Michael, Entry #4, Work Hard, Play Hard

As I'm sure you can tell from the title, this was one of the most action-packed days in lab so far. In addition to running two separate projects today, it was also Jalen's birthday, the fourth one since I arrived here in June.

I got right to work when I got in around 8:15, since I had to run a glucose uptake trial. This trial was my first using the glucose strips and meter which I borrowed from Dr. Johannes' lab yesterday. Today's trial used untransfected cells because I wanted to ensure that the glucose strip protocol worked for me before using the expensive plasmids. I placed a very small amount of media on each glucose strip at intervals of one hour, and saw that there was indeed a decrease in glucose concentration of the media, indicating that the cells were taking up glucose as predicted.

In addition to the glucose uptake trial, I also ran a Western blot today. I was looking for levels of glucokinase, an enzyme that is involved in the early steps of glycolysis and helps convert glucose to energy. Therefore, this enzyme is related to the glucose uptake experiment (my original). I transfected cells with the Hepatitis B Virus (HBV) and the *7 plasmid (HBV genome without the HBx protein). My lab focuses on HBV, specifically HBx, so these two plasmids are widely used. If the HBV plasmid changes the levels of a protein while the *7 plasmid has no change, we can infer that HBx is responsible for this change.

In the middle of the glucose uptake trial and while the gels were running, we decided to eat a five-star lunch at Chik-Fil-A for Jalen's birthday. It's a fairly short walk from the lab to the mall where Chik-Fil-A is located, and we made it back in time to check on our experiments.

Tomorrow is the last day for both Jalen and Sidney. Their program will be in Philadelphia until next Friday, but they have to present early next week, and they aren't required to come back into lab after their presentation. We've had a pretty fun time in lab together, so tomorrow is a bit of a bittersweet moment. Since they are both from Texas, we probably won't see each other much after tomorrow, but we made sure to follow each other on Instagram, so I'm sure we'll keep in touch!

Jay Ha #3 advanced oxidation

I spent the past ten days or so doing number of experiments related to advanced oxidation of OH free radicals. Purpose of these experiments was to understand the kinetics and mechanism of advanced oxidation process that I would be using next week when I am conducting UV disinfection of E. Coli using hydrogen peroxide oxidation processes.

Four experiments were conducted this past week. First one was to find experimental concentration of hydrogen peroxide. Although the concentration of h2o2 can be easily calculated using the equation of 10*d*%/molecular weight, hydrogen peroxide tends to decompose itself after the container is initially unsealed, so it was necessary to find the concentration through experiments. The calculated value was 9.8M while the experimental average was 10.486M so this value was used for further experiments. 

Second set of experiments were the decomposition hydrogen peroxide. Hydrogen peroxide were put to these   equipment containing 6, 4w UVC lamps. I extracted samples at varying time periods to see the rate at which h2o2 is decomposing since as 1 mole of h2o2 decomposes 2 moles of OH radicals are being created. This experiment was done in three varying concentrations of H2O2 solutions and the experiment was conducted twice to evaluate the error bounds.

Third experiment dealt with the production of 4-HBA acid. This was conducted because as produced OH radicals from decomposition of H2O2 combines with benzoic acid to produce 4-HBA and this value enables researcher to estimate the amount of OH radicals that are being produced and reacting to form other compounds. 

Last experiment was the decomposition of methleyne blue dye, and this was conducted because I can visually see the waste material (methleyen blue) being inactivated by OH radicals that decomposed from H2O2. 

This week was more tiring than other weeks in that there were so many data and graphs to work on, but it was kind of fun as well.  

