Rutgers Science Review Volume 2, Issue 1 Fall 2012
Artificial Life: The Next Frontier?
An Electronic SyNAPSE
Table of Contents “Killer Cells�: Miniharpoons in Nature
pg 6
Artificial Life from Synthetic Genomes
pg 9
An Electronic SyNAPSE
pg 12
An Interview With Dr.Yee Chiew
pg 16
Indigo-carmine and its Photophysical Properties
pg 19
Investigating Commissureless Protein Regulation of Robo Localization in the Drosophila Embryonic Heart
pg 21
Eagle Nebula: The Pillars of Creation
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Articles
IPS Cells
Features
“Killer Cells” Miniharpoons in Nature By: Sean Mascarenhas 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
6 | Rutgers Science Review | Fall 2012
Features cnidarians,
and
approximately
25
of the capsule opens, and the tubule
tubule detaches from the capsule. The
different types have been identified.
is immediately deployed in a twisting
entire firing process of the tubule occurs
Nematocysts are incredibly diverse
motion towards the prey. Discharge
within nanoseconds and is one of the
and have a wide array of functions,
of the tubule is also facilitated by an
fastest reactions in nature. There is also
such as defense, feeding, and adhering
intense increase in osmotic pressure
a great deal debate within the scientific
to prey. Although they exist
community concerning whether
within cnidocytes, the stinging
nematocysts fire independently
cells
of
on
jellyfish
tentacles,
any
control
system,
nematocysts are not strictly
provoking questions about the
regarded as organelles. They are
control organisms have over
secretory products of the Golgi
their own tissues and bodies.
apparatus, which modifies and
Once embedded in the prey,
secretes proteins throughout
thousands of nematocysts inject
COcked Nematocyst
the cell in sac-like assemblages
debilitating chemical agents via
called vesicles. Nematocysts
their tubule spines. The type of
are one of the most complex
toxin used, as well the virulence,
secretions of the cell found in
varies
nature, which marks a special
Certain large jellyfish, such
point of interest for the scientific
as
community. After maturation
Portuguese
and secretion from the Golgi
neurotoxins to induce paralysis
apparatus,
and quickly immobilize their
the
nematocyst
complex is exported towards
Fired Nematocyst
its pre-determined firing site. A double-layered capsule,
among
Physalia
cnidarians.
physalis,
the
man-of-war,
use
prey. Additionally, nematocysts can even remain active long after
their
respective
hosts
which has a door-like opening
(the jellyfish) have died. For
called
example, washed-up tentacles
the
operculum,
surrounds nematocysts. Inside
of
the
Portuguese
each capsule is a coiled tubule
war
that is riddled with a vast array
frequently injure beachgoers.
of spines. The deployment of
Due to their nematocyst
the tubule can be triggered by
complexes, certain cnidarians
have
been
man-of-
known
to
a chemical or physical stimulus, such
within the capsule. In association with
are widely regarded as some of the
as prey brushing against a cnidarian’s
the spines, the twisting motion allows
most lethal creatures on earth. The
tentacles.
an
the tubule to penetrate and become
box jellyfish, which inhabits coastal
appropriate stimulus, the operculum
embedded in the prey, after which the
waters around Australia, is considered
Once
triggered
by
Fall 2012 | Rutgers Science Review | 7
Features 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.
8 | Rutgers Science Review | Fall 2012
Features
Artificial Life from Synthetic Genomes By: Apexa Modi Over the past few years, there
(nicknamed Synthia), but also inserted
do so were discovered only as recently
has been rapid growth in the largely
DNA watermarks containing the co-
as 1995. At this time, the Institute of
uncharted field of synthetic biology.
authors’ names, a website, and several
Genomic Research became the first
Synthetic biologists alter organisms’
philosophical quotes, complete with
to sequence the genome of a living
genes and create synthetic biological
punctuation. The watermarks were
organism, the Haemophilus influenzae
parts to engender new functions.
