Duke University | Howard Hughes Undergraduate Program

  So yeah. I guess 24 hours somehow turned into 408 hours in the blink of an eye. Funny how that works. But as I sit here waiting for all of the data I have accrued over the past two months to compile itself onto my lovely, portable desktop, I felt that the world deserved some minor glimpse of my research, since I have done a relatively poor job of explaining it thus far.

With a grand total of 7 days remaing in the program, (one of which will be spent eating and displaying our research before anyone interested) I am finally beginning to realize how little I have done in progressing toward anything remotely close to a conclusion. Simultaneously I am discovering just how much I have learned, about science, about research in a developmental biology lab, about Drosophila, about academia, and about my own potential as a scientist. Instead of being lost in a sea of possible careers, fascinating subjects, and daunting years of schooling, I am now almost able to precieve the mainland through the curtain of dawn's receding mist. That being said, I still cannot describe my future, or rather the future I want for myself, but find myself thirteen steps closer to this achievement.

So, the past 7 weeks have been spent working with dorsal closure, as previously mentioned and explained. The past 4 weeks I have been doing experiments with Drosophila embryos expressing trpa1RNAi and rpkRNAi genes, each knocking out their respective mechanosensing proteins, transient receptor potential A1 and ripped pocket. Our hypothesis was that these embryos would completely fail to complete dorsal closure or at the very least progress far slower than wild type embryos. Through about 100 hours total spent imaging and analyzing these embryos I have discovered that yes, the embryos DO close slower and, despite the rare fluke, not only slower but much MUCH slower. It was definitely a satisfying result to receive, because it means that these two proteins are pivotal in dorsal closure. Additionally, and this is actually more interesting, although many of the embryos were able to complete closure, the often died in the subsequent stages of embryogenesis without these proteins. Ginger, my mentor, has observed a distinct failure in head development and this could definitely result in the death of the little guys. Cool, right? Whatever these proteins are doing, it is not just mediating tension in dorsal closure, but perhaps mediating and directing intercellular signaling throughout the organism. I guess I wish that the embryos had just exploded from too much rogue tension, but you win some and you lose some, right? ; )

I have has begun to develop some side hypothesis from pure observation and instinct, the former of which I am not good at and the latter of which I have not really developed yet, thus I feel like they are purely born out of a natural predilection for seeing extreme possibilities in less than extreme circumstances. So those will have to wait to be pursued and presented at a later date.

My other project, the infamous CLONING PROJECT, has come to a sudden violent stop. Kind of. So the goal of this project was, as you might remember me hinting at, to create a transgenic fly expressing a GFP tagged antibody for phosphorylated tyrosine. This chemically modified amino acid is often found in proteins involved in signal transduction pathways, and thus seems like JUST the thing to be marking in our current course of research. Additionally, although the construct has been synthesized and expressed in other organisms, it has (to my knowledge) not been expressed in Drosophila melanogaster, and thus would be a great contribution to the fly community.

So the specifics of the cloning, the infinite number of recombination reactions and overnight liquid cultures, picking plates and then re-plating bugs, is far from exciting, but until yesterday the results had compensated. We successfully recombined the py20 gene (the phosphorylated tyrosine antibody gene) into a plasmid containing a 6x (6 amino acid) HIS tag to help identify the expression of the antibody in a western blot if necessary. We then transferred this whole construct into an expression clone and have now induced expression over the course of the past 2 days. And have seen very little evidence of a band indicating presence of the antibody. Bummer, right? all that work for nothing. Of course it could easily be due to the conditions of induction, the antibody could be toxic, the auto-induction media could be faulty, the list of possible errors goes on an on. And that is the hard part we now must commit to. Finding/troubleshooting, isolating, and repairing the mistake in induction. YAY for millions of variables shifting constantly! But life goes on, and it is always important to keep the ultimate goal in mind because it is so marvelously cool.

On the fun side of things, I saw watchmen recently and, while not being phenomenal, it surpassed many of my expectations since all I had heard were terrible reviews. I would recommend it to anyone and everyone, just keep an open mind and try to recognize the underlying messages present throughout the film. Plus who doesn’t love to stare at a ripped, naked, floating blue guy for 2 and a half hours?

You know what I hate? When movies end with aliens. Don’t get me wrong, I fully believe in the high probability of some other sentient life form inhabiting another planet in any number of distant solar systems similar to our own. But I just nearly scream with frustration when a decent-to-good movie, for example Indiana Jones and the Kingdom of the Crystal Skull, culminates with the discovery of some ancient, super-human extraterrestrial race that eliminates nearly every degree of believablility from the pre-ceding and post-ceding sections of the film. In my eyes it is a complete cop out by the director as he or she, instead of imagining an ingenious yet believable climax, simply answers the question put forth by the exposition with ALIENS. OOOOOOO!! 