David, Entry #5, Fourth of July Madness

While I was very excited for the 4th since my birthday happens to be on the 3rd, everyone I work with dreads the day. The Fourth is the one day that all humanity at the shore seems to go crazy in a "independence 'Merica" kind of way that leads to people getting drunk and doing stupid stuff on beaches. If they did this in a vacuum all would be fine, I guess, but they don't, so all the stupid things people do during the holiday haze negatively impacts plovers. Each biologist I work with gets assigned a segment of beach to patrol to call the police if anyone is disturbing the plovers. While everyone else in this country is celebrating, these very dedicated biologists are out late into the night protecting birds. I commend them for their dedication. At Belmar a group of guys decided to set a box of firecrackers off mere feet from a colony of terns. At North Brigantine a guy decided to drive around the vehicle barrier and do donuts on the beach, tearing up and destroying sensitive habitat. We also lost chick the day he illegally drove on the beach, so we highly suspect he ran it over. All across the state biologists reported horror stories like this of arrogant people harming wildlife in the name of "freedom." I'll quote spider man by saying, "with great power comes great responsibility." Sadly, the vast majority of people living at the Jersey shore seem to ignore the responsibility part. They destroy the environment they live in and then wonder why their house is flooded during storms. Our earth is deeply connected. You cannot change one thing without changing many many others. In response to the person who drove around the vehicle barrier at North Brigantine, we moved the vehicle barrier a quarter mile further down on the beach. Many town residents drove up to us as we were doing this and very vocally expressed their anger. Dealing with the general public is a very stressful and frustrating part of my work, but it's very important.

Pictured above is the vehicle barrier we built. This took about 2 hours of hard work. The barrier is created by putting large cedar posts in the sand in intervals smaller than the width of a car. Yellow cord with orange flags is strung across. Signs are put on the posts that say "endangered species area, keep out!" 

As I've mentioned in a previous post, studying endangered species has its ups and downs. There is a strong emotional component that has slowly been developing in me. Last week it really seemed to hit me. We were re-capturing chicks at Holgate. We like to give all of our plovers names that we use in the field. The parents of this particular nest are Ross and Paula. The two chicks were Tuna and Noodle. However, when we got to the nest site Emily said something along the lines of, "we need to catch Ross' chick." I really didn't think anything of it until I had Tuna in my hand and realized Noodle was missing. I looked in the cat carrier we put the plovers in while we process them. Empty. Noodle had been taken only a day or two prior by a predator, yet the week before I held Noodle in my hands. I looked down into my palms as if Noodle would suddenly appear, unharmed. I could hear the trill coming from her in my memory. I could see her squirming in between my fingers. Well, I guess that's the whole point of my work: to better protect plovers in the future. If anything, it served as motivation. We work in some pretty bad conditions. There are thousands of insects that constantly bite us all day everyday. We've been caught in a few thunderstorms on the open beach with metal equipment, very very scary, especially because a lighting bolt hit the ground about 20 feet away from us the other day. One thing I've noticed about the people I work with is their extreme devotion to these birds that allows them to work through these tough circumstances. I too am developing such motivation. 

Since I've talked about two sad topics on this post thus far, I'm going to end on a happy note. Enjoy the following pictures of adorable plover chicks! 

Meet the Harry Potter nest! Their names are Serius, Severis, Hedwig, and Dobby (not pictured). We didn't realize until after the fact that we named them all after characters that die...Hopefully there will be some reverse psychology with the predators and they will all live! 

Chicks snuggling with their dad may be the cutest thing I've ever seen! Notice the nano tag on the chick to the far right. That allows us to track the birds 24/7. It's pretty cool technology. 

Ally, Entry #3, I forgot a title last time so I need one this time so don't forget about this

I wanted to start this post off by saying that one day I walked into the lab and Carola was listening to the Wicked soundtrack and I got it stuck in my head and went home and downloaded it, and since then that has been all that I've listened to.

The past weeks has been a mix of busy and waiting for things to do.  One day the maintenance crew was supposed to do work on the fume hood, but never did, so we wasted a day that could have been used to extract FA from tissue samples.  Other than that, it's been pretty busy in the lab.

