Zero Order


A diet to starve cancer?

Posted in Uncategorized by Amy Ross on June 29, 2010

I saw the  title of William Li’s TED talk “Can We Eat To Starve Cancer?”, and I was doubtful about its subject matter.  Sure, there’s plenty of evidence that shows the role of diet in the prevention of cancer.  Eat lots of fiber, limit your red meat intake, and avoid asbestos sandwiches 🙂  On the other hand, there’s a lot of quacks and woo peddlers out there claiming you can cure cancer solely by changing your diet. So where does this talk fall?

What Li says about tumor growth being limited by angiogenesis is absolutely correct.  As he mentioned, a number of anti-angiogenesis drugs such as Avastin have been developed and used successfully to fight cancer. It’s a promising area of research.  As a side note, the enzyme I’m studying in hydrogels,  MMP-2, plays a role in angiogenesis. Its function is to degrade extracellular matrix and create space for new tissue and blood vessels to grow.

Li’s extrapolation of in vitro findings to a definitive clinical outcome is a bit overstated at this point in the research.  Exposing artificially grown blood vessels to a dietary compound and measuring angiogenesis is a good way to begin studying this question, and his results show promise. However, the in vitro environment and actual living beings are two very different things. The  in vivo environment is not only going to include blood vessels, but organs, hormones, signals, not all of which are entirely understood.   In fairness, he cites the tomato consumption-prostate cancer study.  He mentions that the men who did develop prostate cancer had fewer blood vessels at the tumor site, but he doesn’t mention if that was tied to tumor size or clinical outcomes. In addition, the positive effects of lycopene can’t be assumed for every dietary angiogenesis inhibitor. Li says he’s doing more research in human subjects, which is absolutely the right direction to go in.

So can eating foods with anti-angiogenic compounds prevent cancer? We don’t know yet, but it’s at least  plausible and has some decent preliminary research .  I’ll keep checking back on the research every now and then.

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Around the Web

Posted in Uncategorized by Amy Ross on June 26, 2010

I’m working on a couple posts, but it’s going to take me some time to finish them.  In the meantime, here’s some interesting/fun stuff from other sources:

If Sports Got Reported by Science-hilarious and so true. The only thing missing from this football discussion is a personal testimony from someone who was offsides.

From Boing Boing: letter to Cal Tech chemistry  postdoc about his failure to work evenings and weekends. I’m glad my PI doesn’t expect that!  I did go in for a couple hours today, but that was mostly to finish some things with the PEGDA synthesis I talked about in my last post.

PEGDA Synthesis

Posted in Uncategorized by Amy Ross on June 24, 2010

The vast majority of the hydrogels we use in our lab are PEGDA-polyethylene glycol diacrylate.  PEGDA is  biocompatible and easily makes hydrogels, but it’s also very expensive.   The alternative is for a lab to make their own PEGDA, which is what I learned how to do this week.  I asked Mohammed, a former student in our lab, for his help since he had done this many times. Outside of organic chemistry lab, this is the first synthesis I’ve done.

We started with polyethylene glycol (PEG). Basically, the synthesis attaches a new chemical arrangement (or functional group)-acrylate-to each end of a molecular of polyethylene glycol. This makes it functional for the crosslinking of hydrogels. We wanted an end average molecular weight of about 3400, so we started with PEG 3350.

We started by drying the PEG (to remove any acquired moisture from air exposure) by dissolving in organic solvent and using rotary evaporation.  I think rotavaps are neat, so I’m going to talk about them. Basically, a liquid boils when its vapor pressure is equal to atmospheric pressure. To boil it, you can increase the heat and therefore the vapor pressure, or you can lower atmospheric pressure. That’s what a rotavap does. It uses vacuum to lower the pressure inside the flask, boiling off the solvent. The solvent vapor goes through the condenser, becomes a liquid again, and collects in a separate flask. As a bonus, the rotavap  looks like a gun from a 50’s science fiction show:

Rotavap in use

The PEGDA synthesis is not exceedingly difficult, but the setup was time-consuming. There were  ten pieces of glassware that had to be stacked on each other in three levels. Everything has to be well clamped into place and secure.   This was probably the part that took the most time.

The reaction runs under noble gas to prevent any reactions with atmospheric oxygen, so the glassware has to be free of air leaks. Trying to get the right flow rate of the gas was also a challenge. The other hard part is working with nasty chemicals. Several chemicals we used smell a lot or were highly toxic-acryloyl chloride, methylene chloride, and diethyl ether. As is normally the case with such chemicals, we worked in the fume hood  so our whole lab wasn’t exposed to the vapors.  I’m also  a big klutz, so I worry about spilling things or getting the chemicals on me.

