Bowdoin Science Journal

Biology

mRNA: Unpacking the Research that led to the COVID-19 Vaccines and the 2023 Nobel Prize in Medicine

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        By 2023, 81.4% of the United States population have received at least one dose of the COVID-19 mRNA vaccine, and just about everyone has heard of these lifesaving shots (CDC, 2023). But how many of us actually know how they work? In general, vaccinations expose the body to a weakened strain of a virus that initiates an immune response so that the virus can be recognized, and quickly neutralized, in the future (Jain, 2021). This works because of the structure of our immune system, which is split up into innate immunity and adaptive immunity. Innate immunity is the immediate response that our bodies put up when exposed to a pathogen; it sends out macrophages to gobble up infected cells, creates an inflammatory response to kill off the infection, and uses a host of molecules and pathways to destroy the virus. Adaptive immunity involves the presence of B and T cells, which arise in a viral infection and create immunological memory through antibodies and memory cells so that a future infection of the same virus is immediately recognized and shut down. Vaccines work to stimulate this adaptive immunity, while minimizing innate immunity.

        mRNA vaccines use this same basic principle, but instead of injecting a weakened strain of the actual virus into someone, they instead inject a piece of mRNA, or messenger RNA, which codes for a viral protein that stimulates this immune response (Jain, 2021).

Figure 1: The methodology of mRNA vaccines. Adapted from Jain, et. al., 2021

mRNA vaccines are much easier to make than traditional vaccines because they don’t require growing and inactivating a virus, and they don’t carry the risk that live vaccines have of infecting the subject, but up until recently, they weren’t even remotely possible because in vitro mRNA would create a huge inflammatory response when entering the body (Karikó, 2005). However, Hungarian biochemist Katalin Karikó and her colleague Drew Weissman were undeterred, and their research to create mRNA without an inflammatory response paved the way for mRNA vaccines and won them the Nobel Prize in Medicine in 2023 (Karikó, 2005).

        Karikó and Weissman’s research focused on in vitro mRNA’s stimulation of Toll-like receptors (TLRs), which recognize pathogens and activate signaling pathways leading to an inflammatory response (Karikó, 2005). They discovered that mammalian mRNA, which doesn’t stimulate an immune response, contains many base pair modifications, whereas bacterial RNA, which is known to stimulate a response, has no modifications (Karikó, 2005). In vitro mRNA, grown in a lab rather than from mammalian cell cultures, also has no, or very few, modifications. This led the researchers to question whether base pair modifications were how cells distinguished between foreign and non-foreign RNA, and thus which pieces of RNA stimulate an immune response (Karikó, 2005). They discovered that in vitro RNA activates three main TLRs known for activating an inflammatory response, but that nucleoside modifications limit that activation. Most importantly, they discovered that the suppressed immune response is proportional to the number of nucleotide modifications, and that these modifications also increase the stability of in vitro mRNA in the body, another original roadblock in the usage of mRNA for vaccines. (Karikó, 2005).

Figure 2: The use of base modifications to suppress inflammatory response of RNA. Adapted from Nobelprize.org

        Combined with later research also by Karikó and Weissman discovering that modified base-pairs also create increased protein production and other research designing more stable lipid carriers to deposit the mRNA into human cells, mRNA became a promising new vaccine technology, eventually allowing the COVID-19 vaccines to be produced in record times, shortening the pandemic and granting all of us our lives back. But the road to Karikó’s research being recognized, and especially the road to the Nobel Prize, was not predetermined. Karikó’s research was long overlooked, in part because of her identity as a Hungarian immigrant. She was “demoted four times” at UPenn, being told that her research didn’t measure up to their standards, but she continued to persevere, making her both a scientific success story, and a personal one (Shrikant, 2023). The COVID-19 vaccines created by Pfizer and Moderna were the first mRNA vaccines to be released, but future research is focusing on creating a universal flu vaccine and possibly even an HIV vaccine, all using mRNA technologies, which could drastically improve the landscape of viral infections, both in the US and across the globe.  

 

Literature Cited

Karikó, K., Buckstein, M., Ni, H., & Weissman, D. (2005). Suppression of RNA Recognition by Toll-Like Receptors: The Impact of Nucleoside Modification and the Evolutionary Origin of RNA. Immunity, 23(2), 165–175. https://doi.org/10.1016/j.immuni.2005.06.008

 Karikó, K., Muramatsu, H., Welsh, F. A., Ludwig, J., Kato, H., Akira, S., & Weissman, D. (2008). Incorporation of Pseudouridine into mRNA yields Superior Nonimmunogenic Vector with Increased Translational Capacity and Biological Stability. Molecular Therapy, 16(11), 1833–1840. https://doi.org/10.1038/mt.2008.200

 Jain, S., Venkataraman, A., Wechsler, M. E., & Peppas, N. A. (2021). Messenger RNA-Based Vaccines: Past, Present, and Future Directions in the Context of the COVID-19 Pandemic. Advanced Drug Delivery Reviews, 179, 114000. https://doi.org/10.1016/j.addr.2021.114000

 The Nobel Prize in Physiology or Medicine 2023. (n.d.). NobelPrize.Org. Retrieved October 22, 2023, from https://www.nobelprize.org/prizes/medicine/2023/press-release/

 CDC. (2020, March 28). Covid Data Tracker. Centers for Disease Control and Prevention. https://covid.cdc.gov/covid-data-tracker

Shrikant, A. (2023, October 6). Nobel Prize Winner Katalin Karikó was “demoted 4 times” at her old job. How she persisted: “You have to focus on what’s next.” CNBC. https://www.cnbc.com/2023/10/06/nobel-prize-winner-katalin-karik-on-being-demoted-perseverance-.html 

 

The Possibilities of the CRISPR-Cas9 System

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          As humans continue to further explore the fields of science, they deepen their understanding of convoluted subjects through the use of advanced technologies. One of the notable technologies today is the CRISPR-Cas9 system, a highly precise DNA editing tool that allows for genome manipulation in humans, animals, and plants. CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats) works with a CRISPR-associated (Cas) nuclease, which was originally derived from a genome editing system used as immune defense by bacteria. To put it simply, if infected by a virus, the bacteria captures a small piece of the invading viruses’ DNA and inserts their own DNA to create a CRISPR segment (Tavakoli et al., 2021). With this segment, the bacteria can produce RNA segments that will attach to parts of the viruses’ DNA if it attacks again. The bacteria use the Cas9 enzyme to cut the viruses’ DNA apart and incapacitate it. 

          Scientists adapted the system so it could be inputted into the human genome and edit DNA. Researchers created small segments of RNA that have a “guide” sequence which binds to a target sequence in the DNA and attaches to the Cas9 enzyme (Tavakoli et al., 2021). The guide RNA (gRNA) identifies the intended DNA sequence and the Cas9 enzyme cuts the specific location, similar to how the process occurs in bacteria. The cell’s own DNA machinery is then used to add or delete pieces of the genetic material, known as gene knock-in and gene knockout, respectively. However, it can also alter the DNA by replacing segments with more customized sequences. CRISPR activation (CRISPRa) is used to up-regulate the expression of a gene while CRISPR interference (CRISPRi) down-regulates the expression of a cell (CRISPR Guide).

Figure 1. Gene Knockout
Figure 2. Activation of Target  Gene with CRISPRa

                                       Note. Adapted from CRISPR Guide, Addgene, 2022.

          There are many potential applications in cancer immunotherapy, treatment of genetic diseases, improving plant genetic variation, and more. For example, there have been advances towards mitigating the effects of blood disorders such as sickle cell disease (SCD). In those with SCD, the misshapen red blood cells can block blood vessels, thus slowing or stopping blood flow. Effects of this fatal disease are anemia, chronic pain, strokes, and organ damage. In this instance, CRISPR technology is not used to directly rectify the disease-inducing gene variant. Instead of restoring adult hemoglobin, it works to increase the levels of fetal hemoglobin, an oxygen carrier protein that only fetuses make in the womb (Henderson, 2022). Since it’s not affected by the sickle cell mutation, it can be a substitute for the defective adult hemoglobin in red blood cells (Henderson, 2022).

