03/21/21

This past week I started making up a new stock of the in vitro transcribed (IVT) standard. The synthetic sequence-matched plasmid that I used was already transformed into some E. coli so to start, all I had to do was to streak some plates and grow up some colonies in LB media. After I let the bacteria grow for about 16 hours, I isolated the RNA using a Qiagen miniprep kit. Unfortunately, after this step, my mentor and I realized that I didn't have enough RNA plasmid for the next steps so I repeated the growth once more and pooled the products. I then performed an overnight enzyme digest to linearize the circular plasmids. I then performed an IVT to turn the DNA into RNA to mimic the natural intercellular processes. Next week, I am planning on running our IVT products in a bioanalyzer which gives us an estimate of RNA fragment length and concentration. This will tell us whether our IVT digestion and IVT worked as expected and if all our regions are still intact.

I also got some feedback on the sections of the manuscript that I had been working on.

03/14/21

I spent a few hours this week going through previous experiments and data to double-check what we have done and if any of the older experiments have data that we can use for the paper. One key takeaway that this process reinforced for me is the importance of keeping a semi-detailed list or spreadsheet of experiments. As the previous post-doctoral fellow that I was working with left a few months ago, I am now finding that I am missing a lot of dates, notes, and protocols for various early experiments. Additionally, I am having some trouble with matching notes and protocols for experiments that I do have with their results as they have been sorted into different digital folders and physical binders at the lab. I have started making a spreadsheet starting with the first practice experiments that I ran back in the fall of 2019 and I hope to finish this review the next time I go to the lab.

I have also made a plan to make a new stock of the synthetic plasmid standard using some previously transformed E. coli stocks. This will take 3-4 days and I plan on starting the process this upcoming week as I have a few days off school. Finally, We have found a lab - The University of Washington - that has a clinically approved HIV-2 assay that targets a relevant region of the genome for our validation purposes. We are now planning on sending a few samples of our standard in the upcoming week to get a more official quantification of our standards so we don't need to rely on spectrophotometers for estimating concentrations.

3/7/21

Over the past two weeks, I have been adding to and revising my methods. As I have not been able to do much lab work due to my school schedule, my mentors have been giving me feedback and suggestions for the previous draft I had written a few months ago. Some of the helpful suggestions that they have given me are to avoid jargon and to be more conscious of how I organize the subsections of my paper. For example, instead of referring to very specific genomic regions by name, I can first explain their location and function using simpler language. I also learned that the methods can be organized by steps in the project instead of just each individual method used. For example, one subsection of my methods is now "Validations using synthetic genome RNA." This section includes a paragraph describing reverse transcription and another one describing droplet digital PCR. These paragraphs are referenced again in the following sub-sections that use the same protocols.

Another key takeaway that I have had is to use the figures and results to guide the whole paper. Even though I have already drafted my abstract and introduction, my mentor and I are still planning on revising these sections once I have all the data to better format my information in a way to supports my results.

2/28/21

During the past week, I have continued working on my manuscript - specifically the methods section. This part is relatively straightforward for me as I have already used most of my methods in my previous validation experiments. I have also been able to use some of my mentor's recent papers as guides as our projects share many of the same methods.

I also got a chance to workshop with a fellow MARC student. We went over each other's introduction/methods and gave suggestions/feedback. This was helpful as it reminded me to not use too much scientific jargon to make my paper more accessible to others.

2/21/21

This past week I conducted another validation experiment on the ROD standard. I ran 5 of our assays and got relatively consistent/expected results in terms of efficiencies. Poly A and Tat-rev were extremely low but this was expected as they target spliced regions. Long LTR, which I used to get a concentration estimate of the ROD standard with, performed around 100% which meant that our expected input and output were the same. Tar again outperformed all assays which suggests that Tar might have the highest efficiency out of our 6 assays.

After performing this experiment and looking at the data, my mentor and I felt a little more comfortable with planning out and working on my manuscript. There are a few sections and figures which we have isolated to work on for the next few weeks. I will also be taking one or two free days to look through all previous experiments to see what we have and what we can use for our paper.

2/14/21

Unfortunately, I was unable to work in the lab this past week due to some scheduling difficulties. In the meantime, I got to review some of the ethics work from the first year of MARC with the new juniors in the program. In our discussion, we talked about how various ethical lenses can be applied to every situation and how none are of these lenses are perfect. One question that my group talked about is whether or not scientists should have some guidelines or some sort of a procedure to determine the correct ethical approach for a given situation. While we all agreed that scientists should recieve education in ethics, we also felt that there is no perfect solution to this issue.