intended to differentiate the modified
bacteria.1 Since then, many organisms’
In 2010, researchers at the J. Craig
organism from the natural ones and
genomes have been sequenced with
Venter Institute created the world’s
to exemplify the vast possibilities
exponentially less time and cost. It is
first
within
now possible to obtain the sequence
chemically
replicating
synthetic,
organism
–
a
selfmajor
The
genome concept cell
reconstruction. of
creating
(a
cell
a
of all one’s genes for about $10,000
milestone marking the first complete
Frankenstein
whose
– one hundred times less than what
genome replacement. The scientists
“brain,” or genome, has been replaced)
it cost a decade ago. Nonetheless,
not only designed unique techniques
sounds simple enough; however, the
although many genome sequences
to manufacture the synthetic organism
technology and expertise necessary to
have
been
elucidated,
researchers
still do not understand even a single-
Figure 1
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 –
Fall 2012 | Rutgers Science Review | 9
Features the first genome to be created on a
genome remained intact throughout
junctions) were used to screen clones
computer – was based on that of the
the synthesis process (Figure 2),5 as
for the completed genome. Of the 48
M. mycoides strain and was altered to
deviation from sequence design would
clones screened, one (sMmYCp235)
include DNA watermark sequences.
significantly delay project completion
had all 11 desired amplicons, while the
These watermarks, upon translation,
positive wild type control had none.
would produce protein sequences
The results were further verified via a
that spell out words and sentences.
restriction enzyme double digest; with
The
Venter
established
two
Institute major
project
two sites encoded into three of the
objectives:
watermark sequences, yielding unique restriction patterns to characterize the
1. To accurately assemble synthetic
altered M. mycoides genome (Figure 3).1
DNA fragments created de novo
After the genome was successfully
(from scratch)
synthesized, it was transplanted into
2. To jumpstart or “boot up” the
a bacterial cell. The two mycoplasma
synthetic genome, creating a fully
chosen
functional cell
sequence
contained similarity,
91.5%
genome
reducing
the
chance that the recipient cell would
Figure 2
The final genome was reconstructed in three stages (Figure 1).1 First, 100 one
reject this new genome. The recipient cell’s genome was nullified via low
kilobase DNA cassettes were chemically
by hindering synthetic cell survival.
pH conditions that induced nucleotide
synthesized with fragments of the
The 111 10kb fragments were then
starvation and inhibited the cell’s
final genome and inserted into vectors
pooled to produce 100kb assemblies
ability to perform DNA replication.3
consisting of yeast cloning elements.
and extracted directly from the yeast.
The
Each of these cassettes contained an
Multiplex PCR with 11 primer pairs
successfully
80 base pair overlap to enable the
(designed to anneal at the eleven 100kb
appeared blue on X-gal and tetracyline
original fragments to form larger
Figure 3
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 secondstage assembly. These verification steps were performed frequently to ensure that the original synthetic
10 | Rutgers Science Review | Fall 2012
strain
of
M.
Mycoides
transplanted
with
genomes
Features plates.1,4 Initial attempts to transfer the
Figure 4
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, or
transposon insertion, and an 85-bp
this technology has springboarded
duplication. The synthetic sequence
many beneficial projects, including
did not contain any genome from
microbial hydrogen fuel cells (used
“Creation of a Bacterial Cell Controlled by a
the recipient cell, M. capricolum; the
as a source of renewable energy)
Chemically Synthesized Genome.” Science
genome replacement was complete.1
and toxin-degrading or medication-
329.5987 (2010): 52-56.
The protocols described above
other
producing
diseases.6
organisms
Nevertheless,
before they are widely commercialized.
8 new-nucleotide mutations, an E. coli
(Figure
4).7
can now be generalized and are
Other projects are intended to engineer
quickly becoming the fundamental
organisms to for the production of
tools
detergents, cosmetics, and perfumes.
for
many
other
scientists
References: 1.
2.
Gibson, D., John I., Glass, C., et al.
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).
envisioning genome transplants of
4.
