berkeley pharma tech journal of medicine correspondence:� ojeijdiboesbo��!hnbjm�dpn keywords: covid-19 masks� mask mandates public policy sars-cov2 submitted�.bz��� ������� "ccepted�jvof��� ������ 1vcmjtife�+vmz��� ����� full open access creative commons attribution� license 4.0 abstract on march 11, 2020, the world came to a standstill in a truly unprecedented manner. shutdown schools, empty grocery aisles, and barren streets shifted from unfamiliarity to the new normal. perhaps the most significant of these changes was the widespread usage of protective masks to curb the spread of sars-cov2, more commonly known as covid-19. along with the constant influx of changes resulting from the covid-19 pandemic has been an onslaught of controversy regarding whether individuals should be legally required to wear masks. as of march 11, 2022, several states have announced that masks are no longer mandated. from what has been observed, there are two major stances on this subject — those who oppose the lifting of the mandate, and those who are in favor. this has provided a unique opportunity to demonstrate the tenuous relationship between public policy and scientific fact, and how the two are not mutually exclusive. this analysis seeks to dissect these views and present the findings through an evidence-based lens. .btl�.boebuft��5if� $pouspwfstz�$poujovft �ïw�§ßúþß��þ×äúè×ä introduction when the pandemic �rst began, much was unknown about the transmission mechanism of sars-cov2. it was known that the virus was transferred from human to human, but it was not until the fall of 2020 that the cdc reported that sars-cov2 is an airborne virus, meaning that the respiratory droplets of an infected individual can be transmitted to another through actions such as speaking, coughing, and sneezing. an infectious exposure constitutes inhaling viral particles, the deposition of the virus on mucous membranes (i.e. mouth, nose, eyes), or touching one’s mucous membranes with contaminated surfaces1. given that the transmission is respiratory in nature and the face has many points of potential viral entry, experts proposed the implementation of masking around other people to minimize the spread of covid-19. initially, due to resource limitations, cloth masks and bandanas were acceptable for this purpose. as time went on and cases reached record-breaking highs, there was a transition to surgical masks and n95s, which are specially designed to have a high �ltration capacity to block small respiratory particles2. mask controversy as of march 12th, california is one of the many states that have lifted mask mandates. those who are in favor of this decision, alongside even anti-vaxxers, have voiced their opinions as to why it was the right choice. perhaps one of the most commonly discussed reasons is the inconveniences that come with mask use, including di�culty breathing, communicating, and many complaining of the discomfort of wearing a mask for long hours3. there are many voices that believe that requiring masks is an infringement of civil liberties, as it takes away one’s sense of bodily autonomy3. in a survey published by the national panel study of covid-19, it was found that 24% of americans refrained from wearing a mask due to discomfort, while 40% did not wear one because they simply believed it was their fundamental right to decide4. these two factors alone accounted for more than half of the respondents who participated in the study. others believe that masks perpetuate fear. this is especially a topic of concern among minority groups, with african americans reporting the fear berkeley pharma tech journal of medicine | 140 of presumed criminality due to the racial discrimination that still prevails in the u.s. today5. in addition to racial concerns, the mask itself has become a de facto symbol of the pandemic, a visual representation and reminder of the loss and trauma that has ensued, as well as the uncertain future6. many believe that the perception of risk can be equally, if not more, detrimental than the risk of being infected with the virus itself, demonstrating the importance of sociological factors when it comes to understanding the american mask controversy. another salient impact of the pandemic has been that of politics being brought to the forefront of public discussion. while some believe that the decision to wear a mask should be purely based on scienti�c data, the reality is that partisan political a�liation has a signi�cant impact on the matter. according to public opinion polls, those on the more liberal end of the political spectrum are more likely to wear masks compared to conservatives3. published in the journal preventive medicine, a study analyzing covid-19 related risky behaviors and party a�liation found that republicans were less likely to wear a mask for 4 of the 6 activities included in the study7. this is also supported by a probability-based online survey by the gallup panel, reporting that 70% of republicans wear a mask in indoor settings, compared to a rate of 97% in the democrat population8. the opposing view regarding the lifting of the mask mandate is that it is simply too early to make this transition. those with this view have a variety of reasons as to why mask use should be continued, the common thread being safety. as of april 2022, covid-19 transmission has been signi�cantly lower; however, cases and covid-related deaths still occur regularly9. furthermore, despite low transmission rates, certain locations such as gyms, restaurants, and co�ee shops are considered to pose a much higher risk than others10. public transportation also ranks high on this list, with its high risk being associated with the close proximity to others as well as the exposure to frequently touched surfaces11. another point presented is the fact that humans are often asymptomatic carriers of covid, which contributes to the high transmissibility of the virus. in these cases, wearing a mask has been reported to be highly e�ective in mitigating the spread12. berkeley pharma tech journal of medicine | 141 expert opinions indicate that it is likely too soon to eliminate mask usage completely13. according to sources from johns hopkins university, while covid-19 cases and hospitalization rates are declining, there are risks to consider when assessing the removal of the mask mandate. crc vaccinology lead, dr. william moss, describes this as a time to practice “cautious optimism,” the hope being that cases will continue to decrease, and any additional variants will not overcome the immunity that has been acquired through natural infection as well as vaccination13. that being said, it has been shown that around 35% of the population still remains unvaccinated, despite the u.s. having one of the highest covid mortality rates when compared to those of other countries. this discrepancy is exacerbated by the disparity seen in vaccination between younger and older populations (fig 1). medical director of the johns hopkins biocontainment unit, brian garibaldi, expresses particular concern over school-aged children, with reports indicating that only 25% of children between the ages of 5-11 are fully vaccinated; these numbers are even lower in the younger cohort13. considering the fda approval of covid-19 booster shots for all adults in november of 2021, these numbers will likely increase as time goes on18. similarly, other populations of concern are the immunocompromised, those with comorbidities, and the elderly14. those who are highly susceptible to respiratory infections would greatly bene�t from masking, ideally using a kf94 or kn95 mask for maximal protection, despite the recent lifting of restrictions. experts have also vocalized the importance of addressing covid-19 misinformation. this includes myths such as the idea that herd immunity makes vaccination unnecessary, or the claim that heated water supposedly protects one from contracting the virus. many of these erroneous claims have been observed on social media platforms, ultimately adding to the already existing confusion surrounding masking; these issues can be addressed through the thorough reform of online data sharing regulations15. berkeley pharma tech journal of medicine | 142 figure 1. percentages of fully vaccinated people receiving a first covid-19 booster dose. image credits: covid.cdc.gov conclusion based on this analysis, it can be concluded that the decision to wear a mask cannot be adequately addressed using an all-or-nothing approach15. while cases and covid-related deaths have been signi�cantly reduced, the pandemic persists, and the virus remains unpredictable, as it is still evolving into novel variants14. currently, due to relatively low transmission rates, it is the logical choice to provide more autonomy to the public. it is important to note that this does not mean eliminating mask wearing under every circumstance, but rather that the decision should be made individually, after weighing risks and bene�ts. co-director of the national preparedness leadership initiative, dr. leonard marcus, reinforces the idea that low risk is not the equivalent of no risk, which is critical to keep in mind during this period of transition14. certain cohorts that are at an increased risk of contracting the virus, such as immunocompromised individuals or those with underlying health conditions, should ideally continue to mask at this time. vaccination status is also a crucial factor to consider, as increasing these rates could further reduce the necessity of masking. as of now, the us has much room to improve in this area. ultimately, regardless of one’s stance on masking, it is a practice that is proven to be e�ective in mitigating the spread of the virus (fig 2). berkeley pharma tech journal of medicine | 143 figure 2. average of new daily covid-19 cases in non-mask-mandated vs. mask-mandated counties19. image credits: cdc.gov moving forward, it will be important to address covid-19 misinformation which has unfortunately plagued the news media and various other online platforms. moreover, the importance of making decisions based on scienti�c fact rather than political opinion should be emphasized to the public as well. further research is needed to identify novel strategies to combat the spread of false information, with the optimization of online data showing great promise in this regard16. berkeley pharma tech journal of medicine | 144 references 1. scienti�c brief: sars-cov-2 transmission. centers for disease control and prevention. may 7, 2021. retrieved april 9, 2022, from https://www.cdc.gov/coronavirus/2019-ncov/s cience/science-briefs/sars-cov-2-transmission.ht ml 2. use of masks to control the spread of sars-cov-2. centers for disease control and prevention. december 6, 2021. retrieved april 9, 2022, from https://www.cdc.gov/coronavirus/2019-ncov/s cience/science-briefs/masking-science-sarscov2 .html 3. taylor, s, & asmundson, gjg. negative attitudes about facemasks during the covid-19 pandemic: the dual importance of perceived ine�ectiveness and psychological reactance. plos one. february 17, 2021. retrieved april 9, 2022, from https:// journals.plos.org/plosone/article?id=10 .1371% 2fjournal.pone.0246317 4. vargas, ed & sanchez, gr. american individualism is an obstacle to wider mask wearing in the us. brookings. august 31, 2020. retrieved april 9, 2022, from https:// www.brookings.edu/blog/up-front/20 20/08/31/american-individualism-is-an-obstacl e-to-wider-mask-wearing-in-the-us/ 5. howard, j, huang, a. zhiyuan li, & rimoin, a. w. an evidence review of face masks against covid-19. january 11, 2021. retrieved april 9, 2022, from https:// www.pnas.org/doi/10.1073/pnas.2014 564118 6. neilson, s. the surgical mask is a bad �t for risk reduction. cmaj. may 17, 2016. retrieved april 9, 2022, from https://www.cmaj.ca/content/188/8/606 7. schoeni, r. f., wiemers, e. e., seltzer, j. a., & langa, k. m. political a�liation and risk taking behaviors among adults with elevated chance of severe complications from covid-19. national center for biotechnology information. july 16, 2021. retrieved april 9, 2022, from https://www.ncbi.nlm.nih.gov/pmc/articles/p mc8284062/ 8. reinhart, r. j. u.s. face mask usage relatively uncommon in outdoor settings. gallup. august 7, 2020. retrieved april 10, 2022, from https://news.gallup.com/poll/316928/face-ma sk-usage-relatively-uncommon-outdoor-setting s.aspx 9. covid-19 united states cases by county. johns hopkins coronavirus resource center. (n.d.). retrieved april 10, 2022, from https:// coronavirus.jhu.edu/us-map 10. guarino, b., & achenbach, j. these venues are high-risk areas for spreading the coronavirus, model suggests. boston.com. november 11, 2020. retrieved april 10, 2022, from https://www.boston.com/news/ coronavirus/2 020/11/11/these-venues-arehigh-risk-areas-for -spreading-the-coronavirus-model-suggests/ 11. centers for disease control and prevention. requirement for face masks on public transportation conveyances and at transportation hubs. centers for disease control and prevention. february 25, 2020. retrieved april 8, 2022, from https://www.cdc.gov/coronavirus/2019-ncov/t berkeley pharma tech journal of medicine | 145 ravelers/face-masks-public-transportation.html 12. catching, a., capponi, s., yeh, m. t., bianco, s., & andino, r. examining the interplay between face mask usage, asymptomatic transmission, and social distancing on the spread of covid-19. national center for biotechnology information. august 6, 2021. retrieved april 8, 2022, from https://www.ncbi.nlm.nih.gov/pmc/articles/p mc8346500/ 13. donovan, d. (2022, february 11). experts discuss risks of ending mask mandates too soon. the hub. february 11, 2022. retrieved april 6, 2022, from https:// hub.jhu.edu/2022/02/11/experts-wary -of-revoking-mask-mandates-too-soon/ 14. the latest on the coronavirus. harvard school of public health. (n.d.). retrieved april 7, 2022, from https://www.hsph.harvard.edu/news/hsph-in-t he-news/the-latest-on-the-coronavirus/ 15. macmillan, c. is it really safe to end mask mandates in schools? yale medicine. march 1, 2022. retrieved april 7, 2022, from https://www.yalemedicine.org/news/mask-ma ndates-ending-in-schools 16. porter�elder, j. e. q&a: online data is an underutilized piece of the puzzle. johns hopkins coronavirus resource center. march 9, 2022. retrieved april 9, 2022, from https:// coronavirus.jhu.edu/pandemic-data-ini tiative/ expert-insight/q-and-a-online-data-is-anunderutilized-piece-of-the-data-puzzle 17. covid data tracker. centers for disease control and prevention. retrieved april 9, 2022, from https://covid.cdc.gov/covid-data-tracker/#vacci nation-demographics-trends 18. coronavirus (covid-19) update: fda expands eligibility for covid-19 vaccine boosters. u.s. food and drug administration. novermber 19, 2021. retrieved april 9, 2022, from https://www.fda.gov/news-events/press-annou ncements/coronavirus-covid-19-update-fda-exp ands-eligibility-covid-19-vaccine-boosters#:~:te xt=today%2c%20the%20u.s.%20food%20an d,fda%2dauthorized%20or%20approved%20 covid%2d 19. van dyke, me, rogers, tm, pevzner, e, satterwhite, cl, shah, hb, beckman, wj, ahmed, f, hunt, dc, & rule, j. trends in county-level covid-19 incidence in counties with and without a mask mandate kansas, june 1–august 23, 2020. centers for disease control and prevention. november 20, 2020. retrieved april 8, 2022, from https:// www.cdc.gov/mmwr/volumes/69/wr/ mm6947e2.htm berkeley pharma tech journal of medicine | 146 front_page_chandran chandran body berkeley pharma tech journal of medicine correspondence: wojtaram@umich.edu keywords: brain-skin connection hypothalamic-pituitary-adrenal axis (hpa) skin diseases melanoma psoriasis submitted: august 28, 2022 accepted: september 26, 2022 published: december 28, 2022 full open access creative commons attribution license 4.0 abstract the brain-skin connection has been a topic of growing interest in the neuroscience and dermatology communities. research has suggested a bi-directional relationship between the brain and skin. they share a common embryologic origin as both are derived from ectoblast differentiation and the skin has a fully functional peripheral equivalent of the hypothalamicpituitary-adrenal axis (hpa) which allows it to maintain homeostasis and interact with the brain. additionally, serotonin (5-ht), which is a well-known neurotransmitter, has been shown to act as a mediator between the skin and the neuroendocrine system. in the skin itself, 5-ht, which can be produced or metabolized by several skin cells, is involved in vasodilation, inflammation, and immunomodulation. the skin serves a neuroendocrine function through its role in maintaining homeostasis and its communication with the central neuroendocrine system. this connection also establishes that psychological stress can have effects on skin including the development or exacerbation of skin diseases such as melanoma and psoriasis. as our knowledge of the brain and neuroendocrine system grows, so does our understanding of this brain-skin connection. therefore, exploring the role of the brain-skin connection can help establish efficacious long-term therapeutic avenues for the treatment of skin diseases. brain-skin connection: considerations for novel therapies for skin diseases by: magda wojtara berkeley pharma tech journal of medicine | 105 1. introduction recent research has focused on the ‘brain-skin’ axis as a complex interplay between the nervous, endocrine, and immune systems, emphasizing the underlying pathophysiological mechanisms by which psychological stress influences skin homeostasis and clinical applications of this relationship1. perceived psychological stress has become widely known as a force that disrupts the dynamic equilibrium between the nervous, endocrine, and immune systems, thereby triggering and aggravating disease manifestation2. it has also been established in the past decade that the skin has a fully functional equivalent to the hypothalamus-pituitary-adrenal axis (hpa). this finding is crucial because it parallels how other parts of the body utilize the hpa axis to maintain homeostasis. it also shows a potential bidirectional relationship between the neuroendocrine system and the skin. the interplay between this hpa and the other systems can be explained by the schematic in figure 1, which shows how stress impacts the three axes and what downstream effects result from stress. suggested routes of the skin-brain axis include the immune system, hpa axis, and the peripheral and central nervous system3. the skin can be said to be a “diagnostic window into the brain.” the ‘brain-skin’ connection is complex because of the skin’s ability to produce its own serotonin (5-ht) and neurotrophins due to its common embryonic origin with the brain4. berkeley pharma tech journal of medicine | 106 figure 1: schematic representation of the three-stress axis. this includes the sympathetic nervous system, hpa axis, and neurotrophin-neuropeptide axis. our current knowledge of the ‘brain-skin’ axis has been derived from technological advances. graph theory is used to assess coordinated brain activity and estimate the efficiency of information flow5. it has been mathematically applied to psychosocial stress5. salivary cortisol, heart rate, and skin conductance are indices of stress, and psychosocial stress was associated with a decrease in the efficiency of the flow of information within the brain5. functional neuroimaging, especially functional magnetic resonance imaging (fmri), has enabled indirect visualization of brain function, which has been crucial for understanding more about this bidirectional relationship. a recent study has concluded that sensory testing, skin biopsy, and brain imaging show additional promise as pain biomarkers and should be considered for possible inclusion in the design of clinical trials of pain treatments6. another study utilized fmri to demonstrate cerebellar activity and connectivity in skin-picking disorder, which is now known to be a potentially maladaptive emotion regulation strategy7. prior to fmri research, electroencephalography (eeg) was widely utilized, dating back to studies in the 1950s investigating brain activity in syphilis patients. on the other hand, positron emission tomography (pet) allowed scientists to indirectly measure hemodynamic changes. notably, fmri has helped berkeley pharma tech journal of medicine | 107 scientists make strides in establishing their knowledge of crosstalk between the skin and the brain’s hpa axis. many common skin diseases are worsened by stress, and itch (pruritus) is the most well-known symptom associated with inflammatory skin diseases. several candidate molecules of this stress response, including corticotropinreleasing hormone (crh) and mast cells, have been shown to have strong pruritogenic potential1. mast cells, white blood cells that are abundant in the skin, occupy a switchboard position and play critical roles in regulating neurogenic inflammation during stress responses1. furthermore, there has been research showcasing close interactions between the nervous and immune systems in the regulation of peripheral inflammation which links stress with chronic somatic disease and aging8. emerging data suggests that chronic inflammations lead to the pro-inflammatory status, known as inflammaging, which underlies premature aging8,9. it is important to note that dynamic equilibrium disruptions among these systems can also be referred to as maladaptive plasticity. maladaptive plasticity induced by neuroendocrine mediators promotes inflammaging and it is believed that neurotrophins, neurotransmitters, and neuropeptides play a potential role in this process9. 2. mechanism of the brain-skin connection the skin is perhaps most well-known for its role as a physical barrier from the environment which requires it to have precise calibration and a high degree of local autonomy to perform daily functions. highly localized responses are coordinated partly by a skin neuroendocrine system that can reset adaptation mechanisms through either rapid (neural) or slow (humoral) pathways10. these pathways can act on local or systemic levels10. cutaneous responses have a primary goal of protecting, restoring, or maintaining homeostasis and dynamic equilibrium with other systems11. its fundamental functions are believed to have originated from its embryonic origin. however, under evolutionary pressure, it has been suggested that autoregulatory circuitry may have undergone specialization and separation resulting in new functions10. a variety of different neuromodulators involve in this connection, resulting in many proposed canonical and noncanonical pathways10. berkeley pharma tech journal of medicine | 108 one way the skin modifies homeostasis is through direct and indirect stimulation of the adrenal cortex (figure 2). psychological stress induces many downstream effects. for example, stress results in an increase in glucocorticoids which decreases differentiation and proliferation, thereby decreasing lipid synthesis, lamellar body production in the skin and ultimately resulting in abnormal permeability and abnormal stratum corneum integrity and cohesion. skin disorders are sometimes adversely affected by psychological stress and are frequently characterized by this defective cutaneous permeability barrier function10. this is due to the reduced density of lamellar bodies. the barrier to pathogenic microbes is also impacted because psychological stress reduces epidermal amp levels, which are needed to decrease the growth of microbes10. this ultimately results in increased severity of infections due to abnormalities brought on by psychological stress. figure 2: flowchart demonstrating how skin modifies body homeostasis through direct and indirect stimulation of the adrenal cortex. red lines indicate inhibition and green lines indicate activation. il represents interleukins, which aid t cells. tnf is a tumor necrosis factor. urc stands for urotropins and crf for corticotropin releasing factor. acth stands for acetylcholine. pomc is pro-opiomelanocortin, which is a precursor to acth. furthermore, there have been observations suggesting corticotropinreleasing factor (crf) driven responses are another important component berkeley pharma tech journal of medicine | 109 to the mechanism of the brain-skin connection10. these are properties displayed most clearly by neural crest-derived melanocytes and hair follicles which have activation sequences that closely reproduce basic features of the central hpa axis10. crf1 and crf2 are receptors of crf and may have unique characteristics and effects. this is especially interesting to explore as there are differential expressions of isoforms of crf1 resulting in coupling to different sets of signal transduction pathways. many current research projects are investigating the effector functions of crf1 and crf2 agonists and antagonists, specifically in their regulation of keratinocytes, cells that produce keratin, and immunocytes, cells that produce antibodies, activities. skin biomarkers for organ-specific diseases are another area of current research. it has recently been elucidated that alterations of the wnt signaling pathway may predict the aging status of the cardiovascular system, the brain, and bones11. this signaling pathway is well known in the scientific community for its role in cell determination. advanced glycosylation endproducts (age) and wnt pathway proteins are considered laboratory biomarkers for systemic disorders and are believed to be genderindependent11. other intrinsic and extrinsic parameters may also influence aging, but this process is highly variable on an individual basis11. another important contributor is uv energy. as uv light is absorbed by the skin, many downstream effects occur, resulting in mechanisms that defend skin integrity and induces skin pathology such as cancer. exposure to uv radiation is the principal cause of nonmelanoma skin cancer, a process in which serotonin (5-ht) is intimately involved12. the skin is a target for neuroendocrine signals from circulation and via nerve endings. it is believed that the uv touches the brain and central neuroendocrine system to reset body homeostasis13. there are three biologically relevant spectra of uv, including uvc (200-280 nm), uvb (280-320 nm), and uva (320 to 400 nm). among these, uvc is profoundly mutagenic and is absorbed by the stratum corneum13. uv is believed to stimulate both the intracutaneous and the cutaneous hpa (chpa) axis through neural and humoral mechanisms which are wavelength dependent and rely on anatomical structures sensing uv energy14. outside of this function, uva and uvb light also stimulates the opioidergic system, berkeley pharma tech journal of medicine | 110 specifically stimulating β-endorphin levels in the skin and the brain14. uvinduced immunosuppression is triggered via a cascade that begins with cisurocanic acid—an immune modulator produced in the stratum corneum that binds to the 5-ht receptor14. these connections provide another mechanism to explore and therapeutically target. 3. brain-skin connection and melanoma the brain-skin connection is key in explaining how and why psychological stress can impact the skin15. melanoma is a cancer of melanocytes—cells that give rise to skin tone, hair, and even eye color. it is also one of the most aggressive forms of skin cancer16,17. many individuals with melanoma have brain metastases which are severe complications, and the prognosis for patients with metastasis has a median survival time of 6 months after diagnosis18. melanoma cells can proliferate in the brain parenchyma or the meninges, and in cases where the blood-brain barrier is leaky, the lesions are also resistant to chemotherapeutic drugs18. a suggested mechanism involves very late antigen 4 (vla-4), which regulates immune recruitment to inflamed endothelium. vla-4 mediated adhesion of melanoma cells on the blood-brain barrier serves as a cue for melanoma cell intercalation and disruption of the barrier16. 92% of all human melanoma brain metastases stained vla-4 positive16. this finding has further implications for multiple sclerosis as intercellular adhesion molecule 1 (icam-1) and vascular cell adhesion molecule-1 (vcam-1), expressed at high levels during inflammation when vla-4 is expressed, contribute to the development of autoimmune encephalomyelitis (eae)16. recently, a reciprocal relationship has been demonstrated between parkinson’s disease (pd) and melanoma19. this is a relationship wherein pd patients are more susceptible to melanoma and vice versa. the current framework and hypothesis suggest that αsynuclein modulates the aggregation of pmel17, a functional amyloid that serves as a scaffold for melanin biosynthesis 19. research on αsynuclein can help the scientific community develop novel approaches to treating melanoma but also can reveal insights into its role in pd19. berkeley pharma tech journal of medicine | 111 4. psoriasis pathogenesis and antipsoriatic drug development psoriasis is a non-communicable chronic immune-mediated skin disease with pathogenesis derived from both genetic and environmental factors15. it has been previously established that 5-ht is a stress mediator that contributes to the effects of psychological stress on the disruption of skin homeostasis in a variety of skin diseases, including psoriasis15. psoriasis is mediated by t lymphocytes with th1 and th17 profiles20. some of the main triggers of psoriasis include trauma, non-steroidal anti-inflammatories, infections, and of course, psychological stress15. to garner a better understanding of psoriasis, we should first begin by investigating the processes that underlie skin immunity and neuroendocrinology15. psychological stress has been proposed to dysregulate the hpa axis, which thereby exacerbates psoriasis20. this is likely due to the release of proinflammatory cytokines and overactivation of the hpa axis20. sunlight deficiency strongly impacts the severity of psoriasis. nb-uvb, narrow-band uvb, is the only currently accepted form of treatment for psoriasis20. vitamin d deficiency is an important factor in the development and progression of psoriasis, and high doses of vitamin d were found to be efficient in eradicating psoriatic plaques20. this is also a deficiency that is common in both psoriasis and depression, and there may even be a causal relationship whereby vitamin d deficiency may increase the risk of depression20. mouse models provide the scientific community with a basis for studying psoriasis pathogenesis and antipsoriatic drug development20,21. it is not uncommon to utilize animal models to promote the discovery and development of drugs. the mouse model of psoriasis can be divided into spontaneous, genetically engineered (transgenic and knockout), xenotransplantation, and directly induced approaches21. animal models and other research has helped scientists delineate that il-23 mediates the th17 cells, which is a key part of the pathogenesis of psoriasis21. berkeley pharma tech journal of medicine | 112 5. conclusion overall, the scientific community has made significant strides, especially in the past decade, regarding our current knowledge on the ‘brain-skin’ axis. psychosocial stress, brought about by multiple factors, is a force that disturbs the dynamic equilibrium established between the nervous, endocrine, and immune systems, thereby triggering and aggravating disease manifestation1. there are many complexities intrinsic to these connections. currently, our understanding includes the sympathetic nervous system, hpa axis, and neutrophin-neuropeptide axis22. animal models and advancements in fmri technology have contributed to a growing body of knowledge and literature on these mechanisms and their relationships to potential therapeutic applications. this relationship has demonstrated impacts in research on melanoma and psoriasis as well as other diseases and conditions such as stroke and brain trauma23. future therapeutic avenues include the development of drugs that restore homeostasis in terms of the brain-skin connection, such as current research into agonists and antagonists directed toward 5-ht receptors24. berkeley pharma tech journal of medicine | 113 references 1. arck pc, slominski a, theoharides tc, peters em, paus r. neuroimmunology of stress: skin takes center stage. j invest dermatol. 2006;126(8):1697-1704. doi:10.1038/sj.jid.5700104 2. arck p, paus r. from the brain-skin connection: the neuroendocrine-immune misalliance of stress and itch. neuroimmunomodulation. 2006;13(5-6):347-356. doi:10.1159/000104863 3. weiglein a, gaffal e, albrecht a. probing the skinbrain axis: new vistas using mouse models. int j mol sci. 2022;23(13):7484. published 2022 jul 5. doi:10.3390/ijms23137484 4. mueller sm, hogg s, mueller jm, et al. functional magnetic resonance imaging in dermatology: the skin, the brain and the invisible. exp dermatol. 2017;26(10):845-853. doi:10.1111/exd.13305 5. wheelock md, rangaprakash d, harnett ng, et al. psychosocial stress reactivity is associated with decreased whole-brain network efficiency and increased amygdala centrality. behav neurosci. 2018;132(6):561-572. doi:10.1037/bne0000276 6. smith sm, dworkin rh, turk dc, et al. the potential role of sensory testing, skin biopsy, and functional brain imaging as biomarkers in chronic pain clinical trials: immpact considerations. j pain. 2017;18(7):757-777. doi:10.1016/j.jpain.2017.02.429 7. wabnegger a, schienle a. the role of the cerebellum in skin-picking disorder. cerebellum. 2019;18(1):91-98. doi:10.1007/s12311-018-0957-y 8. liezmann c, stock d, peters emj. stress induced neuroendocrine-immune plasticity. dermatoendocrinol. 2012;4(3):271-279. doi:10.4161/derm.22023 9. joachim ra, kuhlmei a, dinh qt, et al. neuronal plasticity of the "brain-skin connection": stress-triggered up-regulation of neuropeptides in dorsal root ganglia and skin via nerve growth factor-dependent pathways. j mol med (berl). 2007;85(12):1369-1378. doi:10.1007/s00109-007-0236-8 10. slominski a, wortsman j, paus r, elias pm, tobin dj, feingold kr. skin as an endocrine organ: implications for its function. drug discov today dis mech. 2008;5(2):137-144. doi:10.1016/j.ddmec.2008.04.004 11. zouboulis cc, makrantonaki e. clinical and laboratory skin biomarkers of organ-specific diseases. mech ageing dev. 2019;177:144-149. doi:10.1016/j.mad.2018.08.003 12. menezes ac, raposo s, simões s, ribeiro h, oliveira h, ascenso a. prevention of photocarcinogenesis by agonists of 5-ht1a and antagonists of 5-ht2a receptors. mol neurobiol. 2016;53(2):1145-1164. doi:10.1007/s12035-014-9068-z 13. slominski at, zmijewski ma, plonka pm, szaflarski jp, paus r. how uv light touches the brain and endocrine system through skin, and why. endocrinology. 2018;159(5):1992-2007. doi:10.1210/en.2017-03230 14. munz b, tretter yp, hertel m, engelhardt f, alzheimer c, werner s. the roles of activins in repair processes of the skin and the brain. mol cell endocrinol. 2001;180(1-2):169-177. doi:10.1016/s03037207(01)00514-7 15. martins am, ascenso a, ribeiro hm, marto j. the brain-skin connection and the pathogenesis of psoriasis: a review with a focus on the serotonergic system. cells. 2020;9(4):796. published 2020 mar 26. doi:10.3390/cells9040796 https://doi.org/10.4161/derm.22023 berkeley pharma tech journal of medicine | 114 16. garcía-martín ab, zwicky p, gruber t, et al. vla-4 mediated adhesion of melanoma cells on the blood-brain barrier is the critical cue for melanoma cell intercalation and barrier disruption. j cereb blood flow metab. 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toxicol methods. 2020;101:106657. doi:10.1016/j.vascn.2019.106657 22. pavlovic s, daniltchenko m, tobin dj, et al. further exploring the brain-skin connection: stress worsens dermatitis via substance p-dependent neurogenic inflammation in mice. j invest dermatol. 2008;128(2):434-446. doi:10.1038/sj.jid.5701079 23. munz b, tretter yp, hertel m, engelhardt f, alzheimer c, werner s. the roles of activins in repair processes of the skin and the brain. mol cell endocrinol. 2001;180(1-2):169-177. doi:10.1016/s03037207(01)00514-7 24. nordlind k, azmitia ec, slominski a. the skin as a mirror of the soul: exploring the possible roles of serotonin. exp dermatol. 2008;17(4):301-311. doi:10.1111/j.1600-0625.2007.00670.x front page wojtara body template_wojtara references_wojtara (2) abstract activation induced cytidine deaminase (aid) is an important enzyme that creates mutations in dna via deamination of a cytosine base into a uracil. aid, also referred to as activation induced deaminase (aicda), plays a crucial part in the human immune response as it is essential for isotype switching and cellular differentiation. however, aberrant expressions in some pathways has been implicated in a plethora of diseases. there is a pressing need for research and comparison of current literature that informs related therapies. previous studies have explored potential mechanisms by which aid works and subsequently ways to target gene therapies based on this information. due to aid’s complexity, there have been many challenges along the path that led to our current understanding of the beneficial and harmful nature of aid. furthermore, a better understanding of the way aid works can aid with the development of more efficacious therapies. although further research on the topic and additional testing in humans and animal models are needed, it is clear that aid may play an important role in the development of therapeutic treatments in diseases like cancer, lupus, and type 1 diabetes. "dujwbujpo�*oevdfe�%fbnjobtf�boe� 1pufoujbm�5ifsbqfvujd�"wfovft �ïw�¦×ýú×�°åàê×è× berkeley pharma tech journal of medicine correspondence:�� xpkubsbn@vnjdi�fev keywords: "*$%" -vqvt (fof�5ifsbqz "vupjnnvof $bodfs 5ifsbqfvujd�"ewbodft submitted�"qsjm 2�, 202�� accepted�juof���, 20��� 1vcmjtife�+vmz��� ����� full open access creative commons attribution�� license 4.0 activation induced cytidine deaminase image by emw own work, cc by-sa 3.0, https://commons.wikimedia.org/w/index.php?curid=8764396 introduction previous studies and research have established that aid regulates secondary antibody diversi�cation. there are many di�erent immunoglobulin (ig) diversi�cation processes, such as somatic hypermutation (shm), class switch recombination (csr), and gene conversion (gc)4. shm allows for b cells to diversify in order to respond to threats to the immune system,5 while csr allows for the generation of di�erent classes of antibodies5. gc is a process in which mutations can occur in the antibody genes5. aid is central to csr/shm and plasma cell di�erentiation and is encoded by aicda and b lymphocyte maturation protein 1 (blimp-1) which is a transcription factor encoded by prdm16. aid and its transcription factors underpin ab and autoantibody responses7 . the deamination results in a change from a cytosine base to a uracil base in ig genes, and this can result in either csr or shm, depending on the deoxyribonucleic acid (dna) repair pathway. aid expression is upregulated by in�ammatory cytokines like interferon-γ and tumor necrosis factor (tnf)-α which induces p53 mutations in in�ammatory or cancer cells. although aid is typically associated with and expressed in b-cells, it can also be expressed, for example, in embryonic germ cells or pluripotent cells like oocytes. aid proteins have been shown to be expressed during early b-cell development in both human fetal liver and adult bone marrow 9. it is important to note that aid is a potent enzyme which instigates genomic diversity for both bene�cial10 and harmful outcomes in humans. this can best be depicted in figure 1, which summarizes much of the following section. aid di�ers from other apolipoprotein b mrrna editing catalytic polypeptides (apobecs) speci�cally due to the size and orientation of its substrate speci�city loop12. it has a larger loop that extends away from the active site and thus can accommodate two purines next to a target c12. despite some sequence similarity to apobec cytidine deaminases, aid’s critical function in ab diversi�cation in csr cannot be substituted by other apobec proteins10. while aberrant deaminase activity can certainly threaten the genome, recent biotechnological e�orts have focused on harnessing and targeting deaminase activity in base editors that are related to aid11. berkeley pharma tech journal of medicine | 108 figure 1: a diagram summarizing some of the beneficial and harmful outcomes of aid aid, a potent dna mutator, must be tightly regulated to prevent any o �-target e�ects which can result in a plethora of problems including mutations in non-ig genes, genomic instability, interchromosomal translocations, and cellular neoplastic transformation13. aid has previously been implicated in the tumorigenic process in b cell tumors potentially through the induction of chromosomal translocations and mutations in tumor suppressor genes and oncogenes14. aid expression has also been implicated in the pathogenesis of human b cell malignancies15. indeed, accumulating evidence suggests aid is pro-oncogenic and induces cancerpromoting mutations or chromosomalrearrangements16. another detrimental impact of aid is the generation of autoimmunity, which can occur after on-target point mutations in variable genes produce antibodies with high a�nity for self-proteins11. these detrimental e�ects are important to consider when choosing to target aid in potential research projects. other studies have proposed more novel functions for aid. for instance, one has suggested that aid functions as an adaptor protein that represses viral transcription, which would have implications for the development of anti-hiv therapeutics and other therapies 17. moreover, aid can exert non canonical functions when aberrantly expressed in epithelial cells and was berkeley pharma tech journal of medicine | 109 long known to lack speci�c inhibitors which prevented therapeutic applications to modulate aid functions18. mechanism historically, the discovery of aid and its essential role in antibody diversi�cation kindled a debate over aid’s nucleic acid substrate, which inspired the dna deamination model and identi�cation of downstream players in the csr pathway such as the dna glycosylase ung19. subsequently, this discovery led to a race to uncover ssdna as the target of aid’s enzymatic activity and its dependence on transcription for deamination19. it is important to note that aid deaminates c→u only on ssdna and does not function on dsdna or rna12. figure 2 depicts this type of deamination reaction. nearly two decades prior to this research, there was an observation that noncoding rna transcripts originated from within the igh locus and could be playing a role in csr19. figure 2: cytosine to uracil deamination reaction wherein an amine group is removed shm generates point mutations in the ig variable regions while csr exchanges the ig heavy chain constant region, and ultimately this gives rise to antibodies with enhanced a�nity and new e�ector functions20. aid initiates both these processes through deamination of cytosine to uracil in ig variable and switch gene regions, and the resultingu:g mismatches are berkeley pharma tech journal of medicine | 110 subsequently processed by uracil-dna glycosylase (ung2) and a pathway that requires the mechanism by which aid works has not been entirely discerned. namely, the mechanism of aid targeting has especially been a long-standing mystery10. currently, there are many di�erent �ndings that are piecing together the puzzle of how exactly aid works. in terms of frequency, the number of molecules containing deamination in both dna strands at the acceptor switch region corresponds to its class switch e�ciency. it has been proposed that the minimal requirement for a dna double-strand break (dsb) formation is as low as only one aid deamination event on both dna strands4. there are also several proposed mechanisms for aid function. aid may target template and non-template strands at similar frequencies and predominantly after r-loops are processed by cellular enzymes that expose dna on both dna strands4. additionally, aid footprints may be distributed evenly across the entire length of the s region, unlike shm, which is not evenly distributed over a distance; thus aid deaminates s and v regions with distinct mechanisms. it has also been suggested that aid-mediated dna demethylation occurs due to the deamination of methylated cytidine residues in single-stranded dna, followed by dna repair7. a long-standing hypothesis on aid targeting, known as the hotspot hypothesis, has recently been under re-evaluation. this hypothesis considered a short sequence motif (agct) conserved in all s regions as functionally important for csr, proposing that it exerts its function via its overlapping aid hotspot structure23. however, an initial weakness of this theory was that these sequences are very common in the genome23. another study determined one of the �rst crystal structures of maltose-binding protein (mbp)-fused aid and its complex with cytidine (c), deoxycytidine (dc), and deoxycytidine monophosphate (dcmp). these structures can help explain the discrimination between dna and rna in aid catalysis and reveal that aid has a bifurcated substrate-binding surface10. this supports the theory that one aid recognizes two adjacent ssdna overhangs from one structured substrate to achieve high a�nity10. g4 structured substrates induce aid cooperative berkeley pharma tech journal of medicine | 111 oligomerization, which could promote clustered mutations in the ig s regions10. overall, the bifurcated substrate binding surface and oligomerization interface are both an essential component of csr and help elucidate recognition of structured substrates as an important aid-targeting mechanism, speci�cally in the ig s regions10. it has therefore been suggested that g4 substrates mimicking ig s regions are preferred aid targets in vitro. this recent �nding is a departure from our previous understanding of aid targeting. this data also posits that aid preference for these substrates is likely due to their bundled ssdna overhangs structure rather than the primary sequence motif, which was long believed to bewhy aid preferred these substrates10. it is important to recognize that a de�nitive complex structure with fully characterized substrate conformation is still lacking and must be developed10. many proposed therapies suggest that selective inhibition of aid may ameliorate the conditions. ultimately,further experimentation and analysis with more sensitive techniques that may eventually be developed is needed to more fully understand the mechanism of aid inhibition. given that the crystal structure of aid has recently been resolved, future e�orts would certainly bene�t from structural modeling approaches10. a more de�nitive structure could serve as a template for potential therapeutic intervention against aid10. progress on aid structure is very timely alongside the growing knowledge about ig class switch region nucleic acid structures, which are supported by functional studies24. already, we are seeing promising results from initiatives focusing on aid. platforms like genevestigator consolidate publicly available studies from microarrays, mrna sequencing, and more under healthy conditions versus diseased states25. using these comparisons is one potential strategy for a comprehensive analysis of the role of aid in the pathobiology of immuneor in�ammatory-based diseases and cancer25. it has also been suggested that we may eventually be able to analyze aid gene signatures to get decisive determinants of patient-speci�c or patient-group-speci�c antiviral response, which could allow us to understand how viruses can impact di�erent individuals25. berkeley pharma tech journal of medicine | 112 estrogen and aid estrogen has been found to reverse the repression of aid, resulting in a subsequent boost in aid expression. this is proposed to occur through the upregulation of hoxc4, which, together with nf-κb, critically mediates aid promoter activation6. there may, however, be additional epigenetic mechanisms at play that serve to regulate aid expression. estrogen reverses hdi-mediated inhibition of aid and csr in ab and autoantibody responses through the downregulation of b cell mir-26a, which targets aid mrna’s 3’utr6. as epigenetic modi�ers, scfa hdis, like mir-26a and mir-125a, inhibit aid expression and csr through the upregulation of select b cell mirnas, which silence aid26. this is interesting as it may provide an explanation for the female bias in autoantibody-mediated autoimmune diseases like lupus2. yet, an experimental and fully functioning in vivo model of the human immune system is needed in order to understand the epigenetic mechanisms relating to the human ab and autoantibody response6. autoimmune diseases cellular reprogramming, broadly, is a mechanism that must be further explored. currently, there are three approaches to induce reprogramming: cell fusion, nuclear transfer, and ipsc14. cell fusion is a great way to understand nuclear plasticity and is a main element of many cancer processes14. nuclear transfer, more commonly referred to as cloning, has potential therapeutic applications, although ethical concerns exist14. ipsc technology is anexcellent option given that it has potential therapeutic applications for clinical use without ethical concerns and can be used to model human diseases and screen potential new treatments27. dna methylation is a major barrier to induced pluripotent stem (ips) cell reprogramming, and putative dna demethylase protein aid can erase dna methylation at pluripotency gene promoters, which will subsequently allow cellular reprogramming14. autoimmune diseases are detrimental to the health and wellbeing of individuals globally. berkeley pharma tech journal of medicine | 113 one example of such a disease is common variable immunode�ciency (cvid), which is a primary immunode�ciency characterized by hypogammaglobulinemia and di�erent degrees of b cell compartment alteration28. we found reduced bcl-2 protein levels in memory b cells from cvid. hypertension is another medical condition where the study of aid can be useful. in the usa, nearly 50% of the adult population has hypertension, and prevalence increases to ~80% at advanced age29. b cell ig production is dependent on a subset of b cells called gc b cells, which are dependent on aid and may play a causal role in the pathophysiology of hypertension. the gc reaction is driven by il-21 and t follicular helper (tfh) cells, which are transcription factors associated with aid and have been demonstrated to play a role in hypertension and hypertensive end-organ damage30. it is possible that b cells and ig contribute to hypertension in speci�c cases as in autoimmune diseases or preeclampsia31. however, future studies should investigate inducible genetic b cell deletion in adult animals to determine if b cells are viable therapeutic targets for hypertension31. multiple sclerosis (ms) is another debilitating chronic disease. b cell depleting therapies are a potential way to ameliorate symptoms in ms given that b cells play a critical role in the ms disease process32. there is a presence of b cells in active lesions and the cerebrospinal �uid of ms patients32. in a recent study, the community was able toglean more information on the role of secondary diversity of the bcr in experimental autoimmune encephalomyelitis (eae) and identify igg class-switched b cells as potential therapeutic targets for the treatment of ms32. aid was also found to presumably still exert some subtle e�ect on rmog-induced (myelin oligodendrocyte glycoprotein) disease trajectory32. arthritis is a debilitating disease that can result in a lot of pain a potential novel treatment for in�ammatory arthritis includes fraxinellone33. the therapeutic e�ect of fraxinellone was associated with the inhibition of cellular di�erentiation and activation. it has been shown to attenuate the clinical and histologic features of in�ammatory arthritis in mice33. there berkeley pharma tech journal of medicine | 114 was a lower expression of aid and blimp-1 following treatment with fraxinellone33. remarkably, it also alleviated synovial in�ammation and osteoclastogenesis in mice33. other drugs such as belimumab, a targeted therapy approved for systemic lupus erythematosus (sle), serve as examples of how targeted therapies that disrupt the aid pathway can be bene�cial34. further investigation is needed to see the side e�ects on normal cells. cancer aid, as previously mentioned, has been largely suggested to induce cancerpromoting mutation. aid is expressed in more than 40% of primary human chronic lymphocytic leukemia (cll) cases, but aid expression can be harnessed for antileukemic e�ect after inhibition of the rad51 homologous recombination (hr) factor 4,4’-diisothiocyanatostilbene-2-2’-disulfonic acid (dids)16. this is a novel antineoplastic role of aid that can be triggered by inhibition of hr, which is a new paradigm to treat aid-expressing tumors and has had proof of principle studies conducted16. this treatment has also been suggested for use in type 1 diabetes6. another avenue that has been considered is the chronic administration of hsp90 inhibitors, which decreases aid protein levels and has been shown to reduce disease severity in a mouse model of acute b cell lymphoblastic leukemia in which aid accelerates disease progression18. this is promising, as a proof-of-concept study has been published that showed hsp90 inhibitors directly target aid in vivo, and endogenous humanaid is sensitive to them 18. yet another study has suggested that targeting aid is bene�cial in the immunotherapy ofaid positive tumors because sirna silencing of aid in plasmacytoma dramatically increases its susceptibility to immunotherapy by cytotoxic t lymphocytes15. overall, aid has shown to be a promising target in the aforementioned instances and more research may yield additional insights. berkeley pharma tech journal of medicine | 115 conclusion although further research will help the scientific community to glean more clear insights, it is clear that elucidating how aid works will help with the development of novel therapeutic strategies for a multitude of diseases. disrupting the aid pathway can have potential therapeutic e�ects. however, it is important to remain cognizant of 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upregulate b cell micrornas that silence aid and blimp-1 expression for epigenetic modulation of antibody and autoantibody responses. j immunol.2014;193(12):5933-5950. doi:10.4049/jimmunol.1401702 29. yamanaka s, blau hm. nuclear reprogramming to a pluripotent state by three approaches. nature. 2010;465(7299):704-712. doi:10.1038/nature09229 30. yewdell wt, chaudhuri j. a transcriptional serenaid: the role of noncoding rnas in class switch recombination. int immunol. 2017;29(4):183-196. doi:10.1093/intimm/dxx027 31. yu k, lieber mr. current insights into the mechanism of mammalian immunoglobulin class switch recombination. crit rev biochem mol biol. 2019;54(4):333-351. doi:10.1080/10409238.2019.1659227 32. zan h, casali p. regulation of aicda expression and aid activity. autoimmunity. 2013;46(2):83-101. doi:10.3109/08916934.2012.749244 33. zhu j, hay an, potter aa, et al. abrogated aid function prolongs survival and diminishes renal pathology in the bxsb mouse model of systemic lupus erythematosus. j immunol. 2020;204(5):1091-1100. doi:10.4049/jimmunol.1900501 berkeley pharma tech journal of medicine | 119 front_page_wojtara_revised_final wojtara body template wojtara reference template-2 berkeley pharma tech journal of medicine correspondence: venkatarepaka@gmail.com keywords: lewy body dementia alpha-synuclein neurodegenerative disorders acetylcholine pathways dopamine pathways submitted: september 12, 2022 accepted: november 28, 2022 published: december 28, 2022 full open access creative commons attribution license 4.0 abstract lewy body dementia (lbd) is a neurodegenerative disorder in which the brain has abundant misfolded alpha-synuclein proteins. while lbd has similar symptoms to other degenerative neurological disorders, such as alzheimer’s disease (ad) and parkinson’s disease (pd), they are not the same medical diagnosis. lbd is characterized by all the motor deficit symptoms of parkinson's disease (pd), but the opposite cannot be said, as pd is more complex and has more underlying symptoms. clinical trials and in vivo experiments have shown that specific genes related to these misfolded proteins are potentially good genetic treatment and therapeutic targets. this review article seeks to provide an overview of the current state of novel therapeutic, genetic strategies to reduce the effects of lewy body dementia. novel genetic therapeutic strategies for lewy body dementia by: venkata repaka, carlota sagarduy and gatik trivedi berkeley pharma tech journal of medicine | 2 1. introduction there are three main goals we would like to achieve with this review paper. the first is to lay out a list of possible and current treatments available for lewy body dementia (lbd) to reduce its effects. next, we aim to understand how lbd can affect other conditions, such as parkinson’s disease (pd) and alzheimer’s disease (ad), and the connection between such diseases. lastly, we seek to understand the molecular mechanism of the initiation of lbd. due to limited research and investigation on the topic, there is currently no cure for lbd. because of the related symptoms among lbd, ad, and pd, some medications used to treat ad and pd can be used for lbd. one medication is an fda-approved drug to treat ad called rivastigmine (exelon). it is a reversible cholinesterase inhibitor that acts on a chemical in the brain critical for memory formation and cognitive thinking1,2. this is a reversible cholinesterase inhibitor that is known chemically as (s)3[1(dimethylamino) ethyl]phenyl ethyl methyl carbamate3. another medication, carbidopa-levodopa, is used to treat pd and could be used for lbd movement-related treatment1,2. levodopa is the precursor of dopamine and can cross the blood-brain barrier to create more dopamine in dopaminedeficient areas in the brain4. some clinical features of lbd include visual hallucinations and parkinsonism. recurrent, complex visual hallucinations occur in up to 80% of patients with lbd and are a frequent clinical signpost to diagnosis5. they are typically well-formed, featuring people, children, or animals, sometimes accompanied by related phenomena including passage hallucinations, sense of presence, and visual illusions. spontaneous parkinsonian features are common in lbd, eventually occurring in over 85% (parkinsonism in parkinson's disease (pd) is defined as bradykinesia in combination with rest tremor, rigidity, or both)5. lbd is defined as a progressive disease in which symptoms worsen as one ages. 1,2 hallucinations are common in the early stages of lbd, as are restlessness, acting out dreams during sleep (called rem sleep disorder), and berkeley pharma tech journal of medicine | 3 movement difficulties5. others may develop urinary urgency and incontinence. unlike ad, memory is usually still fairly intact in the early stages. however, confusion and some mild cognitive changes may be present. as lbd progresses, symptoms develop and more strongly resemble parkinson's disease5. these symptoms include increased problems with motor functions, difficulty with speech, swallowing problems, and greater paranoia and delusions6. cognition also continues to decline, with shorter attention and significant periods of confusion occurring. in the later stages of lbd, extreme muscle rigidity and sensitivity to touch develops. patients need assistance with almost all activities of daily living. speech is often very difficult and may be whispered. some lbd patients stop talking altogether7. both lbd and pd are quite similar and have overlapping characteristics, but there are specific key differences that can differentiate lbd and pd in the spectrum1,2. there is a frequent coexistence of the pathology of amyloid plaques and neurofibrillary tangles in lbd compared with parkinson's disease dementia. amyloid plaque are misfolded proteins in the synaptic clefts of neurons. this would effectively block the transmission of neurotransmitters from neuron to neuron. additionally, the accumulation of the protein tau within neurons are called neurofibrillary tangles. the tau protein works to help the neuron’s microtubules which help guide nutrients and vitamins move in the neuron. particularly, because tau proteins are ‘sticky’ in character they will bind to each other while they are connected to microtubules and create tangles of the microtubules inside neurons. this can harm the synaptic communication and internal cell communication of the neuron31. alzheimer’s differs from lbd clinically because of its unique cognitive profile and its lack of parkinsonian features except in the late stages2. this paper provides an overview of novel therapeutic genetic strategies to reduce the effect of lbd by targeting topics of epigenetics, cognitive enhancers, non-coding rnas, micronutrients, making alterations of central cholinergic (ach) and dopaminergic (da) systems, and looking at current drugs and treatments. this review will also be discussing how factors such as age, diet, and underlying health conditions can affect the intensity of lewy body dementia in a patient. berkeley pharma tech journal of medicine | 4 2. molecular components of lbd while there are many molecular pathways of lbd, research has shown that alpha-synuclein(as)-positive inclusions are the main indicator of lewy body8,9, causing the impairment of protein degradation pathways, including both the ubiquitin-proteasome system and the autophagy-lysosome pathway. the ubiquitin-proteasome pathway (upp) is the main pathway that destroys unneeded proteins and eliminates misfolded or misguided proteins that are in the wrong locations within the cell with the use of a protease or proteasome32. this mechanism works through the formation of a polyubiquitinated protein, which serves as a recognition signal for the eventual action of the proteasome to degrade the proteins into peptides, as shown in figure 1. this is an important mechanism as this can decrease the number of unnecessary proteins and debris in the body that is unneeded. defects in the upp can be detrimental to the homeostasis of the amounts of tau protein in the neuron. a possibility of the amount of extra tau protein in the neuron can be due to a mishap of the upp. berkeley pharma tech journal of medicine | 5 figure 1. the ubiquitin-proteasome pathway (upp). ubiquitin, a 76 amino acid protein, marks the molecule to be degraded by attaching itself to a substrate protein. in this process, e1–the ubiquitin-activating enzyme, e2–the ubiquitin-conjugating enzyme, and e3– the ubiquitin-protein ligase, cause a cascade of reactions, the result of which is the linkage of one molecule of ubiquitin to the protein (mono-ubiquitination). polyubiquitination occurs when additional molecules attach to the seven lysine residues or the n-terminus of the ubiquitin molecule to form a chain. polyubiquitination is the recognition signal for the proteasome to degrade the target protein into peptides. the autophagy-lysosome pathway is another important catabolic pathway that regulates the quality and the number of proteins made within the body’s cells. there are three common mechanisms of lysosomal autophagy which are macroautophagy, microautophagy, and chaperon-protein mediated autophagy. macroautophagy is where an autophagosome is created containing the surplus amount of protein and a lysosome will bind to the organelle-like structure to degrade the particles within it. all these pathways separately are important to degrade an excess number of large proteins. in the case of smaller proteins, a bigger lysosome will use phagocytosis to degrade the need proteins. similarly, chaperon -mediated pathway will utilize a longer way to degrade proteins including another chaperon protein which connects the excess amount of protein to a channel that leads to the lysosome. this lysosomal pathway uses a lysosome to engulf the autophagosome and destroy its contents with enzymes33. these pathways then will degrade the neurons present in the brain. berkeley pharma tech journal of medicine | 6 figure 2. the three different autophagy-lysosome pathways–macroautophagy, microautophagy, and chaperone-mediated autophagy. alpha-synuclein also is a major component of the filamentous glial cell inclusions (gcis) that are abundant in the white matter oligodendroglial cells of multiple system atrophy (msa) brains. accumulation of alphasynuclein into filamentous inclusions could play a mechanistic role in the pathogenesis of several progressive neurological disorders, such as parkinson’s disease, lbd, familial alzheimer's disease, lewy body disease variant of alzheimer’s disease, sporadic alzheimer's disease, and multiple system atrophy10. this path is obscure, but upp and autosomal-lysosomal pathways are the commonly known pathways that lbd is categorized into. lbd specifically affects the thalamus structurally and functionally8,9. additionally, a study suspected there are five specific important and targeted genes from lbd patients: bin1 and tmem175, snca, apoe, and gba 510. this was done by uncovering participant samples from 44 different european ancestry banks; specifically, 17 in europe and 27 across north america. when the sequences from lbd patients and control patients were compared, they found 5 consistent different genes that were among the lbd patients. to confirm their results and conclusions, the researchers also compared those 5 genes to another 970 lbd patients with a new control set of 8,928 control subjects10. berkeley pharma tech journal of medicine | 7 figure 3. the 5 genes gba, bin1, tmem175, snca, and apoe on their respective chromosomes. widespread mitochondrial dysfunction is very closely related to disease development as well. the impairment of protein degradation pathways, including both the ubiquitin-proteasome system and the autophagylysosome pathway, also plays an important role during the development of lewy body diseases11. differential expression changes of isoforms corresponding to genes primarily involved in lewy body formation point to alternative splicing as another important mechanism in the development of dementia with lewy bodies11. 3. epigenetics parkinson's disease and dementia with lewy bodies are deemed similar based on a neurological overlap between them. both pd and lbd are distinguished by an abnormal accumulation and deposition of misfolded and aggregated alpha-synuclein which gives rise to lewy bodies and lewy neurites. the diversity and complexity of lbd make them complex multifactorial disorders. thus, many lbd cases originate from the interaction of multiple genetic and environmental, epigenetic factors. there are many genes of interest that are involved in lbd, one of them being snca, which is a rare gene involved in lbd. it has two distinct profiles within its locus, one pertaining to pd, located in the 3’ snca portion, and the other to lbd, located in the 5’ snca portion12. snca was the first and is one of the most studied genes identified as an lbd-causing gene. sncb, like snca, has been detected in some lbd cases. berkeley pharma tech journal of medicine | 8 the b-syn, from the synuclein family, has shown to be a regulator of the asyn aggregation. common variants, like rare variants, that could increase the risk of developing lbd have been found to be located in genes associated with pd or ad. one of these well recognized risk variants is the allele ε4 of the apolipoprotein e gene (apoe). apoe poses a risk in lbd patients because the allele ε4 accumulates and accelerates the disease which leads to a shorter lifespan for the patient13. on the other hand, apoeε2 has been shown to have protective effects against the development of lbd12. epigenetics regulate gene expression through methods that are independent of the primary dna sequence, even though they can be heritable. without altering the dna sequence, epigenetic mechanisms moderate reversible changes in gene expression and cell phenotype. there are two major mechanisms pertaining to epigenetics modifications, shown in figure 4. the first is dna methylation, a biochemical process by which a methyl group is added to dna, thereby modifying the function of the gene. it occurs at cytosines located 5’ to guanine (cpg), and it is mediated by methyltransferases. in the promoter regions, dna methylation can either repress gene expression, hypermethylated cpg, or increase gene expression, hypomethylated cpg. this mechanism plays a big role in aging and development. the second mechanism is histone tail modification which either loosens or encompasses the tail to turn a gene “off” or “on” or deactivate or activate a gene, respectively. the proteic part of chromatin is referred to the histones which allow the compaction of dna. these two mechanisms have a fundamental part in learning and memory processes because they are dynamically controlled in neurons. epigenetic mechanisms mediate geneenvironment interactions because they can often be provoked by environmental risk factors. this emphasized the importance of certain factors in patients with lbd, such as diet, physical activity, and lifestyle13. berkeley pharma tech journal of medicine | 9 figure 4. the process of the epigenetic mechanisms. the effects of the environment and genetics may have an influence on changes in gene expression and cell behavior. lbd studies have mostly been focused on genetics rather than epigenetics; however, epigenetic mechanisms and regulation have proved to play an important role in the pathophysiology of the disease and genome. even though there are scarce studies on epigenetics in lbd, there is evidence of an overlap of apoe and snca from their genetic and epigenetic regulation (figure 5)13. transcriptomic and proteomic studies analyzing the genetic pathway have shown clinical and neurological correlates to the disorder through the synaptic function, lysosomal processing, and circadian rhythm13. the origin of the cause of these changes has not yet been determined. there are a few limitations in the evidence that exists including small sample sizes, tissue specificity, and lack of information. as more technologies are developed, more will be discovered regarding this topic. berkeley pharma tech journal of medicine | 10 figure 5. genetic association and epigenetic association in lbd. this figure shows the different genes involved in lbd and how they are associated with genetics or epigenetics. as seen, apoe and snca have both genetic and epigenetic associations. with the appropriate information, epigenetics are an important tool and strategy that can be used to battle a variety of different diseases. there are tactics that would delay the onset and progression of neurodegenerative diseases, such as lbd. one of these tactics includes targeting the epigenome14. this can be done using small drugs, such as hdaci, that are able to cross the blood brain barrier14. even though studies are scarce and there are concerns with using this type of drug, there are potential targets for drug development that could have a promising result for potential epigenetic preventive factors for neurodegeneration. as more is discovered about this disease, epigenetics will serve as a fundamental factor in either delaying the onset or helping in the stratification and improvement of the diagnosis. 4. cognitive enhancers cognitive enhancers, also called nootropics, are neuroprotective or extremely nontoxic in consideration to neurodegenerative diseases like lbd. nootropics can be naturally found or synthetically made which can enhance attentional control and memory. there are various mechanisms by which nootropics acts, which are as follows: 1) increasing circulation to the brain, 2) providing precursors to neurotransmitters (chemical messengers in the berkeley pharma tech journal of medicine | 11 brain), 3) improving neuron function, 4) preventing free radical and oxidative damage to brain cells, and 5) providing usable energy to the brain15. nootropics exist in three main categories: supplements, racetams, and stimulants. supplements or dietary sources of nootropics are mainly natural and can be found in fruits and vegetables. natural nootropics come in the form of vitamins like omega-3, iron, antioxidants, amino acids, and caffeine. racetams are positive allosteric modulators of ampa receptors in the brain and include piracetam and nefiracetam. racetams are categorized and claimed as “pharmacologically safe” drugs15. the amnesia reversal effect with racetams is compared with scopolamine, electroconvulsive shock, and hypoxia, which are more invasive strategies considering the safe nature of racetams16. specifically, nefiracetam has been proven to have more affinity for muscarinic receptors at the nanomolar range than other aniracetam and nebracetam. most racetams are safe but can cause adverse reactions to males than women if used incorrectly or in excessive amounts15. lastly, stimulants are another type of smart drug under nootropics that enhance productivity in the brain. some examples include methylphenidate and amphetamines and are specifically known to improve adhd symptoms in patients with adhd15. amphetamine salts contained primarily of dextroamphetamine (d-amp) are known by the trade name adderall that help patients with adhd17. unlike racetams, stimulants can enhance cognitive abilities in impaired or unprescribed patients without causing adverse effects. similar to racetams, they can be found in natural and synthetic forms that affect neurotransmitter levels, neurogenesis, and blood flow to the brain17. some stimulants are known to improve symptoms of lbd and reverse the progression of dementia. additional categories of nootropics include dopaminergics, specifically work to raise levels of dopamine, and serotonergics, specifically work to raise levels of serotonin, which can also raise treatments to reverse symptoms of lbd15. one clinical trial study of nootropics has been done on patients with ad. researchers were particularly interested in comparing the effects of specific nootropics that affected behavioral and psychological symptoms of dementia which is how lbd can also be affected in the study. specifically, the study looked at cholinesterase inhibitors and memantine or a n-methyl berkeley pharma tech journal of medicine | 12 d-aspartic acid (nmda) receptor antagonist. cholinesterase inhibitors of donepezil and rivastigmine increase the concentration of acetylcholine at the neurotransmitter sites. the other cholinesterase inhibitor tested was galantamine which is a double action that not only increases acetylcholine at neurotransmitter sites like donepezil and rivastigmine, but also acts by modulating activity at nicotinic receptors. memantine is a nmda receptor antagonist modulates glutamate in the glutamic system of the brain. the study had selected patients with ad and had given a placebo to some patients and others received a cognitive enhancer. observations were made with the efficacy and reversal of dementia in these patients. this clinical trial has potential to show that more than 100,000 patients are eligible for cognitive enhancers in canada alone as 30% of the ad patients have moderate dementia. these medications have been approved for the treatment of alzheimer’s disease in many countries as well18. another facet to consider is the administration of such effective nootropics. in a clinical trial led by dr. murat emre from istanbul and published in the prestigious new england journal of medicine in 2005 had shown that rivastigmine, as previously mentioned, a cholinesterase inhibitor that enhances ach concentration in neurotransmitter sites was effective in parkinson’s disease (pdd). although this drug is not approved for lbd patients in the dr. emre’s trial19. however, many doctors and providers consider both disorders to have similar enough symptoms for the rivastigmine medication to be effective in both conditions. in this study both the patch, developed and approved for use in 2007, and the pill of rivastigmine were given along with the placebo to patients with pdd. the patch and pill of rivastigmine were proven to be more effective than the placebo and showed the same effectivity. however, one trial of dr. emre’s study declared the patch was more effective than the pill of rivastigmine mainly because of the lower recurrence of side effects of nausea and vomiting in the patch than in the pill. because medications were taken by the skin in comparison to the mouth, gi issues decreased in patients who used the patch19. 5. non-coding rnas as previously described, lbd is very complex and has become an increasing berkeley pharma tech journal of medicine | 13 demand on global health care systems. because it is so complex, there are many factors that need to be researched to create possible therapies or treatments. non-coding rnas (ncrnas) are rna sequences that cannot be translated into proteins. there is a vast variety of different ncrna families; however, the two most valuable to this topic are the mirna and lncrna families. these two families have the capacity to provoke gene regulation across cellular physiological pathways. since mirnas and lncrnas influence disease pathways, it is important to research ncrnas for possible therapies and for more information on the pathogenesis20. mirna and lncrna families have different influences on dementia20. mirna, short for microrna, refers to a small single stranded ncrna molecule. it comprises 22 nucleotides and functions in rna silencing and posttranscriptional regulation of gene expression. mirna inhibits the translation of proteins coded by mrna transcript by acting as a physical obstruction for ribosomal action. there are many studies attributing and demonstrating the effects of mirna defects on multiple forms of dementia, however because lbd has not been deeply studied there are no details on the influence of mirna on lbd21. specifically, lncrna, short for long noncoding rna, refers to a single rna sequence composed of at least 200 nucleotides. lncrna regulates gene expression in several levels such as epigenetics and transcriptional levels. most lncrnas are detrimental to pathways in neurodegenerative diseases21. just as in mirna, there are no studies on the specific effect of lncrnas on lbd. as stated previously, small molecule drugs show potential for the future of this disease. one of the few studies that has been done using ncrnas is the use of small molecule drugs targeting ncrnas as treatments for dementias22. ncrnas could function as therapeutic targets because they are enriched in the central nervous system. a small molecule can bind to ncrna, changing its conformation, to regulate it. there has been extensive work in developing oligonucleotides to target mrnas and ncrnas, however there are many obstacles that have not yet been researched which block the entry, specifically in the blood brain barrier. another tactic that needs further studying is the use of gene therapy. using gene therapy with viral vectors can edit ncrnas and can allow for more durable ncrna modulation. however, this also berkeley pharma tech journal of medicine | 14 comes with obstacles regarding the delivery and expression of the target genes, making the therapy ineffective. more research and knowledge could lead to identifying novel diagnostic procedures and/or drug targets. while these possible therapies or drugs may not be able to cure a patient, they can help with the control of the patient’s mental condition, disease progression, and cognitive decline. 6. other factors in effect 6.1 inflammation as lbd and ad have similar symptoms and neuroinflammatory mechanisms involving the activation of microglia, overexpression of interleukin-1 and other inflammatory mediators, and inflammatory toxicity to neurons. the activation of microglia are also resultants from the overexpression with α-synuclein-containing neurons and glia in pd. these connections are also associated with the microglial associations with neurofibrillary tangle-containing neurons in ad. it is shown that there is a reciprocal induction between α-synuclein and injured neurons on one hand and activated microglia and cytokine overexpression with in vivo and in vitro experiments. this mechanism of inflammation can lead to more injured neurons which can activate microglia and cause a cycle of inflammation beneficial to the neuron which can cause neuronal death. this concept can show the progression and overlap between both ad and lbd25. 6.2 risk factors there are several factors that contribute to a person's risk for developing lbd. a history of high caffeine intake, for example, is associated with a lower risk of lbd. research has suggested that the benefits increase in tandem with the amount of caffeine a person drinks per day and may reduce the risk of lbd by as much as 29%26. genetics and heredity appear to play a role in the risk of lbd as well. generally, if you have a first-degree family member with lewy body dementia or pd, your risk of lewy body dementia increases. additionally, for reasons not entirely clear, a history of depression and anxiety is linked to an increased risk of lewy body dementia27. metabolic berkeley pharma tech journal of medicine | 15 disorders hypertension (high blood pressure) is linked to an increased risk of lewy body dementia. the incidence of hypertension among people with lewy body dementia is roughly 65%. having high blood pressure, in turn, increases the risk of lewy body dementia by 60.5%. type 2 diabetes, a form of diabetes strongly linked to lifestyle, is associated with a 25% increased risk of lewy body dementia. high cholesterol also increases the risk of lewy body dementia by roughly 25%28. 6.3 micronutrients micronutrients are a fundamental part to healthy development, disease prevention, and a good lifestyle. no micronutrients, except vitamin d, are produced in the body therefore they must come from a person’s diet and intake. micronutrients are important for healthy people but even more important for those who have a disease or are genetically prone to developing a disease23. those who do not supply their body with sufficient nutrients can suffer from malnutrition, which has been found to be tied to cognitive function. deficiency of nutrients, specifically vitamin b12 and folate, can lead to decreased cognitive function. in addition, weight loss associated with malnutrition will often occur prior to the onset of dementia23. as the disease progresses, weight loss can also progress and vice versa. while there are several diets that are said to prevent, or help with, cognitive decline and dementia, there are no studies that directly prove one diet to help with lbd24. finding the correct micronutrients that could help patients who have lbd or are predisposed to it could have a huge impact on this area of health. 7. conclusion from the progression of research of lbd in clinical trials, in vivo, and in vitro experiments, the potential of epigenetic therapy should be examined more thoroughly. future directions of the use of deep brain stimulation29 and repetitive transcranial magnetic stimulation (rtms) has shown potential in other neurodegenerative diseases and could be applicable to lbd30. the effects of epigenetics, age, diet, and inflammation within the body can enhance other scientists’ knowledge when designing therapies for lbd in the future. as this article has mentioned many current therapies and potential therapies it is important to note the limitations and drawbacks of the research berkeley pharma tech journal of medicine | 16 presented. one drawback is that many of the clinical trials are research alongside alzheimer's disease and or parkinson’s disease so there is no precise way to isolate lbd research and progression. additionally, many of the clinical trials and experimental data found were said to be inconclusive as the data was little or had little to no value of measurement. protocols on how to measure the data obtained in experimental trials are still being researched upon. to combat these terms, this article took a holistic review of each article and applied most information back to lbd with reason. nevertheless, the research presented is to enhance the knowledge of other scientists researching lbd therapies to give a foundation about the current and probable therapies available. conflicts of interest the authors declare that there is no conflict of interest regarding the publication of this paper. references 1. national institute on aging. what is lewy body dementia? causes, symptoms, and treatments. national institute on aging. published july 29, 2021. https://www.nia.nih.gov/health/what-lewy-bo dy-dementia-causes-symptoms-and-treatme nts 2. schumacher j, peraza lr, firbank m, et al. dysfunctional brain dynamics and their origin in lewy body dementia. 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small non-coding rnas: new class of biomarkers and potential therapeutic targets in neurodegenerative disease. front genet. 22. 2019;10:364. published 2019 apr 26. doi:10.3389/fgene.2019.00364 23. koyama a, hashimoto m, tanaka h, et al. malnutrition in alzheimer's disease, dementia with lewy bodies, and frontotemporal lobar degeneration: comparison using serum albumin, total protein, and hemoglobin level. plos one. 2016;11(6):e0157053. published 2016 jun 23. doi:10.1371/journal.pone.0157053 24. dominguez lj, barbagallo m. nutritional prevention of cognitive decline and dementia. 24. acta biomed. 2018;89(2):276-290. published 2018 jun 7. doi:10.23750/abm.v89i2.7401 25. mrak re, md, w. sue t. gri�n wst. common in�ammatory mechanisms in lewy body disease and alzheimer disease. journ neuropath & exp neur. 2007;66(8):683-686. https://doi.org/10.1097/nen.0b013e31812503e 1 26. brendon p. boot, carolyn f. orr, j. eric ahlskog, tanis j. ferman, rosebud roberts, vernon s. pankratz, dennis w. 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magnetic stimulation (rtms): a possible novel therapeutic approach to dementia with lewy bodies. med hypotheses. 2010;74(5):877-879. doi:10.1016/j.mehy.2009.11.020 31. what happens to the brain in alzheimer's disease? national institute on aging. https://www.nia.nih.gov/health/what-happens-b rain-alzheimers-disease#:~:text=neuro�b rillary%20tangles%20are%20abnormal%20accum ulations,to%20the%20axon%20and%2 0dendrites. accessed august 26, 2022. 32. ubiquitin-proteasome pathway. creative diagnostics. https://www.creative-diagnostics.com/ubiquiti n-proteasome-pathway.htm. accessed august 26, 2022. 33. finkbeiner s. the autophagy lysosomal pathway and neurodegeneration. cold spring harb perspect biol. 2020;12(3):a033993. published 2020 mar 2. 33. doi:10.1101/cshperspect.a033993 berkeley pharma tech journal of medicine | 19 front page repaka et al. body template_repaka et al. references_repaka et al. abstract berkeley pharma tech journal of medicine correspondence: ashleyvaratip@gmail.com keywords: stimulants, non-stimulants, neurotransmission, neurexins 1 (nrxn1), dopamine receptor d3 (drd3), glutamic acid decarboxylase 65 (gad65), patched domain containing 1 gene (ptchd1), norepinephrine transporter (slc6a2) submitted: january 20, 2023 accepted: february 9, 2023 published: june 30, 2023 full open access creative commons attribution license 4.0 issues regarding disorganization and hyperactivity are large burdens on the pediatric population, and the severity of these behavioral disorders, called attention-deficit hyperactivity disorder (adhd), falls on the slow-developing treatments that are unable to fully solve the symptoms of those affected. limiting factors include the heterogeneous responses that many patients have in response to pharmacological treatments, the range of comorbid symptoms and conditions associated with adhd, and the intricacies of the environmental and genetic interactions involved. since adhd has a strong genetic component with up to 80% heritability for the condition, epigenetic and genetic studies offer valuable insight into how future treatments could tackle the issue. in particular, the studies reveal how genes might provide indicators for patients’ response to medication, their symptomatology, and unique risks for comorbidities. this paper outlines the current pharmacological and cognitive treatments for adhd, discusses their limitations, and offers an overview of present genetic risk factors to analyze how they may provide insights for detection, prevention, and responses to treatment. by: ashley varatip, serena z. h. huang, yash kilam, cameron asadi the predictive power of attention-deficit hyperactivity disorder (adhd) genetic risk factors introduction 1.1 introduction to adhd attention-de�cit hyperactivity disorder (adhd) primarily a�ects children and adolescents. as of 2016, there are 6.1 million (9.4%) children aged 2-17 diagnosed with adhd in the united states 1 . along with that, 1 in 20 children in the united states are being medicated for adhd 1 . while present in younger populations, it can persist into adulthood as well. 2.5-4.4% of adults in the united states are diagnosed with adhd and around 2.8% of adults worldwide 2 . the main characteristic behaviors de�ned by this disorder can include hyperactivity, short attention span, and impulsiveness. children and adults can display these symptoms di�erently. hyperactivity and impulsiveness tend to be traits shown more in children while inattentiveness is more persistent in adults 3 . for example, children can exhibit hyperactivity by not being able to sit still. adults on the other hand can show signs of inattention by not being able to sit through long activities or interrupting people’s sentences while they talk. 1.2 causes of adhd the causes and the risk factors of adhd are unknown to this day. however, it is believed that genetics plays a very important role in the development of adhd in individuals 4 . adhd tends to run in the family, transmitted through genes inherited from one’s parents 5 . recent analysis of twin studies demonstrates 80% heritability for the condition, though no speci�c genes have been linked to adhd 6 . along with genetics, some risk factors include brain injury, exposure to environmental risks during pregnancy or at a young age, alcohol or tobacco use during pregnancy, premature delivery, or low birth weight 4 . 1.3 diagnosis of adhd currently, there are no convenient or de�nitive ways to diagnose adhd. children can get diagnosed with adhd by a pediatrician, adult psychiatrist, or quali�ed healthcare professional with training in adhd 7 . a physical examination is run to make sure the symptoms relayed are not caused by something other than adhd. then, an interview can be berkeley pharma tech journal of medicine | 60 conducted with the child and parent. to be diagnosed with adhd, multiple symptoms must be displayed. additionally, the patient must experience these symptoms for over six months and before the age of twelve 7 . this process can be di�erent for adults, though specialists will ask the adult about symptoms that they have. however, an adult will not get a con�rmed diagnosis unless they a�rm that symptoms were present in childhood 7 . all of these diagnostic tests mainly involve speaking to a specialist or doctor because no blood or invasive tests can diagnose adhd 8 . 1.4 current treatments the most popular treatments of adhd include the use of medical stimulants or non-stimulants. medical stimulants aim to correct biochemical imbalances by increasing dopamine and norepinephrine levels to increase attention and focus 9 . stimulants target the brain’s reward system, the mesolimbic dopamine pathway involving the ventral tegmental area of the midbrain, medial prefrontal cortex, and limbic system 10 . in general, stimulant medications have three functions. it could mimic neurotransmitters such as dopamine to increase stimulation of dopamine receptors. it could increase the time in which dopamine stays within the receptors by preventing its degradation. this could occur through blocking reabsorption or enzymatic degradation 10 . the fda was able to approve 29 medical stimulants including adderall, dexedrine, and ritalin, and all twenty-nine of these stimulants have something in common: the use of either the molecule amphetamine or methylphenidate 9 . this is a popular choice of medication for adhd because it is proven to help around 70-80% of children with the diagnosis 9 . although stimulants provide good results for most pediatric patients, there are some downsides of using them. the side e�ects of these stimulants include an increased heart rate, increased blood pressure, decreased appetite, anxiety, and a chance of addiction to medications 9 . along with that, stimulants are not a good long-term treatment for adhd. the need for each dose of the stimulant increases over time and it was found that stimulants may have less e�cacy over time 9 . since there are downsides to using stimulants, non-stimulant medications are a relatively new medical option that was created to reduce the likelihood berkeley pharma tech journal of medicine | 61 for drug misuse 9 . non-stimulants and stimulants are very similar with the main di�erence being the target neurotransmitter. strattera (atomoxetine) was the �rst fda approved non-stimulant medication for adhd with clonidine, guanfacine, and qelbree following not soon after 9 . non-stimulants have fewer side e�ects, but side e�ects like nervousness, sleep problems, fatigue, upset stomach, dizziness, or a dry mouth can still happen 9 . although it seems like non-stimulants are a good option of treatment, it can be less reliable as 20-30% of people with adhd have stated that it does not work for them 9 . another option for treatment for adhd is cognitive behavioral therapy, also known as cbt. cbt is a short-term psychotherapy that focuses on changing a person’s negative perspective of themselves 11 . this could mean changing the way one thinks about themself and their potential. the way cbt works is that each session identi�es situations where a lack of organization creates problems in a person’s everyday life 11 . therefore, these sessions help the person develop coping skills to deal with challenges and obligations. these sessions can also include time for relaxation and meditation as well. there is a recent study that shows the positive e�ect of cbt through a randomized controlled trial 12 . the study primarily focused on determining how a treatment called accessing campus connections and empowering student success (access), a cbt program, a�ects 250 college students with adhd over a course of two semesters. the study assessed primary characteristics associated with adhd such as executive functioning, depression, and anxiety. the results of the study show that cbt reduced these common adhd symptoms. furthermore, a learning curve growth of students was modeled and it showed improvements compared to groups not in the access participants. there were no ascertained changes seen in depression and anxiety with correlations for lower chances in worsening depression and anxiety symptoms. therefore, it provides concrete evidence for access being used as treatment for college students with adhd 12 . berkeley pharma tech journal of medicine | 62 genes of interest as discussed earlier, genes play a primary role in the cause of adhd. currently, no studies have identi�ed a speci�c gene that causes adhd. part of the di�culty lies in the complexity of the phenotypes and relatively small e�ects of genetic variants 13 . this section will lay out and discuss the di�erent genes of interest that could possibly relate to the cause of adhd. gene of interest function in the body relation to adhd neurexin 1 (nrxn1) bind proteins → neurotransmitter release and di�erentiation of synapse mutations in gene → learning or memory problems → adhd dopamine receptor d3 (drd3) controls cognition, impulse control, attention, and sleep mutations in gene → cognition and impulsiveness problems → adhd glutamic acid decarboxylase 65 (gad65) catalyzes the conversion of glutamic acid into inhibitory neurotransmitter γ-amino butyric acid (gaba) → neurotransmission serum of anti-gad65 antibodies in patient → adhd patched domain containing 1 gene (ptchd1) provides thalamic reticular nucleus activity deletion of gene reduces thalamic reticular nucleus activity → attention de�cits and hyperactivity → adhd norepinephrine transporter (slc6a2) primary destruction mechanism of noradrenaline (ne) and involved in the reuptake of dopamine (da) and ne into the presynaptic neuron e�ects treatment of adhd (use of non-stimulants) v figure 1: this table summarizes the �ve genes of interest, their functions in the body, and its relation to adhd. 5.1 gene of interest 1: neurexin 1 (nrxn1) the �rst gene of interest is a family of cell adhesion proteins called neurexins. neurexins are encoded mainly by nrxn1, nrxn2, and nrxn3 genes among others 14 . the main role of neurexins in the cell surface of the neurons is to bind to other proteins and alpha-latrotoxin presynaptic receptors. the binding of these proteins can in return lead to berkeley pharma tech journal of medicine | 63 neurotransmitter release and di�erentiation of synapse 15 . however, the failure to bind together and the miscommunication of the bindings between these proteins is linked to a speci�c neurexin: nrxn1 14 . along with playing an important role in protein binding, another crucial role of nrxn1 is helping many proteins in synaptic transmission. the failure of nrxn1 to carry out these functions within the cell may be a reason for the development of learning or memory, which is a sign of adhd in a patient 15 . there are some in vivo studies that have correlated nrxn1 with adhd. for instance, researchers conducted one study to evaluate the protective e�ect and potential mechanism of nrxn1 on learning and memory in adhd rats 15 . the methods involved grouping the four-week-old rats into two categories: spontaneously hypertensive rats (shrs) and normal sprague dawley (sd) rats. these groups of rats were tested by using a morris water maze on a learning and memory test. moreover, qpcr and western blots were used to analyze the expression levels of nrxn1 at mrna and protein levels. it was concluded that the overexpression and interference of nrxn1 played a role in impairing the ability of the rats to learn and memorize things in both the shrs and sd rats 15 . however, in the experiment, a portion of the sd rats were given treatments with methylphenidate (mph) during the trails. this was shown to make an improvement in the performance of the treated sd rats. the change in nrxn1 in the rats led to a change in other synapse-related genes including psd95, syn1, gap43, and nlgn1 genes. therefore, it can be concluded that nrxn1 de�ciency is associated with the expression of synapse-related genes and adhd pathogenesis and can be a potential therapeutic target for adhd treatment 15 . 5.2 gene of interest 2: dopamine receptor 3 (drd3) another gene of interest is in the dopamine receptor family, which encodes dopamine receptors d1, d2, d3, d4, and d5 16 . dopamine receptors as a family are used in everyday function. it a�ects the brain as it is able to control and process emotions and movement of the body 17 . in particular, dopamine receptor d3 (drd3) a�ects your cognition, impulse control, attention, and sleep 17 . the mutation of drd3 mainly leads to di�erent berkeley pharma tech journal of medicine | 64 diseases such as schizophrenia and tremor. these mutations can be caused by ser-9-gly, a nucleotide polymorphism in drd318. ser-9-gly has a c allele in it which encodes glycine and a t allele which encodes serine 13 . there are quite a few in vitro studies that have tried to correlate drd3 mutations with adhd, but not a lot of them have been successful 18 . however, there is a drd3 gene and adhd pharmco-behavioral genetic study that links the two together 13 . the main goal of the study was to examine multiple types of potential genes related to adhd. this was done using an exploratory analysis with a comprehensive approach. 575 children with adhd aged 6 to 12 were the subjects of the study and were assessed under three of the experimental conditions. the conditions include one week of baseline observation, one week of methylphenidate (mph), and one week of placebo 18 . their parents, teachers, and research sta� evaluated their quantitative behavioral and cognitive dimensions relevant for adhd. the results of the experiment showed a nominal association between the t allele and worse behavioral scores while the subjects were in the mph week. along with that, the t allele demonstrated a nominal association with increased risk for adhd, response to placebo and mph13. the conclusions that could be drawn from this are that drd3, and moreover ser-9-gly, play a role in the cause of adhd and variations in patient’s behavior 18 . 5.3 gene of interest 3: glutamic acid decarboxylase 65 (gad65) glutamic acid decarboxylase 65 (gad65) is another gene to investigate as it has been found in the serum of patients with several neurological disorders. gad65 is an enzyme that is produced primarily by pancreatic islet cells 19 . the primary role of gad65 is that it catalyzes the conversion of glutamic acid into inhibitory neurotransmitter γ-amino butyric acid (gaba) that is present in synaptic vesicles of gabaergic neurons for its release during inhibitory neurotransmission 20 . serum anti-gad65 antibodies can be a common marker of subgroups of patients with autism and adhd as shown in a study that correlated the presence of gad65 antibodies in the serum of children with autism or adhd 20 . in the study, there were 14 normal control patients, 20 patients with autism, and 15 patients with adhd. the gad65 antibodies and berkeley pharma tech journal of medicine | 65 total igg were assessed in the serum of normal patients and patients diagnosed with either autism or adhd 20 . in conclusion, none of the normal patients had gad65 in the serum, 15% of children with autism had gad65 detected, and 27% of children with adhd had gad65 in their serum. with this, 60% of autistic and 53% of adhd patients reacted with purkinje neurons in mouse cerebellum and 20% of adhd patients' serums reacted with the cells in the molecular and granule cell layers and the cells in the vicinity of the purkinje neurons 20 . therefore, it can be concluded that the serum anti-gad65 antibodies are associated with patients with autism and adhd. 5.4 gene of interest 4: patched domain containing 1 gene (ptchd1) the patched domain containing 1 gene (ptchd1) is another gene of interest as many studies and models have displayed this gene’s role in adhd. its deletion reduces the thalamic reticular nucleus activity, a region of the brain that synapses the entire cortex and cerebellum. knockouts of this gene led to symptoms of attention de�cits and hyperactivity due to its involvement with small conductance calcium-dependent potassium currents (sks) within the thalamic reticular nucleus 21 . in one of these experiments, acute injection of the sk positive allosteric modulator 1-ethyl-benzimidazolinone ebio rescued the adhd-like knockout behaviors 21 . by measuring the mouse’s thalamic activity with a �uorescence resonance energy transfer, the sensory-related functions of mice were found to be lower in knockouts but recovered with the injection. this suggests that sk channel dysfunction can be a target without e�ects on aggression, hypotonia, and learning de�cits. it targets only inattention. in another study, ptchd1 ko mice showed drastic changes in kynurenine pathway metabolite concentrations in the serum and the brain, indicating that the activated kp is associated with adhd-like behaviors. this pathway is implicated in generating cellular energy in the form of nicotinamide adenine dinucleotide (nad+). because energy requirements are substantially increased during an immune response, the kp is a key regulator of the immune system. having the ptchd1 gene have a close tie to the kp pathway indicates the association between adhd and the berkeley pharma tech journal of medicine | 66 anti-in�ammatory responses of the immune system. global ptchd1 knockout mice were used to measure neuronal, behavioral, and social function with respect to a gene associated with adhd and asd-like symptomatology. markers of symptomatology can be determined through kp impairments. thus, there is potential for kp to be used as a clinical biomarker when the ptchd1 gene is inactivated 21 . 5.5 gene of interest 5: norepinephrine transporter (net) atomoxetine (atx) is the most commonly used drug in a non-stimulant group in the adhd treatment. it acts by increasing the levels of dopamine (da) and norepinephrine (ne) by inhibition of presynaptic net in the prefrontal cortex 9 . the norepinephrine transporter (slc6a2), which is responsible for the primary destruction mechanism of ne, is found in the plasma membrane of noradrenergic neurons involved in the reuptake of da and ne into the presynaptic neuron. the function of the ne transporter is attributed to multiple allelic variations of the slc6a2 gene. in a recent study, heterozygous genotypes rs12708954 genotypes showed greater side e�ects during treatment than normal genotypes. similarly, in rs3785143 genotypes, side e�ects in heterozygous carriers have been reported more frequently than wt carriers. in only one pilot study, rs3785143 t allele carriers reported a loss of appetite and irritability during atx treatment 22 . this gene polymorphism implies that adhd genetic risk factors not only in�uence its symptomatology, but also modulate the e�ectiveness of stimulant treatment such as atx on the patient’s response. future investigations adhd is a heritable condition, although the inheritance, or rather the likelihood of the disease being passed onto the next generation, is complex. while adhd genetic components are supported by twin studies and slightly less so by gwas studies, genetic studies provide a close estimate to how multiple factors could intertwine and lead to comorbidities. the late-onset form of adhd has not been studied enough. therefore, berkeley pharma tech journal of medicine | 67 con�icting results on genetic studies provide little information about the late-onset of adhd in adults compared to children. understanding heritability, long-term causes, and environment-gene interactions are required in whole-genome sequencing analyses. as of current literature, genetic studies of adhd have been targeted to risk factors. by identifying these genetic underpinnings, it could reveal whether or not people with certain genes have resilience towards adhd given an environmental condition. understanding this interrelationship between genetic resilience and the genetic risk factors is important going forward. conclusion research on adhd and ways to treat it have been researched widely in the past several years. current treatments mainly include the use of stimulants or non-stimulants as medication. also, cognitive behavioral therapy (cbt) is another popular way to help patients deal with adhd. along with this, there is new research that provides evidence that adhd is inheritable and can run in families. with this new information, it can lead to the belief that focusing on gene mutations for treatment is a good next step to �nding a new treatment for adhd. there have been multiple genes that have been discussed including neurexins 1 (nrxn1), dopamine receptor d3 (drd3), glutamic acid decarboxylase 65 (gad65), patched domain containing 1 gene (ptchd1), and norepinephrine transporter (slc6a2). multiple in vivo studies have demonstrated the link between these genes and adhd. while all of this is known, there is still a lot to learn in this area. there is a lack of clinical trials and testing which is essential to determine whether or not these genes can be successfully mutated and provide signi�cant improvements to patients with adhd. berkeley pharma tech journal of medicine | 68 references 1. cdc. national prevalence of adhd and treatment: information on children and adolescents, 2016. centers for disease control and prevention. published september 9, 2020. accessed january 15, 2023. https://www.cdc.gov/ncbddd/adhd/features/national-pr evalence-adhd-and-treatment.html#:~:text=and%20key %20findings-,national%20prevalence%20of%20adhd %20and%20treatment,on%20children%20and%20adole scents%2c%202016&text=cdc%20scientists%20found %20that%2c%20as,is%20similar%20to%20previous%20e stimates 2. adhd statistics: new add facts and research. additude. published october 6, 2006. accessed january 15, 2023. https://www.additudemag.com/statistics-of-adhd/#:~:te xt=adhd%20prevalence%20in%20adults&text=prior 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autoantibodies in the serum of children with autism or adhd. european child & adolescent psychiatry . 2012;21(3):141-147. doi:10.1007/s00787-012-0245-1 21. wells mf, wimmer rd, schmitt li, feng g, halassa mm. thalamic reticular impairment underlies attention de�cit in ptchd1y/− mice. nature . 2016;532(7597):58-63. doi:10.1038/nature17427 22. raymond cm, frantzeskaki n, kabisch n, et al. a framework for assessing and implementing the co-bene�ts of nature-based solutions in urban areas. environmental science & policy . 2017;77:15-24. doi:10.1016/j.envsci.2017.07.008 berkeley pharma tech journal of medicine | 70 � berkeley pharma tech journal of medicine correspondence:� venkatarepaka@gmail.com keywords: thrombin hemophilia factors viii and fix hepatocytes aav vector inhibitors viral vectors submitted may 3, 2022� accepted�june 8, 2022 published july 19, 2022 full open access creative commons attribution license 4.0 abstract hemophilia is an x-linked recessive genetic disorder in which the body has an inability to clot, leading to an increased risk of mortality for individuals if a bleeding episode were to occur. traditional hemophilia treatments, such as prophylactic factor replacement therapy of recombinant factor viii and ix, have been proven to be costly and do not provide long-lasting protection during bleeding episodes. in recent years, the use of adeno-associated viral vectors (aav) gene therapy has been explored as a potential alternative due to efficient gene delivery and tissue tropism, overall safety and efficacy, and longer-lasting effects. however, concerns over inhibitor development persist due to the treatment complications. this review article seeks to provide an overview of the current state of aav-based gene therapy as a treatment for hemophilia. gene therapy for hemophilia �ïw�venkata repakau�daniel lomboy, priscilla castro introduction i. background information hemophilia is a bleeding disorder associated with di�culties in blood clotting. these complications are caused by the low levels of factor viii, associated with hemophilia a, or factor ix, associated with hemophilia b. the amount of clotting factor de�ciency determines the severity of hemophilia for each patient1-3. hemophilia a�ects over 30,000 individuals in the united states. over half ( around 60%) of that population is diagnosed with severe hemophilia while fewer su�er from moderate and mild hemophilia. hemophilia a is four times more common than hemophilia b and is one of the most widely distributed bleeding disorders in the population4. there are three classes of hemophilia: mild, moderate, and severe. the severity of hemophilia is de�ned by the percentage of clotting factor de�ciency. a healthy range of factor viii and ix is between 50-150%factor activity. individuals with mild hemophilia have 5-40% factor activity where bleeding proceeds injury, trauma, or surgery. moderate hemophilia has 1-5% factor activity where minor injuries or spontaneous bleeding occurs. severe hemophilia, the most common, is classi�ed by <1% factor activity where frequent exterior or interior bleeds may physically disable the individual5. current non-gene therapy includes methods such as direct infusion of the exogenous proteins6. these speci�c methods that are non-gene therapeutic do not provide long-lasting e�ects due to inhibitors and antibodies. replacement therapy is e�ective until an alloantibody is formed against the exogenous clotting factors7. ii. emerging technologies when researching gene therapies for hemophilia, there were an abundant amount of other gene therapies shown to be e�ective and helpful. some studies used the method by adding more precursor proteins to make the factors fviii and fix, the liver will be able to produce thrombin and platelets for hemophilic patients. in other words, these studies experimented by adding to the protein pathway rather than the genes of the factors; they added factors to help build the precursors of the factors or necessary stabilizers for the factors. one of these methods is called transgene therapies. there are many transgene therapies that evolved for various diseases as one berkeley pharma tech journal of medicine | 121 study suggests8. one of the transgene therapies is the fix-pauda and robinhood gene therapy which in short is the transfer of fix from a patient who has an abundance of fix, fix-padua proband, to a patient with a low abundance, hb patients. this will help hemophilia patients regain their fix levels. the limitation to this technique is the safety issue of immunogenicity and thrombogenicity as the doses were of concern. including other gene therapies, there are additional useful therapies not using the another method explored was activating more of the existing factor viii if they are turned o� in presence of thrombosis, such as an inducible vector. factor viii is a cofactor in the coagulation cascade and is primarily produced by endothelial cells. a study by alam et. al. observed that because thrombin regenerates itself with the increased amount of platelets and itself in the blood in the coagulation cascade it is important to have thrombin in the body. by endothelial injury the release of thrombin will amplify the activation of factor viii by re-detachment of factor viii from the von willebrand factor and factor viii complex. two patients who had undergone both arterial and venous thrombotic events before observing an increase in factor viii9. von willebrand factor (vwf) is a major contributing factor to the availability of factor viii (fviii) as vwf is a speci�c carrier protein that protects fviii of proteolytic enzymes and from degradation10. this insight of the activation of factor viii suggests that there might be an activation method to make the factor viii protein more readily available to the coagulation cascade and help with blood clotting. this activating more of the existing factor viii if they are turned o� in presence of thrombosis can prove to be a consideration in hemophiliac studies. another study also included alternating the amino acids on factor viii will make it more readily activated. a study by nogami et.al. conducted an in vivo experiment which included altering the 372 amino acid position of an arginine protein on the factor viii protein to a histidine protein to make it more detachable from the vwf-fviii complex. although this is a novel in vivo approach, the study saw little to no change in the cleavage rate with the amino acid change suggesting that there may be other properties associated with the thrombin and the vwf-fviii complex cleavage mechanism11. this inducible mechanism can also be used in the aav vector that is an emerging technology. these mechanisms are sin berkeley pharma tech journal of medicine | 122 vectors which contain an inducible package of proteins that can turn the expression of the vector on and o� determined by a protein presence. particularly the protein would function as an antibiotic such as doxycycline or a small molecule45. furthermore, another method that was proven to be e�ective is site-speci�c bioconjugation which alters the activation of factor vii which triggers the whole cascade of coagulation as suggested by lieser et. al12. another type of therapy that was proven successful by clinical trials of di�erent companies as suggested by a review article by sadiki et. al.. site-speci�c bioconjugation is the concept of aiding the speci�c proteins needed for delivery of drug carriers and moieties of proteins within the body. this technology increases protein circulation, activity, target speci�city, and protein properties as a whole. there are many site-speci�c bioconjugations which include unnatural amino acid incorporation, sortase-mediated ligation, and spycatcher/spytag bioconjugation. all of these methods make alterations to the amino acids present in a particular drug carrier making the permeability of that drug easier in the body. this method is favorable because it changes the way particular factors and proteins are delivered to the body without recombinant engineering13. jivi is an antihemophilic factor (recombinant) gene or a factor viii concentrate. this study focused on reducing the amount of times patients need to have routine prophylaxis by making an on-demand treatment to control the bleeding. they used recombinant dna technology in baby hamster kidney (bhk) cells to generate the recombinant b-domain deleted human coagulation factor viii (bdd-rfviii) to obtain the activated molecule. due to its on-demand property, it can also be used as a perioperative treatment plan in the means of managing bleeding before surgery based on the patient's needs. in terms of hemophilia, this study and product took into consideration the intensity of bleeding of patients with hemophilia and treated people with severe hemophilia (two or more bleeds per week) two times weekly 30-40 iu/kg. patients with a low bleeding tendency were treated every 5 days or twice weekly just as the high intensity patients as weekly treatments did not show e�ciency. at the end of the study, there was a decrease of bleeds per week in patients in total14. in research studies, intensity or severity of hemophilia is de�ned by measuring fviii or fix activity as suggested by a review paper berkeley pharma tech journal of medicine | 123 by samelson-jones et. al.. this is again based on the potency of protein factor products and monitored post-infusion processes. additionally, two methods to test (if this information is going to be included this information, it would be helpful to elaborate on what these tests look like) out this are run through one-stage clotting assays (osas) or chromogenic substrate assays (csas) which can help de�ne or identify the severity of hemophilia in the patient15. lastly, there are a number of non-viral ways to transduct genes. some of those methods include electroporation, cationic proteins, cell-penetrating peptides, nanoparticles, crispr editing genes, and virus-like particles (vlps)45. iii. aav gene therapy the most popular and most successful emerging therapy to treat hemophilia relies on the use of an adeno-associated viral vector (aav) as a vehicle for gene delivery of factor viii and ix. aav-gene therapy is popular due to its e�cacy, relatively low invasiveness, minor side e�ects in animal models, and its ability to provide long-lasting expression of factor viii and ix. despite these advantages, however, the presence of neutralizing antibodies from previous exposure to aav remains a challenge that obstructs this therapy from being widely accepted. with minor concerns over inhibitor development, aav-gene therapy is proving to be a potential treatment for hemophilia patients. aav vector advantages and uses a particular gene therapy that is a gene therapy model that is being explored right now and very successful in various clinical trials. adeno-associated virus (aav) vector is a new technology that is emerging into the scienti�c community as an e�ective method to amend the existing molecular dna sequence of cells, without incorporating the virus’s dna into the host’s dna sequence16. there are many advantages to aav which are used for a plethora of diseases within the body including neurodegenerative diseases, cancer, and genetic diseases as a whole. this gene therapy model is being explored currently in various methods and research papers to observe new berkeley pharma tech journal of medicine | 124 dna appending techniques17. this gene therapy is nonpathogenic and has many uses to express genes of interest. additionally, there are new genetic sequencing methods that allow for the design of vectors/plasmids and target speci�c parts of the body. because of its great amendable characteristics to genetic engineering and repurposing makes it easy to design relative to previous gene therapy techniques suggested by a review by andari et. al.18. this not only makes it easy to design but increases the cell speci�city, cell or organ targeting, and transduction e�ciency. aav has shown to be non-pathogenic and induces a minimal in�ammatory response in mouse models and early human clinical trials. furthermore, aav has fewer biosafety hazards, unlike other gene therapies suggested by a review by aschauer et.al., while having a low immunogenicity and limiting the risk of insertional mutagenesis or other mutational changes when replicating17. figure 1: structure of an aav vector. goi, the gene of interest, target sequence that is to be inserted into the vector. other gene therapies focused on providing the proteins necessary for transduction while we wanted to focus on the core protein that is stopping transduction and see what was lacking in the pathway. the coagulation cascade is a concept that is still being explored as many elements play a role in the triggering events. a general outlook on the cascade is started with a stimulus from the external environment such as a cut or broken tissue increasing the amount of the protein thrombin to form the extrinsic pathway. a set of triggering events will then increase the von willebrand factor and factor viii to interact with factor ix. factor viii and ix are the main factors necessary for transduction which will trigger factor 10 to convert prothrombin to thrombin. thrombin activates factors v, viii, ix berkeley pharma tech journal of medicine | 125 and xi, to promote its own generation. thrombin then will then activate soluble �brinogen to convert into an insoluble version of itself called �brin to make the webbing of the blood clot in the location of the cut or tissue breakage. there is a plethora of gene therapy methods explored that focus on providing the proteins necessary for transduction, and the main protein factors being factors viii and ix. aav gene therapy focuses on promoting factors viii and ix which stop transduction and make up hemophilia19, 20. figure 2: blood coagulation cascades -the intrinsic and extrinsic pathways. by joe d own work, cc by-sa 3.0, https://commons.wikimedia.org/w/index.php?curid=1983833 vector and plasmid design from various clinical trials, we will compose a collection of e�ective aav vectors that can transduce the factors 8/9 into the hepatocytes. aav is a very broad type of vector that has many versions of itself that exist in nature21. the key di�erences between such vectors can include the length of gene it can hold, various repair enzymes, and the type of capsid proteins present for replication. all of these elements make up the speci�c aav vectors’ characteristics of replication speed and the size of the vectors. additionally, the size of the target sequence of the factor needed to insert into the is important to consider42. there is more research done on hemophilia b than hemophilia a due to the smaller size of factor 9 (c. 1.6 kb) and factor 8 (c. 7 kb). the whole size of aav vectors (c. 4.7 kb) limits berkeley pharma tech journal of medicine | 126 the packaging capacity so this is another limitation of clinical studies. a review paper by neinhus et. al. suggested that the recombinant aav2 serotype was used in initial studies because it was the �rst aav vector serotype to be characterized. this serotype was a single-stranded vector and was 7 kb in length21. this length included 145 base pairs of inverted terminal repeats (itrs) which are the regions of the vector that must be preserved for the vector to allow packaging and further sequencing. this virus also included adeno-viral proteins in the form of capsid proteins for packaging and to form viral vectors. another comprehensive study by sands et. al. showed the various types of aav vectors’ serotypes tested through liver-mediated infusion. it was known that the aav2 serotype was very versatile and could pack many genes with other supporting capsid proteins. as aav2 was kept as one type of control, changing the serotype of the vector greatly changed its tropism and e�ectiveness to help the body. vector serotypes 1, 5, and 4 were tested to be more e�cient in transducing cells in the murine nervous system or within the mouse model. because the liver is shown to have more a�nity to particular capsid proteins of di�erent serotypes, vectors 8 and 9 had more a�nity to the liver greater than that of serotype aav 2. aav vector 8 was very readily transduced regardless of the method it was transferred through; it was equally e�cient in both the intraportal vein injection and by intravenous (tail vein) injection. additionally, when comparing the e�ciency of the raav-8 of fix administered through the portal vein injection and tail vein injection in vivo experiments in mice, there was an equal amount of expression of the fix factor in the plasma throughout 20 weeks of observation time. the factor peaked during the 4 weeks mark and gradually decreased its expression by 75% as by the end of the 20 weeks. the vectors were prepared with the help of a native liver cell from the mouse with the double stranded vector and 10 μg of the factor plasmid. in conclusion, this study has resulted in the type of capsid proteins for each serotype making the aav vector serotype have a di�erent a�nity, transduction e�ciency, and tropisms in the body22. limiting factor however, the transduction of aav particles into the liver is limited by the ability for ssdna to be converted into dsdna, as the viral mechanism is dependent on host machinery23. to solve this problem, a study by nathwani berkeley pharma tech journal of medicine | 127 et. al. devised a liver-restricted mini-human factor ix (hfix) expression cassette that allows aav dna to be packaged as dimers24. as a result, hfix expression in mice models produced a 20-fold increase compared to similar single-stranded aav vectors (ssaav). advantage of aav technology is that di�erent serotypes have the capability of targeting speci�c tissues. aav2 serotype has been shown to minimize tissue tropism as its primary transduction comes from hepatocytes. this also was bene�cial in that it allowed researchers to use fewer aav particles in hemophilia patients, thus lowering cytotoxicity. figure 3: stages in aav vector delivery and transgene expression, listed in combination with some resulting proteins involved in the process. gene target sequence after designing and analyzing which aav vector to use for such an experiment, a good gene target sequence is necessary to insert into the aav vector to express the needed vectors. many pharmaceutical companies such as biomarin pharmaceuticals, spark therapeutics, p�zer, and uniqure25. the following companies have already designed and inserted the target fviii or fix gene sequence gene therapy products into the vectors and are in clinical trials evaluated in phase iii studies. a particular review paper by doshi et. al.26 discussed clinical trials that suggested broadening the gene berkeley pharma tech journal of medicine | 128 therapy vector applicability with patients who had pre-existing neutralizing alloantibodies to the vector or in other words the clotting factors. here the vector genome is manipulated so that the needed gene target sequence can replace the vector sequence under a tissue-speci�c promoter to make a recombinant aav vector (ravv). in totality, the completed modi�ed aav vector in order included the itr, promoter, inhibitor, transgene, polya, and itr. the results varied based on the way the aav vector was given via skeletal muscle, liver-directed, and intramuscular trials. aav administration re�ning administration of aav gene therapy so as to optimize “uptake” of vectors, induce expression, provide longer-lasting e�ects, and reduce liver damage/minimize immune system e�ects. the site of infection can greatly increase or decrease the e�ciency of the transduction of aav in the body. intraportal vein injection includes an infusion directly to the portal vein of the liver21. according to a study by nienhuis et. al. from the university of london, raav2 was shown to have the same e�ciency as the portal vein method and the peripheral vein infusion method. initially, it was thought that a portal vein injection was needed to secure the liver transduction in the mice but this was not true. the transduction was largely a�ected by the types of capsid proteins present in the aav vector serotype. based on the study by doshi et. al. suggesting that patients who already had existing neutralizing antibodies (nabs) in the liver—directed gene therapy showed that the aav transduction e�ciency was hindered by pre-existing nabs. (26) although nabs can be present in 30-70% of the population, immunosuppressors were used in subsequent trials involving the liverdirected transduction. contrastingly, the intramuscular trial did not have this e�ect. another method of vector infusion is direct intraparenchymal injection. a study by sands et. al. suggested that a direct injection of an aav2 vector into the liver parenchyma which results in a relatively widespread transduction throughout the liver22. this method also retains the aav gene expression for a longer period of time in the body. berkeley pharma tech journal of medicine | 129 figure 4: the three types of aav administration methods dosage scientists must determine the correct dose of aav vector particles injected into the patients, as a low concentration may not produce a robust amount of expression, while a dosage that is too high may cause an immune response and liver damage. scientists have sought to address this issue by increasing the transduction of the vector into cells such that fewer vector particles are needed and thus limiting host immunity. minimizing immunogenicity there are two ways to decrease immunogenicity; one such measure refers to a process known as site directed mutagenesis. two vector serotypes currently being used in clinical studies, raav-dj and raav-lk03 are chimeric receptors of natural aav serotypes. a study by ran et. al. (2020) showed that site-directed mutagenesis (raav-dj-s269t) yielded higher transduction e�ciency compared to wild-type aav vectors, likely due to evasion of the host immune response27. the second method of re�ning the aav vector for administration is the use of directed evolution28, where a wild-type aav vector is used to generate large mutant capsid libraries and aav2 variants with enhanced properties, such as immune evasion, non-infection of resistant cell types, and tissue transport. inhibitors the immune system develops antibodies to defend against foreign agents. patients with hemophilia sometimes develop antibodies to fviii in response to treatments and medication. polyclonal high-a�nity berkeley pharma tech journal of medicine | 130 immunoglobulin g (igg), or an inhibitor, prohibits clotting factor activity that further promotes bleeding without clot formation. type i and type ii inhibitors vary based upon the extent of inhibition of clotting factors. the development of inhibitors is a widely understood phenomenon that continues to burden treatment e�ciency. its e�ects are being studied through both genetic and environmental factors that contribute to formation. the detection and quanti�cation of fviii inhibitors include the bethesda assay and the nijmegen-modi�ed bethsda assay29, 30. inhibitor formation contains treatment-related risk factors that include age and intensity at �rst exposure, prophylaxis, and the type of treatment (recombinant or plasma derived). the e�ect of age was observed in a population of hemophilia a patients with the onset of fviii therapy within 1 year of age. it was found that patients that started therapy earlier in life are more prone to inhibitor development. however, there is a lack of knowledge on the e�ects of delayed treatment initiation due to the tendency for patients to require therapy in early age31. the intensity of treatment measured by scheduled gaps between series of exposures showed that shorter gaps were re�ective of increased risk of inhibitor development. prophylaxis is a treatment that exposes patients to antigens to minimize the possibility of immune response to additional treatment. it was found that prophylaxis reduces probability for the development of inhibitors32. pre-existing immunity from neutralizing antibodies or inhibitors can be overcome by creating alternate aav serotypes33. the success of serotype-switching is contingent on tropism similarities of the new serotype and the lack of crossreactivity between the serotypes. the criteria has been proven di�cult upon the high rate of crossreactivity in trials that prohibit successful e�orts34. immune tolerance induction (iti) is currently the only proven treatment to eliminate inhibitor development. despite a 60-80% success rate, the high cost creates �nancial di�culties for patients in need of this treatment. there is potential for gene therapy to serve as an iti-type therapy to eradicate the need for iti due to the continual production of clotting factors35. berkeley pharma tech journal of medicine | 131 alternatives to gene therapies while aav-gene therapy has made the most progress in the future treatment for hemophilia, new research has come out in an e�ort to bypass the concerns of aav explained previously. a recent study published by song et. al. (2022) shows a promising alternative to aav gene therapy known as ultrasound mediated gene delivery (umgd), where the plasmid containing the functional factor viii gene is injected directly into hemophilia a patients. umgd of reporter plasmids targeting mice livers achieved high levels of transgene expression predominantly in hepatocytes, while maintaining minimal transaminase levels following injection37. however, these results have yet to be proven in human clinical trials and are still years away. similar to aav vectors, lentiviral vectors do not contain viral genes and promote long-term gene expression which make both vectors strong candidates for gene therapy. despite increased stability upon genomic integration, lentiviral vectors activate the innate immune response to limit its capabilities in increasing clotting factor levels. the adaptive immune system, however, is unresponsive upon cell transduction38. hemostatic agents have been engineered to overcome fviii and fix activity in hemophilic patients as an alternate protein therapy. the hyperactive fviia variant has undergone experimentation as a transgene for aav-based liver-directed gene therapy. this resulted in the usage of a lower vector dose while maintaining a similar response to hemostatic conditions. although clinical trials have proceeded into late stages, it has halted due to immunogenicity speculation. if continued in the future, the fviia transgene may be able to lower the vector dose for patients. protein-engineered bypassing agents have prolonged the half-life of coagulation factors downstream of clotting factors that are designed to resist inactivation via conformational changes to protein structures. this treatment is undergoing early-stage clinical trials and has proven e�cacy as a protein therapy for acute hemorrhagic conditions39. berkeley pharma tech journal of medicine | 132 table 1: comparison between aav, lentivirus, and adenovirus vectors. inhibitors additionally, there are structural, functional, and expression di�erences between the aav vector, lentivirus, and adenoviral vectors to account for. aav is a smaller vector consisting of approximately 25 nm, whereas lentivirus is 80 nm to 120 nm, and adenovector is approximately 95 nm. aav alongside lentiviruses carry single-stranded dna, whereas adenoviral vectors carry double-stranded dna40. double-stranded aav vectors were also formulated by mutation of the inverted terminal repeat (itr). these vectors were shown to have better transduction in many other cell lines including in vivo experiments with hepatocytes. in comparison to the ssaav, the dsaav gene helped maintain better transduction and better dna stability when evaluated 6 months after the initial in vivo experiment41. aav has a lower packaging capacity consisting of approximately 4.7 kb while both lentiviral and adenoviral vectors have approximately 8 kb40. this may be a limitation due to the large gene target sequence of factors 8 and 9. adenoviruses and aav vectors are not enveloped which may cause more e�cient transduction of viral dna. the lentiviral vector is enveloped which keeps it well protected, but concurrently if the envelope is compromised, the virus can disintegrate. additionally, the lentivirus is double-stranded and embeds itself well into the host’s dna. the integration of the viral dna into the host’s dna will prelude long-term transgene expression. the major disadvantage of this mechanism is that it can disrupt the host’s genome by insertional mutagenesis. aav and adenovirus cannot integrate themselves into the host cell’s dna. the adenoviral vector is mostly used for short term uses as it is e�ective in transient expression unlike the persistent gene expression of the aav and berkeley pharma tech journal of medicine | 133 lentiviral vectors. lastly, immunogenicity decreases of each vector adenovirus, aav, and lentivirus respectively40. conclusion the purpose of this review article is to provide an outlook on the current state of gene therapy as a treatment for hemophilia. results of recent clinical trials have proven to be e�ective in restoring clotting ability in hemophilic patients. by identifying key concerns with gene therapy, scientists are taking steps to account for these problems by utilizing the following collection of sources and information to make an impact on their research the �ndings that we make will help other researchers improve upon this research to have a collection and a review composed of aav vectors to use in their clinical studies. results in�uence researchers to use the aav gene therapy because of its e�ectiveness in transducing genes into hepatocytes. just as in hemophilia, aav gene therapy is a useful technique applicable to many other diseases, which can lead to more breakthroughs and for other studies. this results in longer lasting and consistent treatment plans after insertion due to the limited amount of insertional mutability17. predicting or compiling e�ective aav vectors and their key qualities can advance further studies that want to use such gene manipulating technology that is not as biohazardous and feasible scheduled usage as it is e�ective for months17. with a comprehensive collection of data from various clinical trials, researchers in the future will also have a good understanding of challenges of such treatment that requires deliberation before pursuing treatment about options with the type of aav vectors to use and understanding the best way to infuse into the body18. our research can also advise a potential gene sequence for further research to use when developing the precise gene sequence to use in future studies. berkeley pharma tech journal of medicine | 134 references 1. nilsson im, berntorp e, lofqvist t, pettersson h. twenty-�ve years' experience of prophylactic treatment in severe hemophilia. a and b. j intern med. 1992; 232: 25–32. 2. lisowski, l., staber, j. m., wright, j. f., & valentino, l. a. (2021). the intersection of vector biology, gene therapy, and hemophilia. research and practice in thrombosis and haemostasis, 5(6), e12586. https://doi.org/10.1002/rth2.12586 3. what is hemophilia. centers for disease control and prevention. https://www.cdc.gov/ncbddd/hemophilia/fact s.html. published july 17, 2020. accessed march 29, 2022. 4. fast facts. national hemophilia foundation. https://www.hemophilia.org/bleeding-disorde rs-a-z/overview/fast-facts. accessed march 29, 2022. 5. tbh creative info@tbhcreative.com. severity of hemophilia. indiana hemophilia & thrombosis center. https://www.ihtc.org/severity-of-hemophilia. accessed march 29, 2022. 6. peyvandi f, garagiola i, seregni s. future of coagulation factor replacement therapy. j thromb haemost. 2013;11 suppl 1:84-98. doi:10.1111/jth.12270 7. witmer c, young g. factor viii inhibitors in hemophilia a: rationale and latest evidence. ther adv hematol. 2013;4(1):59-72. doi:10.1177/2040620712464509 8. samelson-jones bj, arruda vr. protein-engineered coagulation factors for hemophilia gene therapy. mol ther methods clin dev. 2018;12:184-201. published 2018 dec 31. doi:10.1016/j.omtm.2018.12.007 9. alam a, doshi h, patel dn, patel k, james d, almendral j. myocardial infarction and factor viii elevation in a 36-year-old man. proc (bayl univ med cent). 2021;35(1):93-95. published 2021 sep 15. doi:10.1080/08998280.2021.1973285 10. castaman g. treatment of von willebrand disease with fviii/vwf concentrates. blood transfus. 2011;9 suppl 2(suppl 2):s9-s13. doi:10.2450/2011.003s 11. nogami k, zhou q, wakabayashi h, fay pj. thrombin-catalyzed activation of factor viii with his substituted for arg372 at the p1 site. blood. 2005;105(11):4362-4368. doi:10.1182/blood-2004-10-3939 12. rachel m. lieser, daniel yur, millicent o. sullivan, and wilfred chen. bioconjugate chemistry. 2020 31 (10), 2272-2282. doi: 10.1021/acs.bioconjchem.0c00456 13. sadiki a, vaidya sr, abdollahi m, et al. site-speci�c conjugation of native antibody. antib ther. 2020;3(4):271-284. doi:10.1093/abt/tbaa027 14. protect viii study design. jivi us hcp. https://www.jivihcp.com/en/protect-viii-trial. accessed march 29, 2022. 15. samelson-jones bj, arruda vr. protein-engineered coagulation factors for hemophilia gene therapy. mol ther methods clin dev. 2018;12:184-201. published 2018 dec 31. doi:10.1016/j.omtm.2018.12.007 16. curiel dt, fisher pb, tew kd, kaliberov sa. chapter seven cancer treatment with berkeley pharma tech journal of medicine | 135 gene therapy and radiation therapy. in: advances in cancer research. amsterdam: elsevier academic; 2012:221-263. 17. aschauer df, kreuz s, rumpel s (2013) analysis of transduction e�ciency, tropism and axonal transport of aav serotypes 1, 2, 5, 6, 8 and 9 in the mouse brain. plos one 8(9): e76310. https://doi.org/10.1371/journal.pone.007630 18. el andari, j. and grimm, d. (2021), production, processing, and characterization of synthetic aav gene therapy vectors. biotechnol. j., 16: 2000025. https://doi.org/10.1002/biot.202000025 19. factor viii (fviii). diapharma. https://diapharma.com/factor-viii/. published november 15, 2021. accessed march 29, 2022. 20. fviii: a crucial hemostatic factor to control bleeding. educ8. https://www.educ8-h2020.eu/esrs-blog/fviii-acrucial-hemostatic-factor-to-control-bleeding. accessed march 29, 2022. 21. nienhuis aw, nathwani ac, davido� am. gene therapy for hemophilia. in: molecular therapy. vol 25. 5th ed. cellpress; 2017:1163-1167. 22. sands m.s. (2012) aav-mediated liver-directed gene therapy. in: snyder r., moullier p. (eds) adeno-associated virus. methods in molecular biology (methods and protocols), vol 807. humana press. https://doi.org/10.1007/978-1-61779-370-7_6 23. fisher kj, gao gp, weitzman md, dematteo r, burda jf, wilson jm. transduction with recombinant adeno-associated virus for gene therapy is limited by leading-strand synthesis. j virology. 1996;70(1):520-532. doi:10.1128/jvi.70.1.520-532.1996 24. nathwani ac, gray jt, ng cy, et al. self-complementary adeno-associated virus vectors containing a novel liver-speci�c human factor ix expression cassette enable highly e�cient transduction of murine and nonhuman primate liver. blood. 2006;107(7):2653-2661. doi:10.1182/blood-2005-10-4035 25. breakthroughs in gene therapy for hemophilia. ashpublications.org. https://ashpublications.org/ashclinicalnews/ne ws/4099/breakthroughs-in-gene-therapy-forhemophilia. accessed march 29, 2022. 26. doshi bs, arruda vr. gene therapy for hemophilia: what does the future hold? therapeutic advances in hematology. september 2018:273-293. doi:10.1177/2040620718791933 27. ran, g., chen, x., xie, y., zheng, q., xie, j., yu, c., pittman, n., qi, s., yu, f. x., agbandje-mckenna, m., srivastava, a., & ling, c. (2020). site-directed mutagenesis improves the transduction e�ciency of capsid library-derived recombinant aav vectors. molecular therapy. methods & clinical development, 17, 545–555. https://doi.org/10.1016/j.omtm.2020.03.007 28. maheshri, n., koerber, j., kaspar, b. et al. directed evolution of adeno-associated virus yields enhanced gene delivery vectors. nat biotechnol 24, 198–204 (2006). https://doi.org/10.1038/nbt1182 29. gouw sc, van den berg hm. the multifactorial etiology of inhibitor berkeley pharma tech journal of medicine | 136 development in hemophilia: genetics and environment. semin thromb hemost. 2009;35(8):723-734. doi:10.1055/s-0029-1245105 30. witmer c, young g. factor viii inhibitors in hemophilia a: rationale and latest evidence. ther adv hematol. 2013;4(1):59-72. doi:10.1177/2040620712464509 31. lorenzo ji, lópez a, altisent c, aznar ja. incidence of factor viii inhibitors in severe haemophilia: the importance of patient age. british j haematology. 2001;113(3):600-603. doi:10.1046/j.1365-2141.2001.02828.x 32. gouw sc, van der bom jg, marijke van den berg h. treatment-related risk factors of inhibitor development in previously untreated patients with hemophilia a: the canal cohort study. blood. 2007;109(11):4648-4654. doi:10.1182/blood-2006-11-056291 33. louis jeune v, joergensen ja, hajjar rj, weber t. pre-existing anti-adeno-associated virus antibodies as a challenge in aav gene therapy. hum gene ther methods. 2013;24(2):59-67. doi:10.1089/hgtb.2012.243 34. boutin s, monteilhet v, veron p, et al. prevalence of serum igg and neutralizing factors against adeno-associated virus (aav) types 1, 2, 5, 6, 8, and 9 in the healthy population: implications for gene therapy using aav vectors. hum gene ther. 2010;21(6):704-712. doi:10.1089/hum.2009.182 35. patel sr, lundgren ts, spencer ht, doering cb. the immune response to the fviii gene therapy in preclinical models. front immun. 2020;11. doi:10.3389/�mmu.2020.00494 36. song s, lyle mj, noble-vranish ml, et al. ultrasound-mediated gene delivery of factor viii plasmids for hemophilia a gene therapy in mice. mol ther nucleic acids. 2022;27:916-926. published 2022 jan 10. doi:10.1016/j.omtn.2022.01.006. 37. ultrasound-mediated gene delivery of factor viii plasmids for hemophilia a gene therapy in mice, mol ther nucleic acids, volume 27, 2022, pages 916-926, issn 2162-2531, https://doi.org/10.1016/j.omtn.2022.01.006. 38. vandendruessche t, thorrez l, acosta-sanchez a, et al. e�cacy and safety of adeno-associated viral vectors based on serotype 8 and 9 vs. lentiviral vectors for hemophilia b gene therapy. j thromb haemost. 2006;5(1):16-24. doi:10.1111/j.1538-7836.2006.02220.x 39. samelson-jones bj, arruda vr. protein-engineered coagulation factors for hemophilia gene therapy. mol ther methods clin dev. 2018;12:184-201. published 2018 dec 31. doi:10.1016/j.omtm.2018.12.007 40. tosolini ap and sleigh jn (2020) intramuscular delivery of gene therapy for targeting the nervous system. front. mol. neurosci. 13:129. doi: 10.3389/fnmol.2020.00129 41. wang, z., ma, hi., li, j. et al. rapid and highly e�cient transduction by double-stranded adeno-associated virus vectors in vitro and in vivo. gene ther 10, 2105–2111 (2003). https://doi.org/10.1038/sj.gt.3302133 42. paul batty, david lillicrap, advances and challenges for hemophilia gene therapy, hum berkeley pharma tech journal of medicine | 137 mol gen, volume 28, issue r1, october 2019, pages r95–r101, https://doi.org/10.1093/hmg/ddz157 43. batty, paul; lillicrap, david hemophilia gene therapy: approaching the first licensed product, hemasphere: march 2021 volume 5 issue 3 p e540 doi: 10.1097/hs9.0000000000000540 44. ohmori, t. advances in gene therapy for hemophilia: basis, current status, and future perspectives. int j hematol 111, 31–41 (2020). https://doi.org/10.1007/s12185-018-2513-4 45. james w. larrick, m.d. ph.d., interview, april 27, 2022. berkeley pharma tech journal of medicine | 138 front_page_repaka body berkeley pharma tech journal of medicine correspondence: ygayoung@umich.edu keywords: alzheimer's disease (ad) ad pathology micrornas (mirnas) amyloid cascade hypothesis amyloid beta (aβ) plaques tau hypothesis neurofibrillary tangles (nft) submitted: october 5, 2022 accepted: november 21, 2022 published: december 28, 2022 full open access creative commons attribution license 4.0 abstract alzheimer’s disease (ad) is a progressive, neurodegenerative disease and is the most common cause of dementia. the disease pathology of ad is marked by the accumulation of betaamyloid (aβ) plaques and deposition of phosphorylated tau nfts in the brain. currently, the therapeutic approaches to ad are solely palliative in nature and thus can do little to stop or reverse the disease pathology once it has begun. micrornas (mirnas) are a class of noncoding nucleotides that are critically involved in the regulation of various posttranscription mrna processes, such as synapse plasticity and neuron differentiation. owing to the multifactorial nature of ad, mirnas have garnered much attention in the research field due to their concomitant dysregulation and appearance in several ad pathophysiological processes. this review paper offers insight into the potential use of mirnas as a therapeutic target and diagnostic marker in ad. micrornas as a diagnostic marker and a therapeutic target for alzheimer’s disease by: william ugorji, gayoung yang and prakruti jajoo berkeley pharma tech journal of medicine | 49 1. introduction alzheimer’s disease (ad) is the leading cause of dementia in the world, with a disease burden exceeding more than 5.5 million individuals in the united states and up to 80% of all dementia diagnoses overall1. the two clinical hallmark pathologies of the disease are the beta-amyloid (aβ) plaque and deposition of neurofibrillary tangles (nfts) of hyperphosphorylated tau hypotheses. the beta-amyloid hypothesis suggests that an accumulation of amyloid plaques in the brain triggers a cascade of pathological events that ultimately lead to the clinical presentation of the disease1. isoforms of the aβ peptide are implicated in the beta-amyloid hypothesis, and aβ38, aβ40, and aβ42 exhibit the greatest tendency to cluster and produce the neuronal oligomers that trigger the amyloid cascade1. within this cascade, various biological stressors, such as localized inflammation, glutamate production, and oxidation, are involved. conversely, the tau hypothesis entails the process of hyperphosphorylated tau decoupling from their normal microtubule function to form nfts that disrupt normal neuronal function and transport. while the beta-amyloid and tau hypothesis were initially regarded as two distinct pathophysiological pathways, recent evidence has given credence to the notion that both pathways act interdependently, resulting in the clinical presentation of ad1. symptoms of cognitive impairment and memory loss are the main assessment criteria for ad patients, and differential diagnosis can be made through cerebrospinal fluid (csf) and positron emission tomography (pet) analysis1. these methods, however, suffer from issues such as the high cost of application, invasiveness, and low specificity that combine to limit their accessibility and usage in routine clinical scenarios2. as such, a definitive diagnosis of ad can only be made through post-mortem tissue analysis of suspected ad patients. the current treatment landscape for ad primarily involves palliative therapeutic approaches, such as cholinesterase inhibitors and n-methyl daspartate (nmda) antagonists like memantine. cholinesterase inhibitors include drug classes like donepezil and galantamine and work by stopping the degradation of acetylcholine molecules in the brain3. cholinesterase inhibitors are typically prescribed for ad patients in the mild, moderate, or berkeley pharma tech journal of medicine | 50 severe dementia stages based on the mini-mental state examination score (mmse)4. nmda antagonists function by regulating glutamate action in the brain3. while the current pharmacological approaches for ad can enhance patient quality of life, they are unable to alter disease progression or overall life expectancy for individuals with ad. as such, there exists a great need to develop a non-invasive, cost-effective approach to provide robust early diagnosis and treatment options for this condition in the medical community. micrornas (mirnas) are a class of small, non-coding rna molecules that have the potential to act as therapeutic agents and biomarkers in ad. ranging approximately 22 nucleotides in length, mirnas work on the posttranscriptional level of mrnas and are involved in the regulation of nearly every cell and protein-coding process in the body5. a few of the regulatory facets that mirnas act on include cellular differentiation, proliferation, as well as programmed cell-death mechanisms like apoptosis6. a single mirna can alter up to 200 mrnas and multiple genes, thus making them increasingly valuable targets for research into how changes in their expression levels contribute to disease states such as ad or cancer5. mirnas specifically function by attaching and binding to the 3’ untranslated region (utr) of their associated mrnas, in which the complementarity between the mirna and its target eventually results in the degradation of the mrna targets7. current research shows that approximately 70% of identified mirnas have expression patterns in the brain, which further highlights the dynamic potential of mirna processes in healthy brain function and diseased states7. within the brain and nervous system environment, mirnas are particularly involved in regulating synapse plasticity, dendritic spine morphology, and neurite outgrowth, thus contributing to the modulation of the cognitive and memory functions typically lost in ad pathology through acting upon protein synthesis in synapses8. mirnas are predominantly found in circulatory biofluids, such as blood serum and plasma9. due to its proliferation in the blood serum, mirna has wide potential to be a cost-effective and less invasive diagnostic approach for ad. in this review, we focus on exploring the most recent advances in the berkeley pharma tech journal of medicine | 51 research and development of mirnas involvement in ad, which has broad therapeutic and diagnostic potential that remain to be unlocked. 2. pathophysiology of alzheimer’s disease 2.1. amyloid plaques amyloid pathogenesis starts with altered cleavage of aβ peptide from the internal transmembrane protein amyloid precursor protein (app) by the alpha, beta, and gamma secretase10. two main types of aβ polymers are involved in plaque formation and induced neurotoxicity. aβ40 is more abundant and less neurotoxic than aβ42, which is highly insoluble and neurotoxic. aβ40/aβ42 aggregation blocks ion channels, alters calcium homeostasis, increases mitochondrial oxidative stress, and diminishes energy metabolism and glucose regulation, which promotes neuronal cell death11. under normal conditions, proteolysis of app by either alpha or β-secretases (bace1) cleaves off small nontoxic fragments. however, altered cleavage of app by β followed by γ-secretases results in 42 amino acid peptides (aβ42) which then aggregate to form oligomers that diffuse into synaptic clefts and interfere with synaptic signaling. increased level of aβ42 leads to the polymerization of insoluble amyloid fibrils that causes neuronal toxicity10,11,12. accumulation of these amyloid plaques initially occurs in the basal, temporal, and orbitofrontal neocortex region of the brain and later progresses throughout the hippocampus, amygdala, and cerebral cortex12. sustained elevation and continuous aggregation of dense amyloid plaques can stimulate a chronic response of the innate immune system by inducing astrocytes and microglia recruitment surrounding the plaques. this results in the increased level of local inflammatory response due to the elevated release of inflammation related mediators, such as complement factors, eicosanoids, chemokines, and proinflammatory cytokines, which can disrupt microglial clearance of amyloid plaques, while also increasing microglia mediated neuronal death, damage to axons and dendrites, synaptic loss, and neurotoxicity10,11. consequently, these neuronal dysfunction inhibits cell synaptic communication, contributing greatly to ad pathogenesis. berkeley pharma tech journal of medicine | 52 2.2. neurofibrillary tangles (nfts) tau protein, encoded by the microtubule associated protein tau (mapt) gene, is a microtubule-associated protein that can form insoluble filaments that accumulate as nfts. tau protein facilitates the maintenance of neuronal structure and function. in alzheimer’s, the abundance of aβ plaques in the cell causes the tau protein to become hyperphosphorylated, leading to oligomerization. consequently, the loss of its affinity for tubulin leads to dissociation of tubule subunits that eventually fall apart and form large fragments of tau filaments that can aggregate into nfts. nfts are straight, fibrillary, and highly insoluble patches in the neuronal cytoplasm that are composed of paired helical fragments (phfs) of tau fibrils approximately 20 nm in diameter. they spread throughout the brain as ad progresses, beginning near the entorhinal cortex, leading to abnormal loss of communication between neurons and signal processing and finally apoptosis, cell death, in neurons. in addition, the transfer of hyperphosphorylated tau proteins diffuse to surrounding cells and cause damage to neuronal function, contributing to the onset of ad. genomic studies of brain tissues of ad patients have found significant downregulation of mirna-124 and mir-425-5p, which under normal conditions inhibit the abnormal hyperphosphorylation of tau protein. on the other hand, a significant upregulation of mir-132 and mir-125b in ad patients has been found to induce tau protein phosphorylation and neuronal apoptosis. moreover, mirnas could promote the progression of ad by promoting phosphorylation of the tau protein. mir-483–5p, mir-125b-5p and mir23b indirectly regulates tau phosphorylation by targeting extracellular signal-regulated kinases 1 and 2(erk1/2), protein phosphatase methylesterase-1 (pme-1) and n-acetylglucosaminyltransferase iii (gntiii) signaling pathways, respectively13. amyloid pathology appears to precede that of tau, with nfts only being found in regions where amyloid was already present, but both aβ plaques and neurofibrillary tau tangles are significant factors of the pathogenesis of ad that lead to synapse loss and neuronal atrophy, especially throughout the hippocampus and cerebral cortex11, 12. berkeley pharma tech journal of medicine | 53 2.3. microglial activation microglia are macrophages that reside in the cns and play a significant role in maintaining neuronal plasticity, synapse remodeling, phagocytosis, and clearance of aβ plaques in healthy individuals. the binding of microglia to extracellular aβ plaques via cell-surface receptors such as scara1, cd36, cd14, α6β1 integrin, cd47, and toll-like receptors (tlrs), a class of pattern recognition receptors (prrs) that ignites innate immune response, initiates the endocytosis of aβ oligomers and nfts for degradation by microglial proteases such as neurolysin and insulin-degrading enzymes11. however, under pathological conditions, the phagocytotic functioning of the aβ oligomers by the microglia is weakened, and overexpressed aβ plaques and nfts can lead to microglial activation around the plaque areas and release inflammatory factors such as il-1β and tnf-α, which aggravate neuronal damage and exacerbate ad pathology by giving rise to increased synaptic damage, oxidative stress, and neuroinflammation7. furthermore, insufficient aβ plaque clearance is observed due to elevated levels of localized cytokine concentrations that downregulate the expression of aβ phagocytosis receptors, thus decreasing aβ clearance13. moreover, proliferation and activation of microglia in the brain can affect insulin receptor substrate 1 (irs-1) and block intracellular insulin signaling, which has an important role in neural health11. 3. genetic basis the role of genetics in ad pathogenesis accounts for about 70% of the ad cases. most cases of early onset ad (eoad) are known to be inherited in an autosomal dominant pattern and mutations in the dominant genes including amyloid precursor protein (app), presenilin-1 (psen-1), presenilin-2 (psen-2)14, apolipoprotein e (apoe), clusterin (clu), bridging integrator 1 (bin1), and trem215. 3.1. app app is a transmembrane protein cleaved by α-, β-, and γ-secretase that releases aβ and other proteins and is encoded by the app gene on chromosome 21. twenty-five out of thirty mutations have been found in the app gene to be related to ad, along with the elevated level of insoluble aβ aggregates. km670/671nl mutation in mouse models has shown an increasing level of berkeley pharma tech journal of medicine | 54 amyloid plaques in the hippocampus and cortex. a673v, d678h, d678n, e682k, and k687n mutations have shown cortical atrophy, and e682k has shown hippocampal atrophy15. moreover, a673v mutations have shown a presence of nfts and aβ, activation of microglia and astrocytes, and neuronal loss. other mutations such as t714i, v715a, v715m, v717i, v717l, l723p, k724n, and i716v affect the γ-secretase cleavage site, increasing the aβ42/aβ40 ratio. e693g, e693k, d694n, and a692g mutations affect the α-secretase cleavage site, causing polymorphic aggregates that can disrupt bilayer integrity. meanwhile, one protective mutation (a673t) has been identified, which protects against ad by decreasing aβ, aβ40, and aβ42 secretion15. 3.2. psen1 and psen2 presenilin genes (psen1 and psen2) are part of the γ-secretase family and were also found to be mutated in ad14. it has been found that ad patients may be predisposed to psen1 mutation leading to familial ad at a young age. mutation in the psen1 gene is more common, with more than 200 mutations identified. on the other hand, a rare form with less than 40 mutations was identified in the psen2 gene, causing an increased level of toxic forms of amyloid aβ42 as opposed to non-toxic aβ40, aggravating ad pathology. psen1 plays an important role in the production of aβ from app by activating γ-secretase. knockout studies of psen1 in mice models showed synaptic dysfunction and memory impairment. on the other hand, psen-2 mutations might have a critical effect on the aβ 42/40 ratio, causing familial ad in the presence of normal psen-1 alleles, but some of the psen2 mutations are rare polymorphisms and are not pathogenic mutations and plays a relatively minor role in aβ production15. 3.3. apoe4 apoe protein is a glycoprotein expressed highly in the liver and brain astrocytes and some microglia. it serves as a receptor-mediated endocytosis ligand for lipoprotein particles like cholesterol, which is essential for myelin production and normal brain function. the apoe gene located on chromosome 19 has three isoforms, apoe2, apoe3, and apoe4, which cause changes in the coding sequence15. the apoe4 variant is the most significant genetic risk factor for sporadic alzheimer’s disease (sad). apoe4 berkeley pharma tech journal of medicine | 55 has a multidimensional impact on the pathogenesis of ad, including dysregulation of lipids and lipoproteins, such as apoe plasma levels13. the apoeε4 allele is a strong risk factor for both early onset ad (eoad) and late onset ad (load) compared to apoeε2 and apoeε3 alleles, which are associated with lower risk and protective effect, respectively. apoeε4 plays an important role in aβ deposition as a senile plaque and causes cerebral amyloid angiopathy (caa), which is known as a marker for ad. apoeε4 was also shown to be associated with vascular damage in the brain, which leads to ad pathogenesis15. 3.4. clu and bin1 clusterin (clu) and bridging integrator 1 (bin1) genes are novel risk factors for load. clu gene is reported to be located on chromosome 8. under normal conditions, clu plays a protective role by interacting with aβ and promoting its clearance or a neurotoxic role by reducing aβ clearance. however, in ad patients, clu is upregulated in the cortex and hippocampus. the aβ ratio values determine whether the clu role is neuroprotective or neurotoxic. bin1 is a bin-amphiphysin-rvs (bar) adaptor protein that is involved in the production of membrane curvature and other endocytosis cellular functions. bin1 has several isoforms—some are found in the brain, where they interact with different proteins such as clathrin, synaptojanin, and amphiphysin 1, and others in which they regulate synaptic vesicle endocytosis. recently, bin1 was recognized as the second most important risk factor for load after apoe, where it plays a role in aβ production and as a tau and nft pathology modulator15. 3.5. trem2 trem2 is a transmembrane receptor expressed in cells of the myeloid lineage. it facilitates mediating phagocytic clearance of neuronal debris and binds anionic carbohydrates, bacterial products, and phospholipids to transmit intracellular signals through the associated transmembrane adaptor dap1255 and further phosphorylation of downstream mediators. in ad, a rare mutation of trem2 (r47h) has been reported that plays a potent role in aggravating the risk of developing ad. this mutation leads to an inability of the receptors to clear aβ from the cns, contributing to aβ accumulation and further intensification of pathogenesis in ad patients16. berkeley pharma tech journal of medicine | 56 4. mirna usage in the treatment of ad as a result of their ability to post-transcriptionally regulate many of the genes involved in ad, mirnas have emerged as a valuable potential therapeutic agent for the treatment of ad. various mirnas have been shown to be integral to the production and regulation of amyloid-beta, and thus modification of mirna expression levels could attenuate facets of ad pathology16. therapeutic approaches to ad utilizing mirnas generally function through two pathways. mirnas expression levels may be restored to their normal baseline magnitude or be upregulated through the in vivo transfection of mirna oligonucleotide that mimics endogenous mirna functions. alternatively, harmful mirnas in ad can be downregulated through the administration of complementary antisense oligonucleotides17. subsequently, the most recent studies in the field have demonstrated promising results of mirnas facilitating neuroprotectant and ameliorating cognitive deficits through investigations using in vivo ad animal models and in vitro cell cultures17. in one study, upregulation of the mir-23b-3p molecule via in vitro transfection reduced hyperphosphorylated tau clusters and inhibited levels of neuronal apoptosis in swedish mutant amyloidprecursor protein (appswe) cells via inhibiting the upstream expression of glycogen synthase kinase-3 (gsk-3) apoptotic pathway18. this is significant since mir-23-b-3p has clinical diagnostic potential due to it being greatly downregulated in the blood plasma of ad patients when compared to healthy controls, and further investigations revealed a positive correlation between increased mir-23b-3p and improved measures of cognitive function for individuals with ad18. the mirna-200 family has also been shown to play an innate defensive role against aβ-induced neurotoxicity. a study conducted by higaki et. al. demonstrated that administration of members of the mirna-200 family – such as mir-200b/c – showcased reduced aβ in cellular mediums and in vivo amelioration of aβ-induced cognitive impairments in tg2576 mouse models, thus leading to restoration of spatial memory performance in the barnes maze test19. aβ has been shown to degrade the neuronal memory centers and synapses that act upon the hippocampus and frontal cortex tissue, making the mir-200 family a potential therapeutic target due to their berkeley pharma tech journal of medicine | 57 ability to alter s6k1 levels and modulate extracellular insulin signaling in the brain19. similarly, another study conducted by an et. al demonstrated the differential expression of mir-124 in ad compared to healthy control populations17. mir-124 was revealed to interact with the beta-site amyloid precursor protein cleaving enzyme 1 (bace1) network in ad, which is prominently involved in ad via functioning in the production of aβ from app20. it was found that mir-124 expression was downregulated in ad models while bace1 was simultaneously overexpressed20. subsequently, mir-124 is a potential novel therapeutic target as its administration via transfected mimics could be utilized to regulate and inhibit expression of bace1 mrnas and proteins in affected ad tissues, resulting in lowered aβ and nft burden20. many other mirnas are upregulated in ad pathogenesis and contribute to neurotoxicity, and thus therapeutic approaches that leverage underexpressing these classes of mirnas could potentially alleviate the negative effects on neurons posed by ad. through an analysis study on sh-sy5y cell cultures, it was shown that the pathogenesis of ad triggers dysregulation of mirna-146a, causing significant overexpression of these mirnas in the brain that results in the disruption of the phosphatase pten, thus causing increased hyperphosphorylation of tau protein and nft cluster formation21. the effects of mirna-146a overexpression were most pronounced in the brain regions that are generally most susceptible to tau aggregation in ad and other related dementias – most notably in the hippocampus and temporal cortices, which are fundamental to learning and memory formation21. in this same study, the 5xfad transgenic mice model reacted positively to the downregulation of mirna-146a via a series of mirna-146a antagomir injections, ultimately showing a restoration of endogenous frequencies of phosphatase pten and a rescue of function in hippocampal memory and neural pathways21. while mirnas have traditionally been downregulated in ad pathology, the overexpression of mir-146a has been experimentally shown to be intricately involved in the tau pathology of ad, thus making the selective inhibition of this mirna molecule a viable target for an in vivo therapeutic of the disease. a more extensive list of identified mirnas and their pathophysiologic targets in ad are displayed below in table 1. berkeley pharma tech journal of medicine | 58 the clinical application of mirna in ad can be achieved through diverse pathways and delivery methods. studies have shown that naturally occurring compounds like resveratrol can act upon and regulate specific mirnas in the body and, therefore, can be attuned to modulate mirna expression levels in vivo22. conversely, certain anti-inflammatory therapeutics can exhibit regulatory-like control on the expression of certain mirna molecules, thus providing a treatment option for ad that functions via mirna modulation23. other innovative and emerging approaches involve using exosome vesicles as a targeted vehicle to effectively cross the bloodbrain barrier and deliver mirna into localized regions for the greatest benefit in the diseased brain23. mirna target/pathologic process mir-126 involved in bace1 expression; overexpression suppresses bace1 while under expression increases bace1 proliferation mir-146 observed to have higher levels of expression in mci patients that later manifested with ad; is also associated with apoe4 protein expression mir-26b shown to increase tau phosphorylation and neuronal apoptosis mir-181a associated with an increased aβ concentration in cerebrospinal fluid mir-200b under expressed in experiments with app/ps1 transgenic mice & positively reduced app expression mir-339-5p targets & inhibits bace1; expression levels were shown to be reduced in ad patients mir-16-5p & mir-708-5p differentially expressed molecule in the cerebrospinal fluid cells of ad patients; potential biomarker berkeley pharma tech journal of medicine | 59 mir-93-5p differentially expressed molecule in the cerebrospinal fluid cells of ad patients; potential biomarker table 1: list of the relevant mirnas & the corresponding nervous system targets 5. mirnas functioning as predictive biomarkers of ad 5.1. amyloid cascade hypothesis there is evidence that aβ and its imbalance between production and destruction is a leading cause of ad, as it causes a cascade of events in the bodily systems24,25. app is cleaved by the bace-1 gene first to produce the aβ protein25, 26. some proteases are meant to clear the aβ in the system through proteolytic degradation and receptor-modulated endocytosis24. when this process is not done at an adequate rate, the aβ protein can build up in the blood plasma or csf. some forms of this protein are toxic and are known to cause neurotoxicity, neuron apoptosis, inflammation, and synaptic loss, which all play a role in ad24,25,27. the buildup of aβ protein is deposited as plaque in the systems of the brain and cns, which in turn causes increased production of proinflammatory cytokines. these cause the degeneration of glial cells and their tissue, which causes and progresses alzheimer’s25. 