Figure 3. Hemoglobin Switching in Infants

Note. Adapted from CRISPR Clinical Trials: A 2022 Update, Henderson, 2022, Copyright 2022 by Innovative Genomics Institute.

         As seen in the diagram above, SCD symptoms begin during infancy when levels of fetal hemoglobin decrease. The blood stem cells are harvested from the blood and then the genomes are edited to activate the fetal hemoglobin gene. Next, chemotherapy eliminates the disease-inducing blood stem cells from the person’s body, and the genome-edited stem cells are put back into the bloodstream through an IV, creating a new blood stem cell population that produces fetal hemoglobin (Henderson, 2022). This technique is ex vivo genome editing, where the editing occurs outside the body to prevent the risk of lingering and unwanted CRISPR components in the body. Those treated for SCD in CRISPR clinical trials show normal to near-normal hemoglobin levels with at least 30% as fetal hemoglobin (Henderson, 2022).

          It raises the following questions: is it possible to select certain features in humans that are seen as favorable or desirable? Is it possible to have almost complete manipulation of physical features or to enhance the body’s biological processes to unlock superhuman abilities only seen in movies? These human experimentations would be best measured if implemented during the early stages of life so as to let the body integrate the changes and develop properly. Perhaps we could alter ourselves to increase muscle mass, heighten athletic ability, or augment intelligence. In regard to altering the human genome, there are certain limitations as to what can and can’t be done without violating certain ethical boundaries. 

          This can be seen in the case of He Jianku, a Chinese scientist who used CRISPR to edit two embryos, both of which are now living babies. Though there was little information that only came from He himself and the Chinese government, it has been said that sometime in late 2017, He injected the two embryos with a CRISPR construct that would delete a 32-base-pair in the CCR5 gene on chromosome 3, leading to a non-functional CCR5 protein (Greely, 2019). He sought to achieve humans who could not contract AIDS because they don’t have this functioning protein (Greely, 2019). Though this theory was later proved wrong and the CRISPR construct did not fulfill his intentions, he managed to induce never before seen changes that led to the production of non-functional proteins. His actions prompted the Chinese court to convict him on the basis of deliberately violating medical regulations (Greely, 2019). Could it be argued that pushing the boundaries are necessary for scientific growth and greater human evolution? How far is too far? As CRISPR and other gene editing tools continue to develop, we can keep imagining the unnerving yet exciting possibilities that are awaiting.

 

Literature Cited

CRISPR Guide. (n.d.). Addgene. 

Greely, H. T. (2019). CRISPR’d babies: human germline genome editing in the ‘He Jiankui affair’. Journal of Law and Biosciences, 6(1), 111-183.

Henderson, H. (2022). CRISPR Clinical Trials: A 2022 Update. Innovative Genomics Institute.

Tavakoli, K., Pour-Aboughadareh, A., Kianersi, F., Poczai, P., Etminan, A., & Shooshtari, L. (2021). Applications of CRISPR-Cas9 as an Advanced Genome Editing System in Life Sciences. BioTech, 10(3), 14. 

What are genome editing and CRISPR-Cas9? (2022). MedlinePlus.

The Chronic Lyme Debate

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Second possibly only to mosquitos, ticks are the most reviled insect found in New England nature. Like mosquitos, which are notorious vehicles for viruses such as Zika and West Nile, blacklegged ticks (deer ticks) spread Borrelia burgdorferi infection, resulting in Lyme disease. Affecting over 30,000 people a year in the United States, mostly in the northeastern states, Lyme disease is a bacterial infection which causes both local and global symptoms. Early symptoms include fever, muscle fatigue, swollen lymph nodes, and, mostly notably, an erythema migrans rash. Left untreated, patients may develop facial palsy (partial facial paralysis), arthritis, central nervous system inflammation, and heart palpitations. Diagnosis of Lyme disease is based primarily on symptoms and the possibility of exposure to Lyme-carrying ticks, although laboratory tests of patient serum for anti-B. Burgdorferi antibodies may also be considered. Treatments usually consist of a several week course of antibiotics. 

Although the majority of Lyme disease patients recover after initial treatment, 5-20% of patients continue feeling symptoms such as fatigue, muscle pain, and difficulty concentrating. This disorder, called post-treatment Lyme disease syndrome, falls under a broader category of disorders referred to as “chronic Lyme disease.” Also included in this category are patients without a history of Lyme disease but with Lyme-like symptoms; patients with other recognizable disorders seeking an alternative diagnosis; and patients with positive serological results for anti-B. Burgdorferi antibodies but no past exposure to ticks or other routes of infection. 

The majority of chronic Lyme patients fall into the middle two categories: those with symptoms but no history and the misdiagnosed. Moreover, in clinical trials, traditional treatment routes for Lyme disease such as antibiotics have not been effective in alleviating symptoms. Because many of those diagnosed with chronic Lyme do not match the diagnostic criteria for traditional Lyme disease, most scientists and physicians reject the diagnosis. However, a small but very vocal group of patients and physicians fervently believe in the existence of chronic Lyme disease. They are represented by powerful advocacy groups such as the International Lyme and Associated Diseases Society (ILADS) and the Lyme Disease Association (LDA). These groups have been effective at lobbying politicians and regulators to get protections for chronic Lyme sufferers, even as the physician community become increasingly convinced of its inaccuracy.

Although Lyme disease may seem a relatively simple disease with a clear cause (tick-borne bacterial infection) and treatment plan (antibiotics), the controversy over chronic Lyme disease reveals complexities. And this particular controversy doesn’t seem to be abating. It seems that the concept of chronic Lyme disease, like the disorder it purports to describe, is here to stay.

 

Works Cited:

CDC. (2022, January 19). Lyme disease home | CDC. Centers for Disease Control and Prevention. https://www.cdc.gov/lyme/index.html 

Chronic symptoms. (2018, April 11). Lyme Disease. https://www.columbia-lyme.org/chronic-symptoms 

Feder, H. M., Johnson, B. J. B., O’Connell, S., Shapiro, E. D., Steere, A. C., & Wormser, G. P. (2007). A critical appraisal of “chronic lyme disease.” New England Journal of Medicine, 357(14), 1422–1430. https://doi.org/10.1056/NEJMra072023 

Lantos, P. M. (2011). Chronic Lyme disease: The controversies and the science. Expert Review of Anti-Infective Therapy, 9(7), 787–797. https://doi.org/10.1586/eri.11.63 

Mosquito-borne diseases | niosh | cdc. (2020, February 21). https://www.cdc.gov/niosh/topics/outdoor/mosquito-borne/default.html 

Whelan, D. (n.d.). Lyme inc. Forbes. Retrieved April 2, 2023, from https://www.forbes.com/forbes/2007/0312/096.html 

Mercury, Contaminating Our Oceans and Your Food

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Mercury, you may know it as the solar system’s smallest planet or as the “red stuff” in old thermometers. You may have even heard of its toxic effects on people if exposure occurs. However, you may not know that mercury is significant outside of the realm of toxic thermometers and astronomy. The chemical element mercury is a contaminant that is being pumped into the atmosphere at an alarming rate and is poisoning aquatic environments. Mercury is becoming an increasingly common pollutant in our oceans and lakes and its toxic effects are causing harm to marine life and creating an imbalance in our marine ecosystems.

Before mercury can enter aquatic environments, it is released in large quantities by anthropogenic sources. Mercury can enter the atmosphere through natural sources such as volcanoes or forest fires, but it is primarily released through the burning of fossil fuels and small-scale gold mining (Montes 287). The release of mercury is problematic because it is very easily transported through the atmosphere. Mercury is a volatile element, which means that it evaporates at low temperatures (mercury can even evaporate at room temperature) and easily enters its gaseous state to be carried long distances through the air (Pollet 860).

