Adam Levine and Kelly Clarkson come to celebrate my last week at CHOP!! (kinda)

Overall my time at the Falk lab at CHOP this summer was really enjoyable. Though my stay was longer than expected, I got exposure to a number of techniques and had the opportunity to give a presentation to the entire genetics department at CHOP. It was definitely nerve racking at first, once got into my presentation it was very easy for me to talk about my project. Because I was going to present to  all the other PI's and lab members in the department I gave a few practice presentations to Dr. Falk. After spending a lot of effort of my powerpoint and doing a couple run throughs, I built my confidence. Rebecca and Fred, undergrads in my lab, also gave presentations on their projects. I really enjoyed my time at CHOP and also the commute through Philly since I feel much more comfortable with the city than before. Walking through CHOP everyday was also a humbling and eye-opening experience because I got to frequently see ill children and it reminded me of the difference CHOP makes in so many family's lives. On one of my last days at CHOP, Maroon 5's Adam Levine and Kelly Clarkson came to visit and I got to see them give an interview in the Ryan Seacrest Media Center. I managed to snap a picture too!!

Glowing Worms -Last Week

Hi everyone, this is Rhea researching mitochondrial disease at CHOP.

Because last week, my life span results found that the gas-1 mutant worm had lost its main phenotype, a short lifespan, my PI asked my to perform florescence tests on it to see if it still had chemical differences  from the wild type. Since Fred, an undergrad, has been working with fluoresce for the past few months and perfecting the assay, he taught me what information fluorescence can uncover about the worms being studied. There are three different dyes that the worms are fed, Mito Sox, Mito Tracker Green (MTG), and TMRE. Mito Sox measures the oxidant burden in the animal's mitochondrion which should be higher in gas-1 mutant worms because the sicker animal experiences more oxidative stress in its cells. MTG measures the mito content in the animals cells and should be less in gas-1 worms because the mutant strain is born with less functioning mitochondria. TMRE measures the membrane potential in the animal's mitochondrial complex which should be less in gas-1 worms because their mitochondria are less capable of creating a concentration gradient with H+ ions.

To perform the fluorescence experiment, I first had to prepare enough plates. I had to spread them with OP50 e.coli and then with each dye. After placing about 60 young adults by hand on each plate, I incubated them for 24 hours so that they have time to ingest the e.coli with dye and the dye had time to adhere to the fatty cells in the animal. Then after 24 hours, I handpicked them again and transferred them onto a plate of fresh bacteria. This is done so that the worms can eat the e.coli without dye and clear their gut of the dye. We want only the dye that had penetrated into the cells to fluoresce not the food in the gut itself. After letting them clear their gut for 3-4 hours, I put levamisole (a drug that temporarily paralyzes them) on each plate making sure every worm is submerged in the drug. After giving the worms 30 minutes to fully paralyze, I took the plates to the microscope with a camera. I used different filters on the microscope for each dye, and zoomed into each individual worm and took pictures of each worm's pharyngal bulb.


Here's a good one:

About 2 hours of picture taking later I have to manually circle the pharyngal bulb using a software. This software then counts the pixels and measures the amount of light per pixel and calculates the necessary information. According to the fluorescence experiment, everything seems normal with the gas-1 strain. Nevertheless, my PI has ordered a fresh batch of worms for the lab to work with.  

Problems and Solutions (Week 6-7)

Hey guys! This is Rhea at CHOP, working on Mitochondrial Disease research.

Because I discovered that the mutated worms we're working with are showing less signs of well..being mutated, Julian asked me to preform a lifespan experiment without using FUDR to figure out if that was the cause of the strange results. FUDR is a chemical used to stop cell growth and this prevents the worms being observed to reproduce and infest our plates with hundreds of worm larvae. Julian has been using FUDR for many years now and it has never shown affects on the lifespan of worms but there has been a paper published which reads that FUDR may affect worms. I set up a lifespan experiment without FUDR to check if it was the source of lifespan longevity and when I checked on it the following day it looked like this.

The big clumps on the plate are colonies of contamination (bacteria). 

All of my lifespan plates looked like this and so I had to discard them all (about 25 plates of 20 individually picked and placed worms).

