rutgers science review, fall 2012
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Rutgers Science Review, Volume 2, Issue 1 Fall 2012TRANSCRIPT
Rutgers Science Review
Volume 2, Issue 1Fall 2012
Artificial Life:The Next Frontier?
An Electronic SyNAPSE
Eagle Nebula: The Pillars of Creation
Table of Contents
“Killer Cells”: Miniharpoons in Nature
Artificial Life from Synthetic Genomes
An Electronic SyNAPSE
Investigating Commissureless Protein Regulation of Robo Localization in the
Drosophila Embryonic Heart
Indigo-carmine and its Photophysical Properties
pg 6
pg 9
pg 12
pg 19
pg 21
pg 16An Interview With Dr. Yee Chiew
Wild Bee & Honeybee Forage on Sunflower
AboutThe Rutgers Science Review (RSR) biannually publishes student-written scientific features, opinions, and research papers.
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Features EditorsAlexandra DeMaioDeepak GuptaScott KilianskiArvind Konkimalla
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Faculty Adviser:Dr. Steven Brill
Articles
IPS Cells
6 | Rutgers Science Review | Fall 2012
One of the first things that comes to mind when people
think of jellyfish is their sharp and sometimes deadly sting.
These stingers are characteristic of the phylum Cnidaria,
which encompasses about 9,000 living species around
the planet. In addition to jellyfish, corals, anemones, and
many other aquatic organisms belong to this pylum.
Approximately 200 species of jellyfish are known
to exist. Dispersed throughout the world’s oceans, they
are frequently found in warm tropical habitats. Sizes of
jellyfish range from the one centimeter Irukandji jellyfish
to the great Portuguese man-of-war, whose tentacles can
span more than one hundred feet in length. These creatures,
however, are also very simplistic. They are composed of
approximately 95% water, contain no vital organs, and have
little to no control over the movement of their own bodies.
But looks can be deceiving. The jellyfish, like other
cnidarians, are armed with specialized cells that are
more than capable of paralyzing and killing a wide
range of organisms. Thousands of these stinging nerve
cells called cnidocytes are found on the tentacles of
jellyfish. Within each, a specialized capsule called a
nematocyst contains “coiled springs” that deliver the
sting to prey. These nematocysts are stimulated by
chemicals or neural impulses, and fire at approximately
10,000 times the acceleration of a rocket. These “killer”
cells, likened to miniharpoons, are the primary
defensive and offensive mechanisms of the cnidarians.
Nematocysts are one of the defining features of the
“Killer Cells”Miniharpoons in NatureBy: Sean Mascarenhas
Features
Fall 2012 | Rutgers Science Review | 7
cnidarians, and approximately 25
different types have been identified.
Nematocysts are incredibly diverse
and have a wide array of functions,
such as defense, feeding, and adhering
to prey. Although they exist
within cnidocytes, the stinging
cells on jellyfish tentacles,
nematocysts are not strictly
regarded as organelles. They are
secretory products of the Golgi
apparatus, which modifies and
secretes proteins throughout
the cell in sac-like assemblages
called vesicles. Nematocysts
are one of the most complex
secretions of the cell found in
nature, which marks a special
point of interest for the scientific
community. After maturation
and secretion from the Golgi
apparatus, the nematocyst
complex is exported towards
its pre-determined firing site.
A double-layered capsule,
which has a door-like opening
called the operculum,
surrounds nematocysts. Inside
each capsule is a coiled tubule
that is riddled with a vast array
of spines. The deployment of
the tubule can be triggered by
a chemical or physical stimulus, such
as prey brushing against a cnidarian’s
tentacles. Once triggered by an
appropriate stimulus, the operculum
of the capsule opens, and the tubule
is immediately deployed in a twisting
motion towards the prey. Discharge
of the tubule is also facilitated by an
intense increase in osmotic pressure
within the capsule. In association with
the spines, the twisting motion allows
the tubule to penetrate and become
embedded in the prey, after which the
tubule detaches from the capsule. The
entire firing process of the tubule occurs
within nanoseconds and is one of the
fastest reactions in nature. There is also
a great deal debate within the scientific
community concerning whether
nematocysts fire independently
of any control system,
provoking questions about the
control organisms have over
their own tissues and bodies.
Once embedded in the prey,
thousands of nematocysts inject
debilitating chemical agents via
their tubule spines. The type of
toxin used, as well the virulence,
varies among cnidarians.