5.2. other mirna in pathogenesis of ad astrocytes regulate ion homeostasis in the brain, and therefore, support neuronal function. additionally, astrocytes have been shown to release cytokines which play a crucial role in disease progression and creation25. mirna is an important immunoregulator, and an imbalance in the expression of specific mirnas can cause certain proteins or cytokines to be over or under-produced. for instance, an excess of inflammatory cytokines causes the nearby neural cells and neurons to die25. this process, along with the amyloid cascade hypothesis, has shown that although ad cannot be pinpointed to one specific trigger or cause, the levels of particular mirna play a role in the existence, pathogenesis, and pathophysiology of ad. 5.3. specific mirnas as biomarkers there are many studies that show patterns in the levels of expression of specific mirna segments that relate to ad26. in the serum of ad patients, segments such as mir-149, mir-34a-5p, mir-125b-5p, mir-15b, mir-16, berkeley pharma tech journal of medicine | 60 mir-124, mir-29c and mir-374b-5p were all found in reduced quantities when compared to healthy individuals27. out of these, mir-149, mir-34a5p, mir-16, mir-29c and mir-374b-5p were found to directly target bace127. this relates to the upregulation of bace-1 found in ad patients, which in turn cleaves the app and promotes the production of aβ proteins. there were other mirnas, such as mir-146a and mir-181a, which were found in higher concentrations in ad patients, more specifically in patients who initially had mild cognitive impairment (mci) and later progressed into ad21. in different parts of the body, the expression levels of different mirnas were shown to have similar patterns. the downregulation of mir132/212 and mir-335–5p in the brain tissues were found in ad patients, which caused a higher production of aβ and more plaque buildup in the brain, causing symptoms consistent with the disease26. generally, mirna has shown to be strongly related to the pathophysiology of alzheimer’s and plays a direct role in many of the genes of interest. 6. genes/factors of interest 6.1. app studies have indicated that an increased app level due to genomic locus duplication or mutation in the app regulatory sequences can result in the development of early-onset dementias, including ad. app regulation is of special interest as it provides valuable insight into the genetic basis of ad and its novel therapeutics. a study that tested the regulatory effect of mirnas on the level of app gene expression demonstrated that complementary binding between mirnas hsa-mir-106a and hsa-mir-520c and their predicted target sequences within the 3' utr of app genes results in repression of reporter gene expression, and that over-expression of these mirnas resulted in translational repression of app mrna and significantly reduced app protein levels in human cell lines. these results were the first to experimentally demonstrate the post-transcriptional regulatory role of mirna in the levels of human app28. on the other hand, another study has demonstrated the activity of mir-346, which requires the chelation of an intracellular iron fe, that targets the app mrna 5′-utr to upregulate app translation and aβ production. it has also been found that mir-346 levels are altered in late-braak stage ad. as a result, mir-346 leads to the upregulation of app in the cns, and participate in maintaining app berkeley pharma tech journal of medicine | 61 regulation of fe, which is disrupted in the late stages of ad29. substantial progress towards understanding the role of mirna-induced alterations in the app gene expression and aβ production levels and the multifaceted biological involvement of mirnas in app metabolism is of interest to current research underlying the pathophysiology of ad28,29. 6.2. hyperphosphorylated tau peptide (p-tau) several functional in vivo studies have demonstrated the potential of microrna-23b-3p (mir-23b-3p) in various neurologic disorders30. study results support the potent involvement of mir-23b-3p as a neuroprotectant in ad. functional in vivo studies demonstrated that intracerebroventricular delivery of mir-23b-3p in app/ps1 mice reduced cognitive impairment and p-tau levels, while upregulation of mir-23b-3p resulted in an increased level of p-tau, aβ production, and neuronal apoptosis. these results thereby identify mir-23b-3p as a promising therapeutic target for ad30. several studies have speculated the functional role of tau in promoting microtubule assembly, stabilization, and spacing necessary for axonal transport. moreover, 6 isoforms of tau are present in the central nervous system due to the inclusion and exclusion of exons 2,3, and 10. another study aimed to uncover the role of mirnas in tau metabolism identified mir-16 and mir132 as putative endogenous regulators of neuronal p-tau and exon 10 splicing, respectively, and mutations in tau exon 10 have been associated with neurodegeneration and dementia in the adulthood31. overall, altered levels of mir-16 and mir-132 were associated with tau pathology in human neurodegenerative disorders31. 6.3. amyloid-β (aβ) peptides and bace-1 ad has been associated with increased production of amyloid-β (aβ) peptides and impaired function of its clearance. mirnas targeting the key proteins of the amyloidogenic pathway are of interest to current research on ad therapeutics. a study has shown that mir-31, previously found to be decreased in ad patients, suggests that mir-31-mediated modulation of app and bace1 can become a therapeutic option in the treatment of ad. mir-31 upregulation in 17-month-old ad triple-transgenic (3xtg-ad) female mice simultaneously reduced app and bace-1 mrna levels in the hippocampus, significantly ameliorated deficits in short and long-term berkeley pharma tech journal of medicine | 62 memory, and reduced anxiety and cognitive inflexibility. in addition, lentiviral-mediated mir-31 expression significantly reduced ad neuropathology in the mouse mode with a reduced aβ accumulation in both the hippocampus and subiculum26. mirnas typically inhibit protein expression by binding to its complement mrnas’ 3′-untranslated regions (3′-utr) in a cell-specific manner. however, the mechanisms of the variation of mirna activity remain unknown. another study demonstrated the differing effects of treatment of mir-298 reduced native app and bace-1 translation levels in astrocytic but not in a neuron-like cell line. the variation in its effects on app-3’-utr activity and native protein levels are according to cell type and 3’-utr specificity. researchers postulated that naturally occurring variations in the length of app 3’ utr could account for mir-298 cell-specificity. such novel mir-298 involvement in ad provides valuable insight into the clinically relevant research on tailoring mirna’s effect to a specific cell type in the treatment of ad32. 6.4. apolipoprotein e (apoe4) a study identified the involvement of mir-195 in apoe4-associated pathology in ad disease progression. results demonstrated that reduced levels of mir-195 in the ad brain correlated with early disease progression but not advanced stages of ad. moreover, it has been noted that the apoe4 genotype accelerated mir-195 reduction, leading to increased tau pathology and cognitive decline33. in addition, mir-195 overexpression reduced the expression of its target, synaptojanin1 (synj 1), a brain pip2-degrading enzyme that plays an important functional role in ad pathogenesis in mice models. for example, reduced synj 1 expression is associated with faster aβ clearance via the lysosomal degradation pathway, ameliorating elevated tau hyperphosphorylation levels and rescuing apoe4-associated neuronal impairment33. ongoing research aims to uncover specific mechanisms of mir-195 involvement targeted at apoe4 for future therapeutic purposes. 7. practical/clinical applications and feasibility 7.1. mirna benefits this novel therapeutic has shown promising results in research and laboratory environments. but how are these principles and tools translated and applied to the real world? the basis of the therapeutic is the molecule berkeley pharma tech journal of medicine | 63 mirna. compared to most nucleic acids, mirna is a highly stable molecule, therefore “surviving” in bodily fluids, such as the csf, when observing brain diseases such as ad34. this stability allows for an easier analysis, making it ideal for clinical settings. furthermore, it has been shown that mirna is better amplified in pcr treatments which is a very commonly used method in the scientific and medical field34. another benefit of the mirna is that they constrain sequences that create molecules that have been proven to be directly related to ad. more specifically, mirnas are known to regulate the production of genes like app and the bace-1, which affects the production of aβ32,34. for example, mir-340 can be used to alleviate ad by targeting the aβ and mir342–3p was shown to be in higher concentrations in ad patients, which increased the stress kinase c-jun n-terminal kinase, therefore increased the accumulation of aβ34, 36. the treatment is relatively simple and noninvasive, making it feasible for common use in clinical settings35. lastly, it has proven to be more cost-effective than the current or competing biomarkers for ad34. 7.2. mirna uncertainty a drawback or point of uncertainty in the research of mirna as therapeutics for alzheimer’s is the discrepancy between the different trials and projects, starting from the beginning at the root of the collection34. sometimes the mirnas are extracted from the plasma in the patient’s blood, and other times in the csf. the best example is mir-342-3p, a better biomarker in blood, and mir-127-3p, a better biomarker in csf34. this discrepancy, along with different measurements and amplifications or testing methods, has caused different results from various studies. however, it is unlikely that only one specific approach would be used. it is instead important to standardize each type of treatment or biomarker. some approaches are not limited to difficulties in delivering and targeting these mirna treatments, safety issues, and off-target effects of these treatments37. these mirnas play a role in aβ production through cascades of different molecules. similarly, they have control over other molecules and proteins. so, by changing the levels of specific mirnas apart from the aβ pathway being changed, other molecules and pathways could have residual effects. berkeley pharma tech journal of medicine | 64 7.3. current use the idea of using mirna has already been adapted into some clinical aspects. there are patents in place using the technique of mirna as biomarkers and therapies. one patent that has been filed and granted claims to be an invention that is able to measure the level of one neurite mirnas, specific mirnas that relate to mci, the starting stage of ad. these specific mirnas include mir-7, mir-125b, mir-128, mir-132, mir-874, mir134, mir-323-3p, and mir-38238. another patent in china has published a similar invention in which mirna is used as a biomarker to diagnose alzheimer's in patients. this invention is said to compare mirnas such as mir-191, mir-15b, and mir-142-3p with healthy controls to determine if the given sample has the precursors for ad39. 8. future directions 8.1. mirna delivery one step for the future of this novel treatment is enhancing the delivery method of the mirna therapies. mirna therapy is very targeted; therefore, alzheimer's patients would need the mirna injected directly into the cns40,41. mirna cannot pass through the blood-brain barrier, so conventional techniques would not be adequate40. the system by which the treatment is delivered is important as it affects how feasible and applicable it is in the clinical world as well as its effectiveness. 8.2. mirna studies currently, there is promise in mirna studies; however, there are limited clinical trials that are ongoing that study this potential therapy41. having more of these studies in the future will help the understanding of this therapy and has the potential to make it a more conventional practice in the medical field for diagnosing and treating ad. there are also other issues that need to be addressed, such as the tolerance of the patient’s mirna supplementation or inhibition41. the lag of mirna therapies, compared to treatments of other common or deadly illnesses, decreases the potential of issues like this from being resolved. more research needs to be done to garner a holistic view of mirna therapy. berkeley pharma tech journal of medicine | 65 9. conclusion alzheimer’s disease (ad) is a progressive neurodegenerative disease and is the leading cause of dementia across the globe. ad is frequently characterized by the deposition of aβ plaques and the formation of nfts in the brain. despite continued research into the disease, ad continues to be one of the leading causes of mortality in elderly populations globally, thus highlighting the need for novel and advanced therapies that can produce robust anti-ad effects. current ad treatments are typically invasive, costly, and noncurative, thus highlighting micrornas as an attractive alternative for the care of ad patients. mirnas are associated with several neurodegenerative diseases and play an outsized role in the pathogenesis of ad. several classes and families of mirnas are shown to be either upregulated or underexpressed in amyloid-beta 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references_ugorji et al. inhibitors of apoptosis proteins as potential research targets for decoding the pathological mechanisms of autoimmune diseases by: kezia philip, hoc lan phung and vanloan nguyen berkeley pharma tech journal of medicine correspondence keziaphilip.bpt@gmail.com keywords apoptosis necrosis necroptosis autoimmune diseases caspases submitted june 28, 2021 accepted august 20, 2021 published december 17, 2021 full open access creative commons attribution license 4.0 an artist's rendition of a cell undergoing apoptosis. image credits: kateryna kon / shutterstock.com abstract necroptosis, a type of pathological and inflammatory cell death, resembles necrosis, the termination of function of a bodily tissue, but adopts a unique molecular pathway that is not like apoptosis, resulting in vastly different immunological consequences. until recently, necroptosis was believed to mainly function as a protective mechanism that counteracts the viral barrier of apoptosis. however, mouse model studies have indicated that deficiency in elements of the apoptosis machinery such as caspase-8 or fadd can result in embryonic lethality driven by necroptosis. previous studies using conditional depletion of cellular inhibitors of apoptosis (ciaps) revealed that the necroptosis pathway is triggered under certain stressor conditions. these data support a new approach of targeting molecules within the cell death pathways to identify the origin of autoimmune diseases. hence, distinguishing between these two types of cell death may prove crucial during pathologic evaluations. this review provides a detailed insight into the emerging discussion on the various forms of cell death and the essential roles which certain molecules play in the development and progression of autoimmune diseases. armed with this knowledge, greater efforts can be targeted towards devising more effective treatments for interception of pathological diseases, prior to their uncontrollable progression. introduction over the last decade, advances in cell death research have greatly contributed to our understanding of cell death. dysregulation of cell death has been found to be critically involved in the onset of various human diseases, such as neurodegenerative diseases1, autoimmune diseases2, and cancer3. the three forms of cell death of interest – apoptosis2,4,5,6, necrosis7,8,9, and necroptosis3,10 – have distinct features and activate unique signaling pathways. apoptosis is a caspase-mediated programmed cell death that can be identi ed by chromosome condensation, nuclear fragmentation, and membrane blebbing2. conversely, necrosis is an unregulated, accidental form of cell death, triggered by non-physiological stress inducers and characterized by the expansion of cellular organelles, plasma membrane rupture, and subsequent in ammatory responses caused by the release of the intracellular contents7,8. the recent identi cation of necroptosis has transformed our understanding of regulated cell death. it has become increasingly evident that although the di erent types of cell death have distinctive characteristics, they are ultimately interconnected. thus, the activation or inhibition of a particular signaling molecular pathway under certain conditions determines the regulation or dysregulation of another associated cell death mechanism11,12,13,14. in this review, we focus on the speci c mechanisms involved in each particular type of cell death and the connections between them. by highlighting the pathophysiological relevance of necroptosis and the key roles of certain cell mediators and signaling molecules, we are proposing a new perspective for consequent medical research that investigates the pathogenesis of various diseases across the body, including neurological13,15, cardiovascular16, pulmonary, gastrointestinal, infectious, and autoimmune3,17,18 conditions, all of which have been linked to necroptosis. cell death and autoimmune disorders programmed cell death is an intricate biological element that plays a vital role in several physiological processes, including homeostasis, regulation of the immune system, and disease pathogenesis1. it involves multiple pathways and is regulated by various intrinsic cell death programs. over the past two decades, extensive research has transformed the understanding of programmed cell death and the di erent mechanisms that control it. berkeley pharma tech journal of medicine | 96 initially, apoptosis was believed to be the only form of programmed cell death, whereas necrosis was considered unregulated and resulting from a damaged environmental stress response3. however, emerging experimental evidence reveals a regulated form of necrosis, termed necroptosis, a form of cell death controlled by speci c intrinsic programs. autoimmune disorders, such as rheumatoid arthritis, can result from defects in multiple stages of these forms of cell death, ranging from mutant death receptors and ligands to speci c biochemical changes in death-inducing signaling molecules2. closely examining these complex mechanisms is very essential in gaining a better understanding of the pathogenesis of rheumatoid arthritis and several other autoimmune disorders. table 1: comparison between morphological features of apoptosis, necrosis, and necroptosis apoptosis apoptosis is an intracellular process that occurs during cellular development, providing a homeostatic mechanism to remove damaged cells3,4,5,6. cells undergoing apoptosis display morphological characteristics such as plasma membrane blebbing, chromosome condensation, nuclear fragmentation, formation of apoptotic bodies, and cell shrinkage. in the early stages of the disease, they also exhibit biochemical changes, such as the exposure of phosphatidyl-l-serine on the outer plasma membrane19,20,21. the mechanism of apoptosis involves certain molecules that play an important role in mediating cell death. apoptosis involves an energy-dependent cascade of molecular events that results from three primary pathways: the extrinsic (cell death) receptor berkeley pharma tech journal of medicine | 97 pathway, the intrinsic (mitochondrial) pathway, and the stress-induced pathway in the endoplasmic reticulum22. despite having di erent initiator caspases (caspase-8, caspase-9, and caspase-10), all of these pathways are linked via caspase-3 activation of downstream molecules1. the activation of the death receptor-mediated apoptosis pathway occurs when the fas ligand, tnf-α (tumor necrosis factor α), binds to the corresponding death receptors11,12. the adaptor protein fadd11,23, and the procaspase-8 protein form a complex, the death-inducing signaling complex (disc), in which procaspase-8 is activated by autohydrolysis24,25. the activated caspase-8 then transduces the apoptosis signal through, either the activation of caspase-3, or the cleavage of bid to truncated bid (tbid). tbid translocates to the mitochondria, resulting in conformational changes in bax and bak, as well as their oligomerization for pore formation in the outer mitochondrial membrane25,26. stress inducers such as dna damage, growth factor withdrawal, and oxidative stress activate the mitochondrial-dependent pathway27,28. this intrinsic pathway is controlled by the bcl-2 family of proteins, which regulates the permeability of the outer mitochondrial membrane29,30,31. upon release into the cytoplasm from the mitochondria, cytochrome c combines with apaf-1 to promote caspase-9 activation, which, in turn, activates e ector caspases32,33 to trigger a cascade of proteolytic events. the er-dependent pathway is mediated by an er-resistant caspase (caspase-12). it is activated under the presence of er stresses: the disturbance of calcium homeostasis, excessively unfolded or misfolded protein accumulation, nutrient deprivation, and hypoxia22. activated caspase-12 directly cleaves caspase-9 after its translocation from the er into the cytosol, followed by the activation of caspase-334. similarly, forming a complex with the inositol requiring enzyme-1α-tnf receptor-associated factor 2 (traf2), or by calpains, a family of ca2+-dependent intracellular cysteine proteases, activates the caspase-12 during er stress.7,35. together, these pathways result in phagocytosis of the apoptotic bodies by macrophages, neoplastic cells, or neighboring parenchymal cells24. interestingly, caspase-3 is the common factor linking these 3 pathways together, initiating the apoptosis execution pathway upon activation8. this marks caspase-3 as the potential target for studying the divergence of berkeley pharma tech journal of medicine | 98 each pathway, allowing for the manipulation of apoptosis. understanding the morphological and cellular characteristics of apoptotic mechanisms, as well as identifying the key molecules involved in mediating cell death, is integral in discerning their roles in autoimmune diseases. figure 1: mechanisms of apoptosis table 2: names and abbreviations of proteins in the extrinsic, intrinsic, and er-dependent pathways of apoptosis. berkeley pharma tech journal of medicine | 99 apoptosis and disease the repression of apoptosis increases the possibility of malignancy, as it inhibits tumor cell deaths. on the other hand, uncontrolled apoptosis is associated with various degenerative diseases including acquired immunode ciency syndrome36, cancer2, parkinson's disease37 and alzheimer's disease38. additionally, apoptosis has been correlated with hiv39, type 1 diabetes41,42, autoimmune thyroid diseases, systemic lupus erythematosus, rheumatoid arthritis, and sjogren’s syndrome. controlled apoptosis regulates normal t-cell selection and function. in turn, type 1 diabetes is caused by the insulin-secreting β-cells of the pancreas being attacked by t-cells. the foxp3+cd4+cd25high t-cells (tregs) represent one of the best characterized sub populations of regulatory t-cells that actively suppress e ector t-cells. the increasing evidence of tregs de ciency in various autoimmune diseases3, as well as in type 1 diabetes41,42, suggests a correlation between greater levels of tregs apoptosis and a decline in suppressive potential of these cells. however, the data collected are not always consistent in these studies since the investigations are conducted in di erent phases of the diseases and with various ongoing immunosuppressive therapies. most of these studies also have limitations in terms of distinguishing ntreg cells from activated te cells and characterizing their suppressive function. current studies fail to elucidate the pathways and genes that make tregs sensitive to apoptosis during the progression of the disease. recent evidence supports the involvement of the fas and trail mediated apoptotic pathways in the autoimmune diseases of the thyroid17,18. one of the earliest breakthroughs occurred when giordano et al. (1997) used immunohistochemistry, ow cytometry, and rt-pcr to discover the constitutive expression of fasl (fas ligand) on normal and hashimoto's thyroiditis (ht) thyrocytes43,44. although thyrocytes are known to express the death receptor fas, not much is known about how the expression is modulated. upregulation of fas was also found in the thyrocytes of patients with graves’ disease. discoveries like these strongly support the theory that apoptosis and the proliferation of thyrocytes may be abnormally accelerated in patients with thyroid disease, although the proliferation of thyrocytes may exceed their apoptosis, which would result in hyperplasia. berkeley pharma tech journal of medicine | 100 another autoimmune disease, systemic lupus erythematosus (sle), is linked to apoptosis through an increased number of apoptotic lymphocytes and macrophages observed in patients with sle45. characterized by the declining tolerance of self-antigens, this disease causes the production of antibodies, reactive with multiple self proteins46. apoptosis is crucial for regulating the duration of immune responses and maintaining the diversity of the lymphoid armamentarium. based on considerable statistical data, studies have identi ed that the de ciency of central molecules involved in lymphocyte apoptosis causes lymphoproliferative and autoimmune diseases in mice and humans47,48. in rheumatoid arthritis (ra) tissues in vitro, synoviocytes, synovial t cells and macrophages have been found to express high levels of fas and/or fasl and are highly susceptible to fas/fasl-induced apoptosis. conversely, abnormalities in fas/fasl expression and susceptibility to fas-induced apoptosis are generally not observed in osteoarthritis49. in some studies, invading t cells have been observed to be defective in fasl expression, which could explain the ine ective clearance of activated (fas-expressing) cells. additionally, rheumatoid synovial uid contains high levels of caspase-3 inhibiting nitric oxide. therefore, though these studies indicate that fas induced apoptosis is impaired in ra joints, they do not explain whether these phenomena are a direct result of the initial in ammatory pathways of ra or whether they underlie the disease etiology. apoptosis evidently plays a valuable role in the pathogenesis of several of the autoimmune diseases. the extent of apoptotic regulation dictates the pathological manifestations of these diseases. considerably repressed apoptosis increases the likelihood of malignancy, whereas unregulated apoptosis can directly lead to the initiation and progressions of several autoimmune diseases. the exact molecules and cellular targets determine the type of degenerative disease. thus, studies discovering the functions and mechanisms of cell mediators involved in cell death are crucial to elucidating the pathogenesis of autoimmune diseases. necrosis necrosis is an unregulated or accidental cell death due to internal or external stresses, resulting from the disruption of membrane homeostasis it leads to water imbalance between the extracellular and intracellular environments50,51,52. recently it has been suggested that a programmed berkeley pharma tech journal of medicine | 101 form of necrosis, commonly known as necroptosis, is a critical pathway that is involved in a number of autoimmune diseases, as well as heart diseases. the discovery of necroptosis signi es the need to di erentiate between the mechanisms that induce passive necrosis, and those that stimulate necroptosis, to better understand disease progression8,9. although necrosis and necroptosis share nearly identical characteristics, the implication of necroptosis in several diseases raises a topic of interest in research studying autoimmune diseases. although there are some morphological and mechanistic di erences between apoptosis and necrosis, these two processes overlap in some ways. evidence indicates that necrosis and apoptosis share a biochemical network described as the “apoptosis-necrosis continuum.”53 for example, a decrease in the availability of caspases, or intracellular atp, can convert an ongoing apoptotic process into a necrotic process54,55. the tissue type, the nature of the cell death signal, the physiological make up, and the developmental stage of the tissue all determine whether a cell dies by necrosis, or apoptosis53,56. using conventional histology to distinguish between apoptosis and necrosis proves di cult, as they can occur simultaneously depending on factors such as the availability of caspases, the intensity and duration of the stimulus, and the extent of atp depletion53. one of the primary di erences is that necrosis is an unregulated, passive process that usually impacts a large range of cells. apoptosis is contained, energy-dependent, and can a ect clusters or individual cells. necrosis is initiated by two main mechanisms: interference with the energy supply of the cell and direct damage to cell membranes. on the other hand, there is virtually no in ammatory reaction in apoptotic cells, because they do not release their cellular constituents into the surrounding interstitial tissue and are quickly phagocytosed by macrophages or adjacent normal cells57,58. necroptosis necroptosis di ers from apoptosis in several ways. cellls undergoing apoptosis maintain the integrity of their cell membrane, whereas necroptosis disrupts the cell membrane. although apoptosis and necroptosis share certain triggers, the intracellular signaling pathways that ultimately lead to each cascade di er16. apoptosis is known to be regulated by key mediators called caspases, whereas the main mediators of berkeley pharma tech journal of medicine | 102 necroptosis are receptor-interacting protein kinases (ripks). additionally, apoptosis and necroptosis intersect at several points during the signal transduction process. one of the most well-researched convergence points between the two processes is the role of caspase-8 in inhibiting necroptosis by cleaving necroptosis mediators59,60. in simple terms, necroptosis is a cellular response to environmental stress, triggered by mechanical or chemical injury, in ammation, or infection. the existing knowledge of necroptosis revolves around a key signaling molecular pathway: the tnf-α receptor system — a pleiotropic molecule capable of inducing a survival, apoptotic, or necroptotic response based upon the assembly of various sequential cell death complexes61,62. under some cellular conditions, the binding of the ligand tnf-α to the receptor tnf-ri triggers the formation of complex i (a prosurvival complex that signals through nf-κb). however, in cases where ripk1 is de ubiquitinated, the complex has become an apoptotic complex iia63. furthermore, the absence of caspase 8, in addition to elevated levels of receptor-interacting serine/threonine-protein kinase 3 (ripk3), alter the complex to iib (also called the necrosome). this necrosome contains ripk1, ripk3 and the fas-associated protein with death domain, allowing the cell to undergo necroptosis via direct phosphorylation of mixed lineage kinase domain-like protein (mlkl) by ripk364,65. phosphorylation of mlkl results in a pore-forming oligomer that punctures the plasma membrane and causes subsequent cell death66. figure 2: tnfr1-mediated cell death and survival pathways berkeley pharma tech journal of medicine | 103 a number of proin ammatory cytokines, such as the key mediators of necroptosis, tnf-α and il-1β, as well as in ammatory cells, can cause cell death67. however, the pathological manifestation of in ammation varies according to the type of cell death. apoptosis generates a milder in ammatory response because it maintains the integrity of the cell membrane and avoids the overspill of intracellular contents68. conversely, necroptosis directly activates and regulates in ammatory responses by releasing intracellular contents through the ruptured plasma membrane. the strong connection found between necroptosis and in ammation has been thought to be the primary component of the pathogenesis of necroptosis associated diseases. moreover, ripk1 and ripk3 have been found to incite an in ammatory response regardless of cell death69,70. these distinguishable characteristics of necroptosis draw interest to their implication in the progression of autoimmune diseases. necroptosis in a utoimmune diseases the examination of certain neurodegenerative diseases, such as alzheimer’s disease (ad), multiple sclerosis (ms), and amyotrophic lateral sclerosis (als), have contributed to the study of the implications of necroptosis71. alzheimer’s disease (ad) is a degenerative brain disease, which is characterized by the damage and loss of neurons. a study examining human ad brains and mouse model ad brains72 con rmed the activation of necroptosis73 after observing a major increase in the level of necroptosis markers — ripk1, mlkl, necrosome complex and mlkl oligomers — in ad brains compared to normal. subsequently treating mice with ad brains with the necroptosis inhibitor, cl-o-necrostatin, signi cantly suppressed necroptosis and prevented neuronal loss73. this suggests that inhibiting the function of necrosome components interferes with the activation of necroptosis, highlighting a promising strategy in the treatment of ad. in some reports, apoptotic morphology was not directly observed in any sections of the brain. instead, the cells showed swollen morphologies and were positive for dna fragmentation, implying that ad pathogenesis may not involve apoptosis. others have argued that the apoptosis theory and the clinical manifestations contradict each other. cells directed to apoptosis have shown to die within days, and with great levels of caspase-3 activity, suggesting an acute and massive neuronal loss according to the apoptosis theory. in such cases, the clinical symptoms of ad patients should be observed in the early phase of the disease rather than berkeley pharma tech journal of medicine | 104 assessing the progression of the disease over a decade. amyotrophic lateral sclerosis (als) is a progressive neurodegenerative disease characterized by the loss of motor neurons. compared to healthy control spinal cords, the als spinal cords showed an increase in necrosome components, including ripk1, ripk3 and mlkl, in a mouse model of als15. furthermore, inhibiting ripk1 with nec-1, or knocking-out ripk3, protected against demyelination and prevented the progression of the disease15. thus, through promoting in ammation and cell death, ripk1 and ripk3 induce axonal degradation and act as mediators of als and pd. this further indicates that, since necroptosis is activated in als, inhibition of necroptosis can be a potential therapeutic target for this disease. additionally, a study observing a parkinson's disease (pd) model found that upon inhibition of ripk1 by nec-113 neuronal degradation was reduced by a half, compared to untreated cells. the protective e ect of nec-1 indicates the therapeutic potential of this drug in als and pd. the study used a human ipsc based model to e ectively capture both early pathological events in mutant neural cells and the bene cial e ects of blocking necroptosis, thereby, strengthening the validity of the results. multiple sclerosis (ms) is a chronic neurodegenerative disease characterized by the loss of oligodendrocytes and demyelination. high concentrations of necroptosis components, including phosphorylation of ripk1, ripk3 and mlkl, were detected in pathological samples from ms patients, as well as a prominent increase of mlkl oligomers in ms pathological samples compared to the control40. this indicates that necroptosis is also involved in the pathogenesis of ms40. in the study, oral administration of ripk1 inhibitor 7-cl-o nec-1 diminished oligodendrocyte degeneration and reduced the disease severity in a mouse model of ms 40. these ndings reveal that inhibiting ripk1 speci cally could be of potential therapeutic value in the treatment of ms. although the study does not take into consideration the potential metabolic consequences of orally administering the drug, their results and conclusion are strongly supported by signi cant amounts of experimental data. rheumatoid arthritis (ra) is one of the most common chronic in ammatory diseases that is characterized by joint in ammation and osteoclastogenesis. the key regulators of necroptosis, ripk1, ripk3 and mlkl were detected in signi cantly higher amounts in the synovium of a collagen-induced arthritis mouse model displayed signi cantly higher berkeley pharma tech journal of medicine | 105 amounts of the key regulators of necroptosis, ripk1, ripk3 and mlkl, which emphasized the involvement of necroptosis in the pathogenesis of ra14. in the mouse model, ripk1 inhibitor nec-1 greatly suppressed the expression of these key regulators and the main in ammatory cytokines, il-17, il-1β, il-6 and tnfα74. therefore, similarly to the previous conclusions, this study supports the potential of inhibiting ripk1 as a novel therapeutic approach for the treatment of ra. although these studies provide compelling evidence for targeting ripk1 and mlkl molecules for subsequent therapeutic research, a close examination of another class of proteins that play an important role in necroptosis may shed light on another new approach. inhibitors of apoptosis proteins (iaps) form a family of genetically conserved proteins characterized by the presence of 1-3 baculovirus iap repeat (bir) motifs75. previous studies have identi ed three family members (termed xiap, ciap1, and ciap2) as potent suppressors of cell death. xiap, the most-studied iap, inhibits apoptosis by binding and inhibiting caspases, and it has been broadly assumed that ciap1 and ciap2 block apoptosis through a similar mechanism. however, recent structure function analyses have indicated that these iaps are not direct caspase inhibitors, suggesting that their anti-apoptotic function must involve alternative mechanisms75. cellular inhibitors of apoptosis inhibitors of apoptosis (iap) are proteins that belong to the family of antiapoptotic proteins that prevent cell death, direct cell growth, and participate in cellular signal transduction76. the mechanism of iaps in inhibiting apoptosis involves both the intrinsic and extrinsic apoptotic pathways1. among these iaps, cellular iap1 and 2 (ciap1 and ciap2), the key molecules of the tumor necrosis factor α (tnfα) signaling pathway, are recruited upon tnf receptor (tnf1) activation, along with the other adaptor proteins such as tnf receptor-associated factor (traf), tnfrassociated death domain protein (tradd), receptor-interacting protein kinase 1 (ripk1) and linear ubiquitin chain assembly complex (lubac). these then go on to form the signaling complex i, which activates the nuclear factor kappa b (nf-κb) signaling pathway and promotes cell survival77. the overexpression of the iap protein family has been reported to be associated with cancer development, with x-linked inhibitor of apoptosis protein (xiap) classi ed as the most potent iap family member78. the xiap, ciap1, and ciap2 contain three baculovirus berkeley pharma tech journal of medicine | 106 iap repeat (bir) domains, a ubiquitin-associated (uba) domain, and a newly discovered gene (ring) nger motif, which exhibits ubiquitin e3 ligase activity79. the iaps prevent apoptosis by inhibiting downstream caspases, which are essential proteins of the apoptotic pathways. the cleaving of upstream caspases, such as caspase 8 and caspase 9, leads to the activation of downstream e ector caspases, such as caspase 3, eventually resulting in programmed cell death76. the most recently discovered iap is the mammalian iap ml-iap, which is detectable in embryonic tissue, certain adult tissues and several cancer cell lines80. although ml-iap has only one bir domain, it is reported to inhibit both the initiator caspase 9 and e ector caspases 3 and 7. thus, it inhibits cell death induced through death receptors by overexpression of the cell death pathway proteins fadd, bax, rip, rip3 and dr381. in fact, the most compelling evidence for the regulation of developmental cell death by iaps originates from studies in drosophila, in which loss of diap1 resulted in extensive early embryonic cell death and a corresponding increase in caspase activity82. however, these studies cannot be extrapolated to human models due to obvious anatomical and physiological di erences. an equivalent study in mammals is necessary to establish the role of these proteins in mammalian developmental cell death. recent studies have greatly advanced our understanding of iaps and their part in inhibiting cell death. structurally, it is now evident how iaps interact with caspases, and how this interaction can be regulated by iap antagonists such as diablo. though, further research is still required to de ne the various roles for di erent mammalian iap proteins in the presence of cell-death stimuli. indeed, there could potentially be some redundancy between family members. as a result, it is necessary to generate mice de cient for more than one iap, to establish the role of iaps in mammals. so far, researchers have only found one mammalian iap antagonist, diablo. however, considering that there are three such proteins in drosophila, other mammalian iap antagonists must certainly exist. future research investigating the direct interactions of bir domains from these proteins with other cellular proteins are likely to illustrate the roles of iap proteins in regulating cell death. conclusion apoptosis and necrosis are two of the most known types of cell death that play an essential role in cell development. the strictly distinct berkeley pharma tech journal of medicine | 107 morphological features of apoptosis include chromosome condensation, nuclear fragmentation, and membrane blebbing. necrosis, on the other hand, is characterized by the expansion of cellular organelles, plasma membrane rupture, and in ammatory responses resulting from the release of the intracellular contents. the apoptotic pathway — an energy-dependent cascade of molecular events — is initiated by 3 primary pathways: the extrinsic (cell-death) receptor pathway, the intrinsic (mitochondrial) pathway, and the stress-induced pathway in the endoplasmic reticulum. although these pathways utilize di erent initiator caspases (caspase-8, caspase-9, and caspase-10, respectively), they activate the same downstream mediator molecule, caspase-3. meanwhile, necrosis is classi ed as accidental cell death caused by internal or external stresses. in necrosis, the disruption of membrane homeostasis results in water imbalance between the extracellular and intracellular environments. whether cells undergo apoptosis or necrosis depends on the availability of caspases, and intracellular atp that determine the cell-death signal, signaling duration, tissue type, and the developmental stage of the tissue. in summary, necrosis is an unregulated and passive process that usually a ects a larger region of cells, whereas apoptosis is a contained and energy-dependent process that a ects an individual or smaller clusters of cells. studies have demonstrated that the repression of apoptosis, or the inhibition of programmed cell death, increases the possibility of malignancy. in contrast, uncontrolled apoptosis has been found to be associated with degenerative diseases such as acquired immunode ciency syndrome (aids), cancer, parkinson's disease, and alzheimer's disease. recently, a programmed form of necrosis, necroptosis, has been proposed as an important pathway involved in many diseases. the primary function of necroptosis was believed to be infection control and protection against virus-induced apoptosis. however, like apoptosis, necroptosis has also been found to be associated with certain neurodegenerative diseases such as alzheimer’s disease, multiple sclerosis, and amyotrophic lateral sclerosis. due to the emerging crosstalk between the di ering forms of cell death, these processes are now considered interconnected. thus, di erentiating between apoptotic, necrotic, and necroptotic cellular mechanisms is critical to understanding disease progression. the recent discovery of a family of genetically conserved proteins, known as iaps, provided new insights into the molecules that play a critical role in mediating these cell death pathways. inhibitors of apoptosis proteins (iap) are a type of berkeley pharma tech journal of medicine | 108 antiapoptotic protein involved in both, the intrinsic and extrinsic apoptotic pathways. cellular iap1 and 2 (ciap1 and ciap2) in particular, are the key molecules of the tumor necrosis factor α (tnfα) signaling pathway, which activates adaptor proteins such as tnf receptor-associated factor (traf), tnfr-associated death domain protein (tradd), receptor-interacting serine/threonine-protein kinase 1 (ripk1), and linear ubiquitin chain assembly complex (lubac) to induce the nf-κb signaling pathway and promote cell survival. in short, the iaps prevent apoptosis by inhibiting the normal functions of downstream caspases, including caspase 8, caspase 9, and caspase 3. the impaired function of iap could instruct the cells to undergo necroptosis or apoptosis instead of the nf-κb cell survival pathway. from this step of the cell death process, autoactivation of caspase 8 would result in apoptosis whereas the absence of caspase-8 autoactivation would result in necroptosis. studies have demonstrated how the iaps inhibit programmed cell death. however, further investigation is necessary to clarify the roles of di erent mammalian iap proteins in the presence of various cell-death stimuli. considering their newfound functions, continued research into the characteristics and mechanisms of iap molecules will likely prove an e ective and viable approach to 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82. vucic d, stennicke hr, pisabarro mt, salvesen gs, dixit vm: ml-iap, a novel inhibitor of apoptosis that is preferentially expressed in human melanomas. curr biol. 2000, 10:1359-1366. 10.1016/ s0960-9822(00)00781-8. berkeley pharma tech journal of medicine | 115 berkeley pharma tech journal of medicine correspondence: suhina.sharma@berkeley.edu keywords: major depressive disorder (mdd) antidepressants ketamine psilocybin psychedelics glutamate serotonin submitted: august 12, 2022 accepted: october 31, 2022 published: december 28, 2022 full open access creative commons attribution license 4.0 abstract depression is a complex neurological disorder that has many potential causes and treatment approaches. though there are many conventional antidepressants available in the market, it is becoming increasingly difficult to rely on them as many patients may experience a lack of effectiveness or tolerance to these antidepressants. while current antidepressants increase serotonin and norepinephrine concentrations through reuptake inhibition, novel forms of antidepressants rely on the direct manipulation of receptors, thereby also improving neuroplasticity, which has emerged as a promising approach to treating depressive symptoms. in this literature review, various forms of novel antidepressants are analyzed. the mechanisms studied are psilocybin’s long-term success, ketamine’s rapid action, hormonal treatments’ direct targeted approach, and natural products’ reduction of neurotoxic side effects. understanding how these powerful mechanisms work may allow for the research and creation of more effective antidepressants in the future. novel therapeutic strategies for depression by: suhina sharma, matt gostkowski, jay patel and christina yang berkeley pharma tech journal of medicine | 21 1. introduction within the last few years, major depressive disorder (mdd) has become one of the world’s leading health problems. within the last year, there were over 45,000 deaths, about one death every 11 minutes, by suicide, making it one of the leading causes of death in the united states1. since mdd causes significant impairment in daily life, effective antidepressant treatments are necessary. many current antidepressants focus on increasing serotonin, norepinephrine, or dopamine in patients with moderate to severe depressive symptoms through reuptake inhibition2. however, these conventional forms of antidepressant therapies are often ineffective in patients because there are many types of depression, including stress-induced, anxious depression, and treatment-resistant depression3. this complexity makes it difficult to find therapies that work for each patient. furthermore, there is a lot of uncertainty regarding the long-term effects of conventional antidepressants and the potentially toxic side effects. there have been recent attempts at implementing novel therapeutic strategies for depression that are more effective than conventional treatments. in this literature review, we will analyze ketamine as a shortterm therapeutic option4, psilocybin as a long-term therapeutic option5, other psychedelic-related drugs6, natural depression remedies7, as well as hormonal treatments for depression8. all these novel strategies target depression differently through inhibition and modulation of specific receptors, neurotransmitters, and other biomarkers to significantly reduce suicide ideation and depressive symptoms. these novel antidepressants are researched because of their promising clinical tests and diverse mechanisms. because these antidepressants have different mechanisms of treating depression, it is important to analyze which mechanisms are the most powerful in contributing to the most successful antidepressant, as it can be the baseline for the research and creation of more effective antidepressants in the future. the success of these novel antidepressants is measured based on their ability to prolong the therapeutic effect, increase neuroplasticity, and reduce neural toxicity. berkeley pharma tech journal of medicine | 22 2. pathophysiology of depression currently, many neurobiological theories exist for the pathophysiology of mdd. however, it has been difficult to choose one unified theory due to the clinical and etiological heterogeneity of the disorder9. for example, depressive pathophysiology can change significantly over the course of the illness, and all theories of depression are only relevant to some depressed patients in the population. as each patient is likely unique, it is critical to tailor treatment approaches specifically. this review will discuss a few of the current theories with the strongest empirical foundation, as each can be useful in developing novel therapies. the monoamine deficiency theory posits that the underlying pathophysiological basis of depression is a depletion of the neurotransmitters—serotonin, norepinephrine, or dopamine—in the central nervous system9. depletion of these neurotransmitters happens when presynaptic cells take the neurotransmitters back up before it reaches the receptor or when too little of a specific neurotransmitter is produced. these neurotransmitters are part of the monoaminergic systems, which are involved in important brain functions such as mood regulation, reward processing, circadian rhythm, and attention. in clinical trials, almost every compound that inhibits monoamine reuptake has been an effective antidepressant. this observation is what led to the development of the monoamine deficiency theory. although antidepressants that target the monoamine systems can be initially effective, full, and partial resistance to these drugs and delayed onset of action occurs. these limitations suggest that dysfunctions of monoaminergic neurotransmitter systems in mdd represent effects of other abnormalities9. the monoamine deficiency theory remains the clinically most relevant theory, and new findings on dopamine have shown further potential in producing novel therapeutic strategies for depression. the gabaergic hypothesis of mdd suggests that alterations in gabaergic transmission represent fundamentally important aspects of the etiological sequelae of mdd that are reversed by monoaminergic antidepressant drug action10. gaba is a neurotransmitter predominantly responsible for mediating neural inhibition in the brain. magnetic resonance imaging berkeley pharma tech journal of medicine | 23 studies have shown that in acute depression, there are consistent reductions in total gaba concentrations in the prefrontal and occipital cortex. this observation may be due to acute stress effects or reduction in the density and size of gabaergic interneurons. however, there is also contradictory evidence of the gaba hypothesis9. circadian abnormalities have been hypothesized to be etiologically associated with mdd. the circadian rhythm is part of the body clock that helps regulate body fatigue through day and night11. disruptions to sleep and daytime fatigue is a criterion for depression, which suggests a subgroup of depression patients may have impaired circadian rhythms. in clinical studies, adjustments to the circadian cycle have been shown to have specific effects on subsequent mood and can even have antidepressant effects. based on these findings, this hypothesis suggests that shortened rem latency, the association between phase advance of the sleep-wake cycle and phase advances in nocturnal cortisol secretion, and the effect of antidepressants on circadian rhythms are etiologically associated with mdd9. however, more research is required into the molecular explanation. 3. important receptors involved in depressive symptoms the serotonin hypothesis has dominated the field of depression research for decades. there are multiple receptors that have been implicated, but serotonin-1a (5-ht1a) and serotonin-1b (5-ht1b) are among the most important receptors as deemed by recent and ongoing research projects12. the 5-ht serotonin receptor group has been broadly linked to both anxiety and depression disorders13. the 5-ht receptor is found throughout the central and peripheral nervous systems in the form of either a g proteincoupled receptor or a ligand-gated ion channel. it is acted upon by the chemical serotonin and then proceeds to promote either excitatory or inhibitory neurotransmission. after binding to serotonin presynaptically, the receptor modulates the release of neurotransmitters such as glutamate, gaba, and dopamine and hormones such as oxytocin, prolactin, and cortisol, leading to physiological changes affecting emotion and cognition14. furthermore, a growing and diverse body of evidence support the berkeley pharma tech journal of medicine | 24 involvement of 5-ht1a, the main inhibitory serotonergic receptor, in both mental disorders. this important role of the serotonin receptor was initially discovered through studies of tryptophan depletion, as tryptophan is the molecular precursor to the 5-ht receptors15. recent studies have focused on the direct role of 5-ht receptors. studies have implicated dysregulation of 5-ht neurotransmission as a primary defect in mood and anxiety disorders13,15. complementary research findings suggested that 5-ht may play a role in recovery from mood disorders via selective serotonin reuptake inhibitors (ssris) and other serotonergic agents16. the role of 5-ht receptors in depression has additionally been shown through human studies, 5-ht1a polymorphism research, preclinical pharmacological studies, and preclinical genetic approaches. beyond the 5-ht receptor, other receptors affecting brain chemistry have been indicated to improve outcomes for patients with mdd. nmda antagonists, such as ketamine, have been associated with antidepressant effects in patients suffering from mdd17. the nmda receptor is typically activated by glutamate, which causes an influx of calcium ions and subsequent membrane depolarization for an action potential18. however, antagonists have been proposed to antagonize nmda receptors on gabaergic interneurons and broadly increase levels of glutamate, the major excitatory neurotransmitter in the nervous system. ampa receptor activation functions in a similar manner, except calcium ions, are substituted for sodium ions which allows for faster action potentials. in recent studies, compounds that augment signaling through ampa receptors have been found to promote antidepressant-like behavioral effects in animal models19. 4. molecular mechanisms of antidepressants most current antidepressant medications fall under the category of reuptake inhibitors, namely selective serotonin reuptake inhibitors (ssris) and serotonin and norepinephrine reuptake inhibitors (snris)20. these medications function by preventing neurotransmitters (serotonin and norepinephrine) from being reabsorbed back into neurons in the brain after they are released. the mechanism of these drugs is based on the monoamine hypothesis, which states that depression is caused by a lack of available monoamines. by blocking reuptake and increasing the extracellular berkeley pharma tech journal of medicine | 25 concentration of monoamines, these drugs are thought to enhance and restore connections between neurons, ultimately resulting in an improved mood in patients21. current research in antidepressants, however, is not based on the monoamine hypothesis and reuptake inhibition mechanism, largely due to the inconsistent results and efficacy of the current antidepressant medications. reuptake inhibitors do not directly target the glutamatergic system, which plays a significant role in the brain and is thought to be a vital system regarding depressive symptoms20,21. glutamate is an important neurotransmitter of the glutamatergic system and is released in response to increased 5-ht2a receptor activation22. while ssris and snris indirectly interact with the 5-ht2a receptor by increasing neurotransmitter concentrations, they do not impact receptor activation or functionality22. reuptake inhibitors have instead been found to cause postsynaptic receptor downregulation, which explains the delayed effects that patients often experience with current antidepressants23. ssris have also been shown to disrupt k+ channels that are necessary for glutamate reuptake by astrocytes, which are glutamate transporters. blunted glutamate reuptake has recently been found to be associated with mdd23,24. berkeley pharma tech journal of medicine | 26 figure 1: mechanism of selective serotonin reuptake inhibitors (ssris). when serotonin is released from a presynaptic cell into the synapse it can then bind to 5-ht receptors on the postsynaptic cell. this pathway, as mentioned before, is involved with important brain functions such as mood regulation, sleep, reward processing, and cardiac rhythm. synaptic serotonin can also be reabsorbed by the presynaptic cell through 5-ht transporters, causing a decrease of serotonin available to bind to the postsynaptic 5-ht receptors. as seen in the figure above, ssris block the 5-ht transporter from reabsorbing serotonin, therefore increasing the concentration of serotonin in the synapse that can bind to postsynaptic 5-ht receptors. novel antidepressant therapies currently being researched, such as ketamine and psilocybin, largely function as receptor agonists and antagonists. receptor agonists activate a receptor to produce a response, which results in increased receptor functionality/activation. psilocybin is an example of a receptor agonist, specifically activating 5-ht2a receptors. receptor antagonists are molecules that block receptors to prevent a response, which in turn can increase neurotransmitter concentrations in the synapses and increase the activity of different receptor-mediated pathways, as seen with ketamine and the nmda receptor25,26. while treatments of this nature show promise in effectively treating depression both shortand long-term, much is still unknown regarding the long-term adverse effects and the overall efficacy of repeated exposure to these drugs. the specific concerns and research required for these drugs are discussed below. berkeley pharma tech journal of medicine | 27 figure 2: ketamine as a receptor antagonist. ketamine functions as an nmda receptor antagonist, blocking molecules, such as glutamate, from activating the receptor. when the nmda receptor is activated on inhibitory gaba interneurons (left figure), it blocks the release of glutamate from nearby neurons and from activating ampa receptors in postsynaptic neurons. glutamate and the ampa receptor are important components of the glutamatergic system, which is heavily linked with processes of mood regulation. when ketamine is present (right figure) it functions by blocking the nmda receptor, therefore preventing the inhibitory effect of the gaba interneuron. this increases the amount of glutamate that is released into the synapse and preferentially activates the ampa receptors, which causes a more rapid cascade reaction and antidepressant effect. berkeley pharma tech journal of medicine | 28 figure 3: basic mechanism of receptor agonists. receptor agonists are molecules that can function like neurotransmitters and activate certain receptors, causing a cascade of reactions that ultimately lead to activation of other receptors or expression of specific genes and proteins. psilocybin is an example of a receptor agonist, which is displayed as the red diamond in the figure above. 5. treatments of interest and their efficacy in treating the different forms of treatmentresistant depression 5.1. ketamine as a short-term therapeutic option there has been a lot of recent research utilizing ketamine as a shortterm therapeutic option. ketamine gained popularity with its efficacy for treatment-resistant depression. due to its rapid onset and short duration of action, ketamine has a strong antidepressant effect. as mentioned earlier, ketamine is an nmda receptor antagonist, therefore keeping glutamate levels higher. ketamine affects glutamate levels, essentially targeting a neurotransmitter different from most common neurotransmitters targeted with conventional antidepressants27. increased glutamate levels result in berkeley pharma tech journal of medicine | 29 lessened depressive effects as scientific research confirms the involvement of the glutamatergic system in the processes of mood regulation. in addition, the glutamatergic system is involved in neuroplasticity processes28, such as the formation of synapses and the regulation of memory. however, other nmda receptor antagonists do not have the same fast-acting effects, which suggests something else makes ketamine successful– ketamine metabolites29, depending on its structure and chemistry. ketamine also acts on the mtor pathway30, ampa receptors and even the opioid receptors in the brain. the mtor pathway is involved in cell proliferation, mortality, survival, and protein synthesis, resulting in the growth of new neural connections, and increasing neuroplasticity. clinical trials have been done that show ketamine’s rapid antidepressant effects. in one study, twenty-five males were given one dose of ketamine and its changing effects were measured over time, starting baseline at 1 h and 2 weeks after the last dose of ketamine, and 1 month after the last dose31. the table between the three pairs are shown below. berkeley pharma tech journal of medicine | 30 hamilton rating depression scale mean ± sd pair 1: depression baseline 23.40 ± 5.38 after 1 hr. of ketamine dose 21.20 ± 6.34 pair 2: depression baseline 23.40 ± 5.38 after 2 weeks of ketamine dose 10.25 ± 6.40 pair 3: depression baseline 23.40 ± 5.38 after 1 month of ketamine dose 10.45 +8.47 table 1: effect of ketamine treatment over time: changes in the hamilton rating scale for depression scores. the hamilton depression rating scale is the most used administered depression scale, with a higher rating indicating higher depressive levels in a patient31. from the table, it is clear that the mean reduction of depression scores decreased, with the most notable change being at the two-week mark31. however, after one month of ketamine doses, the numbers do not drop– they stay roughly the same. this data indicates that ketamine’s effects, though powerful and promising, are relatively short-term31. 5.2. psilocybin as a long-term treatment option psilocybin is a naturally occurring hallucinogen that has recently been identified as a potential long-term therapeutic option for depression. psilocybin acts as a 5-ht2a receptor agonist, activating the receptor that is involved with mood, emotions, thoughts, perceptions, etc. 32. by stimulating 5-ht2a receptors on large glutamatergic pyramidal cells, psilocybin also increases the synaptic concentration of glutamate in the prefrontal cortex (pfc) 33. rather than blocking the reuptake of berkeley pharma tech journal of medicine | 31 neurotransmitters to increase synaptic concentrations like current antidepressant medications, psilocybin directly interacts and activates the 5ht2a receptor to induce changes in mood and thoughts32. in december 2016, a randomized, double-blind clinical trial involving psilocybin administration to patients with life-threatening cancer found that treatment with a high dose of psilocybin administered under supportive conditions reduced depressive symptoms in patients as quickly as 1 week, with effects lasting up to 3 months in 12 patients. there was also a decrease in depressed mood in some patients at a 6 month follow-up34. numerous adverse effects were observed, but none were deemed life-threatening. no adverse effects were displayed in patients after the study was concluded34. the table below shows the results of this study, with numerous different outcome measures used to evaluate symptoms of depression, anxiety, and overall mood. each of the outcome measures used is briefly explained in the table. this was a 9month study performed with 56 participants randomly assigned to two groups either receiving high doses or low doses of psilocybin; while the results of this study are promising, there is a need for a larger and more diverse patient population to fully examine the effects of psilocybin as an antidepressant. berkeley pharma tech journal of medicine | 32 measure group baseline post-session 1 post-session 2 6 months grid-hamd-17 (depression)a low-dose 1st 22.32 14.80 6.50 6.95 high-dose 1st 22.84 6.64 6.52 6.23 beck depression inventory (bdi)b low-dose 1st 18.40 12.92 8.17 8.00 high-dose 1st 17.77 7.00 5.80 6.17 hads depressionc low-dose 1st 9.48 6.04 4.57 4.64 high-dose 1st 9.81 3.92 4.28 3.46 ham-a (anxiety)d low-dose 1st 25.68 16.64 8.92 7.95 high-dose 1st 25.73 8.48 7.52 7.04 stai-trait anxietye low-dose 1st 47.46 40.48 35.48 36.83 high-dose 1st 47.73 34.64 34.28 35.32 poms total low-dose 1st 51.72 42.48 21.09 23.50 mood disturbancef high-dose 1st 56.93 18.96 17.14 12.52 brief symptom inventory (bsi)g low-dose 1st 41.76 33.74 26.08 23.50 high-dose 1st 40.19 18.08 16.48 14.35 mqol (overall quality of life)h low-dose 1st 5.69 6.17 6.90 6.88 high-dose 1st 5.32 7.14 7.46 7.65 mqol (meaningful existence)i low-dose 1st 6.03 6.10 7.30 7.29 high-dose 1st 5.43 7.23 7.30 7.62 lot-r optimismj low-dose 1st 13.56 13.60 15.96 16.68 high-dose 1st 14.15 17.23 17.16 17.43 table 2: effects of psilocybin. changes in 10 different outcome measures/scales after session 1, 2, and at a 6month follow-up in two groups receiving different dosages of psilocybin. __________________ a the grid-hamd-17 is a clinician-rated measure of depression, with lower scores correlating to fewer symptoms of depression34. berkeley pharma tech journal of medicine | 33 b,c the bdi and hads tests are self-rated measures of depression, with lower scores indicating fewer depressive symptoms34. d the ham-a is a clinician-rated measure of anxiety, with lower scores signifying less anxiety in patients34. e the stai test is a self-rated measure of anxiety, with lower scores signifying less anxiety34. f the poms test is a self-rated mood measure, with lower scores indicating an overall better mood34. g the bsi score is a self-rated measure of psychiatric symptoms, with lower scores indicating fewer feelings of distress34. h, i the mqol test is a self-rated measure of the overall quality of life, with higher scores indicating a better quality of life34. j the lot-r test is a self-rated optimism measure, with higher scores correlating to more feelings of optimism34. psilocybin also differs from current antidepressants in that limited exposure or administration of the drug has the potential for longer-lasting effects. recent studies also show signs of high transient neuroplasticity after a single dose of psilocybin, which is a possible source of the longer-lasting effects associated with psilocybin33,34. psilocybin targets inflammatory and oxidative stress pathways that lead to an increase in brain plasticity and cognitive flexibility, both of which are important gauges of neuroplasticity33. a study published in november 2021 also found that psilocybin increases the expression of genes related to neuroplasticity (c-fos, junb, dusp1)33,34. this study refers to a clinical trial involving mice in which it finds that mice treated with psilocybin experienced increased density and strength of neural connections by 10 percent33. the same study published in 2021 also finds that suicidal behaviors are associated with neuroplastic dysfunction and that those that die from suicide have low levels of brain-derived neurotrophic factor (bdnf) in certain regions of the brain33. psilocybin’s ability to increase neuroplasticity in patients suggests that it could be used to treat patients at high risk of suicidal behaviors. however, more research is needed to fully evaluate its efficacy for these conditions. while psilocybin has shown promise as a novel therapeutic option to treat depressive symptoms due to its prolonged effects on neuroplasticity, there is still much unknown. many studies and trials performed involved small sample sizes, so their findings cannot be applied to a larger population33. there is a need for a trial involving administration in a clinical setting, which is not as ideal and accessible. berkeley pharma tech journal of medicine | 34 5.3. psychedelic-related drugs we have already seen an example of a popular, effective psychedelic-related drug– psilocybin. other drugs of similar properties will be discussed in this section. ayahuasca is a psychoactive drink shown to have medicinal potential in treating psychological disorders. in recent clinical trials, the drug has been proven to decrease activity in the precuneus and medial prefrontal cortex35. these regions have also been implicated in the default mode network a brain pathway most active during periods of introspection. unsurprisingly, overstimulation of the default mode network has been associated with higher rates of depression and anxiety36. ayahuasca combats stress-induced depression and anxiety through its composition of dmt and b-carboline37. dmt activates the sig-1r receptor, which blocks neurodegeneration and regulates the production of protective antioxidants. b-carboline, meanwhile, has been shown to increase bdnf38. the drug ultimately leads to anti-inflammatory, neuroprotective, and potentially memory-boosting effects because of its focus on promoting nerve cell survival. ayahuasca has added utility compared to traditional antidepressants due to its multifaceted approach to mental disorders: the drug has been shown to combat depression, anxiety, ptsd, and drug dependence39,40. additionally, the psychedelic is faster-acting and lasts longer than other antidepressants that also target serotonin receptors 37. a recent clinical trial focused on ayahuasca’s effects on treatment-resistant depression35. twenty-nine patients with persistent, moderate-to-severe depressive symptoms received either a single dose of a placebo or ayahuasca. the results of the study indicated that when compared to the placebo, ayahuasca generated significant antidepressant effects. statistical analyses proved that the effects were rapid, and the positive outcomes persisted for at least seven days. although ayahuasca is especially promising as an antidepressant medicine due to its rapid effect in combating depressive symptoms, some other key features contribute to its potential. the drug has a rare capability of improving depression in patients resistant to traditional antidepressant therapies35. additionally, ayahuasca’s focus on the default mode network provides a direct focus on reducing the source of depressive berkeley pharma tech journal of medicine | 35 thoughts and instead increases blood flow to areas that regulate emotions and memory. despite these encouraging findings, more studies must be conducted to prove ayahuasca’s efficacy in other mood and behavioral disorders. lysergic acid diethylamide (lsd) is a classical hallucinogen more specifically characterized as an entheogen. some of its characteristic mental effects include distortion of the sense of time and identity, visual hallucinations, and states of euphoria or dysphoria. beyond these short-term effects on the brain, lsd may play an overlooked role in neurogenesis and possible long-term nervous system health. a recent clinical study indicates that the drug may help the brain grow cells and construct novel connections in brain regions that typically exhibit cell death in mental health disorders such as anxiety and depression41. the drug acts on the brain by interacting with the serotonin 5-ht2a receptor in an agonist capacity42. thus, the drug is analogous to serotonin chemically, which has been associated with numerous brain-boosting and physiologically regulating benefits. studies have implicated serotonin receptors specifically in promoting promnesic effects and regulating emotional behaviors43. compared to other traditional antidepressant drugs that focus on inhibiting serotonin reuptake, lsd is both faster-acting and longer-lasting in the human body44. one current research trial presents that lsd promotes social behavior through mtorc1, a protein complex activating the translation of other proteinsin excitatory neurotransmission45.lsd was shown to potential both ampa and 5-ht2a synaptic responses in the mpfc (medial prefrontal cortex) and increase the phosphorylation of protein kinases akt and mtor in a rodent model. this effect on signaling in the mtor pathway was hypothesized as the rationale behind the increase in prosocial behaviors like ketamine. another recent clinical trial utilized the administration of lsd and placebo to 20 healthy volunteers and subsequently measured the emotional behaviors of the participants 2 weeks after administration46. the results indicated that lsd was responsible for the heightened mood, optimism, and trait optimism of the participants. another important ongoing clinical trial will measure the efficacy of lsd administration in patients suffering from mdd. berkeley pharma tech journal of medicine | 36 by targeting the 5-ht2a receptor, lsd functions as a serotonin-like chemical in an agonist role. this may be advantageous to other antidepressants due to its rapid and longer-lasting effects. lsd also affects the mtorc1 pathway, which has been implicated in increasing sociality. although much research was conducted regarding lsd’s therapeutic effects in the 1970s, government restrictions on psychedelic research have curtailed efforts in the past few decades46. the increasing rate of clinical trials, including psychedelics and lsd in psychiatric disorders, must continue to gather evidence of reproducibility and validity. because of the prevalence of “bad trips” and the tendency of the drug to sometimes exacerbate existing psychiatric predispositions, trials testing the drug, in combination with therapy or trials focusing on micro-dosing the drug, may be an optimal focus47. 5.4. natural products natural products encompass many possible treatment options, which all work to cure depression through different mechanisms. most natural products act prophylactically and help to alleviate symptoms of depression while also benefiting internal organ functions7. current natural products investigated as potential depression treatments include traditional chinese medicine formula, herbs or their parts, and natural products that are either extracts or isolated compounds7. there are different types of natural products available for different types of depression, such as stress-induced depression, post-stroke depression, comorbidity depression, and more. some products work well in conjunction with a good diet to prevent depression because some food components, such as omega-3 fatty acids and vitamin l1, can improve mood. traditional herbal medicine has been used with multitargets, multi-levels, and multi-ways48. natural products differ from commonly prescribed antidepressants because they tend to originate from non-western medicine and focus on treating other mechanisms in the brain. for example, suppressing signaling pathways, treating internal heat depression, and protecting brain glial loss. natural products also tend to be produced naturally, compared to most berkeley pharma tech journal of medicine | 37 antidepressants which are produced in a laboratory environment. some natural products have fewer adverse effects compared to conventional depression medication and can also reduce medication load and undesirable side effects49. natural products provide many compounds with antidepressant-like effects, and their therapeutic impacts have been highlighted for a long time. apart from using as-is, natural products are also a great source for future antidepressant drug discovery. there are in vitro, in vivo, nonclinical, and clinical/translational studies looking at the effectiveness of natural products in depression treatment. the potential of tetragonia tetragonioides (ttk) was examined through a trial involving the animal model brain of depression50. glial cells have a protective role within the cns and pns by helping to maintain homeostasis. essentially, glial cells help to hold nerve cells in place so nerve cells can produce their appropriate function. glial cells play a role in depression because the loss of glial functions has been shown to contribute to the pathophysiology of depression50. ttk is commonly called the new zealand spinach and other local names. ttk is edible, both raw and cooked, and looks like a leafy plant51. in the research article, the team was able to protect glial loss (and hence loss of glial function) in the prefrontal cortex of a mouse brain by using tkk52, demonstrating that tkk is a potential candidate for depression treatment. however, further research is necessary. xiaoyaosan, a mixture of dried plants and herbs, was shown to improve depressive-like behavior in rats52. xiaoyaosan works by inhibiting immunoinflammatory activation and reducing the levels of inflammatory cytokines in the colon by suppressing the activation of the tlr4/nlrp3 inflammasome signaling pathway. improvement in depression is likely due to the decrease in levels of various proteins, such as tlr4, tak1, and irak1, which lead to a subsequent decrease in inflammatory cytokines, including il-6, il-1β, and tnf-α. evidence shows that inflammatory cytokines contribute to the development of depression, and reducing the concentration of inflammatory cytokines alleviates depression symptoms52,53. berkeley pharma tech journal of medicine | 38 several natural products are effective in treating depression by reducing the likelihood of depression development, alleviating depressive symptoms, or accompanying conventional western medicine. however, natural products are generally grouped under “alternative treatment," a treatment option used when western medicine proves inadequate54. for natural products to gain more potential as novel therapeutic strategies, it is important for welldesigned, more extensive clinical trials to be carried out. only then more clinical trials using natural products can be carried out to prove the efficacy of natural products in depression treatment. better integration of natural products in western medicinal research is recommended. 5.5. hormonal treatment thyroid, gonadal, pineal gland, and adrenal axis hormones are the four hormonal groups that have garnered the most recent attention in depression research. some hormones play a direct role in the nervous system by acting as neurotransmitters and affecting neuronal signaling8. other hormones regulate the production of proteins that affect both brain cell structure and synaptic neurotransmission. sex hormones have even been proven to stimulate neuroprotection. thyrotropin-releasing hormone, associated with the thyroid hormonal group, has been proven to stimulate locomotor activity, amongst other behavioral effects55. additionally, thyrotropin is a pituitary hormone that stimulates the thyroid gland, and t4 and t3 are hormones secreted from the thyroid gland. research studies have indicated that deficiency of these hormones in the central nervous system can result in fatigue, weight gain, and lack of energy-all common symptoms of depression56. the gonadal steroids estrogen and progesterone, beyond their neuroprotective effects, have been shown to affect brain regions involved in mood and behavior regulation57. furthermore, melatonin, the “sleep hormone,” has been implicated in mediating the circadian rhythm. given the common occurrence of sleep disturbance and irregular sleeping patterns in mdd, melatonin replacement therapies have been gaining popularity58. additionally, a link between hypersecretion of corticotropin-releasing hormone (crh) and depression has been established. higher levels of this berkeley pharma tech journal of medicine | 39 hormone have been shown to reduce free cortisol levels and may be a therapeutic option for stress-induced depression59. hormone therapy targets differ from traditional antidepressants as they focus on replacement therapies that increase levels of chemicals found naturally in the body8. although hormone replacement therapies often have indirect effects on brain function and can thus result in unintended secondary effects, these therapies are much more targeted than novel drug treatments due to their natural prevalence in the human body. furthermore, because such therapies often focus on the replacement of natural chemicals, they are much safer and likely to be approved for funding and distribution. in future research efforts, the three major endocrine systems—the hypothalamic-pituitary-adrenal (hpa) axis, the hypothalamic-pituitarythyroid (hpt) axis, and the hypothalamic-pituitary-gonadal axis—should be studied more to enable hormones as research targets in depression. table 3: summary table that displays all forms of antidepressants discussed– both conventional and novel. berkeley pharma tech journal of medicine | 40 6. practical considerations ketamine and psilocybin are the most thoroughly researched and promising therapeutic options for treating depression. before their widespread implementation, however, greater efforts must be undertaken to reduce stigmas and influence public perceptions surrounding psychedelic treatments. governmental action is needed to ease medicinal restrictions on psychedelic, natural, and hormonal treatments for mental disorders60. reputable scientific journals and agencies should continue to publish and advertise content displaying the efficacy of these novel treatments to change public perception. however, some concerns regarding these treatments are valid—as high doses of certain drugs may cause adverse side effects and set back research efforts60. thus, researchers must continue to prove the reproducibility of their clinical trials and consider the implementation of natural products in conjunction with conventional drugs. such a strategy may prove to make therapeutic options more accessible for patients and more likely to be approved for medicinal use. 7. future directions ketamine and psilocybin have emerged as potential antidepressant therapeutic options through mechanisms that promote increased neuroplasticity. we have seen similar mechanisms in other psychedelic drugs, such as interaction with the 5-ht2a receptor and signaling on the mtor pathway30,32. by better understanding what specifically contributes to psilocybin’s long-term success, ketamine’s rapid action, hormonal treatments’ direct targeted approach, and natural products’ reduction of neurotoxic side effects, researchers can not only guide the development of novel antidepressants with similar properties, but we can even perhaps go into how to personalize antidepressants to meet individual needs. we can also expand this study of depression to other neurological disorders as well, such as post-traumatic stress disorder, which could benefit from similar novel antidepressant mechanisms. berkeley pharma tech journal of medicine | 41 8. conclusion research on antidepressant therapies has significantly advanced in recent years while taking on a new approach to treating depressive symptoms in patients. while current antidepressant medications increase concentrations of serotonin and norepinephrine through reuptake inhibition, new research focuses on the direct manipulation of receptors, specifically targeting and improving neuroplasticity, which has emerged as a promising approach to treating depressive symptoms. ketamine has been shown to rapidly reduce depressive symptoms by targeting glutamate, promoting the formation of synapses and new neural connections. psilocybin has also shown numerous signs of improving neuroplasticity in clinical trials, with the potential for longer-lasting effects. while ketamine and psilocybin are at the forefront of current antidepressant research, much information remains to be known about the impact of long-term and repeated exposure to these drugs, and larger clinical trials are necessary to evaluate their efficacy. psychedelic-related drugs, such as ayahuasca and lsd, display the potential to treat depressive symptoms as well. ayahuasca inhibits default mode network activity to reduce stress-induced depression and anxiety, while lsd functions similarly to psilocybin and promotes cell growth and connections to improve neuroplasticity. however, concerns over side effects and government restrictions have limited research on the psychedelic approach. natural products have also been found to work well in conjunction with current antidepressant drugs and therapies due to reduced side effects and medication loads. while recent clinical trials show signs of antidepressant effects, there is still an overall lack of research surrounding these products and their potential to treat depression. hormonal treatment, which has been used for years to treat a variety of disorders, has recently emerged as a potential antidepressant therapy. replacement therapies involving thyroid, gonadal, pineal gland, and adrenal axis hormones specifically have shown signs of reducing depressive symptoms by adjusting the naturally occurring chemicals to their optimum levels. while hormonal treatment offers a targeted approach due to the knowledge surrounding hormones and their effect on the body, there are concerns about unintended secondary effects that can occur. due to the relative inconsistency and inefficiency of current berkeley pharma tech journal of medicine | 42 antidepressants, many novel therapeutic strategies, and approaches to treat depression have emerged in recent years. larger and more diverse clinical trials are needed for all the potential therapies discussed in this review to 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neurobiological mechanisms and therapeutic implications. neuroscience. 2013;246:199-229. doi:10.1016/j.neuroscience.2013.04.060 54. cleveland clinic. alternative therapies for depression | cleveland clinic. cleveland clinic. published 2013. https://my.clevelandclinic.org/health/treatments /9303-depression-alternative-therapies http://www.cabi.org/ https://www.cabi.org/isc/datasheet/52942 https://my.clevelandclinic.org/health/treatments/9303-depression-alternative-therapies https://my.clevelandclinic.org/health/treatments/9303-depression-alternative-therapies https://my.clevelandclinic.org/health/treatments/9303-depression-alternative-therapies berkeley pharma tech journal of medicine | 47 55. hara j, gerashchenko d, wisor jp, sakurai t, xie x, kilduff ts. thyrotropin-releasing hormone increases behavioral arousal through modulation of hypocretin/orexin neurons. journal of neuroscience. 2009;29(12):3705-3714. doi:10.1523/jneurosci.0431-09.2009 56. shahid ma, sandeep sharma. physiology, thyroid hormone. nih.gov. published march 23, 2019. https://www.ncbi.nlm.nih.gov/books/nbk500 006/ 57. mcewen bs, akama kt, spencer-segal jl, milner ta, waters em. estrogen effects on the brain: actions beyond the hypothalamus via novel mechanisms. behavioral neuroscience. 2012;126(1):4-16. doi:10.1037/a0026708 58. tonon ac, pilz lk, markus rp, hidalgo mp, elisabetsky e. melatonin and depression: a translational perspective from animal models to clinical studies. frontiers in psychiatry. 2021;12. doi:10.3389/fpsyt.2021.638981 29. doi:10.1177/1179573520907397 https://www.ncbi.nlm.nih.gov/books/nbk500006/ https://www.ncbi.nlm.nih.gov/books/nbk500006/ https://www.ncbi.nlm.nih.gov/books/nbk500006/ front page sharma et al. body template_sharma et al. 2. pathophysiology of depression 4. molecular mechanisms of antidepressants 5.1. ketamine as a short-term therapeutic option 5.2. psilocybin as a long-term treatment option 5.3. psychedelic-related drugs we have already seen an example of a popular, effective psychedelic-related drug– psilocybin. other drugs of similar properties will be discussed in this section. ayahuasca is a psychoactive drink shown to have medicinal potential in treating psycholo... 5.5. hormonal treatment 6. practical considerations 7. future directions 8. conclusion references_sharma et. al. � berkeley pharma tech journal of medicine correspondence:� avabagherian@virginia.edu keywords: diabetes encapsulation type 1 diabetes microencapsulation stem cells submitted may 6, 2022 accepted june 9, 2022 published july 19, 2022 full open access creative commons attribution license 4.0 abstract type 1 diabetes (t1d) is one of the most common chronic autoimmune diseases characterized by islet autoimmunity. this is followed by immune destruction of the β cells as t cells attack and destroy insulin-secreting pancreatic β cells, leading to insulin deficiency. currently, life-long insulin therapy is the primary treatment option for the condition with research being centered around islet transplantation to restore glycemic stability. however, this procedure is limited by risks and supply shortages, highlighting the need for a safer, more effective therapy for the approximately 9 million people across the world with type 1 diabetes. this literature review assesses stem cells and their potential as a vast β cell supply towards the treatment of type 1 diabetes. based on current findings, stem cells may differentiate to become a self-renewing β cell line that may reverse type 1 diabetes; however, further studies expanding on ncapsulation techniques, methods whereby living cells are entrapped in semi-permeable membranes for the purpose of disease treatment, are required. with this new horizon of possibilities, targeted efforts towards stem cell manipulation in expressing β cell phenotype can pave the way for a high efficiency treatment for type 1 diabetes. reversing type 1 diabetes: the magnifying potential of stem cell encapsulation �ïw�ava bagherian, anisha jain, darshita prathap and vanloan nguyen introduction type 1 diabetes typically has a sudden onset during childhood or early adolescence, often diagnosed when the child visits the hospital for one of the condition’s symptoms. these symptoms include polyuria, increased thirst, blurred vision, and weight loss¹. if not diagnosed within the �rst few weeks, diabetic ketoacidosis (dka), a condition in which excess blood acids are produced, may develop². this condition is often fatal if left untreated and is marked by abdominal pain, confusion, and nausea. alternatively, hypoglycemia, a condition in which the patient’s blood glucose levels are low, is caused by excess insulin, low levels of eating, or excessive gaps of time between meals³. hypoglycemia and diabetic ketoacidosis are often caused by missing insulin shots, especially if t1d is still undiagnosed. t1d, diabetic ketoacidosis, and hypoglycemia may all be diagnosed through a urinalysis or blood test that reveals abnormally high levels of glucose and blood acids. the onset of t1d is typically sudden, making early diagnosis and treatment integral. stage 1 displays no symptoms, even when a patient’s hemoglobin a1c, a blood test for prediabetes and type 2 diabetes, is tested⁴. however, antibodies have already begun to destroy insulin-producing cells. left unnoticed, stage 2 characterizes itself with increased β cell loss and hence abnormal blood glucose levels as a result. though symptoms may still not appear, the antibodies’ attacks have, at this stage, led to pancreatic damage⁴. it is at this point, stage 3, where symptoms begin due to the great loss of β cells. though the direct causes of these stages are still unknown, certain patients appear pre-disposed to the later stages.⁴ while direct causes of t1d are still unknown, some risks have been identi�ed. environmental risks, such as cesarean section births or the time in life when cows’ milk is introduced into a child’s diet, have been associated with the development of type 1 diabetes⁵. a popular theory linking the environment to t1d links early-life viral infections to the condition’s development⁵. genetics have also been proven to partially cause type 1 diabetes, as the risk of developing the condition ranges between 1%-70% depending on one’s genetic proximity to a family member with type 1 diabetes⁵. additionally, certain drugs and medications can damage β cells, reducing the production of insulin and causing a condition very similar to berkeley pharma tech journal of medicine | 82 type 1 diabetes⁵. in some particular cases, such as a rodenticide introduced in america in 1976 by the name of pyrinuron, the medication can induce the destruction of pancreatic β cells, hence leading to type 1 diabetes⁶. pyrinuron was withdrawn from the american markets after just 3 years; however, other drugs may still lead to pancreatic in�ammation. these risks for and causes of t1d are in need of further research because, as of now, there is no cure for type 1 diabetes. figure 1: damaged cells are unable to create insulin. previous studies have shown that stem-cell therapy may be developed into an e�cient form of cell therapy for type 1 diabetes⁷. the goal of this article is to comprehensively review and outline the most recent data surrounding the bene�ts, risks, and details of stem-cell therapy as it relates to type 1 diabetes treatments. even though treatments have advanced and the quality of life of t1d patients has improved, t1d prevails as a widespread issue. currently, the most prevalent treatments include insulin injections, continuous glucose monitoring devices, utilization of glucose tablets as needed, and a general healthy lifestyle¹. as widespread as these options are, berkeley pharma tech journal of medicine | 83 they hold great drawbacks. insulin injections are a daily maintenance where patients inject insulin subcutaneously in the area between the skin and muscle, typically a location with enough adipose. if injected deeper by mistake, low blood glucose levels combined with increased pain typically occurs. furthermore, injection sites must be rotated to avoid lipodystrophy, a condition where adipose causes indentations that interfere with insulin absorption⁸. continuous glucose monitors, in contrast, involve inserting a minuscule sensor under the skin that measures interstitial glucose levels every few minutes⁹. the data is sent to a monitor, which is sometimes a part of the insulin pump, for the patient to examine when needed. while this option minimizes technically di�cult self-administered shots, it is limited by a high cost that makes the device inaccessible to a large portion of the type 1 population. healthy lifestyles and glucose tablets may be used in combination with traditional blood pricks and glucose monitoring. however, these are not cures, nor do they reverse the e�ects of t1d. due to the prevalence of this disease, more e�cient treatments and therapies are demanded. figure 2: current popular type 1 diabetes treatments. stem-cell transplantation is an encouraging procedure in both cost and safety. stem cells are marked for their capacity to di�erentiate into di�erent berkeley pharma tech journal of medicine | 84 types of cells, creating a large supply of any of the 200+ types of human cells¹⁰, including bone marrow cells, red blood cells, nerve cells, and most importantly for our purposes, islet cells. hematopoietic stem cells, the primary adult stem cells used in medical settings, are found in bone marrow and are utilized to form blood cells¹¹. this allows for stem cells to be used in integral procedures like bone marrow transplants and holds promise for stem cells to be developed into other cells for other procedures. these unspecialized adult stem cells (ascs) can develop into islet cells for islet cell procedures¹². our goal has been to examine this potential procedure that may be the key to a cure of type 1 diabetes. pancreatic cell transplantation is able to stabilize blood glucose levels on its own, however, there is a chronic, nationwide shortage of donors and immunosuppression therapy¹³. islet cell transplantation is an alternative that is less invasive than pancreas transplantation and also proves to be e�ective in reversing complications from t1d but has similar limitations to pancreas transplantation, still su�ering a lack of donors and hindering immunosuppression therapy. ascs and embryonic stem cells (escs) can be di�erentiated into pancreatic islet-like cells to produce insulin in response to change in blood glucose levels¹⁴. this alternative is cost e�ective and involves no donor shortages due to the self-producing cell line. however, transplantation is a potential issue. sourcing of human stem cells requires exploring as new research heavily focuses on umbilical cord stem cells rather than ascs. additionally, immunosuppression when stem cells are implanted is still unclear, leading to the potential bene�ts of encapsulation devices. current strategies for this involve implanted cells shielded from the immune system by a physical barrier. encapsulated stem cell-derived islets may shield β cells from the immune system, ensuring an almost limited supply for islet cells in procedures to cure type 1 diabetes¹⁵. however, encapsulation device strategies need to be improved in order to minimize foreign body response, possibly by targeting di�erent sites. moreover, transplantation of stem cells through encapsulation in minimally invasive areas is still being investigated. nevertheless, this research emphasizes the possible bene�ts within stem-cell therapy based t1d treatment and highlights its need for future research. berkeley pharma tech journal of medicine | 85 current cell therapies current cell therapies in use hold signi�cant value as they can guide scientists to potentially more e�cient and cost-e�ective alternatives. stem cell therapies today include regenerative medicine that causes repair response. a major di�culty within these is that organ donors have to �t restrictive criteria. the di�erent cell types, such as totipotent, pluripotent, and embryonic stem cells, allow for a variety of treatments suited for appropriate diseases¹⁶. it is of utmost importance to carefully select the type of stem cells that are suitable for clinical application. speci�cally pertaining to type 1 diabetes, islet cell transplantations, where islets are taken from the pancreas of an organ donor, contain beta cells that produce insulin and have been used as a treatment for years now. naturally, with this treatment, di�culties such as limited supply of human islets and poor immunosuppression arise¹⁷. other potential treatments to reverse t1d involve mesenchymal stem cells (msc), whose self-renewal potential and ability to di�erentiation into functional cell types can cure diabetes¹⁸. potent strategies combatting these transplantation di�culties have been explored: alternative transplantation sites, novel immune protective agents, and encapsulation techniques. a clinical trial currently run by the university of alberta beginning 2021 evaluates pitfalls of islets but also solutions to islet cell transplantation¹⁹. this highly bene�cial search for higher e�ciency and safety as it pertains to current cell therapies is a major factor as to why islet cells are a great therapeutic treatment method but also why stem cells may be a better option. furthering the msc treatment plan, one stem cell-based clinical trial for diabetes mellitus is the intraportal allogeneic cadaveric islet transplantation. due to the lack of coverage for transplant costs along with the limitations of cadaveric islets, alternatives have been sought, speci�cally regarding mesenchymal stem cells. as a source for newly generated beta cells, such cells have been proven to be e�ective on type 2 diabetes. however, this is not the case for type 1 diabetic patients, as mesenchymal stem cells cannot di�erentiate into beta cells as e�ectively in vitro. in vivo, this di�erentiation does not occur at all. instead, human embryonic stem cells are a plausible treatment method that are being studied as surrogates in replace of cadaveric berkeley pharma tech journal of medicine | 86 islets. immune rejection however is a speci�c issue to this current cell treatment that can be addressed. one potential research route, which will be explained in more depth later on in this article, is the ability to couple hesc derived organoids that produce insulin with microencapsulation technologies. this optimizes the need for vascularization and can create a more bene�cial route in reversing type 1 diabetes²⁰. this research continues to advance in order to ensure safe therapy, as well as develop e�ciency in utilizing adult stem cells, including bone marrow transplants of hematopoietic stem cells. speci�c future research targets involve in vitro studies on the production of functional stem cell-derived β-cells and how they respond to glucose. this will demonstrate how cells respond to di�erent forms of beta cell stress. the protective mechanism exhibited by suppressive immune cells in the pancreas shows promise for future stem cell techniques and potential target sites. similarly, human pluripotent stem cells (induced pluripotent stem cells) serve as alternative beta cell sources for transplantation when there are donor shortages for other treatments. another example of current/pursued research: beta cell replacement through the transplantation of islets of langerhan. the �gure below details stem cell di�erentiation, the process by which stem cells form more specialized functions through signaling mechanisms like dna methylation. the signaling mechanisms are transmitted through nerve cells which generate electrical and chemical signals of action potentials and neurotransmitters to send information. blood cells are one type of specialized cells that can be derived from stem cells, whose specialized function include its self-renewal potential. in a study published in 2015, the generation of sex cells through stem cell di�erentiation is explained, providing new procedures for the e�cient generation of such cells from embryonic stem cells, a speci�c stem cell discussed later. in the study, mouse embryonic stem cells are signaled to di�erentiate into epiblast-like cells and �nally to pgc like cells, or primordial germ cells, a precursor to all germline cells. the versatility of stem cells in di�erentiating into many key specialized cells make them optimal for transplantation as it pertains to diabetes, as the self-renewal potential and personalization of the treatment can prove to be more e�ective and bene�cial²¹. berkeley pharma tech journal of medicine | 87 figure 3: stem cell differentiation. limited donors comprise a portion of the di�culties with stem cell treatments, but economic cost also plays a large role. for example, even costs to create beta cells from stem cells are similar to the cadaveric islet method. both mechanisms, though useful and still developing, require more money, and still immunosuppression issues and autoimmune rejection remain a major factor in their ine�ciencies. so far, only a handful of trials have used human embryonic stem cells in order to regenerate beta cells. while current cell therapies include wearable insulin delivery devices made possible by modern therapy’s increasing normoglycemic ranges—a signi�cant improvement over regular insulin pumps—these are not stem cell therapies and are no closer to reversing the e�ects of t1d. rather, the ability to use stem cells to enhance treatment by coupling it with other alternatives like macro or micro encapsulation can prove to be much more bene�cial in the long run. berkeley pharma tech journal of medicine | 88 combined stem cell alternatives current stem cell therapies have been combined with other studied therapies and/or biomaterials to explore the ability to heal the e�ects associated with t1d or provide reversal treatment altogether. one combined stem cell therapy that has been explored to heal diabetic wounds is through treatment with human umbilical cord-derived mesenchymal stem cell-derived exosomes (hucmsc-exos) and pluronic f-127 (pf-127), which is a medicating hydrogel. pf-127’s unique thermal properties and porous structure allow for the release of therapeutic proteins, hypothesizing that pf-127 can continuously release hucmsc-exos directly onto t1d-a�ected tissues, thus attracting �broblasts and endothelial cells to initiate wound repair22. this treatment was explored through topical application as this delivery was easy, convenient, and non-invasive, with high-e�ciency and low toxicity. the treatment was tested by applying the exosome-hydrogel combination on diabetic rat models, and the researchers observed angiogenesis, cell proliferation, and granulation tissue formation to understand the capabilities of this wound repair mechanism. the diabetic rats were tested in three di�erent groups; one group was treated with hucsmsc-exos only, one with pf-127 hydrogel only, and the last with the combination treatment of hucmsc-exos/pf-127. it was found that after 7 days the wound area was signi�cantly smaller in the combination groups versus the others, and by day 14 the wounds were completely healed for this group²². combination treatment with hucmsc-exos and pf-127 hydrogel allows for the enhanced survival of exosomes and for a controlled release on wound tissue over time which shortens the wound healing time. this study is a notable example of how combined therapies can result in enhanced treatment and accelerate healing time. further alternatives have been explored using insulin producing cell therapies based on stem cells and combined transplantation with mesenchymal stem cells. studies have shown that though mscs prove to be ideal cellular sources due to properties relating to tissue repair and immunomodulatory capacities, further clinical trials have expanded on the idea that its properties are not as e�ective as expected. hence, a study berkeley pharma tech journal of medicine | 89 combining mscs and human type 2 diabetes islets ex vivo and in vivo was performed exploring reverse beta cell dedi�erentiation5. the combination of mscs and islets presents a tempting alternative treatment, particularly as adult mscs su�er fewer ethical issues pertaining to self-renewal and di�erentiation capabilities. although this alternative has been explored with type 2 diabetes islets rather than type 1, it o�ers a novel strategy to reverse dysfunctionality of beta cells, which is crucial given that they secrete insulin. coupling embryonic stem cell therapy with macroand micro-encapsulation devices has the potential to balance the necessity of immune protection. this may serve as an innovative strategy to reverse t1d and in the future may be used to overcome the issue of hosts’ immune responses, improve immunoengineering strategies and encapsulation technologies. a study involved the transplantation of human embryonic stem cell-derived pancreatic progenitors in macroencapsulation devices into diabetic mice. it aimed to create an improved di�erentiation protocol to prevent forming excess tissue in places that weren't targeted, mainly the mesoderm. this method explored how variations of environments could in�uence in vivo pancreatic progenitor development. such cells di�erentiated into pancreatic endocrine tissue in macroencapsulation devices, resulting in a reversal of diabetes within 3 months in the mice²³. this process proved to be successful in generating grafts, necessary to fuel islet cells, in greater than 80% of the endocrine cells. furthermore, 99% of tested mice did not show signs of formed non-endodermal cell populations, providing evidence that an e�cient di�erentiation of human embryonic stem cell-derived pancreatic endocrine cells has potential to couple with a macroencapsulation device, even without direct contact with host environment, ultimately making it capable of reversing diabetes e�ectively. the �gure below illustrates the transplantation possibilities of functional and isolated islet cells in maturation. in a study on the recovery of beta cell de�ciency in type 1 diabetes, it was found that the beta cells regeneration is due to endogenous regeneration or exogenous supplementation. this means that transplantations of certain islets or grafting new beta cells from in vitro cell engineering is a potential alternative through transplantation of islets. mice models have been used through transgenic expression to study berkeley pharma tech journal of medicine | 90 inducible and reversible beta cell destruction. ultimately, studies on immunological mechanisms related to t1d and novel treatment strategies such as grafting beta cells in vitro can prove signi�cant in �nding potential type 1 diabetic drug targets for future clinical trials²⁴. figure 4: islet cell transplantation. encapsulation techniques figure 5: cell encapsulation. berkeley pharma tech journal of medicine | 91 i. macroencapsulation macroencapsulation devices can assimilate islets into semi-permeable membranes that elude typical immune responses while simultaneously allowing for transplanted cells to transport insulin. type 1 diabetes occurs due to an autoimmune response which attacks insulin producing beta cells, yet the complications of immune rejection of islet-like stem cells can be prevented with macroencapsulation devices. immunoprotection is achieved by the selectively permeable membrane that impedes the movement of immune cells and immunoglobulins into the device, while allowing the free di�usion of oxygen, nutrients, insulin, and glucose to and from the encapsulated cells. stem cells are contained within a compartment of the device which allows for selective exchange of nutrients and obstructs antibodies from entering²⁵. when considering macroencapsulation as a potential therapeutic for reversing t1d, it is also crucial to consider implantation sites and shape optimization to maximize the volume of the device within a space. a recent study tested the posterior rectus sheath plane (prsp) as a potential implant site to host the macroencapsulation device. this plane is in between the muscle belly and the fascia of the rectus abdominis muscle and this site is being explored as implantation and retrieval can be performed without invading the peritoneal space. prsp has a large blood supply, allowing for greater di�usion of nutrients, and the encapsulated cells therefore receive adequate amounts of oxygen. in order to maximize the space in the prsp, the best shape for a macroencapsulation device here is presented to be a polygonal-shaped device. these polygonal shapes are ideal as they deliver signi�cantly more cells as compared to device shapes such as circles or rectangles. although a polygon-shaped device presents the most ideal solution, the sharp angles pose challenges with manufacturing and patient comfort. the polygonal shaped devices have favorable interactions with the surrounding environment, and the implantation within diabetic pigs has shown to be a minimally invasive procedure, but the long-term performance of these devices remains untested²⁶. aside from optimizing the site-speci�c sites, a prominent problem with macroencapsulation is being able to supply the encapsulated cells with berkeley pharma tech journal of medicine | 92 enough oxygen. in a clinical trial published in 2018 studying the encapsulation sites for optimal delivery of insulin, the βair device was developed to overcome this obstacle²⁷. the device contained allogeneic human pancreatic islets and was implanted into 4 diabetic patients. two key sites that ensure easy access to minimal surgical intervention are the pre-peritoneal cavity and under the skin. the signi�cance of these sites and the overall use of the βair device is to ensure retrievability and immunoprotection. the results of the trial provided evidence that such a device that utilized macroencapsulation was indeed safe and capable of preventing rejection of implanted cells. however, metabolic control was impacted with the transplanted cells' limited function. potential claims for the ine�ciency of the transplanted cells include hypoxia and hyperoxia which could contribute to a lesser device volume, which is undesirable when needing to deliver insulin and nutrients at a productive rate. macroencapsulation devices must support viability of the transplanted cell at all stages through the maturation process. these devices are bene�cial as they allow for immunoprotection of transplantation islet-like stem cells and can also be retrieved with ease in any circumstance. one limitation present with macroencapsulation devices is accessing a space for implantation. the mechanics of the device are acted upon by di�erent external forces depending on where implantation occurs, which can in turn limit the functionality and lifetime of the device. although recent studies have begun exploring new sites and have been able to successfully implant islet-like stem cells in microencapsulated devices in a minimally invasive fashionthe prsp is a great example of this type of site. another major limitation is that islet cell survival heavily depends on the supply of oxygen; this is a�ected by the devices membrane permeability of oxygen, the rate of oxygen consumption of the encapsulated islet cells, and other factors as well²⁸. one strategy that has been considered to overcome any oxygen de�ciency that sc-islet cells may encounter is oxygen delivery to encapsulated cells through oxygen generating materials. in situ oxygen supplementation with the use of an oxysite disk being placed within the center of the macroencapsulation device has been shown to provide adequate oxygen supplementation and improve the survival of cells²⁹. berkeley pharma tech journal of medicine | 93 unlike macroencapsulated devices, microencapsulated devices avert vessel ingrowth, limiting the supply of nutrients to solely di�use through the selectively permeable membrane. this may also result in a hypoxic environment and therefore requires the need for oxygen delivering technology within encapsulation devices. ii. microencapsulation microencapsulation involves small islet cell mass which favors di�usion of humoral factors and molecules from inside of capsules to the outer environment. the devices are made of thin polymer �lms which tightly adhere to individual islet or cell clusters. they occupy a very limited graft volume and could be eligible for alternative graft sites. issues may still arise from e�ciency of immune barrier competence, the site of implant, and dependence on the �nal microcapsules size. while conformal microcapsules �t a wide array of potential graft sites due to their small size, the issue of their long-term endurance arises. furthermore, microencapsulation requires an adequate oxygen and nutrient supply despite the decreased level of invasiveness regarding the site of the microencapsulation transplantation. in a research paper focused on microencapsulation devices, researchers conducted a clinical trial to understand how an acquired immune tolerance in patients with type 1 diabetes could prevent an autoimmune attack of pancreatic islet beta-cells. researchers studied g3c hybridoma triggering the glucocorticoid-induced tumor necrosis factor receptor-related costimulatory receptor (gitr) to determine if this would promote expansion of tregs¹⁶. the g3c monoclonal antibodies were enveloped in microcapsules which were engineered to allow selective �ow of immunoglobulin m. in the end, researchers observed that long-term gitr triggering did induce treg expansion and prevent diabetes from development in nod micethis clinical trial shows promise that microencapsulation can be a possible treatment for autoimmune diseases. a study conducted in 2019 observed co-microencapsulation of human umbilical cord-derived mesenchymal stem cells (hucms) and pancreatic islet-derived insulin producing cells (hidc), very similarly to what this review proposes as a solution to reversing type 1 diabetes. nod mice were berkeley pharma tech journal of medicine | 94 grafted with microencapsulated hucms and hidc and observed for 180 days. by the end, general health of these mice improved with signs of increase in body weight and the cell co-aggregates were still intact and viable, which proves that there was a su�cient exchange of nutrients³⁰. the microcapsules were easily retrieved, which dismisses one of the main limitations of microencapsulation techniques. once retrieved, there were no signs of pericapsular �brotic overgrowth, and insulin and glucagon were still expressed. this study has shown that co-microencapsulation of hucms/hidc is successful and can o�er a new route of cell therapy of type 1 diabetes. considering the co-microencapsulation was successful through this study, it is likely that microencapsulation of only human mesenchymal stem cells (hucms) would also provide promising results for reversing type 1 diabetes. microencapsulation is a viable method of encapsulating cells as it eliminates the need for immunosuppressant medications by shielding cells from an attack through the host’s immune system. the three-dimensional microcapsule has a semipermeable membrane to allow for the exchange of nutrients and waste, creating an optimal environment for stem cells to survive. one major limitation of microcapsule transplantations is pericapsular �brotic overgrowth due to a foreign body response, yet research has shown that the use of a chemically modi�ed alginate derivative can be e�ective in the prevention of overgrowth as it works to limit movement and clumping. the study conducted with co-microencapsulation mentioned earlier also proved that microencapsulation techniques are viable and can be retrieved without overgrowth occurring. although pericapsular �brotic overgrowth is a major limitation for the long-term e�ectiveness of microencapsulation, few solutions have been found to overcome this obstacle. iii. encapsulation sites diabetic treatment using encapsulated islet cells has continued to grow with new devices such as the aforementioned macroencapsulation and microencapsulation. the importance of �nding a clear and e�ective site is to allow for the e�cient glucose level regulations in an automated, continuous manner. several studies have sought an appropriate site for transplantation berkeley pharma tech journal of medicine | 95 that limits the foreign body immune system response transplantation. for example, the intravascular macroencapsules are particularly integral in achieving a connection between solute transport and di�usion of nutrients³¹. some appropriate sites may include those in the vascular system and may include shunts as well as di�usion chambers. finding an e�cient site limits any detrimental impact of foreign body immune response. such responses are a product of microencapsulation and macroencapsulation sites, and their severity can include in�ammatory responses and hypoxia. hence, many factors play a role in determining adequate sites such as material aspects on porosity, roughness, size of implant, and surface charge. current sites that are explored, however, are generally limited in their space in renal capsules conventional sites, and the respective volume of such sites is necessary in order to create a long-term treatment of such encapsulation devices rather than a temporary solution. microencapsulation sites are ideal as they have increased ease of access for implantation, retrieval, and imaging. furthermore, there is a su�cient blood and oxygen supply. the encapsulation site is still limited as the only site that has been explored so far is the peritoneal cavity for implantation. oher implantation sites that are being explored include epididymal fat pad, skeletal muscle, and subcutaneous tissue. however, transplantation in these alternate sites remains an obstacle. macroencapsulation sites are large transplant masses within a single, well-de�ned 3d device, allowing for device retrieval in case of adverse reaction or failure. most are implanted within the peritoneal cavity and subcutaneous space. while the peritoneal cavity site of microencapsulation allows for intrinsic high vascularity and oxygen tension, graft implantation and monitoring generally requires invasive procedures. subcutaneous space transplantation is less invasive and also provides adequate oxygen which facilitates cell survival. a site for both macroencapsulated and microencapsulated islet-like stem cells must be in close contact with the bloodstream for a su�cient supply for nutrients, yet the liver and spleen (which typically host nonencapsulated berkeley pharma tech journal of medicine | 96 islets successfully) are unable to tolerate the large volumes that are associated with encapsulation techniques³². due to less invasive procedures, most transplantations of encapsulated cells are intraperitoneal, yet this site is not practical as in�ammation and immune responses are associated with intraperitoneal sites. apart from intraperitoneal sites, kidney subcapsular, subcutaneous, and under the skin spaces have also been tested and have shown improved biocompatibility of encapsulated islets and a reduced macrophage recruitment. these sites are being further tested for encapsulation of islet cells to improve survival, engraftment, and function. figure 6: encapsulation devices sites: supply of nutrients and oxygen. current issues involving sites that are more newly explored include death of cells by hypoxia, a condition of low oxygen levels in cells. impaired cell growth and response due to hypoxia results in instability and lack of function through other side e�ects like hyperglycemia and elevated levels of fatty acid³³. other detrimental e�ects that must still be researched include the overgrowth of capsules in the macroencapsulation and microencapsulation devices, since they may inhibit the e�cient transport of nutrients to islets, further causing hypoxia as well as necrosis, the death of body tissue due to too little blood �ow. this may cause delayed berkeley pharma tech journal of medicine | 97 vascularization, or the improvement of nutrient supply, and ultimately lead to the implanted cell destruction and graft failure, which is characterized by the complications of allogeneic hsct or the loss of donor cells. table 1: macroencapsulation vs. microencapsulation macroencapsulation microencapsulation de�nition system where 10^310^6 islets are enclosed in a device that contains a semipermeable barrier. in the context of t1d, acts as a bioarti�cial pancreas, immunoprotection encapsulated beta cells made of thin polymer �lms which tightly adhere to individual islet or cell clusters. bioengineering technique capable of creating an immune-privileged site bene�ts allows the free di�usion of oxygen, nutrients, insulin, and glucose to and from the encapsulated cells. evades the immune response while simultaneously allowing delivery of insulin from transplanted cells occupies a very limited graft volume and could be eligible for alternative graft sites encapsulation sites most implanted within the peritoneal cavity and subcutaneous pace → hypoxic condition & often less vascularized in order to provide optimum nutrient distribution, density of the device should be 5-10% of volume the only site that has been explored so far is the peritoneal cavity for implantation. many other sites are being researched and will soon be explored. berkeley pharma tech journal of medicine | 98 limiting factors contains a limited, cell-loading capacity due to reliance on di�usion. slow glucose stimulated insulin secretion → scaling devices for human application is di�cult e�ciency of immune barrier competence, the site of implant, and the dual dependence on the �nal microcapsules size. microencapsulated devices prevent ingrowth of vessel which limits the nutrient supply to only di�usion through the semipermeable membrane discussion table 2: implications practical implications theoretical implications informs t1d patients/researchers of an alternative to pancreatic transplantation, which can be inaccessible due to high costs and door shortages beta cell transplantation has been regarded as a plausible and e�cient method of reversing type 1 diabetes. no longer limited to ine�ective transport of large/substantial amounts of oxygen as well as nutrients involves reversal potential of mesenchymal stem cells due to their ability to di�erentiate into functional cell types can o�er a proper control of encapsulation devices on reversal of diabetes to prevent foreign body response proposals have the ability to challenge current medical barriers on �nding ideal transplantation sites berkeley pharma tech journal of medicine | 99 poor immunosuppression increases the need for more e�cient strategies, such as exploring alternative transplantation sites and various encapsulation techniques. accessibility may be boosted, breaking down �nancial barriers in the journey to �nd the ideal transplantation sites. selective sites can enhance the compatibility of the devices as it pertains to transplantation and regulating the body responses, and immune barriers can be strengthened encapsulation o�ers optimal sites that help implantation of islet cells. self renewing cells in the future can provide a stable source of insulin whilst also solving donor shortages for future studies, there are immune protective agents that can better stabilize body responses and provide for more e�cient, alternative transplantation sites for islet cells. both macro/microencapsulation provide potential methods to prevent detrimental foreign body response. studies and current proposals lead this research towards reversing t1d in a forward, productive direction. improving characteristics of transport for nutrients and oxygen through the encapsulation devices and coupling it with other current treatments can be an advanced approach to reversing diabetes as research on macro and micro encapsulation continues to develop, potential pitfalls have been examined in order to further the e�ciency of such processes. in a clinical trial held by phd james shapiro, such pitfalls of islet transplantation speci�c to the foreign body response were addressed. the trial induces the growth of new blood vessels and utilizes this foreign body response to its advantage by modifying the target site. the participant must have reduced awareness of hypoglycemia or metabolic instability and be between 18-68 years old. through an angiocatheter tube, a site is made into a viable location, and islet transplantation will occur in the device-less sentinel space and the transplant site is removed from the pocket. this is one of the �rst human studies, and recipients of the islet transplants were berkeley pharma tech journal of medicine | 100 monitored. the outcome measures are to assess implant tolerability or the rate of in�ammation at the site, as well as adverse e�ects on the participants to assess the e�ectiveness of this harnessed foreign body response another ongoing trial from the city of hope medical center aims to assess a safer method of islet transplantation for normal control of blood sugar without needing insulin shots. new onset diabetes is common due to an organ transplant because of certain medications. the treatment is set to transplant human allogeneic islet cells with varying dosage based on patients weight. t1d patients between 18-60 years will receive immunosuppression medication during treatment. the primary outcome measures of this study are to reduce hemoglobin a1c by at least 1 point, as well as eliminate hypoglycemic events after the 1st islet transplant. as detailed before, mesenchymal stromal cells have ample potential to cure diabetes with its ability to di�erentiate into di�erent cell types and for its self-renewal abilities. a clinical trial, cellular therapy for type 1 diabetes using mesenchymal stem cells, through the medical university of south carolina is meant to determine the e�cacy of metabolically active mscs in order to treat the new onset of type 1 diabetes as well as derive its mechanism of protection. currently ongoing, this research uses 50 participants that will receive treatment. group a will receive a single msc infusion, and group b will receive a single infusion of placebo. the primary outcome measure is a 12 month change in c-peptide area under the curve and change in cell beta function. since mscs are e�ective and suppress autoimmunity, the study aims to see msc’s e�ect on insulin secretion rate, change in islet autoantibodies, change in beta cell death measurements, change in t-cell response, etc³⁴. finally, with the introduction of a coupling method between islet transplantation and encapsulation devices, studies detailing the islet cell transplantation process prove to be useful in assessing complications that may arise. in an ongoing clinical trial on improving islet transplantation outcomes with gastrin, sponsored by the city of hope medical center, the e�ectiveness of gastrin treatment with islet transplantation was evaluated. due to the limited supply of donor islets, the study seeks to test if berkeley pharma tech journal of medicine | 101 gastrin, which is a natural gut hormone present in the pancreas in the embryo and a helper of the formation of the pancreas, can be injected to make fewer number of transplanted islets work in a similarly e�cient manner. for this study, participants with frequent hypoglycemic episodes receive treatment with an islet transplant. for the next 30 days, they will be injected with gastrin and continue the process, as well as take anti-rejection medication, which is crucial because foreign body response as talked about before can hinder the e�ects of the study. conclusion type 1 diabetes may be one of the most heavily researched autoimmune diseases; however, the current research into the application and potential of stem cells in pancreatic islet cell transplantation is nominal. the popular, modern-day solutions of relying on cgms, strict lifestyle routines, and daily insulin injections are time consuming and outdated in comparison to the potential of highly e�ective cell research. stem cells can di�erentiate to behave just like pancreatic islets and beta cells and can therefore produce insulin, and this would eliminate the need for daily insulin injections. in fact, stem cell implantation has proven to be one of the leading pathways in transplantation, with current therapies including mesenchymal stem cells, human pluripotent cells, and even embryonic stem cells. however, implantation can prove to have more obstacles than desirable such as cost, limited donors, immunosuppression, and further unwanted foreign body immune responses that may lead to death of the transplanted cells. implantation of stem cells alone is risky as the chances of a host immune response is very likely, encapsulation techniques present a solution. encapsulation methods protect the encapsulated cells from an immune response by the host, which would allow for continuous exchange of nutrients and insulin and increase the life expectancy of these transplanted cells as well. clinical trials in humans have yet to be conducted to explore encapsulation methods, but this solution has proven to be viable in nod mice and has the ability to provide reversal treatment for t1d. details surrounding the most e�cient encapsulation techniques and encapsulation sites are unclear, but it is found that macroencapsulation and microencapsulation provide viable solutions despite their many limitations. berkeley pharma tech journal of medicine | 102 to further stem cell research, a coupling of such macroencapsulation and microencapsulation devices may prove to be bene�cial in the long run when facing technical issues by one device. a strategic employment of combined alternatives such as the devices to embryonic stem cell therapy can be a step towards reducing foreign body immune response as well as o�ering immunoprotective strategies and increasing healthy cell proliferation and nutrient exchange. the clinical trials mentioned before are currently in progress to test out such theories and identify the collective bene�t of using such strategies to reverse diabetes through noninvasive treatment. according to the current state of research surrounding stem cell therapy, the applicability of real-world stem-cell based islet transplantation is ambiguous. once further development in research is made, a conclusion based on the biological, economical, and hazard-based results can be established. the implications of the practical progress involving developing strategies for better immunosuppression techniques and alternatives to current cell transplantation methods set the foundation for diabetic reversal. similarly, theoretical implications including greater accessibility and alternative transplantation sites provide a reliable pathway into the future of not just diabetic reversal but for numerous autoimmune diseases. once that future research is advanced, stem-cell encapsulation may just be the key to clinical cell therapeutics in the e�cient and safe reversal of type 1 diabetes. berkeley pharma tech journal of medicine | 103 references 1. cdc. what is type 1 diabetes? centers for disease control and prevention. published march 25, 2021. https://www.cdc.gov/diabetes/basics/what-is-t ype-1-diabetes.html 2. cdc. diabetic ketoacidosis. centers for disease control and prevention. published january 20, 2021. https://www.cdc.gov/diabetes/basics/diabeticketoacidosis.html 3. dagogo-jack s. hypoglycemia in type 1 diabetes mellitus. treatments in 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doi:10.3748/wjg.v18.i47.6885 33. catrina sb, zheng x. hypoxia and hypoxia-inducible factors in diabetes and its complications. diabetologia. 2021;64(4):709-716. doi:10.1007/s00125-021-05380-z 34. wang h, medical university of south carolina, national institute of diabetes and digestive and kidney diseases (niddk). cellular therapy for type 1 diabetes using mesenchymal stem cells. clinicaltrials.gov. published march 23, 2022. accessed may 10, 2022. https://clinicaltrials.gov/ct2/show/nct04061 746?recrs=a&cond=type1diabetes&cntry=us &draw=3 berkeley pharma tech journal of medicine | 106 front_page_bagherian body �� berkeley pharma tech journal of medicine correspondence:� kblpcpd��!hnbjm�dpn keywords: $01% (fof�uifsbqz (fofujdt 1vmnpobsz�ejtfbtf -voht� %fmjwfsjft� /bopqbsujdmft submitted�.bz��, 202���� "ccepted�jvof��� ������ 1vcmjtife�+vmz��� ����� full open access creative commons attribution� license 4.0 abstract gene therapy is a growing field in research and development that may offer a long-lasting solution to several complex diseases, including chronic obstructive pulmonary disease (copd). copd is characterized by chronic inflammation in the lungs and the airways, leading to respiratory problems. copd includes chronic bronchitis and emphysema. optimizing treatments for gene therapy in copd is of paramount importance given copd's prominence as the fourth leading cause of disease-related death in the united states. we reviewed delivery methods in the current research, including liposomes, nanoparticles, electroporation, adeno-viruses, and adeno-associated viruses (aav). the broad customizability in the diagnostic and treatment methods is evident in the recent studies. in this paper we explore each method and/or biomarker and evaluate several gene therapy avenues for copd. (fof�5ifsbqz�avenues for $ispojd� 0ctusvdujwf�1vmnpobsz�%jtfbtf �ïw�£×ùåø��×âûäúßäûu�¬ûéþ×��×èê×u�£ëúû��þûèüßâéu�¨ã×è�¬×ßú and ¯×äâå×ä�§ýëïûä background chronic obstructive pulmonary disease, also known as copd, is characterized as a chronic in�ammatory lung disease that causes obstructed air�ow from the lungs. it includes multiple progressive lung diseases that a�ect millions of people yearly. in 2018, copd was the fourth leading disease related cause of death in the united states, 6.4% of americans were diagnosed with the disease, unfortunately, this number is likely higher due to undiagnosed cases1. figure 1: diagram of the lungs and the effect chronic bronchitis and emphysema has on them. boxed under chronic bronchitis are images of healthy (a) and unhealthy (b) bronchial tubes, (b) shows the inflammation and increased mucus in the unhealthy bronchial tubes. boxed under emphysema are images of the alveoli, healthy (a) and unhealthy (b). in the unhealthy (b) image there is a membrane breakdown due to inhaled pollutants that causes larger sacs with a decreased surface area. copd is not caused by a single gene or factor, this makes a single treatment for all patients less e�ective, and makes an appeal towards a more personalized treatment1. two of its major contributors include emphysema and chronic bronchitis. chronic bronchitis is characterized by the in�ammation of the bronchial tube lining and excess mucus production, the bronchial tubes are responsible for carrying air to and from the air sacs (alveoli) within the lungs. emphysema is a condition in which the alveoli membranes are destroyed due to chronically inhaled pollutants. the membrane breakdown creates larger air sacs rather than smaller ones, berkeley pharma tech journal of medicine | 59 this decreases the surface area and the amount of oxygen that can di�use into the blood. there are many factors that cause the development and progression of copd, the main factors include cigarette smoke and work-related pollutants2. copd is also considered to be inheritable, as there are genetic components that can play a role in disease development. the �rst gene identi�ed to be associated with copd was seroina, which encodes alpha1-antitrypsin (a1at). de�ciencies in a1at can lead to emphysema, but only 1-3% of copd patients have the de�ciency. additionally, it has been recently discovered that altered mirna expression in the lungs might also play a role in the copd mechanism. the characteristic symptoms that de�ne copd include the following: breathing di�culty, coughing, wheezing, oxidant/antioxidant imbalance, emphysema (alveolar wall destruction), mucus hypersecretion, enhanced cytokines, chemokines, protease, and in�ammation3. current conventional therapeutic strategies of copd utilize antioxidant and anti-in�ammatory drugs3. these drugs tend to be bronchodilators, such as β-agonists and muscarinic antagonists, and inhaled corticosteroids, both of which are only used for short-term management. these treatments only target the patient’s symptoms, and do not stop or reverse damage to the lungs2. bronchodilators, such as β-agonists and muscarinic antagonists work to relax the muscles in the lungs to alleviate coughing and make breathing easier, whereas inhaled steroids reduce airway in�ammation and help prevent exacerbations. working towards a treatment for copd that could prevent or reverse lung damage, would impact the lives of millions. gene therapy is an attractive alternative to current treatments, it is able to deliver medications and therapeutics to speci�c target sites within the lungs. however, it's important to note that there are limitations due to various biological barriers. these limitations can include; o�-target vector e�ects, genetic material, and delivery e�cacy1. due to the variety of infections causing diseases in the respiratory system, there are only 17 fda approved treatments available for patients, none of which treat lung diseases1. abecma is an example of an approved gene berkeley pharma tech journal of medicine | 60 therapy that treats multiple myeloma, a disease where the body’s white blood cells proliferate and cause harmful build ups throughout the body. this speci�c gene therapy modi�es the patient’s t-cells to attack the cancerous white blood cells4. another example includes luxturna, a viral vector gene therapy form that treats congenital blindness. most patients with congenital blindness contain a mutation in the rpe65 gene, which produces the chromophore 11-cis retinal protein that is vital for eye function. luxturna works by delivering functional copies of the rpe65 gene to the retinal epithelial cells5. cystic fibrosis (cf) is a common candidate for gene therapy research and can o�er some insight in the treatment of copd as well. due to defective sodium/potassium ion channels in the ciliated cells, the airway becomes dehydrated and starts secreting more mucus which is a breeding ground for infection-causing bacteria. because of the fatality of the airway obstruction, doctors are researching to replace the cftr gene during the neonatal period to maximize the success of the therapy and the patients’ quality of life6. to deliver the cftr gene into the lung, scientists have experimented with the oral inhalation of aerosolized vectors using a nebulizer rather than a liquid �lled nasal tube. this method of delivery reduces the risk of aspiration and could be useful when treating other lung diseases, including copd6. modulator therapies are also burgeoning methods to treat cf, and each therapy targets a speci�c mutation in the cftr gene. the success of the modulator therapy was shown in a research study when 55% of the subjects experienced signi�cant decrease in p.aeruginosa7. genes that contribute to copd such as serpina1 that causes an α-1 antitrypsin de�ciency would be a good candidate gene for potential modulator therapies with certain mutations in the gene8. recent genetic, biochemical and histological evidence also suggests altered transforming growth factor beta (tgf-β) signaling is associated with copd development and progression. tgf-β regulates the respiratory system and can lead to diseases if it is mutated. subduing altered tgf-β signaling in the airways and alveolar sacs via gene silencing technologies may provide similar berkeley pharma tech journal of medicine | 61 therapeutic outcomes to modulator therapies and other treatment options9. deliveries the development in gene therapy for copd hinges on several factors, one of the most prominent of which is delivery method to ensure e�cacy and precision in use of the particular therapy. delivery methods must optimize performance and transfection as well as safety both for the patient and protection of genetic material. even in common inhalable drug delivery, limitations are present due to the complex defense mechanisms of the respiratory system. these factors are limited in e�cacy and primarily focus on airway obstruction via anticholinergics and dual β2-dopamine 2 receptor antagonists10. techniques used for gene therapy can be separated into 3 major categories: chemical, physical, and viral. chemical techniques entail the non-viral methods for creating materials and particles used to transfect or insert genetic material into the target cells. this can be achieved in several ways whether it be to weaken the cell function or to create new vectors that take advantage of common cellular functions and mechanisms, such as liposomes and nanoparticles (nps). next, physical techniques revolve around mechanical methods to typically achieve new access points or alter the function of the cell slightly without introducing a chemical alteration or manipulation to the equation. increasing the permeability and uptake of genetic material of the cell or direct injection is the most common mechanism in which these methods are able to insert the genetic material. the more prominent of these is a shock treatment to open the cell membrane for a brief period of time known as electroporation (ep). thirdly, viral delivery methods are created by modifying viral genomes to take advantage of viral mechanisms of inserting genetic material. the most common vectors that have extended into the realm of gene therapy for pulmonary diseases are ep, liposomes, viral vectors, and nps. each of the aforementioned methods give way to their own advantages and problems; seen in figure 2, thus validating the need for further research on gene therapy for symptoms of copd. [jg1] longevity and �nding the ideal carrier to inhibit the e�ects of copd using these treatments is necessary to berkeley pharma tech journal of medicine | 62 counteract the lifelong e�ects traditionally associated with it and other commonplace pulmonary diseases. figure 2: modeled from diagram of delivery method choice for gene therapy in the lungs [1]. the more generally observed shortcomings and advantages of each key delivery method to be discussed in detail. individual modifications, especially in the case of liposomes and nps, can be made to better these such as particle coating and more effective targeting mechanisms. non-viral methods liposomes taking advantage of natural lipid bilayers to employ liposomes with genetic material, lipoplexes, for delivery cargo to cells utilizing endogenous functions of the cell membrane is one of the primary strategies in gene therapy for copd. the most glaring functional advantage of lipofection as a technique is the lack of immune response and cytotoxicity upon transfection that has been observed11,12. in addition to this, low cost and ease of use make liposome based gene therapy a preferred treatment. morphology which mimics that of cell membranes’ lipid bilayer can be modi�ed with targeting molecules to adhere to di�erent cells with more precision and limited collateral damage. the accepted but not completely understood mechanism of action takes advantage of random brownian motion of the liposomes upon entrance into the cell to e�ectively disperse the material13. in generic drug delivery this allows for seamless in�ltration and dosing of the cell. however, the speci�city of gene delivery berkeley pharma tech journal of medicine | 63 requires nuclear transport of the contained genetic material; these shortcomings can be observed through endocytosis inhibitors and endosome trackers to visualize delivery sites14. the endogenous nature of liposomes is massively important for pulmonary diseases and speci�cally copd where constant in�ammation and exacerbations take place regularly. a well observed phenomena since the 1970s, liposomal immunogenicity, can be built upon even further to trigger minimal immune responses. for example, liposomes for drug delivery that showed an inverse correlation with immunoglobulin (igm) in the brain were modi�ed to enhance absorption of igm and thus the immune response and overall e�ectiveness of the treatment was improved15. relative to the lungs, clinical trials have utilized lipoplexes in tandem with a plasmid carrying human cftr in inhalable doses to limit mucus problems in patients with cf, a common comorbidity of copd16. the bene�ts of these native characteristics of liposomes are signi�cant but can present their own pitfalls as well which must be monitored. natural mechanisms can act as impediments to treatments such as the bovine pulmonary surfactant alveofact have been shown to weaken berkeley pharma tech journal of medicine | 64 figure 3: image shows the mechanism by which liposomes (and some nps) can introduce genetic information into a cell by taking advantage of endocytosis, a natural cell mechanism, and limiting the immune response– thanks to the endogenous nature of the liposomal structure and makeup. several types of lipoplexes17. overcoming these innate obstacles while maintaining the key bene�ts of lipoplex usage is of paramount importance and needs more research to fully understand the functionality in di�erent cell types within the pulmonary system. speci�c work with cells and the mechanisms of the pulmonary system will yield a better understanding of how lipofection can be used as a widespread treatment. nanoparticles nps (nanoparticles) are a massive and growing �eld of research for a number of possibilities including gene therapy for the lungs. nanoparticles for gene therapy can be characterized by submicron sized particles that berkeley pharma tech journal of medicine | 65 optimize surface to volume ratios while maintaining good performance of biocompatibility and biodegradation3. the customizability of nps for di�erent cell types, degrees of accumulation, and degradation o�er several advantages as carriers for gene therapy treatment in the pulmonary system. taking advantage of endocytosis and permeability factors of cells in similar manner as lipoplex-based techniques, nps can utilize similar lipid based qualities but are ultimately de�ned by size and their diverse makeup which extends beyond liposomes. targeting cells and material within the airways limits the abilities to penetrate into the tissue for many delivery methods. as mentioned, liposomes can encounter problems with penetrating surfactant and mucosal layers due to degradation and dissociation. modi�cations can be made to nps to resist mucoadhesion and trapping or degradation that may otherwise occur. one of the most common methods for this is a coating of polyethylene glycol (peg) polymer to provide a hydrophilic and neutral coating to counteract traditional charge based dynamics used in synthesis18. it has been found that these peg coatings provide varying levels of improvement in penetration of the thick mucus that exacerbates the issue of access to airway epithelial cells in patients with advanced copd; degraded structures such as neutrophils and dense meshes of highly negative charged structures create a less permeable mucus layer19,20. these �ndings further the ability to overcome airway mucus, one of the primary issues in inhalable gene therapy to treat copd and prevent signi�cant worsening of symptoms. plga a candidate in gene therapy using nps is that of poly ((d,l-lactide-co-glycolide) (plga)) based nanoparticles. plga-nps are fda and european medicine agency approved for drug delivery systems, not gene delivery. good results have been demonstrated regarding biocompatibility that exceeds that of common liposome based treatments in transfection of hepg2 cells for gene delivery21,22. surface modi�cations can be made to plga-nps to enhance e�ciency in performance such as the addition of polyethyleneimine (pei) made by bivas-benita et al. to enhance performance in the pulmonary system given its merits as a gene delivery berkeley pharma tech journal of medicine | 66 vector; the particles were seen to escape the endolysosomal envelope and subsequent damage to continue release of material in some instances as well23. dendrimers a relatively unique np structure that has gained exposure in gene therapy treatment given success in diagnostics is dendrimers. these nps are based on the dendrimer polymers that exhibit a radially symmetric pattern of branching chains. this allows for many exposed ends at the surface that can be modi�ed much like other nps to optimize their function as a delivery method for genetic material. hypotheses revolve around the potential for these large amounts of ionizable branches to be modi�ed, most commonly utilizing poly-(amidoamine) and poly-(propyleneimine) as a base, or immediately paired with genetic material for testing29. to help in structure stability and formation the aforementioned peg coating is applied to dendrimer nps prior to material attachment1. given the plentiful terminals, rapid absorption and dissipation of the nps is another problem solved via these peg coatings by enhancing retention by the lung tissue. the majority of the published work on dendrimer nps in lung tissue gene therapy presents them in the context of this ‘pegylation” given the ability to control uptake with a much higher degree of precision than without30,31. dendrimers share in the success of nps as a whole in their continued research and have signi�cant potential for delivery of gene therapy to inhibit copd symptoms provided the state of knowledge continues to advance. electroporation electroporation (ep) di�ers from previously discussed delivery methods in its physical based mechanism of action rather than the chemical basis on which nps and lipoplex therapies rely. it has emerged as one of the few physical methods researched for gene delivery given its safety and e�cacy compared to others. ep takes advantage of an electric �eld applied across cells to alter permeability and allow previously injected genetic material to be more readily endocytosed. transfection e�ciency in tumors using ep has provided the foundation of knowledge for its usage in lung tissues. intense berkeley pharma tech journal of medicine | 67 tissue analysis and/or electric �eld testing is required in most cases to ensure optimal settings given the natural variation in biological tissues32. traditional chemical based approaches make up the vast majority of research due to customizability. however, as these �elds move forward experiments have validated ep as a comparably e�ective method when physiological responses are considered in tandem with pure gene expression amongst cells33. viral methods viral vectors have been researched as avenues for gene therapy for years now given their development in the early 1970s and 80s and ability to take advantage of mechanisms native to the viruses themselves. the primary vector types found in modern gene therapy for copd and lung tissue oriented studies are adenovirus and adeno-associated virus (aav) based approaches. the di�erences in viral vectors and e�cacy is contingent on the exact protein capsid and tissue tropism, the gene of interest for therapeutic purposes, and the alterations that control the gene expression for the vector34. these work together and can be chosen or altered to perfect one’s vector for its desired purpose. common problems brought about in viral vector usage despite modi�cations is the innate immune response. t-cell responses and toxicity within tissue is a cause of viruses that has developed to help the body respond. however, redesign and manipulation of these viral genomes and bodies can still carry markers to inhibit their expression when used as vehicles for gene therapy. adenoviruses with regard to the aforementioned characteristics adenoviruses are a family of icosahedral nucleocapsid viruses. they are unenveloped and hence carry their genetic material, double stranded dna, within this characteristic icosahedral chamber. adenoviruses account for several acute respiratory illnesses and human adenoviruses (hadvs) have a wide range of categorized species based on key characteristics that number over 80 types35. work in gene therapy for copd stems from hadvs’ versatile tissue tropism and prevalence in lung-related diseases and replication36. imaging techniques berkeley pharma tech journal of medicine | 68 have revealed signi�cantly higher carrying capacity for genetic material in adenoviruses than alternative viral vector platforms at around 36 kb pairs while delivering roughly 8 kb pairs. a capacity that can be increased using a newer generation of helper-dependent adenoviruses (hd-advs) by deleting the viral coding sequences1,37-38. such delivery methods have been used in gene delivery to pig airway epithelia to demonstrate delivery of the cftr protein in hopes to reverse mucus production and in�ammation in patients with cystic �brosis and associated lung diseases39. as prominent viruses in everyday life a common phenomena observed in usage of advs for clinical trials is pre-existing immunity 40 which presents the need for research of multiple hadv serotypes to allow for versatility in practical usage as gene vectors. adv types that rely on a singular method of cell entry have limited access points and see more pre-existing immunity. as previously mentioned, hadvs types are plentiful and subcategorized into 7 grand species a-g, the majority of which belong to the hadv-d family40. hadv-d type 49 is one of many hadv types that utilize varying surface molecules to enter the cell and as a result is a prime candidate for research in adv vectors to solve this issue of immunity. the viability of hadv-d49 is compounded by its observed transduction in lung and spleen tissues while showing reduced targeting in liver and other less desirable tissues when looking at in vivo biodistribution41. immune response has also been mitigated by using hd-adv vectors as their stripped down genome removes key markers for the immune system to identify and target. airway basal cells of mice and pigs have been targeted successfully using these hdadvs following intranasal delivery42,43. limiting immune response while maintaining a focus on the e�cacy of adv based gene therapy is the current objective that needs �ne tuning in research for adv vectors to obtain signi�cant clinical success. adeno-associated viruses to build upon the problem of immune response many relatively distant derivatives of adv virus vectors have been created known as adeno-associated virus (aav) vectors. these have more desirable immunogenicity. they evoke a weak in�ammatory response compared to berkeley pharma tech journal of medicine | 69 adv vectors by lacking a staple viral coding sequence and contain a linear single-stranded dna. behavioral di�erences in host integration led to its prevalence in research as an option for gene therapy. carrying capacity in aav vectors is limited in comparison to advs at typically less than 5 kb though reports have seen up to ~9 kb of genome e�ciently incorporated into aav vectors for therapeutics44,45. increased packaging capacity typically comes at a cost of transduction rate and overall e�cacy in aav vectors though research continues to be done to improve the most pressing drawback of the delivery method. researchers at stanford have altered charges in the lumen of aav variants known as aavdj to transduce cells derived from human kidneys better than wildtypes when packed at a higher rate of up to 6.2 kb46. more akin to lung therapies for copd, viral/aav chimeric gene therapy has seen signi�cant research in navigating human epithelial cells. chimeric viral vectors look at combinations of multiple vector methods in hopes to take advantage of favorable characteristics in each and pose grounds for signi�cant advancement in gene therapy for respiratory diseases given local cell selectivity. a type of chimera vector packaging plasmids using aav and human bocaviruses (hbov) been shown to work well in tandem with human airway epithelia, primary human hepatocytes, skeletal muscle cells, and t cells; in addition to good performance in extensive studies in ferret airway models more recently47,48. these aav/hbov combination vectors o�er a novel approach to creating vectors for gene therapy in treatment of copd. potential treatment there are many limitations to modern gene therapies used today caused by many biological barriers. [jg1] most lung-related therapies must pass through multiple barriers such as the mucus, pulmonary surfactant, and local in�ammation24. this could cause a problem since patients with diseases such as asthma and cystic �brosis could also cause a biological barrier due to the airway mucus hypersecretion, which also plays a role in copd, since around 50% of copd patients have airway mucus hypersecretion25. berkeley pharma tech journal of medicine | 70 another limitation is when targeting jg3 cells other than the epithelium cells will cause the epithelium cells itself to become a major barrier24. although there are several limitations to current gene therapies, there are other alternatives to target cells that could be considered in treatments. for example, epithelial cells, alveolar cells, and macrophages all can be accessed through inhalation of nucleic acid containing nanoparticles (nans)26. this method of treatment helps target the lung and not target other cells that could cause biological barriers. although inhalation of nanoparticles seems like a great treatment for lung disorders, it is actually limited in some copd treatments. the inhalation of bibw 2948, which is used for treating copd, helped reduce the internalization of efgr, which plays an important role in epithelial changes in copd, but does not reduce the mucin storage27,28. bambi, which is the bone morphogenic protein and activin membrane-bound inhibitor, plays an important role in indicating if a patient has copd. bambi are expressed stronger in copd patients [28]. also there is a correlation between the levels of bambi and plasma tgf -β1 levels. therefore, the inhibition of tgf-β1 signaling might provide an alternative therapeutic strategy for treating copd28. inhale gene therapy another potential therapeutic treatment for copd is through inhaled gene therapy. this treatment provides direct access to the target of gene therapy for obstructive lung diseases via inhalation. there are limitations that surround inhale gene therapy. in a study conducted by dr. magdalena humenberger from the kepler university hospital in austria concluded that complete adherence to inhaled therapy was only seen in 33.6% and was higher among those with more severe copd, based on these results47. it was shown that mucus poses a barrier for this speci�c treatment. although this is a current problem, other studies have mentioned a di�erent type of gene vector that could be inhaled that would penetrate that mucus barrier. a type of nanoparticle that was introduced by berkeley pharma tech journal of medicine | 71 dr. jung soo suk, from the the center for nanomedicine in the johns hopkins university school of medicine, was the mucus-penetrating dna nanoparticles (dna-mpp), which possess non-adhesive coatings that allow them to rapidly penetrate mucus layers48. the peg coating helps with the dna-mpp treatment since it does not have a dense surface, helping the nanoparticle to penetrate other mucin-based meshwork48. stem cell therapy another potential way of treating copd is through stem cell therapy. this treatment is considered one of the newer treatments that still has a lot of questions �oating around it. although stem cell therapy is not considered safe, it still has potential when it comes to treating disorders such as copd. recently there have been several studies and clinical trials that have focused on stem cell treatment. the studies on copd focused on a speci�c stem cell treatment called mesenchymal stem cells (mscs), which is important for making and repairing skeletal tissues49. in a clinical study, they were infused with expanded allogeneic umbilical cord tissue derived mesenchymal stem cells (mscs) to 20 copd patients and then were monitored for 6 months49. this study concluded a signi�cant improvement in some important outcomes of copd, including mmcr, cat, number of symptoms, and the downregulation of in�ammation49. although this study is considered a pilot study, a small scale clinical study, it provides a unique perspective when it comes to treating copd via stem cell therapy. alpha1-antitrypsin alpha1-antitrypsin (a1at) is a protease inhibitor whose de�ciency is most commonly associated with the zz mutation which causes abnormal folding in the er of hepatocytes during biogenesis, causing its retention within the er. the retention of 90% of the normal a1at levels greatly increases the risk for early onset copd50. in a large lithuanian cohort, out of 1167 patients who had copd ranging from moderate to severe, 3.4% had the mz mutation, 3.3% had the ms mutation, 0.3% had the sz mutation, and 0.7% had the zz mutations. results from the screen indicated that there berkeley pharma tech journal of medicine | 72 was a signi�cant increase in mz, sz and zz genotypes in copd patients; it also supported the concept of using a targeted screen for a1at de�ciency when diagnosing copd51. another study conducted in brazil on 926 copd patients found that 2.8% had a1at de�ciencies and 0.8% had the zz mutation. these results also supported the importance of screening for a1at levels in all copd patients52. mirna mirnas play an important role in lung development, homeostasis, and pathogenesis. they also play a role in the regulation of cellular response to inhaled toxins and in regulation of in�ammatory and anti-in�ammatory processes. mirnas are one of the primary epigenetic modi�ers that can a�ect gene expression through post-translational gene silencing and mrna degradation. their improper regulation can lead to chronic infections and in�ammation. a study was done that obtained lung tissue samples from 15 copd patients and 11 subjects with normal lung function. 12 di�erentially expressed mirna in copd patients compared to subjects with normal lung function were identi�ed which showed to mostly target the nuclear lumen and transcription. of the 12 di�erentially expressed mirnas, mir-28-3p was most signi�cantly down-regulated and mir-212-5p was most signi�cantly up-regulated [53]. more recently, a study showed that two constructed mirna-mrna pathways; mir-126-5p and mir-130-5p-foxo1 could be potential biomarkers for the diagnosis and treatment of copd54. this year, a study showed that mir-126 was higher in copd patients with acute exacerbation compared to stable copd patients and healthy non-copd patients and distinguished the groups. this led to the conclusion that the dysregulation of mir-126 relates to copd susceptibility and acute exacerbation risk, but also is linked to severity and in�ammatory cytokines in copd patients55. berkeley pharma tech journal of medicine | 73 discussion chronic obstructive pulmonary disease provides unique grounds for exploration in new therapies. the compounding factors of several potential diseases or developmental miscues on a cellular level and beyond make it an issue to solve. however, this promotes much more de novo methods and research into the usage of several therapeutic methods in hopes to prevent such a prominent issue that 6.4% of americans were diagnosed with it in 2018. focus on lung tissue dynamics and genetic development can be accomplished in a number of ways through gene therapy is one of the most promising and has great potential in future endeavors[jg5] . as an overarching �eld, gene therapy is on the forefront of pulmonary medicine given its success in diagnosis, treatment, and identi�cation in epidemiology of cystic �brosis, emphysema, and other common issues which all act as compounding factors categorized as copd5-9. gene therapy works as a two pronged tool in lung therapy given the vast possibilities in the �eld between the potential delivery methods and actual genetic and cellular targets. the combination e�ect of these two issues leads to near endless possibilities in potential layouts for therapeutic strategies. in non-viral delivery methods, liposomes, nanoparticles, and electroporation are the more heavily researched aspects and provide a basis for most clinical studies1,3,15. liposome based approaches and many potential nanoparticles limit immune response thanks to their endogenous nature and customizable size to desired scale yet the key di�erence comes when one looks at the e�ciency. liposomes and liposomal nps sacri�ce ease of use and optimal immunogenicity for e�ciency in many cases due to the body naturally developing barriers to them such as nuclear transport of their genetic material and signi�cant degradation before they can have a signi�cant e�ect on the target15,17. non-liposomal nps are the most wideopen area of research given the plethora of potential formulations. plga and dendrimer nanoparticles for lung therapy have seen success given their idealized interaction. yet, price points become an issue when one looks at the extensive research and time that must be put into each individual iteration of a given treatment method22,30. for example, berkeley pharma tech journal of medicine | 74 taking a simple plga coated nanoparticle with targeting sites to deliver to lung epithelia for production in a1at vs. the exact same base particle complemented to target ciliated cells will require entirely di�erent sets of trials and fda compliance before any signi�cant levels of usage can occur. this is obviously necessary for safety and ethical concerns but presents a signi�cant holdup in advancement of np as a therapy for copd. electroporation uses the manipulated electric �elds in cells to increase permeability signi�cantly. targeting speci�city and overall quality of research eludes the �eld but it presents a purely physical method for gene therapy delivery and even assisting other methods should cell access present a signi�cant issue1,33. viral methods for delivering gene therapy are composed of mostly adeno and adeno-associated viruses given the broad spectrum they o�er for behavior and ease of modi�cation35. both o�er one of the more sought after qualities in gene therapy strategies in their integration abilities. the inherent function of viruses makes them ideal vectors that just need to be altered for the desired function. immune response is an issue that can be improved upon but presents one of the only signi�cant issues in their usage as gene vectors44. targets for the readily available delivery methods is where the speci�city of copd comes to the forefront of treatment. inhalable therapies provide a unique avenue for treating pulmonary issues that might not be possible with other groups of diseases, thus it has been explored heavily with iterations of liposomal np and viral vector bodies optimized for breaking through the heavy mucus and surfactant layers in lung tissues17,42-43,48. a variety of cells are still accessible through this minimally invasive gene therapy26. new stem cell therapy for copd focus primarily on mesenchymal stem cells given their limited exhibition of pluripotent properties. one of the most well researched causes and potential targets for copd is that of a1at where gene alteration to the common zz mutation or to improve production in a1at de�cient individuals has been seen to improve both physiological qualities and provided long term expression50-52. the �nal but potentially most broad method for focusing on the issues presented in copd is that of working with mirna using the aforementioned delivery methods. as a primary regulator for gene expression, in depth studies have found disparities in mirna quality in berkeley pharma tech journal of medicine | 75 copd patients53. insertion of nps to change mirna expression for issues like lung irritation have also seen success56. looking at these delivery methods in tandem with targeting goal is the only way to e�ectively evaluate the viability of a therapeutic strategy for copd and look to improve the current state of understanding in treatment for it. conclusion although there are many potential treatment avenues, there needs to be a large focus on personalized medicine as the source of patients’ copd can vary. severity and associated symptoms are plentiful in copd which only serves to necessitate more research into the �eld. making strides towards being able to identify the best suited treatment for an individual will result in the most e�ective outcomes and treatments. the demonstration of e�ectiveness in both speci�c cell targets in vitro as well as delivery methods must come together to formulate idealized gene therapy treatments. issues present themselves in all areas discussed though they remain the primary modes of treatment in modern medicine. understanding the subject and dynamics of both the disease and methods used is necessary to maintain good quality of care and building upon these to improve treatment of copd. berkeley pharma tech journal of medicine | 76 references 1. baliga uk, dean da. pulmonary gene delivery-realities and possibilities. exp biol med (maywood). 