C. Lartigueet al., Creating bacterial
their own. Unfortunately, the Venter
Though it may seem that scientists
Institute’s success was a double-edged
have completely harnessed the powers
sword in the biological community;
of
the benefits of creating synthetic
be subject to the natural evolution
organisms to improve the world
process once they are placed back into
were counterbalanced by the threat
nature, which could potentially render
Biology: Regulating Industry Uses of New
of misappropriation for bioterrorism.
the organisms harmful. It is clear
Biotechnologies. Science 333, 6047 (2011):1254-
Because an organism could potentially
that although biologically synthetic
be altered to acquire any biological
organisms hold great potential for
“Comparative Microbial Fuel Cell evaluations
function, it would be possible to create
a healthier planet, a great deal of
of Shewanella spp.” Electroanalysis. 22.7 (2010):
genomes for new smallpox viruses
additional research must be done
883-894.
evolution,
microbes
may
strains from genomes that have been cloned and engineered in yeast. Science 325, 1693 (2009).
still
Wang, H. “Synthetic Genomes for
Synthetic Biology.” J Mol Cell Biol 2.4 (2010): 178-179 6.
Erikson, B. et. al. Synthetic
1256 7.
Chang IS, Bretschger O, et al.
Fall 2012 | Rutgers Science Review | 11
Features
E S P A N y S c i n o r t c e l E n A King cqueline
ukur, Ja s u P i n a r a h B : y B
to be in a comatose state due to
In the
brain damage,
1960’s, computer
there
processors were analyze
of transistors – the first was the
information, and can be thought of as
calculator, which managed basic
the “brains” of computers. Using digital
information and produced basic
circuits, they perform arithmetic and
computations. As science and the
logical operations. In recent times, it is
human mind evolved, so did the meaning and applications of computer processors. Today, IBM’s SyNAPSE project aims to replicate the raw
are
units
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.
power of a human brain. Processors
no
known remedies. What
constructed with hundreds
are
that
For example, once a person is declared
12 | Rutgers Science Review | Fall 2012
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
and
traumatic
Although
degeneration
brain processors
injury. are
amazing and can be mysterious, the brain is just as intriguing if not more. Not only does the brain store information,
Features 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.
it is also the center of learning and
to
comprehending. Although a patient
new
a human brain. Medically, this could
may have brain damage, he is not
technology has for the future of
be useful for patients whose brains
completely disabled. In fact, he is
computing, as well for neuroscience, is
are damaged in certain areas, (e.g. a
known to be “superabled”. While some
astounding. Replicating brain functions
patient with a damaged occipital lobe
senses may not function, other senses
such as sensation, perception, and
might be able to have the processor and
are heightened and have even been
emotion is a concept just coming to light
artificial SyNAPSEs replace its function
proven to be superior. A brain uses
in the modern age. For instance, IBM’s
and allow the patient to process and
an average of twenty to twenty-five
SyNAPSE is an attempt manufacture
interpret a visual stimulus). In addition
watts a day, which is enough to power
an artificial brain, essentially testing
to mimicking the larger, macro-level
a light bulb. A common misconception
the
power.
of the brain, synthetic stimulants and
about computer processors is that
SyNAPSE makes use of integrated
electric circuits could also be used to
they are faster than the brain because
microprocessors and circuits which
substitute
many computations are made quickly;
replicate
and
however, the brain is much faster
cortexes and pathways in the brain.
and its processing power cannot be
The project can potentially replicate
point in time where we can say our
met.
improving
complex neural pathways of a human
microprocessors
technology, processors could create a
brain such as vision, movement, and
and as efficient as the human brain.
new brain with artificial neurochemicals
autonomous function. In principle, a
Although Moore’s law predicts that
Regardless,
with
increase The
limit
neural
potential
of
the
activity. IBM’s
computing
function
of
various
processor could operate a body just like
specific
neurochemicals
individual We
have
synapses.
not
are
reached
as
a
powerful
Fall 2012 | Rutgers Science Review | 13
Features
the transistor count of integrated
a new industry of artificial brains
circuits doubles approximately every
and processors. Stem cells, brain
IBM. New Ways of Thinking. Retrieved
18 months, the amount of time it would
damage, and neural pathways are all
from http://www.ibm.com/smarterplanet/
take for the processor to work at the
potentially reparable through the use
level of the brain appears to be far in
of microprocessors in combination
the future. Technology is no longer
with synthetic neural pathways –
improving at a steady rate, which
artificial-mechanical
makes it more challenging to predict
may
soon
be
a
transplants real
possibility.
