Problem Set 1 Spring 2024

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Feb 20, 2024

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Problem Set 1 Principles of Cell Biology Spring 2024 Directions : Problem sets will have three types of questions. The " Conventional " type question won't be labeled as such and can be typically answered by referring to your textbook, lecture notes and/or PowerPoint slides. The " Challenge " question (labeled "Challenge") is one that by design will be challenging for you to answer and may require outside sources. Its skill level is designed to be above what is expected in the course. These Challenge questions will be more typical of later problem sets. Finally, the " Opinion " question (labeled "Opinion") often has no right or wrong answer and asks you to think about the science and ethics of the question. Case Study #1 had several of these questions. It may also cite recent articles that you can easily access on-line and are relevant to the lecture presentations. Your TA will give you full credit for each question as long as you make a reasonable attempt to answer the question. The TA will not deduct points if your answer is incorrect. Finally, topics in the discussion section problem sets and case studies that are not in your book or presented in lecture will not be on the exams. 1. Opinion : In a continuation of the theme of last week’s case study, on the very first day of this class (1/16/24) a Genetic Engineering News article ( https://www.genengnews.com/topics/translational- medicine/rhesus-monkey-cloned-via-somatic-cell-nuclear-transfer/? utm_medium=newsletter&utm_source=GEN+News+Highlights+of+the+Week&utm_content=01&ut m_campaign=GEN+News+Highlights+of+the+Week_20240120&utm_id=1100350002&oly_enc_id =2026C0667790F2L ) appeared that celebrated the first successful cloned Rhesus Monkey’s second birthday. While you don’t need to read this article one of the key things that is stated is that cloning (SCNT) of mammalian species is low from 1 to 3% with bovines a bit higher. Do you think this seminal event and birthday should trigger federal laws against human cloning rather than relying on state specific laws given that we are truly inching forward to that possibility? Yes, I think federal laws should be enacted against human cloning. The Rhesus Monkey’s second birthday is a monumental moment in science, since not only does it show that cloning is possible, but it shows that the cloned organism is viable. This will evidently trigger human cloning, which involves ethics and morals. ANSWER: The two lectures on “Cell Biology and Society” had several different themes that we hope you appreciated such as (1) the importance of cell and molecular biology in the medical field; (2) the fact that we can now do experiments in the cell biology arena that may have to be regulated or disallowed by the US government (see question 1 above) and (3) given the diverse nature of cell and molecular biology this discipline offers many post graduate opportunities that include medicine as well as research science and patent attorneys, etc. given the broad strokes of the discipline. The questions below relate to some of these themes. a. As discussed in class several different types of organoids are being generated ranging from intestinal tissue engineered constructs to cardiac organoids. What are two potential medical applications of developing human cardiac organoids?

i. Developing human cardiac organoids can help with heart failure and possibly be used during/for a heart transplant. ii. ANSWER: drug discover, cardiac arythimia. Band-aid while waiting for a transplant b. Many implantable tissue engineered constructs such as human skin and cartilage have been developed but such is not the case for kidneys, heart and liver. Why are decellularized organs important in solving this problem? i. Decellularized organs are important since they can assist with damaged tissue and possibly replace it to a functional form. The decellularized organs can eventually lead to the development of a complete organ. The use of the patient’s own stem cells helps with this process as well. c. Opinion : He Jiankui and Dr. Zhang profiled in class performed experiments related to engineering the birth of humans that could be regarded as “synthetic biology” given that what they both did does not occur in nature. The former was fined and put in jail while Dr. Zhang only received a letter of reprimand from the FDA. What did each do and if you had to make a decision on their “penalty” do you think that both should have received similar reprimands given that both outcomes could be considered the generation of genetically engineered babies? i. He Jiankui assisted with the birth or “creation” or the first CRISPER-Cas9 babies. There were three genetically engineered babies total. Dr. Zhang used nuclear transfer and three parents to genetically prevent mitochondrial diseases in newborns. Both outcomes relate to genetically engineered babies, however, He Jiankui was fined and put in jail for three years. Dr. Zhang’s genetically modified experiment directly said it was to prevent diseases, so maybe that’s why he wasn’t charged or reprimanded as badly. I think genetic engineering is a slippery slope and a lot of it has to do with nature, what’s natural, religion, and ethics. Both should have been reprimanded the same. d. On the other hand, last month (12/2024) a new process/treatment regime using CRISPR was developed called Casgevy which was shown to be immensely successful. This CRISPR clinical trial is regarded as a breakthrough in using cell and molecular biology tools to address complicated diseases for which there have been no past cures. What disease does Casgevy target? i. Casgevy targets sickle cell disease. e. One intriguing question is the following: “If we can totally understand cell aging does this imply that we might be able to slow down or reverse human organismic aging?” In this context list three of the current theories behind cell ageing. i. The shortening of telomeres on the end of chromosomes ii. Release of acetylcholine 1. Treated with botox iii. The expression of vascular endothelial growth factor 1. Treated with RNAi iv. Sertuin genes v. Mitochondria vi. Stem cells vii. Epigenetic patterns