After mercury is transported through the atmosphere and enters aquatic environments, it is transformed into its more toxic state, methylmercury (CH3Hg or MeHg). Mercury is transformed into methylmercury through the process of mercury methylation when Hg incorporates CH3, making it into CH3Hg (or MeHg). In the ocean, methylation of mercury is carried out by bacteria. Essentially, bacteria that are present in aquatic environments absorb mercury and perform the methylation reaction before releasing methylmercury back into the ecosystem (Poulain 1280-1281).

Once organisms ingest methylmercury, they experience detrimental effects on their function and, ultimately, their survival. For example, seabirds with more than 0.2 μg (micrograms) of mercury in their blood per gram of wet weight have observed negative effects on their bodies’ systems and their function. An approximately equivalent concentration can be represented by one person out of the entire state of Alabama, which has a population of 5.04 million. At this level or greater, birds experience detrimental effects on their nervous and reproductive systems as well as changes to their hormonal makeup and trouble with motor and behavioral skills (Pollet 860). 

Another interesting observation of mercury contamination is its differing distribution of concentrations among populations. The methylmercury concentration in seabirds was explored between 2013 and 2019 when egg and blood samples were taken of Leach’s storm petrels along with the GPS tracking of foraging petrels. By comparing measured mercury concentration in blood and eggs to ocean depth of foraging locations, a correlation was found. The study concluded that the water depth had a significant effect on the methylmercury levels measured in Leach’s storm petrels. Storm petrels who foraged in deep waters had higher methylmercury concentrations in their blood than storm petrels who forage in shallow or coastal waters. The positive correlation between ocean depth and mercury concentration is likely due to differences in diet based on foraging location. This could also be related to the fact that mercury methylation is most efficient in deep water (Pollet 860).

These negative effects on seabirds also have broad effects on the entire ecosystem that they are a part of. This is because methylmercury biomagnifies up the food web. As the methylmercury moves up trophic levels in a food chain, its concentration in a given organism increases. This increase in concentration is dramatic. In fact, measured mercury concentrations in predator species can be millions of times greater than the concentration observed in surface waters. The problem of biomagnification is even more dramatic in Maine because of its latitude. While the phenomenon is not entirely understood, ecosystems located at higher latitudes have been observed to be more susceptible to biomagnification than tropical regions (Lavoie 13385-13394). 

Other than affecting our Maine wildlife, mercury contamination could have a negative impact on human health. Mercury is not only a problem for seabirds, but fish are also just as susceptible to contamination. This can become a major problem for commercial fisheries because the seafood they produce for our consumption have the potential for mercury contamination. In fact, there was an incident in Japan in 1956 when people who consumed contaminated seafood became severely ill or died. Over the course of 36 years after the incident, 2252 people were infected 1034 people were killed in relation to the initial methylmercury contamination (Harada 1-24). While this level of contamination is an anomaly, a 2018 study projected that approximately 38% of countries experience some level of human exposure to mercury due to contaminated seafood consumption. There are steps that can be taken to prevent this. For example, setting stricter regulations on mercury content limits, applying proper culinary treatments, or updating fishing practices could all diminish the probability of mercury exposure to humans (Jinadasa 112710). 

That being said, addressing the root of the problem is the only way to most effectively diminish mercury exposure for marine organisms and people. Mercury contamination is a problem that has a dramatic domino effect beginning in our atmosphere and ending in human consumption. It is an issue that is often forgotten in discussions of the environmental impact of burning fossil fuels. However, considering its impact on marine life and eventually human life, it is a byproduct that cannot be overlooked.

 

Works Cited

da Silva Montes, C., Ferreira, M. A. P., Giarrizzo, T., Amado, L. L., & Rocha, R. M. (2022). The
legacy of artisanal gold mining and its impact on fish health from Tapajós Amazonian
region: A multi-biomarker approach. Chemosphere, 287, 132263.

Harada, M. (1995). Minamata disease: methylmercury poisoning in Japan caused by
environmental pollution. Critical reviews in toxicology, 25(1), 1-24.

Jinadasa, B. K. K. K., Jayasinghe, G. D. T. M., Pohl, P., & Fowler, S. W. (2021). Mitigating the
impact of mercury contaminants in fish and other seafood—A review. Marine Pollution
Bulletin, 171, 112710.

Lavoie, R. A., Jardine, T. D., Chumchal, M. M., Kidd, K. A., & Campbell, L. M. (2013).
Biomagnification of mercury in aquatic food webs: a worldwide meta-analysis.
Environmental science & technology, 47(23), 13385-13394.

Pollet, I. L., McFarlane-Tranquilla, L., Burgess, N. M., Diamond, A. W., Gjerdrum, C., Hedd, A.,
… & Mallory, M. L. (2023). Factors influencing mercury levels in Leach’s storm-petrels at
northwest Atlantic colonies. Science of The Total Environment, 860, 160464.

Poulain, A. J., & Barkay, T. (2013). Cracking the mercury methylation code. Science, 339(6125),
1280-1281.

The Contraceptive Brain Drain: How Birth Control Alters Women’s Brains

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There are millions of women taking steroids every day. But how is this possible? Are they just getting really buff? It feels like we always hear stories about how performance-enhancing drugs, namely steroids, are giving world-class athletes the boost they need to beat out their competition. But women across the globe are taking steroids every day as well, in the form of hormonal birth control. Despite their widespread use, side effects of hormonal contraceptives are largely unstudied, or have been until recently. In the last ten years, several studies have come out about the effect of taking a daily dose of steroids on women’s brains and mental health, which until now has been a severely neglected area where lack of knowledge affects millions of people worldwide. 

People take hormonal birth control, or hormonal contraceptives, for a myriad of reasons, from the obvious (preventing pregnancy) to the not-so-obvious (lessening iron deficiency) and everything in between. This type of medication simply refers to methods of pregnancy prevention that act on the endocrine system. The endocrine system controls growth, development, metabolism, and reproduction via signaling molecules called hormones. Two hormones in particular, estrogen and progesterone, control the menstrual cycle and are therefore the major components of hormonal birth control. Types of hormonal contraceptives come in many forms including the pill, the patch, the implant, injections, and hormonal intrauterine devices or IUDs, but despite the wide variety in the forms this medication takes, all of them contain one or both of these two hormones. As steroids, both estrogen and progesterone affect other body systems besides the reproductive system.

To study the impacts of taking a daily dose of steroids on other areas of the body, specifically the brain, Dr. Belinda Pletzer and her colleagues conducted a study in 2010. The brain is particularly susceptible to change due to an influx of synthetic hormones because it contains a very high quantity of hormone receptors. The brain needs to act as a “sponge” for these molecules since it plays an important role in creating the appropriate responses in the rest of the body. Pletzer’s study investigated how the sponginess of the brain would affect changes in its structure by comparing images of the brains of adult men, adult women during different stages of their menstrual cycle, and adult women taking hormonal contraceptives. To perform this comparison they used a technique called voxel-based morphology on MRIs of study participants (Pletzer et al., 2010). Voxel-based morphology measures differences in the concentration of tissue and the size and shape of different areas of the brain.

Overall, they found that women taking hormonal birth control had smaller areas of gray matter, or areas of the brain that have a high concentration of the cell bodies of nerve cells, when compared to “naturally cycling women” in both their follicular and luteal menstrual phases (Figure 1). Pletzer’s study also found interesting gendered differences in gray matter volume. While men had greater gray matter overall, the volume of gray matter in the prefrontal cortex, the pre-and postcentral gyri, and the supramarginal gyrus of both naturally cycling women and women taking hormonal contraceptives was higher than the volume of gray matter in these areas in men (Figure 2). These areas are involved in decision-making and problem-solving, controlling motor function, and emotional responses. However, the higher amount of gray matter in women in these areas was overshadowed by the larger volume of gray matter in men in the hippocampus, hypothalamus, parahippocampal and fusiform gyri, putamen, pallidum, amygdala, and temporal regions of the brain during the early follicular phase (A), mid-luteal phase (B), and in women taking hormonal birth control (C) (Figure 2). Many of these areas of reduced gray matter are ones of high importance for neurophysical ability and mental health.