2021;246(3):260-274. doi:10.1177/1535370220965985 2. kim n, duncan ga, hanes j, 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suk js, cone r, hanes j. mucus penetrating nanoparticles: biophysical tool and method of drug and gene delivery. adv mater. 2012;24(28):3887-3894. doi:10.1002/adma.201201800 48. broekman w, khedoe ppsj, schepers k, roelofs h, stolk j, hiemstra ps. mesenchymal stromal cells: a novel therapy for the treatment of chronic obstructive pulmonary disease?. thorax. 2018;73(6):565-574. doi:10.1136/thoraxjnl-2017-210672 49. broekman w, khedoe ppsj, schepers k, et al. mesenchymal stromal cells: a novel therapy for the treatment of chronic obstructive pulmonary disease?thorax. 2018;73:565-574. https://doi.org/10.1136/thoraxjnl-2017-210672 50. sitkauskiene b, et al. screening for alpha1-antitrypsin de�ciency in lithuanian patients with copd. resp med. 2008;102(11):1654–1658. https://doi.org/10.1016/j.rmed.2008.07.003. 51. russo r, zillmer lr, nascimento oa, et al. prevalence of alpha-1 antitrypsin de�ciency and allele frequency in patients with copd in brazil. j bras pneumol. 2016;42(5):311-316. doi:10.1590/s1806-37562015000000180 52. kim wj, et al. altered mirna expression in lung tissues of patients with chronic obstructive pulmonary disease. mol & cell tox. 2017;13(2):207–212. https://doi.org/10.1007/s13273-017-0022-9 53. zhu m, ye m, wang j, ye l, jin m. construction of potential mirna–mrna regulatory network in copd plasma by bioinformatics analysis. int j chron obstruct pulmon dis. 2020;15:2135-2145. https://doi.org/10.2147/copd.s255262 54. wang c, et al. dysregulated circulating microrna‐126 in chronic obstructive pulmonary disease: linkage with acute exacerbation risk, severity degree, and in�ammatory cytokines. j clin lab ana. 2002;36(3). https://doi.org/10.1002/jcla.24204 55. bobba cm, fei q, shukla v, et al. nanoparticle delivery of microrna-146a regulates mechanotransduction in lung macrophages and mitigates injury during mechanical ventilation. nat commun. 2021;12(289). https://doi.org/10.1038/s41467-020-20449-w berkeley pharma tech journal of medicine | 80 front_page_calendine calendine body template.docx-2 calendine reference template �� berkeley pharma tech journal of medicine correspondence:� nabeen.chu@gmail.com keywords: *tdifnib $bsejbd�ujttvf�sfhfofsbujpo .pe/3" .ftfodiznbm�tuspnbm�dfmmt *oevdfe�dbsejpnzpdzuft $bsejbd�hfoft (fof�uifsbqz submitted�.bz�� ������ accepted�+vof��� ����� published july 19, 2022 full open access creative commons attribution �license 4.0 abstract cardiac disease continues to be among the most prevalent causes of death worldwide. presently, surgeries such as angioplasties, stents, and bypasses pose many risks. to improve outcomes in treating ischemia, researchers have been pursuing minimally invasive, biocompatible treatments such as gene therapies. gene therapies are treatments that enhance or suppress target genes to alleviate illness. there are various applications for gene therapies, including, but not limited to, the treatment of cancers, diabetes, and heart disease. gene and stem cell therapies can regenerate cardiac tissue that is damaged due to ischemia. furthermore, gene therapies intended to evade the immune system may decrease infection risks due to the new tissue being better accepted by the body as it is created from patients’ own cells. while dna treatments show poor results in treating cardiac illness, stem cells, such as mesenchymal stem cells and induced pluripotent cells, can differentiate into cardiomyocytes, and mrna can be modified to express angiogenesis growth factors around the affected tissue. although further research is needed to adapt these techniques for safe clinical use, they show potential for inducing cardiac tissue regeneration in ischemic injury. this paper conducts a review of the emerging techniques and evaluates gene therapy as a potential treatment for ischemic injury. novel methods for inducing cardiac tissue regeneration following ischemic injury �ïw�§×øûûä��þëu�¬þèûï×�£ûï×áëã×èu��åä�¡ûû�¤ååu�®ã×� è×äúþßu�and ¯×äâå×ä�§ýëïûä introduction much research has been done on various approaches to gene therapy1 to treat an ischemic injury, yet few techniques have gone into clinical trials, and these seldom show success. this paper aims to conduct a review of a few major techniques and evaluate the potential of gene therapy as a treatment of ischemic injury. cardiac-related illnesses and deaths have consistently been one of the top causes of death in the u.s. and around the world2, and has also been on the rise since the beginning of the covid-19 pandemic, disproportionately a�ecting minority populations3. thus the importance of researching more e�ective methods of treating and preventing ischemic injury is imperative. the mortality of ischemic heart injury di�ers by demographic, including one’s age, country, and region, with lower-income countries and older people su�ering higher mortality rates, with little to no improvement4. since the rise of gene therapy as a potential treatment method for various illnesses in the 1990s, the �eld has been consistently researched5. the precision of the ability to modify speci�c genes o�ers great potential. however, due to obstacles such as high cost, ethical concerns, and di�culty translating into clinical applications5, there is still much to uncover. cardiac ischemic injury refers to cell damage due to diminished �ow of blood, containing nutrients and oxygen, through the vessel. if ischemic injury is not caught quickly and treated in a timely manner, cardiomyocyte death can occur, with permanent e�ects6. ironically, surgically restoring blood �ow to these regions may actually cause cell death and organ failure7. gene therapy thus presents as a favorable option to instead promote angiogenesis through growth factors such as vegf-a or stem cell therapies to develop new paths of blood �ow rather than risking further ischaemia-reperfusion injury (iri). we have decided to look not only into gene cascades that are often targeted by gene therapies, but various approaches to gene modi�cation, including stem cell research using mesenchymal stem cells (msc), modi�ed mrna (modrna), and induced pluripotent stem cells (ipsc). berkeley pharma tech journal of medicine | 2 mesenchymal stem cells to treat ischemic injury myocardial infarction (mi), more commonly known as heart attack, and related injuries are a leading cause of death globally, and despite the applications of extensive novel cardiac surgery and grafting to lengthen and ameliorate life, long-term cardiac damage may persist8 . the usage and application of mesenchymal stromal cells (mscs) in heart tissue has become an increasingly popular candidate in the treatment of myocardial infarctions, as they exhibit factors that increase cardiomyocyte survivability8. there is an abundance of research aimed toward understanding the implementation of mscs in the prognosis and improved function of heart tissue, due to their desirable trait of being able to di�erentiate into disparate cellular lineages9. it is by far one of the most promising treatments in regenerative medicine. however, it is di�cult to gather results from existing clinical trials due to suboptimal reproducibility and e�cacy outcomes,10 and the exact mechanism of the cell treatment is still largely unknown9. previous and ongoing research has shown that the usage of mesenchymal stromal cells has proved to have signi�cant promise in treating ischemic myopathies11. many clinical trials have shown that the application of mesenchymal stromal cells to infarcted cardiac tissue has been shown to reduce �brosis12, improve regional contractility11, and reduce arrhythmias amongst other outcomes13. in clinical trials with mice, it has been demonstrated that direct application of mscs could aid in angiogenesis and myogenesis of ischemic myocardium in murine post-acute myocardial infarction14. this is a signi�cant e�ect of mscs, since heart failure can be caused by death of large numbers of cardiomyocytes, so the induction of angiogenesis and myogenesis can prove to be a desirable outcome14. clinical studies have also shown that application of autologous bone marrow mscs (bmscs) correlated to reduced scar sizes, reduced infarct sizes, improved left ventricular ejection fraction (lvef), and a signi�cant increase of viable tissue in patients who underwent coronary artery bypass graft surgery (cabg) compared to controls12. one particular study aimed to investigate the direct e�ect of bone marrow-derived mscs on cardiac function immediately after mi by injecting the cell sample into infarcted berkeley pharma tech journal of medicine | 3 intramyocardial tissues15. although the experiment showed that the donor cells minimally remained in the myocardium after implantation, and the exact mechanism is still unknown, there was a signi�cant reduction in infarct sizes, improved cardiac function, expression of pro-angiogenic factors, all occurring in a paracrine manner15. speci�c types of mscs like umbilical mscs, which promote vascular regeneration and cardiomyocyte protection, are accessible and easily expandable in experiments13. however, with the varying stem cell types, there are also many di�erent constraints that have hindered more innovative research into the application of mscs. the application of bone-marrow derived mscs in particular include invasive or unethical harvesting procedures, and decreased proliferation among varying donors13. but in a more promising facet, umbilical-derived mscs demonstrate more safe and feasible qualities, including being more easily attainable, posing less ethical concerns, and undergoing less cellular aging13. safety of msc application is an important facet of the treatment to ensure since there are several potential health concerns surrounding the treatment, including tumor formation, organ toxicity, and ectopic tissue formation in the vasculature16. safety pro�les of intravenous msc application in acute mi patients are successfully present, showing signi�cant results such as reduced tachycardic episodes, improved forced expiratory volumes, and no concerns for ectopic tissue creation in the long term16. however, one study aimed to evaluate the safety of human bone marrow-derived mesenchymal cell application in patients su�ering from acute mi by using a multicenter trial16. in the study, the data safety and monitoring board approved the application of msc dosage in each experimental cohort, and primary safety assessments were carried out to monitor any adverse reactions to the treatment16. the results of this study showed that there was no apparent evidence of increased toxicity with the application of the mscs and there the administration was well tolerated in the cohorts at all doses16. there was also evidence that the arrhythmia ratio was signi�cantly lower in the msc administered patients vs. the placebo group, and also showed improved function in cardiac performance and pulmonary function in comparison with the placebo group16. there was no report of long-term ectopic tissue berkeley pharma tech journal of medicine | 5 formation either, which showed the signi�cant therapeutic bene�ts of the administration of the hmscs overall16. another study aimed to evaluate the safety of intravenous administration of umbilical cord-derived mscs (uc-mscs) as well. the experimental group involved patients with heart failure and reduced ejection fraction, and were presently treated with application of intravenous infusion of allogeneic umbilical-cord derived mscs13. the results of this study showed that the uc-msc treated patients expressed no adverse reactions to the infusion treatment and showed signi�cant improvement in left ventricular ejection fraction over multiple time checkpoints post-administration13. the results of these studies show that the incorporation of mscs as a treatment option is safe and feasible with di�erent types of mscs, and there were no noteworthy deleterious reactions to this treatment. the results described above also showed improvement and potential in recovery and o�ers a potential usage in future medicine and therapy. many questions are still unanswered regarding the mechanism of action of msc treatment9. studies have shown that injection of mscs in a�ected heart tissues reduced infarct size and induced angiogenesis and myogenesis14. one main mechanism of action is injection of bone marrow-derived-mscs directly into infarcted heart tissue to promote cardiac function via expansion of the cells11. this is usually caused by mscs’ ability to expand and di�erentiate into cardiomyocyte-like cells13. one experiment conducted coronary artery ligations in mice and then implemented mscs to note any changes in grafting in the ischemic myocardium and proliferation into cardiomyocytes14. this particular experiment showed results illustrating improved cardiac function after msc implementation through the enhancement of myogenesis and angiogenesis. other experiments involving injection of mscs or bmcs into infarcted tissue have shown some results correlating with improved regional contractibility, and no signs of ventricular arrhythmias, worsening cardiac function, pulmonary embolisms, or cardiac tamponade11. there are still suboptimal results correlating msc transplantation and therapeutic improvement of cardiac function, since the mechanisms of action are quite complex and not fully understood9. but transplantation of mscs in many berkeley pharma tech journal of medicine | 6 recent reviews and studies have proven to be safe13 and the selective study results shown could be bene�cial. mscs have been used as a therapeutic application for many degenerative diseases, mainly due to their various di�erentiation potential , paracrine factors17 and secretory products like angiogenic factors, mitogenic factors, antiapoptotic factors, and growth factors18. in particular msc’s have been frequently involved in experimental models of cardiovascular diseases13 and express attributes that make them desirable for use in cardiac function modulation after myocardial infarction9 including secretions that could prevent cardiac in�ammation and aid in cardiac injury repair19. although the underlying detailed mechanism of msc transplantation is still mostly unknown and incomplete19, and clinical trial results are still suboptimal9, transplantation treatments show promising e�ects15 regarding improvement in myocardial function in experimental models13. additionally, the trajectory of evidence shows that transplanted cells have a bene�cial e�ect on overall cardiac function after myocardial infarction20 and ischemic heart disease18 due to varying di�erent results. transplanted mscs from red bone marrow in adult rat specimens showed di�erentiation into cardiomyocytes by the presence of cardiac proteins like desmin, and also showed to form connections with native cardiomyocytes in the rat sample18. this di�erentiation into both vascular endothelial cells and smooth muscle cells promoted cardiac function via myogenic and angiogenic e�ects14. engrafted cells harvested from a healthy human sample also showed similar results, with the stem cells strongly morphologically resembling host cardiomyocytes and also expressing desmin, myosin heavy chain, actinin, and cardiac troponin amongst others, each of which are native cardiac tissue proteins21. echocardiographic assessment of transplanted bone marrow mscs into post-mi tissues also showed that the cells relieved the restrictive e�ects of left ventricular function and geometry caused by mi20. additionally, intravenous application of umbilical cord mscs showed improved left ventricular ejection fraction and left ventricular end diastolic volume in control groups for patients with systolic heart failure or reduced ejection berkeley pharma tech journal of medicine | 7 fraction13. novel experiments have also shown that direct injection of msc exosomes also reduced infarct sizes, improved cardiac function, and o�ered protection of cardiomyocytes from hypoxia8. fig 1. msc differentiation potential. based on the diagram of differentiation potential for msc terminal phenotypes, we can see how bone-marrow mscs or umbilical cord derived mscs have the potential to differentiate to different cell types including cardiomyocytes, vascular endothelial cells, and skeletal muscle cells amongst other cells. this differentiation into cardiomyocytes explains why the expansion of the cells in cardiac tissue settings is very desirable. the differentiation into cardiomyocytes has been measured by proteins like desmin and troponin, and is also noted to form connections with native cardiomyocytes within the tissue. although much of the application of mscs is still subject to more extensive clinical testing and development15, there is a huge promise of the already performed experiments discussed above to show safety of the clinical application and the therapeutic bene�ts of administration. the studies above have depicted the many potential and con�rmed bene�ts of administration of either bone-marrow derived mscs or umbilical cord-derived mscs, including reduced infarct sizes, overall improved berkeley pharma tech journal of medicine | 8 cardiac function and lvef, cardiomyocyte production and protection, angiogenesis, and myogenesis. safety of administration of bmscs and uc-mscs were both measured with signi�cant results in cohort groups, showing that overall, administration is safe and feasible with generally little to no adverse e�ects13. although these studies presented signi�cant outcomes, it has been suggested that more extensive and larger clinical trials are needed to fully understand the clinical bene�ts of administration of certain mscs13. due to the many measured bene�cial implications of mscs, including overall cardiac function, we can conclude that msc transplantation can be a new therapeutic strategy for treating patients su�ering from myocardial infarction14. modifed rna as a method of gene therapy dna modi�cation comes with many risks, including immunological response, and lack of speci�city regarding its locus of action. consequently, rna therapy is becoming a further researched �eld, including clinical applications for modrna and mirna23. modi�ed mrna (modrna) is becoming a prevalent research topic in many di�erent �elds, such as diabetes, ischemic injury, and the mechanism of a covid-19 vaccine24. in this method of gene therapy, mrna is injected into the heart to enhance target protein translation, which in this case is cardiomyocytes and angiogenesis to repair the heart after massive cell death25. however, mrna is short lived and cannot provide lasting e�ects for cardiomyocyte regeneration. they often trigger an immune response, and are also broken down by rnaases. thus the degree of protein expression is dependent on mrna stability26 and they must be engineered to be more e�cient in their expression in the cell, or be longer lasting. so far, this is achieved through four di�erent methods: altering nucleotides, mrna capping, untranslated region manipulation, and the length of the poly a chain27. understanding the alterations of mrna requires an understanding of mrna synthesis. often, a dna plasmid is transcribed in vitro and the resulting mrna is used for therapy. in this process, substitutions can be made in the nucleotide pairing process. kierzek et al. showed in 2014 the berkeley pharma tech journal of medicine | 9 speci�c e�ects of substituting pseudouridine for uridine with the di�erent nucleic acids, in which ψ-a pairing demonstrated greatest enhanced stability due to hydrogen bonding, at about 0.5kcal/mol. due to the increased stability and because pseudouridines are found naturally in the body, these modrna are able to avoid detection by the immune system, speci�cally by tlr3, tlr7, and tlr828. furthermore, it was found that vegf mrna whose uridines were completely replaced by pseudouridine, did not trigger the immune system and remained locally in the target tissue of cynomolgus monkeys or rats while promoting angiogenesis. in this experiment, the mrna was injected intradermally or intravenously29. mrna caps are methylated guanosine groups attached to the 5’ end by a triphosphate group present in all eukaryotic mrna30. these give identity to the mrna strand as well as signal for degradation when removed31. thus the synthetic mrna must also be capped adequately to increase translation of protein. the untranslated region of rna, called the utr, appears on both the 5’ and 3’ ends of the mrna strand, and plays a role in mrna translation e�ciency and localization32. thus utr of modrna must also be manipulated adequately to optimize translation rate and half-life. for example, the addition of au-rich elements (are) weakened protein expression, and the more stable utr was able to increase transcription stability33. synthesizing utr speci�c to the needs of the modrna is imperative to its e�ective protein synthesis, and also allows for more precise control of the therapy. mrna is modi�ed by extending the polya tail to increase its translation. this modrna is administered through intramyocardial injection to a�ect a large area of cardiomyocyte and non-cardiomyocyte cells23. modrna is able to stay longer in the cell than rna by changing its structure with pseudouridine and evading rnaase34, but is still shorter-lived than dna, thus reducing the risk of mutation and multiple transcriptions over time25. rather, it is described as a “pulse”35, producing a rapid episode of protein synthesis that can be better controlled than dna therapy methods. berkeley pharma tech journal of medicine | 10 currently, due to the temporary nature of modrna, treatments may need to be administered multiple times to demonstrate e�ectiveness, which not only causes trauma on the cardiac tissue but also will become expensive25. modrna is already required in large doses36, thus production or administrative costs must be reduced to make this treatment clinically feasible. furthermore, injecting these genes into the intracardiac tissue is invasive and can be risky for patients who have already su�ered injury to the heart. further research must be done on less invasive gene and drug delivery methods that are still able to target speci�c cells and promote angiogenesis25. one such method is cell-penetrating peptides that have been demonstrated to carry dna, rna, or proteins into the cell via endocytosis37. figure 2. the structure of a modified mrna. the poly a tail increases translation and allows it to remain longer within a cell. despite these intricacies of modifying mrna, it has been demonstrated that modrnas can deliver relatively quick local responses both in vitro and in vivo. mouse hearts were able to retain the e�ects of a cardiac injection of modrna for 24 hours after surgery38. furthermore, modrna coding for vegf-a, a growth factor that promotes revascularization was demonstrated to successfully induce angiogenesis following myocardial infarction39. the modrna injected into the site of injury promoted heart progenitor cells to di�erentiate into vascular cells rather than muscle cells. in this experiment, the mice injected with vegf-a dna and vegf-a rna both formed vessels, but the mice who received the rna injection presented with vasculature that was less permeable and more similar in shape to control hearts. the vessels of vegf-a dna injected mice showed edema, and had higher mortality rates. these di�erences, likely due to the berkeley pharma tech journal of medicine | 11 di�erences in acting speed of rna and dna, display the potential of modrna in the �eld of cardiology. moreover, because vegf-a, a commonly used and researched growth factor, can cause increased vessel permeability at long exposure times, the shorter “pulses” of rna may be more e�ective in treating ischemic heart injury. vegf-a’s results are much more promising with mrna than dna largely due to its rapid and local characteristics29. this e�ect can be further enhanced by moving away from lipid carriers, sometimes called nanoparticles, that are often used to encapsulate proteins or genetic material. removing this layer prevents the mrna from entering circulation and traveling away from the target tissue, and the naked delivery of the modrna increased protein was found to increase translation 53-226 fold38. however, it must be noted that while for ischemic, local injury, removing the nanoparticles improves translation, for drugs that are aimed for general circulation, such as mrna vaccines, the lipid nanoparticles are an essential part of drug delivery40. carlsson et al. (2018)29 demonstrated the e�ects of vegf mrna after further replacing the pseudouridine with 1-methylpseudouridine and injecting naked mrna in a citrate-saline bu�er in swine. the treatment resulted in e�cient and long-lasting protein expression, local e�ects, and improvement in cardiac function at 1 week and 2 months after injection29. improvement here was de�ned as increase in muscle contractility and decrease in �brosis, and were dose-dependent. these �ndings further support and build upon zangi et al.’s �ndings in 201339 but with greater speci�city in the injections’ e�ects, as measured through bioluminescence, and with higher e�ciency. with cardiac angiogenesis in both species of mice and swine, modrna appears to be a strong candidate for carrying out cardiac regeneration after an ischemic injury. however, these studies will have to be carried out over longer periods to ensure whether the modrna must be reinjected, and for any side e�ects. while clinical trials of vegf modrna for cardiac regeneration are scarce, its safety and e�ectiveness were observed in treating patients with type ii diabetes mellitus. the study was conducted in a randomized, double-blind, berkeley pharma tech journal of medicine | 12 placebo-controlled nature, splitting participants into three groups to receive either placebo or vegf-a mrna at various doses41. the results re�ect that of carlsson et al.29 that demonstrated dose-dependent results. vegf-a mrna was able to successfully promote basal skin blood �ow at injection sites of the forearm, and the results were dose-dependent. at 7-14 days after injection, vasodilation and neovascularization were induced, demonstrating the potential for vegf-modi�ed mrna applications in humans41. observed adverse events were of onsite reactions, which occurred in all participants, but with only mild severity. the e�ects of cardiac modrna injection in humans as of yet unknown, though the gene therapy method is considered safe to be researched further in the context of cardiac ischemic injury. however, this clinical trial only had male participants, thus women must also be tested to ensure comparable results, and whether there are sex di�erences in dosing. after the safety and biocompatibility of vegf modi�ed mrna were con�rmed, it was applied in the cardiac �eld for patients undergoing coronary artery bypass grafting (cabg) by astrazeneca in a study called epiccure. this is a very common surgical procedure that aims to revascularize the heart by attaching grafts bypassing the clogged artery. however, there are still many risks, especially for those who have a history of renal disease or stroke. adverse events include infection, atrial �brillation, myocardial dysfunction, which are at higher rates for those who are undergoing dialysis or have other underlying cardiac conditions such as peripheral artery disease or pericarditis42. azd861, a vegf mrna drug in citrate-bu�ered saline, will be administered immediately after cabg before reperfusion at various doses to determine whether angiogenesis will be promoted, and if it will improve outcomes of the surgery36. while no paper published in a scienti�c journal has reported the results, astrazeneca published data on the company’s website and presented it at the american heart association’s scienti�c sessions in 2021. the treated groups demonstrated the biocompatibility and safety of azd8601, and in agreement with the phase 1 clinical trial41, there were no infections or severe adverse events. furthermore, because the injections in this trial were epicardial injections, they better demonstrate that vegf mrna is safe for berkeley pharma tech journal of medicine | 13 human use. but more research must be done to increase e�ciency of the treatment for signi�cant bene�ts in cardiac angiogenesis43. induced cardiomyocytes for cardiac regeneration induced cardiomyocytes (icms) are cardiomyocyte-like cells that are derived from the reprogramming of other cells, the most common being induced pluripotent stem cells (ipscs)44. culturing ipscs with speci�c media allows them to be di�erentiated into the target cell, in this case, icms. ipscs provide a solution to the long-standing di�culty of the lack of regenerative abilities of cardiomyocytes; their ability to proliferate allows treatment to surpass the stagnant number of normal cardiomyocytes in the body, a revolutionary possibility for regenerative medicine44. ipscs also provide a safer alternative to potentially toxic drug treatments and can serve as excellent disease models44, 45. though ipscs remain the most common cell that are used in icm derivation, cardiomyocyte-like cells have also been derived from the reprogramming of �broblasts. derived from direct lineage reprogramming via upregulation of genetic reprogramming factors, several studies have reported the generation of functional icms from cardiac �broblasts in both in vitro and in vivo mouse models46, 47. qian et al. (2012)46 examined the conversion of murine cardiac �broblasts to cardiomyocyte-like cells via the expression of three cardiac-speci�c transcription factors (gata4, mef2c, and tbx5) in vitro and observed a large percentage of cells that were partially reprogrammed. they applied their �ndings to an in vivo model and observed a similar rate of conversion with an even higher level of success in fully reprogramming the �broblasts into cardiomyocyte-like cells46. wada et al. (2013)47 also previously reported conversion using the aforementioned transcription factors to be successful for mouse �broblasts in vitro47. following their in vitro study, they observed the generation of new cardiomyocytes from endogenous cardiac �broblasts with the aid of the same transcription factors, and various improved aspects of overall cardiac function. following these discoveries, the possibility of directly reprogramming human cardiac �broblasts (hcfs) was also studied. while success in the berkeley pharma tech journal of medicine | 14 conversion of hcfs to icms was observed, the process was observed to be more complex than the mouse model, requiring additional reprogramming factors47, 48. the three transcription factors used in the in vitro and in vivo rodent models were not su�cient; the addition of mesp1 and myocd allowed for the generation of human icms in vitro47. however, the icms derived from this process “did not beat spontaneously”47. to di�erentiate into functional, beating icms, “human icms [required] coculture with murine cardiomyocytes,” unlike their mouse counterparts47. finally, the level of authenticity of the replicated icms in comparison to natural cardiomyocytes was unable to be conclusively de�ned47. somatic �broblasts were also found to give rise to icms through upregulation of cardiac gene expression, which can occur both in vitro and in vivo49. further studies involving human cardiomyocytes in vitro and in vivo are needed to understand the speci�c mechanisms required to produce an e�ective treatment based on this technology. while icms are a very promising �eld of cardiac gene therapy, they are, of course, not without their limitations. horikoshi et al. (2019)44 observed that icms derived from ipscs appear to lack the mature characteristics of normal adult cardiomyocytes. based on the natural process of cardiomyocyte maturation, they cultured ipsc-derived icms with fatty acids, the basis for the fatty acid β-oxidation process that supplies mature cardiomyocytes with energy44. their study revealed that a fatty acid culture can promote the maturation process of ipsc-derived icms in order to allow them to function as fully-matured adult icms44. another limitation observed in the �eld is the applicability of icm-based treatment to chronic heart failure, a condition for which regenerative therapy is in high demand48. while ipscs have provided excellent platforms for disease modeling thus far, in vivo cardiac reprogramming studies have been focused on models in the acute stage of myocardial infarction (mi), so its potential to alleviate chronic heart failure has yet to be determined48. however, given the marked improvement of overall cardiac function observed in numerous studies, there seems to be a de�nite potential for chronic heart failure reversal through treatment with icms. berkeley pharma tech journal of medicine | 15 icms themselves have not been observed to proliferate independently, a characteristic which would greatly improve the e�cacy of regenerative therapy48. instead of relying on the proliferation capabilities of the source cells, such as ipscs, development of icms that are able to endogenously proliferate would remove the necessity for repeated administration of icms. certain critical factors have been identi�ed in addition to the possibility of using a third icm derivation source in the form of induced cardiac-progenitor cells (icpcs) that would be converted into icms; however, further study is needed to solidify the technology and translate it to clinical application48. figure 3. common derivation methods of icms. diagram outlining two major sources, ipscs and fibroblasts, used for the derivation of icms. details the processes by which both sources can be used to create icms in vitro that can be inserted for treatment. also touches on how both of the sources can be directly inserted and used to develop functional icms in vivo as part of treatment regimens. berkeley pharma tech journal of medicine | 16 gene cascades to target for cardiac regeneration the manipulation of certain cardiac genes has shown signi�cant progress in improving ischemic heart disease. changes in the expression of certain genes in�uence cardiac cell proliferation and di�erentiation, demonstrating gene therapies’ potential e�cacy in treating cardiac disease. this section examines multiple therapies that concentrate on changing the expression of target genes. two main techniques were investigated, one focusing on the transplantation of bmscs with speci�c gene deletion and the other concentrating on the protein-protein interactions that in�uence a particular gene involved in cardiac cell signaling. recent studies have shown that hepcidin, an iron-regulating protein primarily in the liver, is also present in low amounts in the heart, to regulate iron homeostasis and ferritin content in cardiomyocytes53. injuries in the heart due to ischemic disease result in the destruction of cardiomyocytes, which releases a high concentration of iron into the extracellular space54. the increased iron content outside cardiomyocytes is associated with myocardial �brosis and with the formation of reactive oxygen species (ros)53. these conditions cause an intracellular iron de�ciency that leads to the overexpression of hepcidin proteins55. in e�orts to understand how hepcidin a�ects ischemic heart disease and to study the possibilities of related therapies, the gene coding for hepcidin, hamp, was manipulated in deletion experiments56. despite the lack of knowledge on how cardiac hepcidin functions and a�ects heart injuries, researchers �rst carried out direct gene deletions of hamp in mice to prevent the production of hepcidin and study its e�ect56. nonetheless, this gene therapy demonstrated unpromising results, causing scientists to quickly transition into the transplantation of either mice with bone-marrow-derived cells that are hamp de�cient or with myeloid cells with hamp deletions. the investigation was carried out on mice that were deliberately in�icted with myocardial infarction. those with hamp expression in macrophages were shown to produce high levels of hepcidin as a result of increased interleukin (il-6) production, which is responsible for hamp’s transcription57. berkeley pharma tech journal of medicine | 17 this new study on the e�ect of hepcidin on heart injuries revealed that this protein is related to the secretion of il-4 and il-13 which are each important for inducing cardiac repair56. hamp de�cient mice with myocardial infarction displayed a signi�cant reduction in infarct size and tissue �brosis as well as increased cardiomyocyte renewal. this result was related to the lack of hepcidin that led to improved receptor functions, such as c–c motif receptor 2 (ccr2)+, in macrophages. this improvement enhanced the secretion of il-4 and il-13 from the macrophage increasing cardiac healing as well as cardiomyocyte regeneration after a heart injury56. figure 4. the effect of the hamp gene on hepcidin production. hamp-deficient (-/-) mice reduced hepcidin production and induced the secretion of il4 and il13 responsible for cardiomyocyte renewal, while mice with hamp gene (+/+) produced high levels of hepcidin reducing the release of il4 and il13. while this research demonstrates a new �nding regarding the relationship between hepcidin and il-4/il-13, the mechanisms of how hepcidin in�uences the function of certain receptors and interleukins are still not widely understood. there is limited research on the potential of using this gene therapy due to the researchers’ e�ort to �rst better understand how cardiac hepcidin works in the heart and related injuries54. further berkeley pharma tech journal of medicine | 18 investigation on hepcidin is required to understand how the production of cardiac hepcidin a�ects certain tissues in heart cells before diving into particular therapies53. the study of this protein has great potential of being utilized for improved cardiac function in ischemic heart diseases, particularly the hamp-de�cient bmscs that induced cardiac repair56. although not yet implemented into paradigms of gene therapy, the ongoing in-depth study of protein-protein interactions has led to the possibility of controlling the expression of speci�c genes, essential for signaling pathways involving cell proliferation. the detailed understanding of wnt protein/β-catenin pathways, responsible for cell proliferation and growth, has led to signi�cant improvements in addressing diseases in organs such as the lungs58. in attempts to regulate this pathway for cardiac cell regeneration, recent studies have revealed protein interactions with wnt units that could inhibit injury-induced cardiomyocyte proliferation59. in a study on heart regeneration and injury repair, scientists discovered novel small molecules called cardiomogens (cdmg 1 and 2) that have the ability to inhibit wnt expression and cause β-catenin reduction downstream59. the investigation was carried out using embryonic zebra�sh with surgically-induced heart injuries. cdmgs inhibited wnt by speci�cally targeting β-catenin and tcf/lef-mediated transcription that is needed to initiate the expression of particular genes speci�ed by wnt60. the reduction in β-catenin accelerates the proliferation of damaged heart cells, leading to improved heart function. cdmg1, particularly, was e�ective in healing heart injuries by increased formation of cardiomyocytes and reduction in �brotic scar tissue59. nonetheless, the mechanism of cdmg on particular wnt pathways is still not well-understood. there are a wide variety of wnt proteins and related signaling pathways that a�ect distinct cellular lineages and their functions. therefore, further studies regarding what speci�c wnt proteins cdmg structures inhibit and what speci�c genes are a�ected by these molecules could potentially result in new gene therapies, and ultimately improvements in medicine. in addition to the newly discovered molecules, secreted frizzled-related proteins (sfrp) inhibit canonical wnt signaling pathways responsible for cell berkeley pharma tech journal of medicine | 19 proliferation, while activating non-canonical wnt pathways that lead to di�erentiation61. this research was accomplished by culturing cardiac progenitor cells (cpc) from mice. the binding of sfrp2 to wnt6 reduced cpc proliferation while fostering di�erentiation. inhibition of wnt by sfrp causes an activation of non-canonical wnt/planar cell polarity (pcp) pathway through c-jun n-terminal kinase (jnk)58. this activation encourages the expression of cardiac transcription factors and cpc di�erentiation suggesting that the regulation of wnt proteins is a possible avenue through which clinicians can address ischemic heart injuries61. a signi�cant limitation of this �nding is the lack of comprehension of the particular pathways that the inhibition of wnt6 a�ects, and what factors or genes are involved. figure 5. the role of the wnt pathway in β-catenin production. sfrp2 binds to wnt6 proteins to activate non-canonical wnt pathway that results in cpc differentiation and leads to a reduction of β-catenin responsible for proliferation. the �nding of pathways related to cardiac cell induction and regeneration has demonstrated a possibility of manipulating these processes to address berkeley pharma tech journal of medicine | 20 heart injury. in the case of hepcidin proteins, hamp de�ciency has shown to be associated with iron-homeostasis and the secretion il-4/il-13 that help with cardiomyocyte renewal. cdmg molecules and sfrp proteins were proven to be related to the inhibition of wnt pathways that reduce cell proliferation but result in di�erentiation. however, for both cases, the pathways resulting in improved heart function are still not well-understood and just provide a glance of future research directions. there is, thus, signi�cant room for elaboration; further studies may implicate more speci�c cell or gene therapies for treating ischemic heart diseases. conclusion mesenchymal stromal cells have been a popular and desirable candidate in treating function post-myocardial infarction, as they have shown promising e�ects in clinical trials. the administration of mscs have shown signi�cant therapeutic bene�ts contributing to patients su�ering from myocardial infarction and other pulmonary and cardiac myopathies. although more extensive and larger-scale clinical trials need to be implemented to exactly understand the mechanism and exact bene�ts of msc administration, the existing studies have shown results indicating high e�cacy. studies have shown that through both uc-mscs, patients treated with infusion of mscs have shown outcomes which include increased myogenesis and angiogenesis, improved left ventricular ejection reaction, reduced infarct sizes in heart tissue, cardiomyocyte proliferation, and generally improved cardiac function. the di�erence in msc types and their correlating bene�ts and consequences are also discussed, as well as the safety and feasibility of their administration onto experimental cohorts. these studies also showed little to no adverse e�ects to any of the treatments, and little signi�cant worsening of conditions has been recorded. the exact mechanism of their bene�cial e�ect is still unknown and needs to be more extensively studied, but these studies have shown promising therapeutic bene�ts. modi�ed mrna also demonstrates great potential to be used towards cardiac regeneration, especially expressing vegf, as it has expressed no adverse reactions when used clinically, and successfully promoted angiogenesis. developments have been made in the puri�cation and evading the immune system through rna caps, pseudo nucleotides, and the poly a berkeley pharma tech journal of medicine | 21 tail. however, more research must be done speci�cally on the ischemic injury and cardiac regeneration model for it to be used speci�cally in response to a myocardial infarction. before this, an understanding of the e�ect of vegf on humans, and increasing e�ciency of the treatment is imperative, as there is very little data on optimal dosage concentration or repetitions that carry out the desired e�ects. lastly, for modi�ed mrna to be a viable clinical option, the shelf life and sustainable storage of the puri�ed mrna in bu�er must be researched to ensure minimal degradation over time. thus, modifying mrna shows great potential as a gene therapy mechanism, though more research must be done to adapt it to be used for human cardiac regeneration. icms derived from various sources have been employed in numerous treatment approaches to cardiac illness, and continue to provide many research opportunities in the �eld of regenerative medicine. as observed by the evolution of icm research over time, studies have made their way from animal models to the use of human cell sources for experimentation, suggesting that application of icm-based treatment in clinical trials is not far o�. icms provide a treatment option that has the potential to draw on a abundantly-sourced treatment platform, avoid the negative side-e�ects of toxic pharmacological treatments, and signi�cantly expand the scope of regenerative medicine. continued research into icms seems to be a very promising path to treating ischemic injury induced by cardiac dysfunction. particular genes and protein interactions related to the heart have resulted in a greater insight into cardiac regeneration to address heart injuries. cardiac hepcidin protein reduction through hamp gene de�ciency was shown to be associated with iron-homeostasis and the secretion of il4/il13 from macrophages inducing cardiomyocyte regeneration. the inhibition of pathways related to wnt proteins using cdmg and sfrp molecules also revealed that cardiac progenitor cell di�erentiation is a possible metric with which to gauge improvement in heart injuries. however, these studies are recent research interests that have only been implemented in animal models. the particular genes, reactions, and factors involved in these pathways are still not well-understood. while there is a long way to go for a greater comprehension of how they work in the human heart, the identi�cation of berkeley pharma tech journal of medicine | 22 these pathways and possible molecules provides an insight into future research directions that could potentially result in novel gene therapies. a b c d e berkeley pharma tech journal of medicine | 23 figure 6. overview of amount of research articles pertaining to each therapeutic approach. to obtain a broad overview of how cardiac ischemic injury has been approached in research, a pubmed search of the key terms “gene therapy + cardiac regeneration + ischemic injury” was conducted on may 6, 2022. the first one hundred relevant articles were cataloged and analyzed for the specific research topic that was covered. panel (a) shows the number of research articles that appeared by the topic overall, and panels (b)-(f) show the number of articles per topic by year of publication. “genetic research without specified method” refers to work done with specific genes that did not mention the mscs, modrna, or icms. “other therapies” refers to therapies used to treat cardiac ischemic injury that did not involve genetics, such as cell therapy or drug therapy. “multiple therapies” refers to articles that address more than one form of therapy. research in the �eld of gene therapy to alleviate cardiac ischemic injury has been studied for decades and will continue to be studied in coming years. as the current trends in the 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2015;85:215-225. doi:10.1016/j.yjmcc.2015.06.003 berkeley pharma tech journal of medicine | 29 front_page_bhagwat chu the therapeutic potential of synthetic and natural cannabinoids in treating alzheimer’s disease front page berkeley pharma tech journal of medicine correspondence nidhirkan@gmail.com keywords alzheimer's disease amyloid-beta cannabinoids tau protein hyperphosphorylation ∆9-tetrahydrocannabinol submitted june 28, 2021 accepted july 6, 2021 published december 17, 2021 full open access creative commons attribution license 4.0 abstract alzheimer’s disease (ad) is one of the most prevalent and debilitating neurodegenerative diseases in the world, highlighting the need for research on novel treatments and therapies. previous studies have found that the body’s endocannabinoid system (ecs) interacts closely with its neural system, making it a potential avenue for the treatment of neurological disorders. one hallmark of ad is the accumulation of amyloid-beta (aβ) in the brain and its potentially detrimental effects on cognitive function. cannabis-based drugs have been observed to regulate aβ modifications and inhibit ad progression. furthermore, cannabinoids have been noted to reduce inflammation and neurotoxicity. synthetic cannabinoids were able to rescue memory deficits and neurodegeneration, and reduce immunoreactivity. similarly, natural cannabinoids like ∆9-tetrahydrocannabinol (thc) and cannabidiol (cbd) demonstrate therapeutic potential by interacting with the cholinergic system, and reversing the symptoms of ad. although further research and testing are needed, it is evident that the use of cannabinoids shows promise for future treatment in ad patients. the therapeutic potential of synthetic and natural cannabinoids in treating alzheimer’s disease by: nidhi kanchumarthi, avigail rockland, thejaswini vembar, elaine liu, rithvik kanaad, erick torres hinojosa, kira tiv, benjamin chen, shira hao, sri guttikonda, and ramiro garza i. introduction previous studies have concluded that the endocannabinoid system (ecs) is disrupted by many neurodegenerative disorders (di marzo et al., 2015; fagan, 2014; paloczi et al., 2018). the goal of this paper is to comprehensively review and outline the most recent data around the pathophysiology of the ecs as it relates to alzheimer’s disease (ad), as well as to investigate the e�cacy of natural and synthetic cannabinoids as a therapeutic option. even as medicine advances and life expectancy increases, ad is still a prevalent issue. with increased prevalence comes the need for new and e�ective therapies. the endocannabinoid system shows promise, serving as both a neuromodulator and immunomodulator. cannabis sativa has been studied for its potential neuroprotective qualities and its role in attenuating nociception in several medical contexts, including cancer, psychiatric disorders, and epilepsy. its primary active components, ∆9-tetrahydrocannabinol (thc) and cannabidiol (cbd), interact with the endocannabinoid signaling system through the endocannabinoid cb1 and cb2 receptors. stimulation of these receptors by various agonists has also successfully reduced the chronic neuroin�ammation associated with ad by interfering with the di�erent neural pathways. our goal has been to identify the mechanisms by which the reduction in neuroin�ammation is accomplished. accumulation of aβ peptide and hyperphosphorylated tau protein in the brain play an important role in the onset and progression of alzheimer's disease (ad) (farkhondeh et al., 2020). however, recent research has suggested that aβ is no longer considered the primary cause, which was the accepted belief for many years (cubinkova et al., 2018). nevertheless, the ecs o�ers many therapeutic possibilities for targeting these aβ deposits. many studies support the �nding that elevated levels of the endocannabinoid 2ag, coupled with the modulation of the common cannabinoid receptor cb2, could reduce aβ plaque accumulation (paloczi, 2018). while aβ may not be the primary cause of ad, studies have shown that these deposits are detrimental to healthy synapses (shankar et al., 2008), decreasing long-term potentiation and increasing long-term depression. sometimes, this neural damage is permanent, but the use of cannabinoids to stimulate the ecs has shown some success in preventing this kind of damage. berkeley pharma tech journal of medicine | 2 pathology of the cholinergic system is also associated with ad and thus has been further investigated as a therapeutic target. many cholinergic pathways function in conscious awareness, attention, and working memory, which are areas commonly found to be damaged or dysfunctional in those who su�er from ad. research by k.j. thompson suggests that interacting signals between the endocannabinoid and cholinergic systems may be a way to improve cholinergic signaling and repair synaptic plasticity that is damaged as a result of ad (thompson et al., 2020). we then investigate the inhibitory activity of various receptor agonists of the ecs, both natural and synthetic. this section looks at cbd and thc as receptor agonists and concludes that cbd plays a role in the inhibition of neural apoptosis while thc is active in the inhibition of acetylcholinesterase (ache), which serves to improve the reduced cholinergic signalling associated with ad. both agonists seem to play a part in the reduction of aβ build-up, but the exact pathways still need to be elucidated. some of the most e�ective and well-researched synthetic agonists of the ecs include dronabinol, nabilone, win55,212-2, and jwh-015. dronabinol and nabilone were found to be particularly e�ective in treating the neuropsychiatric symptoms (nps) associated with ad, while the others primarily function in reducing aβ plaques and neuroin�ammation. our review presents published research that supports the e�cacy of both natural and synthetic cannabinoids as a potential treatment for ad. this introduces a potential bias, as studies which conclude positive results and relationships are more likely to be published than those with negative results. nevertheless, this research highlights the potential bene�ts that cannabinoid therapy could provide and recommends that it be studied further. ii. neural pathway a. amyloidogenic pathway accumulation of aβ peptide and hyperphosphorylated tau protein in the brain are the leading sources of the onset and progression of ad (farkhondeh et al., 2020). when amyloid precursor protein (app) is activated, it is cleaved by other proteins, which leads to the production of small aβ fragments. this induces an increased production and aggregation of aβ peptide in limbic and association cortices. as a result, the altered neuronal homeostasis and oxidative injury provoke neuro�brillary tangle formation. these tangles are a characteristic trait of ad brains and lead to berkeley pharma tech journal of medicine | 3 neuronal loss and cognitive impairment (hall et al., 2001; nelson et al., 2012). fig. 1: the process of neuro�brillary tangle formation induced by amyloid plaque buildup. as the amyloid precursor protein (app) is activated, amounts of aβ are produced. this excess aβ leads the neuro�brillary tangle formation, as seen in ad a�ected patients. cbd, a cannabis derivative devoid of psychotropic e�ects, has been shown to interfere with several aβ-triggered neurodegenerative pathways (scuderi et al., 2014). studies found that cbd led to the downregulation of genes linked to ad, including genes coding for the kinases responsible for tau phosphorylation and for the secretases involved in aβ generation. pre-treatment with cbd prevented the expression of proteins potentially involved in tau phosphorylation and aβ production in gingiva-derived berkeley pharma tech journal of medicine | 4 mesenchymal stem cells (gmscs) (libroet al., 2016). in one study, the role of cbd was investigated as a modulating compound of app processing in shsy5y(app+) neurons. in addition, the putative involvement of pparγ was explored as a candidate molecular site responsible for cbd actions. the results indicated that cbd is capable of inducing the ubiquitination —the degradation —of app protein, leading to a decrease in app full length protein levels in shsy5y(app+) and a consequent decrease in aβ production. cbd also promoted an increased survival of shsy5y(app+) neurons by reducing their long-term apoptotic rate (scuderi et al., 2014). in conclusion, research indicates that cannabis-based drugs inhibited the progression of ad by regulating aβ modi�cations (farkhondeh et al., 2020). while the aggregation of aβ plaques has been the leading hypothesis in the onset ofad for several years, recent research has suggested alternative hypotheses. a number of drugs targeting aβ, such as aducanumab — an antibody drug that binds to insoluble and soluble aβ plaques to reduce its levels in the brain — were successful in slowing the rate of cognitive function decline in patients with mild ad, but not in treating the root cause. one hypothesis suggests that ad is a synaptic disease. it has been argued that synaptic impairment is an early event in neurodegenerative processes during ad and that synaptic loss and failure corresponds with cognitive decline in ad as well (cubinkova et al., 2018). recently, it was shown that impaired synapses in ad brains released tau protein in response to potassium chloride (kcl) stimulation, suggesting that it may be involved in the progression of tau in the brain (cubinkovaet al., 2018). in addition to the synaptic hypothesis, some researchers believe that alpha-synuclein acts as a pathogenic modulator in ad. alpha-synuclein is a protein that localizes to the nerve terminal and regulates neurotransmitter release in the presynaptic neuron. it was found that levels of soluble alpha-synuclein are about two-fold higher in the brains of ad patients than in control brains and correlate better with cognitive impairment (cubinkova et al.,2018), suggesting that overexpression of the alpha-synuclein protein may play a role in the onset of ad. even in light of these alternative hypotheses, most research focuses on the e�ects that cannabinoids have on the production of aβ plaques as a measure of treating ad. berkeley pharma tech journal of medicine | 5 b. endocannabinoid signaling the endocannabinoid system regulates brain function as well as immune system activity, thus allowing it to serve as a therapeutic strategy for ad. its dual ability as a neuromodulator and immunomodulator is due to the speci�c locations in which cannabinoid receptors type 1 (cb1) and type 2 (cb2) gather (bonnet & marchant, 2015). the components of the endocannabinoid system primarily include two endocannabinoid signaling molecules, 2-arachidonoyl glycerol (2ag) and anandamide (aea), and their g-protein coupled receptors, cannabinoid receptor type 1 and cannabinoid receptor type 2 (fagan & campbell, 2014). the endocannabinoid system works speci�cally in both the regulation of neuroin�ammation as well as neurogenesis.studies show that the endocannabinoid system is able to reduce chronic neuroin�ammation, a major mediator in neurodegeneration in alzheimer’s patients. ppar𝛾 is a key component involved in the cannabinoid anti-in�ammatory e�ect. this protein consists of a group of hormone receptors involved in gene expression and in�ammation. the use of cannabinoids increases ppar𝛾transcriptional activity in adult rats, thus decreasing neuroin�ammation (fagan & campbell, 2014). fig. 2: illustration and description of cb1 & cb2 receptors, and the endocannabinoid signaling molecules, anandamide (aea) and 2-arachidonoyl glycerol (2ag). an elevation of the 2ag endocannabinoid signaling molecule, coupled with a modulation of the cb2 speci�c cannabinoid receptor, could combat aggregated aβ neuritic plaques (palocziet al., 2018). the cannabinoid receptors play a vital role in allowing the formation of new neurons and neuronal connections to sustain neuronal function. the proliferation of neural precursor cells, both neural stem cells and neural progenitor cells, has been proven to activate cb1 receptors; the proliferation of neural berkeley pharma tech journal of medicine | 6 progenitor cells has been seen to activate both cb1 and cb2 receptors, allowing the production and survival of new neurons (fagan & campbell, 2014). this activation of neurogenesis o�ers promising potential within the endocannabinoid system in reducing brain atrophy in alzheimer patients (fagan & campbell, 2014). a potential therapeutic component of the endocannabinoid system involves the cannabinoid receptors cb1 and cb2. cb1 receptors, the most abundant cannabinoid receptors,are mainly located in the neurons of the central nervous system and work to regulate cognition and memory, motor control, feeding, and pain perception — allowing the endocannabinoid system to serve as a neuromodulator. studies on cb1 receptors have yielded inconsistent results,preventing a correlation between cb1 presence in ad patients and in control patients from being observed (ahmad et al., 2014). a more recent study has indicated a reduction in the number of cb1 receptors in the frontal cortex while no change was observed in the hippocampus. cb2 receptors, on the other hand, are expressed mostly in immune cells and to some extent, in the peripheral system of the brain — allowing the endocannabinoid system to serve as an immunomodulator. as such, cb2 is highly involved in the research regarding the neuroin�ammatory response associated with ad (atakan, 2012). within the endocannabinoid system in an alzheimer’s patient’s brain, studies found an increase in cb2 receptor expression in microglia, phagocytic cells in the brain and spinal cord. this increase correlates with aβ levels and plaque accumulation, suggesting that the activation of cb2 receptors plays a role in stimulating aβ removal (talarico et al., 2019). the use of cb2 receptors has shown to be a promising medium for both a therapeutic treatment and as a marker for the advancement of alzheimer’s disease. in addition, cb2 receptors have continuously been co-localized with aβ plaque buildup (ahmad et al., 2016). despite this, there have been �ndings that demonstrated cb2 receptor concentration is highly expressed within microglia cells surrounding senile plaques — extracellular deposits of aβ — within post-mortem brainsdiagnosed with ad (aso & ferrer, 2016). conversely, studies demonstrated that cb2 receptors may not be the most accurate biomarker for tracking ad advancement, as they are also found on neuronal cells. however, this does not necessarily eliminate them as targets for treatment as they remain largely expressed within immune cells (ahmad et al., 2016). as such, the signi�cant presence of cb2 receptors in microglia cells in brains of ad patients allows for selective activation(stimulating certain cb2 receptors to decrease in�ammatory cell generation) in a�ected berkeley pharma tech journal of medicine | 7 tissues,therefore decreasing the risk of harmful e�ects (ramírez et al., 2005). fig. 3: illustration of the interaction between endocannabinoids and the cannabinoid receptors, cb1 and cb2. endocannabinoids from the postsynaptic neuron travel through the synaptic cleft to the presynaptic neuron, thus attaching to the cannabinoid type 1 receptor. the binding of these two molecules results in a suppression of neurotransmitters altering the neuronal �ring in the postsynaptic cell, thus allowing it to work as a regulator. one avenue of therapeutic treatment is the usage of cb2 activity against in�ammation. within microglia cells, cb2 receptors inhibit neurotoxicity caused by microglia by substantially reducing the production of pro-in�ammatory molecules and by manipulating macrophage migration(aso& ferrer, 2016). "this was supported by experiments utilizing receptor agonists within in vitro experiments containing di�erent species of toxic aβs, such as jwh-015, jwh-133, hu-308, win55,212-2, and hu-210, which observed a reduction of in�ammatory molecules” (aso & ferrer, 2016). it has been postulated that these cb2 receptor agonists aid in inhibiting microglial activation through decreasing intracellular calcium concentration (martín-moreno et al., 2011). in essence, the utilization and subsequent exploitation of the endocannabinoid system provides an essential medium for the therapeutic treatment of alzheimer’s disease. berkeley pharma tech journal of medicine | 8 the endocannabinoid system has shown to reduce in�ammatory response through thepparγ protein as well as facilitate neurogenesis through the activation of both the cannabinoid receptors, when neural stem cells and neural progenitor cells are proliferated. the self-renewal capability and multipotency of stem cells is supported through the complex microenvironment provided through the action of the endocannabinoid system, speci�cally cannabinoid receptors cb1 and cb2 (rodrigues et al., 2019). the prospect of neural stem cells as regenerative therapies is very promising, leading to a possible personalized and e�ective approach (rodrigueset al., 2019). moreover, the various cannabinoid actions on neural stem cells open paths of research to uncover the mechanisms responsible behind cannabinoid e�ects. new avenues of research could provide new knowledge, leading to the development of re�ned therapeutic strategies to alleviate alzheimer’s e�ects. c. synaptic plasticity among the neural pathways negatively impacted by ad are those involved in synaptic plasticity. the brain’s circuitry is heavily a�ected by numerous daily activities ranging from learning new information to engaging in social interactions. these exposures to the environment have the potential to modify the brain’s neural organization and in�uence its activity. synaptic plasticity is the phenomenon that describes these changes in the neural circuits’ structure and function, which result from modi�cations to synaptic transmission. although synaptic plasticity may allow a damaged brain to restrengthen synaptic connections and reverse damage, ad is known to impair this plasticity and leave permanent damage. research has demonstrated that aβdeposition, a widely recognized hallmark of ad pathophysiology, is detrimental to healthy synapses (shankar et al., 2008). soluble dimeric aβ assemblies were found to be strong inhibitors of long-term potentiation —the strengthening of synaptic communication by repeated stimulation (shankar et al., 2008). the soluble aβ assemblies were also found to augment long-term depression causing a decrease in dendritic spine density. dendritic spine density has been found to strongly correlate to the degree of dementia for patients with ad. speci�cally,the research showed that ad brain samples had a 47% decrease in spine density when compared to a control (shankar et al., 2008). the researchers localized various receptors, includingn-methyl-d-aspartate receptors (nmdar), metabotropic glutamate receptors (mglur), and nicotinic acetylcholine receptors(nachr) as among those impacted by aβ and negatively a�ecting berkeley pharma tech journal of medicine | 9 synaptic plasticity in diseased brains. studies con�rmed that the introduction of soluble aβ dimers into rats interrupted their ability to recall learned behaviors, further implicating aβ as the primary culprit responsible for impaired synapse remodeling and plasticity (shankar et al., 2008). fig. 4: in a research study conducted by hughes and herron, e�ects of long-term potentiation (ltp) of cbd was investigated in the hippocampus. treatment of slices with cbd attenuated the aβ-mediated de�cit in ltp. ltp was signi�cantly attenuated in the presence of aβ1–42 (500nm) (112.6±2.9%;n=23) compared to control (148.6±2.4%; n=59; aandc). b application of cbd to slices 30 minutes prior to addition of aβ attenuated theaβ-mediated de�cit in ltp (136.9±5.9%; n=15, p≤0.001) compared to aβ alone. ltp in the presence of cbd and aβ was similar to control (p>0.05). citation: cannabidiol reverses de�cits in hippocampal ltp in a model of alzheimer’s disease. (n.d.). neurochemicalresearch. https://link.springer.com/article/10.1007/s11064-018-2513-z synaptic plasticity can be modulated through the interaction between cbd and the endocannabinoid system. while the exact mechanism must still be elucidated, it is clear that cbd in�uences and interacts with endocannabinoid receptors. various studies have arrived at di�erent conclusions on the actions of cbd, some stating that there is no a�nity between cbd and the cb1/cb2 receptors (mcpartland, 2007). others state cbd binds as a weak antagonist (thomas, 2007), and yet more that claim cbd functions as a negative allosteric modulator for the endocannabinoids 2-ag and delta-9-thc (hughes et al., 2019). cbd enhances neuroprotection by di�erent signal transduction pathways that are controlled indirectly by cannabinoid receptors (li et al., 2020). similarly, cbd treatment enhanced synaptic transmission in mouse models, most likely acting in conjunction with cb1 and cb2 receptors to decrease neurotoxicity and cell death that was the result of aβ deposits. cbd has shown its capability in reversing and preventing cognitive damage due to ad (watt et al., 2017) and precluding the suppression of long-term berkeley pharma tech journal of medicine | 10 https://link.springer.com/article/10.1007/s11064-018-2513-z potentiation (hughes et al., 2019). therefore,modulation of the agonists and antagonists which bind to cb1 and cb2 receptors, such as cbd, has an e�ect on synaptic plasticity in the brain. ad-induced dysfunction of the endocannabinoid receptors negatively in�uences synaptic plasticity. fig. 5: illustration of synaptic plasticity and long-term potentiation. after repeated simulation and long-term potentiation, there is an increase in the number of neurotransmitters in the presynaptic neuron and an increase in the number of receptors in the postsynaptic neuron. this results in a stronger response. pathology of the cholinergic system is also associated with ad. many cholinergic pathways are commonly found to be damaged or dysfunctional in those who su�er from ad, impairing synaptic plasticity. ad has been correlated with decreased cholinergic transmission, especially in the hippocampus where new memories are formed. studies have found ‘crosstalk’ — signals within one system that produce changes in another system — between the cholinergic signaling and the endocannabinoid signaling systems (thompson et al., 2020). past studies speci�cally investigated this communication between the systems in ad. this research found that α-7 nachr are of particular concern because they are highly expressed in the hippocampus and play a signi�cant role in the development of memory and learning abilities (thompson et al., 2020). elevated levels of α-7 nachr have been found to disrupt normal signaling and negatively impact synaptic plasticity (thompson et al., 2020). interestingly, aβ deposition also seems to increase in α-7 nachr rich regions (thompson et al., 2020). while their research did not corroborate a de�nite ‘cross-talk’ between the ecs and α-7 nachr speci�cally, they did �nd that α-5 nachr and α-6 nachr correlated to thc addiction and withdrawal (thompson et al., 2020), which are known to involve the pathways of the berkeley pharma tech journal of medicine | 11 ecs. the researchers concluded that the ecs seemingly plays a role in cholinergic signaling, although not speci�c to the α-7 nachr.this research suggests that indirect crosstalk between the endocannabinoid and cholinergic systems and its modulation may be a way to improve cholinergic signaling and repair synaptic plasticity that is damaged as a result of ad (thompson et al., 2020). iii. receptor agonists a. inhibitory activity by receptor agonists many studies have investigated the variety of proposed therapeutic treatments mediated by endocannabinoid receptor agonists, speci�cally the inhibitory activity of receptor agonists in alzheimer’s disease. one of the primary focuses of cannabinoid receptor agonists is their ability to inhibit neuroin�ammation, a speci�c characteristic signaled by the dysfunction of microglia. cannabinoid agonists increase endocannabinoid availability, which allows activation of receptor agonists to prevent aβ-induced cognitive de�cits (martín-moreno et al., 2011). the synthetic cannabinoid, win 5512-2, inhibits neuroin�ammation induced by aβ through the cb1 and cb2 receptors. in primary cultures, a laboratory procedure in which cell extracts are grown under controlled conditions, win5512-2’s in�ammatory response speci�cally prevented cell death in astrocytes — specialized glial cells that aid in a variety of neurological functions such as water homeostasis and oxidative stress defense (sofroniew, 2010). mice studies have e�ectively demonstrated this by reducing the levels of the proin�ammatory molecules interleukin-1 beta (il-1beta), tnf-alpha, cox-2, and inducible nitric oxide synthase (inos) (aguirre-rueda, 2015) within their system. additionally, two proin�ammatory cytokines, tnf-alpha and il-6, were measured in the cerebral cortex of mice treated with cbd and win5512-2 (martín-moreno et al., 2011). the initial 6-fold increase of il-6 from the aβ was decreased by both cannabinoid agonists, and win5512-2 partially reduced tnf-alpha gene expression (martín-moreno et al., 2011). in a similar experiment, aβ injected mice subject to subchronic administration of win5512-2 or cbd showed better performance in the morris water maze task, an experiment in which a mouse is placed within a circular pool of water and must escape from the water onto a hidden platform whose location is discerned through the use of spatial memory (martín-moreno et al., 2011). berkeley pharma tech journal of medicine | 12 cbd is a cb2 receptor agonist that also prevents aβ neurodegeneration by reducing microglial activation responsible for the release of toxic molecules like nitric oxide (no) and proin�ammatory cytokines (martín-moreno et al., 2011). by inhibiting calcium responses in glial cells, cannabinoid agonists are similarly able to prevent microglial activation. fig. 6: illustration of inhibitory activity by receptor agonists. additionally, cb1 receptor activation causes migration of the n13 (nitrogen-13) microglial cells and primary microglial cells (martín-morenoetal., 2011). migration is a cellular mechanism that ultimately allows for the removal of deposit aβ protein. in a study conducted in aβ injected mice, those that did not receive the cbd treatment of win5512-2 showed signi�cant reduction in their ability to reach a hidden platform (martín-moreno et al., 2011). another strength berkeley pharma tech journal of medicine | 13 pertaining to the inhibitory activity of receptor agonists is the suppression of aβ plaque buildup. aspreviously mentioned, ad has been largely characterized by its substantial aggregation of beta amyloid peptide clusters; thus, focusing further investigation towards the wingless-related integration site (wnt) pathway as it becomes largely impacted due to aforementioned clusters (esposito, 2006). normally, wnt activation triggers the “...inhibition of glycogen synthase kinase-3beta (gsk-3β), a multifunctional phosphorylating serine/threonine kinase and relative accumulation of [unphosphorylated] β-catenin in the cytoplasm” (esposito, 2006). this aggregation allows for the expression of genes that code for homeostasis and neuronal survival (esposito, 2006). within neurons exposed to aβ peptides, however, gsk-3ß is activated through phosphorylation; therefore, wnt signaling experienced signi�cant reduction (esposito, 2006). furthermore, these phosphorylated gsk-3ß lead to the neuro�brillary tangles and signi�cant tau protein hyperphosphorylation witnessed in the brains of patients with ad (esposito, 2006). cbd serves as a rescue of the wnt signaling pathway as it promptly reduces the accumulation of phosphorylated gsk-3ß, consequently inhibiting the rise of neuro�brillary tangles and tau protein hyperphosphorylation (esposito, 2006). furthermore, cbd indirectly inhibits neural apoptosis due to its rescue of the wnt signaling pathway (esposito, 2006). ∆9-tetrahydrocannabinol (thc) is another viable receptor agonist in combating the advancement of alzheimer’s disease via competitive inhibition. thc, the active component of marijuana, competitively inhibits acetylcholinesterase (ache) in addition to preventing acetylcholinesterase-induced aβ plaque buildup (eubanks, 2006). one study, conducted by eubanks et al., demonstrated through computational modeling of thc-ache interaction that thc binds to ache in a critical region involved in amyloid synthesis, ultimately serving an inhibitory function (eubanks, 2006). thc and analogous compounds were found to augment acetylcholine levels by inhibiting aβ aggregation and reducing neurotransmitter degradation(eubanks, 2006).this study proposes a possible mechanism by which thc molecules directly impact the pathogenesis of alzheimer’s disease (eubanks, 2006). through focusing on the cholinergic system, thc was found to be a promising receptor agonist as its potential ache inhibition might be implicated in ad treatment (campbell et al., 2007). multiple drugs — donepezil, edrophonium, galantamine, etc. — inhibit ache for the purpose of increasing acetylcholine (ach) levels in the synapse and increasing cholinergic berkeley pharma tech journal of medicine | 14 transmission. in an investigation of cannabinoids as potential inhibitors of ache, δ-9-thc was found to be a competitive inhibitor for ache, which has the added e�ect of inhibiting aβaggregation (ache has been found to accelerate the aggregation of aβpeptides into complexes, increasing their neurotoxicity) (campbell et al., 2007). nonetheless, more research is needed in order to understand the e�cacies of cbd and thc in the treatment of alzheimer’s disease. as therapeutic treatments for alzheimer’s disease continue to be explored, cbd is a promising candidate. a handful of studies show that thc is a more e�ective inhibitor than cbd in regards to amyloid plaque buildup. while the general consensus of the present community is built upon the e�ects of cannabidiol, the e�cacy of ∆9-tetrahydrocannabinol should also be considered as a viable method of treatment for alzheimer’s. it is nearly impossible to ignore the therapeutic potential of receptor agonists within the endocannabinoid system against the advancement of alzheimer's disease. b. synthetic cannabinoid receptor agonists synthetic cannabinoid receptor agonists target the degenerative e�ects of alzheimer’s through the cb1 and cb2 receptors of the ecs. these receptor agonists can reduce the density of neuritic plaques by inhibiting acetylcholinesterase activity, or increasing expression of neprilysin, an enzyme in the aβ degradation cascade (fernández-ruiz et al., 2015). the syntheticcannabinoids also function as a treatment that blocks the activation of the microglial clustercaused by the deposition of aβ at the senile plaque. this deposition is typically responsible for the prolonged damaging e�ects of the disease, and the presence of cannabinoids alleviates neurodegeneration (ramírez et al., 2005). since these potential neuroprotective e�ects have been discovered, extensive research is being dedicated towards studying the endocannabinoid system. researchers have already found that dysfunction of cb1 and cb2 receptors and in endocannabinoid signaling plays a speci�c role in the pathophysiology of ad (liu et al., 2015). one study investigated the speci�c bene�ts that synthetic cannabinoids could have on neuropsychiatric symptoms (nps) associated with ad. the researchers studied two synthetic analogs of δ-9-thc, dronabinol and nabilone, and their interactions with receptors in the ecs. nabilone and dronabinol were berkeley pharma tech journal of medicine | 15 found to be more potent analogs to thc that act on both cb1 and cb2 receptors and reduced overall agitation in ad patients (liu et al., 2015). participants receiving nabilone experienced no adverse side e�ects. in the dronabinol study, the researchers measured disturbed behavior in study participants using the cohen-mans�eld agitation inventory (cmai), a scale which systematically assesses patient agitation. they found that with administration of the synthetic cannabinoid, disturbed behavior dropped among the participants over the course of the 6-week test period (liu et al., 2015). stimulation of the cb2 receptor by these synthetic cannabinoids increases the removal of aβ deposits by enhancing macrophage activity, thus explaining the improvements in nps seen in study participants. another studyinvestigating the e�cacy of nabilone as a potential treatment for the nps of ad similarly found an overall decrease in agitation, and sometimes aggression as well (ruthirakuhan et al., 2019). the synthetic cannabinoid receptor agonists win55,212-2 and arachidonyl-2-chloroethylamide were also found to decrease aggressive behavior associated with ad and reduce tau hyperphosphorylation and the neuro�brillary tangles (liu et al., 2015). win55,212-2 along with jwh-133 were also found to assist in the removal of aβ deposits, one of the most common pathophysiology associated with alzheimer’s. win55,212-2’s ability to stimulate neurogenesis makes it a great contender as a therapeutic agent. although there are positive outcomes of such drugs on reversing ad-related amnesia, they must be used with caution as there is a risk of exacerbating the neurodegeneration associated with ad (liu et al., 2015). agonists that speci�cally target cb2 activate responses that reduce both in�ammatory and aβ plaque buildup. the cb2 agonist jwh-015 was used to further investigate the function of cb2 with tests done on ad human tissue. these tests demonstrated that jwh-015 was able to reduce plaque buildup dramatically in thp-1 macrophage cells (tolón et al., 2009). however, the u373 astrocytoma cells, which are from the astrocytoma cancer cell line in the human brain, were immune to the e�ects (tolónet al., 2009). thus, the cb2 agonist induced plaque removal was only e�ective in certain cells (the human macrophages). this �nding shows some limits of cb2 agonist use in alleviating symptoms, while narrowing down a line of cells to focus on. cb2 receptors' primary role in plaque removal, with in vitro injection of the antagonist sr144528, showed partial reversal of the jwh-015 aβ removal (tolón et al., 2009), which highlights other factors at play in plaque reduction. these results indicate that jwh-015 can be used berkeley pharma tech journal of medicine | 16 as a speci�c target treatment for ad, however it is not the only option. cb2 receptor agonists have a preventative function in suppressing neuroin�ammation (ehrhart et al., 2005). irregularly stimulated cd40 receptor expression by the signaling pathway ifn-γ is prominent in patients with ad and is known to increase in�ammation (ehrhart et al., 2005). cd40 induction produces in�ammatory cytokines and is correlated with the in�ammation and aβ peptide increase in ad. using the jwh-015 agonists, stimulation of cb2 diminishes ifn-γ-induced cd40 expression in microglial cells (ehrhart et al., 2005). this occurs by the intervention of the jak/stat1pathway (ehrhart et al., 2005), which is a pathway that is involved with immune system response and ultimately decreases ifn-γ-induced cd40 expression. there is further evidence to show thatcb2 agonists have the potential to inhibit in�ammation. jwh-133, a cb2 agonist, was found to rescue spatial memory de�cits in rats injected with okadaic acid (oka), which induces spatial memory impairment and neurodegeneration (çakır et al., 2019). rats injected with oka were found to have higher numbers of degenerative neurons in the cortex and hippocampus, but the addition of jwh-133 ameliorated this damage (çakır et al., 2019). jwh-133 application was found to reduce the level of immunoreactivity caused by oka. caspase-3, aβ, and il-1β immunoreactivity were reduced in both the hippocampus and the cortex, while tnf-αimmunoreactivity was reduced in only the hippocampus (çakır et al., 2019). 1-((3-benzyl-3-methyl-2,3-dihydro-1-benzofuran-6-yl) carbonyl) piperidine (mda7), a highly-selective cb2 agonist, was found to have a neuroprotective e�ect in in vivo and in vitro models (wu et al., 2012). mda7 administration in aβ1–4-induced mice was found to lower immunoreactivity in the hippocampus (wu et al., 2012). the morris water maze test (wu et al., 2012) showed mda7 rescued amyloid �bril-impaired performance. while progress has been made in the development of drugs targeting these receptors, the ones currently available to patients come with many unwanted side e�ects such as nausea, vomiting, and weight loss (wattet al., 2017). while there are bound to be side e�ects to most drugs, synthetic cannabinoids o�er the added bene�t of producing e�ects similar to that which natural cannabis derivatives cause but without the psychoactive side e�ects (campbell et al., 2007). due to the complexity of ad and the pathology of many di�erent pathways involved, it is unlikely that just one drug will su�ce to treat the progression of the disease. rather, berkeley pharma tech journal of medicine | 17 it is more likely that multiple drugs will be needed to treat the multifaceted symptoms of alzheimer’s. iv. potential and e�ective therapies a. natural therapies cannabinoids possess great potential as e�ective natural therapies for ad. previous studies indicate that cannabis use diminishes symptoms associated with ad (suryadevara et al., 2017). when administered in patients with similar neurodegenerative conditions, cannabis use is shown to decrease pain and spasticity in people with multiple sclerosis, decrease tremor, rigidity, and pain in people with parkinson’s disease, and improve the quality of life of amyotrophic lateral sclerosis (als) patients by improving appetite and decreasing pain and spasticity (suryadevara et al., 2017). interestingly, a large portion of the negative side e�ects from cannabis administration in these diseases is absent in alzheimer’s patients (suryadevara et al., 2017). cannabis has been used to alleviate pain and muscular contractions to target the in�amed pathways of the brain in alzheimer’s patients. the perception of pain is atypical in that alzheimer’s patients are more prone to oxidative stress, which is an imbalance between free radicals and the body’s ability to detoxify them. however, studies demonstrate a positive relationship between stress reduction in dementia and medical cannabisuse (aso et al., 2014). the anti-in�ammatory and anti-neurotoxicity properties of cannabinoids help mediate the neuroin�ammation caused by microglia activation, which is characteristic of chronic pain in alzheimer's patients. in addition, further studies have shown that with the use of cannabinoids, alzheimer’s patients have experienced a decrease in altered cognitive ability (aso et al., 2014). research indicates that a "coadministration of cbd and δ9-thc” is necessary in order to deliver the bene�cial elements of cannabiswhile attempting to reduce certain side e�ects of δ9-thc in alzheimer’spatients (giacoppo et al., 2014). cbd and thc, natural components in cannabinoids, provide encouraging results for possible use in alzheimer’s patients. the phytocannabinoid component of cannabis, a naturally occurring component found in the trichomes of this plant, is seen to be particularly bene�cial in the treatment of alzheimer's disease as it lacks psychoactive properties and does not risk further cognitive impairment damage in alzheimer’s patients (karlet al., 2017). in fact, cannabis can can actually help restore cognitive dysfunctions characteristic of alzheimer’s disease (uddin et al., 2020). cannabis is a promising agent with numerous berkeley pharma tech journal of medicine | 18 therapeutic properties that hinder the progression of ad, leading to the investigation of cannabidiol (cbd) therapies. current cbd therapies are seen to bring “very modest symptomatic relief” (karl et al., 2017). cbd treatment is described as being a preventative, multimodal drug strategy targeting the wide variety of symptoms, making it a viable candidate for ad therapy (karl et al., 2017). v. conclusion the features of ad, which consist of in�ammation (atakan, 2012), amyloid-beta plaque buildup (farkhondehet al., 2020), and neuro�brillary tangles (hall et al., 2001; nelson et al.,2012), lead to life-altering cognitive de�cits and other extensive e�ects that impact a signi�cant population of individuals. it is one of the most heavily researched diseases, yet there is still no method to stop its progression. therefore, the injection of cannabinoids provides one promising revenue for scientists to counter the damage. the connection between ad and cannabinoids has been made apparent through many recent studies. investigations into the e�cacy of the ecs as well as natural and synthetic cannabinoids have developed in regards to the treatment and management of ad and its varying pathologies. cannabinoid agonists have presented researchers with neuroprotective qualities as well as the ability to manage the chronic psychiatric pathology associated with ad. this is due to the inherent physiology of the endocannabinoid system. ecs receptors exist both in the brain and parts of the immune system pathways, as a result, controlling its function through cannabinoid agonists and ecs ligands can serve both as a neuromodulator and an immunomodulator. therapeutic use of the ecs and cannabinoids in relation to ad include elevated activation of endocannabinoid receptors to bolster neurogenesis and thus reduce the neurodegenerative e�ects of ad (fagan & campbell, 2014). this process occurs through the stimulation of cb1 receptors by the endocannabinoid signaling molecules and external agonists. cb2 receptors are mainly targeted for their anti-in�ammation properties (tolón et al., 2009); their modulation using receptor agonists — such as jwh-015 (tolón et al., 2009) and jwh-133 (çakır et al., 2019) — indicates a reduction of proin�ammatory molecules,which block proper neurofunction, that are normally present in ad. endocannabinoid-speci�c agonists also inhibit the e�ects of ad by blocking harmful processes in the brain; these processes are microglia activation (martín-moreno et al., 2011), neuro�brillary tangles, and tau protein phosphorylation (esposito, 2006). experimentation with the berkeley pharma tech journal of medicine | 19 synthetic agonist has been seen to further suppress symptoms due to their structural similarity to the natural cannabinoids and have even stronger receptor activation. mice study results identify the synthetic agonists according to what they prevent in the ad injured brain: win55,212-2 is correlated to nerve development (liu et al., 2015), jwh-133 binds to cb2 to reduce rapid in�ammation (çakır et al., 2019), and dronabinol and nabilone treat mostly neuropsychiatric symptoms (liu et al.,2015). in dealing with the synapse, long term potentiation was regained in tests with mice who had amyloid-beta plaque buildup in combination with cbd dosages (hughes et al., 2019). as the major �nding is the revival of neuron activity, restoration of the cholinergic signaling system alleviates and e�ects (thompson et al., 2020). while commonly used as a relief drug, natural cannabinoids thc and cbd derived from the cannabis plant are used in tandem to manage cognitive de�cits (giacoppo et al., 2014). cbd acts as a modulator to thc intoxication and together e�ectively reduces oxidative stress and in�ammation associated with ad (giacoppo etal., 2014). while more studies and clinical trials are required before making any de�nitive statements,the associations discovered thus far indicate necessary future research and possible 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(2009). the activation of cannabinoid cb2 receptors stimulates in situ and in vitrobeta-amyloid removal by human macrophages. brain research, 1283, 148–154. 48. uddin, m. s., mamun, a. a., sumsuzzman, d. m., ashraf, g. m., perveen, a., bungau, s. g.,mousa, s. a., el-seedi, h. r., bin-jumah, m. n., & abdel-daim, m. m. (2020). emergingpromise of cannabinoids for the management of pain and associated neuropathologicalalterations in alzheimer's disease. frontiers in pharmacology, 11, 1097.https://doi.org/10.3389/fphar.2020.01097 49. vallée, a., lecarpentier, y., guillevin, r., & vallée, j. n. (2017). e�ects of cannabidiol interactions with wnt/β-catenin pathway and pparγ on oxidative stress and neuroin�ammation in alzheimer's disease. acta biochimica et biophysica sinica, 49(10), 853–866. https://doi.org/10.1093/abbs/gmx073 50. velikova, m., doncheva, d., & tashev, r. (2020). subchronic e�ects of ligands of cannabinoid receptors on learning and memory processes of olfactory bulbectomized rats. acta neurobiologiae experimentalis, 80(3), 286–296. 51. watt, g., & karl, t. (2017). in vivo evidence for therapeutic properties of cannabidiol (cbd)for alzheimer’s disease. frontiers in pharmacology, 8. 52. wu, j., bie, b., yang, h., xu, j. j., brown, d. l., & naguib, m. (2013). suppression of central chemokine fractalkine receptor signaling alleviates amyloid-induced memory de�ciency. neurobiology of aging, 34(12), 2843–2852. https://doi.org/10.1016/j.neurobiolaging.2013.0 6.003 berkeley pharma tech journal of medicine | 25 article 1_the therapeutic potential of synthetic and natural cannabinoids in treating alzheimer’s disease_publish.pdf 2. front page the therapeutic potential of synthetic and natural cannabinoids in treating alzheimer’s disease (1).pdf 3. body document the therapeutic potential of synthetic and natural cannabinoids in treating alzheimer’s disease.pdf cannabinoids paper reference page (3).pdf berkeley pharma tech journal of medicine correspondence: sydneyworrall@gmail.com keywords: interdisciplinary research clinical medicine syndemics healthcare submitted: april 6, 2023 accepted: july 7, 2023 published: december 30, 2023 full open access creative commons attribution license 4.0 abstract medicine has historically been built upon the discoveries of interdisciplinary research, yet modern clinical medicine has stagnated in its production of such research. this is despite multiple calls for interdisciplinary clinical medicine research from both inside and outside the discipline. complex issues, such as those that physicians are recently facing, require complex solutions that cannot be achieved through the use of a single discipline. in order to promote interdisciplinary research, certain approaches need to change. such changes include the reorganization of university department structures to prioritize interdisciplinary scholarship and create ‘translator’ positions to help bridge the knowledge gap between disciplines. this paper reviews how clinical medicine and interdisciplinary research are intertwined, assesses why there is a lack of interdisciplinary work within clinical medicine, and explores methods to encourage more collaboration between clinical medicine and other disciplines. an overview of interdisciplinary research and clinical medicine by: sydney m. worrall berkeley pharma tech journal of medicine | 2 1. introduction interdisciplinarity refers to the combination of knowledge from many different specialties or disciplines.1 despite interdisciplinarity having a commonly agreed upon meaning, interdisciplinary research is a term that is still being acutely defined. for this paper, the meaning of interdisciplinary research is taken from the institute of medicine, who defines it as a “mode of research by teams or individuals that integrates information, data, techniques, tools, perspectives, concepts, and/or theories from two or more disciplines or bodies of specialized knowledge, to advance fundamental concepts”.2 in short, it is a form of research that involves an integration of distinct disciplines. this differs from other forms of collaborative research, such as multidisciplinary work, because researchers in those projects “remain conceptually and methodologically anchored in their respective fields”.3 there is a distinct lack of integration between the fields in multidisciplinary research. research, particularly science research, is becoming more and more interdisciplinary as time goes on4; yet clinical medicine is an outlier and strikingly uni-disciplinary.5 2. clinical medicine and interdisciplinary research 2.1 interdisciplinary research as stated above, research in the fields of science is becoming more and more interdisciplinary, to the point where papers with a single author are becoming increasingly rare.4 that is not to say that science is only recently becoming interdisciplinary. modern clinical medicine itself is built upon the discoveries from interdisciplinary problems. for example, radiotherapy is founded on the pioneering work of physicist and chemist, marie curie, and engineer and physicist, william roentgen. the ground-breaking work of franklin (chemist), crick (physicist), watson (biologist), and wilkins (physicist) centered upon determining the structure of dna is now secure in the public record.6 the trend of interdisciplinary discoveries fueling medicine stands the test of time. recently, mathematical frameworks have been recognized as being a valuable asset to medicine, and has led to a ‘migration of mathematical physicists into medicine where their skills are being used to advance epidemiological modeling, develop novel clinical trials, and understand tumor dynamics’.6 interdisciplinarity is so integral to medicine that within the last 35 years, 11 of the nobel prize winners for medicine have had a background in fields such as chemistry, physics, or engineering.7 berkeley pharma tech journal of medicine | 3 while interdisciplinary research is important for medicine on a large scale, it is also integral to a patient to patient basis because clinical medicine is constantly facing and researching complex issues. not all the issues that clinicians face are purely medical. when treating a patient, a clinician may have to factor in societal or environmental issues that are impacting the patients’ health. complex problems, such as the ones clinicians face, are not amenable to single-discipline investigations. discoveries are more likely to be found on the boundaries between fields.8 2.2 syndemics theory societal and environmental factors playing into patient health is not a new concept. a theory called syndemics, which has been steadily gaining traction since its original proposal in 1996, is a testament to the intersection between medical conditions and sociological, environmental, and political issues. syndemics, in short, are synergistically interacting epidemics that occur in a particular context with shared drivers.9 the most researched and well-known example of syndemics is the sava model. it refers to the clustering of health and social crises such as substance abuse, violence, and aids into an interrelated complex.10 the sava syndemic takes what seem like three parallel epidemics occurring in a population and connects them into interdependent threats that cannot be wholly separated.10 since these epidemics are interdependent, one cannot be solved without addressing and treating the others. recently, the covid-19 pandemic has been the focus of syndemics, which is increasing attention of the theory in clinical medicine and global health.9 at its core, the syndemics theory is a call for interdisciplinarity for the sake of patient care. it states that you cannot treat a health epidemic without addressing the noxious social conditions that contribute to the severity of it. medical field professionals cannot treat the complex issues involved in syndemics alone; the task involves the work of policymakers, sociologists, public health officials, and a myriad of professionals from other disciplines. while the syndemics theory is a staple in the medical anthropology and global health lexicon, it is not the only call for more interdisciplinary work coming from the field of clinical medicine. clinicians are advocating for a change in treatment models for diagnoses such as musculoskeletal and cardiovascular conditions. patients with cardiovascular disease often experience multimorbidity, meaning that they have two or more chronic medical conditions. these comorbidities cluster into five distinct patterns, each impacting patients’ quality of life and clinical outcomes differently.11 patients require specialized, individualized care depending on which cluster they fall into, as symptoms can be altered by the presence of multiple underlying medical conditions. for example, noncardiovascular comorbidities are associated with a berkeley pharma tech journal of medicine | 4 higher overall symptom burden and the display of more severe symptoms than what cardiovascular comorbidities would cause alone. in addition, cardiovascular comorbidities were more likely to be associated with pain and anxiety rather than symptoms of shortness of breath or fatigue.12 the current treatment models are not meeting individual patients’ needs due to the complex psychosocial and multimorbidity facets of the conditions.13,14 clinicians and researchers are calling for more interdisciplinary research to help improve patient care for complex diagnoses. 3. the lack of interdisciplinary research in clinical medicine 3.1 clinical medicine despite the fact that clinical medicine was founded on the discoveries of interdisciplinary research, and that there is a call from within the house of clinical medicine for more interdisciplinary research, there is shockingly little of it being produced. a citation-based analysis over the years of 2001-2010 found that clinical medicine is strongly uni-discipline, meaning clinical medicine papers rarely cite, and are rarely cited by other disciplines. this is the polar opposite of the health field, which is increasingly interdisciplinary.5 this implies that while the generalized field of health is pushing the boundaries of its discipline, clinical medicine is not. it also shows that there is more that can be done to promote interdisciplinary research within clinical medicine.6 since there is a historical precedent for interdisciplinary research in medicine, and an established need for it within clinical medicine, the question becomes why is there a lack of it being produced? van noorden, the author of the citation analysis from 2001-2010, proposes that clinical medicine is uni-disciplinary because it is a very specialized field of practice. the proposal bears some thought, as clinical medicine is indeed a specialized field with many niche sub-fields. however, it was not always this way. in 1518, when the royal college of physicians was founded, individual physicians 'often embodied interdisciplinarity’.6 the enormous advances in medicine are what led to this stratification of clinical medicine. said stratification can be attributed to what researcher zurubavel calls the “rigid mind”. this rigidity is part human nature, creating an identity involves “lumping supposedly homogenous clusters” together.15 the rigidity, however, is further cemented within academia due to the way “academic libraries, journals, curricula, etc. are organized”.15 this mental categorizing means the structure of academic scholarship is usually envisioned ‘in terms of well-defined, sharply delineated fields’ that are seen as insular entities separated by a large intellectual divide.16 this divide has even spread to fields that are closely related, such as the fields of the biomedical sciences berkeley pharma tech journal of medicine | 5 and clinical medicine; more than 50% of medical practitioners believe that research is only related to advancement of personal careers rather than patient care.17 the rigid delineation of fields of study discourages interdisciplinary work and inhibits creativity. 3.2 ssh study for example, a study interviewed 29 social scientists’ and humanities scholars (ssh), who were working in a medical research environment. the goal of the interviews was to gauge the integration of the scholars into the different research environments. 18 out of 29 ssh scholars reported a partial adaptation in which they altered their research methods to fit into the medical research environment. 3 out of 29 ended up converting wholly to medical research due to the divide between disciplines. another 3 reported alienation and felt marginalized from their peers within the field.18 one interviewee remarked that “i'm creating a new niche for myself, which is actually making me increasingly marginal in my department … i have fewer and fewer colleagues that i can discuss my ideas with. i'm increasingly becoming sort of alienated from my own environment”. the reward these scholars get for pursuing interdisciplinary research is alienation and isolation from their own fields. their peers view the fields of social sciences/humanities and medical research as fields separated by ‘walls’, interviewee’s mentioned the separation of the fields were further pushed when publication of papers came into play. the scholars had the choice of either “watering down” their social theory analysis in order to publish their research in clinical journals or publish the work in social science journals and face a lack the recognition from their medical research environment.18 all of these facets lead to the aforementioned marginalization. 3.3 obstacles rigid thinking is not the only factor inhibiting interdisciplinary research within the field of clinical medicine. there are more practical obstacles compared to philosophical hinderances. ‘the challenges of interdisciplinarity’ by brewer19 discusses such obstacles. practical obstacles often stem from how different fields operate, as each discipline has differing frames of reference, methods and operations, and even ‘languages’ or terminology commonly used in them. institutional workings also pose an impediment due to different funding priorities between disciplinary and interdisciplinary work. there are also professional obstacles concerning hiring, promotions, status and recognition. 19 interdisciplinary work is often incredibly niche, and doesn’t fit into the standard assessments that can aid in decisions such as hiring and promotions. berkeley pharma tech journal of medicine | 6 another possible reason why interdisciplinary research is scarce within the clinical medicine community is that it has a complicated relationship with scientific impact. a study that analyzed all the articles published in the web of science in 2000 found that articles with both the highest levels of interdisciplinarity and the lowest of interdisciplinarity had lower scientific impacts compared to articles in between those extremes.20 finding the right balance between the two extremes can be an intimidating factor that scholars are hesitant to tackle. 4. how to promote more interdisciplinary clinical medicine research 4.1 solutions though there are obstacles that hinder interdisciplinary research within the field of clinical medicine, there are just as many possible solutions. some that are grounded in research, and some that need to be tested further for viability. with part of the lack of interdisciplinarity in clinical medicine being tied back to zerubavel’s rigid mind, a way to combat it is to promote what he calls a ‘flexible mind’.15 a flexible mind is one in which the mental map of academia still features specialty areas, yet not as discrete islands separated from one another by wide intellectual divides. academic fields are seen as nebulous entities which lack sharp contours and are constantly changing their forms and melding into other intellectual nebulae.15 to promote this flexible mindset concerning academia, universities need to enact foundational changes to alter the organizational structure of departments and centers.21 some universities are employing this strategy by modifying the traditional department structure in order to promote interdisciplinary research. this has been achieved by creating, promoting, differentiating, evolving, and consolidating departments and research centers.22,23 to assess the correlation between restructuring academic units and the promotion of interdisciplinary research, leahey and balinger conducted an analytical study that examined 9211 research centers and 12,323 departments across 156 universities. they found that universities that enact structural changes to manifest their commitment to interdisciplinary research produce more interdisciplinary scholarships. not only that, but the universities with a greater structural commitment to interdisciplinary research were observed to have a higher level of scholarship productivity and nih grant activity relative to institutions with less structural commitment.18 this model offers universities a way to adapt to the need for interdisciplinary areas without berkeley pharma tech journal of medicine | 7 demolishing traditional department structures, as no previously existing departments are eliminated.24-26 4.2 strategies as for strategies to specifically target clinical medicine, many potential strategies start in medical school. the general agreement is that “the solution lies in an approach early in a clinical education path that involves training medical students to become junior scientists”.27 the two main strategies involve either introducing research into medical studies at an early stage or introducing clinical rotations for basic researchers in master or phd programs. for example, the berlin brandenburg school for regenerative therapies (brst) promotes interdisciplinarity by offering a “a three-track graduate system embracing clinical scientists as well as biologists and engineers. an essential feature of these tracks is that there is a deliberate strategy to create an incubator model where trainees in the three disciplines work and study together, allowing the cross-fertilization of ideas and cultures”.27 the stanford school of medicine takes the second approach, offering a master of science in medicine; where instead of targeting future clinicians, they aim to get researchers into clinics in order to create a larger group of scientists knowledgeable about human diseases in order to bridge the language and intellect barrier between the field.27 another strategy to increase interdisciplinary research is to implement ways to make interdisciplinary research projects run smoother. this strategy is one that could show very quick results once implemented, as it targets projects that are either in progress or starting out, compared to the earlier models which target professional school structures. targeting the educational systems tackles the problem at the root but would take a longer time to produce results. one proposal on how to help interdisciplinary research is to create a new form of medical practitioner, a sort of translator who assists in the transfer of research findings into clinical practice.28 this would solve the problem of different “languages” between fields. there are a few names for these “translators”, the most being a mentored clinical scientist career path (cscp).27 the downside to this model is that the “translator” would need to have sufficient knowledge in a variety of highly specialized fields, someone with such skill set would most likely be difficult to find. of course these are only a few solutions, and the reality is that more research needs to be done on the inner workings of interdisciplinary research teams to learn how to promote it within other fields. berkeley pharma tech journal of medicine | 8 5. conclusion historically, interdisciplinary research has always been a part of scientific discoveries (especially within the field of clinical medicine), and in modern times we are seeing a rise in interdisciplinary research.4 however, the clinical medicine field is very far behind on that trend, despite calls for more interdisciplinary research due to the increasing complexity of patient diagnoses. physicians are noticing that multimorbidity and socioeconomic conditions are causing patients to require more personalized care plans, ones that they are struggling to meet.13,14 the lack of interdisciplinary research in medicine is due to a variety of reasons. they range from the increasing stratification of academia -leading to the idea that the different disciplines are separated by wide gaps that cannot be crossedto the stark differences in methodology and language between different disciplines. the general agreement on how to promote interdisciplinary work in clinical medicine is to approach it early in a clinical education path that involves training medical students to become junior scientists. there are also strategies that involve master-level and phd-level students to be involved in clinical rotations so that more researchers are knowledgeable on clinical medicine. this paper scratches the surface of interdisciplinary research and clinical medicine, and in order to have more concrete answers on how to encourage this combination, more research needs to be done on how to effectively create teams of professionals in a way that overcomes any departmental differences. references 1. garry d. b. the challenges of interdisciplinarity. springerlink. published 1999. accessed february 21, 2023. https://www.jstor.org/stable/4532473 2. institute of medicine, committee on science, engineering and public policy committee on facilitating interdisciplinary research , national academy of engineering, national academy of sciences. facilitating interdisciplinary research. national academies press; 2004. doi:https://doi.org/10.17226/11153 3. stokols d, hall kl, taylor bk, moser rp. the science of team science.american journal of preventivemedicine. 2008;35(2):s77-s89. doi:https://doi.org/10.1016/j.amepre.2008.05. 002 4. porter al, rafols i. is science becoming more interdisciplinary? 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volk hd, et al. the challenges of modern interdisciplinary medical research.nature biotechnology. 2011;29(12):1145-1148. doi:https://doi.org/10.1038/nbt.2062 28. sherwin t. in search of the clinical scientist. clinical and experimental ophthalmology. 2003;31(4):284-285. doi:https://doi.org/10.1046/j.1442-9071.2003.00 680.x berkeley pharma tech journal of medicine | 11 front page_interdisciplinary research_worrall.pdf blank page �� berkeley pharma tech journal of medicine correspondence:� icibhxbu�cqu!hnbjm�dpn keywords: *tdifnjd�tusplf /fvsphfoftjt (fof�uifsbqz /fvspuspqijd�gbdupst� /fvsp%1 /fvsphfofsbujpo� 7fdupst submitted�.bz�� ������ accepted�+vof��� ����� 1vcmjtife�july��� ����� full open access creative commons attribution� license 4.0 abstract stroke is one of the predominant causes of long-term disability and death, and is characterized by the cessation of blood flow to part of the brain, preventing it from accessing oxygen and nutrients needed for atp production and proper function. ischemic stroke, the most prevalent type of stroke, involves an obstruction such as a blood clot in a blood vessel. current treatments seek to remove the obstruction and restore blood flow, but are time-limited and do not reverse tissue damage once it has occurred. the endogenous response to ischemic stroke involves an increase in neurogenesis but falls short in producing functional recovery. gene therapy that enhances neurogenesis has provided encouraging results in animal models. preclinical studies in this area have utilized neurotrophic factors that can promote migration of cells from the subventricular zone (svz). the transcription factor neurod1 converts reactive glial cells, which increase during ischemia, into neurons that integrate into the existing circuits of the brain. the clinical value of gene therapy depends upon the development of safe and efficient administration methods. in this review, we draw from scientific literature to evaluate the relevant genes and vectors for treating ischemic stroke through neurogenesis, and discuss strategies to overcome current limitations of gene therapy in human patients. /fvsphfoftjt: gene-based strategies for 5sfbujoh�*tdifnjd�4usplf �ïw�¡èßéþßáûéþ��þ×ýí×êu�¬åæþßû��èåäéêûßäu��ä×è��þ×êêu�×äú�¯×äâå×ä�§ýëïûä *ouspevdujpo stroke is a leading cause of death, accounting for 5.5 million deaths worldwide each year1. in the united states, stroke is attributed to 1 of every 19 deaths, and one person will su�er from a stroke every 40 seconds. while 80% of those who su�er from strokes survive, many are impaired with permanent disability. although several types of strokes exist, including ischemic, hemorrhagic and transient ischemic attacks, the majority of research is focused on ischemic stroke, which makes up 80% of cases2. ischemic stroke results from an obstruction of blood vessels that lead to the brain and causes an impairment of function1. this arises from atherosclerosis due to cerebral thrombosis, a blood clot that develops at the fatty deposits, or cerebral embolism, a blood clot that forms in large arteries in the neck, chest or heart and travels into the smaller blood vessels of thebrain3.�based�on� the�duration� that� the�brain� is� deprived� of� blood� and� the� region� that� is� blocked,� strokes� can� bring� about� temporary� or�permanent�disabilities, if not death1. while�stroke�continues�to�be�among�the�most�debilitating�illnesses,�current� treatments� for� stroke�are� limited�by� their�time�of�administration�and�most� notably� cannot� reverse� tissue� damage� once� it� has� occurred.� the� most� prominent�and�vastly�used� ischemic� stroke� treatments�created� thus� far�are� emergency� interventions� given� to� stroke� patients� within� a� few� hours� of� stroke� onset,� and� are� limited� in� safety� and� availability.� these� include� thrombolysis,� the� chemically� induced� breakdown� of� blood� clots,� and� thrombectomy,� the� surgical� removal�of� the�blood�clots�with�a�catheter4.�a� stroke� patient� must� receive� the� standard� thrombolytic� treatment,� tissue� plasminogen�activator�(tpa),�within�4�½�hours�of�onset,�and�patients�above� 80�years�of�age�must�receive�tpa�within�3�hours5.�moreover,�tpa�comes�with� the�risk�of�hemorrhage,�neurologic�decline�and�orolingual�angioedema6,�and� its�positive�outcome� rate� is�estimated�to�be�only�43%5.�thrombectomy�is�a� more�versatile�option�because�of�its�ability�to�treat�patients�with�a�proximal� occlusion,� unlike� thrombolysis.� still,� it� is� restricted� by� a� six-hour� window� from� the� onset� of� symptoms� and� is� associated� with� risks� such� as� hemorrhage, reocclusion and cerebral edema7,8. berkeley pharma tech journal of medicine | 31 neurogenesis a strategy for treating ischemic stroke by gene therapy involves increasing neurogenesis, the process by which neurons are formed by neural stem cells (nscs). neurogenesis is the basis of cognitive function and allows the brain to respond to new stimuli9. through constant re�ning and moderation of these neurons, the brain is able to adapt to cognitive, environmental, and pathophysiological demands. during prenatal and embryonic stages of human development, the majority of neurons are formed, but the process is continued through adulthood at a decreased rate in localized zones10. during prenatal neurogenesis, the central nervous system (cns) consists of nscs, speci�cally radial glial cells (rgcs), that generate neurons11. the rgcs occupy a majority of the prenatal ventricular zone and move from symmetric to asymmetric cell division. as asymmetric division and proliferation occurs, the rgcs separate, allowing for the creation of neural progenitor cells (npcs) and intermedial neuronal precursors. after further separation, new neurons are formed12. as these neurons migrate through the embryo, they generate neural circuitry and increase in maturation. the rate of this prenatal neurogenesis and the type of neurons formed depend on genetic and molecular factors, although prenatal neurogenesis in humans typically occurs at gestational week 5 and is thoroughly developed by week 3413. although the majority of neurogenesis occurs before a human is born, the neurogenic processes that occur throughout the lifespan continue to play a signi�cant role in brain function9. in contrast to prenatal neurogenesis, which occurs throughout the entire cns, neurogenesis during adulthood occurs primarily in the subventricular zone (svz) and subgranular zone (sgz)15. stem cells formed during adulthood do not spontaneously activate, but instead are dormant in the brain and only activate in response to new cognitive or environmental signals16. along with the lack of spontaneous growth and decreased proliferation of neurons during adulthood, the density, plasticity, and ability to repair neurons also diminishes over time17. these factors all contribute to a decreased ability of berkeley pharma tech journal of medicine | 32 the brain to recover after injury. because neurological impairments, including those caused by ischemic stroke, are more prevalent among older populations, the creation of healthy neurons after neurological injury is rarely su�cient to induce functional recovery18. pathophysiology of ischemic stroke in ischemic stroke, the obstruction of blood vessels to the brain limits cerebral oxygen and glucose supply, causing tissue damage. this reduction in oxygen and glucose due to inadequate cerebral blood �ow initiates biochemical cascades that lead to cell death19. oxygen and glucose are required to produce atp, the key source of energy in cells, and as a result of ischemia, atp levels decline20. this prevents the function of ion pumps that rely upon atp to maintain ionic gradients. failure of the na+/k+ atpase pump, which normally transports k+ into the cell and na+ out of the cell, causes excess intracellular na+ and extracellular k+. intracellular ca2+ levels rise because of ca2+ pump failure21, and the binding of ca2+ to synaptotagmin stimulates glutamate release to the synapse. excess glutamate leads to the hyperexcitation of nmda glutamate receptors, providing positive feedback for intracellular ca2+ levels22. in a phenomenon known as excitotoxicity, excess glutamate sets the stage for mechanisms of cell death. excess intracellular ca2+ levels activate endonucleases, lipases, phosphatases and proteases23, which contribute to cell membrane disruption, cytoskeletal breakdown, dna fragmentation, free radical production and mitochondrial damage. these processes lead to cell death by apoptosis or necrosis and irreversible tissue damage19. berkeley pharma tech journal of medicine | 33 figure 1: pathway from ischemic stroke to tissue damage in the brain. obstruction of blood vessels leading to the brain reduces oxygen and glucose supply, which are required for atp production. ca2+ and na+/k+ pumps rely on atp and their failure in ischemic conditions causes ion imbalances. excess intracellular ca2+ leads to glutamate excitotoxicity and cell death. ischemic stroke additionally results in neuroin�ammation. inadequate blood supply activates microglia, leading to in�ammation through cytokine upregulation, disruption of the blood-brain barrier (bbb) and the production of reactive oxygen species. in astrocytes, in�ammatory factors like glial �brillary acidic protein (gfap) result in reactive gliosis and glial scar formation, and failure of the na+/k+ atpase pump leads to the swelling of astrocytes. ischemia obstructs the connection between astrocytes and endothelial cells at the bbb, allowing peripheral in�ammatory cells to breach24. the glial scar—primarily made up of astrocytes and microglia—forms to protect the brain after stroke but prevents the regeneration of neurons. in berkeley pharma tech journal of medicine | 34 ischemic stroke, astrocytes undergo morphological and functional changes, proliferating and overlapping with each other during reactive gliosis while gfap is upregulated. the scar has neuroprotective e�ects, such as providing a barrier between healthy and damaged tissue and limiting the spread of tissue damage25,26. however, it impedes the restoration of neural function in stroke25 because axons physically cannot cross the scar, and reactive astrocytes produce growth-inhibiting factors that inhibit axon extension. while eliminating reactive astrocytes increases tissue damage, due to their bene�ts15, strategies to mitigate the obstructive e�ects of the scar and restore long-term function would be favorable in treating stroke. neurogenesis increases as an endogenous response to ischemic stroke, but this is insu�cient to restore function after stroke27. this process is driven by nscs in the svz, which proliferate and di�erentiate due to an upregulation of neurotrophic factors28. the mechanism behind nsc proliferation and di�erentiation in post-stroke neurogenesis is reported to be induced by h19, a long non-coding rna29. stroke patients experience a level of functional improvement in the �rst few months following stroke due to self-repair30, so enhancing neurogenesis would be a promising approach to improve function after stroke. in vivo animal models are typically used for preclinical research of ischemic stroke and allow researchers to replicate the pathophysiology of the condition in humans with far more complexity than in vitro models31. the standard method to model focal ischemic stroke in animals is through the occlusion of the middle cerebral artery (mca)—which is easily accessible and the most common site of stroke in humans—by mechanical devices, blood clots or pharmacological agents32,33. mca occlusion is often induced by inserting an intraluminal thread into the internal carotid artery via the external carotid artery to block the mca. this leads to signi�cant damage in local brain tissue within three hours but can be lacking in precision, despite its non-invasiveness. more direct access to the mca requires craniectomy, the surgical opening of the skull, which exposes the brain to the atmosphere and poses a risk for cerebrospinal �uid leakage, infection and unintended damage34. berkeley pharma tech journal of medicine | 35 gene therapy to enhance neurogenesis to induce neurogenesis by gene therapy, genes are typically administered through a vector. to successfully deliver a gene in vivo, we would have to identify the appropriate transgenes for the local recruitment of endogenous stem cells. this technique could be used to slow the spread of neural decline or generate a neural niche in regions that did not already express the necessary signals or factors35. vectors are preferred over the delivery of naked dna because of their greater e�ciency, and viral vectors take advantage of viruses’ evolved ability to alter gene expression in cells they infect36. viruses deliver genetic information by infecting the cell directly, but in gene therapy, they are modi�ed so they are unable to transfer unwanted information and result in pathogenicity. delivery of vectors is carried out in two ways: in vivo, where the gene is delivered directly into the patient, and ex vivo, where cells are removed from the patient before gene delivery and then inserted back into the patient37. figure 2: schematic of gene therapy methods. in vivo gene therapy often involves direct delivery of a vector into the body to alter gene expression. ex vivo gene therapy is performed by removing cells from the body, delivering the vector into cells and transplanting altered cells into the body. berkeley pharma tech journal of medicine | 36 each viral vector o�ers unique advantages in gene therapy but has particular drawbacks. a class of vectors that is rising in popularity in gene therapy utilizes the adeno-associated virus (aav)38 because of its low immunogenicity and thus its propensity for an undesired immune response. integration of aav into the host genome depends on the presence of the rep gene, which exists in wild-type aav. recombinant aav (raav) does not contain rep and therefore helps limit side e�ects39. a key disadvantage of aavs that do not integrate into the host genome is that subsequent doses, if needed, would have a reduced e�cacy due to an adaptive immune response. to combat this, methods to remove anti-aav antibodies from the bloodstream, including plasmapheresis and igg-cleaving endopeptidases, have been developed and shown e�ectiveness37. serotypes of aav have also been engineered to improve its permeability to the bbb, which is necessary for peripheral administration in treating cns disorders like stroke40. for example, aav-php.eb has the ability to transduce most neurons in the cortex and striatum and e�ciently promote gene expression in the cns41. another viral vector being investigated for use in ischemic stroke models is the adenoviral vector, which o�ers greater capacity and transfection e�ciency. a study of adenovirus-mediated gene transfer to an ischemic brain model found that this vector provided e�ective expression of transgene at the nonischemic and peri-ischemic areas42. however, adenoviral vectors are limited by their high immunogenicity, and high doses can lead to in�ammation43. lentiviral vectors, derived from the retrovirus family, have lower immunogenicity than other retrovirus-based vectors and most notably integrate into the host genome. this ability makes them useful for stimulating stable long-term expression. at the same time, genomic integration puts target cells at risk for insertional mutagenesis44. unlike lentiviral vectors, retroviral vectors have limited use in gene therapy to promote neurogenesis because they only transduce dividing cells, and neurons are non-dividing cells45. all viral vectors have advantages and drawbacks and should be chosen on a case-by-case basis, but raav would generally be favorable to enhance neurogenesis in humans. low immunogenicity should be prioritized from a therapeutic standpoint, and this is a de�ning characteristic of raav. berkeley pharma tech journal of medicine | 37 although less common than viral vectors, another area of interest in gene therapy for stroke is the delivery of genes by non-viral means. in general, non-viral vectors limit the risk of cytotoxicity, immunogenicity and mutagenesis, making them an attractive alternative to viral vectors for therapeutic use in humans, while being less costly. their e�ectiveness is hindered by their relatively low transfection e�ciency, speci�city and duration of expression46. the conventional method of non-viral gene delivery is polyethylenimine (pei), a polymer that is associated with cytotoxic risks due to its high positive charge density47. conjugation of pei with deoxycholic acid to carry the anti-in�ammatory gene heme-oxygenase 1 can help to overcome this challenge and reduce infarct size from ischemic stroke48. despite the potential of non-viral vectors, their viral counterparts remain the most prevalent type of vector in research and existing drugs because of their e�ciency, diversity and adaptability49. neurotrophic factors in gene therapy neurotrophic factors promote the growth and survival of neurons and are the primary group of molecules that can be overexpressed through gene therapy to induce neurogenesis. they serve an array of other functions in treating stroke50. neurotrophic factors play roles in the development of axons, dendrites and synapses51, and are endogenously upregulated in response to stroke52. neurotrophins, a family of neurotrophic factors, target transmembrane receptors trk and p75-ntr. neurotrophins bind, causing dimerization and transphosphorylation of trk. this activates plcγ, which produces ip3 and dag to enhance intracellular ca2+ release and promote synaptic plasticity via protein kinase c. the growth of axons and dendrites is mediated by a separate pathway featuring trk, ras and erk. the pi3-k pathway activates serine/threonine kinase to drive neuronal survival53. several neurotrophic factors that have been investigated in gene therapy research pertaining to ischemic stroke, and their bene�ts and limitations, will be assessed in this section. berkeley pharma tech journal of medicine | 38 the �rst neurotrophic factor to be discovered and the one that is most well-understood is nerve growth factor (ngf)50, a neurotrophin that promotes both neurogenesis via upregulation of growth-associated protein 43 (gap-43) and angiogenesis via upregulation of vascular endothelial growth factor (vegf)54. serum ngf levels are inversely correlated with post-stroke function, potentially indicating a therapeutic link55. pseudolentiviral gene therapy with β-ngf, a subunit of the factor with very similar e�ects, in the rat hippocampus increases neurogenesis and reduces apoptosis following ischemic stroke, as reported by cao et al. (2018). cognitive functional recovery was demonstrated by improved morris water maze performance of rats that received β‐ngf56. non-invasive routes of ngf administration have been investigated as well. in a study by zhu et al. (2011), intranasal delivery in rats improved survival in the striatum and svz but failed to enhance cell proliferation in those regions57. ngf has been explored in relation to non-viral vectors, in part to overcome the impermeability of ngf to the blood-brain barrier. in vitro carbon nanotube delivery showed that ngf treatment promotes neuron survival in a sustained manner, although its in vivo applications remain unclear58. albumin nanocarriers successfully promoted neurite growth in vitro. however, in vivo delivery of ngf in combination with u0126, which inhibits the mek pathway involved in ischemia, did not signi�cantly reduce infarct size compared to u0126 alone, suggesting that this treatment is lacking in e�cacy59. ngf delivery with exosomes in the ischemic cortex of mice resulted in long-lasting neuroprotection and indications of neurogenesis, but functional recovery was not tested60. clinical trials of ngf in alzheimer’s disease and parkinson’s disease patients revealed negative side e�ects, such as weight loss and back pain due to nociceptive activation by ngf. the e�ect of ngf on nociception must be mitigated and methods for e�cient transport across the bbb must be developed to make it an e�ective therapeutic option61. brain-derived neurotrophic factor (bdnf), the most abundant neurotrophin, is another factor that induces neurogenesis and helps prevent apoptosis. overexpression of bdnf protects neurons from glutamate excitotoxicity and has been looked at as a potential therapeutic option in stroke patients62. at the same time, excess bdnf in the forebrain can have berkeley pharma tech journal of medicine | 39 negative e�ects on learning and memory63. as with ngf, stroke is associated with a reduction in bdnf levels in the bloodstream64. aav-bdnf gene therapy targeting the svz, a key location of adult neurogenesis, of rats two weeks before contralateral mca occlusion increases the migration of npcs from the svz and contributes to behavioral recovery. it failed to reduce infarct size signi�cantly, contradicting the �ndings of previous studies into the factor65. gene therapy utilizing bdnf has also been used to reduce symptoms of stroke in preclinical research, including post-stroke pain and depression. this suggests that bdnf plays roles in both enhancing neurogenesis and reducing in�ammation66,67. a clinical trial conducted in 1999 tested the e�ectiveness of bdnf, administered subcutaneously, in patients with amyotrophic lateral sclerosis, a neurodegenerative disease. although side e�ects were minimal, the trial was unable to establish a signi�cant bene�t of bdnf treatment. still, it had success in patients with early respiratory impairment and altered bowel function, a side e�ect of the treatment, indicating a need for further research68. future trials with bdnf would likely bene�t from more re�ned methods of gene transfer into subjects. neurotrophin-3 (nt-3) is part of the same family as ngf and bdnf and plays a distinct role in neurogenesis during development53. it is involved in the survival, proliferation and di�erentiation of neurons, as well as in the plasticity and regeneration of glutamatergic neurons. nt-3 is a contributor to revascularization and has anti-in�ammatory properties69. duricki et al. (2016) investigated the e�ectiveness of nt-3 in gene therapy treatment for stroke. aav-mediated intramuscular delivery of the factor in adult and elderly rats 24 hours after stroke resulted in neuroplasticity, as observed through the sprouting of corticospinal axons, and behavioral recovery. neuroprotection was not displayed due to the delay in treatment, and rats experienced minor in�ammation from aav injection70. nt-3 binds to trkc receptors, which are not found on adult nociceptors, making it advantageous over the pain-regulating ngf. duricki et al. (2019) con�rmed this bene�t in the forepaws of adult and elderly rats. furthermore, human clinical trials in patients with neuropathy have not revealed signi�cant side e�ects71. nt-3 appears to be a safer alternative to other members of the berkeley pharma tech journal of medicine | 40 neurotrophin family, and future research into its e�ect on neuron survival could reinforce a need for clinical studies in stroke patients. neurotrophic factors that are not part of the neurotrophin family have also attracted the attention of researchers in gene therapy. among these is ciliary neurotrophic factor (cntf), an important contributor to endogenous neurogenesis that occurs after stroke. cntf promotes proliferation of cells from the svz72. its continuous administration into the lateral ventricle for four weeks after mca occlusion in rats had the capacity to reduce infarct size of the cortex and degeneration of the thalamus, while improving spatial learning73. maclaren et al. (2006) found that cntf has protective e�ects on ganglion cells, following intravitreal administration of aav-cntf74. although not directly related to ischemic stroke, this study reveals the therapeutic applications of cntf in preventing tissue damage from cns injury that occurs in ischemic stroke. a clinical trial by chew et al. (2019) using cntf in patients with macular telangiectasia, a neurodegenerative condition of the retina, had success in promoting retinal neuroprotection75. while this trial showed minimal side e�ects, it is important to note in future human applications that cntf and its neurogenic e�ects are linked to satiety and weight loss76. glial cell-derived neurotrophic factor (gdnf) promotes neurogenesis in striatal neurons and is involved in synaptic plasticity. it primarily acts on dopaminergic neurons, stimulating the release of the neurotransmitter dopamine in the striatum77. dopamine plays a major role in sensorimotor function, and research shows that ischemic stroke contributes to motor de�cits through the loss of dopaminergic neurons78. beker et al. (2022) reported that intracerebral lentiviral delivery of gdnf 10 days before mca occlusion led to an increase in the expression of the dopaminergic neuron transcription factor nurr1 and reduced degeneration of neurons in mice. the treatment did not appear to increase the number of striatal dopaminergic neurons following ischemic stroke77. beker et al. (2020) observed that lentiviral gdnf delivery in mice induces both neurogenesis and angiogenesis and reduces the glial scar in the peri-infarct region. in addition, motor recovery was demonstrated79. clinical trials using gdnf to berkeley pharma tech journal of medicine | 41 treat parkinson’s disease have not shown a signi�cant improvement in motor function compared to a placebo group. trials in which gdnf was delivered directly to the putamen had greater success, indicating that it could be useful through more e�cient routes of administration80. insulin-like growth factor 1 (igf-1) contributes to neurogenesis and angiogenesis. low levels of igf-1 are associated with more severe tissue death in stroke patients, and it is produced as part of the endogenous response to stroke. zhu et al. (2009) intracerebrally delivered aav-igf-1 after mca occlusion, which was shown to promote neurogenesis by enhancing proliferation and migration of nscs from the svz. its ability to increase vascular density promotes cerebral blood �ow81. okoreeh et al. (2017) reported that aav5-igf-1 six to eight weeks before mca occlusion was unable to reduce infarct size by targeting astrocytes. behavioral recovery and a reduction in neuroin�ammation were still observed82. the e�ect of igf-1 on neurogenesis to treat diseases like stroke is yet to be clinically researched. future preclinical studies are required to investigate its neuroprotective abilities before application in humans. vascular endothelial growth factor (vegf) primarily induces angiogenesis but is also known to enhance neurogenesis in the svz and sgz. wang et al. (2007) shed light on the connection between vegf overexpression and neurogenesis. liposomes, a non-viral vector, were used to transfer vegf into the lateral ventricle of the ischemic rat brain, increasing striatal neurogenesis and reducing the size of infarction83. markosyan et al. (2020) tested adenoviral vectors carrying vegf in combination with gdnf and neural cell adhesion molecule as a preventative option for ischemic stroke in rats. intrathecal delivery reduced infarct size and brought about functional recovery of neurons, though neurogenesis was not observed84. acute upregulation of vegf is associated with disruption of the bbb, which must be considered for clinical trials and could be mitigated by combining vegf with other angiogenic factors85. berkeley pharma tech journal of medicine | 42 conversion of glial cells to neurons a related approach to promote the formation of neurons after stroke involves neuroregeneration through the conversion of reactive glial cells, which proliferate in the post-stroke brain, into neurons. the endogenous response to ischemic stroke features the formation of the glial scar, largely made up of reactive astrocytes and microglia, which is driven by gfap25,26. considering the hindering e�ects of the scar and the low auto-regenerative abilities of the cns, it would be favorable to convert components of the scar into neurons. inducing neurogenesis through the conversion of reactive glial cells would both reduce the size of the glial scar and potentially restore neuronal function. the neural transcription factor neurogenic di�erentiation 1 (neurod1), which plays a major role in prenatal neurogenesis, has been the primary focus of gene therapy research aiming to convert glial cells into functional neurons as a treatment for stroke86. neurod1 binds to regulatory elements of neuronal development genes to promote neuronal migration and transcription via conversion of heterochromatin to euchromatin, a process that resembles the maturation of neurons seen in the prenatal stage86,87. although several other transcription factors have been identi�ed, including neurogenin-2, olig2 and ascl1, and have been experimented in di�erent combinations, they do not display as much e�ciency or e�cacy as neurod1 alone88. in vivo neurod1-based gene therapy studies have shown that the conversion of reactive glial cells to neurons promotes functional recovery from ischemic stroke. a study by ge et al. (2019) using non-human primates (nhps), which model the human brain more closely than rodents, showed that the overexpression of neurod1 in reactive astrocytes of the cortex successfully led to their conversion into neurons. after stroke induction, it was revealed that neurod1-aav increased neuronal density and synaptic and dendritic markers, while protecting parvalbumin interneurons, in treated areas. a 90% reduction in targeted astrocytes was observed, as well as decreases in reactive microglia and macrophages. these changes were initiated between 10 and 30 days after the onset of stroke and lasted between two months and one year, suggesting that neurod1-mediated gene therapy is long-lasting and has a more generous berkeley pharma tech journal of medicine | 43 time window than existing treatments89. jiang et al. (2021) reported that lentiviral delivery of neurod1 into the peri-infarct region of mice, one week following stroke onset, resulted in 66% of infected cells being marked as mature neurons, reduction in astrogliosis, and increases in synaptic plasticity and sensorimotor function88. a study of neurod1-aav in mice by chen et al. (2020) showed the ability of this factor to promote functional recovery from ischemic stroke, with 30% to 40% of neurons in the motor cortex being regenerated after neurod1 was expressed in reactive astrocytes90. altogether, preclinical research into neurod1 has been encouraging, because it accomplishes two critical objectives in reversing tissue damage from stroke: reducing the obstructiveness of the glial scar and increasing neurogenesis. nonetheless, gene therapy with neurod1 must be performed in a regulated manner, because eliminating the glial scar leads to adverse outcomes15. practical considerations while preclinical in vivo studies have shown success in promoting functional recovery from ischemic stroke through gene therapy, several limitations still exist that prevent moving forward with treatments in humans. there have been few clinical trials in humans, many of which have produced inconclusive or discouraging results. although studies involving rodents and nhps serve as an important basis for our understanding of gene therapy, the di�erences between their genomes from humans could create unsuccessful results in the clinical stage. one of the main obstacles in using gene therapy to treat cns disorders like stroke in humans is the bbb. the vast majority of drugs including the factors discussed cannot pass through this barrier, but to minimize the invasiveness of gene therapy, vectors expressing therapeutic genes must be able to cross in an e�cient manner. therapeutic options that bypass the bbb require direct injection into the brain or high peripheral doses and present a signi�cant risk to the patient40,91. high doses of aav to treat x-linked tubular myopathy led to liver dysfunction, sepsis and the deaths of two patients in a 2020 clinical trial92. intravenous administration is a non-invasive route, though it requires genes to cross the bbb. it additionally berkeley pharma tech journal of medicine | 44 lacks speci�city, increasing the likelihood of o�-target e�ects on peripheral organs93. the development of vectors that can e�ciently cross the bbb would improve the feasibility of intravenous gene therapy. research in this area has largely focused on serotypes of aav, such as aav-php.eb, although these variants reduce the already small packaging capacity of the vector91,93. in more recent strategies, vectors cross the bbb by paracellular transport through disruption of the barrier or by attaching to a transport receptor94. intranasal delivery has received interest as a minimally invasive method of bypassing the bbb95. ngf has been administered intranasally in rat stroke models and had success in promoting neurogenesis54. another aspect that must be considered is the risk for viral-mediated gene therapy treatment to provoke an immune response. gene therapy with raav is promising for in vivo treatment of cns diseases such as stroke because it is associated with low immunogenicity and no integration into the host genome39. however, this lack of integration is also a key drawback of raav, because anti-aav antibodies are produced by the adaptive immune system, attenuating subsequent doses37. existing approaches to remove these antibodies from the bloodstream, as outlined earlier, would make raav gene therapy far less practical in humans. inducing neurogenesis through gene therapy may additionally target unwanted cells, allowing for potentially harmful results. vectors that can integrate into the host genome, such as lentiviral and wild-type aav vectors, present the greatest risk for insertional mutagenesis44. therefore, the use of vectors that do not integrate may presently be the safest option. safety of gene therapy in humans requires that side e�ects are minimized. the target cells of genes must be as restricted as possible, which is dependent on how well the promoters used are able to target desired cells. cell-type speci�city of promoters remains a challenge, especially for intravenous administration93. gene therapy that enhances neurogenesis to treat stroke aims to target neurons in the hippocampus and lateral ventricles, the two primary locations of adult neurogenesis. finneran et al. (2021) reported that the promoters camkiiα and human synapsin 1 allow high-level gene expression speci�c to neurons, including those in the berkeley pharma tech journal of medicine | 45 hippocampus, and prevent peripheral expression when used with aav. use of camkiiα resulted in lower aberrant expression in the heart, lung and muscle compared to human synapsin 196. for gene therapy utilizing neurod1, it would be bene�cial to target reactive glial cells to optimize their conversion into neurons. the standard promoter used for expression in astrocytes, the main component of the glial scar, is gfap, but its large size is unfavorable for aav, which has a small capacity. gfaabc1d is a smaller promoter that also targets astrocytes but research into its speci�city has been inconclusive97. taschenberger et al. (2017) investigated the microrna124 (mir124) target sequence, which is speci�c to neurons, as a means of reducing o�-target e�ects. the study demonstrated that mir124 greatly reduces o�-target gene expression in neurons, although the level of expression was less than desired98. future directions although the factors examined in this review have generally shown promise in gene therapy for stroke in the preclinical stage, minimal clinical research has been conducted in relation to stroke speci�cally, and success in humans has been limited. key areas of future research that would help open the door for gene therapy in humans include vectors that can e�ciently cross the bbb and minimize aberrant gene expression. improving the transfection e�ciency of non-viral vectors could potentially make them a more practical option than viral vectors for gene therapy in humans, and thus should be an area of future research interest. non-viral vectors are far lower than viral vectors in immunogenicity, cytotoxicity and mutagenesis. to make them a more practical option in gene therapy to treat stroke, strategies to improve their speci�city and transfection e�ciency must be further researched46. to gain a fuller understanding of ischemic stroke and the e�ectiveness of gene therapy as a treatment option, animal stroke models must be re�ned. the standard model for focal cerebral ischemia, intraluminal mca occlusion, can lead to hyperthermia by blocking hypothalamic blood supply and subarachnoid hemorrhage if the suture is not inserted properly99. laser doppler �owmetry has attracted attention as a way to accurately guide the berkeley pharma tech journal of medicine | 46 suture, but studies into its bene�ts for modeling mca occlusion have had mixed results100,101. alternative models must also be investigated so that new treatments encompass less common forms of ischemic stroke. only 50.8% of ischemic stroke arise from the mca but the vast majority of in vivo models used to evaluate gene therapy use mca occlusion. small-vessel and brainstem strokes, for example, combine to make up 24.2% of cases. developing techniques to access deeper regions of the brain would help overcome this disproportion102. further research into stroke models would improve the likelihood of translating preclinical successes in gene therapy into viable treatments for humans. gene therapy using neurotrophic factors and neurod1 must be carefully controlled in its duration to avoid the development of tumors. accumulating evidence has implicated them in tumor neurogenesis, the formation and proliferation of cancer cells. ngf is overexpressed in breast, gastric, liver, lung, ovarian, pancreatic, skin and thyroid cancers. cancer cells secrete ngf and contribute to tumor neurogenesis by acting on trk receptors on nerves. moreover, ngf is linked to heightened pain experienced by cancer patients103. other neurotrophins, including bdnf, are upregulated in various types of cancer104. studies have indicated that neurod1 is expressed in small cell lung and colorectal cancers105,106. however, the risk of cancer from these factors in gene therapy for stroke is yet to be thoroughly investigated. finally, the enhancement of neurogenesis through gene therapy must be further investigated in relation to symptomatology. preclinical studies have focused on histological markers like growth of axons and dendrites and proliferation of neurons. although previous research has indicated that adult neurogenesis from the hippocampus contributes to learning and memory107, the direct e�ects of neurogenesis on cognitive recovery from stroke must be studied in greater detail, and the implementation of new measures would be bene�cial. the factors discussed in this review had a wide range of e�ects in reducing the symptoms of ischemic stroke, so the direct role of neurogenesis remains largely unclear. berkeley pharma tech journal of medicine | 48 conclusion as one of the most common causes of death and disability around the world, ischemic stroke has become the focus of a growing number of studies related to gene therapy. where current treatments may fail, gene therapy can reverse the pathophysiology of stroke and greatly extend the period in which patients can be treated after stroke and still experience functional recovery. treatment can be administered to increase neuron proliferation and growth. vectors expressing genes for neurogenic molecules have shown the capacity to augment the endogenous response to stroke, which involves low-level neurogenesis, in preclinical studies. overexpression of neurotrophic factors, including the neurotrophins ngf, bdnf, nt-3, as well as cntf, gdnf, igf-1 and vegf, may have the potential to enhance neurogenesis and aid in recovery. however, clinical trials have uncovered side e�ects that should be explored in greater detail. from a safety perspective, raav is the most favorable viral vector for stroke gene therapy due to its low immunogenicity, and methods for cell-type speci�city should be re�ned. an alternative approach to neurogenesis is the conversion of glial cells to neurons. this tactic would employ neurod1 to reduce the size of the glial scar while simultaneously working to restore neuronal function. to progress further from preclinical in vivo studies to treatment in humans, further research is required to investigate less invasive methods for e�cient gene therapy. a formidable obstacle is the bbb, which is extremely di�cult to cross with minimal invasiveness. more research is needed into routes of administration and side e�ects to ensure success in human clinical trials. berkeley pharma tech journal of medicine | 49 references 1. donkor es. stroke in the 21st century: a snapshot of the burden, epidemiology, and quality of life. stroke res treat. 2018;2018:3238165. published 2018 nov 27. doi:10.1155/2018/3238165 2. boehme ak, esenwa c, elkind ms. stroke risk factors, genetics, and prevention. circ res. 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front_page_bhagwat bhagwat_galley references the role of the gut microbiome in neuromodulation therapies as a potential treatment adjunct for multiple sclerosis abstract berkeley pharma tech journal of medicine correspondence: jwlarrick@gmail.com keywords: chloroquine, hydrochloroquine, autophagy, aging, geroprotection submitted: march 10, 2023 accepted: april 6, 2023 published: june 30, 2023 full open access creative commons attribution license 4.0 autophagy, the turnover of cellular components including organelles, declines with age. thus, enhancement of this characteristic process is hypothesized to improve health and extend lifespan. two recent papers present data indicating that contrary to expectation, chloroquine (cq), a nominal inhibitor of autophagy, extended the lifespan of middle-aged mice and rats by ~10%. details of these studies provide a cautionary tale regarding traditional reagents or “tool compounds” of “established” mechanisms often used in cellular biological research. however, these and earlier studies support a deeper investigation of cq or its more commonly used clinical analog, hydroxychloroquine (hcq), as potential drugs to increase health span and slow the aging process. chloroquine defeats aging? by: james w. larrick, m.d. ph.d. and jasmine w. larrick introduction 1.1 autophagy autophagy, literally “self-eating," was �rst described by christian de duve, who shared the 1974 nobel prize for his discovery of lysosomes. autophagy is an evolutionarily conserved catabolic process wherein lysosomes degrade various cellular components to maintain cytoplasmic quality control and thus cellular homeostasis. several distinct forms of autophagy include macroautophagy, microautophagy, and chaperone-mediated autophagy (cma). dysregulation of this critical process has been associated with many pathological processes including infectious, metabolic, and neurodegenerative disorders, as well as cancer and autoimmunity. reduced autophagy was identi�ed by lópez-otín et al. (2013) as a so-called "hallmark of aging". 1.2 chloroquine native peoples of south america have long used an extract of the bark of the cinchona tree ( cinchona officinalis ) as a remedy for fevers. by the 1600s, this herbal medicine was being used in europe for treatment of malaria. quinine was isolated from the bark in 1820. seeking an alternative to quinine, bayer chemists discovered chloroquine, a synthetic analog with a similar mechanism of action. a related analogue, 3-methyl-chloroquine, was utilized by the germans in world war ii. material captured by the americans in north africa was analyzed by the united states government, resulting in corroborative studies and subsequent clinical approval in 1947 of chloroquine as a prophylactic treatment for malaria. while chloroquine and its derivative hydroxychloroquine are widely used in rheumatology (sle, ra, etc.) 1 , chloroquine-resistant malaria has mandated the development of newer antimalarials such as pyrimethamine, artemisinin and me�oquine 2 . chloroquine inhibits hemozoin formation from the heme released by parasitic digestion of erythrocyte hemoglobin during plasmodium infection. the free heme then lyses membranes and leads to parasite death 3, 4, 5 . chloroquine passively di�uses through cell membranes and into berkeley pharma tech journal of medicine | 2 endosomes, lysosomes, and golgi vesicles which become protonated, trapping the chloroquine in the organelles and raising the surrounding ph. the raised ph in the endosomal compartments (a so-called lysosomotropic e�ect), inhibits viral infectivity and various cellular processes such as autophagy. geroprotective activities of chloroquine li et al. reported the geroprotective e�ects of low-dose cq on aged rats 6 . these studies were inspired by a perspective article published at the height of the covid-19 pandemic by sargiacomo et al. in 2020 7 . this british group sought to explain the considerably higher mortality rate in covid-19 patients with advanced chronological age prior to the development of safe vaccines. they pointed out that the proposed sars-cov-2 viral receptors, cd26 and ace2, were associated with senescence 8, 9 . furthermore, at the time, azithromycin, quercetin, and hcq were proposed as "o�-the-shelf" treatments for sars-cov2 infection 10 . quercetin has a senolytic activity 11 and hcq is known to inhibit beta-galactosidase, a marker of senescence 12 . although early and questionable trials demonstrated some bene�t of hcq 13 for sars-cov2, subsequent well-controlled trials demonstrated limited, if any, bene�t 14, 15 . however, it is important to note that investigation of the role of the autophagy pathway in viral pathogenesis continues 16 . to investigate the geroprotective e�ects of hcq, li et al. treated 24-month-old sprague dawley male rats (n = 9) with cq (0.1 mg/kg, in drinking water) twice weekly for 5 months 6 . compared to controls (n = 13), the treated animals exhibited a 6% increase in medial survival and 13% increase in maximum lifespan (p = 0.02) 6 . related to the work of li et al., doeppner et al. (2022) also reported a chloroquine-mediated increase in the lifespan of rodents, in this case mice 17 . work by fifan et al. 18 and others 19, 20 demonstrated that the polyamine spermidine increased the maximum life span of c. elegans and the median life span of mice (~10%). because spermidine increases autophagy, they hypothesized that treatment with chloroquine, an inhibitor of autophagy, would shorten the lifespan of mice. remarkably, addition of chloroquine (50 mg/kg) to the drinking water extended overall lifespan of middle-aged berkeley pharma tech journal of medicine | 3 male nmri mice (n = 28; treatment initiated at age 500 days) by 11.4% (786 days) compared to control mice (n = 28; 689 days, p = 0.0002). median life span of the middle-aged mice increased by 11.4%. studies of chloroquine by these two groups and other data dating back a couple decades suggest the e�ect is real, although the precise mechanism is not clear. mechanistic studies carried out by both groups suggest a variety of mechanisms, though this "ball of yarn" is far from being unraveled. mechanism studies as noted above, cq treatment was initiated at age 500 days when the mice weighed on average ~35 gm. over the next 100 days, the control animals gained weight, reaching a maximum of 45 gm. in contrast, the cq-treated animals did not gain signi�cant weight over the duration of the experiment (almost 800 days). while the control animals consumed more liquid (p = 0.002), the food consumed by both groups was equal (~4 gm/day) ruling out the idea that cq somehow reduced food intake through an arti�cial "calorie restriction" e�ect. previously, cq (at 60 mg/kg) was reported to impair autophagosome-lysosome fusion rather than a�ecting the acidity and/or degradative activities of lysosomes 21 . in addition, hcq treatment was associated with "autophagy-independent" disorganization of the golgi and endo-lysosomal systems, with predominant golgi disorganization seen in kidney and intestinal tissues. the microtubule-associated protein i light chain (lc3) family of proteins (lc3a, b, c) 22 is the major structural protein family of autophagosome membranes. lc3-ii is generated by the conjugation of cytosolic lc3-i to phosphatidylethanolamine on the surface of nascent autophagosomes. doeppner et al. showed that the cq-treated mice exhibited a dose-dependent increase in lc3b-ii as well as p62 in the liver and heart, as con�rmed by transmission electron microscopy 17 . curiously, the treated animals exhibited increased liver glycogen and reduced serum insulin growth factor binding protein 3 (igfbp3), though no di�erence in igf-1, irs, or growth hormone levels 23, 24 . cq treatment elicited a decrease in glycogenolysis in the liver. berkeley pharma tech journal of medicine | 4 igfbp3 binds igf1 and igf2 to modulate their binding to the igf-1 receptor. while igfbp3 levels, like igfs, are regulated by gh, expression in the liver is gh-independent 25 . igfb3 mediates a plethora of other activities via its binding to a number of proteins and its interaction with various cell surface and nuclear signaling pathways. several labs have demonstrated a bene�cial role for reduced igfb3 in senescence 26, 27, 28 . li et al. found that low-dose cq (0.1 mg/kg given twice a week) extended the lifespan of aged rats when given in late middle age 6 . this dose is ~100-fold lower than doses typically given as a putative autophagy inhibitor in cancer 29 . qian et al. attributed the geroprotective e�ect of cq to atm activation leading to enhanced dna damage repair within their c. elegans and progeria mouse strain models 30 . others have shown that activation of atm by cq can slow atherosclerosis, improve insulin sensitivity, and rescue glucose intolerance in type 2 diabetes (t2d) 31, 32, 33 . li et al. studied the transcriptomes from cq-treated and control old rats to de�ne cq-induced, di�erentially expressed genes so-called “cq degs” across multiple tissues. for example, 40% of kidney-speci�c aged genes, 30% of small intestine-speci�c aged genes, and 20% of liver-speci�c aged genes were "rescued" by cq treatment. these changes were consistent with the reduced �brosis and improved histology of the kidneys compared to other tissues. contrary to expectation, cq treatment augmented a number of genes associated with various cardiac diseases (e.g. heart failure, cardiac arrhythmias, ischemic cardiomyopathy (caps2), hypertrophic cardiomyopathy, and cardiac arrhythmias (myh7). thus, cq treatment may actually mediate pro-aging activity in certain tissues 6 . summary a number of labs have studied several animal species to generate data suggesting that cq can mediate a pro-longevity e�ect. mechanistic studies indicate that multiple mechanisms are at play, each addressing a hallmark of aging 34 . while cq and hcq are familiar to cell biologists as prototypic berkeley pharma tech journal of medicine | 5 lysosomotropic e�ect tool compounds, numerous diverse studies highlight the plethora of other activities mediated by cq. isolated papers over the past 20 years have identi�ed non-autophagy-mediated e�ects of cq. for example, cq improves vasculogenesis 35 , insulin metabolism 31 , �brosis 36 , reduces oxidative stress 37 , modulates autophagic �ux 21 , boosts dna repair via stm 30 , and attenuates atherogenesis 33 to name a few. when we began looking into this story, we sought a unifying mechanism, pathway, target, etc. to explain the increased lifespans. unfortunately, at the present time, descriptive studies nominate many parallel mechanisms. the detailed transcriptome studies of li et al. show quite variable tissue speci�c patterns, with some that are bene�cial for health span and others that are consistent with cardiac diseases 6 . of course, investigating the geroprotective e�ects of cq using horvath or other methylation clocks might lead to interesting �ndings. medical implications as noted above, cq and hcq are widely used drugs 38 . the studies of li et al. and doeppner et al. support further, possibly human studies of these drugs. on october 8–13, 2013, a workshop entitled ‘interventions to slow aging in humans: are we ready?’ was held in erice, italy 39 . at the time, a number of interventions were deemed worthy of human clinical trials. since then, some of these ideas have been put to the test or are being tested . with regard to development of cq/hcq, suitable biomarkers will need to be validated. at low doses, cq or hcq seem to qualify for evaluation in humans. however, the safety pro�le of this class of drugs will need to be considered. for instance, periodic eye exams and cardiac monitoring are routine 40 at doses commonly used in rheumatology 38 . clearly these anti-malarials have come a long way from the rainforests of south america! berkeley pharma tech journal of medicine | 6 references 1. plantone d, koudriavtseva t. current and future use of chloroquine and hydroxychloroquine in infectious, immune, 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proteins: serum levels and hepatic messenger ribonucleic acid responses in humans1. the journal of clinical endocrinology &amp; metabolism . 1999;84(2):553-560. doi:10.1210/jcem.84.2.5466 26. hong s, kim m-m. igfbp-3 plays an important role in senescence as an aging marker. environmental toxicology and pharmacology . 2018;59:138-145. doi:10.1016/j.etap.2018.03.014 27. hong s, kim m-m. igfbp-3 plays an important role in senescence as an aging marker. environmental toxicology and pharmacology . 2018;59:138-145. doi:10.1016/j.etap.2018.03.014 28. yamada pm, mehta hh, hwang d, roos kp, hevener al, lee kw. evidence of a role for insulin-like growth factor binding protein (igfbp)-3 in metabolic regulation. endocrinology . 2010;151(12):5741-5750. doi:10.1210/en.2010-0672 29. solomon vr, lee h. chloroquine and its analogs: a new promise of an old drug for e�ective and safe cancer therapies. european journal of pharmacology . 2009;625(1-3):220-233. doi:10.1016/j.ejphar.2009.06.063 30. qian m, liu z, peng l, et al. boosting atm activity alleviates aging and extends lifespan in a mouse model of progeria. elife . 2018;7. doi:10.7554/elife.34836 31. emami j, gerstein hc, pasutto fm, jamali f. insulin-sparing e�ect of hydroxychloroquine in diabetic rats is concentration dependent . canadian journal of physiology and pharmacology . 1999;77(2):118-123. doi:10.1139/y98-146 32. schneider jg, finck bn, ren j, et al. atm-dependent suppression of stress signaling reduces vascular disease in metabolic syndrome. cell berkeley pharma tech journal of medicine | 8 metabolism . 2006;4(5):377-389. doi:10.1016/j.cmet.2006.10.002 33. razani b, feng c, semenkovich cf. p53 is required for chloroquine-induced atheroprotection but not insulin sensitization. journal of lipid research . 2010;51(7):1738-1746. doi:10.1194/jlr.m003681 34. lópez-otín c, blasco ma, partridge l, serrano m, kroemer g. the hallmarks of aging. cell. 2013;153(6):1194-1217. doi:10.1016/j.cell.2013.05.039 35. maes h, kuchnio a, peric a, et al. tumor vessel normalization by chloroquine independent of autophagy. cancer cell . 2014;26(2):190-206. doi:10.1016/j.ccr.2014.06.025 36. he w, wang b, yang j, et al. chloroquine improved carbon tetrachloride-induced liver �brosis through its inhibition of the activation of hepatic stellate cells: role of autophagy. biological and pharmaceutical bulletin . 2014;37(9):1505-1509. doi:10.1248/bpb.b14-00297 37. shen h, wu n, wang y, et al. chloroquine attenuates paraquat-induced lung injury in mice by altering in�ammation, oxidative stress and �brosis. international immunopharmacology . 2017;46:16-22. doi:10.1016/j.intimp.2017.02.020 38. schrezenmeier e, dörner t. mechanisms of action of hydroxychloroquine and chloroquine: implications for rheumatology. nature reviews rheumatology. 2020;16(3):155-166. doi:10.1038/s41584-020-0372-x 39. longo vd, antebi a, bartke a, et al. interventions to slow aging in humans: are we ready?. aging cell . 2015;14(4):497-510. doi:10.1111/acel.12338 40. wozniacka a, cygankiewicz i, chudzik m, sysa-jędrzejowska a, wranicz jk. the cardiac safety of chloroquine phosphate treatment in patients with systemic lupus erythematosus: the in�uence on arrhythmia, heart rate variability and repolarization parameters. lupus . 