Certain large jellyfish, such
as Physalia physalis, the
Portuguese man-of-war, use
neurotoxins to induce paralysis
and quickly immobilize their
prey. Additionally, nematocysts
can even remain active long
after their respective hosts
(the jellyfish) have died. For
example, washed-up tentacles
of the Portuguese man-of-
war have been known to
frequently injure beachgoers.
Due to their nematocyst
complexes, certain cnidarians
are widely regarded as some of the
most lethal creatures on earth. The
box jellyfish, which inhabits coastal
waters around Australia, is considered
COcked Nematocyst
Fired Nematocyst
Features
8 | Rutgers Science Review | Fall 2012
to be one of the deadliest marine animals in the world. Its
tentacles, which can grow nine feet long, harbor approximately
500,000 nematocysts filled with enough venom to kill an
adult human in less than three minutes. Venom absorbed
into the body can cause several different systemic effects:
labored breathing, necrosis of the skin, loss of consciousness,
scarring of tissue, cardiac arrhythmia, and cardiac arrest.
In the past decade, the Box jellyfish has claimed
approximately one hundred lives; however, with correct and
timely intervention, there are methods to relieve the effects of
jellyfish stings. Vinegar is one such treatment. Its acidity denatures
the proteins in nematocysts, causing them to lose their initial
conformations and disrupting cellular activity. Because high
temperatures can also denature proteins, a hot water bath may
be able to ease jellyfish stings. It is clear that jellyfish, although
simply designed, can be incredibly dangerous creatures.
Works Cited
Aaseng, Nathan. “Sea Creatures With Stinging Cells.”
Poisonous Creatures. 11. n.p.: Lerner Publishing Group, 1997.
Science Reference Center. Web. 21 Oct. 2012
Cnidarians: Simple Animals With a Sting!. eLibrary
Science. Web. 21 Oct 2012.
Comprehensive Information About Cnidarians. eLibrary
Science. Web. 21 Oct 2012.
“Jellyfish.” Magill’s Encyclopedia of Science: Animal Life.
2001. eLibrary Science. Web. 21 Oct 2012.
“Tentacles and stings.” DK Eyewitness Seashore. 2004.
eLibrary Science. Web. 21 Oct 2012.
Features
Figure 1
Fall 2012 | Rutgers Science Review | 9
Over the past few years, there
has been rapid growth in the largely
uncharted field of synthetic biology.
Synthetic biologists alter organisms’
genes and create synthetic biological
parts to engender new functions.
In 2010, researchers at the J. Craig
Venter Institute created the world’s
first chemically synthetic, self-
replicating organism – a major
milestone marking the first complete
genome replacement. The scientists
not only designed unique techniques
to manufacture the synthetic organism
(nicknamed Synthia), but also inserted
DNA watermarks containing the co-
authors’ names, a website, and several
philosophical quotes, complete with
punctuation. The watermarks were
intended to differentiate the modified
organism from the natural ones and
to exemplify the vast possibilities
within genome reconstruction.
The concept of creating a
Frankenstein cell (a cell whose
“brain,” or genome, has been replaced)
sounds simple enough; however, the
technology and expertise necessary to
do so were discovered only as recently
as 1995. At this time, the Institute of
Genomic Research became the first
to sequence the genome of a living
organism, the Haemophilus influenzae
bacteria.1 Since then, many organisms’
genomes have been sequenced with
exponentially less time and cost. It is
now possible to obtain the sequence
of all one’s genes for about $10,000
– one hundred times less than what
it cost a decade ago. Nonetheless,
although many genome sequences
have been elucidated, researchers
still do not understand even a single-
celled organism’s genes “in terms of
their biological roles.”1 To address this
issue, a Venter Institute team led by
Daniel Gibson set out to craft a cell that
would contain only genes essential for
function – a minimalist cell. In a project
that cost $40 million and took over
twenty scientists ten years to complete,
Gibson and his team were able to
successfully transplant a synthetic 1.08
Mb Mycoplasma mycoides genome into
a Mycoplasma capricolum recipient cell.
Mycoplasma were chosen
for their rapid growth rate and
minimalist genome composition.2 The
sequence of the synthetic genome –
Artificial Life from Synthetic GenomesBy: Apexa Modi
Features
10 | Rutgers Science Review | Fall 2012
the first genome to be created on a
computer – was based on that of the
M. mycoides strain and was altered to
include DNA watermark sequences.
These watermarks, upon translation,
would produce protein sequences
that spell out words and sentences.