Mike and Mario went out to Tuckerton to fish for the blue crabs and caught around 85-90 which they brought back.  To go into more detail, we're using the crabs to find a baseline for the FA levels in blue crabs based on 2 different diets (black sea bass and clams).  Again, FA are used as dietary trackers to construct a food web.  The concentration of FA in prey will dictate the FA concentration in predators, and by knowing those specific concentrations, a food web can be constructed.  That's what the crabs will be used for in the gulf project which as a reminder is basically to construct a food web in the Gulf of Mexico to see if the 2010 Horizon oil spill affected it in any way.

Anyway, Steph, Carola, and I prepped for the crabs while Mike and Mario were catching them.  Mike had bought tupperware that holds around 1.5 liters of water, what the crabs are being held in.  I labelled 88 of those with a square of plain making tape to give a number to the crab, and with either red or blue tape for  respectively a sea bass or clam diet.  We rigged the tubing for the bubblers to aerate the sea water.  The crabs are fed every day, and the water changed every weekday.

The sea bass we prepared already for the crabs to eat, as mentioned in the previous entry.  On friday, Mike, Carola, Steph, and I prepared the clams, and let me tell you it was the most disgusting think I've done in a while.  The clams were relatively fresh, Mike bought them the previous day and they had been caught around Monday of Tuesday, but unlike fresh fish, fresh clams still smell very very bad.  For almost 3 hours, Mike and I shared a cutting board and minced the clams with filleting knives while Steph and Carola did the same except with scissors, and we packed the goop into ice cube trays.  Standing and in a constant state of nausea for an extended period of time isn't fun, and I wouldn't recommend it to those with a queezy  stomach.  I couldn't be in marine biology for the smell alone, although Mike tells me that a lot of the field is working with models and computers, not as much lab work as you would think, and I've gotten to do most of the dirty work.

Steph and I also did a lot of errands during our lunch breaks.  Things like picking up markers and a white board for the lab, etc.  However, since Mario killed all the little fish in his aquarium in his office, Steph and I went to Petco and got 2 more guppies, a yellow one I named Owen Wilson, and an orange one.  We asked the cashier what we should name the orange one, and he said creamsicle,and since his name is Joe we named the fish Creamsickle Joe.

Mrs. Terhaar is visiting tomorrow, so I'll have to be doing something more exciting than cleaning test tubes.

Michael, Entry #3, Multitasking

Since my last blog post, I've changed the methods for my original project and also added a new project. For the original, which sought to measure how the Hepatitis B Virus regulates glucose uptake, Dr. Bouchard and I decided that it would be more productive and efficient to measure the glucose concentration of the media rather than going through the entire process of collecting the cells and using the complicated Guava machine. In order to accomplish this, I will use glucose strips (as used by diabetics to measure blood sugar), which one of Dr. Bouchard's colleagues currently uses and feels work better than the Guava.

The other project I will be doing will be to measure the levels of glucokinase in HBV-infected cells and determine whether HBV regulates the production of this enzyme, which is involved in the early steps of glycolysis. Dr. Bouchard also wants me to look at the levels of the Glut transporter proteins, which Jalen did some work on (but the results have not yet been confirmed). In order to do this, I will be running a Western blot. I have ran multiple Western blots so far (both for practice and while helping out other lab members), so I am expecting the procedure to go well.

Monday will be a busy day for me. I am slotted to present at lab meeting, along with my fellow high school students, Jalen and Sidney. They are in the same program (STEM Prep), in which they have to present to their peers and mentors in early August, so this will amount to a warm-up round for them. Jalen worked in the Bouchard Lab last year, and assures us that we have nothing to worry about at lab meeting. All of the lab members have been extremely kind and helpful, so I'm sure Jalen is right.

