Matt Boyle, Entry #5, Horse Round 2 and Last Day

After some thorough research, I found that equine IL-4 is nearly 5 times less effective of a protein than canine IL-4.  In the first round of experiments, I had been putting an amount of IL-4 into the wells equivalent to that of the dog experiment (2ml IL4/ml of media). However, if the equine protein is less effective, a much higher concentration of IL-4 must be added to the cells in order to induce a similar response.  We also decided on a new formula for growth media, consisting of horse serum as opposed to the original fetal calf serum, and pen strep as an antibiotic as opposed to what we used the first time (gentamycin), for there are published papers about people culturing equine B Cells using pen strep.

We are also adding a component to this experiment.  Some of the B Cells are going to be CFSE labeled.  This will tell us if cells are not proliferating at all, or if they divide and then die off immediately.  CFSE dies the cells a dark green and with each division of the cells the intensity of the green is cut in half.  Therefore we can flow the cells and the machine will tell us how intense the green is in relation to that very dark shade.  This will tell us if we are not getting any proliferation at all, or conversely, is the cells proliferate but their living conditions are such that the cells cannot survive.

New blood arrived on the Monday of my last week and again the PBMCs were again isolated and plated with and without feeder cells expressing CD40L.  They have been cultured with the new growth media and received a more concentrated dose of IL-4 so we will see how this one goes!

The Mason lab is embarking on a new project. Led by the PhD student Kazim, the first infusion of CAR-T Cells will be infused into a German S

heppard with lymphoma named Lady.  The CAR T Cell project is really remarkable as it uses the HIV Virus to infect healthy T-Cells that become “serial killer cells” that seek out and kill tumor cells.  Carl June, a revolutionary researcher at Penn, has done some incredible work with CAR T cells and you should really watch this video if you have 5 minutes. Its so cool.

https://www.youtube.com/watch?v=h6SzI2ZfPd4

Ms. Cozine came to visit me last week which I thought was pretty funny because the last time she saw me in a science setting was when I was in her freshmen chem honors class! I taught her how to count cells via hemacytometry, we made LB Agar plates, and even made some more 70% EtOH (ethanol) solution before meeting everyone for lunch.

 

Hemacytometry -- a 10ul sample of the cells in their growth
media is put in this little glass frame containing a tiny tiny grid that looks
like this under the microscope--you count all the cells within the boxes and
plug that (when multiplied by the volume of the media in the vial)
number into a big equation that gives you the total cell count in
the media.

A picture of my building I snagged on my way home on my second to last day.

Wednesday, my last day, was incredibly sad.  We all went out to dinner at Mad Mex and my friends from the lab gave me presents, including a necklace made from the exact vial from the liquid nitrogen freezer that once contained a type of cell that I mistakenly thawed and cultured.  Pretty funny.  Everyone has tried to convince me to go to Veterinary School while I have been here and they even gave me a Penn Vet T-shirt.  EXP has easily been the coolest experience I have had in any summer and I could not have asked for better people to work with, a cooler topic to study, or cooler city to do EXP in! From Phillies games to Cheesesteaks to the Palestra, and meeting my fellow EXPers in the city, it was all pretty cool.See you all in September!

Met Bridgid for a Phillies game.

Matt Boyle, Entry #4, Do We Only Cure Dogs? "Nayyyyy."

After all this talk about CD40 and K562s that are irradiated and culturing the cells, I realized I have never explained to you guys what CD-40 B Cell activation is.  My bad.   So here we go.

Cd40 B Cell activation is a method by which B Cells become antigen presenting cells.  The binding of CD40 to CD40 Ligand activates the cells, thereby increasing the ability of the cells to present foreign antigen to the immune system.  In the, the PBMCs of an organism are extracted and cultured with these feeder cells (from the K562 cell line) expressing CD40L.  In the presence of the cytokine interleukin 4 (IL-4) the activated cells proliferate, allowing for a large number of cells to be grown from a small number extracted from a blood sample.  This is extremely advantageous because, as each vaccine is specific to the host the blood was taken from, just a small amount of B cells from a small blood sample can grow into a huge amount of antigen presenting B cells that will increase the immune-response to the cancer.

The problem is (and the efforts of my project are based around) the feeder cells we have in the lab express HUMAN CD40L.  Canine CD40 recognized the human CD40L but we do not know if the horse CD40 will.  I performed a blast search on the homology of canine and equine CD40 and the proteins are approximately 70% similar so nothing is guaranteed.  Should the CD40 of the equine cells bind with the human CD40L, the cells will be activated and their proliferation will be sustained in the lab until B-Cell count reaches a threshold of viable cells able to be infused back into the horse.  Then the cells will be electroporated with the RNA from a tumor sample of the horse removed via a biopsy.  The B Cells will then present the tumor RNA as an foreign antigen to the immune system, and upon re-infusion into the horse (the same horse that the B Cells came from) its immune system will recognize the tumor as foreign, thereby educating it to recognize and kill the rest of the tumor cells.

An animation of the CD40 B Cell Activation. The little orange things on the sides is RNA being electroportated into the cells before re infusion of the B Cells into the patient 

The science behind the protocol is truly fascinating but that is only half of the reason I am enjoying EXP so much. The other half is because of the people I get to work with.  Dr. Mason Is truly hilarious; for example, when the horse blood came in she was walking around saying “NAYYY.”  Dr. Mason also just got a full-time vet to help her with the trials in the clinic (her name is Martha and she just graduated from Cornell’s Vet School).  We all went out to dinner in Philly for Martha’s first day where Dr. Mason was telling us about how she was invited to Richard Lichter’s dog’s birthday party on Saturday.  Richard Lichter is a multi-millionaire who owns Mel Gibson’s old estate in Greenwich Connecticut with 16.5 bathrooms) and his goal in life is to cure equine lymphoma.  He is an enormous benefactor for Penn’s veterinary research and has a party date set (April 16) for the celebration of Dr. Mason having cured canine lymphoma.  Upon hearing this, Dr. Mason decided we must get to work as cancer is certainly not cured yet. We sat outside at dinner and Dr. Mason was directing a girl to parallel park next to our table when a group of people came out of the restaurant in a huge fight.  People were screaming at each other and Dr. Mason did not miss a beat as she stood there like one of those people on the runways at airports helping this girl parallel park.  It was probably the funniest moment from my time in the lab.