The Venter Institute project
established two major objectives:
1. To accurately assemble synthetic
DNA fragments created de novo
(from scratch)
2. To jumpstart or “boot up” the
synthetic genome, creating a fully
functional cell
The final genome was reconstructed
in three stages (Figure 1).1 First, 100 one
kilobase DNA cassettes were chemically
synthesized with fragments of the
final genome and inserted into vectors
consisting of yeast cloning elements.
Each of these cassettes contained an
80 base pair overlap to enable the
original fragments to form larger
10kb fragments. Cassette and vector
assemblages were then recombined in
yeast and transferred to E. coli to obtain
greater DNA yields. All of the fragments
were sequence verified, and any
errors were corrected before second-
stage assembly. These verification
steps were performed frequently to
ensure that the original synthetic
genome remained intact throughout
the synthesis process (Figure 2),5 as
deviation from sequence design would
significantly delay project completion
by hindering synthetic cell survival.
The 111 10kb fragments were then
pooled to produce 100kb assemblies
and extracted directly from the yeast.
Multiplex PCR with 11 primer pairs
(designed to anneal at the eleven 100kb
junctions) were used to screen clones
for the completed genome. Of the 48
clones screened, one (sMmYCp235)
had all 11 desired amplicons, while the
positive wild type control had none.
The results were further verified via a
restriction enzyme double digest; with
two sites encoded into three of the
watermark sequences, yielding unique
restriction patterns to characterize the
altered M. mycoides genome (Figure 3).1
After the genome was successfully
synthesized, it was transplanted into
a bacterial cell. The two mycoplasma
chosen contained 91.5% genome
sequence similarity, reducing the
chance that the recipient cell would
reject this new genome. The recipient
cell’s genome was nullified via low
pH conditions that induced nucleotide
starvation and inhibited the cell’s
ability to perform DNA replication.3
The strain of M. Mycoides with
successfully transplanted genomes
appeared blue on X-gal and tetracyline
Figure 2
Figure 3
Features
Figure 4
Fall 2012 | Rutgers Science Review | 11
plates.1,4 Initial attempts to transfer the
genome failed because the recipient and
donor mycoplasma shared a common
restriction enzyme system. This issue
was overcome by either methylating
the DNA with purified methylases
or by disrupting the recipient cell’s
restriction system. In the final step, one
successful transplant of the sMmYCp235
genome was sequenced to expose
any alterations that the cell may have
undergone. The sequence matched
the intended genome design with the
exception of known polymorphisms,
8 new-nucleotide mutations, an E. coli
transposon insertion, and an 85-bp
duplication. The synthetic sequence
did not contain any genome from
the recipient cell, M. capricolum; the
genome replacement was complete.1
The protocols described above
can now be generalized and are
quickly becoming the fundamental
tools for many other scientists
envisioning genome transplants of
their own. Unfortunately, the Venter
Institute’s success was a double-edged
sword in the biological community;
the benefits of creating synthetic
organisms to improve the world
were counterbalanced by the threat
of misappropriation for bioterrorism.
Because an organism could potentially
be altered to acquire any biological
function, it would be possible to create
genomes for new smallpox viruses
or other diseases.6 Nevertheless,
this technology has springboarded
many beneficial projects, including
microbial hydrogen fuel cells (used
as a source of renewable energy)
and toxin-degrading or medication-
producing organisms (Figure 4).7
Other projects are intended to engineer
organisms to for the production of
detergents, cosmetics, and perfumes.
Though it may seem that scientists
have completely harnessed the powers
of evolution, microbes may still
be subject to the natural evolution
process once they are placed back into
nature, which could potentially render
the organisms harmful. It is clear
that although biologically synthetic
organisms hold great potential for
a healthier planet, a great deal of
additional research must be done
before they are widely commercialized.
References:
1. Gibson, D., John I., Glass, C., et al.
“Creation of a Bacterial Cell Controlled by a
Chemically Synthesized Genome.” Science
329.5987 (2010): 52-56.
2. C. A. Hutchison IIIet al., Global
transposon mutagenesis and a minimal
Mycoplasma genome. Science 286, 2165 (1999).
3. C. Lartigueet al., Genome
transplantation in bacteria: changing one species
to another. Science 317, 632 (2007).
4. C. Lartigueet al., Creating bacterial
strains from genomes that have been cloned and
engineered in yeast. Science 325, 1693 (2009).
