Jay Ha #1 first week at yale

After enjoying lovely three weeks of summer in Korea I came back to US.

As soon as I came to the new haven area, first thing I had to do was to check out the house I rented. It was nice, had clean bedroom, kitchen, and bathroom. House owners had two cute pugs. The house was about ten minutes away on foot from my lab and I liked everything except the internet was kind of laggy at times. The professor came by to the house I am staying, said hello, and he brought me to the Korean marketplace nearby so that I can shop some foods to cook at home at times. I bought mostly instant foods since I can't cook well. 

On our ride back to the house, professor told me something quite interesting yet unexpected. I had spent a summer two years ago in Georgia Tech with Dr. Kim regarding cerium oxide nanoparticle and its acute toxicity, and both myself and Dr. Kim were expecting me to work on expanded version of the same research. However, Dr. Kim informed me that he had recently started a cooperative research with Korean condo company regarding surface disinfection using UV lights, and that he wanted me to take part in this along with his other graduate student, Stephanie, instead of the nanoparticle research. Having been kind of "not very excited for this summer" since I already kind of knew exactly what I would be doing and because I knew that acute toxicity research with cerium oxide often involves very tedious and boring repetition of counting microorganisms under microscope, I was actually very happy to hear the news.

Well, all the works I have done in the spring term EXP may not be useful for the new research with UV disinfection. But, life's tough. And strong falcons deal with it. 

The day after, I went to the Mason laboratory, and met Stephanie. She was grad student who just came to yale last year after finishing her science works in Toronto university. She was very kind and nice and gave me many research papers and other background information to read about UV disinfection processes. She briefly described the research with Korean condo company she is working with under Dr. Kim to me. The project itself seemed kind of commercially-evident, in that the company is trying to get some data approval of renowned university research team that their condo is safe from bacterias and stuff, but the research itself and the field of disinfection using UV lights seemed quite interesting so I was getting excited. I was not able to enter the lab because I was supposed to get some laboratory safety training stuff so she toured me around the building, but I could not really enter the labs themselves. 

I spent many hours reading numerous research papers under each of these categories:"Introduction to light and the electromagnetic spectrum," "Introduction to pathogens in the environments," "Using UV light for disinfection," and "Advanced Oxidation." Some of the concepts I did not really understand originally as I am reading these materials for the first time, but some parts I got it and I asked those parts aI did not get to Steph later on. 

I received the lab training couple days later, and Steph referred to this delay as Yale's bureaucracy saying that they are not good at handling stuffs as quickly as possible. Meanwhile, I learned a lot from Steph, Dr. Kim, and the readings about the research that I would be conducting over summer.

After the training was complete, I was eventually allowed to enter the actual labs. There were one huge lab space in the second floor of the building, small tiny lab space for myself to work on in the third space, and I was using couple other facilities in the third floor such as the autoclaving machine or incubator and shakers that were in other labs of the third floor but I would be using at times. 

The actual lab works started with making different solutions that would be used to cultivate the bacteria, E Coli. I made phosphate buffer solution and LB broth following after the procedures Steph taught me. E Coli culture was prepared and incubated for later extraction of samples, and I prepared forty plates containing solidified broth solution for the E Coli bacteria to dwell on. I was told that I would be growing E Coli and try to understand the correlation between time v. CFU, time v. OD measurements, and CFU v. OD measurements. 

Aside from the lab stuffs, I got to enjoy good foods in the new haven area and exercised daily in the second largest gym facility in United States. 

First week was full of unexpected, yet fun and fun stuffs. 

Ally, Entry #2,

Since the last blog entry there's been a lot more to do in the lab.  We're not really just working on one project, but there are a couple going on at once.  Carola an I finished packing all the SI samples, and went through the FA (fatty acid) training.  Since I can't work with a lot of the chemicals, I can't really participate much in the first day of extracting the FA from the tissue sample, but I can do day two.  The process involves plenty of pipetting some clear fluids that can knock you out from a bottle to a test tube to another test tube to then be centrifuged and evaporated.  A lot of similar steps that no one would ever be able to remember without the instructions, but basically biotech all over again. The end result of 2 days of this is a graph which spits out numbers that dictate the FA levels in each sample.

Besides that, I've perfected my technique in washing glassware.  Fun stuff right there.

Probably the most exciting thing I've done yet is help fillet sea bass, which it turns out I am very good at.  I've found my calling.  Who needs marine biology, I'll work in a fish market filleting fish, or as a sushi chef.  My knife skills are pretty top notch, after all.  But the fish are being cut up and frozen in ice cube trays to feed crabs, part of the main project to establish the FA baseline of crabs on a certain diet.  That will help in establishing the food web project, but I won't be in the lab when it comes to the meat of  the experiment.

