Julius Sim, Entry #2, Week 2 at UPenn

This is Julius, and I've just finished my second week at UPenn, working in the chemistry department at the Ballatore Lab (working on organic synthetic chemistry targeted at Alzheimer's drugs).

To give this week an overview, I was able to begin to run parts of my own reaction , under the supervision of Anne-Sophie, with the goal of being able to independently synthesize possible compounds in the future. In addition, I was joined in the lab by another high schooler, a girl named Maya, who worked in the lab last year, who will be staying at the lab with me for around 3 weeks.

So, last/this week, I've started to work on my own reaction to produce:

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

The name of the compound is rather complicated, so we often simply refer to it in the lab as the "pyrimidine", the family of compounds that this specific analogue belongs to. While I'm working to create this specific pyrimidine, Maya will be working to create a slightly different one, with the same basic structure but a different functional group attached to the NH.

So in planning the reaction, we needed to react (get ready) 5,7-dichloro-6-(2,4,6-trifluorophenyl) [1,2,4] triazolo [1,5-a] pyrimidine with 2,2,2-trifluoroethylamine. Since both of these reagents are solids we also needed a solvent, utilizing DiEA (N,N-diisopropylethylamine) to dissolve the pyrimidine reagent. Last, we added DMF (dimethylformamide) to the reaction mixture, allowing the reaction mixture to stir for >13 hours. This was all according to a procedure outlined in a 2007 article published in J. Med. Chem. by Zhang, et. al. To run this reaction though, we needed to first create the pyrimidine reagent in a reaction that by itself requires several steps and reagents.

The reaction to create the reagent pyrimidine (used in the final reaction) requires two important steps, a reaction synthesizing trifluorophenyl malonate, then a subsequent reaction synthesizing the reagent pyrimidine. While trifluorophenyl malonate is actually commercially available (meaning we can purchase it online), it is a very expensive chemical compound, meaning that synthesizing it ourselves in the lab would help the budget (commercial factors are consideration in science too!).

After synthesizing the trifluorophenyl malonate product AND purifying it, we utilized it to create the reagent pyrimidine. Then, utilizing the crude mixture of the pyrimidine (meaning we didn't purify it and there were still impurities present), we went through the reaction as outlined above.



While the steps taken in each reaction involved different reagents, the general steps were very similar: reaction set-up, reaction, "work up", column chromatography, TLC/NMR/LC-MS, purification, drying/Rota Vapor. While the first two steps are relatively self-explanatory, the next four are a bit more complicated (and constitute a large portion of work in the lab). "Work-up" refers to a very basic isolation of the desired product into a "crude mixture"from the rest of the "reaction mixture", a combination of reaction by-products and remaining starting material. The next two steps, TLC/NMR/LC-MS and purification, involve identifying the specific product within the "crude mixture" and utilizing knowledge of the compound's polarity to isolate it (>95% purity). The last step, drying/Rota Vapor, only applies if the desired product is a solid; since it will be solution after purification, this step simply involves evaporating the solvent and leaving the solid.

 "Allez La France" (Come on France!). Dates to the 2014 World Cup. My Post-doc Anne-Sophie is French. 

Example of reaction in progress; note the nitrogen balloon. Many of our reactions are air/moisture sensitive, meaning that the flasks must be completely filled with nitrogen before the reaction. 

Azza Entry #1 Befriending Bacteria

My first week at the UCSF lab was great and went extremely well! Upon my arrival, on Monday, my mentor explained the 2 projects that I would be working on over the course of the next few weeks. One project is based on an inherited heart disease called ARVC and the other is based on the study of the Calsequestrin protein. I have been working on both of these projects simultaneously over the course of my first week here at the lab.

As part of the ARVC project, I’m still in the process of creating a nuclease called Cel I which has the ability of basically cutting up DNA at a specific target and ultimately detecting heterozygosity. I had to extract CEL-I from celery juice and purify it using a protein purification protocol my mentor gave me to follow. CEL-I, as I mentioned, is a nuclease the lab is using to detect heterozygosity. Since ARVC is autosomal dominant, CEL-I helps prove that an organism has successfully taken in the disease by running their DNA onto a gel and seeing cut up pieces of DNA at a specific location.

The second project I have been working on is the Calsequestrin project. I think of the CASQ2 project as climbing a ladder. Because it deals with understanding the structure and function of a mutant protein, we first begin with trying to understand the chemistry and physics of the mutant protein, and this can be done in the test tube. Then we ask biology questions about the protein, but in vitro. In vitro is learning about the protein in a cell line that is not part of an animal. Afterwards we can look at what the protein does in cell culture using mouse skeletal muscle cells that have been modified to grow individually. Lastly we make an animal model and look for a phenotype that shows evidence of the disease in vivo. I learned that this is how any scientist would proceed for any project involving a mutant protein. You start with the basic characterization up to the in vivo experiments.  So far in this project, I have successfully ligated our target DNA into a strain of bacteria and allowed a culture of bacteria to grow overnight.  The next day I transformed the plasmid into competent cells called XL-1 cells and miniprepped 5 different bacteria colonies and sent them for sequencing.

My lab bench

It’s only been a week here at the lab, and I’ve already learned so much! I’m looking forward to the next couple weeks!