Most of the apparatus for PEGDA synthesis. One additional piece-the DriRite tube-goes on top of the condenser (the tall tube on the left)

The reaction ran overnight, and we precipitated the PEGDA this morning. I like watching precipitation reactions.  Pour one liquid into another and poof!  There’s a white solid. After precipitating, we filtered out the solvent and dried.

The final product is still drying, but from the appearance, the PEGDA looks pretty good. The yield wasn’t very high, but it’s not bad for a first try. We’ll confirm the success using IR spectroscopy and NMR spectroscopy.

Even in a week where my experiments don’t go well, if I make PEGDA, I can feel like I accomplished something!

Classroom vs. Research

Posted in Uncategorized by Amy Ross on June 20, 2010
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I was talking to my friend Joe, who is working on his MBA in finance. He asked “Do you consider your lab classes research?”

I said no. They’re helpful in teaching rudimentary techniques, things like how to properly measure using a graduated cylinder, or how to connect an  operational amplifier without destroying it. But no, they’re not research, because you’re only following instructions to acheive a predetermined result.

He remarked that he didn’t feel like his classes prepared him enough for doing research or for real life.  I feel the same way. Granted there is no class offered that could have trained me for the work I’m doing, but very little of my classwork has prepared me for doing research. For the most part, it’s been the collection of facts and understanding of concepts. Don’t get me wrong, it’s important to have the background knowledge, but there’s just so much you’re not prepared for, like experiments that don’t work…again and again. And in pretty much any graduate program, you’ll be doing research.

I could go on at length about the inadequacies in the current education system, but that’s a topic for another post. I do have a few examples of assignments or designs I or people besides me found helpful, and are worth incorporating into curricula:

-Learning instrumentation. In organic chemistry lab, we analyzed our products using IR spectroscopy and on one occasion, NMR spectrscopy. We didn’t do the NMR ourselves, but we prepared the samples and interpreted the results. The other day in my lab, we ran NMR on a chemical. Even though I hadn’t run NMR myself, I was familar with how it worked and understood how to interpret the data.

-Replicating real life scenarios. For a course in Drug Delivery, we had a group project to prepare a mock NIH proposal. That meant we had to invent a hypothesis related to drug delivery and explain how we would go about testing it.  The project made us think about how we would design experiments in order to confirm all the things we said our drug delivery system would do, what results we expected, and problems we might have. It took a lot of literature review, discussion, and revision to formulate good experiments.

-Troubleshooting and problem solving skills. I went to a Bioengineering alumni panel a few months ago. The panelists were in a variety of jobs, but they all said Bioinstrumentation Lab was one of the most helpful courses they took. Your circuit isn’t working? Well, let’s check the connections, examine the components, check settings.

While I wouldn’t expect the classroom to replicate actual research, I’d like to see the above-especially the problem solving-incorporated into other classes. These are small steps that can be taken without a drastic overhaul of American education.

Why do Women Leave Science and Engineering?

Posted in Uncategorized by Amy Ross on June 15, 2010
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Over the years, I’ve read a number of articles and blog entries about why women are don’t enter the STEM disciplines. This one looks at why women leave. The article cited women’s main concerns regarded pay and lack of promotions, and noted that women exited fields at high rates when men made up a large percentage of the workforce.

I liked this article because the author actually looked at data about women leaving science and engineering, and compared it to women in non-STEM fields. Other articles I’ve read often cite statistics and maybe an anecdote or two about women in science. Anecdotes are emotionally potent and useful for illustration, but they are not data. I often wonder how much of the information is based on facts, and how much is based on the author’s beliefs and biases.

The authors concluded the women’s complaints were due to discrimination. I think that’s a bit of a jump, but I can definitely see how a woman feel isolated in a mostly male field. As a student, I don’t feel the lack of women as potently as others in the field. Bioengineering has the highest percentage of women (about 40%) of all engineering subspecialties.  The department in which I do my research,  Biopharmaceutical Sciences, the majority of students are women. In fact, the vast majority of my lab is female.  I’ve gotten good support from both the men and women I work with. So far, I’ve been fortunate in that regard.

The Immortal Life of Henrietta Lacks

Posted in Uncategorized by Amy Ross on June 12, 2010

Rebecca Skloot’s book The Immortal Life of Henrietta Lacks

is the story of  HeLa, the first immortal cell line,  the woman from who the cells originated, and the consequences this had on her family.

Henrietta Lacks was diagnosed with cervical cancer in 1951. She was diagnosed and treated at Johns Hopkins hospital, the only hospital in her area that would treat black patients at that time. A Hopkins researcher was trying to grow a human cell line, and was requesting samples from a variety of patients. Lacks’ cervical cancer cells were the first cells that grew indefinitely, revolutionizing the field of in vitro research.