In addition, I will be preparing for both the glucose uptake and glucokinase projects on Monday. I will have to split all of my cells for my three stock plates and three six-well plates, which will be transfected with the HBV plasmid (among others) on Tuesday. For the rest of the week, I will be measuring the glucose uptake by the new process (which worked downstairs and should work for me) and completing the Western blot. I look forward to getting some meaningful results. Since Jalen, Sidney, and I are working on similar topics, we are hoping to collaborate and piece together a more comprehensive understanding of the relationship between HBV and glucose once we all complete our projects.

Kelsie, Entry #6, A Full Circle Ending

Tuesday so far has not gone as planned.  I was expecting to be able to start by putting the primary on my one blot but the problem I faced was that the antibody had not come in yet.  Julie ordered it before she left so it should have arrived but no one received any antibody packages and no one can find it. I’m hoping that the company is running behind or hasn’t shipped it yet instead of us receiving it and getting lost in the lab.  Anyway, Julie told me to skip it and go to the next primary.  So I put on the PSD95 and hopefully the Axin1 antibody will show up.  With the other blot I did a FastGreen to see the total protein on the blot.  I had to make more of the destain so I got to use the methanol and acetic acid so I was happy to do that.  Then while the primary was incubating, I worked on my blog post and my poster.  Julie had said that my intro looked very good and made sense and was accurate so now all I really have to work on is the method because we ended up doing something different than I was told and the actual results.  I went out to lunch with the now Dr. Venanzi and Ms. Cozine along with Matt, Emma, and Julius.  That was a lot of fun, we went to Wahoo’s which I had never heard of before.  Then afterwards, there were cornhole boards outside so we started tossing some bean bags while Dr. Venanzi and Ms. Cozine were taking pictures.  The trek back was long and hot but Matt and I did it safely.  Tomorrow, Dr. Venanzi and Ms. Cozine are coming in again and we are going to a place called the White Dog, a couple of the others were saying it’s really good so I’m looking forward to that.  I finally got back to the lab and the paper for the Axin1 delivery was on my bench!  That was very exciting because now I don’t have to worry about not having it and what will happen once I leave.  So I got back and then began the washes on the blot I had put the primary on earlier.  It was later than I thought and I was thinking about what I should do so that I don’t keep my mom waiting for too long outside.  I finished the washes and went down to the dark room to develop my film.  Luckily the signal was very strong and I only had to lay the film on for a couple of seconds and the bands showed up well.  I got back upstairs, put the Axin1 antibody on and set it in the cold room overnight.

Wednesday started with a nice ride in with Dr. Venanzi and Ms. Cozine.  We were talking about a lot of different and interesting things and laughed a lot.  Once I got into the lab I started to run a gel.  I had a couple questions to ask Eddie and Anna but it ended up working out well.  I loaded the gel and let that run while I prepared for the transfer.  I set the transfer up after the gel ran and let that run while I went out to lunch with all the other EXPers.  The lunch was a lot of fun because I got to talk to everyone and the food was really good as well.  We all walked back to our respective labs and once I got back the transfer was still running but I had set it to run longer than I needed to make sure that it was ok when I came back.  I took that out and blocked it with BSA.  I started the washes on the other gel I had set in the cold room the night before.  When I was done with the first set of washes and had to put the secondary on, I didn’t realize I didn’t have enough BSA to both block and make the secondary.  Luckily the blocking was finished at just about the same time as the other blot so I could reuse the BSA that was used for blocking and then make the secondary.  While that incubated, I scanned in the films I had done the previous few times.  I hadn’t had time to scan them in when I completed them so luckily I had time today to get them into the system and quantify them.  When I was done with the second set of washes, I went down to the dark room and developed the films.  Once I was finished I came back up and put Axin2 on because the last time we tried it, it ended up being dark and difficult to quantify.  Unfortunately, the same band has been showing up on the previous three blots so that one, synaptophysin, must have a really strong signal.  I put the Axin1 back on the blot and hopefully if I expose it for longer the other bands will appear.