Victor, Entry #5: Farewells and the Laser Capture Dissection Microscope

Hi everyone,

Friday was my last day in Baltimore working for the Goggins lab that focuses on researching early detection of pancreatic cancer. My last week at the Goggins lab was particularly interesting. This week my lab gave me the opportunity to experience more then preforming experiments and really familiarize myself with what goes on in the hospital. In addition to being able to observe a Whipple procedure like I talked about in my previous post, I was able to shadow my PI in the hospital and observe him there. I was able to observe him preforming colonoscopies and was also able to watch other another physician preform an ERCP or an endoscopic retrograde cholangio-pancreatography. In an ERCP an endoscope is placed through the mouth and into the duodenum. Once into the duodenum, a cannula or small plastic tube is inserted in a small opening within the duodenum. Then, contrast material is injected into the tube and x-rays indicate where the contrast goes. This test is most commonly used to diagnose conditions within the pancreas and bile ducts. X-rays can easily recognize stricture within the pancreatic ducts so this is a very useful procedure. Unfortunately, pancreatitis can develop in the patient because of irritation due to the contrast material, but it is a risk that the physicians and the patient both consider before the procedure.

In addition to observing certain procedures, I have also been given access to the laser capture dissection microscope. A laser dissection microscope uses a laser to cut and isolate desired tissues. The lab wanted to isolate PanIN tissue for certain experiments and in order to do that I needed to use the laser capture dissection microscope. PanINs are the most common precancerous lesions within the pancreas. The Goggin's lab is very interested in investigating PanINs because they could play a vital role in future early detection methods and because currently there is no way to detect PanIN lesions in patients.

The microscope cutting and isolating a
pancreatic duct. The laser is the blue light.

Laser Capture Dissection Microscope

Overall I had a great experience researching at Johns Hopkins. I learned how to preform many different experiments and procedures like DNA isolation and Western blots, but most importantly I got to meet many different extraordinarily smart people. I got to make many friends and experience living on my own. I would like to thank everyone that made this experience possible include my PI Dr. Goggins, my graduate student mentor Andro, and of course I would like to thank Dr. Peretz and Dr. Crider. See you in September!

Amber #Entry 5 A Great Ending

Hellos,
   The last week at the lab was a great ending to my summer experience at Lopez lab. According to the result from the previous data, a new mutant 3P0/5P0 showed a great immunostimulatory ability. In order to confirm the stimulatory ability of this candidate, this week's experiment aimed to do a triplicate repeat and determine whether 3p0 tail or 5P0 is responsible for this behavior. After troubleshooting with my techniques last time, I am now more confident with running the experiments by myself.
  Alongside the main project, I also get to dig deeper about a side project I am helping my mentor with this week. The purpose of the project is to design a recombinant MF49 vaccine in vitro system. Even though the recombinant of MF49 with DVG has shown a great immunostimulatory ability in vivo, the manufacture of inexpensive, optimal vaccine depends on the success in vivo system. After reading a paper on MF49 vaccine, I had a talk with my PI about the development of vaccine in America right now. Currently, this vaccine is broadly applied in Europe but because of the censorship in America, the use of MF49 is limited. Dr.Lopez said instead of creating vaccine, the lab is trying to use MF49 as a delivery adjuvant to increase the immunostimulatory ability of vaccine.  The experiment is set up to test whether the combination of DVG and MF49 will increase the production of interferons comparing to only using one of them. In this week's experiment, different cells were cultured to knock out some immune pathway (such as interferon, ISG56 OR IL29). And the cell genome was dyed with fluorescent blue, DVG was dyed with green and viral genome was dyed with red. The data was recorded at the time point 6H, 12H, 24H after infection.

A fluorescent-detected microscope with three different channels.

Besides learning to use the microscope to take pictures, I also learned to use the program Velocity. Velocity is designed to process the cell images and do the quantifications.

The day before I left, i had dinner with all the people at my lab. Not only did they become my teachers at lab, but also my friends who I can grab lunch together and play tennis with. It has been a great experience at Upenn for these past six weeks. This was also my first chance living in an apartment alone during summer. These six weeks provided me a better understanding of how research lab works and a great chance of applying all the techniques to do real experiments! I hope everyone can enjoy the rest of the summer and see you guys in Fall! 

Bridgid, Entry #3, Something Fishy is Going On Around Here

Last week was my third week in the lab. Just like any other week, it was nothing short of exciting and eventful.  I made big strides on my project this week, and in addition I unexpectedly helped out Abby, the other college student in the lab, and Patrizia with her project when they ran into some trouble in the middle of the week. Let me explain.