As for the horse cells, they look horrendous. But not really.   If you look through the microscope at the K562 Wells in relation to the KtCD40L Wells, there is significant more clumping in the CD40L wells, leading one to believe the B Cells were indeed interacting with the human CD40 protein.  However, upon analysis, the B Cell populations were almost completely dead (as you can see in the flow graph).  Despite this failure, the way the cells looked through the microscope was very promising.  The cells seem to be interacting with the protein but not proliferating, which could be very closely related to an insufficient amount of the cytokine IL-4 that causes proliferation or perhaps there is insufficient serum, insulin, or other component of the growth media.

The K562 (left) and KtCD40L (right) wells as they appeared through the microscope.  There is obvious clumping going on in the KtCD40L wells; significantly more than in the K562 wells, leading one to believe that the equine CD40 is indeed interacting with the huamn CD40L.

 These the flowcytometry graphs pertaining to B Cell population in one horse (R600). You can see from the day the bloods arrived (left) that the B Cells made up 17% of the living PBMCs, but on day 7 when the cells were flowed again, the percentage of B Cells had shrunk to only 3.12% of the population.  This is an opposite result to what we thought was the case by looking at the cells through the microscope.

The plan from here is to get new horse blood and determine which components we will change from the media or in what is added to the wells to initiate B-Cell proliferation.  Now I am staying for an extra week to have some time to see round two through to completion.

Matt Boyle, Entry #3, Working Like a Dog (or should I say horse)

The plan was to get samples of horse blood last Wednesday until Dr. Mason realized that we did not have enough irradiated k562 cells (irradiated means the membranes are sort of stuck in time but the cells are still alive) for the control of the experiment.  The problem was that irradiated K562 cells expressing CD40 Ligand had been in the liquid nitrogen for several months and when we irradiated the K562 cells we wanted to put them in the liquid nitrogen as well to make the conditions as similar as possible to the CD40L expressing cells; who knows the effects that freezing a group of cells down to          -150⁰ dos  to the viability of cells.  Therefore, we postponed the blood delivery to next Monday I had to go into the liquid nitrogen and thaw out a vial of K562’s, grow them to a total of three million, irradiate them, and freeze them down.  By the weekend, they were in the liquid nitrogen and had been in identical conditions to the CD40L expressing cells.

Speaking of the irradiator, going to use that machine is an intense process.  The irradiator is located in the basement of my building which also happens to be the rat facility (that you can smell every day from the stairwells).  Once you get downstairs you need to put on little boot things and a hairnet and gloves and then at another station you must put on a sterile gown that looks like you are scrubbing in to perform a surgery.  Then you must stand in front of a machine that does a retina scan that allows you to open the door with a separate key.  I told you it is intense.   Dr. Mason took me down once and I wasn’t even allowed in the room.  She scanned her retina, entered the room, and I had to watch from outside.

Dr Mason at the irradiator, in her sterile gown and hair net, as I look on through the small window in the door.

 On Monday morning, the blood of three horses was pulled at New Bolton Center and shipped to Dr. Mason’s lab.  Dr. Mason made sure it would be “hand delivered” as to ensure optimal condition of the blood.  The members of the lab that are participating in the horse lab (myself, Dr. Mason, lab tech Josephine, and PhD student Kazim) had a quick meeting to make sure everyone knew what their role was and how the bloods would be handled.

The three horses all had labels to identify them by (R600, R665, and R289) however their given names were also written on the tubes of blood – Wise guy, Class Fantacy (spelled like that), and Idol – which I thought was hilarious.

The vials in which the horse blood arived.  You can see that each horse had an ID number as well as a given name that was usually pretty funny.

I was in charge of extracting the PBMCs by using a protocol that I had discovered was successfully used in a lab in 1978 to extract equine PBMCs over ficoll.  We did not know if this 1978 procedure would be more effective than the protocol the lab uses for the dogs.  We had 20ml of blood from each horse so we used a single sample of 10ml to decide which protocol we would use.  We got decent yield of PBMCs from the 1978 protocol which was around three million.  We attempted PBMC isolation with another 10ml of blood, this time with Dr. Mason’s protocol, and got a yield of approx. 8 million cells and therefore used her protocol on the remaining 20 ml. 

The equine PBMCs are pretty visible -- they are the clear carpet-like layer right below the yellow (which is serum)

The PBMCs were then cultured under three conditions.  For each horse, a given number of their cells would be left alone to see if the B Cells proliferate on their own (obviously they will not, this is just a formality to say we evaluated this).  The next set of wells contains The PBMCs cultured with K562 feeder cells.  This is the control for the following row in which the PBMCs are cultured with K562 cells that express CD40 Ligand.  The only difference between the last two rows is the expression (or lack thereof) of CD40 Ligand on the surface of the feeder cells.  Therefore, if there is a difference in growth of B cells among these rows, it HAS to b

This is a diagram of the plate that the cells were cultured in.  For the three different horses, there were three different conditions that the PBMCs were cultured in (by themselves, with plain feeder cells, with feeder cells that express human CD4

We will check the cells on days 2,4, and 7 and add new growth media and cytokines so by next week

we will be able to stain flow them (mark with antibodies and computer analyze) to determine the content of all the wells.  

Julius Sim, #5, Summary about Last Week at Penn

I finished my last week at my lab at UPenn under the supervision of Dr. Carlo Ballatore, working on microtubule-stabilizing agents as Alzheimer's disease therapeutics.

Azza- Entry #5

I have learned an incredible amount while I was at the Lab. It was definitely a great experience! I had the opportunity to transform, transfect and ligate bacteria. Grow small scale and large scale bacterial cultures. Work with mice and observe the work other lab members spent with zebrafish. I performed many minipreps and midipreps. I became super comfortable with PCR and gel electrophoresis. I also got to learn how to make and use protein gels and interpret the results .I also got to grow crystals, which an essential step in understanding the biophysics and biochemistry of the mutant protein or wild-type portein that you are researching.  I walked into UCSF knowing the basics such as PCR and gel and I left with a much greater understanding and appreciation for lab work. My graduate student taught me so much. I got to work with him while we focused on the main project of the lab and he also assigned me solo projects to work on. It was a fantastic experience

, and I would love to do it again!!!

