A day in the life

When I first walk into lab, I check my email to see if there are any updates from my mentor on my project. Then I prepare PCR to test the primers for Shank1 and Dlg4, as I am finding that I have to adjust the master mix to minimize dimers that appear during this PCR, Once I stick that day’s PCR in the thermocycler, I then run a gel for PCR that I did overnight. After doing that, I consolt with my mentor about what the gel means, and what further alterations to the mastermix I should make. After this, I take my lunch break where I read protocols and the literature. After lunch, I spend a few more hours experimenting with optimal conditions for Shank1 and Dlg4 primers in preparation for pyrosequencing.

Once I am done optimizing these primers, it will be time to pyrosequence to test for methylation, and then use RT-PCR to test for gene expression.

Life in the Lab

I usually get to the lab quite early, in order to get a head start on my experiments before going to the usual BSURF meeting. Many of the experiments we run, such as Western Blots and RT-PCRs, require a lot of preparation and busy work (including a ridiculous amount of pipetting). A procedure can take up most of a day, if not multiple days, and you cannot simply stop and start at whatever point you like. There are set points in the procedure that you have to reach before calling it a day, and getting started early is therefore crucial, if you want to avoid having to stay late. For example, the membrane in a Western Blot has to be washed overnight in primary antibody, so that it can be developed the following day. Failing to set up a primary antibody wash before leaving would mean waiting a whole additional day for your results. For other procedures, the intermediates we work with are easily degraded, and must be converted to something else before being stored. RNA is particularly fragile, and after being isolated from cells must quickly be converted to cDNA. Therefore, you must perform the necessary reverse transcription reaction before finishing up for the day.

Another regular task is cell maintenance. After several days of growth, cells become overly “confluent” – crowded on their plate – and their growth media becomes depleted. In order to keep our cancer cell lines growing and healthy, we have to “passage” them every few days. This involves taking a fraction of the cells that were growing in culture and transferring them to a fresh plate with new growth media. A substantial amount of work goes into simply ensuring that you have cells to perform experiments on, let alone performing the experiments themselves!

This past week has been particularly busy, as I have been doing a time course of GNF5 (ABL kinase inhibitor) treatment – essentially, I treat cells with the drug at a number of different time points over a period of 72 hours. This has involved coming to lab several times a day, often at odd hours, ranging from 7AM to 11PM. The LSRC is a somewhat eerie building that late at night!

An entire experiment, from plating cells out for a drug treatment to producing presentable data by developing your Western Blot or analyzing your RT-PCR, takes at least several days. It is therefore important to enjoy the procedures themselves, and not only be in it for the results  – otherwise you are likely to find yourself bored and frustrated most of the time.

A Day in the Lab

Every day I spend in the lab is different. So it’s hard to choose a prototype of my day. The protocol for my lab work usually runs on a weekly basis and each day is an incremental step of completing a western blot in combination with optimizing previous western blot procedures and troubleshooting problems with antibodies and film resolution. I start my morning by doing prep-work, usually getting ice, labeling tubes, or turning on centrifuges, and thinking through what I have to get done for that day. On our lighter days of the western protocol, we conduct additional experiments that analyze K48 ubiquitination from different approaches. On these days, a lot of coordination is required to keep the timing correct on both experiments.

When people have a break in their work, around 12 PM, everyone in the lab goes for lunch together. Having lunch together is very important in my lab. They want to facilitate a culture that encourages us to just hang out outside of the lab in a more casual setting. It’s one of my favorite parts of the day. Besides the fact that by noon I’m starving and in need of my third coffee, I like to get a chance to talk to the members of my lab about their weekend and random topics without the immediate distraction of experiments in the background. The dynamics of the lab are great. Since it’s a relatively new lab, there are only 5 people in it: my PI, the lab manager, a postdoc, another summer undergraduate research assistant, and me. This has allowed us to get to know each other better due to such close contact. Some days we’ll take a break and go out for coffee and watch the world cup game that’s on. In the Silva lab, it’s not just about developing the ability to work with one another, but also developing relationships between lab members.

