Tag: Chemistry

Integrated Pain Solutions: Day 2 + Making Balms!

Another day at Integrated Pain Solutions in Southern Pines. We started the day off by reviewing our notes from yesterday. We all enjoyed getting to draw the structures (as shown by JR’s thumbs up). A highlight of the day was watching the formulation process for making balms and other serums. From precisely measuring out each ingredient to pipetting each extract, the balms were perfectly made so they will be effective in mitigating pain for patients. After, we discussed why each ingredient is added and how it contributes to the overall effect of the balm. We enjoyed learning not just about the balm process, but about future career paths and collegiate studies from Ryan. In addition, we spent lots of time researching different types of pain killers and drugs to understand how the urine analysis works on site. The structures all had very unique shapes and lots of benzene groups!

Integrated Pain Solutions: Orientation and Preliminary Knowledge

 

After a very fun, early morning, caffeine-hyped, carpool drive to Southern Pines (Thanks to JR for not hitting the giant snapping turtle on the road), we had an orientation at Integrated Pain Solutions. We thoroughly enjoyed learning, visualizing, and experiencing how their cannabinoid products are made from the plant to the serum to the bottles to the store within the warehouse.  We loved seeing the machines used to bottle and formulate all the products. A highlight of the day was learning about the chemical structures of cannabinoids and how they work in the body to attach to pain receptors; as well, we got to apply some of Mr. Rushin’s Adv Chem curriculum to real-life by analyzing the cannabinoid structures and how the disconnected Oxygen off the Benzene on the THC makes it different than CBD. Shout out to Alekh for helping JR navigate the Chick-fil-a line.

ILS Day 3

After only shadowing for the last couple of days, Om and I were itching for a hands-on experience. Today was that day. In the morning, we worked with Adam in his lab, where he processes patient samples from Dr. Taylor’s clinic. Adam guided us through prepping patient samples, and we carefully followed his instructions as we pipetted urine samples, control solutions, enzymes, and buffers in various order. We were nervous, and our shaky hands caused the occasional cross-contamination. Fortunately, we weren’t testing on real patient samples, which reassured us a little. Before injecting the enzyme and buffer mixture, we heated the samples in an incubator for 35 minutes. After adding all the constituent parts together, we spun the wells in a centrifuge at a mind-blowing 4000 rounds per minute for 15 minutes. Finally, we booted up the mass spectrometer and let that instrument have a go at analyzing the sample wells. We haven’t figured out how to navigate the complex software utilized to control the mass spectrometer, so we left that part up to the experts. We’ll find out the results of the mock drug screenings tomorrow, and I couldn’t be more excited.

We took a quick lunch break, and the Chick-Fil-A cookies and cream milkshake was a great way to cool down from the lab environment.

In the afternoon, another lab technician (who prefers to remain anonymous) led us into a different lab, which processes samples from clients, such as providers, hospitals, and clinics not under the wing of Dr. Taylor. Again, we performed the mass spectrometer drug screening test, but in contrast, we utilized proficiency testing samples this time. These are samples of urine donated from hundreds, if not thousands, of individuals and combined together to be averaged into a “normal” urine. Then, the state testing agency spikes the urine with certain drugs and drug metabolism products, such as oxycodone. The reference lab is only allowed to continue operations if it can correctly detect these drug presences within test samples. The process was much the same as it was also a mass spectrometer test, but we got to be mix (dangerous) chemicals such as methanol, which was exhilarating.

I can’t believe that tomorrow is going to be my last day at Integrated Laboratory Solutions. I certainly have had a great time so far, from conversing with the diverse community of employees even within a lab workplace, to the great lunches. Without a doubt, my last day tomorrow will be just as interesting.

Day 1: Toly Who?!

Today was a very exciting day in the lab! I started my day at around 9:30 with Jessi, the graduate student for Dr. Lindsey at NC State. We first began by learning how to use various micropipeters and proper lab procedures when dealing with bacteria. Besides wearing safety glasses and a lab coat, one of the most important safety procedures when dealing with bacteria is making sure each flask that will be reused is exposed to a flame- this is to ensure no external contamination. After learning these procedures, Jessi informed me about the project she and Dr. Lindsey have been working on: extracting Tolyporphin from cynobacteria. This project is of crucial importance because currently scientists are unaware of the various functions inside the DNA of Tolyporphin and its origins inside the cyanobacteria. To truly understand this concept, Jessi taught me how to read a cell map and design a genetic sequence that can be inserted into a bacteria. Moreover, one of the preliminary experiments today was preparing solutions of old Tolyporphin bacteria cultures and placing them into new base antibiotic solutions. This experiment was an application of the micropipeting skills Jessi taught me earlier, with a focus on precision and minimizing error. For Jessi, this procedure is important so that the bacteria in the flasks are able to continue for longer periods of time and not die out.

At around 12:00, Jessi, her friend, a post-doc at duke, and I went to go eat lunch at a nearby restaurant- the food was amazing! I remember Jessi recounting to me how most days graduate students in her lab would grab lunch here and stop at the nearby coffee shop next door.



I honestly can not thank Jessi and Dr. Lindsey enough for this amazing experience. It is truly fascinating to see all the hustle and bustle of scientists throughout the university, in the labs, and across the offices - ground breaking research really is at every corner.