Azza- Entry #4

One of my side projects was getting to make more of an enzyme called phusion that is used in PCR. Phusion is similar to the Taq polymerase were more familiar with, but the difference is that phusion is a lot more heat stable because it was first discovered in hot springs and therefore can withstand high annealing temperatures. It was definitely not easy trying to purify phusion with the FPLC, but once I was finally able to the amount of phusion I was able to replicate was worth up to $168,000! Taq is available for commercial purchase, but it’s also very expensive, so most labs (if they have the time) prefer to make more phusion on their own. The process is very straight-forward. You began with a phusion stock that is stored in the -80 degree. You use a pipette tip and scrap some phusion out and place it in a 25mL starter culture of broth (either LB or 2XYT). Because phusion has a kanamycin and chloramphenicol resistance gene, we used 25 uL of each to have a selection component. The next day we use the starter culture to induct a larger culture of 750 mL of bacterial broth. Then we spin down the larger culture and suspend the pellet in lysis buffer and sonicate and heat it up and finally run it on the FPLC. The FPLC is a machine basically composed of anion and cation exchange tubes that allow for the purification of phusion. To test if we’ve purified phusion we run the sample on a protein gel. The image below are my results after performing the protein gels. The different wells are different concentrations of phusion which were diluted with nuclease-free water. The last well is the commercial phusion we used to compare the one I made to. You can see how concentrated the phusion I made, which shows that they can now start using the phusion I made! 

 

Azza- Entry #3

Since I've come into the Lab, I was given my own personal project to work on. The goal of this project is to clone a plasmid responsible for creating Cas9. Cas9 is a protein that is a part of the CRISPR/Cas9 method of cleaving out around 20b.p of DNA and inserting foreign DNA into a cell line or an organism in order to observe a mutation of a targeted gene. Cas9 is an RNA guided DNA endonuclease enzyme. To clone CAs9 we are using a plasmid created by Martin Jinek, who once worked for my PI here at UCSF. Martin Jinek was also the student working for Jennifer Doudna when she discovered CRISPR/Cas9. Jinek is also the first author on some very famous papers that introduced the world to CRISPR/Cas9. The way I started off with this project was to convert the word document I've received from Martin Jinek for the coding of the MJ922 plasmind to an application called ape which lets you annotate certain regions of the plasmid. Then we got the plasmid from a postdoc student in the lab and transformed it using Rosetta cells. Rosetta cells are probably the most versatile cells used for transfection and transformation. Once we transformed the cells, we grew them at large scale. Then we spun them down and created large cultures and then extracted the  protein from the pellet using the FPLC machine. I only got so far with this project as time was running out and I wanted to stick with a different project of cloning and purifying more Phusion. 

this is what the annotation of the plasmid looked it, just to familiarize myself with what was being transformed.

Amy Qian #5: Last day here

I went to the museum again to collect data last Sunday and finished coding and uploading them on the last day. We still don't know the results yet and my mentor told me she is going to see if we have enough data later and email me about if we need more data. I asked my mentor and professor how this project is going to benefit our understanding on pedagogy contexts and how can we use this information in more practical areas and my professor told me to think about it myself and emailed her about what I think. 

Today is my last day at Stanford :( My lab manger organized the whole lab to have a picnic together. I brought food that I bought from local supermarket and we played frispi together. We had a lot of fun and I am truly going to miss my lab buddies :( 

My professor and lab manager bought me a Stanford mug and thanked me for helping them this summer. I had a lot of fun and learnt a lot in the past 6 weeks and hopefully I will see all of them again in the future! 

Thank you all for a great six weeks experience! 

Amy Qian #4: Data Collecting!

Again, I collected data for most of the week and coded the videos. In addition, I went to Bing Nursery School and helped my mentor for her another study. Basically, in this study, she is trying to see if children are more likely to tell the person who doesn't see them win the game that they win the game. In this study, children are presented a card games four times and they were asked to pick a card. If they pick a card that has a sticker on it, they receive a sticker. But if they pick a card that doesn't have a sticker on it, they don't get a sticker. For the first trial, all children are designed to get the sticker. For the second and third trial, they recieved a sticker card and a non sticker card once with two different people watching. Then, they played the game again and got stickers. Finally, the children were shown two pictures of the two people who watched the game and were asked to choose one to tell her the result. Hypothetically, professor predicted that children would choose the person who saw them did not win the game to tell the person that they won the game. 

Moreover, I went to the poster presentation of my lab members, Andrew and Grace. The poster presentation was a lot of fun and it reminded me of the poster I presented back in Peddie. There is one poster regarding how taking pictures on iPhone and sharing them on social media affected our memories that I found particularly interesting. The results show that people who took pictures and shared them on social media had better memories of the object that those of who did not share them on social media. However, taking photos while observing the object in general decreased person's memory on this object. 

Time passed fast and I can't believe it is my last week soon. I am going to miss this place a lot. 

Amy Qian #3 August 15th

Grace left at the end of this week for MA. Our lab members went to a spicy Chinese restaurant to eat dinner to congratulate Grace for finishing her summer internship. The dinner was great and I told my lab members to try all the "disgusting" Chinese food (such as organs) and they did! :) 

This week is particularly challenging for me because I went to the museum by myself for the first time. I had to do both recruiting and consenting. The first day was a big bomb. I got rejected six times in a roll and I felt so destroyed. In the end of the day, I only tested 2 children. My PI told me it was completely normal and everyone had good days and bad days. Because I went to the museum in the afternoon, most of the people were leaving and it made sense that people would reject me. 

I spent most of my time collecting data and coding my videos because my mentor and I figured that we are actually short of enough samples of 4 year olds. So my job this week is mostly collecting data. 

