at the
Kwansai Gakuin University in Sanda, Hyogo Japan.

7. Research implementation and results under the program