2006;15(8):521-525. doi:10.1191/0961203306lu2345oa berkeley pharma tech journal of medicine | 9 berkeley pharma tech journal of medicine correspondence: tanviy@berkeley.edu keywords: amyotrophic lateral sclerosis pathogenesis of als mesenchymal stem cells neurodegenerative disease sod1 riluzole astrocytes submitted: august 12, 2022 accepted: october 24, 2022 published: december 28, 2022 full open access creative commons attribution license 4.0 abstract amyotrophic lateral sclerosis (als) is a neurodegenerative disease caused by the degeneration of motor neurons in the brain and spinal cord, leading to muscle weakness, paralysis, and eventually death. exactly how and why motor neurons degenerate is still not clearly known, but certain pathogenetic mechanisms could explain the process. current treatments and interventions, including pharmacological approaches, are not highly targeted and only offer short-lived benefits. no cure for the disease exists to date. however, mesenchymal stem cells, which are adult stem cells that are highly accessible and can differentiate into different types of tissue, are currently being studied. this review offers insight into some of the pathogenetic mechanisms, potential treatments, and how mesenchymal stem cell treatment could be the potential cure for als. administration of mesenchymal stem cells as a therapeutic for amyotrophic lateral sclerosis by: tanvi yalamanchili, saahil mohta and donna tran 1. introduction als is a progressive neurodegenerative disease caused by the loss of brain connectivity to the muscles. as motor neurons degenerate or die, the muscles weaken, eventually leading to paralysis1. motor neurons are specialized brain cells located in the brain and spinal cord and control the motor function of the body. there are two types of motor neurons: upper motor neurons, which originate in the brain, and lower motor neurons, which send signals from the brain to the spinal cord and then to the target muscles2. the disease leads to the degeneration of upper motor neurons in the primary cortex and the degeneration of lower motor neurons in the brainstem and the spinal cord1. the word “amyotrophic” comes from greek roots that mean “without nourishment to muscles” and refers to the loss of signals nerve cells normally send to muscle cells. therefore, an important e�ect of als is the reduction of the blood-brain and blood-spinal cord barriers. these barriers are responsible for brain homeostasis, regulation of in�ux/e�ux transport, and protection from damage caused by the debris in the bloodstream7. als was �rst discovered in 1869 by the french neurologist jean-martin charcot but became more commonly known as lou gehrig’s disease after the famous baseball player lou gehrig was diagnosed with als at the age of 36. after being diagnosed, the yankee player retired soon after as he experienced symptoms such as loss of strength, and loss of coordination, making him un�t on the �eld3. as a progressive disease, als causes motor neuron degeneration to worsen until the patient passes away1. symptoms in the early stages of the disease include fatigue, poor balance, slurred speech, and tripping due to fasciculations (spontaneous muscle contractions/twitches) and atrophy (loss of muscle tissue)4. as some muscles paralyze, fasciculations continue, and joints start to become rigid and painful. walking, eating and breathing start to become di�cult. some people have uncontrolled crying or laughter. in the late stages of the disease, the patients’ voluntary muscles become paralyzed, and the muscles that help expand and contract the lungs for air are compromised, leading to respiratory failure5,6. berkeley pharma tech journal of medicine | 71 https://www.zotero.org/google-docs/?41igcv https://www.zotero.org/google-docs/?xobdid https://www.zotero.org/google-docs/?rk7qzn https://www.zotero.org/google-docs/?otzlqz https://www.zotero.org/google-docs/?mhiodq https://www.zotero.org/google-docs/?xylydq https://www.zotero.org/google-docs/?ur2rcy https://www.zotero.org/google-docs/?wna0u2 https://www.zotero.org/google-docs/?o2bl6i around 5000 people are diagnosed with als every year, and someone dies from the disease every 90 minutes. once diagnosed, the average life expectancy is two to �ve years. although it is most common for the disease to develop at ages greater than 60 years, it can occur at younger ages as well6. there are two types of als cases: familial als (fals) and sporadic als (sals)7. familial als occurs due to genetic inheritance, and accounts for only 5-10% of als cases. it has an earlier age of onset compared to sals6,7. it is known to primarily take form as a result of gene mutations such as sod1 mutations. over 20 gene mutations have now been identi�ed that play a role in als disease progression. silence superoxide dismutase 1 (sod1) is a major gene correlated with als. sporadic als on the other hand, although phenotypically indistinguishable from fals, is not genetically inherited. as the most common form of als, sals accounts for 90-95% of all cases6,7. although the cause for als is unknown, the growing evidence points to the possibility of disease occurrence in humans due to gene-environment interactions. exposure to certain toxic substances, viruses, or physical trauma could cause the disease6. certain genes, known as “susceptibility genes,” can trigger a neurodegeneration cascade upon interaction with certain environmental stimuli and/or factors. unfortunately, no de�nitive environmental risk factors have been identi�ed for als that can be replicated8. however, possible associations, based on clinical studies, have been identi�ed between autoimmune pathology, neuroin�ammation, head injury/trauma, metabolic disease, and als—speci�cally, the pathogenesis of als8. there are four main pathogenetic mechanisms that may explain the degeneration of motor neurons; these include mitochondrial dysfunction, glutamate excitotoxicity, oxidative stress, and in�ammation. current treatments include fda-approved drugs such as riluzole and edaravone9. in addition, many treatments are being tested to reduce the e�ect of these mechanisms, including but not limited to, sod1 gene therapy, and astrocyte transplantation9. however, the treatments mentioned above are berkeley pharma tech journal of medicine | 72 https://www.zotero.org/google-docs/?wa8ikg https://www.zotero.org/google-docs/?broken=48p26t https://www.zotero.org/google-docs/?broken=h5vp4v either short-lived or only target one of the pathogenetic mechanisms. of the treatments being studied, mesenchymal stem cells (mscs) stand out as a potential cure for als. mscs not only target the pathogenetic mechanisms to replace dead cells with healthy, di�erentiated cells but they also repair damaged cells10. stem cells are unique in their ability to migrate to damaged tissues and/or sites, stimulate tissue repair and regeneration, and di�erentiate in response to extracellular signals. mscs are adult stem cells that contain properties of self-renewal, immunosuppressive potential, and potency for trans-di�erentiation into motor neurons10. in particular, mscs have been shown to be a promising therapeutic for als in preclinical and clinical trials. mscs have the ability to stimulate tissue repair by di�erentiating into motor neuron cells, repair damaged neurons, and rebuild the brain-to-muscle connectivity10. this review o�ers insight into the di�erent pathogenetic mechanisms of als, current treatment options, and why mscs might o�er a cure. 2. the pathogenetic mechanisms of als 2.1. mitochondrial dysfunction there are a few pathogenetic mechanisms that may explain the disease progression. of those, mitochondrial damage is an important pathogenetic mechanism to consider. mitochondria is the major organelle responsible for many cellular processes such as producing energy, conducting cellular respiration, and maintaining calcium homeostasis8. this organelle is also known to play a key role in the process of apoptosis—a type of cell death activated by a series of molecular steps known as the caspase cascade. this pathway is often used by the body to remove unneeded or abnormal cells11. because the mitochondria play a key role in regulating apoptosis, damage to this organelle within motor neurons can alter its ability to regulate the process. in als patients, the mitochondria that are present in their spinal motor neurons, skeletal muscles, and intramuscular nerves are swollen and vacuolated. speci�cally, these swollen mitochondria activate the apoptotic caspase cascade, causing the death of the motor neurons12. berkeley pharma tech journal of medicine | 73 https://www.zotero.org/google-docs/?yu8gsj https://www.zotero.org/google-docs/?qetopm https://www.zotero.org/google-docs/?f2vwug https://www.zotero.org/google-docs/?zb1ptl additionally, deposits of the misfolded sod1 enzymes and altered protein expression in the mitochondria further alter the physiological function of the organelle9. a de�cit in atp production a�ects the cell’s energy homeostasis and causes a defective energy metabolism. alterations in the energy metabolism weaken the cell’s health. mitochondria with an altered structure or biochemical imbalance are prone to triggering apoptosis, thus leading to neuronal degeneration. mitochondria are responsible for regulating calcium levels as well. in als models, scientists have reported dysregulation of intracellular ca2+ in motor neurons. chronic elevations of cytosolic ca2+ due to neuronal hyperexcitability elicit an increase in mitochondrial ca2+ uptake and a decrease in mitochondrial ca2+ e�ux. when this condition is combined with altered mitochondrial calcium regulation, excitatory mitochondrial toxicity occurs, leading to dendritic mitophagy, mitochondrial depletion, and eventually dendritic atrophy and neurodegeneration13. overall, ca2+ dysregulation in motor neurons is at the cornerstone of disease progression of als13. 2.2. glutamate excitotoxicity glutamate excitotoxicity is another pathogenetic mechanism that can provide an explanation as to how motor neurons degenerate in als patients. glutamate is a principal excitatory neurotransmitter in the central nervous system (cns) and plays a key role in triggering action potentials14. neurotransmitters are chemical messengers that send signals from one nerve cell to a target cell, which could be a nerve cell, a motor cell, or a gland15. glutamate sends signals within the cns, which is made up of the brain and spinal cord. this neurotransmitter is originally generated in the presynaptic cleft terminal, exits through exocytosis, then moves across the synaptic cleft in order to activate postsynaptic receptors in the dendrites of postsynaptic motor neurons, which triggers action potentials16,17. the synaptic cleft is the space neurotransmitters travel through to send messages from one neuron to the next. glutamate reuptake transporters remove glutamate from the synaptic cleft to regulate the concentration of the neurotransmitter in the synaptic cleft, berkeley pharma tech journal of medicine | 74 https://www.zotero.org/google-docs/?hsgu91 https://www.zotero.org/google-docs/?iqrupn https://www.zotero.org/google-docs/?qbb4hd https://www.zotero.org/google-docs/?tz405s https://www.zotero.org/google-docs/?809vuj getting rid of excitatory stimuli along with it16. high glutamate concentration in the synaptic cleft leads to excitotoxicity, which can either result from increased synaptic levels of glutamate or greater glutamate sensitivity in the postsynaptic terminal9. overall, excitotoxicity occurs due to prolonged activation of glutamate receptors, which eventually leads to the degeneration and death of the motor neurons involved, induced through excessive neuronal �ring18. als patients reported having lower levels of glutamate transporters, which leads to an increased concentration of this neurotransmitter in the synaptic cleft and an overactivation of glutamate receptors, determining the level of excitotoxicity within the neurons7. as a key regulator of neuronal activity due to the role it plays in apoptosis and neurotransmitter release, calcium plays an important role in excitotoxicity19. the activation of glutamate receptors opens calcium channels, allowing calcium to enter the cell. the calcium-bu�ering proteins in motor neurons make motor neurons sensitive to excitotoxicity. if intracellular calcium levels get too high, mitochondrial damage may result and lead to the activation of biochemical processes that a�ect neuronal degeneration13. additionally, motor neurons are especially sensitive to excitotoxicity due to the high calcium permeability of these cells13. with more calcium able to enter the cells, intracellular calcium levels get high and lead to cell damage that induces the degeneration of motor neurons. therefore, increasing the concentration of glutamate transporters would decrease the synaptic glutamate concentration and reduce excitotoxicity levels to help prevent degeneration13. 2.3. neuroin�ammation and glial cells neuroin�ammation is an immune response in the cns to neuronal damage and plays an important role in the pathogenesis of als. an immune response is the body’s way of �ghting foreign substances to heal damage, but this can become harmful when accompanying a disease20. neuroin�ammation is characterized by overactivated microglia, reactive astrocytes, and in�ltrating immune cells such as lymphocytes into the site of neuronal injury9. as the �rst line of defense within the cns, microglia are activated and respond to signals released by damaged cells, more speci�cally berkeley pharma tech journal of medicine | 75 https://www.zotero.org/google-docs/?bi37br https://www.zotero.org/google-docs/?ua3ebt https://www.zotero.org/google-docs/?vjlwju https://www.zotero.org/google-docs/?2xfvqn https://www.zotero.org/google-docs/?i63i3p https://www.zotero.org/google-docs/?zyqh6h https://www.zotero.org/google-docs/?i63i3p https://www.zotero.org/google-docs/?knvyc7 https://www.zotero.org/google-docs/?p46f10 injured motor neurons21. glial cells refer to the cells present in large quantities in the cns and function as mediators of neuronal activity by surrounding and separating neurons; they can also play a part in regenerating damaged neurons22. astrocytes are a type of glial cells that are closely associated with motor neurons. however, when the damage becomes worse, astrocytes and motor neurons release mutated sod1 proteins, which activate pro-in�ammatory microglia. there are two kinds of microglia: m1 type microglia and m2 type microglia. m1 microglia are proin�ammatory and secrete ros and other neurotoxic molecules, which contribute to neuronal death9. by contrast, m2 microglia are anti-in�ammatory and secrete neurotrophic factors—molecules that enable neurons to maintain connections with other neurons23. during the early stage of the disease, microglia with an m2 phenotype are present24. as the disease progresses, however, astrocytes and motor neurons activate microglia that present with an m1 phenotype9. these m1 microglia release ros and other neurotoxic molecules that contribute to neuronal degeneration. additionally, astrocytes that are supposed to support neurons by using glutamate receptors to maintain low synaptic glutamate levels present di�erently in als patients who have astrocytes with the mutant sod1 gene, which instead induces motor neuron degeneration. they downregulate the glutamate transporters, which in turn limit the amount of glutamate to be uptaken by the receptors out of the synaptic cleft9. they also secrete in�ammatory molecules, and this in�ammatory response further contributes to the alteration and degeneration of motor neurons16. while neuroin�ammation is not the initial cause as to why motor neurons degenerate, it is a response to the damaged neurons that further exacerbates degeneration20. reducing the in�ammatory e�ects caused by overactivated microglia and reactive astrocytes could potentially be used to reduce neuroin�ammation and slow neuronal degeneration. 2.4. sod1 and oxidative stress oxidative stress is a cellular state motor neurons undergo that progresses and contributes to als pathogenesis. als patients have been shown to berkeley pharma tech journal of medicine | 76 https://www.zotero.org/google-docs/?ajduxz https://www.zotero.org/google-docs/?x8u5uf https://www.zotero.org/google-docs/?3iih5k https://www.zotero.org/google-docs/?wfrcoo https://www.zotero.org/google-docs/?ylvly8 https://www.zotero.org/google-docs/?pgk8rm https://www.zotero.org/google-docs/?fyu7me https://www.zotero.org/google-docs/?oxcbfp https://www.zotero.org/google-docs/?95hnmr have an increased level of oxidation, meaning they are in oxidative stress. oxidative stress occurs when reactive oxygen species (ros) accumulate within the cell or are being produced at a greater rate than they are removed, meaning the cell is unable to repair the stress25. this causes permanent damage to cell structures and macromolecules. the term ros embodies superoxide, ozone, and all other reactive species that contain oxygen26. sod1 is a major enzyme used to prevent oxidative damage28. most neurodegenerative diseases are characterized by the cellular accumulation of misfolded proteins. in als mutant sod1 proteins aggregate and alter normal cell function. the original function of the sod1 enzymes is to reduce the reactive oxygen species (ros) released from the mitochondria. they do this by catalyzing the conversion from superoxide into oxygen and hydrogen peroxide. this process plays an important role in the antioxidant defense of motor neurons16. however, in als, the mutant sod1 enzymes no longer function as important molecules for antioxidant activity, but instead create oxidative stress. they convert antioxidants into superoxide by donating electrons from antioxidants to molecular oxygen. in this case, oxidative stress is not caused by a decrease or loss in the function of sod1 but rather a new toxic function of the enzyme9. not only is oxidative stress linked to the progression of als, it also contributes to the other pathogenetic mechanisms as well. when mutant sod1 enzymes are present in the mitochondria, the mitochondria produces an abnormal amount of ros, which, in turn, exacerbates oxidative stress16. furthermore, this also determines excitatory damage and therefore motor neuron degeneration. berkeley pharma tech journal of medicine | 77 https://www.zotero.org/google-docs/?eimuju https://www.zotero.org/google-docs/?3w9fkd https://www.zotero.org/google-docs/?oiih9t https://www.zotero.org/google-docs/?cqzxx2 https://www.zotero.org/google-docs/?nlhgje https://www.zotero.org/google-docs/?ytaeys figure 1. the four pathogenetic mechanisms of als: mitochondrial dysfunction, glutamate excitotoxicity, inflammation, oxidative stress. 3. novel cell therapies 3.1. pharmacological approaches pharmacological therapies for als target neurodegeneration by stagnating or impeding the death of motor neurons. riluzole is the �rst fda approved drug for als after it initially proved to be successful in clinical trials in the 1990s28. common brand names of riluzole are exservan, rilutek, and tiglutik. since its successful administration, new drugs including edaravone and amx0035 have been studied to evaluate their e�cacy against predecessors. riluzole functions by protecting motor neuron cell lines from glutamate stress. glutamate is an essential neural excitatory neurotransmitter which facilitates proper signal propagation throughout the central nervous system29. imbalances in glutamate levels can a�ect nerve health and contribute to cell death. like many neurodegenerative therapeutics, riluzole prevents excitotoxicity and slows degeneration of motor neurons. the most prominent clinical trials involving solely riluzole as a therapeutic for als are from the late 20th century where the drug was initially found to be a successful treatment. in a 1994 clinical trial, conducted before the fda approval of riluzole in 1995, riluzole slowed als progression by inhibiting berkeley pharma tech journal of medicine | 78 https://www.zotero.org/google-docs/?id0erx https://www.zotero.org/google-docs/?xmwe24 disease processes in the cns30. factors including death rate and deterioration of muscle strength were higher for the placebo group than those treated with riluzole in a one year period. the study also noted that a subsequent experiment on dosage should be conducted to which researchers in 1996 tested the impact of riluzole dose-ranges on als patients to determine that the prescription amount with the best bene�t-to-risk ratio is a 100 mg dose, further con�rming the success of riluzole. while a complete understanding of riluzole’s mechanism of action has not been established, properties including its ability to inhibit glutamate release and inactivate voltage-gated sodium channels associated with damaged neurons are attributed to its e�ectiveness for als31. neuroprotective drugs like riluzole function by binding to voltage-gated sodium (nav) channels to keep them in an open and inactivated conformation32. it also inhibits na+ currents which regulate excitability imbalances to prevent neuronal damage. a common brand of riluzole is rilutek® which o�ers the medication in the form of a prescription pill. in 1995, it became the �rst drug to receive fda approval for a treatment of als as it proved to “extend survival and/or time to tracheostomy” in clinical trials33. in the original clinical trials, it was estimated that the patients treated with riluzole have longer survival times of two to three months. however, a retrospective statistical analysis on population studies of als compared the mean survival rate between treated and non-treated patients to suggest that riluzole may even extend survival by six to 19 months, longer than the original expectancy34. although riluzole is a favorable option, advances in pharmacology have been geared towards new drugs and additive treatments to improve e�cacy. in may of 2017, the fda approved the second targeted drug intended to slow als progression: edaravone. the most common formulation is radicava iv which has shown signs of selective e�ectuality35. unlike riluzole which targets excitotoxicity, edaravone is an antioxidant– a compound inhibiting lipid oxidation to prevent the production of free radicals and chain reactions that may induce cell damage36. by reacting with radical species, this antioxidant captures unstable reactive oxygen species because a large concentration of these molecules contributes to the berkeley pharma tech journal of medicine | 79 https://www.zotero.org/google-docs/?qtliq5 https://www.zotero.org/google-docs/?zcoayd https://www.zotero.org/google-docs/?4izppz https://www.zotero.org/google-docs/?pwqioz https://www.zotero.org/google-docs/?ei1suf https://www.zotero.org/google-docs/?wankcq https://www.zotero.org/google-docs/?zfweia degeneration of motor neurons. an active phase 3 clinical trial is currently evaluating the safety and e�cacy of oral edaravone for als patients based on the als functional rating scalerevised (alsfrs-r) score which observes lifestyle changes such as independence, speech, and mobility37. the �rst version of this drug was administered via intravenous infusion, a drug-delivery method administered directly into a vein using a cannula, which received fda approval on may 5, 2017. the doses are given in slow and set treatment cycles to observe for signs of hypersensitivity. the infusion can take up to an hour and must be performed by a healthcare provider. on may 12, 2022, radicava oral suspension was fda approved, which grants the user more �exibility in administering treatment. prior to radicava’s oral formulation, di�erences between the two treatments such as absorption and distribution were more observable38. while riluzole is in a more convenient tablet form, injections of edaravone do not face issues with absorption, and there may be more transport across the brain blood barrier (bbb), a semipermeable membrane facilitating the transport of molecules and substances from the bloodstream to the brain. cohort studies have also observed the e�ects of using intravenous edaravone in conjunction with riluzole, but there was no improvement in als in comparison to standard riluzole therapy39. since the issue of convenience is no longer a dividing factor, new research is geared towards testing the variation between both oral forms in e�cacy for als treatment. another novel drug that is currently being tested in clinical trials is amx0035, used as an oral combination therapy of sodium phenylbutyrate (spb) and taurursodiol (turso) to block cell death mechanisms37. amx0035 was developed by amylyx pharmaceuticals, inc., and on september 29, 2022, the fda approved this drug as the newest therapeutic for als41. it will be sold to patients in the u.s. under the name relyvrio, and it proved successful in clinical trials, with patients reporting less als symptoms under the treatment in comparison to a placebo40. in the brie�ngs between the company and the fda, it was also noted in the results of their clinical trials that patients who were randomly assigned to receive amx0035 also showed statistically signi�cant increases in survival duration of around �ve months longer than the placebo. this value is comparable to berkeley pharma tech journal of medicine | 80 https://www.zotero.org/google-docs/?o3bfwg https://www.zotero.org/google-docs/?imtdv2 https://www.zotero.org/google-docs/?07j7p8 https://www.zotero.org/google-docs/?zjvu0o https://www.zotero.org/google-docs/?qszaxt the survival bene�ts of riluzole and edaravone which are three and six months, respectively. after nearly a decade of developing a new combination drug for als, amylyx’s co-founders justin klee and josh cohen became successful at providing the als community with newfound hope for an improved lifestyle. this drug targets two of the various mechanisms contributing to disease propagation: er stress and mitochondrial stress. spb alleviates toxicity from er stress, increasing the number of heat-shock proteins (hsps) which provide neuroprotection42. likewise, turso reduces mitochondrial stress by increasing the mitochondrial selectivity and cell's threshold for apoptosis. in a clinical trial conducted by the northeast amyotrophic lateral sclerosis consortium (neals) and amylyx pharmaceuticals, patients received a 2:1 ratio of spb to turso dissolved and taken orally or through a feeding tube. after the six month period of daily medication, there was a reduction in the rate of dysfunction based on the alsfrs-r. als patients involved in this trial were still taking riluzole, edaravone, or both, but researchers did not observe notable di�erences in e�cacy. in the near future, more information on amx0035 for als treatment may be obtained if it receives fda approval and becomes accessible to als patients as a viable option alongside riluzole and edaravone. each als therapy has its own bene�ts and caveats, and due to the diversity in als types and als patients, one form may prove to be more e�ective in reducing neurodegeneration than other. as the �rst drug for als, riluzole remains more e�ective with extensive results from clinical trials to prove its e�cacy. advancements in the development of newer drugs such as edaravone and amx0035 provide increased con�dence in the possibility of slowing the rate of als progression. similarly, stakeholders are increasingly interested in new therapies for increased access and improvements on the als rating scale. while none of the drugs approved for als are a complete cure because they solely function to delay disease propagation, new approaches are still being tested for factors including an increase in survival probability, safety, and long-term consequences, riluzole still remains the most prevalent treatment. berkeley pharma tech journal of medicine | 81 https://www.zotero.org/google-docs/?koqact table 1. a comparison table of the drugs riluzole, edaravone, and amx0035 for als 4. gene therapy since pharmacological therapeutics have yet to eradicate and cure als, gene therapy approaches have also been explored as a potential solution. gene therapy can be divided into two types: (1) non-viral gene therapy where nucleic acid sequences are delivered to the host cell for pathological modi�cation; and (2) viral gene therapy in which genetically-modi�ed viruses are used as viral vectors to deliver sequences. antisense oligonucleotide (aso) drugs belong to category one and are used in pre-clinical trials of sod1-fals. superoxide dismutase 1 (sod1) is the �rst of 30 als-related genes currently discovered. oxidative damage is a result of an abundance of ros such as superoxide in the cell, making it unable to prevent cellular stress. sod1 prevents oxidative damage and reduces superoxide leakage from mitochondria by catalyzing the conversion of superoxide into oxygen and hydrogen peroxide. mutations in the sod1 gene contribute to the pathogenesis of familial als and accounts for 15–20% of fals cases, and begin with the misfolding of the gene which causes it to be degraded by the ubiquitin/proteasome system (ups)18. however, once a large concentration of misfolded proteins accumulate, this disrupts proteostasis–the regulation of proteins from synthesis to degradation–and retriggers autophagocytosis to increase the number of autophagosomes for the removal of dysfunctional cellular constituents16. over time, an accumulation of mutated sod1 proteins induces cell stress responses, resulting in disease pathogenesis. a buildup of misfolded mutant sod1 proteins in the mitochondria can adversely a�ect physiological function of the organelle. speci�cally, studies have noted irregularities in the berkeley pharma tech journal of medicine | 82 https://www.zotero.org/google-docs/?bokk7b https://www.zotero.org/google-docs/?3w43hl production of atp and ros which signal cell survival and cell death, energy and calcium homeostasis, and apoptosis in mitochondrial transport along axons9. an aso used to target sod1 mrna is tofersen (biib067), previously ‘ionis-sod1rx.’ it relies on ribonuclease h (rnase h), an endoribonuclease enzyme that cleaves the rna from the rna/dna hybrid, to degrade sod1 mrna and prevent the production of toxic sod1 proteins18. in a phase 1/2 clinical trial, als patients with a sod1 mutation received multiple doses of tofersen, and results showed that patients treated with the greatest concentration of the aso had lower sod1 protein levels by 36%. also, a decline in the reduction in the alsfrs-r score was observed. however, since the drug was delivered via an intrathecal lumbar puncture, adverse e�ects including headache and back pain were common consequences. in another approach, researchers relied on a lentivirus encoding for an rna silencing (sirna) gene that catalyzes the selective degradation of sod1-mrna. the injection of this virus in the muscle or directly in the spinal cord of transgenic mouse models of als showed reduced sod1 expression delaying the neurodegeneration, but data about the slowing down of the disease progression and survival is controversial and requires further study9. gene therapy targeting the many als-related genes can prevent disease proliferation by directly preventing mutations at the source. sod1 mutations can lead to excitotoxicity, mitochondrial dysfunction, and er stress which increases the likeliness of motor neuron degeneration. the appeal of this form of treatment is that the di�erent genes can be individually suppressed by aso’s like tofersen to reduce the degree of toxic variants. to optimize gene therapy for als, the pathological mechanisms and roles that the genes play must be well understood and develop a barrier for further propagation of neurotoxic factors. the limitations and challenges associated with this approach are that it is mostly used to treat fals cases and very few sals cases. berkeley pharma tech journal of medicine | 83 https://www.zotero.org/google-docs/?jcbxsl https://www.zotero.org/google-docs/?i7urgl https://www.zotero.org/google-docs/?xjpnwz figure 2. pathways linking sod1 mutations to als pathology 5. astrocyte transplantation the most abundant glial cells within the cns are astrocytes, a form of brain cell that regulates neural functions through signal di�usion. correspondingly, they are necessary in the cns for neuronal survival. any changes including stress or injury to the cns cause astrocytes to transition from healthy astrocytes to a1-type neurotoxic astrocytes which promote degeneration of motor neurons43. the progression in toxicity of healthy motor neurons causes a loss in homeostatic functions or gain of toxic functions. astrocytes of als patients are characterized as a1-type, and targeted therapies to control the proliferation of neurotoxic astrocytes are currently being tested for als. astrocyte-targeted therapy involves restoring the function of neurotoxic astrocytes through the transplantation of healthy astrocytes43. while this decreases the concentration of misfolded proteins and aids in neuroprotection, there is also the risk that these healthy astrocytes can undergo the transition to a1-type astrocytes when they are introduced to the cns of als patients. this new environment can be toxic to the new cells due to the large accumulation of misfolded proteins. although it has berkeley pharma tech journal of medicine | 84 https://www.zotero.org/google-docs/?mkli6i https://www.zotero.org/google-docs/?ibxc2b been deduced that astrocytes secrete neuroprotective factors that di�use to the motor neurons, later mechanisms including their attachment and long-term survival need to be further explored. in a phase 1/2a trial, researchers from cedars-sinai medical center are hoping to �nd a long-lasting solution to als by testing the e�ectiveness of their engineered neural progenitor cells which di�erentiate into astrocytes inside the body to secrete the gdnf protein, promoting astrocyte survival44. cns10-npc-gdnf are progenitor cells which will release paracrine factors inside the body. as they release gdnf, this neuroprotective factor is also transferred to dying motor neurons in the motor cortex. two doses of adult stem cells will be delivered to the cortical area of the human primary motor cortex (m1) as researchers observe for signs of repaired upper motor neurons and improvement in hand function. signs of potential e�cacy will be determined by comparing the di�erence in rate of loss of hand strength between the two sides. studies relating als-genes to astrocyte therapies noted that msod1-expressing astrocytes induced selective death of spinal motor neurons, so a potential solution could be the transplantation of healthy astrocytes or selective silencing of the msod1 gene in astrocytes. while many hypotheses are developed, it is believed that transplanting healthy astrocytes will generate a better environment for the motor neurons. therefore, the molecular pathology of astrocytes in als patients needs to be further studied. once the healthy cells are delivered, the pathway it takes for di�usion and speci�c function remains unknown45. while this treatment is appealing because it has the potential of restricting the propagation of toxic cells and slowing degeneration, transplanted cells can still transform into a1 reactive astrocytes once they are introduced to a more neurotoxic environment. under excitotoxic conditions, als neurons and astrocytes can be more sensitive to neuromodulation, so it is pivotal to developing novel cell therapies for als. 6. stem cells because the focus of this paper is the application of mesenchymal stem cells (msc) as a therapeutic for als, it is imperative to broadly consider stem berkeley pharma tech journal of medicine | 85 https://www.zotero.org/google-docs/?6icqrh https://www.zotero.org/google-docs/?4zrbtd cells, their capabilities, and their advances in pertinence to regenerative medicine (a process by which healthy cells are introduced in order to replace cells that have been adversely impacted by disease) and disease therapeutics46. stem cells are cells from which di�erentiated daughter cells, or cells with a specialized function, can be engendered within the human body. daughter cells are cells that result from the mitotic or meiotic reproductive division of a singular cell, and possess a speci�c function; examples include epithelial cells, adipose stromal cells, and smooth muscle cells. furthermore, these cells are undi�erentiated and are typically sourced from adult, fetal, and embryonic tissue, in addition to di�erentiated somatic cells47. there are four main forms of stem cells: adult, embryonic, induced pluripotent, and mesenchymal48. 6.1. adult stem cells adult stem cells are commonly regarded as tissue-speci�c or somatic stem cells, and are found in a wide range of tissues, including the brain, heart, liver, and bone marrow. they can facilitate the creation of various cell types upon injury, but the degree to which this di�erentiation can occur is limited in comparison to embryonic stem cells. adult stem cells are multipotent, or limited to di�erentiating into the specialized cell type in the tissue or organ of residence. they can also be utilized for general maintenance of tissue and organs and have the capacity to maintain and repair organs and bodily tissue, which can give rise to cell regeneration, thus indicative of the role stem cells can play within regenerative medicine49, 50. 6.2. embryonic stem cells embryonic stem cells (escs) are conventionally sourced from embryos three to �ve years of age and are more versatile in comparison to adult stem cells because they are pluripotent, or capable of giving rise to a variety of cell types not limited to their organ or tissue of residence. as a result, they are more versatile and have the capability of tissue/organ regeneration and repair51. however, embryonic stem cells have a higher probability of being rejected by the host, and their use is ethically controversial by virtue of the berkeley pharma tech journal of medicine | 86 https://www.zotero.org/google-docs/?gwlwit https://www.zotero.org/google-docs/?kmvrcs https://www.zotero.org/google-docs/?3upw7h https://www.zotero.org/google-docs/?4lcndl https://www.zotero.org/google-docs/?gvqryq https://www.zotero.org/google-docs/?sknnii fact that the destruction of human embryos is necessary in order to obtain escs52. 6.3. induced pluripotent stem cells induced pluripotent stem cells are a class of pluripotent cells that are commonly derived from blood or skin cells and have been genetically engineered into an embryonic-like state in order to behave as escs53. mesenchymal stem cells, the subject of this paper, are a subgroup of adult multipotent stromal cells that are derived from adipose tissue, umbilical cord tissue, amniotic �uid, and bone marrow, and have the capacity to di�erentiate into numerous cell lines54. stromal cells comprise the connective tissues that surround bodily tissues and organs and are separate from the stroma. table 2. a comparison of adult, embryonic, induced pluripotent, and mesenchymal stem cells stem cells are especially signi�cant and have been the subject of intensive study due to the fact that they are the only cell that can naturally develop and di�erentiate into varying cell types primarily through the autocrine production of immunomodulatory molecules, bioactive molecules stored within extracellular vesicles, and growth factors such as the vascular endothelial growth factor (vegf)47. as a result, stem cells could, theoretically, limitlessly participate in cell division in order to replace cells within damaged tissue55. they have the unique ability to di�erentiate in response to the microenvironment in which they have been directed berkeley pharma tech journal of medicine | 87 https://www.zotero.org/google-docs/?xaaej1 https://www.zotero.org/google-docs/?gmrvek https://www.zotero.org/google-docs/?hgkexh https://www.zotero.org/google-docs/?dpwwmn https://www.zotero.org/google-docs/?avsmik toward56. it is also important to consider, though, that stem cells need to be carefully selected according to their biological capacity to survive within the host organism (where the cells are transplanted), and their ability to di�erentiate and migrate to various tissues. the upsides and downsides of each form of stem cells must be evaluated upon consideration for disease treatment or regeneration. on an interdisciplinary scale, however, because the treatment options for neurological diseases remain heavily limited and drug approval rates remain poor in comparison to other therapeutic areas, the implementation of stem cells carries immense potential for patients a�icted with disorders such as als. furthermore, numerous degenerative diseases involve an improper functioning or loss of specialized cells within the body and there is an imbalance between the supply and demand of potential donors and their cells within their organs or tissues. speci�cally, the supply of donors is currently unable to ful�ll the demand of replacement cells. as such, stem cells could serve as a solution, continuously producing viable cells that could replace damaged or injured tissues and thus address a number of a�ictions55. to date, stem cells have been implemented for the treatment of diseases such as amyloidosis, germ cell tumors, and certain cancers through autologous (obtained from the same individual) and allogenic (obtained from a source outside the individual, such as a donor) transplantation. with cancers, speci�cally, stem cells have been implemented to replace cells that have been adversely impacted by the cancer pathogenesis itself or chemotherapy. while stem cells are continually being utilized for the treatment and maintenance of a myriad of diseases, further research is necessary to fully elucidate the pathogenetic mechanisms by which stem cells function, to properly isolate and identify stem cells to avoid immunological rejection to the greatest extent possible, and to address the predisposition of induced pluripotent stem cells to tumorigenesis, or the formation of tumors in vivo57, 55. 6.4. mesenchymal stem cells mesenchymal stem cells (mscs) are a class of stem cells multipotent and non-hematopoietic stromal cells which are conventionally derived from adult and fetal tissue, such as skeletal muscle, the placenta, blood, adipose, and the umbilical cord7. mscs have been demonstrated to take residence berkeley pharma tech journal of medicine | 88 https://www.zotero.org/google-docs/?m9egga https://www.zotero.org/google-docs/?bopw39 https://www.zotero.org/google-docs/?evkrhb https://www.zotero.org/google-docs/?chmphx https://www.zotero.org/google-docs/?efbkl6 within most connective tissues in the human body. this high availability within human tissues makes mscs accessible as a disease therapeutic. since mscs are multipotent, they have a high di�erentiation plasticity, or a capability of trans-di�erentiating into multiple specialized cell types58. transdi�erentiation is the process by which a cell can be made to di�erentiate from one cell type into another through genetic reprogramming. for instance, they are able to di�erentiate into nerve, heart muscle, liver, and endothelial cells, in addition to the germ layers the ectoderm, mesoderm, and endoderm. furthermore, mscs are capable of di�erentiating into neural, glial, and astrocyte-like cells, which can be signi�cant in pertinence to their utilization as a treatment for als and similar neurodegenerative diseases as this indicates their ability to e�ectively function as motor or dopaminergic neurons. this in-vitro di�erentiation potential of mscs to generate a myriad of cell types that could replace lost cells within injured tissue has been a signi�cant justi�cation for the use of mscs themselves59. mscs possess several advantages which increase their potential of being implemented in disease therapeutics, such as an ability of self-renewal (the process by which stem cells continually divide to produce additional undi�erentiated stem cells), their ease of isolation, and a decreased susceptibility to tumorigenesis (formation and accumulation of malignancy and malignant properties within cells). in addition, there is no speci�c immunosuppressive treatment that is necessary or otherwise warranted upon the transplantation of mscs, as demonstrated by the fact that studies have indicated that mscs are immunosuppressive both in vivo and in vitro9,60. typically, mscs employ this immunosuppressive e�ect on t cells, which are a major component of the adaptive immune system. most signi�cantly, however, the therapeutic e�ects of mscs are largely associated with their immunomodulatory e�ects, which include mscs’ regulation of lymphocytes that are associated with the innate and adaptive immune system8. immunomodulation is the modulation or alteration of the immune system, as determined by the activation or suppression of immunomodulating berkeley pharma tech journal of medicine | 89 https://www.zotero.org/google-docs/?r2qmvd https://www.zotero.org/google-docs/?udimnl https://www.zotero.org/google-docs/?znilkj https://www.zotero.org/google-docs/?fga3bi https://www.zotero.org/google-docs/?yxjjze agents such as cytokines, vaccines, and monoclonal antibodies. speci�cally, mscs are able to regulate t cell proliferation and have the potency to upregulate regulatory t cell function, thus simultaneously highlighting their immunosuppressive abilities because the regulatory t cells function to suppress the body’s immune response, allowing for a maintenance of homeostasis. for this reason, there have been proposals for the application of mscs to the treatment of autoimmune diseases, which are a category of diseases in which healthy cells are attacked by the body’s immune system. mscs also regulate their immunomodulatory function in accordance with micro environmental and in�ammatory conditions, indicative of the high level of therapeutic plasticity of mscs (they have a potential of being applied in order to treat a myriad of medical conditions). mscs function upon transplantation through various methods. one such method is cell fusion, which is the process of a cell interacting with nearby cells in order to form a multicellular assemblage with a universal function. for instance, the fusion of mscs with rodent purkinje cells was observed, and these fused cells were implemented to improve the therapy of neurodegenerative disorders and disorders involving the cerebellum56. furthermore, mitochondrial transfer is a unique mechanism of action of mscs, and is the process in which cells transfer mitochondria to neighboring injured cells (thus replacing damaged mitochondria and aiding in the restoration of cellular function). additionally, the secretion of extracellular vesicles (exosomes) by mscs is a similarly important mechanism of action. msc exosomes are secreted into the extracellular space with multivesicular bodies within mscs fused with the cellular membrane59. extracellular vesicles have the potency to transport essential macromolecules such as genetic material to neighboring cells through endocytosis to maintain physiological homeostasis, and as such, carry a potential therapeutic application. studies have demonstrated that the exosomes secreted by mscs possess anti-in�ammatory and regenerative properties in pertinence to traumatic brain injury, stroke, perinatal brain injury, and wound healing61, highlighting that msc-derived exosomes are crucial to the proper function and therapeutic properties of mscs as a whole. berkeley pharma tech journal of medicine | 90 https://www.zotero.org/google-docs/?vgfg1q https://www.zotero.org/google-docs/?pqc90s https://www.zotero.org/google-docs/?wi3wxm there are certain characteristics of mscs that aid in their applications within regenerative medicine56. mscs have the ability to form colonies, which can be measured by the term “colony forming unit” (cfu). a cfu is a measure of the number of viable cells within a sample that are able to multiply through binary �ssion. this is one of the primary functional characteristics of mscs, which highlights their ability to proliferate upon transplantation and which would reasonably eliminate (1) the need for consecutive transplantations and (2) possible supply shortages associated with the availability of mscs. in addition, while it has been discussed above that mscs have a high di�erentiation plasticity as compared to other stem cells, mscs have the unique capability of undergoing trilineage di�erentiation into adipocytes (fat cells which store energy as fat), chondrocytes (key component of cartilage tissue), and osteocytes (bone cells which make up approximately 90-95% of cells within bone tissue). furthermore, the telomerase activity of mscs is signi�cant as their activity is typically augmented within mscs. telomeres are the end caps of chromosomes which shorten with cell replication and division, and similarly, telomerase is the enzyme responsible for the region of the telomeric region at the 3’ ends of chromosomes. telomeres also protect chromosomal ends from degradation and improper dna recombination (which can result in apoptosis). as such, an increased telomerase activity is directly correlated with a longer cell lifespan. since the telomerase activity is augmented within mscs, mscs have a longer period of survival. the telomerase activity and the telomere lengths can also be analyzed as a quality control measure to aid in the selection of mscs for therapeutic applications. studies have demonstrated that mscs have the capability and potential of being trans-di�erentiated into neuron-like cells; however, the speci�c protocols for inducing this di�erentiation involve toxic chemicals and thus cannot directly be utilized within humans. as such, further research and study of the di�erentiability of mscs is warranted when considering their potential as a viable therapeutic for als and more broadly, neuron degeneration. berkeley pharma tech journal of medicine | 91 https://www.zotero.org/google-docs/?iadymz 7. mesenchymal stem cell treatment for als mesenchymal stem cells could be the potential cure for als not only due to its ability to stimulate tissue repair by di�erentiating into motor neurons but also by repairing damaged cells within the body. mscs are especially advantageous compared to other stem cells due to many of its properties. one of these advantages include its great availability—they can be derived from adult tissue such as bone marrow tissue, skeletal tissue, or adipose (fat) tissue or can also be derived from fetal tissue such as the placenta or umbilical cord. because of these speci�c tissue sources, mscs can be found in abundance, especially due to the medical waste created from these tissues. for example, adipose tissue can be obtained from liposuction while the placenta is delivered and most of the time gotten rid of. however, bone marrow tissue—the soft, spongy tissue found inside the bone and contains stem cells62—can be obtained in the most abundance and is even possible to be extracted from the patient itself to mitigate the chance of rejection by the host. although mscs are multipotent, they can exhibit pluripotent properties63. there is growing evidence that mscs can transdi�erentiate into motor neurons. one study shows that a cocktail of basic �broblast growth factor (bfgf) and retinoic acid (ra) with human mscs (hmsc) provide the most e�ective and e�cient transdi�erentiation of msc into neural cells64. two days after injection of these cells, these cells expressed glial markers, and 12 days after injection, 90% of the hmscs di�erentiated into cells that expressed neuronal markers, which also include transcription factors linked to aid the development of the di�erentiated neurons65. this con�rmed the ability of mscs to be able to di�erentiate into neurons, providing a method to stimulate tissue repair. after being injected intrathecally, in order for the mscs to di�erentiate and develop in the correct area, als allows for an e�ective stimulus. a key advantage of mscs over similar forms of stem cells is their ability to migrate towards in�ammatory foci through the expression of chemokine receptors, and as a pathogenetic mechanism of als, in�ammation provides mscs the knowledge of the damaged site66. chemokines are a family of cytokines which stimulate white blood cell migration to sites of infection and thus berkeley pharma tech journal of medicine | 92 https://www.zotero.org/google-docs/?17ayui https://www.zotero.org/google-docs/?wftqzr https://www.zotero.org/google-docs/?8teuq4 https://www.zotero.org/google-docs/?flgw3j https://www.zotero.org/google-docs/?zsm4np play an integral role in the homeostasis of the immune system67. chemokine receptors interact with the chemokines, themselves, in order to manage the function of the immune system; it is thus evident that the expression of chemokine receptors by mscs plays a key role in the reduction of in�ammation to maintain equilibrium within the immune system. figure 3. mesenchymal stem cells are extracted from bone marrow and are intrathecally injected into the lateral spinal cord, where they can transdifferentiate into motor neurons. mscs not only stimulate tissue repair by di�erentiating into motor neuron cells but can also repair damaged cells and prevent motor neuron degeneration from taking place to cure the disease. human and animal mscs showed promising results in regard to using these stem cells for neural repair and not just to replace the damaged motor neurons. one way mscs do this is by producing trophic factors, which can repair or regenerate tissue68,69. trophic factors are helper molecules that allow neurons to develop and maintain connections with their neighbors. they help establish the connection and allow motor neurons to communicate with their target cell, skeletal muscle cells. they help maintain and support motor neurons and also aid in repairing damaged motor neurons back to a healthy condition23. however, although animal trials were successful in proving the healing properties of these factors, human trials did not produce the same success and need to be further researched due to the factors’ inability to reach the target cell but large amounts being too harmful. another way mscs help repair damaged motor neurons is through mitochondrial transfer. the use of mitochondrial transfer can aid in tissue regeneration and repair, and can also restore the bioenergetic needs of damaged cells70. this transfer primarily occurs through the formation of intracellular nanotubes, gap junctions, and microvesicles for cell-cell berkeley pharma tech journal of medicine | 93 https://www.zotero.org/google-docs/?6l8e8b https://www.zotero.org/google-docs/?xiwwfe https://www.zotero.org/google-docs/?xvmwjo https://www.zotero.org/google-docs/?sycupg https://www.zotero.org/google-docs/?wrre0q communication. numerous studies have demonstrated that there has been an upregulation of mitochondrial respiration and atp levels with a simultaneous reduction of oxidative damage upon the culturing of mscs with injured tissue, indicative that mitochondrial transfer is an important means by which cellular functions and energies can be restored. there is also growing evidence to suggest that mscs have anti-apoptotic e�ects, which limits the extent of damage to improve tissue healing. although more research has to be done in this area, exosomes are used to release factors that indirectly have this e�ect69. the secretion of exosomes is an important property of als, which allows for the reduction of the e�ect of pathogenetic mechanisms in order to heal damaged cells. exosomes have the ability to cross the blood-brain barrier, which is a unique ability and is signi�cant for neurological disorder therapeutics/treatment. they secrete therapeutic factors like cytokines that limit in�ammation as well as trophic factors that help maintain brain and spinal cord connectivity to muscle cells. additionally, studies have shown that msc derived exosomes mediate angiogenesis8, and this enhancement of angiogenesis and myogenesis promotes muscle regeneration, which is crucial for als treatment in which the motor neurons are implicated. studies have indicated that mscs have successfully improved the pathological features and development of als and display a high level of therapeutic plasticity7. therefore, mscs would be a promising cure as it allows for di�erentiated motor neurons to replace damaged cells while also providing mechanisms for the damaged cells to repair themselves. 8. practical applications there have been numerous studies conducted regarding mscs and their applications as a therapeutic for als, many of which have o�ered a greater insight into potential applications of mscs in a clinical setting. clinical trials have indicated that msc cells, upon their transplantation, are capable of inducing a substantial upregulation of neurotrophic factors, some of which include the glial-derived neurotrophic factor and the basic �broblast growth factor. neurotrophic factors are crucial with respect to the consideration of potential treatments for als, since it has been berkeley pharma tech journal of medicine | 94 https://www.zotero.org/google-docs/?vvsgzt https://www.zotero.org/google-docs/?m5mjza https://www.zotero.org/google-docs/?inkv8k demonstrated that they prolong motor neuron survival in als. a uni�ed delivery of various neurotrophic factors has been shown to have a synergistic e�ect on the pathogenesis of als itself, and mscs induced with neurotrophic growth factors are demonstrated to display protective e�ects in many neurodegenerative disease models71. in a similar clinical trial, a medium based msc induction process was devised in which mscs were induced with neurotrophic factors, which possessed the ability of an enhanced secretion of gdnf, vegf (vascular endothelial growth factor), the hepatocyte growth factor, and brain-derived neurotrophic factors71. upon the transplantation of these neurotrophic factor induced mscs, no signi�cant adverse e�ects were noted, and a higher secretion of neurotrophic factors was observed, which directly correlated with a substantial improvement in the monthly rate of decline of als pathogenesis. similarly, it has been found that when mscs have been cocultured with healthy tissues, there is a reduction in oxidative damage, an upregulation of mitochondrial respiration (indicative of proper cellular function and mitochondrial transfer), and an upregulation of atp levels70. in general, though, the practical and clinical applications of mscs are attributed to their di�erentiation plasticity, their capability of the secretion of bioactive compounds as dependent on the microenvironmental condition (playing a role in immunomodulation, reduction of in�ammation, and regeneration), their ability to migrate to sites of injured tissue upon intravenous injection, and their immunomodulatory capacities72. 9. future directions mscs do present a few challenges with regard to their transplantation and application as a reliable disease therapeutic that need to be further researched before use. for example, challenges can arise in the characterization of mscs, because after they are isolated and selected, it must be ensured that the mscs are pure, active, undi�erentiated, and functional for their clinical application. in addition, when mscs are utilized in cell delivery treatments such as in neurodegenerative disorders, it must be ensured that the microenvironment of the host meets the requirements of the speci�c type of cell56. also, although msc therapies are berkeley pharma tech journal of medicine | 95 https://www.zotero.org/google-docs/?nsak57 https://www.zotero.org/google-docs/?myofjo https://www.zotero.org/google-docs/?8lidxr https://www.zotero.org/google-docs/?yinhrb https://www.zotero.org/google-docs/?bgmoyd generally considered to be safe as documented in various clinical studies, the utilization of a relatively large number of living mscs carries certain risks, such as occlusion within arterioles, capillaries, and venules, a transformation of transplanted cells into inappropriate cell types such as those displaying malignancies, and proarrhythmia. similarly, the transplantation of living cells with the ability to replicate is inherently high-risk, because the grafted cells cannot be completely removed if there is an adverse response to treatment or if the disease is ultimately resolved59. moreover, it has been demonstrated that the functioning of mscs declines with age, and this degradation of function can be implicated in the loss of tissue integrity and homeostasis8. interestingly, the functional activity of mscs typically declines during and as a consequence of cell senescence a mechanism through which the aging of a cell occurs and cellular replication ceases, with an absence of cell death73. this can have a signi�cant adverse impact on msc function when utilized for the regeneration of injured tissue and therefore, strategies to prevent or delay senescence must be evaluated in order to yield a longer bene�cial impact and increased msc quality8. it is relevant to note that a comprehensive study of the mechanisms of di�erentiation of stem cells can aid in the current understanding of how the brain and spinal cord can be individually targeted to stimulate their repair74. furthermore, the speci�c pathogenetic mechanisms of exosomes, and their role in therapeutic applications through their capacity of cellular repair should be further examined. the advantages of msc derived exosomes/extracellular vesicles (ev) have been elucidated in this paper; for instance, although these evs have demonstrated bene�cial e�ects in the treatment of malignancies, tumor cells through evs have the potency to function as either tumor promoters or suppressors. speci�cally, msc-derived exosomes have been reported to be involved with tumor growth, angiogenesis, and metastasis. however, there does exist a discrepancy between the behaviors of msc exosomes, which may be associated with the sources of mscs, genotypic characteristics of tumors, and the stages of tumor growth8. thus, the side e�ects and implications of msc-derived evs must be further investigated. berkeley pharma tech journal of medicine | 96 https://www.zotero.org/google-docs/?sgt1ox https://www.zotero.org/google-docs/?t1nezd https://www.zotero.org/google-docs/?ycqqyb https://www.zotero.org/google-docs/?zznzbq https://www.zotero.org/google-docs/?ccyomy https://www.zotero.org/google-docs/?mvbxmk most signi�cantly, the protocols for inducing cellular di�erentiation into mscs cannot be directly incorporated within human beings due to their incorporation of toxic chemicals75. this warrants further study into the development of induction methods that can be safely executed within patients. overall, though, the mechanisms of mscs, in addition to their capacity as a universal therapeutic within a clinical setting for als and similar degenerative diseases needs to be examined to a greater extent, in order to gain an increased understanding of the current state and application potential of mscs, as well as of methods that could be implemented to circumvent the potential risks associated with the administration of mscs. 10. conclusion amyotrophic lateral sclerosis is a rare and progressive neurodegenerative disease with no cure to date. while the causes of als remains unclear, current theories for als pathogenesis include mitochondrial damage, glutamate excitotoxicity, neuroin�ammation, and oxidative stress. existing treatments to slow the degeneration of motor neurons and extend survival rates and/or time to tracheostomy include therapeutics like riluzole and edaravone, gene therapy to target als-related gene mutations, and astrocyte transplantations to reverse neurotoxicity in the cns. however, the mechanisms for these therapies are also not well-understood, and they are not a long-term solution. they mainly target one or two pathogenetic mechanisms or do not provide a solution to treat all als cases, so msc therapy may be the answer to a cure for als. mesenchymal stem cells have been shown to be a promising therapeutic for als, based on preclinical and clinical trials, due to their ability to stimulate tissue repair by di�erentiating into muscle cells as well as providing for means to repair damaged tissue. challenges associated with msc therapies such as the high risk and irreversibility deem it essential for further research on the administration of msc treatments for als. berkeley pharma tech journal of medicine | 97 https://www.zotero.org/google-docs/?gkkvkt con�icts of interest the authors declare that there is no con�ict of interest regarding the publication of this paper. berkeley pharma tech journal of medicine | 98 references 1. what is als? amyotrophic lateral sclerosis | the als association. accessed july 23, 2022. https://www.als.org/understanding-als/what-is-als 2. about motor neurons | columbia university motor neuron center. accessed july 23, 2022. https://www.columbiamnc.org/about-us/about-motorneurons 3. 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doi:10.1186/s13619-015-0026-7 berkeley pharma tech journal of medicine | 103 front page yalamanchili et al. body template_yalamanchili et al. references_yalamanchili et al. berkeley pharma tech journal of medicine correspondence: wojtaram@umich.edu keywords: telemedicine telehealth google trends obstetrics and gynecology surgical telehealth submitted: april 14, 2023 accepted: july 24, 2023 published: december 30, 2023 full open access creative commons attribution license 4.0 abstract the coronavirus (covid-19) pandemic resulted in a plethora of healthcare challenges. to adapt, many health systems implemented telehealth interventions. this retrospective study using data from march 1, 2021 to march 31, 2022 evaluates the relationship between the daily reported number of new covid-19 cases and corresponding changes to search volume on obstetrics and gynecology telehealth visits. google trends™ outputs were compared to covid-19 case data for the time period and region as provided by the world health organization (who). spearman’s correlation coefficient (ρ) was used to determine the strength of the relationship between new cases and relative search volumes (rsvs) related to obstetrics and gynecology telehealth. globally, there was a significant positive strong correlation between public interest regarding telehealth in obstetrics and gynecology and new covid-19 cases (ρ=0.986, p-value<0.001). however, the united states and mexico demonstrated non-significant poor correlations. brazil exhibited a positive fair correlation. based on this retrospective study, there was a steady rise in public interest in telehealth usage for obstetrics and gynecology throughout the pandemic. increased telehealth intervention in the field of obstetrics and gynecology (ob-gyn) has shown promising initial results. there are numerous considerations for utilizing telehealth for surgical specialties such as ob-gyn. interest in telehealth for obstetrics and gynecology during the covid-19 pandemic by: magda wojtara, simran athwal, kehinde dorcas anuoluwapo adebogun, and maira elahi berkeley pharma tech journal of medicine | 13 1. introduction the covid-19 pandemic had a large impact on the healthcare system globally. it prompted a shift to utilizing digital health solutions such as through telehealth or telemedicine. while some specialties were utilizing telehealth interventions prior to the pandemic, others, especially surgical specialties, had less uptake until the onset of the pandemic.1 a recent systematic review found that telehealth interventions not only improved obstetric outcomes, but also decreased the need for high-risk obstetric monitoring office visits.2 considerations for surgical specialties include better patient preparedness for surgery with preoperative telehealth calls.3 healthcare providers are refocusing and adopting information and communication technologies (icts) such as telemedicine as a benefit tool for real-time, online consultations.4 there has been a significant reduction in the prevalence of covid-19 in north, central, and south america because of the success in addressing vaccine hesitancy and vaccine uptake.5 telehealth systems played a crucial role in decreasing the spread and number of covid-19 cases. telemedical alternatives ensured the safety of the healthcare workers by allowing them to maintain the continuity of care to patients remotely.6 it limited the spread of the pandemic by offering virtual visits from the comfort of a patient’s home, allowing symptomatic patients to receive expert medical advice, and reducing the number of visitors to hospitals. telemedicine usage increased during the covid-19 pandemic as the pandemic also triggered patient demand for virtual healthcare services.6 telemedicine incorporates the use of sound and video technology to allow for remote patient health visits, which were more sought out during the pandemic.6 even though telehealth cannot replace in–person visits, especially surgical procedures which cannot be performed remotely, it does lessen risks of unnecessary exposure and offers a secure way of providing testing and services. telehealth interventions were notably effective in reducing the burden of healthcare systems during the pandemic.7 while telehealth has been utilized in primary care and some specialties, telehealth uptake has not been as prevalent amongst surgical specialties. telehealth usage for surgical specialties remains higher than pre-pandemic levels. it is important to consider this emerging dimension of healthcare delivery and the rapid adoption of new technologies in obstetrics and gynecology. a recent study found that maternal-fetal medicine obstetrical patients and providers were satisfied with telemedicine because it improved access to healthcare providers, saved time on traveling, and provided safety from any physical contact during the time of the covid-19 pandemic.8 for prenatal care, telehealth virtual visits consolidated the in-person visits for prenatal berkeley pharma tech journal of medicine | 14 screening and surveillance, alongside minimizing covid-19 exposure and patient travel.9 google trends™ is a dynamic tool that offers insights into the popularity of search terms and topics on the world's most widely used search engine, based on data from billions of searches that google processes every day. its ability to display relative search volumes for a given keyword compared to the total search volume on google over a specific period and location is one of its key features.10 the timing of searches on google trends™ can be highly correlated to events such as a spike in cases of a particular disease or health condition, as individuals often rely on the internet for information about their health.11 during the outbreak of covid-19, there was a surge in searches for symptoms, testing, and treatment options, and the global google trends™ index peaked on march 12, 2020 when covid-19 was proclaimed a pandemic. as such, it was shown that rsv indices can be used to track the spread of an outbreak like the present covid-19 pandemic.12 a populationbased study demonstrated a strong correlation between search interests in ‘covid19’, ‘covid pneumonia’, and ‘covid heart’, and the covid-19 daily new cases and new deaths. this study provided evidence that the trends in covid-19 daily new cases and new deaths in the usa are substantially connected with searchinterests relevant to covid-19.13 in another study, the strongest correlations were observed between ‘face mask’, ‘lysol’, and ‘covid stimulus check’ among the 10 keywords analyzed from google trends™ when looking at the united states as a whole, with r values of 0.88, 0.82, and 0.79, respectively.14 google trends™ is a research tool that provides information on the frequency at which specific search terms are entered into google relative to the total search volume regionally or globally. this tool allows for data collection to provide insight into the trends related to the public interest in different healthcare subjects using keywords.15 the output of google trends™, relative search volume (rsv), reflects changes in the magnitude of online public interest in a particular subject.16 this association between rsv and public interest permits the primary objective of this study, which is to examine the association between the daily reported number of new cases during the covid-19 pandemic and corresponding changes in rsv of topics in obstetrics and gynecology telehealth in selected north, central and south american countries on march 1, 2020 march 31, 2022. countries were selected based on data availability. 2. materials and methods 2.1 study tool berkeley pharma tech journal of medicine | 15 google trends™ is a tool to analyze the public’s interest via analyzing the number of web searches for a given term. google is the most utilized search engine; therefore, the prevalence of web searches on google represents public interest in a topic. google trends™ allows any user to extract data from a selected time period and selected region or country. these searches can be further localized and are indicated in order of prevalence by country and city. the selection of appropriate keywords for utilization in google trends™ is crucial for obtaining an accurate idea of public interest in a topic. the “+” feature allows users to search multiple related or synonymous keyword terms. this allows users to create a combination of keywords that accounts for different possible searches on the same topic. the output of a google trends™ search is displayed as relative search volume (rsv), which expresses the search volume that corresponds to the number of searches for a given keyword. it is displayed on a numerical scale in a visual and downloadable data file form from 0 to 100, where 100 is the peak of the search term for the selected duration and geographic location whereas 0 indicates an almost negligible amount of searches for the term given those parameters.17 google trends™ is a good approximation of searches and public interest; however, it does filter out some types of searches. these types of searches include: (1) searches made by very few people appear as “0” (2) duplicate searches from the same person in a short period of time are eliminated (3) queries with apostrophes and other special characters are filtered out from the trends.17 2.2 study design this was a retrospective study conducted to determine the relationship between the new covid-19 cases and the public interest towards obstetrics and gynecology telehealth. to get a longitudinal analysis, a time range was selected for the study from march 1, 2020 to march 31, 2022. peaks in hospitalizations vary by country and region, so this study included a larger time range to provide better insight into any relationship between covid-19 cases and public interest towards obstetrics and gynecology telehealth. 3. selection criteria the world health organization operates a covid-19 tracker which allows for surveillance of global covid-19 data since the start of the pandemic. this tracker reports the official count of total confirmed cases and the number of new cases in each country. it is important to note that testing for covid-19 has been berkeley pharma tech journal of medicine | 16 challenging in many countries with the strain on already struggling healthcare infrastructures. additionally, the number of tests performed has dropped as cases have declined and the vaccine uptake has increased. as a result, there may be discrepancies between the reported number of confirmed cases at a specific time. we chose to determine the relationship between rsv from google trends™ and the number of new cases reported per day. 3.1 new covid-19 cases countries with the highest number of total confirmed cases of covid-19 in north, central and south america as of march 1, 2023 were selected to be part of the study. based on this criterion, the following countries were included: united states of america, mexico, and brazil. the data for these countries and the worldwide data for new cases were extracted from reports published on the who website from march 1, 2020 to march 31, 2022.18 3.2 rsv data from google trends™ the initial combination of keywords selected for our study was “ob-gyn telehealth”, “telemedicina obstetricia y ginecológica”, and “telemedicina obstetrícia e ginecologia” to reflect english, spanish and portuguese searches. these keywords were used with the “+” feature of google trends™. these keywords were selected on the basis that they are most utilized by the public and interchangeable with other variants that differ in the ordering of the terms. the data for these search terms were downloaded from the google trends™ website. the following filters were selected before extracting the data: “3/1/2020 to 3/31/2022” as time range; “all categories” for the category, and “web search” for the type of search. the parameter “region” was changed to either the united states, mexico or brazil respectively to obtain data for each country. the worldwide rsv for these parameters was downloaded from the site in order to compare public interest in obstetric and gynecology telehealth globally during the pandemic. 3.3 statistical analysis the data extracted from the world health organization and google trends™ were entered and analyzed using the statistical package for social sciences (spss) version 29.0 (ibm corp., armonk, ny). the rsv data for each country for the search terms from 3/1/2020 to 3/31/2022 were plotted against the number of new covid-19 cases reported for that country. the data were visually plotted as a histogram using the feature of the bell-shaped curve, and then tested by the shapiroberkeley pharma tech journal of medicine | 17 wilk test. given that the data was aberrantly distributed, spearman’s rank-order correlation test was performed to analyze the association between the rsv and number of new covid-19 cases worldwide and in each of the selected countries. the spearman’s correlation coefficient which is denoted by ‘ρ’ was used to determine the strength and direction of the relationship between two variables. this coefficient’s value lies between -1 to +1 with a “-” sign indicating an inverse relationship, a “+” sign indicating a positive correlation, and “0” demonstrating no correlation between the variables [19]. a “-1” value indicates a perfect negative association whereas a “+1” value indicates a perfect association. the value of ρ ≥ 0.8 was used to depict a very strong relation, ρ < 0.8 and ≥ 0.6 for moderately strong relation, ρ < 0.6 and ≥ 0.3 for fair relation, and ρ < 0.3 for a poor relation between the variables [19]. a p-value of < 0.05 was considered statistically significant for this study. 4. results 4.1 relative search volume data 4.1.1 worldwide the global interest regarding telehealth in obstetrics and gynecology was assessed during march 1, 2020 to march 1, 2022. as shown in table 1, a mean of the global search volume regarding this type of telehealth was calculated to be 13.23 ± 22.34 . based on the study’s selection criteria, countries from north, central and south america were selected due to their high amount of total covid-19 cases: the united states, mexico, and brazil. table 1. means of google trends rsvs related to obgyn telehealth during the covid-19 pandemic country/region mean standard deviation worldwide 13.23 22.34 united states 21.89 14.58 mexico 0* 0* brazil 0** 0** https://pubmed.ncbi.nlm.nih.gov/14770254/ https://pubmed.ncbi.nlm.nih.gov/14770254/ berkeley pharma tech journal of medicine | 18 * searches in spanish for ob-gyn telehealth yielded no significant google trend results ** searches in portuguese for ob-gyn telehealth yielded no significant google trend results the selected time range was march 1, 2020 to march 31, 2022. during january 2022, it was observed that new covid-19 cases rose sharply. one of the highest number of cases was the week of january 10thjanuary 17th, 2021 when the total number of confirmed cases rose 4,583,915 to a total of 23,309,763. another notable peak was the week of december 19-december 26, 2022 when cases rose 29,381,144 to a total of 44,265,843 confirmed cases. the maximum rsv (rsv=100 for our search terms was recorded on january 17, 2021. interestingly, the rsv slope illustrated a no discernable trend after january 17, 2021. it would peak several times including july 18, 2021 (rsv=90 and january 16, 2022 (rsv=74. a period from september-november 2021 showed a minimum rsv of 0. 4.1.2 united states of america in march 2020, the mean rsv for “ob-gyn telehealth” was calculated to be 17.60 ± 17.59. in march 2021, the mean rsv was calculated to be 18.00 ± 22.33. in march 2022, the mean rsv was calculated to be 0 ± 0. interest in telehealth for obstetrics and gynecology during the covid-19 pandemic was also plotted against new cases reported worldwide per day. this was illustrated for march 1, 2020-february, 2023 in figure 1. berkeley pharma tech journal of medicine | 19 4.1.3 mexico due to a lack of any google trends results for ob-gyn telehealth in english or spanish, we instead monitored the rsv of the terms “telemedicina” and “obstetricia” separately. in march 2020, the mean rsv for “telemedicina” was calculated to be 5.60 ± 2.19. in march 2021, the mean rsv was calculated to be 3.00 ± 2.16. in march 2022, the mean rsv was calculated to be 6.75 ± 2.75. in march 2020, the mean rsv for “obstetricia” was calculated to be 56.40 ± 12.62. in march 2021, the mean rsv was calculated to be 58.00 ± 5.29. in march 2022, the mean rsv was calculated to be 80.75 ± 9.21. 4.1.4 brazil due to a lack of any google trends™ results for ob-gyn telehealth in english or portuguese, we instead monitored the rsv of the terms “telemedicina” and “obstetricia” separately. in march 2020, the mean rsv for “telemedicina” was calculated to be 42.00 ± 31.34. in march 2021, the mean rsv was calculated to be 47.00 ± 6.78. in march 2022, the mean rsv was calculated to be 26.75 ± 2.99. figure 1. berkeley pharma tech journal of medicine | 20 in march 2020, the mean rsv for “obstetricia” was calculated to be 21.50 ± 6.95. in march 2021, the mean rsv was calculated to be 17.25 ± 1.50. in march 2022, the mean rsv was calculated to be 24.25 ± 0.50. 4.2 spearman’s correlation analysis between google trends™ and new covid-19 cases the spearman’s correlation coefficient which is denoted by ‘ρ’ was used to determine the strength and direction of the relationship between rsv and new covid-19 cases during the selected time frame. this coefficient’s value lies between -1 to +1 with a “-” sign indicating an inverse relationship, a “+” sign indicating a positive correlation, and “0” demonstrating no correlation between the variables.19 a “-1” value indicates a perfect negative association whereas a “+1” value indicates a perfect association. the value of ρ ≥ 0.8 was used to depict a very strong relation, ρ < 0.8 and ≥ 0.6 for moderately strong relation, ρ < 0.6 and ≥ 0.3 for fair relation, and ρ < 0.3 for a poor relation between the variables.19 a p-value of < 0.05 was considered statistically significant for this study. table 2. spearman’s correlation of rsvs and new covid-19 cases region/country spearman’s correlation coefficient (ρ) p-value worldwide 0.986e <0.001a united states 0.181c 0.533 mexico* “telemedicina”: -0.231b 0.426 “obstetrica”: -0.227b 0.435 brazil* “telemedicina”: 0.330d 0.249 “obstetrica”: -0.205b 0.482 berkeley pharma tech journal of medicine | 21 *mexico and brazil spearman’s correlation was performed between “telemedicina” and “obstetrica” rsv separately with new covid-19 cases a indicates p-value < 0.05 which was considered statistically significant; b indicates negative poor correlation; c indicates positive poor correlation ; d indicates positive fair correlation e indicates a positive strong correlation based on these standards and the calculated spearman’s correlation coefficient, a few interesting findings emerged. worldwide there was a significant positive strong correlation between rsv for “obgyn telehealth” and new covid-19 cases march 1, 2020-march 31,2022. in the united states, there was a non-significant positive poor correlation between rsv and new cases for the same keyword and timeframe. in mexico, there were non-significant negative poor correlations between rsv for “telemedicina” or “obstetrica” and new covid-19 cases march 1, 2020march 31, 2022. in brazil, there was a non-significant negative poor correlation between rsv for “obstetrica” and new covid-19 cases whereas there was a non-significant positive fair correlation between rsv for “telemedicina” and new covid-19 cases march 1, 2020march 31, 2022. 5. discussion as our previous findings have indicated, telehealth interventions have brought forth advantages during the covid-19 pandemic that shifted the viewpoint on telehealth as the prominent care delivery mode. however, there were certain barriers for telehealth uptake for surgical specialties. not all health care providers are interested in utilizing telehealth interventions or are equipped with the technological infrastructure to do so.20 during the covid-19 pandemic, there was a rise in the utilization of telehealth in various countries due to the multiple advantages it provided compared to in-person visits.21 with the help of telehealth care, patients could still consult with doctors even during self-quarantine; patients with either suspected or confirmed symptoms could be closely telemonitored using a telehealth cloud-based form, allowing for the maintenance of minimal patient and care-provider exposure.22 additionally, physicians quarantined due to exposure to covid-19 could still deliver healthcare services to non-exposed patients using teleintake and care for patients remotely.23 furthermore, implementing telemedicine during the pandemic decreased the likelihood of the further spread of covid-19 through remote care of patients; it also reduced the rate of emergency room visits.24 several categories of patients benefited from telehealth during the covid-19 pandemic, including elderly patients and patients with chronic conditions such as diabetes mellitus and hypertension.25 for example, a cross-sectional study conducted in ontario before and during the pandemic found a significant increase berkeley pharma tech journal of medicine | 22 in telemedicine visits in patients with chronic disease conditions and patients of older age groups in rural areas during the pandemic.25 telemedicine would be particularly helpful to these groups of people, especially in rural regions, due to a decrease in the cost of traveling and time spent traveling. other benefits include reduced hospital readmission and increased availability of inpatient beds for patients requiring critical care.25 several measures were put in place to facilitate the integration of telehealth care during the pandemic; for example, due to the increasing patient load during the pandemic, several countries expanded access to telehealth using various methods such as relaxing previously strict laws regarding telehealth, which had been put in place due to ethical concerns, concerns about patient data privacy, and concerns about accountability.21 similarly, the lack of insurance coverage for telehealth is another factor that initially limited the incorporation of telehealth into healthcare systems before the pandemic; however, insurance companies have begun reimbursing patients' costs incurred due to telehealth care delivery.21 it is evident that covid-19 has caused a global crisis. this subsequently resulted in a paradigm shift within the healthcare and public health systems, the loss of millions of lives, and economic disruption. healthcare systems across the world developed a new framework encompassing public health functions such as testing, contract tracing, disease surveillance, telehealth, and non-pharmaceutical public health interventions.26 despite differences in healthcare infrastructure across north, central and south america, these regions all actively considered telehealth to meet the increased health needs of their populations. in north america, there is a focus on providing the public with a mix of public and private programs. in the united states, there is medicaid, a public program, offered for those who fall within the category of being low-income and medicare is a public program for older patients, and some younger individuals with disabilities. within the united states, however, private insurance costs are often exorbitant. canada, on the other hand, has a decentralized, universal and publicly funded health system called canadian medicare.27 private insurance is held by ⅔ of canadians and covers services excluded under universal health coverage such as vision and dental care, rehabilitation services and outpatient prescription drugs.27 mexico also has both public and private healthcare sectors. the private sector has increased in prevalence over time, however, only 7% of mexicans have private insurance.27 due to this, private insurance to cover out-of-pocket expenses is considered to be linked to mexicans with higher socioeconomic status.27 public healthcare is fully or partially subsidized by the federal government through insabi for individuals without employment, imss for those employed, and the issste for public employees.28 brazil utilizes a national health system, sistema único de saúde, providing its citizens with berkeley pharma tech journal of medicine | 23 decentralized, universal health coverage which is delivered at the state and municipal levels.28 north american nations utilize a mix of public and private programs, while central and south america primarily concentrate on public programs that are fitted to meet the health needs of their populations. the covid-19 pandemic has distributed all fields of healthcare. this has led to the postponement or cancellation of elective procedures such as cervical cancer screenings and clinic closures for reproductive healthcare. telehealth has emerged as a crucial tool for accessing reproductive healthcare, particularly in areas where clinics have closed or reduced services.21 the pandemic has also intersected with political and social issues correlated to reproductive healthcare,21 namely stigmas surrounding ob-gyn procedures that continue to hinder women's access to critical services. i.e., reproductive surgeries such as termination of pregnancy or surgical sterilization were not initially prioritized by many national organizations, excluded from insurance coverage, and not permitted by healthcare institutions due to sociopolitical views and stigma surrounding unintended pregnancy and termination.29 a study indicates that society associates reproductive healthcare with sexual relations, shaping stigmas that place limitations on unmarried women for using such services. stigma regarding abortion is seen as a negative attribute that marks individuals as inferior to ideal womanhood and is based on a shared understanding that abortion is morally wrong or socially unacceptable.29 additionally, there emerges the potential for internalized stigma as women report self-blaming and feeling shame for their hpv and/or cervical cancer diagnosis.29 in the united states, abortion continues to be stigmatized and has led to state-level restrictions. moreover, mexico’s population's religious values have led to a similar stigmatization as traditional beliefs about women’s role and sexuality render it difficult to access contraception or seek reproductive healthcare. however, as of september 2021, mexico's supreme court ruled that it is unconstitutional to punish abortion as a crime, a watershed ruling that clears the way for the legalization of abortion across the country.27 conversely, brazil maintains that abortion is illegal except for certain legal exceptions. the country enforces comprehensive sexual and reproductive health policies according to national laws. exacerbated by political polarization and push for restrictions, brazil’s reproductive health systems have been further made difficult due to the inaccessibility of critical services. while google trends™ is a helpful tool to determine relative interest in a topic over a period in a region, there are intrinsic limitations to its usage. it does not accurately represent the entirety of internet search traffic for a topic because it is only harvesting data from google searches. furthermore, relative search volume (rsv) which is measured on a scale from 0 to 100 only demonstrates relative interest in a berkeley pharma tech journal of medicine | 24 topic. there is also potential for bias since not everyone can access technology or stable internet to make searches. specifically, these individuals may also not be searching about telehealth at all due to this lack of access. another consideration is that different countries may utilize different search languages and subsequently different terminology and communication channels. this makes it difficult to directly compare relative interest in a topic in different countries across different regions. the implementation of telehealth in obstetrics and gynecology is promising. it introduces new avenues to increase patient education before procedures, follow-up visits, and other opportunities. the incorporation of telemedicine for pregnant women has been associated with numerous advantages compared to exclusively inperson care. for example, a retrospective observational cohort study on pregnant women who received exclusive in-person and alternate (telemedicine and in-person) care showed that women who received alternated care had similar maternal and perinatal outcomes to women who had received exclusively in-person care.30 interestingly, these women were also more likely to be admitted into antenatal follow-up programs earlier and obtain a more significant number of evaluations resulting in increased maternal satisfaction and decreased adverse outcomes such as perinatal mortality.30 in addition, they also experienced reduced unnecessary exposure to the hospital environment during pregnancy.30 the incorporation of telehealthcare also allows for increased utilization of web-based informational programs, an effective tool for patient education. these web-based programs empower patients to participate more effectively in medical decision-making, resulting in a higher likelihood of adherence to treatment plans. increased preoperative knowledge can help to reduce anxiety levels before surgery. furthermore, telehealth reduces several barriers which pregnant women might face as it may make care more affordable and a convenient method of healthcare delivery. telehealth care allows patients to interact with physicians remotely regardless of time and day thereby increasing access.22 with the promising nature of telehealth, addressing barriers regarding the integration of telehealth as a prominent part of healthcare systems in countries is essential. such barriers include concerns about the lack of data privacy and security and the lack of rules and regulations concerning telehealth utilization.22 addressing these issues by improving data privacy and security, outlining clear rules and regulations by the government concerning the use of telehealth and upgrading the infrastructure of telemedicine in hospitals and clinics will go a long way in advancing the capacity for integration of telehealth care.22 another strategy that can facilitate telehealth incorporation is the reformation of medical school curricula to include berkeley pharma tech journal of medicine | 25 telehealth and telehealth care delivery components. current medical practitioners can also be encouraged to participate in continuing medical education and continuing professional development to improve their knowledge and level of comfort in delivering telehealth to patients; this would keep them abreast of the rapid advancement in this system.21 6. conclusion telehealth has been of increased interest during the covid-19 pandemic due to its role in helping the ill and immunocompromised receive care without concerns of further spreading the virus. this is a novel way to enable high-quality supportive care for a variety of patient populations. using google trends™, our study found a significant rise in worldwide interest in obstetrics and gynecology and telehealth. despite this, many physicians and patients have concerns due to regulatory, legal, reimbursement and privacy barriers. this has largely hindered the widespread adoption of telehealth. previous studies have shown that there are many barriers to telehealth uptake specifically for surgical specialties and generally due to the lack of adequate healthcare infrastructure in many countries. telehealth in surgical specialties also varies from other specialties as its primary use would likely be for preoperative assessment as well as evaluation and follow-ups post-surgery. this study strongly highlights the interest in telehealth for obstetrics and gynecology and potential of telehealth applications for surgical specialties globally. references 1. chao gf, li ky, zhu z, et al. use of telehealth by surgical specialties during the covid-19 pandemic. jama surgery. 2021;156(7):620-626.10.1001/jamasurg.2021. 0979 2. denicola n, grossman d, marko k, et al. telehealth interventions to improve obstetric and gynecologic health outcomes: a systematic review.obstet gynecol. 2020;135(2):371-382.10.1097/aog.00000000 00003646 3. halder ge, white ab, brownhw, et al. a telehealth intervention to increase patient preparedness for surgery: a randomized trial. int urogynecol j. 2022;33(1):85-93.10.1007/s00192-021-04831-w 4. almathami hky,win kt, vlahu-gjorgievska e. barriers and facilitators that in�uence telemedicine-based, real-time, online consultation at patients’ homes: systematic literature review. journal of medical internet research. 2020;22(2):e16407.10.2196/16407 5. sallamm. covid-19 vaccine hesitancy worldwide: a concise systematic review of vaccine acceptance rates.vaccines. 2021;9(2):160.10.3390/vaccines9020160 6. kapoor a, guha s, kanti das m, goswami kc, yadav r. digital healthcare: the only solution for better healthcare during covid-19 pandemic? indian heart journal. 2020;72(2):61-64.10.1016/j.ihj.2020.04.001 7. akintunde ty, akintunde od,musa th, et al. expanding telemedicine to reduce the burden on the healthcare systems and poverty in africa for a post-coronavirus disease 2019 (covid-19) pandemic reformation.glob health j. 2021;5(3):128-134.10.1016/j.glohj.2021.07.006 8. tozour jn, bandremer s, patberg e, et al. application of telemedicine video visits in a maternal-fetal medicine practice at the epicenter of the covid-19 pandemic.american journal of obstetrics & gynecologymfm. 2021;3(6):100469.10.1016/j.ajogmf.2021.100469 9. aziz a, zork n, aubey jj, et al. telehealth for high-risk pregnancies in the setting of the covid-19 pandemic. am j perinatol. 2020;37(8):800-808.10.1055/s-0040-1712121 10. rovetta a. reliability of google trends: analysis of the limits and potential of web infoveillance during covid-19 pandemic and for future research. frontiers in researchmetrics and analytics. 2021;6. accessed april 11, 2023. https://www.frontiersin.org/articles/10.3389/fr ma.2021.670226 11. satpathy p, kumar s, prasad p. suitability of google trendstm for digital surveillance during ongoing covid-19 epidemic: a case study from india.disastermed public health prep.:1-10. 10.1017/dmp.2021.249 12. e�enberger m, kronbichler a, shin ji, mayer g, tilg h, perco p. association of the covid-19 pandemic with internet search volumes: a google trendstm analysis. int j infect dis. 2020;95:192-197.10.1016/j.ijid.2020.04. 033 13. yuan x, xu j, hussain s, wang h, gao n, zhang l. trends and prediction in daily new cases and deaths of covid-19 in the united states: an berkeley pharma tech journal of medicine | 26 https://www.frontiersin.org/articles/10.3389/frma.2021.670226 internet search-interest based model. explor res hypothesis med. 2020;5(2):1-6. 10.14218/erhm.2020.00023 14. kurian sj, bhatti a ur r, alvi ma, et al. correlations between covid-19 cases and google trends data in the united states: a state-by-state analysis.mayo clin proc. 2020;95(11):2370-2381. 10.1016/j.mayocp.2020.08.022 15. schootmanm, toor a, cavazos-rehg p, et al. the utility of google trends data to examine interest in cancer screening. bmj open. 2015;5(6):e006678. 10.1136/bmjopen-2014-006678 16. ali sa, arif tb, maab h, et al. global interest in telehealth during covid-19 pandemic: an analysis of google trendstm. cureus. 2020;12(9). 10.7759/cureus.10487 17. faq about google trends data trends help. accessed april 11, 2023. https://support.google.com/trends/answer/43 65533?hl=en 18. who coronavirus (covid-19) dashboard. accessed april 11, 2023. https://covid19.who.int 19.chan yh. biostatistics 104: correlational analysis. singaporemed j. 2003;44(12):614-619. 20. chang je, lai ay, gupta a, nguyen am, berry ca, shelley dr. rapid transition to telehealth and the digital divide: implications for primary care access and equity in a post-covid era. milbank q. 2021;99(2):340-368.10.1111/1468-0009.12509 21.doraiswamy s, abraham a,mamtani r, cheema s. use of telehealth during the covid-19 pandemic: scoping review. j med internet res. 2020;22(12):e24087.10.2196/24087 22. garfan s, alamoodi ah, zaidan bb, et al. telehealth utilization during the covid-19 pandemic: a systematic review. comput biol med. 2021;138:104878.10.1016/j.compbiomed.2021.1 04878 23. hollander je, carr bg. virtually perfect? telemedicine for covid-19.nengl jmed. 2020;382(18):1679-1681.10.1056/nejmp20035 39 24. bokolo aj. exploring the adoption of telemedicine and virtual software for care of outpatients during and after covid-19 pandemic. ir j med sci. 2021;190(1):1-10.10.1007/s11845-020-02299-z 25. chu c, cram p, pang a, stamenova v, tadrous m, bhatia rs. rural telemedicine use before and during the covid-19 pandemic: repeated cross-sectional study. j med internet res. 2021;23(4):e26960.10.2196/26960 26. haldane v, de foo c, abdalla sm, et al. health systems resilience in managing the covid-19 pandemic: lessons from 28 countries.natmed. 2021;27(6):964-980.10.1038/s41591-021-01381-y 27. juan lópez m,martínez valle a, aguilera n. reforming the mexican health system to achieve e�ective health care coverage.health systems & reform. 2015;1(3):181-188.10.1080/23288604.2015.10589 99 28. castro r. health care delivery system: mexico. in: cockerhamwc, dingwall r, quah s, eds. thewiley blackwell encyclopedia of health, illness, behavior, and society. johnwiley berkeley pharma tech journal of medicine | 27 & sons, ltd; 2014:836-842.10.1002/9781118410868.wbehibs 101 29. bruno b, shalowitz di, arora ks. ethical challenges for women’s healthcare highlighted by the covid-19 pandemic. j med ethics. published online october 2020:medethics-2020-106646.10.1136/medethic s-2020-106646 30. escobar mf, gallego jc, echavarria mp, et al. maternal and perinatal outcomes in mixed antenatal care modality implementing telemedicine in the southwestern region of colombia during the covid-19 pandemic. bmchealth serv res. 2023;23(1):259.10.1186/s12913-023-09255-4 berkeley pharma tech journal of medicine | 28 berkeley pharma tech journal of medicine correspondence: kellyyan@berkeley.edu keywords: autism, gene therapy, neurodevelopment, genetic factors, epigenetics submitted: december 11, 2022 accepted: january 27, 2023 published: june 30, 2023 full open access creative commons attribution license 4.0 abstract autism spectrum disorder (asd) is a range of developmental disorders characterized by impaired traits associated with three distinct domains: communication, social interaction, and stereotypic repetitive behavior. although the etiology of asd depends on various components, current research mainly focuses on the genetic factors that contribute to the development of asd and its effects. a big treatment consideration is gene therapy, which has disease-modifying potential. this article provides insight into the foundation of asd and the leading gene therapies that aim to address impaired neurological behavior. we will discuss how certain genetic factors can have large contributions to asd development and how scientists can go about targeting these factors for potential remedies. gene therapy as a promising approach for the underlying causes of autism spectrum disorder by: kelly an, fadel batal, sonya svyatskya, vanloan nguyen autismspectrumdisorder introduction autism spectrum disorder (asd) is a neurodevelopmental condition that a�ects communication and social interaction. it is a complex condition with a range of symptoms and severity levels, and the exact causes are still not fully understood. 1 however, genetic factors are believed to play a signi�cant role in the development of asd. 2 symptoms of asd, which typically appear in early childhood, include impaired social interaction and communication, repetitive behaviors and interests, and sensory processing issues. these symptoms range from mild to severe and may a�ect an individual's ability to function in daily life. 1, 3 at a brain developmental level, asd is thought to result from abnormal development, particularly in regions of the brain involved in social interaction and communication. some studies have suggested that individuals with asd may have di�erences in brain structure and function, such as altered connectivity between di�erent regions. 4 at a genetic level, asd is believed to be caused by a combination of genetic and environmental factors. while the exact causes of asd are still not fully understood, research has identi�ed a number of genetic variations that are associated with an increased risk of developing the condition. these genetic factors may interact with environmental factors to in�uence brain development, ultimately resulting in asd. 1 autism is one of the most heritable neurodevelopmental disorders, a�ecting 78 million people or 1.5 % of the world’s population. 5 despite its ubiquitous existence, there are aspects of the disorder that science has yet to unveil. with the etiology of autism still in question, scientists have been able to conclude that at least 40% of neurobehavioral disorders within the autism spectrum are the result of genetic abnormalities. 6 the current treatment for asd falls into two categories: those that target core symptoms which include impaired communication, social interaction, and repetitive behaviors; and those that target secondary or consequential symptoms such as adhd and irritability. gene therapy, a treatment that targets core symptoms, focuses on mending genetic building blocks. it aims berkeley pharma tech journal of medicine | 11 to alter abnormalities in hopes of preventing domino-e�ects in the body that result in the neurobehavioral de�cits which characterize asd. using viral vectors such as recombinant adeno-associated viruses, gene therapy is able to introduce new genetic material to counteract the establishment of asd symptoms. 7 this paper will be discussing studies and their adaptation of gene therapy to varying pathophysiologies of asd in order to o�er e�ective treatment methods. this paper will cover the underdevelopment of neurons, impaired neural migration, impaired synaptogenesis, and dendritic morphogenesis. 8 etiology of autism spectrum disorders although the details of the origin of asd have not yet been discovered, scientists have come up with multiple theories answering the question of “how” asd comes to be in individuals. one such theory on the pathophysiology of asd is linked to neural connectivity. in a typical human body, an individual develops a surplus of neurons where over time, non-functional and unnecessary neurons are removed through various mechanisms. in patients who have asd, the mechanism that targets the elimination of underdeveloped neurons is damaged. as a result, the excess neurons impair the shaping and �ne-tuning of neural circuits. 9 another theory focuses on neural migration. similar to neural connectivity, neural migration also contributes to well-formed neural circuits which aid in proper communication and other neural behaviors. in asd patients, the misplacement of neurons as a result of faulty migration during development increases the thickness of the cortex and “smudges” the boundaries of white matter. this mostly a�ects the frontal and temporal lobes: key elements in processing language and emotion—functions commonly found to be impaired in asd patients. 9 an additional theory that de�nes the mechanism by which asd is established focuses on impaired synaptogenesis and dendritic morphogenesis. the normal development of synapses and dendrites entails excess formation followed by the purging of faulty expression. in patients with asd, the suppression mechanism responsible for the expulsion of berkeley pharma tech journal of medicine | 12 faulty signaling factors is impaired. the overwhelming number of defective synapses and dendrites damages the pathway of signals within the body, resulting in the core symptoms associated with asd. 9 key genetic factors the build-up of faulty factors as a result of impaired mechanisms stems from mutations present in the genetic factors that regulate such processes within the human body. the existence of these mutations is what has led scientists to link asd to genetic abnormalities. in typical human physiology, genetic factors work in a factory-line fashion to facilitate proper operations in the formation of neurons, synapses, and dendrites as well as key neural factors. the malfunction of any genetic factor in the factory line creates a “domino e�ect” that leads to forms of neurobehavioral impairment associated with asd. although there are many di�erent factors that contribute to proper neurobehavior, this paper focuses on the mutation of three in particular: tcf4, reln and mecp2. 10, 11, 12, 13, 14 studies: linking etiology of asd and mutations of ‘key genetic factors’ 4.1 tcf4 gene study transcription factor 4 (tcf4) is responsible for encoding a helix-loop-helix transcription factor expressed most often during brain development. an alteration in tcf4 causes pitt-hopkins syndrome (pths): a disorder characterized by profound cognitive and motor disabilities and de�ned under the umbrella de�nition of autism disorders. patients with pths have mutations that either knock out the functional tcf4 gene, eliminate its essential dna-binding domain, or impact one of its transcriptional activation domains. 10 the university of california san diego medical school sought to understand how a mutated tcf4 gene results in this form of autism. 11, 12 to berkeley pharma tech journal of medicine | 13 explore this idea, scientists used pluripotent stem cells (ipsc), adult somatic cells that have been reprogrammed to be in an embryonic state, of 5 patients with pths. 12 to guarantee a controlled experiment, mutated tcf4 was the only chromosomal abnormality present in the experimental pths ipcs lines. these ipsc lines were used to generate neuron progenitor cells (npcs), the precursors of most of the neurons that make up the central nervous system. the pths npcs, produced by the pths ipsc lines, presented a reduction in tcf4 expression. this resulted in neurons with altered expressions of cntnap2 and kcnq1, which are usually regulated by the tcf4 gene. 10,11 for further analysis, scientists used the control and pths ipscs line to create brain cortical (cto) organoids, an arti�cially manufactured tissue resembling the functionality and structure of the human brain. 11 in comparison to the control organoid’s normal spheroid form, the pths cto organoid presented an abnormal structure and size. through single cell rna sequencing, it was concluded that the pths cortical organoid’s diminished size and characteristic factors were the result of a loss of cellular diversity. to make certain that this loss was not a result of mispatterning, the uc san diego team performed single cell transcriptomic testing on the organoids. the telencephalic marker, foxg1, expression analysis in combination with the single cell rna sequencing concluded that the loss of cell diversity in the pths cortical organoid was due to a higher percentage of underdeveloped neurons, known as neural progenitor cells (npc). the e�ects of the mutated genetic factor tcf4 on the brain correlates to the neural connectivity theory. this theory posits that a surplus of de�cient development of neurons due to impaired progenitor proliferation, alters the function and structure of the brain and its neural circuits responsible for functions such as communication and behavior. this excess of underdeveloped neurons prevents the idealized function of neural circuits. 11 with an understanding of the �rst domino in the developmental pathway of pths, the uc san diego team focused on abolishing its molecular and cellular characteristics by correcting the expression of the tcf4 gene. the �rst method used two viral vectors and a crispr based transepigenetic berkeley pharma tech journal of medicine | 14 strategy. all three cassettes worked together to enhance the transcription of the tcf4 gene, ultimately correcting the downstream targets. 11 figure 1: crispr-based transepigenetic strategy for correction of abnormally low tcf4 expression; a complex comprising of grna targeting promoters of tcf4, a transcriptional activation complex (mph) and dead cas9 however, the use of two viral vectors created another problem: a decrease in cellular aggregation in the brain organoids. the scientists attempted another method called virus-mediated overexpression in which a wild-type copy of tcf4 was overexpressed in hopes of overriding ectopic expression of the mutation. ptsh organoids exposed to overexpression-type gene therapy showed improvements in two key regions that marked the downstream corrected function of the tcf4 gene: increased �ring rates and number of network electrical bursts. the combination of these two methods, provided that the impaired proliferation of neurons from a mutated tcf4 can be genetically corrected, could prove to lessen the symptoms of other genetic autism disorders. 11 4.2 reln study reln is a gene that encodes for a reln glycoprotein in the extracellular matrix of gabaergic (gamma-aminobutyric acid) neurons. these cells perform important functions in neural migration and cortical lamination. 13 neural migration is an important process that occurs in mammalian nervous system development. 14 cortical lamination is the layering of cells of berkeley pharma tech journal of medicine | 15 the outer regions of the brain’s cerebrum, helping to maintain neuronal cytoskeletal stability. reduced reln levels have been shown to be associated with di�erent psychological disorders including bipolar disorder, alzheimer's disease, and autism spectrum disorder. studies have shown signi�cantly lower levels of reln in asd patient’s superior frontal cortex, parietal cortex, cerebella and plasma. this could potentially be because of reln promoter hypermethylation that occurs in gabaergic neurons, as seen in seizure disorder patients. 13 in addition, sex hormones can also play a role in methylation of the reln promoter, which has shown to increase asd-associated behaviors. after postmortem cerebellar studies of asd patients, it was found that lower reln mrna levels are associated with higher mecp2 (another essential protein for nerve cells) binding, increasing gene regulator 5-hmc at the reln promoter and in turn decreasing transcription and protein levels. such reln mutations that cause disruptions in signaling pathways are connected to asd disorders. one example is the loss of purkinje cells, regulated by reln, which increases the risk of cognitive delay and epilepsy. this is a phenomenon also seen in asd patients. 15 investigating the role of reln on neuronal signaling and asd development can produce promising leads for potential treatments of asd. increasing levels of reln protein can alleviate behavioral symptoms of reln that asd patients also experience. however, although there is much evidence for reln’s in�uence on asd, it is not the sole factor of asd development. for the diagnosis of asd, there are usually secondary genetic or environmental factors that contribute to the disorder’s development. 16 4.3 mecp2 gene study many studies have shown mecp2 protein’s role in brain development and regulation, such as expression of the brain-derived neurotrophic factor (bdnf) gene and regulation of synaptic homeostatic plasticity. mecp2 is a part of the methyl-cpg-binding protein family which regulates gene expression by modifying chromatin, a protein complex of dna and histone proteins that package dna in chromosomes. mecp2 performs regulation of dna methylation via recruitment of histone deacetylases. 17, 18 berkeley pharma tech journal of medicine | 16 figure 2: mecp2 protein structure figure 3: the mecp2 gene mecp2 is found in high concentrations in neurons. the gene locus is located on the long (q) arm of the x chromosome in band 28 (“xq28”). mecp2's main role is "repressing" or "silencing" other genes, preventing transcription and translation when they are not needed. recent studies have suggested that mecp2 can act as an activator, but this is still a new and controversial theory. mecp2 represses gene expression by recognizing and binding to methylated cytosine residues in dna called 5mecyt; regions enriched with a/t neighbor bases. mecp2 is also able to bind to hydroxymethylated dna called 5-hydroxy methylated cytosine. 18 berkeley pharma tech journal of medicine | 17 figure 4: the varied molecular functions of mecp2 protein the relationship between autism and mecp2 mutations that alter typical mecp2 function will lead to severe neurodevelopmental disorders such as rett syndrome (rtt) and asd. more than 95% rtt patients carry mutant mecp2, and some mecp2 mutations have been reported in asd patients. asd and rtt overlap in certain phenotypes, including stereotypical body movements, social avoidance, and anxiety. 17, 18 in a study conducted by zhu wen at school of life sciences in peking university, whole-exome sequencing (wes) was performed on 120 asd cases. this experiment was able to identify three mutations in the coding regions of the mecp2 gene. they found that the mecp2 gene was linked with a host of neuropsychiatric disorders and neurological phenotypes. 18 5.1 methods: 120 han chinese families were selected with probands diagnosed with asd from 2013-2015 in the department of child and adolescent psychiatry in shanghai mental health center. the range of age of the patients was between 2 to 18 years old and included 18 females and 102 males. the study excluded patients with severe somatic disorders rrt. 18 berkeley pharma tech journal of medicine | 18 in terms of clinical assessment, comprehensive pro�les of each patient were collected in the case report form. researchers classi�ed the symptoms into 4 categories: social interaction, language, repetitive behaviors, and functional impairment. 2–3 μg of genomic dna was extracted from each patient and libraries in order to prepare cluster generation and sequencing. based on the patients’ wes experiment, families within probands carrying mecp2 variations were selected for sanger sequencing to determine if the variations were de novo or inherited. the wt mecp2 gene (the e2 isoform of rat mecp2 cdna) and other mutated plasmids such as mecp2-p152l, mecp2-r294x, and mecp2-p376s were used to detect asd probands. 18 on days 15–16 of mouse embryo development, cortical neurons and hek-293 cells were individually cultured and electrophoretically transfected into each group at 0 days in vitro individual. hek-293 cells and mouse cortical neurons were collected after 3 days in vitro for western blot and immuno�uorescence analysis, respectively. 18 5.2 results: three mutations of mecp2 were detected among 120 asd patients via wes. p.p152l (c.455c>t) and p.p376s (c.1162c>t) were missense mutations and p.r294x (c.880c>t) was a truncating mutation. sanger sequencing showed that p.p152l and p.r294x were de novo mutations, but p.p376s was inherited maternally. they did not �nd any of these mutations in gnomad, indicating that they were rare mutations. 18 in terms of clinical features, poor social interaction and functional impairment were major symptoms for all three mutations, but language use and repetitive behavior di�ered greatly between them. also noted were abnormalities of dendritic and axonal growth found after autism-related mecp2 mutants were expressed in mouse cortical neurons, suggesting that autism-related mecp2 mutations impair proper development of neurons. 18 the results strongly suggest that mecp2-p152l, mecp2-r294x, and mecp2-p376s a�ect the proper physiological function of the mecp2 protein and may contribute to the pathogenesis of autism. 18 berkeley pharma tech journal of medicine | 19 mecp2 gene therapy methods in addition to these studies, there have been a number of experiments utilizing gene therapy for the treatment of asd. the majority of these trials have utilized a viral vector to deliver a modi�ed version of the mecp2 gene to patients. the results of these trials have been promising, with patients exhibiting improved social interaction, better communication skills, and an improved quality of life. 6, 19 recombinant adeno-associated virus (raav)-delivered gene substitution has been shown to improve behavior in several investigations employing monogenic animal models. for example, a recent study has shown that systematic administration of a raav9-mecp2 vector su�cient for 10% cns transduction (of primarily neuronal cells) in an rs animal model resulted in modest behavioral improvements. 6 comparatively, 25% cns transduction at a 6-fold higher vector dose led to noticeable behavioral and phenotypic bene�ts. 19 a promising mecp2 gene therapy strategy is using antisense oligonucleotides. antisense oligonucleotides are small, modi�ed nucleic acids that can selectively hybridize with m-rna from a target gene and silence it. mecp2 duplication syndrome has been associated with autism, intellectual disability, motor dysfunction, and anxiety, and is one of the most common genomic rearrangements seen in male patients. the cause of these de�cits is the over-expression of methyl-cpg-binding protein 2 (mecp2). this syndrome poses a challenge to traditional therapeutic approaches. 19 some symptomatic mice models of monogenic loss of function neurological disorders, including the loss of mecp2 in rett syndrome, have demonstrated reversal of phenotypes, indicating that the molecular correction of the underlying dysfunction could potentially restore typical physiology. the study focused on the restoration of normal mecp2 levels in mecp2 duplication syndrome using mice models. 17, 19 berkeley pharma tech journal of medicine | 20 figure 5: methods used in sztainberg et al. trials testing use of humanspeci�c antisense oligonucleotides (asos) to normalize necp2 levels in mouse models the contributors generated a conditional mecp2 overexpression mouse model and showed that the correction of mecp2 levels e�ectively reversed the molecular, electrophysiological, and phenotypic de�cits using antisense oligonucleotides (aso). it was found that antisense oligonucleotide treatment resulted in broad phenotypic rescue in symptomatic transgenic mecp2 duplication mice and corrected mecp2 levels in lymphoblastoid cells in a dose dependent manner. 19 experimental results suggest that delivery of asos to cns could prove to be promising for treating mecp2 duplication syndrome, with potential applications in asd and rett syndrome. future directions should focus on testing di�erent aso dosages, determining safety margins of mecp2 levels, and screening mecp2 asos for o�-target e�ects. 19 antisense oligonucleotides (asos) and short interfering rnas (sirnas) are two methods for sequence-speci�c suppression of mrna transcripts which can then be used to mute gene expression. in a conditional mecp2-overexpressing mouse model of mecp2 duplication syndrome, it was demonstrated that halving mecp2 expression restored cellular function and phenotype postnatally. the same study found that intraventricular delivery of asos speci�cally directed against mecp2 caused widespread aso dispersion throughout the cns. this resulted in e�cient knockdown of mecp2 to levels close to wildtype and a long-lasting phenotypic reversal. both studies suggest gene replacement and rna knockdown of mecp2 gene may be a method worth exploring as a possible remedy for asd. 19 berkeley pharma tech journal of medicine | 21 future directions in addition to the aforementioned genetic in�uences on asd cases, there are other factors that can impact the pathophysiology of asd. for example, many autism patients have reported gastrointestinal disorders (gi). this has prompted interest in possible relationships between the gut microbiome and autism. 7 up to 70% of autism patients have reported dealing with gi disorders. these gi disorders can be a result of dysbiosis, an imbalanced gut microbiome, characterized by reduced microbial diversity and increased micro�ora in gut microbiota. the gut microbiome generates a large portion of metabolites, and is responsible for energy conversion, signaling, and, most importantly, epigenetic signaling. an imbalanced gut microbiome can create an excess formation of s-adenosyl-methionine, the main methyl group in a microbiome, which results in increased methylation. the activation and deactivation of dna strands through dna methylation could potentially lead to the gene impairment/variation that results in asd. further studies into this phenomenon may have implications for treatment development if methylation events in�uencing asd development can be targeted and altered. future research studying the gut microbiome’s ability to interfere with methylation patterns can be used to inform development of gene editing in treating asd pathogenesis. 1 conclusion while there is currently no cure for asd, there are various approaches to managing its symptoms and improving quality of life. these include behavioral therapy, medication, and support services. research into the causes and potential treatments of asd is ongoing, and new developments are constantly emerging. 2 gene therapy is one promising approach that holds signi�cant potential for treating the underlying causes of asd, rather than just managing its symptoms. however, more research and clinical trials are needed to fully understand the safety and e�ectiveness of gene therapy for asd. in the last berkeley pharma tech journal of medicine | 22 �ve years, clinical research has shown that tcf4, reln, and mecp2 are linked to asd, providing scientists with a basis to explore the possibilities of gene therapy. the crispr strategy and viral overexpression in relation to the tcf4 gene, mecp2 levels in relation to the reln gene, and raav-delivered transgenes to alter gene expression all show promise in altering the behavioral phenotypes associated with autism spectrum disorder. 20 however, there are also challenges and limitations to the use of gene therapy for asd. gene therapy is a complex and expensive approach that may not be accessible to all individuals with asd. in addition, there are ethical considerations surrounding the use of gene therapy, such as the potential for unintended e�ects on other genes. 2 overall, gene therapy holds signi�cant potential for the treatment of asd, but further research and clinical trials are needed to fully understand its safety and e�ectiveness. if successful, gene therapy could o�er a promising new approach for treating the underlying causes of asd and improving the lives of individuals with the condition. berkeley pharma tech journal of medicine | 23 references 1. national institute of mental health. autism spectrum disorder. www.nimh.nih.gov. published march 2022. https://www.nimh.nih.gov/health/topics/autism-spectr um-disorders-asd 2. american psychiatric association. diagnostic and statistical manual of mental disorders . 5th ed. american psychiatric association; 2013. 3. ha s, sohn ij, kim n, sim hj, cheon ka. characteristics of brains in autism spectrum disorder: structure, function and connectivity across the lifespan. experimental neurobiology . 2015;24(4):273. doi:10.5607/en.2015.24.4.273 4. autism spectrum disorder fact sheet | national institute of neurological disorders and stroke. www.ninds.nih.gov. published july 25, 2022. https://www.ninds.nih.gov/autism-spectrum-disorder-fa ct-sheet 5. benger m, kinali m, mazarakis nd. autism spectrum disorder: prospects for treatment using gene therapy. molecular autism . 2018;9(1). doi:10.1186/s13229-018-0222-8 6. alharthi a, alhazmi s, alburae n, bahieldin a. the human gut microbiome as a potential factor in autism spectrum disorder. international journal of molecular sciences . 2022;23(3):1363. doi:10.3390/ijms2303136 7. capra e, godfrey a, loche a, smith j. innovation in viral-vector gene therapy: unlocking the promise | mckinsey. www.mckinsey.com. published may 17, 2021. https://www.mckinsey.com/industries/life-sciences/our insights/gene-therapy-innovationunlocking-the-promise -of-viral-vectors 8. singer e. finding new treatments for autism. spark for autism . published august 25, 2017. https://sparkforautism.org/discover_article/�nding-new -treatments-for-autism/ 9. yenkoyan k, grigoryan a, fereshetyan k, yepremyan d. advances in understanding the pathophysiology of autism spectrum disorders. behavioural brain research . 2017;331(0166-4328):92-101. doi:10.1016/j.bbr.2017.04.038 10. mlynaryk n. gene therapy reverses e�ects of autism-linked mutation in brain organoids. today.ucsd.edu. published may 22, 2002. https://today.ucsd.edu/story/gene-therapy-reverses-e�ec ts-of-autism-linked-mutation-in-b rain-organoids 11. papes f, camargo ap, de souza js, et al. transcription factor 4 loss-of-function is associated with de�cits in progenitor proliferation and cortical neuron content. nature communications . 2022;13(1):2387. doi:10.1038/s41467-022-29942-w 12. bioinforx. products. alstem. accessed december 10, 2022. https://www.alstembio.com/web/product_list.php?cate gory=human_ips_cell_lines. 13. lópez-bendito g, cautinat a, sánchez ja, et al. tangential neuronal migration controls axon guidance: a role for neuregulin-1 in thalamocortical axon navigation. cell . 14. frotscher m, chai x, bock hh, haas ca, förster e, zhao s. role of reelin in the development and maintenance of cortical lamination. j neural transm (vienna) . 2009;116(11):1451-1455. doi:10.1007/s00702-009-0228-7 15. zhubi a, cook eh, guidotti a, grayson dr. chapter six epigenetic mechanisms in autism spectrum disorder. sciencedirect. published january 1, 2014. berkeley pharma tech journal of medicine | 24 https://www.sciencedirect.com/science/article/pii/b978 0128013113000068?via%3dihub 16. lammert db, howell bw. reln mutations in autism spectrum disorder. front cell neurosci . 2016;10:84. doi:10.3389/fncel.2016.0008 17. ehrhart f, coort slm, cirillo e, smeets e, evelo ct, curfs lmg. rett syndrome – biological pathways leading from mecp2 to disorder phenotypes. orphanet journal of rare diseases . 2016;11(1). doi:10.1186/s13023-016-0545-5 18. wen z, cheng tl, li g, et al. identi�cation of autism-related mecp2 mutations by whole-exome sequencing and functional validation. molecular autism . 2017;8(1). doi:10.1186/s13229-017-0157-5 19. sztainberg y, chen h, swann jw, et al. reversal of phenotypes in mecp2 duplication mice using genetic rescue or antisense oligonucleotides. nature . 2015;528(7580):123-126. doi:10.1038/nature16159 20. kaufmann we, kidd sa, andrews hf, et al. autism spectrum disorder in fragile x syndrome: cooccurring conditions and current treatment. pediatrics . 2017;139(supplement 3):s194-s206. doi:10.1542/peds.2016-115f berkeley pharma tech journal of medicine | 25 the role of the gut microbiome in neuromodulation therapies as a potential treatment adjunct for multiple sclerosis berkeley pharma tech journal of medicine correspondence: pipertonne@gmail.com keywords: gut-brain axis gut microbiome immune system multiple sclerosis neuromodulation submitted june 28, 2021 accepted july 20, 2021 published december 17, 2021 full open access creative commons attribution license 4.0 abstract gut microbiome research has surged in popularity over the past decade. these studies have found local and distal effects of micro organisms such as fungi and bacteria on human physiology, including the nervous, immune, and endocrine systems. a number of studies have demonstrated the potential for gut microbiota to combat classical diseases such as clinical depression and autism spectrum disorder. the impact of gut-produced metabolites on the secretion of various cytokines has presented a new-found opportunity for future disease therapy through these micro-organisms. this review examines recent evidence for the use of gut bacteria in neurological rehabilitation, specifically for multiple sclerosis (ms) patients. available data has shown overwhelming support for microbiota-based ms therapy, but the lack of comprehension regarding the specific physiological mechanisms of these microbiota suggests that clinical trials may be far off. furthermore, there has been minimal research investigating the consequences of using microbiotic therapy in tandem with current therapies such as neurostimulation or drug therapy. factors including the mechanisms and restorative capability of specific species of microbiota must be studied in depth in order to successfully manipulate the gut microbiome for the treatment of neurological disorders. the role of the gut microbiome in neuromodulation therapies as a potential treatment adjunct for multiple sclerosis by: piper tonne, deondre do, nabeeha hasan, sravya gundapaneni, ariya reddy, saadhana sridharan, and sierra richardson the axon of the neuron, pictured on the right side, begins to degrade following the onset of multiple sclerosis. image credits: n/c introduction in recent years, the accessibility and prevalence of research regarding the gut microbiome has increased tremendously. it is common knowledge that the human gut plays an important role in regulating health. the question that remains to be asked is, just how much in�uence does the gut microbiome hold? newfound curiosity regarding the e�ects of manipulation of the bacterial population residing in the gut prompts the scienti�c community to expand research on ways to harness the power of the gut microbiome. research on the gut microbiome has advanced considerably since its discovery decades ago with the development of technologies such as 16s rrna sequencing to assist in mapping the human microbiome (choileain, 2020; jangi, 2016; takewaki, 2020; zuo, 2018). furthermore, with advancements in microbiome-related engineering and technology, researchers have come far closer to understanding the speci�c genes and mechanisms behind microbial functions in the gut and beyond (azad, 2018; cani, 2018). however, with a collective human genome including tens of thousands of genes, discovering speci�c causation from gut-related studies has proven to be exceedingly challenging. a large number of papers studying the human microbiome within the past decade have covered its immense potential to impact human health. however, none have been able to specify the mechanisms, much less the genes behind these microorganisms, which mainly consist of bacteria (cani, 2018; han, 2018; strandwitz, 2018). in this review, recent evidence showing the impact of general probiotics and speci�c bacterial implants on human neurophysiology will be examined. the various e�ects of the gut microbiome on the neuroimmune system will be discussed along with a scrutinization of the current neuromodulatory therapies. this paper explores the possibility of using gut therapy speci�cally for the clinical treatment of multiple sclerosis (ms) patients. before jumping into this discussion, it is imperative to know the background of this disease and the organisms that may assist in its treatment. the gut microbiome the term gut microbiome refers to the trillions of microbes living within the tract of humans and other animals. although incredibly small, the microbes in the gut play a huge role in human metabolism, physiology, and immune berkeley pharma tech journal of medicine | 65 system development (azad, 2018). the gut microbiome encompasses a large scope of symbiotic functions in the body including vitamin synthesis and gi hormone release, the latter of which protects the body from pathogen colonization (collins, 2012). one aspect of this symbiosis investigated in this review is the e�ects on the human central nervous system (cns), where alterations of neuronal signaling by the gut, especially in the brain, have piqued scholarly interest. particularly, there has been extensive research on microbial impact on the gi tract, as well as its impact on human physiology. within the past decade, research on the gut microbiome has seen an exponential increase due to the metagenomic revolution the study of genomes of bacterial species in a speci�c environment rather than pure laboratory cultures. one reason the gi tract is an area of interest is due to its key role in the complex mechanisms of immunoregulation (joscelyn, 2014; kirby, 2018; mangalam, 2017; ochoa-repáraz, 2014,2018; shahi, 2019; velasquez-mano�, 2015). at �rst, interest in the role of the microbiome led researchers to look for any associations regarding innate and adaptive immunity within the human body. for example, gut-associated lymphoid tissue (galt) represents almost 70% of the entire immune system, and the gi tract hosts around 80% of plasma cells, such as immunoglobulin a (iga)-bearing cells (vighi, 2008). this research has since dramatically expanded with the upsurge of discoveries correlating bacteria, such as akkermansia muciniphila or prevotella copri, with various disease pathologies including: obesity, type ii diabetes, and multiple sclerosis (cani, 2018). for example, researchers now understand that the function of the overall human immune system is deeply in�uenced by bacteriophages, viruses that parasitize bacteria by infecting them and reproducing inside them. research indicates that bacteriophages, such as caudovirales, can be manipulated in order to provide relief to patients su�ering from clostridium difficile infection through successful fecal microbiota transplantation. by administering fecal matter from a donor into the intestinal tract of a recipient, the recipient experiences relief of infection symptoms as a result of the change in their gut microbial composition (zuo, 2018). furthermore, with the increasing interest in elucidating the potential manipulation of the gut microbiome, there has been an increase in research highlighting its connection with the central nervous system, and its role in maintaining homeostasis (ochoa-repáraz, 2009; wang, 2014; wang & kasper, 2014; winter 2018). however, although research is extensive, there berkeley pharma tech journal of medicine | 66 is an inadequate understanding of the speci�c mechanisms of the gut on human physiology. it is widely accepted that the development of the gi microbiome begins at birth. the gi tract is rapidly colonized after childbirth and is impacted throughout life by various external factors such as: illness, antibiotic treatment, and changes in diet (rodríguez, 2015). it is imperative to acknowledge the consequences of certain lifestyle choices on the gut microbiome, as any shifts in one’s microbial genome could a�ect their entire physiology (figure 1). diet is a major lifestyle choice that in�uences gut health. both short and long-term alterations in diet can impact microbial pro�les, and infant nutrition may have lifelong consequences through microbial modulation of the immune system (harmsen, 2000). in conjunction with the prevalence of malnourishment, with the most common representation being obesity, diet is an ever-growing area of concern within medical research (manichanh, 2006). gut microbes produce a large number of bioactive compounds that can be bene�cial, such as vitamins. bacteria, such as bifidobacterium, can generate crucial vitamins such as: vitamin k, b12, biotin, folate, and thiamine (nicholson, 2012). in addition, digestion is in�uenced by many enzymes produced by microbes. the microbial diversity in the human gut is berkeley pharma tech journal of medicine | 67 figure 1. the gut microbiome plays an important role in maintaining host immunity and homeostasis via the gut-brain axis. since the axis is bidirectional, factors such as hunger, anxiety, stress, and depressive disorders, in addition to lifestyle choices such as diet and exercise, can reshape the gut bacteria's composition and exert an influence on immune function and health. attributable to the spectrum of microbial enzymatic capacity needed to degrade nutrients, particularly the many forms of complex polysaccharides that are consumed by humans (cantarel, 2012). nourishing the body properly through a balanced diet is the best way of maintaining a healthy gut microbiota population. the gut microbiome is a complex ecosystem that cycles nutrients between the microbiota and their host cells. this cycling of nutrients dictates the body’s immune response to foreign invaders (azad, 2018). however, the bidirectional nature of this relationship should be noted. changes in chemical, nutritional, and immunological pathways of the body have also been shown to in�uence the density and composition of the gut microbiome (thursby, 2017). research has already shown that the gut microbiome is largely dominated by rapidly growing probiotic bacteria because they have the capability to survive in harsh conditions. some of the most common genera of gut bacteria in adults are bifidobacterium, lactobacillus, bacteroides, clostridium, escherichia, streptococcus, and ruminococcus (conlon, 2014). in order to protect the integrity of the gut, some researchers have explored gut modulation by probiotic species, which may be able to improve and restore the gut �ora if certain bacterial species of the gut were ever eradicated (azad, 2018). two crucial microbial strains discovered for gut microbiome regulation were lactobacillus and bifidobacterium. these quintessential probiotics are target bacterial groups made from short-chain nondigestible carbohydrates (inulin-type fructans, fructo-oligosaccharides [fos], and galacto-oligosaccharides [gos]) (loo, 2009). as it stands, gut regulation is essential in isolating speci�c bacterial species for clinical therapy, especially for neurological disorders like multiple sclerosis (ms), whose causes are still unknown. multiple sclerosis ms has cell-mediated pro-in�ammatory e�ects (also known as type iv hypersensitivities) that result in demyelination of neurons and autoimmune pathogenesis of the disorder, leading to disruptions in brain-body communication. in ms patients, autoreactive t and b lymphocytes enter the cns, induce in�ammation, and undermine the blood-brain barrier (bbb) via cytokine secretion (ghasemi, 2017). these cytokines begin a signaling cascade that results in oligodendrocytic death and thus, the destruction of neuronal myelin sheaths in the cns (ghasemi, 2017). the resultant cns lesions disturb proper communication between neurons, berkeley pharma tech journal of medicine | 68 and lead to various cortical dysfunctions within ms patients such as modulations to resting motor threshold, short interval cortical inhibition, and central motor conduction time (ghasemi, 2017). although microbial therapy holds promise, its implementation as a universal ms therapy remains far o� because of the lack of understanding behind the speci�c mechanisms of gut-brain interactions and early research into this �eld (figure 2). while the speci�c origin of ms still eludes scientists, most postulate that a combination of genetic and environmental factors plays a signi�cant role in pathogenesis. environmental elements such as geography, vitamin d de�ciency, obesity, diet, smoking, and physical or emotional stress have been shown to be relevant in ms progression (gianfrancesco, 2016; ochoa-repáraz, 2014; rosso, 2019; sintzel 2018). when these factors collaborate with adverse genetics, the health of ms patients can quickly deteriorate. one of the most salient genetic factors is sex, with ms diagnosis showing an astounding 2:1 male-to-female ratio (reynolds, 2018). women are also diagnosed with an irreversible disability at older ages than men (confavreux, 2006). another important genetic factor is the hla-drb1*15 gene haplotype. the dr2 haplotype hla-drb1*15 gene encodes a protein important in t-lymphocyte reactivity and is also associated with ms disease progression, age at onset, and atrophy of subcortical gray matter (isobe, 2016). berkeley pharma tech journal of medicine | 69 figure 2. a neuron is made up of a cell body, an axon, and dendrites. the protective coating of the axon, the myelin sheath, are damaged in patients with multiple sclerosis. the immune system attacks the myelin sheath and causes injury and inflammation. neural deficits ensue. types of multiple sclerosis ms patients are categorized into four subtypes including: relapsing-remitting (rrms), secondary progressive (spms), primary progressive (ppms), and progressive-relapsing (prms). rrms is the most common subtype, a�ecting approximately 87% of the patient population (ghasemi, 2017). autoimmune in�ammatory attacks on the nervous system among patients of this subtype occur months or years apart followed by a period of remission, leading to a progressive increase in irreversible disability. this neural damage eventually manifests as vision loss, muscle weakness, and impaired coordination (weiner, 2008). during periods of remission, patients revert to relatively normal neurological activity (høglund, 2014). this subtype of ms shows the greatest promise of therapy because intervention is possible during remission periods. unfortunately, within the population of rrms patients, around 70% develop spms (kirby, 2018). however, what facilitates the progression from rrms to spms remains unclear. spms is characterized by a minimal to complete lack of relapse activity, leading to a constant increase in irreversible neural damage (høglund, 2014). although the underlying mechanisms of the progression from rrms to spms are currently unclear, most likely a combination of environmental and genetic factors, spms di�ers from rrms in that demyelination is restricted to short lengths of disrupted myelin located in aggregates of microglial cells (prineas, 2001). patients in this category have a mean age of about 44-63 years at spms onset (confavreux, 2006). ppms patients see similar e�ects, but their pathogenesis is slightly di�erent. ppms patients compose approximately 10-15% of all ms patients and tend to have less brain atrophy but increased spinal cord atrophy (ghasemi, 2017). in ppms, regression does not occur. patients instead experience a steady increase in the debilitating e�ects of ms (reynolds, 2018). lastly, prms is the subtype of ms with the fastest increase of disability over time due to periodic immune system attacks along with a steady increase in disability. prms is the most devastating subtype, and it comprises approximately 5% of the ms population (ghasemi, 2017). ms symptoms are similar among patients, with many resulting from plaque formation after demyelination. these symptoms are notoriously di�cult for physicians to predict due to di�ering plaque locations between patients leading to di�erent symptoms (kister, 2013). however, common symptoms berkeley pharma tech journal of medicine | 70 in those a�ected include vision loss, decreased mobility, bowel/bladder dysfunction, sensory loss, impaired coordination, decreased energy, and spasticity (kister, 2013). patients often experience chronic pain from conditions such as trigeminal neuralgia (pain triggered by oral activity), dysesthetic pain, back pain, and painful spasms (solaro, 2004). these conditions are often comorbid with mental health disorders such as depression, further confounding diagnostic e�orts (chwastiak, 2007; kister, 2013; vattakatuchery, 2011). thus, it is critical to pinpoint the speci�c causes of ms in order to establish a framework for the development of e�ective microbial therapies. multiple sclerosis pathogenesis it is still unclear whether an overactive immune system and acute in�ammation are the incipient causes of ms, or if pathogenesis stems from a radically di�erent cause. similarly, there is still ambiguity regarding autoreactive immune cell leakage into the cns. the current literature demonstrates that t helper (th) lymphocytes and various cytokines play a primary role in ms pathogenesis (williams, 2020; choileain, 2020; berer, 2011). th17 is the most studied t helper cell, and it is considered by many to play a large role in the in�ammation observed in ms patients. (choileáin, 2020; reynolds, 2010; mangalam, 2017; tahmasebinia, 2017). many studies support the role of th17 in producing pro-in�ammatory interleukins (il) such as il-17, il-17a, il-17f, il-21, il-22, and il-26 (ghasemi, 2017; xu, 2020; reynolds, 2010). this increased in�ammatory response is aggravated further when the normal negative feedback response becomes impaired. ms patients have displayed a reduced ability to produce t regulatory cells, which normally mediate the in�ammatory e�ects of th cells that occur as a result of leakiness of the blood-brain barrier (bbb) (cekanaviciute, 2017). the blood-brain barrier is a semipermeable border composed of endothelial cells (ecs), pericytes, astrocytes, and an extracellular matrix (abbott, 2010). recent studies indicate that ms patients have circulating factors and the bbb engages in crosstalk that is mediated by endothelial cells (ecs) and adjustment of astrocytic expression (williams, 2020; setiadi, 2019; zivadinov, 2016). ms patients are further characterized by lymphocytes berkeley pharma tech journal of medicine | 71 undermining bbb permeability by inducing oxidative stress in ecs and enhancing leukocyte transmigration by producing various facilitative proteins: p-glycoprotein, intercellular cell adhesion molecule 1(icam-1), and vascular cell adhesion molecule 1 (vcam-1) (sheikh, 2020). vcam-1 and icam-1 expression is directly correlated to levels of il-17 and tnf-α, suggesting another mechanism of the enhanced in�ammation seen in ms patients (gao, 2017). by modifying bbb immuno-tra�cking, autoreactive cells are readily able to cross into the cns and cause further damage. this leakage of immune cells is exacerbated as ms lesions begin increasing b cell transcription ten-fold through promoting the production of b cell-activating factor (baff) (krumbholz, 2005; kannel, 2015). furthermore, once pathogen-associated molecules bind to toll-like receptors (tlr) on these lymphocytes, autoreactive t cells are signaled to produce cytokines that induce additional cell di�erentiation (reynolds, 2010). ms patients reportedly possess high levels of tlr2 and tlr4 among others, with tlr2 being associated with the defective remyelination seen in ms (hasheminia, 2014; wasko, 2020). microbial manipulation of the immune system gives scientists hope for gut-based therapies, albeit speci�c causation has not been established. current treatment options for ms due to its complex neurological pathogenesis, ms is currently an incurable disease. treatment options for patients with ms are divided into three categories: acute relapse management, slowing of disease progression, and treatment of related symptoms (hart, 2016). glucocorticoids are the drugs of choice for an acute attack because they downregulate molecules associated with in�ammation in the body, such as cytokines and chemokines, and upregulate anti-in�ammatory proteins. therapy with glucocorticoids such as high-dose methylprednisolone should be considered in patients whose relapse is of moderate to severe severity (doshi, 2016). ultimately, glucocorticoids are used to shorten the duration of a relapse or acute attack. it has become increasingly important to focus on slowing disease progression in order to improve the quality of life of ms patients. disease-modifying treatments (dmts) have become a key component of comprehensive ms care for this reason (hart, 2016). rrms is the most berkeley pharma tech journal of medicine | 72 treatable subtype of ms. drugs such as beta-interferons, glatiramer acetate, teri�unomide, dimethyl fumarate, and �ngolimod have moderate e�cacy but are a safer option in patients with rrms. the human body already produces beta-interferons as a natural response to in�ammation. the man-made drug further aids in the downregulation of in�ammation and decreases the damage to the nerves in the body. however, since other dmts can have life-threatening adverse e�ects, it is necessary to monitor clinical conditions and conduct frequent mri scans. alemtuzumab and natalizumab, targeted cancer drugs, have a higher e�cacy but present serious side e�ects such as progressive multifocal leukoencephalopathy (pml). pml, an illness that leads to brain damage, severe disabilities, and death, can develop in patients that are on natalizumab treatment for more than two years with a prior history of chemotherapy or immunosuppression (doshi, 2016) (figure 3). it is recommended that dmts are started as soon as possible to increase the likelihood of slowing the progression of the disease before severe neurological de�cits can arise. unfortunately, there is not enough current research indicating that dmts can aid in slowing progressive ms and there is still a lack of cohesive understanding about dmts in the research community. more studies are necessary in order to understand the e�cacy and safety of drugs while simultaneously slowing the progression of the disease. berkeley pharma tech journal of medicine | 73 figure 3. the mechanism of action of the dmt ifnβ. this drug targets immune cells. it decreases t cell activation, b cell proliferation and promotes antiinflammatory cytokine production. symptomatic treatment of ms is just as important as slowing the progression of the disease or treating an exacerbation. patients often complain of fatigue, depressive symptoms, urinary frequency, sexual dysfunction, constipation, pain, ataxia, and more (doshi, 2016). not all symptoms necessarily require medication. however, it is important that physicians recognize the heterogeneity of this disease when creating a treatment plan for their patients. ms can largely impact one’s functionality, and patients typically struggle with adapting to the changes in their daily life. thus, patients are often recommended to start cognitive therapy alongside their medication. pain and depression are symptoms that have long been overlooked in individuals with ms. one study showed that more than 88% of people with ms experience pain in more than one bodily area (gromisch, 2020). another study reported that 22.8% of patients with ms struggle with lifetime major depression (wang, 2000). studies have shown that using cognitive behavioral therapy as an adjunct treatment for ms can improve mental health and quality of life in patients (gromisch, 2020). neurostimulation technologies are recent medical advancements that are rapidly being researched as potential symptomatic treatment options for neurologic and psychiatric disorders. an increased understanding of neural circuitry and neurotechnologies has shown promising therapeutic results in patients with neuropsychiatric conditions. these therapies include invasive and noninvasive approaches that target a speci�c nerve or anatomical region in the body. some examples include brain temperature control, magnetic stimulation, deep brain stimulation, spinal cord stimulation, and vagus nerve stimulation (edwards, 2017). research has shown that gut bacteria communicate with neurons of the enteric nervous system to send signals to the brain via the vagus nerve (galland, 2014). currently, an fda-approved implantable vagus nerve stimulator called livanova can treat drug-resistant epilepsy. in addition, vagus nerve stimulation has been used to treat drug-resistant depression (edwards, 2017). researchers at the texas biomedical device center have developed a technology called the repair system that rewires neural circuitry through stimulation of the vagus nerve, focusing on targeted plasticity therapy. the �rst human trials involved stroke patients and focused on improving upper and lower limb motor berkeley pharma tech journal of medicine | 74 de�cits following a brain injury or disease (darrow, 2020). symptoms of ms often include muscle weakness and motor imbalance, leading researchers to believe that this therapy has the potential to change how physicians approach the treatment of ms and other neurological disorders. another clinical trial tested vagus nerve stimulation in three ms patients in an e�ort to reduce cerebellar tremor and dysphagia. symptoms were improved over a period of two to three months and the involvement of the nucleus tractus solitarius, a key visceral component of the vagus nerve, was further studied (marrasu, 2007). to date, the underlying mechanisms of neuromodulation therapies are not well understood, which is why they are not primary treatment options for most neuropsychiatric diseases. however, a better understanding of neural circuitry in the human body has led to promising technological advancements that may signi�cantly reduce symptoms in patients su�ering from various neurological conditions. microbial links to human physiology lactobacillus is a genus of bene�cial gut bacteria that have gained an increasing amount of scholarly attention (esber, 2020; zhou, 2015). speci�c species of lactobacillus including l. acidophilus, l. casei, l. rhamnosus, and l. helveticus have been linked to the prevention of disease in humans and animals (azad, 2018). lactobacillus can alter the population of microorganisms in the gut microbiome by producing lactic acid, preventing harmful bacteria from colonizing the intestines. a study using a mouse model of hyperlipidemia explored the impact of modulating the gut microbiome by introducing probiotic feeding of lactobacillus into the mice’s diet. signi�cant changes in the microbiota composition were found, including an increased abundance of bacteroidetes and verrucomicrobia and a reduced ratio of firmicutes (chen, 2014). l. acidophilus has even displayed the ability to maintain a homeostatic concentration of in�ammatory cytokines, th17, and regulatory t (treg) cells (park 2018). furthermore, l. acidophilus suppressed proin�ammatory cytokines such as il-6, tumor necrosis factor-α (tnf-α), and il-1β in colon tissues. similar to lactobacillus, there has been a research focus on the probiotic genus bifidobacterium. bifidobacterium assists the human body in performing essential functions such as digestion, improving gastrointestinal barrier integrity, preventing harmful bacterial colonization, and suppressing proin�ammatory cytokines (ganz, 2002). it maintains immune homeostasis berkeley pharma tech journal of medicine | 75 by altering the function of dendritic cells in order to protect against foreign bacteria and pathogens (azad, 2018). moreover, bi�dobacteria increase the proportion of bene�cial bacteria in the gut microbiota through cross-feeding, allowing other bacteria to live o� of their metabolic products. bifidobacterium bifidum has shown signi�cantly increased metabolic activity when cocultured with bifidobacterium breve (turroni, 2015). this co-culture of probiotic bacteria a�ected the metabolic shift in the gut microbiota by increasing the production of short-chain fatty acids, suggesting bifidobacterium could play a role in cognitive function via hormonal signaling (savignac, 2013). this can lead to improved memory function, including the growth of brain-derived neurotrophic factor (bdnf) and n-methyl-d-aspartate receptor expression (savignac, 2013). the prophylactic role of this bacterial species among other probiotic strains was further explored in a mouse model of β-lactoglobulin allergy, �nding that the administration of bifidobacterium longum subsp. infantis (b. infantis) la308 for 3 weeks modi�ed the composition of the gut microbiota, signaling a connection between probiotics and the diversity of gut microbiota (azad, 2018). there was a signi�cant change in forkhead box p3 (foxp3), transforming growth factor-beta (tgf-β), and il-10 ileal gene expression, as well as plasma metabolomic alterations in the tryptophan (trp) pathway. the study concluded that probiotic introduction to the human body led to alterations in immune responses, tolerogenic energy induction, and anti-in�ammatory responses (esber, 2020). human gut-immune interactions the immune system plays a pivotal role as the intermediary between the gut microbiome and cns, especially in the regulation of autoimmune responses. the presence or absence of several components of the gut microbiota regulates t and b cell activation within the brain, leading to the enhanced in�ammatory response associated with autoimmune diseases like ms. for instance, the presence of gut-residing bacteria faecalibacterium prausnitzii caused various symptoms in patients with crohn’s disease, an autoimmune disease characterized by heightened in�ammation in the digestive tract (velasquez-mano�, 2015). patients who lacked this bacteria experienced in�ammatory bowel syndrome and asthma, while those possessing them displayed a negative correlation with in�ammatory berkeley pharma tech journal of medicine | 76 autoimmune diseases (velasquez-mano�, 2015). the potential therapeutic applications of this bacteria's anti-in�ammatory nature are currently under research for autoimmune disorders (figure 4) (velasquez-mano�, 2015). human-gut immune interactions metabolites further studies on gut-immune interactions have emphasized modi�cations to in�ammatory cytokine pathways. these interactions vary from cytokine-speci�c to stimulant-speci�c responses or sometimes, both concurrently. studies revealed that 10% of the variability of cytokine responses associated with in�ammation can be accounted for by variations in the gut microbiome (schirmer, 2016). moreover, the interaction between the bacterial component of the gut microbiome and immune cells does not occur directly, but through metabolites, small molecules referring to the intermediates or end products of a metabolic pathway (wirthgen, 2018). one such pathway leads to the formation of tryptophol, a metabolite and common inhibitor of tnf-α. the degradation of the essential amino acid trp to tryptophol is negatively associated with the interferon gamma (ifnγ) in�ammatory pathway (schirmer, 2016). several other trp-derived metabolites, particularly the tryp-6 neuroactive compounds kynurenine, quinolinate, serotonin, indole, and indole derivatives, may have a critical role in bacterial signaling (kaur, 2019). low levels of these compounds are associated with disorders like major depression, autism spectrum disorder, berkeley pharma tech journal of medicine | 77 figure 4. the presence of bacteria f. prausnitzii in the gut maintains the integrity of the epithelial gut lining and inhibits the t cell inflammatory response. the absence of this materia creates leaky cell junctions in the gut lining, which allow for opportunistic microbes and toxins to enter and activate the inflammatory response. and parkinson's disease. researchers have seen extensive modulation of these metabolic pathways from several genera of gut bacteria which break down trp like clostridium, burkholderia, streptomyces, pseudomonas, and bacillus (kaur, 2019). in addition, it is believed that gut microbiota contribute to the availability of trp and the neurotransmitter 5-hydroxytryptamine, which regulates neuroendocrine signaling (martin, 2018). 5-hydroxytryptamine, also known as serotonin, is important in combating the comorbidities that often accompany ms. namely, major depression and anxiety disorder (kelly, 2016; zheng, 2016; winter, 2018). however, there are a multitude of additional disorders that may be associated with gut microbiota including obesity, diabetes mellitus, schizophrenia, and autistic disorders (evrensel, 2015). changes in gut microbiota are relevant to mood states due to their contribution to the production of neurotransmitters (mittal, 2017). regulation of this pathway appears to be extremely important for the development of gut-based ms therapy. however, while the formation of trp metabolites is a key pathway, there are various other important metabolites that should be considered. secondary bile acids (2bas) are strongly in�uenced by microbial activity and activate the intestinal l cell’s surface g protein-coupled bile acid receptors (martin, 2018). 2bas derived from spore-forming bacteria of the gut regulate a signi�cant percentage of 5-ht synthesis and release from enterochroma�n cells, introducing another intriguing connection between gut metabolites (yano, 2015). short-chain fatty acid (scfa) synthesis is another metabolic pathway that is a�ected by gut bacteria, often through bacterial fermentation or host protein glycosylation. microbial fermentation (primarily by bacteroidetes) leading to scfa production in the colon and blood is believed to play a critical role in immunoregulation (choileáin, 2020). furthermore, the biochemical conversion of nutrients into scfas and other amino acid-derived metabolites like 5-ht are often conducted by intestinal microbes (hemarajata, 2013). these peptides function as immunomodulators through mechanisms including (park, 2019): 1. metabolic integration, or the integration of two or more metabolites 2. microbiota regulation 3. histone deacetylase (hdac) inhibition, which is involved in epigenetic berkeley pharma tech journal of medicine | 78 or non-epigenetic regulation of cancer cells 4. g-protein coupled receptor (gpcrs) activation that plays a role in the cellular signal transduction pathway the proin�ammatory cytokines tumor necrosis factor-alpha (tnf-α) and il-1β even see an increased level after scfa induction of the gut-immune system (galland, 2014). scfas have been shown to upregulate or downregulate primary immune cells such as cd4+ e�ector cells and il10+ tregs in mice with experimental autoimmune encephalitis (eae), a disease with pathogenesis similar to ms (höftberger, 2015). some scfas can contribute to the anti-in�ammatory response by stimulating il-10 production while others, for example, the g-protein coupled receptors gpr41 and gpr43, may initiate a pro-in�ammatory response. however, further research must occur to determine the exact relationship between scfas and related immune cells, as well as the pathways through which this in�ammation occurs (park, 2019). gut-immune interactions proteins an important part of gut-immune interactions is the e�ect that they have on toll-like receptors (tlrs). a few studies have suggested there is a connection between tlr2, and its signaling pathway, that ties ms to the microbiome (wang, 2014; wasko, 2020). tlr2 can be stimulated by bacterial lipopeptides. in murine models of ms, microbial injections inducing tlr2 tolerance have shown inhibition of cns in�ammation while improving remyelination (wasko, 2020). another protein, tlr4, has also displayed responses to lipopolysaccharides (lps) from gram-negative bacteria (park, 2009). in practice, lack of exposure to lps from gut bacteria resulted in a lack of tlr4 tolerance, resulting in de�cient regulation of innate immune tlr responses and enhanced autoimmunity (wasko, 2020). gut-immune interactions cytokines when researching the gut-brain axis relationship, it is important to understand how each micro or macromolecule a�ects both sides of this a�liation. with the importance of cytokines in the formation of ms lesions, these proteins are particularly important in understanding how the gut can impact the brain. numerous studies have found that dysbiosis of the gut may induce proin�ammatory responses, suggesting a potential avenue for gut-based ms treatment (choileáin, 2020; galland, 2014; berkeley pharma tech journal of medicine | 79 monteleone, 2011; martin, 2018; adamcyzk-sowa, 2017; shahi, 2017). proin�ammatory interleukins such as il-6 and il-17 have shown evidence of being a�ected by microbial modi�cations in the gut. interestingly, mesenteric lymph nodes (mln) of antibiotic-treated animals produced less il-6 while signi�cantly increasing levels of the anti-in�ammatory il-13 and il-10 compared to controls (ochoa-repáraz, 2009). furthermore, research regarding the gut-brain axis has shown that arti�cial activation of aryl hydrocarbon receptor (ahr) ligands, a part of the tryptophan metabolite pathway, decreased the concentration of ifn-γ while up-regulating proin�ammatory il-22 in the gut of in�ammatory bowel disease patients (monteleone, 2011). these gut-based cytokine alterations are only compounded with the modi�cations to peripheral blood mononuclear cells (pbmcs) within the body. gut-immune interactions-peripheral blood mononuclear cells pbmcs such as t cells among others all see concentration adjustments due to the gut. exposure of healthy pbmcs to parabacteroides distasonis, a common gut bacteria, signi�cantly increased the percentage of il-10 expressing cd4+cd25+ t cells and il10+foxp3+ tregs within the cd4+cd3+ population (cekanaviciute, 2017). furthermore, treatment of mice with prevotella histicola also showed an increase in cd4+foxp3+ tregs in addition to a decrease in proin�ammatory th1 and th17 immune cells (mangalam, 2017). these correlations display bacterial importance in the regulation of cd4+ and cd8+ t cells; which have shown an increased expression of cxcr3+ in ms patients, contributing to greater leakage in the blood-brain barrier (choileáin, 2020). protection of this barrier will be crucial in the development of gut-based ms therapies in order to combat the reduced expression of regulatory proteins seen in rrms patients such as occludin and vascular endothelial cadherin (sheikh, 2020). while t lymphocyte regulation is extremely important in achieving this goal, so are other pmbcs like nk cells and b cells, the latter of which is believed to be involved with ms because of their association with immunoglobulin presence (høglund, 2014). ms usually involves the depletion of b cell numbers and an increase in t lymphocytes (krumbholz, 2012). nk cells are important in their role of target cell lysis and can also be a therapeutic target due to their role in cytokine and chemokine secretion (høglund, 2014). additionally, they have the ability to modify or lyse t berkeley pharma tech journal of medicine | 80 cells, an interaction that can be investigated in the future to improve the understanding of the dysregulation of immune systems in ms patients (høglund, 2014). the gut-immune relationship is a two-way street. in the future, research must take into account that the gut does not a�ect human physiology unidirectionally. while the composition of the gut microbiome heavily in�uences the autoimmune response by regulating the interactions between immunoregulatory cells and metabolites, the immune system is also critical in cultivating healthy bacteria and destroying harmful bacteria in our gut (velasquez-mano�, 2015). the key players in shaping the gut microbiome are nucleotide-binding and oligomerization domain-like receptors (nlrs or nod-like receptors). for instance, the nod2 bacterial sensor regulates in�ammation caused by the growth of the commensal bacteroides vulgatus (ramanan, 2014). this regulation promotes epithelial stem cell survival and regeneration in the gut. furthermore, nlrs that assemble into multiprotein complexes, known as in�ammasomes, activate in�ammatory caspases and regulate microbial diversity in the gut. the nod-, lrr (leucine‐rich repeat)and pyrin domain-containing 6 complex (nlrp6) is a type of in�ammasome co-modulated by microbiota-derived metabolites that is linked with intestinal homeostasis, intestinal antiviral innate immunity, and the regulation of epithelial il-18 secretion and amp expression pro�les (zheng, 2020). to fully grasp the intricacies around this bidirectional relationship and the pathways that associate the two, researchers continue to explore this vital frontier. discussion it is clear that the gut-brain axis has an impact on human physiology, so taking advantage of this non-invasive approach will be the crucial next step to treating and/or preventing ms. there has been prior evidence showcasing the relevance of gut microbiota in the treatment of neurological and motor disorders similar to ms including major depression and parkinson’s disease (bremner, 2020; haney, 2018; han, 2018; o'reardon, 2006; tian, 2020; winter, 2018; zhou, 2015; martin, 2018). yet, even though extensive research has been conducted over the past decade, the speci�c mechanisms of the gut-brain axis in relation to ms have yet to be found. however, a few notable considerations for these mechanisms include (1) systemic cytokine activation, (2) neurotransmitter synthesis, and/or (3) neuronal circuitry alterations (galland, 2014). one could interpret this as berkeley pharma tech journal of medicine | 81 academia leaning toward investigations on the nervous, immune, and endocrine systems. researchers of the nervous system believe the vagus nerve, the cranial nerve that controls the signaling between the brain and gi tract, plays a key role in the interactions of the gut-brain axis (figure 5). one study found that the vagus enhances neural plasticity (post-stroke) with improvements in both cognitive and motor function, both of which are largely diminished in ms patients (liu, 2016). the vagus nerve is also important for neurogenesis through the modulation of brain-derived neurotrophic factor (o’leary, 2018). further research also correlates neuroelectrical stimulation of the vagus nerve with a decrease in symptoms of various neurological disorders in mice (zhou, 2015; haney, 2018). as it stands, gut-brain signaling appears to be relatively dependent on vagus nerve activity, but this may only be true in certain experimental systems (bercik, 2011). impacts on chemical communication in the brain have also illustrated the value of the gut-brain axis in ms treatment. it has been noted that more than 90% of the body’s 5-hydroxytryptamine (5-ht), or serotonin, is produced in the gut (yano, 2015). 5-ht receptors are critical in the berkeley pharma tech journal of medicine | 82 figure 5. the vagus nerve is the tenth cranial nerve and extends from the brainstem through the neck and the thorax down to the abdomen. it carries signals from the digestive system and organs to the brain and vice versa. in addition, it modulates inflammation, maintains homeostasis, and regulates many body sensations. mediation of gut-brain axis activity in ms patients (malinova, 2018). gut microbiota have also shown success in the alteration of host serotonin levels through the mediation of small molecules like scfas or secondary bile acids (2bas) (yano, 2015). scfas in particular are important in mediating host-microbe communication via enteroendocrine and enterochroma�n cells, the latter of which also play a role in tryptophan metabolism (figure 6) (martin, 2018). although the gut-brain axis shows great potential for ms therapy, research on the functions of speci�c bacterial species on the gut-brain axis is limited. there is signi�cant evidence to suggest that few genera such as bacteroides and firmicutes are a�ected by mental and physiological stress, but their speci�c e�ect on ms pathology is unclear (tian, 2020; choileáin, 2020). studying the e�ects of general probiotics versus speci�c bacterial species on ms symptoms is necessary to determine the direction of future research on ms therapy via the gut-brain axis. furthermore, future research directed toward the integration of current ms therapies and their e�ect on the gut-brain axis is needed. for example, in regards to neuromodulation technology, the development of closed-loop adaptive systems which use predictive models of neural circuitopathies to alter neurostimulation parameters without clinical supervision is a promising avenue of research (drew, 2019; edwards, 2017; lozano, 2019). another promising berkeley pharma tech journal of medicine | 83 figure 6. stimulation of the vagus nerve has shown efficacy in modulating 5-ht receptors, scfas, and 2° bas in the gut microbiome. development is the creation of minimally invasive, wireless neuromodulation technology to alter biological parameters in real-time (tanabe, 2017; iodice, 2017). in recent years, there has been an increase in clinical trials exploring the connection between the vagus nerve and neurological de�cits. for example, a current clinical trial is exploring how stimulating the transcutaneous vagus nerve could improve cognitive function (university of ostrava, 2019). the clinical trial is using non-invasive stimulation provided by a transcutaneous electrical nerve stimulation device for four hours a day at 25hz, 250 μs pulse width placed on the tragus. this novel area of preventive medicine could provide an interesting perspective on ms therapies. if the technological device results in clinically signi�cant applications, it could improve executive neurological function and potentially help prevent demyelination. conclusion there has been minimal research regarding many areas of concern such as the e�ects on the blood-brain barrier by b cells, the speci�c mechanisms of lymphocytes and cytokines on the gut-brain axis, and the association between ms and the microbiome. it is unclear whether or not 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(2014). di�erential expression of mir-125a-5p and let-7e predicts the progression and prognosis of non small cell lung cancer. cancer investigation, 32(8), 394-401. doi:10.3109/07357907.2014.922569 berkeley pharma tech journal of medicine | 94 artlcle 3_ms gut microbiome_publish_20211217.pdf front page the role of the gut microbiome in neuromodulation therapies as a potential treatment adjunct for multiple sclerosis.pdf 3. body the role of the gut microbiome in neuromodulation therapies as a potential treatment adjunct for multiple sclerosis.pdf the role of the gut microbiome in neuromodulation therapies as a potential treatment adjunct for multiple sclerosis (reference page template).pdf traumatic brain injury-induced parkinson's disease: an analysis of the potential correlation �ρॸ��ǧƺκƺǿƺ�^ȗǔǔȓƺǐॹ�eƺǹǩǧƺ�?