The past two weeks reading groups have been very interesting and the topics are on pedagogy contexts and learning. Moreover, I got a chance to present the paper with other undergrads. This is very exciting and new to me: I presented power point and my understanding of the paper in front of everyone. We examined whether being altruistic is human nature or not from both a social and neuroscience perspective.

I was a bit nervous at first because I was afraid that I might make some mistakes. However, my lab members are really nice and understandable and told me that I did a great job.

Amy Qian #2: First time testing!

This week I got to go with Grace and Mika to test in a local museum at Stanford. It was a really interesting experience. Our lab has a testing room in the local museum and we go to the museum and look for children of the right age and collect data from them by playing lab designed games. 

For the first couple times in the museum, I was piloting for Grace and Mika and did not get to the actual testing part myself.  Before testing, researchers need to recruit children and give parents consent forms. Mika sent me an powerpoint before hands on what kind of problems I would encounter during the recruiting and consenting process and how I should answer them. Since the experiments require video tape or audio tape, we need to be really careful in making sure parents  are okay with that. 

I got to test on my own project (VPT) on the third time going to the museum. Basically, in the experiment, the children are presented with a puppet show called Emma and her friends (laptop in a fancy box to create a feeling that this is a magic show). And then, the children are shown with two shows: 1. There is an orange on the table and Emma asks what fruit that is. The guy on the left says it is a banana and the guy on the right says it is an orange. 2. There is a number 6(from Emma's perspective) on the table. Based on different conditions, Emma either knows or doesn't know about the number. Then she asks what the number is. The guy on the right says either 6 or 9 and the guy on the left says the other. Then, we ask children couple questions such as "who did a better job answering Emma's question" The game takes about five minutes 

It was really awkward at first because we had to go up to parents and asked them if they were willing to let the children play games with us. Sometimes, even when parents agreed to participate, children rejected us. 

The first day of my experiment went well overall. I coded the videos and sent the videos to my mentor. My mentor gave me suggestions on my voice tone since we have to be careful when we worked with kids.  

We didn't have lab meetings and reading group this week because some lab members went to LA for a convention. 

Also, on the weekend, my friends and I went to a concert together at Stanford! Another great week. One third there! 

Amy Qian #1 First week at Stanford

I arrived at Stanford last week on July the 15th and the weather is great in California. I talked to and met my PI, Professor Gweon and our lab manager, Mika on the first day and briefly talked about what I hoped to accomplish in my next six weeks. There are four other people who worked in the same room with me. Two of them are Stanford undergrads, one is Wellesley undergrad and another one is a high intern as well. They are all very nice and welcomed me to the lab. 

On the first couple days, I helped the lab members with their ongoing block project and made blocks. Mika and Grace explained the project to me. This project is related to rarity. Basically, the project is trying to figure out whether children between the age of two to five have a preferences on things that are rare. Two different colors red and green blocks are shown to children and children are asked which block they want to play with. Because adults tend to choose something rare over common, researchers are trying to figure out whether infants have the basic instincts to choose the rare one as well. Meanwhile, I learnt how to code videos and helped coding videos for VPT (that I collect data for the rest of the summer) on the first couple days.  Mika and my mentor Xuan explained to me once again what my project is about and I realized some of my understandings before were wrong and I had a more thorough understanding after watching the videos. 

In order to make undergrads and high school interns learn more about psychology, my lab assigned papers for us to read and held reading group every week. This week, the reading group topic is preferences. In addition, there is a lab meeting every week for lab members to present ongoing projects.

In terms of living location, I lived in a graduate student dorm which is 20 minutes walking distance from my lab, which is in Main Quad Jordan hall.  Since it is not a far walk, I walked to campus every day instead of biking. 

The first weekend was awesome and I went to San Francisco with my friend. We went to Fisherman Wharf and China town. It was a lot of fun. 

It's been a great week and looking forward to next week. :) 

Matt Erman #5- Finally Got Data!

Hi Everyone! I know it has been about a month since I last posted to the blog, but that was because I was awaiting data from the experiment that I was working on. Guess what? I just got the data from the experiment I was working on this summer. Just to remind you about my experiment, I was screening a specific gene in yeast cells, checking to see how yeast cells with different deleted genes were affected by a wide range of genes, all of which are related to the C9orf72 gene, which is a gene that is being studied to see its effect on the progression of ALS in people. Thanks to the screen, we were able to find multiple genes (24 in all) that caused yeast cell death, with many of the genes causing different reactions in the cells to cause the cell death. 

During my last week at the lab, I was really happy that I had the opportunity to see the person I was working with, Matt, have his thesis defense. It was really cool to see how much work he has put in over the years to earn his PhD. I think the best part of his presentation was how he talked about how his research evolved over the years, in that how much more in-depth his interest and research became as the years past. It was cool to me how his interest in the subject developed, from something that he was kind-of interested in, to something he wanted to spend the rest of his life studying. What made this cool to me was that if his interest in the topic blossomed into a desire to find out as much information as possible about ALS, I can only imagine what my future could be, whether my interest can turn into a PhD like Matt's did. 

All in all, I am so happy that I had the opportunity to work out in California. It taught me how to survive on my own, to work in a lab, and showed me how what research is really like. I was so lucky to end up in such a great lab with such great people, and was able to work on something that really interested me with ALS and neurodegenerative disease. I want to thank all of the people in my lab for being so helpful, nice, and just good people to be around. Thanks also to Dr. Peretz and Dr. Crider for giving me the opportunity to have this great experience this summer. Last of all, I would like to thank all of the other EXPer's (if that's a thing) for writing about what they have been doing this summer too and giving me something good to read throughout the summer. See you all soon!

Katie Entry #5: Last Week in Lab

Sorry I haven't posted in awhile, but these past few weeks have been hectic!