Because now I won't have time to restart my lifespan, I've decided to take on a fluoresce project which I've watched Fred do a few times. He grows worms in plates with different dyes which illuminate the pharyngeal bulb and he takes pictures of them under a special microscope. I'll be analyzing pictures to see if the mutated gas-1 strain compares to previous results to make sure the gas-1 are truly gas-1... addressing what I had found with my lifespan. 

The Real Deal Week 4-5

After a few weeks of waiting and disappointments, we're finally on a roll again. The last few weeks have been slow because we had a major contamination problem... meaning the plates we use to grow our worms had other bacteria and/or fungi growing in them. It took a while to pinpoint the source but after making batches of fresh plates, flasks of fresh E. coli and after taking extra precaution we finally have a stock of contamination free plates. However that was only the beginning of what’s to come…

The practice lifespan I started during my first few weeks in the lab is now coming to an end and the results show that the mutant worm is living much longer than it should be. The gas-1 strain (mutant strain) is supposed to have a median lifespan of about 8-10 days but my lifespan experiment shows that they are living 18-20 days…almost as long as the wild type (normal strain). Though I had begun prepping for my actual lifespan using drugs, my PI said that no experiments could be conducted with an animal that has lost its main phenotype. What she means is that the worm’s life span is a major phenotype that we use for experimentation and if this phenotype is not apparent in the animal we’re working with, then any experiments we do using this animal are worthless. All of the experiments in the lab that have  been working on has also been affected by this discovery. The mutant worm could be living longer than usual for a few reasons. 1. A random beneficial mutation may have occurred in one of the worms and then outlived the mutant worms and reproduced (in a sense survival of the fittest)

2. A wild type worm may have somehow crawled onto the mutant worm plate and then outlived the mutant worms (again survival of the fittest)

So long story short, my original drug lifespan has been put on hold until we find a solution. 

The Wait Finally Ends

Hey guys, it's Katie from the Children's Hospital of Philadelphia again.

So last week I received some news that I thought would be good, our probe was ordered and should arrive any day!  On the day Dr. Peretz and Dr. Crider came to visit my PI told me that even if it didn't arrive I would be able to start my project.  Unfortunately, that didn't end up happening and I was told to wait for delivery.  Almost a week later, we found out that the probe would not be arriving at all and I was given the go ahead to start my project.  With only a week to do a month's work of work I haven't had much down time.  Our project includes extracting, plating, and analyzing over 200 patient samples for HSV 1 and HSV 2.  The samples are run through a real time PCR thermocycler for amplification and the results are presented on a computer system.  I can't show you on the blog because I don't have access to my pictures yet but I will come poster time.  Anyway, the plate we are using has 96 wells all of which are filled with 50 mL of liquid.  Picture a tear drop in a miniature thimble and you kind of get the idea.  Our trial includes 4 different assays, two of which are type common (don't differentiate between HSV1 and HSV2) and the others are type specific (they tell you which one it is).  Each assay has a different target sequence so we are comparing the sensitivity and specificity of each.

My days now consist of arriving at the lab, retrieving my samples from the refrigerator, and moving to the amplification area.  Plating one run takes about an hour because you have to wait for your samples and master mixes to thaw, spin down your master mix, and then proceed to the actual aliquoting  of sample and master mix.  I generally get 2-4 plates done a day and I am down to my final run today.  By the end of today I should have my data ready to be analyzed and I will begin the long process of condensing a binder full of material into four spreadsheets.  Sounds fun right?

Overall I am enjoying my time in the lab.  Although I had more downtime and waiting time than anyone originally expected my project is coming along and things are looking pretty good. 

PROJECT!!!!

Hey it's Katie again from the Children's Hospital of Philadelphia.  Finally after 7 long months of waiting I finally have my project.  In Dr. Hodinka's lab, the primary goal of research is to improve their clinical outcomes.  With that in mind, he has assigned me to two new projects.  The one which has taken up the majority of my time is the HSV (Herpes Simplex Virus) comparison study.  Mike (a med-tech) and I are comparing 5 different HSV assays (three type specific and two type independent assays).  Mike and I are waiting for one more probe (a PCR agent that emits florescence when replication is occuring) before we can officially begin the study.