Additionally, a study done by Rush University Medical Center showed an association between higher levels of gray matter and better cognitive function (“Everyday Activities Associated with More Gray Matter in Brains of Older Adults”). These findings suggest that taking birth control, and the associated decrease in gray matter, could be directly causing some of the symptoms women on hormonal contraceptives experience, such as brain fog, mood changes, and even anxiety and depression. For example, a smaller hypothalamus, one of the areas of decreased gray matter, is associated with heightened irritability and depression symptoms (“Study Finds Key Brain Region Smaller in Birth Control Pill Users”). Pletzer’s research and the work of others after her on the impact of birth control on structures of the brain represent important first steps in proving a causative relationship between birth control, symptoms associated with it, and structural changes in the brain.

Although this research has made some crucial preliminary steps into researching how taking a daily dose of steroids affects the brains of women taking hormonal contraceptives, the highly complex nature of the brain and its relationship with the regulation of the rest of the body means that further research is necessary. The sheer number of people that this issue affects means that it is essential to continue researching the impacts of this widely used drug. More importantly, knowing the potentially serious negative side effects enables millions of people to make more informed decisions concerning their health and their bodies.

 

Works Cited

Rush University Medical Center. (2018, February 14). Everyday activities associated with more gray matter in brains of older adults: Study measured amount of lifestyle physical activity such as house work, dog walking and gardening. ScienceDaily. Retrieved March 11, 2023 from www.sciencedaily.com/releases/2018/02/180214093828.htm.

Lewis, C. A., Kimmig, A. C. S., Zsido, R. G., Jank, A., Derntl, B., & Sacher, J. (2019). Effects of hormonal contraceptives on mood: a focus on emotion recognition and reactivity, reward processing, and stress response. Current psychiatry reports, vol. 21, no.11, 2019, p 115. PubMed Central, https://doi.org/10.1007/s11920-019-1095-z.

Meyer, Craig H., Kinsley, Elizabeth A. “Women’s Brains on Steroids.” Scientific American, https://www.scientificamerican.com/article/womens-brains-on-steroids/. Accessed 7 Mar. 2023.

Nemoto, Kiyotaka. “[Understanding Voxel-Based Morphometry].” Brain and Nerve = Shinkei Kenkyu No Shinpo, vol. 69, no. 5, May 2017, pp. 505–11. PubMed, https://doi.org/10.11477/mf.1416200776.

Pletzer, Belinda, et al. “Menstrual Cycle and Hormonal Contraceptive Use Modulate Human Brain Structure.” Brain Research, vol. 1348, Aug. 2010, pp. 55–62. ScienceDirect, https://doi.org/10.1016/j.brainres.2010.06.019.

Sharma, Rupali, et al. “Use of the Birth Control Pill Affects Stress Reactivity and Brain Structure and Function.” Hormones and Behavior, vol. 124, Aug. 2020, p. 104783. ScienceDirect, https://doi.org/10.1016/j.yhbeh.2020.104783.

“Study Finds Key Brain Region Smaller in Birth Control Pill Users.” ScienceDaily, https://www.sciencedaily.com/releases/2019/12/191204090819.htm. Accessed 7 Mar. 2023.

What Causes Aging? An Epigenetics Study From The Sinclair Lab May Have an Answer

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Dr. David Sinclair, A.O., Ph.D. Photo from the Sinclair Lab, Harvard Medical School (2023).

Aging, also known as “senescence,” is an inevitable process in all living things. Organisms small and large eventually break down, accumulating enough wear and tear in their cells that ultimately causes the body to stop functioning as a whole. While medicine and lifestyle improvements stave off aging, identifying its fundamental causes has been more challenging. In January 2023, scientists in Dr. David Sinclair’s lab at Harvard Medical School published a paper with experimental evidence supporting what Sinclair calls the “Information Theory of Aging,” where damage to the epigenome can cause aging.

After receiving his Ph.D. in molecular genetics from the University of New South Wales, Sinclair completed his postdoctorate at MIT where he co-discovered the role of sirtuin enzymes in limiting age-related cellular damage in yeast. In addition to teaching genetics and translational medicine at Harvard Medical School since 1999, Sinclair authored the popular book Lifespan: Why We Age – and Why We don’t Have To (2019). His breakthroughs in the science of aging have earned him a great deal of attention from the public eye, resulting in appearances on several popular media outlets, including CBS’s “60 Minutes” and TIME magazine (The Sinclair Lab, 2023).

Sinclair’s newest discovery, published as “Loss of epigenetic information as a cause of mammalian ageing” in January 2023, focused on the role of epigenetics in aging. The title specifies that epigenetic information loss, rather than genetic information loss, is a cause of aging. Genetics refers to the raw molecular information sequences stored in cells as deoxyribonucleic acid (DNA), which is physically condensed inside the nucleus into pairs of chromosomes. The material inside a chromosome is known as “chromatin.” The central dogma of molecular biology states that information in DNA sequences is read by the cell in the form of messenger RNA (mRNA) through transcription, and then ribosomes in the cell read this mRNA to make proteins through translation. DNA sequences that correspond to the production of a specific molecule are genes. The prefix epi- means “on top of,” so “epigenetics” refers to mechanisms that function “above” the molecules of DNA themselves, including reading DNA sequences, regulating gene transcription, and repairing mutated DNA. Like the DNA sequence, epigenetic changes are inheritable from parents to offspring.

The differences between genetics and epigenetics influence cellular reaction to damage. Damage to genes causes mutations, which are changes in the sequence of the DNA of that gene. Cells have mechanisms of repairing mutated DNA, but failure of these mechanisms can lead to cell death, or worse, cancer. Some DNA mutations, like those where a nucleotide is deleted, are irreversible.

By contrast, epigenetic mechanisms are more easily reversed. One epigenetic mechanism is DNA methylation, where a methyl group (-CH3) is added to a cytosine nucleotide by the DNA methyltransferase enzyme. This extra functional group blocks transcription factors from binding to promoter regions nearby the methylated cytosine, in effect “silencing” the gene as it cannot be transcribed into mRNA. DNA methylation is important for differentiating cells into specific cell types by enabling cells to only express the most pertinent genes while still containing the entire genome (Moore et al., 2012). DNA methylation is reversible with the help of TET dioxygenase enzymes (Wu and Zhang, 2014). Geneticists have found DNA methylation to be a way to assess molecular aging in cells as a sort of “epigenetic clock”. By analyzing methylation patterns of the genome (the “methylome”), scientists can find the biological age as well as the rate of aging in an organism’s cells (Hannum et al., 2012).

Figure 1: Methyl groups attached to cytosine bases in a gene block the enzyme RNA polymerase from binding to the promoter region of a gene, preventing transcription. Adapted from BOGOBiology (2017)

To investigate how changes to the epigenome affect aging in mice, Sinclair used a mouse system with induced changes to the epigenome (ICE). The genetically modified mice had a higher frequency of double-strand breaks (DSBs) in the DNA, which cause changes in the epigenome as cells are required to use their mechanisms of DNA repair more often . Sinclair’s method reduced the frequency of mutations by breaking the DNA strands in a way that left more whole unpaired nucleotides in the severed strand, making it more difficult for the cell to repair the DNA strand with a different sequence than before. While ICE mice had no significant difference in mutation frequencies versus control mice, DSBs in specific locations of the genome were observed as expected (Yang et al., 2023). Thus, any apparent changes in aging in the ICE mice of this study could be ascribed to the epigenetic DSB changes rather than mutations.

Figure 2: ICE treated mice after 10 months appear to have the physical hallmarks of aging, such as hair loss and reduced body mass, early compared to CRE control mice (Yang et. al. 2023).