During my last week in the lab, I tried transduction again using the phage lysate that I had made the previous week to move the mutant regions from two cold-sensitive mutant S. aureus strains to the wild type S. aureus strain.  However, this time I used a different protocol which included more bacteria and a longer incubation period.  Fortunately, more bacteria colonies grew on the plates containing erythromycin.  Next, I grew these colonies in liquid media at different temperatures, took their ODs every hour, and calculated the doubling time of each colony at each temperature to determine if the resulting colonies showed cold sensitivity similar to the mutant strains. For the first few days, the colonies did not exhibit any cold-sensitivity.  Later in the week, the colonies tested showed moderately impaired growth in cold conditions, but they were not as cold-sensitive as the original two mutant strains. 

Throughout the week, I also worked on my final presentation that I would give at the end of the week on all of the work that I had done during the past six weeks. While working on the powerpoint, I went through my lab notebook and read a ton of primary research articles (which luckily I had a lot of practice with from EXP class) to highlight background information, what I had done, my results, and what was left to do.  I was a little nervous going into the presentation because I had an hour to talk, but it ended up going pretty well and there were a lot of questions and discussion so I didn't end up talking for the whole time.  It was really interesting to see how everyone in the lab confers and figures things out together. We also had pizza and an ice cream party on my last day in the lab, which was really fun.  

Overall, I had an incredible time in the Cheung Lab.  I learned so much about microbiology, lab technique, and research skills and everyone in the lab was so welcoming and nice. I had an amazing experience and I can't wait to hear about everyone else's lab when school starts.

Jay Ha #5

It's been a while since I left the lab, and I am fully enjoying my vacation in Korea. Occasionally, I miss being at the lab, emerging myself into the entertaining world of science. But soon, when I get back to Peddie, AP bio would be waiting for me, so yeah. 

I am keeping in touch with some of the post docs of my PI's research group, and am really glad that I was given an opportunity to discuss science with truly brilliant scientists. 

Since my research topic had unexpectedly yet drastically shifted from toxicology of nanoparticle to UV- disinfection, great chunks of my poster needed to be adjusted. So, I just started to give major retouches on my poster starting yesterday, and hopefully, I will choose not to procrastinate and get it done before school starts. 

I am also reading a book called "Survival of the sickest" for my AP bio summer reading, and the book is much more interesting than I expected. Books easy to read and eye-catching. If any one of you guys are wondering what to do with final days of your summer vacation, reading this book may be one of attractive choices. 

Well, see you guys all soon.

Arnob #5 - Final Day and My Presentation

It's been about three weeks since I finished my lab. Within that time, I visited colleges, went to Chicago for my grandfather's 90th birthday, and then came back home and slept and did school work/college apps.
To sum up my experience in the lab in a couple words: "Extremely challenging, but interesting and rewarding". Everything from learning MATLAB to my PI Mike challenging me to think like a graduate student was difficult. But as I got more and more into the rhythm of my open-ended research, I started to slowly conquer these challenges.
I presented to my lab members on my last day at the weekly lab meeting. There was about 15 people attending my presentation, most of them post-docs. I was expecting my presentation to be no more than 10 minutes, but my 8 slide presentation ended up being 45 minutes long. This occurred because many of the lab members (mostly post-docs) were constantly asking me questions and seemed genuinely interested in my research. This made me happy but I also had be very focused and calm during my presentation. In the end it was a a great success (exceeding even my own expectations). Mike (PI) was going to bring everyone out to dinner to celebrate me leaving the lab (as he does with everyone) but he had to catch a flight to visit an ill family member. But nonetheless, my last day ended strongly and I'm glad I got this experience over the summer.

Soo Entry #5, Heading Back to School

My two-month-long internship in Dr. Bassett's Research Lab has ended, and now I am getting ready to head back to Peddie. I have learned so much working with her and it was an unforgettable, valuable experience. For future EXP students, I have made PowerPoint slides summarizing my EXP experience, and here is a glimpse of it:

I'm excited to share my story with my friends who are getting ready for their own EXP experience next summer :)

Jenny - Entry #5 at CHLA :(

I am sad to say that this will be my last week here in the lab and CA before I go back to school. I basically spent my whole summer here in the lab and in Mongolia with other CHLA staff, so I will definitely miss it! This past week has actually been pretty slow in terms of actual work. I technically finished my work for the 3D surface rendering, and all we had to do was see it on Imaris (3D imaging program) this week and see how it turned out. The final product turned out to look pretty awesome and I think we were all pretty happy with how it looked! There were some parts where the bronchi were all interconnected where they were not supposed to be (the program probably highlighted blood vessels accidentally) so we had to call an Imaris specialist to see where we can cut certain parts of the rendering.

Anyway, outside of my main project, I've been catching up on reading and work in the lab. Denise and Soula have been extremely busy lately because there is a lot of reading, writing, and experiments to do. I've just been helping out by making some solutions when they ask, and doing some washes for lung samples if they were busy. 

I also made a new friend which was pretty nice! In the last few days, I started talking to another girl who just graduated from USC, and although I've seen her around in the lab, we sat at computers that were in completely different areas, so I was never able to see her or talk to her. But we got along really easliy and went out for lunch a couple times after that - I wish I had talked to her sooner! I will really miss her when I'm gone because it can get pretty lonely and boring in the lab when you're alone doing the same thing over and over again.

On the last day, I was sad to leave, but I definitely had a fun summer with a super amazing team and project. I am planning on visiting whenever I am in California again and might even come back next summer depending on how everything goes. Words cannot describe how grateful I am to have experienced such a patient and kind lab. I hope everyone else has a great rest of their summer (which is almost over :(...) and I'm excited to see everyone back at school.

Bridgid, Entry #5, Getting Off the Hook

Hi Fellow EXPers!

Sorry it has been a while since my last post, it’s been a crazy few weeks since my time ended in the lab. (college visits, summer work, hopefully I'm not alone here......) I officially finished my time in the lab the first week in August.  The last 2.5 weeks in my lab were great, frustrating at times, but I ended up getting results for my project, which is so awesome to think about since I was only there for 6.5 weeks!