Works cited:
us/en/business_analytics/article/cognitive_
computing.html.
IBM(Researcher). (2011). DARPA funded IBM SYnAPSE program [Computing], Retrieved 11,27,2012 from: http://www.
when, and if, we can ever replicate
wired.com/
a human brain. Although the task of
blogs/wiredscience/2011/08/synapse-
creating a microprocessor to work at
development-darpa-ibm.jpg.
images_
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
System/Processor Human Brain IBM Power A2 ARM Cortex-A15
Power (Watts) 20-25 55 2
Operations/Second 10^13-10^16 10^11 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.
14 | Rutgers Science Review | Fall 2012
Real ideas. Real research.
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Interview An Interview With:
Dr.Yee Chiew Conducted by Brian Schendt 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.
How did you get a start in Chemical Engineering, and how has
derground water. We wanted to know what happened to
the field changed since you started?
these undesirable chemical compounds. If there is a leak
Let me tell you a little bit about my background. I gradu-
somewhere else, how would those [hazardous chemicals] be
ated with my PhD in Chemical Engineering in 1984. Now
transported to different places? It would be absorbed into the
traditionally, Chemical Engineering is an area that started
soil—but how long would it stay there? Those were the kinds
as a field in Applied Chemistry. We worked with oil refiner-
of problems that I looked at.
ies, converting petroleum into gasoline and other chemical products. So when you’d drive along the NJ Turnpike in
What is the most interesting task you’ve encountered during
the old days, you’d see those distillation plants being used
your work as an engineer?
to separate mixtures into isolated chemical compounds. In
My research area has to do with the properties of materials in
the past 10 or 20 years, the type of research that we chemi-
fluids—it is in the field of Applied Thermodynamics, which
cal engineers do has expanded. In addition to the traditional
deals with the physical chemical properties of compounds in
petrochemical type of problems, now we are looking at
different types of materials. For example, I look at the solubil-
materials, biotechnology, biomolecular engineering, and
ity of drug molecules in different solvents. That’s important
pharmaceutical engineering. For example, in a tablet that we
because typically a pharmaceutical molecule is created to
make, the actual amount of active pharmaceutical ingredient
have therapeutic functions, and so therefore, when you
is very low--in milligram range--so how do you make sure it
manufacture that, there is a chemical process. In the manu-
is in that range? It’s harder than you think.
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
How did you get into Environmental Thermodynamics, and
interested in understanding the physical properties of this
what kind of research have you done in the field?
compound in different environments. Now, why is that im-
Ah, that was something that I did years back. The problem
portant? When you’ve taken the tablet, it’s now in the stom-
that I had looked at was the solubility of some hazardous
ach, and that environment is very different—aqueous and
materials in water. That has to do with the water table—un-
acidic—so you need to know the solubility of this compound
16 | Rutgers Science Review | Fall 2012
Interview 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,
Submit to the RSR!
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
We’re interested in your article proposals, editorials, research papers, art, and photography.
sometimes there is some fear of living overseas—it depends on the person. With some exposure, such as studying abroad,
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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.
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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.
Fall 2012 | Rutgers Science Review | 17
DNA Microarray
Research
Research
Indigo-carmine and its Photophysical Properties By: Parabjit Kaur
Indigo-carmine, also known as Indigotine, is one
it takes a lower-energy level electron and places it in a
of the most integral dyes in the food industry for coloring
higher energy-level orbit. This process is called excitation.
blue food products. Whether it’s blue cotton candy that
The higher the frequency of the electron, the more energy
we eat seasonally at local fairs or the more ubiquitously
the beam of light carries. It requires a specific amount of
enjoyed blue M&Ms from a candy bag, indigo-carmine is
energy to excite a certain molecule, moving one atom’s
undoubtedly present in many of our diets. The producers
electron from a lower to a higher energy level state; this
of blue cotton candy and blue M&Ms decided to use
amount of energy can be used to calculate the wavelength
Indigotine because it is a relatively harmless synthetic and
of light necessary to excite an electron. After a certain
convenient dye which is commonly referred to as Blue-2.
excitation wavelength hits the sample of Indigotine, it
absorbs energy of certain wavelengths and emits others.