On Monday, since Patrizia was in the fish room injecting, Abby and I spent the majority of the morning reading articles that had to do with the expression of lmo2 at different stages of development in the zebrafish and also how to sort cells, which is what Abby’s main focus for the week was.  In the afternoon, I got to have first hand experience in what Patrizia says is one of the most dreadful tasks in working with the fish—imaging.  Imaging is done after the in situ is finished and the embryos were kept in PFA to freeze them in the desirable stage of development.  For my experiment, I started out by imaging the mutant lmo2 18hpf embryos that I performed the in situ on.  Because the 18hpf is in the middle of the other two stages of development that I have, (8-10 somites and 24hpf) Patrizia wanted me to start with these to see if I noticed any difference in the staining and then if needed I could image before and after this time point. After starting with 25 embryos for the in situ, I was able to recover 19 that I was able to image.  I took one picture of each embryo, making sure to focus on the posterior end staining because that was the area of interest in which the gene lmo2 is normally expressed at 18hpf.  Although it may not seem like a lot, between getting all 19 good embryos in position on a mini gel under the microscope and then using the computer and the microscope to find the right exposure and focus for each embryo, it took the remainder of the day on Monday and the beginning part of the day Tuesday to image all 19 embryos.  It got a little frustrating towards the end when it was hard to find the right focus for the picture, but it was so cool to notice that between just these 19, we were already seeing differences in the amount of staining or gene expression in embryos. Although we were seeing differences, they didn’t mean anything yet because I was doing the imaging blind, which means that I didn’t know the genotypes of each embryo so that I am not biased to how I interrupt or take the images based upon the result that we would like to see.  After a long day of switching between staring in a microscope and at a computer screen, it was so nice to grab dinner in the city!

Tuesday morning I spent almost the whole morning finishing my images for the 18 hpf lmo2 embryos.  After I was finished imaging, I started the DNA extraction for the first 8 emryos that I imaged.  This was the first time that I started the DNA extraction from single embryos, which was pretty cool! I felt like I was really doing it all by myself, which is a great experience and test in responsibility and concentration.  That being said, because it was my first time doing the DNA extraction starting from the single embryos, Patrizia and I both thought that it would be a good idea to start with just the 8 samples.  Once I put my samples in the water bath to sit over night, I prepared and ran the PCR for the mutant rescue experiment that Abby and I have been switching off working on.

Wednesday was quite the day in the lab! I started in the morning by finishing up the DNA extraction for my 8 embryos and then once the DNA was extracted, I quantified the DNA on the Nanovue to determine the concentration and purity of the DNA.  I then used that DNA to prepare and run PCR on my samples.  Even though DNA extraction and PCR is pretty cool (especially for me), the real agenda for the day was to help Abby with her project.  Today was the big day where we planned on sorting the lmo2 cell line with and without the mutated gene, MLL-GAS7.  Abby and Patrizia spent the whole morning sorting the embryos based upon their type and color of their fluorescence in order to make sure that we sorted the cells correctly.  After walking through the maze of building between Penn and CHOP, we finally arrived at the sorter. The cell sorter this is huge machine that sorts cells based on certain parameters that you set- for example, what types of cells you would like to keep to analyze, how many cells (or events) per tube, and the percentage of cells expressing a the desirable trait that you are looking for, which in our case was a certain type of fluorescence.  Because we had never sorted this cell line before, no one had any idea what to expect in terms of the number of cells we would be getting.  Everything seemed to start out well, after all the controls were in place, things were going as planned, until we started to run the actually samples. The first sample, our control with only one variable, sorted fine, ending with a total of 5 tubes with 9,000 cells in each.  The real problem started when we went to run the mutant sample.  Even though we started out with more embryos in the sample, we ended up only filling 3 tubes with 9,000 cells each and had a lower percentage of expression of the gene that we were looking for, the exact opposite of what we expected.  Because we didn’t know what to expect, we just went with it and Abby and Patrizia went the cytosine in the Main Hospital, where the machine is supposed to produce slides of cells from the culture that you started with so that you can look at them under the microscope.  Except that didn’t happen.  When they came back to the lab, we noticed that not one slide at a single cell on it.  There was absolutely nothing on every slide. 

We were stumped, and spent the remainder of the afternoon trying to figure out what went wrong and why did we not have any cells at all.  Ti was a frustrating afternoon, just because so much of the experiment was machine based that we had no idea where to start troubleshooting. We all tried to think of something, but by the end of the day, we only had one hypothesis—maybe there was something wrong with the sorter, but none of us knew where on earth to start looking for that answer.