Wang, H. “Synthetic Genomes for
Synthetic Biology.” J Mol Cell Biol 2.4 (2010):
178-179
6. Erikson, B. et. al. Synthetic
Biology: Regulating Industry Uses of New
Biotechnologies. Science 333, 6047 (2011):1254-
1256
7. Chang IS, Bretschger O, et al.
“Comparative Microbial Fuel Cell evaluations
of Shewanella spp.” Electroanalysis. 22.7 (2010):
883-894.
Features
12 | Rutgers Science Review | Fall 2012
In the
1960’s, computer
processors were
constructed with hundreds
of transistors – the first was the
calculator, which managed basic
information and produced basic
computations. As science and the
human mind evolved, so did the
meaning and applications of computer
processors. Today, IBM’s SyNAPSE
project aims to replicate the raw
power of a human brain.
Processors are units that
analyze
information, and can be thought of as
the “brains” of computers. Using digital
circuits, they perform arithmetic and
logical operations. In recent times, it is
thought that they can even potentially
be used to replace parts of or perhaps
the entire human brain, which could
be useful in a multiplicity of situations.
For example, once a person is declared
to be in a
comatose
state due to
brain damage,
there are no
known remedies. What
if processors could change this
fate? Processors could be parts of the
brain acting as stem cells, and could
have multiple functions to help the
brain maintain stability even in the
aftermath of neural degeneration
and traumatic brain injury.
Although processors are
amazing and can be mysterious, the
brain is just as intriguing if not more. Not
only does the brain store information,
An Electronic SyNAPSEBy: Bharani Pusukur, Jacqueline King
Features
Fall 2012 | Rutgers Science Review | 13
it is also the center of learning and
comprehending. Although a patient
may have brain damage, he is not
completely disabled. In fact, he is
known to be “superabled”. While some
senses may not function, other senses
are heightened and have even been
proven to be superior. A brain uses
an average of twenty to twenty-five
watts a day, which is enough to power
a light bulb. A common misconception
about computer processors is that
they are faster than the brain because
many computations are made quickly;
however, the brain is much faster
and its processing power cannot be
met. Regardless, with improving
technology, processors could create a
new brain with artificial neurochemicals
to increase neural activity.
The potential IBM’s new
technology has for the future of
computing, as well for neuroscience, is
astounding. Replicating brain functions
such as sensation, perception, and
emotion is a concept just coming to light
in the modern age. For instance, IBM’s
SyNAPSE is an attempt manufacture
an artificial brain, essentially testing
the limit of computing power.
SyNAPSE makes use of integrated
microprocessors and circuits which
replicate the function of various
cortexes and pathways in the brain.
The project can potentially replicate
complex neural pathways of a human
brain such as vision, movement, and
autonomous function. In principle, a
processor could operate a body just like
a human brain. Medically, this could
be useful for patients whose brains
are damaged in certain areas, (e.g. a
patient with a damaged occipital lobe
might be able to have the processor and
artificial SyNAPSEs replace its function
and allow the patient to process and
interpret a visual stimulus). In addition
to mimicking the larger, macro-level
of the brain, synthetic stimulants and
electric circuits could also be used to
substitute specific neurochemicals
and individual synapses.
We have not reached a
point in time where we can say our
microprocessors are as powerful
and as efficient as the human brain.
Although Moore’s law predicts that
The DARPA funded
IBM SyNAPSE project
is attempting to
develop artificial
neural pathways
and create an
autonomous body
able to replicate
human brain function,
including higher levels
of cognition.
Features
14 | Rutgers Science Review | Fall 2012
the transistor count of integrated
circuits doubles approximately every
18 months, the amount of time it would
take for the processor to work at the
level of the brain appears to be far in
the future. Technology is no longer
improving at a steady rate, which
makes it more challenging to predict
when, and if, we can ever replicate
a human brain. Although the task of
creating a microprocessor to work at
an entire human brain’s level appears
to be daunting, the rewards outweigh
the obstacles. There is the potential of
finally creating an autonomous being
that not only talks and sees just as
we do, but also thinks and expresses
dynamic human emotions fluidly.
Though SyNAPSE is just starting
out, it has the capability to innovate
a new industry of artificial brains
and processors. Stem cells, brain
damage, and neural pathways are all
potentially reparable through the use
of microprocessors in combination
with synthetic neural pathways –
artificial-mechanical transplants
may soon be a real possibility.
Works cited:
IBM. New Ways of Thinking. Retrieved
from http://www.ibm.com/smarterplanet/
us/en/business_analytics/article/cognitive_
computing.html.