Lacks didn’t know the tissue sample was taken from her during treatment. She died nine months after being diagnosed.  Her husband and children didn’t find out about the cells until years later, when Henrietta’s sons and daughter were asked for blood samples.

Although the first part of the book goes into a lot of detail about HeLa, the story’s focus is on the Lacks family. Despite the fact a business had been made of selling He La, the family never received a dime. They lived in poverty for decades.  They became highly distrustful of the medical profession and anyone making inquiries about the cells.  And from hearing this story, who can blame them? The researchers were not upfront with the Lackses, and the family was well aware of how blacks had been treated by the health care system. After reading what they had been through, I wouldn’t have trusted the doctors either.

Skloot writes a compelling narrative that balances the significance of He La with the human story of the family. Normally, I dislike when authors put themselves in narrative nonfiction. I consider it an ego trip on the part of the author. However  for this book, I think it was essential. Skloot reached out to the Lackses and spent years earning their trust. Part of the story is the Lackses understanding and coming to terms with HeLa, and SKloot was instrumental in this process.


I’d recommend this book if your work involves tissue culture, or if you’re just interested in learning more about the field.








Adventures in Cell Seeding

Posted in Uncategorized by Amy Ross on June 10, 2010
Today I’m going to share a tale of employing scientific methodology and the sometimes frustrating nature of research.

For the past month or so, I’ve been working on cell seeding of hydrogels. In order to demonstrate penetration of my hydrogel system by MMP-2, I have to place cells on the top layer of my hydrogel. But cells do not like to grow on hydrogels. When placed in a tissue culture plate, cells will do fine growing on the bottom of the well, but not on the hydrogel surface.

To improve cell attachment, I’ve added a short peptide sequence, RGD, to the top (polyacrylamide) layer of my hydrogels. The letters RGD stand for the one letter initials of the pertinent amino acids: aRginine, Glycine, and aspartic aciD. Basically, it works like this:

We all know the human body is made up of cells. In addition to cells, there ares other proteins in the spaces between the cells known as the extracellular matrix (ECM). Collagen is the best-known example of an ECM protein, but there are many others.  Cell membranes have proteins called integrins that attach to the ECM. The RGD peptide is the attachment site for cell integrins.  The cells will bind to the RGD and attach to the hydrogel surface.

That’s the idea, at least.

I first tried seeding my hydrogels with U-87 MG cells, a line of human glioblastoma cells. I put cells on three groups of hydrogels: 1) Hydrogels with RGD, 2) Hydrogels with the peptide DGR (same amino acids, different sequence), 3) Hydrogels with no peptide. The purpose of the DGR group is to ensure it’s the specific RGD sequence that has the effect, not just the presence of a peptide. I assessed cell viability using an MTS assay. I won’t go into all the details of this, but the pertinent facts are that it’s commonly used for to test cell viability, and it doesn’t give an exact count of living cells but is useful for providing comparisons between groups.

My results were not promising. Either there was no difference between hydrogels with RGD and without RGD, or in some cases, the hydrogels without RGD had greater viability. I repeated this a couple times to make sure it wasn’t a problem with my technique, but saw no improvement in results. So, I wondered, what was the problem?

It was suggested that I look at published scientific papers to see if anyone else had grown these type of cells on polyacrylamide hydrogels. A search of PubMed showed no results for U-87 MG cells. So now the question was: were my cells not growing on the hydrogels because there was a problem with the hydrogels (or my technique), or was there something unfavorable about this type of cell growing with polyacrylamide?

To rule out a problem with the hydrogels, I tried seeding them with a different type of cell: NIH/3T3. The 3T3s are human fibroblast cells. They’re not cancer, but there are several published papers successfully demonstrating 3T3s on polyacrylamide hydrogels with RGD. I tried this last week, and the results were much better. I still need to make a few modifications to this system, but I expect this portion of my project will be done soon, and I’ll be able to progress. It’s a good feeling when your creation works!

Hydrogels

Posted in Uncategorized by Amy Ross on June 7, 2010
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The term hydrogel may be unfamiliar, but you’ve probably encountered its oldest and most successful commercial use: the soft contact lens. In order to understand hydrogels, first it’s necessary to explain a few things about polymers.

Polymers are material composed of long chains of repeating molecules. The molecule chains can be as long as tens of thousands of repeating units.  The properties of the polymer depend on the repeating unit and the circumstances under which it is made.  A variety of materials-plastic, rubber, and nylon to name a few-are made of polymers.