f. Galleri and Theranos are/were both biotechnology companies that proposed to use a simple blood sample to diagnose disease. Galleri has been very successful whereas the CEO and CFO of Theranos are both serving prison sentences for misleading their investors and the public as a whole. What type of disease(s) does Galleri test in humans and how does it work? How does liquid biopsy relate to this test? i. Galleri tests for cancer in humans. It detects DNA methylation patterns from the blood. Depending on how the protein folds, it could mean that cancer is present or occurring. It analyzes cfDNA. 1. Can detect over 50 diff cancers ii. Galleri focuses on liquid biopsy, which is quicker than a tissue biopsy where it takes days instead of weeks. The blood is tested in a liquid biopsy. 1. CSF or urine too g. Challenge : Monoclonal antibodies (mAbs) were developed in the 1970s as tools to identify and label proteins of interest in cells and tissue. (1) What are the Fc and Fab regions and why are they important to the function of mAbs? (2) What is the difference between a native mAb produced in the laboratory, versus a “conjugated mAb” used to treat cancer and a bispecific trifunctional antibody used to treat cancer? i. Fc regions are responsible for binding cellular receptors and Fab regions are the antigen binding fragment. They are important to mAbs since they communicate with surrounding cells. A native mAb is a regular antibody that targets any cell, while a conjugated mAb is one that specifically targets a cancer cell due to an attachment. A bispecific trifunctional antibody is one that is able to manipulate the T-cells in the body to attack the cancer cells. 1. Conjugated = standard, Fc region 2. Native: 2 FAb 2. Personalized medicine has been a theoretical concept for decades but now is a reality. a. Define personalized medicine and explain why the concept of the “$1000 genome” is important to this topic. i. Personalized medicine is when a treatment is tailored to the individual’s genome, so its more effective. The $1000 genome concept is important since a company needs your genetic information in order to personalize the medicine, and they can then steal the information or keep it in their system for later. ii. Cohort of pts with the same exact disease can have four diff outcomes for the same medication iii. Can analyzise our own b. Why was J Craig Venter’s research in year 2000 important to the emergence of personalized medicine? i. Venter was the first one to sequence the human genome in 2000. This made personalized medicine possible, proving that genetic information can be processed and used. c. How does GINA impact and relate to personalized medicine? i. GINA prohibits genetic information from being used as a criterion in your employment or medical insurance. For instance, if you are predisposed to cancer or rare diseases, health insurance companies are not allowed to deny you coverage.

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This relates to personalized medicine since its more expensive and often not covered by insurance. ii. Protects data from being used against you d. What is the difference between how 23 and Me and Genesight use DNA sequencing in this arena? i. 23 and Me originated as a company that analyized the genome in order to create a family tree of the individual. They were not authorized to give diagnostics or health reports. Genesight originated as a company that used your genome to assist with which medication would work best for the individual specifically through a cheek swab. They were authorized to do this. e. Opinion : Seventy years ago HeLa Cells were grown in culture at Johns Hopkins University marking the first time that any human cells had been successfully established in culture. For 50 of these years the descendants of Henrietta Lacks who unwittingly donated these cells 70 years ago, have unsuccessfully sued biotech companies for royalties of using these cells for the development of biologics, the polio vaccines, general research etc.. Finally, however, on August 1, 2023, the Lacks family sued and settled with Thermo Fisher, a large biotech company based in Boston, for an undisclosed sum – the first known settlement of this type for the Lacks family. Let’s assume that you are the lawyer representing Thermo Fisher. What type of arguments against any settlement might you use to win your case against the Lacks family? i. I would say how the cells have saved the population from rapidly declining because of diseases. We were able to get a head of a terrible outbreak, such as polio, and we have been able to keep it at bay thanks to the cells from Henrietta Lacks. Families were saved money from treatments and suffering due to one shot that has drastically improved the quality of life. f. Opinion : One of the problems of genetic tests is that very few are regulated (approved and tested) by the FDA. Do you think all genetic tests should be approved by the FDA before release in the United States? Why or why not? i. No, all genetic testing should not be approved without many reviews. A test result could come back positive for cancer or a gene that pre-disposes someone to cancer, and it can cause issues if wrong or inaccurate. It’s better to be cautious and give out right information than to worry someone for no reason. 3. Stem cells and cancer are one of the “threads” that we use in the course to give it some type of continuity. Yet we actually don’t discuss either of these themes formally until the last two weeks of the course. a. How would you define a stem cell? i. A stem cell is able to adapt and specialize in any cell function since it is at the most basic level. ii. Divide or differentiate into a diff cell b. What is the difference between cancer stem cells and circulating tumor cells (CTCs) and why are cancer stem cells problematic when considering cancer treatment?