This minor qualm I have with the film industry was once again realized in the movie “Knowing” with Nicholas Cage, which I watched only 3 days ago. I was entranced by the first 80 minutes of the movie, even when the plot twisted nearly beyond belief and the only reasonable solution seemed undeniably outlandish. The acting was good, the logical puzzles that the protagonist, John, solves are fascinating, and even the CGI was not half bad. I even enjoyed John’s ubiquitously pessimistic philosophy of life and the randomness of events, his argument for spontaneity over determinism. But then my opinion did a 180. Enter the Whisperer People! Aliens! Ahhhhh that’s the solution! Suddenly, the aliens swoop in, save random children across the globe and the earth is engulfed in an anomalous, explosively hot emission of thermal energy from the sun. By the Hammer of Zeus! What is humanity to do? Luckily, the children saved by the abnormally white Whisperer People are put onto a new planet harboring a hospitable environment to re-cultivate the Human species and begin life anew. So, although I still believe that the movie was worth watching, I would have loved to see a more creative, less abrupt, and more believable dénouement to the film.

But on to life at Duke and his outlying provinces. I had the pleasure of going home last weekend, July 4th included, to a fun-filled 3 days of nothing, nothing, and more nothing. It makes one realize how, despite the difficulty of lab work and the knowledge the unending stream of responsibilities outside of lab to maintain a degree of sanity and health in life, that to be busy and productive and learning is truly an ideal situation. Even though it is truly a relief to have someone to cook your meals and clean your clothing while you relax in the land of doldrums, there is something to be said about harvesting the rewards of independence, bringing home a pay check, and simply standing on your own two feet for once.

Speaking of giving speeches, this past weak served witness to the Mid-Program Chalk talks of the HHRF, a 4 day extravaganza of blissful learning. We were each tasked with an 8 minute talk about our research up to this point which was subsequently followed by 2 minutes of questions. My initial goal going into the presentation was to do a generally good job of explaining dorsal closure, and then spend only a fraction of the time integrating this information into the experiments I am actually doing. I valiantly drew some pictures, and spoke what I thought was English, and probably made a more garbled, convoluted, series of explanations that I thought previously possible. So all in all, it went pretty well! I will update the blog with my research in under 24hrs, but until then please try not to die of anticipation.

Last, let me urge you never to forget one simple fact, that “Moisture is the essence of wetness, and wetness is the essence of beauty. “

 

Once again we return to our scheduled blog programming with another epic installment of life during the HHRF with your host, Julian Genkins! This past Thursday marked the beginning of a new era, an era that will surely change our world forever. What happened? Well, put simply, Michael Jackson died of cardiac arrest. Simultaneously one of the most lauded and mocked celebrities of our time, none can deny the impact of his seraphic falsetto crooning whether as a cute young boy while part of the Jackson 5, or as the bizarrely transformed plastic man of the past couple decades. No matter what, his life was a Thriller, and his death reminds us that nobody could Beat It like Mike. His heart must have stopped because it had had enough, and whatever the case, I know that his will be missed by Billie Jean and the rest of the world. Now, whenever I look at the Man in the Mirror, I will forever think of Michael Jackson and the bizarre, yet world-changing legacy he left behind. 

Speaking of bizarre, who would have thought that Gandalf could die to AIDS? I mean, the guy is a pretty skilled wizard; one would think that he could save himself or at least prevent immediate immune system collapse. I always figured that Tolkien create the Balrog as a symbol for something, what I never deduced was that it represented AIDS. And considering he wrote the Lord of the Rings during the 30’s and 40’s, it is incredible that he already had knowledge of the disease. But if you consider the two, one a retrovirus that invades and compromises your immune system leaving your body open to all sorts of nasty opportunistic infections, and the other a big fiery monster, I get the feeling that Tolkien was grasping at straws and didn’t yet have a complete understanding of the disease. Or maybe Ian McKellen is only an actor and not really Gandalf. I think the latter is more likely though. I guess we will have to wait for the sequel to “And the Band Played On” to find out if he returns as Gandalf the White or not. The anticipation is really killing me, especially since there has been no evidence of a sequel nor would it make logical sense with the storyline. But hey, if band plays on, who says it has to stop with just one movie.