ǧƺǿॹ�[ȅǉǧǔǹǹǔ�^ǧƞȣƞǩǿॹ�^ȅйƺ�xǩǔǐȗƺйƞƺেkȗƞǩφॹ� tejaswini govindaraman, neha ramachandran, danielle mcmillion, ariana samimi and diego sandoval abstract research findings on the correlation between traumatic brain injury (tbi) and the onset of parkinson’s disease (pd) are inconclusive. with pd affecting thousands of individuals, it is imperative to determine a potential causal link. the purpose of this literature review is to under-stand the validity and extent of the relationship between tbi and pd by examining population demographics, biological, sociological, and cognitive factors, as well as therapeutic treatments. previous research has found that the incidence of tbi can occur during the prodromal period of pd, which is the period when symptoms of the disease begin to show, though the directionality of the correlation between tbi and the onset of pd requires a more in-depth investigation. other findings include the overaccumulation of alpha-synuclein in the substantia nigra—a protein that regulates vesicle trafficking—which is prominent in both patients with tbi and those with pd. the presence of tyrosine hydroxylase from tbi has also been proven to contribute to the decrease in dopamine seen in pd. hippocampal damage is present in patients who experience either pd or tbi, which leads to cognitive decline. these findings indicate a positive correlation between tbi and the subsequent onset of pd, and confirm the need for additional research to develop new therapies for pd. berkeley pharma tech journal of medicine correspondence: bhavana.sreepad@gmail.com keywords: parkinson’s disease traumatic brain injury alpha-synuclein tyrosine hydroxylase dopamine submitted july 7, 2021 accepted august 28, 2021 published december 17, 2021 full open access creative commons attribution license 4.0 image credits: n/c introduction parkinson’s disease (pd) a ects movement and is the second most common progressive neurodegenerative disorder after alzheimer’s disease. symptoms include slow movement, rigidity, shu ing gait, postural instability, and imbalance [1]. this neuronal imbalance is followed by the degeneration of dopamine neurons in the substantia nigra of the basal ganglia tissue in the brain, and the development of lewy bodies, which interfere with brain activity [2]. pd was rst described by james parkinson in 1817 [3]. the cause of neurodegeneration associated with pd has not been scienti cally proven and, only a small percentage of pd diagnoses are attributed to genetic factors. however, pd has been explored in association with non-genetic risk factors such as traumatic brain injury. traumatic brain injury (tbi) typically results from blunt external force to the skull, which may result in cognitive and behavioral disruptions that could lead to brain pathologies [4]. current research remains divided regarding the strength of the correlation between tbi and the onset of pd. as such, this review analyzes population demographics, biological and cognitive factors to elucidate the nature of this correlation. studies on the risk factors for tbi and parkinson’s disease tbi is traditionally organized into clinical categories that distinguish the levels of severity and each category’s potential to lead to neurodegeneration. yet, most research serves to analyze the impact on only severe tbi. insu cient research has been conducted on patients with moderate tbi, proving to be a challenge for researchers [6]. however, through research of mild and severe tbi, it has been determined that the majority of tbi cases in the united states result from falls and motor vehicle accidents. researchers analyzed demographics and the causes of injury using registry data from the national trauma data bank (ntdb) between 2007 to 2014 for patients over the age of 18 with mild tbi. it was determined that falls and motor vehicle-related injuries accounted for 43% and 34% of tbi cases, respectively. furthermore, assessing tbi outcome severities demonstrated that adults in the 45-to-64-year age range had about a two-fold increased risk, while those over 80 years old had nearly a ve-fold increased risk of experiencing poor outcomes. researchers de ned poor outcomes as in-hospital mortality, hospice, and long-term care. similarly, a cross-sectional analysis conducted by majdan et al. identi ed 1,375,974 berkeley pharma tech journal of medicine | 27 tbi-related hospital discharges and found the most prevalent causes of tbi to be falls and tra c incidents [5]. in recent years, however, there has been a shift from tra c accidents to falls as the most frequent cause of tbi [8]. on the other hand, there are many risk factors associated with the development of pd — one of which is genetic risk. a genome-wide linkage and sequencing study conducted from 2008 to 2017 aimed to identify genes involved in the onset of pd. the researchers rst conducted a genome-wide analysis on an italian family whose members had pd through dominant inheritance. the identi ed gene was then analyzed and compared in a representative sample of international patients diagnosed with pd. quadri et al. identi ed a mutation on lrp10 on chromosome 14 to be involved in the onset of pd. out of the 11 patients identi ed with this mutation in the international cohort, 10 had a family history of pd. the lrp10 mutation was also found in the dna of nine out of 10 relatives of the international cohort. these results suggest that there is a hereditary aspect of the increased risk of developing pd [9]. other risk factors of pd include the consumption of dairy products, cancer, and exposure to pesticides [7]. researchers conducting an observational study on nutritional factors and pd found that the higher intake of dairy was associated with an increased risk of pd development, with a larger risk in men compared to women [10]. an increased prevalence of malignant melanoma and skin carcinoma is also associated with a higher risk of pd. an epidemiological study in denmark found the prevalence of malignant melanoma was 0.57% in pd patients compared to 0.4% in control patients, while skin carcinoma prevalence was 4.24% in pd patients compared to 3.4% in controls [11]. researchers have found similar patterns of increased relative risk when investigating the e ect of pesticides on patients who su ered from pd and/or tbi [12]. a controlled study researched the e ects of both tbi and paraquat exposure, which causes damage to the digestive system organs, in patients with pd. researchers estimated a two-fold increase in the risk of developing pd in patients who experienced tbi, further supporting the correlation between the two diseases. in contrast, when examining the combined e ects of both paraquat exposure and tbi, researchers noticed a three-fold risk increase [13]. a study in rats further substantiated these ndings by demonstrating that tbi and paraquat exposure led to a loss in dopaminergic neurons and berkeley pharma tech journal of medicine | 28 appleofmyeye cross-out concurrently increased alpha-synuclein accumulation and in ammation in the brain, which are common characteristics of pd [14]. demographics age and gender have proven to be signi cant factors in the incidence of tbi and in the development of pd [16, 17]. when researching the demographic data relating to these diseases, studies have been geared towards researching the demographics of tbi and pd individually rather than on their relationship to each other. a study analyzing the characteristics of patients with pd found that the age-adjusted incidence rate of pd was higher in men than in women, at 19.0 per 100,000 persons and 9.9 per 100,000 persons, respectively. incidence rates and the male to female ratio increased rapidly after 60 years of age. the mean age of diagnosis was 70.5 years for both men and women, yet women were diagnosed over a larger age range (31 to 93 years) than men (38 to 91 years). further analysis found incidence rates of 0.50 per 100,000 persons in the 30-to-39-year group and 119.01 per 100,000 persons in those over 89, suggesting that the risk of being diagnosed with pd increases dramatically with age. researchers also sought to distinguish the prevalence of idiopathic pd among di erent racial groups. non-hispanic whites were found to be diagnosed with pd at an older age than hispanics and asians/paci c islanders, and a slightly older age than african americans. when adjusting for age and gender, incidence rates of pd were highest among hispanics, followed by non-hispanic whites, asians/paci c islanders, and african americans. pairwise comparisons among di erent groups found this data to be somewhat statistically signi cant. prevalence of pd in the non-hispanic white cohort compared to the asian/paci c islander cohort was 13.6 and 11.3 per 100,000 persons (p = 0.07) respectively, while the non-hispanic white cohort compared to the african american cohort exhibited incidence rates of 13.6 and 10.2 per 100,000 persons (p = 0.11). researchers also noted that in every group besides the asian/paci c islander cohort, the incidence of pd was higher in men than in women by two-fold [18]. researchers have found increasing evidence pointing to gender as an important factor in the development and expression of pd [19]. a case study conducted by haaxma et al. found women to be 2.1 years older than men at their age of symptom onset, the mean ages being 53.4 and 51.3, respectively [20]. at symptom onset, women presented tremors, a symptom of pd, more frequently than men (67% and 48%), regardless of their age of berkeley pharma tech journal of medicine | 29 onset. however, men presenting tremors at the age of onset were, on average, 3.6 years older than women. from this research, it was concluded that gender-based di erences in pd patients were signi cant. women tended to be older at symptom onset, presented higher striatal dopamine a nity and tremor dominant symptoms, and experienced slower disease progression compared to men. other studies have shown that women diagnosed with pd tend to present more benign phenotypic symptoms, but later tend to have an increased risk of treatment complications compared to men, further pointing to the biological and phenotypic di erences between male and female patients with pd [21]. reviewing demographic factors relating to tbi reveals similar trends regarding data on the age and gender of patients. a cross-sectional study analyzed data from the national hospital ambulatory medical case survey (nhamcs) and collected information on demographic characteristics of older patients who sustained mild tbi in the united states. the average age of patients diagnosed with mild tbi was 79.1 years of age, 87.8% of whom were white, and 64.3% of whom were female. emergency department (ed) visits were also observed to increase with age. in patients aged between 65 to 74 years old, the rate was 386 per 100,000 persons, while patients 85 years or older had an incidence rate of 1,205 per 100,000 persons. since the rates for those 85 and older triple the rates for those between 65 to 75 years of age, tbi has been found to be more prominent in older age groups. researchers found that women visited the ed for tbi at a rate of 706 per 100,000 persons, while men visited at a rate of 516 per 100,000 persons. another characteristic that researchers investigated was geographic region, nding that visits related to diagnosed tbi were more common than visits related to possible mild tbi in the northeastern and western regions of the united states. the diagnosis of tbi was more prevalent on the west coast and less prevalent in the south and midwest, compared to the northeast regions of the united states. this data, however, should be interpreted with caution, as some have suggested that the cdc codes for detecting mild tbi have poor sensitivity and mild tbi can be underdiagnosed [22]. a retrospective cohort study analyzed data on older adults from the ontario association of community care access centers (oaccac) home care database. in this experiment, incidence of tbi among patients of varying education levels, marital status, sex, race, and age groups were observed over a ten-year period from 2003 to berkeley pharma tech journal of medicine | 30 2013. investigators found that those who had sustained a tbi had an equal sex distribution. overall, positive associations were found between sustaining tbi and several groups, including males, patients of aboriginal origin, increasing age, education level, being widowed, and having pd [23]. in another study on the medical data of californian patients 55 years or older who were diagnosed with tbi but not pd, patients with tbi were slightly older, more likely to be male, have higher income, and higher severity scores when compared to patients with non-tbi trauma (ntt) [24]. moreover, patients with tbi were more likely to be diagnosed sooner with pd than patients with ntt. the average time until pd diagnosis was 3.1 years for tbi patients versus 3.3 years for ntt patients, and 66% of the trauma for both the tbi and ntt group was caused by falls. patients with pd had an average age of 76 years old, with 59% being female and 68% being white. data showed that incidence rates of tbi among racial groups was highest among whites, followed by hispanics, asians, and african americans. cases of tbi were found to increase with wealth; there were 10,276 incidents of tbi in the lowest quartile, while there were 14,132 cases of tbi in the highest, wealthiest quartile. researchers also noted that tbi was associated with a 44% increased risk of pd diagnosis after having adjusted for demographic variables (age, sex, race, income, etc.) this data suggested an increased risk of pd associated with tbi compared to ntt. similarly, researchers found that the risk of being diagnosed with pd increases with the severity and frequency of tbi, providing support to a more causal association [24]. prevalence of tbi in patients with pd contradicting evidence exists regarding the statistical signi cance of the association between tbi and the risk of developing pd [25]. tbi-related emergency visits have increased in recent years in the united states, with age-adjusted rates showing increases from 534.4 per 100,000 persons in 2007 to 787.1 per 100,000 persons in 2013 [26]. savica et al. similarly cites an increase in pd incidence in recent decades. with these increasing rates, it becomes even more pertinent to assess whether there is a correlation between pd and tbi [27]. recent epidemiological studies have cited a lack of correlation between the development of pd and severe head injury. one study analyzed the medical berkeley pharma tech journal of medicine | 31 records of the danish population 20 years and older who were hospitalized for head trauma from 1981 to 1993. of the 8769 cases requiring hospitalization, 107 patients developed pd, in which 55 of those cases were male and 52 cases were female. the mean age to sustain the injury was 39.7 for males and 49.2 for females [28]. regarding the severity of tbi, raj et al. found that the mean age of sustaining moderate-to-severe tbi (39 years) was less than that of patients diagnosed with mild tbi (46 years) [29]. in a study by spangenberg et al., the expected number of participants who developed pd without sustaining a severe head injury was 112.14 per 100,000 persons [28]. due to these results, the study concluded that there was no correlation between pd risk and severe head injury among adults. similarly, raj et al. found no signi cant pattern of correlation between tbi history and pd risk in the adult finnish population [29]. conversely, other studies have observed a correlation between pd risk and tbi. a case control study consisting of twins found that head injuries were associated with a three-fold increase in pd, while head injuries with amnesia and loss of consciousness were more strongly associated with the onset of pd. these results suggest that mild‐to‐moderate closed head injury may increase pd risk. it was also observed that the risk for pd increased with the frequency and severity of the head injury [30]. similarly, a study by bower et al. corroborated this as the researchers found that only moderate-severe diagnoses of tbi were correlated with an increased pd risk [31]. similarly, white et al. found a 2.69-fold and 3.70-fold increase in pd risk in veterans a airs healthcare facilities’ patients diagnosed with mild tbi and moderate tbi, respectively, indicating a correlation between increased incidence rate and the severity of tbi [32]. bower et al. and white et al. also observed a possible association between pd risk and tbi in their studies [31, 32]. during our literature review regarding the prevalence and demographics of pd and tbi, some studies noted the possibility of attaining tbi due to pd. in a study conducted in denmark, patients were found to have a 50% higher risk of developing pd if they sustained a head injury less than 10 years before the diagnosis, and an even higher risk if the tbi was sustained three months prior to pd diagnosis. due to this lack of correlation over the l0-year study, rugbjerg et al. concluded that there was no correlation between head injury and pd. in fact, rugbjerg et al. mentioned the possibility of pd-induced tbi due to pd patients’ tendency to have a slower reaction time [33]. delayed reaction time makes it di cult for pd berkeley pharma tech journal of medicine | 32 patients to break their fall with their arms, resulting in a more severe injury which could lead to tbi. it is possible that head injury in the months preceding pd is not a cause of diagnosis, but rather a consequence due to decreased control of motor function. this could serve as a plausible explanation for the observed association between tbi and the subsequent pd diagnosis in the previous study. this reverse relationship could be mistaken for an association between tbi and increased pd risk and should be researched further to assess the nature and directionality of this association [33]. cognition pd and tbi can greatly a ect a patient's cognitive function. the association between the two suggests that cognition can be a ected from both physical and biological standpoints. cognitive dysf unction in pd and in tbi cognitive dysfunction appears as a common symptom among those diagnosed with pd, and numerous studies have examined the extent to which cognition is a ected by pd. in one study, 115 participants newly diagnosed with pd and 70 healthy control participants were given a neuropsychological assessment, which included tests for psychomotor speed, memory, language, attention, and executive and visuospatial functions. pd patients performed signi cantly worse than healthy controls for most of the cognitive measures. the results indicated that pd patients were cognitively impaired due to their dysfunctional performance on at least three neuropsychological tests. 24% of pd patients were determined to be cognitively impaired, whereas only 4% of healthy controls were cognitively impaired. the de cits in pd patients appeared in memory and executive functions. apparent de cits may be misrepresented by the type of test given, as the tests may have favored assessment of immediate memory rather than cognitive dysfunction as a whole [34]. further analysis is warranted to discern factors such as tbi and falls that may have contributed to performance. another study consisted of a population-based, case-control examination of cognitive function in early pd patients. the study consisted of 46 patients who were 65 years or older that presented pd within ve years. all these patients were enrolled in the neurological disorders in berkeley pharma tech journal of medicine | 33 central spain (nedices) and matched with 138 controls for comparison. the mini-mental state examination, a cognitive ability examination of the elderly, was administered and pd patients performed worse (p = 0.04) than controls. subjective memory complaints were observed in 58.7% of pd patients compared to 37% in controls (p = 0.010). results indicated that pd patients who were diagnosed within the last ve years did not perform well on global cognition tests, verbal uency tests, and memory tests. additionally, 16 of the 46 pd patients with early pd were previously undiagnosed [35]. in addition, a study following cognitive change for ve years in newly diagnosed patients was done to explore the extent of cognitive dysfunction in pd patients. at zero, three, and ve years, a sample of pd patients (n = 59) and a sample of healthy controls (n = 40) were given neuropsychological assessments in which six cognitive domains were tested. in all the domains except the assessment of attention, patients with pd showed a greater decline than the healthy controls over time. individually, 53% of pd patients showed a greater decline in cognition than controls. overall, cognitive impairment with varying levels of severity seems to be prevalent among pd patients, with memory and psychomotor speed being the most a ected [36]. similarly, patients with tbi commonly exhibit cognitive impairment. furthermore, studies have researched areas of the brain in which cognitive impairment occurs in patients with pd. one study assessed the areas in which brain atrophy occurred in pd patients who also had mild cognitive impairment (mci), a cognitive impairment that does not interfere with daily activities. in comparison to pd patients without cognitive impairment, pd patients with cognitive impairment had hippocampal atrophy, a potential biomarker for initial cognitive decline in patients with pd [37]. as with pd, cognitive dysfunction in patients appears to be a common characteristic among patients with tbi. to better understand the association between cognitive dysfunction and tbi, a study was conducted in 2008 to investigate cognitive impairments ten years after participants experienced tbi (time range of initial injury was limited to the years of 1992 and 1995). 60 tbi participants were gathered and compared to a group of 43 demographically similar participants. these groups underwent a series of tests that measured functions such as attention, processing speed, memory, and executive function. results showed that the group of patients who had a tbi 10 years prior scored signi cantly lower than the control berkeley pharma tech journal of medicine | 34 group on the symbol digit modalities test and the digit symbol coding subtest, both of which measured information processing speed. these results strongly suggest that patients who su ered a tbi may experience long-term cognitive dysfunction [38]. in addition to long-term e ects, there appears to be a short-term e ect on cognitive function in tbi patients as well. a study from the university of texas at dallas in 2016 examined whether a history of tbi was associated with an increased risk of earlier onset of mild cognitive impairment (mci). 3,187 subjects with mci and 3,244 normal-cognition subjects were selected from the national alzheimer's coordinating center database and categorized based on severity and demographics. the results demonstrated that patients with mci were diagnosed 2.3 years earlier in the tbi positive group than the subjects without tbi, showing tbi as a potential risk factor for mci. limitations are vast, as this association could be due to other factors such as gender or mental state. however, this opens the door for further studies to discover whether tbi and mci can be associated with an increased risk of neurodegenerative diseases such as pd [39]. tbi and pd cognition studies cognitive impairment is a shared characteristic among patients with tbi and pd. further exploration of cognitive impairment in recent studies has shown that there may be greater cognitive impairment in pd patients who experienced tbi, compared to pd patients who have not experienced tbi. hence, there is a great need to investigate the role of tbi in inducing or exacerbating pd. a research team at ucsf conducted a study to investigate the relationship between head injury and pd phenotype. data collected from 267 patients in the parkinson's progression markers initiative (ppmi), it was found that individuals who experienced head injury prior to being diagnosed with pd had higher non-motor symptoms. pd patients with any report of head injury received a mean score of 7.73 on the movement disorders society-uni ed parkinson's disease rating scale, where lower scores indicate milder symptoms. by comparison, pd patients with no history of head injury had a mean score of 6.19 on the same scale (p = 0.035). those with severe head injury had scores of 8.29, and those without a head injury report had a score of 6.19 (p = 0.051). additionally, motor symptoms were berkeley pharma tech journal of medicine | 35 higher in those who had a severe head injury (score of 8.35) on the mds assessment, whereas patients without a head injury history had a score of: 6.19 (p = 0.042) [40]. figure 1: pd with severe head injury has the highest score (8.29) on the movement disorders society-unified parkinson’s disease rating scale. a separate longitudinal study took place over two years and consisted of cognitive testing on pd patients with and without a history of tbi. the study found that patients with a history of tbi faced more signi cant cognitive decline over the two years. after taking the mattis dementia rating scale test, which measures attention, initiation-perseveration, construction, memory, and conceptualization, pd patients without tbi had improved scores after two years, whereas pd patients with tbi had a decline in scores. however, both faced a similar decrease in motor function and a similar increase in depressive symptoms. the pd group with a history of tbi fare worse in both memory and initiation/preservation subscales [41]. this study demonstrated a potential correlation between tbi and pd symptoms. another cross-sectional cohort study was conducted to investigate whether a patient's history of tbi could be associated with pd and cognitive, motor, and neuropsychiatric symptoms. 120 patients between the ages of 60 to 85 were assessed for demographic data, neuropsychological tests, motor evaluation, neuropsychiatric questionnaires, and a brain injury screening questionnaire. of the 120 patients, 69 had pd and 51 were healthy controls. results from this study concluded there was a signi cant negative correlation between the number of tbis in pd patients and mean z-scores for global cognition (rs (69) = −0.338, p = 0.004) [42]. berkeley pharma tech journal of medicine | 36 neurodegeneration in pd and tbi and its e ects on cognition the e ects of tbi on the brain may increase the severity of cognitive impairment by exacerbating the neurodegeneration present in pd. weintraub et al. [37] conducted a study to assess regions in which brain atrophy occurred in patients with pd with normal cognition, pd with mild cognitive impairment, and dementia-level cognitive decline. 84 pd patients (61 pd normal cognition, 12 pd mild cognitive impaired, and 11 dementia-level cognitive impaired) and 23 healthy controls were assessed using magnetic resonance imaging of the brain. results indicated that individuals who had pd and normal cognition did not have signi cant brain atrophy compared to the healthy controls. when comparing the pd patients with normal cognition and patients with mild cognitive impairment, pd patients with mild cognitive impairment demonstrated hippocampal atrophy (β = −0.37; p = .001), and pd patients with dementia had hippocampal (β = −0.32; p = .004) and medial temporal lobe atrophy (β = −0.36; p = .003) [37]. pd patients with mild cognitive impairment had a similar pattern to the patients with both dementia and pd. a correlation between memory-encoding performance and hippocampal volume was found in the pd groups not exhibiting dementia. from this study, it was concluded that hippocampal atrophy may serve as a biomarker of initial cognitive decline in pd [37]. pd patients with mild cognitive impairment also showed a faster rate of cortical thinning. according to the montreal cognitive assessment, signi cant thinning of the temporal and medial occipital lobe was correlated with a decline in cognitive function [43]. to understand the genetic nature of cognitive impairment, researchers from north china university conducted an experiment to analyze the dysregulated expression of microrna144 (mirna) and its role in the pathogenesis of tbi in a rat model tbi. according to the researchers, mir-144 overexpression was a common characteristic of neurological diseases, as mir-144 could alter gene expression in the hippocampus. after analyzing the mir-144 gene in tbi patients and tbi in rats in vivo and in vitro, researchers found that the inhibition of the gene led to a better neurological outcome after tbi in vivo and improved cognitive de cits. the researchers also found that overexpression of the mir-144 gene led to an inhibition of the adam10 expression, which can modulate beta-amyloid formation, a common berkeley pharma tech journal of medicine | 37 protein involved in the development of cognitive de cits. these results demonstrate the potential relationship between cognitive de cits in tbi and gene expression in the brain, which could lead to neurological diseases such as pd [44]. previous research has investigated areas of the brain responsible for cognitive dysfunction commonly observed in tbi, pd, depression, and dementia. after analyzing magnetic resonance imaging (mri) data from participants, researchers found frontoparietal and fronto-occipital networks and temporoparietal, inferior frontal cortices were the primary systems responsible for information processing speed and stimulus-driven attention of the brain [45]. figure 2: the medial temporal lobe, highlighting the hippocampal area affected by tbi and pd. biological factors current literature has largely focused on the role of biological molecules such as alpha-synuclein, a presynaptic neuronal protein that regulates the tra cking of synaptic vesicles and the release of neurotransmitters, and enzymes such as tyrosine hydroxylase. additionally, biological phenomena such as oxidative stress and neuroin ammation are possible conditions that may contribute to the loss of dopaminergic neurons. berkeley pharma tech journal of medicine | 38 alpha-synuclein the neurodegenerative e ects caused by tbi and pd have been correlated with speci c proteins in the brain. to understand pathological correlations between pd disease and tbi, the structure and signi cance of alpha-synuclein must be discussed. a dominant pathological characteristic of pd is the abnormal folding and accumulation of alpha-synuclein in protein deposits known as lewy bodies. aggregation and overexpression of alpha-synuclein have been shown to disrupt the cell membrane of dopaminergic neurons, causing neuronal damage [46]. alpha-synuclein has di erent structural forms, such as the oligomer and brillar forms, which have varying e ects on pd pathology. a study conducted by froula et al. investigated the stable β-amyloid–sheet oligomer form (secondary protein structure) of the alpha synuclein protein in comparison to the brillar form to identify their varying abilities to induce characteristics of pd [47]. the various well-de ned forms of alphasynuclein were injected into the striatum of mice, with a monomeric alphasynuclein injection serving as the control of the experiment. neuropathological outcomes were monitored by performing immunohistochemistry using an antibody for alpha synuclein, phosphorylated at serine 129. results showed that in comparison to the monomer-injected mice, brils, cellular components that provide stability, produced ~30% loss of dopaminergic neurons in the substantia nigra compacta and loss of dopamine terminals in the striatum. injection of bril alpha-synuclein also yielded a higher concentration of alpha-synuclein– positive inclusions. in contrast, injection of the β-sheet oligomer resulted in a smaller loss of dopamine neurons in the substantia nigra compacta but showed no signs of inclusion formation. in addition, the brillar form of alpha-synuclein led to notable motor defects, while oligomer-injected mice did not exhibit a decline in motor behavior. it was concluded that the brillar form of alpha synuclein had a more signi cant toxic impact than the β-sheet oligomers due to the brillar protein’s ability to recruit alphasynuclein monomers and spread more quickly in vivo. overall, this research concluded that the brillar form of alpha-synuclein plays an important role in the induction of pd-related phenotypes, and interestingly, alphasynuclein has proven to be a characteristic of tbi. in an in vivo study by acosta et al., male rats underwent cortical impact to simulate tbi [46]. sixty days after tbi surgery, the brain tissues of these berkeley pharma tech journal of medicine | 39 rats were harvested for testing. characterization of alpha-synuclein in tbi-induced brains was studied through immuno uorescent staining of sections of the substantia nigra. performing this experiment showed that brain tissue exposed to tbi had an increased accumulation of alpha-synuclein in the ipsilateral substantia nigra pars compacta. tbi resulted in a three-fold upregulation of alpha-synuclein density around the soma and neurites of dopaminergic neurons, which triggered dopaminergic cell death. researchers concluded that the alpha synuclein overexpression in tbi-exposed brains led to a loss of dopaminergic neurons and served as a connection between tbi and the development of pd pathology [46]. furthermore, a study from impellizzeri et al. con rmed this nding. this study analyzed alpha synuclein levels in male mice brains 30 days after the mice were exposed to induced tbi. through dopamine transporter and alpha-synuclein staining, signi cant increases in alpha-synuclein expression and signi cant decreases in dopamine transporters were observed in comparison to controls. impellizzeri et al. also found a reduction in neurotrophic factors after 30 days, which are biomolecules that support the growth and development of neurons [48]. overall, ndings from these two studies suggest that alpha-synuclein may serve as a notable pathological link between tbi and parkinson’s disease. tyrosine hydroxylase in addition to alpha-synuclein, tyrosine hydroxylase, an enzyme that plays a key role in the synthesis of dopamine, has been researched as another biological marker in tbi and pd. shin et al. investigated tyrosine hydroxylase levels in rat brain tissue to determine this enzyme’s e ect on dopaminergic activity. for this study, a set of rats was rst exposed to tbi using a controlled cortical impactor. thirty days after exposure to tbi, the brain tissues of these rats were harvested for testing. western blotting techniques were utilized to detect the phosphorylation of tyrosine hydroxylase. between one and four weeks after exposure to tbi, tyrosine hydroxylase levels decreased signi cantly at the serine 40 sites. in addition, analysis of the striatal tissue revealed a decrease in potassium-evoked dopamine release. it was concluded that lower tyrosine hydroxylase levels after tbi negatively impacts dopamine concentration in the brain, suggesting a correlation between tbi and the development of pd pathology [49]. berkeley pharma tech journal of medicine | 40 figure 3: location of the substantia nigra--the site of the dopamine-producing neurons. a decrease in these neurons is implicated in the development of parkinson's disease. oxidative stress oxidative stress is a phenomenon in which neuronal damage can occur due to the overproduction and accumulation of reactive chemical species that cannot be readily detoxi ed [50] [51]. typically, these chemicals are reactive oxygen species (ros) that take on the form of oxygen containing radicals [50]. this may be because brain cells require and receive large amounts of oxygen. current literature has discussed oxidative stress in the context of tbi and pd independently, and the similar pathology of both suggests that this is one biological aspect that may link these two a ictions. several studies have implicated oxidative stress in tbi pathology through a variety of mechanisms. hill et al. observed, within hours of injury, an increase in ros production and the accumulation of reactive aldehydes acrolein and 4-hydroxynonenal, which are indicative of oxidative damage. in addition, hill et al. observed impairment of complex i and ii activity [50]. these e ects were more pronounced in the synaptic mitochondria compared to those that were nonsynaptic. another study focused on monitoring the synergistic e ects of mechanical strain induced-tbi and paraquat pesticide exposure. research has been carried out through an in vitro model of undi erentiated sh-sy5y cells, which are often used to model neuron-like activity in experiments. cells that underwent a moderate degree of strain experienced greater and longer-lasting mitochondrial membrane depolarization and increased ros mitochondrial berkeley pharma tech journal of medicine | 41 production, ultimately resulting in the death of dopaminergic neurons. these e ects were ampli ed when combined with exposure to paraquat [52]. together, these studies suggest that oxidative stress plays a unique role in further propagating neuronal damage from tbi at the cellular level, likely by interfering with the cellular respiration processes in the mitochondria. gene-regulated expression may also play a role in oxidative stress. wang et al. examined the role of protein-disul de isomerase-associated 3 (pdia3) regulated oxidative stress in augmenting tbi damage [53]. though this study focuses on the expression of ros and antioxidant enzymes as a metric for gauging the level of oxidative stress rather than mitochondrial activity, it appears to con rm the ndings of hill et al. while mitochondrial dysfunction and interference with neuronal function appear to be unifying themes connecting tbi and oxidative stress in these studies [50] [52] [53], further research is required to elucidate the exact mechanism to determine a causal relationship. additional research has sought to characterize the potential role of oxidative stress in the development of pd — more speci cally, its impact on the decline of dopaminergic neurons. paul et al. aimed to evaluate the e cacy of melatonin as an antioxidant capable of providing neuroprotective bene ts to mitigate the development of pd. researchers showed that rats experiencing oxidative stress, as the result of a homocysteine injection in the brain, exhibited a decrease in the number of functioning dopaminergic neurons in the substantia nigra region [54]. the study concluded that melatonin was an e ective antioxidant and could prove promising as a treatment. oxidative stress and the overexpression of alpha synuclein together have been found to possibly cause a synergistic e ect in inhibiting the function of cholinergic neurons in the vagus nerve [51]. this study, however, noted that not all cholinergic neurons within the brain were a ected by oxidative stress, suggesting that speci c regions of the brain and nervous system may be a ected di erently. a similar pattern has been observed for dopaminergic neurons, with the substantia nigra and striatum being the main regions of focus [54] [55] [56]. a causative relationship has to be made cautiously between oxidative stress and pd due to limited information. if the aforementioned observations involving oxidative stress have any signi cant overall impact on dopaminergic neurons, then there may be a causal relationship between tbi and pd. researchers from purdue berkeley pharma tech journal of medicine | 42 university used a mild-tbi rat model, immuno uorescence staining, and western blot analysis to demonstrate acrolein’s role in inducing aggregation and modi cation of alpha-synuclein, further corroborating the ndings from hill et al. [55]. this study is one of the few that discusses the role of oxidative stress in the potential pathological link between tbi and pd. however, additional research is needed on the e ects of oxidative stress and how its association with tbi can in uence the development of pd. speci cally, the mechanism of mitochondrial dysfunction and the accumulation of aldehyde byproducts in relation to its interaction with alpha-synuclein requires further research to determine its relation to the development of pd-related symptoms. figure 4: general trends associated with oxidative stress and its potential role in pd neuroin ammation neuroin ammation has been researched as another biological link between pd and tbi. as dopaminergic neurons are easily susceptible to in ammation, neuroin ammation has been found to be an important characteristic of pd. impellizzeri et al. conducted a study in 2016 analyzing neuroin ammation processes in the brains of male mice with berkeley pharma tech journal of medicine | 43 induced tbi. western blot analysis was utilized to analyze midbrain tissue of mice 30 days after exposure to tbi using iκb-α and nf-κb speci c antibodies. performing this analysis showed a signi cant reduction in iκb-α expression and an increase in nf-κb translocation in mice exposed to tbi. nuclear κb translocation plays a critical role in the transcriptional stimulation of pro-in ammatory target genes such as inducible nitric oxide synthase (inos) and cyclooxygenase-2 (cox-2). inos and cox-2 are both neurotoxic markers that can damage dopaminergic neurons. evaluating inos expression revealed that there was a prominent increase in inos in the tbi-induced brains in comparison to the control brains. in addition, cox-2 expression increased in the midbrain of mice that su ered tbi [48]. interestingly, increased cox-2 expression was also detected post-mortem in the substantia nigra of parkinson's patients [57]. based on these ndings, researchers concluded that neuroin ammatory processes resulting from tbi can lead to the development of pd pathology in the midbrain. in a 2018 study, yu et al. examined the role that the expression of the transcriptional factor early growth response-1 (egr-1) played in inducing neuroin ammation and neurodegeneration in mouse models of pd [58]. mice were injected with 1-methyl-4 phenyl-1,2,3,6-tetrahydropyridine (mptp), induced expression and upregulation of egr-1 in the substantia nigra pars compacta. upon egr-1 activation, researchers also noted an increase in interleukin 1-beta (il-1b) and tumor necrosis factor-alpha (tnf-a), which are cytokines involved in in ammatory responses. conversely, using egr-1 knockout mice models, researchers noted that the extent of the in ammatory response and dopaminergic neuronal death was less than in non-knockout models, suggesting that egr-1 could play a role in linking neuroin ammation to pd pathology. the researchers con rmed these ndings by administering mithramycin a, an antibiotic known to displace transcription factors such as egr-1 from their binding sites. yu et al. also noted that the protective e ects of egr-1 knockout was largely limited to the substantia nigra, and that this protective e ect was not observed in the striatum. thus, further investigation needed on the speci c molecular pathways in the substantia nigra and the striatum. other studies have focused on how the presence of pro-in ammatory cytokines a ects pd patients. karpenko et al. quanti ed the prevalence of di erent cytokines present in blood samples from pd patient groups at various disease stages using immunosorbent assays. in particular, the berkeley pharma tech journal of medicine | 44 researchers examined samples from the blood serum and cerebrospinal uid to determine if the presence of tnf-a and interleukin (il)-type cytokines correlated with the severity and progression of pd [59]. researchers found that higher levels of il-1b in the serum and tnf-a in the cerebrospinal uid were correlated with increased severity and rapid disease progression, but the correlation between other cytokines such as il-6 and pd was still unclear [59]. another study in 2018 conducted by li et al. employed similar methods to evaluate the correlation between cytokines in the blood plasma and pd-related pain found increased levels of il-1 in the blood serum of pd patients [60]. because il-1b is contained within the il-1 family, these ndings appear to corroborate the ndings of karpenko et al., further suggesting that proin ammatory factors may play a role in pd progression, though the exact role remains unclear. while there is evidence that neuroin ammation, oxidative stress, alpha-synuclein, and tyrosine hydroxylase may provide a link between tbi and pd, these ndings are speculative and require further research to prove a causal relationship. such research could prove useful in the development of new pharmaceutical treatments. treatment and therapeutic targets medical treatment of chronic disease is crucial in ameliorating symptoms, improving disease management, and prolonging life. current research focuses on prescription drugs and non-invasive treatments for the management of pd, with a particular concentration on relieving motor symptoms such as lack of balance, slowness, or parkinsonian gait, which is a distinctive change in the walk of a patient with pd [61]. medications used to treat tbi, regardless of their extremity, have the goal of stabilizing complications that arose from the injury, preventing further symptoms, and minimizing pain to maximize the patient’s well-being. medication pharmaceutical drugs serve as a powerful, successful treatment strategy in combating the detrimental e ects of tbi and pd, despite the high prevalence of side e ects. unlike non-invasive treatments such as exercise and behavioral therapy, prescription drugs work biologically to trigger body responses that mitigate and prevent the progression of symptoms. berkeley pharma tech journal of medicine | 45 rascol et al. discuss medications that have been proven to e ectively prevent the worsening of pd symptoms. some treatments include levodopa (in combination with carbidopa), pergolide, pramipexole, ropinirole, and selegiline [62]. levodopa is the strongest and most e ective treatment used in patients to delay disease progression, lessen persistent and long-term side e ects, and improve well-being. levodopa is also vital in managing acute symptoms of pd relating to body movements such as sti ness, tremors, and slowness of motion. the development of these motor symptoms stems from the lack of dopamine in certain brain areas of patients with pd. levodopa acts as a dopamine replacement agent to manage motor issues in patients [63]. conversely, many have speculated that levodopa can potentially be harmful and directly provoke motor complications. studies have shown that levodopa can be noxious to dopaminergic neurons, triggering the advancement of neuron degradation in the substantia nigra [63]. however, there have been few in vivo studies that support these ndings in pd patients. nonetheless, the bene ts of levodopa in mitigating pd symptoms may outweigh the negative controversies that have arisen regarding its e cacy. much like most existing pharmaceutical drugs on the market, there are numerous side e ects of levodopa, including drowsiness, nausea, and hallucinations. however, gandhi and saadabadi discuss that the key in administering levodopa to parkinson's patients is to integrate it with a decarboxylase inhibitor like carbidopa [64]. carbidopa is commonly prescribed with levodopa, as it minimizes nausea and blocks levodopa from being transformed to dopamine prior to moving into the brain, preventing side e ects such as vomiting. furthermore, pergolide, pramipexole, ropinirole, and selegiline are dopamine agonists that function by emulating the actions of dopamine. studies have shown that dopamine agonists are e cacious as both monotherapies and adjunct therapies in treating motor abnormalities and dyskinesia, known as involuntary muscle disorder [62]. however, there is still insu cient research on the performance of these dopamine agonists in levodopa-naive patients and a lack of veri cation that these promote neuroprotective mechanisms. rascol et al. discuss that for many patients, dopamine agonists other than levodopa have proven to be more e ective. however, as with carbidopa, co-prescribing dopamine agonists has proved to signi cantly mitigate symptoms compared to monotherapies like levodopa alone [65]. in their study, rascol et al. examined the e cacy of both levodopa and ropinirole in alleviating pd berkeley pharma tech journal of medicine | 46 symptoms. a patient's risk of developing dyskinesia was found to be almost three times lower while taking ropinirole alone, as opposed to levodopa alone. ropinirole and levodopa taken together more e ectively reduced the likelihood of worsening pd symptoms [65]. numerous pharmaceutical medications serve to ease the pain of patients who have experienced tbi. unlike pd, which is characterized by a speci c set of side e ects common to each patient, tbi symptoms are diagnosed on a case-by-case basis [37]. therefore, diagnostic procedures di er with each patient and prescription dosages directly correlate to the severity of the tbi [66]. for milder forms of tbi such as brain damage that are blast-related (caused by air pressure changes), focal (caused by contact), di used (caused by force), or from concussions, the process of diagnosis involves examining solely the history of the incident and direct cause of injury [66]. however, for acute brain injuries such as skull fractures, bruising of brain tissue known as cerebral contusions, or hematomas, prognosis is determined via analysis of imaging tests and a patient’s clinical presentation. in most mild cases, recommended medications include over-the-counter pain relief drugs such as naproxen and acetaminophen [67]. other prescription medications directly targeted to reduce both physical pain and emotional trauma experienced by tbi patients include anticonvulsants, antidepressants, antipsychotics, and medications relieving motor complications. anticonvulsant medication is utilized in patients who experience epileptic episodes due to their tbi and works to prevent the occurrence of seizures [68]. antiepileptic drugs, such as gabapentin and topiramate, inhibit excessive neuronal excitability and thus prevent the escalation of existing seizures. used to treat many forms of neurological and mental illnesses, antidepressants and antipsychotics are common treatments for patients who su er from both pd and tbi. as both pd and tbi directly in uence the levels of neurotransmitter signaling in the brain, antidepressants and antipsychotics serve to combat any psychiatric disorders that may arise, such as depression and hallucinations [69]. amantadine, an antiviral medication that was rst prescribed to combat the in uenza a virus, has increasingly been proven to signi cantly reduce prevalent symptoms of pd such as sti ness, tremor, and slowness of movement known as bradykinesia [70]. amantadine has also been shown to stimulate the functional recovery of those who su er from motor dysfunction related to tbi. furthermore, chang and ramphul point out berkeley pharma tech journal of medicine | 47 that the primary advantage of amantadine is its low side e ect pro le, which allows it to properly function without excessive harm. though amantadine has been prescribed to patients su ering from pd and to patients su ering from tbi, few studies have examined the e ect of amantadine in treating pd and tbi simultaneously [70]. thus, further research is needed to determine the drug’s e cacy in patients with both disorders. acosta et al. demonstrate the possibility for a molecular mechanism that could be harnessed to create new medication for pd [55]. newer and more advanced medications can target acrolein in the body to impede and moderate oxidative stress. with the existing research on the e ectiveness of prescription medications, many classes of drugs can be utilized to target symptoms of pd and tbi both individually and simultaneously. figure 5: mechanism behind levodopa, a pd dopamine agonists non-invasive treatment (cognitive therapies /exercising) non-invasive treatments for pd and tbi have demonstrated e cacy in many studies. such treatments center on physical and cognitive rehabilitation to ameliorate many of the common symptoms found in both pd and tbi, including motor weakness and stability. a common appearance of motor weakness in pd and tbi patients is an abnormal gait [71]. an abnormal gait is characterized by an asymmetric rhythm that a ects the ability of the patient to walk e ectively and can manifest as shu ing steps, gait initiation failure, or freezing of gait [72]. another appearance of motor weakness is a lack of balance, which can be characterized by an unusually increased number of falls [73]. many studies berkeley pharma tech journal of medicine | 48 have been successful in their focus on exercise as a possible method of improving gait, balance, mobility, and overall motor symptoms of patients with parkinson’s disease [74]. furthermore, basic treadmill exercises have markedly improved most motor symptoms in comparison to other forms of movement, such as dance and yoga [75]. to maximize therapeutic bene t, other studies have coupled exercise with other forms of stimulation. a new study found that combining rhythmic auditory stimulation (ras) and treadmill training greatly improved the overall gait performance of the participants. ras was implemented in this study through the use of music to capture their innate internal timing process and improve their gait parameters. all participants in the trial reported no side e ects during the entirety of the rehabilitation process. overall, it was concluded that the addition of rhythmic auditory stimulation to treadmill exercise o ers complementary advantages to pd patients’ balance, stride count, and overall gait performance [76]. due to the loss of rhythmic movements that characterize gaits in pd patients, rhythmic auditory stimulation provides compensation for this loss. a few studies have also researched the loss of rhythm in the movements of patients with pd and its correlation with the appearance of abnormal gait. a study conducted in 1997 focused on the e ect of rhythmic auditory stimulation on the gait velocity and cadence of pd patients. the study found that a faster ras resulted in a signi cant improvement in gait velocity and cadence, indicating that rhythmic auditory facilitation could be a successful technique in gait rehabilitation [77]. another study incorporated rhythmic auditory cues of a metronome beat to analyze the overall e ect pd patients’ performance on single and dual-motor tasks. no signi cant change in gait performance was observed when the metronome beat was implemented as a standalone; however, signi cant improvement was observed in patients who experienced a combination of a rhythmic auditory cue and an attentional cue (e.g., requiring patients to focus on taking big steps) [78]. this study corroborates the claim that some type of incorporation of rhythmic auditory stimulation can have a signi cant positive change in gait performance of parkinson’s disease. nevertheless, the topic of ras and treadmill training as a therapeutic strategy to improve gait appearance lacks recent research and requires further investigation. other forms of exercise, such as aquatic exercise, may also provide therapeutic bene t to pd patients. a randomized controlled trial studied berkeley pharma tech journal of medicine | 49 the e ects of aquatic exercise therapy on the improvement of parkinsonian gait. the experimental group performed various exercises in a community pool setting for two 45-minute sessions per week for a total of six weeks, whereas the control group received their normal treatment, which only consisted of medication. both the experimental and control groups demonstrated an improvement in gait, yielding inconclusive evidence for the bene ts of aquatic exercise as a potential therapeutic technique [79]. another study sought to compare the e ectiveness of both on-land gait training and aquatic gait training to mitigate peripheral neuropathies, a common occurrence for pd patients. the study concluded that the improvements from the inclusion of aquatic-based gait training in conjunction with traditional on-land gait training were similar to the improvements of the control group that did not undergo any aquatic-based gait training [80]. despite original postulates that aquatic-based therapies would provide bene t to pd patients, current research does not provide durable support for this claim. as with pd, cognitive and physical therapy have received attention as a potential therapeutic avenue. alberto esquenazi et al. focused on speci c methods of locomotor therapy that targeted the gait velocity and spatiotemporal symmetry of tbi patients. researchers tested the e ects of an end e ector robot, a robotic exoskeleton, and treadmill training with manual assisted partial-body weight-support (lokomat). the lokomat training method included a harness and a robotic orthosis, which were used on a treadmill. the partial-body weight-supported treadmill training method used a litegait body weight support system in tandem with a treadmill. esquenazi et al. found signi cant improvement in the participants’ self-selected velocity with all three methods of intervention, as well as a signi cant improvement in maximal velocity with the exclusion of the group that used an end e ector robot [81]. overall, improvements in gait speed, gait symmetry, and walking endurance were observed. similarly, peters et al. researched the impact of intensive mobility training on balance and gait speed of chronic tbi patients. though various subjects reported fatigue and pain throughout the experiment, the researchers noted signi cant improvement within the three months of the trial. thus, peters et al. concluded that the high consistency with these pain and fatigue reports prove the possibility of using intensive mobility training to continue to be a possible therapeutic technique for the tbi patients [82]. while the aforementioned studies analyzed a variety of di erent techniques, both found a signi cant berkeley pharma tech journal of medicine | 50 improvement in gait performance, pointing to their possible e ectiveness as a therapeutic intervention for patients with tbi. figure 6: berg balance scale comparing the following locomotor therapy interventions: partialbody weight-supported treadmill, lokomat, and gait velocity and spatiotemporal symmetry using an end effector robot (g-eo) training in patients with tbi. with the intersection between technology and medicine, additional studies have focused on the use of di erent forms of virtual reality simulations to improve the balance and overall motor symptoms of patients with parkinson’s disease. when comparing virtual reality rehabilitation to conventional physical therapy, hao feng et. al. concluded that the use of virtual reality by patients with pd resulted in a greater improvement in their balance and gait. the study consisted of a 12-week rehabilitation program in which the experimental group received balance and gait training using virtual reality (vr) technology. while the experiment demonstrated that balance and gait signi cantly increased with vr therapy, the study did berkeley pharma tech journal of medicine | 51 not control for variables such as age, time of onset, and level of interest with the vr therapeutic modality [83]. despite this, the study demonstrated that the inclusion of exercises that use vr technology is a possible therapy to improve parkinson’s balance and mobility. another study researched vr-based balance rehabilitation in tbi patients. through a randomized controlled trial, virtual reality therapy was compared to the traditional home-based exercise programs currently utilized to help improve the balance in tbi patients. there were 6-week, 12-week, and 24-week follow-ups, where both the experimental group and the control group saw signi cant improvements in balance [84]. despite the improvement in the balance of tbi patients who participated in vr-based rehabilitation, this improvement did not exceed that of the home-based exercise programs. the conclusion of both studies suggests that virtual reality may be a plausible therapeutic technique that improves balance for both pd patients and tbi patients. although many of the studies mentioned above solely focused on either pd or tbi, these non-invasive treatments and therapies include a combined improvement of symptoms of both pd and tbi. additionally, other treatments and therapies that may not be speci cally marketed towards pd or tbi, but include an improvement in their symptoms as well. as previously mentioned, many prescription medications that are used to combat symptoms of pd and tbi include mental health drugs, such as antipsychotics and antidepressants. targeted to alleviate internal psychological disorders, these classes of medication aid in minimizing the mental pain and su ering that occurs as an accompaniment to neurological disorders such as tbi and pd. symptoms of both often include psychosis, depression, anxiety, and personality disorders [85]. furthermore, a recent study found a correlation between depression and other psychiatric symptoms and increased motor severity [86]. research done by conrad et al. illustrates that the appearance of depression, anxiety, and various personality-related disorders have been statistically proven to be more prevalent in those who have pd and tbi. many types of treatment, both non-invasive and in the form of prescription medication, such as psychotherapy, antidepressant drugs, and neuromodulation, are utilized to minimize the e ects of depression and anxiety that appear in both pd patients and tbi patients. makio takahashi and colleagues conducted a multicenter randomized study in which they analyzed the e ectiveness of pairing duloxetine, a serotonin and berkeley pharma tech journal of medicine | 52 norepinephrine reuptake inhibitor (snri), with paroxetine and escitalopram, which are selective serotonin reuptake inhibitors (ssris), to improve depressive symptoms, apathy, and gait freezing in pd patients. the study concluded that the combination of snris with ssris improved both the freezing of the gait and the depressive symptoms in pd patients. however, the study did not indicate any signi cant change in feelings of apathy in the participants [87]. nevertheless, treatments that provide a combined improvement for both pd patients and tbi patients must continue to develop and improve. conclusion while the incidence of traumatic brain injury has demonstrated a positive correlation with the onset of parkinson’s disease, further research is required to determine the biological and cognitive mechanisms of this association. the incidence of tbi, and thus pd, was found to disproportionately a ect certain age and gender groups, being more prevalent in men than women, with women experiencing slower disease progression and older age at symptom onset. studies regarding the in uence of gender and prevalence of tbi showed contradictory results, pointing to the importance of further research. when studying patients with both pd and tbi, researchers found the risk of developing pd and/or head injury to increase greatly with age [5]. research has correlated pd with genetic and environmental risk factors (paraquat exposure, malignant melanoma, skin carcinoma, and increased dairy consumption) [10] [11] [13]. further research is required to understand the impact of tbi on the onset of pd in greater depth. furthermore, pd patients are more likely to experience tbi during the prodromal period of their disease. these results could indicate that with a decline in motor function due to aging, individuals are more likely to experience tbi from falling, which could increase their chances of developing pd. while the incidence of tbi can lead to an increased chance of developing pd, the prodromal period of pd can increase an individual’s chances of experiencing a tbi from incidents such as falls. therefore, the direction of the correlation is unclear, and further research is required. berkeley pharma tech journal of medicine | 53 when understanding the biological correlation between tbi and the onset of pd, it can be concluded that tbi leads to overaccumulation of alpha-synuclein in the substantia nigra; these accumulations are also a prominent characteristic of parkinson’s disease. decreased levels of tyrosine hydroxylase serve as another pathological link between tbi and pd. the presence of alpha-synuclein has been observed in patients with tbi as well as patients with pd, and thus serves as a potential link between both conditions. signi cant decreases in tyrosine hydroxylase in tbi can result in a decrease in dopamine and potentially initiate the onset of parkinson’s disease, which is also associated with a loss of dopaminergic neurons in the brain. while tyrosine hydroxylase and alpha-synuclein are good biological markers for understanding the link between tbi and the onset of pd, further experimental research is needed to validate their clinical value. further research is also needed to supply the currently insu cient research on the role of oxidative stress and neuroin ammation as a biological correlate between tbi and the onset of pd. research on the cognitive e ects of tbi and pd demonstrate that pd patients who su ered from tbi had greater cognitive decline compared to patients who did not su er from tbi. furthermore, hippocampal damage, a region correlated with subsequent cognitive decline, was present in patients who experienced either tbi or pd. however, it is unclear whether tbi could aggravate the symptoms of prodromal pd or directly lead to pd. the discussion on therapeutic targets and prescriptions determined e ective medications that treat the symptoms of pd and tbi independently. these include antidepressants, muscle relaxants, and even over the counter treatments. however, more research is needed to understand medications and therapeutic targets that can alleviate symptoms of tbi-induced pd. in addition to therapeutic targets, non-invasive treatments have been identi ed to potentially target symptoms from both pd and tbi. both pd patients and tbi patients present similar motor symptoms; therefore, non-invasive treatments such as the incorporation of treadmill activity and virtual reality therapy can potentially target these symptoms for improvement. the combination of invasive and non-invasive treatments could possibly treat patients who experience tbi-induced pd. therefore, while tbi can be understood as a potential non-genetic risk factor for the berkeley pharma tech journal of medicine | 54 development of pd, more research is required to understand the extent of this correlation and the implications of current research. understanding the causal link between traumatic brain injury and the incidence of parkinson’s disease would allow for more precise treatment plans that could potentially prevent the onset of pd. berkeley pharma tech journal of medicine | 55 references 1. beitz, janice m. 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(2019). results from a randomized controlled trial to address balance de�cits after traumatic brain injury. archives of physical medicine and rehabilitation, 100(8), 1409–1416. https://doi.org/10.1016/j.apmr.2019.03.015 85. reijnders, j. s. a. m., ehrt, u., weber, w. e. j., aarsland, d., & leentjens, a. f. g. (2008). a systematic review of prevalence studies of depression in parkinson’s disease: the prevalence of depression in pd. movement disorders, 23(2), 183–189. https://doi.org/10.1002/mds.21803 86. nagayama, h., maeda, t., uchiyama, t., hashimoto, m., nomoto, n., kano, o., takahashi, t., terashi, h., hamada, s., hasegawa, t., hatano, t., takahashi, t., baba, y., sengoku, r., watanabe, h., inoue, m., kadowaki, t., kaneko, s., shimura, h., & kubo, s. (2017). anhedonia and its correlation with clinical aspects in parkinson’s disease. journal of the neurological sciences, 372, 403–407. https://doi.org/10.1016/j.jns.2016.10.051 87. takahashi, m., tabu, h., ozaki, a., hamano, t., takeshima, t., & for the reborn study group. (2019). antidepressants for depression, apathy, and gait instability in parkinson’s disease: a multicenter randomized study. internal medicine, 58(3), 361–368. https://doi.org/10.2169/internalmedicine.1359-18 berkeley pharma tech journal of medicine | 63 article 2_traumatic brain injury-induced parkinson's diseasean analysis of the potential correlation_publish.pdf parkinson's paper_bptjm (reference page template).pdf body paragraph_fang et al._diabetes-related cancer.docx berkeley pharma tech journal of medicine correspondence: robertfang17@gmail.com keywords: metabolic-reprogramming obesity diabetes metformin fasentin panitumumab submitted may 5, 2023 accepted july 7, 2023 published december 30, 2023 full open access creative commons attribution license 4.0 abstract cancer is a condition characterized by the uncontrolled growth and spreading of certain cells within the body. the formation of malignant tumors necessitates a substantial amount of energy to sustain the abnormal rate of cell division. this process leads to a significant alteration in the primary metabolic pathway, transitioning from mitochondrial respiration to aerobic glycolysis, particularly in cancers associated with diabetes. this shift creates an opportunity for less invasive treatment options that can limit cancer growth by targeting specific transporters and enzymes crucial for energy production. this article focuses on the biological functions of fasentin and metformin, exploring their effectiveness in constraining cancer development. the discussion delves into their roles in regulating metabolism and highlights how these drugs can be instrumental in impeding the progression of cancer. effective reprogramming strategies for treating diabetes-related cancer: a focus on fasentin, metformin, and panitumumab therapies by: heyi fang, hailey guinto, and harshita pinneboina 1. introduction since its initial discovery, cancer has been one of the most formidable diseases. though its early-stage symptoms are less virulent and the rate of recovery can be as high as 90%, advanced stages are faced with a high mortality rate. during intermediate and advanced stages, common treatments are invasive and seek to eradicate all cancer cells -though recurrence is possible. if the cancer progresses to advanced stages, however, the mortality rate will quickly rise to 70% over the span of �ve years.1 there are various causes and risk factors for di�erent types of cancer, but chronic diseases2, high body mass index, (bmi) and unhealthy lifestyles appear as general components in all types. diabetes mellitus, one of the most common chronic diseases, has proved to be positively correlated with the progression of cancer. diabetes mellitus refers to a group of diseases that a�ect how the body uses blood glucose with regulations by insulin, adiponectin, leptin, and glucose transporter groups (gluts) that manipulates the metabolic �ux of metabolites between bloodstream and peripheral tissues with energy needs. type i diabetes will disable the pancreas from producing insulin and type ii diabetes is characterized by insulin resistance, which heavily impairs the patient's ability to utilize insulin for the regulation of sugar. insulin is a hormone secreted by the pancreas and is used extensively for the metabolism of glucose and adipose tissue after food intake, especially for the synthesis of glycogen. glucose is the major energy source and upon the damage of insulin's function, the homeostasis for glucose and glycogen is broken. insulin also serves as a negative feedback signal for glycogenolysis, which transforms stored glycogen to free glucose. moreover, more insulin circulating in the bloodstream will inhibit the synthesis of various proteins in di�erent tissues, interfering with normal physiological functions throughout the body, especially in the digestive system. in discussion of diabetes and the risk of faster cancer progression, diabetes provides extra energy supply to the uncontrolled dividing cancer cells, �lling the gap with an abnormally high consumption of energy. heavily depending on the intake of energy, the shifted metabolic mechanism is a novel interest for the treatment of diabetes-related cancer. the complex energy transfer process including the berkeley pharma tech journal of medicine | 102 use of insulin, glucose transporter, and the change-of-function of many metabolism-related cells in mitochondria are hypothesized as new aims with a non-invasive treatment: treatment with no need of physical insertion of instruments into the patient’s body. in this article, the relationship among the chain of obesity, diabetes mellitus, and cancer development researched by di�erent articles regarding to distinct diabetes-associated cancer types is reviewed, the potential metabolic reprogramming pathways of glucose uptake and digestion by cancer cells are studied, and the mechanisms of inhibitory functions on process of reprogramming by fasentin, panitumumab, and metformin are possessed for the possibility as drug treatments of diabetes-associated cancer. 2. obesity and its correlation to diabetes development obesity is considered a common risk factor for many diseases3. regarding the accumulation of excess adipose tissue, a body mass index (bmi) of over 30 kg/m2 is utilized as an indicator. obesity leads to metabolic disturbance, the severe impairments of the ability to regulate the synthesis of adipocytokines and the conversion between glucose and glycogen. researchers have revealed that common metabolic disturbances like dyslipidemia and hyperinsulinemia are the causal factors of cancer development. many adipose cytokines are critical regulators for the maintenance of homeostasis between glucose and energy supply with some of them impacting a range of metabolic pathways to other organs. two of the most intensively studied adipocytokines are adiponectin and leptin. while both are heavily impacted by the change in adiposity, the change in concentration and distribution of adipose tissue4 impacts the body’s response to metabolites. adiponectin is secreted by adipose tissue into the bloodstream, with a function of mediating glucose metabolism and fatty acid oxidation process5. study conducted by reneau, james et al reveals that the linkage between the accumulation of adipose tissue and secretion of adiponectin shows a negative correlation6. therefore, the increased adiposity inhibits the production of adiponectin, disabling its function to regulate gluconeogenesis6. leptin, another cytokine secreted by adipose tissue, has an opposite e�ect to adiponectin. acting on the central nervous system, namely the hypothalamus7,8, leptin serves to decrease the sense of hunger while inducing berkeley pharma tech journal of medicine | 103 the activity of beta pancreatic cells that produce insulin. it is shown that leptin production is positively correlated with the increased adiposity, with a positive feedback loop involving increased circulating insulin and the resulting increase of adipose tissue. both researched adipokines promote the risk of getting diabetes. furthermore, insulin also plays a critical role in the hydrolyzation of fatty acid, which serves to increase the rate of glucose uptake and fatty acid synthesis, as well as decrease the rate of fatty acid decomposition9. the tumor, upon receiving bene�cial signals from the adipose tissue, also feeds back with inhibitory signals on the decomposition of fats. therefore, a positive feedback loop is observed among obesity-high blood insulin-tumor development, as shown here in �gure 1. figure 1. the positive feedback of adipose tissue accumulation and cancer development. for summary, the abnormally high bmi level indicates the obesity level of an individual, resulting in the �uctuation of production for critical metabolic regulators, which leads to the increasing level of insulin circulating in the body and subsequently, will cause type ii diabetes mellitus by inducing insulin resistance. over time, the disturbance of homeostasis can generate enough energy to promote the growth of cancer, particularly in organs with high concentrations of adipose tissue. 3. type ii diabetes mellitus (t2dm) and its correlation to cancer progression berkeley pharma tech journal of medicine | 104 the compilation of the mentioned negative impacts from the previous section will lead to the development of t2dm. establishment of insulin resistance (ir) by tissues will be disabled to respond normally to the hormone insulin or downregulate insulin receptors in response to hyperinsulinemia10. the detailed development of ir remains unclear, but the hypothesis is that decrease in insulin sensitivity with adjustments in the pi3k/akt/mtor signaling pathway11. hyperinsulinemia boosts the advancement of cancer by providing access to excess glucose from the bloodstream12. normal functioning cells, upon receiving glucose, will degrade it for energy with the assistance of oxygen in the process of glycolysis, citric acid cycle, and oxidative phosphorylation, producing approximately 34 atp per glucose used13. however, during the pre-malignant expansion stage of tumor, the development of peripheral tumor microenvironment (tme) will separate the interior of tumor cells farther away from the local bloodstream14, which is the carrier of oxygen-rich hemoglobin. the result of this isolation is the leveled average partial pressure of oxygen in tme around 5 mmhg, which is only 12.5% in average of the venous oxygen pressure15. this shift of oxygen level disallows cancer tissue to utilize oxygen for the mitochondrial respiration process and instead, it switches its dependence for energy to the warburg e�ect, the metabolic reprogramming e�ect done by tumor cells to rely on the energy provided by aerobic glycolysis. this is only e�ective when there is an excessive supply of glucose and an anaerobic environment, for the production of energy is only 4 atp per glucose consumed, rather ine�cient compared to the normal process16. therefore, the t2dm with its in�uence on tissues incapable of utilizing glucose e�ciently, the extra circulating glucose in the bloodstream will be recruited and made use of by the tumor complex17. on the other hand, t2dm also provides conveniences for the progression of cancer by the hyper-expressed insulin. insulin serves as a mitogen in the human body18. with high levels of insulin expressed, cells are excited by the signal from mitogens to proceed with more mitosis. besides insulin, insulin-like growth factor 1 (igf1) also serves as an important mitogen. however, igf1 is not induced by the increased level of blood glucose, but the excess circulating insulin serves to compete with igf for the constant amount of igf binding berkeley pharma tech journal of medicine | 105 protein 3 (igfbp3). as igf1 is a mitogen as well, the imbalance between igf1-igfbp3 ratio leads to excess proliferation of cancer cells. on top of that, igfbp3 is considered a negative regulator of cancer as a low-penetrance tumor suppressor gene19. 4. the role of glucose transporter (glut) and treatment with fasentin and panitumumab the two mentioned changes negatively impact the in vivo microbiological environment for all cancer types, because energy is a common restriction for the development of all tumors. this has led the discussion to the molecular metabolic pathway of energy generation, especially the relationship of blood glucose and insulin level, as well as the mechanism of how glucose enters into tissues. in order for the intake of glucose to peripheral tissues from the bloodstream for further mitochondrial oxidation and atp production, mammalian cells have developed a family of glucose transporter proteins for the transportation of sugar through the plasma membrane20. the abnormal blood glucose level within the t2dm patients will induce a stronger expression of glut families, especially in the energy-demanding tissue like adipose tissue and breast cells, the emphasis of our paper. there are 3 classes of glut, and the responsible group here is class i glut, which contains glut1-4 and glut1421. di�erent glut types are responsible for the intake by di�erent tissues, and our emphasis will be on glut4, the one that controls uptake of glucose by skeletal muscle, cardiac muscle, and most importantly for cancer development, adipose tissue22. the expression of glut4 is heavily regulated by the level of presenting insulin. stored in intracellular vesicles, glut4 is released vesicular fusion once insulin binds with the insulin receptors expressed on the plasma membrane23. increased glut4 availability to glucose in the bloodstream causes more glucose uptake by fat. this is thought to be the e�ort against the development of insulin resistance, although it also promotes the creation of a positive feedback loop for fat accumulation and development of diabetes22. this has led berkeley pharma tech journal of medicine | 106 the research interest to a potential drug that restricts metabolic e�ciency of cancer cells by inhibiting the process of glucose intake. there are two potential targets in regard to this possible map. first, the insulin receptors are receptor tyrosine kinase (rtk), which requires the process of dimerization and phosphorylation to the transduction of the chemical substances from ligands to intracellular electric signals24. this is not unique because many signal regulation pathways utilize rtk as well, and they can also be seen as targets. therefore, a potential therapy is to block or terminate the phosphorylation process of rtks with pharmaceutical interventions. there are already drugs developed for cancer treatments via this pathway, namely the panitumumab25. panitumumab is an agent serving to restrict progression of colorectal cancer on epithelial level26. epithelial cancer is a critical cancer type that is a�ected by t2dm, for the transformation of normal epithelial to cancerous cells requires enormous amounts of energy consumption. panitumumab inhibits the function of epithelial-growth factor receptor (egfr), which after cancerous lesion, starts the process of uncontrolled replication27. panitumumab, by binding to the extracellular receptor of egfr and outcompeting the essential nutrients for cell proliferation, e�ectively inhibits the progression. recently, clinical trials were made with panitumumab on its pharmaceutical e�ect on cancer types other than colorectal cancer. records show that it can also repress the development of head cancer and neck cancer28. secondly, the drug can also target the intake of glucose by the direct blockage of glut4. fasentin serves 2 functions for the suppression of tumor establishment. firstly, fasentin is a direct inhibitor of glut4. by outcompeting the glucose on binding a�nity represented by a higher ic50 value29, fasentin e�ectively reduces the glucose uptake by cancer-surrounding tissue and therefore, an inhibitory e�ort is made to reduce energy production. secondly, fasentin presence serves as a stimulatory signal for the activation of fas-directed apoptosis process of cancer30. therefore, intake of fasentin can both directly, by inducing cancer cell death by apoptosis and indirectly, by restricting the amount of glucose in�ow. berkeley pharma tech journal of medicine | 107 5. treatment incorporating monocarboxylate transporter 4 and cannabinoid receptor 2 the distorted glucose in cancer cells results in the upregulation of glycolysis in cancer cells. this induces a high amount of lactate production, and consequently, its accumulation in these cells. blocking upregulation of aerobic glycolysis has been ine�ective, such as using 2-dg as an anti-cancer agent. mere inhibition of glycolysis is insu�cient for the eradication of cancer cells due to the reason that cancer cells have the potential to adapt their metabolism to their environmental conditions. upon glycolytic suppression in multiple types of tumor cells, intracellular energy metabolism is reprogrammed in an autophagy-dependent manner to ensure cellular survival. rather, a possible treatment method for these cancer cells is aggravation into a hyper-glycemic condition, followed by blocking products from tca cycle, resulting in high amounts of lactate production. this lactate export is then blocked, causing intracellular acidi�cation and consequently cell death. the high amount of lactate can cause a strong acidi�cation process, in which most of the normal cell functions are inhibited, including division. the high amount of lactate can cause a strong acidi�cation process, in which most of the normal cell functions are inhibited, including division. in addition, lactate released from tumor cells through monocarboxylate transporter 4 (mct4) is enough to stimulate angiogenesis and tumor growth. increased lactic acid can in turn enhance glycolysis in cancer cells, causing a vicious cycle. however, this high lactate content causes normal cell functions to be inhibited, including the process of replication and division. using this manner, a treatment of poisoning the cancer cells is feasible by inducing over-production of lactate to decrease ph level. however, research shows that a highly acidic environment surrounding the tumor is responsible for the development of chemotherapy resistance. thus, two needs that must be met are to increase lactate formation and block the lactate from exiting the cancer cell. a potential pathway of this treatment is the utilization of monocarboxylate transporter 4 (mct4) along with the cannabinoid receptor 2 (cb2).31 berkeley pharma tech journal of medicine | 108 monocarboxylate transporter 4 (mct4) is highly expressed in metastatic tumors and at in�ammatory sites, referentially in glycolytic muscle �bers and facilitating the lactate e�ux. mct4 is responsible for the bidirectional transport of lactate across the plasma membrane. the cb2 receptor modulates immune cell functions. cannabinoid receptors (cb1 and cb2)– g-protein coupled receptors, inhibit adenylate cyclase activity in response to psychoactive cannabinoids32. the activation of cb2 receptors does not appear to produce psychotropic e�ects, and therefore, it may also be helpful in treating diseases that have a neuroin�ammatory or neurodegenerative component, such as multiple sclerosis. mapks are enzymes involved in a wide variety of important signaling cascades in many cellular responses– cell proliferation, migration, transformation, and cell death. mapk activation by a nonselective cb2 receptor agonist (d9-thc) was found to have a proapoptotic e�ect in the jurkat human leukemia cell line (herrera et al., 2005) and cytotoxicity in j774-1 macrophages; in the same cells, there was also a c-jun n-terminal kinase–mediated cytoprotective e�ect mediated by δ9-thc activation of cb2 receptors, displaying the same cb2 receptor ligand can activate multiple mapks, each with di�erent outcomes33. the activation of cb2 receptors by natural or synthetic ligands favors a range of receptor conformations that can variably a�ect di�erent signaling pathways as the following procedure– inhibition of adenylyl cyclase, decreased camp (production, and less activation of camp-dependent protein kinase (pka), inhibiting a-type potassium channels as well as speci�c gene expression. this is followed by activation of akt/protein kinase b– stimulating cell survival, migration, and growth. proceeding activation of the mitogen-activated protein kinase (mapk) cascade favors cell survival and modulates gene expression. in addition, there is inhibition of speci�c calcium channels and enhanced opening of g protein–gated inwardly rectifying potassium (girk) channels. lastly, stimulation of de novo synthesis of ceramide and inhibition of the mapk cascade promotes apoptosis34,35. recruitment of b-arrestin to the activated cb2 receptor results in desensitization and/or internalization of the receptor and potential activation of arrestin-speci�c signaling. decreased pka activity increases raf-1 to stimulate the mapk cascade, positively regulating the berkeley pharma tech journal of medicine | 109 expression of many genes and indicating activation of a pathway by cb2 receptor agonists. functional selectivity of cb2 receptor agonists must be considered during the therapeutic development of cb2 agonists, which increases the possibilities for developing drugs targeting cb2 receptors. by forcing glycolysis with metformin and a nf-kb inhibitor lowers the ph of a cell, our treatment has a minor e�ect on normal cells and most e�ective in cancer patients with any hyperglycemic stages, including. as an example, nk-kb inhibition causes increased lactate secretion from breast cancer cell line mcf-736,37. the e�ect of this metabolic reprogramming strategy was observed by checking oxygen consumption and extracellular acidi�cation rates. this resulted in reduced ocr and blocked ecar, essentially leading to an accumulation of lactate. fluorescent bcfl-am was used as a probe for detecting phi. therefore, the reprogramming did not decrease viability in (normal) mcf-12a cells. it did, however, a�ect mcf-7, t47d, and mda-mb-231 (breast cancer cells), demonstrating signi�cantly inhibited migration and invasion ability38. consequently, in high glucose cancer cells, a higher glycolysis rate is present, allowing the reprogramming strategy to further promote the process and raise intracellular lactate, permitting cancer cells to poison themselves. essentially, there is limited cytotoxic e�ect on non-subject cells, reducing the chance of untargeted damage. cb-2 might bind to a pocket of mct4 composed of ser156, phe243, tyr332, gln339 and glu363. the combination of metformin and cb-2 exerts a deleterious e�ect on breast cancer cell viability and exhibits synergistic antitumor e�ects. results of the combination treatment showed a 63% inhibition of cell viability in mda-mb-231 breast cancer cells39. in addition, moderate e�ect on cell viability was observed in normal mcf-10a human mammary epithelial cells. disrupting mct4 function leads to an accumulation of intracellular lactate and a decrease in intracellular ph which may rapidly damage a cell thereby inducing necrosis, apoptosis or growth arrest. metformin increases glycolysis thereby increasing the buildup of intracellular lactate, thereby accelerating the e�ects of the mct4 inhibitor. this can be used to treat high glycolytic rate/mct4-expressing malignancies. another possible combination is the mct4 inhibitor cb-2 and a gls1 inhibitor cb-839 to reengineer cancer metabolism40. this combination solves berkeley pharma tech journal of medicine | 110 the problem of increasing ammonia production to neutralize lactate via restricting the metabolic �exibility of these cancer cells. 6. metformin to target cancer cells’ glucose metabolism and mtor pathway the collection of genetic alterations in cancer cells causes interference with the regular cellular signaling pathways, this then leads to cell growth for cancer. despite the di�erent current treatment plans that are o�ered, many recoveries fail due to the drug resistance and its adverse side e�ects. though in recent studies, metabolic reprogramming has served as a possible cancer therapy. with this, we want to identify the glucose metabolism of cancer cells to alter so it causes glucose-lowering agents like metformin to be a possible treatment in cancer cells. one change we can see in cancer metabolism is known as the warburg e�ect. the metabolic adaptation shifts their energy production from oxidative phosphorylation in the process of aerobic glycolysis. metformin is a commonly prescribed drug used for type 2 diabetes, which also displays anticancer properties in inhibiting mitochondrial complex i,41,42 activating ampk, a regulator for energy metabolism, and reducing insulin and insulin-like growth factor 1 (igf-1), which performs anti-tumor functions. in addition, metformin has shown ability to inhibit the mtor pathway, which is involved in the protein synthesis and cell growth process. there are two types of forms of mtor: complex 1 (mtorc1) used in growth factors, glucose, and helps with protein synthesis; and complex 2 (mtorc1) used for regulating cell survival and metabolism43. metformin inhibits mtorc1 which then activates ampk and phosphorylates the tsc2 protein, the negative regulator of mtoc1. this inhibition can occur both dependent and independent of amp-activated protein kinase (ampk) activation, leading to the decrease in protein synthesis and cell growth. metformin is able to inhibit mtorc1 independently when ampk is activated as it is binding to the complex.43 this dual mechanism causes a positive treatment for the host, since metformin has multiple strategies to repress pathways critical for cancer cell growth. berkeley pharma tech journal of medicine | 111 recent studies with mice given metformin after being exposed to carcinogen show a reduction of lung tumor burden by up to 53%. however, only modest e�ects presented as mtor was inhibited in lung tumors 45. the researchers then inject mice with metformin to assess whether this method would improve mtor inhibition. the result shows that plasma levels of metformin were higher after injection than oral administration. on the other hand, metformin also activates ampk and inhibits mtor in liver tissue, but it only inhibits phosphorylation process of igf-ir/ir, akt, erk, and mtor in lung tissue. this suggests that metformin indirectly inhibited mtor in lung tissue by decreasing activation of igf-1r/ir and akt upstream of mtor45. a follow-up study showed that intraperitoneal administration of metformin decreased tumor burden by 72%, which correlates with decreased cellular proliferation and marked inhibition of mtor in tumors, as shown in �gure 2. figure 2. as the intake ofmetformin treatment inhibits plasma igf-1 level in all tissues. as mentioned, themetformin inhibition also has specificity in lung and liver tissue. metformin swords decrease with the phosphorylation of igf-1 and the insulin receptors in lung tissues. the drug is known for reducing the levels of hormones presentation in patients. with oral administration of metformin, study shows a decrease of 1 or 5 mg/ml of circulating igf-1 by approximately 20% and the insulin by 20% and 35%44. berkeley pharma tech journal of medicine | 112 there was an injection of metformin intraperitoneally to assess its inhibitory e�ect on mtor pathway. however, the intraperitoneal injection did not decrease the igf-i levels signi�cantly. instead, the levels of igf-1 inside the mice shows a signi�cant reduction from the wild type comparison group. this can indicate that stress in daily injection can alter the masked inhibitory e�ect of metformin in circulating the levels of igf-1. this supports the hypothesis that metformin can be used in mtor pathways with cancer patients, since it will decrease the levels of circulating igf-1 (insulin) in preventing the nnk-induced lung tumorigenesis45. in addition to its ability to inhibit the mtor pathway, metformin triggers a decrease in cap-dependent translation. a study utilizes mcf-7 cells, metformin treatment and led to a maximal inhibition of 40% in cap-dependent translation45. the polysome pro�le analysis shows how the metformin treatment of mcf-7 cells leads to a shift of mrnas from heavy to light polysomes and how concomitant increased 80s ribosomes46. this suggests that metformin can be a treat and cause signi�cant impact on the translation of speci�c mrnas and leads to alteration of protein expression in cancer cells. the change in polysome pro�les towards lighter polysomes indicates a reduction in the translation e�ciency of speci�c mrnas. this can help with further implication for cancer cells that rely on increased protein synthesis for their survival and proliferation47. the increase of 80s ribosomes indicates that metformin treatment may also a�ect the biogenesis of ribosomes, which is essential for protein synthesis. this decreases the rate of protein synthesis within cancer hosts, which can then be added to the growing body of evidence supporting the potential of metformin as a therapeutic agent for cancer. in mcf-7 breast cancer cells, metformin has been shown to inhibit translation initiation by activating smp-activated protein kinase (ampk) through its upstream kinase, liver kinase b1 (lkb1)48. this results in the inhibition of the mammalian target of mtorc1. although in contrast, in mda-mb-231 breast cancer cells, didn’t express lkb1 mrna, metformin had no e�ect on its protein synthesis, con�rming the requirement of lkb1 for inhibition of translation by metformin in mcr-7 breast cancer cells49. this con�rms the requirement of lkb1 for inhibition of translation by metformin in mcf-7 berkeley pharma tech journal of medicine | 113 breast cancer cells. it is an important biomarker for predicting the response to metformin treatment. 7. conclusion as discussed, the development of diabetes is a result of imbalance between glucose metabolism and anabolism. this distorted metabolic system can be caused by abnormally increased glucose intake and the subsequent development of insulin resistance, causing t2dm. furthermore, the establishment of t2dm, along with an anaerobic environment that is bene�cial for the cancer progression, will reshape the metabolic dependence of tumor from mitochondrial respiration to aerobic glycolysis. extensive supply of glucose can be advantageous for cancers with high energy demand to ful�ll its need of rapid division and proliferation. therefore, a new target for non-invasive cancer treatment emerges. by limiting the energy in�ux to tumor tissue with either direct blockage on membrane receptors or indirect pathways that induce speci�c cell killing, three of the potential candidates are available for consideration, which 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treatment and therapies, pre-clinical studies, alzheimer's disease, stem cell therapy, neural stem cells, mesenchymal stem cells, embryonic stem cells submitted august 9, 2023 accepted march 15, 2024 published june 28, 2024 full open access creative commons attribution license 4.0 abstract alzheimer's disease (ad) is a neurodegenerative disorder, a form of dementia commonly affecting people aged 40-65. growing more prevalent in society, approximately 6.2 million americans aged 65 and older live with ad. ad is a progressive, long-term neurological disorder that worsens cognitive skills, memory, and communication abilities, leading to a performative decline in daily tasks. characterized by the accumulation of extracellular amyloid beta (aβ) plaque and neurofibrillary tangles (nfts) of tau in the central nervous system. ad accounts for neuronal death in the brain. unfortunately, even with its detrimental impacts, clinical trials of therapeutic drugs are still to be tested and not available to the public. current research on stem cell transplantation has shown to alleviate neuropathology and is explored as a prospective treatment for ad. this literature review assesses the important uses of stem cell therapy for ad patients to provide a new clinical approach for future treatment. further clinical research should be conducted on the long-term outcomes of stem cell therapy for deeper analysis of its therapeutic effects for ad. stem cell therapy as a target for alzheimer’s disease by: jasmine segal 1. introduction alzheimer's disease (ad) is a form of dementia a�ecting memory and behavioral capabilities, resulting in the deterioration of cognitive function. as of 2023, this neurodegenerative disease has impacted around 50-75% of the united states population.1 symptoms of ad are characterized by impairments in memory, which interfere with daily activities and other cognitive �elds—this is linked through a reduction in brain volume of such individuals.2 the risk of ad increases with age, which doubles about every �ve years above the age of 65. with increasing age, the hippocampus’ cognitive function declines, a common trait of neurodegenerative disorders and ad. patients with ad tend to live within 5–12 years of the onset of ad symptoms due to declining performance of basic functions and brain incompatibilities.3 the hippocampus serves as the main part of the brain responsible for memory. the cerebral cortex and hippocampus are closely associated with cognitive function and neurogenesis (neuronal formation) in the brain.4 reduction of brain mass is often indicated through neural death and synapse degradation of the hippocampus. it has been commonly noted that hippocampal degeneration is an indication of ad.2 diagnosis of ad is characterized in the brain by an increase in the buildup of proteins between neurons.5 ad can be examined through chronic neuroin�ammation with neuronal loss of the extracellular senile plaques (sps), amyloid-β peptide (aβ) deposits, and tau proteins that form neuro�brillary tangles (nfts), which lead to extensive metabolic dysfunctions. nfts (abnormal production of tau protein neurons) contribute to neural death, inducing progressive deterioration of memory and cognitive ability.6 in ad, degeneration occurs with the accumulation of β-amyloid (aβ) and tau proteins in the brain. senile plaques of protein fragments beta-amyloid (aβ) induce the build-up of tau proteins leading to ad patients’ nerve cells shrinking and dying, spreading to the entire brain. neural networks of ad patients are impaired due to a lack of the transmitter acetylcholine, which plays an important role in intercellular signaling.7,8 in the gene encoding aβ precursor protein (app), mutations of this gene can cause hereditary cerebral hemorrhage, leading to aβ build-up.9 berkeley pharma tech journal of medicine | 2 hereditary cerebral hemorrhage with amyloidosis (aβ buildup) showed that app mutations could cause an abnormal amyloid deposition, albeit mainly outside the brain parenchyma.10 altered app processing and aβ accumulation predate tau. the mechanism for the aggregation of ad is characterized by the “amyloid cascade hypothesis” (figure 1). according to this hypothesis, the extracellular deposition of aβ is a critical and central event in the disease's progression, leading to the formation of neuro�brillary tangles, causing neuroin�ammation, cell death, and dementia.11,12 abnormal processes or mutations of processing app and aβ peptides hold signi�cant development for ad. yet, it is suggested that targeting app processing to treat ad can result in tumor development.13 the accumulation of aβ peptides and tau are caused by disturbances of homeostasis from app degradation. the clinical manifestations of ad are progressive and clinical trials are still ongoing. figure 1: summary sequence of events of ad development via the amyloid cascade hypothesis, presenting the signi�cance of aβ plaque buildup in ad symptoms. as previously discussed, app genes encode amyloid precursor proteins that are the main building block of aβ plaques. ps1 and ps2 genes express presenilin-1 and presenilin-2 proteins respectively. mutations in the aforementioned genes lead to exempli�ed aβ plaque production, leading to neuronal cell dysfunction and death. berkeley pharma tech journal of medicine | 3 stem cells are immature cells with self-renewal capabilities and the ability to di�erentiate into various cell types. stem cell transplantation can mobilize endogenous stem cells in adult brains and provides a promising form of therapy for other neurodegenerative diseases, such as parkinson's disease.14,15 for cells to be labeled as stem cells, they must have unlimited self-renewal and encompass specialized cell types.16 pluripotent stem cells are common regeneration approaches in their abilities to di�erentiate into all cell types, which naturally don’t last before di�erentiating to a specialized stem cell.16 they di�erentiate into cells of all germ layers, making these types of cells e�ective for stem cell therapies through autologous or allogeneic transplantation.17, 18 in this instance, stem cells can repair neural damage through cell division, which the most common approach to stem cell therapy. 2. discussion ad develops as a process of many factors by high neuropathological diversi�cations. therapeutic stem cells can di�erentiate into other body cells, which brings a promising approach to stimulating neurogenesis circuitry. to develop stem cell therapy, a suitable cell source must be determined. during literature searches, common stem cell types were noted: brain-derived neural stem cells (nscs), mesenchymal stem cells (mscs), and embryonic stem cells (escs) (figure 2). figure 2: the most common stem cell types are brain-derived neural stem cells (nscs), mesenchymal stem cells (mscs), or embryonic stem cells (escs). transplantation of these berkeley pharma tech journal of medicine | 4 https://www.ncbi.nlm.nih.gov/pmc/articles/pmc4104807/ cells has been investigated as a prospective therapeutic approach for neurodegenerative disease, including cases with ad.35 2.1 neural stem cells nscs are multipotent and self-renewing cells capable of di�erentiating neurons in the brain’s hippocampus.13 ad generates approximately half the rate of neurogenesis compared to normally aging individuals. nscs can ameliorate ad symptoms by regenerating lost or damaged cells.6 mechanisms of improved cognition involving enhanced hippocampal synaptic density are mediated through brain-derived neurotrophic factors (bdnf). several studies found that the transplantations of nscs lead to di�erentiation into neural types, promoting hippocampal neurogenesis and elevating bdnf levels. in mouse models, nsc transplantation rescued cognitive performance in ad mice.7 ager, davis, agazaryan, benavente, poon, la ferla, and blurton-jones found that with human cells and models, human nscs can improve cognition of ad pathogenesis and hippocampal neural loss.20 studies showcased that nsc injections rescued cognitive functions in transgenic mice, exhibiting advanced ad pathology. nsc-derived cells elevated hippocampal bdnf, which led to the increased synaptic density of the hippocampus and restored cognition abilities.21 figure 3: modi�ed from chang, kim, joo, ha, and suh,3 the amyloid cascade hypothesis illustrates the accumulation of aβ derived components of ad, including neuronal and synaptic loss, neuro�brillary, and cognitive dysfunction (blue arrows). while drugs target the initial accumulation of aβ, stem-cell-based therapies intervene at all stages. stem cell berkeley pharma tech journal of medicine | 5 therapies could treat ad by targeting ad pathogenesis. nsc (green arrows) provides synaptic plasticity and neurotrophic activity. 2.2 mesenchymal stem cells mscs can regenerate and di�erentiate under appropriate conditions, such as standard cultured solutions.4,23 in particular, mscs can be isolated from brain regions. a recurring cell in literature reviews was found to be human umbilical cord blood-derived mscs (hucb-mcss) and bone marrow-derived mscs (bm-mcss). hippocampal transplantation of (hucb-mcss) showed rescuing of memory de�cits and cognitive abilities in ad mice by reducing neuronal apoptosis, which increased brain volume.24-27 bm-mcss were transplanted in theapp mouse model of ad, , resulting in a reduction in microglial numbers without altering amyloid plaques, the baseline of the development of ad.27 yet, some studies also show a signi�cant decrease in amyloid plaques after two months of injection.28 transplantation of hucb-mscs and bm-mcss into the hippocampus through multiple studies has shown to be feasible, safe, and tolerated to improve cognitive function and lower amyloid plaques in the brain.4,29 2.3 embryonic stem cells escs from inner masses of blastocysts have shown to be pluripotent and thus capable of cell di�erentiation and self-renewal into primary germ layers.2,6 for cell replacement therapies, escs provide pluripotency-regulated neural lines. several reports have explored esc’s roles in mitigating ad in rodent models. here, esc progenitor and cultural cells transplanted into the hippocampus of rat models di�erentiated neuron cells, increased synapse regulation, and improved memory de�cits of ad pathologies.26 neuron-derived escs also di�erentiated into mature cholinergic neurons and restored the cognitive performance of ad transgenic mice.16,28 nevertheless, there are controversial issues with escs, such as immune system rejection, ethical concerns, and risks of teratoma (a germ cell tumor) upon transplantation.6,30 due to the low number of literature papers on escs, more research should be conducted to explore the role and e�ects of ad as an alleviating factor. berkeley pharma tech journal of medicine | 6 table 1: summary of ad model results of stem cell therapy. 3. limitations and future research clinical �ndings have advanced in recent decades, generating promising results in stem cell technology. yet, technological challenges remain on stem cell therapy use in ad. even with the possibility of regenerative medicine, the future of stem cell therapy for regeneration remains unclear. research concerning stem cell therapy and ad analysis are based on animal models, which may not demonstrate favorable results in humans.14,36,37 stem cell therapy results in di�erent cognitive e�ects based on animal models transfected with ad, proving that it can improve learning and memory de�cits.18,29,32 . the current ad research primarily used transgenic mice models to reveal cellular alterations through disease progression. these �ndings support the amyloid cascade theory of the accumulation of tau nfts causing neurodegeneration.30 yet, the progressive nature of ad requires longitudinal studies to assess the lasting e�ects and safety of pro�le treatments. regarding the high aggregate quantities of aβ plaques and tau in the brain of patients or experimental models with ad, , transplanted cells can typically generate nonneuronal cells or unexpectedly die based on speci�c environments. this approach does not aid the therapeutic approach, creating a possible discrepancy with the results of experiments.7,38 a study berkeley pharma tech journal of medicine | 7 by bae, jin, lee, richardson, and carter indicated a drastic decrease in aβ plaques in the brain’s hippocampus region after two months.27 on the other hand, naaldijk, jaeger, fabian, leovsky, bluher, rudolph, and stolzing found no di�erence in terms of aβ plaque concentration but noted improvements in cognitive functions in ad models.39 based on recurring inconsistent results, further research on stem cell therapy is necessary to produce a more robust conclusion. stem cell therapy aims to promote the regeneration of tissues with inhibition of in�ammation, reduction of apoptosis, stimulation of angiogenesis (blood vessel formation), and cell di�erentiation, bringing a considerable amount of attention to stem cell therapy as a feasible way of treating ad patients.14.27 additionally, issues with the procedure and basis of stem cell therapy remain unresolved, such as long-term safety of transplantation delivery systems, cell source, and donor cell responsiveness to ad-pathogenic environments.19,40 even so, stem cell therapy will become an e�cient candidate for ad treatment due to promising results in neuron regeneration. it is important to note that rather than multiple databases, the literature search used only three: pubmed, google scholar, and the national library of medicine. this may have limited the information represented in this paper, leading to fewer referenced sources and greater variability in results. though more current studies were implemented, eliminating those older than 1991 allowed analysis of more recent data and research advances since the beginning of stem cell technology. further research would be ideal to analyze the e�ectiveness of stem cell therapy over a longer duration and to ensure the therapeutic does not lead to tumors through excess cell di�erentiation. 4. conclusion while the cause of ad is only partially understood, hereditary genes are thought to play a signi�cant role in determining who contracts it. hypotheses have tried to explain the cellular events leading to inheritance of ad, with the focus lying on extraneous app accumulation. however, it has been determined that vaccines removing app buildup do not reverse ad in patients.31 berkeley pharma tech journal of medicine | 8 more clinical trials employing stem cell therapy are ongoing for ad, focusing on its capability to di�erentiate into neuronal and glial cells and alleviate ad symptoms or curation.2 this paper analyzes the speci�c stem cells of nsc, msc, and esc to foresee possible treatment for ad pathogens (table 1). in recent years, stem cells have become a promising alternative to conventional methods for ad research. as a multifactorial disorder, ad creates barriers to alleviating its adverse e�ects, particularly in the brain’s hippocampus. to combat such e�ects, stem cell therapy uses a multitargeted approach through cell di�erentiation upon transplantation. stem cells have been tested for e�ective modi�cation in slowing neuronal death and preventing tau accumulation in wad. recent data from quin, wang, zhang, bai; and shin, park, kim, oh, bae, ha, and lee indicated that transplantation of stem cells alleviated neuropathology and cognitive de�cits in several animal models with ad.32-35 no current drugs demonstrated an improvement in ad symptoms and e�ects.16 research has provided promising results for stem cell transplantation improving memory and learning abilities as a potential treatment for ad. therefore, stem cell therapy should be considered a healthy and suitable regeneration of cell function to alleviate cell death and hippocampus damage. experts convey that stem cell therapy for ad has bene�cial outcomes, displaying a clinical e�ciency of approximately 82.2%.41 hence, treatments that enhance or improve cognition, especially in the presence of aggregate plaque and tangle pathology, are urgently needed. stem cells are being actively studied for their potential to replace dead or diseased cells.21,42-44 regardless of the recent breakthrough of stem cells, advancements in technology con�rm the therapeutic as a potential treatment for ad. in the future, a thorough evaluation of implanted cells with ad pathogens should be conducted to con�rm their successful therapeutic e�ects. berkeley pharma tech journal of medicine | 9 references 1. srivastava s, ahmad r, khare sk. alzheimer’s disease and its treatment by di�erent approaches: a review. eur jmed chem. 2021;216:113320. doi:10.1016/j.ejmech.2021.113320. 2. vasic v, barth k, schmidt mhh. neurodegeneration and neuro-regenerationalzheimer's disease and stem cell therapy. int j mol sci. 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embryonic stem cell-derived neural precursor cells improves cognitive function in alzheimerian rats.differentiation. 2009;78(2-3):59-68. 26. mousaei ghasroldasht m, seok j, park hs, liakath ali fb, al-hendy a. stem cell therapy: from idea to clinical practice. int j mol sci. 2022mar 5;23(5):2850. doi: 10.3390/ijms23052850. pmid: 35269990; pmcid: pmc8911494. 27. bae js, jin hk, lee jk, richardson jc, carter je. bone marrow-derived mesenchymal stem cells contribute to the reduction of amyloid-β deposits and the improvement of synaptic transmission in a mouse model of pre-dementia alzheimer's disease. curr alzheimer res. 2013;10(5):524-531. 28. si z, wang x. stem cell therapies in alzheimer’s disease: applications for disease modeling. j pharmacol exp ther. 2021;377(2):207-217. berkeley pharma tech journal of medicine | 11 29. bissonnette cj, lyass l, bhattacharyya bj, belmadani a, miller rj, kessler ja. the controlled generation of functional basal forebrain cholinergic neurons from human embryonic stem cells. stem cells. 2011;29(5):802-811. 30. hentze h, soong pl, wang st, phillips bw, putti tc, dunn nr. teratoma formation by human embryonic stem cells: evaluation of essential parameters for future safety studies. stem cell res. 2009;2(3):198-210. 31. langford k. anatomy 101: frommuscles and bones to organs and systems, your guide to how the human bodyworks. adamsmedia; 2015. 32. qin c, wang k, zhang l, bai l. stem cell therapy for alzheimer's disease: an overview of experimental models and reality. animalmodel expmed. 2022 feb;5(1):15-26. doi: 10.1002/ame2.12207. epub 2022 jan 29. pmid: 35229995; pmcid: pmc8879630. 33. rao yl, ganaraja b, murlimanju bv, joy t, krishnamurthy a, agrawal a. hippocampus and its involvement in alzheimer's disease: a review. 3 biotech. 2022 feb;12(2):55. doi: 10.1007/s13205-022-03123-4. epub 2022 feb 1. pmid: 35116217; pmcid: pmc8807768. 34. rodriguez-jimenez fj, ureña-peralta j, jendelova p, erceg s. alzheimer’s disease and synapse loss: what can we learn from induced pluripotent stem cells? j adv res. published online january 2023. doi:10.1016/j.jare.2023.01.006 35. shin jy, park hj, kimhn, oh sh, bae js, ha hj, lee ph.mesenchymal stem cells enhance autophagy and increase β-amyloid clearance in alzheimer’s disease models. autophagy. 2014 jan;10(1):32-44. doi: 10.4161/auto.26508. epub 2013 jan 1. pmid: 24149893; pmcid: pmc4389879. 36. delacourte a, buée l. modélisation de la maladie d'alzheimer: un parcours semé d'embûches [animal models of alzheimer's disease: a road full of pitfalls]. psychol neuropsychiatr vieil. 2005 dec;3(4):261-70. french. pmid: 16316817. 37. fitzsimons cp, van bodegraven e, schoutenm, lardenoije r, kompotis k, kenis g, et al. epigenetic regulation of adult neural stem cells: implications for alzheimer’s disease.mol neurodegener. 2014;9(1):1-21. doi:10.1186/1750-1326-9-1. 38. biehl jk, russell b. introduction to stem cell therapy. j cardiovasc nurs. 2009;24(2):98. 39. naaldijk y, jaeger c, fabian c, leovsky c, blüher a, rudolph l, et al. e�ect of systemic transplantation of bone marrow-derived mesenchymal stem cells on neuropathology markers in app/ps 1 alzheimer mice.neuropathol appl neurobiol. 2017;43(4):299-314. doi:10.1111/nan.12339. 40. duncan t, valenzuela m. alzheimer’s disease, dementia, and stem cell therapy. stem cell res ther. 2017;8:111. doi:10.1186/s13287-017-0567-5. 41. kumar l. engrafted stem cell therapy for alzheimer's disease: a promising treatment strategy with clinical outcome. j control release. 2021;338:837-857. 42. bruni ac, bernardi l, gabelli c. from beta amyloid to altered proteostasis in alzheimer's disease. ageing res rev. 2020;64:101126. doi:10.1016/j.arr.2020.101126. 43. caplan ai. mesenchymal stem cells. j orthop res. 1991;9(5):641-650. berkeley pharma tech journal of medicine | 12 44. harach t, jammes f, muller c, duthilleul n, cheatham v, zu�erey v, et al. administrations of human adult ischemia-tolerant mesenchymal stem cells and factors reduce amyloid beta pathology in a mouse model of alzheimer's disease.neurobiol aging. 2017;51:83-96. doi:10.1016/j.neurobiolaging.2016.11.009. berkeley pharma tech journal of medicine | 13 article 1_segal_galley segal_references body paragraph_ghazzawi et al._wound healing.docx berkeley pharma tech journal of medicine correspondence: jghazzawi24@gmail.com keywords: burn wound injury lnc rna xist il-33 mir-19b wound healing stages inflammatory pathways submitted: may 5, 2023 accepted: june 30, 2023 published: december 30, 2023 full open access creative commons attribution license 4.0 abstract as the largest organ of the body, it is crucial that the skin, and all it protects, remain healthy and functioning. however, burn injuries may result as a serious threat to the skin, possibly forming greater health issues depending on the severity of the issue. burn wound injury is characterized as damage to the skin or other organic tissue originating from sources such as heat, friction, radiation, radioactivity, electricity, and/or chemical contact. different degrees of burn wounds make it increasingly more difficult to treat the more severe the wound is. for this reason, it is crucial to study how skin can not only heal itself via its own equipped healing related pathways, but also what treatments exist outside of the ongoing biochemical mechanisms in order to accelerate healing and repair the body’s greatest defense. more standard forms of treatment that currently exist adopt a more holistic approach, treating at the surface level, while treatments targeting the molecular level are less well studied. this review paper assesses several novel therapeutic alternatives including, but not limiting to, targeting lncrna xist, il-33, and mir-19b through analyzing the molecular pathways in which wound healing is performed. advancing wound healing: innovative pathway strategies for enhanced recovery by: jowana ghazzawi, crystal yu, and maria favela 1. introduction according to the world health organization, approximately 180,000+ deaths per year are a result of burns, with the majority taking place in low and middle income countries. the leading cause of morbidity is non-fatal burn injuries, leaving both infants and older adults at the greatest risk for burn injury4. according to johns hopkins, the leading causes of burn injury for adults are smoking and open �ame, while the leading cause of burn injury for children is scalding⁵. depending on the cause of the burn injury itself, there are four de�ned degrees of burn wounds that increase in severity6. though treating �rst or second degree burns may be more manageable and not require intensive therapies, third and fourth degree burns may cause trauma to both the external surface and the internal environment⁷. therefore, it is important to learn more about molecular pathways that currently exist in order to treat burn wound injuries at all levels. current therapeutics focus on topical treatment and management, especially when it comes to keeping the injury clean and reducing the severity of scars as a result. in terms of assessing whether novel treatments are e�ective, there are several factors to identify that have been proven to indicate e�cient burn wound repair. these factors include the reduction of healing rates and in�ammatory in�ltration, scar scores, an increase in collagen deposition, and other markers that will de�ne successful treatment execution. novel targets of therapy including lncrna xist, il-33, and mir-19b, are proven to be e�cient mechanisms of accelerating burn wound healing through the enhancement of cell proliferation, extracellular matrix synthesis, and the inhibition of �broblast apoptosis. several clinical trials were reviewed with their respective results contributing to the understanding of novel approaches in treating burn wound injury⁸. scienti�c literature was necessary in order to gather more information on the background of burn wound injury, its pathological course of events, and current treatment e�ectiveness. 2. pathophysiology of burn wound injury berkeley pharma tech journal of medicine | 66 burn wounds can be classi�ed into four stages: hemostasis, in�ammation, tissue proliferation, and tissue maturation/remodeling⁹⁻¹¹. each stage involves a complex interplay of molecular mechanisms, growth factors, and signaling pathways. hemostasis is the �rst stage of the wound healing process, which stops bleeding and prevents further damage¹². this stage is composed of several processes that occur simultaneously. primary hemostasis involves the formation of a platelet clot, where platelets attach to damaged tissue and activate to attract more platelets. vasoconstriction, the narrowing of blood vessels by small muscles, slows blood �ow. secondary hemostasis involves the activation of coagulation factors in the blood, which amplify the clotting e�ect. fibrin is then formed, which acts as a stable blood clot. fibrin clot remodeling or �brinolysis occurs when the temporary seal is removed, and the blood clot is remodeled into the tissue that was there before the injury. growth factors involved in this process include platelet-derived growth factor (pdgf), epidermal growth factor (egf), transforming growth factor-beta (tgf-β), insulin-like growth factor-1, and platelet factor-iv. the second stage of wound healing is in�ammation, where platelets aggregate to block bleeding and release a chemoattractant to activate in�amed cells for wound healing. edema, or swelling and in�ammation, occurs in the �rst phase after the secretion of histamine/leukotrienes. vasodilation and increased capillary permeability lead to �uid leaking from blood vessels. the second phase involves �uid penetration through capillaries and activation of cytokines, enhancing cellular immune response. white blood cells are driven into the interstitial space surrounding the wound, and protease enzymes degrade dead cells. macrophages defend against dead cells and bacteria. growth factors stimulate the generation of new capillaries and promote the synthesis of �broblasts¹³. the third stage of wound healing is tissue proliferation, where cells of the epidermis and dermis migrate to the wound site. fibroblasts migrate to the wound site and synthesize collagen and elastin. collagen causes the wound to adhere, and keratinocytes cause re-epithelialization. angiogenesis occurs, and berkeley pharma tech journal of medicine | 67 new blood vessels form within the healing tissue. burns that a�ect deep layers of the skin heal slower because they don't have as many helper cells, and new skin can only grow after the dead tissue is removed. new blood vessels initially form densely, but later reduce to levels similar to the surrounding skin. the �nal stage of wound healing is tissue maturation/remodeling, which is the formation of new epithelium and scar tissue. fibroblasts degrade protein and realign collagen �bers. apoptosis of �broblasts and myo�broblasts prevent excessive scarring. realignment of collagen �bers transforms the initial collagen matrix into a highly organized collagen matrix whose structure mimics that of the native tissue. new tissue with a high tensile strength and a minimal number of cells and vascularization is formed. a visual diagram of the four burn wound stages is displayed in figure 1. figure 1: the four stages of wound healing. (a) hemostasis: clotting factors such as platelets aggregate to form a blood clot. (b) inflammation: white blood cells like neutrophils and macrophages fight off potential infection by clearing bacteria. they also prepare the area for fibroblast proliferation. (c) tissue proliferation: fibroblasts begin the process of epithelialization. (d) remodeling: there is a build up of collagen and scar tissue becomes visible while the new tissue is stronger and flexible. 3. molecular mechanisms of treatment wound healing is a complex process involving numerous molecular mechanisms. recently, long non-coding rna (lncrna) xist, interleukin-33 (il-33), and microrna-19b (mir-19b) have been identi�ed as key regulators in wound healing-related pathways. this section will discuss berkeley pharma tech journal of medicine | 68 how lncrna xist binds to mir-19b, which binds to il-33 and activates m2 macrophages in burn injury healing in human skin �broblast (hsf) cells (figure 2)¹⁴. it will also explore how these molecules contribute to the proliferation, migration, and extracellular matrix (ecm) production of hsfs, and their role in the regulation of wound healing. figure 2: molecular pathways of lncrna xist and mir-19b. in the process of wound healing, lncrna xist binds and inhibits mir-19b, which in turn binds and inhibits il-33. the binding of lncrna xist to mir-19b decreases the amount of mir-19b that is available freely, leading to an increase in il-33. thus, lncrna-xist indirectly activates il-33, leading tom2macrophage activation berkeley pharma tech journal of medicine | 69 figure 3: relative expression of lncrna xist & mir-19b. in the process of wound healing, regulator expression is time dependent and varies throughout the process. lncrna xist & il-33 increase while mir-19b decreases. lncrna xist and il-33 expression are proportional because xist activates il-33. lncrna xist is a key regulator in wound healing-related pathways¹⁵. studies have found that lncrna xist expression increases during the healing process after a burn injury in a time-dependent manner¹⁴. lncrna xist contributes to the proliferation and migration of hsfs by inhibiting mir-19b and enhancing �broblast ecm production by promoting the transformation of macrophages into the m2 phenotype. in this way, xist can promote the repair of the injured dermis. moreover, lncrna xist targets mir-29b-3p/col1a1¹⁶ and can also inhibit mir-29a and promote lin28a expression, e�ectively contributing to the synthesis of hsfs¹⁴,17. finally, xist is also crucial in alleviating pain behavior as it suppresses cytokines that cause in�ammation14,18. these results suggest that lncrna xist may be a promising therapeutic target for promoting wound healing. il-33 is an immune cytokine that plays a critical role in wound healing. il-33 participates in the pathological process of many diseases and acts as an alarm to alert the immune system when released by epithelial barrier tissues during injury, e�ectively it acts as a link between the skin and the immune system14,19. berkeley pharma tech journal of medicine | 70 a study shows that il-33 is not detected in healthy, undamaged human skin cells, but its expression increases when there is a wound and the immune system is alerted. it is not normally produced and is only synthesized when needed. il-33 promotes the repair of skin damage and enhances wound healing through mucosal healing and epithelial restoration and repair14,20. it further plays a role in the regulation of wound healing by upregulating the expression of m2 macrophages, which are the proliferative, anti-in�ammatory form compared to the m1 macrophages. these �ndings suggest that il-33 may be a potential therapeutic target for wound healing21. mir-19b is a microrna that has been implicated in wound healing-related pathways. studies have found that mir-19b expression decreases during the healing process after a burn injury in a time-dependent manner14. it can be concluded that mir-19b has roles that are more important at the beginning of the healing process. studies show that mir-19b increases the proliferation and migration of cardiac �broblasts14,22,23 and inhibits the apoptosis of endothelial cells24. mir-19b also promotes the activation of m1 macrophages, tlr3-mediated nf-κb activation by targeting shcbp1, and reducing the production of in�ammatory chemokines and cytokines by keratinocytes. to account for the decrease in expression, studies have shown that overexpression of mir-19b may inhibit the expression of cgtf25, which is a connective tissue growth factor. cgtf plays a crucial conducive role in wound healing so its decreased expression due to the overexpression of mir-19b is detrimental to the healing process. these results suggest that targeting mir-19b may be a promising therapeutic pathway for promoting wound healing. 4 .treatments of interest andmeasurement of e�cacy the treatment of patients who experience di�culty and obstacles during burn wound healing is a signi�cant hurdle in clinical practice26. therefore, gaining a deeper comprehension of the molecular mechanisms underlying burn wounds can facilitate the development of more e�cacious treatments, which can ultimately enhance patients’ life quality. the main ways lncrna xist, mir-19b, and il-33 have been used treatment-wise is through expression modulation via gene knockdowns, small molecule inhibitors, and rna interference27. berkeley pharma tech journal of medicine | 71 the measurement of e�cacy of lncrna xist can be assessed through several methods, including in-vitro and in-vivo studies. in-vitro studies can be executed using hsf cells or other appropriate cell lines to assess lncrna xist e�ects. these in-vitro studies use techniques like western-blotting, quantitative polymerase chain reaction (qpcr) and immuno�uorescence to detect changes in gene and protein expression28. for example, in a clinical trial involving 25 burn patients16, total rna was extracted from hsf cells and reversely transcribed to cdna followed by qpcr. a western blot was then applied for protein identi�cation. using these methods, the researchers were able to demonstrate that upregulation of xist expression boosts �broblast proliferation and migration via mir-29b-3p/col1a1 pathway16, which regulates collagen synthesis, resulting in improved wound healing. other in-vitro studies that use qpcr and western blotting include a study where results show that overexpression of lncrna xist in hsf cells signi�cantly increased cell proliferation and migration with a decrease in apoptosis, suggesting a possible for xist in burn injury wound healing28. fewer studies have been conducted in-vivo to investigate the role of lncrna xist in burn wound healing. these studies use animal models of burn injury to evaluate the e�ects of lncrna xist on wound healing and scar formation. these studies use techniques such as immunohistochemistry, histological analysis, and mechanical testing to evaluate changes in wound healing parameters29. for example, in a study involving mice6, burn wounds were induced and the wound tissues were processed for histological analysis, immunohistochemistry, and qpcr data. the results reveal that lncrna xist targets the il-33/mir-19b axis to promote wound healing. results from other in-vivo experiments include seeing an increase in hsf proliferation and ecm synthesis when xist is overexpressed7 and inhibition of xist leads to a decrease in wound healing acceleration29. through in-vitro and in-vivo studies, il-33 has been shown to accelerate wound closure, help reduce scar formation in animal models, and aid in �broblast proliferation, which are cells that help form new tissue30-32. in addition to the techniques mentioned in the previous paragraphs, cytokine and chemokine levels are measured to evaluate il-33’s role in regulating berkeley pharma tech journal of medicine | 72 in�ammation and immune response. some examples are using histological analysis on wound tissue samples to evaluate the degree of in�ammation and tissue repair and qpcr to measure gene expression levels, in which it was found that il-33 recruits group 2 innate lymphoid cells (ilc2s) to promote wound healing via re-epithelization30. using a full-thickness skin wound model on mice, where the wound extends below the epidermis and dermis layers, researchers assessed wound healing at four time points (days 0, 1, 3, and 14)31. they also researched how autophagy, or the clearance of damaged or old cells, is involved in wound closure rate by looking at the yap/il-33 pathway. the yes-associated protein (yap) has been shown to regulate in�ammation33, speci�cally, it increases the articulation of proin�ammatory cytokines34. the researchers utilized an autophagy inhibitor (3-ma) as well as vertepor�n, a yap inhibitor, and anti-il-33, an il-33 inhibitor to observe wound healing results. their results reveal that with autophagy inhibition, there is an upregulation of il-33 along with a suppression of tumor necrosis factors. vertepor�n decreased the expression of yap while increasing the expression of il-33, in which the upregulation of il-33 contributed to wound healing in both cases. anti-il-33 downregulated il-33 leading to no promotion of wound healing. thus, il-33 can increase wound closure and help decrease wound size. there is limited research speci�cally investigating the role of mir-19b in burn wound healing, however, some studies show that mir-19b could be a potential therapeutic target35-37. in an in-vitro experiment, the researchers investigated the potential of exosomes derived from human adipose-derived mesenchymal stem cells (adscs) containing mir-19b35. their results reveal that treatment with mir-19b enhanced cell proliferation and migration, and in a mouse model, mir-19b increased the deposition of collagen and blood vessel formation. in a clinical trial, 18 human samples were collected and results show that mir-19b is an important factor in wound healing by reducing in�ammation while an absence of mir-19 reduces wound healing capabilities. their methods involve qpcr, western blotting, mir-19 antisense inhibitors and luciferase reporter assay36. berkeley pharma tech journal of medicine | 73 5. practical applications in the case of lncrna xist, il-33, and mir-19b, although they show promising burn wound healing results in preclinical studies, more research is needed to determine the safety and e�cacy of these molecules in practical settings. they are currently not commonly used as a clinical treatment for burn wounds, but ongoing clinical trials are exploring its potential use38,39. for more severe injuries, like third and fourth degree burns, more intensive treatment is required, particularly at the molecular-level, to restore healing related pathways40. however, for minimal burn wounds requiring repair at a super�cial level, such as �rst degree burns, minimally invasive treatments may be more appropriate, including holistic applications. holistic treatments for burn injuries include a combination of traditional and medical treatments and alternative therapies to promote physical, mental and emotional healing. some holistic approaches that have been explored include: acupuncture, eating a good and healthy diet, massages, and herbs41,42. acupuncture helps decrease pain and shock after a burn injury and may also decrease infection43,44. as for a diet, with the right amounts of vitamins and nutrients, as well as su�cient protein and calorie intake, the body can promote new skin and tissue in a timely manner37. in a review paper, the authors suggest that burn patients may have increased requirements for certain micronutrients, and that supplementation with these nutrients may help improve outcomes45. like acupuncture, massages help decrease pain and itching46, 47. several herbs have been traditionally used for their potential wound healing and anti-in�ammatory properties, for example, aloe vera and calendula can help reduce pain and in�ammation in �rst and second-degree burns48, 49. 6. prevention strategies burns are preventable, especially when considering the majority of burns take place in the home and workplace. in order to strategize prevention plans, it is important to address impactful factors that heighten/lessen burn wound injuries. these factors include knowledge regarding the hazards related to speci�c burn injuries, access to education for vulnerable populations, and �rst aid training in case of an emergency. it is insightful to study which populations berkeley pharma tech journal of medicine | 74 are most a�ected, especially as it was mentioned previously that the majority of burn wound injuries occur in low and middle income countries. after considering the following factors, an e�ective prevention procedure can be produced and include several initiatives. initiatives such as heightened awareness within the community, e�cient policy enforced in order to safely prevent burn wounds if possible, prioritized research endeavors committed to learning more about not only prevention plans but also treatment options available, and the revaluation of current burn care therapeutics in order to ensure success alongside verifying new treatments being studied currently are all necessary to research when prevention strategies are organized50. as initial strategies have targeted more physical and pathophysiological consequences, research has also suggested signi�cant mental health outcomes, especially for the parents of burn wound victims51, that should be studied further in order to provide the necessary resources. according to stanford children’s health, burn injuries and fatalities have signi�cantly decreased throughout the last 20 years52. this is promising when addressing the e�ect that increasing public awareness may result in more adequate resources, further pushing the importance of �re safety and burn wound injury research. though these statistics suggest the harm is not as intense as it might have been in the past, it is still just as important to continue studying both the medical and sociological factors to enhance burn wound injury healing. 7. future directions lncrna xist, il-33, and mir-19b all contribute to burn wound healing repair and their results thus far should permit them to be treatment options to be further studied. it is still important to compare the e�ectiveness between these novel therapeutic options and other standard treatments for burn wound healing in order to identify any gaps or contrasting ideas that might exist and inhibit e�ciency. by studying the molecular pathways in which these treatment options interact with, other factors that are also present in the repair mechanism may also come to light in terms of promising targets, as well as targeting other related or unrelated biological pathways. studying the association between lncrna xist in particular, and burn wound repair presents promising results, though an exact mechanism is unclear. by studying berkeley pharma tech journal of medicine | 75 its role in depth, it is possible to speci�cally investigate the network of cell proliferation and extracellular matrix synthesis, accelerating repair further. 8. conclusion the investigation of novel therapeutic options for burn wound injury has advanced in the last 20 years, and continues to produce promising results the more the scienti�c literature is discussed. targets of therapeutic strategies including lncrna xist, il-33, and mir-19b as well as more standard and holistic approaches to burn wound healing have proven to be e�ective options for the acceleration of burn wound repair as it results in cell proliferation, extracellular matrix synthesis, and an inhibition of �broblast apoptosis, all resulting in overall healing on a molecular and more surface level. the combination of both topical and standard products that serve as treatments for less severe wounds, as well as the incorporation of molecular acceleration via targeting key receptors collectively aid repair. due to the lack of substantive results, more diversi�ed and inclusive trials are necessary to solidify their role as viable treatment options for long-term use. berkeley pharma tech journal of medicine | 76 references 1. swann g. the skin is the 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nsclc personalized medicine prognosis submitted june 29, 2021 accepted july 6, 2021 published december 17, 2021 full open access creative commons attribution license 4.0 abstract biomarkers are a range of biological signals that measure the presence and severity of the disease. this literature review assesses circulating dna (ctdna) and microrna (mirna) biomarkers detected in liquid and tissue biopsies and their importance in the prognosis, outcomes, and treatments for non-small cell lung cancer (nsclc). these biomarkers have the potential for clinical use; however, further studies with requisite data and sufficiently large trials are required to refine our understanding of their applicability. the prognostic significance of ctdna and mirna biomarkers in nsclc care has demonstrated that liquid biopsy and molecular diagnostic testing may provide a feasible and noninvasive method for tailoring treatment plans to the specific mutational landscape of diverse nsclc patients. however, further testing must be conducted to analyze the significance and benefit of ctdna and mirna biomarkers in larger cohorts and substantiate the standardization of liquid biopsy in clinical practice. circulating nucleic acids as promising biomarkers: a new frontier of personalized medicine by: carolyn skotz, samrat thapa, mary nowak, hoc lan phung, akshitha mamidi, hetu patel and mabel ho circulating dna and microrna are useful biomarkers in non-small cell lung cancer prognosis and treatment. image credits: n/c introduction despite representing 13% of all cancer diagnoses, lung cancer accounts for 24% of all cancer deaths in the united states each year. among the two subsets of lung cancer (non-small cell and small cell), nsclc accounts for 80% of all lung cancers. with only 19% of the diagnosed patients surpassing the ve-year survival rate, there is an urgent need for early detection, diagnosis, and prognosis. recently, studies have shown that these predictions for nsclc can be assessed via biomarkers (howlader et al., 2020). a biomarker is broadly de ned as an indicator of the presence and severity of a disease state. it can refer to a range of biological signals, including pulse, blood pressure, blood tests, and tissue tests. hundreds of novel biomarker-related articles are published each year, but only a few biomarkers are currently used in practice (rinaldi et al., 2011). notably, the current approach relies primarily on the use of tissue biopsy-based biomarker testing. tissue biopsy refers to an invasive procedure in which solid matter from the body, usually coming directly from the tumor or bone marrow, is sampled and assessed for the presence of clinically actionable biomarkers. although this method is deemed the gold standard for nsclc testing, it is time-intensive, invasive, and often fails to capture tumor heterogeneity (rijavec et al., 2019). liquid biopsy is an emerging, minimally invasive process that can address this demand for early disease monitoring. via this method, clinicians can quantify biological components circulating in bodily liquids, such as tumor cells and nucleic acids, including dna and mirna. circulating dna (ctdna) is derived from dna and released by cancerous cells and tumors into the bloodstream. as a tumor grows, newer cells replace older cells. the dead cells are broken down into their main components, releasing dna into the bloodstream (mader et al., 2017). microrna (mirna) are single-stranded, non-coding rnas in the blood and function as antisense rna to regulate the target genes. multiple mirnas can target the same gene (wang et al., 2018). the pathways of ctdna and mirna are depicted in figure 2 and figure 3, respectively. there are three main classes of biomarkers. predictive biomarkers interact with speci c treatments to a ect the outcome, prognostic biomarkers are berkeley pharma tech journal of medicine | 117 associated with particular outcomes regardless of the course of treatment, and treatment-focused biomarkers are any biomarkers that in uence the selection of a speci c course of treatment (stein, n.d.). this paper outlines several ctdna and mirna biomarkers detected from liquid biopsies and uses previous data to assess their value in delineating nsclc prognosis, outcome prediction, or treatment strategies. studies regarding these biomarkers were reviewed according to sample size, procedure, and clinical relevance. the speci c ctdnas and mirnas addressed in this paper are summarized in figure 1. figure 1: list of nsclc ctdna and mirna biomarkers. this list is not comprehensive; it serves as a brief summary of the ctdna and mirna biomarkers discussed in this paper. common methods utilized for circulating nucleic acid assessment: several primary technologies have been used to assess circulating nucleic acid biomarkers. 1. fluorescence in situ hybridization (fish): fish utilizes a dna sequence probe with a uorescent dye attachment to locate and label a speci c complementary dna strand on a chromosome. this uorescent marker can be visualized through microscopy. this method is applicable to blood, cytology smears, and bone marrow to detect duplications, deletions, and total chromosomal loss or gain (green). 2. immunohistochemistry (ihc): during ihc, xed tissue is exposed to antibodies, which enter and adhere to antigenic determinants. once this antibody-antigen substrate is created, it catalyzes an oxidation reaction to form a visible colorized marker. this can berkeley pharma tech journal of medicine | 118 be used as a tool for measuring protein and nucleic acid expression (schildhaus et al., 2020). 3. polymerase chain reaction (pcr): pcr allows for the rapid ampli cation of small dna fragments. first, the sample dna is denatured at a high temperature to form two single-stranded dna fragments. second, taq polymerase, a thermostable enzyme, attaches complementary dna strands (known as primers) to each exposed strand. this process results in the duplication of the original dna strand once elongation has been completed. millions of copies of a speci c dna strand can be produced after several cycles of this process (waters & shapter, 2014). 4. next generation sequencing (ngs): ngs refers to the parallel sequencing of genomic segments. there are three main kinds of ngs: a. illumina sequencing: in illumina sequencing, the dna bases are identi ed by their unique uorescent signals. b. roche 454: roche 454 sequencing relies on the release of visible uorescent pyrophosphate that occurs upon nucleotide addition to a new strand of dna. c. ion torrent: ion torrent sequencing measures the release of protons due to the incorporation of nucleotide bases in the dna. circulating dna biomarkers figure 2: an illustration of the ctdna pathway. at the top, the tissue cells are shedding into the bloodstream. an immune response occurs in order to eliminate the tumor cells entering the bloodstream and to eliminate any ctcs. as part of this elimination, many tumor cells are berkeley pharma tech journal of medicine | 119 phagocytized, thus increasing the concentration of ctdna in the bloodstream, which can be analyzed through liquid biopsy (delmonico et al., 2020, bettegowda et al., 2014). ercc1 excision repair cross-complementation group 1 (ercc1) is a part of the nucleotide excision repair (ner) pathway that acts as a mechanism for dna repair, and also removes the cytotoxic elements from genomic dna. for example, platinum is a cell-damaging agent that causes apoptosis, therefore regulating cell growth and death in the body. overexpression of ercc1 can cause lower platinum absorption, resulting in decreased cell death and subsequent increased cell growth and carcinogenesis. underexpression of ercc1 can cause carcinogenic factors to enter genomic dna and hinder dna repair mechanisms because ercc1 typically repairs any dna damage done by platinum (li et al., 2016). hence, ercc1 expression level directly correlates to platinum sensitivity (hamilton et al., 2018). programmed death receptor-1 is an immune-regulatory protein with two ligands, pd-l1 and pd-l2, that is expressed by immune cells and tumor cells. in a study done by buderath et al., pd-l1, pd-l2, and ercc1 levels were measured in 83 patients with epithelial ovarian cancer through liquid biopsy. results showed that higher ercc1 expression was associated with lower pd-l2 levels. lower sp-l2 levels were found to be associated with platinum resistance (p < 0.0001), which supports the results from previous studies (li et al., 2016; hamilton et al., 2018). higher pd-l2 levels were associated with lower ercc1 expression and reduced progression free survival (pfs) and overall survival (os), while lower pd-l2 levels were associated with platinum resistance and higher ercc1 expression (buderath et al., 2019). this demonstrates that ercc1 expression levels detected through liquid biopsies can be used to predict the outcomes of multiple cancers, including nsclc and epithelial ovarian cancer. met met is a proto-oncogene involved in cell growth, wound healing, and post-physical injury response. it encodes the receptor tyrosine kinase c-met, for hepatocyte growth factor, which causes c-met dimerization and autophosphorylation. this results in the activation of mitogen-activated protein kinases (mapk), phosphatidyl-inositol-3’ phosphate kinases (pi3k), viral oncogene homologs, and signal transducers berkeley pharma tech journal of medicine | 120 and activators of transcriptional signaling pathways. mutated met initiates the growth of cancer cells. unusual met expression has been shown to correspond with nsclc, as well as gastrointestinal cancer and hepatocellular carcinoma. if the met receptor is overexpressed via genomic ampli cation, mutation, or alternative splicing, cellular degradation of met can occur (mo et al., 2017). c-met receptor activation is associated with egfr-tki resistance and has poor prognosis. a trial consisting of 167 patients examined the prognosis, determined by pfs, after treatment with met inhibitors, erlotinib and tivantinib. 84 patients were randomly assigned to receive oral erlotinib and tivantinib (et group), and 83 patients were randomly assigned to receive erlotinib plus placebo (ep group). the average pfs was 3.8 months for et and 2.3 months for ep, which is statistically signi cant (p =.24) (sequist et al., 2011). deng et al. also studied nsclc patients with met ampli cation and egfr mutation. for one patient, a combination of crizotinib and osimertinib was administered, which greatly improved his overall health. results showed a 20-40% partial response rate to dual therapy with and without prior treatment with tkis. unfortunately, met mutations have occasionally been shown to cause resistance to crizotinib treatment (deng et al., 2018). in a study done by bardelli et al., 7 patients with metastatic colorectal tumors who had responded to panitumumab or cetuximab-based treatment and later relapsed were analyzed. met expression was higher in post-relapse patients compared to rst-occurence patients. in relapse patients with kras mutations, met expression was low or undetectable. met ampli cation was seen in the blood before relapse, indicating that met monitoring could be used to predict relapse. met ampli cation is detected in 5-20% of egfr-mutated lung cancers (bardelli et al., 2013). in metastatic colorectal tumors and nsclc, met can be used for predictive outcomes and may help direct the course of treatment. ntrk neurotrophic tyrosine kinase receptor (ntrk) mutations are found in many solid malignancies, including nsclc. trk receptors include three transmembrane proteins: trka, trkb, and trkc, which are encoded by ntrk1, ntrk2, and ntrk3, respectively. in carcinogenesis, trka, trkb, and trkc undergo changes that lead to activation of signaling berkeley pharma tech journal of medicine | 121 pathways involved in cell growth and proliferation. mutations to ntrk1, ntrk2, and ntrk3 have also been observed in 2-3% of nsclc patients. for example, overexpression, in-frame deletions, and alternative splicing of ntrk1 have been demonstrated to be potential oncogenic mechanisms. several ntrk inhibitors have been developed, but only loxo-101 and entrectinib are under clinical evaluation (ricciuti et al., 2017). of 1,378 patients with locally advanced or metastatic solid tumor malignancies with ntrk1, ntrk2, ntrk3, ros1, or alkmutations, two showed ntrk1 gene rearrangements on anchored multiplex pcr. one patient, a 45-year-old male with a history of smoking, was treated with 400 mg/m2 oral entrectinib daily. ct scans of his lungs 26 days after beginning the treatment showed no tumor growth. after 155 days, ct scans showed a 77% overall tumor reduction (farago et al., 2015). cdkn2a the cdkn2a mutation is uniquely present in nsclc. studies have suggested that the cdkn2a mutation is associated with the increased expression of the extracellular matrix and metabolic gene sets in nsclc cell lines (kim et al., 2015). this heightened expression has been suggested as an indicator of increased mortality in lung, breast, and gastric cancers (gilkes, semenza, and wirtz, 2014). as a result, early detection of cdkn2a mutations is critical. the e cacy of liquid biopsy for mutation detection has been suggested in breast cancer studies, but further testing is needed to determine whether the detection of cdkn2a alterations via liquid biopsy would yield signi cant clinical bene t for nsclc patients possessing alterations in this gene (veldore et al., 2018; delmonico et al., 2019). cdkn2a alterations in nsclc can be categorized into several discrete classes, including copy number changes, hyperphosphorylation, and deletion (liu et al., 2020). a study by wen et al. suggests that the relative frequency of cdkn2a alterations exhibits signi cant variability among di erent racial groups, with prevalence ranging from 5.1 to 21.5 percent in lung adenocarcinomas and 23.2 to 43.6 percent in squamous cell lung carcinomas. percentages in this study were drawn fromngs assessment of 1200 chinese nsclc patients and the cancer genome atlas (tcga) data for american and european cohorts (wen et al., 2019). liu et al., also assessed data from tcga to pinpoint important genes implicated in lung cancer tumor biology related to cdkn2a. a549 and h322 cell lines were berkeley pharma tech journal of medicine | 122 cultured and evaluated through real-time pcr, western blot analysis, 2,5-diphenyl tetrazolium bromide (mtt) assay, and cell counting, invasion, wound healing, and migration assays. the researchers observed a marked decrease in survival among patients with cdkn2a depletions and found a direct correlation between cdkn2a knockdown and increased cell invasion, migration, and proliferation in experiments in a549 and h322 cell lines. a tcga provisional and pan-cancer atlas survival analysis and further studies on other diseases have suggested decreased survival rates and other adverse e ects in patients with cdkn2a depletion. (reis et al., 2015; zeng et al., 2018; dacic et al., 2008). while cdkn2a loss is suggested to have a negative in uence on prognosis and survival outcomes, more research is necessary to understand cdkn2a’s speci c implications in lung cancer (liu et al., 2020). with cdkn2a’s potential to be a clinically actionable biomarker, testing and genomic panels with this gene have been bene cial and often included in biomarker testing. recent advances in assay techniques have established ctdna as a viable biomarker for detection. one study suggests that cdkn2a ctdna, along with the ctdna of several other cell-cycle related genes, exhibit only an 81.6% concordance rate to their corresponding solid tumor biopsies (mao et al., 2017). studies analyzing the concordance of cdkn2a mutation in both liquid and tissue biopsies from breast cancer patients also revealed di erences in mutations detected through cell-free dna analysis and tissue biopsy, although overall mutational prevalence was quite similar to frequencies observed in tissue samples (delmonico et al., 2020). hence, the impetus for further exploration of ctdna’s clinical value is essential to develop increasingly accurate, noninvasive, and timely methods of detection to ensure that cdkn2a mutations are subject to earlier interventions, which may ultimately lead to a better prognosis for nsclc patients. ret rearranged during transfection (ret) activating fusion mutations are found to occur in approximately 1% to 2% of nsclc patients and have been suggested to show heightened prevalence in patients who never/lightly smoked and who are younger (kohno et al., 2013; kato et al., 2017). ret codes for a receptor tyrosine kinase that recognizes growth factors for the neurotropic factor family, which is derived from glial cell lines. fusion of the ret proto oncogene can activate the receptor tyrosine kinase in the berkeley pharma tech journal of medicine | 123 absence of its ligand. such an activation can have downstream e ects on other pathways such as those including pi3k and mapk (kato et al., 2017). the existence of fda-approved therapies for ret-altered cancers such as vandetanib and cabozantinib underscores the value of early ret detection to improve prognosis for patients with nsclc presenting with ret-altered cancers. clinical trials with vandetanib and everolimus have demonstrated the potential of these drugs to serve viable treatments for ret-altered cancers that have metastasized to the brain (subbiah et al., 2015). in addition, kodama et al. assessed the e cacy of alectinib in ret fusion-positive ccdc6-ret and kif5b-ret genes. the researchers determined that alectinib can preclude phosphorylation of ret, thus providing substantial inhibitory control. furthermore, alectinib demonstrated substantial e cacy in treating v804l and v804m mutated cancers compared to cabozantinib and vandetanib. the suggested basis of this di erence is supported by alectinib’s structure, which is not in uenced by the steric e ects of the v804l and v804m gatekeeper mutations (kodama et al., 2014). mao et al. found that plasma samples of ret ctdna from patients with histologically con rmed lung cancer demonstrated 96.2% concurrence with solid tumor tissue dna samples. concordance rates in this study were determined by an analysis of 40 tissue and plasma samples collected from participants aged 18-80 who did not qualify for rst-line surgical treatment (mao et al., 2017). similar studies have also demonstrated high concurrence rates between tissue and liquid biopsies, although the concurrence percentage was shown to drop signi cantly in one study when analysis was limited to speci c subtypes of genomic alterations, such as copy number variations, which demonstrated concordance rates as low as 3.5%; however, sample size in this cohort was limited to 45 patients (chae et al., 2017). the variability in concordance rates between ctdna and tissue biopsy shows that further analysis is required to determine whether ctdna is a viable standalone method of detection for ret-altered cancers, given that ret mutations are included in the national comprehensive cancer network’s recommendation for genomic targets in nsclc (yang et al., 2018; thompson et al., 2016). erbb2 (her2) erb-b2 receptor tyrosine kinase 2 (erbb2), human epidermal growth factor receptor 2 (her2), has become widely acknowledged as a potential berkeley pharma tech journal of medicine | 124 target for therapy (kirs et al., 2015; chuang et al., 2017). while alterations in erbb2/her2 have primarily been studied in breast cancer, recent research has assessed the potential for targeted therapies in treating erbb2/her2positive lung cancers (de melo gagliato et al., 2016; chuang et al., 2017; veatch et al., 2019). some studies have shown that her2 commonly presents in female patients with adenocarcinoma histology with no history of smoking (garrido-castro and enriqueta felip, 2004). erbb2/her2 receptors can also be phosphorylated to form heterodimers with other epidermal growth factor receptors (egfr), such as her1, and play a critical role in signaling pathways that regulate transcription, cell proliferation, and prevention of apoptosis (de melo gagliato et al., 2016). approximately 1-5% of lung cancers were found to harbor erbb2/ her2 aberrations, but some studies have demonstrated that the percentage for the nsclc subtype likely lies between 1-2% (kris et al., 2015; chuang et al., 2017). regarding detection methods, mao et al. demonstrated that erbb2, along with other driver genes, exhibits a 96.2% concordance between tissue and liquid biopsy methods (2017). chuang et al. performed a retrospective assessment with nine patients, seven of whom presented with erbb2 mutations and advanced nsclc between 2013-2016 and two of whom met similar criteria from a 2014-2015 cohort. all patients were treated with a regimen of paclitaxel, vinorelbine, and trastuzumab. to pinpoint erbb2 mutations, ngs-based methods solid tumor actionable mutation panel, pcr-based erbb2 sizing assay, geneseq assay, or ngs-based foundationone assay were executed on tissue samples, while ngs-based pro ling using deep sequencing was performed on ctdna samples. therapeutic response was assessed according to response evaluation criteria in solid tumors (recist) version 1.1. the results of this study demonstrated that 44% of the patients exhibited response to targeted therapies. although the sample size was small, clinical indications and availability of targeted therapies for erbb2/ her2 may warrant the use of these treatments. the authors caution that genotypic di erences in erbb2 mutations may result in di erential e cacy of targeted therapies. trastuzumab in conjunction with chemotherapy has demonstrated therapeutic promise in clinical settings, although it has not been shown to penetrate the blood-brain barrier, rendering it less useful for metastases to the brain and spinal cord. other potential therapies include dacomitinib and neratinib (nct01953926) (chuang et al., 2017). berkeley pharma tech journal of medicine |125 in conjunction with targeted therapies, detection remains a critical factor in prognosis. early detection is optimal, but tissue sampling can be di cult at certain periods of cancer development. as such, ctdna analysis may provide a reliable baseline for determining tumor mutational landscape. in other cancer types, such as cholangiosarcoma, ctdna analysis resulted in the successful detection of erbb2 (her2) mutations. liquid biopsy was selected in one case study in lieu of tissue biopsy due to challenges in tissue acquisition. as a result of erbb2 (her2) mutation detection via liquid biopsy, the patient was able to begin dual anti-her2 pertuzumab/trastuzumab therapy as an o -label treatment regimen (yarlagadda et al., 2019). another study in 29 colon cancer patients demonstrated that proper clinically veri ed circulating free tumor dna analysis may provide a method of detection for her2 copy number changes (concordance with tissue equaled 96.6%), allowing clinicians to identify patients who may bene t from anti-her2 therapies. in addition to high concurrence rates, ctdna has also been suggested to surmount the issue of tumor heterogeneity. nevertheless, more studies are required to determine whether the observed concordance rates are a ected by di erential release of tumor dna into circulation (siravegna et al., 2019). brca1/2 accurate dna replication is integral to genomic integrity and protection against deleterious mutations. the brca1/2 genes work sequentially to ensure that this integrity is maintained, speci cally by assisting in dna lesion repair. despite deriving its name from its frequent association with breast cancer, brca1/2 genes have also been implicated in nsclc (hu et al., 2019; remon et al., 2020; ji et al., 2020). with regards to targetability, germline brca-mutated (gbrcam) cancers have demonstrated promising responses in poly-(adp ribose) polymerase (parp) clinical trials and other studies (hu et al., 2019; ji et al., 2020). in a study by remon et al., analysis was performed on advanced nsclc patient specimens negative for alk rearrangements and positive for activating egfr mutations. ngs and single nucleotide polymorphism (snp) arrays were performed on the samples in order to assess genomic features in patients new to targeted therapy. using molecular data, the authors determined the prevalence of pathogenic mutations in brca genes, recist-assessed response rate to platinum-based chemotherapy, and characteristic berkeley pharma tech journal of medicine | 126 clinicopathological features of brca mutated patient subgroups. reported brca prevalence without respect to mutation type was 5.3% (20 out of 379 patients), although pathogenic mutations in brca1/2 constituted only 2.1% of advanced nsclc patients. the results of ramon et al.’s study demonstrated that there may not be a signi cant link between brca mutation in nsclc and improved response to platinum based chemotherapy; a similar inconclusiveness on brca’s role in predicting treatment outcomes was noted in another study on tumor samples that exhibited positive ihc staining for brca1 (ramon et al., 2020; watchers et al., 2005). based on study data, it appeared that brca variants of unknown signi cance (vus) exhibit longer os (p=0.07), whereas tumor protein p53 (tp53) mutation status in brca-positive subgroups did not yield a statistically signi cant di erence in prognosis (p=0.3). although the authors found that biallelic mutations in brcamay warrant further study to determine their prognostic signi cance, they noted that data gathered in their patient cohort did not yield su ciently robust evidence to support implementation of speci c targeted therapies in monoallelic brca1/2 patients (remon et al., 2020). in contrast to the data presented by ramon et al., ji et al. also analyzed brca1/2 in determining e cacy of targeted olaparib (parp inhibitor) therapy. assessment of data demonstrated that brca1 and brca2 depleted cells are more susceptible to olaparib. similar to its role in breast cancer cells, olaparib was found to exhibit a similar apoptosis-inducing function in brca-mutated nsclc cells (ji et al., 2020). moreover, other studies have demonstrated that heightened brca1 expression correlates with lower overall survival rates (joerger et al., 2011). while a common concern with the use of ctdna assays is their ability to provide an accurate indication of a patient’s mutational landscape, recent studies have assessed the utility of a brca1 selective polyethylene glycol, gold nanoparticle-infused biosensor. this biosensor showed substantial selectivity and a brca1 lower limit of detection of 1.72 femtomolar (wang et al., 2015). these results suggest that biosensor-based detection of speci c biomarker sequences may serve as a viable method of detection to address the issue of sensitivity in liquid biopsies (wang et al., 2015; yang et al., 2016). berkeley pharma tech journal of medicine | 127 pten phosphatase and tensin homolog (pten) serves as a phosphatase for phosphatidylinositol-(3,4,5)-triphosphate (pip3). pten decreases pip3 concentration, which in turn serves to downregulate phosphorylated akt (p-akt). in healthy cells, pten serves as an indirect downregulator of p-mapk and p-akt, resulting in lower rates of cell division and increases in apoptosis (teresi et al., 2006). patients carrying egfr mutations have reaped signi cant bene t from egfr-tkis; however, resistance to these tkis poses a signi cant obstacle in treating patients. pten loss represents one mechanism of resistance to tkis such as ge tinib and erlotinib, and continuing studies have set out to discover methods to circumvent the e ects of this resistance mechanism (to et al., 2018). peroxisome proliferator-activated receptor agonists (ppars) are implicated in the development and di erentiation of cells, tumorigenesis, and metabolism. studies have demonstrated that pten loss is correlated with resistance to ge tinib (to, wu, and loong, 2018; lee et al., 2006). increases in apoptosis were observed in cell lines treated concurrently with pparγ agonists and ge tinib (p<0.017) (to et al., 2018). hence, pparγ agonists in conjunction with egfr-tkis may serve as a viable treatment strategy to circumvent egfr-tki resistance in patient subsets that exhibit pten loss (to, wu, and loong, 2018; ni et al., 2017; lee et al., 2006). although future studies require further analysis of pten’s ctdna and solid tumor concordance rates in nsclc, comparative analysis of liquid and tissue biopsies in prostate cancer patients revealed an 88.9% agreement between individual copy number calls of pten (wyatt et al., 2017). despite the higher concordance rate observed in some studies, others caution that pten copy number alterations, when present in low circulating concentrations, can go undetected (vandekerkhove et al., 2019). thus, a predominant factor in the standardization of ctdna assessment is the development of su ciently sensitive assays that have lower limits of detection, which may preclude the aforementioned issue. braf (v600e and non-v600e) braf (v-raf murine sarcoma viral oncogene homolog b) is a serine/threonine kinase involved in the ras/raf/mapk pathway. braf mutations can be categorized in three classes. braf v600/e/r/k berkeley pharma tech journal of medicine | 128 mutations are class i mutations that result in strong activation of braf’s kinase activity and mapk pathway. the mechanism of v600e mutations is that it can form salt bridges with k507 and the c-terminal of the alpha-c helix. they signal as monomers but mimic the dimerization process and respond to braf inhibitors (dankner et al., 2018). class ii and class iii mutations are non-v600e mutations. class ii mutations have high or intermediate kinase activity and serve as rasindependent mutations, which signal as dimers and can respond to mek and pan-raf inhibitors. class iii mutations show low kinase activity and are also called ras-dependent mutant braf-wild-type. class ii and class iii non-v600 braf mutants dimerize with braf or craf (dankner et al., 2018). a study by lin et al. was performed to determine the frequency of braf mutations in chinese individuals. the study determined that 1.2% to 4.2% of nsclc patients in the chinese population had the braf mutations. authors also determined that only 30% of patients with braf v600e mutation bene ted from braf inhibitors. furthermore, lin et al. also reported that the class i mutation is predominantly found in females, while class ii and iii mutations co-occur with the kras mutation. this study’s impact is limited because of its analysis of the braf mutation in speci c races and ethnicities, and thus the ndings cannot be readily extrapolated to other groups (lin et al., 2019). in the clinical setting, detection of braf mutation via liquid or tissue biopsy appears equally reliable, as one study demonstrated that assessment of braf alteration yields a 96.2% concordance rate between liquid and tissue biopsies (mao et al., 2017). nf1 nf1 is a tumor suppressor gene that shuts down activity of ras protein via a protein called neuro bromin, which under normal physiological conditions stimulates gtpase activity of ras and converts active ras-gtp complex to its inactive ras-gdp complex. this inactivation can lead to the shutdown of ras associated gtpases (i.e. kras, nras, mras, hras, rras, and rras2). in patients with the nf1 mutation, there is a loss of functional neuro bromin, which can lead to accumulation of active ras gtpases. this results in excessive cellular proliferation and tumor formation which can lead to the development of additional mutations, apart from those a ecting ras activity. beau-champ et al. berkeley pharma tech journal of medicine | 129 found that loss of nf1 has shown response to dasatinib in dasatinib-resistant lung cancer (2014). furthermore, low levels of nf1 with map-erk expression can be sensitive to erlotinib and reverse the erlotinib resistance (tao et al., 2020). tki resistant nf1 mutations develop by the following mechanisms: 1. activation of heat shock factor 1 2. inhibition of tumor cell apoptosis 3. promotion of epithelial-mesenchymal transformation 4. promotion of sustained angiogenesis chian et al. performed a case-control study in the blood samples of 187 patients with nsclc and 310 non-cancer patients. authors used quantitative pcr to identify age and gender-based biomarkers (cpeb4, dusp6, eif2s3, grb2, mcm4, mdm2, nf1, poldip2, rnf4, stat2, and wee1). the majority of the population was comprised of males 66 years and older, as well as smokers. participants who never smoked expressed nf1 at lower levels. nf1 also had protective e ects compared to mdm2 gene. nf1 was shown to be an age-dependent marker (or: 0.16, ci: 0.14 to 0.88, p-value: 0.0255). pik3ca hyperactivation of the pi3k/akt pathway is common in nsclc patients. this pathway is involved in interfering with various cellular mechanisms such as proliferation, migration, invasion, and resistance to therapy. mutations in akt, the catalytic subunit of pik3ca, or pten downregulation can activate the akt pathway. sawa et al. determined a relationship between pik3ca mutation and lung cancer in patients with copd. the researchers extracted dna from the surgical specimens of 197 patients and analyzed using deep sequencing techniques. the frequency of pik3camutation was signi cantly higher in the copd group than in the non-copd group (10.4% vs. 1.7%, p = 0.015). the pik3ca gene mutation can a ect the prognosis of nsclc through several mechanisms. a systematic review and meta-analysis performed by wang et al. showed that pik3ca expression status was not an independent risk factor for os of nsclc patients (hr = 0:80; 95% ci: 0.58-1.12; p = 0:193), which is consistent with the results of 3 included studies. berkeley pharma tech journal of medicine | 130 ros1 ros1 is a receptor tyrosine kinase similar to alk. it is a single-pass transmembrane protein with an intracellular c-terminal tyrosine kinase domain and an extracellular n-terminal domain. ros1’s extracellular domain has similar amino acid sequences to that of cell adhesion molecules and the extracellular matrix. typically, ros1 is expressed more in the kidneys than in the lungs. the mechanism of ros1 mutations is currently unknown. in general, ros1 rearrangement occurs in the absence of other known oncogenic drivers (egfr mutations, kras mutations, alk rearrangements). there are some exceptions as well, which exclude it to be useful in screening. ros1 can be identi ed via the fish and rt-pcr techniques, but rt-pcr sometimes yields false negatives. ihc is another technique used to detect mutations of ros1 (luk et al., 2018). concordance rates for detecting ros1 alteration is 96.2% between liquid and tissue biopsies, according to analysis by mao et al. (2017). a multicenter prospective cohort study by mezquita et al. was conducted on 128 patients to determine liquid biopsy’s clinical usefulness in detection of alk/ros1 fusion and mutation to extrapolate with e cacy of tyrosine kinase inhibitors. patients aged 18 years and older with alk and ros1-fusion-positive were enrolled during october 2015 and august 2018. samples were collected at diagnosis and during radiation therapy. blood samples were collected, and ct-dna analysis as well as fusion load were evaluated. absent mutation showed better os. the median os was 58.5 months in patients with alkmutations (95% ci, 26.9 to not reached [nr]) while 44.1 months in patients with non-alk mutations (95% ci, 21.7 to nr) and it was found to be 105 months in patients who were detected negative for ctdna (p = .001). also, patients who had complex alk mutations showed poor os (median: 26.9 months; 95% ci, 13.9 months to nr) compared to patients with single alkmutations (median: nr; 95% ci, 57.0 months to nr; p = .003). the median pfs was 20.7 months (95% ci, 6.3 to nr) in the negative ctdna group while it was found to be 2.8 months (95% ci, 1.2 to nr) in the patients with one or more alk mutations (p = .03). berkeley pharma tech journal of medicine | 131 alk alk is most common in nsclc patients, presenting itself in about 3-5% of cases per year. alk alteration is not hereditary and is most commonly present in younger patients (55 years old or below) or patients who have never smoked. ihc or fish remain the most common strategies to identify this biomarker. alk normally encodes for a protein that plays a role in neural development. alterations in this gene are caused by chromosomal translocations and are almost always mutually exclusive to egfr and kras. alkmutations are most common among patients with a never/light smoking history, an adenocarcinoma histology, of a younger age, of the female gender, and in tumours with the wild type for egfr and kras. the most common rearrangement of alk in nsclc is eml4-alk (echinoderm microtubule-associated protein-like 4 (eml4)alk). this mutation leads to uncontrolled proliferation, migration, and tumorigenesis. increasing research in fusion genes such as elm4-alk show that alongside coding for proteins, they generate non-coding rnas that contribute to tumor progression (camidge et al., 2010). in 2011, the fda approved the rst tki for alk mutations, crizotinib. since then, researchers began studying alk as a predictive biomarker. for instance, vincent et al. outlined biomarkers involved in clinical practice in 2012, indicating the importance and extensive research that has been done on alk over the past couple years. currently, ihc combined with crizotinib is considered a gold standard for the detection and treatment of alk-positive nsclc (shaw et al., 2011). eml4-alk fusion variant 3 (v3) serves as a new potentially targetable biomarker for higher-risk cases. evidence shows that the presence of the v3 protein in elm4-alk leads to acceleration of disease, failure at early stages of treatment, and a poorer os. elm4-alk v3 is also associated with shorter pfs, even after non-tki treatments and chemotherapy (christopoulos et al., 2018). this suggests the di erences between the v3 variant and other more common variants, such as eml4-alk v2, are clinically distinguishable due to v3’s stark di erences (p<0.001) in pfs regardless of previous treatment to other alk tumor types. v3 variants progress faster in response to tki treatments, which is likely due to the developments of tki resistant mutations. a study conducted by madsen et al., analyzed ctdna of nsclc patients with the alk gene. the results showed the presence of ctdna before treatment initiation is associated with inferior pfs as well as the presence of ctdna shortly after the start of alk tki treatment. this study berkeley pharma tech journal of medicine | 132 emphasizes the value of genomic pro ling through ctdna during all stages of the treatment. although there are multiple targeted therapies for alk, fusion variants of alk that show poorer pfs and os still need to be studied (madsen et al., 2020). kras kirsten rat sarcoma viral oncogene homolog (kras) is the most frequent oncogenic mutation in western countries for nsclc (liu et al., 2020). it is involved in the ras/mapk pathway and codes for the k-ras protein (gtpase), the most commonly mutated gene in its pathway. mutations in the kras gene impair the gtpase activity, resulting in a constantly active gtpase, which leads to cell survival, proliferation, and di erentiation. the most common nucleotide mutation in kras is the g12c mutation, which has been directly linked to smoking. further research is necessary to establish a relationship between variant kras types and prognosis. cox regression and multinomial logistic regression were used to di erentiate the e ect of kras mutation subtype on os. g12c subtype was associated with poor os (p = 0.021), compared to g12d (aredo et al., 2019). data was extracted from patients in the thoracic malignancies cohort (tmc) to compare clinical features and os of the wild type kras to kras g12c, amongst other mutated types. 