The ride in on Thursday didn’t go quite as planned.  My mom’s car started to give her trouble when she ran out to get something and so we had to go get my grandmother’s car which was north.  Then we got on the turnpike but she started to head north instead of south.  Luckily, we were able to turn around at the next exit and go south.  Once I got in, I started on the washes for both blots.  I asked Anna and Eddie a couple questions about my project but the main problem is that they are not familiar with this project and therefore can’t accurately answer my question unless they have more information, information I don’t know.  Hopefully, I’ll be able to figure it out and Julie can help me but I’ll do my best.  I put the secondary on both blots and I had to be careful that I put the correct one on the correct blot.  I did that and did it successfully.  I finished up with the washes and then went down to the dark room.  The blot that has been giving me trouble still gave me trouble, the protein I wanted, Axin1, didn’t show up only the synaptophsyin did.  But the other blot, that I ran the gel yesterday, worked really well and the PSD95 showed up nicely.  I was very happy with that and it made me feel better that one actually worked.  Then there was a meeting that I didn’t have to go to so I was able to leave early. 

Friday.  My last day as of the moment.  I brought in donuts today because I am planning on today being my last day.  I will be done with everything Julie gave me to do after she left so unless Kelly needs me to do something else, I’m finished.  I came in and started to work immediately.  I grabbed my blot from the cold room that was incubating in primary and I started the washes.  For the other blot, I decided that no matter what I’m doing the same protein is showing up so I ran a FastGreen to determine what the total protein is and make sure I’m not going crazy.  I put the secondary on while the destain was on the other blot.  I’m hoping the FastGreen worked decently well or that it shows the only protein that’s prominently on there is the synaptophysin that continues to show up.  I then started to look for the file that has all the pictures I took a while ago and it wasn’t on the computer I needed it to be but it was still on the computer in the microscope room.  I asked Anna if she could help me and she asked Cagla if we could use her flash drive which she let me use it.  So I’ll have all those images on the proper computer so I can then analyze the neurites.  I did the last three washes on the remaining blot and then went downstairs to the dark room.  Luckily, the antibody worked well and the bands showed up nicely.  When I got back upstairs, I didn’t have any more proteins to probe for on this blot so I decided to run a FastGreen to determine the total protein concentration.  While I was waiting I got a response from Kelly and we will meet next Tuesday or Thursday to discuss my results and what I’ve learned and what my career plans are.  While the blot I FastGreened dried, I counted neurites on the neurons I had taken images of a little while ago.  Seems like an appropriate way to end my time in the lab, I started with counting neurites and I ended with counting neurites.  Things really come full circle.

Post #3, EXP Fun Fun

I have around 2 more weeks left at the Ballatore Lab at UPenn, and so far I've had a very good experience. Although my lab is small, with only 4 people working at the moment, there is a very unique dynamic at play, and always an opportunity to learn something new (or make a mistake). 

Goals

So, as I explained in my previous blog post, we completed the reaction to create: 

5-Chloro-6-(2,4,6-trifluorophenyl)-N-(2,2,2-trifluoroethyl)-[1,2,4]triazolo[1,5-a]pyrimidine-7-amine (C₁₃H₆ClF₆H₅)

Overall, the yield was 100mg, or above around 30% yield, which was the goal for this reaction. Since the compound is a possible drug candidate, the goal was to produce >100mg, the amount required to conduct advanced drug testing. Our lab is unique in that Dr. Ballatore is a professor in both the Department of Chemistry and the Department of Pathology and Laboratory Medicine; this means that there is close collaboration between the Ballatore Lab (Chemistry) and the Center for Neurodegenerative Research (Drug Testing/Biology) in the Perelman school of Medicine. Whereas labs elsewhere sometimes spend months collaborating in order to mediate drug synthesis and testing, compounds created in our lab are able to be quickly tested for pathological "activity" (whether they display stabilization of tubulin, a key factor in Alzheimer's research), with results within a week or two. 