2/6/21

This week I moved forward with testing my other assays on the ROD HIV-2 standard. However, despite last week's positive results, the new data was not in line with what I was expecting. Long LTR, which was last week to quantify the ROD standard, detected around 5 times as many copies as my expected input while Readthrough only detected around 1/100th of expected copies. As such, my mentor and I are planning on checking the aliquot of the sample which I used as an input. We found no issues with the dilution scheme that I used so we are guessing that there might have been some variation in the original volumes which I used.

1/31/21

I conducted a preliminary experiment to quantify the concentration of our lab's ROD HIV-2 standard this past week. After referring back to some old experiments, my mentor and I determined that two of our HIV-2 assays - GAG and Long LTR - had around 100% efficiency on the ROD standard. I ran duplicates of a dilution series for both of these assays in hopes that this would yield consistent results. The procedure for my experiments was similar to most of my previous experiments with a reverse transcription step followed by the droplet digital PCR and reading the samples. My results were quite similar between both assays and the dilution series - undiluted, 1e^4*, 1e^3*, and 1e^2* copies (* based on nanodrop calculations) - showed the expected pattern. My mentor and I have decided that we can move ahead next week with testing all of our assays on the standard to get a rough idea of their efficiencies.

1/15/21

After reviewing my calculations for the previous experiment, I found that there was a slight error. With updated expected values, the assays performed with between 32%-60% efficiency with a bias towards the 3' end of the HIV-2 genome. However, I am still unsure about our newly made standard's exact concentration as I have been relying on a measurement from a nanodrop spectrophotometer instead of a clinical assay or molecular quantification. My mentor is exploring the possibility of using an official HIV-2 clinical assay to quantify one of the virion standards that we have. In the meantime, I will run a few experiments on an old stock of HIV-2 ROD (a full-length lab virus standard). Two of our assays (Gag and Long LTR) have yielded consistent and expected results in previous experiments. I plan first to get an exact quantification of the ROD standard using Gag and Long LTR. Then I can measure the other assays on this standard to get a better general approximation of their efficiencies. However, my mentor believes that we will need to run a clinical assay at some point to get a usable quantification for our paper.

12/23/20

The previously mentioned experiment gave us unexpected results that were much lower than what we had calculated. Thus, we decide to in vitro transcribe a new standard using some left-over HIV-2 plasmid. However, this RNA standard gave us surprisingly low concentrations again. Because of both of these results, we decided to change our dilution scheme as we believed that specific steps in our previous serial dilutions (5ul:495ul) could lead to dilution errors, which would lead to significant differences between expected and actual resulting concentrations. As such, we conducted another experiment comparing both the original IVT HIV-2 standard and the new one. However, this time we diluted in much more gradual steps (10ul:90ul). This experiment yielded much more consistent results than our calculations, and I will now be moving forward with the final few experiments. Steve and I also decided that it would be good to run our assays on a clinical assay to get a more exact quantification of our standard.

Lastly, on 12/23/20, Arion Bio's rapid antigen tests, which I helped validate, have received emergency approval in Europe for public use.

10/25/20

I was able to start lab work on my HIV-2 project this past week. The first thing that I did was check the synthetic standard that Nitasha and I had made back in March. Measuring our stock with a NanoDrop Spectrophotometer gave an output of around 62.5 ng/ul, converting to around 4.07E10 RNA molecules/ul. This result was well within a usable range, so I could start planning my last few experiments.

Steve and I decided that it would be best to conduct one practice reverse transcription droplet digital PCR (RT-ddPCR) to help me regain some muscle memory. Sushama Telwatte, another post-doctorate fellow in Steve's lab, kindly volunteered to help me with the planning and practice. On Friday, I performed serial dilutions of the standard and an RT for both our HIV-2 and negative control (isolated RNA from donor PBMCs) samples. I plan to use them for a simple 16-sample test next Thursday to get some practice as well as a more exact quantification of our standard. If the results look good, I will finish the sensitivity and background validation experiments for the methods paper.

Steve has also gotten an invitation to submit a paper to Elsevier's Methods Journal. The loose deadline for submission is around mod-December, so Steve and I decided that this would be an excellent opportunity to publish the HIV-2 assays if we can get the paper together in time.

10/16/20

Today is the last day of quarter one which means that I will not be meeting regularly with my MARC class until next year. Despite this, I am still going to continue my internship and my MARC project.

This week I finished the clinical data collection for Arion Bio's rapid antigen tests. The tests should be ready for an FDA EUA application after a brief write-up and analysis.