Despite the incredibly intriguing nature of discovering Gandalf’s true sexual preferences as well as hearing him speak gibberish (more than likely it was just an arcane language which we fail to recognize as aliens to the land of Middle Earth), I guess that the true purpose of watching this film was not to explore the viability of Tolkien characters on true Earth. Instead the motive behind the movie was to expose the unethical methods used by Dr. Robert Gallo to gain renown for the discovery and identification of the HIV virus, which others such as the CDC and the Pasteur Institute had truly identified and described. His bending of trust and falsification of information exemplifies unscrupulous scientific research, an omnipresent problem in the scientific world. The dispute between Gallo and the head of the Pasteur Institute team, Dr. Luc Montagnier, although settled comfortably in the late 80’s and 90’s, was truly put right by the awarding of the Nobel Prize to Montagnier last year. Although it took 3 decades, this result is evidence that trying to falsify science, to gain credit for discoveries that you have not actually made does not pay and is easy enough to see through. This is also seen in the number of retracted “scholarly” papers presenting fabricated data or stolen information.

But bad research is no fun. Good research is where all the excitement is, therefore it will be much more interesting if I discuss my own. So finally, after 3 weeks, I have completed the preparation for my true investigation of drosophila Embryos and will begin it next week. I have established an extensive portfolio of GFP and RFP marked embryos, showing that their rate of closure is unaffected by the combination of markers. And now the fun begins. Ginger, my mentor if you forgot, has established 3 lines of flies with RNAi genes that, when expressed, effectively knockdown or knockout three proteins responsible in some way for mechanosensation in Drosophila. These three genes, RPK, TRPA1, and nompC, are all thought to be pivotal in the tension detecting processes that mediate dorsal closure. Whether they are actually the gated channel mechanosensors themselves, or involved in the transduction of the mechanical signal beyond the cell membranes is uncertain, and that is where I come in. Well, at least that is the ULTIMATE goal of this experiment, to identify and categorized the major role of the proteins coded by these 3 genes in the process of mechanosensation. Thus, next week I will begin imaging RNAi expressing, GFP/RFP marked embryos and analyze their rate of Dorsal Closure. Although the phenotypes have been varied (at best) in the RNAi flies, and it is difficult to identify the mutant phenotypes because of how irregular they look, I have high hopes for the stock currently being crossed and cannot wait to report on the results of the imaging.

Additionally, besides embarking on a more intriguing voyage into mechanosensation, I have also been charged to finish a cloning project begun by Ginger a couple months ago. It, once again, involves the creation of a new drosophila capable of being imaged live while marked with GFP at the sites of phosphoylated tyrosine (phosphotyrosine). But just to keep you on the edge of your seats I will leave it to my next entry to discuss this project. Until Then!
 

Stay classy, San Diego

All is well in the lab lifestyle, as I search for answers to the great mysteries of humanities in the tiny, thoughtless embryos of my favorite insectoid friends. My tiny subjects, despite being fated for a short career in modeling before their ultimate, timely end, continually strike me with their embryonic beauty. I find myself rooting for each miniscule drosophila as I place them onto slides, cheering for their successful expression of GFP and RFP such that I can immortalize their dorsal openings forever. Some might identify these feelings as Lima syndrome, the inverse of Stockholm syndrome, and some might just be correct. But I still like to imagine that, as I shoot lasers through the oblong, white creatures, they are looking back at me and smiling, grateful to be contributing to the advancement of science!

So here we are, back for our second installment of news from Blogwarts. After two weeks on a bicycle, I am finally figuring out how to work the pedals, and this one time I successfully turned left. I was so excited, not in the Zoolander sense of a turning inability, but instead in the sense that I had only been able to turn a few degrees off of straight before this momentous event. The art of going up curbs still eludes me, and this inability has already made for multiple embarrassing encounters. So if you ever see me riding my bicycle near a curb, and somehow I end up falling and throwing myself at you, please do not get the wrong idea. It was just an accident, I promise. Although, come to think of it, I could probably use my cycling handicap to my advantage! I just can’t wait until I actually learn how to ride a bike!

Which leads smoothly into my expectations for this summer and the research I am attempting to do during my 8 week behind a microscope. I would love to learn to ride a bicycle like Lance Armstrong. I also want to increase the size of my heart to become superhuman like him. Although this has yet to be related to my research, I feel that I am coming closer and closer to bridging the gap between dorsal closure in drosophila melanogaster and forced, rapid microevolution in vascular tissue. Or maybe not. I also am hoping to learn how to glide, in the dancing sense of the word, so that I can move everywhere without actually LOOKING like I am moving. Gliding is harder than it looks though, as any large bird or those sail plane things can tell you.