Azza J

Conor, Entry #1, Ready For Takeoff

Today is a little different from the typical day. Right now it is 11:20 am and I am not the first one from my team in the lab. The other day no one from my team showed up until 12:30. I had already left for lunch and came back. The first day of work, I asked what my daily hours should be, and Jon, the phd student I am following replied with, "Show up whenever you want." With my abnormal sleeping habits, I could definitely get used to this. 

I have the PR2 humanoid robot staring me down always to make sure I stay on task

But do not interpret it like we are not doing work, we definitely are. We have deadlines to match, and as long as we meet that quota, we can get there by any means possible, which justifies sleeping later every day. Our current project is to get our quadcopter to navigate itself in an indoor environment using a variety of sensors and methods, and to ultimately work in tandem with our segbot. The segbot is actually a very scary machine. It is around 200 pounds and can run at speeds of 25 mph. When Jon was giving me a tour of the lab, he showed me all the holes and dents in the walls where the segbot ran into. Just in case the segbot gets out of control, Jon said to either push the giant red buttons that they installed on the robot, or to just run. However the segbot is pretty much done, as the project was completed a while ago. Our big hurdle now is to allow visual odometry with our quadcopter. What this pretty much is from looking at known QR markers, the quadcopter is able to judge distances and locate itself on a small scale, such as a room.

Under the hexacopter is the segbot. Like I mentioned it is a 200 lbs and 25 mph behemoth.

This is the quadcopter my team and I are working. It looks innocent but it potentially has the power to chop off your pinkie finger.

The first week of the lab I was getting used to the lab setting and going over ROS (Robot Operating System) and C++ tutorials, as I will likely be using them in the coming weeks. I have also assisted with flight tests, and I will post a video soon. But recently my project has been to model and 3D print parts for the quadcopter. That fall term project in EXP class to 3D print something came in uber handy! So far, I have made landing gear, blade guards, and parts to mount sonar and infrared sensors. 

It is now the start of my third week. So far so good, and I am optimistic about the weeks to come!

Katie, Entry #1: Great Times with MRSA!

My first two weeks at the Cheung Lab were great!  My first day started with a lab meeting where I got to meet everyone in my lab and learn about their research.  My lab has 6 members: my PI, 3 post docs, and 2 graduate students.  I am primarily working with Dhana, one of the post docs. 

During my first week, I learned more about my experiment and the techniques that I would be using.  I am looking for genes necessary for cold shock response in S. aureus. I am working with mutant S. aureus strains that have one gene that is interrupted by a transposon, inhibiting the function of that gene.  To determine whether we would conduct the experiment by growing the mutant strains in solid or liquid media, I grew the strains in both and monitored the growth.  I grew mutant and wild type strains on agar plates at different temperatures and checked their growth daily.  I also grew the strains in liquid media and took the optical density, the measurement of light absorbed by an object, using a spectrophotometer to measure the growth of the bacteria cultures (as cells divide and replicate, the optical density increases).  I measured the optical density until the cultures reached the stationary phase of growth and made a growth curve using the measurements. I did this for a few days to make sure that I got accurate results. I also helped another post doc with his research by randomly picking colonies of S. aureus, which he will later analyze. 

The spectrophotometer

Over the weekend, Dhana and I went to a nearby hot air balloon festival and while I did not get to see any of the hot air balloons take off (bad weather), I got to see the beautiful Quechee Gorge.   

The Quechee Gorge

During my second week at the lab, I grew1,920 mutant S. aureus strains on agar plates at different temperatures, observed which strains had difficulty or could not grow in cold conditions, and made a log of those strains and the genes that were interrupted by transposons in each strain.  Next, I began growing some of these potentially cold-sensitive strains at different temperatures in liquid media, observing their growth using the spectrophotometer, and making more growth curves.  I also helped Dhana with some of her other research by preparing and loading gels for gel electrophoresis.  At the end of the week, I went to a barbeque at my PI’s house and had a lot of fun!

My desk/lab bench

Overall, I am enjoying my lab and am excited to learn more in the upcoming weeks.

Bridgid, Entry #1, Everything is going Swimmingly at CHOP

After 2 weeks of waiting, my time in the lab has finally begun! Unfortunately, I could not start exactly when I was supposed due to large amount of clearances I needed to complete before being able to work at CHOP.  Although it was hard to wait everyday until 4pm when the mail came, only to realize that my authorization didn’t come that day, it only made last Friday feel even more like Christmas when I got my special blue piece of paper, certifying that I was clear and ready to go!

Despite not being able to work in the lab until this Monday, I had been given an article to read relating to the project that I would be doing this summer. When I arrived on Monday, I then presented the article and led the discussion for our lab’s Journal Club.  The Journal Club, kind of of like a book club, is something that my post doc created for a few of us in the lab to get together and discuses articles having to do with the work that each of us would be doing this summer. I am so thankful that thanks to this term, reading an article like this didn’t scare me as much as it did in March! I was nervous going into it, especially because how well I could explain this article to a group of people that specialize in this field would be my first impression, but I’m glad that I had to opportunity to do so. Just in that short hour I already felt like I learned so much!

