Happy New Year everyone! I rang in the New Year with a performance of A Klingon Christmas Carol at the Greenhouse Theater in Chicago. It was a lot of fun, although I’ve never sang “Auld Lang Syne” in Klingon before.
The New Year is off to a busy start. I’ll be leaving tomorrow to travel for an interview. In the meantime, here’s an article I ran across recently:
Teenager wins $100,000 prize for designing a controlled drug delivery system. Angela Zhang’s nanoparticles have components of gold and iron oxide and are designed to specifically target cancer stem cells. Unfortunately, I wasn’t able to find able to find more information about the specifics, but I can talk about some general concepts regarding the design of her system
A number of research groups have worked with gold nanoparticles (GNPs). GNPs are inert in humans, nontoxic, and are easily chemically modifiable, allowing for the attachment of drugs or targeting molecules. Drug release from GNPs can be activated by light. Both gold nanoparticles and iron oxide nanoparticles can be used for imaging. The nanoparticles are injected intravenously and can be visualized through medical imaging. In the case of iron oxide nanoparticles, MRI is highly effective. Imaging can be used in conjunction with drug delivery by locating the area with imaging, and then activating the nanoparticles by heat, light, or ultrasound. In the case of Zhang’s drug delivery system, the nanoparticles are activated by a laser. This localizes the drug’s effects on the affected area, and prevents adverse drug effects elsewhere in the body.
Finally, a word or two about cancer stem cells, the target of Zhang’s nanoparticles. Cancer stem cells (CSCs) are malignant cells that have properties of stem cells; namely, CSCs are able to differentiate. They are believed to be capable of differentiating into multiple types of malignant cells accounting for the heterogeneity of cells found in tumors. There is interest in specifically targeting CSCs to prevent tumors from recurring or metastasizing. The study of cancer stem cells is still young, and we have a lot to learn before we can precisely target the cells for destruction.
Zhang has been working on this project since 2009, but I wasn’t able to find out what stage her project is in. Still, it’s fantastic to see a young person with great ideas about controlled drug delivery, and I wish her the best of luck in her future endeavors!
Boisselier and Astruc “Gold nanoparticles in nanomedicine: preparations, imaging, diagnostics, therapies and toxicity.” Chemical Society Reviews 2009 38(6): 1759-82
Ghosh et. al. “Gold nanoparticles in delivery applications“, Advanced Drug Delivery Reviews, 2008; 60(11): 1307-1315
There’s been a lot of debate about the recent announcement that the U.S. government advisory boards asked Science and Nature to censor information about the alteration of a bird flu virus.
The virus in question, subtype A H5N1 caused a scare in mid 2000s because of its high human fatality rate (60%). Remember when people feared bird flu would kill millions? ABC even aired a mediocre movie about what might happen in a worldwide flu epidemic.The virus’ damage was limited in that it could not be transmitted from one human to another; the few hundred affected individuals contracted the virus from birds. Researchers at University of Wisconsin-Madison and the Erasmus Institute in the Netherlands created a strain of H5N1 that was easily transmissible-and fatal-among ferrets. The US National Science Advisory Board for Biosecurity recommended the methods sections of the papers be withheld to prevent use of this information in bioterrorism.
I understand the rationale for not wanting this information to fall into the wrong hands, but I don’t like the idea of censoring research. I really don’t. I worry about sacrificing liberties in the name of national security. There’s been a lot of criticism of the board’s decision, arguing that censorship of research is not in the spirit of scientific inquiry. The board has stated they will make the methods available on a need to know basis. How they determine need to know, and how long will the process take? We don’t know. Also, the board’s recommendation can’t prevent the methods from being published in other ways. The reseachers could easily publish their technique in a European journal.
I also find it ironic that the news about the papers being censored brought a lot more attention to the story than if the paper had been quietly published. Perhaps the board’s decision will have the opposite of its intended effect!
Those are just my thoughts. More later if there are further developments.
I recently read The Cure, Geeta Anand’s account of John Crowley’s effort to develop a treatment for the rare debilitating disease affecting his children. The story was the basis for the 2010 movie Extraordinary Measures.