But beyond gliding and biking, I also came into this summer with both ambitious and critical expectations for my lab experience. Just as I tell everyone who asked me a year ago, and those who continue to ask me now, I am wholly uncertain as to what I want to do in my future. So, why not try research? My hope is that, with the multitude of experiences I will accumulate over the course of the HHRF program, I can decide if I want to pursue research in life beyond the dream world of undergraduate college. I want to discover if I can love being immersed in solving scientific enigmas for 8 hours or more a day. So far, it hasn’t been too bad. In a broader sense, I also want to see if my true academic love is biology, if it is truly the subject I will be most happy pursuing in school and in a career. Most importantly, and most generally, I just want to learn. I want to fill every nook and cranny within my gray matter with knowledge. And I think after two weeks, it is about maxed out, partially due to my distinct lack of brain, but also the sheer amount of facts and information I have absorbed just working with the other scientists in the Kiehart lab.

So far, I have yet to fulfill any of these expectations completely enough to draw conclusions, but we are getting there. And last week, my research got much, much more interesting. After two days of work on a microscope worth more than my life, I was able to create beautiful pictures like this.

What you are seeing that picture is the dorsal opening in an ~24 hour old drosophila embryo. The RFP is Moesin and it highlights the actin/myosin filopodia throughout the embryo. The GFP is cadherin, which is a protein located in the intercellular adhesive regions in the amnioserosa. Thus, generally speaking, the red you see is the lateral epidermis of the embryo, and the green is the amnioserosa. As described in last week’s blog, this opening will eventually close as the green tissue is forced beneath the pinching “purse string” of the lateral epidermis. Voila, dorsal closure in color!

Besides the microscopy, I have also been learning significantly more about Ginger’s research, but I am going to find out more before pretending to know what I am talking about! Let me just leave my faithful readers (who actually don’t exist) with the cliff hanger that it is going to involve gated channels, mechanosensation, and RNAi mediated knockout of RPK and PPK proteins!

Live long and prosper!
 

 You know what I have always wanted? A baby with a face that looks like a cross between a hockey mask, a disco floor, and the matrix. And this week that life goal came closer to being realized upon the release of the Black Eyed Peas new Album, The E. N. D. This acronym, surprisingly enough, does not stand for the Epic of Newborn Drosophila, instead it is expanded to "The Energy Never Dies."

The Album is categorized by tradition BEP beats, will.i.am's fearless, lyrical rapping, and Fergie's angelic swooning. But really, the music, although lacking meaningful lyrics in most songs, is slowly growing on me, and songs like "I Gotta Feeling" and "One Tribe" are truly masterpieces. If you like the BEP, this CD is definitely worth the money, if you havent heard of them and like a good beat then this is still a good buy. Plus anyone who incorporates lechiem or mazel tov into their music wins my award of approval

But anyways, on to the stuff I am actually supposed to be writing about. Hi bloglodytes! My name is Julian, and I am a PC. Err I mean I am a lucky member of the group of Duke students chosen to participate in the Howard Hughes Research Fellowship over June and July. Here is a picture of me outside my castle.

It needs a little bit of work, but I find the dependable hardness of the stone and protection afforded by the crumbling towers to be a constant comfort. And roofs are definitely overrated. Although it was tough to leave behind this security, I am glad to be back at Duke. And I brought my longbow just in case. For the next two months, and hopefully longer, I will be working in the Kiehart lab on the third floor of the FFSC. My PI is Dan Kiehart, a benevolent, intelligent, and generally excellent man. Although he has been gone for most of the week I have been here, I can already tell that he works very hard, seeming to have all the answers while knowing all the questions.

This is Ginger. Ginger is my mentor. But more importantly, Ginger is amazing. She is a grad student here at Duke and knows just about all there is to know about Dorsal Closure in Drosophila. Which leads me to discussing my research!

The Kiehart lab focuses on a biological process in embryonic drosophila melanogaster known as Dorsal closure. Basically, this process is the closure of an epidermal opening on the dorsal side of the embryos and the subsequent apoptosis of amniserosal tissue that composes this opening. They use many types of imaging and time lapse photography to study the mechanism of cell structure and shape change for morphogensis in this process. The Kiehart lab actually works together with phyisics and math labs as well (not sure which ones) to apply mathematical models to this dorsal closure process and discover correlations that help us to better understand the underlying mechanisms. Dorsal closure itself is very similar to wound regeneration, and thus the ultimate goal of this research is to extrapolate the knowlege gained from observing embryonic drosophila to larger eukaryotes. Here is a link to a video showing the time lapse photography and how we use it to record the progress of dorsal closure.

And if the awesome people and fascinating research were not enough, they also have tea time! I mean, what could be better. And look at the acclectic assortement of tea avaliable for general consumption. It is absolutely fantastic, no wonder everyone in the lab seems so pleasant all the time.

 Happy Trails!

-Julian