After lunch, we pick up where we left off on our experiments. I’m usually kept really busy while in the lab, running around mixing solutions and processing samples. More often than not, Latin music is being played in the lab while we work and sometimes big world cup games are streamed over the speaker. It’s a really good environment to work in and I’m really happy to be there.

A Day in the Lab

In my lab, not every day is the same. Depending on what is going on with our project, some days may be busy days, and some days may be “waiting” days, when we are waiting for our experiments to run. As I stated in my chalk talk, the main thing my mentor and I are working on is isolating exosomes and looking at exosomal biomarkers.

When I walk into lab, I ask my mentor what the plan is for the day and we discuss any bench work that needs to be done. These past few weeks, my mentor has been running trial experiments to isolate the exosomes (exosomes are kind of hard to isolate) so we can send them off to another lab for further analysis on the proteomics and RNA sequencing. As a result, on any given day, my mentor could be running an ultracentrifugation, a  bicinchoninic acid assay, doing a western blot, or passaging the cell lines for the project, and I just try to assist wherever I can.  On days where we are waiting for one of these experiments to conclude (i.e. ultracentrifugation  or western blots), we usually try to find someone else in the lab who is working on something that I want to learn about. For example, last Friday I ran my first PCR for one of my lab member’s project, so that was pretty exciting! Overall, I will say that I have learned a lot and I really admire every one of my lab members and the projects they are working on. I definitely wish I could contribute more to the lab, but I guess with time comes experience!

Episode 5- A Day in the Life (in the Lab)

For me in the Wray lab, everyday is pretty close to the same (so long as there are sea urchins in stock). As soon as I get to the lab, I check on the sea urchins I injected the day before if I did injections. Then, I check the dry erase board to see if there’s anyone planning to spawn an urchin before 1 since any time after that is too late. If there isn’t anything written on the board, I go ahead and make the injection mixtures I’m going to be doing that day and set them on ice. Then, I grab the urchin tray and bring it to the urchin room.

Once I’ve spawned some eggs, I have to wash them several times to remove the jelly so the eggs don’t stick together and dirty the water. While waiting for the eggs to settle during the wash, I set up what I need to row the eggs on some plates. After the wash,  put the eggs in a glass dish, swirl them into the center, and place them under a microscope to begin collecting them.

Using a mouth pipette, I suck up some eggs and then blow them out gently onto a plate filled with sea water and PABA (Para-Aminobenzoic Acid) which keeps the eggs soft enough to inject. If done right, the eggs stick to the bottom and don’t roll around much from where they were placed. Then, I add some sperm to each plate and swirl them around to make sure the eggs get fertilized. Now, they’re ready for injection.

To set up for the injection, I take a needle and place it into a nozzle and place a plate on the microscope stage. After a minute or two of adjusting the needle and stage positions, I can see the needle and inject the fertilized eggs. Once injection is finished, all the plates are placed in a plastic case and into an incubator to develop.

Four to five hours later, I can take the embryos out of the incubator and begin transferring them to a 12 well plate. I use the mouth pipette again to select the glowing embryos since those were the ones injected and I move them to different plates depending on the treatment and how brightly they are glowing. Afterwards, all the plates go back into the incubator to develop more overnight. The embryos have to be transferred because spending too much time in the PABA makes them sick and develop poorly.

The following morning, I check the results and see how many of the embryos/larvae have developed and if there appear to be any significant differences in the way the injected versus control animals have developed. After that, I wash and throw out the plates and begin another batch of injections.

Week 5 – Fecal Samples and Iodine

As I mentioned in my past post about my project, I am determining the thyroid concentration in fecal samples of a wild population of male baboons using T3 radioimmunoassay (RIA).