Crowley’s children Megan and Patrick were diagnosed with Pompe disease, a genetic metabolic disorder that causes muscle weakening, heart enlargement, and severe respiratory difficulties. Both children are ventilator dependent by the age of two. In Pompe disease, alpha-glucosidase, an enzyme that breaks down glycogen is deficient or absent. Glycogen is a complex carbohydrate molecule that stores energy for use in the body. Alpha glucosidase is found in the lysosomes of cells. Lysosomes are the “garbage bins” of cells, primarily responsible for processing cellular waste. Anyway, since the enzyme is deficient, glycogen builds up in cells and causes muscle weakening.
Megan and Patrick have a life expectancy of about five years. Crowley, desperate to save his kids, begins meeting with Pompe researchers. He eventually becomes CEO of Novazyme, a company formed to manufacture the missing enzyme. He eventually negotiates the sale of Novazyme to Genzyme and continues to work with the Pompe team to get the enzyme in clinical trials.
Crowley is stubborn, charismatic, and absolutely determined to get his kids in clinical trials before they succumb to the disease. I’d like to address a couple issues I had with his behavior in the book.
1. Indiscriminate research grants. Crowley’s initial tactic is to form a nonprofit, the Children’s Pompe Foundation, and raise research funds. The foundation impressively raises $750,000 in eight months. However, he makes all the decisions about awarding grant money himself, and solely based on what the researchers say about their expediency to be approved for clinical trials. Crowley, a director of marketing at Bristol-Myers Squibb, acknowledged science is not his strong suit. I certainly didn’t expect Crowley to retrain as a biochemist, but he should have had a scientific advisor in the Foundation to assess research feasibility. The Komen Foundation and the Tourette’s Syndrome Association award research grants, and both have scientific advisory boards.
Granted, the field of Pompe researchers is small, but his ignorance about the research specifics cause problems for Crowley when he becomes CEO of Novazyme. He takes the job fully believing the company is his best chance into getting a Pompe treatment into clinical trials. At a meeting with venture capitalists, the potential investors point out the planned manufacturing process would never be approved by the FDA-something Crowley didn’t realize.
2. Conflict of Interest. In order to motivate the Novazyme staff, Crowley brings in Pompe patients and hosts lunchtime seminars on the disease. A number of venture capitalists question Novazyme’s ability to practice good science since Crowley is far from objective in the outcome of Novazyme’s drug development. Being in the senior management of Novazyme (and later Genzyme) actually is detrimental in getting Megan and Patrick in clinical trials, and I have mixed feelings on Crowley getting involved as intimately as he does getting his children treated. He wasn’t well qualified to be a CEO and had a steep learning curve. Taking the Novazyme job undoubtedly gave him a sense of control. I did like the lunchtime seminars. I think it gives a sense of urgency to the research being done. It’s easy to focus on the small details of a project on a day to day basis-this technique isn’t working, the cells are contaminated-that it’s easy to forget the end goal, and that people’s lives are depending on what you are able to accomplish.
Overall, I enjoyed the book. Anand tells the story well, and it took large amounts of willpower to not skip to the end and find out if Megan and Patrick were treated. I also learned a lot about venture capital investment!
Although it’s a couple days since Solstice and the start of Haunakkah, I hope you all have a great holiday!
Discovery News reported last week on an artificial intestine intended to treat short bowel syndrome in children. I thought this was pretty snazzy and looked up the original publication on PubMed.
Tissue engineering, while an exciting field with sexy goals, is fraught with challenges, starting with the scaffold. What scaffold materials can be used that can be implanted in a patient without adverse affects and encourage functional tissue development? What characteristics should the scaffolds have? Hydrogels are useful scaffolds because their physical and mechanical properties resemble soft tissue. A hydrogel also allow water and small molecules to diffuse throughout its structure, allowing nutrients to reach growing tissues. Cells can be encapsulated either within the hydrogel at the time of polymerization, or added to the surface after polymerization; the choice depends on a variety of factors including the application and the type of cells.
The researchers who have developed this artificial intestine employed the principle that replicating the cells’ environment as closely as possible will provide the most functional engineered tissue. One of the challenges in replicating small intestinal structure was to be able to create villi, or fingerlike protrusions from the surface of the intestine. The presence of villi increases the small intestine’s surface area and allows a greater amount of nutrients to be absorbed. The villi are small structures-on the order of millimeters in a human-and conventional molds would either not be able to make the villi small enough or allow the hydrogel to emerge intact from the mold. The researchers solved the problem by making two molds. The first is made of PDMS, a hard polymer with the villi projections. A mold of calcium alginate was cast on the PDMS. The sodium alginate mold is filled with the hydrogel. Once the hydrogel is polymerized, the calcium alginate is dissolved, leaving just the hydrogel with the projections for villi. The villi formed were 400-500 microns (millionths of meters) in height, or 0.4-0.5 millimeters.