My lab bench

            Then, my post doc Patrizia, who I will be working closely with, showed me around the lab and introduced me to everyone.  Along with the two of us on the zebra fish project, there is a college student, Abby, working in the lab this summer.  In addition to the zebra fish team (which in my opinion has the most fun) there is another post doc, Karen, who does MLL target therapy work, and James, the lab assistance to my PI, who works with the MLL translocations.  Shortly after I got to meet everyone, Patrizia, Karen, Abby, and I all went to a seminar presented to the entirely oncology department at CHOP.  The seminar focused on T-ALL and the ground breaking clinical trials that are currently taking place in attempts to find targeted therapies to cure the cancer.  Although some of it was a little over my head, being able to be exposed to that kind of research at such a high level was so cool and gave me lots of hope that with all of the new technology we have and are developing, we can make huge strides in terms of precision medicine to treat pediatric cancer

patients.  For the rest of the day, I took care of some clerical stuff, including getting my ID, and set up for the experiment that we would be doing on Tuesday.

            On Tuesday, my day started with a 1.5 hour mandatory research safety training meeting After learning every hazardous chemical known to man and memorizing the safety procedure for what too do if it spills or if it is taken into the body in any way, I finally headed up to the lab to begin my work! My project with Patrizia this summer focuses around the MLL mutant.  We will be testing to see if injecting one of the transcription factors, pu 1, down stream of the MLL gene, that normally goes down in quantity when the mutation occurs, we are able to rescue the colony of hematopoietic cells back to forming regular blood cells. In order to do this, we need to do an RNA extraction from the zebra fish embryos to eventually look at gene expression. Relating to this, I had two main projects for the week, RNA extraction and cDNA.  Because it was my first time performing the protocol and Patrizia wasn’t sure when I would be starting with the clearances, in addition to the fact that the embryos are extremely hard to work with and we only get a certain number per breeding pair, I did the same RNA extraction protocol along side of Patrizia, except I did it with REH (human leukemia cell line) cells, instead of the zebra fish.  I started by dividing the cells into two separate tubes, harvesting them by centrifugation and then using a PBS solution to make sure that the mixture was homogenous.  I then added two solutions, TRIzol LS Regent and chloroform, which allows the mixture to separate into 3 phases: a pink organic phase at the bottom containing proteins, a white interphase with DNA, and a transparent aqueous phase containing our RNA.  We then completed the RNA isolation procedure, finishing with a RNA precipitation to end up with an RNA pellet at the bottom of the tube.  Then, we used the Nanovue to measure the quality and quantity of the RNA that we extracted.  I was so excited to see that for my two samples, I had concentrations of 334 ug/ ml and 1088 ug/ml and that my purity ratios (260/280 ratio) was a 1.9/2.0.  We then ran my RNA on a gel to look at the concentration of the two types of RNA 18S and 28S that were concentrated in my solution. 

            My other project for the week was creating cDNA. Starting using 1 ug of the RNA that we extracted earlier in the week (in my case the REH cell line and Patrizia’s case the zebra fish embryo RNA), we added water until we reached a final volume of 10 ul.  Then we added random primers, which anneal throughout the RNA molecule and give a good representation of the entire RNA molecule, and the dNTPs.  After rounds of PCR, then we added buffer, DTT (denaturing agent), and RNase OUT.  Again after a round of PCR, we added the reverse transcriptase enzyme (superscript II).  Finally we added RN-ase H, which breaks down the RNA so that we are left with only the DNA. 

            Since both of those projects were moving along nicely, Patrizia also had me start on DNA extraction from the zebra fish embryos.  Because the embryos have to be incubated over night, we prepared the embryos on Thursday to sit overnight and then when I came in on Friday we did the extraction.  The extraction mainly consisted using special tubes with a membrane to store the DNA on top and in the membrane, along with 3 to 4 washes with buffer and ethanol and spinning in the centrifuge.  Once we had out DNA, we ran PCR, which we will analyze on Monday.  Then using the PCR, this coming week, we are genotyping 160 embryos to look for the mutants.  On Friday afternoon, we had a lab meeting with my PI, where each of the summer students presented on what they had done so far this summer.  I was nervous since I had only been there for a week, but getting up and being able to explain what I have done in a very short amount of time to my lab was actually pretty cool (and great public speaking practice)!

            In addition to working with Patrizia, in my down time when Patrizia has post-doc meetings or is working her in situs, I have gotten to work with the college student Abby.  Although she is working with the zebra fish, she is working with a transgenic line, in which she is using a transgene in a vector inserted into the zebra fish to try and express the MLL-GAS7 mutation in the fish and mark it by fluorescent to prove that it is a gene that causes leukemia in zebra fish, along with humans.  I was able to work with her on her imaging of the zebra fish at 33 hpf (hours post fertilization). Through this imaging we found that the fish with MLL-GAS7 had a significant amount of more red fluorescence in the hematopoietic cells than the control.  This is ground breaking because now this is possibly a way to create

Zebrafish MLL-GAS7 imaging 

leukemia in a fish and allows the fish to become a model for MLL in identifying how the cancer occurs.

            Overall, my first week was awesome! Everyone is so friendly and knowledge that I feel like I have already learned so much! I can’t wait to see what the rest of the summer has in store!!