First, here’s a bit more information about how RIAs work: In a T3 RIA, an unknown amount of T3 thyroid hormone competes with a known amount of radioactively labeled T3 for binding sites on an antibody-coated tube. The antibody has equal affinity to the radioactive T3 tracer solution (in which the T3 is labeled with 125I) and the T3 present in the fecal samples. Once the T3 in the fecal sample competes with the T3 tracer solution for the binding sites, the antibody-coated tubes can be placed in a gamma counter that will determine radioactivity of the sample, which is measured in counts per minute (cpm). Seven standards (A-F and B ½) that contain different, known concentrations of T3 are analyzed each time a RIA is performed so that a standard curve can be generated. Therefore, the radioactivity of the fecal samples can be compared to the standards, and the concentration of T3 can be extrapolated from the curve.

After five weeks of working on my research project, I have performed 22 RIAs on about 1,500 fecal samples. Since the results of my project heavily rely on how many samples my mentor and I analyze, so far my days in the lab have followed a fairly predictable routine.

When I arrive in the morning I first collect the pre-selected 68 samples sitting in the freezer and prepare them for analysis the following day. To prepare them, I warm the samples to about 37°C in a water bath, vortex them for 10 seconds (more often than not there are solid components at the bottom of the test tube and we need those to be evenly distributed throughout the solution), transfer 2.5 mL of each sample into test tubes, and then dry them in the evap-o-rack. All the steps leading up to the evap-o-rack can take about 1.5 to 2 hours to complete. While the amount of time it takes for the samples to dry can vary, once the samples start drying then the morning phase of my day is over and onward I progress to the next stage of my day: performing the RIAs. (An important note: once the samples are completely drying sometime during the afternoon, I store them in the freezer to be analyzed the following day.)

In the second, longer part of my day, I retrieve the dry samples prepared the prior day and add 250 μl of Standard A (which contains no T3) to every test tube. I then vortex all the samples for 30 seconds, transfer them into Eppendorf tubes, and obtain the antibody-coated tubes. 100 μl of each standard, three controls, one pool, and the samples are added to two tubes as outlined below, ultimately resulting in 160 tubes that will be put in the gamma counter. But before I reach that step, I add 1 ml of the T3 tracer to all the tubes, vortex them for 10 seconds, incubate the samples for 1 hour at 37°C, aspirate the tubes, add 3 ml of deionized water to them all, then aspirate again. After the tubes are aspirated for the second time, I place them into the gamma counter and voilà: my day in the lab is over.

Before I leave, I like to label the test tubes and Eppendorf tubes to prepare for the next time I’m in the lab, which helps me save some time during the process. In the first couple of weeks, I often would end up leaving lab after 7:00. By week 4, I got use to the procedure and became more efficient, which helped me settle in a regular schedule of work, have a snack, work, have lunch during the hour wait, then finish up for the day and leave around 6:00. The only exception to this flow is on Mondays when I join my lab for the weekly lunch meetings. I may end up leaving the lab after 7:00 on Mondays, but it’s well worth it to spend the time with members of the Alberts lab and my mentor’s two dogs: Cyclone and Twister.

Life in the lab

For me, there are things that I do on a daily basis, but there are also things that I do that depend on how a previous day’s work turned out.

Most days, I arrive at the lab I check in with my mentor Brittany and plan out the day’s work.  Then we usually check on our cells to see if we need to split them, how to split them (1:10, 1:20, etcetera), or if the cells just need a media change.

Once we make a plan I get to work. On a given day, I might be making gels, and running gels, to get ready to develop a western blot the next day.  I might also be collecting cells, and lysing the cells to either do an Immunoprecipitation (IP) or a normal western blot.  Additionally, I will sometimes treat the cells, or transfect the cells in order to delete a certain gene or express a construct so that I can see what is happening to expression levels when I treat my cells.  Finally, I may also step away from the cells and attempt to do a mutagenesis reaction.  In order to do this, I will first set up the mutagenesis reaction (it is similar to a PCR reaction but with a different primer), and run the mutagenesis reaction.  The next day (day 2), I will do a DPN1 to get rid of the original template (so that you have your mutated DNA).  Then I will transform the new DNA into bacteria, and plate the bacteria.  The next day(day 3), I will collect various colonies and grow them out.  Then on day 4, I will isolate the DNA from the bacteria and send it off for sequencing.  On day 5, I will receive the sequence and go from there.