Using collagen as their hydrogel of choice, the researchers were able to grow Caco-2 (colon cancer cells) on the hydrogel. The cells were able to grow on the scaffold, and the cells that grew on the projections resembled villi.
Although this model of a human intestine is pretty awesome, I wouldn’t consider artificial intestines to be “near reality”. The Discovery News article states their next step is to grow a larger intestine model (the published data was on a very small scale), and then to implant the intestine in mice. Will the engineered intestine function absorb enough nutrients to sustain a living being? Is it possible to vascularize the newly developed tissue? Will cells from the patient be able to grow as well in the hydrogel as the Caco-2 cells? There are a lot of steps between these experiments and a working intestine for humans.
J.H. Sung et. al. “Microscale 3-D Hydrogel Scaffold for Biomimetic Gastrointestinal (GI) Model” Lab Chip, 2011, 11, 389
I haven’t written anything since the beginning of March, although it appears I’m still getting some traffic. Here’s what I’ve been up to:
- I graduated! I finished the bulk of my research by the end of June, and defended my thesis in mid-August. My thesis took about four months to write, off and on, and ended up running nearly 150 pages. This is a bit long for a Master’s, but I did have a lot of figures. The thesis defense went very well. I expected my committee would ask me a lot of tough questions, but it wasn’t as bad as I thought it would be. I got the bound copies back last month, and gave one to my parents. Mom got to page one and asked if there was an audio version 🙂
- I became an experienced presenter of scientific data. I presented my research at multiple regional conferences, and actually won second place at the Midwest Biomedical Engineering Career Conference in April. I presented at my first national meeting at the AAPS National Biotechnology Conference last May. The poster presentations were great preparation for both my thesis defense and discussing my research with potential employers for two reasons. One, I gained experience explaining my research to people who weren’t in my field. Two, I had practice thinking on my feet when questions were asked.
- I have two publications in progress. I wrote a paper on my thesis that has been submitted to a pharmaceutical journal. The other is a previous M.S. student’s project whose paper I helped rewrite.
Now I’m looking for a job. I’ve had several interviews this fall, but no offers yet. I’ve also been doing volunteer work and doing lots of reading. A few nonfiction titles of interest:
- Vaccinated! by Paul Offit-some history of vaccines and the development of multiple vaccines at Merck
- The Xeno Chronicles by G. Wayne Miller-about xenotransplanation research at Harvard
- Every Second Counts: The Race to Transplant the First Human Heart by Donald McRae-Christiaan Barnard, who performed the first successful human heart transplant, was actually the underdog among four competing physicians.
- Ether Day by Julie Fenster- about the discovery of ether as a surgical anesthetic.
Since I’m not actively doing research, I’d like to rebirth this blog into my take on current events in science, thoughts on reading, and adventures in job hunting! Stay tuned.
I recently completed my first poster presentation. It was at College of Pharmacy Research Day, an annual event with seminars in addition to the poster session.
I spent a lot of time working on my poster, and going over the details with my PI. One of the big challenges was how to represent the concepts and data pertaining to my project clearly using as little text as possible. Even though people may look at your poster, they may not read everything! We discussed effective schematics for showing the cleavage of the MMP substrate peptide. This is something that people have misunderstood in the past. I decided to show the two parts of the peptide with a double-sided arrow, and labeling which part is released from the hydrogel.
Another hard part was using Microsoft Equation Editor, but this was mostly due to my inexperience with it. My poster had several lengthy, complicated looking equations for calculating hydrogel mesh size. I initially wrote all the equations in MS Word, but it took me forever to figure out how to change the font! Plus, I was using a Powerpoint slide as my poster template, and the equations were pasted in as pictures. Later I figured out how to use Equation Editor in Powerpoint, but that meant typing all the equations…again.
Overall, I think the presentation went well, for a first experience. The format was the presenters would stand in front of their poster for two hours. Three judges were assigned to each poster. For each judge, the presenter walks them through the content and answers questions. For anyone else in attendance, same thing, but not scored.
I picked up quickly from the first two judges that there were things I wasn’t explaining. What disease my drug delivery system was for, how is it put in the body. The other big questions I got were what are my next steps, and how would it be tested in animals. Fortunately, one of the other members of my group was presenting on how the system would be implanted in animals, so we had discussed it a bit before the presentation.