Kelsie, Entry #3, A Farewell to Kyle with Amazing Gelato

The third week has begun with a slow morning as Kyle came in a little after me and Julie had to drop her husband off so she was running late as well.  But eventually we were all here and we prepared ourselves for a busy day.  My job for the day was to prepare the cells we are going to look at under the microscope.  I had to use the aspirator multiple times in taking the liquid out of the wells.  I washed them with PBS and PBSt then blocked and permeablized the membrane to allow the indicators to enter the cells.  While that sat for 40 minutes, Julie, Kyle, and I harvested another set of cells that were already treated.  It was the same as last week where we washed off the media and added PBS, then a protease inhibitor and a lysis buffer.  Once the cells were placed in the small tubes they were placed in the centrifuge, which only resulted in a small pellet at the bottom.  During that time as well, we attempted to make more TBS-t, which ended up not being correct.  We started to make 20x TBSt when we needed to use the current stock solution of 20x TBSt and dilute it to the proper concentration of 4x.  Luckily that ended up working out and we made more of the stock as well as the concentration we needed.  Once the 40 minutes were up, I washed the cells again with PBSt and then added the two indicators to the correct wells.  We used GAP43 and PSD95 as the indicators.  Once they were incubated for an hour I took off the antibodies and then did the same procedure again but I put on the secondary antibody which is what will fluoresce when it is under the microscope.  While waiting during one of the washes, one of the other people in the lab, Wade, was in today and I learned that he is from Charleston which is where my brother is living.  It was nice to talk to him about it and Julie as well who just returned from Charleston. 

Tuesday was busy as well.  In the morning, I continued with the cells I was treated yesterday.  I did a couple more washes and put on a secondary antibody again.  While waiting during the longer time period, Julie, Kyle, and I went in to talk to Kelly about what we’ve been doing.  That was a cool conversation because she was telling us where she’s hoping this project will be in the next 5-10 years.  She was also discussing other projects that have proposed other mechanisms for HIV entering the brain and the possibility of those being accurate.  One of the long-standing controversies is if T-cells actually contain the HIV virus or if they are just the transport into the brain.  The seemingly obvious solution would be to stop the flow of T-cells into the brain but if we were to do that then the patient will most likely develop PML, which is fatal.  Therefore that’s a problem in and of itself.  Once the washes were finished, the cells were ready to be mounted on the slides.  At first it seemed pretty difficult since the coverslips were in these tiny wells and so the coverslips were very small themselves but I was able to move all of my designated ones without breaking any.  Supposedly, as Eddie said I can probably take the title of the best newcomer to mount slides.  Then we took the membrane Kyle had been working on and we went down to the dark room to develop the film.  Our first one happened to be the best so luckily we got that.  When we returned to the lab room we ran a Fast Green analysis which is for a total protein count.  While waiting for that to dry, Julie had begun to teach me how to analyze the western films but the Kelly came in and told us there was another microscope demonstration that would be starting in a few minutes.  We all headed over and that was really cool to watch all the capabilities of the microscope especially the parts that could help me with the counting of the cells. While we were waiting for the microscope to realign itself, the presenter asked me how I liked working in the lab and he asked if I was on a rotation, I responded with I really like it so far and that actually I’m in high school.  He was very shocked I was so young and working in a lab and he was saying what a great opportunity this is for me and I couldn’t agree more. 

Wednesday began a little rough as the storm from the night before left a lot of roads closed and therefore many more people were on the highway causing there to be a little more traffic than usual.  Once I got in, Julie, Kyle, and I went into the dark room with the microscope and were attempting to look at the cells I mounted the day before, unfortunately the microscope needs to be serviced so we couldn’t see much.  After that we got protein ready to be run on a gel by first making a Bradford assay to determine how much protein was in each sample so we could make sure that each had the same amount of protein once we added the water and loading dye.  We used a spectrophotometer which I have used at school but this was more intricate than ours since there was a special program to compare it against the standard curve and then it’s called a nano drop because you can put a small amount of DNA or RNA on a small plate and it will tell you how much is in that sample.  After we got the solution prepared to be loaded onto the gel, Julie allowed me to set up the gel electrophoresis and while that ran the three of us went down to the dark room to develop the membrane Kyle has been working on.  That ended up being really overexposed so we had to do a couple more washes.  By then the gel was almost done running so Julie and I got the membrane and all the things needed for the transfer ready.  Then I got to take out the gel and make the “sandwich” to transfer the protein from the gel onto the membrane.  While the membrane was washing Julie took Kyle and I out for gelato since it was Kyle’s last day and Eddie tagged along.  It was AMAZING.  Supposedly it’s the best in the world or the best in the country, I don’t remember which one, but I got dark chocolate and hazelnut which was one of the hardest decisions to make.  We sat outside and talked for a while, which was very nice and relaxing. Afterwards, Kyle went back to Julie’s apartment since he was staying with her and her husband and that was the last I’ll see him.  He is going to Vietnam this coming Friday, the 26 to teach English which is incredible.  Then we walked back to the lab and took out the membrane that was in the transfer and got that in the BSA to wash and the other membrane sat in TBSt.  During this entire time, Eddie has been planning parts of his wedding so that has been entertaining for everyone else in the lab, so we had all been helping him plan his rehearsal dinner.  Once the washes were finished we tried one more time to see if there was protein on the older of the membranes but the film didn’t show anything, we used Fast Green and even then there wasn’t much total protein on the membrane.  We put the other one in the primary antibody and let that sit in the cold room overnight. 