Some days, the science does not go as you hoped it will and some experiments have to be done again, some might have to be changed, or you might have to stop the experiment altogether.  All in all, the days in the lab in my experience is slightly different each day and builds upon the previous day.  Each day, I learn something new and it has been a great experience.  I am learning to be patient and realize it is okay if things don’t go as well as you hoped… more on this on next weeks blog where I will talk about the highs and lows of my project.

 

 

What do I do?

Through the past couple of weeks my routine in lab has kind of been all over the place. Depending on the goal for the day, I alternate between the third, fourth, and ultimately the fifth floor (where my lab is located) of the French Family Science Center.

So why does my daily routine potentially have me going all over the place?

I always start my day in the fifth floor, leaving my backpack and checking what I have to do for the day. Sometimes I will have to check in with my grad students to see if they have something they want me to do for them. but usually, I will have some kind of plan before-hand for what I could be doing. If it’s a protein purification, I get started immediately on thawing the pellets in ice (from the fourth floor) and preparing my buffers. Protein purifications generally take 3 days so, when I need to start them, I will come into lab at 8 am in order for the sample to lyse, clean, and spin down to be injected into the chromatography system at a reasonable time. (Because spinning down the sample takes such a long time, I usually will load the sample on the second day). If it’s a protein expression, I’ll come into lab at around 10am, start making the medium, and then deliver it to the fourth floor to be autoclaved. After being autoclaved, I inject the sample and wait for incubation, finally harvesting them once incubation is done.

While I wait for incubating, centrifuging, or autoclaving (I’m usually waiting for one of these things) I’ll be at my desk area reading up on papers that I’ve been given or surfing the internet. Sometimes I’ll go on a walk. (Recently, though, I’ve been working on some image analysis for one of my grad students) Once my waiting is done, I will usually be at the bench finishing up preparation. Sometimes, I will need to set up a chromatography system or gel running in the background.

So far, though, I’ve only covered the fourth and fifth floors. Where does the third floor come in? Well, the Nano ITC I’m using is in the Hargrove Lab on the third floor so throughout my day, I go back and forth cleaning the ITC and then loading samples to run and doing my work in lab on the fifth floor. I’ll usually run the ITC while I am doing other experiments and check in on it after an hour and a half.

My daily routine varies depending on what I’m doing. I rotate between floors, wait a lot, and run different kinds of experiments. Overall, I love what I’m doing and working in lab.

My favorite Chalk Talk from Last Week

I really enjoyed all of the chalk talks from last week. To be frank I felt a little proud of everyone because from what I could understand everyone had fairly complicated subjects and projects. To see that everyone could effectively explain what their projects were about and what they were doing was impressive despite out lack of experience. In addition, I thought everyone had demonstrated effective public speaking even though I noticed some were nervous (I was nervous, too). However, I really enjoyed Christine’s chalk talk because it was so different from everyone else’s.

Most if not all of us are working at the microbe or molecular level but Christine’s project has to do with quanifiying the amount of stress disctributed amongst different ranks amongst baboons. When I was first listenin to the premise of the study, I was first thinking that it was obvious that the alpha and lower-ranking baboons would have the most stress but then I realized that this was assumption based on bias and observations. Christine’s work is actually quantifies the stress by measuring the amount of stress hormones excreted by the animals. I also liked that the types of stress expressed by the varying ranking baboons were identified. Alpha baboons were marked with energetic stress, having to defend or fight for their position in the group. Low-ranking baboons were marked with psychological stress, meaning their stress originated mainly from their inability to access resources and isolation due to their percieved weakness. I guess I enjoyed her chalk talk the most because it involved a combination of behavioral science and molecular biology. Moreover, (she stressed that this was a stretch)I found it interesting about the implications that this project has on Homo sapiens. I am primarlily interested in microbiology/infectious disease but I do have a keen interest in society and how socioeconomic status affects people in an abundance of ways because the two topics interconnect in several ways.

Three Days in the Lab!