Lucky for me, these last two weeks were really the only time that I experienced some frustration in my project.  Since I had already extracted the DNA from all of my 18hpf and 24hpf samples, the only step left was to run a good PCR so all the bands were visible on the gel so that they could be cut and sent to be sequenced, with the ultimate goal of getting clean sequences back to genotype the fish.  Seems simple, right? Yeah that's what I thought, but Not exactly.  From the start there are a few things about this process that make it not exactly a piece of cake- 1. Because I started with single embryos that weren’t that developed to start with, there is limited DNA that I could extract in the first place which means that the PCR could easily be faint on the gel and hard to amplify, and 2. The entire process of running the PCR, making a gel, then running the samples, cut the bands, and preparing the sequencing reaction takes much more time that you think, so having to start from the beginning of the 2 hour PCR reaction every time is a bit of a pain, but nonetheless is very doable.  

It seemed that just about every time I ran the process, something went wrong- the bands were too faint to cut, the samples were some how contaminated, or worst of all I would get through the whole process, send the purified PCR off to be sequenced and have the sequences stop 150 base pairs before the mutation.  Between the last two weeks, I probably ended up running that whole process about 5-6 times (which is a lot, trust me).  The whole time, Patrizia and I continued to troubleshoot and figure out why we couldn't get clean sequences; adding more eth. brom. to stain the gel, running the gel longer, and even changing the sequencing core to Penn and not CHOP, and still nothing, for any of the samples.  We had just about tried everything, when Patrizia had one last idea- try centrifuging for longer during the purification to make sure to elute the DNA. So I ran the PCR one more time, cut the bands, and centrifuged for double the amount of time to make sure we had all the DNA we could possibly extract and sent the PCR off to CHOP (NAPCORE) for sequencing.  Both of us were so anxious about getting the sequences back, but when we opened the files, almost all the sequences worked for both time points, which meant that we could genotype the fish!!! It was really so exciting to finally know that I had a result and that I was able to help Patrizia confirm what she thought for her paper!! After looking at the pictures I took before and comparing the different straining of the 3 genotypes, we concluded that there was really no difference in the expression of the transcription factor, lmo2, in the mutant, heterozygous, or the wild type, which means that the mutation must affect something else further downstream.  Patrizia then helped me gather some pictures and graphs for my poster, which was so nice and extremely helpful!

Aside from my main project with lmo2, while we were waiting for the sequencing results, I started another project.  My second project involved going into the fish room, which so cool in itself and required me to cross an mll mutant fish with the new transgenic line to look at the expression of the hematopoietic stem cells.  On Wednesday night, Patrizia, Abby, and I all went to the fish room, where I learned how to prepare the breeding tanks.  Then, the next morning, I came in early to learn how to heat shock the fish (we heat shock them in order to make transgenic line expression MLL-GAS7, which has a heat shock promotor upstream from the gene).  I also learned how to sort the fish after the heat shock, based on the presence of a green heart (how we know that the fish are expressing MLL-GAS7).  Early the next week I helped Patrizia start the WISH, and although I didn’t get to finish it, she said that she would finish it and send me the results so that I could include them in my poster.  

Overall, my experience in the Felix Lab this summer was probably one of the best experiences I’ve ever had.  I learned so much in such a short amount of time and everyone in the lab was so welcoming and always there to help or answer any question whenever I needed.   I thought that my project was great, it was just the right balance between giving me freedom and feeling like I was doing something on my own, but at the same time, I never felt overwhelmed with not knowing where on earth to start or what to do next.  My post doc was a fabulous mentor and never really made it feel like I was working. Six weeks goes by amazing fast, especially when the days don't even feel like work. I don’t think I could have asked for a better lab team or environment to spend 2 months of my summer in!

I hope that everyone had a great time in their labs! Can't to hear about everyone's summers in person in just a few short weeks!! See everyone soon! :)

Emma, Blog #5, A close to the summer

After ten weeks in the lab, my summer at the Siegel lab has officially come to a close. It is so weird to be saying those words, as the last ten weeks have flown by so quickly.

I ended up finishing my two projects, one with the other high school student Meghan doing immunohistochemistries on ketamine and saline treated brain slices, and another running TMazes (a working memory paradigm) on mice with a Dexras1 abscission.

The results from both of my projects endeed up yielding publishable results which I am ecstatic about. I will not be an author of either of the two papers my results will be featured in, but I will be acknowledged in the reference section. The first paper I expect to be sent out for review will be the one about Dexras1 abscission's in mice, and whether this specific genetic manipulation cause schizophrenic-like endophenotypes. Below is the final report I gave to the post-doc writing the paper on what I found out about working memory in mice with the Dexras1 abscission.
Note: I couldn't paste the graphs I made on Statistica into this blog because they are not compatible.

T Maze Report

Dexras Mice

12 mice total

Wildtype Mice

14 mice total

Possible point of interest for future studies (?): Dexras mice on average ran 8.33 trials in T-Maze across 3 days, whereas the wildtype ran 10.067 during the same time period.

95% confidence interval for % of correct choices for Dexras mice: (.5, .65)

95% confidence interval for % of correct choices for WT mice: (.58, .72)

Null hypothesis: The % of correct choices is the same for Dexras and Wildtype mice.

Alternative Hypothesis: The % of correct choices is different for Dexras and Wildtype mice.

P-value: .104

With an alpha level of .05: Because the p-value is so high, we fail to reject the null hypothesis in favor of the alternative. Evidence to suggest that there is no difference in the % of correct choices in the T maze between Dexras and Wildtype mice.

This data supports the claim that there is no difference in working memory between Dexras and Wildtype mice.

My second project was about whether or not the number of neurons (measured by density and area) were different between the brains of ketamine treated mice and saline treated mice, This relates to schizophrenia because ketamine is an NMDA antagonist and produces symptoms in people similar to those that schizophrenics experience. We were in effect, trying to figure out if the cause of these schizophrenic like symptoms had to do with a difference in neurons in the prefrontal cortext. 

Immunohistochemistry Report

Ketamine Mice: 3429 (6 sections), 3425 (6 sections), 3418(6 sections). 18 sections total

Saline Mice: 3422 (5 sections), 3413 (6 sections), 3412 (6 sections). 17 sections total

Two ways we measured whether or not the amount of neurons differ in ketamine or saline exposed mice: Density & % Area of neurons in brain slices

Density: 

95% confidence interval for density of neurons in ketamine brain slices: (~.00018, ~.00026)

95% confidence interval for density of neurons in saline brain slices: (~.00016, ~.00023)

Null hypothesis: Density of neurons will be the same between ketamine and saline treated mice.