I have also set up a meeting with Steve and another postdoctorate fellow next week to start planning the rest of my project. I will most likely need to spend a day or two getting reoriented in the lab before I can tackle the final two experiments for my methods paper. I hope to start lab work on my project by November.

10/11/20

I was not able to go to Arion Bio this past week due to school work and swimming. However, Steve informed me that the VA is allowing HIV and other non-COVID-19 research to start again with research labs at 25% - 50% capacity. I will be meeting via Zoom with him sometime this week to work out the details of my last few projects. Hopefully, I will be able to complete all my remaining lab experiments during the one week break between MA's first and second academic quarters.

10/03/20

The tests I helped complete from the first day of my internship with Arion Bio confirmed that the cartridges are working as expected. Yesterday I started performing some of the required tests for the FDA EUA approval of Arion Bio's rapid antigen testing strips. The first step is to collect 30 negative and 30 positive correlating tests using previously collected patient nasopharyngeal specimens. What this means is that we have to show, for 30 negative and 30 positive each, that Arion Bio's colloid gold and fluorescent test strips perform at a comparable level to standard PCR assays.

I used samples from a local hospital that were tested using PCR. I loaded the samples straight from their transfer media tubes to the testing strips to maximize the accuracy. Arion Bio provides a dilution buffer which breaks apart the viral particles in a sample to expose the proteins that the testing stips detect. I did not use the dilution buffer for the tests because I reasoned that the viral capsids were already broken. I did this is because all the specimens I used had already undergone multiple freeze/thaw cycles which tends to fragment any viral capsids.

My results were similar to what I found on my first day. The fluorescent test required the most time to analyze but also provided the most accurate results for all viral load levels. On the other hand, the colloid gold strips were only able to detect strong infections. Additionally, I continued to notice that the color of the initial transfer media had a great effect on the readability of the test. Clear media worked best while pink/red colored media often caused positive markers (pink glow for the fluorescent and purple line for colloid gold) to blend into the absorptive testing filament.

09/28/20

I have started lab work for my internship with Arion Bio. Today I ran two pre-collected patient samples on antigen rapid-test cartridges. The company is testing two different types of rapid-tests, fluorescent and colloid gold. Both the cartridges have a loading well for liquid samples and a paper-like filament that absorbs the specimen and provides the test result. The fluorescent cartridges require a special light to read the test result while the colloid gold provides visible indicators. Both cartridges have one control line and one test line. The control marker should always be visible while the presence or the lack of a test line indicates a positive or negative result respectfully.

The two samples I tested were both from positive patients. I did a serial dilution (up to 1 in 100,000) for both specimens to measure the sensitivity of the rapid-test cartridges. So far, the cartridges all worked as intended and have demonstrated a relatively good level of sensitivity.

09/20/20

I have been in contact with Steve who has updated me that the SF VA is still not allowing HIV research. However, he did offer me the opportunity to join in on one of the ongoing COVID-19 projects in his lab. We are still working out the details but hopefully I will be able to get back into the lab within the next few weeks.

I am also planning on starting a new paid internship at a new local biotech company called Arion Bio which is developing point-of-care COVID-19 testing kits to serve large groups such as schools and sports teams. I will be helping with clinical data collection and analysis.

Lastly, I have decided to set a strict deadline for myself regarding my HIV-2 project. If I am unable to start lab work by the end of 2020, I plan on focusing my time on an HIV-2 review article as my MARC final project.

09/11/20

Along with helping me land a Business Development Internship at DiaCarta, my COVID-19 Breakthrough Junior Challenge Video has scored in the top 10% of all submissions internationally and has also been adopted by labtestsonline.com which is a digital resource for millions of researchers and physicians around the world.

I have also started looking at possible journals for a methods paper or possible review article that I might write during my senior year. I have identified one called eLife which my mentor has submitted similar assay development methods paper to before. Unfortunately, I am still waiting for an update on when I can get into the lab to finish my last few experiments.

9/4/20

I am currently still trying to figure out a plan with Steve regarding how and when I will be able to finish the last few in-lab experiments I need to complete before I can write my methods paper.

In the meantime, I am considering other options incase I am not able to get back into the lab or for the remainder of the senior year after my assays are finnished. I am currently considering COVID-19 testing and HIV-2 latency as possible topic for a review paper as I have had a lot of experience with both subjects over the past few years. I also think that both topics are relavent and important to furthering the understanding of retroviruses given that we are now in a coronavirus pandemic.

8/25/20

Today was the first day of my senior year. Although I was really looking forward to starting in-person classes again, it appears that MA will maintain online learning for at least another month and a half.