So far through this summer, I have discovered that the stereotypic perception of the routines of a researcher is mostly false. Each day there is usually a new challenge, concept, or method to try, creating an experience very much different than the monotonous routine everybody is seemingly attaches to research. It’s true that a simple task of pipetting or waiting for a culture to grow may seem repetitive, but all these things ultimately build up to contributing towards a larger goal which makes even a daily routine exciting. As I move further into my project, each day is new as there is a new step to be taken in ultimately solving a structure for my protein of interest. The things done in the lab are often determined by the results received the day before, as I have had a wide range of different days in the lab. However, here is a slight look into a possible day in the lab.

If I was just starting a new experiment or starting from the very beginning to express a protein and eventually solve a structure, I usually start off with checking my bacteria cultures from the night before. After checking the cell density of these cultures, most times I dilute them and wait till they grow toward the preferred density for the experiments of the day. While that is happening, depending on the day, I will either prepare needed solutions or samples needed for the rest of the day. After the bacteria has grown to preferred density, I usually will conduct an inducing experiment and attempt to express the protein of interest in different conditions. Often times there are time conditions involved in these different conditions so some of the day does consist of waiting. After waiting (this could be actually be done the next morning, depending of the conditions), I run an electrophoresis gel to see the results of the conditions. Analyzing these results will give me indication for what my next few days will look like. If there is significant protein expression, the next few days will consist of creating large cultures of the same strain of bacteria with the protein of interest in order to attempt a purification process. The purification process is generally consists of lysing the cells and running a column to ultimately elute the cells. If not, then it is back to the drawing board, trying new strains or methods in order to express the protein needed. I would create new cultures and grow them overnight to once again begin this process all over again.

Often times it seems like my “day” is actually three days in total as these three days is what it takes to usually complete a cycle of experiments (growth, expression, gel/purification). With results, my following day would be quite different than the preparation days. In addition, because I am now helping with a project analyzing the protein MprA, my days now also consist of ITC and other purification methods. The exact details of a day are often driven by the results of my previous experiments, leaving every day up in the air and adding an element of excitement.

 

Till next time,

Luke Sang

A Day in the Lab

I normally start my day at the lab habituating mice to a head-fixation device connected to a treadmill. Mice that are considered habituated will groom and run (and sometimes vocalize a little bit spontaneously) instead of displaying freezing behavior. Before placing them back to their home cages, I usually give the mice some sweet treats for having put up with the treadmill training that day. The goal of this habituation is to get the head-fixed mice to vocalize. Since male mice vocalize most reliably during courtship, I run behavior sessions occasionally during or after treadmill to give the males opportunities to spend time with females.

Depending on the exact content of mice training, I might do histology during or after the habituation sessions. Sometimes, the afternoon is spent learning or performing surgeries (which I’m still very slow at…).

While everyone in the lab has a busy schedule and tries the best to be the most productive every day, one lesson I learned so far is that you can never force behaviors to happen naturally. If the mice refuse to vocalize…mind-blowingly, they just don’t vocalize. If the mice dislike the treadmill…well, I haven’t found the magic wand yet to make them relaxed right away. Working in the lab has been an incredible learning experience beyond just the technical aspect. I’m learning to be patient and flexible, to troubleshoot, and to look positively at the reality that things don’t always work! 🙂

A Day in the Lab with Claire

A normal day in the lab usually begins with checking on my 293T cell lines to see if they are confluent enough to passage to a new plate. If the cells are 80-90% confluent, then I will thaw my media and trypsin, prepare the cell culture hood and then passage 10% of my cells to a new plate so that they don’t become overconfluent and die. I usually need to split my cell lines every other day.

After I do this, sometimes I will have a mini prep or a midi prep of bacteria with my designed gRNA plasmids culturing in the incubator overnight, so I will take that out of the 37° shaker. I will begin the protocol by centrifuging the tubes with my transformed bacteria for 10 minutes, so during this time I check in with my mentor. We will go over the procedures I will need to do that day and why they are necessary for my project. This is usually a good time for me to ask questions about the various protocols for the day’s tasks and be sure I understand each step. After this check-in, I carry on with the mini prep, or whatever procedure I need to complete first for the day.