i. Cancer stem cells are within the tumor that reproduce and renew, and circulating tumor cells travel through the blood. Cancer stem cells are problematic since chemotherapy does not kill them. They are still able to survive. ii. Cancer stem cells give rise to tumors, can be metastatic, can give rise to extra tumors, can be dormant than rise c. The Cynvenio and Vortex systems were described in class as two different laboratory devices that can enumerate (count) CTCs. How do they differ and why is counting CTCs important in the medical field? i. Cynvenio uses liquid biopsies to count CTCs while Vortex focuses on the unique characteristics of CTCs (their speed compared to WBCs and RBCs) in order to capture them. Counting CTCs is important since it can help diagnosis cancer and see how far along it is. ii. Vortex: CTCs are larger therefore slower d. Opinion and Challenge : There are over 1000 different worldwide clinical trials that use a variety of stem cells including Phase I and II trials using human embryonic stem cells (hESCs) derived from the inner cell mass of a blastocyst. Strict rules on the origin of these human blastocysts apply such as they cannot be procured from women who are paid to generate them, etc – a set of extensive rules put in place when Baraq Obama took office. They are all acquired as extra blastocysts from in vitro fertilization clinics that would be otherwise discarded. What are the various types of stem cells currently used in clinical trials? What is the basis for the arguments that hESCs should be used for clinical trials and do you think it is unethical to use these cells clinically? Also, why is the distinction between autologous and allogeneic important in this arena? i. Blood stem cells are used in clinical trials. It can either be stem cells from the individual themselves or a donor. hESCs are controversial since they’re from a blastocyst, the early stages after an egg is fertilized, but they’re “leftovers” from IVF. I don’t think its unethical since they were all fertilized and would be put to waste otherwise. The difference between autologous and allogenic is important since the cells are either from the patient themselves or from the clinic. e. Why are stem cells preferred by tissue engineers who are fabricating organoids and other tissue engineered constructs and models? i. Stem cells are preferred because they’re less likely to be rejected since they’re from the participant/ recipient. 4. There are a variety of techniques, tools and probes available to cell and molecular biologists. By this point of time in class we will have covered many associated with visualizing cells. There are a number of experimental scenarios presented below. Match each one to an appropriate microscope, device, tool etc. a. Analyzing the location and distribution of actin fibers in cells using a primary monoclonal antibody labeled with a rhodamine-labeled secondary antibody. i. Fluorescence microscopy b. Analyzing some human endothelial cells using a microscope that is capable of generating stereo images of the these cells and can optically section them as well in the X, Y and Z axes.

i. Confocal microscopy c. Using hematoxylin and eosin to analyze 15  m sections of a liver tumor. i. Bright Field/ Compound Microscope d. Visualizing a group of unstained living cells in culture generating contrast by relying on the differences in the refractive indices of cell organelles to generate contrast. i. Phase microscopy e. Doing single cell electrophysiology of a neuron in culture be relying on generating a 3-D image of this cell i. Differential and intereference contrast f. Analyzing dead versus living cells using Propidium Iodide and Calcein-AM i. Vital Fluorescence Microscopy (live-dead assay) ii. Confocal iii. Fluorescence plate reader g. Measuring the lateral mobility of a fluorescently tagged outer cell membrane protein in a living cell by photobleaching the fluorescent probe. i. FRAP: Fluorescence Recovery After Photobleaching

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Problem Set 1 Spring 2024

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