LIJ Cardiology Internship: Week 1 & 2

It is impossible to write about everything I have learned because I have never learned so many interesting things in one week! My nerves ran high the first day due to the fact that I thought I was out of my comfort zone in a department where I barely knew anyone. Looking back now, it is humorous. Everyone is so welcoming and eager to teach and explain. I am starting to see that the hospital environment is completely within my comfort zone.

Every few days there is a morning conference at LIJ, and every Wednesday there is a lunch conference. They discuss things like interesting cases from over the weekend and unique EKG's that might be on the board exams. Sometimes presentations are given about the histories of certain procedures and the risk/benefit of one's that exist. The main thing I have observed is that there is no step by step guideline for the doctors to follow. Every patients situation brings it's own complications and the cardiologist has to factor everything together like age, allergies, chronic disease, family history, previous visits, and so on for each decision they make. It might seem like an implantable cardioverter defibrillator (ICD) should be introduced to someone because of frequent fribrillations, but they have a history of dementia and live alone in a trailer park. Some doctors might not see this as a ethical thing to do because the patient might not understand what is being put in them. There are so many decisions the cardiologists have to make in such intense situations.

My first week I spent on the cardiology consult service with one of the first year (now second year) fellows. This is a service where a few of the patients are directly under your care (primaries) and the rest are under the direct care of another physician and you are examining them only in terms of cardiology (consults). It is a very busy service, with a lot of patients, but each patient gets the time they need to be examined. After my week on the service, I was able to see the routine the fellow undergoes everyday which is to meet with the resident and get the vitals of each patient, and then go through each patients case with the attending physician to make a plan for the day. Once that was completed, we were off to visit each patient. Sometimes you would go to see a patient and they might be getting an Echocariogram or be in the Catheterization lab and you would have to come back. Sometimes they would be asleep and you would have to wake them up. At other times we would be going in to recommend procedures like valve replacement surgeries and pacemakers. Each patient has a story and a reason why they are there, and that needs to be understood before entering the room.

Dr. Slotwiner who organized this internship with Dr. Shanies, has been nothing but nice, helpful and motivating. Spending the week with him in the EP lab (electro-physiology) was a great experience. I got to stand in on an EP study and the implantation of a pacemaker device, which was one of the most amazing things I have ever seen. It is unbelievable how something so small can do so much! Not only can it pace the atrium and the ventricles separately, it understands threshold limits, and has a memory for when the heart has fribrillations and palpitations. I also was able to see an ablation, where an ectopic beat is corrected non-invasively!

My experiences thus far in the LIJ cardiology department have been nothing but excellent. I look forward to the weeks to come and to the new things I will learn.
-Stephanie Lombardi

Week 3

Results from the Wicking Technique

The beginning of the week was focused on developing western blots from last week's wicking transfer. I followed the standard procedure for developing westerns, but only the IEF markers appeared. Furthermore, the markers were found on only one of the membranes, instead of both as expected. Dr. Clendening and I thought that the proteins may have been retained in the agarose gel, so we preformed a coomassie stain. Sure enough, there were proteins in the gel. We decided not to use agarose gels in the future.
In the middle of the week I tested the effects of the formaldehyde application. Last week's results indicated that the formaldehyde might have reacted with the extraction buffer from the NucBuster kit. To test this hypothesis, I isolated a nuclear fraction using a different protein extraction kit (presumably with different extraction buffers). This fresh sample was to be compared with a nuclear fraction from the previous week (NucBuster extraction kit). I aliquoted samples of the two nuclear fractions and gave them either the standard formaldehyde and glycine treatment or nothing at all. All of the samples were incubated on ice for 15 minutes. Upon examination, it was clear that the nuclear fraction from the NucBuster kit reacted with the formaldehyde/glycine application and formed a precipitate. The fresh sample, that was isolated from the other kit, did not appear to react. Dr. Clendening and I decided to continue with our test the next day and load samples from both extraction kits on a protein gel.

Yet, when I came into lab, there was a slight change of plans. Dr. Clendening wanted me to isolate nuclear fractions from fresh tissue using the NucBuster extraction kit. She also wanted me to isolate nuclear fractions from not only brain tissue, but also the adrenal/kidney tissue; the gel would hopefully tell us if one tissue was preferable over the other. I preformed the isolation steps and added the formaldehyde solution to half of the samples. But, when I was about to add the glycine, I noticed that it contained mold. Dr. Clendening thought that the mold might have caused the precipitate we had seen earlier in the week, so we made a new glycine solution and added it to the formaldehyde samples; it was unclear whether or not these samples formed any precipitate. We proceeded to load the samples into a 20-40% polyacrylamide gel and I successfully transferred the proteins to a nitrocellulose membrane later that day.