100% of the patients with the kras g12c subtype were active smokers. results showed that the treatment and survival of kras mutant subtypes were similar (cui et al., 2020). however, kras g12c in particular proved to be an important predictive biomarker compared to the other kras mutant types, such as wildtype kras and mutated kras (p = 0.74) (cui et al., 2020). although there are no current kras inhibitors on the market, it has been marked as a clinically applicable biomarker in a paper by vincent et al. in 2012. subsequently, research conducted since then corroborates the potential for kras to serve as a potential predictive biomarker in nsclc. data collected from plasma based liquid biopsies by ngs showed that 36 (18.6%) out of the 194 liquid biopsies were a kras g12c variant. since tissue biopsies are often rejected (17%) due to insu cient cellularity, kras detection in ctdna may bene t from clinical implementation. tsc1/2 the tsc1-tsc2 complex is a key regulator of the mtorc1 (mammalian target of rapamycin complex 1), which controls cell growth. hamartin (tsc1) and tuberin (tsc2) are the mutations of genes found in the berkeley pharma tech journal of medicine | 133 tuberous sclerosis complex (tsc). inhibition of tsc1/2 results in inactive mtorc1, leading to uncontrolled cell growth. fuchs et al., determined the inverse correlation between hamartin and p-mtor (the catalytic subunit of two distinct protein complexes which controls cell proliferation) expression in nsclc and sclc samples. fish and western blot analysis were performed on 166 nsclc and sclc cell lines. furthermore, cytoplasmic hamartin expression was expressed in greater than 50% of nsclc cell lines (fuchs et al., 2014). fuchs et al., concluded that activation of lung cancer cell lines is caused by the inhibition of the mtorc by the tsc. unlike most other nsclcmutations, tsc1/2 may be caused by egfr expression, but is not dependent on it. wang et al. utilized 144 nsclc patients to study the characteristics of tsc1 and tsc2 in nsclc. the researchers found 27 of the 144 nsclc patients exhibited tsc1 mutation and 40 exhibited tsc2 mutation. it must also be noted that most patients with tsc mutations expressed other oncogenic gene alterations as well. the expression of tsc1/2 was slightly more common in nsclc compared to sclc (53.7% vs. 43.6%) (wang et al., 2020). furthermore, the tsc1 mutation had a median os of 14.1 months, whereas patients with tsc2 mutation had a median os 110.6 months; however, the di erence was not statistically signi cant (p = 0.201). wang et al. concluded that tsc1/2 de nes a unique nsclc population and often coincides with other mutations such as tp53. in an experiment conducted by rulli et al., liquid biopsies were used to measure the number of circulating tumor cells (ctcs), the quantity of cell free tumor dna (cftdna), and the mutational pro le of dna from ctcs (ctcdna) and cftdna in early stage breast cancer patients. ngs of ctcdna and cftdna showed that 52% of the patients expressed mutations in multiple genes including tsc1 (rulli et al., 2020). in addition to the signi cance of tsc in nsclc prognosis and treatment, initial research shows strong promise for it to be implemented via liquid biopsies. currently, there are no targeted therapies on the market for tsc1/2, although pd-1 and/or ctla-4 inhibitors show success in clinical trials for tsc1/2 associated tumors (liu et al., 2018). fgfr1/2 fibroblast growth factor receptor 1 and broblast growth factor receptor 2 are members of the broblast growth factor receptor family. fgfr1 and fgfr2 span the cell membrane and interact with broblast growth factors outside the cell. this results in a cascade of downstream signaling within berkeley pharma tech journal of medicine | 134 the pi3k and mapk pathways, which control embryonic development, cell proliferation, di erentiation, and migration (theelen et al., 2016). in a study conducted by theelen et al., 653 early stage nsclc samples were assessed. the levels of expression in fgfr1 and fgfr2 directly correlated to clinicopathological features. fgfr1 expression was associated with light smoking (p = 0.02), fgfr2 correlated negatively with age for the whole cohort (p = 0.27), and there was a negative correlation between tumour stage and fgfr2 expression (p = 0.002). fgfr1 was expressed in 10.6% and fgfr2 in 12.9% of all nsclc tumor samples (theelen et al., 2016). protein expression of fgfr is related to worse os. moreover, while fgfr1 is associated with light smoking, fgfr2 is more common in females and younger age patients (age 31 to 53 years). the study by theelen et al. demonstrated an abundance of fgfr mutations present in nsclc, suggesting that they play a role in growth and malignant progression of nsclc. a study by santiago-walker et al. demonstrated a 63% concordance rate for detecting fgfr mutations in temporally unmatched blood and tissue, which supports the potential for patient selection with blood-based testing. clinical di erences were observed between ctdna-fgfr positive and negative patients, but they were not statistically signi cant (santiago-walker et al., 2019). furthermore, fgfr1’s indication of a worse os suggests that it may serve as a prognostic biomarker (theelen et al., 2016). egfr epidermal growth factor receptor (egfr) mutations are observed in 10-35% of all patients with adenocarcinomas. egfr-targeting tkis (egfr-tkis) have shown higher objective response rates and longer pfs. egfr-tkis are considered more successful than chemotherapy for egfr mutated patients. unfortunately, about 30% of patients develop resistance to these egfr tkis. a study done with 502 egfr-mutated nsclc patients was conducted between 2003 and 2014 to examine the relationship between egfr mutations and tp53 mutations. tp53 is a tumor suppressor that is commonly mutated in many cancers including nsclc. tp53 mutations are found in 35-55% of nsclc patients. of the 502, 43 had both tp53 and egfr mutations. those with mutant tp53 had relapse-free survival (rfs) for a median of 42.2 months whereas those with wildtype had rfs for a median of 37.7 months (p=0.59). 60 patients received egfr-tkis as treatment. of these 60 patients, 24 had mutant tp53 and 36 had wildtype tp53. the results of the treatment were not berkeley pharma tech journal of medicine | 135 statistically di erent (labbe et al., 2017). egfr can be used to direct the course of treatment. rrm1 ribonucleotide reductase catalytic subunit m1 (rrm1) is a biomarker involved in tumor proliferation, invasiveness, and metastasis. in a study done by mlak et al., 60 nsclc patients’ rrm1 expression was measured through use of liquid biopsy. all of the patients had not been treated for nsclc yet. high rrm1 expression was associated with higher risk of oral mucositis, but it was not signi cantly associated with disease-free survival (dfs) or os shortening (mlak et al., 2018). in another study done by zhang et al., rrm1 and ercc1 levels were measured through liquid and tissue biopsy in nsclc patients using pcr. patients with lower level rrm1 expression, as seen in the liquid biopsy, had longer median os. their os was 18.5 months, as opposed to patients who had higher level expression whose os was 13.0 months (p = 0.043). higher level rrm1 was also associated with prolonged pfs. patients with low expression level had a median pfs of 6.0 months whereas patients who had lower expression level had a median pfs of 4.0 months (p = 0.044) (zhang et al., 2012). rrm1 levels, measured through use of liquid and tissue biopsy, can be used to predict outcomes such as os and pfs. wip1 overexpression of wildtype p53-induced phosphatase 1 (wip1) is commonly found in many types of tumors and is associated with poor prognosis. in a study by zhao et al., 117 nsclc patients were examined and wip1 expression was determined through ihc. the patient cohort had a mean age of 56.9 years, of which 87 were male and 30 were female. in normal lungs, wip1 is not expressed; the results of this study showed that wip1 was expressed in 69.3% of the nsclc patients. wip1 overexpression was observed more in lung adenocarcinomas than other subtypes of nsclc. wip1 negative patients survived for a longer time than wip1 positive patients. after 80 months, the survival of wip1 positive patients was less than 20% but the survival of wip1 negative patients was over 40% (p=0.014) (zhao et al., 2016). in another study, 84 patients, comprising 46 males and 38 females, were examined and treated. all of them had lung adenocarcinomas. wip1 levels were examined through ihc. patients were considered wip1 positive if 10% or more cancer cells within the tumor were strongly stained for wip1 after ihc. wip1 expression was positive in berkeley pharma tech journal of medicine | 136 64.3% of the patients. the survival of wip1 negative patients was signi cantly greater than wip1 positive patients. the os rate was around 50% for wip1 positive patients 100 months after surgery whereas the os rate was around 90% for wip1 negative patients (p = 0.0099). these results demonstrate that wip1 expression correlates with negative prognosis (satoh et al., 2011). wip1 can be used as a predictive outcome biomarker in order to foresee the prognosis of the patients. mirna based biomarkers figure 3. this figure depicts the mirna pathway. 3a: mirna genes are transcribed to primary mirna (pri-mirna). 3b, 3c: pri-mirna is processed to precursor mirna (pre-mirna). 3d: pre-mirna is exported from the nucleus to the cytoplasm, maturing into mirna duplex. 3e: mirna duplex is cleaved to produce mature mirna. 3f: mature mirna participates in the regulation of cellular pathways (kwak et al., 2010). let-7 the mir let-7 family regulates components of cellular development and di erentiation. although typically a tumor suppressor, some cases have been recorded in which let-7 acts as an oncogene (chirshev et al., 2019). xie et al. examined the role of let-7 in detecting time of acceleration re-proliferation, a key contributor to radiotherapy failure. the study enrolled 19 eligible patients and via qrt-pcr and statistical analyses found a high level of serum let-7 was associated with a better os rate (p=0.024), suggesting that serum let-7 could re ect the proliferation of tumor tissue (xie et al., 2016). another study tested let-7 prognostic capability as part of a mir panel consisting of let-7e, 125a-5p, mir-30a, mir-30e, and berkeley pharma tech journal of medicine | 137 mir-30-3p. the study found nsclc dedi erentiation was associated with reduced expression of mir-125a-5p, let-7e and mir-30e (p=0.038); the loss of expression of let-7e and mir-125a-5p was associated with shorter postoperative survival in nsclc patients (p=0.007) (zhu et al., 2014). dedi erentiation is a process required for tumorigenesis, occurring when specialized cells become less specialized and acquire tumor cell plasticity or self-renewal ability (friedmann-morvinski & verma, 2014, p. 245). a more recent study performed qrt-pcr analysis on 120 nsclc patients and 360 healthy controls and found plasma let-7c and mir-152 expressions were lower in nsclc patients. furthermore, receiver operating characteristic curves displayed an association between low let-7c level and poor di erentiation status (p<0.001), cancer metastasis (p=0.021), and advanced stage classi cation (p=0.013). lastly, comparing postoperative and preoperative plasma from 96 nsclc cases showed an increase in let-7c expression level post-operation: suggesting the potential of let-7c as a prognosis biomarker for nsclc (dou et al., 2015). mir-21 mir-21 is an extensively studied oncogenic mirna due to its association with tumor suppressor genes that regulate proliferation, apoptosis, and invasion (feng et al., 2016) more recently, researchers have sought to utilize the presence of mir-21 as a predictive marker of nsclc. wang et al. utilized serum samples of 88 nsclc patients and 17 healthy controls obtained from jiangsu province people hospital. the researchers found that the 3-year os in nsclc patients with high serummir-21 expression was lower (39.8%) compared to 58.2% in patients with low serummir-21 (p<.001) (wang et al., 2011). liu et al. reported consistent results with the prior study regarding os, but noted that the mir-21 levels in serum and tumor mir-21 levels had no signi cant correlation (2012). mir-21 levels have also been used to predict nsclc tumor recurrence in patients post surgical resection. munagala et al. studied cancer recurrence in mouse models and found that mir-21 was upregulated in recurrent tumors compared to primary tumor tissue, and more notably, serum mir-21 also mirrored the tumor pro le tumor mirna results (munagala et al., 2011). dejima et al. con rmed this observation by examining mir-21 levels in 195 nsclc patients and 30 healthy patients; nding that exosomal mir-21 levels showed signi cant increase in nsclc patients with recurrent tumors (p<0.01) (dejima et al., 2017). in the clinical berkeley pharma tech journal of medicine | 138 setting, han-bo le et al. found that mir-21 levels in post-operative patients’ serum samples was lower than pre-operative levels, which further suggests mir-21’s role as predictive biomarker for tumor recurrence. the use of mir-21 to evaluate treatment options has also been investigated. wei j et al. followed up with 53 patients who received platinum-based chemotherapy to treat nsclc. they found that patients with partial response to the therapy had mir-21 plasma levels several fold lower than patients with stable orprogressive disease (wei et al., 2011). gao et al. noted similar results via tumor mirna analysis of 58 patients, with high levels of mir-21 expression correlating with increased platinum resistance (gao et al., 2012). mir-30 the mir-30 family serves in a regulatory capacity during tissue and organ development. the mir-30 family possesses tumor suppressor abilities, which include its roles in the pathogenesis of cancers, including breast cancer, thyroid cancer, colon cancer, and lung cancer (mao et al., 2018). a study investigated the prognostic value of plasma mir-30b and mir-30c from egfr-mutated lung cancer patients undergoing erlotinib treatment. blood qpcr analyses from 29 erlotinib-treated, egfr-mutated lung cancer patients demonstrated associations between low plasma levels of mir-30b and mir-30c and increased e cacy of erlotinib in egfr-mutated nsclc patients as indicated by pfs (p<0.05 for both mirnas). the study concluded that mir-30b and mir-30c may serve as potential biomarkers to predict erlotinib e cacy in egfr-mutated nsclc patients (hojbjerg et al., 2019). another study on 104 lung cancer cases with benign lesions and 20 healthy controls from west china hospital investigated the prognosis value of mir-30a-5p. through qrt-pcr, high plasma mir-30a-5p levels correlated with increased tumor size (p=0.02), advanced tumor di erentiation (p=0.03), and advanced tumor node metastasis (tnm) stage (p=0.0001). the plasma level of mir-30a-5p was also found to signi cantly decrease post-surgery (p<0.0001) (liang et al., 2019). through kaplan–meier survival analysis, low mir-30a-5p expression was associated with longer survival compared to high mir-30a-5p expression (p=0.0001). kaplan– meier survival analysis is an approach to measure the portion of berkeley pharma tech journal of medicine | 139 samples surviving post-treatment after a certain time period (kishore et al., 2010). finally, cox multivariate regression analysis con rmed the correlation between mir-30a-5p, advanced tnm stage, and os, signifying the prognostic value of mir-30a-5p in nsclc (liang et al., 2019). mir-125b the dysregulation of mir-125b is a common feature across many cancers that a ects tumor cell proliferation, di erentiation, invasion, migration, drug resistance, and tumor immunity. due to its tendency to act as both tumor-suppressor and oncogene, depending on the cancer type and molecular contexts, mir-125b has not been utilized for clinical purposes. recently, however, several studies have noted mir-125b’s potential as a prognostic biomarker for advanced nsclc patients. cui et al. compared serum mirna-125b across 260 inoperable advanced nsclc patients and 260 healthy patients. utilizing qrt-pcr to measure circulating mir-125b, and evaluating e cacy of chemotherapy in accordance with the radiologic recist, the study found that 99 patients (38%) responded to chemotherapy with partial or complete response; 161 (62%) patients were not responsive, but instead exhibited stabilization or disease progression. mir-125b was signi cantly associated with chemotherapeutic response, with nonresponsive patients exhibiting signi cantly higher expression levels than responsive patients (p=0.003). the authors postulate that mir-125b may act as a viable biomarker to predict chemotherapy resistance. con rming this observation, shi et al. measured the relative expression of mir-125b in 74 patients with advanced nsclc preand post chemotherapy using rt-qpcr and noted that sensitivity to chemotherapy in nsclc patients with high expression of mir-125b was lower than those with low expression of mir-125b (p<.05) (2020). the utility of mir-125 to predict survival outcomes has also been studied. yuxia et al. compared mir-125b serum levels in 193 patients with varying stages of nsclc, following surgery and therapy. patients with mir-125 expression levels lower than 2.79 were put into the low-expression group, and those above, in the high expression group. kaplan-meier survival curves revealed that high expression signi cantly correlated with poor survival (p<. 00001). multivariate cox hazard analysis also showed mir-125b to be an independent prognostic marker on nsclc (yuxia et al., 2012). berkeley pharma tech journal of medicine | 140 mir-145 mir-145 has been reported to have decreased levels in many di erent cancers, such as pancreatic cancer, prostate cancer, breast cancer, and colorectal cancer (liu et al., 2018). mir-145’s tumor suppressive abilities and its prevalence in many types of cancer makes it a potential biomarker for prognosis. a study on immune responses and cancer progression assessed 345 nsclc patients and focused on a 5 mirna panel, which consisted of mir-191, mir-28-3p, mir-145, mir-328 and mir-18a. through the use of liquid biopsy and pcr, these mirna levels were measured; a high expression level was de ned as greater than 80%. the median survival time for those with high expression of all 5 mirnas was 3.6 to 5.7 months shorter than those with low expression. out of the mirnas investigated in the study, mir-145 demonstrated higher expression in normal patients than in nsclc patients. nevertheless, higher levels of mir-145 in nsclc patients is associated with poorer survival. high mir-145 expression had an 88.4% 3 year death rate as opposed to low mir-145 expression which had an 79.2% 3 year death rate (p = 5.21e−03). mir-145 is associated with ras, mitogen-activated protein kinase, atm and estrogen receptor signaling pathways, which are closely associated with resistance to chemoradiotherapy and/or targeted therapy in advanced nsclc (zhang et al., 2019). mir-155 mir-155 is overexpressed in many diseases and is signi cant in carcinogenesis. it is most commonly overexpressed in solid tumors and hematopoietic malignancies. its overexpression in nsclc has foreshadowed its potential as a biomarker for prognosis and predictive purposes. mir-155 has exhibited high expression in nsclc patients and has been seen to induce proliferation of nsclc cancer cells. in a study with 180 total nsclc patients and 80 control patients, mir-155 and mir-21 expression levels were compared between one group of 68 newly diagnosed patients and 112 patients with recurrent or metastasized nsclc. the expression of mir-155 and mir-21 was higher in those with nsclc when compared to the control group (p < 0.01), while expression of both mir-155 (p < 0.05) and mir-21 (p < 0.01) was higher in those in the recurrence group than those in the newly diagnosed group. higher levels of these mirnas were found to a ect the prognosis negatively, with a mortality rate of 91.96% and a 19 month median survival time in the berkeley pharma tech journal of medicine | 141 recurrence group compared to a 57.35% mortality rate and a 28 month median survival time in the newly diagnosed group (p < 0.05) (xu et al., 2019). in combination of a three-mirna signature panel of high level mir-155-5p, high level mir-223-3p, and low level mir-199a-5p, there was a mean dfs of 46 months in 52 resectable nsclc patients (san orenzo et al., 2013). in three independent cohorts of nsclc patients in maryland, norway, and japan, mir-155 was concluded in addition with mir-17 and mir-21 in association with mortality rates and dfs (saito et al., 2011). the prognosis impact of mir-155 expression is in uenced by other factors as well. in another study, 335 nsclc patients were examined, and it was discovered that squamous cell carcinoma expressed higher levels of mir-155 than adenocarcinomas. higher mir-155 expression has a negative role in disease speci c survival (dss) prognosis in patients with adenocarcinomas, but a positive role in dss in patients with squamous cell carcinomas. the median survival for those with high mir-155 expression was 84 months whereas the median survival rate for those with low mir-155 expression was 190 months (p=0.43). thus the prognostic impact of mir-155 di ers by histological subtype (donnem et al., 2011). hana et al. also found that in 52 patient serum samples high mir-155 expression level correlated with poor prognosis, and is correlated with a median survival (ms) of 69 days (p=0.034) in adenocarcinomas and a ms of 58 days (p=0.023) in patients with positive egfr gene mutations (hana et al., 2020). mir-486 while mir-486 is widely controversial, with many studies reporting con icting results in mir-486’s role as an oncogene or tumor suppressor, it has been proven to be a signi cant, non-invasive biomarker for prognosis. sromek et al. measured mir-486 levels in patients that underwent tumor resection and found increased mir-486 levels expressed in their plasma samples (n=14) one year after surgery (sromek et al., 2017). a meta-analysis conducted by jiang et al. among 7 studies demonstrated that there was no increased risk of poor outcome with lower expression of mir-486 (jiang et al., 2018). poor outcome was de ned using 3 factors: os, pfs, and rfs. conversely, in a study by gao et al. high levels of mir-486 in 140 nsclc patients was directly correlated with shorter os, speci cally in stage i nsclc cancer (gao et al., 2020). when using berkeley pharma tech journal of medicine | 142 mir-486 in combination with mir-30d, mir-1, and mir-499 on a 4 signature mirna signature panel, high serum levels of mir-486 also showed similar results of unfavorable survival. more speci cally, it was shown that within the 60 patient cohort, those that carried two or more of the high-risk mirna would have signi cantly shorter survival (p all < 0.001) compared to those who had none or one high-risk mirna (hu et al., 2010). in patients that showed down-regulated mir-486 levels in plasma after surgical removal of tumors, the recurrence free survival (rfs) was higher than in those that had stagnant levels of mir-486, the un-reduced group, that did not have reduced mir-486 levels or mir-486 levels did not change. the group with down-regulated mir-486 had a median unreached survival, while the un-reduced group had a median survival of 19 months (p=0.056) (li et al., 2015). in studies that focused on clinical phenotype of metastasis, it was shown that mir-486-5p suppressed migration and invasion abilities of nsclc cells, preventing cancer progression and metastasis. in one study, mir-486-5p downregulated nsclc through arhgap5, a protumorigenic gene. mir-486-5p had a statistically signi cant inverse relationship with arhgap5 (p=0.0156), which correlated with further cancer progression (n=76, frozen nsclc samples) (wang et al., 2014). moreover, in correlation with the p13k-akt signaling pathway, mir-486-5p and the p13r1 gene had regulatory e ects. in a549 and h1299 mir-486-5p transfected nsclc cell lines, inhibited a549 and h1299’s abilities of migration and invasion after 48 hours (p < 0.05), showed higher apoptosis rates after 72 hours (p < 0.05), and inhibited cell proliferation through mir-486-5p suppression of p13r1 (p < 0.001) (tian et al., 2019). furthermore, it was found that mir-486-5p was an e ective therapeutic agent for cisplatin resistant nsclc treatment. mir-486-5p improved the susceptibility to cisplatin through mir-486-5p’s suppression of twf1, twin lin actin binding protein 1, a gene that plays a role in tumor invasion and chemotherapy resistance. an in vivo investigation of cisplatin resistance showed that mir-486-5p expression correlated with reduced tumor size in nude mice (p < 0.001) (jin et al., 2019). conclusion in the literature about mir, we found several articles that indicate signi cant promise for their utility as a biomarker for prognosis, outcome prediction, or treatment strategies. first, recent studies regarding mir berkeley pharma tech journal of medicine | 143 biomarkers have begun to diversify from general metrics such as os. more speci c metrics now include predicting tumor recurrence, resistance to treatments, medication performance, and much more. second, the articles we reviewed have indicated mir testing to be feasible in most clinical settings. we found the measurement of mir typically involves some variation of pcr procedure performed on plasma, serum, and saliva samples. lastly, while we failed to observe standardization in the studies we reviewed, each independent study demonstrated signi cant promise for mir utility as a biomarker for prognosis, outcome prediction, or treatment strategies. these studies consisted of large sample sizes with little error indicated in the observed results. if these studies are veri ed, they present great potential for mir biomarkers that would assist clinicians in determining treatment strategies. similar trends were found in the ctdna literature. our review paper noted that many ctdna biomarkers indicate predictive outcomes including pfs and os. many ctdnas are used for directing the course of treatment, including met, brca, pten, and egfr. this is often because abnormal expression of these biomarkers can lead to resistance, which requires the use of alternate therapies. furthermore, while comparing ctdna from liquid biopsies with tissue biopsies, we found high concordance rates between liquid biopsies and tissue biopsies. there is a 96.2% concordance rate between liquid and tissue biopsies for ret and ros1 and an 81.6% concordance rate for cdk2na. high concordance rates between tissue and liquid biopsies furthers the case for clinical implementation of liquid biopsy as a means to detect genetic alterations in ctdna. unlike standard tissue biopsy options, ctdna and mirna are obtained through less invasive methods (usually via a simple blood test). however, the question remains whether ctdna or mirna will serve as viable standalone methods in the detection of actionable or prognostic biomarkers. based on current literature, circulating nucleic acid biomarkers appear to hold promise in indicating targeted treatment plans for nsclc patients presenting with speci c genomic pro les. with the increasing number of emerging biomarkers, it is critical to analyze the clinical utility of these biomarkers in light of their prognostic value and clinical indications. clinical actionability remains a signi cant factor in determining biomarker value. berkeley pharma tech journal of medicine | 144 ease of implementation and invasiveness of testing procedures are important considerations in developing assay methods that ease patient discomfort without diminishing speci city and accuracy of detection. the impetus to ensure reliable and streamlined testing measures is paramount, as the current literature suggests that early detection of speci c biomarkers can provide a remarkable clinical bene t. moreover, in an e ort to ensure equal access to advanced cancer care, the nancial burden of testing should be assessed. ultimately, the prognostic signi cance of ctdna and mirna biomarkers in nsclc care has demonstrated that liquid biopsy and molecular diagnostic testing hold promise for the future of personalized cancer care. further testing must be conducted to analyze the signi cance of ctdna and mirna biomarkers in larger cohorts and to determine the precise clinical bene t of their 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(july 9, 2018). bi-allelic loss of cdkn2a initiates melanoma invasion via brn2 activation. cancer cell, 34(1) : 56-68.e9. https://doi.org/10.1016/j.ccell.2018.05.014. 105. zhang, g. b., chen, j., wang, l. r., li, j., li, m. w., xu, n., wang, y., & shentu, j. z. (2012). rrm1 and ercc1 expression in peripheral blood versus tumor tissue in gemcitabine/carboplatin-treated advanced non-small cell lung cancer. cancer chemotherapy and pharmacology, 69(5), 1277–1287. https://doi.org/10.1007/ s00280-012-1834-x 106. zhang, y., roth, j. a., yu, h., ye, y., xie, k., zhao, h., chang, d. w., huang, m., li, h., qu, j., & wu, x. (2019). a 5-microrna signature identi�ed from serum microrna pro�ling predicts survival in patients with advanced stage non-small cell lung cancer. carcinogenesis, 40(5), 643–650. https://doi.org/10.1093/carcin/bgy132 107. zhao, m., zhang, h., zhu, g., liang, j., chen, n., yang, y., liang, x., cai, h., & liu, w. (2016). association between overexpression of wip1 and prognosis of patients with non small cell lung cancer. oncology letters, 11(4), 2365–2370. https://doi.org/10.3892/ol. 2016.4245. 108. zhao w, zhao jj, zhang l, xu qf, zhao ym, shi xy and xu ag. (2015). serum mir-21 level: a potential diagnostic and prognostic biomarker for non-small cell lung cancer. int j clin exp med., 8:14759–14763. 2015 109. zhu, w., luo, b., an, j., he, j., chen, d., xu, l., . . . zhang, y. (2014). di�erential expression of mir-125a-5p and let-7e predicts the progression and prognosis of non small cell lung cancer. cancer investigation, 32(8), 394-401. doi:10.3109/07357907.2014.922569 berkeley pharma tech journal of medicine | 155 2. front page circulating nucleic acids as promising biomarkersa new frontier of personalized medicine (2).pdf 3. body pages circulating nucleic acids paper (body template).docx (1).pdf circulating nucleic acids paper (reference page template) (1).pdf the role of the gut microbiome in neuromodulation therapies as a potential treatment adjunct for multiple sclerosis berkeley pharma tech journal of medicine correspondence: ftobomanu@uchicago.edu keywords: neuroinvasive infections sars-cov-2 central nervous system (cns) neurological complications viral pathogenesis submitted december 13, 2023 accepted august 19, 2024 published december 27, 2024 full open access creative commons attribution license 4.0 abstract infectious diseases have had significant threats to human health, with neurological complications as a challenging frontier in the realm of medical science. neuroinvasive infections are caused by viruses, bacteria, fungi, or protozoa and can lead to various neurological problems such as meningitis, encephalitis, abscesses, myelitis, and neuromuscular dysfunction. among these pathogens, viruses, including dengue, west nile, and most recently sars-cov-2, have emerged as potent human pathogens capable of inflicting severe damage to the central nervous system (cns), culminating in hemorrhagic diseases. exploring the intricacies of neuroinvasive infections: a comprehensive analysis of sars-cov-2’s impact on the nervous system by: fantasia obomanu, emily woo, sunanya adoni, verdasri surada 1. introduction 1.1 the multifaceted world of neuroinvasive infections the pathogenesis of neuroinvasive infections is characterized by the remarkable ability of these pathogens to infiltrate the nervous system. they exploit a mechanism known as axonal transport, which utilizes the physical structures of axons– the elongated projections of nerve cells—to move within the host (infected person). by doing so, they evade immune responses focused on targeting them in other parts of the body. the inflammatory responses triggered by these infections resemble those observed autoimmune disorders, involving microglial proliferation (responses of immune cells in the brain), blood-brain barrier dysfunction (a protective barrier in the brain), and immune cell infiltration. the intricate interplay between pathogens, the nervous system, and the resulting neurological consequences forms the core of our exploration.the intricacy lies in the multifaceted ways these pathogens can engage with the nervous system, potentially leading to a spectrum of neurological consequences. this dynamic relationship is not a unidirectional process; rather, it involves a reciprocal influence where the pathogen can impact the nervous system, and vice versa. understanding this intricate interplay is crucial for researchers delving into the mechanisms of neuroinvasion and subsequent neurological manifestations. the covid-19 pandemic serves as a great example, illustrating the practical implications of the complex relationship between pathogens and the nervous system. sars-cov-2, the virus responsible for covid-19, has not only demonstrated the ability to infect respiratory cells but has also revealed a neurotropic potential. the virus can enter the central nervous system, leading to a range of neurological complications, from mild symptoms like loss of smell to more severe conditions such as encephalitis. this manifestation underscores the intricate nature of the interplay between pathogens and the nervous system. the unprecedented scale and global impact of the covid-19 pandemic further emphasize the urgency of berkeley pharma tech journal of medicine | 108 comprehending these complex interactions for effective medical research and intervention. in tandem with these neuroinvasive infections, the covid-19 pandemic has presented a global health crisis of unprecedented proportions. the novel coronavirus, sars-cov-2, has unveiled the complex landscape of neurological complications, thrusting the nervous system into the forefront of medical research. figure 1. model of external transport and neuron-neuron or neuron non-neuron cells propagation. (a) murine primary mixed neuron cultures (pmnc) grown in xonachip microfluidic compartmentalized chambers. these devices allow fluidic isolation of axons. (b) electron microscopy images of infected pmnc (c) model of hcov oc-43 propagation 1.2 purpose of the review the purpose of this review is to investigate the potential therapeutic interventions aimed at mitigating the neurological damage inflicted by the sars-cov-2 pathogen, with a particular focus on the prevention of neurological consequences resulting from an infected host. the evolving landscape of neuroinvasive infections and the continuous battle against the repercussions of sars-cov-2 on the nervous system beckon us to chart a course toward effective therapeutic strategies that can offer relief and hope to those suffering from the consequences of covid-19. berkeley pharma tech journal of medicine | 109 2. significance of the global impact & damage of sars-cov-2 the global impact of sars-cov-2 has been staggering, with approximately 600 million people affected by covid-19 as of 2022.1 sars-cov-2, akin to neuroinvasive infections, can cause a wide array of neurological problems. these problems include anosmia, ageusia, headaches, confusion, delirium, strokes, encephalitis, guillain-barre syndrome, and ischemic stroke.2 this disease affects multiple organs and systems in the body, leading to severe and long-term complications.3 the mechanism through which sars-cov-2 gains access to the central nervous system (cns) is a subject of ongoing investigation. it is suggested that the virus may exploit multiple routes, including crossing the protective blood-brain barrier (bbb) or interacting with the ace2 receptor, which is expressed in the brain. the direct assault of sars-cov-2 on the cns, coupled with the abnormal immune response within the cns, contributes to the complex nature of the disease. beyond its respiratory manifestations, covid-19 has revealed its capacity to inflict neurological deficits at different stages of the infection. neurological deficits associated with covid-19 include headache, changes in sense of smell and taste, muscle pain, mood disturbance (depression, anxiety), weakness of the limbs, loss of consciousness, seizure, confusion, vision changes, acute encephalopathy, cerebrovascular events, acute inflammatory syndromes, and seizures. these deficits can occur in both adults and preterm infants with low birth weight born to covid-19 affected mothers. neurological sequelae after critical covid-19 disease can result in substantial deficits in activities of daily living and reduced health-related quality of life. moreover, it can leave a lasting imprint on the nervous system, causing post-infectious complications and long-term effects that are yet to be fully understood. the emergence of sars-cov-2 variants, characterized by their high mutation rates, has posed further challenges to global health. the diversity berkeley pharma tech journal of medicine | 110 in the presentation of covid-19 symptoms, coupled with the complexities of diagnosing the disease, has added layers of difficulty to its management.4 3. neurological damage caused by sars-cov-2 the evidence of neurological damage caused by sars-cov-2 is becoming increasingly clear, as various studies have uncovered potential mechanisms underlying this damage. autopsy analyses of covid-19 patients, observed using electron microscopy and fluorescence microscopy, have shown the presence of sars-cov-2 proteins in vital organs, including the brain, indicating the potential for neuroinvasion.5,6 additionally, the isolation of sars-cov-2 from the cerebrospinal fluid (csf) in some patients further supports the notion of neuroinvasion.7 furthermore, numerous covid-19 patients have reported anosmia (loss of smell) and various other neurological symptoms, highlighting the neurological impact of the virus.8,9 studies using human brain organoids have revealed clear evidence of sars-cov-2 infection in cortical neurons, accompanied by metabolic changes in infected and neighboring neurons.10 these studies have also found the presence of ischemic damage and microinfarcts in post mortem brain samples of covid-19 patients, indicating the potential for severe neurological consequences.11 in in-vivo studies using mice, similar to previous reports of sars-cov, researchers observed increasing viral titers in the brain following intranasal administration of sars-cov-2, demonstrating the virus's neurotropic characteristics.12,13 several mechanisms of neuroinvasion have been proposed. sars-cov-2 may directly infect vascular endothelial cells, potentially allowing the virus to cross the blood-brain barrier (bbb).14 another entry route to the central nervous system (cns) may involve the olfactory nerve, where the virus can travel from the nasal passages to the brain.15 damage to lung blood vessels can lead to viral entry into the bloodstream, facilitating spread to other organs, including the brain.16 additionally, sars-cov-2 can induce systemic inflammatory responses, which have the potential to disrupt the bbb and permit the virus or infected immune cells to reach the brain.17 furthermore, sars-cov-2 may utilize certain entry proteins berkeley pharma tech journal of medicine | 111 expressed in the brain, such as ace2, nrp1, and bsg, to facilitate its invasion.18 to address this growing concern, strategies for early detection and intervention are vital. early detection involves monitoring covid-19 patients for neurological symptoms like anosmia or ageusia.19,20 using brain organoids and in vivo models can help simulate and study the neuroinvasive potential of the virus.21 for early intervention, research efforts should focus on potential treatments that block the pathways sars-cov-2 uses to invade the cns. exploring treatments that reduce inflammation or restore the integrity of the bbb is crucial to prevent sars-cov-2 entry into the brain.22,23 the efficacy of brain-penetrant antiviral drugs like sofosbuvir in treating sars-cov-2 cns infection should be investigated.24 further study of ace2, nrp1, and other receptors in the human brain is essential for developing targeted interventions.25 figure 2. evidence of sars-cov-2 invasion in autopsy samples. autopsy analysis of (a) cov antigen detected by anti-sars-cov protein antibodies (brown), (b) sars-cov-2 rna ish showing intense signals in the mucus layer and cells (arrows) of the epithelium, (c-f), ultrastructural images of cov-particles (c, arrows) attached to kinocilia (c, white asterisks) and intracellular cov particles (d-f), (f) high magnification of cov particles (black arrows). berkeley pharma tech journal of medicine | 112 figure 3. potential pathophysiological mechanisms underlying cerebral vascular involvement in covid-19. a schematic representation of hypothesized mechanisms linking covid-19 to cerebral vascular damage, focusing on endothelial injury, coagulopathy, and inflammation contributing to neurological manifestations. 4. neuroprotective strategies for managing neurological damage neurological damage mitigation is a complex and multifaceted endeavor, necessitating a comprehensive approach that delves into diverse strategies aimed at minimizing harm to the delicate nervous system. among these crucial strategies, three stand out prominently: the utilization of anti-inflammatory drugs, the deployment of antiviral agents, and the preservation of the blood-brain barrier 5. strategies for neurological damage mitigation in the realm of neuroprotection, the role of anti-inflammatory drugs is paramount, functioning as key players in the effort to minimize neuronal damage. these drugs operate with precision by targeting the reduction of inflammation, a critical factor in the pathophysiology of various neurological complications. specifically, their primary objective is to down-regulate proinflammatory cytokines such as interleukin-6 (il-6) and tumor necrosis factor-alpha (tnf-α).26 this targeted approach aims to berkeley pharma tech journal of medicine | 113 mitigate the severity of associated complications, emphasizing the importance of managing inflammatory responses in the intricate landscape of neurological health. another pivotal strategy in the arsenal against neurological challenges involves the use of antiviral agents. these agents act as guardians by inhibiting viral activity, a crucial aspect in the preservation of neurological well-being. their mechanisms extend beyond mere suppression, encompassing the impediment of virus entry into the central nervous system and the downregulation of virus receptors. by curbing viral activity, these agents make significant strides in reducing neurotoxicity, fortifying the defense against potential neurological damage. the preservation of the blood-brain barrier emerges as a linchpin in the safeguarding of neurological integrity. functioning as a protective barricade, this barrier regulates the passage of substances between the bloodstream and the brain. the maintenance of this barrier is not only fundamental but also integral in preventing neurological damage. a breach in this defense could potentially expose the delicate neural environment to harmful agents, emphasizing the critical role of a robust blood-brain barrier in neurological health. within this intricate landscape, the exploration of histone deacetylase inhibitors (hdaci) emerges as a significant avenue.27 hdaci holds promise in the amelioration of neurological damage through a multifaceted approach. these inhibitors demonstrate efficacy by down-regulating proinflammatory cytokines, thereby curbing the inflammatory response. additionally, hdaci plays a pivotal role in impeding virus entry and replication within the central nervous system, adding another layer of defense against potential neurological threats. 6. ongoing research and clinical trials for novel therapeutic solutions the ongoing pursuit of effective interventions for managing and mitigating neurological damage caused by viral infections is a dynamic field that berkeley pharma tech journal of medicine | 114 continually expands through comprehensive clinical trials. these trials encompass a diverse array of therapeutic options, ranging from traditional pharmaceutical interventions to cutting-edge approaches like cell therapy. in-depth exploration into convalescent plasma therapy, monoclonal antibodies, immunoglobulin therapy, and cell therapy provides a nuanced understanding of their potential efficacy in addressing neurological complications arising from viral infections.28 out of the ongoing clinical initiatives, approximately 86% focus on examining the efficacy of small molecules or antibodies, either in isolation or in conjunction with immunomodulators. the remaining approximately 14% of clinical endeavors are directed towards assessing vaccines and therapies based on convalescent plasma to alleviate symptoms associated with the disease.29 vaccination, as a proactive measure against the spread of viruses with neurological implications, remains a crucial focus. emerging technologies, such as nanoparticle vaccines, present a promising avenue. these vaccines, designed to combine spike proteins from multiple coronaviruses, hold the potential to offer broad protection against diverse strains. the exploration of these innovative vaccination strategies adds a layer of anticipation to the ongoing efforts in preventing and minimizing neurological complications associated with viral infections.30 furthermore, investigations into prominent drugs and therapies broaden the spectrum of our understanding. protease inhibitors, rna-dependent rna polymerase inhibitors, immunomodulatory treatments, and gene-editing techniques like crispr represent the frontier of antiviral research.31 by targeting various aspects of viral infections, these interventions aim to disrupt the replication and progression of viruses within the body. a comprehensive grasp of the specifics of these trials, including the drugs investigated and the precise aspects of viral infections they address, enhances our comprehension and sets the stage for potential breakthroughs in neuroprotection. berkeley pharma tech journal of medicine | 115 7. impact on patient care and healthcare systems the exploration and application of prospective therapies hold immense promise in significantly elevating the standard of patient care, presenting a diversified arsenal of treatment options and preventive measures. the direct targeting of neurological impacts stemming from viral infections through these therapies marks a crucial advancement in the medical landscape. however, a comprehensive understanding of the challenges that may arise during the implementation of these interventions is imperative for a nuanced approach to patient care enhancement. effective therapies not only promise to ameliorate the specific neurological complications associated with viral infections but also have the potential to address broader healthcare challenges. by reducing the severity and incidence of these complications, these interventions could alleviate the burden on healthcare systems. this, in turn, paves the way for more efficient and streamlined patient care, fostering an environment where resources are optimized, and healthcare professionals can focus on delivering comprehensive and timely treatments.32 the public health implications of incorporating prospective therapies into the broader healthcare framework are profound. the necessity of widespread vaccination campaigns takes center stage, emphasizing the importance of preventing the spread of viruses, particularly sars-cov-2, which has demonstrated significant impacts on neurological health. concurrently, the establishment of early detection protocols emerges as a critical strategy in mitigating the overall impact on public health.33 addressing the challenges associated with implementing these therapies becomes a focal point for healthcare systems aiming to optimize patient care. by proactively tackling barriers to access, affordability, and dissemination of these prospective therapies, healthcare systems can better navigate the complexities of neurological health management. this holistic approach not only benefits individual patients affected by viral infections but also contributes to the overall resilience and adaptability of healthcare infrastructures.34 berkeley pharma tech journal of medicine | 116 8. conclusion navigating the complex terrain of sars-cov-2 neurological impact in the wake of the covid-19 pandemic, this comprehensive review has ventured into the intricate landscape of neuroinvasive infections, focusing on the unprecedented global impact and neurological consequences of the sars-cov-2 pathogen. from exploring the multifaceted world of neuroinvasion mechanisms to unraveling the evidence of neurological damage, this journey has aimed to shed light on the complexities that define the interplay between pathogens and the nervous system. the significance of sars-cov-2’s global impact becomes increasingly apparent as we navigate the neurological spectrum it presents, from mild symptoms to severe conditions. the virus’s ability to infiltrate the central nervous system adds layers of complexity to the ongoing battle against its repercussions, urging researchers and clinicians to adapt and devise effective therapeutic strategies.35 this review has highlighted the importance of robust therapeutic interventions that address the diverse manifestations of neurological damage caused by sars-cov-2. strategies for early detection and intervention have been highlighted as key components in mitigating the severity of neurological consequences, the evidence of neuroinvasion, gleaned from autopsy analyses, human brain organoids, and in vivo studies, forms a foundation for future research directions and targeted interventions. as we navigate the intricate terrain of sars-cov-2’s impact on the nervous system, the pursuit of effective therapeutic solutions is emphasized. ongoing research and clinical trials offer hope to explore novel approaches ranging from traditional pharmaceutical methods to cutting-edge techniques, like cell therapy. the dynamic field of neuroprotection continues to expand, providing a nuanced understanding of potential breakthroughs in managing and mitigating neurological damage caused by viral infections. berkeley pharma tech journal of medicine | 117 as we stand at the intersection of scientific inquiry and practical application, this review propels us forward into a future where adaptability, resilience, and comprehensive understanding pave the way for advancements in neuroinvasive pathogenic research. the journey to unravel the mysteries of sars-cov-2 continues and will be guided by the collective efforts of the scientific community in the pursuit of relief and hope for those affected by the consequences of covid-19. berkeley pharma tech journal of medicine | 118 references 1. sips gj, wilschut j, smith jm. neuroinvasive flavivirus 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human and mouse brain. j expmed. 2021;218(3):e20202135. doi:10.1084/jem.20202135. berkeley pharma tech journal of medicine | 121 https://www.nih.gov/news-events/nih-research-matters/research-context-progress-toward-universal-vaccines https://www.nih.gov/news-events/nih-research-matters/research-context-progress-toward-universal-vaccines https://www.nih.gov/news-events/nih-research-matters/research-context-progress-toward-universal-vaccines https://www.nih.gov/news-events/nih-research-matters/research-context-progress-toward-universal-vaccines https://www.nih.gov/news-events/nih-research-matters/research-context-progress-toward-universal-vaccines https://www.nih.gov/news-events/nih-research-matters/research-context-progress-toward-universal-vaccines https://www.nih.gov/news-events/nih-research-matters/research-context-progress-toward-universal-vaccines https://www.nih.gov/news-events/nih-research-matters/research-context-progress-toward-universal-vaccines berkeley pharma tech journal of medicine correspondence: hasset1@gmail.com keywords: ptpn digestive cancer phosphatase lung cancer tumor promoter submitted: may 10, 2023 accepted: june 30, 2023 published: december 30, 2023 full open access creative commons attribution license 4.0 abstract protein phosphorylation and dephosphorylation are pivotal in regulating protein activity. two key players, protein tyrosine kinases and protein tyrosine phosphatases (ptpn), especially non-receptor ptpns, exert opposing influences in this process. while all ptpns dephosphorylate substrates, their impact on different cancers varies. some act as tumor suppressors in specific cancers, while in others, they may function as tumor promoters. this review focuses on comprehending the roles of ptpns in lung and digestive cancers. notably, lung cancer ranks as the third most common cancer in the us, with around 200,000 new cases reported annually. despite declining rates in the united states, stomach cancer remains a major cause of cancer-related deaths worldwide. the objective of this review article is to elucidate the functions of ptpn1, ptpn2, ptpn3, ptpn6, ptpn11, ptpn12, and ptpn13 in lung and/or digestive cancers. emphasis is placed on exploring their potential as prognostic markers or therapeutic targets. exploring the therapeutic potential of ptpn families in lung and digestive cancers by: hasset yishak, kareem halwah, and jiyun rhim berkeley pharma tech journal of medicine | 83 1. introduction cancer is a leading cause of death all over the world, and the complications associated with the disease makes it difficult to efficiently treat. various types of cancer exist, such as lung cancer, gastric cancer, breast cancer, lymphoma, prostate cancer, and kidney cancer. lung cancer is the second most common cancer in the united states,1 with approximately 80% of lung cancers being nonsmall lung cancer (nsclc). furthermore, lung cancer accounts for the greatest number of deaths stemming from cancer in men with a 5-year survival rate ranging from 10 % to 20%.2 digestive cancer encompasses different types of cancer, such as gastric and colorectal cancer. gastric cancer, also known as stomach cancer, significantly affects 1 in 96 men and 1 in 152 women.1 known as the fifth most common cancer in the world, gastric cancer’s 5-year survival rate is 32%.3 current cancer treatments include surgery, chemotherapy, hormone therapy, radiation therapy, hyperthermia, immunotherapy, and targeted therapy.4,5 despite the variety of treatments, these treatments are not fully effective as millions of people continuously die from cancer annually. thus, there is a strong drive to develop alternative treatments by exploring the molecular mechanisms of various proteins involved in the progression or inhibition of cancer. 1.1 introduction to ptpn with current experimentation being conducted in regards to novel therapeutics in lung and digestive cancer, this review paper explores, as well as summarizes, the existing body of scientific literature on the family of protein phosphatases known as non-receptor protein tyrosine phosphatases (ptpn). phosphorylation and dephosphorylation are highly used mechanisms by the cell to regulate signaling within and between cells. importantly, ptpns affect tumor progression by dephosphorylating proteins in order to activate or inhibit potentially oncogenic pathways.6 this creates a strong potential for ptpn families to be used as a prognosis marker and/or therapeutic target in treating lung and digestive tract cancers. therefore, this literature review characterizes the importance of several ptpns, ptpn 1, 3, 6, 11, 12, 13, by summarizing their roles as a tumor promoter, tumor suppressor or both as well as exploring their molecular mechanisms in digestive and/or lung cancer [figure 1]. berkeley pharma tech journal of medicine | 84 figure 1: an overview of the role of each ptpn in lung or digestive cancer. ptpns marked in green have tumor suppressing capabilities, red as tumor promoting, and orange as having both tumor suppressing and promoting capabilities. 2. ptpn1 ptp1b, also known as protein tyrosine phosphatase non-receptor 1b, is encoded by the ptpn1 gene. its uncontrolled growth is shown to be correlated with growth of digestive cancer, demonstrating ptpn1b’s potential oncogenic role in digestive cancer.7 for crc (colorectal cancer), ptp1b was associated with crc patients’ low overall survival; additional studies also support the relationship of ptp1b overexpression and late stage tumors.8 therefore, ptp1b expression level can be utilized as a possible biomarker for prognosis of late stage tumors, including crc.9 2.1 gastric cancer likewise, according to the rt-pcr assay, ptp1b was found to be overexpressed in gastric cancer tissues relative to normal gastric cells. amplification of ptp1b was associated with poor survival of gastric cancer patients.9 therefore, the amplification of ptp1b can be both used as a biomarker for gastric cancer and indicative for poor survival rate, revealing ptp1b’s oncogenic role as a tumor promoter in gastric carcinogenesis.10 furthermore, inhibition of ptp1b in gastric cells was associated with hindrance of gastric cancer cell growth in both vitro and vivo. furthermore, its inhibition changed genome-wide expression of genes related to cell growth; thus, ptp1b can be directly used as cancer therapy to slow down digestive cancer growth.11 despite there being evidence of ptp1b functioning as a tumor promoter, contrasting ideas in the literature suggest it can also function as a tumor suppressor depending on the cellular context.12 for example, when egf (epidermal growth factor) binds to its receptor egfr, it activates the shc-grb2-sos interaction. at the end of interaction, ras activates mek/erk pathway’s raf and ends with erk berkeley pharma tech journal of medicine | 85 entering the nucleus to activate fgf2 (fibroblast growth factor 2) which causes uncontrolled growth of the cancer.13 if ptp1b does not interfere in the process, fgf2 promotes cancer growth. however, ptp1b can inhibit the process by interacting at the beginning or end of the shc pathway and/or prohibiting the entry of erk into the nucleus ptp1b’s inhibition of signaling pathways leads to decrease in cancer growth13 [figure 2]. figure 2: effect of ptp1b on the pathway of cancer growth. when ptp1b prohibits shc-grb2-sos interaction, the growth of cancer is prohibited due to a decreased expression of fgf2. oppositely, when ptp1b does not stop the pathway, the cancer cells will be able to proliferate and duplicate itself. 2.2 non-small cell lung cancer (nsclc) in regards to nsclc, ptp1b levels increase with the progression of the cancer, highlighting how increased ptp1b levels are associated with poor survival of patients with nsclc.14 additionally, when ptp1b expression was downregulated, berkeley pharma tech journal of medicine | 86 there was a decrease in cell proliferation and metastasis in vitro. likewise, downregulation of ptp1b in mice transfected with nsclc cells showed a significant decrease in tumor size compared to control mice that had unaltered ptp1b levels. these results provide evidence for ptp1b’s role as a tumor promoter in lung cancer. mechanistically, ptp1b activates the oncogene, src (protooncogene c-src), which promotes nsclc proliferation and metastasis. therefore, designing an inhibitor of ptp1b could be promising in treating nsclc due to its suggested tumor suppressing capabilities.14 in lung adenocarcinoma (luad), ptpn1 is downregulated according to comprehensive bioinformatics analysis. its upregulation has been conferred with overall higher survival rates in patients, demonstrating the overexpression of ptpn1 as a potential therapeutic target for treating luad patients. as well, decreased expression can be a prognostic biomarker for lung cancer progression.15 3. ptpn2 in crc tumor cells, ptpn2 levels are enhanced, with greater ptpn2 gene expression correlated with reduced t cell activity, recruitment, and cytotoxicity.16 it is also inversely correlated with low immune checkpoint molecule expression.16 ptpn2 negatively regulates ifn-γ signaling, a pathway involved in the upregulation of immunity related genes, by dephosphorylating proteins involved in the signaling pathway. mouse models with tumors deficient in ptpn2 had increased activation of the ifn-γ receptors by the cytokine, ifnγ, which resulted in the phosphorylation of the signal transducer and transcription protein, stat1 [figure 3]. stat1 is able to homodimerize and enter the nucleus to drive transcription of genes involved in immunity related processes such as mhc-1, possibly pd-1, and others [16-8]. this results in the activation of cd4+ th1 cells and increases the cytotoxicity of cd8+ t cells, which inhibits the growth of the tumor.16 cd4+ th1 cells is a type of t helper cell that releases cytokines in response to inflammation and is also involved in the activation and growth of cd8+ t cells, otherwise known as killer t-cells. therefore, tumors deficient in ptpn2 have increased ifn-γ signaling, which results in a significant reduction of tumor size as well as greater mrna expression of chemokines, like cxcl9/10/11 and ccl5.16 this suggests that the inhibition of ptpn2 through a small molecule might prove to be an effective potential therapeutic drug. while additional experimentation would need to be conducted to better understand the kinetics involved with the inhibitors, a recent paper by zhu et al.19 has identified various small noncytotoxic molecule inhibitors that were able to inhibit ptpn2. this resulted in successful upregulation berkeley pharma tech journal of medicine | 87 of genes involved in ifn-γ signaling, and the sensitization of the crc tumor to treatment. 3.1 kras gene approximately 40% of patients with crc have a missense mutation in the kras (kirsten rat sarcoma viral oncogene homologue) gene.20 those with the mutation in the kras gene tend to have a relatively poorer prognosis compared to crc patients with wild type kras.20 kras encodes for the protein k-ras, which is part of the signaling pathway of ras/mapk, a pathway that induces cellular proliferation, migration, and cell growth. mutations in kras are thought to be the most common oncogenic gene driver in human cancer, especially in pancreatic cancer, crc, and nsclc.21 these mutations lead to a continued active state of kras that result in continuous proliferation of tumor cells by upregulating the ras/mapk pathway. ptpn2 has been identified as a key regulator of kras due to its ability to dephosphorylate kras, activating the kras-mediated mapk pathway.22 therefore, it is hypothesized that inhibition of ptpn2 could suppress cancer by no longer activating kras, presenting itself to be a novel therapeutic target.22 figure 3: ifn-γ signaling pathway in ptpn2 deficient tumor cell. stat1 remains phosphorylated and homodimerizes in order to enter the nucleus and induce the transcription of immune related genes. this ultimately leads to an increase in chemokines as well as cd8+ cells that reduce the size of the tumor. berkeley pharma tech journal of medicine | 88 4. ptpn3 nonsense and frameshift mutations in ptpn3 that hinder its phosphatase activity have been found in lung cancer tissue.23 consequently, overexpression of ptpn3 results in reduced lung cancer cell growth and migration, indicating that it might have tumor suppressor capabilities.24,25 ptpn3 is capable of suppressing lung cancer cell invasion by dephosphorylating the protein, src, at tyr416, which inhibits srcmediated phosphorylation of tyr652 on another protein known as dishevelled associated activator of morphogenesis 1 (daam1).24 tyrosine phosphorylation of daam 1 at tyr652 by src is needed for daam1 dimerization.24 in the absence of ptpn3, daam1 is able to dimerize, leading to long and thick actin, which improves cancer cell migration. ptpn3 knockdown cells moved 30% faster than the control which had normal expression of ptpn3.24 4.1 egfr additionally, ptpn3 is able to target egfr for lysosomal degradation inhibiting proliferation of cancer cells. ptpn3 is capable of dephosphorylating eps 15, which promotes the endocytosis of egfr, given that egfr is bound to its ligand, egf.25 when egfr is internalized via lipid raft-mediated endocytosis, it is either recycled back to the cell surface, targeted to the lysosome for degradation, or internalized to subcellular compartments.25 thus, since ptpn3 is capable of causing egfr to be degraded via its effects on eps 15, ptpn3 functions as a tumor suppressor. overexpression of ptpn3 resulted in a decrease of egfr levels when stimulated by egf.25 this confirmed dephosphorylation of eps15 by ptpn3 is capable of suppressing tumor growth. 5. ptpn6 5.1 crc tissue ptpn6 was highly expressed in crc tissue as demonstrated by qpcr, cck-8, clone formation assay, and other assessments.26 the overexpression of ptpn6 in malignant colon cancer cells was associated with poor prognosis in colon cancer patients. conversely, the inhibition of ptpn6 restrained migration, invasion, and clonogenics of crc tissues.26 therefore, ptpn6 amplification can potentially be used as a biomarker for crc and its progression. ptpn 6 is a possible tumor promoter due to its ability to promote proliferation and migration of tumor cells. additionally, ptpn6 interacts with egfr, a receptor known to induce pathways berkeley pharma tech journal of medicine | 89 involved in proliferation, migration, and adhesion. increased expression of both ptpn6 and egfr resulted in the greatest cancerous proliferation as compared to cells with only ptpn6 or egfr overexpressed. thus, either targeting ptpn6 or the ptpn6-egfr complex with an inhibitor could be a potential therapeutic. however, other research suggests ptpn6 might have tumor suppressing capabilities by decreasing the levels of the protein sp1 (specificity protein 1). mapk pathway is typically activated by sp1; however, due to ptpn6’s inhibition of sp1, mapk pathway is consequently suppressed. ptpn6 inhibition promotes enhanced chemosensitivity within crc cells. yet, more research needs to be conducted in order to further understand the various effects of ptpn6 within crc.27 6. ptpn11 6.1 shp2 ptpn11 is a gene that encodes for shp2, a protein consisting of two n-terminal src homology (sh2) domains, a catalytic ptp domain, and a cterminal tail with tyrosyl phosphorylation sites.28 shp2/ptpn11 is involved in promoting signaling pathways such as ras/erk, ras/mapk, jak/stat, as well as kras signaling within the tumor microenvironment.28 increased expression of shp2/ptpn11 is associated with a 5.34 fold increase in risk for gastric cancer and a 2.95 fold increase in risk for lung cancer.29 furthermore, other studies have found shp2 to be highly expressed in 60.78% of gastric cancer and 70% of nsclc tissue samples.29 patients infected with helicobacter pylori are at a greater risk for gastric cancer.30 a virulence factor of h. pylori known as cytotoxin associated antigen (caga) is able to interact with the epithelial gastric cells, leading to the src-dependent tyrosine phosphorylation of caga.30 the phosphorylated caga binds with shp2, forming a complex that allows for the transition of shp2 into its active form. in its active form, shp2 is capable of inducing oncogenic properties, such as neoplasia, gastric atrophy, and increased migration of gastric epithelial cells.30 in contrast, in many cases of lung cancer, a missense mutation in the ptpn11 gene leads to a dysfunctional shp2 protein that results in inappropriate activation of various signal transduction pathways.31 due to the proto-oncogenic nature of ptpn11, various strides have been made in developing small molecule inhibitors which are capable of binding to the catalytic site of shp2. one potential inhibitor, shp099, is able to bind to the n-terminal, cberkeley pharma tech journal of medicine | 90 terminal and ptp domain on shp2, resulting in increased immune system activity such as greater ifn-γ signaling. this induced greater transcription of cytotoxic tcell related genes within a mice model for lung cancer treatment.31 while most of shp2 inhibitors are in the preclinical study stages, the potential of ptpn11 as a therapeutic drug target is a promising prospect in treating many types of cancers, including gastric and lung cancer. 7. ptpn12 ptpn12 is a tumor suppressor that normally has an inhibitory effect on the ras/mek/erk signaling by dephosphorylating the protein shc.32 however, it was hypothesized that a missense mutation in ptpn12 would leave shc phosphorylated, resulting in hallmarks of cancer, such as cellular proliferation and increased migration.33 for example, researchers identified that a variant of ptpn12 (rs3750050 g allele) increased the risk of crc by 19%.33 conversely, upregulation of ptpn12 has been correlated with incidences of esophageal carcinoma, stomach adenocarcinoma and colorectal cancer, making it a highly favorable candidate for biomarker.34 the researchers acknowledge that these findings are seemingly in contradiction with previous literature that suggests ptpn12 to have tumor suppressing capabilities.34 another study showed that ptpn12 might be a favorable prognosis marker for nsclc in patients due to higher expression levels associated with higher 5-year survival rates, especially within the subgroup with non-squamous cell carcinoma.35 due to the non-definitive role of ptpn12, further research still needs to be conducted.33-35 8. ptpn13 like many of the other ptpns previously discussed, ptpn13 has been shown to have both tumor suppressive and tumor promoting roles depending on the cancer being examined.36 ptpn13 was shown to act as a tumor suppressor in breast cancer37 and high grade serous ovarian carcinoma.38 likewise, in lung cancer, ptpn13 has been shown to have tumor suppressor capabilities by acting on various pathways.38-42 ptpn13 is downregulated in lung cancer mainly due to a loss of at least one copy of the ptpn13 locus at chromosome 4q.40 40% of cases are not accounted for by this mechanism, however, and further research is needed to understand how it is downregulated in those cases.40 various studies have shown that ptpn13 berkeley pharma tech journal of medicine | 91 downregulation results in increased proliferation of nsclc [39-41] in addition to greater tumor cell size.40 to further confirm the role of ptpn13, ptpn13 expression was restored to ptpn13 knockdown cells and this resulted in slower proliferation of nsclc.40 ptpn13’s tumor suppressing capabilities stem from it inhibiting various oncogenic pathways. this is explored through the use of micrornas which are known to have different effects on luad proliferation. for example, mir-36143 and mir-34044 inhibit luad cell growth while mir-48345 and mir-22446 promote luad cell growth. microrna-30e-5p (mir-30e) is of interest to this review paper due to its ability to downregulate ptpn13 [figure 4b]. knockdown of mir-30e suppresses luad growth, suggesting that the presence of mir-30e is indicative of poor prognosis.39 upregulation of ptpn13 counteracts the tumor promoting effects of mir-30e by inhibiting egfr/akt signaling.39 ptpn13 was also shown to inhibit the src/erk/yap1 signaling pathway, further contributing to its tumor suppressive properties in lung cancer. yap1 promotes the proliferation of nsclc cells, classifying it as an oncoprotein.42,47 yap1 has also been shown to activate the mek/erk pathway by promoting the expression of fgf239 [figure 4a]. nuclear yap1 levels were increased in ptpn13 knockdown cells, indicating that ptpn13 might act to inhibit yap1. it is also thought that yap1 could be upregulated via the mek/erk pathway when ptpn13 is suppressed.42 thus, another pathway by which ptpn13 acts as a tumor suppressor is by inhibiting the mek/erk pathway, which suppresses the upregulation of yap1. various cancers, including lung cancer, have been shown to have higher than normal levels of her2, indicating that upregulation of her2 leads to tumor growth and poor prognosis. ptpn13 downregulates her2 activity by dephosphorylating the cytoplasmic domain of her2, possibly decreasing the metastasis associated with her2-overactive nsclc tumor cells.41 ptpn13 was also found to dephosphorylate egfr.40 downregulation of ptpn13 increased egf-stimulated egfr and her2 phosphorylation, leading to increased activation of mapk and akt dependent pathways.40 berkeley pharma tech journal of medicine | 92 figure 4: overview of ptpn13’s effect on various pathways. ptpn13 has been shown to work by affecting the mek/erk pathway in different ways. a) ptpn13 can downregulate egfr and her2 receptors by dephosphorylating them [40]. yap1 levels decrease in the presence of ptpn13, which could be due to ptpn13 inhibiting the mek/ erk pathway. b) mir-30e is capable of downregulating ptpn13, resulting in increased cell growth in luad by promoting egfr/akt signaling. 9. current clinical trials there are ongoing clinical trials exploring the efficacy of small molecule inhibitors on ptpn11, like jab-3068 and jab-3312, in patients with advanced solid tumors. it is hypothesized that these small molecules will prevent various oncogenic phenotypes associated with hyperactivation of shp2 mediated signal transduction pathways. jab-3068 is currently in phase 1/2a and is recruiting patients with advanced solid tumors of nsclc, head and neck cancer, esophageal cancer, and other metastatic solid tumors.49 the experiment will consist of oral administration of jab-3068 every morning after a six hour fast.49 a pharmacokinetics (pk) analysis will be taken which will be used to monitor the drug as it reacts with the body. similarly, the small molecule inhibitor jab-3312 is in phase 1 study and is also recruiting patients with advanced solid tumors of nsclc, crc, pancreatic ductal carcinoma, esophageal squamous cell carcinoma, head and neck squamous cell carcinoma, breast cancer, and other solid tumors. the experiment will consist of daily oral administration of the drug in treatment cycles of 21 days.50 9.1 matched targeted therapy another ongoing clinical trial is utilizing matched targeted therapy (mtt) in order to test the efficacy of trametinib on ptpn11 and other proteins involved in berkeley pharma tech journal of medicine | 93 cancer progression. specifically, trametinib is a kinase inhibitor that blocks the abnormal protein signals that cause cancer cell multiplication. the drug will be consumed orally with two mg per day. the trials are expected to be completed by 2026. some measures that researchers will be noting are short-term and long-term progression, duration of response, survival, adverse effects, and more.51 10. future directions & conclusion this article provided a review of various ptpn protein families in regards to lung and digestive cancer with the goal of providing researchers foundational information in their efforts to develop therapeutics that target ptpns. many studies have investigated the roles of ptpns in tumor progression, outlining how ptpns act on different pathways and thus affect tumor progression differently. this review paper focuses on the molecular mechanisms that underlie the effects of ptpns, as it could aid in determining if ptpn therapeutics are worth exploring. many of these ptpns can be used as biomarkers and/or prognosis markers as overexpression or underexpression of certain ptpns are associated with each cancer [figure 5]. in regards to digestive cancer, ptpn2 and ptpn11 were shown to have tumor promoting capabilities while ptp1b, ptpn6, and ptpn12 were shown to have both tumor promoting and tumor suppressing capabilities. in lung cancer, ptpn3, ptpn12, and ptpn13 were shown to have tumor suppressing capabilities while ptpn11 had tumor promoting capabilities. ptp1b was seen to have both tumor suppressing and tumor promoting capabilities depending on the cellular substrate. given these ptpns and their varying effects on each cancer, therapeutic targets that act to either promote or suppress various ptpn have great potential for cancer treatment. to suppress ptpns with tumor promoting capabilities, researchers could look into developing inhibitors that bind to the ptpn proteins. in contrast, in order to promote ptpns with tumor suppressing capabilities, researchers could look at developing transcription factors that enhance ptpn gene expression. therefore, additional clinical trials and research need to be conducted in order to better understand how to mechanistically target ptpns to develop effective treatments for lung and digestive cancer. berkeley pharma tech journal of medicine | 94 ptpn member lung cancer digestive cancer ptpn1/ptp1b biomarker: increased levels of ptp1b in nsclc; decreased levels of ptpn1 in luad therapeutic: inhibition of ptp1b in nsclc biomarker: increased levels of ptp1b in crc, gastric cancer therapeutic: inhibition of ptp1b blocks shc-grb2-sos pathway and mek/erk pathway contrasting: tumor suppressor qualities based on cellular context ptpn2 insufficient research biomarker: increased levels of ptpn2 across all stages of crc therapeutic: inhibition of ptpn2 which could increase ifn-γ signaling pathway and suppress krasmediated mapk pathway ptpn3 biomarker: decreased levels of ptpn3 in lung cancer tissue therapeutic: upregulation of ptpn3 to allow for the dephosphorylation of src and targeting of egfr for lysosomal degradation insufficient research ptpn6 insufficient research biomarker: increased levels of ptpn6 in crc therapeutic: inhibition of ptpn6sp1-mapk pathway to increase chemosensitivity of tumor cells; inhibition of ptpn6-egfr complex berkeley pharma tech journal of medicine | 95 ptpn11 biomarker: increased levels of ptpn11/shp2 in 70% of nsclc tissue samples therapeutic: small molecule inhibition of ptpn11/shp2 which prevents shp2 mediated signal transduction pathways that are hyperactivated biomarker: increased levels of ptpn11/shp2 in 60.78% of gastric cancers therapeutic: small molecule inhibition of ptpn11/shp2 which prevents shp2 mediated signal transduction pathways that are hyperactivated ptpn12 prognosis marker: increased levels of ptpn12 are associated with higher 5year survival rates in nsclc biomarker: increased levels of ptpn12 are correlated with incidences of digestive cancers contrasting: other research has shown ptpn12 as a tumor suppressor that normally has an inhibitory effect on the ras/mek/erk signaling by dephosphorylating the protein shc ptpn13 biomarker: decreased levels in nsclc therapeutic: upregulation of ptpn13 to inhibit the src/erk/yap1 signaling pathway, counteract the effects of mir-30e, and to downregulate her2 activity insufficient research figure 5: summary of ptpn families and their potential as a biomarker, prognosis marker, and or/ therapeutic target in lung and digestive 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(2020). ptpn2 regulates the activation of kras and plays a critical role in proliferation and survival of kras-driven cancer cells. journal of biological chemistry, 295(52), 18343–18354. https://doi.org/10.1074/jbc.ra119.011060 23. jung, y., kim, p., jung, y., keum, j., kim, s.-n., choi, y. s., do, i.-g., lee, j., choi, s.-j., kim, s., lee, j.-e., kim, j., lee, s., & kim, j. (2012). discovery of alk-ptpn3 gene fusion from human non-small cell lung carcinoma cell line using next generation rna sequencing. genes, chromosomes and cancer, 51(6), 590–597. https://doi.org/10.1002/gcc.21945 berkeley pharma tech journal of medicine | 97 24. li, m.-y., peng, w.-h., wu, c.-h., chang, y.-m., lin, y.-l., chang, g.-d., wu, h.-c., & chen, g.-c. 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(2020). progress in the correlation between ptpn12 gene expression and human tumors.medicine, 99(24). https://doi.org/10.1097/md.00000000000204 45 33. shen, n., li, l., xu, w., tian, j., yang, y., zhu, y., gong, y., ke, j., gong, j., chang, j., zhong, r., &miao, x. (2019). a missense variant in ptpn12 associated with the risk of colorectal cancer by modifying ras/mek/erk signaling. cancer epidemiology, 59, 109–114. https://doi.org/10.1016/j.canep.2019.01.013 34. chen, j., zhao, x., yuan, y., & jing, j.-jing. (2020). the expression patterns and the prognostic roles of ptpn family members in digestive tract cancers. https://doi.org/10.21203/rs.3.rs-19689/v1 35. cao, x., chen, y.-z., luo, r.-z., zhang, l., zhang, s.-l., zeng, j., jiang, y.-c., han, y.-j., &wen, z.-s. (2015). tyrosine-protein phosphatase non-receptor type 12 expression is a good prognostic factor in resectable non-small cell lung cancer.oncotarget, 6(13), berkeley pharma tech journal of medicine | 98 11704–11713. https://doi.org/10.18632/oncotarget.3588 36. mcheik, s., aptecar, l., coopman, p., d’hondt, v., & freiss, g. (2020). dual role of the ptpn13 tyrosine phosphatase in cancer. biomolecules, 10(12), 1659. https://doi.org/10.3390/biom10121659 37. hamyeh, m., bernex, f., larive, r. m., naldi, a., urbach, s., simony-lafontaine, j., puech, c., bakhache, w., solassol, j., coopman, p. j., hendriks, w. j. a. j., & freiss, g. (2020). ptpn13 induces cell junction stabilization and inhibits mammary tumor invasiveness. theranostics, 10(3), 1016–1032. https://doi.org/10.7150/thno.38537 38. d’hondt, v., lacroix-triki, m., jarlier, m., boissiere-michot, f., puech, c., coopman, p., katsaros, d., & freiss, g. (2017). high ptpn13 expression in high grade serous ovarian carcinoma is associated with a better patient outcome.oncotarget, 8(56), 95662–95673. https://doi.org/10.18632/oncotarget.21175 39. zhuang, l., shou, t., li, k., gao, c.-l., duan, l.-c., fang, l.-z., zhang, q.-y., chen, z.-n., zhang, c., yang, s.-t., & li, g.-f. (2017). microrna-30e-5p promotes cell growth by targeting ptpn 13 and indicates poor survival and recurrence in lung adenocarcinoma. journal of cellular and molecularmedicine, 21(11), 2852–2862. https://doi.org/10.1111/jcmm.13198 40. scrima, m., demarco, c., de vita, f., fabiani, f., franco, r., pirozzi, g., rocco, g., malanga, d., & viglietto, g. (2012). the nonreceptor-type tyrosine phosphatase ptpn13 is a tumor suppressor gene in non–small cell lung cancer. the american journal of pathology, 180(3), 1202–1214. https://doi.org/10.1016/j.ajpath.2011.11.03 8 41. zhu, j.-h., chen, r., yi, w., cantin, g. t., fearns, c., yang, y., yates, j. r., & lee, j.-d. (2007). protein tyrosine phosphatase ptpn13 negatively regulates her2/erbb2 malignant signaling.oncogene, 27(18), 2525–2531. https://doi.org/10.1038/sj.onc.1210922 42. wang, j., li, s., zhang, x., zhu, n., yiminniyaze, r., dong, l., li, c., gulinuer, w., xia, j., li, j., zhou, d., liu, x., zhang, y., zhang, y., & li, s. (2022). protein tyrosine phosphatase ptpl1 suppresses lung cancer through src/erk/yap1 signaling. thoracic cancer, 13(21), 3042–3051. https://doi.org/10.1111/1759-7714.14657 43. chen, w., wang, j., liu, s., wang, s., cheng, y., zhou, w., duan, c., & zhang, c. (2016). microrna-361-3p suppresses tumor cell proliferation and metastasis by directly targeting sh2b1 in nsclc. journal of experimental & clinical cancer research, 35(1). https://doi.org/10.1186/s13046-016-0357-4 44. fernandez, s., risolino, m., mandia, n., talotta, f., soini, y., incoronato, m., condorelli, g., ban�, s., & verde, p. (2014). mir-340 inhibits tumor cell proliferation and induces apoptosis by targeting multiple negative regulators of p27 in non-small cell lung cancer.oncogene, 34(25), 3240–3250. https://doi.org/10.1038/onc.2014.267 45. song, q., xu, y., yang, c., chen, z., jia, c., chen, j., zhang, y., lai, p., fan, x., zhou, x., lin, j., li, m., ma, w., luo, s., & bai, x. (2014). mir-483-5p promotes invasion and metastasis of lung adenocarcinoma by targeting rhogdi1 and berkeley pharma tech journal of medicine | 99 alcam. cancer research, 74(11), 3031–3042. https://doi.org/10.1158/0008-5472.can-13-2193 46. cui, r., meng, w., sun, h.-l., kim, t., ye, z., fassan, m., jeon, y.-j., li, b., vicentini, c., peng, y., lee, t. j., luo, z., liu, l., xu, d., tili, e., jin, v., middleton, j., chakravarti, a., lautenschlaeger, t., & croce, c. m. (2015). microrna-224 promotes tumor progression in nonsmall cell lung cancer. proceedings of the national academy of sciences, 112(31). https://doi.org/10.1073/pnas.1502068112 47. vichas, a., riley, a. k., nkinsi, n. t., kamlapurkar, s., parrish, p. c., lo, a., duke, f., chen, j., fung, i., watson, j., rees, m., gabel, a. m., thomas, j. d., bradley, r. k., lee, j. k., hatch, e. m., baine, m. k., rekhtman, n., ladanyi, m., … berger, a. h. (2021). integrative oncogene-dependency mapping identi�es rit1 vulnerabilities and synergies in lung cancer.nature communications, 12(1). https://doi.org/10.1038/s41467-021-24841-y 48. zhang, y., wang, y., zhou, d., wang, k., wang, x., wang, x., jiang, y., zhao, m., yu, r., & zhou, x. (2021). radiation-induced yap activation confers glioma radioresistance via promoting fgf2 transcription and dna damage repair.oncogene, 40(27), 4580–4591. https://doi.org/10.1038/s41388-021-01878-3 49. a �rst-in-human study of jab-3068 (shp2 inhibitor) in adult patients with advanced solid tumors in china full text view. full text view clinicaltrials.gov. (n.d.). retrievedmarch 24, 2023, from https://clinicaltrials.gov/ct2/show/nct03565003 ?term=ptpn11&cond=non-small%2bcell%2bl ung%2bcancer&draw=2&rank=3 50. a �rst-in-human, phase 1 study of jab-3312 in adult patients with advanced solid tumors full text view. full text view clinicaltrials.gov. (n.d.). retrievedmarch 24, 2023, from https://clinicaltrials.gov/ct2/show/nct0404 5496?term=ptpn11&cond=non-small%2b cell%2blung%2bcancer&draw=2 51. a study evaluating the activity of anti-cancer treatments targeting tumormolecular alterations/characteristics in advanced / metastatic tumors. (megamost) full text view. full text view clinicaltrials.gov. (n.d.). retrievedmarch 24, 2023, from https://clinicaltrials.gov/ct2/show/study/nct041 16541?term=protein+tyrosine+phosphatases+non -receptor+type&draw=2&rank=2 berkeley pharma tech journal of medicine | 100 berkeley pharma tech journal of medicine correspondence: holstoncalifornia@gmail.com keywords: autism spectrum disorder, neuroinflammation, mesenchymal stem cell therapy, oxytocin, resveratrol, sulforaphane, combination therapy submitted august 11, 2023 accepted april 5, 2024 published june 28, 2024 full open access creative commons attribution license 4.0 abstract one of the defining factors of autism spectrum disorder (asd) is neuroinflammation, which may be targeted to find effective therapies in managing asd symptoms. some promising treatments include mesenchymal stem cell (mscs) therapy, cxytocin (ot), sulforaphane (sfn), and resveratrol (rsv). mscs are located in many parts of the body that can reduce secondary neurodegeneration and neuroinflammation while promoting neurogenesis and angiogenesis. ot is a hormone that moderates social and emotional communication, bonding, and social learning, while also having profound antiinflammatory effects. sfn is a naturally occurring compound in cruciferous vegetables, such as broccoli and sprouts, and activates a transcription factor which regulates anti-inflammatory and antioxidant genes. rsv is found in plants, such as grapes and berries, and helps stabilize the central and peripheral immune response and oxidative stress markers, subsequently reducing neuroinflammation. all of these treatments have shown promising potential, but it is abundantly clear that further research is needed in addition to combined therapies. since asd is a spectrum, not every case can be treated the exact same way. by targeting neuroinflammation, we can address the root cause of asd rather than the symptoms. anti-inflammatory interventions for autism spectrum disorder by: haily holston, avani karvat, simran lallian, pingju wu 1. introduction asd a�ects 1 in 36 children as of 2020,1 but the cause of the disorder is still unknown. while there is no clear consensus in the scienti�c community on the speci�c mechanism that leads to the development of asd, some theories include genetic predispositions or environmental factors.2 however, neuroin�ammation has been consistently found in individuals diagnosed with asd. some of the key characteristics of asd are poorly developed social skills, di�culty with expressive and receptive communication, and the presence of restrictive and repetitive behaviors.3 there is no current cure for asd which poses a signi�cant call for action and attention to novel treatment. there are many pharmacological therapies prescribed for asd such as antipsychotics, hormones, cns stimulants, and antidepressants to name a few.4 however, these interventions only target the symptoms of asd instead of neuroin�ammation, and out of the symptoms only one aspect of asd such as aggression, repetitive behaviors, hyperactivity/inattention, or social behavior. it is very di�cult to �nd treatments that cater to all the symptoms, for they mainly focus on accommodating symptoms and limitations associated with asd such as speech, language, and learning de�cits. 75% of individuals with asd also su�er from other associated disorders such as attention-de�cit hyperactivity disorder (adhd), anxiety, bipolar disorder, depression, and many others.5 non-pharmacological therapies that mediate symptoms of asd include behavioral management treatment, cognitive behavior therapy, social skills training, and speech and language therapy. however, the focus of this review will be on novel forms of anti-in�ammatory treatments such as mesenchymal stem cell therapy, oxytocin, sulforaphane, and resveratrol, which have been successful in reducing neuroin�ammation in animals and humans, providing synaptic protection, and relieving symptoms of asd. 2. pathophysiology of asd and neuroin�ammation a single cause of asd is yet to be found, but there are a large range of possible factors that play a signi�cant role in its onset, like genetics, environmental in�uences. berkeley pharma tech journal of medicine | 61 neuroin�ammation is found to be an underlying factor of asd and other neurodevelopmental disorders. although this correlation is apparent, it is still unclear whether neuroin�ammation is a symptom of asd or if asd is a result of neuroin�ammation.6 neuroin�ammation refers to an in�ammatory response taking place in the central nervous system (cns). at low levels, neuroin�ammatory signaling is critical for learning and memory functions, but at higher or chronic levels from cns injuries, it can be a causing factor of neurodegenerative diseases and even aging.6 the neuroin�ammatory response involves microglia cells in the cns and their polar nature. in asd patients, neuroin�ammation has been observed in the cerebellum in early developmental stages and continues to the later stages of their lives.6 neuroin�ammation can be detected through an increase in microglial cell density and somal volume in the white matter of the brain.6 once microglia are activated, there are two in�ammatory pathway options:the pro-in�ammatory response in the m1 phenotype and the anti-in�ammatory response in the m2 phenotype.6 pro-in�ammatory cytokines that commonly increase in patients with asd are il-6, tnf-ɑ, gmcsf, and il-8, while those that decrease are tgf-β and il-10.7 furthermore, chronic glial activation of the pro-in�ammatory pathway leads to the overall in�ammatory response being altered.8 additionally, transforming growth factors (tfgβ 1, 2, 3) play vital roles as regulators in the immune system and general homeostasis including the regulation of in�ammation. for instance, asd patients with decreasing behavioral measures have been found to have decreased tfgβ plasma.7 an environmental factor, maternal immune activation (mia), is one of the most signi�cant factors linked to asd in children. mia triggers in�ammation of the placenta and neuroin�ammation in the mother.9 mia can increase il-17a expression, which leads to neuron cell death and thus hinders normal social behaviors as found in embryonic mouse brains.9 other asd symptoms are found to be induced by mia including anxiety-related repetitive behaviors.9 neuroin�ammation caused by mia has also been correlated to oxidative stress, another factor in causing asd. oxidative stress triggers negative feedback leading to unnatural and berkeley pharma tech journal of medicine | 62 disrupted brain development, which is a key factor asd and other neurodevelopmental disorders.9 figure 1: the in�ammatory response in the central nervous system and brain is mediated by genes and cytokines.10 the increased tnf-alpha gene expression leads to the release of pro-in�ammatory cytokines in the pns. 3. anti-in�ammatory interventions 3.1 mesenchymal stem cell therapy mscs are stem cells that can be found in any postnatal tissue, including the brain, it can perform a variety of functions, such as reducing secondary neurodegeneration and neuroin�ammation, or promoting neurogenesis and angiogenesis.11 the popularity of msc therapy is due to its ability to be harvested from various locations in the body, being easy to culture in lab, having little ethical considerations, and being well-tolerated when implanted into patients.6 in addition, there are no signi�cant safety concerns during infusion or after.12 the way mscs function is through bidirectional immunomodulatory e�ects, caused by direct contact that increase in�ammation when the immune system is under-active and decrease in�ammation when it is over-active.6 secondly, mscs secrete extracellular vesicles (evs), growth factors, chemokines, and cytokines.6 evs are known to be essential for berkeley pharma tech journal of medicine | 63 regulating the in�ammatory response, mediating cell communication, and transmitting mediators during in�ammation to ensure the anti-in�ammatory response.13 growth factors are hormone-like proteins such as nerve growth factors (ngfs) in the brain, that can help suppress in�ammation and switch the immune response to anti-in�ammatory.14 cytokines and chemokines are proteins that help regulate the immune response; when they are not in control it can lead to neuroin�ammation, neurodegeneration, and demyelination of the cns and pns.15 pre-clinical studies demonstrate the potential of msc therapy. an in vitro study demonstrated that mscs modulate neuroin�ammation through signi�cant reduction of mrna expression of proin�ammatory cytokines in microglia.16 mscs were harvested from male mouse tibia and �bula, then tested with bv2 and primary microglia isolated from mouse pup brains.16 another study utilized in vitro culture exosomes from mscs derived from human umbilical cords (huc), which proceeded to be administered intranasally into mice. there was improved sociability and decreased repetitive behaviors in mice treated with valproic acid, which closely mimics asd.12 a clinical study was conducted on 37 children with asd from ages 3-14 years old, during which umbilical cord-derived mesenchymal stem cells (ucmsc) in conjunction with human cord blood mononuclear cells (cbmncs) were tested. the subjects were divided into three groups in a non-randomized, open-label, single center phase i/ii trial, which are most suitable for establishing medication dosages with the highest e�cacy.17 the cbmnc group consisted of 14 participants, who all received a transfusion of cbmncs and rehabilitation therapy. the 9 participants in the combination group received both cbmnc and ucmsc transfusions, as well as rehabilitation therapy (9 subjects). the control group, which also had fourteen participants, received only rehabilitation therapy. the cbmnc group had signi�cant results in comparison to the control group, however the combination group had the most signi�cant results based on three scales: the aberrant behaviour checklist (abc), clinical global impression scale (cgi) and childhood autism rating scale (cars), with no signi�cant safety issues.17 this study demonstrated the e�ectiveness of bothmsc therapy and the impact of dual therapy. berkeley pharma tech journal of medicine | 64 another clinical trial observed the impact of intravenous (iv) infusions of human cord tissue mesenchymal stromal cells (hct-mscs) in 12 children with asd from ages 4 to 9 through an open-label, phase i study.18 the hct-mscs were provided by a third party manufacturer, and each child underwent one, two, or three doses with 2 month intervals in between. clinical and laboratory assessments were conducted in person initially and at the 6-month mark for a baseline, then remotely again at the 12-month point following the last infusion. upon the end of the study, 50% of all the children across all groups showed signs of improvement in at least 2 asd measures. the tests used to gauge e�cacy were the vineland adaptive behavior scale, pervasive developmental disorder-behavior inventory, and clinical global impression scale. it is uncertain whether this was only due to the treatment, but it was concluded that hct-mscs improves communication and socialization in asd patients. once the trial treatment was also determined to be well-tolerated and safe for children, hct-mscs were successfully manufactured.18 however, it is important to note further trials are necessary to con�rm the long-term e�ects and safety of the treatment for the general population.18 in summary, the bene�ts of msc therapy is limited to improvement of symptoms without reversing the condition itself, making it a promising supplementary treatment for managing asd.12 more research on a larger scale is necessary to understand the full extent of the treatment’s e�cacy in managing varying degrees of asd symptoms and characteristics. 3.2 oxytocin oxytocin (ot), or the “social hormone”, is a neuropeptide naturally produced by the human body. it is most widely known for its ability to moderate social and emotional communication, bonding, and learning in the human brain. oxytocin is produced in the brain’s hypothalamus and is released into the bloodstream by di�erent regions of the brain, such as the pituitary gland, and the spinal cord. berkeley pharma tech journal of medicine | 65 figure 2: map of the key pathways by which oxytocin modulates social functioning in the brain. oxytocin in�uences the modulation of sensory input (olfactory system in rodents), social learning through interactions with serotonin systems in the nucleus accumbens, and amygdala and limbic circuit activity. it also directly a�ects the brainstem nuclei to promote bonding, trust, and social interactions.19 oxytocin has profound anti-in�ammatory e�ects in multiple organ systems, including but not limited to the inhibition of the immune system’s activation of in�ammatory cells, signi�cant reduction of nadph oxidase and ros production, and the lowering of pro-in�ammatory cytokine production and neurotoxicity in the nervous system.20 a 2008 study researching the role of oxytocin’s anti-in�ammatory properties conducted trials with ot and lipopolysaccharide (lps) treatments through both individual and combination therapies. the results found that in 10 healthy men who received the treatments, through individual or combination therapies, there was long-term reduction of endotoxin-induced macrophage in�ammatory protein-1-alpha, macrophage in�ammatory protein-1-beta, cortisol, and vegf levels, among other proteins responsible for generating in�ammatory responses in the body.21 most notably, ot had a signi�cant e�ect on decreasing neuroendocrine cell and cytokine expression; these are two components that play a vital role in cell signaling between the nervous system and other organs that coordinate the body’s in�ammatory immune response. oxytocin’s moderation of the release of these proteins has been berkeley pharma tech journal of medicine | 66 shown to relieve in�ammation in the body. in conclusion, oxytocin demonstrates noteworthy therapeutic potential for asd not only because of its anti-in�ammatory properties, but because of the vital role the hormone plays in the development of complex social behaviors.22 in mouse models of asd, oxytocin exhibits an increase in social recognition and restoration of social behavior. rodents, like most animals, rely on e�cient extraction of sensory information using their olfactory system to shape their behaviors and perceive the world around them. a study was conducted to test whether ot would trigger the main olfactory system in female adult wistar rats to process social odor cues. the experiment found that after invoking the release of endogenous ot in mice with an olfaction-ot social recognition task, the mice that released endogenous ot had longer anogenital investigation events in comparison to the control rats. in rodents, anogenital investigation is vital for olfactory sampling and social recognition; the rats with prompted endogenous ot release took more time to investigate and draw social conclusions about their surroundings.23 another study observing the restoration of social behavior in a cntnap2 mouse model of asd after exogeneous ot release concluded that the treated mice with previously low sociability demonstrated stronger preference to interact with other mice after ot treatment.24 therefore, studies tests on mouse models demonstrated how both endogenous and exogenous ot release prompts heightened social engagement and learning. clinical trials testing the role of ot in relieving social de�cits in patients with asd have been vital to our understanding of ot's capacity as a potential therapy. one of such studies was a randomized, double blinded, and placebo controlled clinical trial observing probiotic and oxytocin combination therapy in patients with asd aged 3-20 years old. the patients were separated into two groups: 1 placebo group and 1 probiotic group. the groups were observed for 28 weeks and at week 16, ot was introduced to both. the results indicated trends of improvement in scores based on the study’s aberrant behavior checklist, social responsiveness scale, and stereotypic behavior score in the probiotic and ot combination group, as well as signi�cant cgi improvement.25 berkeley pharma tech journal of medicine | 67 another clinical trial explored the e�ect of ot nasal spray on social interaction de�cits observed in young children with autism. the study administered the ot nasal spray in the morning and night over a 5-week trial period, and concluded that it was both well-tolerated by children and improved caregiver-rated social responsiveness in comparison to the children in the placebo group.26 this study noted that children with asd found this method preferable and signi�cantly more tolerable, providing valuable insight into the future testing of exogenous ot in younger patients. though the success of oxytocin in both relieving in�ammation throughout the body and promoting social processing and engagement is apparent, one of the limitations of oxytocin as a therapy for asd is the inconsistency of studies on oxytocin treatment in humans. not all clinical trials show signi�cant behavior changes or relief from repetitive behaviors and social de�cits in individuals with asd.27 3.3 sulforaphane sulforaphane (sfn) is a naturally occurring compound abundant in cruciferous vegetables, such as broccoli and sprouts, and recent research has demonstrated its e�cacy in alleviating symptoms associated with asd.28 sfn's therapeutic e�ects stem from its ability to activate a master transcription factor known as nuclear factor erythroid 2 related factor (nrf2). nrf2 plays a pivotal role in regulating anti-in�ammatory and antioxidant genes, making it crucial for the body's defense against oxidative stress and in�ammation.29 while the exact underlying mechanism remains uncertain, evidence from a rodent-model study suggests that sulforaphane's activation of nrf2 is likely to address elevated th17 immune responses and oxidative stress observed in individuals with asd.30 as a result, this correction of immune dysfunction and oxidative stress is expected to lead to a reduction in asd symptoms. several clinical trials have been conducted to explore the e�ects of sfn treatment in asd, yielding encouraging results. in one placebo-controlled, double-blind, randomized trial done by singh et al., 29 young men with moderate to severe asd received daily oral doses of sfn for 18 weeks.31 the results demonstrated substantial and reversible improvements in behavior, berkeley pharma tech journal of medicine | 68 as quanti�ed by widely accepted measures completed by parents/caregivers and physicians. sfn's mechanism of action lies in its ability to upregulate genes that protect cells against oxidative stress, in�ammation, and dna damage, all of which are prominent characteristics associated with asd. even though two participants encountered unprovoked seizures following treatment, it's essential to emphasize that sfn exhibited minimal toxicity, making it a comparatively safe option for therapeutic use. another clinical trial investigated the e�ects of adjuvant treatment with sfn and risperidone in alleviating irritability in 60 children with asd.32 the combination of sfn and risperidone led to greater improvements in irritability and hyperactivity/noncompliance symptoms compared to the placebo group. these results support the safety and e�cacy of sfn as an adjuvant treatment for behavioral improvements in children with asd. additionally, a larger randomized clinical trial with 108 subjects in china further substantiated the potential of sfn treatment.33 clinician-rated scales showed a signi�cant improvement in the sfn group, with one-third of participants experiencing a signi�cant decrease in scores after 12 weeks of treatment. sfn was well-tolerated across all age groups, including young children, and its e�ects appeared to be greater in participants over 10 years of age. however, inconsistencies between caregiver and clinician-rated scales indicate the need for more clinical trials to con�rm and re�ne the �ndings. while sfn treatment holds promising potential for addressing asd symptoms, it also has limitations. not all clinical trials have yielded statistically signi�cant improvements in behavior, and the e�ects may vary depending on age groups and assessment methods. in some studies, sfn treatment did not show signi�cant clinical improvement in the behavioral outcome measures evaluated in children with asd.34, 35 moreover, the sample sizes in some trials were limited, which may impact the generalizability of the �ndings. further research with larger cohorts is necessary to better understand sfn's e�ects and determine the optimal dosages and treatment duration for di�erent age groups and severity levels of asd. the need to validate the responses to sfn treatment in asd has led to studies exploring potential biomarkers. these candidate molecular markers, berkeley pharma tech journal of medicine | 69 associated with asd in three physiological pathways, include cytoprotective enzymes, heat shock proteins, and pro-in�ammatory markers.36 ex vivo experiments using peripheral blood mononuclear cells (pbmcs) from healthy subjects showed that all markers exhibited quanti�ability, accuracy, and reproducibility after sfn treatment. when administered orally to asd patients, sfn led to an increase in cytoprotective enzymes and heat shock proteins, while pro-in�ammatory markers decreased. these encouraging results indicate that these markers have the potential to be utilized as guidance for the development of sfn interventions for asd. in a nutshell, the promising potential of sfn treatment in asd o�ers hope for the development of mechanism-based therapeutic approaches. sfn's ability to modulate oxidative stress and in�ammation highlights its relevance in addressing the underlying pathophysiology of asd. nevertheless, additional clinical trials along with biomarker establishment are required to address the limitations and validate the consistency and robustness of sfn's e�ects on asd symptoms. 3.4 resveratrol resveratrol (rsv) is a polyphenolic stilbenoid acting as a phytoalexin that has been found to reduce common symptoms of asd in animal models due to its anti-in�ammatory and anti-oxidative properties.37 rsv is naturally made in plants, such as grapes and berries, as they respond to pathogen attacks.37 rsv can decrease neuroin�ammation by inhibiting activation of the pro-in�ammtory pathway and the proin�ammatory cytokine release.38, 39 tnf-ɑ and mmp-9 levels signi�cantly decrease in the presence of rsv, therefore reducing neuroin�ammation39. mmp-9 stimulates proin�ammatory cytokines as well as processes the nlgn3 (neuroligin) and nrxn1 (neurexin) genes that are linked to asd39. microrna-155 (mir-15) also increases microglia’s in�ammatory response, which is decreased by rsv to reduce neuroin�ammation.29 rsv has shown its e�ectiveness in reducing neuroin�ammation in several studies di�ering in models used, dosage amounts, and methods of administration. rsv admitted orally (5, 10, 15 mg/kg) was found to berkeley pharma tech journal of medicine | 70 decrease pro-in�ammatory cytokine concentrations such as il-6 and tnf-ɑ, which in turn counteracts neuroin�ammatory markers.37 in a study performed with rsv being injected at 0 hours, 8 hours, and 18 hours, rsv was found to increase the anti-in�ammatory m2 phenotype polarization and reduce the m1 pro-in�ammatory response.29 the valproic acid model (vpa), a fatty acid used as an antiepileptic drug and mood stabilizer, is often used to study asd.37 vpa is also a potent teratogen, leading to abnormalities in embryonic development which can induce asd.38 vpa exposure during pregnancy has been associated with asd in o�spring and can cause developmental neurotoxicity in the child’s central nervous system.37 in a study with rsv administration of 3.6 mg/kg for 12-13 days, there was a signi�cant prevention and reduction of social de�cits of asd in the vpa model37. in this model, rsv decreased negative e�ects of asd to the nest-seeking behavior of the rats; however, rsv didn’t have an e�ect on latency to decision making37. in a separate study with the vpa model with rats, rsv was found to prevent impairments in reciprocal social interaction40. nose-to-nose sni�ng habits of rats were also signi�cantly di�erent when treated with rsv40. decreased decision accuracy caused in the vpa model was also prevented with rsv41. however, rsv couldn’t prevent food preference changes or the behavior of repetitive self-grooming as it was a�ected by vpa exposure40. in addition to rsv’s success in the vpa model, in the btbr model rsv reduced persistent self-grooming, a repetitive habit in rats with asd, with doses of 20-40 mg/kg37. in human patients with asd, these repetitive behaviors include �dgeting or sni�ng. in the btbr mice, ccr and cxcr, chemokine receptors related to in�ammation, were signi�cantly higher, but decreased with rsv treatment37. in the propanoic acid (ppa) model, studies found the association of mmp activation to in�ammatory cytokines and mitochondrial dysfunction39. with this ppa model, rsv has proven to improve modi�cations of rats berkeley pharma tech journal of medicine | 71 with asd because of its properties, speci�cally being anti-tnf-ɑ and anti-mmp-939. further studies and clinical trials can help to further investigate rsv’s e�ectiveness against neuroin�ammation and in turn, symptoms of asd. additional research will also be able to �nd how to utilize rsv while minimizing side e�ects such as fetal abnormalities37. 4. practical applications 4.1 mesenchymal stem cell therapy there is a large potential for msc therapy because it is very easy to obtain through bone marrow, adipose tissue, placenta, skin, umbilical cord blood, umbilical cord perivascular cells, umbilical cord wharton’s jelly, amniotic �uid, breast milk, and more42. mscs are easy to isolate and expand, and are unique due to their self-renewal and di�erentiation properties42. these cells are also able to cross the blood-brain-barrier which helps it migrate to sites of tissue injury and in�ammation. although there are numerous proof of concept studies, there is a signi�cant shortage of clinical trials and other therapies researching practical use of mscs. further investigation of the mscs as a potential therapy for asd is necessary to determine the e�ectiveness on patients with varying severities. since most clinical trials have studied young children, study participants within a wider range of age groups would signi�cantly increase our understanding of mscs and adult patients with asd. furthermore, the e�cacy of this therapy based on mscs di�erent tissue types and varying administration methods has yet to be explored42. researchers are unsure whether systematic delivery (ex: intravenous) is enough to reach the brain, compared to direct implantation, or intranasal administration which is non-invasive42. additionally, it is unclear whether mscs obtained from di�erent sources in the body will have di�ering or similar therapeutic e�ectiveness42. berkeley pharma tech journal of medicine | 72 asd is also associated with a myriad of other autoimmune conditions such as autoimmune thyroiditis, rheumatoid arthritis, ulcerative colitis, celiac disease, and type 1 diabetes42. there is potential to investigate the use of mscs in helping manage these conditions alongside symptoms of asd, however many of these are either in the pre-clinical or early clinical trial phases. although asd still remains without a cure, msc therapy emerges as an intervention with great potential as mscs can bene�t synaptic health and have the potential to target tissue damage, regeneration/repair, in�ammation, and ultimately aid in addressing the underlying pathology of neuroin�ammation42. mscs can be transplanted directly with no genetic modi�cation, or pretreatment, can di�erentiate itself, and don’t have any signi�cant side e�ects such as tumors42. 4.2 oxytocin ot has tremendous therapeutic potential for asd speci�cally because of its role in promoting social learning and bonding, as well as its anti-in�ammatory properties in various systems in the body22. the neuropeptide has been highly studied throughout the past few decades to investigate how it may relieve one of asd’s most notable characteristics: social de�cit. studies have shown that in both animals and humans, ot has the capacity to heighten social problem solving skills and increase social engagement in both patients with asd and rodent models of autism. although there are a plethora of successful clinical trials exploring ot and the social changes it creates in asd patients, further research of ot in humans is necessary in order to develop useful therapies. while ot shows great promise for increasing sociability in patients with asd, many clinical trials in humans show inconsistent results in treatments using ot alone27. most notably, there are many unsuccessful clinical trials investigating ot where there have been no signi�cant increases in social engagement or a decrease in repetitive behaviors in patients with asd27. the strength of ot lies in combination therapy. when used with probiotics simultaneously, ot treatment has shown drastic increases in the patients’ caregiver rated sociability and decreases in repetitive behavior berkeley pharma tech journal of medicine | 73 patterns25. further research, especially exploring successful combinations of ot and other potential therapies, is necessary to better understand how to target and relieve speci�c symptoms of asd. lastly, ot demonstrates therapeutic capability for asd due to its method of administration. ot is produced in the brain’s hypothalamus, it is one of the body’s naturally occurring neuropeptides. exogenous ot treatments are commonly distributed intranasally, and this method has been reported to be tolerable by children and show low levels of uncomfortability in patients. the human body’s ot can also be evoked to collaborate in treatment methods26. some limitations of the trials using ot are that only healthy men were included as participants, and no women were involved.21 additionally, the proposed half-life of ot was only around 20 minutes in the mammalian brain25, the small sample size limited the ability to e�ectively analyze subgroups, population heterogeneity led to subjects with varying treatment responses, and cultural and language barriers potentially in�uenced behavioral assessments26. overall, ot shows great potential in helping patients with asd boost social awareness, increase social engagement and social behaviors and decrease repetitive behaviors patterns. the neuropeptide’s capability to collaborate with other treatment methods such as probiotics in combination therapy must be further researched to determine the most useful amalgamation to relieve the most notable characteristics revolving around the social de�cits of asd. 4.3 sulforaphane further research is essential to determine the most suitable dosage of sfn tailored to individuals with varying body weights. the emerging evidence regarding sfn's favorable e�ects on alleviating symptoms associated with asd is undeniably promising, underscoring the need for additional exploration to unveil the most e�ective dosing regimen that maximizes its therapeutic bene�ts while minimizing potential adverse e�ects. berkeley pharma tech journal of medicine | 74 studies conducted thus far have reported encouraging outcomes in individuals with asd who have consistently integrated sfn into their dietary routine31, 32, 33. this naturally occurring compound, abundantly found in various food sources such as broccoli, brussels sprouts, and cabbage, provides a convenient and readily accessible method of supplementation28. given its ubiquitous presence in everyday foods, sfn represents a safe option for oral consumption, enhancing its appeal as a potential treatment for asd. sfn possesses multifaceted attributes that position it as an exceptionally promising candidate in the search for e�ective therapies for asd. beyond its easy availability, sfn boasts various pharmacological properties that contribute to its therapeutic potential. as a potent antioxidant, sfn combats oxidative stress, a common feature associated with asd43, 44. additionally, it exhibits anti-in�ammatory e�ects that can alleviate neuroin�ammation, frequently observed in individuals with autism8, 45, 46. furthermore, sfn's impact on the body's detoxi�cation mechanisms is noteworthy. by enhancing phase ii detoxi�cation enzymes, it facilitates the elimination of harmful substances and toxins, further promoting the overall health and well-being of individuals with asd47, 48. sfn's versatility reaches beyond its capacity as a standalone therapy. considering that individuals with asd can derive bene�ts from personalized combinations of treatments and services, there is a signi�cant opportunity to synergize sfn with other compounds or medications33, 34. such integration has the potential to enhance sfn's therapeutic impact on asd symptoms, presenting a promising pathway for future therapeutic interventions and optimizing its overall e�cacy. while these developments are encouraging, it is crucial to acknowledge that research in the �eld of autism is continuously evolving. delving deeper into the complexities of asd and exploring the precise mechanisms through which sfn exerts its e�ects will enable us to re�ne treatment protocols better, catering to individual needs more e�ectively. overall, sfn's potential as a therapeutic agent for asd shines brightly. its accessibility, presumed safety, and bene�cial e�ects on alleviating symptoms berkeley pharma tech journal of medicine | 75 have garnered signi�cant attention from the scienti�c community. as further investigations are conducted to establish ideal dosages and explore potential synergistic e�ects with other treatments, the path towards harnessing the full potential of sfn in enhancing the lives of individuals with asd becomes closer. this dedicated pursuit represents a crucial step towards o�ering comprehensive and e�ective solutions to those living with this neurodevelopmental condition. 4.4 resveratrol because rsv can cross the brain-blood barrier, studying its e�ects is helpful in investigating neurodevelopmental disorders37. due to this characteristic, rsv has low concentrations in the brain and high concentrations in the blood, meaning rsv isn’t very e�ective when administered orally with only a 1% bioavailability due to how easily it is absorbed and excreted49. this low bioavailability makes rsv more bene�cial while being used in combination with other therapies49. polyphenols, including rsv, aren’t naturally synthesized in animals, and thus plant-rich diets are a more e�ective way to achieve its e�ects49. foods with high amounts of rsv include grapes, peanuts, and plums in addition to several other recommended options31. even with rsv’s high potential to be used as an additional therapeutic agent to reduce and improve the symptoms of asd on a biochemical, molecular, and behavioral scale, more research must be done to e�ectively utilize its abilities39. more trials and research can help to better understand rsv’s mechanisms of action in various conditions50. it is important to note the participants of most rsv studies are predominantly male animals, leaving room for questions regarding how females may experience di�erent interactions with rsv for asd symptoms37. there have been studies with evidence of di�erences in levels of estrogen receptors in asd patients and with limited knowledge of how sex hormones play a role in rsv, this is a future area if study necessary to further prove rsv’s e�ectiveness37. berkeley pharma tech journal of medicine | 76 a notable negative e�ect of rsv’s use is particularly on pregnant women because of fetal birth defects as shown in a study with japanese macaques37. the risk to pregnant women may also be accompanied with symptoms of diarrhea and nausea31. further study of these e�ects is bene�cial to provide better care with less risk. rsv shows promising results to patients with asd particularly because of its ability to reduce neuroin�ammation. with the goal of alleviating the symptoms of asd these patients face, further research of rsv in combination with other therapeutic options would be extremely bene�cial. even with this further research, it is crucial to acknowledge the diversity of asd patients and their symptoms, leading to evolving information about this treatment. 5. future directions msc, ot, sfn, and rsv are all anti-in�ammatory treatments that have demonstrated their individual capacities to relieve in�ammation in the body, namely neuroin�ammation, one of the hallmark underlying factors of asd. each of these treatments have their own strengths and weaknesses, for example ot shows signi�cant promise in increasing social engagement and social problem solving in patients but has inconsistent results in humans when not used in combination therapy. one discrepancy to note is that ot has shown consistent measurable increases in sociability when administered intranasally in mice, yet when administered intranasally in children, there is great variation in results. in future, more research with other methods of distribution of ot may lead to more consistent results and higher levels of sociability in human patients with asd. further research to investigate the capacity of each of these therapies in combination must be explored in order to gauge their combined therapeutic potential. combination therapies that utilize the strong points of msc, ot, sfn, and rsvmust be studied in order to eventually create individualized therapies for asd patients that are speci�cally designed for that patient’s symptoms and de�cits. since asd is a spectrum, individualized combination treatment therapy holds immense promise in alleviating each patients’ most notable berkeley pharma tech journal of medicine | 77 symptoms. currently, there is no literature speci�cally exploring mscs, ot, rsv, and sfn in combination to alleviate symptoms of asd. exploring these therapies in combination with current treatments, especially probiotic treatments, is necessary to create e�ective and tolerable treatments to reduce neuro-in�ammation in patients with asd. 6. conclusions research of potential therapies for autism spectrum disorder has signi�cantly advanced in recent years as scientists explore new remedies for the symptoms and underlying causes of asd. however, current treatments focus on alleviating common symptoms of asd rather than targeting the foundational causes of asd such as neuroin�ammation. although there is no cure for asd, treatments targeting both neuroin�ammation and critical symptoms of asd such as mesenchymal stem cell therapy (msc), oxytocin (ot), sulforaphane (sfn), and resveratrol (rsv) show immense therapeutic potential. most notably, asd is a spectrum. no individual therapy studied thus far has the capacity to be e�ective in all individuals with asd. potential interventions serve to resolve some of the overarching consequences of asd, particularly neuroin�ammation, and social de�cits such as repetitive behavior patterns, poorly developed social skills, and di�culty with expressive and receptive communication. it is imperative that further research studying the e�ciency of individualized combination therapy of msc, ot, sfn, and rsv be explored to create the most e�ective medicinal combinations for individuals with asd. berkeley pharma tech journal of medicine | 78 references 1. maenner mj, et al. prevalence and characteristics of autism 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h, yang hj, an yj, et al. enhanced phase ii detoxi�cation contributes to bene�cial e�ects of dietary restriction as revealed by multi-platform metabolomics studies. molecular & cellular proteomics: mcp. 2013;12(3):575-586. https://doi.org/10.1074/mcp.m112.021352 49. sawda c, moussa c, turner rs. resveratrol for alzheimer's disease. annals of the new york academy of sciences. 2017;1403:142-149. https://doi.org/10.1111/nyas.13437 50. malaguarnera l. in�uence of resveratrol on the immune response. nutrients. 2019;11(5):946. doi:10.3390/nu11050946 berkeley pharma tech journal of medicine | 82 56-scientific review paper-573-1-11-20240510 (1).pdf reference page_holston et al..pdf the role of the gut microbiome in neuromodulation therapies as a potential treatment adjunct for multiple sclerosis berkeley pharma tech journal of medicine correspondence: roshni.nandwani@emory.edu keywords: brain-machine interface neuroengineering paralysis communication disabilities eeg submitted july 07, 2024 accepted july 08, 2024 published december 27, 2024 full open access creative commons attribution license 4.0 abstract brain-machine connections and neuroengineering are technologies that can be used to act as a direct communication pathway between the brain’s electrical activity and an external device. this paper explores the realm of brain-machine interfaces (bmis) and neuroengineering technologies, focusing on their potential to address cognitive and mobility impairments. this comprehensive literature review delves into the current state of bmi technology, emphasizing its applications in stroke rehabilitation, paralysis management, prosthetics, memory enhancement, and communication solutions for speech impairments. various methodologies, from invasive to non-invasive techniques, are examined for their impact on patients’ quality of life. while these advancements hold great promise, they also pose ethical and practical challenges, including the need for further clinical testing, algorithm refinement, and affordability concerns. the future of bmis is envisioned considering the role of artificial intelligence in expanding their possibilities and reflecting on the ethical considerations in deploying these technologies. this review provides valuable insights into the current landscape of bmis, their potential for enhancing human capabilities, and the critical issues that must be addressed as the field progresses. cognitive and mobility enhancement through brain-machine interfaces and neuroengineering by: roshni nandwani, srilakshmi palanikumar, sahaj sidhu singh and jehan balading 1. introduction the brain is often considered one of the most important organs in the human body because of the wide range of functions it performs. it is also extremely complicated, with over 100 billion neurons.1 equal numbers of neuronal and nonneuronal cells make the human brain an isometrically scaled-up primate brain.2 as a result, managing diseases and impairments of the brain presents significant challenges. modern science has begun to move away from looking at individual neurons, as it did in the past. now, scientists are pivoting towards observing neural networks and neural ensembles. bmis emerged from this direction of research. a bmi is a computer-based system that acquires brain signals, analyzes them, and translates them into commands that are related to output devices. these commands then carry out desired actions, aiding the user to do things they previously would not have been able to.4 berkeley pharma tech journal of medicine | 23 figure 1. a decoding process using an invasive method of eeg recording to yield motor function. the eeg recording decodes the brain’s intentions, resulting in motor control of prosthetics and other external devices. research on brain-machine interfaces and brain-computer interfaces began as early as the 1970s. tests were initially conducted on primate subjects until they were considered safe enough to be tested on humans in the 1990s.5 as this technology evolved, ethical concerns increased in importance. like with other kinds of medical procedures, finding a balance between repeated procedures on a highly sensitive organ and being able to have the bmi be as effective as possible poses various ethical concerns. additionally, because bmis are often used in treating cognitive afflictions, it is important to consider the patient’s agency and consent.6 furthermore, the concerns related to data privacy and the potential for misuse of neural data upon conducting continuous monitoring of brain activity further raises questions regarding bmi technology ethics. the high amount of interest from the scientific community comes from the potential for bmis to aid the rehabilitation of devastating diseases that are difficult to manage. in the past, they have shown capability in in regard to helping patients who are afflicted by strokes, chronic pain, paralysis, and other cognition and mobility-related problems.7 while current bmis already offer significant amounts of aid in helping patients manage neurological disease, there are many open avenues for research in this field. for instance, artificial intelligence could bring various new possibilities to the applications of bmis. additionally, predictive models are believed to be able to bring function back to amputees and more severe cases of paralysis.8 in this review, though, we focus on the bmi and neuroengineering technologies that currently exist and have the potential to resolve various cognitive and mobility impairments. we review current scientific literature to gauge the potential of this technology in enhancing quality of life through bmi in memory, mobility, and home improvement technologies. we also look into potential areas of future research including the use of artificial intelligence with bmis. finally, we discuss the role that clinical berkeley pharma tech journal of medicine | 24 trials have played in the current research in human subjects and their success/areas of improvement going forward. 1.2 methods in this review, we conducted a literature search of the sources, key studies, and findings describing issues and conditions related to bmi and cognitive and mobility impairments. our literature review focused on the current technologies that exist to resolve and enhance the quality of life of those with mobile and cognitive impairments. studies included in table 1 met the criteria of being a recent study, published within the last 10 years, as well as fitting the keywords of bmi, neuroengineering, eeg, and the name of the condition or improvement technology. our sources were collected from globally acknowledged databases, such as pubmed and researchgate. prominent journals include the american physiological society, science, frontiers neural interface, and journal of neuroengineering. the information provided in the selected recent studies (ie. purpose of study, results of scientific findings, and impact of paper’s outcome) were carefully evaluated and discussed in the following section. 2. literature review 2.1 mobility conditions 2.11 stroke stroke is a sudden interruption of blood supply to the brain, causing damage to brain tissue. there are two primary types of strokes: ischemic and hemorrhagic, and both can be highly dangerous.9 they often lead to motor deficits, communication impairments, and cognitive challenges.10 current stroke rehabilitation approaches include physical therapy, speech therapy, and pharmaceutical interventions. however, due to the complexity of stroke recovery, innovative methods like bmi are being explored as potential game-changers in rehabilitation.11 berkeley pharma tech journal of medicine | 25 bmis designed for stroke recovery work by decoding neural signals from the brain and translating them into meaningful actions.12 this technology’s application in stroke rehabilitation is a beacon of hope, particularly for those who have limited mobility or communication abilities. by allowing individuals to interact with the external world and control assistive devices directly with their thoughts, bmis offer a new frontier for enhancing independence and recovery post-stroke. there are two types of bmi based approaches to stroke rehabilitation: invasive and noninvasive. a popular technique associated with both of these approaches is signal acquisition. invasive techniques use intracranial microelectrode arrays, while noninvasive techniques use electroencephalograms (eeg).13 both of these methods collect brain signals, which are then processed and decoded. a long-standing issue at this point is noise in signal decoding. they have been tested in clinical settings, and the findings are promising; they solidify the presence of bmis as a method of rehabilitation for mobility conditions. for example, a study exists that shows that bmi-based rehabilitation shows long-term improvements in the motor function of people with severe hand paresis. they found that bmi-based therapy, specifically targeting movements via eeg-triggered robotic assistance, led to lasting improvements in motor function six months after the intervention.14 similarly, another study was conducted in which 28 patients with motor deficits were treated with motor imagery (mi) training, and only half of them received bmi-based rehabilitation in addition. fugl–meyer assessments showed that bmi-assisted rehabilitation was significantly more effective than pure mi treatments.15 despite their potential, bmis for stroke recovery face several challenges that must be addressed to fully recognize their benefits. one significant challenge is the need for more extensive clinical testing to validate the efficacy and safety of bmi interventions across diverse patient populations. though initial studies have already indicated that bmi-assisted rehabilitation compared to motor imagery training alone have led to better berkeley pharma tech journal of medicine | 26 outcomes, these findings need to be replicated in a larger, more diverse setting in order to ensure generalizability and further reliability. another critical issue is the fine-tuning of algorithms used in bmis. the accuracy of such fine-tuning systems is dependent on their ability to decode neural signals precisely. advances in machine learning and signal processing are imperative to create more robust and adaptive algorithms that can learn from patient data and improve performance over time. moreover, affordability is a major concern, as the high cost of developing bmi technology limits its widespread adoption. there are significant costs associated with intracranial surgeries and proper training and equipment, limiting accessibility to a broader patient population. figure 2. functional near-infrared spectroscopy (fnirs) in action. fnirs is placed on the patient’s head. the detector collects the data from the light emitter and then the data is transported and decoded (a computer). this data is used to control basic motor functions in a prosthetic. 2.12 paralysis bmis have revealed potential in aiding individuals with paralysis resulting from amyotrophic lateral sclerosis (als), a disease that leads to complete berkeley pharma tech journal of medicine | 27 loss of motor function and speech. in particular, bmis have been instrumental in helping individuals communicate when they are in a completely locked-in state (clis). there are various types of bmis, including eeg-based bmis for als patients with functioning vision and eye control, and fnirs-based bmis (functional near-infrared spectroscopy) that offer spatial and temporal resolution, making them suitable for bedside applications. while eeg-based bmis have achieved success in als patients with some motor function, they are limited in clis patients. on the other hand, fnirs-based bmis have facilitated communication for individuals in the most severe stages of als, providing hope for enhanced quality of life. the electroencephalography (eeg) based bmi’s main task is to restore communication and control for individuals with als. these bmis employ eeg to locate and interpret brain signals associated with movement. through decoding these signals, the bmis enable the signal to output desired physical actions. these signals allow one’s mind to control assistive technology, proving autonomous control over assistive technology. this technology enhances the independence of individuals with als. the cognitive abilities needed to interact with the environment are restored. meanwhile, functional near-infrared spectroscopy (fnirs) based bmis work through decoding neural activity. they achieve this through measuring changes in blood oxygenation in the brain. these are non-invasive bmis, unlike eeg. they work by shining near-infrared light into the scalp, which penetrates the skull and interacts with oxygenated and deoxygenated hemoglobin in the brain's blood vessels. through detecting variations in light, the bmi maps brain activity. finally, this information is decoded and used to control external devices such as prosthetics or computer devices. fnirs-based bmis are promising for individuals with motor disabilities or conditions like paralysis as they serve to regain control and improve quality of life through thought-based communication and control. berkeley pharma tech journal of medicine | 28 one of the downsides of fnirs is that they provide lower spatial resolution compared to other invasive bmis like eeg since they only capture visual information from outside the brain. however, since they are non-invasive, the technology is more accessible and safer to implement. figure 3. eeg-based brain-machine interfaces for mobility and communication. combination for emplace electrodes and eeg based bmis not only provide mobility through a wheelchair. this also connects with compatible tablets and computers giving autonomous control over the software. 2.13 prosthetics prostheses are substitutes for parts of the body lost due to accidents, amputation, or birth defects. types of prosthetics include arm and leg prostheses that fit over joints, artificial eyeballs, and hearing aids. the way bmi is incorporated to develop new prosthetics, specifically neural prosthetics, is by recording signals from neuron populations and decoding the signals through mathematical algorithms. they then translate the intended action into physical limb movement.15 specific feedback calls berkeley pharma tech journal of medicine | 29 like somatosensory feedback are used to provide a more natural sensation for prosthetics. somatosensation is a system of biological organs that respond to inputs and body states. the basis of this paradigm makes it so that the prosthetic user can use patterns of intensity and frequency of stimulation that are critical to perceived sensation. benefits to using such a system is true embodiment of the limb; normal motor function; decrease in negative psychological impact of limb loss.14,15 2.2 cognitive impairments 2.21 memory while bmi technology has traditionally focused on restoring or enhancing sensory perceptions and muscular abilities, scientists are now venturing into a new realm of bmis. these emerging technologies expand mobile conditions and incorporate implanted computers to amplify the formation and retrieval of memories within the brain.16 following aging or memory related diseases such as dementia and alzheimer’s, certain regions of the brain become slower due to a reduction in both neurotransmitters and blood flow to these areas.17 following this trajectory, developers of bmi have utilized non-invasive bmis.18 electromagnetic stimulation and biofeedback techniques as a part of a rehabilitation strategy to adjust brain activity. in addition, by focusing on specific brain wave patterns in the alpha and theta band regions, scientists can predict memory performance and enhance episodic memory. this usage of the technology is especially helpful as episodic memory the long-term memory associated with previous experiences and associated emotions19 is the memory system that is most rapidly affected in response to both normal and unhealthy aging. furthermore, electrophysiological signals could be connected to particular behavioral conditions whereby altering the timing of specific stimuli could enhance memory encoding. in other words, if these signals can serve as a “memory enhancement” just before engaging in specific tasks, elderly patients could give themselves a cognitive boost before remembering where they parked their car or berkeley pharma tech journal of medicine | 30 comprehending their physician’s instructions for medication use. such technologies have the potential to allow patients with pathological memory loss the ability to perform daily life activities. the use of bmi and neuroengineering technologies to resolve memory decline serves almost as a cognitive prosthesis, which is specifically useful for patient populations that need restoring their ability to form new long term-memories, especially after the natural process of aging or post-alzheimer’s disease. 2.22 communication and speech impairments individuals affected by neurological conditions like brainstem stroke or als (4.2 paralysis) often encounter profound difficulties with both their mobility and their speech functions.20 for the latter, in some instances, these patients may experience a total inability to communicate verbally, a condition known as locked-in syndrome. to address this condition, researchers and engineers have worked on speech-based bmi programs to resolve such speech-based cognitive issues and ultimately improve communication disabilities. one such example of this speech-based bmi program is a technology called a neuroprosthesis, which is an approach that decodes words and sentences directly from the cerebral cortical activity of impaired individuals. this technology involves surgically placing a high-density electrode on the outermost layer of the sensorimotor cortex, which is the part of the brain most responsible for controlling speech and having the patient attempt to speak with computer models connected.21 as the participant attempts to articulate words from a list of basic vocabulary words, advanced deep-learning algorithms and computer models recognize and categorize words based on patterns in the brain activity. using these computational models and a natural-language model that predicted the likelihood of the next word based on the words preceding it, the participants’ words are translated into complete sentences.22 berkeley pharma tech journal of medicine | 31 figure 4. advanced neuroprosthetics for speech communication. invasive bmi systems record accurate speech data through eegs which are converted to words via umabci (university of málaga-brain-computer interfaces). voice assistants like google, siri and alexa are used for voice commands. though this technology is limited to vocabulary that is not very advanced, future directions in the field are working on mitigating this and expanding current technologies to include vocabulary lists with over 150,000 words with less than 9% error.23 additionally, when looking at these technologies that are directly related to the brain and speech, the implications of their value from an ethical standpoint must be considered. though neuroprosthetics for speech decoding have the potential to inform consent and capacity allow for agency and consent to medical treatment this technology, for the same reason, may cause harm.24 neurotechnology-mediated communication can pose a risk to privacy, and in the case of defamation, threats, or harassment that is recorded from the participant, poses the question of whether or not they should be held responsible.25 berkeley pharma tech journal of medicine | 32 these technologies, as powerful as they are in showing feasible paths for restoring rapid communication with those with communication disabilities, must be tweaked to account for both ethical and practical concerns as the technology advances. 2.3 life-enhancing technologies 2.31 home improvement and life enhancing bmi technologies in order to help mitigate the effects of these impairments on the populations they affect, developers and businesses have incorporated artificial intelligence (ai) technology and electronics with home improvement tools to ease daily life functions. whether its voice controlled speakers, ai cellphone assistants, or clap activated lights, the use of bmi and neuroengineering take home improvement advancements to a new level. building on this foundation of technological innovation, btc (brain to thing communication) represents a significant leap forward. btc is a system that establishes direct connection from the human brain to outside devices such as tv’s and other smart objects. this development has been helpful, as it can improve the lives of those disabled or advance home improvement in general. the features of the btc system consists of nearby smart objects that will be embedded with systems running sensing and actuation services. this makes the system aware of where the subject is around the house and adjusts accordingly. in fact, users can access the system throughout their daily lives no matter what and can remotely send brain commands via the application. in other words, patients with paralysis can perform different applications (control a wheelchair, nursing bed, lights, tv, and phone) inside a room through bmi based berkeley pharma tech journal of medicine | 33 on biological signals (ie. left eye blink, right eye blink, eyebrow raise).26 nevertheless, these technologies do come with various challenges. first, real-time eeg signal processing can pose difficulties whereby all commands should be processed in real time in order for the system to validate the action request. secondly, while previous studies have demonstrated the accuracy of these prototypes, it is crucial to acknowledge that the system still has limitations in distinguishing specific patterns that differentiate user actions and requests. this is an area that requires significant improvement in the technology in the years to come.27 figure 5. enhancing mobility with assistive exoskeletons. assistive exoskeleton is one of the examples of life-enhancing technologies. with the use of non-invasive bmi’s the suit increases mobility in the elderly and heavily reduces the risk of critical falls. berkeley pharma tech journal of medicine | 34 2.4 literature review summary table 1. literature review study examples of related work for bmi applications organized by mobile, cognitive, and home improvement technologies. the table provides an overview of key findings and their implications for advancing bmi research. 3. discussion 3.1 key findings and implications of research in terms of stroke rehabilitation, bmis allow individuals with limited mobility and communication abilities to regain independence. for those berkeley pharma tech journal of medicine | 35 with full paralysis, eeg-based bmis provide communication and control options for als patients, while fnirs-based bmis offer potential for those in a completely locked-in state. for this reason, both invasive and non-invasive approaches have shown improvements in motor function, but challenges remain, including the need for further clinical testing, algorithm refinement, and affordability concerns. from the cognition standpoint, emerging bmi technologies are targeting memory improvement, especially for individuals dealing with memory-related diseases like alzheimer's. non-invasive approaches, such as electromagnetic stimulation and biofeedback, aim to enhance episodic memory and cognitive function. meanwhile, speech-based bmis have the potential to restore communication for individuals with severe speech disabilities. these technologies can decode words and sentences directly from brain activity, allowing patients to express themselves. however, ethical concerns related to privacy, consent, and accountability must be addressed as these technologies advance. lastly, btc systems enable direct communication between the brain and external devices, offering the potential to both enhance and improve the lives of individuals with disabilities and advance home automation. 3.2 limitations as mentioned, bmis hold great potential to revolutionize the way we interact with technology and understand the human brain. however, bmis are not without their limitations, which span economic, ethical, and scientific domains. first, bmis are intricate systems that depend on knowledge of the human brain, as well as the creation of incredibly sensitive hardware and software. the need for better signal processing algorithms to increase the accuracy and reliability of bmis is one scientific challenge among many. furthermore, significant obstacles to research studies are posed by the variability of brain activity and the possibility of signal noise. concerns about potential risks and side effects are also raised due to the fact that the berkeley pharma tech journal of medicine | 36 long-term effects of brain-computer interfaces are still not fully understood.28 second, a primary limitation of bmis is their cost. developing and deploying bmi technology is expensive, making it inaccessible to many individuals and institutions. the economic barriers limit the widespread adoption of bmis, particularly for medical purposes. patients with neurological disorders who could benefit from bmis often struggle to afford the technology, creating a disparity in access to potentially life-changing treatments. addressing these economic challenges through cost reduction and better funding mechanisms is a pressing concern for bmi development.29 third, ethical dilemmas surrounding bmis are significant and multifaceted. for instance, bmis raise significant ethical issues related to defending the agency of patients who may not have full cognitive ability or the capacity to provide informed consent. protecting the rights and dignity of individuals with cognitive impairments is a crucial concern. ensuring that bmis are used to enhance the quality of life of these patients without infringing on their autonomy is paramount. developing ethical guidelines and regulations that prioritize the welfare and agency of vulnerable populations is essential.30 another ethical concern arises when considering individuals' perspectives on invasive procedures. invasive techniques have more associated risks because of the requirement for surgery, as well as the possibility of scarring.31 despite these challenges, the development of bmis continues to progress. efforts are underway to reduce costs, establish ethical guidelines, and advance scientific research. 4. future directions bmis hold great potential for enhancing the quality of life for individuals with cognitive or mobility-based impairments, motivating sustained innovation in this field. bmis are expected to witness substantial enhancements in terms of control precision, communication speed, and berkeley pharma tech journal of medicine | 37 versatility. these advancements will likely involve the integration of cutting-edge artificial intelligence (ai) techniques for more accurate signal processing and decoding algorithms, offering individuals with disabilities more practical and intuitive ways to interact with the external world. moreover, ongoing research will continue to focus on neurorehabilitation, where ai-powered bmis can revolutionize the recovery process for individuals affected by neurological disorders. personalized rehabilitation programs, guided by ai and responsive to patients' unique needs and progress, could significantly improve the effectiveness of recovery exercises. additionally, the development of neuroprosthetics, including limbs and sensory devices, will drive future bmi research. these devices aim to replicate the natural functions of missing or damaged body parts, and ai will play a pivotal role in making them adaptable and responsive to users' movements. moreover, as bmis expand beyond medical applications, they have the potential to transform human cognition. researchers are exploring ways to use bmis, enhanced by ai, for cognitive augmentation, memory improvement, and knowledge transfer, opening up new horizons for human potential. overall, the future of bmis is not only exciting but also transformative, offering unprecedented possibilities to enhance human lives, both in healthcare and daily interactions. 4. conclusion the field of bmis and neuroengineering holds great power for improving the lives of individuals with cognitive and mobility impairments. continued research and development are essential to address technical challenges, refine algorithms, and improve the affordability and accessibility of bmi technologies. the integration of 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doi:10.1186/s12910-017-0220-y. 30. bergeron d, iorio-morin c, bonizzato m, et al. use of invasive brain-computer interfaces in pediatric neurosurgery: technical and ethical considerations. j child neurol. 2023;38(3-4):223-238. doi:10.1177/08830738231167736. 31. fiani b, reardon t, ayres b, cline d, sitto sr. an examination of prospective uses and future directions of neuralink: the brain-machine interface. cureus. 2021;13(3):e14192. doi:10.7759/cureus.14192. berkeley pharma tech journal of medicine | 42 berkeley pharma tech journal of medicine correspondence: nethra.srinivasan22@gmail.com keywords: precision medicine, alzheimer’s, schizophrenia, ipsc, pharmacogenomics, genetic profiling, neurological disease submitted february 9, 2024 accepted march 29, 2024 published june 28, 2024 full open access creative commons attribution license 4.0 abstract there exist over 600 neurological conditions, each characterized by unique pathologies tailored to individual patients. over the past two decades, advances in biotechnology have propelled the field of neurogenetics forward. this progress has illuminated therapeutic targets and methodologies tailored to the specific needs of each patient. current treatment options primarily encompass therapies and conventional medications like cholinesterase inhibitors for alzheimer’s disease and antipsychotics for schizophrenia. however, these treatments often address symptoms or general targets rather than the precise underlying causes. precision medicine has emerged as a promising approach in both animal and human clinical trials. examples include the identification of specific genetic variations linked to alzheimer's risk and progression, as well as the application of multigenic pharmacogenomics-guided therapies for schizophrenia patients. this review paper delves into the role of precision medicine in neurogenetics, focusing on neural stem cells, induced pluripotent stem cells (ipscs), genetic profiling, and pharmacogenetics within the contexts of alzheimer’s disease and schizophrenia. by evaluating current achievements alongside existing challenges, this paper underscores precision medicine as a pivotal strategy for effectively targeting neurological disorders. neurogenetics: precision medicine-based approaches to neurological disorders with an emphasis on addressing alzheimer’s disease and schizophrenia by: nethra srinivasan, eshaan mehra, sriya dommaraju, ethan kakavetsis 1. introduction as we delve into the secrets of the human mind, one thing becomes abundantly clear: our genes hold the key to understanding the most profound mysteries of consciousness. neurogenetics is employing the evolving �eld of genomics–the study of our genes and genetic variations–to understand factors contributing to the structure and function of the nervous system.1 speci�cally, neurogenetics also seeks to understand how alterations in genes can lead to neurological diseases and conditions. as researchers continue to uncover various genetic factors for each disorder, precision medicine has started to address neurological diseases. precision medicine is an approach to therapeutics that uses an individual’s genomic, environmental, and lifestyle information to form decisions about their treatment.2 often informally termed as personalized medicine, precision medicine can be used to create a more precise approach to diagnose, prevent, or treat a disease. the basis behind precision medicine is substantial because the human genome consists of about 3 billion base pairs of dna, and no two humans are genetically identical, with genetic variation of about 0.1 percent.3 this means that about 6 million base pairs di�er, making it necessary to understand genetic variation. oftentimes, these variations occur as single-nucleotide polymorphisms, or single-base pair di�erences.3 to target these various genetic risk factors, techniques of precision medicine such as drugs tailored to genomic or metabolomic targets and induced pluripotent stem cells (ips) are promising methodologies. while understanding the hallmark genes of neurological disorders through neurogenetics studies may help create therapeutics for these genes, many studies tend to exclude non-white individuals. research involving diverse populations reveals varying prevalence rates of risk genes for opioid use disorder (oud) based on ethnicity, with speci�c genetic variations being linked to susceptibility in caucasian individuals but not in african-american or hispanic populations.4 this evidence suggests that there are larger implications in other neurogenetic diseases such as alzheimer’s disease and schizophrenia. there exist multiple polymorphisms in a diverse society, making neurogenetics a complicated �eld. we ought to berkeley pharma tech journal of medicine | 15 https://www.ncbi.nlm.nih.gov/pmc/articles/pmc9630880/ https://www.genome.gov/genetics-glossary/precision-medicine https://www.ncbi.nlm.nih.gov/books/nbk20363/ https://www.ncbi.nlm.nih.gov/books/nbk20363/ https://www.ncbi.nlm.nih.gov/pmc/articles/pmc7457418/#:~:text=studies%20that%20include%20diverse%20populations,or%20suds%2c%20and%20vice%20versa. adopt precision medicine-based management through analyzing ancestry-based genetic information to provide the best precision-guided therapeutics. variance in neurogenetics presumes the need for precision medicine to target genetic risk factors with tailored medicine speci�c to the patient with neurological diseases, such as alzheimer’s disease and schizophrenia. we present our evaluations by analyzing scienti�c literature on varied neurological disorders and diseases and their basis in neurogenetics, reviewing the scienti�c literature for recent advances in precision medicine, and examining clinical trials to understand the potency of precision medicine techniques in neurological conditions. 2. variance in neurogenetics 2.1 alzheimer’s disease alzheimer’s disease (ad) is a progressive and irreversible neurological disorder that a�ects the brain, leading to a decline in memory, thinking, and ability to carry out simple tasks.5 this decline in cognitive ability hasn’t been attributed to a speci�c cause due to the complexity of the genetic and environmental causes of this disease. 