Thursday was a good day.  I got in at 9 and Julie and I started with the membrane.  We began the washes and while that was going, the proteins for another gel were thawing.  I prepared the Bradford assay to determine the protein concentration in the samples, same as yesterday.  After we used the spectrophotometer and determined the proper amounts for the components to reach the same protein concentration, I mixed everything together.  At first I had read the pipette wrong and instead of putting 23ul and 28ul, I put 2.3ul and 2.8ul.  Luckily that was an easy fix and I just had to add more but it’s a good thing I caught my mistake before we added the protein.  The secondary antibody was added to the membrane that was washing and the protein was heating up.  While we were waiting for that to finish, back in the “office” Eddie, Anna the newest addition to the lab, Julie and I were talking about lab efficiency and got on the topic of diapers in lab.  That was very funny and then the case of the woman who drove across the country in a diaper to take revenge on a woman came up and we were cracking up about that.  Being able to openly talk about weird stuff like that is so much fun.  Then Julie got all the stuff out I would need for the afternoon because she had an interview she had to go to.  I’ve been doing this on my own which is pretty exciting, I feel very independent but I also have many others in the lab like Eddie and Cagla if I need to ask a question.  I finished the washes and put the substrate on the membrane then got everything ready and Eddie and I went down to the dark room.  I developed a couple different films in hoping that the background would fade and the contrast would be greater but I needed to do a couple more washes.  Unfortunately, when I went back down, nothing really showed up so I went back up and put the next primary antibody on and set it in the cold room.  There was a lab wise meeting at 3 but I didn’t need to go to that and luckily my mom had to pick me up then anyway so everything worked out perfectly. 

Friday began well.  The drive in was so easy, no traffic whatsoever.  When I got to the lab, Julie had already started the washes on the western and running the gel.  I put the secondary antibody on the western and let that sit for an hour.  I cut the paper and the membrane for the gel, afterwards.  Once the gel was finished running, we made the “sandwich” with the sponges, papers, gel, and membrane and set that to run for an hour.  We began the second set of washes for the western and while we were waiting for all of that to finish, Julie and I were talking about the group of students that will be attending our lunch next week and began talking about Peddie in general and all of the international students.  Then college came up and boarding life which was interesting to hear her opinion.  The membrane was finished transferring so I took apart the setup and extricated the membrane then put it in BSA to block it.  Blocking is when you cover the membrane so that only the proteins show up when developed, otherwise the antibodies would stick to anywhere on the membrane because it’s sticky.  That sat for an hour so we ate lunch.  That was fun, there was a group of us talking about vets and dentists.  Once that was done, we set the first membrane to go through the next set of washes before developing it and the most recent one was placed in the fridge in TBSt.  Julie had to leave to go to a wedding so I was on my own for the last two hours of the day, but it was something that I’d already done numerous times before and had no problem by myself.  The only problem I ran into was that when I developed the film, it was either extremely dark where you couldn’t differentiate between anything or extremely light where it just didn’t show up.  This was quite frustrating since it occurred no matter what time I laid the film on for or if I had a filter.  I went back upstairs and Julie said to just put it in the fridge with TBSt and she’ll add the primary antibody on Sunday.  For the last couple minutes of the day I just relaxed and read some more of the grant proposal Kelly had given me.  Finished with my third week already, half way done and it flew by.

                                                                                                                                               

Amber #Entry 2 one of many failures and successes

Hellos,

The second week at my lab was exciting and reflecting. I became more independent at my work right now in terms of all the techniques and lab settings. Moreover, my mentor is starting to take classes starting from July, I will take more responsibility in proceeding her project. The gradual independence at my lab work also came with the price with failures and mistakes.

 My first task alone is to culture the LLCMK2 cells. The procedure can be simply broken down into washing the flask with PBS and adding medium and trypsin. The importance of splitting cells is to prevent the cells from clumping together. This is one of the basic techniques in a molecular biology lab since all the lab work will be based on using the cells. After watching the postdocs split the cells for a few times, I was prepared for this task. However, the bad news came on the weeknd when my mentor told me the cells actually died. Even though my mentor told it’s not a very important sample, I was frustrated because the tissue culture SEEMED so easy. Later we went over the steps again and concluded that I might pippet too hard on the walls that the cells died. It’s really hard to tell at that time because it’s impossible to visualize the cells without microscope. After this failure, I became extra careful with every single step at lab because any error can be amplified in the result. My next failure was ABRT. ABRT is using RNA as a template to make the cDNA. The procedure mainly consist of making the master mix of enzyme and buffer and dilute RNA. However, I put the primer without the ice in RT for 1 minute and that’s probably the reason it failed. I didn’t give that many thoughts at that time because I put them back onto ice again. Now I learned all these things I need to be extra careful with during my lab work. I feel really grateful of working at this lab because everyone here is so encouraging and friendly-- they are always willing to explain things for me and helping me find the mistakes.

  My accomplishment came at the success of my RNA extraction. After a week of practicing and learning, I did my own RNA extraction on Friday. The fact that my mentor said these samples will directly affect the grant due next Wednesday made me even more nervous. The 260/280 (a way to test the purity of extracted RNA) was really good and my mentor said I really helped her with the grant project. I was happy to hear the contribution I made to the lab.

Besides that, I am learning the program PRISM to process data derived from the qCR results. It mainly uses matrix and excel sheet to process the raw data. And I helped other postdocs with virus harvesting and gel extraction. Besides the work at lab, I started taking tennis lessons at UPenn tennis center. The food festival near spring garden was a lot of fun too.

 I am excited to continue my life at Philly and lab!