Today was also my first formal MARC II class and we are now starting to discuss research methods and skills as my classmates and I are all now working on our projects. In today's reading, we covered the importance of organization and planning in the research process. Many of the things were covered in the readings aligned very closely with the advice which my mentors have been giving me. Fortunately, I have already begun keeping detailed notes and documentation of my project in a lab notebook as well as in the shared drive at my mentor's lab. I plan on continuing this approach as it has worked for my mentor and me without any issues so far.

In the upcoming month, I plan on coordinating with the post-doctorate fellows at my lab so I can make sure that I can start my experiments as soon as HIV research is given the green light at my hospital. This comes as one of my mentors, Dr. Nitasha Kumar, is moving labs which means that I will need additional supervision in the lab. Fortunately, we have already planned out all the remaining experiments for validating our assays which means that I can get to work immediately. We have also already written a large portion of our methods paper which means we will only need to fill in the results and discussion.

8/20/20

After uploading my COVID-19 testing video to YouTube as a part of my submission to the Breakthrough Junior Challenge, it was noticed by a senior VP at a local BioTech company called DiaCarta. He reached out and offered a business development internship at his company. I happily accepted and have been working closely with his business development and marketing team to create presentations and educational materials for clients. I also spent a lot of time working directly with clients where I would present general information about DiaCarta and it's COVID-19 testing services; set up company and client accounts; and assist with issues and concerns. More recently, I have also helped with the design and development of a new e-commerce website dedicated to our COVID-19 testing kits.

5/26/20

After my first year in the MARC program, I have learned a lot. Everything from research planning, methods, and structure to networking and communication skills. Although the COVID-19 pandemic and subsequent shelter in place orders impacted my experience, I still was able to accomplish many new things. I finished the school year with a poster presentation (via Zoom) and taught a lesson about RNA viruses at my school. Looking forward, I have finished recording the segments for my Junior Breakthrough Challenge and I am getting ready to head back to the lab. I plan on spending a large portion of this summer working on my MARC project and video and hope to make up for some of the lost time before school starts again in August.

5/18/20

While preparing my video for the Junior Breakthrough Challenge, I have been learning a lot about the novel coronavirus (SARS-CoV-2) and the science of pandemics. I have been working with one of my MARC mentors (Nitasha Kumar, Ph.D. UCSF) who is giving a zoom presentation series on the new virus. I have learned about the definition of pandemics, different types of tests, and the science behind the virus. I am also learning how to use Adobe After Effects and Illustrator which I plan on using in the future as well.

5/4/20

As everyone has started adjusting to the stay at home orders, I have started finding ways to contact and meet with my MARC mentors online. I have been communicating via zoom and have finished a rough draft of my formal introduction for future research papers and presentations. I have also begun writing the materials and methods for the assay validation process. This step is what I will be working on once I can get back into the lab.

For now, I am working on planning experiments and my Junior Breakthrough challenge video. I have considered a couple of different ideas but ultimately settled on the science of pandemics and the role which testing plays in helping countries get back on track. I have finished a draft script which I am now editing to fit the three-minute time cap.

4/21/20

I am currently working from home on my project. I have decided to create a three-minute video for the 2020 Junior Breakthrough Challenge on the science behind the novel coronavirus. I plan on working with my mentor as well as the other post-doctorate fellows I work with. Along with the video, I am working on a formal introduction as well as planning for my assay validation experiments which I plan to carry out as soon as the SFVA opens research labs again. I am hoping to get back to lab work by mid-May but everything is still uncertain.

3/20/20

Marin Academy and the Yukl Lab at the SFVA have been closed due to the recent outbreak of the Covid-19 Coronavirus in the Bay Area. I am not able to do any lab work during this time so I have decided to start writing a formal introduction for my Science Symposium Poster and any future papers. The HIV-2 plasmid has also arrived at the lab and is ready for assay validation experiments. Because of this, I will also be planning my background, sensitivity, and false-positives experiments during my time off so I can begin lab work as soon as the shelter-in-place order is over.

3/1/20

I recently presented an introductory presentation to both my MA classmates as well as the researchers at the SFVA Yukl Lab. I first presented to my mentor and the post-doctorates fellows at the VA. They gave me a lot of feedback which I incorporated into my presentation to my classmates and teachers at school. Some of the key points they touched on were understanding my audience (i.e. not using jargon) and being more specific on important points. Their advice allowed me to create an understandable and clear presentation that succeeded in explaining the background and goals of my project to an audience of a different field.