IBM(Researcher). (2011). DARPA funded
IBM SYnAPSE program [Computing],
Retrieved 11,27,2012 from: http://www.
wired.com/ images_
blogs/wiredscience/2011/08/synapse-
development-darpa-ibm.jpg.
System/Processor Power (Watts) Operations/SecondHuman Brain 20-25 10^13-10^16IBM Power A2 55 10^11ARM Cortex-A15 2 10^10
Figure: Various processors are shown with their corresponding power usage and operations per second.
Compared to the brain, the IBM processor uses double the power of a human brain while calculating
fewer operations per second. Although the ARM Cortex-A15 uses significantly less power than both the
Human Brain and the IBM Power A2, it is limited in regards to the number of operations per second it can
perform. This limiting factor is another problem that many modern, powerful yet efficient processors face.
Features
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16 | Rutgers Science Review | Fall 2012
How did you get a start in Chemical Engineering, and how has
the field changed since you started?
Let me tell you a little bit about my background. I gradu-
ated with my PhD in Chemical Engineering in 1984. Now
traditionally, Chemical Engineering is an area that started
as a field in Applied Chemistry. We worked with oil refiner-
ies, converting petroleum into gasoline and other chemical
products. So when you’d drive along the NJ Turnpike in
the old days, you’d see those distillation plants being used
to separate mixtures into isolated chemical compounds. In
the past 10 or 20 years, the type of research that we chemi-
cal engineers do has expanded. In addition to the traditional
petrochemical type of problems, now we are looking at
materials, biotechnology, biomolecular engineering, and
pharmaceutical engineering. For example, in a tablet that we
make, the actual amount of active pharmaceutical ingredient
is very low--in milligram range--so how do you make sure it
is in that range? It’s harder than you think.
How did you get into Environmental Thermodynamics, and
what kind of research have you done in the field?
Ah, that was something that I did years back. The problem
that I had looked at was the solubility of some hazardous
materials in water. That has to do with the water table—un-
derground water. We wanted to know what happened to
these undesirable chemical compounds. If there is a leak
somewhere else, how would those [hazardous chemicals] be
transported to different places? It would be absorbed into the
soil—but how long would it stay there? Those were the kinds
of problems that I looked at.
What is the most interesting task you’ve encountered during
your work as an engineer?
My research area has to do with the properties of materials in
fluids—it is in the field of Applied Thermodynamics, which
deals with the physical chemical properties of compounds in
different types of materials. For example, I look at the solubil-
ity of drug molecules in different solvents. That’s important
because typically a pharmaceutical molecule is created to
have therapeutic functions, and so therefore, when you
manufacture that, there is a chemical process. In the manu-
facturing phase, you need to isolate it into a pure compound,
crystallize it into a solid form, and process it into a tablet. I’m
interested in understanding the physical properties of this
compound in different environments. Now, why is that im-
portant? When you’ve taken the tablet, it’s now in the stom-
ach, and that environment is very different—aqueous and
acidic—so you need to know the solubility of this compound
An Interview With:
Dr. Yee Chiew
Dr. Yee Chiew is a Professor and Chair of the Department of Chemical and Biochemical
Engineering at Rutgers University. His research involves predicting the thermophysical
properties of materials in fluids.
Conducted by Brian Schendt
IntervIew
Fall 2012 | Rutgers Science Review | 17
in the new environment versus the processing environment.
This is some of the work I do.
How important is it for engineering students to get
international experience by studying abroad?
It is very important. We now live in a globalized world. The
playing field is different; we have to compete in a global
arena. Let me give you an example. One of our PhD students,
two years ago, was looking for jobs, and he couldn’t get an
interview. In his application, he checked a box—he said
he was willing to work overseas. Immediately, he got an
interview in Beijing with a multinational company, and
they offered him a job. Now, he didn’t accept the job...
[laughs]—I’m not sure why and you’d have to ask him…but
sometimes there is some fear of living overseas—it depends
on the person. With some exposure, such as studying abroad,
it makes it easier—if one chooses to. So I encourage students
to participate if at all possible. A lot of companies have
overseas branches, and at this point, growth is much faster
in other countries than in the United States. The market is
there—companies will go there, and they need engineers that
can perform in multicultural environments comfortably work
with those who are different from themselves.
Do you have any advice for current students?
For undergraduate students, they should make sure that they
have a very broad education—not limited to just engineering.