I was nervous going in to the presentation, but I didn’t feel nervous while I was doing it, even though I do get nervous when giving a talk. Perhaps it was because it was more of a one on one conversation and not presenting to a group.
One disappointment was despite the time spent working on my peptide cleavage schematic, I talked to three people who misunderstood it. I’ll have to work on a better graphic.
One of the fun parts of being a grad student in the sciences is giving presentations. Lots of presentations. After doing them for classes and group meetings, I’ve gotten a lot of practice. Recently, I’ve created a presentation using Keynote, and gave it using the iPad.
My presentation approach:
-First and foremost: I HATE outlines! Scientific presentations have a standard, predictable format. You don’t need to explain it, especially in a short (ten minute) talk.
-Words to a minimum. I make an exception for explaining procedures. Pictures instead of words whenever possible.
-Dark text, light background. I try to avoid white because the I usually do black test, and the contrast between black text and white background is too stark. I’m partial to light neutral colors, like beiges or greys.
Since Keynote on the iPad has limited functionality, I did most of the presentation on my husband’s Mac, then transferred it to the iPad using Dropbox. I also used Dropbox to transfer all my images, Excel spreadsheets and such from my PC. I had a lot of fun playing in Keynote, and I found it easier to use than Powerpoint. Sadly, no version of Keynote supports error bars, rendering any charts I made useless! I had to redo them all as images. And if I thought “Hmmmm, the font size on the axis should be larger”, I had to redo the whole image. The iPad keyboard only does punctuation symbols, so I had to install a separate app (Easy Symbols) to include Greek letters and plus-minus signs.
I also had some fun playing with the animations in Keynote, but tried not to be overly obnoxious with it. I gave my presentation for a few members of my group on Thursday, and all went well.
I’m debating if I want to do future presentations in Keynote. I think the “Oh look! Shiny toy!” gambit only works once, and it was a lot of extra work. I might pull it out the next time I need to impress someone.
Did something pretty stupid in lab la week. A few weeks ago I talked about gel zymography. I had some initial difficulties learning the technique, but I feel reasonably competent now that I’ve done it enough times. Or so I thought.
I keep all my buffers and solutions on a shelf above my bench. To run a gel, you fill the electrophoresis cell (a plastic box that holds the gel and apparatus) with running buffer. So I pulled the jar of running buffer off the shelf, on to my bench, and filled the cell. Then I closed the cell, applied the electrical field, and let it run. Normally, I see well-defined blue bands travel down the gel as it progresses. But the bands were blurry, faded, and the ladder (prestained protein markers) was smudged and bent. I’d had some issues with gels with “frowny” bands about a month ago. I had done some Googling and concluded the problem was my sample buffer had gotten old. I made new sample buffer and resumed running gels without issue. So I didn’t think that was the problem this time.
Then I looked at the bottle on my bench.
It wasn’t running buffer. Crap.
Things I did wrong:
-I have no distinct labeling system for my bottles. I use purple tape for everything. I should do different colors for solutions that have no other distinguishing characteristics (i.e., color). I might switch to different colors, or use a numbering system like hair dye.
-Obviously, I didn’t read the label before pouring. Kind of important.
Things I did right:
-I figured out what was causing the problem.
-I didn’t throw away my leftover samples, making it easier to set up another gel.
-I used a ladder. The ladders haven’t been showing up when I photograph the gels. I’m only looking at one molecular weight, and I’ve never had a problem with the MMP-2 bands showing up in a range other than between the 50 and 85 kDa bands on the ladder (active MMP-2 has a molecular weight of 66-72 kDa, so the bands are showing up in the expected position). Despite this, I’ve continued using one every time. In this case, it was valuable as an indicator that something was wrong. It only takes a few minutes to defrost and inject the solution, and ladders aren’t expensive. I think the benefits outweigh the costs.
I developed and stained the failed gel just to see how it turned out. The bands were present, but bent. The bands were also higher than their normal position, but I attribute that to my stopping the run when I realized my mistake. The run time was about 45 minutes, rather than the usual 60-75 minutes.
If you’re interested, I found the SDS-PAGE Hall of Shame helpful and amusing. I may set up my own “Hall of Shame” to help future lab members troubleshoot their gels!