My other project is developing a new corona virus assay.  Corona virus is a respiratory virus that is most known for the SARS outbreak a few years ago.   However, this outcome is pretty rare and coronavirus usually results in the common cold.  Its name originated from the sun shaped rash that can sometimes develop on the patient's skin. The assay I will be developing tests for 4 strains of the virus.  So far this project has involved pulling past respiratory samples and doing a lot of paper work.  At this point I have over 700 patient samples on file, and that is just from one month!  Once we get a little deeper into the project I will fill you guys in on more details but I just wanted to check in!

$3,600 Responsibility and Mock Life Span Experiment! -Week 2

(Rhea - CHOP - Mitochondrial Disease Lab)

Now that I am much more acquainted with the lab members and the ways in which the lab runs, I'm being given a lot more responsibility. After learning to pour plates and spread bacteria, Fred (an undergrad) tested my skills by asking me to pour and spread my own plates without supervision. Taking great precaution, I made sure to follow the protocol correctly (ex. didn't blow up the autoclave machine, didn't let the agar solidify in the flask I prepared it in). Fred meticulously checked my final products and informed me that my plates were completely contamination free (that's a huge deal!!). Now if the lab is ever running out of plates or solutions, I can be called upon to help them out. Just the other day during my free time, I made 2 liters of S. Basal, a salt solution used to maintain the worms, and 75 new plates for Zsoka to use. Later in the week, Julian taught me about the biochemistry work in the lab. We test carbon-13 labeled worms under different conditions for their organic acids, amino acids and protein abundance by using gas chromatography/ mass spectrometry and high performance liquid chromatography. To make the samples for each type of testing, the worms have to be grown synchronously (all at the same life stage) and under the specified conditions (ex. with drug, without drug), and then collected into viles. Once in the viles, they need to be centrifuged, ground up with a little drill, and treated with certain chemicals to ensure the best results. After showing me how to make samples with the first two viles, Julian left me to do the rest saying "you probably have a steadier hand and a better eye than me anyway" in his thick Russian accent. He also reminded me that every one of the samples cost us $150 to put through testing so I made sure to be careful with each and most importantly I made sure not to mix up the order of the samples...otherwise we wouldn't know which results corresponded to which sample! Overall my second and a half week in the lab has been even more exciting than the first and the best part is that my PI, Dr. Falk, told me that at this rate, I could be starting my actual life span project next week! Woo hoo!

Added Responsibility - Week 2

Finally, a little more responsibility.  The end of my first week in lab finished with a bang as I was invited to take the validation of a new assay to completion.  Because this lab processes active patient samples, each test and modality used needs to be validated before hand.  This involves running the new and old test side by side and comparing results, specificity, and sensitivity.  Because all samples used in a validation are from previous patients, not current ones, I was able to run some of the tests.  The new assay we were testing was for HepB, both the initial antibody testing and the confirmatory assay.  In total we ran about 500 samples (at least 3 times each) and ended up with a 14 page excel sheet to prove it.  This project was a major improvement over the beginning of my week and offered some hope for the future.

My expectations were met toward the beginning of my second week.  Having observed most of the benches and tests in the lab, my PI Dr. Hodinka decided that I should get more hands on experience.  One of the tests he wanted me to run was a Western Blot HIV confirmatory assay.  This test detects proteins in a sample (HIV antibody in this case) by binding them to a strip.  Each band on the strip represents a different protein and the intensity of each band can indicate the severity of the virus present.  The blot that I ran compared two lots of controls; a high positive, low positive, and negative control.  Because this test is slightly outdated, it is preformed over night and is very labor intensive.  I had to mix my reagents and manually add them to my plate.  

HIV Western Blot      

The most exciting part of the week in my opinion was the indication that my project would begin sometime next week.  Because most advances made in the field of clinical virology in the future will be in the molecular testing area, Dr. Hodinka will most likely give me a project involving PCR of some kind.  After months of having no clue what I would be doing in the lab, this little inkling has me pretty excited.  Being the "gold standard" of molecular testing, RT-PCR (real time polymerase chain reaction) is highly specific and is a relatively efficient way of identifying and diagnosing viruses present in sample.  Differently from traditional end point PCR, real time PCR can detect amplification as it is happening and accurately report how severe an infection is based on the number of cycles it took a positive sample to pass a threshold.  This is a very useful piece of information for clinicians when making a diagnosis.

Overall I am very excited about the remainder of my time in the lab.  The people who work here are very friendly and always willing to explain something or offer insight when I need it.

