Fourth Week; Aarav Dubey

My sophomore year, from courses like AP Biology and Chemistry, I had been excited by the cellular cities underlying our existence. Intrigued by the interconnectedness of this city’s parts, I began pondering questions about deviations from the norm. I yearned to learn more about the intricacies of our existence, but unfortunately had no means of doing so. As such, I saw the CIRM SPARK program as a place to finally use my toolbox of knowledge to problem solve in the real world.

Unfortunately, the program this year was online. However, I am fortunate to have my amazing mentor who found ways to give me experiences that would benefit me despite being online. The initial weeks of this program were in preparation for this week.After intensive learning in the first few weeks about the pathology of AKI and immune cells like macrophages, and also about various semi-quantitative and semi-qualitative techniques, I have now begun to apply my scientific knowledge confidently in designing the basic lab experiments with proper controls and interpretation of the data.

My journey is made even more rewarding with the thoughtful homework assignments that Dr. Aggarwal has assigned me. This week I applied the tools and techniques I have learned to design a mock-experiment. I was given two different macrophage specific mice lines, one Lyz;tdt/+ which marks all the F480+ macrophages and another Argyfp which marks specifically Arginase expressing (M2) macrophages and asked to design an experiment to interpret macrophage infiltration in the kidney after injury. I find this kind of experiential learning very beneficial, as there is no “correct” way of approaching the problems, and I can independently think of different ways to go about solving it.

            Week 4 has been amazing so far, and I am really grateful that, despite the program being online, it is still developing knowledge and problem-solving skills that can aid me tremendously in higher level research. Though I am not able to be in the lab, I am able to vicariously learn through Dr. Aggarwal. Each western blot and q-pcr, as Dr. Aggarwal would often remind me, are parts of the bigger puzzle we had hoped to solve. The purpose behind the methodology gave me a far deeper appreciation of the research and clever tools it utilized. While I have learned about newer scientific techniques, I am more proud of being able to appreciate the science behind these processes and understand how to utilize them properly in lab.

Fourth Week; Eamon Bashiri

As I reflect on all of the impactful moments from the first of half of my time with the Cedars-Sinai CIRM SPARK Program, it is hard to pick just one memorable moment. From learning about the impact of microRNA 10b (miR-10b) on stem cell homeostasis to learning about correct immunofluorescence procedures to visualize specific proteins, I have gained so much knowledge and experience the past four weeks. But for me, learning how to conduct a Western Blot and analyze the results on Image Studio has proved not only to be immensely valuable, but also extremely exciting. Thanks to the guidance of Dr. Adam Poe, I was able to visually gain an understanding of the numerous steps involved in preforming a Western Blot. The first involves running the proteins through a gel electrophoresis to separate them based on their size followed by transferring the proteins onto a membrane. After blocking nonspecific sites and washing the membrane with a wash buffer such as tris-buffered saline (TBS), primary/secondary antibodies are used to illuminate the specific protein of emphasis. These skills are essential, and I look forward to using them as I work with my mentor and other members of the Saghizadeh Ghiam Lab on our current project of exploring the effects of microRNA 146a (miR-146a) on the inflammation pathway following trauma to the eye. In this study, western blot analysis allows us to quantify the prevalence of inflammatory mediators, such as IRAK1 and TRAF6, in the inflammatory response. I cannot wait for the second half of my internships and continue learning and working alongside some of the brightest minds at Cedars-Sinai.

Third Week; Angela Lee

The day after the Observed Independence Day Holiday, we started off with a tour of the Vivarium at Cedars-Sinai. A vivarium is an animal research facility that is designed to accommodate controlled environments for the care and maintenance of experimental animals. We were shown many different animals, including rodents and pigs! Dr. John Young explained how the animals are cared for, precautions that are taken for the wellbeing of both the animals and researchers, and the use of each animal. It was fascinating learning how much care goes into ensuring psychological and physical wellbeing for all animals—environment and diet are carefully considered for each animal. Dr. Young also shared how researchers handle allergies they may have to the animals. Special precautions, including wearing personal protective equipment (PPE) and sanitization procedures, are made to ensure all researchers are safe.

The last two weeks, I learned about Western Blot, immunostaining, and wound healing. Western Blot and immunostaining are similar in the use of specific antibodies to detect a single target protein of interest. However, Western Blot is used to separate and identify proteins based on molecular weight while immunostaining is used to detect the distribution and localization of a protein within cells or tissues.