It may be alarming to hear that we still do not
know many of the physical and photochemical properties
Emission is the process in which the previously
of Indigo-carmine, and yet, it is the most common blue
excited electron returns to its lower-energy orbit position after
dye used in food industries. Dean Ludescher’s lab aims to
emitting of energy in the form of light. The wavelengths that
understand some of these potentially important properties.
are emitted back become the color visible to the eye, in this case,
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
Fall 2012 | Rutgers Science Review | 19
Research 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 indigocarmine 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
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. photochemical
is change in indigo-carmine’s photophysical properties when placed in a fairly acidic solution. Because of this
of
indigo-carmine
can
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: 1. Trovaslet, M., Dallet-Choisy, S., Meersman, F., Heremans, K., Balny, C., & Legoy, M. D. (2003). Florescence and
FTIR study of
pressure-induced structural modifications
of horse liver
alcohol dehydrogenase (HLADH). Eur. J.
Biochem.,
discovery, the next research focus should include creating
270, 119-128.
variant pH levels in a solution and adding a sample of
2. McGown, L., & Nithipatikom, K. (2000). Molecular Fluorescence and
indigo-carmine to it. The spectrophotometer will then be used to discover any significant changes in intensity
Phosphorescence. Applied Spectroscopy Reviews, 35(4), 353-393. 3. Guilbault, G. (1990). Practical Fluorescence. (2nd, Revised and Expanded ed.). New York, NY: Marcel Dekker, Inc.
of emission and excitation wavelengths in the dye.
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.
Understanding the properties of indigo-carmine
can be very useful to food industries. If there is significant
20 | Rutgers Science Review | Fall 2012
be
discovered that might add to the appeal of indigo-carmine
viewing the change in intensity of excitation and emission wavelengths. Recently, it has been discovered that there
properties
Other
Toxicology and Applied Pharmacology, 8(1), 29-36.
Research
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 Genetics University of Medicine and Dentistry of New Jersey, Department of Pathology
The Kramer lab is currently involved in the study of the transmembrane protein Commissureless (Comm), which
Introduction Drosophila Melanogaster: a Model Organism
is a powerful negative regulator of Robo proteins. If the
Although the Drosophila heart consists of only a single
expression of Comm is decreased, then Robo protein is
tube, many cells must work together to enable normal
overexpressed in the CNS, causing defects. This semester,
heart function. Because the development of human and
the Kramer lab will investigate the role of Comm in
Drosophila heart tubes is similar, it is essential to learn
regulating Robo during Drosophila heart development.
the functions of the involved cells and their roles in tube
To examine the heart in Comm mutant embryos, Whole-
formation in order to better understand the human heart.
Mount and which
Embryo
confocal are
Fixation,
microscopy
standard
Immunohistochemistry
were
protocol
in
performed the
(all
Kramer
of lab).
Drosophila studying
is
a
embryological
useful
model
development
organism
for
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
Fall 2012 | Rutgers Science Review | 21
Research less lumen formation. Roundabout (Robo) is also localized
replaced with a new agar plate with yeast paste on it. The
in the Central Nervous System of Drosophila Melanogaster.
previous plate has embryos collected on it; this plate of
Comm is a protein that is localized in the central
embryos is now ready to go through the process of fixation.
nervous system. Its function is to ensure that Robo
First, distilled water is squirted in the plate and a small
expression is limited so that the CNS appears normal.
brush is used to lift the embryos and mix them in the water.
Because Robo is also present in the heart, we hypothesized
The water then is poured in this tube that has a mesh cover
that altering Comm levels would affect the expression of
and a cap on one side with the other side open. This is done
Robo in the heart and thereby modify heart development.