By the time I am done with the day’s first assignment, it is usually time for lunch. Some days, the lab will all go out to lunch or someone will order pizza for everyone. These are the best days because I get to hear about the cutting-edge research they do and also get to know them outside of the lab setting. After lunch, I will complete the rest of my tasks, whether that includes transfecting cells, changing media, harvesting cells, PCR, restriction digests, surveyors or designing new guides for my project.

Before I leave for the day, I will transform one of my plasmids into competent cells or start a mini prep from bacterial plates that I have already prepared. This is done at the end of the day so that the bacteria can grow overnight. Overall, the lab is a relaxed, collaborative environment where I don’t feel afraid to ask questions.

Stage 4: A Quick Intermission

Although a life of research often leads to an extremely narrow field of expertise, reading and learning about new research and knowledge from other, sometimes completely unrelated, fields can be both exciting and intriguing. One such example is Ayana’s project on Cryptococcus neoformans, specifically on searching for links between the BZP4 gene and their virulence in humans.

C. neoformans is a very common yeast, often found clinging to the dirt and other plants and animals. While widespread, infections from the fungus is rare, as most people’s immune systems are capable of defending against the pathogen. However, occasionally, they opportunistically infect the lungs, which can then spread to the central nervous system where the fungi can cause meningitis or encephalitis- two very dangerous conditions. Therefore, Ayana’s lab is researching the pathology and potential factors that could help the development of some form of prevention or cure. Currently, her project revolves around the curious BZP4 gene within C. neoformans, as the gene has been previously known to fluctuate in gene expression levels in different conditions and upon knockout, the virulence of the fungi disappears.  Thus, she has decided to investigate its relationship to the virulence of the fungi by directly interfering with it function.

In Ayana’s first aim, she hopes to confirm the link between virulence and BZP4 in C. neoformans, by taking a BZP4-knockout strain and blasting it with the BZP4 gene to observe for a recovery in lethality. This is cool primarily because it involves the use of a gene gun, which is just a really awesome machine that exists apparently, transforming cells with DNA by blasting them with a gene-coated bullet- and it works! At some point in my life, I need to devise a transformation experiment that requires that machine just to see it in action. Furthermore, her work would further pinpoint a site of target for treatments of C. neoformans infections, which is a monumental success in the world of disease prevention and care. Additionally, it could demonstrate a stronger link between virulence and the BZP4 gene, as the gene’s interactions and expression post-transformation could be identical to pre-knockout levels which would indicate greater independence between the gene’s mere presence and virulence over the possibility of gene expression interactions and post-translational activity with nearby genes being the source of virulence.

In Ayana’s second aim, the goal is to determine if there a competitive advantage given by the BZP4 gene. To study this, she will be inoculating a BZP4+ strain of C. neoformans and a BZP4- strain in close proximity, and measuring their growth and interactions upon reaching one another. This will perhaps give some insight in the purpose of the BZP4 gene, as not much is known beyond the excitatory effects it has on melanin production, which at most can be suspected to boost tolerance to environmental oxidants such as UV radiation. This is also important because the results can be used to determine if virulent C. neoformans growth can be stymied by the introduction of nonvirulent BZP4- strains into important sites of infection. However, something I wondered in this component of the project but did not remember to ask was the activity of BZP4 in various environments. As antioxidants are so versatile and diverse, many function differently in different conditions, so I wonder if there are any environments where BZP4 activity is optimized, boosting the vitality of C. neoformans in that environment. Similarly, which environments is activity dampened in? This could perhaps explain what BZP4 is specialized for (or what the melanin is meant to do) and what pathway exactly that it manipulates in the body to cause so much damage. Studies like these fascinate me and remind me of just how essential research is to the field of medicine, even in straight biology settings. While not at all related to my research in Alzheimer’s disease, this project gave me some energy and exciting plans to bring to my work.