The haggard appearance of the ICE 10 month old mice confirmed the early aging effects of the epigenetic changes (Figure 2). At the molecular level, analysis of DNA methylation at genome sites associated with age showed that ICE cells were approximately 1.5 times “older” than the control cells. With this artificially increased age came physiological consequences. ICE mice showed diminished short-term memory retention compared to Cre-control mice, as assessed by fear conditioning tests, and the ICE mice performed half as well in a Barnes Maze test as control, indicating decreased long-term memory. Additionally, ICE mice had decreased muscle mass and grip strength after 16 months. The authors attributed the cause of this accelerated aging to increased “faithful DNA repair” from the induced DSB breaks in the ICE mice, meaning that there were not significant mutations in the repair of DSBs.(Yang et al., 2023). During DSB repair, chromatin modifying factor proteins activate and move within a cell in a process known as “relocalization,” with repeated activation of this process known to cause epigenetic changes that silence genes normally expressed in young mice. Sinclair’s lab hypothesized that the relocalization of chromatin modifiers that occurs from repeated DSB repair associated with induced epigenetic changes lead to a gradual loss of cellular function associated with aging (Yang et al., 2023).

It may seem ironic that DNA strand break repair, a process meant to keep cells functioning when critical genes are damaged, is part of what ultimately causes the death of organisms. The fact that the mechanism implemented is epigenetic rather than genetic suggests that the effects of ageing may be reversible, like epigenetic mechanisms. In fact, Sinclair has shown that it is possible to partially undo the damaging effects of aging: after inducing OSK expression, which is a set of proteins known as “Yamanaka factors,” ICE mice exhibited some signs of rejuvenation in their eyes, kidneys, and muscles. Yamanaka factors like OSK are important in the fields of aging and regenerative medicine because they are keys to the synthesis of induced pluripotent stem cells, where somatic cells can become stem cells and potentially re-differentiate into other cell types (Takahashi and Yamanaka, 2006). The OSK treatment decreased the expression of age-associated markers in the kidney and muscle cells of the mice (Yang et al., 2023).

Sinclair’s experiments have shown the epigenome to be a key front in the investigation of aging, as the reversibility of changes to the epigenome can allow it to be a more accessible interface for scientists to interact with. The plasticity of the epigenome as demonstrated from the ability of Yamanaka factors to reverse the molecular indicators of aging mice show that there may be hope for science to bring this phenomenon to human epigenomes. Indeed, the news of reversing aging in mice made rounds in the media when Sinclair’s paper was published earlier this winter, and for good reason.

Works Cited

Al Aboud, N. M., Tupper, C., & Jialal, I. (2022). Genetics, Epigenetic Mechanism. In StatPearls. StatPearls Publishing. http://www.ncbi.nlm.nih.gov/books/NBK532999/

Bannister, A. J., & Kouzarides, T. (2011). Regulation of chromatin by histone modifications. Cell Research, 21(3), Article 3. https://doi.org/10.1038/cr.2011.22

BOGObiology (Director). (2017, October 11). Epigenetics: Nature vs. Nurture. https://www.youtube.com/watch?v=Q8BMP6HDIco

CDC. (2022, August 15). What is Epigenetics? | CDC. Centers for Disease Control and Prevention. https://www.cdc.gov/genomics/disease/epigenetics.htm

David Sinclair | The Sinclair Lab. (n.d.-a). Retrieved March 5, 2023, from https://sinclair.hms.harvard.edu/people/david-sinclair

Fernandez, A., O’Leary, C., O’Byrne, K. J., Burgess, J., Richard, D. J., & Suraweera, A. (2021). Epigenetic Mechanisms in DNA Double Strand Break Repair: A Clinical Review. Frontiers in Molecular Biosciences, 8, 685440. https://doi.org/10.3389/fmolb.2021.685440

Gilbert, S. F. (2000). Methylation Pattern and the Control of Transcription. Developmental Biology. 6th Edition. https://www.ncbi.nlm.nih.gov/books/NBK10038/

Hannum, G., Guinney, J., Zhao, L., Zhang, L., Hughes, G., Sadda, S., Klotzle, B., Bibikova, M., Fan, J.-B., Gao, Y., Deconde, R., Chen, M., Rajapakse, I., Friend, S., Ideker, T., & Zhang, K. (2013). Genome-wide Methylation Profiles Reveal Quantitative Views of Human Aging Rates. Molecular Cell, 49(2), 359–367. https://doi.org/10.1016/j.molcel.2012.10.016

Jin, B., Li, Y., & Robertson, K. D. (2011). DNA Methylation. Genes & Cancer, 2(6), 607–617. https://doi.org/10.1177/1947601910393957

Kulis, M., & Esteller, M. (2010). 2—DNA Methylation and Cancer. In Z. Herceg & T. Ushijima (Eds.), Advances in Genetics (Vol. 70, pp. 27–56). Academic Press. https://doi.org/10.1016/B978-0-12-380866-0.60002-2

Molecules discovered that extend life in yeast, human cells. (n.d.). EurekAlert! Retrieved March 5, 2023, from https://www.eurekalert.org/news-releases/664233.

Moore, L. D., Le, T., & Fan, G. (2013). DNA Methylation and Its Basic Function. Neuropsychopharmacology, 38(1), 23–38. https://doi.org/10.1038/npp.2012.112

Offord, Catherine. Two research teams reverse signs of aging in mice. (n.d.). Retrieved March 14, 2023, from https://www.science.org/content/article/two-research-teams-reverse-signs-aging-mice.

Takahashi, K., & Yamanaka, S. (2006). Induction of Pluripotent Stem Cells from Mouse Embryonic and Adult Fibroblast Cultures by Defined Factors. Cell, 126(4), 663–676. https://doi.org/10.1016/j.cell.2006.07.024

What is Epigenetics? The Answer to the Nature vs. Nurture Debate. (n.d.). Center on the Developing Child at Harvard University. Retrieved March 5, 2023, from https://developingchild.harvard.edu/resources/what-is-epigenetics-and-how-does-it-relate-to-child-development/

Wu, H., & Sun, Y. E. (2009). Reversing DNA Methylation: New Insights from Neuronal Activity–Induced Gadd45b in Adult Neurogenesis. Science Signaling, 2(64), pe17–pe17. https://doi.org/10.1126/scisignal.264pe17

Wu, H., & Zhang, Y. (2014a). Reversing DNA Methylation: Mechanisms, Genomics, and Biological Functions. Cell, 156(0), 45–68. https://doi.org/10.1016/j.cell.2013.12.019

Yang, J.-H., Hayano, M., Griffin, P. T., Amorim, J. A., Bonkowski, M. S., Apostolides, J. K., Salfati, E. L., Blanchette, M., Munding, E. M., Bhakta, M., Chew, Y. C., Guo, W., Yang, X., Maybury-Lewis, S., Tian, X., Ross, J. M., Coppotelli, G., Meer, M. V., Rogers-Hammond, R., … Sinclair, D. A. (2023). Loss of epigenetic information as a cause of mammalian aging. Cell, 186(2), 305-326.e27. https://doi.org/10.1016/j.cell.2022.12.027

 

Teplizumab: A New Breakthrough in the Treatment of Type 1 Diabetes

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     In Ancient Egypt, a diet of whole grains was prescribed to patients with frequent urination and emaciation (History of diabetes, 2020).  This condition, similarly documented by physicians in Ancient Greece, was likely first called “diabetes” by Apollonius of Memphis in the third century BCE. By the fifth century CE, type 1 diabetes had been differentiated from type 2, and in 1776, English physician Matthew Dobson confirmed the presence of excess glucose in the urine of diabetic patients. Canadian physician Frederick Banting and his colleagues successfully used insulin injections to treat a diabetic patient in 1922; this remains the predominant method of treatment today. Another milestone was reached this past November, when the drug Teplizumab (under the name Tzield) gained FDA approval. Teplizumab showed potential in delaying the onset of clinical type 1 diabetes in adults and pediatric patients 8 years and older who have not yet developed the condition (Commissioner of the FDA, 2022). 

     Type 1 diabetes, otherwise known as juvenile diabetes or insulin-dependent diabetes, is a form of diabetes mellitus in which a deficiency of insulin causes hyperglycemia (high blood sugar). It is referred to as “juvenile diabetes” because the symptoms of the condition typically appear in adolescence and is the result of an autoimmune disorder. The onset of type 1 diabetes depends on environmental factors that interact with predisposing genes to induce a long-term autoimmune attack against the pancreatic β cells, the insulin-producing cells of the pancreatic islets of Langerhans (de Beeck and Eizrik, 2018). 