Thursday morning I ran by PCR for the first 8 lmo2 samples on a gel, cut the bands from the gel, and then prepared the sequencing reaction.  By the time the afternoon rolled around, although we didn’t have an answer to what went wrong with the cells yesterday, we did have an alternative way look at the cells.  One way that we could try and see the blood cells was to take a fish that was about 2 days old and cut its major blood vessel. Because the fish was already 2 days old, the blood cells were already in circulation, so we would cut the fish and then let it bleed onto our slide.  After the cells were on the slide, we removed the dead fish and then stained the slides in various purple dyes to highlight the cells that would hopefully be on the slide.  Since we weren’t completely sure that this technique would be entirely successful, we only tried it on wild type fish so that we knew what the cells were supposed to look like.  After spending the rest of the day on Thursday and a large part of the day on Friday attempting to perform to the staining in order to have cells to look at, we ended with about 3 or 4 good slides were the cells were visible.  Although we ended with some slides, this method was extremely time consuming and the cells still weren’t the best to analyze. Luckily, by Friday morning, Patrizia had figured out what went wrong with the experiment on Wednesday.  She found out that the speed in which the cell sorter was sorting the cells was too fast for the types of cells that we had so the cells were most likely destroyed in the process of the sorting.  Since we now believed that this was the reason for the failure, we booked a time on the Influx, a gentler machine, for next Tuesday to determine if we would have cells at the end of that sorting process.  Friday afternoon concluded with a few more staining and then a lab meeting, at which both Abby and I presented on the work that we had done in the past two weeks.  

Soo, Entry #3, More Codes

During the past couple days, I've continued working on my codes to efficiently make multiple boxplots (description about the boxplots is in entry #2). For the code, I mainly used MatLab, but since I am more familiar with java, I sometimes used java to write a draft version of the code, then changed the format to run the code with MatLab. 

These (lines below) are parts of my code:

rowThick = 50;

UT = triu(ones(rowThick, rowThick),1);

[ib] = find(UT>0);

randomX = randperm(numel(ib));

[i,j] = ind2sub([rowThick,rowThick],ib(randomX(1:20)));

-> This code picks out position of 20 random data pairs out of 2500.

for count = 1:20;

  Z(count,1) = zrand(thick(i(count),:), thick(j(count), :));

end

-> This code calculates the zrand score of the picked 20 data pairs, and stores the result in the matrix Z

scatter(x(:), y(:), 10, 'black', 'filled');

hold on;

boxplot(y,x,'labels', {});

hold off;

-> This code uses matrix x and y to plot boxplots and data points (scatter plot).

Victor, Entry #4: In the Operating Room and Cryosectioning

Greetings people,

Hello everyone, so far it has been a very interesting last week at the lab! So far this week I have been given the opportunity explore and observe what goes on in the clinical aspect of the research that we do as well as what goes on in the hospital itself. Occasionally, in the lab I would get a blood sample from a patient that has just undergone a Whipple procedure. On Monday I actually got the amazing opportunity to observe a Whipple procedure. A Whipple procedure is the partial or whole resection of the pancreas, and in most cases they also remove the duodenum because it is so intertwined with the pancreas that it is hard to take out the pancreas without also resecting the duodenum. The Whipple procedure is the most common operation preformed to treat pancreatic cancer or even precancerous lesions. Not every patient with pancreatic cancer is able to go to the operating room. Because the pancreas is so close to important blood vessels it is common for a cancer to have arterial involvement and therefore be unresectable. When this is the case a patient is usually given chemotherapy and radiation in the hope that the cancer will shrink and become resectable.

The operation is 6 hours long and depending on how much of the pancreas is removed, the patient may become diabetic. I had to be in the building and with scrubs on by 7:45 am because the procedure started at 8:00 am. Watching the surgery was a really cool experience. All throughout the 6 hour surgery the surgeon, assisting surgeon, nurses, anesthesiologist, and medical technicians all worked together like a well oiled machine. It was really something to watch! Although at first I felt somewhat queasy and I had to fight the urge to pass out, I somehow managed to pull through and act like the procedure didn't faze me. I think that it helped that I skipped breakfast that day. After I got used to all of the blood and it became a lot more interesting and it started to turn into a really cool experience. When the pancreas and the duodenoum were removed they were taken to a different place in order to be examined. Slides of the pancreas tissue were made and looked at to ensure that no cancerous cells are left in that particular area. In this particular case the patient did not have pancreatic cancer, but had multifocal IPMNs which are precancerous lesions and would become cancerous given a few years. The pathologists dissected the pancreas and measured and took note of each IPMN within the pancreas. Seeing these cysts was a unique experience and I am glad to have been able to watch them do their work. This was really good for me the see where the blood that I process comes from and witness what goes on in the hospital.

In addition to being in the hospital, my lab had me do some cryosectioning. Cyrosectioning is just a fancy term for making slides. I made many slides from pancreatic tumor tissue and normal pancreatic tissue. I also stained the slides in order to make them easier to see under the microscope.

A pic of the cyrosectioning station