Weekly Updates

“A piece of my gel fell on the floor once and I didn’t notice, so renovations re-varnished the lab. Now it’s forever imprinted on the floor.”-Stuart Sundseth detailing one of many Western fails

“I accidentally threw my gel into the sink once.”-Stuart Sundseth

“I’ve torn a gel before”-Stuart Sundseth an hour before tearing another gel

“Have fun at the beach this weekend!”-Christine O’Connell
“Wow, you’re going to the beach again?”-Dang Nguyen
“Yeah, I left some potatoes in the cupboard”-Joan Wilson

“I feel…so dizzy…I think…I might fall over”-Hui Fang as she aggressively hoses the floor with liquid nitrogen

“And now I’ll just seal up this bad boy and throw him in the cold room for the-” *throws dead spider in box at Dang* “-night and let him blot since I can’t find the antibody.”-Stuart Sundseth
*graphic screaming*-Dang Nguyen

Episode 4-The Phenotype approach

For this past week, I have been listening to my peers present chalk talks on theirs works, providing me with insight into other areas of biological research. Of these projects, Sweta Kafle’s  phenotypic analysis of transgenerational seed stability in Arabidopsis thaliana could act as an indicator for the stability of transgenerational affects in these plants. These transgeneraiontal affects would be induced by  placing Arabidopsis thaliana  F0’s in hot or cold conditions, then either keeping these conditions constant throuighout the F2 generation, or alternting this patern of hot and cold until the F2 generation, Or altering the hot and cold conditions then keeping those conditions constant ( Hot cold cold, cold hot hot) until the F2 generation..The reason that her project was so interesting to me is while we are analyzing a similar phenomenon, we are taking 2 very different approaches. While S’weta is testing for phenotypic changes and then going on to test for epigenetic ones, I am doing the exact opposite. This talk helped provide me with insight into how the next steps for my own project should be to analyze the phenotypic affects( behavioral, physiological) that an epigentic insult has on my F1 rats. But  coming back to S’weta’s project, I really liked the way that Sweta provided a comphrehensive look into both the flaws and strengths of her study, and it in turn provided a wonderful experience.  I am looking forward to the results of Sweta’s study, and any further insight that future related projects have.

 

Until Next Time

Sea Urchins, Baboons and Mice, Oh My!

Over the past week, myself and other members of the BSURF program had to give chalk talks; 8-minute talks about our project with nothing but a whiteboard and a marker. Even though I was nervous about giving my talk, I was looking forward to the talks. I had previously talked to some of the other students about their projects, but I wanted to know more about everyone’s project. Hearing about the diverse research opportunities gave me the opportunity to expand my knowledge on topics including Alzheimer’s and sea urchin embryos.

One talk that I found fascinating was entitled “Neural Circuitry for Vocalization in Mice”. The chalk talk was given by Alina Xiao, who is working in the Mooney Lab. Her project involves looking at a specific area in the brain to see if it is activated when male mice vocalize, whether it be for defense purposes or courtship. The project got my attention because humans vocalize all the time, yet I rarely think about how and why other animals vocalize. Seeing how mice use this trait compared to humans is interesting. I also enjoy learning about how how the activation of different pathways illicit different responses in the body. Besides being fascinated by the project, I was also impressed with the quality Alina’s talk. It was apparent that she had thoroughly rehearsed her talk and she enjoyed what she was doing. I cannot wait to see the results for her project at the end program.

Overall, everyone gave an amazing chalk talk. Even though we have four more weeks until we present our results (we hit the halfway point!!), I am excited to see how everyone’s projects turn out.

Diverse cellular responses to oxidative stress

This past week, everyone had the opportunity to present their project to the other BSURFers through a chalk talk.