2.1.1 amyloid beta (aβ): formation of amyloid plaques βamyloid proteins are known to be one of the main culprits of alzheimer’s disease. they collect between neurons and inhibit neuronal function, and some molecular forms of these defunct proteins form plaques that are extremely detrimental to neuronal activity.6 2.1.2 tau protein: formation of neuro�brillary tangles neuro�brillary tangles are clusters of abnormal proteins called tau which accumulate within nerve cells.6 in healthy neurons, microtubules provide internal support for the function of transporting nutrients and molecules from the cell body to the axon and dendrites.6 tau usually binds to and stabilizes these microtubules; however, in ad, chemical changes cause tau to detach from microtubules and adhere to other tau molecules, forming threads.6 these amalgamate to form tangles within neurons, disrupting the neuron’s transport system and impairing synaptic communication.6 berkeley pharma tech journal of medicine | 16 https://www.nia.nih.gov/health/what-alzheimers-disease https://pubmed.ncbi.nlm.nih.gov/24493463/#:~:text=the%20soluble%20building%20blocks%20of,%2denriched%20microtubule%2dassociated%20protein. https://pubmed.ncbi.nlm.nih.gov/24493463/#:~:text=the%20soluble%20building%20blocks%20of,%2denriched%20microtubule%2dassociated%20protein. https://pubmed.ncbi.nlm.nih.gov/24493463/#:~:text=the%20soluble%20building%20blocks%20of,%2denriched%20microtubule%2dassociated%20protein. https://pubmed.ncbi.nlm.nih.gov/24493463/#:~:text=the%20soluble%20building%20blocks%20of,%2denriched%20microtubule%2dassociated%20protein. https://pubmed.ncbi.nlm.nih.gov/24493463/#:~:text=the%20soluble%20building%20blocks%20of,%2denriched%20microtubule%2dassociated%20protein. evidence has shown that these plaques and tangles are associated with the occurrence of alzheimer’s disease, as represented in figure 1, and that the occurrence of either plaques or tangles can cause the formation of the other.6 figure 1. a visual representation of alzheimer’s disease manifests in patients because of genes that are prime targets for precision medicine techniques. the result of mutations or rare variants in certain genes, such as apoe4, can have many harmful e�ects that manifest as alzheimer’s disease.9 2.1.3 glial cell interaction with aβ microglia act as immune cells that monitor the brain for signs of damage, infection, or foreign substances.7 when threats are detected, they activate and engulf via phagocytosis, cellular debris, aggregated proteins, etc.7 microglia have the ability to detect the presence of aβ in the brain.8 when encountered with aβ plaques in healthy cells, they become reactive, indicating they are responding to abnormal protein accumulation.8 microglia in which aβ has a direct interaction expresses a range of berkeley pharma tech journal of medicine | 17 https://pubmed.ncbi.nlm.nih.gov/24493463/#:~:text=the%20soluble%20building%20blocks%20of,%2denriched%20microtubule%2dassociated%20protein. https://www.ncbi.nlm.nih.gov/pmc/articles/pmc6463297/ https://www.frontiersin.org/articles/10.3389/fncel.2018.00488/full https://www.frontiersin.org/articles/10.3389/fncel.2018.00488/full https://www.mdpi.com/2073-4409/11/21/3421 https://www.mdpi.com/2073-4409/11/21/3421 structurally diverse molecules, including trem2, cd33, cd35, etc.8 these receptors are responsible for recognition and response to aβ.8 2.1.4 current target genes apoe4: this gene variant is a risk factor for alzheimer’s disease. apoe expression produces a protein that helps move cholesterol in the bloodstream.9 while the exact mechanism of apoe4 is not fully known, di�culties with a brain cell’s ability to process fats may play an important role in alzheimer's disease. app: this gene encodes amyloid precursor protein, the precursor for amyloid-b peptides.10 the cleavage of app forms amyloid beta plaques which aggregate in the brain in ad. psen1 and psen2: these genes encode for a large component of the protein y-secretase, which is responsible for splicing and processing of app to form amyloid b peptides.11 the accumulation of amyloid beta plaques is prominently seen in those with ad. trem2: (triggering receptor expressed on myeloid cells 2): this gene provides instruction for a cell surface receptor found on microglia, immune cells in the brain. variations in trem2 have been associated with an increased risk of developing ad. trem2 is responsible for various functions in microglia including cell survival, phagocytosis, etc.12 reports indicate that trem2 de�ciency results in the decreased presence of microglia around aβ plaques.12 due to the plethora of mutations within the trem2 gene resulting in the progression of ad, precision medicine can help target speci�c genetic subgroups. by catering to these speci�c trem2 mutations, a more e�ective treatment plan can be constructed. 2.1.5 current treatments for alzheimer’s disease the current treatments for alzheimer’s include cholinesterase inhibitors (donepezil and galantamine), antipsychotics (brexpiprazole), and disease-modifying immunotherapies (lecanemab and aducanumab).13 besides the immunotherapies, these medications address the symptoms of alzheimer’s disease rather than attacking the disease at its source. meanwhile, the immunotherapies help remove amyloid plaques, but do not berkeley pharma tech journal of medicine | 18 https://www.mdpi.com/2073-4409/11/21/3421 https://www.mdpi.com/2073-4409/11/21/3421 https://www.ncbi.nlm.nih.gov/pmc/articles/pmc6463297/ https://www.ncbi.nlm.nih.gov/pmc/articles/pmc5453386/ https://pubmed.ncbi.nlm.nih.gov/24927704/#:~:text=presenilin%201%20(psen1)%20and%20presenilin,formation%20of%20amyloid%2d%ce%b2%20peptides. https://molecularneurodegeneration.biomedcentral.com/articles/10.1186/s13024-018-0247-7 https://molecularneurodegeneration.biomedcentral.com/articles/10.1186/s13024-018-0247-7 https://www.nia.nih.gov/health/how-alzheimers-disease-treated stop the plaques from accumulating or the amyloids from forming. as revealed by extensive research, the pathophysiology of ad is incredibly nuanced with various factors. current treatments are not a cure-all; more targeted treatments personalized to the root causes for each patient are necessary for prevention and mitigation. 2.2 schizophrenia schizophrenia, unlike neurodegenerative diseases like alzheimer’s disease, is a neurological disorder commonly known as a mental illness impacting how a person acts, feels, and behaves, thus not only a�ecting themselves, but also the individual’s relationships with those around them.14 coined by eugen bleuler in 1908, schizophrenia in more professional terms is a functional psychotic disorder characterized by the experience of delusional beliefs, hallucinations, and disturbances in thought, perception, and behavior.15 schizophrenia can be diagnosed through classi�cation systems, of which the diagnostic and statistical manual of mental disorders 4 (dsm-4) and international classi�cation of diseases (icd-10) are most commonly used. for the dsm-4: two or more of the following symptoms must be present for a signi�cant portion of time during a one-month period16: ● delusions ● hallucinations ● disorganized speech ● grossly disorganized or catatonic behavior ● negative symptoms. there must also be social/occupational dysfunction. continued negative symptoms and disturbances must last for at least six months, with at least 1 month of active-phase symptoms described above. on the other hand, the icd-10 has sub-categories for schizophrenia based on presenting symptoms: berkeley pharma tech journal of medicine | 19 https://www.nimh.nih.gov/health/topics/schizophrenia https://www.ncbi.nlm.nih.gov/books/nbk539864/ https://www.ncbi.nlm.nih.gov/pmc/articles/pmc3181977/ one of the following symptoms, for a period greater than or equal to a month: ● thought insertion, echo, broadcast, or withdrawal ● delusions of control, in�uence, or passivity ● hallucinatory voices providing a running commentary of the patient ● persistent delusions that are culturally inappropriate or implausible or, at least two of the following symptoms for a period greater than or equal to a month16: ● persistent hallucinations in any modality when accompanied by �eeting delusions ● breaks of interpolations in thought resulting in incoherence or neologisms ● catatonic behavior ● negative symptoms ● signi�cant and consistent transformation in the overall quality of behavior manifesting as anhedonia and social withdrawal these detailed criteria allow for further characterizing schizophrenia into paranoid schizophrenia, hebephrenic schizophrenia, catatonic schizophrenia, undi�erentiated schizophrenia, post-schizophrenic depression, residual schizophrenia, and simple schizophrenia.16 schizophrenic symptoms can be classi�ed as positive symptoms, including hallucinations, delusions, and formal thought disorders, and negative symptoms such as anhedonia, poverty of speech, and lack of motivation.15 2.2.1 pathophysiology berkeley pharma tech journal of medicine | 20 https://www.ncbi.nlm.nih.gov/pmc/articles/pmc3181977/ https://www.ncbi.nlm.nih.gov/pmc/articles/pmc3181977/ https://www.ncbi.nlm.nih.gov/books/nbk539864/ the neurochemical abnormality hypothesis credits imbalance in dopaminergic, serotonergic, and alpha-adrenergic hyperactivity or glutaminergic and gaba hypoactivity.15 another hypothesis includes the involvement of the four main dopaminergic pathways with the highs resulting from hyperactive d2 receptors via the mesolimbic pathway. this theory also attributes motor symptoms to low dopamine levels in the nigrostriatal pathway due to how the extrapyramidal system is a�ected.15 reduced mesocortical dopamine levels in the mesocortical pathway result in negative symptoms.17 research has demonstrated the exacerbation of positive and negative symptoms in schizophrenia from nmda receptor antagonists, shining light on the potential role of glutamatergic hypoactivity.18 neuroanatomically, a ventricle enlargement and reduction of gray matter volume is typical in patients with schizophrenia.17 the genes neuregulin (ngr1) and dysbindin (dtnbp1), which are involved in glutamate signaling and glutamate release, have been implicated respectively, as well as a gene polymorphism that regulates dopamine function known as catecholamine o-methyltransferase (comt).15 higher levels of the immune protein c4 are also linked to an increased risk of developing the condition.19 interestingly, other factors such as advanced paternal age and the association with auto-immune diseases have been recent epidemiological �ndings for increased risk of schizophrenia.20 2.2.2. current treatments for schizophrenia mainstream medications for schizophrenia include an oral second-generation antipsychotic (sga) such as aripiprazole, olanzapine, risperidone, quetiapine, asenapine, lurasidone, sertindole, ziprasidone, brexpiprazole, molindone, iloperidone, etc.14 benzodiazepines such as diazepam, clonazepam, or lorazepam may also be prescribed to control behavioral disturbances and non-acute anxiety.14 apart from medication, cognitive behavioral therapy (cbt) can be used to address anxiety, depression, speech disabilities, and psychosis.14 however, while these medications aim to target speci�c neurotransmitters or certain symptoms, they fail to target the root issues that may emerge in di�erent ways for each patient. with multiple di�erent factors, neurological conditions such as schizophrenia cannot be addressed with a blanket solution. advances in berkeley pharma tech journal of medicine | 21 https://www.ncbi.nlm.nih.gov/books/nbk539864/ https://www.ncbi.nlm.nih.gov/books/nbk539864/ https://www.nature.com/articles/mp201247 https://www.ncbi.nlm.nih.gov/pmc/articles/pmc4159061/ https://www.nature.com/articles/mp201247 https://www.ncbi.nlm.nih.gov/books/nbk539864/ https://www.cell.com/stem-cell-reports/fulltext/s2213-6711(22)00551-3 https://www.ncbi.nlm.nih.gov/pmc/articles/pmc2727721/ https://www.nimh.nih.gov/health/topics/schizophrenia https://www.nimh.nih.gov/health/topics/schizophrenia https://www.nimh.nih.gov/health/topics/schizophrenia research uncovering schizophrenia pathophysiology reveal modulated receptors and neurotransmitters as possible targets for precision medicine through various methods, such as gene editing, pharmacogenomics, and ipsc models. 3. precision medicine precision medicine (pm) stands as a transformative paradigm, especially in neurology where diseases can exhibit dynamic and ever-changing characteristics. neurogenetic disease, in�uenced by the diverse genetic, environmental, and stochastic factors, eventually leads to aberrant biological pathways that unfold at varying rates. this variability is further compounded by compensation mechanisms, resulting in subtle changes in physiological functions and behavioral performance. in the realm of neurology, pm has the potential to revolutionize healthcare by shifting the focus from diagnostic work-ups and therapeutic interventions after clinical manifestation to proactive prevention and health prolongation serving as a screener, changing expectations toward prevention, and prolonging health.21 it should be recognized that current applications have limitations in regard to diversity that exists within the human genome. future integration of genome databases that re�ect the global population could change how precision medicine is administered universally and remove some of the barriers that currently exist within the �eld.21 rather than intervening therapeutically only when diseases manifest clinically, pm enables time-sensitive detection and diagnosis. treatment strategies can be tailored based on an individual's unique clinical, genetic, and biological characteristics, minimizing the risk and cost of unnecessary medical interventions, as demonstrated in figure 2. molecular changes, often the target of treatment, can be speci�cally addressed through pm, providing a more e�ective and e�cient healthcare approach. the proposed work�ow for pm in neurology involves a comprehensive approach to data collection, integration, and clinical decision-making.22 system biology and systems neurophysiology serve as major data sources, incorporating information from various modalities, including mri, pet, genomics, cytomics, and proteomics. digitally enabled data collection berkeley pharma tech journal of medicine | 22 https://www.ncbi.nlm.nih.gov/pmc/articles/pmc9086532/ https://www.ncbi.nlm.nih.gov/pmc/articles/pmc9086532/ https://www.cell.com/trends/neurosciences/fulltext/s0166-2236(22)00258-2#:~:text=precision%20medicine%20(pm)%20approaches%20in,biological%20characteristics%20and%20risk%20factors systems and large-scale population genomic analyses, such as genome-wide association studies (gwas), contribute to a holistic understanding of neurological diseases and are part of the model. digital data, gwas, systems biology, and systems neurophysiology are then integrated using the three key steps–data generation, data integration, and clinical approach–that form the foundation of pm in neurology. ai plays a crucial role in facilitating time-dependent analysis, longitudinal tracking, and clinical decision-making, addressing the complexity and diversity of neurological diseases. by analyzing this data on a population scale, researchers can identify patterns, correlations, and genetic markers associated with neurological diseases. this comprehensive approach allows for a deeper understanding of the complex interplay between genetics, environment, and disease manifestation. moreover, it enables researchers to uncover potential biomarkers, risk factors, and novel therapeutic targets, thus providing a more holistic view of neurological disorders beyond just their symptoms or genetic components. the data system operates by collecting, storing, and analyzing vast amounts of genomic and clinical data from diverse populations. through sophisticated algorithms and analytical techniques, researchers can identify genetic variants associated with neurological diseases, understand their underlying mechanisms, and predict disease risk in individuals. this knowledge can inform personalized treatment approaches tailored to a patient's genetic pro�le, lifestyle, and environmental factors, leading to more e�ective diagnoses and treatment plans for neurological conditions. berkeley pharma tech journal of medicine | 23 figure 2. a diagram showing how precision medicine di�ers from traditional medicine by designing treatment plans that are individualized to target the speci�c patient. the use of general procedures versus targeted treatments is at the discretion of the physician and the availability of treatment plans for that particular patient, as the �eld advances, targeted treatment plans are expected to be more prevalent, providing a more holistic approach to addressing neurological disease. 4. challenges and limitations of precision medicine despite the promises of pm, there are notable challenges and limitations. “black box” medicine refers to untransparent computational models in healthcare decision-making.23 traditional approaches rely on explicit understanding of biological mechanisms such as through clinical trials, whereas “black box” medicine utilizes large-scale datasets and sophisticated algorithms to uncover complex and often hidden relationships among multiple patient characteristics. unlike traditional personalized medicine, which relies on explicit understanding of simple relationships, “black box” medicine delves into intricate networks of variables, such as observable factors like age and sex, as well as less obvious ones like genomic markers. “black box” medicine represents a shift towards leveraging the power of big data and advanced algorithms to improve healthcare outcomes, but it requires careful navigation of technical, ethical, and regulatory complexities. berkeley pharma tech journal of medicine | 24 https://jolt.law.harvard.edu/articles/pdf/v28/28harvjltech419.pdf first, the “black box” issue in the underlying ai models raises immense concerns about the lack of transparency and understanding of model outputs.23 current ai approaches may reveal systems complexities without elucidating the underlying reasons, making interpretation and clinical decision-making di�cult. data standardization and curation challenges can also arise, particularly in large-scale, multimodal data collection and monitoring. addressing the complexities of algorithms in precision medicine necessitates a multifaceted approach. transparency and interpretability are crucial for understanding algorithmic decisions, even in black-box systems. rigorous validation and evaluation procedures ensure reliability and e�ectiveness, while ethical considerations guide fair and equitable outcomes. regulatory oversight, continual monitoring, and interdisciplinary collaboration further enhance algorithmic development and deployment. by navigating these challenges thoughtfully, stakeholders can harness the potential of algorithms to advance precision medicine while safeguarding patient interests and promoting public health.23 additionally, the transition from gene-gene association analyses to multi-omics analyses in systems biology poses di�culties that must be addressed for the e�ective implementation of pm in neurology.23 furthermore, the successful implementation of precision medicine encounters several other hurdles, encompassing ethical considerations, the impact of stigma, and potential issues related to cost-e�ectiveness. ethical components crucial to the correct application of precision medicine involve con�dentiality and privacy concerns, particularly regarding the analysis of massive datasets.24 this requires the development of a robust ethical-legal framework to ensure secure data sharing. both public and self-stigma could in�uence the acceptance of precision medicine, potentially a�ecting public health policies and patient participation.24 the problem of cost-e�ectiveness in precision medicine lies at the intersection of evolving medical technologies and economic evaluations. while precision medicine holds the promise of tailoring treatments to individual patients, the economic viability of these interventions faces multifaceted challenges. economic evaluations, typically assessing the cost per quality-adjusted life year (qaly) gained, encounter di�culties in capturing the true value of precision berkeley pharma tech journal of medicine | 25 https://jolt.law.harvard.edu/articles/pdf/v28/28harvjltech419.pdf https://jolt.law.harvard.edu/articles/pdf/v28/28harvjltech419.pdf https://jolt.law.harvard.edu/articles/pdf/v28/28harvjltech419.pdf https://www.ncbi.nlm.nih.gov/pmc/articles/pmc7186890/ https://www.ncbi.nlm.nih.gov/pmc/articles/pmc7186890/ medicine.25 the scarcity of robust clinical and cost data, particularly in real-world settings, poses a signi�cant hurdle. additionally, the lack of standardized willingness-to-pay thresholds and the varying perspectives adopted in economic analyses contribute to the ambiguity surrounding the cost-e�ectiveness of precision medicine.25 this all raises questions about the long-term sustainability of these approaches in public healthcare. 5. speci�c strategies and methods 5.1 ipsc with rapid and advanced-paced technology, new avenues have opened to treating neurogenetics disorders with stem cell treatment. for example, induced pluripotent stem cells (ipsc) are a type of stem cell derived from adult cells that have the potential to develop into di�erent types of cells in the body.26 these cells are created through a process called reprogramming which involves the introduction of speci�c genes into a specialized cell to reprogram it back to a state similar to that of an embryonic stem cell. because these ipscs are derived from the patients themselves, genetic information and characteristics unique to the individual can be transferred to the newly reprogrammed stem cell. by personalizing therapies, scientists can work toward creating treatments targeting speci�c underlying causes. an ongoing clinical trial derives ipscs from somatic cells from individuals with neurological disease in hopes of creating a line of cells for modeling diseases and drug discovery.27 5.1.1 alzheimer’s disease (ad) studies show ipscs have enormous potential for treating neurological diseases such as alzheimer’s disease and schizophrenia.28 in one study ipscs derived from autologous, mouse skin �broblasts injected into subiculum resulted in the decrease of alpha beta plaques deposition and beta/gamma secretase activity. these mouse models have immense impact in understanding the underlying mechanisms for ad; however, while mice are evolutionarily similar to humans, stark di�erences remain due to only 50% of microglial genes being identical between the two species.29 due to such limitations of modeling age-related neurodegenerative aspects of ad in dividing cells of mice, ongoing e�orts focus on optimizing protocols for berkeley pharma tech journal of medicine | 26 https://www.ncbi.nlm.nih.gov/pmc/articles/pmc6867980/ https://www.ncbi.nlm.nih.gov/pmc/articles/pmc6867980/ https://www.intechopen.com/chapters/85399 https://clinicaltrials.gov/study/nct00874783?cond=alzheimers%20disease&intr=ipsc&rank=1 https://www.ncbi.nlm.nih.gov/pmc/articles/pmc8869146/#:~:text=a%20number%20of%20studies%20demonstrated,into%20glial%20cells%20upon%20implantation https://www.nature.com/articles/s41380-019-0468-3 the creation of induced pluripotent stem cells (ipscs) tailored for human applications. 5.1.2 schizophrenia one study used human induced pluripotent stem cells (hipsc) from individuals with high genetic risk for schizophrenia and those without, and found di�erences in how these cells behaved and functioned in people with schizophrenia. these di�erences in cell behavior matched some of the main symptoms seen in people with schizophrenia.30 the identi�cation of neurophysiological measures via hipsc associated with the patient’s personal clinical characteristics is signi�cant for generating novel therapeutics. 5.2 genetic pro�ling genetic pro�ling involves studying an individual's dna to identify speci�c variations (mutations or polymorphisms) in genes associated with neurological disorders.31 identifying speci�c genetic variations associated with the disease allows for personalized risk assessment. 5.2.1 alzheimer’s disease speci�c genetic variations associated with alzheimer's risk or progression can provide valuable information about a person's susceptibility to alzheimer's and their risk of developing the disease, allowing for a personalized approach to treating the neurodegenerative disease. for example, certain variations in genes like apoe and mutations within the ps-1, ps-2, and app genes are known to increase the risk of developing alzheimer's in families with multiple individuals a�ected by ad. studying these variations helps estimate an individual's likelihood of developing alzheimer's based on their genetic makeup, allowing for implementation of proactive measures and lifestyle changes that may help reduce the risk or delay the onset of alzheimer's. an example of this approach can be seen through genetic linkage analysis, where the researchers investigated the in�uence of the apoe, app, ps1, and ps-2 genes on the increased risk of ad in families where multiple individuals were already diagnosed with the disease.32 berkeley pharma tech journal of medicine | 27 https://www.pnas.org/doi/epdf/10.1073/pnas.2109395119 https://www.ncbi.nlm.nih.gov/pmc/articles/pmc4157398/ https://clinicaltrials.gov/study/nct05010603?cond=alzheimers%20disease&intr=genetic%20testing&rank=3 5.2.2 schizophrenia schizophrenia is believed to have a complex genetic component.33 by studying an individual's genetic makeup, researchers aim to identify speci�c genetic markers or variations that may increase the risk of developing schizophrenia. this information can be used to provide personalized risk assessments, allowing for early preventative measures. one study used rna sequencing (lcm-seq) on a speci�c area called the granule cell layer in the hippocampus, which is important for memory.34 this area mainly has a type of brain cell called granule neurons, and they make up a small part of the whole hippocampus. problems with these neurons have been linked to conditions like bipolar disorder and schizophrenia. the results posed a clear picture of which gene variants pose a risk for schizophrenia compared to more general methods. 5.3 pharmacogenomics pharmacogenomics involves studying how an individual's genetic makeup in�uences their response to medications.35 genetic variations can a�ect how drugs are absorbed, metabolized, and utilized in the body. tailoring drug choices maximizes e�ectiveness and minimizes side e�ects. this method helps identify drugs that may not be well-tolerated by a speci�c individual due to their unique genetic makeup. 5.3.1 alzheimer’s disease in the context of alzheimer's, pharmacogenetics is crucial for optimizing medication selection and dosages. studies have shown how an ad patient’s biological response to drugs depends on speci�c gene clusters that in�uence how drugs are received in the body.36 pharmacogenetic research indicates that the e�ectiveness of drug therapies for alzheimer's disease (ad) varies depending on genetic makeup, which is closely linked to gene clusters associated with pharmacogenetic processes. in the course of the study, a signi�cant proportion of ad cases exhibit an accumulation of 15 to 26 defective pharmagenes, a�ecting the metabolism of drugs through enzymes. pharmacogenetics holds promise in enhancing drug development and maximizing the e�ectiveness of available therapeutic options for ad by optimizing patient care and therapeutic outcomes.37 berkeley pharma tech journal of medicine | 28 https://www.ncbi.nlm.nih.gov/pmc/articles/pmc3433970/ https://www.nature.com/articles/s41593-020-0604-z https://www.nigms.nih.gov/education/fact-sheets/pages/pharmacogenomics.aspx#:~:text=pharmacogenomics%20(sometimes%20called%20pharmacogenetics)%20is,or%20she%20responds%20to%20medications. https://www.alz.org/alzheimers-dementia/treatments/medications-for-memory https://link.springer.com/protocol/10.1007/978-1-0716-2573-6_13#abs1 leveraging pharmacogenetics can enhance the e�ciency of drug development and maximize the constrained therapeutic resources dedicated to alzheimer's disease (ad). this approach enables the personalized utilization of anti-dementia medications, both independently and in combination with other drugs tailored to address concurrent disorders. 5.3.2 schizophrenia schizophrenia can be treated through utilizing pharmacogenomics to analyze how genetic variations in�uence an individual's response to medications commonly used to treat the disease, such as antipsychotic drugs. this enables the selection of drugs and dosages that are most e�ective while minimizing potential side e�ects. while antipsychotics are the main treatment for schizophrenia, many have adverse reactions towards these medications and the alternative is to use a trial and error method to �nd an e�ective treatment plan. the drawbacks to this are delays in treatment and overall worsening the disease condition. one study aimed to research the therapeutic e�cacy of multigenic pharmacogenomics-guided treatment in patients with schizophrenia. the results displayed that patients treated with mpct had greater symptom improvement than their counterpart, patients treated with the standard 6-week treatment. this highlights the e�ectiveness of pharmacogenomics as a potential treatment plan for those with schizophrenia.38 6. conclusion/future directions precision medicine is the future to address neurological diseases due to addressing the genetic variance in patients that cause similar symptoms but require di�erent treatment plans. personalized care through a variety of treatment options that are based on the patient’s genetic pro�le provides an opportunity to drastically improve their quality of life by treating the disease on a molecular level rather than only the symptoms. currently, the accessibility and cost of precision medicine are limiting factors for it to be a realistic treatment plan for the masses. however, as the �eld continues to advance through continued research in pharmacogenomics, metabolomics, and novel treatments such as ipscs, precision can become normalized as a berkeley pharma tech journal of medicine | 29 https://jamanetwork.com/journals/jamanetworkopen/article-abstract/2810261 more e�ective strategy to cure neurological diseases, and costs will presumably be reduced as the technology becomes more widespread. berkeley pharma tech journal of medicine | 30 references 1. traynor bj, al-chalabi a. the neurogenetics collection: emerging themes and future considerations for the �eld in brain. brain. 2022;145(5). doi:10.1093/brain/awac120 2. precision medicine. genome.gov. accessed december 6, 2023. https://www.genome.gov/genetics-glossary/precision-m edicine. 3. understanding human genetic variation nih curriculum supplement ... accessed december 7, 2023. https://www.ncbi.nlm.nih.gov/books/nbk20363/. 4. abijo t, blum k, gondré-lewis mc. neuropharmacological and neurogenetic correlates of opioid use disorder (oud) as a function of ethnicity: relevance to precision addictionmedicine. current neuropharmacology. 2020;18(7):578-595. doi:10.2174/1570159x17666191118125702 5. national institute on aging. what is alzheimer's disease? national institute on aging. https://www.nia.nih.gov/health/what-alzheimers-disease. accessed december 6, 2023. 6. bloomgs. amyloid-β and tau. jamaneurology. 2014;71(4):505. doi:10.1001/jamaneurol.2013.5847 7. bachiller s, jiménez-ferrer i, paulus a, et al. microglia in neurological diseases: a road map to brain-disease dependent-in�ammatory response. frontiers in cellular neuroscience. 2018;12. doi:10.3389/fncel.2018.00488 8. busch l, eggert s, endres k, bufe b. the hidden role of non-canonical amyloid β isoforms in alzheimer's disease. cells. 2022;11(21):3421. doi:10.3390/cells11213421 9. kunkle bw, grenier-boley b, sims r, et al. genetic meta-analysis of diagnosed alzheimer’s disease identi�es new risk loci and implicates aβ, tau, immunity and lipid processing.nature genetics. 2019;51(3):414-430. doi:10.1038/s41588-019-0358-2 10. tcw j, goate am. genetics of β-amyloid precursor protein in alzheimer’s disease. cold spring harbor perspectives inmedicine. 2016;7(6). doi:10.1101/cshperspect.a024539 11. delabio r, rasmussen l, mizumoto i, et al. psen1 and psen2 gene expression in alzheimer's disease brain: a new approach. journal of alzheimer’s disease. 2014;42(3):757-760. doi:10.3233/jad-140033 12. zhong l, wang z, wang d, et al. amyloid-beta modulates microglial responses by binding to the triggering receptor expressed on myeloid cells 2 (trem2). molecular neurodegeneration. 2018;13(1). doi:10.1186/s13024-018-0247-7 13. national institute on aging. how is alzheimer's disease treated? national institute on aging. https://www.nia.nih.gov/health/how-alzheimers-disease -treated. accessed december 6, 2023. 14. national institute of mental health. schizophrenia. national institute of mental health. https://www.nimh.nih.gov/health/topics/schizophrenia. accessed december 6, 2023. 15. schizophrenia statpearls ncbi bookshelf. accessed december 7, 2023. https://www.ncbi.nlm.nih.gov/books/nbk539864/. 16. jablensky a. the diagnostic concept of schizophrenia: its history, evolution, and future prospects.dialogues in clinical neuroscience. 2010;12(3):271-287. doi:10.31887/dcns.2010.12.3/ajablensky 17. miyamoto s, miyake n, jarskog lf, fleischhacker berkeley pharma tech journal of medicine | 31 ww, lieberman ja. pharmacological treatment of schizophrenia: a critical review of the pharmacology and clinical e�ects of current and future therapeutic agents. molecular psychiatry. 2012;17(12):1206-1227. doi:10.1038/mp.2012.47 18. patel kr, cherian j, gohil k, atkinson d. schizophrenia: overview and treatment options. p t. 2014;39(9):638-645. 19. rapino f, natoli t, limone f, et al. small-molecule screen reveals pathways that regulate c4 secretion in stem cell-derived astrocytes. stem cell reports. 2023;18(1):237-253. doi:10.1016/j.stemcr.2022.11.018 20. messias el, chen cy, eatonww. epidemiology of schizophrenia: review of �ndings and myths. psychiatr clin north am. 2007;30(3):323-338. doi:10.1016/j.psc.2007.04.007 21. martschenko do, young jl. precisionmedicine needs to think outside the box. front genet. 2022;13:795992. published 2022 apr 26. doi:10.3389/fgene.2022.795992 22. hampel h, gao p, cummings j, et al. the foundation and architecture of precisionmedicine in neurology and psychiatry. trends in neurosciences. 2023;46(3):176-198. doi:10.1016/j.tins.2022.12.004 23. crouch, dennis, black-boxmedicine, 28 harv. j.l. & tech. 419 (2015). https://jolt.law.harvard.edu/articles/pdf/v28/28harvjl tech419.pdf 24. manchia m, pisanu c, squassina a, carpiniello b. challenges and future prospects of precisionmedicine in psychiatry. pharmgenomics pers med. 2020;13:127-140. published 2020 apr 23. doi:10.2147/pgpm.s198225 25. kasztura m, richard a, bempong ne, loncar d, flahault a. cost-e�ectiveness of precision medicine: a scoping review. int j public health. 2019;64(9):1261-1271. doi:10.1007/s00038-019-01298-x 26. kizub i, rozhok a, bilousova g. induced pluripotent stem cells: advances and applications in regenerative medicine. intechopen. 2023. doi: 10.5772/intechopen.109274. 27. 1. ctg labs ncbi. accessed december 7, 2023. https://clinicaltrials.gov/study/nct00874783?cond=a lzheimers+disease&intr=ipsc&rank=1. 28. yefroyev da, jin s. induced pluripotent stem cells for treatment of alzheimer's and parkinson's diseases. biomedicines. 2022;10(2):208. published 2022 jan 19. doi:10.3390/biomedicines10020208 29. penney j, ralvenius wt, tsai lh.modeling alzheimer’s disease with ipsc-derived brain cells.mol psychiatry. 2020;25:148-167. doi:10.1038/s41380-019-0468-3 30. page sc, sripathy sr, farinelli f, et al. electrophysiological measures from human ipsc-derived neurons are associated with schizophrenia clinical status and predict individual cognitive performance. proc natl acad sci u s a. 2022;119(3). doi:10.1073/pnas.2109395119 31. abul-husn ns, owusu obeng a, sanderson sc, gottesman o, scott sa. implementation and utilization of genetic testing in personalized medicine. pharmacogenomics pers med. 2014;7:227-240. https://www.ncbi.nlm.nih.gov/pmc/articles/pmc4157 398/ 32. ctg labs ncbi. accessed december 7, 2023. https://classic.clinicaltrials.gov/ct2/show/nct0501060 3 33. salleh mr. the genetics of schizophrenia. malays j med sci. 2004;11(2):3-11. berkeley pharma tech journal of medicine | 32 https://www.ncbi.nlm.nih.gov/pmc/articles/pmc3433 970/ 34. ja�e ae, hoeppner dj, saito t, et al. pro�ling gene expression in the human dentate gyrus granule cell layer reveals insights into schizophrenia and its genetic risk. nature neuroscience. 2020;23(4):510-519. doi:10.1038/s41593-020-0604-z 35. u.s. department of health and human services. pharmacogenomics. national institute of general medical sciences. https://www.nih.gov/about-nih/what-we-do/nih-turnin g-discovery-into-health/promise-precision-medicine/pha rmacogenomics. accessed december 6, 2023. 36. medications for memory, cognition and dementia-related behaviors. alzheimer’s disease and dementia. https://www.alz.org/alzheimers-dementia/treatments/m edications-for-memory. accessed december 6, 2023. 37. cacabelos r, et al. pharmacogenomics of alzheimer’s disease: novel strategies for drug utilization and development. in: yan q, editor. pharmacogenomics in drug discovery and development.methods inmolecular biology. vol 2547. new york, ny: humana; 2024. p. 275-310. available from: https://doi.org/10.1007/978-1-0716-2573-6_13. 38. kang z, qin y, sun y, et al. multigenetic pharmacogenomics–guided treatment vs treatment as usual among hospitalized men with schizophrenia. jamanetwork open. 2023;6(10). doi:10.1001/jamanetworkopen.2023.35518 berkeley pharma tech journal of medicine | 33 galley 2_srinivasan et al..pdf 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(1).pdf chang references berkeley pharma tech journal of medicine correspondence: yoeu@ucsd.edu keywords: neurovascular coupling dementia alzheimer's disease parkinson's disease vascular dysfunction published july 31, 2025 full open access creative commons attribution license 4.0 abstract cognitive decline—including dementia, alzheimer’s disease, and parkinson’s disease—is a growing global health concern. these neurodegenerative disorders affect approximately 50 million people worldwide. in the united states, nearly two out of three seniors experience some form of cognitive impairment. since the 1990s, the prevalence of mild cognitive impairment has been rising in the u.s. and is projected to continue increasing for future decades. these alarming trends underscore the urgent need for more effective methods of early detection and treatment that address the physical, emotional, and cognitive needs of affected individuals. emerging evidence suggests that neurovascular coupling (nvc)— the critical process linking brain activity to dynamic blood flow—plays an essential role in regulating brain energetics and function, and may contribute to cognitive dysfunction when impaired. this review delves into the characteristics of nvc and the neurovascular unit (nvu), examines early detection strategies targeting nvc-related biomarkers, and discusses current and investigational treatments for nvc-associated cognitive disorders. the pulse of cognition: investigating neurovascular coupling's role in cognition and therapeutic promise for alzheimer’s, parkinson’s, and dementia by: irene eu, cindy lu, kalp soni and evan lee 1. introduction 1.1 cognitive decline cognitive decline is an escalating public health concern, driven in part by the aging global population. data show that nearly 20% of adults aged 50 and older worldwide experience some degree of cognitive decline.¹ in the united states, approximately two out of three adults aged 70 or older have some form of cognitive impairment, while one in three individuals aged 85 or older suffers from more severe conditions such as alzheimer’s disease.2,3 these statistics highlight the urgent need for comprehensive interventions. effective strategies include pharmacological treatments, nutraceutical approaches, and vascular-targeted therapies aimed at addressing both the underlying causes and the consequences of cognitive decline. figure 1: dynasim projection of adults with cognitive impairments from 2020 to 2060 cognitive decline encompasses a range of conditions, including common neurodegenerative diseases such as alzheimer’s disease (ad), parkinson’s disease, and various forms of dementia. ad, for example, involves impairments across multiple cognitive domains, affecting memory, reasoning, language, coordination, mood, and behavior.4 similarly, parkinson’s disease presents a range of non-motor and motor symptoms, including a diminished sense of smell, digestive issues, and drooling, berkeley pharma tech journal of medicine | 122 alongside difficulties in executing normal movements, experiencing tremors, and maintaining a stable walking posture.5 dementia is a broader term that refers to the progressive deterioration of memory and other cognitive functions, significantly interfering with an individual’s ability to perform daily activities. 6 collectively, these conditions underscore the multifaceted nature of cognitive decline and emphasize the need to address these diseases. as cognitive decline progresses, impairments in neuronal activity can lead to inadequate perfusion of vital brain regions, one of the main causes of neuronal damage and cognitive impairments. therefore, understanding the interplay between cognitive decline and neurovascular coupling is crucial for developing effective strategies to preserve brain function and slow the progression of neurodegenerative diseases. 1.2 neurovascular coupling nvc refers to the process by which the metabolic demands of active neurons trigger localized changes in cerebrovascular blood flow (cbf). this mechanism ensures that the brain receives adequate oxygen and nutrients during periods of increased neural activity, thereby maintaining optimal perfusion in response to fluctuating neuronal demands.7 1.2.1 neurovascular unit the regulation of nvc is coordinated by the neurovascular unit (nvu), a complex assembly of neurons, glial cells, and vascular cells. glial cells—such as astrocytes and microglia—play essential roles in supporting and protecting neurons. they also contribute to vasculogenesis (the formation of new blood vessels from endothelial progenitor cells) and the development of synapses.8 vascular cells, including endothelial cells, help maintain the blood-brain barrier (bbb), sense mechanical and hormonal signals to regulate cbf. these cells also release substances, like proteoglycans and glycoproteins, to adapt blood flow accordingly. vascular smooth muscle cells adjust cbf by modulating vascular tone and capillary diameter in response to signals from endothelial cells. likewise, pericytes influence cbf by altering capillary diameter in response to neurotransmitters such as noradrenaline and glutamate. this interconnected network ensures the delivery of essential nutrients, such asglucose and oxygen, to sustain the berkeley pharma tech journal of medicine | 123 energy-demanding brain activity, while also maintaining cerebrovascular homeostasis.9 figure 2: neurovascular coupling mechanism. stimulation triggers glutamate secretion, activating neurons and astrocytes to release nitric oxide (no), potassium (k+), adenosine (ado), epoxyeicosatrienoic acids (eet), and prostaglandins (pge2), leading to arteriolar vasodilation. astrocytes also release arachidonic acid (aa), causing vasoconstriction, and pericytes are stimulated separately, leading to capillary dilation. berkeley pharma tech journal of medicine | 124 figure 3: the neurovascular unit. the neurovascular unit (nvu) consists of endothelial cells, pericytes, astrocytes, neurons, and the extracellular matrix. the nvu balances the brain microenvironment, regulates cerebral blood flow, facilitates immune cell movement, produces growth factors for cell survival, and aids in clearing harmful brain byproducts, crucial for brain health. berkeley pharma tech journal of medicine | 125 figure 4: the blood-brain barrier. vascular stresses affect the blood-brain barrier, which regulates brain perfusion. conditions like hypertension damage the endothelium, disrupting the blood-brain barrier, causing microhemorrhages, inflammation, and neuronal injury. this process contributes to cognitive decline and dementia. 1.3 purpose of study building upon our review of cognitive decline, we propose that nvc plays a critical regulatory role in neurovascular diseases associated with cognitive impairment. this study aims to identify early signs of cognitive decline by targeting relevant biomarkers and to explore treatment options for conditions linked to nvc dysfunction. specifically, we seek to understand how changes in neuronal activity affect blood vessel function and cerebral blood flow (cbf) in cognitive disorders such as ad, parkinson’s disease, and dementia. 2. histopathological features of nvc-related diseases histopathological markers of nvc-related diseases, including alzheimer’s and vascular dementia, can be observed through tissue imaging techniques. particularly, the well-known biomarkers of neurofibrillary tangles and β-amyloid plaque is associated with alzheimer’s, while neuronal tissue death and myelin breakdown is associated with vascular dementia.10 it is berkeley pharma tech journal of medicine | 126 hypothesized that vascular abnormalities contribute to the early progression of cognitive disorders and precede the emergence of these histopathological features.11 for instance, one study found that patients with vascular dementia have significant hippocampal neuronal loss due to microvascular pathology.12 these findings support a correlation between vascular dysfunction and development of key features of cognitive disorders. 2.1 associated biomarkers 2.1.1 pericyte detachment pericytes are instrumental in maintaining the nvu, as they are positioned in the unit between endothelial cells, astrocytes, and neurons. they contribute not only to the structural maintenance of blood vessels but also to the regulation of molecular waste clearance, cell signaling, and cerebral blood flow. for example, pericytes influence the activity of proteins that preserve the tight junctions of the blood–brain barrier (bbb) and modulate the transport of fluid-filled vesicles across it.12 in the cases of higher blood pressure, which can stretch the vein and weaken it, pericyte detachment from the unit is promoted. pericyte detachment from around the circumference of the vein can lead to bbb permeability, neuroinflammation, and issues with neuronal communication, ultimately resulting in cognitive dysfunction. additionally, recent studies have implicated pericyte detachment with hypoxia and loss of myelination, which leads to the loss of essential connections within the brain.13 2.1.2 genetic factors the apoeε4 allele, located on chromosome 19, has often been described as the greatest genetic risk factor for alzheimer’s. although its exact role in disease pathology remains incompletely understood, it is known to be involved in lipid transport within the brain and the production of β-amyloid and tau proteins—key biomarkers of alzheimer’s disease. emerging research suggests that apoeε4 also disrupts neurovascular regulation and exacerbates cognitive decline. individuals carrying this allele exhibit marked pericyte deficiency—approximately 50% fewer pericytes compared to individuals without the allele—and increased blood–brain barrier breakdown.14 the correlation between apoeε4 with protein berkeley pharma tech journal of medicine | 127 aggregate formation and nvc regulation supports our hypothesis that the nvc contributes to cognitive decline.15 2.1.3 vascular dysfunction given the nvu’s intimate relationship with brain function, vascular dysfunction as a whole can act as a root for brain dysfunction. the “2-hit vascular hypothesis model”, proposed by berislav v. zlokovic, implicates vascular dysfunction in exacerbating cognitive decline.16 the “first hit” are risk factors such as aging, atherosclerosis, hypertension, diabetes, or stroke, all of which jeopardize or breakdown vascular integrity and alter cerebral blood flow. the “second hit” refers to the increased aβ protein levels, inflammation, and tau protein levels that result, which causes even further decline. this model describes a positive feedback loop between vascular and brain dysfunction: injury to the vascular system can accelerate neurodegeneration, while neuronal damage can, in turn, worsen vascular health.17 consequently, vascular markers may serve as valuable early indicators for the prevention and early-stage intervention of neurodegenerative diseases. berkeley pharma tech journal of medicine | 128 figure 5: 2-hit hypothesis. 2-hit hypothesis model shows that vascular factors and genetic factors discussed before serve as predisposing factors to ‘hit 1 vascular damage,’ which causes bbb breakdown and ‘hit 2’: physiological manifestations of cognitive decline such as protein aggregation and inflammation. these hits cause significant neurodegeneration and also (not pictured) a positive feedback loop of brain and vascular damage. 2.1.4 blood-brain barrier breakdown the blood-brain barrier (bbb) regulates the exchange of compounds between the blood and the brain by selectively transporting solutes through active transport mechanisms and passive diffusion. these include organic compounds such as vitamins, hormones, and macromolecules necessary for brain neuron functions. different levels of stress such as hypertension or vascular recanalization can cause the breakdown of the barrier. not only does the breakdown of the barrier result in the loss of its functions, but it also allows for the release of neurodegenerative compounds such as protein aggregates and pathogens that are absorbed by the blood to the brain which leads to neurovascular coupling-related diseases. bbb breakdown is also associated with aging, berkeley pharma tech journal of medicine | 129 which progressively alters its permeability. given the bbb’s close functional relationship with pericytes, astrocytes, and neurons, its failure is often accompanied by dysfunction in these associated cells—providing a broader pathological context for detection and diagnosis. 3. novel neuroimaging techniques recent advances in medical imaging have paved the way for the detection of biomarkers and early disease indicators. neuroimaging plays a critical role in diagnosing neurodegenerative diseases, enabling clinicians to detect early signs of cognitive impairment, abnormalities in cerebral blood flow, and evidence of stroke, while also facilitating ongoing disease monitoring. this section explores several state-of-the-art techniques, including in-vivo photon imaging, dynamic vessel analyzer (dva), multimodal imaging platforms, fmri, and fnirs. 3.1 in-vivo photon imaging in-vivo photon imaging is a powerful technique that allows for deep-tissue visualization beyond the superficial layers of the dorsal brain. it has been instrumental in identifying correlations between external factors—such as dietary salt intake—and internal biomarkers, like elevated tau protein levels, a potential indicator of neurodegeneration. two-photon imaging is a specific type of in-vivo photon imaging technique. this specific technique uses two photons of lower energy to excite a fluorescent molecule. compared to single photon imaging techniques, it allows deeper tissue penetration and provides high-resolution images with less photodamage. researchers employ two-photon microscopy to track changes in blood vessel diameter, blood flow, and oxygenation levels in response to neuronal activity.18 berkeley pharma tech journal of medicine | 130 figure 6: schematic of a 2pf microscope. a two-photon fluorescence microscope utilizes a femtosecond laser to send light through xy-scan mirrors, scan and tube lenses, focusing on a sample with an objective lens. this induces fluorescence in the sample, which is then collected back through the objective lens, filtered by a dichroic mirror, and detected by a photomultiplier tube, ultimately translating into an image by a computer. 3.2 dynamic vessel analyzer in one study, patients with varying degrees of alzheimer’s disease were compared to controls by assessing the retinal blood vessel response to flickering lights. retinal vasculature is a valid proxy for cerebral blood flow since they share anatomical and physiological features that stem from a shared embryonic origin. to assess these responses, scientists used the dynamic vessel analyzer (dva)—a novel imaging tool that captures microvascular dynamics in the eye. as a non-invasive proxy for cerebral vasculature, the dva provides valuable insights into neurovascular function. results revealed that patients with moderate or mild dementia from alzheimer’s had emphasized arterial and venous dilation, as well as delayed arterial reaction to the flickering lights. these findings suggest that increased and delayed retinal neurovascular coupling, perhaps caused by damaged feedback loops or excessive activity of retinal neurons, is associated with alzheimer’s. as a non-invasive and accessible diagnostic tool, the dva berkeley pharma tech journal of medicine | 131 holds promise for monitoring retinal and vascular changes linked to alzheimer’s disease and may aid in tracking disease progression.19 figure 7: static analysis performed by the dynamic vessel analyzer. the technology gives valuable glimpses into arterioles (red segments) and venules (blue segments) 3.3 multimodality imaging platforms traditional high-resolution microscopy offers excellent detail but is limited by its small field of view. to overcome this limitation, researchers in one study employed a multimodality imaging approach to investigate the role of calcium ion (ca²⁺) fluctuations in astrocytes, key glial cells involved in maintaining the nvu. using electrical stimulation in mice, researchers observed that astrocytes exhibited a delayed response to stimulation, while cerebral blood flow increased rapidly. this finding suggests that astrocytes contribute to the coupling between neuronal activity and blood flow. additionally, the stimulation induced vasoconstriction—the narrowing of blood vessels—indicating that astrocytes may regulate blood flow not only during periods of high neural activity but also at rest. by integrating multiple imaging modalities, the study provided a more comprehensive berkeley pharma tech journal of medicine | 132 understanding of astrocytic function in cerebral perfusion and cognitive maintenance.20 3.4 fmri (functional magnetic resonance imaging) fmri is a widely used, non-invasive technique for visualizing brain activity by measuring changes in blood oxygenation levels. it relies on bold (blood-oxygen-level-dependent) contrast, which detects variations in the magnetic properties of oxygenated and deoxygenated hemoglobin. fmri is repeatable, widely available, and offers superior spatial resolution compared to other neuroimaging methods. it enables in-depth, whole-brain analysis of functional activity. however, fmri has limitations: it is contraindicated for individuals with metal implants or devices from prior medical procedures, and its sensitivity to movement makes it less suitable for use during physical activity or with populations such as infants. 3.5 fnirs (functional near-infrared spectroscopy) although fmri is considered one of the most advanced neuroimaging modalities, functional near-infrared spectroscopy (fnirs) has emerged as a promising alternative or complementary technique. like fmri, fnirs is non-invasive and measures hemodynamic responses in the brain, but it does so using near-infrared light instead of magnetic fields. fnirs is relatively inexpensive, portable, and tolerant to motion, making it suitable for dynamic settings and use in young or mobile populations. however, it has limitations in terms of probe placement and depth of measurement, typically capturing data only from the frontal cortex and superficial cortical areas. berkeley pharma tech journal of medicine | 133 figure 8: functional near-infrared spectroscopy. functional near-infrared spectroscopy (fnirs) is used for non-invasive monitoring of changes in oxyand deoxyhemoglobin levels within the brain. it involves a configuration of fiber optic-based light sources and detectors arranged on a flexible head cap worn by the participant. 4. neurovascular coupling targeted market therapeutics exploring new treatments for brain health is essential in addressing cognitive decline. this article introduces several options, including cocoa flavonoids (cf), nicotinamide mononucleotide(nmn), i-no pericyte relaxant, inorganic nitrate supplementation, and nicotinamide adenine dinucleotide (nad) supplementation. each offers potential benefits, but future research is required to confirm their safety and effectiveness. 4.1 cocoa flavonoids (cf) cocoa flavonoids (cf) are recognized for their neuroprotective attributes, antioxidant and vasodilatory properties, and their ability to inhibit cholinesterase and tau formation. despite these promising attributes, their effects appear limited in scope. current evidence suggests that cf primarily increases bold (blood-oxygen-level-dependent) responses in specific brain regions—namely the supramarginal gyrus of the parietal lobe and the inferior frontal gyrus.21 the effects of cf intake are not well understood, and clinical studies are required to further investigate their impact. berkeley pharma tech journal of medicine | 134 4.2 nicotinamide mononucleotide (nmn) nicotinamide mononucleotide (nmn) is another treatment option that enhances endothelial no-mediated vasodilation and serves as an nad+ intermediate. it is also recognized for improving spatial working memory and movement coordination. however, most research has focused on its vascular effects in large arteries, such as the aorta, leaving its broader systemic and neurological interactions underexplored. moreover, some clinical trials have reported adverse effects, raising concerns about the safety and long-term efficacy of nad⁺ intermediates like nmn. these findings underscore the need for further investigation to better understand its therapeutic viability.22 4.3 i-no pericyte relaxant i-no pericyte relaxant demonstrates potential as treatment by opening constricted blood vessels, becoming neuroprotective in other mammals, and assisting in recanalization and pretreatment. while showing strength as a neuroprotective agent and in aiding blood flow, this presents certain challenges. for example, its efficacy can be compromised by interactions with other chemicals such as adenosine triphosphate, norepinephrine, angiotensin ii, and intracellular reactive oxygen species. these chemicals can potentially reduce its therapeutic benefits. additionally, there are reported side effects that could be detrimental to patients. given these issues, it is clear that extensive human clinical trials are required to verify the treatment's safety and to ensure its therapeutic efficacy is consistent and reliable. 4.4 inorganic nitrate supplementation inorganic nitrate supplementation is a non-pharmaceutical approach used to support cerebral vascular health. it is available over the counter in supplement form and naturally occurs in dietary sources such as beetroot juice. inorganic nitrates are known to reduce blood pressure, enhance endothelial function, and potentially improve athletic performance. while generally well-tolerated and being a non-pharmaceutical option, some individuals may experience side effects such as stomach discomfort, changes berkeley pharma tech journal of medicine | 135 in bowel movements, or colored urine. a notable concern is the potential for drug interactions, particularly with medications that affect nitric oxide pathways, which may increase the risk of conditions like methemoglobinemia. despite its benefits, the long-term effects of inorganic nitrate supplementation remain unclear. further research is necessary to fully understand its efficacy and safety for prolonged use. 4.5 nicotinamide adenine dinucleotide (nad) supplementation nicotinamide adenine dinucleotide (nad) supplementation has emerged as a promising strategy to mitigate age-related cognitive decline. nad is a coenzyme essential for cellular metabolism and energy production, but its levels naturally decline with age, which may contribute to reduced brain function and vascular integrity. supplementation typically involves the use of nad precursors, such as nicotinamide riboside (nr) and nicotinamide mononucleotide (nmn), which are intended to boost intracellular nad⁺ levels. this, in turn, may enhance mitochondrial function and promote neurovascular health. however, side effects such as nausea, fatigue, headaches, and diarrhea have been reported. although these supplements are generally considered safe at recommended doses, the long-term effects remain uncertain. further clinical trials are needed to evaluate the full therapeutic potential and safety profile of nad supplementation for cognitive health. currently, researchers are conducting two ongoing significant clinical trials, nct03617302 and nct05483465, related to the neurovascular coupling in cognitive decline. these studies aim to understand how changes in the interaction between neuronal activity and cerebral blood flow contribute to cognitive impairments associated with aging. by investigating different interventions, these studies aim to identify interventions that may slow or mitigate cognitive decline in older adults. 4.6 inorganic nitrate supplementation on cerebrovascular aging and arterial stiffness the first ongoing clinical study "inorganic nitrate supplementation on cerebrovascular aging and arterial stiffness" focuses on examining the effects of inorganic nitrate supplementation, particularly from beetroot berkeley pharma tech journal of medicine | 136 juice, on aging cerebrovascular systems and arterial stiffness.23 launched on november 1, 2018, the study is expected to conclude on june 30, 2024. this randomized, double-blind, crossover study enrolled 53 community-dwelling older adults, randomly assigned to either an experimental or control group. the experimental group received nitrate-rich beetroot juice, while the control group consumed nitrate-depleted beetroot juice. the primary outcomes measured were the acute changes in carotid artery stiffness, assessed through ultrasonography and applanation tonometry, and changes in cerebral blood flow which were measured using 4d pc vipr mri technology. the 4d pc vipr mri is an advanced imaging technique that generates detailed, dynamic images of blood flow in cerebral arteries, allowing researchers to visualize circulation patterns throughout the brain. carotid artery stiffness is a critical biomarker of cerebrovascular aging, as reduced compliance in these vessels can impair cerebral perfusion. this study underscores the potential role of dietary nitrate supplementation in supporting vascular function and mitigating cognitive decline associated with aging. 4.7 the effect of nad supplementation on brain vascular health in aging the second study "the effect of nad supplementation on brain vascular health in aging," delves into its effects on brain vascular health in aging individuals.24 the objective is to ascertain whether nicotinamide riboside, a precursor to nad, can enhance brain health and memory in older adults by replenishing nad levels. the trial began on may 3, 2023 and is scheduled for completion in december 2027. this randomized, double-blind, placebo-controlled parallel study involves 214 community-dwelling older adults with normal cognitive function. participants in the experimental group receive oral nicotinamide riboside (1 gram per day) for 8 weeks, while the control group receives an indistinguishable placebo. primary outcomes include changes in neurovascular coupling, assessed using functional near-infrared spectroscopy, and alterations in neuronal activity, measured through eeg. eeg provides spectral data through power spectral density analysis, enabling researchers to assess brain activity before and after treatment, reported as a percentage change from baseline. this study is particularly berkeley pharma tech journal of medicine | 137 significant as it explores the direct impact of nad⁺ replenishment on brain vascular health, with the potential to prevent or delay cognitive decline in the aging population. 5. future directions future directions of research on nvc-associated cognitive decline could include conducting longitudinal studies spanning 5-10 years to track the efficacy of interventions targeting nvc in individuals with cognitive impairments. this could also include the development of combination therapies, such as medicinal treatments and lifestyle interventions to investigate the benefits of improving overall well-being on brain health. in addition, genetic factors play a critical role in cognitive decline and warrant deeper investigation. while the apoeε4 allele is a well-known genetic risk factor, emerging evidence suggests that other genes—such as cldn5, which is associated with blood–brain barrier integrity—may also be implicated in nvc dysfunction.21 future research could explore epigenetic approaches, including gene expression modulation, as a potential therapeutic strategy for nvc-related diseases. 6. conclusion as discussed, the growing prevalence of cognitive decline, including alzheimer’s, parkinson’s, and dementia, presents a significant challenge to public health systems worldwide. this review highlights the important role of nvc in regulating cerebral blood flow and brain health, both of which are essential in mitigating cognitive decline and managing neurodegenerative diseases. innovations in biomarkers–pericyte detachment, apoeε4 gene expression, vascular dysfunction in accordance with the 2-hit hypothesis model, and bbb breakdown–and advanced neuroimaging techniques–in-vivo photon imaging, dynamic vessel analyzer, multimodality imaging platforms, fmri, fnirs–can be the key for the early detection and combating of nvc-related diseases. additionally, nvc-targeting market therapeutics provide a look into future treatment. substances like cocoa flavonoids and inorganic nitrate supplementation are being explored for their potential benefits in improving brain vascular health and cognitive function. with berkeley pharma tech journal of medicine | 138 continued research, these market therapies could be developed into effective treatments for both early and late stages of nvc-related symptoms. ongoing research is necessary to refine these therapies to ensure both effectiveness and safety across various stages of disease progression. although many of these interventions are still in early-stage research and suffer from limited funding and clinical validation, they show promise for future therapeutic development. in summary, the integration of advanced neuroimaging techniques with targeted biomarker detection holds considerable promise for improving the early diagnosis, monitoring, and treatment of cognitive decline. focusing on the neurovascular unit as a therapeutic target may pave the way for more effective and personalized interventions in the fight against neurodegenerative diseases. berkeley pharma tech journal of medicine | 139 references 1. hale jm, schneider dc, gampe j, mehta nk, myrskylä m. trends in the risk of cognitive impairment in the united states, 1996–2014. epidemiology. 2020;31(5):745–754. doi:10.1097/ede.0000000000001219 2. pais r, ruano l, carvalho op, barros h. global cognitive impairment prevalence and incidence in community-dwelling older adults: a systematic review. geriatrics (basel). 2020;5(4):84. doi:10.3390/geriatrics5040084 3. hale jm, schneider dc, mehta nk, myrskylä m. cognitive impairment in the u.s.: lifetime risk, age at onset, and years impaired. ssm popul health. 2020;11:100577. doi:10.1016/j.ssmph.2020.100577 4. cleveland clinic. alzheimer’s disease: causes, symptoms, treatment & stages. accessed april 26, 2024. https://my.clevelandclinic.org/health/diseases/ 9164-alzheimers-disease 5. cleveland clinic. parkinson’s disease: what it is, causes, symptoms & treatment. accessed april 26, 2024. https://my.clevelandclinic.org/health/diseases/85 25-parkinsons-disease-an-overview 6. centers for disease control and prevention. what is dementia? published april 5, 2019. accessed april 26, 2024. https://www.cdc.gov/aging/dementia/index.html 7. feng l, gao l. the role of neurovascular coupling dysfunction in cognitive decline of diabetes patients. front neurosci. 2024;18:1375908. doi:10.3389/fnins.2024.1375908 8. das a, mcguire pg. retinal and choroidal angiogenesis: pathophysiology and strategies for inhibition. prog retin eye res. 2003;22(6):721–748. doi:10.1016/j.preteyeres.2003.08.001 9. stackhouse tl, mishra a. neurovascular coupling in development and disease: focus on astrocytes. front cell dev biol. 2021;9:702832. doi:10.3389/fcell.2021.702832 10. shabir o, berwick j, francis se. neurovascular dysfunction in vascular dementia, alzheimer's and atherosclerosis. bmc neurosci. 2018;19(1):62. doi:10.1186/s12868-018-0465-5 11. sweeney m, ayyadurai s, zlokovic b. pericytes of the neurovascular unit: key functions and signaling pathways. nat neurosci. 2016;19:771–783. doi:10.1038/nn.4288 12. kril jj, patel s, harding aj, et al. patients with vascular dementia due to microvascular pathology have significant hippocampal neuronal loss. j neurol neurosurg psychiatry. 2002;72:747–751. 13. wang s, tang c, liu y, et al. impact of impaired cerebral blood flow autoregulation on cognitive impairment. front aging. 2022;3:1077302. doi:10.3389/fragi.2022.1077302 14. sienski g, et al. apoe4 disrupts intracellular lipid homeostasis in human ipsc-derived glia. sci transl med. 2021;13(583):eaaz4564. doi:10.1126/scitranslmed.aaz4564https://investor .jazzpharma.com/news-releases/news-release-detail s/jazz-pharmaceuticals-announces-fda-acceptanceberkeley pharma tech journal of medicine | 140 new-drug 15. zlokovic bv. neurovascular pathways to neurodegeneration in alzheimer’s disease and other disorders. nat rev neurosci. 2011;12:723–738. doi:10.1038/nrn3114 16. nelson ar, sweeney md, sagare ap, zlokovic bv. neurovascular dysfunction and neurodegeneration in dementia and alzheimer’s disease. biochim biophys acta. 2016;1862(5):887–900. doi:10.1016/j.bbadis.2015.12.016 17. roy rk, althammer f, seymour aj, et al. inverse neurovascular coupling contributes to positive feedback excitation of vasopressin neurons during a systemic homeostatic challenge. cell rep. 2021;37(5):109925. doi:10.1016/j.celrep.2021.109925 18. decroix l, van schuerbeek p, tonoli c, et al. the effect of acute cocoa flavanol intake on the bold response and cognitive function in type 1 diabetes: a randomized, placebo-controlled, double-blinded cross-over pilot study. psychopharmacology. 2019;236(12):3421–3428. doi:10.1007/s00213-019-05306-z 19. hashimoto y, greene c, munnich a, campbell m. the cldn5 gene at the blood-brain barrier in health and disease. fluids barriers cns. 2023;20(1):1. doi:10.1186/s12987-023-00424-5 20. hwang e, song s. possible adverse effects of high-dose nicotinamide: mechanisms and safety assessment. biomolecules. 2020;10(5):687. doi:10.3390/biom10050687 21. kotliar k, hauser c, ortner m, et al. altered neurovascular coupling as measured by optical imaging: a biomarker for alzheimer’s disease. sci rep. 2017;7:12906. doi:10.1038/s41598-017-13349-5 22. gu x, chen w, volkow nd, et al. synchronized astrocytic ca2+ responses in neurovascular coupling during somatosensory stimulation and for the resting state. cell rep. 2018;23(13):3878–3889. doi:10.1016/j.celrep.2018.05.091 23. clinicaltrials.gov. inorganic nitrate supplementation on cerebrovascular aging and arterial stiffness study. accessed april 26, 2024. https://clinicaltrials.gov/ct2/show/study/ nct03617302 24. clinicaltrials.gov. the effect of nad supplementation on brain vascular health in aging. accessed april 26, 2024. https://clinicaltrials.gov/ct2/show/nct0548346 5 berkeley pharma tech journal of medicine | 141 copy of eu body paragraph.docx.pdf copy of eu et al reference page.pdf berkeley pharma tech journal of medicine correspondence: mayumimagdalena@gmail.com keywords: gut microbiome autism spectrum disorder neurodevelopment gut-brain-axis dysbiosis microbiota transfer therapy blood-brain-barrier submitted august 11, 2023 accepted march 15, 2024 published june 28, 2024 full open access creative commons attribution license 4.0 abstract the gut microbiome, a diverse community of microorganisms in the gastrointestinal tract, is vital for human health and has a symbiotic relationship with the host. autism spectrum disorder (asd), a complex condition affecting social interaction, speech, and behavior, manifests early in life and affects 1 in 36 children globally. research shows a significant link between the gut microbiome and asd symptoms, with dysbiosis observed in individuals with asd compared to neurotypical populations. pathogenic gut microbiota can produce toxins that increase gut permeability, impair the intestinal barrier, and activate the immune system. the vagal nerve, influencing central nervous system (cns) activity, can release peripheral cytokines inducing depression-like behaviors. thus, dietary changes and treatments targeting the gut microbiota, such as microbiota transfer therapy (mtt), offer promising methods for treating asd symptoms. current research focuses on dietary therapies like gluten-free and casein-free diets, ketogenic diets, and probiotics/prebiotics supplementation to modify the gut microbiota and enhance asd treatment outcomes. however, more research is needed to fully understand the gut microbiota-asd connection and establish evidence-based interventions. individualized approaches are crucial for the safety and effectiveness of therapeutic strategies for asd. exploring the gut microbiome's role in asd offers promising avenues for novel treatments and improved understanding of the disorder. exploring the gut microbiome-autism spectrum disorder connection: implications for therapeutic interventions and future directions by: mayumi schaepers-cheu, mansi patel, gizell lien, amaaya arora 1. introduction autism spectrum disorder (asd) is a complex developmental disability characterized by distinct neurobiological variations in the brain.1 while the exact causes of asd are not yet fully understood, some cases are associated with speci�c genetic conditions. asd is characterized by a diverse array of behavioral and developmental symptoms, which typically manifest early in childhood, often before the age of three years. the term "spectrum" encapsulates the wide range of symptoms and the varying degrees of impairment that individuals with asd may exhibit, making each case unique with its own set of challenges and strengths. two primary categories of symptoms are commonly observed in individuals with asd: di�culties in social communication and engagement, and the presence of restricted and repetitive behaviors.2 in the realm of social communication, individuals with asd often struggle to interpret and respond to social cues, which can include challenges in maintaining eye contact, recognizing facial expressions, and understanding nonverbal gestures.2 these di�culties can hinder their ability to initiate and maintain meaningful interactions with others, leading to potential feelings of isolation and social disconnect. additionally, individuals with asd may exhibit a wide array of restricted and repetitive behaviors.2 these behaviors can manifest as repetitive movements, such as hand-�apping or body-rocking, intense and narrow interests in speci�c topics or objects, and a strong preference for adhering to rigid routines.2 these behaviors may serve as coping mechanisms for individuals with asd, providing them with a sense of comfort and predictability in an otherwise overwhelming world. it is crucial to recognize that asd is a spectrum disorder, encompassing a broad range of symptoms and levels of functioning.4 some individuals with asd may have exceptional abilities in certain areas, often referred to as "splinter skills," while facing signi�cant challenges in others. the degree of impairment can vary widely, and early intervention, therapy, and support can play a crucial role in fostering developmental progress and enhancing the quality of life for individuals with asd. as the understanding of the disorder continues to evolve, ongoing research e�orts are working to elucidate the underlying causes and identify e�ective interventions. by recognizing and embracing the unique strengths and challenges of each individual with asd, it is berkeley pharma tech journal of medicine | 35 possible to foster a more inclusive and compassionate society that promotes understanding for all. due to its impact on health and disease, the human gut microbiome is a dynamic and complex ecosystem that has attracted signi�cant interest. numerous studies have shown that the biome plays an important role in a wide range of physiological functions, including immunological, metabolic, nutrition, and even neurological function. the gut microbiome is a colony of trillions of bacteria, viruses, fungi, and other microbes. the most prevalent and well-studied of the microbes are bacteria. however, due to a complex interaction of variables including genetics, nutrition, age, geography, lifestyle, and exposure to environmental e�ects, the variety and relative abundance of microbial species can di�er signi�cantly from one person to another. in a symbiotic connection with the human host, the gut microbiome plays a crucial part in digestion and nutritional absorption. it allows for the breaking down of �bers and complex carbohydrates that our systems cannot digest on their own, which supports energy absorption and the creation of nutrients and vitamins. additionally, the gut microbiome supports a healthy immune system by defending against dangerous infections and fostering immunological tolerance to advantageous microbes and food antigens.14 a bidirectional communication connection between the gut and the brain has been revealed by recent studies, which have also given light on the gut brain axis (gba) (figure 1). the gut microbiota can a�ect not only physical health but also mental health and cognitive performance, according to this complex connection. changes in the gut microbiota have been linked to a number of neurological and psychiatric illnesses, including anxiety, asd, and depressive and phobic disorders.15 however, dysbiosis—disturbances in the delicate equilibrium of the gut microbiome—have been connected to a variety of health issues and disease states. antibiotic usage, dietary changes, infections, stress, and other environmental e�ects are some factors that may cause dysbiosis. dysbiosis itself can result in in�ammation, metabolic problems, and an elevated risk of infection ( figure 3). therefore, the gut microbiome plays a crucial role in human health, explaining the growing interest in researching therapeutic approaches that target the gut microbiota to enhance general health and treat a variety of medical diseases.12 healthy gut microbiota can berkeley pharma tech journal of medicine | 36 be restored and maintained through a variety of methods, including probiotic supplementation, prebiotic intake, and dietary changes. it is expected that as research on the gut microbiome develops, so will understanding of how the biome a�ects human health and disease. utilizing this knowledge can result in cutting-edge customized medicine strategies and the creation of targeted medicines to treat a variety of health issues related to dysbiosis, opening up a promising, new area for enhancing human health and well-being. the central and enteric nerve systems communicate in both directions through the gba, which connects the brain’s emotional and cognitive regions to peripheral intestine processes (figure 1). the gba is a complex and dynamic network of communication that is extremely important in controlling di�erent facets of our physical and mental health (figure 1). the investigation of the connection between the gut microbiome and asd is driven by several compelling rationales. each highlights the potential signi�cance of the gba and its impact on neurodevelopment and behavior in individuals with asd. a high proportion of individuals with asd experience gastrointestinal (gi) issues, such as chronic constipation, diarrhea, abdominal pain, and in�ammation. these gi disturbances suggest a potential connection between gut health and asd, prompting investigations into the role of the gut microbiome in contributing to or exacerbating these symptoms. studies have revealed that individuals with asd often exhibit alterations in the composition and diversity of their gut microbiota compared to neurotypical individuals.11 these di�erences have sparked curiosity about whether these changes could in�uence asd symptoms. the gut microbiome is known to in�uence neurodevelopment and brain function through its impact on neurotransmitters and immune responses3 (figure 1). given the neurological and behavioral nature of asd, exploring the potential link between the gut microbiome and asd has become a signi�cant area of interest and could pave the way for personalized therapeutic strategies.3 if speci�c gut microbiome patterns and biomarkers are associated with certain asd symptoms, it could lead to targeted interventions tailored to individual needs, potentially improving treatment outcomes.3 the emerging research in this �eld holds promise for berkeley pharma tech journal of medicine | 37 developing more e�ective and individualized therapies for individuals with asd, providing new avenues for enhancing their health and quality of life. 2. gut microbiome in asd the gba is a bidirectional communication network that links the cns with the enteric nervous system (ens) of the gastrointestinal (gi) tract (figure 1). this sophisticated and dynamic axis plays a crucial role in regulating various aspects of our physical and mental health. the gba’s relevance to asd has garnered increasing attention in recent years.8 figure 1: gut-brain axis communication network many individuals with asd also experience gi disturbances, such as chronic constipation, diarrhea, and in�ammation, suggesting a potential link between gut health and asd. the gut microbiota, a crucial component of the gba, plays a signi�cant role in modulating brain function and behavior through the production of neurotransmitters, microbial metabolites, and immune signaling molecules.14 this suggests that disruptions in the gut microbiome could in�uence neurodevelopment. the gba is involved in the regulation of immune responses, in�ammation, and neural signaling, which are all processes implicated in asd pathogenesis.9 the bidirectional communication between the gut and the brain can impact neurodevelopmental processes, social behaviors, and cognitive functions, which are frequently a�ected in individuals with asd.1 berkeley pharma tech journal of medicine | 38 understanding the relevance of the gba to asd holds immense potential for the development of new therapeutic strategies. targeting the gut microbiome through interventions like probiotics, prebiotics, and dietary modi�cations may o�er novel approaches to managing asd-related symptoms and improve the overall well-being of a�ected individuals. overall, the gba represents a complex and promising area of research in the context of asd. investigating the interactions between the gut microbiota and the cns may pave the way for innovative treatments and personalized interventions.15 figure 2: blood-brain-barrier and gastrointestinal barrier two separate physiological barriers in the human body, the blood-brain barrier (bbb) and the gi barrier, play crucial, protective roles (see figure 2). the intestinal mucosa is where the intestinal barrier is in the digestive system.5 it controls the entry of bene�cial molecules, poisons, and berkeley pharma tech journal of medicine | 39 pathogens into the body while regulating the transit of nutrients and water, among other molecules, into the bloodstream.5 the lining consists of mucous membranes that trap hazardous chemicals, epithelial cells that provide a protective barrier, and tight junctions that close cell gaps. the intestinal barrier supports immune defense, promotes nutrition absorption, and preserves healthy gut microbiota.5 moreover, the bbb is a highly selective protective barrier that regulates the passage of substances between the bloodstream and the brain tissue to maintain a stable and controlled environment within the cns.7 endothelial cells, astrocytes, and pericytes play a role in maintaining the integrity of this barrier, which safeguards the brain by controlling the passage of chemicals across the bbb.7 the bbb protects the brain from toxins and keeps the brain's environment steady for healthy neuronal activity.7 it may, however, make it more di�cult to transport medications to the brain. the intricacy of the body's defense mechanisms is shown by the importance of both barriers for maintaining general health and protecting the brain from harm. unidenti�ed erysipelotrichaceae, faecalibacterium, and lachnospiraceae have been shown to be positively linked with asd severity.10 notably, three microbial indicators (faecalitalea, caproiciproducens, and collinsella) were found in a random forest model as potential biomarkers that distinguish between healthy controls and individuals with asd based on gut microbiome pro�les. this current study provides evidence for a link between gut microbiota and asd, with the data implying that gut microbiota may play a role in symptomatology.15 as a result, modulating the gut microbiome can serve as a novel therapeutic method for asd.10 3. mechanism of the gut microbiome and asd certain compounds have been the center of attention in studies aiming to uncover a correlation between the gut microbiome and the onset of asd. vast amounts of evidence connecting changes of maternal gut microbiome pro�les to the di�erences present in brain development patterns of o�spring have been found through experimental methods.16 speci�cally, research involving the in�uence of cesarean section deliveries, and the use of mice as animal models, hint at these links.17 multiple studies have found berkeley pharma tech journal of medicine | 40 that newborns delivered through cesarean sections have a higher risk of developing asd in their lifetime.16 since it has been proven that the composition of the microbiota is di�erent amongst newborns delivered through a vaginal delivery compared to those born through cesarean delivery, the in�uence of the microbiota is a potential theory for the development of asd.17 this theory is strongly supported by the fact that 70-80% of individuals with asd experience gi symptoms (constipation, bloating, diarrhea, and nausea, among others).18 multiple theories relating the e�ects of maternal microbiome environments on fetus’ development have also been studied with mice. one particular study focused on brain metabolomic pro�les and gene expression.4 this study demonstrated that mice that were induced with maternal obesity factors (ex) produced o�spring with social behavioral de�cits linked to the mesolimbic reward system.19 moreover, subsequent transfer of gut biome from the study’s control mice into the o�spring of the induced mothers completely corrected these de�cits.19 thus, this was a clear sign that the gut microbiome played a signi�cant role in in�uencing the neurodevelopmental pathways of o�spring, and that it can be in�uenced by the surrounding mother’s gut biome.19,20 3.1 e�ect of short-chain fatty acids on neurodevelopment short-chain fatty acids (scfas), primarily acetate, propionate and butyrate, have been found to play a role in the development of asd. scfas are produced by the breakdown of dietary carbohydrates and amino acids.21,22 to study whether there is a clear di�erence in the level of microbiome scfas in individuals with asd compared to individuals without asd, wang et al. analyzed concentrations of scfas in participants’ fecal samples. to prevent diet from interrupting results as a confounding variable, all subjects were given similar diets with equivalent amounts of protein, sugar, starch, and �ber.21 it was found that the total scfa (primarily propionic acid, acetic acid, and butyric acid) concentration was signi�cantly higher in the asd group (136.6 mmol/kg compared to 111.1 mmol/kg).21 providing further evidence for the role of altered scfa levels on the onset of asd-like symptoms, studies with rats have shown that the administration of propionic acid induced asd-like berkeley pharma tech journal of medicine | 41 behaviors and produced neuropathological changes reported with asd in the rats.21 moreover, the previous studies have discovered that increased intestinal permeability is reported in asd cases. since acetic acid is suggested to play a role in the gut epithelial layer, there is a link that has appeared between the altered acetic acid levels and asd symptoms.21,22 subsequent analysis also found that ammonia levels were highly elevated in the fecal samples of those with asd compared to the samples of those without asd (42.7 mmol/kg compared to 32.3 mmol/kg).21 thus, this study indicates that there is a clear elevation in these microbiome compounds in individuals with asd, emphasizing an association between the onset of asd and altered microbiome scfas. 3.2 e�ect of lipopolysaccharides on neurodevelopment another focus of scientists working to establish the link between the microgut and the onset of asd is lipopolysaccharides (lps), an endotoxin that can mimic a gram-negative bacteria infection.23 a study using wistar rats between the age of 12 and 14 weeks highlighted the speci�c e�ects of induced lps solution in the pregnant rats.23 although this research group previously found that prenatal exposure to lps induces behaviors similar to those caused by asd. these symptoms were measured with t-maze spontaneous alternation tests, which were impaired in mice that had prenatal lps exposure.23,24,25 these tests are designed to measure social de�cits, repetitive behaviors, and cognitive in�exibility in the asd-context.23,24,25 in addition to these results, the researchers identi�ed reduced maternal levels of magnesium, selenium, manganese, and zinc following inducement of lps.8 more interestingly, prenatal zinc treatment prevented o�spring from demonstrating such autistic-like behaviors.23,24,25 a follow-up study by the same group of researchers illustrated the immune and in�ammatory e�ects of lowered lps.23 lps was shown to signi�cantly increase the production of proin�ammatory cytokines in the o�spring.23 these cytokines sequestered zinc and caused maternal as well as fetal hypozincemia.23 another study highlights a theory for the alteration of lipopolysaccharide (lps) levels within the biome of individuals with asd by demonstrating that 36.7% of children with asd exhibit modi�ed intestinal permeability, a marked contrast to the change observed in less than 5% of non-asd children.26 berkeley pharma tech journal of medicine | 42 3.3 e�ect of serotonin on neurodevelopment serotonin has also been shown to be associated with the development of asd due its connection with the gut-microbiome axis.27,28 the fact that serotonin is observed to have intense activity during the early stages of development gives the neurotransmitter great importance when discussing neurodevelopmental disorders.28 scfas are able to cross the bbb and modulate the production of serotonin, in�uencing early brain development.29 about 90% of serotonin in the body is produced by enterochroma�n cells in the gi epithelium. therefore, alterations in the gut microbiome can result in the deregulation and changes in serotonin production. the main mechanism through which serotonin can play a role in mediating asd-symptoms is by acting as a pro-in�ammatory molecule. goeden et al. discovered that increasing maternal in�ammation midway during pregnancy results in an upregulation of placental tryptophan (trp) metabolism.30 since trp is converted into serotonin, there is increased serotonin present in the fetal forebrain, linking the maternal microbiome composition to the fetal biome, disrupting some serotonin-dependent neurodevelopmental processes.30 mild immune activation in the dams used in the study also presented similar results of increased serotonin in the fetal forebrain.30 this study o�ers a strong direction for uncovering the speci�c mechanisms through which serotonin in�uences fetal brain development in the early stages of life. positron emission tomography (pet) and post mortem studies also indicate that the pattern of serotonin synthesis in non autistic children resembles closely with the pro�le of synaptic density in their frontal cortex, revealing a somewhat global abnormality of serotonin synthesis and synaptogenesis.28 lastly, the processes of high brain serotonin synthesis and synaptogenesis during the preschool years has been shown to be highly disrupted in autistic children.28 although serotonin serves as a vital link between the gut microbiome and the development of asd, abnormal levels of other metabolites are associated with asd-like symptoms.31 mass spectrometry studies have enabled the observation of the gut composition as a whole, revealing di�erences in steroid hormones between individuals with and without berkeley pharma tech journal of medicine | 43 asd.31,32 speci�cally, many metabolites within the pregnenolone and androgen pathways were found to be elevated in individuals with asd.32 this �nding further demonstrates that pathways of downstream cholesterol metabolism are altered in individuals with asd.32 moreover, studies have indicated that levels of di�erent metabolites are altered in mothers with children born with asd compared to mothers of children without asd.32,33 through blood tests, it was found that carnitine conjugated molecules were present in lower levels in the mothers with asd children.33 this �nding hints at an underlying mechanism that this metabolite takes part in which can a�ect the presence of asd symptoms. in addition to carnitine-conjugated molecules, b12, cis-4decenoylcarnitine, catechol sulfate, 7-methylxanthine, and tiglyl carnitine were found to be in levels signi�cantly di�erent between both mother groups.33 the results of the study’s statistical analysis provides researchers with an idea of how changes in a wide variety of microbiome metabolites in�uence a developing baby. instead of focusing on examining the relation of altered levels of one molecule, developing methods of analyzing how altered levels of one metabolic cell can induce changes in the levels of other molecules can provide a more inclusive view on the e�ects of the microbiome on the development of asd. 4. therapeutic studies there is compelling evidence from previously conducted research about a link between the gut microbiota and the nervous system. the bi-directional gba includes signaling from the gut microbiota to the brain via neural, endocrine, immune, and humoral pathways.34 bacteria, including commensal, probiotic and pathogenic, in the gi tract are involved in the activation of neural pathways and cns signaling systems.35 emerging data support the role of microbiota in in�uencing stress related to anxiety and depression disorders. gut microbial dysbiosis, frequently observed in asd patients, is associated with mood disorders and disruption of the gba34 (figure 3). a large registry based study showed an association between increased autism severity with higher probability of having gi problems.36 berkeley pharma tech journal of medicine | 44 figure 3: eubiosis vs. dysbiosis 4.1 probiotics and prebiotics probiotic and prebiotic supplements have been proven to improve gut health via host-microbe symbiosis and growth/ maintenance of good bacteria. probiotic bacteria and dietary prebiotics can cause distinct changes in the composition of the gut microbiota which can improve peripheral (gastrointestinal) and central (psychological) symptoms37 (figure 3). in the double-blind placebo-controlled study by schmidt et al.,38 forty-�ve healthy volunteers aged 1845 years were administered one of two prebiotics (fructooligosaccharides, fos, or bimuno® galactooligosaccharides, b-gos) or a placebo (maltodextrin) daily for 3 weeks. b-gos intake was shown to signi�cantly reduce salivary cortisol awakening response as well as increase processing of positive versus negative attentional vigilance as compared with the placebo. this indicates the potential of b-gos supplementation in the treatment of stress-related disorders and suggests that it may modulate hpa activity. however, this study did not account for gender and no e�ect of prebiotics on subclinical anxiety or perceived stress were observed. another randomized control study used a double-blind research method to divide 86 healthy, young adults into �ve groups as per colony-forming unit (cfu) and bacterial species count to assess the e�ect of probiotics on anxiety and related factors.39 the high cfu group reported to have a signi�cant decrease in panic anxiety and worry as well an increase in passive a�ect and anxiety berkeley pharma tech journal of medicine | 45 control compared to the low cfu group. probiotics were overall observed to improve panic anxiety, neurophysiological anxiety, negative a�ect, worry, and increase negative mood regulation. this study did not account for a diverse and large sample size and was reliant on self reported results, both of which posed limitations on the experiment. the use of over-the-counter probiotics with di�erent compositions could have also led to skewed results. the study by akkasheh et al.40 included 40 patients, aged 20 to 55 years, diagnosed with major depressive disorder (mdd) based on dsm-iv criteria who were randomized to receive probiotic supplementation for 8 weeks. experimental results showed a signi�cant decrease in beck depression inventory scores, serum insulin levels, homeostasis model assessment of insulin resistance and serum hs-crp concentrations in patients who received the probiotic supplementation as compared with the placebo, indicating that probiotic administration can have bene�cial e�ects for mdd patients. the study’s limitations include a relatively short duration of intervention and insu�cient information about the speci�c bacterial strain responsible for the treatment results. 4.2 gluten imbalance in the gut microbiota is a common occurrence in individuals diagnosed with asd. incompletely digested peptides acting as opioid agonists have been hypothesized to cross the bbb by entering the bloodstream.41 accumulation of these peptides a�ects brain function, speci�cally brain maturation, social communication, and learning, and can reduce pain sensitivity while increasing the severity of autism related behaviors.42 elimination diets, particularly a gluten-free casein free (gfcf) diet, have been adopted to serve as an alternative treatment of asd. a parental report of strict diet implementation involving the elimination of all foods containing gluten and casein has shown to be associated with an improvement of asd behavior, physiological symptoms and social behaviors as compared to partial elimination interventions.43 parents who implemented this diet for more than 6 months reported greater improvements in asd behavior and symptoms as compared with a lesser implementation duration. these �ndings suggest that strict adoption, compliance, and length of diet implementation are all important factors in optimizing the e�ectiveness of gfcf diet in children with asd. in a study by knivsberg et al.,44 15 children with asd were recommended dietary berkeley pharma tech journal of medicine | 46 interventions due to pathological urine patterns. the experiment reported that the gfcf diet helped reduce urinary peptide levels, indicating a reduction of opioid e�ects, which corresponded with an improvement in autistic behaviors, and non-verbal, cognitive and motor issues. however, it is important to consider the role of a placebo e�ect in this study due to the absence of a control group. another randomized, controlled clinical trial found that a gfcf diet has a signi�cant bene�cial e�ect on autistic and related behaviors of prepubescent asd patients at 8, 12 and 24 months of diet intervention.45 these results imply that adoption of the gfcf diet can have a positive e�ect on the developmental outcome for some children with asd. the exclusion of a double-blind or placebo factor may have posed limitations on this study. the nutritional de�ciencies that could arise from the gfcf diet are an area of high concern. long-term implementation of such dietary interventions can cause a lack of proper supplementation that could have adverse e�ects on bone health. children with asd commonly have picky eating behaviors associated with speci�c taste, colors or appearances of food due to which it is relatively di�cult to implement nutrition plans with adequate macro and micronutrients.46 this can cause the implementation of a speci�c diet such as gfcf to become even more di�cult. the high healthcare costs associated with this diet could also place a burden on the families. to overcome such challenges, appropriate nutrition plans should be curated for children with asd and the families should be provided with proper information and training. fecal microbiota transplantation (fmt) has also been identi�ed as a potential therapeutic approach to treat asd (see figure 4). fmt can be used to change the composition of the gut microbiota and improve gi and neurobehavioral symptoms in asd patients.47 the fermentation of undigested food along with the production of bioactive compounds like scfas can a�ect the health of the large bowel.45 protein fermentation by-products can also severely impact host health by increasing in�ammatory response, tissue permeability, and colitis severity in the gut.48 in a study by wang et al.,49 researchers found higher fecal concentrations of large bowel fermentation products, speci�cally scfas and ammonia, in children with asd as compared with controls. since these fermentation berkeley pharma tech journal of medicine | 47 products can impact the health of the large bowel, the observed higher concentrations could be a reason for alterations in gi health and function in asd patients.49 a clinical trial evaluating the e�cacy of fmt in asd patients performed fmt by fecal and oral routes in asd children experiencing gi disorders.50 although the study was limited by a lack of a control group and the use of participants from the same geographical location, the results demonstrated that fmt could improve gi symptoms and autism related behaviors while signi�cantly changing the serum levels of neurotransmitters.50 5. microbiota transfer therapy mtt, a modi�ed fmt protocol, has emerged as a viable treatment option for asd. a shotgun metagenomics study showed that the relative abundance of �ber consuming, and bene�cial microbes increased after 10 weeks of mtt in children with asd, thereby normalizing levels that were relatively low as compared with typically developing (td) children.51 the �ndings also indicated that mtt leads to an initial improvement in the metabolic pro�les of children with asd, but that a booster or longer treatment time would be required for retention of the bacteria.51 a further open-label clinical trial evaluated the impact of mtt on gut microbiota composition and gi and asd symptoms of asd-diagnosed children52 which will be later discussed. in essence, mtt alters a recipient’s current gut microbiome by replacing it with a donor’s healthy gut microbiome.53 this is done by transferring fecal samples from the donor to the gi tract of the recipient54 (figure 4). this method aims to have therapeutic bene�ts that can target a wide range of health issues.53 the knowledge on the scope of mtt e�cacy and safety is, however, limited due to it being a newly emerging therapy.55 studies have been fully executed to test whether mtt can alleviate asd symptoms, as mtt has already been tested with other psychiatric diseases.56 there is foundational evidence that mtt should aid in alleviating asd symptoms, as children with asd have gi problems or gut microbiota related issues, such as bloating and constipation.56 the gut berkeley pharma tech journal of medicine | 48 microbiome is also vastly di�erent in children with asd, as they have less bene�cial bacteria like bifidobacterium spp., and instead, greater amounts of pathogenic bacteria, such as desulfovibrio and clostridium.56 figure 4: fecal transplant therapy methodology one foundational study examined the e�ects of mtt on fecal metabolite pro�les of children with asd and gi symptoms, in comparison to children that are td.57 the researchers analyzed 669 biochemical compounds in the children before, during, and after mtt. they found that fecal metabolite pro�les of the asd group became more similar to the td group after treatment, suggesting the positive impact of mtt on fecal metabolites.57 in another clinical trial, mtt was used with 18 children with asd and resulted in a signi�cant reduction (approximately 80%) in gi symptoms. this persisted for 8 weeks after treatment and behavioral asd symptoms also improved and remained after treatment.52 in addition, the microbiota of the donors appeared to be at least partially engrafted in the recipients which helped to shift the gut microbiota of children with asd towards that of td children. however, this study was limited as it had a small sample size and was also not placebo controlled, blinded, or randomized. a similar study in china also found fmt therapy to improve gi issues, asd symptoms, and serum neurotransmitter levels after an 8-week follow-up.47 berkeley pharma tech journal of medicine | 49 in terms of greater long-term e�ects, there are minimal studies and still much to uncover. however, one study followed up with 18 participants with asd two years after mtt therapy.58 the improvements in gi symptoms and autism-related symptoms observed during the initial treatment were largely maintained and even improved after the treatment ended.58 the study also found persistent positive changes in the gut microbiota, including increased bacterial diversity and the increases of bene�cial bacteria abundance, such as for bifidobacteria and prevotell.58 there are also two prevalent ongoing clinical trials, as well, exploring fmt as an e�ective long term therapy. one of the trials, estimated to be completed in 2024, focuses on children with asd and gi issues. in this trial, some children receive mtt treatment, while others are assigned to a placebo group to compare the e�ectiveness in alleviating gi symptoms and managing asd symptoms.52 another trial to be completed in 2024 follows the same methodology, but with adults with asd and gi issues. they will undergo oral administration of full spectrum microbiota for a speci�ed duration to understand longer lasting implications of mtt as a therapy.59 mtt or fmt as a therapy has side e�ects and limitations. in general, it is safe, however, it can also lead to side e�ects such as gi discomfort, vomiting, nausea, diarrhea, and spotty stools.54 a study with a two-year follow-up shows the safety of fmt in reducing gi and neurobehavioral symptoms in children with asd. no adverse side e�ects associated with fmt were found, suggesting its overall safety in both the short and long-term.47 the implementation of treatments such as fmt and mtt in clinical settings involves navigating various challenges and opportunities for standardization and personalization. barriers to standardization include regulatory hurdles, donor variability, and the need to establish consistent treatment protocols while ensuring e�cacy and safety. [gr1] personalization strategies for these treatments involve microbiome pro�ling to tailor interventions based on individual patient characteristics, stratifying patients for personalized treatment selection, and implementing follow-up monitoring to adjust treatment protocols over time. berkeley pharma tech journal of medicine | 50 to understand the limitations of mtt more speci�cally, further studies are needed to clarify the exact contributions of each factor in mtt to gut microbiota changes in asd. many of the previously mentioned studies include a mixed group of participants with various gi issues, and a more homogeneous test group is needed.60 the open-label design of some studies also introduces potential placebo e�ects and longer observation periods in future trials would enhance the understanding of long-term safety and bene�ts.60 6. future directions and implications so far, it has been established that gut microbiota imbalances have been observed in individuals with asd and other psychiatric disorders61. the gut microbiome a�ects the production of speci�c molecules, immune function, and gi integrity, all in�uencing asd, and all areas where therapies can be developed.62 for the future, asd research and therapies are centered around microbial-mediated therapies, including probiotic therapy, prebiotic supplementation, and fmt.63 these therapies have growing potential in addressing asd symptoms and gi issues that commonly supplement asd.62,63 fmt therapy is gaining substantial acceptance, although there are concerns on long term impacts, standardization and infection.63 due to these concerns, synthetic stool samples are being researched, which consist of predetermined bacterial populations and could be customized to enhance the personalized therapy's e�cacy.64 personalized dietary treatments, based on the gut-immune-endocrine-brain axis, have also been explored.64 these methods consider dietary factors, immune responses, hormonal regulation, and brain function connections.65 by screening for in�ammatory responses to dietary proteins such as gluten and casein, health care providers can construct patient-speci�c dietary plans.16 these diets can be used in tandem with prebiotic or probiotic supplements to restore a healthy gut microbial balance, thereby alleviating asd symptoms.65,66 various studies have shown that speci�c bacterial strains, such as those of bacteroides fragilis and berkeley pharma tech journal of medicine | 51 lactobacillus species, hold potential in reversing autistic behaviors and improving gi symptoms in both mouse models and autistic children. however, further research is necessary to optimize the formulations.66 biomarkers have also served to assess the implementation of probiotic supplementation.13 microbiota composition and in�ammatory markers can demonstrate how to improve gi issues and thus how to manage asd symptoms. this is also a non-invasive diagnostic tool for asd that may be developed more and can serve as early intervention and prevention strategies. both single strain and multi-strain probiotics have been e�ective in modulating these biomarkers and these �ndings highlight biomarker-guided probiotic therapy as a future option for managing asd.13 these approaches show potential in improving both gi symptoms and behavioral irregularities of asd. however, further research, standardization of procedures, and optimization of treatment regimens are necessary to fully evaluate their long-term e�ectiveness.63 7. conclusion the research on the gut microbiome-asd connection has provided valuable insights into the way mechanisms of the gut and brain intertwine. numerous studies have consistently shown di�erences in the gut microbiomes of individuals with asd, characterized by dysbiosis and reduced microbial diversity.12,34,61 the discovery of gut-brain communication pathways has further underscored the relevance of these microbial communities in in�uencing brain development and asd symptoms.14,15,34 these �ndings highlight the importance of ongoing research in this �eld, as discoveries may pave the way for novel and targeted treatments that leverage the gut microbiome to manage asd more e�ectively. by exploring the intricate relationship between the gut microbiome and asd, researchers can aim to unlock promising therapeutic avenues that could improve the quality of life for individuals living with these complex neurodevelopmental disorders. berkeley pharma tech journal of medicine | 52 references 1. fattorusso a, di genova l, dell'isola gb, mencaroni e, esposito s. autism spectrum disorders and the gutmicrobiota.nutrients. 2019 feb 28;11(3):521. doi: 10.3390/nu11030521. pmid: 30823414; pmcid: pmc6471505. 2. garcia-gutierrez e, narbad a, rodríguez jm. autism spectrumdisorder associatedwith gut microbiota at immune, metabolomic, and neuroactive level. front neurosci. 2020 oct 8;14:578666. doi: 10.3389/fnins.2020.578666. pmid: 33117122; pmcid: pmc7578228. 3. 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sci. 2020 jun 10;21(11):4159. doi: 10.3390/ijms21114159. pmid: 32532137; pmcid: pmc7312735. berkeley pharma tech journal of medicine | 58 54-scientific review paper-598-1-11-20240625.pdf schaepers cover page schaepers body.docx (2) reference page_schaepers-cheu et al..pdf berkeley pharma tech journal of medicine correspondence: gonzaga.patriciaangelica@gmail.com keywords: tumor heterogeneity, immunotherapy, cancer, dual/ multi ex vivo armed t cells (eats), nanornps, car-t cell therapy submitted august 11, 2023 accepted april 5, 2024 published june 28, 2024 full open access creative commons attribution license 4.0 abstract tumor heterogeneity refers to the phenomenon when tumors possess a medley of cell types, and it is one of the central challenges facing cancer treatment as it is associated with drug resistance and worse prognosis. each cell type responds to treatment in a unique way, thus tumors with high levels of heterogeneity are not often fully treated by typical cancer therapies. there are a variety of novel treatments being developed that aim to eliminate the obstacle that heterogeneity poses by utilizing more personalized approaches. this review assesses immunotherapy, combination therapy, dualor multi-ex vivo armed t cells, and a nano-cas9 ribonucleoprotein system as treatment strategies. although further research is required to ensure their clinical safety, these treatments show their potential to overcome the challenges posed by tumor heterogeneity in cancer development. this paper also discusses circulating tumor cells as a way to test therapeutic drugs and determine treatment progress. tumor heterogeneity and therapeutic challenges: exploring approaches and future directions by: patricia gonzaga, ammabel tukiman, noelie macmullan, jason park, m.s. 1. introduction cancer, a disease in which cells grow uncontrollably, was one of the three leading causes of death in 2022, with 607,790 deaths in the u.s.1 in 2023, an estimated 609,820 out of roughly 2 million people in the u.s. were predicted to die of cancer.2 however, treating cancer is rather complicated. cancer is not merely one disease but rather a group of diseases that consist of various classi�cations, with each cancer generally named after the body part where the abnormal cell growth begins. the various types of cancers share certain hallmarks, such as invasion and metastasis, tumoral angiogenesis, and the evasion of apoptosis. the most common cancers in the u.s. are breast, prostate, and lung cancer, but it can also occur in skin cells or bone tissues.3 the problem is that no two cancers are exactly alike, even if they are classi�ed as the same type or occur in the same body tissue. each individual’s cancer contains unique combinations of genetic changes, which can a�ect a patient’s response to speci�c treatments.4 researchers from the wellcome trust sanger institute analyzed 4,938,362 mutations from 7,042 cancers, investigated the genomic dynamics of tumors undergoing exogenous and endogenous mutational processes, and extracted more than 20 distinct mutational signatures.5 these statistics highlight the vast possibilities of genetic alterations a cancer cell could undergo, leading to more variation between and within tumors. factors such as family history and lifestyle habits, especially smoking, also contribute to the uniqueness of each individual’s tumor. hence, this individuality of cancer, also known as tumor heterogeneity, complicates its treatment. tumor heterogeneity refers to the disparities between tumors of the same type in di�erent patients, between cancer cells within a single tumor, or between primary and secondary tumors. intratumor heterogeneity (ith), which refers to the presence of a diverse cell population within a single tumor, poses the greatest challenge to cancer treatment today. it can be further distinguished by genetic and phenotypic properties, which vary and a�ect the behaviors of di�erent tumor cell populations. one system of tumor cell analysis is the clustering, classi�cation, and sorting tree (ccast), which aims to target speci�c characteristics that can vary widely berkeley pharma tech journal of medicine | 84 within malignant cells. it analyzes the genetic and phenotypic variations within tumor cells by distinguishing homogeneous subpopulations within a mixed group of single cells. for example, ccast was applied to a breast cancer cell line and identi�ed at least �ve distinct cell types, which helped elucidate which tumor cell subpopulations warranted further investigation.6 thus, utilizing such diagnostic guidelines to identify variations in genetic and phenotypic properties is bene�cial as a prognostic indicator to better predict tumor response and guide therapeutic precision strategies. associated with poor prognosis, outcome, and overall survival, ith is thought to be a signi�cant factor in causing therapeutic resistance and treatment failure.7 tailoring e�ective cancer treatments is also challenging due to the unique response patterns exhibited by individual patients. thus, understanding tumor heterogeneity is crucial in treating cancer and overcoming therapeutic resistance. initially, the primary source of tumor heterogeneity was thought to be genetic or epigenetic alterations as scientists were beginning to understand the impact of tumor heterogeneity.8 while this understanding holds some truth, more studies in recent years have observed tumor heterogeneity of varying types, such as metabolic, cellular, spatial, and more. however, despite the e�orts of current common cancer treatments to address tumor heterogeneity, there is still a signi�cant risk of cancer cells resisting treatments and therapies through mutations, selective pressure, and other adaptation processes, setting back the development of more e�ective cancer treatments. the latest endeavors have focused on developing novel strategies to overcome the challenges posed by tumor heterogeneity and combat treatment resistance and disease progression. some of these treatments target unique features of the tumor, allowing for a more precise and personalized approach. we reviewed recent scienti�c literature for an overview of tumor heterogeneity, along with current and potential cancer treatments that surmount its challenges. in addition, we examined recent and ongoing clinical trials to analyze the purpose and design of the studies and gain more information on the signi�cance of their results. through this review, we hope to provide insight into the causes and negative e�ects of berkeley pharma tech journal of medicine | 85 tumor heterogeneity on individual cancer treatments, along with potential strategies that could overcome such negative implications. 2. tumor heterogeneity 2.1 overview of tumor heterogeneity tumor heterogeneity has countless causes that complicates its prevention methods and treatment, with two of its leading causes being genetic and environmental factors. table 1 expands on the causes of tumor heterogeneity, including genetic mutations, clonal evolution, microenvironmental factors, and phenotypic plasticity. factors genomic instability clonal evolution microenvironmental factors phenotypic plasticity de�nition the higher tendency of cells to obtain genetic alterations9 the process by which di�erent subclones within a tumor change over time11 factors (e.g. oxygen, nutrient availability, immune cell in�ltration) and interactions with surrounding stromal cells in the tumor microenvironment the ability of one genotype to cause di�erent phenotypes to arise in response to di�erent environments15 causes anything that causes more genetic mutations (e.g. limitless replicative potential10, dna repair defects9) typical evolutionary processes12 in�uenced by many factors, such as the tumor microenvironment, metabolism, growth factors, and mutation collection of tumor cells that combine to create the tumor microenvironment13 consists of the extracellular matrix, immune cells, and stromal cells13 epithelial-tomesenchymal transition (emt) and certain transcription factors inducing emt (e.g. zeb1, twist)15 genetic mutations and epigenetic berkeley pharma tech journal of medicine | 86 rate12 modi�cations e�ects results in genetically distinct subpopulatio ns of di�erent cells → genetic heterogeneity leads to the expansion of speci�c populations of tumor cells11→ cellular heterogeneity promotes angiogenesis and an environment for cancer cells to grow14 prevents immune cells from in�ltrating the tumor14 in�uences surrounding cells and cancerous cells14 protects metastasized cancer cells from ferroptosis, a type of cell death16 give rise to subpopulations of tumor cells with distinct functional properties16→ cellular heterogeneity table 1. factors contributing to tumor heterogeneity. this table summarizes the de�nition, causes, and e�ects of the di�erent factors that contribute to tumor heterogeneity–from left to right: genomic instability, clonal evolution, microenvironmental factors, and phenotypic plasticity. there are four types of tumor heterogeneity: intratumor, intermetastatic, intrametastatic, and interpatient. all four types cause unique complications in the clinical process. this review focuses on intratumoral heterogeneity, which refers to phenotypic or genetic variation in cells within a single tumor. the other three types of tumor heterogeneity o�er additional context to the complexities of tumor heterogeneity. for instance, intermetastatic heterogeneity is the variety between two metastatic tumors within the same patient; metastatic tumors are two or more tumors that di�er from one another. intrametastatic heterogeneity is ith within a metastasis – there is variation within that single lesion. lastly, interpatient heterogeneity is the di�erence in tumors between di�erent patients possessing the same type of cancer. interpatient heterogeneity is the main reason for the need of personalized treatments, regardless of the level of variation observed within a single tumor.17 berkeley pharma tech journal of medicine | 87 figure 1. types of tumor heterogeneity. the di�erent-colored dots represent di�erent subpopulations of cells in a tumor – in other words, tumor heterogeneity. the top left circle represents interpatient tumor heterogeneity between two patients with tumors in their liver, where despite having the same cancer, their tumors have unique subpopulations of cancer cells. the top right circle shows intermetastatic heterogeneity in a single patient. the diagram illustrates how di�erent subpopulations of cancer cells from the liver metastasize to the lung and the brain, resulting in the presence of tumor heterogeneity between the di�erent metastatic lesions. the bottom left circle displays intrametastatic heterogeneity, where di�erent subpopulations of cancer cells are present within a lung metastasis. lastly, the bottom right circle shows intratumor heterogeneity in the liver of a single patient, where the primary tumor contains di�erent subpopulations of cancer cells. 2.2 intratumoral heterogeneity across di�erent types of cancers intratumoral heterogeneity can re�ect genetic alterations in the tumor, which can a�ect disease progression and treatment response. for example, mutations in genes like brca1, brca2, tp53, and her2 in breast cancer were identi�ed as risk factors in cancer development and can impact treatment outcomes.18additionally, in lung cancer, mutations in epidermal growth factor receptor (egfr) and kras, along with alk and ros1 berkeley pharma tech journal of medicine | 88 rearrangements, are signi�cant determinants of tumor behavior and therapeutic choices.19,20 identifying these genetic variations allows physicians to accommodate treatment plans to tumor-particular genetic structures, resulting in more e�ective treatments and improved patient outcomes. one method that targets these genetic mutations is crispr-cas9-mediated genome editing, which can be utilized to target speci�c alleles, such as the tp53 gene in khos and khosr2 cell lines of osteosarcoma. this technology can hinder tumor proliferation and migration, as well as modify drug sensitivity in cancer treatment.21 cellular heterogeneity in ith refers to the numerous subpopulations of cells inside a tumor, each with precise traits and sets of behaviors. for instance, cell types in breast cancer include luminal cells, basal-like cells, and her2-positive cells, all of which impact general tumor behavior due to di�erent signaling pathways and levels of receptor expression. 22,23 understanding cellular heterogeneity helps researchers and clinicians identify potential targets for therapy since di�erent cell populations may respond di�erently to various treatments. targeting particular cellular subpopulations would disrupt a tumor’s growth and development more e�ectively. table 2 provides a comprehensive overview of the genetic and cellular heterogeneity observed in key cancer types. cancer type genetic heterogeneity cellular heterogeneity breast cancer brca1, brca2, tp53, her2/neu mutations18 luminal cells, basal-like cells, her2-positive cells22,23 lung cancer egfrmutations, kras mutations, alk rearrangements, ros1 rearrangements19,20 adenocarcinoma cells, squamous cell carcinoma cells24 colorectal cancer apc, kras, tp53, braf mutations25 stem-like cells, di�erentiated cells, tumor-in�ltrating lymphocytes26 berkeley pharma tech journal of medicine | 89 prostate cancer pten, tp53, ar alterations27 adenocarcinoma cells, neuroendocrine cells, cancer stem cells28,29 bone cancer (osteosarcoma) tp53, rb1, p16ink4a variations30 osteoblastic cells, chondroblastic cells, �broblastic cells31 table 2. genetic and cellular heterogeneity of di�erent types of cancers. this table illustrates genetic and cellular heterogeneity of di�erent types of cancers. column 1 identi�es cancer types, column 2 indicates genetic heterogeneity, and the last column shows cellular heterogeneity. the presence of ith across these di�erent types of cancers emphasizes the signi�cance of personalized treatment approaches. understanding a tumor’s unique genetic and cellular landscapes can guide the selection of appropriate therapies that target speci�c cell populations or overcome resistance mechanisms. integrating technologies like single-cell analysis and spatial pro�ling techniques allows for a comprehensive assessment of ith and aids in the development of tailored treatment strategies.37 2.3 determining the degree of tumor heterogeneity a tumor’s degree of heterogeneity is correlated with tumor prognosis, genomic instability, tumor advancement, and immunosuppression. higher levels of heterogeneity (i.e. a tumor with a greater cell diversity) suggests that the patient is more likely to experience worse treatment outcomes and drug resistance. knowing the level of heterogeneity a tumor possesses permits researchers to begin planning specialized treatments since more diversity in the cell population demands a more personalized treatment. this knowledge also permits studies to be done on groups with high and low levels of heterogeneity in order to determine the impact of ith on a speci�c treatment.36 research has determined that three methods are the most accurate and e�ective regarding how closely correlated they are with heterogeneity berkeley pharma tech journal of medicine | 90 outcomes: depth38 , depth239 , and tith40 , which all have comparable performances. these newer algorithms di�er from those used in previous studies in that they implement rna sequencing as opposed to dna sequencing. experiments using depth, which stands for deviating gene expression pro�ling tumor heterogeneity, were conducted for over 25 cancer types alongside 10,000 samples of tcga pan-cancer. depth showed stronger correlations between tumor prognosis and anti-tumor heterogeneity than dna-based algorithms such as absolute, expands, math, and phylowgs.38 depth2 showed similar correlations to the original depth program, but it had the advantage of being applicable to more gene expression pro�les as it does not reference normal controls like other mrna or dna-based algorithms.39depth2 was used in a recent 2022 study to separate groups into high and low intratumor heterogeneity groups. these groups were then compared in a study to determine a correlation between heterogeneity and chemotherapy response in patients with colon adenocarcinoma.41 finally, transcriptome-based ith (tith) involves de�ning a network and determining the distance between typical genetic sequences and cancer sequences. pathway-tith is de�ned using the genes from one speci�ed pathway. one study demonstrated that in 255 out of 291 pathways, genomic ith was strongly correlated with pathway-tith, supporting earlier �ndings that overall genetic diversity impacts variation in certain pathways.40 table 3 below displays information on the dna-based algorithms used in earlier studies, as well as the more recent rna-based algorithms primarily discussed in the paper. algorithm name description features year created absolute42 pro�les dna from heterogeneous cell populations to determine cellular copy number and identify variant alleles identi�es alterations in cancer cells evaluates tumor ploidy estimates 2012 berkeley pharma tech journal of medicine | 91 math43 measures intratumor genetic heterogeneity based on mutant-allele fraction correlates ithwith mutations in tp53 and hpv status 2013 expands44 characterizes coexisting tumor subpopulations using copy number and allele frequencies estimates tumor purity and predicts clonal subpopulations quanti�es genetic ith 2014 phylowgs45 combines somatic mutation and copy number information for subclonal reconstruction provides complete subclonal reconstruction 2015 tith40 models gene relationships and measures network disruptions to assess ith shows positive correlation with tumor progression and worse survival 2016 depth38 calculates ith based on gene expression pro�les from rna sequencing data is associated with genomic instability, worse survival, and decreased antitumor immunity 2020 depth239 calculates ith based on disruptions of gene expression pro�les without reference controls is associated with worse survival and more aggressive cancer subtypes 2022 table 3. algorithms used to assess tumor heterogeneity. this table lists the algorithms that are used to assess tumor heterogeneity in order of the year it was created. it also explains how each algorithm works and what it assesses. single-cell rna sequencing (scrna-seq) has also shown promise in helping develop personalized treatment plans. scrna-seq works by isolating the tumor cells and running mrna reverse transcription and berkeley pharma tech journal of medicine | 92 cdna ampli�cation before sequencing. as recent as 2020, single-cell rna sequencing e�ciently analyzed thousands of cells at once, making it more useful for treatment than in previous years. a study found that scrna-seq could adequately determine clusters of cells associated with poor clinical outcomes and �nd targets for immunotherapy treatments in an analysis of triple-negative breast cancer (tnbc). knowing the di�erent types of cells in a tumor will become essential in the personalized treatment for high-degree heterogeneous tumors, which will need a combination of therapies in order to be e�ectively treated. there are, however, some limitations to this technology, including cell integrity and viability, its relatively high cost, and its integration with other genomic and protein information. further research to advance scienti�c and technological developments, such as gentle extraction and data analysis methods, is expected to overcome these challenges.46 2.4 complications of tumor heterogeneity di�erent cell types respond to di�erent treatments, which is the reason behind the utilization of combination therapies for tumors with high degrees of heterogeneity. using a single treatment can lead to a relapse of cells that are more resistant to typical therapies. the �rst round of treatment might e�ectively eliminate one type of cell, making the tumor appear smaller. however, over time, the cells that were unresponsive to the treatment will become the dominant cell type, which makes the tumor drug-resistant and more challenging to treat further. the term for this phenomenon is called selective pressure.10 a study observed 20 patients with tnbc treated with neoadjuvant chemotherapy (nac) to combat highly heterogeneous cells, a characteristic of tnbc. they determined that this type of resistance can be acquired and adapted. additionally, they found evidence that the patients who experienced relapses, as opposed to cancer elimination, had cells with genetic markers for that chemoresistance. the researchers were able to determine potential treatments for the cells that had genetic resistance using single-cell rna and dna sequencing. these treatments included emt signaling, p13k inhibitors, and hypoxia inhibition using hif-1 inhibitors.47, 48 berkeley pharma tech journal of medicine | 93 another study investigated the role of tumor heterogeneity in the resistance to egfr-targeted treatment in colorectal cancer cells. three cetuximab-resistant derivatives of lim1215, oxco-2, and difi cell lineages were utilized in next-generation sequencing, immunohistochemistry, and proliferation assays to identify the mechanisms of drug resistance in tumor cells. the results in the cell proliferation assays showed that colorectal cancer cells with developed resistance to cetuximab and panitumumab secrete transforming growth factor alpha (tgf-α) and amphiregulin. these secreted growth factors protect the encompassing sensitive cells from egfr blockade by sustaining egfr/erk signaling in sensitive cells. the results showed that tgf-α and amphiregulin binding to egfr caused a longer retention time of the receptor on the surface of the plasma membrane and redirected egfr to the recycling pathway rather than to proteasomal degradation. this can potentially enhance the pro-proliferating e�ect of the protective microenvironment.49 3. treatments of interest 3.1 immunotherapy cancer immunotherapy utilizes the body’s immune system against cancer. some patients possess immune system components that naturally �ght the cancer cells, called tumor-in�ltrating lymphocytes (tils), while others do not. despite a patient possessing tils, the immune system still has di�culty �ghting the cancer cells since they are, by de�nition, abnormal and lack certain processes that normal cells should have, such as missing proteins.49 immunotherapy includes various treatment options, including immune checkpoint inhibitors, adoptive cellular therapy (act), monoclonal antibodies, treatment vaccines, and immune system modulators.50,51 one type of immunotherapy that has shown great promise in recent years is chimeric antigen receptor (car)-t cell therapy, a type of act. unfortunately, it is only approved for the treatment of blood cancers, and research into its e�ects on solid tumors is ongoing. car-t cell therapy uses either analogous or allogeneic donated t cells that can be found as part of the immune system. these cells are then genetically modi�ed to express berkeley pharma tech journal of medicine | 94 cars that target a speci�c antigen found on the surface of the intended cancer cells. this therapy is designed to boost the immune system and provide a speci�c target. figure 2 summarizes the steps of car-t cell therapy. a disadvantage of this therapy is that it can lead to selective pressure since t cells only target one antigen at a time. selective pressure refers to the eventual resistance of a heterogeneous tumor with multiple antigens to car-t cell therapy. this process is due to the elimination of all of the cells within the target antigen, leaving only the cancer cells that do not have the target antigen and therefore do not respond to the therapy.51elimination of the target antigen is also known as antigen loss or escape. many ongoing clinical trials seek to determine if car-t cells are e�ective treatments for cancers with solid tumors, given that it is an approved treatment for blood cancers. trials, even those without reliable results, are relevant to conversations about treatments of interest because it demonstrates the researchers’ belief that these therapies have great potential to be e�ective. until current research shows promising results, the success of the treatment will remain unknown. one clinical trial run by fred hutchinson cancer center from 2016-2021 investigated the use of modi�ed car-t cells to �nd ror1 proteins on cancer cells from di�erent cancer types, including but not limited to non-small cell lung cancer (nsclc) and tnbc, both of which present as solid tumors. participants with either nsclc or tnbc had unsuccessfully undergone chemotherapy and other traditional treatments. upon undergoing the clinical trial, all patients displayed adverse e�ects with mixed responses to the therapy. one out of three patients in dosage level 2 experienced either complete or partial remission, one out of six patients in dosage level 3 experienced progression-free survival after one year, and, across all dosage levels, there was a 38.89% overall survival rate.52,53 a second study funded by the national natural science foundation of china and conducted at nanjing normal university analyzed ways to enhance car-t cell therapy. knowing that tigit was a suppressor of anti-tumor processes and that msln was highly expressed in breast, prostate, and ovarian cancers, researchers combined an anti-ɑ-tigit with mlsn car-t cells as a treatment. this combination signi�cantly berkeley pharma tech journal of medicine | 95 enhanced the anti-tumor properties of mlsncar-t cells. the number of car-t cells positive for tigit was initially 18.3% but dropped to 1.81% after the addition of anti-ɑ-tigit, which led to a more e�cient mlsn car-t cell. 53additionally, a third phase i trial is ongoing to treat prostate cancer with car-t cells modi�ed for psca. the city of hope medical center began this study in 2019 and it is estimated to complete in late 2023/early 2024. no current results are available.53,55 figure 2. car-t cell therapy procedure. in car-t cell therapy, blood is �rst removed from the patient to obtain their t cells. car-t cells are then engineered and grown in the laboratory before being infused back into the patient. in the patient, the car-t cells would target and bind to speci�c antigens present on the tumor cells, killing them. in the long term, however, the tumor could acquire resistance to this therapy through antigen loss. a more common type of immunotherapy treatment is immune checkpoint inhibitors (icis). these checkpoints are part of the immune system and prevent immune cells from reacting too aggressively and attacking bene�cial cells.50 however, these checkpoints can also prevent the immune system from e�ectively dealing with cancer cells. hence, by blocking these checkpoints, scientists are able to permit the immune system to start berkeley pharma tech journal of medicine | 96 treating the cancer cells fully. the most common immune checkpoints targeted are cytotoxic-t-lymphocytes-associated proteins (ctla-4), programmed cell death 1 (pd-1), and programmed cell death ligand 1 (pd-l1).56figure 3 shows the mechanism of this checkpoint blockade. there are a variety of clinical trials that involve icis. many of them use combination therapy by combining one of the seven fda-approved ici treatments with either another approved ici treatment or with another therapy like chemotherapy.56 a clinical trial run by bristol-myers squibb from 2016-2023 tested the combination of ipilimumab and nivolumab (treatment a, immunotherapy) against pemetrexed and cisplatin or carboplatin (treatment b, type of chemotherapy) in malignant pleural mesothelioma (mpm). treatment a had an overall survival that was, on average, four months longer than treatment b. the median disease control rates for treatment a and treatment b were 76.6% and 85.1%, respectively.57 in a three-year minimum follow-up, the trial showed overall survival rates of 23% and 15% for treatment a and treatment b respectively. moreover, at three years, 28% of patients had an ongoing response to treatment a while treatment b had 0% patients with ongoing response.58 these results demonstrate how the combination of nivolumab and ipilimumab continued to provide long-term survival bene�t over chemotherapy, supporting this combination of icis as a �rst-line treatment for unresectable mpm. berkeley pharma tech journal of medicine | 97 figure 3. mechanism of immune checkpoint inhibitors. (a) an antigen-presenting cell (apc) displays an antigen bound by the major histocompatibility complex (mhc). t cell receptors (tcr) recognize the antigen, causing interaction between the t cell and the apc or tumor cell. a co-stimulatory signal caused by cd80/86 binding to cd28 results in t cell activation and proliferation, allowing the t cell to kill the cancer cell. (b) when ctla-4 is present on a t cell, it binds to cd80/86 in place of cd28. pd-1 on t cells also bind to pd-l1 that are present on tumor cells. both of these interactions lead to an inhibitory signal that blocks the t cell from killing the cancer cell. (c) anti-ctla-4 binds to ctla-4, blocking its interaction with cd80/86. this allows for binding of cd28 to cd80/86, producing a co-stimulatory signal. similarly, anti-pd-1 binds to pd-1 while anti-pd-l1 binds to pd-l1, blocking pd-1 and pd-l1 interactions. thus, through immune checkpoint inhibition, t cells are reactivated and can kill tumor cells. 3.2 combination therapy combination therapy involves using multiple types of treatment. it can be a combination of drugs, immunotherapies, chemotherapy, radiation, and other cancer treatments. since cells respond to di�erent therapies, a highly heterogeneous tumor likely needs multiple treatments in order to eliminate berkeley pharma tech journal of medicine | 98 all tumor cells from the body. however, too many drugs can strain the body and be highly toxic. combination therapy thus seeks to maximize e�ciency while minimizing toxicity. by using existing treatments, it can also be cheaper and more time-e�ective to research than developing a new drug or treatment.59 one study on mice, published in 2018 and funded by the canadian cancer society, used oncolytic viruses and the hdac inhibitor ms-275 to prevent relapse from act. the cd8+ t cells targeted a speci�c antigen on the tumor. with only act, there was a signi�cant improvement in tumor size; however, selective pressure had occurred until only antigen-negative tumor cells were remaining, signifying that the t cells could no longer target those tumor cells. the use of oncolytic viruses andms-275 was shown to prevent any relapse and change tumor-in�ltrating myeloid cells into pro-in�ammatory cells, allowing for better recovery.60 in a phase ii clinical trial funded by bristol myers squibb that ended in 2018, researchers found that patients with melanoma responded favorably to a combination of melphalan (chemotherapy) and the approved ctla-4 blocker ipilimumab (immunotherapy). based on the data provided, 85% of patients had observable responses to the treatment, and there was a 58% progression-free survival (pfs) rate after one year, with no increase in toxicity at the site of the treatment.61 an ongoing clinical trial (2018-2024 estimate) by medimmune llc is testing various dosages of oleclumab and osimertinib in the treatment of nsclc. the percentage of patients with disease control (complete response, partial response, or stable) and overall survival for each dose is as follows: oleclumab 1 + osimertinib 1: 80%, 21.9 months oleclumab 2 + osimertinib 1: 81%, 24.8 months none of the patients that received the above doses had any dose-limiting toxicities within twenty-eight days of the �rst treatment. however, within ninety days of the last dose, all patients had adverse e�ects, such as infections and infestations like cystitis and pneumonia, as well as nervous system disorders like cerebral infarction and spinal cord compression. even so, only berkeley pharma tech journal of medicine | 99 one patient who received the oleclumab 1 + osimertinib 1 doses had abnormal vital signs that were considered severe.62 this shows the importance of �nding the right amount of dose to give to the patient, especially in the case of combination therapy. 3.3 dual/multi ex vivo armed t cells (eats) dualor multi-eats are t cells armed with two or more bispeci�c antibodies (bsabs). generally, t-cell-engaging bispeci�c antibodies (t-bsabs) bind speci�cally to a tumor-associated antigen (taa) and a cd3 subunit that forms a complex with the tumor cell receptor (tcr). t-bsabs can thus link tumor cells and t cells together, activating t cells and leading to tumor death. moreover, cd3 engagement stimulates the t cells’ immune response, which redirects host immunity toward tumors. hence, t-bsabs are a promising antibody therapy for various cancers.64a phase i/ii clinical trial was conducted on epcoritamab, a t-bsab that targets cd3 and cd20. this targeting redirects and activates t cells to kill cd20-expressing malignant cells in relapsed or refractory large b-cell lymphoma (lbcl). among 157 patients, the overall response rate (orr), de�ned as the proportion of patients who have a partial or complete response to therapy, was found to be 63.1%.64an orr value greater than 60% is grade 3 and is considered a high value, showing the high e�cacy of epcoritamab.65 the complete response (cr) rate was 38.9%, with the median time to cr being 2.7 months. responses with epcoritamab were also shown to have transitioned from partial response (pr) to cr at the later assessments in nine patients. these results suggest there is an added bene�t in certain patients with continuous treatment using this t-bsab.64another clinical trial found that abbv-383, a b-cell maturation antigen x cd3 t-bsab, could treat patients with relapsed or refractory multiple myeloma with an orr of 68% at ≥ 40 mg dosage, showing the t-bsab’s promise in treating already heavily-treated patients at that dosage amount.66 eats are similar to t-bsabs in that they are also able to crosslink tumor cells and t cells together, activating the subsequent immune response. however, in eats, the bsabs are already attached to the t cells, which makes them similar to car-t cell therapy. the only di�erence is that they berkeley pharma tech journal of medicine | 100 are armed with bsabs instead of cars. with multiple bsabs attached, dualor multi-eats can target a wider variety of taas, thus helping to overcome tumor heterogeneity. a study by park and cheung tested the e�cacy of dual-antigen targeting strategies using di�erent kinds of eats, including pooled-eats (eats with unique speci�city administered simultaneously), alternate-eats (eats with unique speci�city administered in an alternating schedule), dual-eats, triab-eats (t cells armed with a bsab speci�c for two targets besides cd3), and multi-eats, with gd2 and her2 as target antigens. among these, they found that dualand multi-eats had the most potential in overcoming tumor heterogeneity and target antigen loss, both of which are challenges to current t cell immunotherapies. dual-eats, armed with gd2and her2-bsabs, and multi-eats, armed with gd2-, her2-, cd33-, psma-, and steap1-bsabs, had induced stronger cytotoxicity against a mixed lineage of cancer cells than mono-eats armed with only one type of bsab. this stronger cytotoxicity resulted in a more potent anti-tumor response and dualand multi-eats exceeded the e�cacy of mono-eats, signi�cantly improving tumor-free survival. they, along with alternate-eats, were also successful in inducing tumor regression, giving rise to long-term survival. furthermore, dualand multi-eats exerted a synergistic anti-tumor e�ect when they encountered multiple antigens simultaneously, which played a signi�cant role in preventing antigen loss.67 however, experiments have only been done in mouse models. even though no additional toxicities that could cause serious or fatal e�ects upon infusion of car-t cells or bsabs were observed, the same results might not be reproduced in humans. the bsabs also hold speci�city for human antigens, not mouse antigens, so using a mouse model fails to mimic human diseases and their therapeutics perfectly. although the t cells, tumors, and bsabs were of human origin, the tumor microenvironment contained cells of mouse origin, which included tumor-in�ltrating myeloid cells, �broblasts, vasculature, and lymphatics. these could interact with one another and a�ect tumorigenesis and anti-tumor response. despite these limitations, dualand multi-eats have potential to overcome tumor heterogeneity and cancer resistance.67 moreover, they could potentially be berkeley pharma tech journal of medicine | 101 used in more targeted and personalized treatments by arming the t cells with bsabs that target speci�c taas found in a patient’s tumor. 3.4 nanornps with a combination of single guide rnas (sgrnas) in the early stages of integrating nanotechnology into cancer therapy, profound strides were made in improving existing therapies. one such advancement has been the development of nanotechnology-mediated drug delivery systems to enhance their delivery to tumor sites and reduce systemic toxicity.68 meanwhile, nano-cas9 ribonucleoprotein (nanornp) with a combination of sgrnas harnesses the full potency of nanotechnology and gene editing for precision treatment. in a study by liu et al., a nanornp system that could carry any combination of sgrnas was demonstrated to achieve targeted gene disruption and e�ective suppression of heterogeneous tumors. nanornp has a core-shell structure linked by ca that degrades under acidic conditions. this way, it maintains a stable structure in blood circulation and normal organs but detaches its shell when in the acidic tumor microenvironment. this action facilitates tumor accumulation, cell internalization, and eventual gene editing by the cas9/sgrna complex in its core. with cas9, nanornps can disrupt the targeted gene sequence under acidic conditions, signi�cantly downregulating the expression of the target genes. when nanornps carry a combination of sgrnas, they simultaneously disrupt the expression of multiple target genes, and this could potentially overcome the genetic heterogeneity that causes treatment resistance in cancer.69 in a heterogeneous tumor model at ph 6.5, liu et al. expressed the target genes stat3, which increases tumor cell proliferation, survival, and invasion while suppressing immunity towards tumors, and runx1, whose increased levels correlate with cancer cell proliferation, tumoral angiogenesis, and metastasis. the nanornp carrying a combination of sgrnas, nanornp-stat3+runx1, disrupted the expression of both genes, inhibiting the proliferation of the tumor cells. it also increasingly induced cell apoptosis in the tumor. in contrast, nanornps carrying a berkeley pharma tech journal of medicine | 102 single type of sgrna led to the reduced expression of only one of the genes, resulting in partial growth inhibition in the heterogeneous tumor. however, through an analysis of gene disruption on stat3 and runx1, it was discovered that complete reduction of the target genes could not be achieved by the nanornp, even if it carried multiple di�erent sgrnas. even so, nanornps carrying a combination of sgrnas could simultaneously suppress the proliferation of multiple tumor cell subpopulations, showing their potential to overcome tumor heterogeneity.69 similar to dualor multi-eats, this treatment strategy can be used in a more personalized approach, wherein the nanornps could carry the sgrnas required to disrupt the speci�c target genes expressed in a patient’s tumor. 3.5 circulating tumor cells (ctcs) ctcs, rather than a treatment, are better described as a research methodology. they are cells in the blood that come from a tumor, and they have the potential to become the primary way to test and develop new drugs, as well as to test for the progression of cancer. more traditional methods have various problems. for instance, 2d cultures lack the complexity of a tumor structurally on the genetic and physical level but can be tested in high volumes at high speeds. patient-derived xenografts �x the structural problems of 2d cultures, but they are unable to be used in high-throughput screenings, which signi�cantly reduces research speed. ctcs maintain the level of heterogeneity and the tumor’s structure, which allows for high-throughput screenings. these advantages allow for new and more personalized treatments as these therapies can be tested on an accurate model that poses zero risk for the patient.70 the greatest challenge to utilizing ctcs is accurately isolating them from other cells in the blood since ctcs are very rare in the blood and little is known about their genetic structure.71,72 ctcs can be isolated based on di�erences in their physical properties, such as density, size, deformability, and electrical properties. however, these methods are very ine�cient as they lack purity and speci�city. for this reason, researchers usually use ctc-related technologies based on biological properties, particularly berkeley pharma tech journal of medicine | 103 techniques dependent on the epithelial cell adhesion molecule (epcam), a marker positively enriched in ctcs.72 ctcs are also indicators of prognosis and a non-invasive method of determining whether a treatment is working. through liquid biopsies70 , doctors can determine the ctc count in a patient’s blood.70 in a study that analyzed blood samples from 59 patients with esophageal squamous cell carcinoma, ctc levels were found to be correlated with overall survival (os) and pfs rates. researchers found that the overall and progression-free survival rates were signi�cantly better for patients with a ctc count of less than three. the mortality rates for the patients with either >0, >5, and >7 ctcs per 7.5 ml were 65.2%, 78.4%, and 87.5% respectively.73 a lower ctc count after treatment is also associated with an excellent prognosis.71,72 there are also limited studies that suggest ctcs can be used for early cancer detection, although this has only been shown in mouse models.71 4. future directions dualor multi-eats and nanornps with a combination of sgrnas have strong potential to confront therapeutic resistance, making them promising improved treatment strategies for cancer. these strategies would also provide more insight into the design of more advanced and e�ective cancer therapies. however, they have only been studied in mouse models and heterogeneous tumor models respectively, both of which may not represent a human system perfectly. hence, before these treatments can undergo clinical trials and be used as personalized approaches for tumor heterogeneity, more research must be conducted to ensure that side e�ects, such as o�-target toxicities, are minimized.67,69 on another note, further research on optimizing models to better imitate human diseases would undoubtedly be useful for preclinical testing of the safety and e�cacy of drugs and treatment strategies. ctcs, in turn, are promising as a way to determine the progress and clinical e�ciency of a treatment. the ctc count can be used as an indicator that a treatment is no longer e�ective, triggering a change in the type of therapy a patient is receiving. since a key challenge is the rarity of ctcs, more research has to be done to �nd ways to identify and isolate ctcs. this berkeley pharma tech journal of medicine | 104 could then contribute to a larger sample size that can be used in studies, improving their reliability.70 the emerging technologies and methodologies for assessing tumor heterogeneity are clear implications of the advancement of personalized treatment and precision targeting for heterogeneous tumors. currently, novel targets and therapeutic strategies for overcoming tumor heterogeneity are critical in cancer research. cancer research is extremely complicated and unique because each type of cancer for every individual is di�erent, one factor of which is tumor heterogeneity. this poses a major barrier to producing a generalized approach and treatment for all individuals su�ering from this disease. because tumors possess the capability to overcome therapeutics and treatments, scientists and medical researchers are constantly faced with new challenges in producing a cure for cancer. on the other hand, novel techniques that analyze individual cells and their distribution, such as single-cell sequencing and spatial transcriptomics, have transformed our predictive value and comprehension of the complex cellular organization.74,75 all of the mentioned novel technologies and ongoing clinical trials pave a clearer path toward �nding a more target-speci�c treatment for cancer. once tumor heterogeneity is addressed, research can focus on other complex aspects of cancer treatment. 5. conclusion tumor heterogeneity is the largest barrier in developing ground-breaking and target-speci�c cancer treatments. with therapeutic resistance forming among all forms of cancer, tumor heterogeneity highlights the need for novel treatments that will overcome this barrier. current therapies that can limit cancer disease progression include immunotherapy, particularly car-t cell therapy, and combination therapy. there are also novel strategies, such as dualor multi-eats, nanornps with a combination of sgrnas, and ctcs. each strategy presents their own respective challenges that limit them to mouse model studies or insu�cient target gene reduction. ongoing clinical trials exist to reduce such limitations and challenges and discover crucial information for the treatment of humans. since no single treatment has yet been discovered to completely overcome berkeley pharma tech journal of medicine | 105 tumor heterogeneity, medical research in developing personalized treatment and precision targeting is thus vital in saving lives. berkeley pharma tech journal of medicine | 106 references 1. ahmad 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https://my.clevelandclinic.org/health/diagnostics/23992-liquid-biopsy mythili et al. body template.docx berkeley pharma tech journal of medicine correspondence: mynair@ucsc.edu keywords: oral contraceptives (oc) depression estrogen progesterone synaptic pruning prefrontal cortex hypothalamic-pituitary-adrenal (hpa) axis submitted december 11, 2023 accepted july 14, 2024 published december 27, 2024 full open access creative commons attribution license 4.0 abstract adolescence is a critical stage of development marked by profound physical, emotional, and psychological changes. during this period, many adolescents turn to contraceptives for various reasons, including birth control, menstrual regulation, and addressing medical conditions. however, the potential impact of contraceptives on the mental health of adolescents remains a topic of significant concern and debate. this paper serves as a review of current literature on oral contraceptives (ocs), which are the most commonly prescribed form to adolescents, and concentrates on research that correlates contraceptives with heightened depression symptoms. the synthesis of findings in this review aims to inform healthcare professionals and researchers about the importance of further research and to help young individuals acquire a deeper comprehension of the long-term mental health implications associated with oc use during adolescence. long-term effects of hormonal oral contraceptives on adolescents’ mental health by: mythili nair, isabelle pappas, and sahithi lingala 1. introduction this paper aims to delve into the pharmacological and psychological mechanisms of oral contraceptives and their role in the onset or exacerbation of depression in adolescents. it will examine scientific reviews and primary articles to discuss how oral contraceptives work and alter adolescent physiology. additionally, it will provide insights into what depression is and how ssris and oral contraceptives can interact. this paper serves as an information guide for adolescents, raising awareness about the effects of birth control and emphasizing their increased vulnerability to depressive symptoms. it also serves as a call to action, advocating for further research to create effective contraceptive methods with a reduced risk of mood disorders. contraceptives encompass a diverse range of devices and drugs designed to prevent pregnancy, and they also serve several purposes like menstrual regulation, pain management, and addressing medical concerns such as endometriosis. in today's market, contraceptives are broadly categorized into two main groups: hormonal and non-hormonal. hormonal birth control includes emergency contraception (typically in pill form), oral contraceptives, implants, the vaginal ring, and estrogen/progestin patches. hormonal contraceptives function by introducing synthetic hormones that mimic the body's naturally occurring sex hormones, estrogen, and progesterone. oral contraceptives are taken daily by mouth, while implants, rings, and patches are placed inside the body and slowly release hormones into the bloodstream.1 non-hormonal birth control methods consist of condoms, spermicides, and natural family planning/fertility awareness. condoms, which can be made of latex, polyurethane, or lambskin, act as a barrier to prevent sperm from entering the uterus. spermicides are chemical agents that kill or immobilize sperm, often used in conjunction with other barrier methods. family planning involves cycle tracking and avoiding intercourse during ovulation to prevent unwanted pregnancy. additionally, intrauterine devices (iuds) are a popular choice, available in both hormonal (progestin iud) and non-hormonal (copper iud) variations. the progestin iud functions berkeley pharma tech journal of medicine | 2 similarly to other hormonal contraceptives, while the copper iud creates a toxic environment for sperm, effectively killing them before fertilization.1 in 2018, a study unveiled that 38.7% of adolescents utilized contraception for medical purposes and to prevent unwanted pregnancies. among the various options, oral contraceptives stood out as the most frequently prescribed for young women due to their effectiveness and user-friendly nature.2 typically, the combination pill, incorporating both estrogen and progesterone, constitutes the most prevalent type, administered over a 28-day cycle comprising 21 active pills followed by a week of placebos, inducing menstruation. doses are personalized by physicians upon prescription. these pills work by mimicking a pregnancy-like state, fooling the pituitary gland into halting hormone release for ovulation, thus preventing pregnancy. progesterone, meanwhile, thickens cervical mucus, hampering sperm access to the uterus, and alters the uterine lining, impeding egg attachment. while crucial for maintaining stable hormone levels and aiding in addressing medical conditions, the introduction of synthetic hormones can disrupt the body's natural equilibrium, leading to various side effects. it is especially vital to discuss these potential effects with adolescents, given their heightened susceptibility to hormonal and bodily changes.3 among these side effects, the deterioration of mental health and worsening of mood disorders is one of the most studied. over the years, multiple studies have explored the correlation between oral contraceptives and depression, particularly in adolescents.4 depression has been linked to low serotonin, dopamine, and norepinephrine levels, influenced by genetic predisposition rather than solely inheritance. to address depression, selective serotonin reuptake inhibitors (ssris) are commonly prescribed, enhancing serotonin transmission by blocking its reabsorption into neurons. it is worth noting that ssris can also impact estrogen and progesterone levels due to their interaction with the body's hormones, potentially affecting those using both antidepressants and birth control. in adolescents, the onset of depression is associated with increasing levels of gonadal hormones, including estrogen and progesterone. contraceptive pills introduce synthetic sex hormones into the body, potentially elevating berkeley pharma tech journal of medicine | 3 these hormone levels and worsening depression symptoms. the connection between depression and oral contraception remains a subject of ongoing research.5 figure 1. a visual representation of the different types of contraceptives. the figure highlights the distinctions between oral contraceptives, implants, iuds, and barrier methods, emphasizing their mechanisms and clinical applications. this visualization underscores the diverse choices available for managing reproductive health. 2. literature review 2.1 studies show that the link between oc use and depression in women is not entirely straightforward the link between oral contraceptive pill (ocp) use and depression is not entirely straightforward and has been a topic for debate for decades since the conception of the pill. more recent studies (from 2020 onward) tend to agree that oral contraceptive use can cause depressive symptoms in those with no history of psychiatric illness as well as exacerbate symptoms in those with a history of mental illness. according to a pilot study conducted by jayashri kulkarni, depressive symptoms that result from oral contraceptive use was the single most reason for discontinuation of the pill.6 however, there are several earlier studies that show no correlation between ocp use and depressive symptoms, suggesting that our knowledge about the link between the two is limited. berkeley pharma tech journal of medicine | 4 b. bottcher et al. analyze several studies from 1960-2005 that examine the effects of ocp use on mood. bottcher et al. contribute the misattribution of ocp use to depression with “the inconsistent use of the term ‘depression’ and the large number of combined contraceptives which vary in their compositions.”7 according to the dsm, “a major depression is not equivalent to negative mood changes, tension, irritability, anxiety, or sadness,” and, as a result, the results of the studies analyzed in this article do not necessarily indicate ocp use as a cause of depression.8 bottcher et al. also claim that depression is more common in women than it is in men, and the “median age for diagnosis was 31, which is within the reproductive age.”9 thus, it may not be entirely true that ocp use directly causes depression but rather that women are more likely than men to show signs of depressive symptoms. additionally, the majority of women who use oral contraceptives are likely to be of reproductive age, which is the most common age at which depression is diagnosed in both women and men. ultimately, b. bottcher at al. concluded that there is no significant correlation between ocp use and depressive symptoms. that being said, bottcher et al. also recognize the limitations of their analysis, as “the composition of hormonal contraceptives has also changed dramatically– the dose of ethinyl estradiol has been consistently reduced over the years and is less than half of that used in the early years of hormonal contraception.”10 the researchers analyzed studies from around the late 20th century, and the compositions of present-day ocps have changed significantly since then. this change may have something to do with the conflicting results in later studies that often find a correlation between ocp use and depression in women. another study conducted by t. johannson et al. examined the long-term mental health effects of birth control on women and found that ocp use is “causally” related to risk of depression. the study utilized medical information from more than 250,000 women in the uk biobank (ukb) and looked particularly at the risk of depression in adolescents and adults during and after ocp use. the study found that “the first 2 years of oc use were associated with a higher rate of depression compared to never users.”11 even more, their findings “showed that women who used ocs during adolescence remained at a heightened risk even after they discontinued, berkeley pharma tech journal of medicine | 5 whereas such a risk was not apparent among adult oc users.”12 johannes et al. explain the results of their findings by suggesting that adolescents are more sensitive to hormonal changes, putting them at an increased risk for depression with ocp use. according to johannson, sensitivity to hormonal changes is greatest “during crucial developmental periods that affect the organization of the brain structures and may lead to long-lasting changes.”13 this article confirms the causal relationship between ocp use and an increased risk of depression in adolescents. however, the researchers’ discussion of their results suggests that adolescents are inherently vulnerable to depressive symptoms due to puberty and environmental changes and that ocp use may only exacerbate these symptoms. while t. johannson et al. discovered a causal relationship between an increased risk of depression and ocp use, a study conducted by anouk e. de wit et al. (2021) suggests that it is not clear whether the relationship is causal or not. the researchers conducted a meta-analysis of clinical trials consisting of over 5000 women who were given either a hormonal contraceptive pill or a placebo pill. only one trial consisted of adolescent women; thus, the researchers concluded that they “cannot rule out that first-time use is a risk factor for experiencing depressive symptoms with hormonal contraceptive use.”14 the hormonal contraceptives that were given to the women included combined oral contraceptive pills, progestin-only pills, contraceptive patches, combined injectable contraceptives, progestin-only injectable contraceptives, intrauterine devices, and more. thus, the pill was not the only hormonal contraceptive method that was studied here but also other forms of hormonal contraception, like the patch and injectables. the results of the study suggest that not a single form of hormonal contraception showed a greater risk of depression over another or the placebo. this conclusion is in direct opposition to that of several observational studies, which have found a significant increase in incidence of depression in ocp users and particularly in adolescent ocp users.15 a year earlier, the same researcher anouk e. de wit co-authored a different article that looked at the association of oral contraceptive use with depressive symptoms among young women and adolescents. the berkeley pharma tech journal of medicine | 6 researchers “did not find support for an overall association between ocp use and depressive symptoms among young women. however, 16 year old girls using ocps did report higher concurrent depressive symptom scores compared with their nonusing counterparts.”16 16-year-old users of ocps reported a greater incidence of crying, hypersomnia, and eating problems, which symptoms are more associated with adolescent depression as opposed to adult depression. the researchers also discuss several confounding variables that may explain this trend. anouk de wit et al. suggest that there may be a higher prevalence of depression in 16-year-old ocp users not only due to the hormonal changes that the drug itself induces but also because “treatment with ocps is standard care for cycle-related mood problems and for polycystic ovarian syndrome, which is associated with depressive symptoms.”17 thus, the increased prevalence of depression in 16 year-old ocp users as compared to nonusers of that same age group might be related to the fact that those 16 year-olds who use ocps are more likely to have suffered from depression and other mood disorders even before they started the pill. in an article published in 2004, stephen a. robinson et al. set out to determine whether psychological or pharmacologic mechanisms were responsible for the adverse emotional side effects of the pill in oc users. the researchers conducted a study in which half of the women were given oc pills and the other half placebo pills. the results of study show that women on the placebo pill “experienced a similar side effect profile of ocp users.”18 psychiatrist johan culberg proposed that the psychological side effects of oc use were due to the “symbolic property of contraception:” “it is something that prevents sexual intercourse from being followed by pregnancy and an unwanted child.”19 additionally, the blood levels of estrogen and progesterone in the ocp users were measured, and researchers found no significant correlation between hormone blood levels and depressive symptoms in these women. this is to suggest that the adverse mental and emotional side effects from oc use are largely due to psychological, not pharmacologic, mechanisms. robinson et al. conclude, “it is not the pharmacodynamics that primarily impact the individual’s psyche and subsequent emotions and behavior, but rather the belief that one is contracepting is causing such a phenomenon.”20 additionally, the berkeley pharma tech journal of medicine | 7 authors introduce the idea that “contraception leads to a complete dissociation from the pleasurable/ relational and procreational components which, in turn, places a different value on sexuality itself.”21 it turns out that the use of oral contraceptives is not the only factor that changes the “value of sexuality.” in fact, the “value of sexuality” has been changing since the sexual revolution of the 60s and, even more so, with the advent of the modern hook-up culture. according to an article published by justin r. garcia et al. in the national library of medicine, “hook-ups” are defined as “uncommitted sexual encounters [which] are becoming progressively more ingrained in popular culture, reflecting both evolved sexual predilections and changing social and sexual scripts.”22 since sexual encounters are becoming more non-committal, and, thus, more frequent, the use of products such as oral contraceptives to prevent lifelong consequences is much more prevalent today than it was even only a decade ago. as such, there have been several articles published within the last four years that aim to discuss both the physical and mental-emotional side effects of oral contraceptives in women, particularly adolescents. perhaps this is why more recent studies, by dewit et al. in 2020-21 and by johannson et al. in 2023, are largely focused on the effects of oc use in adolescent women or first time users as compared to older women or women who have been using ocs for more than two years. as it turns out, all scientific inquiries are influenced by the social phenomena that plague the particular period of time in which they are made. figure 2. a graphical representation of the correlation between oral contraceptive use and the prevalence of depression in adolescent users.oc users have a higher depression symptom score from approximately ages 16 to 21 than do non-users. a higher depression symptom score indicates more depression symptoms berkeley pharma tech journal of medicine | 8 overall. the graph highlights the higher risk during the first two years of use, providing evidence of increased vulnerability in younger populations. 