     When left untreated, juvenile diabetes poses serious health risks. The buildup of sorbitol—a sugar alcohol that is manufactured from glucose—within the eye can result in cataracts and blindness (CDC, 2022).  Excess sorbitol is also associated with blood vessel lesions and gangrene. Other significant health risks for individuals with juvenile diabetes include ketoacidosis, the buildup of acidic ketones that can cause diabetic comas and diminished brain function, and hypoglycemia, otherwise known as “insulin shock,” resulting from an overdose of insulin or the failure to eat and potentially causing nerve damage and death (CDC, 2022). Approximately 1.6 million Americans live with type 1 diabetes, including 200,000 youth. Furthermore, despite significant developments in the treatment of type 1 diabetes, desired glycemic targets are rarely achieved in patients, who continue to face a higher risk of complications and death because of their condition (Herold et. al., 2019). 

     In those who are genetically susceptible to type 1 diabetes, there are two asymptomatic stages prior to the development of overt hyperglycemia, the clinical disease which requires insulin treatment. Stage 1 is characterized by the appearance of autoantibodies targeting pancreatic cells and stage 2 involves dysglycemia, an abnormality in blood sugar stability (Herold et. al., 2019). In this case, metabolic responses to high levels of glucose could be impaired, but other metabolic indexes, such as the level of glycosylated hemoglobin, are normal. During these stages, insulin treatment is not required. The goal of Teplizumab is to delay the development of clinical (stage 3) diabetes in those currently in stages 1 and 2 (Herold et. al., 2019). 

     Teplizumab is an Fc receptor–nonbinding anti-CD3 monoclonal antibody that modifies CD8+ T lymphocytes, which are part of the body’s adaptive immune response and thought to be important effector cells that kill β cells in the pancreas (Herold et. al., 2019). Evidence indicates that type 1 diabetes is initiated by both CD4+ and CD8+ T cells (Li and Qin, 2014). Autoreactive T cells differentiate into effector (CD4+) cells by engaging β-cell antigens on local antigen-presenting cells; these effector CD4+ T cells stimulate other immune cells to target β cells, whereas the cytotoxic CD8+ T cells can directly kill β cells via cell-to-cell contact (Gearty et. al., 2022). The CD3 protein complex is involved in activating both the cytotoxic and helper T-cells (Yang et. al., 2005); thus, the anti-CD3 properties of Teplizumab can inhibit the T cell–mediated damage to β-cells. 

     In the Teplizumab trial, which was conducted over a 7-year period and led by Dr. Kevan Herold of Yale University, patients were randomly assigned to a single 14-day course of Teplizumab or placebo. Seventy-two percent of the participants were under the age of 18, and the majority had siblings with clinical type 1 diabetes, meaning they were at a high risk of developing the condition themselves (Herold et. al., 2019). Furthermore, of the 55 patients who were under 18, 47 had a confirmed dysglycemic oral glucose-tolerance test, one of the hallmarks of stage 2 type 1 diabetes, before undergoing randomization in the trial. Follow-up for progression to clinical type 1 diabetes was performed via oral glucose-tolerance tests every 6 months. The study indicated that a 2-week course of treatment with Teplizumab delayed the diagnosis of clinical type 1 diabetes in high-risk participants: following the completion of the trial, 57% of individuals in the Teplizumab trial group were diabetes-free compared to 28% in the placebo group, with a median delay in the diagnosis of clinical diabetes of 2 years (Figure 1). These results also reinforced prior findings that type 1 diabetes is a T-cell mediated condition and showed that immunomodulation before the onset of clinical type 1 diabetes is a promising development in the treatment of this disease (Herold et. al., 2019).

 

Figure 1. From the time of randomization until the clinical diagnosis of type 1 diabetes, the recipients of the Teplizumab infusion experienced a longer median time until diagnosis than those in the placebo group (adapted from K. C. Herold, et. al.). 

     Given the prevalence of patients with clinical type 1 diabetes, these results of this study are highly promising and a significant step forward in the mitigation of the harmful side effects of this condition. However, there are several areas which, in an effort to make the benefits of this trial widely applicable, require further research. Since the participants in the study were all relatives of patients with type 1 diabetes, it is currently unknown whether the findings can be applied to those who seem to be at risk for the type 1 diabetes and lack first-degree relatives with the condition (Evans-Molina and Oram, 2023). Furthermore, while patients can be carefully screened for the immunological or metabolic markers of preclinical type 1 diabetes in the research setting, a lack of infrastructure prevents a larger-scale screening of the general public and high-risk populations are typically the only groups surveyed for these symptoms (Evans-Molina and Oram, 2023). Finally, despite the promising results of the newly-approved drug, the current cost of treatment presents a significant barrier regarding access to care: one vial of Tzield costs $13,850, amounting to $193,000 over the 14-day infusion (Rodriguez, n.d.).  The exorbitant sum is not surprising, given the high cost of insulin medications and their widely-reported underuse (Herkert et. al., 2019). As a result, further efforts must be taken to ensure that those who are eligible for this new and potentially life-saving medication are able to access it. 

 

Sources: 

CDC. (2022, November 3). Prevent diabetes complications. Centers for Disease Control and Prevention. https://www.cdc.gov/diabetes/managing/problems.html 

Commissioner, O. of the. (2022, November 18). FDA approves first drug that can delay onset of type 1 diabetes. FDA. https://www.fda.gov/news-events/press-announcements/fda-approves-first-drug-can-delay-onset-type-1-diabetes 

de Beeck, A. O., & Eizirik, D. L. (2016). Viral infections in type 1 diabetes mellitus—Why the β cells? Nature Reviews. Endocrinology, 12(5), 263–273. https://doi.org/10.1038/nrendo.2016.30 

Dolgin, E. (2023). How a pioneering diabetes drug offers hope for preventing autoimmune disorders. Nature, 614(7948), 404–406. https://doi.org/10.1038/d41586-023-00400-x 

Evans-Molina, C., & Oram, R. A. (2023). Teplizumab approval for type 1 diabetes in the USA. The Lancet Diabetes & Endocrinology, 11(2), 76–77. https://doi.org/10.1016/S2213-8587(22)00390-4 

Gearty, S. V., Dündar, F., Zumbo, P., Espinosa-Carrasco, G., Shakiba, M., Sanchez-Rivera, F. J., Socci, N. D., Trivedi, P., Lowe, S. W., Lauer, P., Mohibullah, N., Viale, A., DiLorenzo, T. P., Betel, D., & Schietinger, A. (2022). An autoimmune stem-like CD8 T cell population drives type 1 diabetes. Nature, 602(7895), 156–161.  https://doi.org/10.1038/s41586-021-04248-x 

Herkert, D., Vijayakumar, P., Luo, J., Schwartz, J. I., Rabin, T. L., DeFilippo, E., & Lipska, K. J. (2019). Cost-related insulin underuse among patients with diabetes. JAMA Internal Medicine, 179(1), 112–114. https://doi.org/10.1001/jamainternmed.2018.5008 

Herold, K. C., Bundy, B. N., Long, S. A., Bluestone, J. A., DiMeglio, L. A., Dufort, M. J., Gitelman, S. E., Gottlieb, P. A., Krischer, J. P., Linsley, P. S., Marks, J. B., Moore, W., Moran, A., Rodriguez, H., Russell, W. E., Schatz, D., Skyler, J. S., Tsalikian, E., Wherrett, D. K., … Greenbaum, C. J. (2019). An anti-cd3 antibody, teplizumab, in relatives at risk for type 1 diabetes. New England Journal of Medicine, 381(7), 603–613. https://doi.org/10.1056/NEJMoa1902226 

History of diabetes: Early science, early treatment, insulin. (2020, June 17). https://www.medicalnewstoday.com/articles/317484 

Li, M., Song, L.-J., & Qin, X.-Y. (2014). Advances in the cellular immunological pathogenesis of type 1 diabetes. Journal of Cellular and Molecular Medicine, 18(5), 749–758. https://doi.org/10.1111/jcmm.12270 