Day 5: The Case of the Festering Fun Fungi Felon

One word: contamination. It’s like muttering the dark lord’s name. It’s like cursing in front of your mother. You say it and the whole world crumbles around you. The E. Coli flasks that Dr. Masoudi, Dr. Li Yin, and I had put together did not show growth over the weekend, meaning that some sort of alien substance had ousted our benign bacterium. Initially, Dr. Masoudi thought that the unwelcome stranger was Phage, a horribly persistent virus that would require a thorough bleaching of our workbench, but something had caught his eye before we went to bleach Dr. Li Yin’s station. At the bottom of our LB medium, a solution that we evenly poured in each of the E. Coli flasks, there grew an innocuous speck of fungi that competed for nutrients against our bacteria. It was this seemingly minor contaminating agent that forced us to pour 12 liters of prepared bacterial solution down the drain.

After washing our flasks and going over protocol again, we had to prepare new bacterial colonies that will house our precious Nb6B9 protein. We made two separate solutions: the first solution contains our LB medium and agar, a solidifying agent, while the second solution only has LB. The first solution was poured into about twenty petri dishes that serve as the houses for our bacterial colonies; we followed this by adding in the antibiotic kanamycin. Colonies will form overnight once we introduce E. Coli cells that have a particular plasmid, a set of DNA that is both resistant to our antibiotic and has the target gene for Nb6B9 production. We ensure that the plasmid enters the cell envelopes of the E. Coli with a half-hour cooling process followed by a prompt “heat-shock” that loosens up the cell membrane, increasing it’s permeability. Antibiotic exists in the petri dishes to weed out the bacteria that won’t produce Nb6B9 at our desired capacity, leaving the successful colonies that we will grow and eventually harvest. It’s a long process, but it’ll be rewarding once we extract our precious protein.

In the meantime, we checked up on the results from our Western. The membrane that has our protein imprint was taken to a special scanning device that emits different wavelengths of light to expose the presence of different bio-molecules. For example, DNA is detected using short-wave ultraviolet radiation. We had proteins though, and we forced the molecules to react with a substrate that leaves a bio-luminescent product. We turned the scanner from an ultraviolet machine to something like a photographer’s “dark room” so that we could see the faint bio-luminescence. What was found was a single strip of protein between 7 and 12 kDa that matches the identity of our Nb6B9 protein, indicating that Nb6B9 didn’t form any dimers (combinations of itself) and can be filtered out when using carboxypeptidase (the enzyme that we used to differentiate Nb6B9 from its constituent peptide chains).

Day 1: Introduced to the Lab

Getting to the “CARL” building where my supervising post-doc works was a pain this morning. I had actually come to the Duke Medical Campus in Downtown Durham earlier in the year, so I was acquainted with the tortuous paths, high-rising brick buildings, and construction sites. Needless to say, I still got lost. Knowing that I’d get lost, I came to the lab one hour early, wandered around for thirty minutes, and (surprisingly) ran into my supervisor, Dr. Masoudi. Promptly, he directed to me to the lab’s manager with whom I signed some confidentiality paperwork. The lab is tucked at the top level of the building, where a plethora of groups are working on biochem projects. My specific lab, the Lefkowitz lab, deals with G-Protein Coupled Receptors that act as intercellular communication devices in eukaryotes (animal cells). These critical receptors come in thousands of different forms but they all work by wrapping around the cell membrane seven times. A “binding site” occupies the end of the receptor outside of the cell while a G-protein – a protein composed of three primary parts that can be ejected to communicate intracellular messages – is connected to the receptor on the inside of the cell. Whenever a unique body binds to the receptor outside of the cell, a “conformational” change occurs where the positioning of the macromolecule slightly alters and releases the G-protein. This complex process is the same process that cells undergo for nearly 40% of our prescribed medicine. The G-protein coupled receptor plays a colossal role in human health, and I can’t wait to get started in the lab!

Today’s labwork began with the expression of beta-2 adrenergic receptors in insect cells. The cells were placed in a solution that expedites receptor formation. The flasks holding these cells were put in a massive centrifuge – a device that spins vessels at high speeds to separate insoluble particles. We poured out the solution, extracted the cells while stabilizing their pH with a buffer solution, and labelled each of our solution-filled flasks. Dr. Masoudi emphasized that everything in the lab must be labeled. We used a smaller centrifuge (going at 4000 rpm!) to separate the insect cells from the buffer solution. Using ethanol and dry ice (the lab ran out of liquid nitrogen), we flash-froze our vessels and later placed our rack of flasks in a massive freezer. The cells in these flasks will eventually be used when the receptors are needed for X-ray crystallography. After our lunch break, Dr. Masoudi and I went over to a lab-wide meeting where different project leaders explicate the results from the past week. Besides X-ray crystallography, other researchers only a couple doors down use cryogenic electron microscopy to better understand the structures of these receptors.

Everyone in the lab is incredibly kind. One researcher called “Bullet” gave up his set of pipets so I could use them later on in the internship. Dr. Masoudi works with another researcher, Li Yin, who was kind enough to give up her work bench and desk so I could use it for labwork. Overall, the lab’s atmosphere is positive, but what I found to be most surprising is that this space is highly diverse; a large majority of the researchers here are first-generation immigrants. Dr. Masoudi just so happens to be a first-generation Iranian immigrant like my mom! It’s been such a phenomenal experience so far, and I’m so grateful for the opportunity to witness some crucial work in the field of biochemistry. Can’t wait for tomorrow!

 

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