On Friday, I developed the western blot. Every sample showed up! Thankfully, we were able to learn many things from this gel. For one, the adrenal/kidney tissue contained more SF1 than the brain tissue. Secondly, the samples with formaldehyde/glycine were less visible than the untreated samples. They did show up though, so presumably the cross-linking experiment was feasible. Third of all, the isolated SF1 and DAX1 samples appeared, indicating that we had useful positive controls. Moreover, these samples could be used in later application to more accurately depict the interactions between SF1 and DAX1. Though, they would need to be concentrated, because they produced a faint band. Last of all, and perhaps most importantly, we recognized several bands of SF1 in the nuclear fraction. These bands indicated that SF1 was bound to some additional protein(s) or other nuclear component(s). Since our research was based upon the hypothetical binding of SF1 and DAX1, two different proteins, this evidence was rather encouraging.

Weeks 1 and 2

Week 1

Most of week 1 was spent dissecting turtle embryos. The eggs, for the most part, had reached the temperature-sensitive period (TSP). Embryos were removed from the eggs and the brain, gonadal, and adrenal/kidney tissues were collected. These tissues were stored in a sodium/phosphate buffer and placed in the freezer at -80 degrees celsius.

Week 2

Week 2 was comprised of a variety of activities, all focused on determining the interactions between DAX1 and SF1. Like the previous week, a great deal of time was spent on collecting the necessary tissues. Eggs were dissected and the brain, gonadal, and kidney/adrenal tissues were stored in sodium phosphate buffer at -80 celsius. In addition, a nuclear fraction was extracted from a stored sample of brain tissue. This fraction was split into multiple aliquots, which were to be incubated and used in an isoelectric focusing (IEF) gel (since the fraction presumably contained both DAX1 and SF1).

Samples were incubated at 26, 31, and 0 degrees celsius. After 30 minutes, a formaldehyde solution was added to fix any protein-protein interactions that had taken place. Incubation continued for an additional 15 minutes, before a solution of 1.25 M glycine, pH 7.5, was added to stop the fixation process.

The IEF gel was loaded with the samples and run according to the gel electrophoresis recommendations. Proteins were transferred to a nitrocellulose membrane using acetic acid transfer and developed using the western blotting technique. Unfortunately, the results were not satisfying.

Only the IEF markers and the positive control (isolated SF1) appeared on the developed membrane. We were not sure why the others samples did not appear, but we did have a couple of predictions. First of all, we had never preformed an acetic acid transfer. The procedure was new to us and there were a number of things that could have gone wrong. Secondly, none of the samples treated with formaldehyde appeared. We had seen a white slurry form on top of the formaldehyde containing samples before loading, which we believed may have interfered. Our initial conclusion was that a salt had formed between the sodium phosphate buffer in the formaldehyde and the elution buffer in the nuclear extract.

To resolve our conflict we decided to test the transfer technique and make a new solution of formaldehyde. An agarose gel was made up this time and loaded with samples following the same conditions as before. The only difference was that the formaldehyde was made up in a tris-HCl buffer instead of sodium phosphate. The gel was run and transferred to nitrocellulose again, but the transfer conditions were altered. This time we decided to use the wicking method. We cut the gel in half, placed one half in acetic acid buffer, and placed the other half in saline-sodium citrate buffer (SSC).

Project Overview

The focus of this research project is to determine the temperature-sensitive sex determination (TSD) factor in Chelydra serpentina, the snapping turtle. In most species of vertebrates the initial trigger for sex determination is genetic and, in a much smaller number of species, the sex determination relies on environmental factors. In the snapping turtle sex determination is temperature dependant; eggs incubated at 26 degrees (celsius) produce all male hatchlings, while eggs incubated at 31 degrees (celsius) produce only females. The exact mechanism that regulates sex determination is unknown, but it is believed to depend on the expression of a series of sex determining genes.

I am focusing my work on the protein-protein interactions of DAX1 and SF1. Evidence suggests that these genes may work together to regulate the downstream expression of SOX9 and AMH, eventually leading to testes differentiation. DAX1, is a promising candidate for the "master switch," because it appears to regulate the expression of both the male and female sex determining genes. That's why I'm trying to figure out if DAX1 has different binding capabilities to SF1 at 26 and 31 degrees. My hypothesis is that DAX1 binds to SF1 at 31 degrees and inhibits the downstream expression of male sex determining genes. Conversely, at 26 degrees, DAX1 may be incapable of binding to SF1, resulting in the expression of the female sex determining genes. I will be performing experiments this summer in an effort to test this hypothesis.

*Getting Down to Business!*

I completed the experimental design for the greenhouse portion of my research project. I have also been using the scientific papers from my literature searches to locate companies that sell activated carbon and have been contacting them. Activated carbon will be used in two treatments of the experiment and it is often used to reduce the interference of allelopathic chemicals in the soil. It does this because it has a high affinity to organic compounds and a weak affinity to inorganic nutrients (Prati and Bossdorf 2004). The seeds for my experiment also came in and they will stratify for a month in cold, moist conditions at about 5 degrees. I’ve been watering them every other day with deionized water.