I found Julia’s talk particularly interesting, as her work on Oxidative Stress relates to what I am working on in the Pendergast Lab. Reactive Oxygen Species (ROS) cause cellular damage. By oxidizing amino acid residues, ROS can alter protein structure, causing protein unfolding, aggregation and plaque formation. There are two general mechanism by which the cell responds to such damage: damaged proteins are either degraded or repaired. Julia’s project focuses on a process called ubiquitination. It is the cell’s method of flagging damaged proteins for degradation. K48 ubiquitins are secondary ubiquitin molecules that are attached to the primary ubiquitin’s position 48 lysine (K48) residue. K48 ubiquitination is not understood as well as other forms of ubiquitination. Julia’s project focuses on identifying which enzymes flag proteins with specifically K48 ubiquitins. She is also interested in whether K48 accumulation on proteins following oxidative stress is the result of reduced proteasome activity, or reduced deubiquinating activity.

My project looks at ROS from a somewhat different angle. I focus on system xCT, which is a membrane antiporter responsible for importing cystine into the cytoplasm. Cystine is an important starting molecule in an oxidative stress response pathway. Through a number of intermediates, cystine is converted to glutathione, which quenches harmful reactive oxygen species through conversion to glutathione disulfide. While my project looks at how cells quench ROS before they can damage cellular components, Julia’s project focuses on how cells deal with proteins that have been damaged. It was nice to discover a connection between my project and that of another BSURFer, and I would be interested to see whether anyone in Julia’s lab studies the glutathione synthesis pathway.

Similar Research from a Different Perspective!

 

I enjoyed being able to here about the various research projects going on by my fellow B-Surfers. It was interesting hearing the different problems/questions posed and seeing the ways and which these research questions were being answered. I found it intriguing how some of us were looking at similar topics or ideas but the ways in which we tackled the problems and the methods we used were completely different. With that being said I found Ayana’s project particularly interesting.

Although me and Ayana are both focusing on Cryptococcus neofromans and the ways by which we can treat this fungal pathogen, her project project looks at this opportunistic human fungal pathogen from a different perspective. While my project focuses on controlling sexual reproduction of Cryptococcus by regulating its essential gene expression Ayana’s project is focusing on a transcription factor of Cryptococcus that could play a role in its virulence. Tests have shown that when you delete the BZP4 transcription factor in a host, the fungal pathogen is no longer able to kill that host which is a very interesting finding. She is current running more experiments to confirm this finding but if this is true then its a major breakthrough in finding an efficient method of treating illness caused by Cryptococcus neoformans. 

 

A Different Approach…

My week was highlighted by great chalk talks from every member of the BSURF program. I definitely learned a lot about various topics and projects. Further, these chalk talks spark my interest in new subjects and has gotten me talking about things I normally would have never even thought about! This week was a great lesson in the communication aspect of science and research: having to prepare a chalk talk, listen to presentations from others, and explore other topics through challenging question has certainly helped me to see the utmost importance communication and discussion has in research. Something to be noted is a question asked during my own chalk talk as given me a new idea in which to approach my project, something that might actually help move along my project objective.

That being said, a talk that particularly stood out was the one presentation by Simeon. Simeon gave a chalk talk on the study of essential genes involvement in cryptococcus neoform sexual reproduction expression.

He began with a bit of background information, presenting the case that cryptococcus is a fungi that can cause brain damage to those suffering immunodeficiencies. Cryptococcus is specifically hard to target and create successful drugs to combat it as through its sexual reproduction process different and various types of the cryptococcus fungi are formed and some of these will ultimately carry drug-tolerance. Therefore, going into his project, Simeon noted that he is targeting two essential genes involved in sexual reproduction. Using different constructs, the goal is to test three different approaches and see which conditions will ultimately repress sexual reproduction the most. Having more information on what successfully alters the reproduction expression within these fungi could build toward antifungal treatments in the future.

I found this presentation to be particularly interesting due to the way in which they approached this drug-tolerance issue. Approaching this issue from the perspective of altering sexual reproduction creates an opportunity to then create drugs to target specific types of cryptococcus to combat this fungus. Other studies focus on functional proteins within the fungi or creating drugs that will be undeterred by such tolerance, leaving this approach quite interesting. As this fungi issue is a big clinical problem around the world, this different approach could ultimately begin to lead toward a solution to this problem.

I am very much looking forward to see the information or conclusions gathered by this project and others at the end of the program!

 

 

Luke Sang