A hydrogel is a type of polymer that is  crosslinked and can absorb water. In crosslinking, the polymer chains  are bound together by another compound at regular intervals, creating a “mesh”. The molecular structure looks kind of like a fishing net:

Hydrogel structure at the molecular level

Hydrogels are useful in drug delivery because a drug placed in the hydrogel will diffuse out when placed in an aqueous environment. The rate of diffusion can be controlled by the mesh size (the size of the space bordered by the polymer chains and crosslinking compounds).

From "Matrix metalloprotease selective peptide substrates cleavage within hydrogel matrices for cancer chemotherapy activation". J. Tauro, B. Lee, S. Lateef, and R. A. Gemeinhart. Peptides, Volume 29, Issue 11 (2008): 1965-1973

The system I’m working with takes this idea one step further. It’s designed to treat glioblastoma multiforme, a particularly nasty form of brain cancer. The hydrogel is implanted at the tumor site at the time of resection. The drug in question is attached  to the polymer via a peptide. The peptide is the cleavage site for the enzyme MMP-2. MMPs are a class of enzymes that are produced in normal cells , but are overproduced in cancer cells. The MMP-2 enters the hydrogel and cleaves the peptide, thereby releasing the drug (see Figure).

My specific project is to demonstrate the MMP-2 actually penetrates the hydrogel, and doesn’t just cleave peptides on the surface. To accomplish this, I’ve made a two layer hydrogel. The bottom layer is polyethylene glycol diacrylate (PEGDA) and includes the MMP cleavable peptide.  The top layer is made of polyacrylamide.  Cells will be placed on the top layer. If the peptide is cleaved, we know that the MMP penetrated through the top layer and diffused through the bottom.

I’ve been working on this project for the past four months. I’ve created and optimized the hydrogel bilayer. In the past month, I’ve started cell experiments. I’m also working on some characterization of the hydrogels.

In the next few entries, I’ll be discussing some of the techniques I’ve been utilizing and challenges I have faced in the course of my research.

Drug Delivery-not a visit from your pharmacist

Posted in Uncategorized by Amy Ross on June 4, 2010

Drug delivery refers to the area of altering the way drugs are released in the body. In many (but not all) cases, this refers to the way the active ingredient is “packaged”, and typically doesn’t involve changing the drug’s molecular structure. Drug delivery encompasses:

Timing-changing the amount of time it takes for a drug to be consumed.  Let’s say there’s a great drug for migraines, but it’s eliminated from the body in two hours. Taking a pill every two hours is going to be very inconvenient. Can we change it to 12 or 24 hours?

Route-changing the way a drug can be adminstered. Can we make an oral version of a drug that’s currently given IV only?

Solubility-A great example of this is the cancer drug paclitaxel (Taxol). It has good antitumor activity, but it’s highly insoluble in aqueous solvent. How can we deliver it to the (aqueous) human environment?

Targeting-Can the drug only target the area of disease, (e.g., cancer cells), and leave healthy cells unaffected?

The approaches used in drug delivery and accompanying details are too numerous to discuss in a single blog post, but I’ll discuss a few.  One strategy is to encase the active ingredient in a biocompatible material. Depending on the material’s properties, it may release the drug either through diffusion or degradation. This can be used to address solubility, route, and timing issues.

Another strategy involves designing a delivery device. Subcutaneous implants and skin patches fall into this category.

Targeting can be achieved through passive means such as a material that degrades at a specific pH, or it can be done by active means. Active targeting is typically accomplished by having a compound that binds to a specific cell receptor.

The type of work I do involves a class of polymers called hydrogels, which I’ll discuss more in the next post.

What is Bioengineering?

Posted in Uncategorized by Amy Ross on June 1, 2010
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Typical conversation had with someone outside STEM (science, technology, engineering, and math) academia:

Them: What do you do?

Me: I’m a graduate student in bioengineering

Them: Bio-wha?

Not a lot of people are familiar with the field. So here’s an introduction.

Though it sounds a bit “duh”, bioengineering can best be described as the intersection between biology, medicine, and engineering. There’s a wide variety of things we do:

-Design, testing and maintenance of medical devices

-Design and implementation of medical implants

-Development of biological treatments for illness and injury (e.g., tissue engineering)

-Development of imaging systems

-Review and evaluation of medical devices and biologicals

-Genome analysis (this falls under the subspecialty of bioinformatics)

I chose bioengineering because I was interested in working in the health care field in an area without direct patient contact. Perhaps the most unique thing about BioE is that it is highly collaborative. My classmates are doing research in a variety of departments on campus, from Chemistry to Surgery to Mechanical Engineering. I’m excited because it’s a young field with a variety of opportunities.

For more information, check out the Biomedical Engineering Society’s FAQ about the field.