Cold Stratifying Seeds

Seeds of Eurbia divaricata That Are Stratifying

For the past two weeks I have been participating in a research project with Dr. Aronson that observes the response of Hempstead Plains’ prairie plants in urban environments. We have been conducting this project on a field plot on campus. Hofstra’s Public Relations crew has interviewed all of the participants and created a video on this exciting research. Last week, I continued to measure the initial growth of these plants as well as label and maintain each specific plot.

Dr. Aronson and I have also started another research project with Dr. Sanford looking at the dispersal rates of the bulbils that are produced by the invasive species that I am working with, Ranunculus ficaria. I am excited about this project because it takes my research a step further and I will be able to make a direct correlation to its dispersal rates and its invasiveness. This plant is a major ecological problem in the Northeast as well as the Pacific Northwest, therefore, I am eager to gain some understanding to how it is dispersed and how far the bulbils can travel so that native plant conservation can be managed as well as provide some insight on other invasive species.

I have done literature searches on seed dispersal in water because this plant usually grows along streams. These papers have helped me a great deal in organizing an experimental design for this project. In order to start this project, we need to find the buoyancy of each bulbil. Therefore, I have weighed over 200 bulbils and will begin to find the volume of each. Each bulbil weighs approximately 0.1 grams. Dr. Aronson and I were not exactly sure how to find the volume of an object this small so we reached out to the chair of the Physics Department, Professor Hastings. He is also excited about the project and is prepared to assist us in any questions we might have. This process makes my research experience richer with all of the possibilities and ideas that we have collectively discussed for the rest of the summer and upcoming year with these projects. I am honored to be a part of such a profound cause and it gives me a sense of fulfillment to know that my research will significantly contribute to the control and management of this species.

Bulbils produced by Ranunculus ficaria

In the lab weighing bulbils

June 4

Last day of the week and it was the most successful mammal trapping we’ve had yet! Dry Bay was our first site and we found 3 mice had run into the Sherman traps last night. There were two girls and 1 boy and all were deer mice (Peromyscus maniculatus). Like all the other mice we have caught so far, they had no ticks that we could tell. These mice were so cute until they started to squirm the first two were biters and we discovered that they really like to bite the rubber part of a mechanical pencil. It was a great distraction for them and they stayed relatively still. The third mouse was more of a squirmer then a biter, plus her fur was wet so she was quite slippery. But I did learn how to give them an ear tag. It’s simple enough you just need a steady hand, a pair of pliers and the ear tag. I gave the last two mice their ear tags.
With high hopes for more mammals we headed over to Long Tin. All of the Sherman traps were empty and we were about to check the last pitfall and last medium mammal trap of the array when our luck hit again. In the pitfall trap we found a house mouse (Mus musculus). This girl took us a while time to key out because this species is usually found in buildings but sure enough her home is in the woods. Like the other mice she had no ticks. Then we realized we caught a big one. In the Tomahawk trap was a male opossum (Didelphis virginiana). He was just laying in the back of the cage very calm, he barely even showed his teeth when approached him. The protocol for handling medium size mammals requires us to use a sedative by injection. He barely even flinched when the needle pierced his skin. Within ten minutes he had dozed off. Since I’ve never handled anything larger than a mouse Russ and Kaety processed him but I paid careful attention because after the next trip I will be handling everything with Kaety because Russ will no longer be coming down. The opossum had 6 engorged Lone-star ticks on the back of his ears. He was having a rough week, besides being caught in a cage he had recently been in a fight. He had cuts on his head and back and the padding on his front paw had a hole that looked like it was infected. The poor boy was then released and by released I mean he was carried into the woods and laid on a comfy bed of leaves to wake up on. All in all a great way to end the week, until next time folks.
The first picture is of the opossum in the cage playing dead.
The second picture is of the opossum's front foot were he has a big cut.

June 3

Today was the first day that we did not have to run around everywhere trying to get things done. Unfortunately we did not find any mice and we only found an anolis (Anolis carolinensis), which was not in any of our traps. But we did find a cotton rat (Neotoma floridana) this morning. It managed to get into a Sherman trap, which it barely fits into. Russ and Kaety we able to maneuver her out of the trap by collapsing the cage while everything was in a bag. The hold to pin down a mouse or rat is typically to grab the tail, which is what we did. Then the outer layer of skin of the tail slid off the bone. Surprisingly she did not bleed nearly as much as you would expect. Once we had her in a good hold she calmed down and did not seem too distraught. Like all the other mammals we caught there were no ticks as far as we could tell. The anolis we caught also had no ticks but this species is not known for carrying ticks. It rained last night so the amphibians came out. We caught 15 frogs and toads, most were the southern leopard frog (Rana utricularia) and we also had pickerel frogs (Rana palustris), which are morphologically extremely similar. Our two biggest toads were the Southern toad (Bufo terrestris) and the Woodhouse’s toad (Bufo woodhousei woodhousei). Both of these toads were as big as my palm. I never thought I would counting warts on a toads to id what species it is but they were the cutest toads.
The first picture is of an Anolis.
The second picture is of the rat being checked for ticks.