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
Materials
the mesh, that contains the embryos, is placed into a vial
For this experiment, flies with less than the normal
that has a solution that contains heptane, formaldehyde
amount of Comm are required. As the flies mate, their
solution, and water. Once the embryos are in the vial, the
embryos are collected and stained with two primary
mesh is removed, and the vial is put on the shaker for 20
antibodies (alpha Spectrin and BP102). As a result, the
minutes. This process removes the vitelline membrane
CNS and the heart cells of the fly embryo are also stained.
of the embryos, which is an exoskeleton that protects the
An epiflourescent microscope is used to further select for
embryos while they are developing. After the 20 minutes
specifically stained embryos. Embryos with two parallel
on the shaker, the bottom layer in the vial is removed and
lines (and no horizontal lines) in their CNS are the mutants,
methanol is added in the vial. Then, the vial is vortexed for
these are the ones that are selected to be processed and
60 seconds. Now, this time, bottom layer is saved because
imaged. These embryos are then analyzed via high-resolution
that is where the embryos are, they are transferred to an
imaging. The images of modified and unmodified subjects
ependorf tube. Then immediately after that, methanol
will be compared to identify deformities and abnormalities.
washes are performed at least three times. Lastly, methanol is added and stored the tube at -20째 C, alternately, it can
Methods Embryo Fixation After the 20 hours, the cage is taken out and then
22 | Rutgers Science Review | Fall 2012
be used right away if the embryos need to be stained.
Research Staining Embryos
Analyzing Embryos
The staining process is important because it marks
It is important to select the embryos in the proper stage,
certain areas in the embryos that are of importance.
which is around 16-17. This is because at this stage, the heart
The embryo is stained depending on what the region of
tube formation is complete and can be analyzed properly
importance is. For the experiment, the main focus is the
for this project. In order to analyze the stained embryos, the
heart and more specifically the region where there is lumen
process of whole mount is used. A whole mount slide has
formation because that is where Roundabout is located.
embryos dorsal side up. Then, they are viewed under the
First, methanol is pipetted out from the ependorf tube
confocal microscope, which uses high resolution to display
and two PT washes are performed for five minutes each.
images of the heart by projecting light in the embryo itself
Then a 30 minute PT wash is performed. After the PT washes,
refracting through the ectoderm. Furthermore, embryos are
500 microliters of PT+NGS is added and the ependorf tube
viewed in cross-section rather than whole mount. This process
is placed on the shaker for 30 minutes. An aliquot of the
requires that the embryos are cut one third of the way from the
primary antibody is made; 50 microliters of the primary
anterior side. This allows them to stand vertically on a slide
antibody and 450 microliters of the PT+NGS are added.
so the images on the confocal can be taken at a vertical angle.
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
Results and Discussion
a stir plate. The next step is to recover the primary antibody
Some embryo dorsal view images suggest that
for another use if needed. Sodium Aizde can be added the
changes in Comm expression affect Robo expression and,
antibody to prevent any unwanted bacterial growth. The
in turn, alter the appearance and formation of the heart.
embryos are washed three times with PT for five minutes
When there is an overabundance of Robo, gaps in the
each. Following the 3 five minute washes are 4 30 minute
heart result. The images that are processed display heart
PT washes. Shortly after that, 500 microliters of PT+NGS
deformities such as twisting, gaps between cardioblasts, and
is added and incubated for 10 minutes. Then there is an
atypical cardioblast shape. Investigation is still underway.
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
References Santiago-Martinez, E., Soplop, N.H., Patel, R., and
stir plate. After the incubation, the embryos are washed with
Kramer, S.G. (2008). Repulsion by Slit and Roundabout
PT for five minutes once and then for 30 minutes four times.
prevents Shotgun/E-cadherin-mediated cell adhesion
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
during Drosophila heart tube lumen formation. J Cell Biol 182, 241-248. Developmental Cell, “Axon Targeting Meets Protein
added and the embryos are able to settle at the
Trafficking: Comm Takes Robo to the Cleaners” Mark
bottom of the Eppendorf tube. This takes a few hours.
Rosenzwei Nature Neuroscience Volume 8, Number 2, “Comming across the midline” Catherine Krull
Fall 2012 | Rutgers Science Review | 23
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