I think our curriculum lends itself to actually training
students for the profession rather than for a particular
industry. Know the fundamentals very well, so that you
may learn very well on your own. Students also need to
develop practical skills beyond technical academic skills;
communication, oral and written, is important. You need
to communicate with people who are different than you—
whether you like it or not—because you will deal with that
in your professional life. That, and developing leadership
abilities, will become extremely important.
We’re interested in your article proposals, editorials, research papers,
art, and photography.
For more information:
Email [email protected]
On the Webthersr.com/submit
Submit to the RSR!
IntervIew
DNA Microarray
Research
Fall 2012 | Rutgers Science Review | 19
Indigo-carmine, also known as Indigotine, is one
of the most integral dyes in the food industry for coloring
blue food products. Whether it’s blue cotton candy that
we eat seasonally at local fairs or the more ubiquitously
enjoyed blue M&Ms from a candy bag, indigo-carmine is
undoubtedly present in many of our diets. The producers
of blue cotton candy and blue M&Ms decided to use
Indigotine because it is a relatively harmless synthetic and
convenient dye which is commonly referred to as Blue-2.
It may be alarming to hear that we still do not
know many of the physical and photochemical properties
of Indigo-carmine, and yet, it is the most common blue
dye used in food industries. Dean Ludescher’s lab aims to
understand some of these potentially important properties.
Despite Indigo-carmine’s popularity,
it has been a fairly under-researched
dye, and there are not many academic
resources to confirm earlier findings
that it is relatively harmless. However,
repetition of trials will provide more
definitive answers to the questions
that still remain about Indigotine’s
physical and photochemical properties.
A little background information
will be needed to understand the
research on Indigotine: when a photon
of light hits an atom, the atom is excited
to a higher energy state in which
it takes a lower-energy level electron and places it in a
higher energy-level orbit. This process is called excitation.
The higher the frequency of the electron, the more energy
the beam of light carries. It requires a specific amount of
energy to excite a certain molecule, moving one atom’s
electron from a lower to a higher energy level state; this
amount of energy can be used to calculate the wavelength
of light necessary to excite an electron. After a certain
excitation wavelength hits the sample of Indigotine, it
absorbs energy of certain wavelengths and emits others.
Emission is the process in which the previously
excited electron returns to its lower-energy orbit position after
emitting of energy in the form of light. The wavelengths that
are emitted back become the color visible to the eye, in this case,
Indigo-carmine and its Photophysical Properties
By: Parabjit Kaur
ReseaRch
20 | Rutgers Science Review | Fall 2012
an indigo blue. In the search of indigo-carmine’s properties,
such as optimal emission and excitation wavelengths, light
spectroscopy is used. The emission wavelength of indigo-
carmine is yet to be discovered, along with the variance in
emission wavelength in different environmental conditions.
In light spectroscopy, a sample of indigo-carmine
is placed into a spectrophotometer. Through a software
application that controls the spectrophotometer, random
intervals of emission and excitation are chosen to observe any
peaks in emission for indigo-carmine. The spectrophotometer
shoots a beam of light at the sample of indigo-carmine in a
solution of water, and displays the results in graphical form
on the computer. Graphs of the results show the intensity of
an emission wavelength for certain intervals of excitation.
Using this process, we are able to discern any significant
emission wavelengths from Indigotine under varying
factors. The photochemical properties of indigo-carmine
are important because the light that it emits (and so the
color that we see) is the reason why it is so useful as a dye.
There are many factors affecting indigo-carmine
that can be researched by placing the dye in different
solutions of varying pH and chemical composition,
as molecules will behave differently when placed in
different environments. The effects on an indigo-carmine
sample can be tested through the spectrophotometer by
viewing the change in intensity of excitation and emission
wavelengths. Recently, it has been discovered that there
is change in indigo-carmine’s photophysical properties
when placed in a fairly acidic solution. Because of this
discovery, the next research focus should include creating
variant pH levels in a solution and adding a sample of
indigo-carmine to it. The spectrophotometer will then
be used to discover any significant changes in intensity
of emission and excitation wavelengths in the dye.
Understanding the properties of indigo-carmine
can be very useful to food industries. If there is significant
change in the emission of light (color) of indigo-carmine
under certain conditions, food industries will be able to
use such knowledge to their advantage to either enhance
or protect their products from such factors. Food industries
utilize dyes to create visual appeal for their products
and attract more consumers. To protect the appeal of
the product and indigo-carmine’s charming blue color,
it must be protected from all environmental factors that
can potentially disrupt its attractive visible hue. Other
photochemical properties of indigo-carmine can be
discovered that might add to the appeal of indigo-carmine
dyes in addition to how it behaves in varying pH. There
is no dearth of interesting and applicable information
that can be found by researching the photochemical and
photophysical properties of indigo-carmine. The next time
we eat blue jelly beans, we’ll have some food for thought.