Alternative hypothesis: Density of neurons will be different between ketamine and saline treated mice.

P-value: .285

With an alpha level of .05: Because the p-value is so high, we fail to reject the null hypothesis in favor of the alternative. Evidence to suggest that there is no difference in neuronal density between ketamine and saline treated mice.

This data supports the claim that ketamine does not alter the amount of neurons in the brain in the long term (in this case 6 months after receiving treatment).

% Area:

95% confidence interval for % area of neurons in ketamine treated brains: (~5.2, ~9.5)

95% confidence interval for % area of neurons in saline treated brains: (~3.1, ~7.75)

Null hypothesis: % Area of neurons will be the same between ketamine and saline brain slices

Alternative hypothesis:  % Area of neurons will be different between ketamine and saline treated brains.

P-value: .195

With an alpha level of .05: Because the p-value is so high, we fail to reject the null hypothesis in favor of the alternative. Evidence to suggest that there is no difference in % Area of neurons between ketamine and saline treated mice.

This data supports the claim that ketamine does not alter the amount of neurons in the brain in the long term (in this case 6 months after receiving treatment).

Jenny - Entry #4 at CHLA

Hello everyone, I'm back for my fourth blog post. This past week and a half was pretty ordinary. I've still been working on the imaging of the lungs, and it's taking much longer than I thought it would. The process is pretty tedious, as I'm basically sitting at my desk,staring at 283 slides of similar images on the computer for hours. I'm not complaining, though! I am super grateful that I am able to help out my lab so that they can use this for their grants in the future - and hey, someone has to do the work, right? My PI has been super busy writing her grant and going to conferences and I've pretty much been working alone.

Last Friday, though, I was pretty so close to finishing up my slides (only about 10 more out of 283) and then the worst possible thing happened - the program crashed. I thought that it wasn't a big deal because I had been saving so frequently, but unfortunately, when I opened up the file, nearly all my data from the week was gone. At first I was kind of in disbelief that basically all my work had been erased, and then I just got very annoyed. Not only did we never figure out why the program crashed, but I never retreived the data back. But no matter how upset I was, I reazlied that this is what research is about. Not everything works out the first time, and you have to keep trying and troubleshooting to get results. Since it was the end of the week, I was just told to go home and rest so that we could figure it out the next week.

Thankfully when I came back to the lab, the situation was looking a little better. Although my data would never be recovered, our imaging specialist came up with another method so that the process would go by much faster. Instead of me individually circling each cell, he manipulated the image so that nearly everything, even dimly stained parts, of the lung would be highlighted. All I had to do was erase the "fuzzy" spots, since a lot of the marks were not significant and would just be background noise in the final product. This method was much more efficient than before, and I was able to finish it in 3 days instead of a whole week! I just had to keep going at it, with Spotify by my side.

And as I mentioned before, I had been testing out a new protocol for clearing human lung tissue for antibody staining. A 15 week sample was mostly cleared so the goal by the end of the week was to mount it in primary antibody. Unfortunately, because my PI was not directly here with me and her assistant, we were unsure of exactly what antibody she wanted and there was a lot of confusion. Eventually, everything was figured out and I washed and blocked the sample, while the assistant mounted it in the primary antibody. Next week will be my last week at the lab, and even though I haven't even left yet, I know I'm going to miss everyone so much! I hope everyone's having a nice summer!

Julius, Post #4, Turn up for Biology

The sample tested was a new compound (described in previous post), which I had synthesized the day before. Part of the "triazolopyrimidine" family of compounds, it is structurally similar to previously tested compounds. We tested the compound for its "MT (microtubule) stabilizing" ability. A common symptom in Alzheimer's patients is the destabilization of neuronal cell microtubules. This leads to cell death, as the microtubule plays an important role in intracellular transport. Thus, compounds with the "MT stabilizing" properties are at the focus of Alzheimer's research.

When I got to the lab in the morning, I met Jane. Jane had been a post-doc at the lab for a year and a half, and had done her graduate work at Temple on cocaine drug studies. The first thing she did was show me around the lab, which was a pretty big surprise to me. The lab was only 3 years old, compared to our 42 year old chemistry lab, with a beautiful view of the campus and spacious, clean work spaces Despite my love for our chemistry lab, I had to admit that the medical lab seemed much nicer, an opinion that my other lab-mates agreed with. 

While Jane showed me around the lab, she explained the steps of the experiment. In her words, we were to "test the microtubule stabilizing" effects of the compound on "QBI-293" HEK kidney cells through an "ELIZA test". In order to do that though, we would first have to conduct a "BCA protein assay" in order to determine the concentrations of the proteins in advance. I did not understand anything she was talking about, until she explained it to me in laymen terms.
"So basically", she said, "we're adding the compound to the kidney cells, then using a lysis buffer to open the membrane and collect the proteins."
(Wait...so the cells die in the process)
"Yeah, the cells die when you add the buffer. That way it prevents degrading and accumulation of protein."
"Then we'll add BSA to the cells, which will help change the color of the cells for the BCA protein assay.
(Wait, what's BSA).
"Bovine Serum Albumin. Write that down. It's important for determining protein concentration."
(Got it).
"So, using a BCA protein assay, we can find the concentrations of the proteins. Basically, depending on the concentrations of protein, the cells will change color. So by measuring the color change of the cells, we can then quantitatively determine their protein concentration."
(And that's it?)
"No, that's actually the preparation for the ELIZA test itself. We need to know the concentrations of protein to conduct the ELIZA. You'll have to come in tomorrow to finish that."

So, for the rest of the first afternoon, I shadowed Jane as she conducted the preparation for the ELIZA test. It being my first time in a biology lab,  I had a lot of  questions for her, which she answered patiently. There was a lot of precise pipet work and centrifuging involved, but I was just waiting a large portion of  the time. I was definitely surprised by how much down time there was between the different tests and assays. (In the chemistry lab, there are often several different reactions and purifications occurring at once, meaning that we're at work more frequently).