David, Entry #3, Holgate: Plover City

Holgate is a 3.5 mile stretch of pristine beach that is part of the Edwin B Forsythe National Wildlife Refuge. Located at the southern end of LBI, Holgate is designated by the federal government as a Wilderness Area, which means no human activity is permitted. My lab has special permission from the government to include this area in its plover study. As a result I am one of only a few individuals who are lucky enough to ever see this beautiful area. The absence of humans means the habitat is near perfect with no disturbance, which means it's great for plovers. As the name of this post suggests, Holgate is one of the best plover nesting sites in NJ. There are 23 pairs this year, about a dozen of which are part of our study. The map above shows Holgate. Each marker represents a nest (the colors correspond to the nest status) and each yellow square represents a predator survey plot. We have a good sense of the locations of all the nests, but use GPS to find the predator plots in the field, although I'll go into that in another post.

Holgate is bitter-sweet. It's sweet because it's awesome plover habitat, but bitter because it involves a strenuous 7 mile hike through soft sand to access. However, the sweet far out ways the bitter. It's a pretty awesome place. Pictured above are the people from my lab. From left to right is Emily (biologist with Conserve Wildlife Foundation), Michelle (grad student from SUNY who is leading the study), and Kashi (biologist with the NJ Endangered Species Program). We caught a total of 14 chicks today from 5 different

 nests. Pictured to the left is photo taken from a GoPro on Kashi's head. It shows how we catch the chicks. Pillow cases are useful, because kind of like dogs, birds stop moving in dark spaces. However, in the strong winds of the beach they aren't always effective. In this case the chick escaped the pillow case but ran right into my hand. Catching chicks is risky and we need complete focus. One wrong move could kill a chick. You can see in the photo below how we split up to catch all the chicks at once. Emily and Kashi (foreground) are chasing one and Michelle and I (background) are getting the second chick. Look closely and try to find the chick. It blends in very well with the sand!

One of the efforts to this study is to learn where chicks go after fledging (being able to fly) and what predators are taking chicks. Attaching radio transmitters to their backs (pictured left) is one of the many ways we collect data that will help to answer those questions. We can actually figure out what type of predator took a chick (as sad as it is) by the speed at which the dead chick is carried away (using the transmitter). By understanding the types of predators that pose the biggest threat we can better protect this species in the future.

Our work may seem a bit comical from the chick capture photos, but it's serious business. Since piping plovers are endangered we cannot make any mistakes. The lives of these birds are literally in our hands. There are some people who think banding birds is bad and do not like what we do. We know that catching the chicks does not negatively effect them (we've been chick catching at Holgate for weeks and haven't lost a single one yet). However, in order to save this species we need to know what's wrong. Banding birds is an essential part of solving this issue, it allows us to calculate survivor-ship among other things. That's all for now, thanks!

Victor Leo, Entry #1: Fun Beginnings!

Hello fellow EXPers!

I have been working at my lab for about two weeks now and it has been a really interesting experience. My lab focuses on the early detection of pancreatic cancer, which encompasses the screening of the pancreas as well as the search for viable biomarkers that pertain to pancreatic cancer. My main job around here is to help out with the CAPS 5 study. The CAPS 5 project is one of the three main projects that this lab is currently pursuing. This CAPS 5 study is an on-going project and is an acronym that stands for Cancer of the Pancreas Screening Study (don't know why there is an A in the middle of the acronym). The CAPS study has been ongoing for 15 years now and it is now in its fifth form. The CAPS study (as its name suggests) screens patients and takes patient's blood samples from people who are at high risk for pancreatic cancer as well as people that are not at high risk (controls). Patients that are at high risk usually means that they have strong familial connection to pancreatic cancer but it could also mean that they exhibit certain mutations that are known to promote risk for pancreatic cancer such as a mutation in either of the BRCAs or P16 genes. Additionally people with Peutz-Jeghers syndrome are also considered at high risk for pancreatic cancer and are invited to participate in the study while control samples are taken for the purpose of comparison. The main hope of this study is to further determine what exactly causes the risk of pancreatic cancer in certain families and to better understand what can lead to pancreatic cancer in order to create countermeasures to that. I help out in this study mainly by processing the blood that we receive from patients and by going through EUS (endoscopic ultra sound) reports that are given by the doctor and entering data that is provided by the reports into an online database. The exact procedure of the processing of the blood depends on which study the blood is for but for the most part the blood that we receive needs to first be centrifuged a number of times and then the plasma needs to be aliquoted into smaller tubes with protease inhibiter. For EUS reports certain information needs to be drawn from the surgical reports and put into the shared database. Things like the number of lesions and whether they exhibit thick or thin septations as well as other thinks like lobularity and calcification are considered necessary information.

My Building! (CRB2, Cancer Research Building 2)

I am glad to say that everyone is the lab is extremely friendly and are very welcoming and understanding. In my lab, I work directly under a very nice graduate student and work together with 3 helpful undergraduates. We are all assigned with mainly helping out with the CAPS study and it has been a pleasure to work with them. I could not ask more from my workmates as everyone in the lab was very understanding when I continuously got lost on the way to lab all throughout the first week and when I messed up the samples on my first day. Although I do not get to see my PI very much because he is very busy and always in and out of the lab (he also wasn't there for the first week) my grad student mentor has taught me a lot and I greatly appreciate everything that he does for me. I will have more pictures of my experiments next time!

Arnob Dam #1 - Starting out in my lab

I was introduced to my lab last week as well as a new life in the city for two months. My lab is situated in part of the top two floors of the College of Physicians and Surgeons in the Columbia Medical Center Campus. There are six main rooms to the lab. The lab almost gives off an office-like feeling because most of the rooms are just filled with desks and computers (besides the lounge and kitchen). It is a Neuroscience lab, but the actual experiments are computer based with a big emphasis on MATLAB.  I am at the lab from about 9:30 to 4, and multiple hours of the day is spent analyzing the data of experiments on MATLAB.