WORKS CITED:
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C., & Legoy, M. D. (2003). Florescence and FTIR study of
pressure-induced structural modifications of horse liver
alcohol dehydrogenase (HLADH). Eur. J. Biochem.,
270, 119-128.
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Phosphorescence. Applied Spectroscopy Reviews, 35(4), 353-393.
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Expanded ed.). New York, NY: Marcel Dekker, Inc.
4. Hansen, W. H., Fitzhugh, O. G., Nelson, A. A., & Davis, K. J. (1966).
Chronic toxicity of two food colors, brilliant blue FCF and Indigotine.
Toxicology and Applied Pharmacology, 8(1), 29-36.
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Fall 2012 | Rutgers Science Review | 21
The Kramer lab is currently involved in the study of the
transmembrane protein Commissureless (Comm), which
is a powerful negative regulator of Robo proteins. If the
expression of Comm is decreased, then Robo protein is
overexpressed in the CNS, causing defects. This semester,
the Kramer lab will investigate the role of Comm in
regulating Robo during Drosophila heart development.
To examine the heart in Comm mutant embryos, Whole-
Mount Embryo Fixation, Immunohistochemistry
and confocal microscopy were performed (all of
which are standard protocol in the Kramer lab).
Introduction
Drosophila Melanogaster: a Model Organism
Although the Drosophila heart consists of only a single
tube, many cells must work together to enable normal
heart function. Because the development of human and
Drosophila heart tubes is similar, it is essential to learn
the functions of the involved cells and their roles in tube
formation in order to better understand the human heart.
Drosophila is a useful model organism for
studying embryological development because the
species mates quickly and controllably. For example,
one can physically collect a male and a female fly of
different phenotypes, and place them in a vial to mate.
Project Background
The heart tube in the Drosophila forms when two
cardioblasts come together with pericardial cells on each
side. As they come together, central lumen is formed as
some sites attract, leading to adhesion, while others repel,
leaving a gap. The e-cadherin protein from each cardioblast
comes together at the top and bottom and creates a gap in
the middle (shown above). This phenomenon is due to Slit
and Roundabout signaling. When Slit binds to Roundabout,
repulsion occurs, and e-cadherin is negatively regulated
in those sites. If the Roundabout function ceases, Slit and
Roundabout do not interact, therefore e-cadherin is no
longer negatively regulated in the lumen. As a result,
e-cadherin adheres throughout the cardioblast sites, causing
Investigating Commissureless protein regulation of Robo localization in the Drosophila embryonic heart
By: Krishna Parikh, Frank Macabenta, Dr. Sunita Kramer
Rutgers, the State University of New Jersey, Department of GeneticsUniversity of Medicine and Dentistry of New Jersey, Department of Pathology
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22 | Rutgers Science Review | Fall 2012
less lumen formation. Roundabout (Robo) is also localized
in the Central Nervous System of Drosophila Melanogaster.
Comm is a protein that is localized in the central
nervous system. Its function is to ensure that Robo
expression is limited so that the CNS appears normal.
Because Robo is also present in the heart, we hypothesized
that altering Comm levels would affect the expression of
Robo in the heart and thereby modify heart development.
Materials
For this experiment, flies with less than the normal
amount of Comm are required. As the flies mate, their
embryos are collected and stained with two primary
antibodies (alpha Spectrin and BP102). As a result, the
CNS and the heart cells of the fly embryo are also stained.
An epiflourescent microscope is used to further select for
specifically stained embryos. Embryos with two parallel
lines (and no horizontal lines) in their CNS are the mutants,
these are the ones that are selected to be processed and
imaged. These embryos are then analyzed via high-resolution
imaging. The images of modified and unmodified subjects
will be compared to identify deformities and abnormalities.
Methods
Embryo Fixation
After the 20 hours, the cage is taken out and then
replaced with a new agar plate with yeast paste on it. The
previous plate has embryos collected on it; this plate of
embryos is now ready to go through the process of fixation.
First, distilled water is squirted in the plate and a small
brush is used to lift the embryos and mix them in the water.