Masharani, U. B., & Becker, J. (2010). Teplizumab therapy for type 1 diabetes. Expert Opinion on Biological Therapy, 10(3), 459–465. https://doi.org/10.1517/14712591003598843 

Rodriguez, A. (n.d.). FDA approves first treatment that delays Type 1 diabetes. Why it could be “game changing.” USA TODAY. Retrieved April 2, 2023, from https://www.usatoday.com/story/news/health/2022/11/18/fda-approves-teplizumab-delays-onset-diabetes/10721707002/ 

Yang, H., Parkhouse, R. M. E., & Wileman, T. (2005). Monoclonal antibodies that identify the CD3 molecules expressed specifically at the surface of porcine γδ-T cells. Immunology, 115(2), 189–196. https://doi.org/10.1111/j.1365-2567.2005.02137.x 



The Battle of the Medications: The Connection Between Antidepressants and Antibiotic Resistance in Bacteria

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         The consumption of antidepressant medications has skyrocketed in recent decades, reaching more than 337 million prescriptions written in 2016 in the United States alone (Wang et. al. 2019). For many individuals, these drugs are critical to maintaining everyday health as they treat many life-threatening psychiatric disorders. While their exact mechanisms differ, these medications travel in the bloodstream to the brain where they are able to influence the release of chemicals known as neurotransmitters that generate emotional states. However, while their intended target is the brain, these drugs continue to circulate throughout the body, thereby interacting with other organs and structures (Wang et. al. 2019).

         In their 2019 study, researchers led by Iva Lukic used data indicating the presence of antidepressants in the digestive tract to investigate the effect of these medications on the gut microbiome. After treating mice with different types of antidepressants, the team noticed a change in the types of bacteria present within the gut when compared to controls (Lukic et. al. 2019). This discovery that antidepressants could impact the types of bacteria present within the body ultimately led researcher Jianhua Guo to question the additional effects that these medications could have on bacteria. As antibiotics have also been shown to affect the composition of the gut microbiome, Guo began by investigating if the antidepressant fluoxetine could help Escherichia coli cells survive in the presence of various antibiotics. After finding that exposure to this medication did increase E. coli’s resistance to antibiotic treatments, Guo decided to expand his hypothesis to examine the overall connection of antidepressant usage with antibiotic resistance in bacteria.

        Collaborating with researchers Zue Wang and Zhigang Yue, Guo’s lab began by choosing five major types of antidepressant medications: sertraline, escitalopram, bupropion, duloxetine, and agomelatine. These medications differ in the ways that they prevent the reuptake of serotonin and norepinephrine in the brain, thereby allowing the researchers to examine the effects of various types of antidepressants that may be prescribed to patients. Then, E. coli bacteria were added to media containing varying concentrations of these five antidepressants. Once these cells were treated with antidepressants, the researchers began to test the cells’ resistance against antibiotics. In order to accurately reflect antibiotic use in the real world, the tested antibiotics covered the six main categories of antibiotic medications available on the market. The antidepressant-treated bacteria were then swabbed onto plates containing one of the tested antibiotics to observe cell growth. Based on the growth present on these plates, the researchers were able to estimate the incidence rate of bacterial resistance of E. coli bacteria treated with different antidepressants.

         Through this experiment, the lab observed that E. coli cells grown in sertraline and duloxetine, two antidepressants that inhibit the reuptake of serotonin, exhibited the greatest number of resistant cells across all the tested antibiotics (fig. 1). They also noted that E. coli cells exhibiting resistance to one antibiotic often demonstrate some level of resistance to other antibiotics as well. After detecting a correlation between antibiotic-resistance development and exposure to antidepressants, the lab tested the concentration dependence of this effect. While lowering the concentration of antidepressants seemed to decrease the amount of resistant E. coli cells, resistant cells continued to appear on the plates over time, suggesting that lowering antidepressant dosages only prolongs the process of developing antibiotic resistance.

Figure 1: These graphs showcase the change in the number of antibiotic-resistant E. coli cells after exposure to antidepressants over sixty days. The title of each graph indicates the tested antibiotic while the colored trend lines on the graph represent one of the five, tested antidepressants. On the y-axis of each graph, the fold change measurement is used to describe the change in the number of resistant cells that develop over time. As demonstrated by the purple and yellow trend lines, duloxetine and sertraline are associated with the greatest development of resistant cells to each of the four represented antibiotics. (Adapted from Wang et. al. 2019)

         After analyzing this data, the researchers were confronted with a question: what about anti-depressants led to the development of antibiotic resistance in bacteria? To examine this question, the lab used flow cytometry to examine what was happening within the bacterial cells. This lab technique uses a fluorescent dye that binds to specific intercellular target molecules, thereby allowing these components to be visualized. After applying this dye to resistant cells grown on the antibiotic agar plates, the researchers noticed the presence of specific oxygen compounds known as reactive oxygen species (ROS). Unstable ROS bind to other molecules within a cell, disrupting normal functioning and causing stress. Elevated cellular stress levels have been shown to induce the transcription of specific genes in bacteria that produce proteins to help return the cell to normal functioning (Wang et. al. 2019).

         ROS molecules have been shown to induce the production of efflux pumps in bacteria, leading the lab to investigate if these structures were involved in the antibiotic resistance of E. coli cells. Efflux pumps are structures in the cell membrane of a protein that pump harmful substances out of the cell. The lab mapped the genome to look for activated genes associated with the production of this protein. According to the computer model, more DNA regions in resistance bacteria coding for efflux pumps were active than in the Wild Type. The researchers then concluded that efflux pumps were being produced in response to antidepressant exposure. These additional efflux pumps removed antibiotic molecules in resistant E. coli, thereby allowing them to survive in the presence of lethal drugs.

         The antibiotic resistance uncovered in this study was significant and persistent. Even one day of exposure to antidepressants like sertraline and duloxetine led to the presence of resistant cells. Furthermore, the team demonstrated that these antibiotic-resistant capabilities often do not disappear over time; rather, they are inherited between generations of bacteria, leading to the proliferation of dangerous cells unsusceptible to available treatments. The next logical step towards validating the connection between antidepressants and antibiotic resistance would include studying the gut microbiomes of patients taking anti-depressants to look for antibiotic-resistant bacteria.

         This study reveals a novel issue that must be attended to.  In 2019, 1.27 million deaths worldwide could be directly attributed to antibiotic-resistant microbes, a number expected to grow to 10 million by the year 2050 (O’Neill 2023). These “superbugs” present a dangerously growing reality. If the correlation between antidepressant use and antibiotic resistance is left uninvestigated, superbugs will likely continue to develop even as antibiotic use is regulated and monitored to battle them. Only by taking this connection seriously will researchers be able to fully grapple with and battle the growing antibiotic resistance trends, thereby preventing common infections from becoming death sentences. 

 Sources:

CDC. (2022, July 15). The biggest antibiotic-resistant threats in the U.S. Centers for Disease Control and Prevention. https://www.cdc.gov/drugresistance/biggest-threats.html

Drew, L. (2023). How antidepressants help bacteria resist antibiotics. Nature. https://doi.org/10.1038/d41586-023-00186-y

Jin, M., Lu, J., Chen, Z., Nguyen, S. H., Mao, L., Li, J., Yuan, Z., & Guo, J. (2018). Antidepressant fluoxetine induces multiple antibiotics resistance in Escherichia coli via ROS-mediated mutagenesis. Environment International, 120, 421–430. https://doi.org/10.1016/j.envint.2018.07.046 

Lukić, I., Getselter, D., Ziv, O., Oron, O., Reuveni, E., Koren, O., & Elliott, E. (2019). Antidepressants affect gut microbiota and Ruminococcus flavefaciens is able to abolish their effects on depressive-like behavior. Translational Psychiatry, 9(1), 1–16. https://doi.org/10.1038/s41398-019-0466-x

O’Neill, J. (Ed.). (2016). Tackling Drug-Resistant Infections Globally: Final Report and Recommendations. The Review on Antimicrobial Resistance. https://amr-review.org/sites/default/files/160518_Final%20paper_with%20cover.pdf

Thompson, T. (2022). The staggering death toll of drug-resistant bacteria. Nature. https://doi.org/10.1038/d41586-022-00228-x

Wang, Y., Yu, Z., Ding, P., Lu, J., Mao, L., Ngiam, L., Yuan, Z., Engelstädter, J., Schembri, M. A., & Guo, J. (2023). Antidepressants can induce mutation and enhance persistence toward multiple antibiotics. Proceedings of the National Academy of Sciences, 120(5), e2208344120. https://doi.org/10.1073/pnas.2208344120

Ferroptosis-Related LncRNAs Found in Colon Cancer

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Colon cancer is the malignant growth of tumor cells in the large intestine. It is the third most common cancer and has the fourth highest death rate out of all types of cancer. Current treatments are limited, as they can be painful for the patient by killing healthy cells alongside cancer cells, and there is no guarantee that the treatments will completely eliminate all cancer cells.