With this in mind, Dr. Carlo decided that the next step would be to 

1.Create a new, previously un-synthesized compound

2. Visit the Perelman School of Medicine and observe drug testing

Reaction Overview

Although the reaction sounds confusing, it was almost identical to the previous reaction I previously finished; the only difference was the amine that we used, the functional group that was added to the starting compound in the substitution. (The products of both reactions belong to the same family of chemical analogues known as "imidazoles"). Another minor difference was the scale of the reaction. Since the reaction had not been done before, we used approximately 4x less reagent than in a normal reaction, in order to minimize possible loss of compounds.

The new compound I would synthesize was 5-Chloro-6-(2,4,6-trifluorophenyl)-N-[1,3-Dimethylbutylamine][1,2,4]triazolo[1,5-a]pyrimidin-7-amine (C₁₇H₆ClF₆) through a "substitution reaction", a reaction where "one functional group in a chemical compound is replaced by another functional group" (Wikipedia). Simplified, this meant I would remove a functional group off the starting compound (a chlorine atom) and replace it with an amine group, in this case "1,3-Dimethylbutylamine". 

1.     5,7-dichloro-6-(2,4,6-trifluorophenyl) [1,2,4] triazolo [1,5-a] pyrimidine (starting compound)
 2.     1,3-Dimethylbutylamine (amine group)
3.      5-Chloro-6-(2,4,6-trifluorophenyl)-N-[1,3-Dimethylbutylamine][1,2,4]triazolo[1,5-a]pyrimidin-7-amine (final compound)

The process of synthesizing the compound was relatively straightforward, as I had already successfully completed a very similar reaction. The steps were exactly the same: dissolution of the starting compound in solvent (DMF), followed by the quick addition of the amine and base (DiEA), before leaving the reaction to complete overnight. 

LC-MS (It takes samples from the test tubes)

Afterwards, we took the reaction mixture and did an LC-MS scan on it (Liquid Chromatography-Mass Spectrometry) in order to determine whether the reaction had been completed or not. 
(Mass-Spectrometry reveals the molecular weight of the compounds present in a sample and based on the molar weight of the predicted compound, we can tell whether the reaction is successful or not). 
Since we saw a large peak of the predicted compound, and no peaks for the reagents, we concluded that the reaction has concluded and began the purification, first completing a "workup" to purify the reaction mixture into a still relatively crude mixture, then using the HPLC (High-performance liquid chromatography) machine. 





Quick Digression (HPLC Summary):

HPLC (It expels the constituent compounds into test tubes)

The machine works according to the basic principles of liquid chromatography, the same way that a TLC or column chromatography would work. By running a certain "eluent" (a liquid mixture of a specific ratio between a polar and non-polar solvent) literally through a sample, the constituent compounds of the sample will separate, as they "adsorb"/adhere to the "eluent" at different times, based on their differing polarities. The HPLC machine, therefore, is a very precise and effective way to purify a compound (>95%), but usually takes a very long to conduct. In order to be sure of the precision of the machine, each trial/run can only handle up to 50mg of a sample, with each trial taking between 10-15 minutes (not including setting up the machine, throwing away tubes, etc). Since the scale of the reaction this time was relatively small though, this time the HPLC was quick and painless :). 

Finishing the HPLC, we were collected the tubes that contained the desired product (based on computer) and collected them in a single vial. Since the product was still dissolved in the eluent, we used a combination of Rotovapor (a machine that simply dissolves solvent) and the freeze-dryer (low pressure-vacuum) to get rid of the excess solvent and completely purify the product. 

Plans:
Having successfully conducted the reaction, the next step will be to bring the sample to the Perelman School of Medicine (Center for Neurodegenerative Disease Research) and see the drug tested on cells, specifically an ELISA test that will test for the sample's tubulin stabilizing abilities. Whether or not the compound will undergo further testing (transgenic mice, clinical testing) will be based on the results of the test (!).