The water then is poured in this tube that has a mesh cover
and a cap on one side with the other side open. This is done
several times to ensure that all of the embryos are collected
in the mesh tube. Once the embryos are in the tube, bleach
is squirted in the tube and is allowed to remain there for
three minutes. After this step, it is important to remove all
of the remaining bleach properly because it could interfere
with the rest of the fixation process. To remove the bleach,
continuous washing of the embryos with water is required
and to check if the bleach is removed, a paper towel is used.
If the paper towel turns pink when the tube is placed on
there then there are still traces of bleach present. After
removing all of the remaining bleach, the cap is opened and
the mesh, that contains the embryos, is placed into a vial
that has a solution that contains heptane, formaldehyde
solution, and water. Once the embryos are in the vial, the
mesh is removed, and the vial is put on the shaker for 20
minutes. This process removes the vitelline membrane
of the embryos, which is an exoskeleton that protects the
embryos while they are developing. After the 20 minutes
on the shaker, the bottom layer in the vial is removed and
methanol is added in the vial. Then, the vial is vortexed for
60 seconds. Now, this time, bottom layer is saved because
that is where the embryos are, they are transferred to an
ependorf tube. Then immediately after that, methanol
washes are performed at least three times. Lastly, methanol
is added and stored the tube at -20° C, alternately, it can
be used right away if the embryos need to be stained.
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Fall 2012 | Rutgers Science Review | 23
Staining Embryos
The staining process is important because it marks
certain areas in the embryos that are of importance.
The embryo is stained depending on what the region of
importance is. For the experiment, the main focus is the
heart and more specifically the region where there is lumen
formation because that is where Roundabout is located.
First, methanol is pipetted out from the ependorf tube
and two PT washes are performed for five minutes each.
Then a 30 minute PT wash is performed. After the PT washes,
500 microliters of PT+NGS is added and the ependorf tube
is placed on the shaker for 30 minutes. An aliquot of the
primary antibody is made; 50 microliters of the primary
antibody and 450 microliters of the PT+NGS are added.
This is added to the ependorf tube and incubated for 1 to 2
hours. It could also be placed at -4° C with gentle rocking on
a stir plate. The next step is to recover the primary antibody
for another use if needed. Sodium Aizde can be added the
antibody to prevent any unwanted bacterial growth. The
embryos are washed three times with PT for five minutes
each. Following the 3 five minute washes are 4 30 minute
PT washes. Shortly after that, 500 microliters of PT+NGS
is added and incubated for 10 minutes. Then there is an
addition of 1 microliter of secondary antibody diluted in 499
microliters of PT+NGS; this is incubated for two hours. It can
also be placed in the -4° C freezer with gentle rocking on a
stir plate. After the incubation, the embryos are washed with
PT for five minutes once and then for 30 minutes four times.
Following all of the washes, the embryos are
lastly washed with PBS (1X) for one minute. Right
after the wash, 500 microliters of 60% glycerol is
added and the embryos are able to settle at the
bottom of the Eppendorf tube. This takes a few hours.
Analyzing Embryos
It is important to select the embryos in the proper stage,
which is around 16-17. This is because at this stage, the heart
tube formation is complete and can be analyzed properly
for this project. In order to analyze the stained embryos, the
process of whole mount is used. A whole mount slide has
embryos dorsal side up. Then, they are viewed under the
confocal microscope, which uses high resolution to display
images of the heart by projecting light in the embryo itself
refracting through the ectoderm. Furthermore, embryos are
viewed in cross-section rather than whole mount. This process
requires that the embryos are cut one third of the way from the
anterior side. This allows them to stand vertically on a slide
so the images on the confocal can be taken at a vertical angle.
Results and Discussion
Some embryo dorsal view images suggest that
changes in Comm expression affect Robo expression and,
in turn, alter the appearance and formation of the heart.
When there is an overabundance of Robo, gaps in the
heart result. The images that are processed display heart
deformities such as twisting, gaps between cardioblasts, and
atypical cardioblast shape. Investigation is still underway.
References
Santiago-Martinez, E., Soplop, N.H., Patel, R., and
Kramer, S.G. (2008). Repulsion by Slit and Roundabout
prevents Shotgun/E-cadherin-mediated cell adhesion
during Drosophila heart tube lumen formation. J Cell Biol
182, 241-248.
Developmental Cell, “Axon Targeting Meets Protein
Trafficking: Comm Takes Robo to the Cleaners” Mark
Rosenzwei
Nature Neuroscience Volume 8, Number 2, “Comm-
ing across the midline” Catherine Krull
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