Ferroptosis is a type of programmed cell death caused by high intracellular iron levels, which in turn activates cell death pathways. It differs from traditional cell death, apoptosis, since it is triggered by high iron intracellular concentrations. Rather than affecting the cell’s genetic material or plasma membrane, ferroptosis causes cell death through shrinking mitochondria and increasing mitochondrial membrane density. 

Long noncoding RNAs (lncRNAs) are a type of RNA that does not code for protein synthesis. While they don’t code for protein, lncRNAs have other functions, such as controlling gene regulation through unwinding chromatin for transcription and consequent translation and RNA processing. In regards to cancer, lncRNAs have been proven to contribute to proliferation, metastasis, and reproduction of malignant cells, and can therefore be indicators of the disease and its prognosis. Ferroptosis-related lncRNAs (FRLs), which influence the titular cellular process, in particular have been identified as possible indicators of cancer prognosis, yet not much is known.

The purpose of Wu et al (2022)’s study was to determine the molecular functions of FRLs in colon cancer. In this experiment, RNA sequencing data and genes related to ferroptosis were obtained from databases. In addition, human intestinal epithelial cells and various human colon cancer cell lines and colon cancer cell samples taken from patients at the Gastrointestinal Surgery Department of Xiangya 3rd Hospital were tested for cell composition via CIBERSORT, and had their RNA extracted for qRT-PCR and analysis. Malondialdehyde (MDA), Fe2+, reactive oxygen species (ROS), and IC50 levels of various drugs were also tested in these cells, as they all have a role in controlling ferroptosis and the consequent cell death. It was found that 26 different FRLs had some relationship to colon cancer, most of them being risk genes, genes specifically associated with the onset of cancer. Two lncRNAs, AP003555.1 and AC005841.1, had a significant relationship to colon cancer, as seen by the increased MDA, Fe2+, and ROS levels in cells with those two lncRNAs silenced and their knockout inhibiting cell proliferation.

 

Figure 1: Construction and validation of the ferroptosis-related lncRNA signature model in the training cohort, validation and overall groups. (A–C) The distribution of the risk scores and the distributions of overall survival status and risk score in the training, validation and overall groups. (D–F) The Kaplan–Meier curves for survival status and survival time in the training, validation and overall groups. (G–I) The receiver operating characteristic (ROC) curve shows the potential of the prognostic ferroptosis-related lncRNAs signature in predicting 1-, 2-, and 3-year overall survival (OS) in the training, validation and overall groups. (J–L) AUC of ROC curves comparing the prognostic accuracy of the risk score and other prognostic factors in the training, validation and overall groups.

 

Sources

Li, Jie, Feng Cao, He-liang Yin, Zi-jian Huang, Zhi-tao Lin, Ning Mao, Bei Sun & Gang Wang (2020), Ferroptosis: past, present and future, Cell Death and Disease, Volume 11, Issue 2, Page 88

 

Mármol, Inés, Cristina Sánchez-de-Diego, Alberto Pradilla Dieste, Elena Cerrada, and María Jesús Rodriguez Yoldi (2017) Colorectal Carcinoma: A General Overview and Future Perspectives in Colorectal Cancer, International Journal of Molecular Sciences, Volume 18, Issue 1, Pages 197. 

 

Qian, Yuchen, Lei Shi, and Zhong Luo (2020) Long Non-coding RNAs in Cancer: Implications for Diagnosis, Prognosis, and Therapy, Frontiers in Medicine

 

Wu, Zhiwei, Zhixing Lu1, Liang Li, Min Ma, Fei Long, Runliu Wu, Lihua Huang, Jing Chou, Kaiyan Yang, Yi Zhang, Xiaorong Li, Gui Hu, Yi Zhang, and Changwei Lin (2022) Identification and Validation of Ferroptosis-Related LncRNA Signatures as a Novel Prognostic Model for Colon Cancer, Sec. Cancer Immunity and Immunotherapy, Volume 12

 

Yao, Run-Wen, Yang Wang & Ling-Ling Chen (2019) Cellular functions of long noncoding RNAs, Nature Cell Biology, Volume 21, Issue 5, Pages 542-551

 

Yu, Haitao, Pengyi Guo, Xiaozai Xie, Yi Wang, and Gang Chen (2017) Ferroptosis, a new form of cell death, and its relationships with tumourous diseases, Journal of Cellular and Molecular Medicine, Volume 21, Issue 4, Pages 648–657

 

Zhang, Kaiming, Liqin Ping, Tian Du, Gehao Liang, Yun Huang, Zhiling Li, Rong Deng, and Jun Tang (2021) A Ferroptosis-Related lncRNAs Signature Predicts Prognosis and Immune Microenvironment for Breast Cancer, Frontiers in Molecular Bioscience

Targeting the MYC Proto-Oncogene, BHLH Transcription Factor (MYC) interaction network in B-cell lymphoma via histone deacetylase 6 inhibition

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According to the World Health Organization (WHO), in 2020, cancer was responsible for the deaths of almost ten million people worldwide. Such statistics place cancer as a leading cause of death worldwide, second to heart disease. Cancer is when a series of mutations occurs in a cell, resulting in uncontrollable cellular division that eventually leads to interference in the function of vital organs. One of the more common types of cancer is lymphoma, the malignant growth of tumor cells of the lymphatic system. Current treatments for lymphoma include radiation therapy and chemotherapy, but these treatments can have drawbacks: they can be painful for the patient by killing healthy cells alongside cancer cells, and there is no guarantee that the treatments will completely eliminate all cancer cells. With a treatment that specifically targets the malignant cells, we can better treat lymphoma as well as other types of cancers.

MYC is a gene that when expressed in moderation, is responsible for maintaining cellular functions such as the cell cycle, apoptosis (programmed cell death), and protein production. It does so through “recruiting” enzymes such as histone acetyltransferases p300/CBP or the histone deacetylases (HDACs) to regulate expression of other genes. However, dysregulation of MYC expression can cause these cell functions to lose control as HDACs will have no means of regulation, genes to aid in the increase in cellular processes and pathways that would lead to the cell to become cancerous. MYC has also been found to be overexpressed in other types of cancers, such as uterine leiomyosarcoma.

The purpose of this project was to determine the effect of HDAC6 inhibitor Marbostat-100 (M-100) on oncogenic MYC expression levels in mice with MYC-induced aggressive B-cell lymphoma. In this experiment, mice with B-cell lymphoma as well as human B cell lymphoma cells were treated with various concentrations of M-100. It was found that all experimental concentrations of M-100 caused HDAC inhibition and reduction of MYC expression and protein levels, consequently inducing apoptosis in the murine and human cancer cells and statistically significantly increasing the mice’s survival rates. Therefore, MYC inhibition could be a possible therapeutic treatment for cancers like B-cell lymphoma.

Sources

https://www.nature.com/articles/s41388-022-02450-3

https://www.who.int/news-room/fact-sheets/detail/cancer

https://www.cancer.gov/about-cancer/treatment/types