Soooooo..in a comment to my last entry, I remarked that not only was it possible my cells weren’t producing enough active MMP-2 to produce a measurable release of peptide in the hydrogels, I thought it was likely. It’s a hypothesis I’ve been pursuing for about a month. As we’ve talked about before, cell lines in culture don’t always behave the same way as their primary cell counterparts in vivo. They’re different genetically, they don’t produce MMP while living on tissue culture plastic…there could be any number of reasons.
I started looking at papers on MMP-2 expression, and how other groups boosted theirs. One option is to use chemicals to convert the inactive form of the enzyme to the active form. I found one paper that used plates coated in extracellular matrix proteins boosted their MMP-2 presence.
This was a fairly easy hypothesis to test. I ran two experiments. In one, I did an in-vitro peptide release study, and saved the media at each time point. In the second, I collected media after 48 hours of exposures to my cells, and exposed hydrogels to this media (hereafter referred to as conditioned media). I ran gel zymography on the samples from the in vitro study and the conditioned media study.
The results? Unsupportive of my hypothesis. The gel showed plenty of active MMP-2 production by the 48 hour mark in the in vitro study and in the conditioned media study. However, there was no significant difference in peptide release compared to the controls (in both cases, hydrogels in media only).
Next hypothesis: the MMP-2 may be there, but is it active? Testing this at the moment with mixed results. Hopefully I”ll have more to talk about later!
In other news, my wonderful husband gave me an iPad for Christmas. I’m thinking of downloading the Keynote app and doing my group meeting presentations from the iPad. One of our rotation students from last semester did some nice work with Keynote. I think incorporating some more of the sophisticated animations could be useful without being obnoxious.
I’ve still been working on MMP-2 mediated peptide release from hydrogels. I did another study with just MMP-2 in buffered saline, and it worked nicely. The MMP group had much greater peptide release than the non-MMP group. So why was it, when I used MMP-2 producing cells, that there was no difference in the group with cells and the control group?
One hypothesis I had was that the cells were either a) not producing MMP-2, or b)not producing it in the presence of hydrogels. To test this, I had to learn a technique called gelatin zymography. It’s similar to SDS-PAGE, gel electrophoresis technique is to separate and identify proteins. Here’s how it works.
Gel Electrophoresis in Two Minutes
If you want to identify your sample has a specific protein, gel electrophoresis can help you identify it.* Samples (and controls) are put in a buffer with sodium dodecyl sulfate (SDS) and dye. The SDS partially denatures the protein (unfolds it, thereby rendering it nonfunctioning, but easier to move through the gel!) and gives it a negative charge. All the samples are loaded into a polyacrylamide gel. The gel is placed in an electric field, with the negative charge at the loading end, and a positive charge at the opposite end. Now negatively charged, the proteins are attracted to the positive end and start travelling through the gel.
However, the smaller the protein is, the faster it can travel through the gel. Threfore, the proteins sort themselves by size in each sample, with the smallest being towards the bottom, and the largest towards the top. The dye in the buffer is used to track how far the sample has moved. When the dye is most of the distance through the gel,it is removed from the electric field and stained. The proteins show up as bands on the gel.
How is Gelatin Zymography Different?
In a zymography gel, an enzyme substrate (in this case, gelatin) is added. After removing from the electric field, the gel is placed in a renaturing solution, to let any present enzymes refold into their functional conformations. The gel is incubated at 37 degrees C, which allows the enzymes to degrade any substrate in their vicinity. Then the gel stained. Unlike an SDS-PAGE, since protein is present throughout, the whole gel stains and the bands are clear. The presence of enzyme is indicated by a lack of protein.The downside to this technique is it’s not quantititative. You can confirm the presence or absence of active enzyme, but you can’t translate it to an amount of protein or a concentration. There are other techniques to do this for proteins such as ELISA or a Western Blot, but they can’t be used for MMP-2, for reasons I’m not going to go into here.
What were my results?
Well, it took a few tries to learn this technique. Everyone had to help me with some aspect of it, even our visiting high school student! But after 5 or 6 gels, I felt like I had the hang of it. I have confirmed:
-The cells I’m using are producing active MMP-2
-The cells produce active MMP-2 in the presence of hydrogels
So what was the next step for my cell studies? I’ll leave that for next time.
*Gel electrophoresis is also used to identify DNA segments. Since DNA is negatively charged already, there’s no need to add SDS. DNA gels are done in a material called agarose.