My usual work-screen.

The work I do on MATLAB is very challenging and can be both exciting and frustrating. For the past couple months I have been learning how to use the program, but within the first hour of my first day of my lab I started learning new stuff on MATLAB. I enjoy using it because it takes many problem solving skills which keeps me very occupied. When I get stuck my graduate student Yul is always ready to assist me which is reassuring.

 My specific research entails piloting an experiment for my PI, Mike. The experiment's hypothesis is 'changes of mind has a higher probability of leading to the correct solution than leading to the incorrect solution'. The experiment is conducted when a human subject plays this very simple computer game (made in MATLAB) where he or she selects which way a collection of dots on the screen is moving. It is more challenging than it sounds. The subject is also hooked up to a machine which tracks his or her eyes to make sure the subject is focusing on the dots while making the selection. 

My lab space, including the equipment that tracks eye movement (on the right)

This cool staircase in the lab

All in all, it has been a challenging but fun first week. I am close to learning all the MATLAB that I will need to successfully pilot the experiment and I am looking forward to actually starting doing trials. For my next blog post, I think I will write about how living by myself in New York has been as well as my impressions of my other lab members.

Kelsie Sirak, Entry #2, Microscopes and Harvesting

I’m just closing out my second week here at the Jordan-Sciutto lab and it’s been very enjoyable.  I came in on Tuesday later than usual because I was working with Eddie, since Julie was still away and Kyle was shadowing in another lab.  It was a relatively slow day but I got to learn about Eddie’s project which is more on the pharmacological side of the lab.  He’s working on a pathway with PERK that may lead to neurodegeneration because of unfolded or misfolded proteins.  I did however get to watch him plate neurons as well as treat a set of his older plates with Ritonavir and Darunavir to then observe the effects of the drugs the next day. 

When I got in the next day, I went straight to work with Kyle.  We started the next western and during the down time for that, I was watching and working with Eddie.  He was harvesting the cells we incubated the day before.  I watched him do a few and he offered for me to scrape the cells and then pipette them.  That was pretty cool in knowing that they were the cells that were going to be used in a gel and then a western.  Once all of the cells were harvested, we centrifuged them to pellet the unnecessary cell debris.  While that was going we went over to watch a microscope demonstration.  Dr. Jordan-Sciutto was determining if she wanted to purchase this new microscope, and we were all allowed to watch the demo.  It was awesome.  Everything was automatic, choosing the dyes that were shown and then overlaying them to view it all together.  Then it did something called Z stacking with a potato sample, since it’s too thick to focus all at once, the machine takes pictures of each of the levels and then stacks it so all of the layers are in focus in one image.  We popped in and out of the demo to work on our experiment but for a couple minutes they were analyzing a cross-section of a brain.  I went back to working with Kyle and  during the second set of washes for the western we went down to the dark room real quick to make sure the machine was on and ready for when we needed it.  After finishing up the washes and gathering everything we needed to develop the film, we headed down to the darkroom.  This time I did all of it.  I transferred the protein signals onto the film and then placed it in the machine and we got some really good results.  Once we finished that, we got to use the microscope to view previous samples Kyle and Julie had been working with.  We were working and listening to some country music so that was very enjoyable.  To finish out the day we analyzed these new pictures we took with the microscope and were playing around with Excel to help with the standard error and then graphing the data. 

Thursday Julie was back from her trip.  For the first part of the day Julie and Kyle were making a PowerPoint presentation for the lab meeting that was taking place later that day.  I sat in on that and it was interesting to listen to them talk about the article.  Then I went back to counting.  This time I was using a different image to count to neurites, which was more difficult than before because with this one you have to adjust the settings to be able to even see the dendrites.  But we ate lunch and talked which was very nice.  At 2, the three of us headed over to the library so they could practice their presentation.  The library was amazing.  It was huge and had so many nice resources to use and it’s somewhere I could spend my whole day.  Since we had some time to kill before the lab meeting, Julie took us to the Fine Arts Library to look around.  That was incredible.  The building itself was quite interesting and then inside it was very elaborate.  Finally it was closing in on 3, so we started to walk over to the building where the meeting was taking place and we stopped at a Wawa on the way to grab some gum.  When we got to the meeting room there were some people from another lab there and they were all very friendly and welcomed me to the journal meeting.  It ended up that there were a lot of new people in attendance.  I was able to listen to Brigid, a grad student working in my lab as well as another lab on campus, about her most recent project which she’s hoping to get published soon.  She talked about oligodendrocytes and oxidative stress and its effects on the cells.  Unfortunately, I wasn’t able to stay to listen to Julie and Kyle’s presentation, but I’m sure they did great!

Friday was slower.  We continued with another western to test for synaptophysin.  During the downtime, Julie showed both Kyle and I how to analyze the film we developed.  Then the majority of the time I was counting more neurites.  Once all the washes were done we went down to the dark room once more to develop this most recent western.  The rest of the day was spent counting the neurites, which wasn’t the most exhilarating but it’s important.  Then I got a call form Matt right as I was finishing up counting and he asked if I wanted to come down and talk for a little bit.  We ended up walking down to the Ben & Jerry’s down the street and just relaxed outside on a nice Friday afternoon.  It was a good close to my second week.