My New Worm Pets! -Week 1

My first day at CHOP started out soggy and gloomy, but once I reached the tenth floor of the Abramson building and entered Dr. Falk's lab, I met two other friendly students working in Dr. Falk's lab for the summer (just to remind you I'm working in Dr. Falk's lab who works with Mitochondrial Disease at CHOP). Anyway, where was I? ...oh yeah so Fred, an undergrad at Penn, had been working in the lab during the school year as well and has a lot of experience with techniques and material in the lab. Rebecca, an undergrad at Notre Dame, very recently arrived at the lab and was in my shoes exactly a week ago. Before I could get my hands dirty in the lab, I had to learn the basics about mitochondria and about the subject of our experimentation, C. elegans. Essentially C. elegans is a 1 millimeter long, transparent worm which feeds on bacteria, typically E. coli. Fred told me that I will be working with two strains of C. elegans, N2 and Gas-1. The N2 strain is the wild type meaning it is the normal, healthy strain which has nothing wrong with it. The Gas-1 strain has a malfunctioning Complex 1 (a part of the electron transport chain in the inner membrane of the mitochondria). He also taught me how to pour agar plates (on which the worms and bacteria grow) and how to spread bacteria on the plates (the food for the worms) as I watched Rebecca do both. Fred also taught me how to use the dishwasher and the autoclave in case I ever ran out of clean glassware during my experiment. Then Julian, with whom I will doing my project since he is the life-span expert (the focus of my project), gave me my own worms to play with. He asked me to try to transfer them from plate to plate using his pick, which is essentially a rod with an extremely thin platinum wire at the end. From experience Julian told me that beginners usually kill their worms within a few days and tear holes in their agar plates but after a few hours of practicing I got the hang of it.

Here's a picture of my first ever plate:

The little lines you see are actually the ridges I made with the pick by accident when I tried to pick up and put down the worms, don't get too excited! The worms are much smaller and are very difficult to see with the naked eye...especially in this picture... sorry! Maybe if you zoom in?

My third plate:

You can see this one is much clearer and less damaged in comparison to my first plate. Woo hoo!! I made progress! I'm not a worm-killer anymore! The big blob in the middle is bacteria which the worms eat.

By the fourth day in, I have my own bench space, my own box of worms in the incubator with my name on it (yay!!), a lot more knowledge about C. elegans and the Philadelphia mass-transit system (phew!) and some awesome "colleagues" as Zsoka calls us :)

I hope everyone else at their lab is having fun and learning a lot too!

-Rhea 

The First of Many 12 Hour Days - Week 1

At 5:00 AM on the dot a sharp ring echoed through my room signaling the beginning of my time working in The Children's Hospital of Philadelphia (CHOP).  Two hours, two train rides and a taxi ride later I walked through the doors of the hospital.  Immediately the impact this experience would have on my life began to show.  All around me hung posters and images of the patients and doctors involved in this life saving institution.  Through the EXP program I was now a part of CHOP, which I think is pretty cool.

Because my lab is not soley research based there are a few unique protocols I had to follow before I started any work.  I was trained in the safetly regulations for the hospital and I took a tour through the different sections of the lab.  As the main Virology lab for the hospital, technicians are constantly running samples and testing patients who are waiting for their diagnosis.  As if that weren't enough work, several of the techs are responsible for validating new diagnostic modalities and equiptment that will be implemented in the future.  And just in case that didn't cover it, they were (are) now responsible for a 17 year old high school student from Peddie (me). 

After my first three days in the lab I have seen several steps of the process of diagnosing patients.  From processing to analysis each step must be done with care and accuracy EVERY TIME.  If not the results could be dire.  As a minor and not an official employee of CHOP it will be a while before I am cleared to do substantial work in the lab.  With that being said, there has not been a moment in the lab where I haven't been learning something.  Each member of Dr. Hodinka's team is about as knowledgable in their field as it gets and they always have something new to share. 

After my two week introduction to the lab is over I will recieve my project for the remaining four weeks. SO far the name of the game has been patience... learning how to take my time and STAR (the background of every computer in the lab) S-top T-hink A-ct R-eview: make sure all of your actions are well intended and appropriate.  I am excited for the remainder of my time in the lab and what new challenges await me.