So the next day, I showed up early for the ELIZA test.
"So today, we'll be conducting the ELIZA test."(Alright).
"By any chance, do you know what it might stand for?"(No, I'm not really sure).
"Alright, thats fine. It stands for Enzyme-Linked immunosorbent assay. What we've doing is we're adding capture and reporter antibodies to the kidney cells from before. These antibodies will bind to two types of microtubulin, and have HRP "tags" that will change color depending on the protein concentration."(So kind of like the BSA?)
"Yeah"(Alright, and why is this important?)
"Well, that's what this experiment is all about. This color change allow us to detect the concentrations of two substances: acetylated tubulin and alpha tubulin. And after we collect this quantitative data, we can use it to see the microtubulin levels, to see how effective a stabilizer the compound is".(Ahhh).(By the way, what does HRP stand for?)
"It stands for horseradish peroxidase. You might want to write that down".

Similar to the BCA assay from before, the ELIZA test worked based on color change and concentration. The two different antibodies changed color based on tubulin concentration, thanks to the HRP tag; this meant that by measuring this color change, using a specialized machine, we could quantify the microtubulin levels.

To sum the entire test up, we added the compound to the cells, first checked the protein levels using the BCA assay (which was needed to set up the ELIZA test), then used the ELIZA test to find the final concentration of microbutulin present in the cells. Gathering this data then, we were able to determine whether the compound was a potent microtubulin stabilizer based on the level of acetylated microtubulin present and whether the compound was harmful towards microtubulin levels in general (which is a negative characteristics), based on alpha tubulin levels.


When we finally performed the statistical analysis on the data (Dunnett Test, Nova test), we were able to see that the cells treated with the compound had a statistically significant increase in levels of acetylated tubulin (indicating strong microtubulin stabilizing properties) and no decrease in levels of alpha tubulin (indicating that it is not harmful to tubulin in general), meaning that the compound shows promise for further testing. 

Jenny - Entry #3 at CHLA

Hey all, I hope everyone's having a fun, research-filled summer in their labs. I got back from my trip to Mongolia with a few doctors and girls associated with the guild that fundraises for the research institute in CHLA. It was probably the best experience in my life - I learned so much not only about global healthcare and medicine, but different cultures and lifestyles! Although I am sad the trip is over, I'm glad to be back in California and continuing my project. I am in the middle of my fifth week and there have actually been a few changes in the lab when I came back. Before my trip I mainly worked with my PI, Denise, and another undergraduate, Emily. When I came back, Emily couldn't come to the lab anymore, but instead there were two other research observers, Alex and Winnie. Although we don't work together, it's nice having more people walking around on our side of the lab because it's usually so quiet! I am also working with Soula, who is like Denise's sidekick. Everyone is super friendly like I remembered and I felt right at home when I came back!

Me, Emma (a medical student from Holland), and Dr. Warburton (my lab director) sipping on tea in our Mongolian gear!

My project of imaging the human lung continues! Before I left, my main task was to wash and stain the samples. I did this for about 10-12 lung samples, and the whole protocol took a while. We were able to get 2D images images under the fluorescent microscope which was pretty cool, but the main goal was to get a 3D image where we can trace cells and see how they branch and grow. We were able to get that done with outside help so this is where I step in! (I'm sorry about the lack of pictures from the lab - I don't think I'm allowed to share them just yet!)

The 3D imaging is basically a video with around ~280 frames of the whole lung lobe that we had stained beforehand. Although just the images themself look amazing, the whole goal is to trace cells to get a better understanding of how lungs develop. Focusing on one particular antiody, our imaging specialist had already used a computer program to highlight very prominent stains. However, there are a bunch of other cells that have been stained, just not picked up by the program. So for the past few days, I have basically been tracing these undetected cells with ImageJ. It's not the most exciting thing ever, just because there are 280 frames and they all look pretty similar. I don't mind too much, however, because in the end, someone has to do it. 

On the side, I've also been testing out a new protocol on clearing tissue. Although the method to clear our lung samples was effective, some of the solution actually had melted the plastic and leaked out onto one of the microscopes - eek. When clearing tissue, methods that are shorter are usually more effective, but I am testing out the CLARITY clearing method, which takes a few weeks. I am currently waiting on an embryo and lung. I hope this method actually works! 

Michael, Post #6, A Glimpse of the Future?

Due to some misfortune (aka contaminated cells), I could not complete my final experiment by last Friday, my original last day at Drexel. Instead of calling it quits, I decided to come in for a few days this week to finish up one last experiment. Although I haven't had a ton of individual lab work over the past two days, there have been other important things going at the Bouchard Lab.

Today was Jason's thesis defense. It was the first (and last) one during my time in lab this summer, and it was interesting to see how the Ph.D process concludes. Jason came in to lab today dressed up in his suit, and at 1:00, the entire lab filtered downstairs to meet him in the Geary auditorium for his thesis presentation. Also attending were members from other labs on our floor, as well as Jason's family and friends. The talk lasted until 2:15, and he did a great job summarizing all of his work into that period of time, though his heavy use of bioinformatics was a little above my level of expertise (Dr. Bouchard even commented that he learned many new techniques as a result of Jason's work).

After the talk, Jason met with the five members of his thesis committee (including Dr. Bouchard), who questioned him while he defended his thesis. This process was supposed to last until 3:00, at which point there would be a celebration upstairs.

We set up for this party while we waited for the defense to come to an end. We hung streamers and decorations from the conference room where lab meeting is normally held and set up the food. However, we had to wait until 3:45 for Jason and the thesis committee to conclude their meeting and for the party to begin. Dr. Bouchard made a toast while Nikhil and I stood there with no champagne in our hands. The food was pretty good, and there was pizza, salad, and cake from all different places in Philadelphia.

Setting up for the party

Jason will still be in lab for a few months to finish up some work, but tomorrow should be my last day. I plan on running my final experiment tomorrow, assuming my transfection went well today. It's been a long ten weeks in lab, but I'm excited to see what results I get tomorrow!