Matt Erman Blog Post 1: Welcome To Cali

I would like to preface my blog post by saying that I have the worst luck in the world, and as a result of that luck, I am always sick. Sadly, I cannot even escape that terrible luck out in California, as on the first day of working in the lab, I nearly killed myself. By eating a cashew. 

Sadly, the whole situation could have been avoided. It was one in the afternoon, and the entire lab was going to lunch at some cafeteria. I tagged along, and when we got to the cafeteria, I realized there was not much selection in food, so I just picked whatever chicken they had that day. I paid for it, went to sit down with the other people in my lab, and I took a bite of lunch. Uh oh. Apparently a sign near the chicken said "CONTAINS NUTS" (I am allergic to some nuts) in big, red letters, but my clearly attentive self missed the sign, and as a result, I ate the chicken. Well, right as I took a bite, I knew that I was in trouble, but I also knew that if I stopped eating, I probably would be able to survive the rest of the day. I lasted about two hours until I realized that I needed to get back to my apartment and take some medication. Luckily for me, the person I was shadowing for the day (Joseph, not the person I am working with) came in on Sunday and finished his work early, so I was able to leave, get home, take lots of allergy medication and then fall into a sixteen hour benadryl induced coma. What a great first day! 

Thankfully, the rest of my week has been much better than that first day. On Tuesday, I was able to meet Matt, the grad student who I will be working with for the next few weeks. He was able to show me what he was working on. He was working with yeast that had mutated human genes that were found in patients with ALS integrated into its genome, and he was trying to see if the genes caused the yeast cells to die. The reason that Matt, and most of the lab, uses yeast genes to test for mutations in ALS genes is because of the similarities in yeast and the cells of the brain and the spinal cord. Matt was growing these cells in a nutrient rich broth and with specific anti-bodies that would attach to specific proteins translated by the genes Matt inserted into the cells. 

Matt also has his thesis due soon for his PhD, so he has spent much of the week writing. As a result, I have kind of bounced around people in the lab trying to see and help with as much as possible. I was able to watch someone in the lab dissect a mouse in order to test on the eye and optical nerve, which was really cool, as the optical nerve about the size of a hair and in order to dissect it it needs to be done under a microscope. I have also tried to help out others in the lab, and today, I started my 'own' project with an undergrad student, Olivia. We will be running synthetic genetic arrays on samples of yeast cells with the C9orf72 gene inserted into it (a known ALS causing gene), along with other genes that may or may not help in decreasing the C9orf72 affect on the cell. Today, we began by growing the yeast cells that we will be running the array on.

So far, my time in California has been great (minus the first day), and I am really happy I came out here. And while the lab has been great, it has also been really cool just exploring the area. I really love San Francisco and the rest of the Bay Area, especially all of the nature around here. It's really cool that I can be walking through San Francisco one minute and then through a Redwood Tree forest an hour later. All and all, a great week at Stanford.

Jenny Lee - Entry #2 at CHLA

Hey guys! I'm finishing up my third week here at CHLA and about to head off to Mongolia with a few lab members tomorrow! Although most of my days I have quite a lot of downtime, today was pretty exciting because Dr. Peretz visited! (Thank you for visiting!) The weather was great and we had a nice Italian lunch with my PI and another person that works in the lab. Unfortunately, the lab director and one of the girls I work with already left for Mongolia, but I was able to show her the human lung samples I am working with. Currently I am sitting here as the lungs are being washed in the cold room. (There is a lot of preparation that goes into attaching antibodies on these samples. Today, I am washing them 5 times with a solution for one hour each.)

For the past one and a half weeks, I've basically been doing the same things as I have mentioned in my first blog post. I don't think specific details would be any fun or informative, but here's the big picture. Although there are a lot of studies and papers that focus on the development and cells of the mice lungs, there really isn't much knowledge on the human lungs. My PI (Dr. Al Alam, but we just call her Denise) decided that if we're ultimately trying to find cures for human lung diseases, it only makes sense to study the human lung! We were fortunate enough to get human fetal lung samples, ages ranging from 10 weeks to 16 weeks. We are staining the lungs with antibodies so that we can get pictures and models that show the progression and development of the human lung. Right now we are following a protocol that has previously been used to stain mice - but it works fantastic on human tissues! Fixing, washing, mounting, and photographing the lung samples take about a week in total, although the techniques itself aren't very complicated. 

Outside of the Dr. Peretz's visit, today was also a great day because we were able to 3D image a lung sample! We were previously looking and taking pictures of the lungs under a dissecting scope, but it is often difficult to get clear images because some of the samples are too big. And because we want to study the development of the human lung, a 3D image would be helpful in analyzing that. It looked amazing - and although it will take some more practice and patience to 3D image all of our current samples, it will all be worth it.

I've also been doing other things besides just sitting and washing the lung samples. I went to a PhD defense, a couple more lab seminars, a meeting on RNA sequencing technology, and I brought donuts one day (which everyone really enjoyed). There is also another research observer working with us - Victor. It's cool having Victor and Emily around because they're both super smart, and company is always nice, especially on the more quiet days.

Overall, my first three weeks have been great. I've gotten closer to my PI and the people I work with. The lab director (Dr. Warburton) has been really pleased with our work so far, and I hope that we can continue to make some great discoveries in August. I am slowly getting ready for Mongolia and will definitely upload some pictures of my time there! Sorry about the lack of pictures right now!