Diabetic corneal disease causes the abnormalities of epithelial wound healing. Last week, I learned about corneal epithelial wound healing. Scratch wounds are created in normal and diabetic limbal epithelial cells (LEC) using P200 pipette tip and the closure of wounds is monitored over 24 hours. I was given the task by Dr. Shah to calculate the open area of the scratch wound of diabetic and normal LEC as they heal over time.

Thanks to CIRM SPARK and Cedars-Sinai, I have the opportunity to gain access to multiple resources and work on my very own, first research project as a high school student with the help of an experienced mentor. I am very grateful to be an intern of the Ljubimov Research Lab under the supervision of Dr. Ruchi Shah who has been patiently guiding me through the program. Although only three weeks have passed since the start of the CIRM SPARK Internship program, my interest in the eye and diabetic eye diseases has skyrocketed. I learned so much over the past few weeks and I am excited to gain even more knowledge over the next weeks as well as apply my knowledge to my research project!  

Third Week; Anton Wagner

After studying tons of research papers and procedures, exchanging with Dr. Jiang in one-on-one meetings, and attending a series of presentations, demos, and virtual tours by Cedars-Sinai experts, two key success factors for cutting-edge research stand out to me at this point: 1) interlinked, multidisciplinary approaches, and 2) state-of-the-art resources.

1) While our lab aims to develop stem cell-directed therapies for back pain resulting from intervertebral disc (IVD) degeneration, we face a huge puzzle which requires tackling lots of different pieces and interdependencies, and then drilling down deep into one bit at a time. My own path to absorbing knowledge in this field led me from stem cell basics and reprogramming, back to the biology of our target, namely the physiology and morphology of healthy intervertebral disc (IVD) cells, that is nucleus pulposus (NP) and annulus fibrosus (AF) cells. Then, I moved to degeneration characteristics, and eventually to the complexity of causes. Beside natural aging (senescence) and environmental factors, the current understanding is that IVD degeneration is influenced by genetic factors, many of which are unknown. So, while Dr. Sheyn has established the utility of human iPSCs for reducing degenerated IVD tissue in vivo in a previous study using a porcine model, our lab currently focuses on the genetic disposition to develop IVD degeneration, with an emphasis on the role of pain. We are undertaking a study supported by next generation sequencing (NGS) of relevant human patient samples that investigates genetic expressions. Based on this research direction, my learning has transitioned into genomics, currently studying up on genes mostly associated with IVD degeneration, such as Collagen. It was very interesting when Dr. Jiang showed me steps of analysis of the performed single-cell RNA sequencing (scRNA-seq). The gene expression profiling of NP cells he shared illustrated molecular signatures in differentiated expression clusters segmented by the phenotypes we are looking into (healthy, degeneration with pain, and asymptomatic degeneration). The next step for me is to analyze and establish connections of a set of identified expressed genes. Overall, the Sheyn Lab’s work showed me that many disciplines, such as (stem and somatic) cell biology, molecular biology (specifically genomics), bioinformatics, and biophysics need to come together to advance the research.

2) The tours through Cedars-Sinai research facilities our intern group was invited to attend this week, specifically, the Vivarium and the Research Imaging Center, were impressive and showed me how important these resources are to be able to conduct leading research. I was surprised to see how large the Vivarium was, with many species-specific rooms holding animals, ranging from small rodents to large animals. Although the thought of sacrifice is not easy to accept, in vivo animal models are an essential step on the way to human clinical trials. Cedars-Sinai takes the ethical responsibilities with maintaining an appropriate environment and quality veterinary care for the animals very seriously, and I was relieved to hear that the dogs get released and adopted after having served in research. When Dr. Tawackoli led us through the imaging center, I was extremely impressed. This truly is an excellent facility, which has all the newest imaging technology and instrumentation for both, human and animal studies. The instrumentation we have seen included human MRI systems, such as the Siemens Magnetom Vida 3T, small animal MRI, such as the Bruker BioSpin 9.4T, CT scanner, Optical Imaging, Fluorescence Microscopy, and X-Ray. It was particular intriguing to observe an imaging process of a pig for a heart study in the Siemens Biograph mMR PET-MRI machine, which is one of the newest, most advanced diagnostic tools. The astronomical cost for all this technology, for example about $8 million for the PET-MRI, explains why research grants are so highly selective.

