Rutgers Science Review, Fall 2012

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

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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


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


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


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


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


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


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


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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Real ideas. Real research.


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


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


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


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:

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.,

270, 119-128.

2. McGown, L., & Nithipatikom, K. (2000). Molecular Fluorescence and

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.

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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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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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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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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Rutgers Science Review, Fall 2012

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