Second Week; Hassan Samiullah

Last Friday, I got to witness surgery… on mice. They were injected with anesthesia, shaved, and placed on a ghastly-looking stand (stereotaxic apparatus), complete with an arm holding a syringe filled with 10,000 GL261 glioblastoma cells (a cell line that just so happens to originate from mice). After the mice had their scalp cut, a small hole was drilled through the skull and the cancerous cells were injected.

When many of us think of scientists, we think of crazy people performing crazy procedures in a lab. While I won’t try refuting the first part, the crazy procedures can actually be very consequential to society at large. What is now common knowledge was once found in the discussion section of a research paper. The therapies we will use to treat cancer tomorrow are being tested in labs today, even if they’re being injected into mice brains. 

We were often told as kids that sharing is caring, and scientific research is no exception. One of the ways that scientists share their data is by publishing their studies publicly. I’ve been reading a lot of papers from labs all over the world (and even one from the Breunig Lab, where I’m currently interning) to learn about what models and techniques researchers have used when studying glioblastoma. Knowing this information is crucial because it can help guide the direction of our project, especially because we’re focusing on glioblastoma’s interactions with the immune system, a topic that extracts knowledge from so many different disciplines of biology and medicine. 

Now that I’ve completed about one-fourth of the CIRM SPARK, I’m excited to see the direction our project will take. Cedars-Sinai is offering great resources to help guide the research projects, and even though I don’t understand everything I hear at lab meetings or training sessions, I’m thankful for the opportunity to experience scientific excellence at great depths.

Second Week; Kasey Afshani

Last week, Dr. Lall and I had a conversation about the environmental contributors to ALS and she said that “It’s not so much about if you’ll develop a neurodegenerative disorder, but about which you will develop.” Since then, my views on disorders and brain health have completely changed. While your environment cannot cause ALS, the foods you eat, your smoking habits, your job, and so many other factors can help contribute to ALS or help prevent ALS.

After gaining background on ALS, I began to learn about iPSCs, neurons, glial cells, and different models that are affected in ALS. I studied the different functions that each of them has in the brain and the ways that they are impaired in neurodegenerative disorders. One of the factors that characterize some ALS cases is the aggregation of toxic proteins which, under physiological conditions, are required for normal functions of the body. Once aggregated, these proteins can become toxic that can kill the neurons.

            Two things caught my eye as I was reading about different models to study ALS—the methods that scientists use to get animals to mimic ALS symptoms, and the increased use of Zebrafish to model ALS. Cycad, a seed plant, is epidemiologically linked to ALS. Because of this, scientists feed mice some flour made from cycad seeds, causing the mice to exhibit progressive motor neuron degeneration. This mimicked ALS in humans both phenotypically and pathologically. Although this is just one way that scientists cause mice to mimic ALS symptoms, I thought that this was the most interesting because it exhibits the effect of environmental exposure to ALS contribution. This environmental exposure derives from a natural substance, not through genetically modified substances or cell culture, which I enjoyed reading about.

Moreover, ALS is a disease that affects both upper and lower motor neurons in humans. Because zebrafish aren’t mammals and do not have upper motor neurons, I was surprised to learn about their growing popularity as an animal model for ALS.

            At the end of the week, I began to learn about the blood-brain barrier (BBB) and the ways that it inhibits treatment for some neurodegenerative disorders, including ALS, Alzheimer’s, and Parkinson’s. Our brains want to protect themselves from bacteria and possible toxins, which they do with the BBB. Endothelial cells line the interior of all blood vessels and, in the brain, form tight junctions with one another, preventing any bacteria or toxins from entering the brain. However, it also prevents drugs that could be used to help fight neurodegenerative disorders from entering the brain as well. Currently, there are two ideas of how to open the BBB for treatments. One of them is to “trick” the BBB by securing the drug to something that is allowed to pass through the BBB via transport protein. This is called the “Trojan Horse Approach.” The other method is through a combination of inserting microbubbles into the patient’s bloodstream and using ultrasound on the brain to open the barrier. I’m looking forward to applying the background that I’m learning on ALS, stem cells, and the brain to researching new treatment methods for the disease. Stay tuned!