2.2 chemistry/ pharmacology of oral contraceptives associated with depressive symptoms in adolescent users oral contraceptive pills (ocps) are a commonly used form of birth control, and while they are generally considered safe and effective, there is growing concern regarding their potential link to mood disorders, particularly in adolescent users. to understand the plausible chemical and pharmacologic explanations for depression in adolescent ocp users, it is essential to examine the intricate relationship between the hormones present in these pills, namely estrogen and progesterone, and their impact on neurochemistry and brain function. these hormones exert their effects through specific receptors, with estrogen acting primarily through estrogen receptors (er)-alpha and er-beta, and progesterone through progesterone receptors alpha and beta, distributed throughout the brain. er-alpha is prominently found in the hypothalamus, hippocampus, amygdala, and brainstem,34 while progesterone receptors alpha and beta are most abundant in the amygdala, cerebellum, cortex, hippocampus, and hypothalamus.35 estrogen, in particular, has been associated with neuroprotection in various regions of the brain, such as the hypothalamus, hippocampus, amygdala, and brainstem. several studies have suggested that estrogen can safeguard the brain against neurodegenerative diseases, such as alzheimer’s and parkinson’s disease, cognitive decline, and affective disorders.36 functional brain imaging studies have revealed that estrogen plays a role in regulating the activation of brain regions involved in emotional and cognitive processing, such as the amygdala and dorsolateral prefrontal cortex.37 additionally, in animal studies, estrogen has been shown to modulate several neurotransmitters, including serotonin, dopamine, and noradrenaline, all of which have significant implications for mood regulation and depression. estrogen can also influence the release of adrenocorticotropic hormone, further impacting stress responses and mood.38 in contrast to estrogen, progesterone does not exhibit the same neuroprotective properties. in fact, evidence suggests that progesterone can berkeley pharma tech journal of medicine | 9 exacerbate mood symptoms and potentially contribute to the development of mood disorders. plausible mechanisms for this include progesterone's augmentation of gaba-induced inhibition of glutamate transmission and its ability to increase the concentrations of monoamine oxidase. these actions can lead to decreased serotonin levels, which are often associated with depressive symptoms.39 moreover, a study has shown a positive association between the use of levonorgestrel-containing intrauterine devices (iuds) and the development of depression, anxiety, and sleep problems in women who did not have these conditions before using the iud.40 levonorgestrel is a synthetic progestogen, and the two formulations of progestogen-releasing iuds, containing 19.5 mg and 52 mg of levonorgestrel, may have varying effects on mood.41 the former, which releases smaller amounts of levonorgestrel, may be more tolerable in terms of mood disturbances. however, it is crucial to note that there is a lack of comprehensive data regarding the relationship between this specific iud and the development or exacerbation of depression. selective serotonin reuptake inhibitors (ssris) and oral contraceptives can have notable interactions when used concurrently, which is essential to consider for individuals who are prescribed both medications. ssris, commonly used to treat depression and anxiety disorders, work by increasing the levels of serotonin in the brain, a neurotransmitter that affects mood and emotional state.42 on the other hand, oral contraceptives contain synthetic hormones, typically a combination of estrogen and progestin, which are used for birth control and to regulate menstrual cycles.43 when taken together, these medications can influence each other's metabolism and effectiveness. for instance, some ssris can increase the levels of estrogen in the body by inhibiting enzymes responsible for estrogen metabolism. this elevation could potentially increase the risk of side effects associated with oral contraceptives, such as blood clots, especially in individuals with other risk factors. conversely, certain oral contraceptives can affect the metabolism of ssris, potentially altering their efficacy and leading to either increased or decreased levels of the antidepressant in the bloodstream. this interaction might necessitate adjustments in ssri dosages to maintain therapeutic effectiveness and minimize side effects.44 it's crucial for individuals taking both ssris and oral contraceptives to have close berkeley pharma tech journal of medicine | 10 monitoring by healthcare professionals to manage these potential interactions effectively. figure 3. molecular structures of estrogen and progesterone, the primary hormones in oral contraceptives. the figure demonstrates their chemical composition and structural differences, helping to explain their unique roles in contraceptive efficacy and potential effects on mood regulation. 3. discussion 3.1 higher risk of depression in adolescents adolescence is a period marked by a higher risk of depression, a risk deeply rooted in biological factors. firstly, the ongoing development of the adolescent brain plays a crucial role. during this stage, the brain undergoes synaptic pruning, a process that eliminates unnecessary connections to increase efficiency. this phase involves the removal of neural connections that are less frequently used, while strengthening those that are more active. this selective pruning helps in optimizing brain efficiency and functionality. however, it is important to note that this process is not uniform across the brain. different areas of the brain undergo this pruning at different times, which can lead to imbalances in cognitive and emotional processing during adolescence.23 additionally, the prefrontal cortex, a part of the brain responsible for higher-order functions like decision-making, impulse control, and emotional regulation, is one of the last areas to mature. this delayed development can contribute to characteristic adolescent behaviors. adolescents might struggle with impulse control, making them more prone to risk-taking behaviors. they might also experience challenges in decision-making, leading to choices that seem irrational or poorly berkeley pharma tech journal of medicine | 11 thought out from an adult perspective.24 the combination of an underdeveloped prefrontal cortex and ongoing synaptic pruning can lead to heightened emotional experiences in adolescents. they might face difficulties in managing their emotions, leading to mood swings or intense emotional reactions to situations that adults might find more manageable. this heightened emotional sensitivity is often compounded by hormonal changes that occur during puberty, further complicating the emotional landscape of adolescence. the adolescent phase marks a significant surge in sex hormones, primarily estrogen in females and testosterone in males. these hormones do more than just drive physical development; they profoundly influence the brain's chemistry, particularly the neurotransmitter systems.25 a key player in this scenario is serotonin, a neurotransmitter that plays a critical role in regulating mood and emotional well-being. the surge in sex hormones during adolescence directly impacts serotonin levels in the brain. serotonin is known for its role in feelings of happiness and emotional stability, and its fluctuating levels can significantly affect an adolescent's mood. these hormonal changes can make adolescents more vulnerable to mood disturbances. for instance, a dip in serotonin levels can lead to feelings of sadness or a depressed mood, contributing to the increased incidence of depressive symptoms observed in this age group.26 notably, these hormonal fluctuations also interact with the adolescent's stress response system. the stress response system, which includes the hypothalamic-pituitary-adrenal (hpa) axis, becomes more reactive during adolescence. this heightened reactivity, combined with fluctuating serotonin levels, can make adolescents particularly sensitive to stress. they may experience more intense reactions to stressful situations, and their ability to cope with these stressors may be compromised due to the ongoing developmental changes in their brains. moreover, these biological factors do not function independently. they are intricately connected to and influenced by the adolescent's environment, including their social interactions, family dynamics, and life experiences. stressful or negative environments can exacerbate the impact of hormonal changes on mood and stress response, leading to a higher risk of developing mental health issues berkeley pharma tech journal of medicine | 12 such as anxiety or depression.27 the intricate relationship between the hypothalamic-pituitary-adrenal (hpa) axis and adolescent development plays a pivotal role in understanding the increased vulnerability to stress and depression during this critical life stage. the hpa axis, a central part of the body's stress response system, becomes highly sensitive during adolescence. this sensitivity is largely due to the significant hormonal changes occurring during this period, which interact with and can potentially dysregulate the hpa axis. typically, the hpa axis helps the body manage stress by releasing cortisol, a hormone that prepares the body to handle stressful situations. however, during adolescence, this system can become overactive or remain active for longer periods due to the heightened hormonal fluctuations. this dysregulation of the hpa axis in adolescents leads to an enhanced stress response, making them more reactive to stressors that might seem manageable to adults. this heightened stress reactivity is not just a momentary experience; it can have prolonged effects on the adolescent's emotional and psychological well-being. the continual overexposure to cortisol and other stress hormones can lead to various adverse outcomes, one of the most concerning being an increased susceptibility to depression.28 adolescents, already dealing with various physical, social, and emotional changes, may find this added stress reactivity overwhelming, pushing them towards depressive states. moreover, this heightened vulnerability is not just a matter of increased emotional sensitivity; it has biological underpinnings that can lead to long-term patterns of stress response and emotional regulation. the ongoing stress and potential for depression during adolescence can, in turn, affect the brain's development and functioning, particularly in areas responsible for mood regulation and stress management. this creates a feedback loop where dysregulation of the hpa axis exacerbates stress and depression, which further affects the hpa axis's functioning.29 importantly, these biological processes do not exist in isolation; they are closely intertwined with the social and environmental factors that adolescents face. peer relationships, academic pressures, family dynamics, and the quest for self-identity all influence an adolescent's emotional well-being.30 a biological predisposition to depression refers to the inherent berkeley pharma tech journal of medicine | 13 tendency, often influenced by genetic and physiological factors, that makes certain individuals more susceptible to developing depression compared to others. genetics indeed play a significant role in this predisposition.31 research has identified several genes associated with an increased risk of depression, although it is important to note that no single gene causes the disorder. instead, depression is a complex trait influenced by multiple genes, each contributing a small effect. these genes often affect neurotransmitter systems in the brain, such as serotonin, dopamine, and norepinephrine pathways, which are crucial for mood regulation.32 in adolescents, the interplay between these genetic predispositions and the rapid physiological changes they undergo becomes particularly impactful. the brain is still developing during adolescence, and hormonal fluctuations are significant. these hormonal changes can influence the expression of genes related to mood regulation and stress response, potentially triggering or exacerbating depressive symptoms in those with a genetic predisposition.33 understanding these intricate biological underpinnings is essential in developing effective interventions and support systems to help adolescents navigate this challenging period and reduce the risk of depression. 3.2 practical applications the following section discusses the practical applications of the research that attempts to demystify the mental health effects of oral contraceptives on adolescents. since the sexual revolution in the 60s, the stigma surrounding sexual health and pleasure has declined significantly. the advent of a hook-up culture in more recent years means that women are having sexual experiences at a younger age today than ever before. of course, the value of sex has changed over time as well, as more women choose to engage in sex acts not only for procreative purposes but also for pleasure. patentors of birth control packaging should make a clear list of not only the physical but also the mental-emotional side effects of the pill on its packaging. with the recent rise in abortion bans across the us, administering safe and effective forms of birth control to adolescent women is more important now than ever before.1 more recently, the fda approved a non-prescription, over-the-counter birth control pill the opill, to be released in early 2024.2 this means that birth control will be much more berkeley pharma tech journal of medicine | 14 accessible to women of all age groups. adolescents may benefit most from the opill as neither a prescription nor parental consent will be required to obtain birth control. since adolescents will soon have easier access to hormonal contraception, it is particularly important to educate adolescents on the mental and emotional side effects of the pill. additionally, before prescribing birth control pills to their patients, healthcare providers should take into account the psychiatric history and age of their oc candidates. both parties, the prescriber and the patient, need to be informed of the physical and mental side effects of the oral contraceptive they choose to administer and take, respectively, as if it were any other drug. knowledge about the ways in which both prescription and non-prescription medication can affect our emotional-well-being is paramount to using hormonal contraceptive safely and effectively. figure 4. the opill, a progestin-only birth control pill now available over the counter. this marks a major step forward in making contraception more accessible and empowering individuals to take charge of their reproductive health. 4. future directions scientists are engaged in extensive research and development efforts to address the risk of depression and mood disorders associated with oral contraceptive pills. one promising avenue involves fine-tuning the hormone formulations in these contraceptives. by modifying the types and quantities of hormones, researchers aim to create pills with reduced mood-altering berkeley pharma tech journal of medicine | 15 effects while maintaining their effectiveness in preventing pregnancy. for instance, scientists are working on progestins with a lower propensity to affect mood.47 additionally, the use of transdermal patches or hormonal intrauterine devices (iuds) is being explored as alternative delivery methods that might limit systemic hormone exposure and, consequently, mood-related side effects. personalized medicine is emerging as a key strategy. researchers are investigating ways to identify genetic, hormonal, and psychological factors that make certain individuals more susceptible to mood disturbances when using oral contraceptives.48 this approach allows healthcare providers to match women with contraceptives that are less likely to negatively impact their mood based on their unique profiles. to delve deeper into this issue, scientists are conducting extensive research into the mechanisms underlying the connection between hormonal contraceptives and mood disorders. this includes studying how these medications affect the brain, neurotransmitter systems, and the endocrine system. by understanding the exact mechanisms at play, researchers can develop more targeted interventions and, ideally, identify biomarkers that can predict which individuals are at a higher risk of experiencing mood-related side effects. furthermore, the future of contraceptive care is set to encompass behavioral and psychological support. integrated counseling, resources, and strategies will be provided to women both before and during contraceptive use, empowering them to manage mood changes and mental health concerns more effectively. this support can include coping strategies, stress management techniques, and interventions to address any mood disturbances that may arise.49 long-acting contraceptives are gaining attention as alternatives for some women. hormonal iuds and implants release hormones in a more controlled and localized manner, potentially reducing the systemic impact on mood. researchers are working on expanding the availability and improving the safety of these long-acting options.50 in this digital age, telemedicine and digital health tools are becoming invaluable. they enhance access to healthcare providers and mental health resources for women using oral contraceptives. real-time monitoring and digital platforms can facilitate early identification of mood changes and enable timely interventions, contributing to improved mental well-being during contraceptive use. berkeley pharma tech journal of medicine | 16 4. conclusion in conclusion, this review paper has addressed the critical issue of the long-term effects of hormonal oral contraceptives on adolescents' mental health. the research highlights the complex and multifaceted relationship between oral contraceptives and mood disorders, particularly in adolescent users. while the topic remains a subject of debate, it is clear that various factors, both biological and psychological, can contribute to the development or exacerbation of depressive symptoms. this paper has shed light on the biochemical processes at play, elucidating how hormones like estrogen and progesterone interact with the brain, potentially impacting mood regulation. it has also explored the role of psychological mechanisms, demonstrating that beliefs and societal attitudes about contraception can influence emotional well-being. furthermore, the increased vulnerability of adolescents to mood disorders, owing to ongoing brain development, hormonal fluctuations, and heightened stress reactivity, has been discussed. the interplay between biological factors and the social and environmental challenges that adolescents face underscores the need for comprehensive support and interventions to mitigate the risk of depression during this crucial stage of development. looking ahead, scientists and healthcare providers are actively pursuing strategies to minimize the potential risks of hormonal contraceptives, emphasizing personalized medicine, alternative delivery methods, and the integration of behavioral and psychological support. long-acting contraceptives and digital health tools are emerging as promising options to enhance mental well-being for contraceptive users. as we move into the future, it is imperative to continue research in this field to gain a more comprehensive understanding of the implications of oral contraceptive use on adolescents' mental health. the goal is to provide young individuals with safer and more effective contraceptive choices while prioritizing their emotional and psychological well-being. with an informed approach and tailored interventions, we can empower adolescents to make educated decisions about their reproductive health, ultimately leading to improved mental health outcomes. berkeley pharma tech journal of medicine | 17 references 1. mayo clinic. birth control options: things to consider.mayo clinic. 2022 feb 16. https://www.mayoclinic.org/healthy-lifestyle/b irth-control/in-depth/birth-control-options/ar t-20045571. accessed 2024 dec 5. 2. centers for disease control and prevention (cdc). products data briefs number 388. 2020 dec 8. accessed 2023 dec 10. https://www.cdc.gov/nchs/products/databriefs /db388.htm. 3. nhs. combined pill.nhs.uk. 2017 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psychol. 2012;16(2):161. doi:10.1037/a0027911. 23. spear lp. adolescent neurodevelopment. j adolesc health. 2013;52(2):s7–s13. doi:10.1016/j.jadohealth.2012.05.006. 24. arain m,mathur p, rais a, et al. maturation of the adolescent brain.neuropsychiatr dis treat. 2013;9(9):449–461. doi:10.2147/ndt.s39776. 25. peper js, dahl re. the teenage brain. curr dir psychol sci. 2013;22(2):134–139. doi:10.1177/0963721412473755. 26. montoya er, terburg d, bos pa, van honk j. testosterone, cortisol, and serotonin as key regulators of social aggression: a review and theoretical perspective.motiv emot. 2011;36(1):65-73. doi:10.1007/s11031-011-9264-3. 27. spear lp. heightened stress responsivity and emotional reactivity during pubertal maturation: implications for psychopathology.dev psychopathol. 2009;21(1):87-97. doi:10.1017/s0954579409000066. 28. romeo rd. the teenage brain: the stress response and the adolescent brain. curr dir psychol sci. 2013;22(2):140-145. doi:10.1177/0963721413475445. 29. karin o, raz m, tendler a, et al. a new model for the hpa axis explains dysregulation of stress hormones on the timescale of weeks.mol syst biol. 2020;16(7). doi:10.15252/msb.20209510. 30.twenge jm.why increases in adolescent depression may be linked to the technological environment. curr opin psychol. 2019;32:89-94. doi:10.1016/j.copsyc.2019.06.036. 31. shadrina m, bondarenko ea, slominsky pa. genetic factors in major depression disease. front psychiatry. 2018;9(334). doi:10.3389/fpsyt.2018.00334. 32. lohoff fw. overview of the genetics of major depressive disorder. curr psychiatry rep. 2010;12(6):539-546. doi:10.1007/s11920-010-0150-6. berkeley pharma tech journal of medicine | 19 33. jiang s, postovit l, cattaneo a, binder eb, aitchison kj. epigenetic modifications in stress response genes associated with childhood trauma. front psychiatry. 2019;10(808). doi:10.3389/fpsyt.2019.00808. 34. paterni i, granchi c, katzenellenbogen ja, minutolo f. estrogen receptors alpha (erα) and beta (erβ): subtype-selective ligands and clinical potential. steroids. 2014;90:13-29. doi:10.1016/j.steroids.2014.06.012. 35. brinton rd, thompson rf, foymr, et al. progesterone receptors: form and function in brain. front neuroendocrinol. 2008;29(2):313-339. doi:10.1016/j.yfrne.2008.02.001. 36. koszegi z, cheong ry. targeting the non-classical estrogen pathway in neurodegenerative diseases and brain injury disorders. front endocrinol. 2022;13. doi:10.3389/fendo.2022.999236. 37. hara y, waters em,mcewen bs, morrison jh. estrogen effects on cognitive and synaptic health over the life course. physiol rev. 2015;95(3):785-807. doi:10.1152/physrev.00036.2014. 38. mu e, kulkarni j. hormonal contraception and mood disorders. aust prescr. 2022;45(3):75-79. doi:10.18773/austprescr.2022.025. 39. standeven lr, mcevoy ko, osborne lm. progesterone, reproduction, and psychiatric illness. best pract res clin obstet gynaecol. 2020;69:108-126. doi:10.1016/j.bpobgyn.2020.06.001. 40. slattery j, morales d, pinheiro l, kurz x. cohort study of psychiatric adverse events following exposure to levonorgestrel-containing intrauterine devices in uk general practice.drug saf. 2018;41(10):951-958. doi:10.1007/s40264-018-0683-x. 41. hidalgom, bahamondes l, perrotti m, diaz j, dantas-monteiro c, petta c. bleeding patterns and clinical performance of the levonorgestrel-releasing intrauterine system (mirena) up to two years. contraception. 2002;65(2):129-132. doi:10.1016/s0010-7824(01)00302-x. 42. stahl sm.mechanism of action of serotonin selective reuptake inhibitors. j affect disord. 1998;51(3):215-235. doi:10.1016/s0165-0327(98)00221-3. 43. cooper db, mahdy h, patel p. oral contraceptive pills. pubmed. published november 24, 2022. https://www.ncbi.nlm.nih.gov/books/nbk43 0882/. 44. berry-bibee en, kimmj, simmons kb, et al. drug interactions between hormonal contraceptives and psychotropic drugs: a systematic review. contraception. 2016;94(6):650-667. doi:10.1016/j.contraception.2016.07.011. 45. foster dg. new abortion bans will increase existing health and economic disparities. am j public health. 2022;112(9):1276-1277. doi:10.2105/ajph.2022.306993. 46. fda. fda approves first nonprescription daily oral contraceptive. fda. published july 13, 2023. berkeley pharma tech journal of medicine | 20 https://www.ncbi.nlm.nih.gov/books/nbk430882/ https://www.ncbi.nlm.nih.gov/books/nbk430882/ https://www.ncbi.nlm.nih.gov/books/nbk430882/ https://www.fda.gov/news-events/press-annou ncements/fda-approves-first-nonprescription-d aily-oral-contraceptive. 47. sivasankaran s, jonnalagadda s. advances in controlled release hormonal technologies for contraception: a review of existing devices, underlying mechanisms, and future directions. j control release. 2021;330:797-811. doi:10.1016/j.jconrel.2020.12.044. 48. personalised and precision medicine for contraception.damahealth. published august 17, 2022. https://damahealth.com/the-science/. 49. dehlendorf c, krajewski c, borrero s. contraceptive counseling. clin obstet gynecol. 2014;57(4):659-673. doi:10.1097/grf.000000000000005 50. bahamondes l, fernandes a, monteiro i, bahamondes mv. long-acting reversible contraceptive (larcs) methods. best pract res clin obstet gynaecol. 2020;66:28-40. doi:10.1016/j.bpobgyn.2019.12.002. berkeley pharma tech journal of medicine | 21 https://damahealth.com/the-science/ berkeley pharma tech journal of medicine correspondence: sanchithabpt1@gmail.com keywords: tumor microenvironment immunotherapy cytotoxic t lymphocytes pd-1 ctla-4 lactic acid non-small cell lung cancer (nsclc) submitted august 11, 2023 accepted may 23, 2024 published june 28, 2024 full open access creative commons attribution license 4.0 abstract the tumor microenvironment (tme) surrounds the tumor and includes blood vessels, immune cells, fibroblasts, signaling molecules, and the extracellular matrix. this review examines the cellular components and pathways within the tme, highlighting their potential as targets for immunotherapy. it also covers recent advances from the past several years in tme, immunotherapy, and combination therapy. the review emphasizes the role of cd8+ t cells in the tme and their relevance to immunotherapy. it discusses various t cell-targeted treatments, including pd-1/pdl1, ctla-4, vegf, interferon-ɣ, lag-3, and er stress-xbp1. recognizing the complexity and uniqueness of each tumor's immunotherapy network, the review aims to understand and compare the tmes of different cancers and the respective immunotherapies. cancers covered include non-small cell lung cancer (nsclc), pancreatic ductal adenocarcinoma (pdac), cervical cancer, chronic lymphocytic leukemia, and gastric cancers. the review also addresses future research directions and applications of immunotherapies, aspiring to advance tme understanding and research. decoding the interplay: exploring immunotherapy resistance in the tumor microenvironment by: sanchitha kannabran, akshaya ajan, leanne lui, zaden yet 1. introduction the tumor microenvironment (tme) constitutes the immediate surroundings of a tumor, forming a complex ecosystem inclusive of nearby blood vessels, immune cells, �broblasts, signaling molecules, and the extracellular matrix (ecm).1 within the tme, there exist hypoxic and acidic conditions, alongside altered expression patterns of ecm proteins. these conditions foster the presence of resident and in�ltrating immunosuppressive cells, trigger the expression of immune checkpoint proteins, and promote the exclusion and exhaustion of cytotoxic t lymphocytes (ctls).2 such circumstances serve as the foundation for immunotherapy targeting the tme. while contemporary interest in tme immunotherapy appears recent, investigations into utilizing immune cells for combating tumors date back to the 1900s.3 new studies are directed towards pinpointing cellular pathways within the tme with the use of immunotherapy components to hinder growth. among the primary pathways are lactic acid accumulation, metabolic processes, and diverse signaling routes within the tme. 5,6,7 all these pathways represent potential targets for immunotherapy interventions. moreover, treatments are geared towards enhancing the immune system's ability to eradicate cancer cells and impede their proliferation. to achieve this goal, two primary strategies are employed: immune checkpoint inhibition and adoptive cellular therapy (act).4 immune checkpoint inhibition involves obstructing certain proteins, known as checkpoints, to enhance the responsiveness of immune cells.4 adoptive cell therapy (act) entails administering t cells to patients to bolster their ability to combat tumor cells.4 in this discussion, our focus lies on t cell-based immunotherapies, including anti-pd-1/pd-l1, anti-ctla-4, vegf, interferon-ɣ (ifn-ɣ), lag-3, and er stress-xbp1. however, the complexity of immunotherapy arises from the variable responses of individual cancers to di�erent immunotherapeutic approaches. this review delves into the distinctive attributes of cancers such as non-small cell lung cancer (nsclc), berkeley pharma tech journal of medicine | 114 melanoma, pancreatic cancer, cervical cancer, and chronic lymphocytic leukemia. 2. immune cell overview the extracellular matrix (ecm) is comprised of collagen, �bronectin, elastin, and laminin, serving as the structural framework within the tumor microenvironment (tme). within this environment, various immune cells including t cells, b cells, natural killer cells (nk), macrophages (m1 and m2), stromal cells, endothelial cells, and cancer-associated �broblasts (cafs) play pivotal roles in either promoting or suppressing tumor growth.8 t cells, equipped with t-cell receptors, target speci�c antigens on tumor cells, halting angiogenesis—the formation of new blood vessels—and leading to tumor cell destruction. however, t cells can become in�ltrated and dysfunctional within the immune system.8,9 b cells contribute to antibody production, antigen presentation, and cytokine secretion. 8,9 in the context of lymphocytes, both t and b cells, particularly tumor-in�ltrating lymphocytes (tils), are crucial in research. while tils e�ectively eliminate tumor cells, they can be recruited by the tme, compromising immune response e�cacy.4 regulatory t cells (tregs) maintain immune response balance, but when recruited to the tme, they hinder t cell formation, posing a danger to immune function. consequently, immune mechanisms originally aimed at protecting the body now facilitate tme growth.10 nk cells surveil the bloodstream for tumor cells, inhibiting metastasis.8,9 macrophages regulate immune responses by phagocytosing pathogens and presenting antigens. notably, increased macrophage in�ltration in tumors, particularly the m2 phenotype, and cytokine secretion, support tumor growth.8,9 berkeley pharma tech journal of medicine | 115 figure 1: this figure illustrates the distinct contributions of various immune cells to the tumor microenvironment (tme). it delineates the overall immune response attributed to each mentioned immune cell type: t cells, b cells, macrophages (m1 and m2), natural killer cells (nk), tregs, and cd8+ cells. the upper side represents the positive immune response, while the lower side depicts the autoimmune suppressive response. stromal cells, including endothelial cells, �broblasts, adipocytes, and stellate cells, play crucial roles in tumor development. tumor cells recruit these supporting cells from nearby tissue to aid in tumor formation. stromal cells secrete various factors that in�uence processes like angiogenesis, proliferation, invasion, and metastasis.8,9 endothelial cells, for instance, are pivotal in orchestrating blood vessel formation and signi�cantly contribute to cancer progression by promoting cancer cell migration, invasion, angiogenesis, and metastasis. they transition into cancer-associated �broblasts (cafs), facilitating communication between tumor cells and the tumor microenvironment (tme).1 this interaction often leads to the disruption of cell connections and detachment. cafs can derive from various immune cells, but within the tme, they become berkeley pharma tech journal of medicine | 116 signi�cant producers of extracellular matrix (ecm), tumor growth factors, cytokines, and other essential components. each stromal cell type contributes uniquely to the tme, and targeting their speci�c functions presents an opportunity for novel immunotherapeutic approaches.1 the immune response involves numerous intricate processes, among which angiogenesis stands out—a phenomenon where endothelial cells proliferate and migrate, forming new blood vessels.11 this process is pivotal as it grants tumors the capability to metastasize, spreading to other locations within the body. conversely, inhibiting angiogenesis can e�ectively curtail tumor growth and metastasis.11 for instance, research conducted by chen et al. demonstrates that the administration of anti-malarial drugs like dihydroartemisinin and artesunate resulted in a reduction in the size of cell lines. these drugs were found to hinder the growth factors crucial for cancer cell proliferation, thereby impeding angiogenesis.11 anti-angiogenic (aa) therapy plays a signi�cant role in remodeling the extracellular matrix (ecm), altering the distribution of cell types and populations.12 this therapy redistributes pericyte proteins along blood vessels, increasing coverage and eliciting a pro-aggressive tumor response. notably, glycosylation of these proteins is speculated to contribute to malignant resistance to aa therapy.12 however, a complication arises as resistance can develop post-aa treatment, fostering tumor cell migration and invasion. tumor cell receptors possess mechanisms to detect aa-induced alterations in their environment, prompting a remodeling of the entire tumor microenvironment (tme) to facilitate tumor growth once more.12 3. t cell based immunotherapy this section of the review will delve into several key surface cell receptors found on endothelial cells, tumor cells, and t-cells. it will explore their roles in promoting tumor growth, strategies to overcome resistance, and the berkeley pharma tech journal of medicine | 117 underlying mechanisms of resistance. emphasis will also be placed on the critical functions of in�ltrating tumor lymphocytes and cytotoxic t cells. within the tumor microenvironment (tme), various surface cell receptors recognize speci�c antigens, initiating signaling cascades that ultimately impact downstream events. these cascades often lead to the inactivation or impairment of e�ective t-cells, which are typically responsible for targeting and eliminating tumor cells. the tme, being highly adaptable, fosters conditions conducive to tumor cell growth, proliferation, and survival.13 several immune response cells contribute to immune resistance, including myeloid-derived suppressor cells (mdscs), regulatory t cells (tregs), tumor-associated macrophages (tams), tumor-associated neutrophils (tans), and endothelial cells. the secretion of negative regulatory factors by these cells can induce t-cell exhaustion, dysfunction, and apoptosis.13 figure 2: a. vascular endothelial growth factor-a (vegf-a) binds to vascular endothelial growth factor receptors (vegfrs) situated on endothelial cell surfaces, initiating a series of reactions that stimulate angiogenesis. b. the interplay between proteins located on t cell surfaces and their corresponding ligand surface cell receptors on tumor cells contributes to t cell exhaustion. berkeley pharma tech journal of medicine | 118 one e�ective therapeutic approach, immune checkpoint inhibition, has demonstrated success in rejuvenating the cytotoxic function of ctls and t-cells. this therapy works by alleviating the suppression of antitumor immunity, freeing ctls from exhaustion.13 the fda has approved antibodies targeting programmed cell death 1 (pd-1/pd-l1) and cytotoxic t lymphocyte-associated protein 4 (ctla-4) for treating various cancers. additionally, ongoing clinical research is targeting other immune checkpoints like lag3, cd39, cd73, and cd47, all showing promising potential in advancing cancer immunotherapy.14 3.1 pd-1/pd-l1 immune checkpoint inhibition stands out as an e�ective therapeutic approach in reviving the cytotoxic capabilities of ctls and t-cells. by alleviating the exhaustion-induced suppression of antitumor immunity, this therapy unleashes ctls to combat tumors.13 the fda has approved the use of antibodies targeting programmed cell death 1 (pd-1/pd-l1) and cytotoxic t lymphocyte-associated protein 4 (ctla-4) as treatments across various cancer types.13 numerous other immune checkpoints are currently under investigation in clinical research, including lag3, cd39, cd73, and cd47, all of which show promising potential in advancing the landscape of cancer immunotherapy.14 among these, pd-1 is a pivotal protein implicated in t cell apoptosis, or programmed cell death. it is predominantly expressed on the surface of activated immune cells such as macrophages, dendritic cells, b cells, and t cells.14 notably, pd-1 expression is particularly elevated on exhausted t cells, where it plays a role in inhibiting their normal immune function. when pd-1 interacts with its ligand receptor, pd-l1, on tumor cells, it initiates a series of signaling events that directly impede the response of activated cells by enhancing local evasion mechanisms.14,15 these immune checkpoint inhibitors (icis) interfere with t cell functionality, leading to berkeley pharma tech journal of medicine | 119 an increase in exhausted t cell populations, both of which contribute to adaptive resistance against immune checkpoint blockade therapy.16 researchers conducted clinical trials investigating the interaction between pd-1 and pd-l1 on cytotoxic t cells in mice. they observed accelerated tumor growth, which was mitigated when treated with an anti-pd-l1 antibody or through pd-1 knockout, resulting in reduced tumor growth. similar �ndings were noted in human trials.17 for instance, qian et al. demonstrated in a study focusing on glioma cancer that tumor-in�ltrating t cells become activated and can increase pd-1 expression, leading to t cell dysfunction mediated by immune checkpoint inhibitors (icis). consequently, the researchers suggested that anti-pd therapy could impede glioma cancer progression.17 another instance illustrating the e�ectiveness of immune checkpoint inhibition therapy in microsatellite instability/de�cient mismatch repair (msi/dmmr) tumors comes from the ongoing trial conducted by imhotep, which showcases a group of 120 patients bene�ting from anti-pd therapy.18 while anti-pd therapy has shown success in certain patients, others have exhibited poor responses due to both primary and acquired resistance mechanisms working against the treatment. e�ective anti-pd therapy hinges on the precise blockade of the pd-1 and pd-l1 pathways. however, if either protein lacks expression, the treatment proves ine�ective against the tumor cells—a direct primary resistance mechanism observed in some cases.15 furthermore, certain cancers lack tumor-in�ltrating t cells or pd-l1, rendering them unresponsive to anti-pd therapy.15 to combat this adaptive resistance, combination therapy involving both immunoand chemo-therapies has been proposed as a more e�ective approach to treating cancer compared to immunotherapy alone. 3.2 ctla-4 ctla-4, a protein associated with cytotoxic t lymphocytes, plays a role in dampening the anti-tumor immune response by regulating the activation of ctls, thus hindering an e�cient immune reaction.14 unlike pd-1, which manages programmed cell death, ctla-4 dictates whether a t cell berkeley pharma tech journal of medicine | 120 undergoes activation or remains in a state of rest termed exhaustion. it is present on the surface of stimulated functional t cells, and activation occurs upon stimulation from a t cell receptor.14 ctla-4 competes with cd28 for binding to the t cell ligand, blocking the crucial costimulation signals required for activation.14 due to its higher a�nity for the ligand, ctla-4 binds more e�ectively, leading to t cell exhaustion and inhibiting activation. using an antibody that selectively targets and binds to ctla-4 can restore t cell activation by eliminating the competition for t cell ligand binding with costimulation signals.14 additionally, ctla-4 is prominently expressed on the surface of tregs, which play a role in immune response suppression.14 research indicates that ctla-4 plays a pivotal role in regulating the function and production of tregs,14 which in turn directly suppress the activation of target immune cells by increasing ctla-4 expression.19 utilizing ctla-4 blockade therapy has emerged as an e�ective strategy for enhancing anti-tumor immune responses.14 the fda-approved monoclonal anti-ctla-4 antibody, ipilimumab, has signi�cantly enhanced overall survival rates among patients with malignant melanoma.4 it's worth noting that while anti-ctla-4 antibodies show e�cacy in late-stage melanoma, their e�ectiveness in other tumor types is limited. consequently, clinical trials are exploring combination therapies to address this limitation.4 3.3 lag-3 lymphocyte activation gene 3 (lag-3) serves as an immune checkpoint receptor, capable of dampening responses orchestrated by t and nk cells, thereby fostering a hyporesponsive condition, which aids tumors in escaping immune surveillance. typically found on activated and exhausted t and nk cells, b cells, dendritic cells, and tregs, lag-3 signaling inhibits t cell proliferation, cytokine generation, and cytolytic activity.20 additionally, its presence on tregs contributes to immunosuppression.20 an illustrative instance of this phenomenon is evident in individuals a�icted with chronic lymphocytic leukemia (cll), where there is signi�cant dysregulation of lag-3. both nk cells and t cells exhibit berkeley pharma tech journal of medicine | 121 heightened lag-3 expression, and elevated levels of lag-3 or soluble lag-3 (slag-3) are associated with adverse cytogenetics and unfavorable outcomes among cll patients. consequently, treatment involving the application of an anti-lag-3 blocking antibody called relatlimab to peripheral blood mononuclear cells (pbmcs) has demonstrated e�cacy in reducing leukemic cell counts and restoring nk cell and t cell mediated responses.20 as a result, interventions aimed at reinstating t and nk cell mediated responses through lag-3 blockade have emerged as promising therapies for hematological malignancies such as cll, follicular lymphoma (fl), di�use large b cell lymphoma (dlbcl), and acute myeloid leukemia (aml).20 3.4 er stress/ire1a-xbp1 a fresh angle in cancer therapy involves focusing on the er stress pathway, with a particular emphasis on the xbp1 gene.21 in a 2022 study conducted by zundell et al., investigating ovarian clear cell carcinomas (occc), it was discovered that the arid1a gene, responsible for epigenetically regulating gene expression via the swi/snf chromatin remodeling complex, is mutated in over 50% of occcs. this mutation results in inadequate expression of arid1a protein, leading to advanced-stage disease and early recurrence.21 this element plays a vital role by transcriptionally inhibiting the ire1𝛂-xbp1 pathway of the er stress response. their research using mouse models indicates that disabling xbp1 and inhibiting the ire1𝛂/xbp1 pathways signi�cantly enhances the survival rate of mice with occc, as it suppresses the growth of arid1a mutant occc cells.21 in the face of er stress, the ire1-𝛂 component, a type of unfolded protein response (upr), undergoes a structural change, splicing the mrna encoding the xbp1 transcription factor. this splicing facilitates the translation of xbp1, which in turn aids cancer cell survival by resolving er stress.21 er stress itself fosters the survival of cancer cells by activating adaptive programs through the unfolded protein response (upr) once detected. berkeley pharma tech journal of medicine | 122 consequently, inhibiting the upr is emerging as a therapeutic strategy for cancers characterized by heightened er stress response.21 in this study, the e�cacy of a therapeutic intervention targeting the ire1𝛂 rnase, b-i09, was examined. by inhibiting ire1𝛂 rnase activity, b-i09 e�ectively suppressed the growth of arid1a-activated cells in ovarian clear cell carcinomas (occcs), indicating the potential of targeting the ire1𝛂-xbp1 axis of the er stress response as a promising approach for treating arid1a mutant occcs.21 a study by ma et al. illustrated the role of cholesterol, the primary sterol distributed throughout the human body, in the er stress-xbp1 pathway.22 through staining techniques to observe cholesterol levels in tumor-in�ltrating t cells in mice, researchers found that elevated cholesterol content triggers cd8+ t cell exhaustion via the er stress-xbp1 pathway.22 inhibiting the xbp1 pathway or reducing cholesterol levels in cd8+ t cells restored normal antitumor function, while increased cholesterol was consistently linked to elevated pd-1 expression on tumor-in�ltrating t cells.22 similar patterns were observed in human colon cancer and myeloma samples, underscoring the signi�cance of cholesterol content in t cell exhaustion.22 3.5 vegf/vegf-a: vascular endothelial growth factors (vegfs) are signaling proteins crucially involved in the microvasculature of tumors, the suppression of immune cells, and the promotion of immunosuppression, both locally and systemically, in cancer.19 these proteins are predominantly secreted by the endothelial cells lining blood vessels, with additional production occurring in immune cells within the tumor microenvironment (tme).19 vegfs play a pivotal role in fostering tumor development and progression by interacting with receptors on tumor cells through autocrine and paracrine signaling pathways. among them, vegf-a primarily binds to vegfr-2, triggering the proliferation and migration of endothelial cells, thereby facilitating angiogenesis.19 berkeley pharma tech journal of medicine | 123 vegf-a boosts the expression of both pd-1 and ctla-4, dampening the body's ability to �ght tumors and reducing the activity of cd8+ t cells by transitioning them from an active state to an exhausted one, impairing their normal function. blocking vegf with anti-angiogenic agents can reverse the immune checkpoints' inhibitory e�ects.19 in a phase i trial led by daniel chiu et al., a combination of anti-pd-1 therapy and antiangiogenic agents was used to treat glioblastoma, a challenging form of brain cancer known for its resistance to ici therapy. the �ndings revealed that this combination treatment was well tolerated, safe, and did not lead to any previously unreported adverse events. furthermore, the results showed decreased levels of vegf-a and other angiogenic factors in the tumor microenvironment, underscoring the relationship between vegf and pd-1 expression.16 4. overcoming resistance to immune checkpoint blockade although the immune checkpoint blockade strategy has shown e�cacy in reviving the antitumor response within tumor-in�ltrating lymphocytes (tils) and cytotoxic t cells, the tumor microenvironment (tme) employs adaptive resistance mechanisms against this therapy. speci�cally, the upregulation of immune checkpoints such as pd-1 and ctla-4 in response to targeted antibodies can foster this adaptive resistance.14 moreover, the tme utilizes evasion tactics, such as enhancing pd-1 expression in response to t cell attacks, leading to the functional impairment of tils.13,17 additionally, the di�erentiation of cd8+ t cells poses a signi�cant challenge, as tils develop resistance to antitumor treatments, resulting in a diverse cd8+ t cell subset.17 researchers are exploring combination therapies, pairing immunotherapy with chemotherapy or radiation therapy, to address malignant tumors. in a study by feng y. et al., a dual gene therapy approach was investigated to counter adaptive resistance to immune checkpoint blockade therapy in both cd4+ and cd8+ t cells. the scientists developed a dual gene delivery system aimed at eliminating tumor adaptive resistance and restoring t cell function by modulating the expression of vegf-a and pd-l1 proteins.14 gene-silencing techniques were employed to decrease vegf-a expression in the tme, reducing angiogenic activity and blocking pd-l1 immune berkeley pharma tech journal of medicine | 124 checkpoint. the �ndings demonstrated an increase in the number of cd8+ t cells expressing granzymes b, leading to inhibited tumor growth.14 furthermore, increasing vegf-a gene silencing e�ectively alleviated tumor hypoxia. however, it's crucial to regulate the dosage of each treatment, as excessive pshvegf-a antibody treatment can exacerbate tumor hypoxia by overly depleting tumor vessels. upon treating tumor-infected mice with the dual gene therapy, scientists observed substantial inhibition of tumor growth and prolonged overall survival rates compared to mice treated with monotherapy.14 this dual gene therapy not only countered immune checkpoint blockade-induced adaptive resistance but also reversed the immunosuppressive tumor microenvironment simultaneously. this dual gene delivery system represents a promising avenue for immunotherapy across various tumor types, though further research is warranted 4.1 lactic acid in the tumor microenvironment one of the key areas of exploration for scientists has been the manipulation of lactic acid within the tumor microenvironment (tme). lactic acid, a byproduct of altered metabolism, plays a signi�cant role in shaping the tme. its acidic nature fosters various processes including angiogenesis, metastasis, and drug resistance. a 2020 study highlighted that heightened lactic acid production can detrimentally a�ect anticancer immunity by suppressing immune responses due to the low ph environment it creates.7 lactic acid impedes the di�erentiation of monocytes into dendritic cells, leading to a reduction in antigen-presenting functions. additionally, it hampers the anti-tumoral activities of immune cells like natural killer cells and cytotoxic t cells.7 consequently, the targeting of lactate and lactic acid has emerged as a compelling area of focus within contemporary immunotherapy research. scientists have been investigating the neutralization of lactic acid as a potential method. by bu�ering the tumor microenvironment using oral bicarbonate and coupling it with anti-pd-1 immunotherapy and adoptive t-cell transfer, there's potential for enhancing survival rates.5 furthermore, research has shown that v-domain ig suppressor of t cell activation (vista) can inhibit t cells in an acidic ph environment. consequently, berkeley pharma tech journal of medicine | 125 blocking vista in conjunction with pd-1 blockade, and the development of ph-speci�c antibodies against vista, have demonstrated e�cacy in tumor rejection.5 another signi�cant focus lies in targeting the lactate-producing enzyme ldh, which shows promising anti-cancer e�ects. however, its impact on immune cells remains largely unexplored, as ldh inhibition can decrease t cell levels5. an alternative approach involves inhibiting lactate transporters. notably, researchers identi�ed slc4a4 as the predominant bicarbonate transporter in pancreatic ductal adenocarcinoma (pdac). inhibiting slc4a4 in pdac cells reduces tme acidi�cation by accumulating bicarbonate in the extracellular space and lowering lactate production. combining slc4a4 targeting with immune checkpoint blockades has been found to overcome immunotherapy resistance.6 targeting lactic acid holds signi�cant promise for advancing immunotherapy in the tumor microenvironment, as it plays a pivotal role in tme acidi�cation and subsequent immune response suppression. 4.2 targeting metabolism in the tumor microenvironment focusing on metabolism to enhance the tumor microenvironment for cancer immunotherapy holds signi�cant promise. cancer cells exhibit heightened metabolic activity, fueling their rapid proliferation. by zeroing in on metabolism, scientists can slow down cancer cell spread and impede their function. the realm of immune metabolism o�ers potential metabolic targets to bolster anti-cancer immunity. the dynamic metabolism of immune cells signi�cantly in�uences their functions as well.25 a primary approach involves targeting amino acid metabolism, where speci�c amino acids such as glutamine, tryptophan, and arginine have emerged as key targets for inhibiting tumor progression and enhancing immunity. glutamine, in particular, plays a vital role as a nutrient for cancer cells. enzymes like glutaminase (gls) facilitate the conversion of glutamine to glutamate, making gls a prime target to curb cancer cell metabolism and glucose utilization. however, targeting gls poses challenges as cancer cells employ alternative methods to boost glucose metabolism. additionally, directing gls inhibition towards immune cells, particularly t cells, a�ects berkeley pharma tech journal of medicine | 126 their di�erentiation, survival, proliferation, and e�ector functions.5 this presents a signi�cant hurdle, as it necessitates reducing glutamate levels in cancer cells while ensuring adequate glutamine for proper t cell function. consequently, a strategy has been devised to broadly block glutamine metabolism alongside anti-pd-1 immunotherapy. this combined approach enhances anti-tumor e�ects by dampening tumor metabolism while maintaining robust glucose metabolism in t cells.5 furthermore, researchers have shown keen interest in targeting arginine and nitric oxide, delving into amino acid metabolism. arginine plays a pivotal role in cancer and immune cell functions, particularly in proliferation, survival, and protein synthesis. the metabolic pathways of arginine heavily rely on enzymes like arginase (arg) and nitric oxide synthase (nos). however, given arginine's signi�cance to both cancer and immune cells, its potential as a therapeutic target presents complexities. while arginine depletion proves e�ective in certain scenarios, it can hinder anti-tumor t cell responses and bolster the population of myeloid-derived suppressor cells (mdscs), thus exacerbating arginine depletion.5 yet, inhibiting arg o�ers promise by restoring arginine levels, leading to tumor regression and enhancing t cell functionalities.5 targeting arginine and nitric oxide in myeloid cells could potentially enhance immunotherapy, but further research is needed to understand its impact on other immune cells. tryptophan is another signi�cant amino acid being investigated for immunotherapy due to its role in cell growth and maintenance. adaptive immune cell subsets such as tregs, tolerogenic dendritic cells (dcs), and myeloid-derived suppressor cells (mdscs) exhibit high levels of indoleamine 2,3-dioxygenase (ido), the enzyme that converts tryptophan to kynurenine.5 kynurenine has immunosuppressive e�ects on t cells, so targeting ido activity could not only mitigate these immunosuppressive e�ects on t cells but also inhibit these adaptive immune cell subsets.5 lipid metabolism has emerged as a recent target in immunotherapy. cancer cells exploit lipid metabolism to support their rapid proliferation, survival, migration, invasion, and metastasis.26 reprogramming lipid metabolism can berkeley pharma tech journal of medicine | 127 prevent e�ector t cell senescence, suggesting that lipid metabolism plays a role in regulating senescence development in t cells.27 fatty acid metabolism is crucial for the di�erentiation of immune cells, and the upregulation of fatty acid oxidation (fao) can enhance the functions of cd8+ t cells. reprogramming fatty acid metabolism in immune cells could promote an inhibitory immune microenvironment.28 it is often used in combination therapies with other immunotherapeutic treatments, such as immune checkpoint blockades (icbs).28 recent studies have shown that targeting fatty acid metabolism in t cells can improve the e�cacy of icbs. for instance, a study found that combining beza�brate with pd-l1 antibody treatment yielded better results in llc xenograft mouse models. beza�brate treatments increased the expression of fao-related genes in cytotoxic t lymphocytes (ctls), such as pgc-1α, cpt1a, and lcad, and maintained the survival and function of ctls.29 scientists have explored manipulating iron metabolism as a strategy for treating tumors, given iron's crucial role in tumor proliferation. iron metabolic dysfunction directly impacts cancer pathophysiology, with the availability of iron regulating the aggressive phenotypes of tumors.30 many tumor microenvironment (tme) cells rely on iron to thrive and function properly. for instance, activated m2 tumor-associated macrophages (tams) can disrupt iron homeostasis within tumor cells by exporting high amounts of iron and increasing the production of other iron-related proteins.30 it has been observed that iron chelation, the bonding of ionic molecules to metal ions, can reverse the iron-exporting phenotype of m2 tams.30 blocking transferrin receptor 1 (tfr1), a receptor protein that recognizes transferrin-bound iron, has proven e�ective in suppressing tumor growth, as tfr1 is often upregulated in cancer cells to enhance their iron supply.30 thus, cutting o� a tumor's iron supply appears to be an e�ective method to reduce tumor growth. berkeley pharma tech journal of medicine | 128 figure 3: the interactions between tryptophan and kynurenine. the enzyme ido converts tryptophan into kynurenine, which exerts an immunosuppressive effect on t cells. blocking ido would not only eliminate the immunosuppressive effect on t cells but also suppress adaptive immune cell subsets, including mdscs, tregs, and dcs. 4.3 targeting signaling pathways di�erent signaling pathways, such as oxygen and nutrient sensing pathways, have signi�cant potential for reducing the glucose metabolism of cancer cells. in low-glucose environments, amp-activated protein kinase (ampk) becomes activated, and in high-glucose environments, ampk is promoted, which can lead to the persistence of immunosuppressive cells.5 high levels of ampk can inhibit t cells, but disabling ampk may result in increased glycolysis in cancer cells. metformin is one drug that targets ampk by activating its signaling. studies have shown that metformin reduces cancer risk in both mice models and humans. ampk activation is believed to reduce tumor burden by slowing tumor growth, supporting the growth of tumor-in�ltrating lymphocytes (tils) in the tumor microenvironment (tme), and blocking certain glycolytic enzymes necessary for cancer cell berkeley pharma tech journal of medicine | 129 proliferation. however, ampk activation can also increase the number of immunosuppressive cells while promoting tumor regression.5 it is crucial to examine the e�ects on both cancer cells and the phenotypic ratios of immune cells. another crucial nutrient pathway is the mtor pathway, which is activated by high levels of glucose and amino acids. in t cells, mtor supports glycolysis, di�erentiation, and e�ector functions. however, because mtor enhances memory formation in cd8+ t cells while low mtor levels boost treg activity, it is vital to balance mtor levels to enhance memory formation without promoting treg activity.5 like ampk, the mtor signaling pathway has a complex nature, necessitating further research to develop nuanced methods that induce bene�cial immune responses without creating a tumor-protecting immune environment.5 two critical targets of immunotherapy are the immune checkpoint pathways pd-1/pd-l1 and ctla-4. anti-ctla-4 antibodies can deplete treg cells from the tumor microenvironment (tme), leading to successful tumor rejection in animal studies.31 pd-1/pd-l1 are involved in signaling mediated by antigen recognition through t cell receptors.31 antibodies that block the pd-1/pd-l1 checkpoint have shown signi�cant therapeutic e�ciency.31 these immune checkpoints mainly function by suppressing the metabolic reprogramming of immune cells, inhibiting glycolysis, and increasing lipolysis. blocking these pathways promotes anabolic metabolic pathways and glycolysis, thereby restoring the e�ector function of tumor-in�ltrating lymphocytes (tils). antibodies against ctla-4, pd-1, and pd-l1 reverse the glycolysis restrictions on t cells, essentially restoring t-cell glycolysis and interferon production.6 another signaling pathway that has been extensively studied is the csf-1/r pathway, which is expressed in myeloid cells. this pathway regulates the in�ltration, phenotypic and functional di�erentiation, and survival of myeloid cells, including tumor-associated macrophages (tams). blocking the csf-1/r pathway promotes the tme by facilitating the in�ltration and reactivation of cytotoxic t lymphocytes (ctls).32 consequently, studying berkeley pharma tech journal of medicine | 130 various signaling pathways can pave the way for developing nuanced cancer immunotherapy treatments. as discussed, the tme consists of various components, and understanding these di�erences in tme is crucial for developing e�ective treatments and immunotherapy approaches for di�erent cancers. identifying and targeting the most relevant aspects of the tme for each speci�c disease is key to creating successful therapies. 5. non-small cell lung cancer non-small cell lung cancer (nsclc) is the leading cause of cancer-related death and has become a primary focus for the development of tme-based immunotherapy. similar to other cancers, nsclc features an immunosuppressive tumor microenvironment (tme) and ctl exhaustion. however, it also has distinct characteristics such as variable blood �ow, areas of acidosis and hypoxia, and increased expression of hypoxia markers. these features can lead to metastasis and elevated resistance to treatment.32,33 consequently, past therapies targeting the tme have focused signi�cantly on addressing hypoxia and angiogenesis.33 a recent study conducted by zhao et al. adopts a similar strategy focusing on the tumor microenvironment (tme). the study delves into a phase iii trial combining bevacizumab, atezolizumab, and chemotherapy for metastatic non-squamous non-small cell lung cancer (nsclc). bevacizumab, an igg antibody that obstructs the vascular endothelial growth factor (vegf) pathway by binding to vegf-a, has demonstrated e�cacy in treating nsclc. the vegf pathway is a prime target due to its overexpression in nsclc, correlating with tumor recurrence, low survival rates, metastasis, and mortality. a recent study conducted by zhao et al. adopts a similar strategy focusing on the tumor microenvironment (tme). the study delves into a phase iii trial combining bevacizumab, atezolizumab, and chemotherapy for metastatic non-squamous non-small cell lung cancer (nsclc). berkeley pharma tech journal of medicine | 131 bevacizumab, an igg antibody that obstructs the vascular endothelial growth factor (vegf) pathway by binding to vegf-a, has demonstrated e�cacy in treating nsclc.19 the vegf pathway is a prime target due to its overexpression in nsclc, correlating with tumor recurrence, low survival rates, metastasis, and mortality.19 apart from bevacizumab, other antibodies like ramucirumab and nintedanib have demonstrated enhancements in overall survival (os), along with tyrosine kinase inhibitors (tkis) capable of inhibiting the vascular endothelial growth factor (vegf) pathway.19 however, the vegf pathway primarily addresses the angiogenic aspect of tumors, prompting its utilization in combination therapies with chemotherapy and immunotherapy, which have proven e�ective. in a similar vein, this phase iii trial combines bevacizumab with atezolizumab, an immune checkpoint inhibitor targeting the pd-l1 ligand (anti-pd-1), alongside chemotherapy. this multifaceted therapeutic approach to non-small cell lung cancer (nsclc) aims to enhance progression-free survival and os.32 5.1 nsclc combination therapy the primary treatment modality for non-small cell lung cancer (nsclc) typically involves immune checkpoint inhibitors (icis) like pembrolizumab, an anti-pd-1 antibody. pembrolizumab has emerged as the �rst-line treatment for metastatic nsclc due to its notable response rates and sustained tumor regression.14 however, despite its e�cacy, many patients encounter resistance to these treatments or su�er from immune-related adverse events.14 consequently, combination therapy has emerged as a pivotal focus for advancing cancer treatments. a recent study conducted in 2022 by reckamp et al. explored combination therapy involving icis and vascular endothelial growth factor (vegf) receptor inhibition in nsclc patients. patients were administered a combination of either ramucirumab (vegf inhibitor) and pembrolizumab (ici), or the investigator’s standard of care, such as docetaxel, gemcitabine, or pemetrexed.34 among the 136 eligible patients, those who received ramucirumab and pembrolizumab exhibited signi�cantly higher overall berkeley pharma tech journal of medicine | 132 survival (os), suggesting another promising combination therapy avenue for treating nsclc.34 in september 2022, casarrubios et al. conducted a study focusing on a speci�c form of combination therapy called neoadjuvant chemoimmunotherapy. this approach utilizes both chemotherapy and immunotherapy to reduce tumor size before more targeted treatment aimed at tumor removal. through bulk rna sequencing, the researchers analyzed samples from 41 patients with stage iii non-small cell lung cancer (nsclc) who underwent three cycles of nivolumab (an anti-pd-1 drug) and chemotherapy. their �ndings revealed downregulation of tumor and proliferation markers, along with genes associated with ifnγ signaling.35 additionally, they observed the regulation of numerous genes such as ifng and nkg7, distinguishing between complete pathological response (cpr) and non-cpr tumors before and after treatment. this study lays the groundwork for future personalized immunotherapy approaches based on predictive biomarkers of cpr, in conjunction with neoadjuvant immunotherapy.35 5.2 melanoma anti-pd-1 therapy represents a standard immunotherapy regimen for melanoma, a type of skin cancer. however, this treatment modality is only e�ective in providing long-term clinical bene�ts to approximately 40% of patients with advanced melanoma.36 to address this challenge, davar et al. conducted a study involving the administration of pembrolizumab, an anti-pd-1 drug, alongside a fecal microbiota transplant (fmt) in 16 patients with advanced melanoma who did not respond to anti-pd-1 therapy alone.36 this combined approach of fmt and pd-1 blockade resulted in the reprogramming of the tumor microenvironment (tme), overcoming resistance to anti-pd-1 therapy. notably, the treatment led to the upregulation of cd8+ t cells, a decrease in the percentage of naive cd8+ t cells, and an increase in activated and di�erentiated cd8+ t cells. responders also exhibited downregulation of cytokines and chemokines associated with anti-pd-1 resistance, along with upregulation of biomarkers linked to improved outcomes.36 berkeley pharma tech journal of medicine | 133 a recent approach in melanoma treatment involves categorizing the tumor microenvironment (tme) to anticipate the cancer's responsiveness to immunotherapy. while combining di�erent immune checkpoint inhibitors (icis) like anti-pd-1 and anti-ctla-4 may appear promising, trials on advanced melanoma patients showed severe immune-related adverse events (iraes) such as hypophysitis, pneumonitis, and thyroiditis, likely due to the distinct mechanisms of t cell inhibition—ctla-4 acting on t cell activation, while pd-1 intervenes later.14 a recent study identi�ed 29 functional gene expression signatures (fges) delineating major functional and cellular components of melanoma tumors, classifying them into four subtypes: immune-enriched/�brotic (elevated angiogenesis and cancer-associated fibroblast activation), immune-enriched/non-�brotic (more immune-active tme), �brotic (low lymphocyte in�ltration, high �broblast activity), and immune-depleted (immune-desert).37 this classi�cation aids in determining treatment e�ectiveness and alternative approaches. for instance, responses to anti-ctla-4 treatment varied across subtypes, with 82% of immune-enriched subtypes responding compared to only 10% of �brotic subtype, suggesting appropriate utilization of ici immunotherapy.37 5.3 other cancers (novel treatments/combination therapies) pancreatic cancer (pdac) poses a signi�cant challenge for immunotherapy due to its low t cell in�ltration, low tumor mutational burden, and highly suppressive tumor microenvironment (tme), making it the fourth leading cause of cancer-related deaths.44 combination therapy emerges as a promising approach, as demonstrated in a recent study by padron et al. this study investigated the administration of sotigalimab and/or nivolumab (both anti-pd-1 drugs) alongside chemotherapy.38 the results revealed that combining nivolumab with chemotherapy was associated with a less suppressive tme, increased numbers of activated and circulating t cells, and achieved the primary endpoint of one-year overall survival (os). although sotigalimab combined with chemotherapy did not meet the os endpoint, it led to greater in�ltration and di�erentiation of cd4+ cells. interestingly, administering all three components together did not yield additional improvements.38 berkeley pharma tech journal of medicine | 134 cervical cancer (cc) ranks as the fourth most prevalent cancer, with concurrent chemoradiotherapy (ccrt) serving as the standard treatment.39 however, individuals with advanced-stage disease often experience diminished long-term outcomes, prompting investigation into combination therapies. a recent study by huang et al. in august 2022 explored the utilization of adoptive cell therapy (act), a form of immunotherapy involving the introduction of tumor-in�ltrating lymphocytes (tils) to evoke an immune response, following ccrt and its impact on survival.39 among the 27 patients receiving act alone, 20 saw successful expansion of tils. subsequently, 12 patients received tils post-ccrt, with 75% experiencing complete response and a disease control duration ranging from 9 to 22 months. treatment-related adverse events were minimal, with only one patient encountering severe toxicity, underscoring the potential of post-ccrt act in managing cc.39 e�ective combination therapy entails strategic timing of treatments. another study focused on determining the optimal timing for immune therapy concerning ccrt revealed the bene�t of administering immune checkpoint inhibitors (icis) before ccrt. this strategy capitalizes on maintaining tumor-speci�c immune response, as it was observed to diminish following ccrt. post-ccrt, reduced numbers of cytotoxic t lymphocytes (ctls), decreased t cell receptor (tcr) diversity, and increased regulatory t cells (tregs) signi�ed a weakened antitumor immune response.40 researchers are investigating new treatment avenues for chronic lymphocytic leukemia (cll) that focus on targeting lag-3, a protein involved in inhibiting the function of natural killer (nk) cells and cd8+ t cells. in cll, lag-3 expression on leukemic cells is associated with poorer outcomes and decreased response to treatment.20 relatlimab, an igg4 antibody that blocks lag-3, has shown promise in preclinical studies. administering relatlimab increased the proliferation of nk cells and cd8+ t cells without a�ecting the growth of leukemic cells. this suggests that lag-3 blockade can reverse the inhibition of nk and t cells, potentially restoring their anti-leukemic activity. 20 berkeley pharma tech journal of medicine | 135 moreover, combining relatlimab with lenalidomide, an angiogenesis inhibitor, led to increased t cell numbers and enhanced anti-leukemic activity. this combination therapy approach may o�er a synergistic e�ect in treating cll by targeting both lag-3-mediated immune suppression and angiogenesis. overall, these �ndings provide a promising direction for the development of novel immunotherapies for cll, particularly for patients who have not responded well to pd-1 or ctla-4 blockade. 20 cancer often spreads to the liver, complicating treatment with immunotherapy. liver metastases attract activated cd8+ t cells, which then undergo apoptosis, resulting in a signi�cant reduction in e�ective t cells. 41 a study comparing patients with metastatic melanoma and nsclc who received immunotherapy and targeted therapy, or immunotherapy and chemotherapy respectively, revealed that liver metastases were linked to reduced response to immunotherapy but not targeted therapy. 41 similarly, in nsclc cases, immunotherapy had limited e�cacy compared to chemotherapy, while radiotherapy showed promising results. these �ndings suggest that liver metastasis may be more e�ectively managed through nonimmunotherapeutic methods, informing future treatment strategies.41 6. practical applications/discussion numerous innovative combination therapies extend beyond traditional chemotherapy and immunotherapy, delving into the modulation of the tumor microenvironment (tme) to bolster immune responses. for instance, one approach involves delivering a fecal microbiota transplant (fmt) via colonoscopy alongside immunotherapeutic anti-pd-1 drugs.36 this strategy e�ectively reshapes the tme, overcoming primary resistance to anti-pd-1 in advanced melanoma patients. following treatment, there's an increase in the proportion of cd56+cd8+t cells, indicating heightened circulation of active and di�erentiated cd8+ t cells.36 responders exhibit decreased levels of certain cytokines and chemokines linked to anti-pd-1 resistance, while biomarkers associated with improved survival are upregulated.36 notably, variations in patient response may occur due to factors like tumor immunogenicity or unsuccessful colonization by berkeley pharma tech journal of medicine | 136 fmt, necessitating further investigation for its establishment as a standard treatment.36 another method, as explored by ribas et al., involves combining anti-pd-1 therapy (speci�cally pembrolizumab) with intratumoral administration of an oncolytic virus, such as talimogene laherparepvec, in advanced melanoma patients. initially, tumor size may transiently increase post-injection; however, this approach boosts cd8+ t cell in�ltration and peripheral cd4+ t cell presence.48 pembrolizumab promotes the proliferation of cd8+ t cells, leading to a comprehensive shift in the tme and an augmented immune response compared to anti-pd-1 therapy alone. importantly, t cells expressing pd-1 encounter tumor cells expressing pd-l1, a dynamic that, alongside pd-1 blockade, moderates the antitumor e�cacy of the administered virus.48 as previously discussed, extensive research has already been conducted on pd-1, pd-l1, ctla-4, and vegf/vegf-a as targets for treating various cancers. scientists are now focusing on identifying new potential biomarkers to better predict patient responses to speci�c treatments, given that the existing biomarkers are not perfect indicators of a patient’s response to immune checkpoint inhibitor (ici) therapy.49 vanhersecke et al. observed that the tertiary lymphoid structure (tls) within tumors is associated with the population of cd8+ t cells. higher tls densities correspond to increased densities of cd8+ t cells. moreover, the presence of more mature tls was linked to improved survival rates, higher objective response rates, and longer progression-free survival. notably, mature tls emerged as the most signi�cant predictive factor for an objective response, independent of pd-l1 expression and cd8+ t-cell in�ltration. patients with tumors containing mature tls had better outcomes compared to those without mature tls.49 studying the tls in tumors opens a new avenue for identifying biomarkers to predict patient responses to speci�c ici treatments. two ongoing clinical trials are exploring recent advances in immunotherapy and tumor microenvironment (tme) mediated treatments, focusing on combination therapies involving lag-3, anti-pd-1, and vegf signaling. berkeley pharma tech journal of medicine | 137 the �rst trial is investigating the e�ects of anti-lag-3 (urelumab) alone and in combination with nivolumab (an anti-pd-1 drug) in patients with recurrent glioblastoma. the study aims to determine which treatment regimen is more e�ective at killing tumor cells. currently, 63 patients are enrolled, and the trial is expected to conclude by november 2023.50 the second trial, entering phase ii, is evaluating the e�cacy and safety of a combination of anti-pd-l1/vegf drugs with traditional chemotherapy in treating non-small cell lung cancer (nsclc). the primary objectives are to measure the objective response rate to the treatment and progression-free survival, which involves monitoring the time from the start of treatment until the cancer progresses or the patient dies from any cause.51 other monitored outcomes include overall survival rate, disease control, response duration, treatment-related adverse events, and the correlation between pd-l1 expression and antitumor e�ect. the clinical trial aims to enroll 374 patients and is expected to be completed by december 2025.51 7. conclusion the tumor microenvironment (tme) is a promising target in immunotherapy. due to its variability and adaptability, further research is needed to e�ectively target di�erent tme components. current techniques include immune checkpoint blockades like pd-1 and ctla-4, and 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multiple sclerosis berkeley pharma tech journal of medicine correspondence: vukellin@gmail.com keywords: genetic editing crispr-cas9 ethical considerations mental health treatments public medical influences submitted july 14, 2024 accepted august 19, 2024 published december 27, 2024 full open access creative commons attribution license 4.0 abstract genetic editing has become a pivotal biotechnology tool in healthcare, enabling precise manipulation of dna through insertions, deletions, modifications, or replacements. its applications have expanded from treating monogenic diseases to addressing polygenic conditions, including potential mental health therapies. this study examines public sentiment and ethical considerations surrounding genetic editing systems like crisprcas9, zinc-finger nucleases (zfns), and transcription activator-like effector nucleases (talens). analyzing a decade of medical literature, the research highlights a growing public acceptance of these technologies despite ethical concerns, such as off-target effects and unintended consequences. positive sentiment trends indicate that social acceptance may support the integration of genetic editing in mental health treatments, driving advancements in healthcare. addressing risks, including long-term impacts and ethical dilemmas, remains critical for responsible implementation. as public attitudes continue to improve, genetic editing may become a widely accepted therapeutic approach, fostering innovation in healthcare. future research should focus on mitigating risks and enhancing the safety and efficacy of genetic editing to ensure its sustainable development and adoption. exploring ethical frontiers: a content analysis of perceptions surrounding genetic editing in mental health treatments over time by: kellin vu 1. introduction genetic, or genome, editing is reshaping modern medicine as one of the latest advancements in the field, where improvements in genetic modification technology are now being applied to polygenic disorders rather than just monogenic disorders. genome editing uses genetic engineering to manipulate dna sequences from one nucleotide in length to large fragments, allowing the modeling for individual cells to complete organisms.1 genome editing tools are molecular strategies for modeling and correcting errors in mutated genes; thus, genome editing could change the human genome to treat or develop gene-based diagnoses and therapeutics for a number of diseases and disorders.2 bioengineering is a constantly progressing field, with genetic editing as a relatively new development in healthcare and medicine as a tool for treatment. nevertheless, genetic editing has little insight into the progress of genome editing and its application to the polygenic nature of psychiatric disorders is still in its infancy.3 with this remarkable progress in biotechnology, genetic editing presents promising applications for mental illness treatments in healthcare. the rapid advancement of genetic editing technology engenders the question: what are the potential ethical considerations and public perceptions that arise from the implementation of genetic editing technology in healthcare as a treatment for polygenic diseases and mental health? exploring the ethical frontiers of genetic editing is pivotal for scientists and medical professionals to understand its transformative potential and limits in medicine. the insights gained from examining this field of inquiry will play a vital role in defining the ethical framework and societal acceptance of this new advancing technology. in order to establish a connection between the perceptions and patterns in attitudes supporting or against the application of genetic editing technologies and the ethical considerations of this form of healthcare treatment, it is appropriate to hypothesize that the overall trend will exhibit a positive correlation in sentiments of various polygenic diseases as time progresses over the last decade. berkeley pharma tech journal of medicine | 78 2. literature review 2.1 current landscape of mental health treatments mental health poses a significant challenge in contemporary society and continues to steadily escalate as a prevalent issue. a study found that “approximately half the population can expect to develop one or more mental disorders by age 75.”4 according to the national institutes of health, a mental illness is “a health condition that changes a person’s thinking, feelings, or behavior (or all three) and that causes the person distress and difficulty in functioning.”5 presently, the main traditional forms of treatments for mental health include psychotherapy, which encompasses counseling and cognitive-behavioral therapy, and pharmacotherapy, involving the administration of medications like antidepressants or antipsychotics. psychotherapy has been defined as an effective psychological intervention for a multitude of psychological, behavioral, and somatic problems, symptoms, and disorders and thus rightfully considered as a main approach in mental and somatic health care management.6 yet, a study by sarah cook and her team challenge this idea, suggesting that psychotherapy to be difficult to apply to individuals of complex multimorbidities, specific sociodemographic groups, and minority populations.7 this study highlights that psychotherapy often overlooks individual factors influencing a patient’s health, potentially leading to reduced efficacy and negative outcome.8 another form of mental health treatment is pharmacotherapy, the use of medicines for combating a disease or its symptoms that allow for the alleviation of symptoms and improvements in the health and quality of life of patients.9 nonetheless, witczak and her team reveal that the frequent errors in prescription of medicine and medicine administration due to the complex and multifaceted nature of the pharmacotherapy process present alarming risks.10 brucker and faucher complement this finding, as they argue that pharmacotherapy introduces several disadvantages, such as drug resistance that makes pharmacotherapy inefficient as a long-term treatment option, adverse drug reactions, and drug-to-drug interactions that can cause further negative side effects that may be unpredictable and be more damaging than beneficial.11 these studies ultimately imply that traditional approaches may berkeley pharma tech journal of medicine | 79 present challenges and limitations such as partial efficacy, side effects, and variability in individual responses. this growing understanding of the biological basis of mental disorders emphasize the importance of considering biological interventions in mental health treatments. for instance, “scientists have identified genes as a factor that plays a role in determining whether someone develops a mental illness, specifically illnesses that are most likely to have a genetic component such as autism, bipolar disorder, schizophrenia, and adhd, where the interaction of several genes may trigger mental illness in contrast to a change in a single gene”12 this further highlights the concept of precision medicine in mental health, as “precision medicine is an approach for clinical treatment that uses genomic, lifestyle, and environmental information to provide targeted, individualized care that is tailored to patients’ specific needs.”13 these studies insinuate and introduce the need for new innovative forms of mental health treatment, such as gene therapy. as a potential approach for more precise mental health intervention, gene therapy addresses genetic components associated with mental disorders and illnesses to affect the overall treatment efficacy for individuals. 2.2 genetic editing technology gene therapy, defined by sherkow is “the intentional, expected permanent, and specific alteration of the dna sequence of the cellular genome, for a clinical purpose.”14 this signifies the potential of gene therapy to cure hereditary diseases like mental illnesses by manipulating gene sequences. “this therapy became possible through the advances of genetics and bioengineering that enabled manipulating vectors for delivery of extrachromosomal material to target cells.”15 within these advances include some of the most prominent approaches to genetic editing, which include the clustered regulatory interspaced short palindromic repeats (crispr) / cas9 (crispr-associated protein 9) system, zinc-finger nucleases (zfns), and transcription activator-like effector nucleases (talens). the crispr/cas9 system, one of the most significant gene editing technologies in the development of gene therapies, consists of “two essential berkeley pharma tech journal of medicine | 80 components: a guide rna [ribonucleic acid] to match a desired target gene, and cas9—an endonuclease which causes a double-stranded dna [deoxyribonucleic acid] break, allowing modifications to the genome.”16 the crispr/cas system “affords the requisite recognition selectivity necessary to ensure single-site specificity in complex genomes, allowing the crispr system to provide acquired immunity against invading foreign dna through rna-guided dna cleavage.17 this insight implies how this form of technology holds the ability to address complexity in gene sequences and proposes this genome editing tool as a treatment approach for polygenic diseases. this development contrasts with earlier genetic editing technologies, such as zfns and talens, which relied on specially coded proteins to recognize key dna sequences.18 this process required “complex, labor-intensive development processes that limited their practicality and efficiency in terms of time, cost, and efficiency (e.g., limited specificity and target recognition, off-target effects).”19 this denotes how these developing technologies present several barriers in their application to medicine, suggesting how these advanced tools still hold disadvantages and restrictions that depict possible ethical considerations to take notice of. talens is an earlier genetic editing technology that comprises of a “non-specific dna-cleaving nuclease fused to a dna-binding domain that can be easily engineered so that talens can target essentially any sequence.”20 “talens are similar to zfns in that they can generate dsbs [double-strand breaks] at a desired target site in the genome and so can be used to knock out genes or knock in mutations in the same way.”21 zinc-finger nucleases are more specifically defined as targetable dna cleavage reagents, in which zfn-induced double-strand breaks are subject to cellular dna repair processes that lead to both targeted mutagenesis and targeted gene replacement at remarkably high frequencies.”22 this further signifies the capabilities of these gene editing tools, revealing the numerous methods and approaches available to potentially act as an effective mental health treatment. hence, these new advancements in genetic editing technology found by recent medical experiments and studies portray the possibilities of this biotechnology and how it may revolutionize healthcare. berkeley pharma tech journal of medicine | 81 2.3 research gap as i examine the notable gene editing technologies and their applications in healthcare, it becomes evident that the use of genome editing for mental illnesses is still a treatment in its infancy. despite the recent advancements in genetic editing technologies, there is a notable lack of research that discusses the ethical considerations and perceptions associated with the use of genome editing for mental health treatments. since the introduction of gene editing in the 1970’s, much research has focused on the application of genetic editing technologies toward preventing and treating certain inherited disorders or monogenic diseases.23 however, as the capabilities of these technologies advance and expand to the domain of polygenic diseases, such as mental illnesses, it remains unclear what the ethical implications and surrounding perceptions are in using genome editing technology as a form of treatment for mental illnesses in healthcare. therefore, the purpose of this study was to predict the potential ethical considerations and public perceptions towards the use of genetic editing for mental disorders through analysis of public sentiment and add to the conversation of existing literature on the function and perceptions of genetic editing in the medical field. 3. methods 3.1 introduction over the course of 120 days, i conducted a study to construe trends of the sentiment expressed from ethical considerations towards gene editing as a medical treatment to interpret the possible implications and perceptions of genetic editing utilized for a mental health treatment. to achieve this objective, a content analysis was necessary to streamline the principal ethical considerations associated with the implementation of genetic editing in healthcare and facilitate the range of sentiments within significant academic, medical publications. this was essential to create a generalized comprehension of the ethical implications in contemporary society towards genetic editing and clarify an existence of a trend within the attitudes correlated with the implementation of this technology in healthcare, berkeley pharma tech journal of medicine | 82 whether it is positive, negative or neutral, to predict the perceptions of genetic editing technology for mental health treatments. a correlational analysis was also conducted based on the time intervals and the sentiment scores connected with the attitudes toward the implementation of genetic editing as a medical treatment, as well as with the ethical implications and perceptions towards the application of gene editing technology for mental health treatments to ascertain the most prevalent ethical considerations. 3.2 instrumentation this study employed a content analysis to deduce the major considerations of genetic editing in healthcare and utilized a sentiment analysis to discover the perceptions that will develop from these implications. in order to conduct this method of study, a semantic differential scale was used, shown in table 1. the semantic differential scale is a 7-point rating scale that presents pairs of bipolar adjectives at the endpoints of the scale. this scale was processed with opposite adjectives that indicate extremely strong negative sentiment, very strong negative sentiment, moderate negative sentiment, neutral sentiment, moderate positive sentiment, very strong sentiment, and extremely strong positive sentiment. with a set of bipolar adjectives on the endpoints, each of these codes were assigned a value to measure the attitude toward a specific negative or positive sentiment, ranging from the sentiments of extremely strong negative sentiment at -3, very strong negative sentiment at -2, moderate negative sentiment at -1, neutral sentiment at 0, moderate positive sentiment at +1, very strong positive sentiment at +2, and extremely strong positive sentiment at +3. this study underscores the notable distinction between negative and positive sentiments to facilitate easier differentiation of the perceptions towards genetic editing for healthcare treatments. a statement within academic articles or journals will be categorized as moderate sentiment if stance is primarily neutral with a slightly skewed viewpoint, regardless of denunciation or commendation. for a classification of very strong on the scale required such intense words like damaging, great, bad or beneficial. for a classification of extremely strong on the scale required such extreme words like extraordinary, catastrophic, remarkable or disastrous. these words emphasize and reflect the pronounced opinions and attitudes of genetic berkeley pharma tech journal of medicine | 83 editing in healthcare. for this study, statements or paragraph fragments from medical papers were analyzed and applied to the semantic differential scale. the primary data sources used for my method of study were medical journals and studies retrieved from credible databases, such as pubmed or sciencedirect. this study utilized the delve tool website to administer this process for qualitative analysis and coding these medical journals. this website contains coding software that generates transcripts of the medical journals and the code of sentiment applied. consequently, the paper is averaged across the various sentiment scores, charted in table 2 below. table 2 reflects the averages of the sentiment analysis, employing a given range of values. the table comprises of a 7-point scale, where within each is a range of assigned values with an extremely strong negative at the spectrum of any value less than -2.0, to very strong negative at a range of -2.0 to -1.0, to moderate negative at a range of -1.0 to less than 0, neutral at 0, to moderate positive being between 0 and 1.0, to very strong positive being between 1.0 and 2.0, and extremely strong positive at any value more than 2.0. as qualitative research, this study utilized the collected data along with a ti-84 calculator to compute the correlation coefficient and values. 3.3 content analysis i performed a content analysis, a detailed and systematic examination of the contents of a particular body of material for the purpose of identifying patterns, themes, and biases.24 the content analysis classified thirty medical journals and studies, concluding the paper’s data to express the article’s general sentiment. using medical journals and studies, i conducted a comprehensive analysis of the perceptions concerning the implementation of genome editing in healthcare, followed by an examination of the attitudes towards various polygenic diseases, to determine the potential ethical considerations and perceptions regarding the use of genetic editing technology as a viable mental health treatment. berkeley pharma tech journal of medicine | 84 in order to ensure that the medical journals and studies would be relevant and representative of how gene editing technology is presently used as a treatment for polygenic disorders, criteria were established for the medical journals and studies analyzed. the medical journals and studies were chosen due to their relevance in healthcare as each indicate the use of one of the foundational gene editing technologies, including the crispr/cas9 system, talens, or zfns.25 furthermore, due to the rapidly evolving aspect of the medical field, all medical studies utilized have been conducted in the past decade. i analyzed medical journals across various polygenic diseases in order to gain a deeper comprehension on the extensive perceptions that exist with gene editing as a healthcare treatment overall, especially for more complex diseases. to analyze sentiment, this study used a consistent search engine within medical journal databases pubmed and sciencedirect. once the study found all suitable medical journals and studies, the study applied the qualitative code to words, adjectives, and paragraph fragments in the medical journals that correlate to positive, neutral, or negative sentiment. these sentiments were evaluated on the 7-point semantic differential scale to deduce a more comprehensive range of sentiment. the thirty medical journals this study analyzed and fit the criteria were transcribed into the delve tool website, where a trend became prevalent. after all the papers were coded through delve tool, the study calculated the sentiment score and how the sentiment varied across different types of polygenic diseases. the polygenic diseases were separated into three categories: neurological diseases, cancer, and other polygenic diseases. these categories will help highlight and clarify any significant variance in attitudes towards a specific type of polygenic disease to address if the perceptions towards all types of polygenic diseases can be used to predict the attitudes of genetic editing for mental health treatments. since this study is only utilizing medical journals and studies published in the last decade due the rapid nature of the medical field and its advancements, separate sentiment scores were also averaged for articles published in the first five years compared to the last five years in the last berkeley pharma tech journal of medicine | 85 decade to denote if attitudes have increasingly become more positive, negative, or neutral towards genetic editing technology. 3.4 correlational analysis using a sentiment analysis to discover the current attitudes towards genetic editing, i used a correlational analysis in order to determine which considerations were most prevalent for medical professionals in their studies. therefore, this study used the pearson correlation coefficient, or “r,” to establish the existence of an association between the number of ethical considerations and the sentiment score of genetic editing technology as a healthcare treatment. the pearson correlation coefficient was used to determine the strength of the correlation and to accept or reject the null hypothesis that a relationship does not exist between sentiment towards genetic editing and polygenic diseases. 4. findings after the study averaged the sentiment scores, general sentiment scores were evaluated for the different types of polygenic diseases that have been tested with genetic editing technology as a healthcare treatment. for neurological diseases, the attitudes toward genetic editing technology treatment were characterized with a positive sentiment at 0.295 for the first five years of the last decade and a positive sentiment of 0.507 for the last five years. this revealed an increase in positive sentiment over time, with a change of 0.212 in positive sentiment, and a total sentiment score resulting in a positive sentiment of 0.4234. for types of cancer, the attitudes toward genetic editing technology treatment were also characterized with a positive sentiment starting at 0.350, compared to the concluding sentiment of 0.950 with a change of 0.60, and total sentiment score of 0.900, which can be seen on table 2. for other types of polygenic diseases, the perceptions also started with a moderately positive sentiment of 0.518, however experienced a slight decrease of 0.09 in positive sentiment, producing a moderately positive final sentiment score of 0.428 and total sentiment of 0.4574. this trend displays how the average sentiments over time had an increase in positive sentiment for cancer and neurological diseases, as genetic editing berkeley pharma tech journal of medicine | 86 technology strengthened as viable treatments for these polygenic diseases over time. in contrast, for medical journals concerning other polygenic diseases, the average sentiment score had encountered a small decline in positive sentiment over time, yet the total average sentiment still remained moderately positive which portrays that there was an increase of negative sentiment. this implied that more concerns emerged as perceptions toward the application of gene editing treatment became more negative. after this study marked a trend of increased positive sentiment in the attitudes towards genetic editing technology, the study utilized the sentiment score and the number of ethical implications that were considered and reflected in the perceptions of the medical journals to establish if there is a relationship between the ethical implications most prevalent and what implications signify advantages or disadvantages of genetic editing. although the study could not establish an accurate correlation between all ethical implications across all polygenic diseases that have a form of genetic editing treatment, the two quantitative variables of sentiment score and number of ethical considerations could be utilized to represent a correlation for each type of polygenic disease. for this study, the pearson correlation coefficient used the number of ethical considerations as the x values, or independent variable, and the sentiment score as the y values, or dependent variable, in order to convey if the prevalence of ethical implications has a true influence on the sentiment of attitudes towards genetic editing technology as a healthcare treatment for polygenic diseases. the study analyzed the perceptions across the past ten years to indicate if the sentiment and number of ethical implications affected any correlation or trend of positive, negative, or neutral sentiment. the pearson correlation coefficient or “r” had resulted with r=0.4673, which expresses that there is a moderately strong relationship between the number of ethical implications and the sentiment score reflecting the public’s attitudes, as seen on table 3. table 1 depicts the 7-point semantic differential scale that this study utilized to calculate the sentiment scores, in which each scale value number is assigned to how strong a statement or paragraph fragment found in the medical paper is, in order to average the sentiment score on a scale of negative to positive. berkeley pharma tech journal of medicine | 87 table 1. semantic differential scale for sentiment analysis. this table defines the 7-point semantic differential scale used in the study. it outlines how statements from medical journals were rated based on sentiment polarity, ranging from extremely negative to extremely positive, for precise sentiment classification. figures 1, 2, and 3 represent the number of phrases or paragraph fragments observed for each level of sentiment for genetic editing technology in healthcare treatments across each category of polygenic diseases in the past decade. these polygenic diseases, labeled figures 1, 2, and 3, were respectively categorized as neurological diseases, cancer, and other polygenic diseases to highlight the three main categories of polygenic diseases that have used genetic editing technology as a form of treatment. each of these three figures are also separated into two 5-year time periods, 2016-2020 and 2021-2024, to differentiate changes in the observed sentiment polarity over time, as healthcare treatments experience rapid changes in its evolving field with new advancements daily. berkeley pharma tech journal of medicine | 88 figure 1. sentiment of genetic editing treatments in healthcare across neurological diseases. this graph illustrates the changes in sentiment regarding genetic editing treatments for neurological diseases over the past decade. the data shows a notable increase in positive sentiment from 2016 to 2024, reflecting growing acceptance of this technology in treating neurological conditions. figure 2. sentiment of genetic editing treatments in healthcare across cancer. this graph highlights the increasing positive sentiment toward genetic editing technologies berkeley pharma tech journal of medicine | 89 in cancer treatment from 2016 to 2024. the sharp rise in sentiment demonstrates growing confidence and optimism in using genetic editing as a therapeutic approach for oncology. figure 3. sentiment of genetic editing treatments in healthcare across other polygenic diseases. this graph shows sentiment trends for genetic editing technologies in polygenic diseases other than cancer and neurological conditions. while sentiment remains moderately positive overall, the slight decline in positivity over time suggests increasing ethical and practical concerns. table 2 portrays the average sentiment scores and the various interval scales that classifies a score as extremely negative, very strong negative, or moderately negative, neutral, moderately positive, very strong positive, or extremely strong positive. these scores are calculated using the number of phrases or paragraph fragments sorted as negative, neutral, or positive with the scale value numbers outlined in the semantic differential scale, shown by table 1. the table conveys the average sentiment score for each category and each 5-year time period to express any relevant changes in public sentiment observed over time. a total sentiment score is also calculated for each category of neurological, cancer, or other polygenic diseases, so the average value of sentiment overall can compare against the different categories. berkeley pharma tech journal of medicine | 90 table 2. average sentiment score. this table presents the calculated average sentiment scores for each polygenic disease category over two time periods, 2016–2020 and 2021–2024. the data highlights an overall positive trend in sentiment, with significant growth in cancer and neurological disease treatments. note: values are averages. table 3 illustrates the correlational data between the number of ethical considerations and sentiment score. the number of ethical considerations is the ethical implications observed when coding the medical journal’s words and phrases with negative sentiment. this is compared to the sentiment score of each category of polygenic diseases, neurological, cancer, and other, to establish how strong the existing relationship between the number of ethical considerations and the sentiment score is. table 3. ethical considerations correlation data. this table shows the correlation between the number of ethical considerations and sentiment scores for genetic editing technologies. it provides insights into how ethical implications influence public attitudes across different categories of polygenic diseases. figure 4 illustrates how the average sentiment of genetic editing technology changes over the past decade. the sentiment score was calculated for 5-year periods, between 2016-2020 and between 2021-2024, to determine the difference of sentiment score over the past decade. figure 4 illustrates the increase in positive sentiment, indicating how attitudes towards the berkeley pharma tech journal of medicine | 91 implementation of genetic editing technology significantly becomes more positive as the technology continues to advance over the years. figure 4. average sentiment over the past decade. this chart compares the average sentiment scores for genetic editing technologies across different categories—neurological diseases, cancer, and other polygenic diseases—over two five-year intervals. it underscores a consistent rise in positive sentiment, particularly in cancer and neurological applications, reflecting advancements in genetic editing technology. 4. analysis thirty medical journals extending across the last decade were annotated and analyzed for their sentiment, exemplifying the existence of a positive trend of sentiment over time for the application of genetic editing technologies for polygenic diseases in healthcare. since the correlation coefficient between sentiment scores and ethical implications was r=0.4673, it concluded that a moderately strong relationship was present between sentiment and the number of ethical implications presented. in similar regards with other polygenic diseases, the results demonstrated a moderately positive correlation with the total sentiment of genetic editing technology as a healthcare treatment overall, yet a minor negative trend did occur over time on contrary to the positive trend that arose for cancer and neurological diseases. therefore, the polygenic diseases reject the null hypothesis because there is an existence of a trend between time and sentiment across all three categories of polygenic diseases. this study can conclude that there is a berkeley pharma tech journal of medicine | 92 positive correlation between the implementation of genetic editing technology as a treatment for polygenic diseases within the domain of cancer and neurological diseases and their sentiment over time in the past decade but a slight negative correlation under other polygenic diseases. in addition, this study identified a pattern of which positive sentiment was often associated, behind context, that the specific benefits of genetic editing technology include its efficiency and effectiveness in modifying specific genes. 38 percent of the coding in positive sentiment was correlated with how effective and precise genetic editing technology is for polygenic diseases. in contrast, this study observed a pattern of negative sentiment that was frequently connected to the ethical consideration that genetic editing technology has the significant risk of negative unpredictable effects and off-target effects, which entails genome editing affecting other parts of the target dna that were unintentional. 36 percent of the coding in negative sentiment was related to this risk, and thus indicates the distinct danger and ethical consideration that genetic editing presents. this overall portrays how the attitudes towards the use of genetic editing technology as a form of treatment for polygenic diseases has increasingly become more positive over time as more advancements are being made in this type of treatment. this indicates that as genetic editing is presently used for mental health disease and psychiatric treatment, it will be met with a more positive response according to the trend of positive sentiment reflecting public attitudes and perceptions of the use of genetic editing technology as a healthcare treatment. this study found a consistent positive correlation of positive sentiment over time in polygenic diseases such as cancer and neurological diseases, reflecting how attitudes and perceptions towards the use of this technology in healthcare will likely follow this positive trend. although other polygenic diseases experienced a slight decrease in positive sentiment within its perceptions, it concluded with a moderately positive sentiment, indicating that genetic editing for mental health diseases will be received with positive attitudes. this is significant because the public acceptance of the new medical technology can substantially affect the ability of genetic editing technology developing further. opposition against the development of genetic editing technology to be implemented into healthcare as a treatment would reflect that the berkeley pharma tech journal of medicine | 93 perceptions of gene editing focus on negative ethical considerations and concerns. if negative sentiment prevailed to be most prominent, it would conclude that relevant perceptions reject the proposition of gene editing based treatment for polygenic diseases in healthcare. nevertheless, across the various polygenic diseases, the total sentiment concluded as moderately positive, suggesting acceptance of the application of genetic editing technology in healthcare. hence, this reinforces my initial assumptions that attitudes toward genetic editing technology in polygenic diseases will increasingly become positive, as a positive correlation indicates a growth in positive perceptions and establishes a total moderately positive sentiment across all polygenic diseases in the past decade. this shows how polygenic diseases are met with moderately positive attitudes and regarded with positive perceptions, further supporting the advancement of genetic editing technology to be utilized in polygenic diseases’ treatments. although a moderately positive sentiment is reflected throughout the last decade, the correlation between the sentiment over time and relevant ethical considerations cannot be concretely proven, even though a general trend of sentiment is apparent. there is no definite methodology to confidently prove the correlation of data across time, as the patterns of sentiment are not a clear catalyst due to existing external variables. therefore, this study can only absolutely establish that there is a possible larger trend that exists. 4. conclusion 4.1 implications the trend of total sentiment resulting in moderately positive across the polygenic diseases disclose how genetic editing technology-based treatments are often met with positive perceptions and attitudes. this implies that the public will hold similar sentiments toward the application of genetic editing for other complex polygenic diseases, specifically mental health diseases. “in psychiatry, gwas [genome-wide association studies] have uncovered a high degree of polygenicity underlying mental illnesses and related complex phenotypes.”26 this indicates how mental health diseases are very complex berkeley pharma tech journal of medicine | 94 polygenic diseases, as multiple genes must be considered in the treatment of mental health illnesses. hence, the positive sentiment observed over time towards the application of genetic editing technology for the treatment of various polygenic diseases signifies how public perceptions and attitudes will likely follow a similar trend of positive sentiment. with a trend of positive sentiment, it insinuates that public attitudes and perceptions will likely be supportive of the implementation of genetic editing in mental health treatments over time, and this social acceptance will empower the advancement of genetic editing technology in healthcare treatments. 4.2 limitations this study utilized a coding process in order to rate the sentiment of words, phrases, and paragraph fragments in the medical journals analyzed; nonetheless, the subjective aspect of coding the sentiment is a crucial consideration as a limitation. in this particular study, the content analysis consisted of identifying words that are associated with a positive or negative connotation and the extremity of the connotation, where the scale values were rated from extremely strong, very strong, moderately strong, and neutral. this can cause varying results of the content analysis in replicability because it is subjective in what the coder defines a word or phrase with regards to how extreme of a positive or negative connotation exists. to maintain as much objectivity as possible, the study ensured to emphasize the english language’s definitions and connotations. additionally, this study analyzes and codes thirty medical journals, which is a small sample in total; therefore, this limited sample size can restrict the study’s total reflection on the perceptions towards genetic editing technology for polygenic diseases. due to the small sample size, this study will conclude general data for the trend of sentiment that is insufficient to be utilized as a measure for how other trends will appear for genetic editing technology in healthcare. moreover, it is necessary to acknowledge that since genetic editing technology is encompassed within the rapidly evolving medical field, time brings forth change in how people perceive the information presented in medical journals and studies. significant events that have occurred in the past decade, such as the coronavirus pandemic, can incite bias towards how berkeley pharma tech journal of medicine | 95 the medical journals’ information and studies are received or accepted by the public. the lack of focus on analyzing the trend of sentiment for only one specific polygenic disease also presents as a limitation since it is difficult to accurately identify if a certain sentiment is only present for a specific disease. however, this study examines various polygenic diseases, categorized as neurological, cancer, and other, in order to efficiently conclude a general trend of data for the sentiment that reflects the public’s attitudes and perceptions towards the implementation of this technology in healthcare overall. in addition, the 7-point semantic scale is a limitation, as the scale restricts the ability to rate the public perceptions present in the medical journals past the sentiment extremity of positive, neutral, and negative. yet, the scale is optimized as an efficient method to classify the general data of public attitudes and perceptions towards genetic editing. this is a prime research method to identify any existing trends of sentiment towards genetic editing over time due to the lack of direct public surveys that analyze the perceptions and ethical considerations of the public. this is the best methodology to predict the sentiments of the public’s perceptions towards the implementation of genetic editing technology in mental health diseases and treatments due to the absence of medical studies with a focus on genetic editing technologies utilized as a mental health treatment. 4.3 future directions the genetic editing technologies of zfns, talens, and the crispr/cas9 system present a new revolution in the healthcare field as it reflects the significant advancements developed in technology and medical devices. nonetheless, this widely-used genetic editing tool still has rising concerns associated with its ethical implications, demonstrated by the slight decrease in positive sentiment analyzed under other polygenic diseases, but specifically shown by this correlational analysis that negative off-target effects are the most relevant ethical implication. hence, future research should draw a focus on how to prevent these negative off-target effects associated with the application of genetic editing technology. the accuracy and precision of this technology was notably recognized throughout the berkeley pharma tech journal of medicine | 96 medical journals, contributing to a significant percentage of the positive sentiment coded; therefore, it is imperative that additional research addressing this concern is investigated in order to maintain the observed trend of positive sentiment which insinuates that public sentiment and perceptions support the implementation of genetic editing technology in mental health illness treatments and other healthcare applications. another concern that arises when analyzing the negative sentiment of medical journals is the challenge of unpredictable long-term effects and consequences caused by genetic editing technology. to address this issue, future research should analyze the long-term effects of genetic editing technology for polygenic diseases, as this study only examined medical journals regarding the last decade. thus, these guiding topics can help further the discourse about the implementation and advancements of genetic editing technology as viable treatments in healthcare for various diseases, with the consideration of the ethical implications and public perceptions surrounding the subject, and this research can serve to enable and revolutionize the efficiency of mental health treatments through this biotechnology. berkeley pharma tech journal of medicine | 97 5. appendix a: sentiment code ratings each number indicates the number of words or phrases that were coded and correlated with that sentiment extremity of positive, neutral, or negative within the medical journal. berkeley pharma tech journal of medicine | 98 berkeley pharma tech journal of medicine | 99 each number indicates the number of words or phrases that were coded and correlated with that sentiment extremity of positive, neutral, or negative berkeley pharma tech journal of medicine | 100 5. appendix b: representative quotations of sentiment score berkeley pharma tech journal of medicine | 101 berkeley pharma tech journal of medicine | 102 berkeley pharma tech journal of medicine | 103 berkeley pharma tech journal of medicine | 104 references 1. gutiérrez-rodríguez a, cruz-fuentes cs, genis-mendoza ad, nicolini h. crispr/cas9 genome editing approaches for psychiatric research. braz j psychiatry. 2023. doi:10.1476260/1516-4446-2022-2913. 2. gutiérrez-rodríguez a, cruz-fuentes cs, genis-mendoza ad, nicolini h. crispr/cas9 genome editing approaches for psychiatric research. braz j psychiatry. 2023. doi:10.1476260/1516-4446-2022-2913. 3. hoffmann a, ziller m, spengler d. focus on causality in esc/ipsc-based modeling of psychiatric disorders. cells. 2020;9(2):366. doi:10.3390/cells9020366. 4. mcgrath jj, al-hamzawi a, alonso j, et al. age of onset and cumulative risk of mental disorders: a cross-national 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26. wendt fr, pathak ga, tylee ds, goswami a, polimanti r. heterogeneity and polygenicity in psychiatric disorders: a genome-wide perspective. chronic stress. 2020;4:247054702092484. doi:10.1177/2470547020924844. berkeley pharma tech journal of medicine | 106 https://eric.ed.gov/?id=ed594592 https://eric.ed.gov/?id=ed594592 https://www.ncbi.nlm.nih.gov/pmc/articles/pmc5131771/ https://www.ncbi.nlm.nih.gov/pmc/articles/pmc5131771/ https://www.ncbi.nlm.nih.gov/pmc/articles/pmc5131771/ berkeley pharma tech journal of medicine correspondence: jpopovich@luc.edu keywords: ptsd, post-traumatic stress disorder, hyperbaric oxygen therapy, stem cell therapy, stellate ganglion block, topiramate, immunotherapy submitted: december 11, 2022 accepted: january 27, 2023 published: june 30, 2023 full open access creative commons attribution license 4.0 abstract post-traumatic stress disorder (ptsd) is a psychological disorder that affects about 12 million americans every year. the main treatments for ptsd are selective serotonin reuptake inhibitors (ssris) and serotonin and norepinephrine reuptake inhibitors (snris). although ssris have been shown to produce a response rate of about 60% in patients with ptsd, the complete remission rate is only about 20% to 30%. the ssris sertraline and paroxetine hydrochloride are the only two fda-approved ptsd treat-ments, though they are highly outdated. ptsd is a widely misunderstood disorder that extends far beyond its classification as solely a psychiatric disorder. ptsd has been correlated with elevated levels of gene expression, an overactive immune system, and elevated levels of norepinephrine (ne), all of which contribute to physical and psychological symptoms. this systematic review aims to evaluate novel therapeutic approaches which can be used in concert with psychotherapy to further improve the symptoms of ptsd compared to current treatments. novel therapies for post-traumatic stress disorder: a systematic review by: jillian popovich, atoosa heidari-bigvand, emma son and vanloan nguyen introduction 1.1 post-traumatic stress disorder post-traumatic stress disorder (ptsd) is a psychological disorder that people often experience after a traumatic event, including but not limited to sexual assault, combat, child abuse, and natural disasters 1 . the psychological response to enduring this type of event is not solely panic. the most common lasting e�ects include insomnia, hyperarousal and hypervigilance, panic attacks, night terrors, depression, anxiety, and physical pain 2 . the �rst line of treatment for these symptoms, once a diagnosis has been documented, is the use of selective serotonin reuptake inhibitors (ssris) and serotonin and norepinephrine reuptake inhibitors (snris) along with psychotherapy and harmful benzodiazepines 2 . the pathophysiology of ptsd is complex and includes several neurobiological systems. the hypothalamic-pituitary-adrenal (hpa) axis is a focus of ptsd research because of the involvement of the neurotransmitters in associated pathways 1 as well as the decreased hypothalamus size commonly associated with ptsd. the hpa axis is an intricate neuroendocrine pathway responsible for the stress response and the link between the cns and endocrine system 1 . berkeley pharma tech journal of medicine | 27 1.2 current interventions for ptsd the interventions approved for ptsd show low success rates compared to more novel therapeutic approaches. these are commonly prescribed simultaneously with psychotherapy, exposure therapy, or cognitive processing therapy (cpt) 3 . 1.3 novel therapeutic approaches novel therapeutic approaches that have been proven to be e�ective are greatly underutilized in part because they are not fda-approved for ptsd treatment, meaning that many of these promising treatments are o�-label. o�-label use of medication complicates access for individuals looking for therapy as the medication or treatment is often much more expensive. with the expense of o�-label medication and the limited fda-approved treatments, individuals with ptsd are limited in their options. hyperbaric oxygen therapy (hbot) is an approach that is often used for healing large wounds. hbot has recently been tested on individuals with ptsd without a history of traumatic brain injury (tbi) 4 . among other brain structures, hbot has been found to improve hippocampal activity which is responsible for fear extinction, a process those with ptsd often exhibit lower levels of. hbot can also improve mitochondrial function, induce hyperoxia, and assist in resurfacing memories for those with trauma 4 . stellate ganglion blocks (sgbs) are injections of local anesthetic, commonly lidocaine or ropivacaine, inserted into the stellate ganglion nerve bundle. studies have indicated several immediate e�ects of sgbs, most notably reduced hyperarousal related to the oversensitive �ght-or-�ight response 5 . stem cell therapy for ptsd only has one in vitro study testing the e�cacy of the treatment, conducted with rats 6 . though the research is limited, the use of stem cell transplantation for reducing the symptoms of ptsd is promising and warrants further research. no controlled clinical studies have been completed to evaluate the e�cacy of immunotherapy for ptsd speci�cally 7 . still, research has been conducted in pain management clinics to track the results and correlation between physical pain from an overactive immune system and the severity of berkeley pharma tech journal of medicine | 28 the psychological e�ects of ptsd 7 . the use of glucocorticoids and other non-steroidal anti-in�ammatory drugs (nsaids) aims to reduce pain, and in ptsd patients, the severity of the psychological symptoms is remediated along with physical pain when treated with anti-in�ammatory drugs 7 . topiramate is an anticonvulsant commonly used for epileptic seizures and migraines. though its widespread use is for neurological disorders, it has also been shown to be e�ective for psychological disorders such as ptsd and alcohol use disorder (aud) 8 by acting as a gaba agonist. 1.4 mechanisms of interventions the mechanism of hyperbaric oxygen therapy and its therapeutic e�ects have been the focus of research now more than ever since hbot’s �rst use in 1662. hbot works to induce hyperoxia by forcing 100% oxygen into the bloodstream and surrounding cells 4 instead of the 22% oxygen that is normally perfused under standard atmospheric conditions. the unusual hyperbaric pressure of 100% oxygen on cells can activate or deactivate both oxygen-sensitive and pressure-sensitive genes, which can lead to the e�ective regulation of gene expression and inhibition of more damage by ptsd 4 . the elevated levels of dissolved oxygen in body tissues contribute to restored mitochondrial function, proliferation and maturation of neural stem cells, and increased neuroplasticity multiple years after injury 4 . it also works to promote anti-in�ammatory biological properties. hbot has been used as a treatment for a variety of disorders and has shown to be e�ective in many di�erent aspects. however, a main risk of hbot is oxygen toxicity 8 . neurological status must be observed throughout the course of treatment because of this possibility. oxygen toxicity induced by hbot can manifest in several ways such as tonic-clonic convulsions or oxidative cellular damage 8 . a stellate ganglion block is a single injection of local anesthetic into the stellate ganglion nerve bundle in the cervical spine. this location in the cervical sympathetic trunk has been described as an “anatomical funnel” because it allows the passage of all sympathetic nerve �bers that lead to the thorax as well as to structures of interest in the neck and head 5 . these ganglia provide a connection to the central nucleus of the amygdala and hypothalamus, two of the main nervous structures altered by ptsd. the berkeley pharma tech journal of medicine | 29 sgb injection aims to block the typical hyperarousal during the �ght-or-�ight response and other high-stress situations 5 . the mechanism by which sgbs function is still unknown, though it may indirectly work by altering levels of norepinephrine (ne) in the brain 5 . norepinephrine is a key neurotransmitter in the hyperarousal and hypervigilance observed in individuals with ptsd. ne �oods the brain when signals arrive in response to sympathetic stimulation and increases the heart rate, alertness, arousal, and amount of energy available to the body. this further contributes to the stress response reported by those with ptsd. topiramate is typically used as an anticonvulsant medication though it has recently been tested for its e�cacy in reducing ptsd symptoms 9 . topiramate is an agonist of gaba, an inhibitory neurotransmitter involved in creating inhibitory postsynaptic potentials (ipsps). another notable fact is that topiramate blocks glutamate binding 8 . glutamate is an excitatory small-molecule neurotransmitter that increases action potential �ring when it binds to ligand-gated receptors kainate and ampa. dysregulation of ampa receptors (ampars) can in�uence mental health and its e�ects 10 . ampars are dynamic and changes in the quantity of ampar at a synapse alter the e�cacy of neurotransmitters completing synaptic transmission 10 . kainate receptors, which share a similar structure and function with ampa receptors, partly make up nociceptive signaling pathways 11 . kainate receptor antagonists that target a single subunit (gluk1) have been shown to produce analgesic e�ects for migraines, indicating their potential use for other painful chronic disorders 11 . their stimulation not only leads to chronic pain but also increased severity of ptsd symptoms. because topiramate inhibits glutamate signaling, learning and memory function are often transiently impaired. over the duration of treatment, however, this impairment is nearly restored 9 . immunotherapy as an intervention for ptsd is not common practice. the research done on this topic related to ptsd is extremely limited and no relevant controlled clinical trials have been conducted. because ptsd is classi�ed as a psychological disorder, the main symptoms that are well-de�ned are behavioral and psychological, not physical. many individuals with ptsd exhibit elevated levels of several in�ammatory markers such as c-reactive protein (crp), tumor necrosis factor-α, and berkeley pharma tech journal of medicine | 30 interleukin-6 10 . additionally, autoimmune disorders are often comorbid with ptsd. though numerous factors can contribute to in�ammation, the elevated levels of in�ammatory markers indicate a correlation between ptsd and the immune system. since in�ammation can alter neural circuits in brain regions that regulate emotions such as anxiety and fear, a hyperactive immune system causing in�ammation can have a detrimental e�ect on those with ptsd 10 . however, glucocorticoids and nsaids have been shown to indirectly reduce ptsd symptom severity through pain relief, providing a basis for further research. aiming to reduce in�ammation, which can restore neural circuits and neurotransmitter activity to normal levels, can help regulate the stress response and overall severity of symptoms 10 . stem cell therapy is a therapeutic approach that is being investigated for several di�erent disorders, both physical and psychological. for ptsd, there has been one study with stem cells di�erentiated in vitro that investigated the use of induced human pluripotent stem cells di�erentiated into neural progenitor cells (ipsc-npcs) 6 . the ipsc-npcs are di�erentiated in a laboratory before being transplanted into the rat model brain. once they are transplanted as neural progenitor cells, symptom severity can be evaluated throughout the course of treatment. as cells mature in the brain, they can reverse hippocampal tissue damage, promote regeneration, and induce motor function recovery in rat models of ptsd 6 . adapted assessments were performed on the rat models that showed similar responses to stimuli as humans with ptsd. at the end of treatment, it was found that ipsc-npcs can reduce symptom severity, speci�cally the fear response, and reverse the physical neural damage that ptsd causes 6 . 1.5 outcomes the clinician-administered ptsd scale for dsm-5 (caps-5) is a 30-item interview that can give clinicians and clinical researchers more information about an individual’s past history of ptsd, present symptoms, symptom severity, and more 12 . questions aim to assess the impact of ptsd symptoms on daily activities, including one’s social and occupational life. the scale can also classify a subject within the dissociative subtype as well as assess the overall ptsd severity and symptom improvement 12 . for the studies berkeley pharma tech journal of medicine | 31 included in this review, the caps-5 is a common outcome used to evaluate the e�cacy of an intervention. another outcome assessment includes the brief symptom inventory-18 (bsi-18) which questions the subject on three symptom scales: depression, anxiety, and somatization. furthermore, the beck depression inventory-ii (bdi-ii) is a widely used and accepted psychometric test to assess the severity of depression in a subject and includes 21 multiple-choice questions along with a self-reported inventory about recent feelings, emotions, and well-being 13 . in addition to these tests, several imaging techniques were used including fmri and di�usion tensor imaging-fractional anisotropy (dti-fa), which evaluates the white matter microstructures in the brain. for non-imaging tests, higher scores are associated with more severe symptoms 12 . what warrants signi�cant changes in caps-5 scores changes with the study, though it is commonly a change of 10 points that indicates signi�cant improvement or deterioration. 1.6 objective the objective of this paper is to qualitatively analyze the e�cacy of these �ve interventions for ptsd. this analysis is based on several clinical trials, randomized control trials, in vivo and in vitro studies, and one review paper. 1.7 method this systematic review follows the standard principles for search strategies. a comprehensive search using pubmed was performed on september 16th, 2022. another search was performed on october 12th, 2022 to ensure that all relevant articles are being utilized. this search included published manuscripts and abstracts. the two abstracts provided useful information pertaining to these therapeutic approaches, warranting their inclusion. the pubmed search included the following terms: post-traumatic stress disorder[mesh], ptsd[mesh], stellate ganglion block[mesh], hyperbaric oxygen therapy[mesh], topiramate[mesh], immunotherapy[mesh], and stem cell[mesh]. the boolean operator used in these search terms is ‘and’. included are all primary research studies that are published. there are �ve interventions with both in vivo and in vitro studies being considered. all included studies are relevant to the topic and published in english. this systematic review is from the latest studies published related to ptsd and its interventions. each study is screened by berkeley pharma tech journal of medicine | 32 two group members. the key elements of the study design were assessed and reported for each study, including clinical trials, randomized clinical research studies, and in vivo studies. the snowballing technique was also used to acquire additional articles through citations in previously included articles. after applying the search strategy stated above, 9 stem cell studies, 3 stellate ganglion block studies, 1 topiramate study, 1 immunotherapy study, and 11 hyperbaric oxygen therapy studies were found. after applying the snowball technique, 14 studies have been included in this paper based on relevance and inclusion criteria. this includes 1 stem cell study, 1 immunotherapy study, 6 stellate ganglion block studies, 4 hyperbaric oxygen therapy studies, and 2 topiramate studies. endpoints include the e�ects of hyperbaric oxygen therapy, stellate ganglion blocks, topiramate, immunotherapy, and stem cell therapy on ptsd symptoms severity. berkeley pharma tech journal of medicine | 33 results 2.1 hyperbaric oxygen therapy a us military-sponsored, randomized clinical trial was conducted in 2018, testing the e�ects of hyperbaric oxygen therapy (hbot) on 71 participants with ptsd and/or a traumatic brain injury (tbi) 14 . the participants were randomized to either the treatment group (n=36) or the sham group (n=35), and at baseline, 49% of participants met ptsd criteria. the primary outcome of this study was the neurobehavioral symptom inventory which was assessed at baseline, 13 weeks, and six months, in addition to several other indicators of ptsd symptom severity and neurocognitive tests 14 . this study administered daily one-hour sessions, 5 days a week, for 40 sessions total. the treatment group received >99% oxygen at 1.5 atmosphere absolute (ata) while the sham group received regular air at 1.2 ata (25% oxygen). the sessions were completed within 12 weeks. the participants in the hbot group were all older with more combat deployments, exhibiting worse anger control and more frequent traumatic axonal injury 14 . though these may indicate that the treatment group encountered worse brain injuries, the baseline post-concussive and ptsd symptom scores were similar to the sham group. the rivermead post-concussion symptom questionnaire (rpq) total, rpq-13, and ptsd checklist-civilian version hyperarousal scores showed more severe symptoms in the treatment group at baseline 14 . at 13 weeks, univariate and longitudinal analysis was used to assess symptoms based on the outcomes. the rpq-3 domain, which includes headaches, dizziness, and nausea, was improved compared to the sham group (p=0.01). the neurobehavioral symptoms inventory total score and a�ective domain change scores indicated success in the treatment group compared to the sham group, but only using univariate testing. at 13 weeks, several tests indicated an improvement in anger subscores with both univariate and longitudinal assessment but the results were not statistically signi�cant 14 . additionally, at 13 weeks and 6 months, 19 out of 36 participants in the hbot group reported feeling the bene�ts of hbot versus 10 out of 33 participants from the sham group. at 6 months, 19 berkeley pharma tech journal of medicine | 34 participants from hbot and �ve participants from the sham group reported the same e�ect. other neuropsychological tests were performed, with improvements for the hbot group at 13 weeks, but most did not reach statistical signi�cance. however, the sham group never exhibited statistically signi�cant results in any total or subscore assessments at 13 weeks 14 . with all of these analyses, participants who had ptsd had larger improvements than those without ptsd. furthermore, participants without ptsd had no signi�cant improvements in both the treatment and control groups 14 . this indicates that hbot may work better as a treatment for ptsd as opposed to tbi. by 6 and 12 months, any distinct di�erences between groups had diminished, with improvements at six months no longer being signi�cant as they were at 13 weeks. both post-concussive and ptsd symptoms were worse at 12 months post-hbot compared to baseline for participants in both groups. hbot participants improved on six out of seven california verbal learning test-ii subtests compared to the sham group at 13 weeks, and two subtests reached statistical signi�cance in univariate and longitudinal assessments. by 6 months, most score di�erences between groups were no longer statistically signi�cant. sleep issues within the hbot participants exhibited improvement at 13 weeks, but the results between groups were not signi�cant. both groups also reported improved sensory organization scores. in addition to these outcomes, participants with ptsd who received hbot were able to walk further in the six-minute walk test after treatment at 13 weeks 14 . these results indicate that hbot may be better utilized as a treatment for ptsd rather than post-concussive symptoms or tbi symptoms. furthermore, this study exhibits �ndings that support long-term treatment of hbot to keep symptoms reduced for longer. though many of the �ndings were not statistically signi�cant, those with ptsd showed more improvement than participants without the disorder, warranting further research on the use of hbot for ptsd. this study was quite small with about an equal number of participants in both groups. additionally, all of the participants were military personnel which limits generalizability to other populations of individuals with ptsd. further berkeley pharma tech journal of medicine | 35 analysis needs to be done on hbot for ptsd because of the lack of statistical signi�cance in this study though it did show to alleviate ptsd symptoms more than tbi symptoms. a randomized case-control study published in 2022 completed a hyperbaric oxygen therapy study on 29 veterans with ptsd 4 . compared to the control (n=15), the treatment group (n=14) showed signi�cant improvement in caps-5 scores by the end of the trial. the study focused on male veterans aged 25-60 who had treatment-resistant ptsd with debilitating symptoms for at least four years prior. participants underwent baseline evaluation which included brain imaging and psychological interviews by clinicians. the participants also completed an evaluation three months after hbot or control exposure. the hbot was administered in concert with the subjects’ pre-trial psychotherapy. the participants underwent 60 daily sessions, �ve days a week. during each hbot session, the treatment group was exposed to 100% oxygen at 2 ata with a �ve-minute break every 20 minutes. the primary objective of this study was a change in caps-5 score compared to baseline evaluations. the secondary objective included the bsi-18 and bdi-ii questionnaires. other outcomes included imaging data acquisition which measured gradient-echo blood oxygen level-dependent contrast berkeley pharma tech journal of medicine | 36 sequences. a functional task design, in which participants had to perform a memory recall test, was also used to test their results. table 1 indicates the signi�cance of results in the treatment arm (p<0.0001) compared to the control group, which didn’t improve in any subscores or total score 4 . similarly, bsi scores (p=0.024) as well as bdi scores (p=0.01) signi�cantly improved 4 . in the hbot group, there were improvements in group-time interactions in frontal white-matter �ber bundles that connect the thalamus and frontal lobe. there were also improved clusters found in parietal white matter and the anterior limb of the internal capsule and cerebral peduncle. the same patients in both groups were used for task-related functional imaging, and there were no signi�cant functional di�erences between the fmri images except for improved activity after hbot in several regions of the brain including the hippocampus 4 . this study is limited by its size with only 35 randomized patients. additionally, there was no blinding for either group. the results of this study support the fact that hbot is highly e�ective in treating ptsd symptoms based on caps scores and brain imaging. there is evidence that this treatment should be more widespread, though it is limited by the fact that hbot is a historically di�cult treatment to be covered by insurance and it requires specialized physicians to perform and oversee the treatment. hyperbaric oxygen therapy has revealed the pressure-sensitive and oxygen-sensitive genes involved in the etiology of ptsd. these genes could berkeley pharma tech journal of medicine | 37 be further investigated and possibly manipulated with gene therapy, though this has not been done yet. in addition to these trials, an observational cohort study was conducted to investigate any persistent post-concussive symptoms (pcs) in participants from two completed united states military trials of hyperbaric oxygen therapy (hbot). individual changes varied widely, ranging from -23 to +28 points 15 . 2.2 stellate ganglion block stellate ganglion blocks were �rst used to treat ptsd in 2010 16 . they have been used to treat dysautonomia, vasomotor symptoms, pain in the upper extremities, and most recently, psychiatric disorders. the �rst multisite, randomized clinical trial of stellate ganglion block e�ects on ptsd symptoms severity was published in february 2020 16 . this trial pulled participants from three us army interdisciplinary pain management centers which included active-duty service members as the �rst inclusion criteria. the participants were randomized 2:1 to a sham group (n=39) or treatment group (n=74), and baseline characteristics were similar in both groups based on mean caps-5 scores 16 . participants were administered a right-sided sgb at 0 and 2 weeks, and they were assessed at 0 and 8 weeks post-injection. the injections were performed using ultrasonography. 7-10 ml of 0.5% ropivacaine was administered to the treatment group and the sham group received 1-2 ml of normal saline at the same anatomical location, the c6 anterior tubercle 16 . measures were taken so as to not unblind participants due to the di�erence in medication and medical instruments, and conversations were scripted for medical personnel who were not blinded. the primary outcome of this trial was a decrease in caps-5 total symptom severity scores (caps-5 tsss), indicating improvement. this assessment ranges from 0-80 points, with higher scores indicating more severe ptsd symptoms and a 10-point change per individual indicating the results as signi�cant and clinically meaningful 16 . berkeley pharma tech journal of medicine | 38 table 2 highlights the overall trend of improvement for the treatment arm as well as the sham group. though both groups improved by some amount, the treatment group had a greater overall decrease in symptom severity scores, though not statistically signi�cant. however, there were clinically signi�cant improvements in subsets of ptsd-symptom assessments, including but not limited to depression and distress 16 . this study heavily supports the use of sgb injections for those with ptsd as a way to signi�cantly reduce the most common and detrimental symptoms of ptsd. limitations of this study include unblinding of the participants in the treatment arm due to the onset of horner’s syndrome. this is a side e�ect and indication of a successful injection in which there is a disrupted nerve pathway going from the brain to the face, resulting in a smaller pupil on the a�ected side, a drooping eyelid (ptosis), and a reduction in sweat 16 . the medical personnel performing the procedure on the participants were also unblinded to ensure the safety of the participants, though their interactions were limited and scripted. other limitations include generalizability, as this study only included active-duty military personnel with only 10 females and 3 females in the treatment and sham groups respectively. because of the limited studies done on this intervention as a treatment for ptsd, there is little evidence of whether a right-sided sgb or left-sided sgb is more e�ective with the use of �uoroscopy versus ultrasonography, or if they produce the same results. in 2021, however, a cadaveric study was performed and published testing vertebral body spread of the injectate based on sidedness 17 . ten soft-cured human cadavers were administered both �uoroscopic and ultrasound-guided injections each. the injection was a berkeley pharma tech journal of medicine | 39 mixture of 7 ml of omnipaque and methylene blue and was used as the injectate on both sides. the cadavers were then dissected to visualize the staining of the sympathetic trunk associated with methylene blue 17 . the primary outcome was the staining of methylene blue at the cervical sympathetic trunk upon dissection. the secondary outcome was a craniocaudal spread of the dye. after performing two injections on each cadaver for a total of 20 injections, they were dissected. fluoroscopic guidance of sgb injections had a successful stain rate of 60%, six out of ten cadavers 17 . for the ultrasound-guided sgb, the successful stain rate of the sympathetic chain was nine out of ten for a 90% success rate 17 . the failed injections were because the injections were administered in the wrong location, such as the carotid sheath and within or beneath the longus colli muscle, instead of the stellate ganglion nerve bundle at the c6 anterior tubercle 17 . the study found no statistically signi�cant di�erences in approach using �uoroscopy versus ultrasonography, though there was greater staining using ultrasonography. limitations of this study include the fact that it was performed on cadavers which may not accurately represent the spread of injectate in living subjects. the fascial layers play a large role in the spread of injectate, and the compositional properties of the fascial layers in cadavers are altered compared to living humans. in addition, no functional outcomes can be assessed on cadavers. a study examining clinical endpoints in people with ptsd was completed in may of 2013, analyzing 1,462 data points from the ptsd checklist-military (pcl-m) 18 . this study analyzed the data points of 26 patients in a military clinic, all of whom had several combat deployments that consisted of receiving and returning direct �re. the subjects were all male ranging in age from 29 to 45. the participants were administered 7 ml of 0.5% ropivacaine injected over two minutes, and a successful injection was indicated by the presence of horner’s syndrome. the primary outcome, pcl-m score, was tested at baseline and at two follow-up appointments 18 . the 16-point decrease in mean pcl-5 score is signi�cant (p<0.001), but the mean score at the second follow-up did not show signi�cant improvement from 1-week post-injection. berkeley pharma tech journal of medicine | 40 all three symptom cluster scores were signi�cantly improved (p<0.001) one-week post-injection, except for one item in a single cluster 18 . these results support the use of sgb injections for military personnel who su�er from combat-related ptsd. the limitations of this study are similar to others: many studies for ptsd generally test on active-duty personnel or veterans only, as well as speci�cally men of certain age ranges and ethnicities. this limits generalizability for other ptsd-a�ected populations including women, children, and civilians who have never served in the military or have experienced combat. in addition to the cadaveric study and in vitro trials, an in vivo study done on rats was conducted and published in 2021 19 . this study di�ers in that it tested the e�ects of ptsd-related sleep deprivation on memory dysfunction and hippocampal injury. sleep deprivation is a common symptom of ptsd because of insomnia and night terrors. sleep deprivation can detrimentally a�ect the brain and the body and lead to a cascade of other symptoms. it has been found that sleep deprivation is associated with cognitive dysfunction, which is mediated through melatonin 19 . stellate ganglion blocks have been found to exhibit similar e�ects as those of therapeutic interventions with melatonin in that they prevent the breaking of cervical sympathetic preganglionic �bers, reduce sympathetic nerve tension, and help regulate the balance in multiple body systems, including the autonomic nervous system, endocrine system, and immune system. sleep deprivation has also been shown to impair hippocampal coding activity, cause hippocampal mitochondrial dysfunction, neurodegeneration, microglia activation, and neuronal apoptosis in the hippocampus 19 . furthermore, there has been con�rmation of increased white blood cell counts and in�ammatory factors, including c-reactive protein (crp), il-1β, il-6, and tnf. there are also decreased serum melatonin levels 19 . berkeley pharma tech journal of medicine | 41 this study tested whether alleviating the hippocampal tissue damage can improve spatial learning and memory dysfunction caused by sleep deprivation in a rat model of ptsd. 65 male sprague dawley rats were randomized to four di�erent groups. these groups were control, sleep deprivation (sd), sgb, and sgb + sd (n=16). the sgb and sgb + sd groups were administered one right-sided stellate ganglion block once per day. the sd and sd + sgb groups were subjected to a multi-platform water environment for 96 hours to induce sleep deprivation. their body weights were also analyzed at this time. the morris water maze was used to detect the spatial learning and memory function of rats. the escape latency, the time it took from the rats entering the water to �nding the security platform, was measured as an outcome. after the water maze, eight rats each had 3 ml of inferior vena cava blood samples analyzed for serum melatonin content. the sgb and sgb + sd groups were given a right-sided sgb six days prior to sleep deprivation induction up until the end of the day, with one sgb of 0.2 ml of 0.2% bupivacaine per day. the onset of horner’s syndrome indicated that the injection dissipated into the correct anatomical area. rats in the sd group were given the same volume of normal saline, and the control group was not treated. after the injections were administered, the berkeley pharma tech journal of medicine | 42 escape latency time of the sd group was signi�cantly longer compared to the control group. the sd + sgb group was shortened, and the escape latency in the sgb and control groups tended to be consistent 19 . the number of rats that crossed the platform was much lower in the sd than in the control group (p<0.05), and those in the sd + sgb was signi�cantly higher compared to the sd group (p<0.05) 19 . the platform was removed and resident time and frequency of crossing the platform were recorded. the sd + sgb group showed higher crossing and resident times in the test compared to the sd group. the body weight of the rats in the sd group was reduced compared to the control group. the sd + sgb group showed signi�cantly increased body weight after injection compared to the sd group. in comparison with the control group, there was no signi�cant distinction between il-6, il-1β, and serum mt content in the hippocampus of the rats in the sgb group. however, there were elevated levels of il-6 and il-1β content in the hippocampus of sd rats compared to the control. a western blot of the relative expression of caspase-3 protein in sd rats’ hippocampus was signi�cantly higher, and the relative expression of the same protein in the hippocampus of sd + sgb rats was signi�cantly lower with no di�erence in the protein’s expression between sgb and control groups 19 . histological analysis of hippocampal tissue showed that the stress damage and the berkeley pharma tech journal of medicine | 43 number of hippocampal neurons that were injured in the sd group were very elevated, most prominent in the vertebral neurons in the hippocampal ca3 region. the vertebral cells were decreased in size, irregular, loose, and fuzzy. the neuronal damage in the sd + sgb group was signi�cantly improved compared to the control group, and the cells were more ordered with a clearer structure. in addition, the serum mt levels decreased with more sleep deprivation. the serum mt content signi�cantly improved in the sd + sgb group, indicating that a right-sided sgb can alleviate the decrease in serum mt secretion secondary to sleep deprivation. the elevated il-6 and il-1β levels caused by sleep deprivation are pro-in�ammatory cytokines, indicating that right-sided sgbs can inhibit the initial in�ammatory reaction after trauma exposure 19 . they can also be used to inhibit the excessive in�ammatory reaction after tbi, preserving brain function. the caspase-3 protein that was found in elevated levels in sd rats was reduced after sgb, further indicating that the excessive apoptosis can be alleviated as evidenced by the fact that ca3 protein levels were reduced in the sd + sgb group 19 . furthermore, the degree of stress damage on hippocampal neurons was signi�cantly reduced in the sd group. though these are all post-injury alleviating factors, the behavioral platform used in this experiment showed that prophylactic administration of sgb can provide preventative treatment for spatial learning and memory dysfunction 19 . the results of this experiment make it highly evident that sgb injections produce several signi�cant bene�ts such as increased melatonin production and secretion, alleviation of hippocampal apoptosis due to decreased caspase-3 protein levels, spatial learning and memory dysfunction improvement, and hippocampal neuron regeneration after damage. given that this is a non-invasive, generally safe treatment, sgbs should be further investigated and utilized based on this experiment done in the rat model. in addition to these studies, a cohort of behavioral clinicians was interviewed about their thoughts on the e�cacy of sgb for trauma-related disorders 20 . approximately 50 mental health providers were sent an 18-item survey to assess their experiences with sgb as a treatment for trauma-related berkeley pharma tech journal of medicine | 44 conditions. of the 27 clinicians that responded, 23 had input that was used in this study (10 psychologists, 7 psychiatrists, 4 licensed clinical social workers, and 2 psychiatric nurse practitioners). experience with sgb varied between providers. the survey showed that 95% (22 out of 23) of providers would recommend the use of sgb to a colleague while 65% said this procedure is very bene�cial 20 . 30% indicated sgb as somewhat helpful and over 80% were likely or very likely to refer a patient to get an sgb procedure. 96% of the providers also responded that sgb is most helpful for arousal and reactivity compared to other symptoms 20 . this cohort responded that sgb is at least as bene�cial as other heavily used interventions, with 100% responding that sgb is very bene�cial or somewhat bene�cial. none of the respondents said that sgb is harmful or not helpful 20 . furthermore, sgb was overall favored over each of the 8 interventions endorsed for ptsd in the clinical practice guideline 20 . both qualitatively and quantitatively, the overall results of studies assessing the e�cacy of sgb as an intervention for ptsd support their use for widespread treatment. sgbs are highly bene�cial with minimal risks and side e�ects. the main side e�ect is horner’s syndrome which subsides a couple of hours post-procedure when the local anesthetic has worn o� and also acts as an indicator to clinicians that the injection was successful 16 . this, in addition to the fact that this is non-invasive, quick, and has immediate bene�cial e�ects, can help increase treatment adherence rates, thereby also improving the number of those with ptsd who can bene�t from this treatment over others. because of the sheer number of veterans and active military personnel that su�er from this disorder, all of the conducted studies have been limited to veterans, mainly caucasian men aged 18-65. this limits generalizability for others that ptsd a�ects including sexual assault victims, those who have experienced natural disasters or other non-combat-related trauma, women, children, and people of other ethnicities. furthermore, the department of defense is the main source of funds for these clinical trials. this forces the trials to only include those who are serving or who have served in combat. due to this, funding outside of the dod needs to be identi�ed to carry out trials on non-military civilians who su�er from ptsd. berkeley pharma tech journal of medicine | 45 future applications of sgbs are limited given these trials. in order to make this treatment more mainstream and widespread, there can be increased and more diverse populations in clinical trials, more in-depth research, training of physicians outside of military hospitals and clinics to perform this procedure, and better analysis of complete data sets. overall, this is a highly e�ective treatment that is limited by its research and the training of physicians as well as funding for trials and insurance coverage for patients. it has been shown to increase melatonin levels to combat insomnia, regulate gene expression, decrease hippocampal apoptosis, reverse hippocampal damage, improve spatial learning and memory, and decrease depression, anxiety, hyperarousal, and hypervigilance [5, 16, 18, 19, 20] , making this a target for further application. the numerous bene�cial e�ects of sgbs warrant further research and application, especially for those outside of the military. sgb can be used to target the expression of caspase-3 protein, il-1β, and il-6. targeting these proteins can aim to reduce highly damaging hippocampal tissue reduction and neuronal degeneration, along with reducing in�ammation in the brain and body immediately following a traumatic event because of the pro-in�ammatory properties of il-1β and il-6 genes. further research on the e�ects of sgb on gene expression is warranted because of these proteins’ roles in ptsd symptom severity. western blot assays should be utilized in further studies to assess this as this has only been done in a rat model. 2.3. topiramate topiramate, a medication commonly used to treat migraines and epilepsy, is a second-generation anti-epileptic drug 21 . though the precise mechanism is unknown, it is known that the drug blocks voltage-gated sodium channels, which are thought to control depolarization during seizures. it reduces membrane depolarization with ampa and kainate receptors [10, 11] while also enhancing gaba receptor activity. additionally, it is known to be an inhibitor of carbonic anhydrase and nmda receptor activity, allowing for partial regulation of seizures in the brain 21 . berkeley pharma tech journal of medicine | 46 an in vivo study was conducted with 30 veteran patients su�ering from ptsd and alcohol use disorder, in which patients were placed in two groups, treatment or placebo (negative) 9 . there was a 12-week double-blind treatment administered in which half the patients (randomized) were given increasing doses of topiramate from 25 mg on day 1 up to 300 mg a day by week 6, and constant doses through week 12. participants in the negative control group were given a placebo treatment. the primary hypothesis of the di�erence between the tested topiramate group and placebo group was tested using a statistical t-test, which produced a p-value=0.019 that indicated statistically signi�cant results in the di�erence between these two groups and their drinking days per week 9 . the main e�ect of the treatment for those who received it was reduced standard drinks per week, which decreased by 55% for the topiramate group along with a 61% reduction in drinks per day 9 . an additional in vivo study analyzed e�ciency of topiramate treatment on post-traumatic stress disorder patients from the clinic of the violence program of federal university são paulo city 22 . this was another 12-week double-blind treatment test where 35 patients were randomly assigned to the treatment group or negative control group. the clinician administered berkeley pharma tech journal of medicine | 47 post-traumatic stress scale (caps) and beck depression inventory (bdi) were used as primary indicators of results 22 . the mean topiramate dose administered was 102.95 mg a day. the study yielded 82.35% of patients with signi�cant improvements in their caps-b score, seen with improvements in re-experiencing symptoms such as �ashbacks, intrusive memories, and nightmares of the trauma (p = 0.04) 22 . there was also a signi�cant improvement in avoidance/numbing symptoms associated with trauma, social isolation, and emotional numbing in caps-c scores (p = 0.0001). additionally, there was a signi�cant decrease in caps total score for the experimental group when compared with the placebo group (p = 0.0076) 22 . the �rst study analyzed a high ratio of male participants with barely any female subjects. this made results highly biased towards male patients, and further research may have to be done to gauge if the results can be generalized to female patients due to di�erences in ptsd manifestation between genders. however, the second study saw similar improvements in caps scores with a majority of the participants being women, at 21 out of 35 patients. additionally, the study focused on ptsd patients that were veterans. we cannot generalize these e�ects to other populations struggling with post-traumatic stress disorder since the studies were concentrated on veterans su�ering from symptoms. furthermore, the majority of participants in the �rst study were white. more research concentrating on di�erent races can help broaden the spectrum of participants that gain positive results following the treatment of topiramate. for both studies, only 30-40 patients were observed, and with such a small sample size, the results are less accurate. more studies conducted with greater sample sizes will increase the signi�cance of the results. though already being widely used as a drug to treat neurological illnesses such as epilepsy or migraines, further studies regarding the dosage of topiramate to treat post-traumatic stress disorder are proven to produce positive results. the main risk is topiramate may also produce adverse e�ects when taken in larger doses, with the most common side e�ects in migraine berkeley pharma tech journal of medicine | 48 patients being insomnia and drowsiness. however, it is known as a relatively safe drug. it should be avoided in patients prone to experiencing metabolic acidosis due to being a carbonic anhydrase inhibitor 21 . with the already signi�cant results seen in topiramate decreasing post-traumatic stress disorder indicative diagnosis, including caps score, bdi score, and ptsd symptom severity scores, further studies conducted on a larger and more diverse sample group can give support in popularizing this method for post-traumatic stress disorder patients. 2.4. stem cell therapy stem cell therapy and transplantation have been used widely since their �rst application in 1957 23 . stem cell transplants are mainly used to target damaged cells from chemotherapy or diseases usually related to cancer. stem cell therapies are of particular interest because of the vast results they have the potential to produce. the application of stem cell therapy for trauma-related disorders such as ptsd has not yet been explored in humans. the only research done on this topic consists of a single in vivo study published in 2021 6 . the 2021 study used induced pluripotent stem cells (ipscs), generated by reprogramming somatic cells by inserting certain genes. ipscs can be di�erentiated into any cell type 6 . among the various types of cells, induced pluripotent stem cell-derived neural progenitor cells (ipsc-npcs) can replace lost neurons that have degenerated in a highly speci�c region of the brain. they can replace the loss of neurons in the hippocampus and regenerate impaired hippocampal structure, and there is accumulating evidence that ipsc-npcs can support tissue repair and functional recovery after neurogenic injuries 6 . in this study, male sprague dawley rats were used as subjects. cell culturing of ipscs was done in an embryonic stem cell medium until typical human embryonic stem cell morphology was present. the stem cell colonies were then dissociated. the rats were randomized to six groups (n=8) on days 7, 14, and 21. on day 7, the groups included control, ptsd, ptsd + pbs, and ptsd + ipsc-npcs. on days 14 and 21, the group was ptsd + ipsc-npcs. 14 days after the initial injury, ipsc-npcs were transplanted berkeley pharma tech journal of medicine | 49 into the hippocampal site 6 . at di�erent points in time, the rats’ brains were dissected for analysis. the open �eld test was used to evaluate outcomes related to locomotor activities. the fear conditions test was used to subject the rats to a tone-foot-shock pairing. the induced pluripotent stem cells were allowed to develop for 24 days in the laboratory to ensure di�erentiation. seven days after transplantation, there was no signi�cant di�erence between the control and transplant groups in residence time and behavior modi�cation 6 . this indicates that short-term transplantation does not a�ect locomotor activity or anxiety. long-term treatment was shown to be e�ective 14 and 21 days after treatments, with increased residence time and behavior modi�cations indicating lowered levels of anxiety and depression 6 . this also supports that long-term treatment improves and regulates cognitive dysfunction. furthermore, neuronal nuclear protein (neun) was used as a marker of mature neurons in immuno�uorescence assay. this suggested that ipsc-npc transplantation can increase neurogenesis in the damaged hippocampus found in ptsd 6 . in addition, immunostaining indicated that transplantation can increase astrogliosis, thereby minimizing damage to cns injuries. in another western blot analysis, there was a reduction in bdnf expression which was reversed after 14 and 21 days in the transplant group. this suggests that transplantation supports functional recovery through upregulating bdnf 6 . berkeley pharma tech journal of medicine | 50 the results of this study indicate that ipsc-npc transplantation after npc treatment in vitro can promote neurogenesis and functional recovery, reduce anxiety and depression, and repair damaged hippocampal tissue and neurons 6 . the limitations of stem cell transplantation as an intervention for ptsd are numerous. this is the only study that has been done on this intervention for ptsd, and it is only in a rat model, not in humans. the results of long-term transplantation indicate that further research is needed before it can be considered a viable treatment option. stem cell transplants are also limited because of the high risk of developing neoplasms, so the cells must be treated carefully before transplant. though this may seem like an intense treatment for ptsd, there is promise that it can be bene�cial in reducing symptoms and physiological damage. 2.5 immunotherapy ptsd is often comorbid with a number of in�ammatory diseases and autoimmune disorders 7. though many factors can play a role in in�ammation unrelated to trauma, there are elevated rates of immune-related conditions in those with ptsd. a review published in 2022 assessed the use of anti-in�ammatory drugs for reducing ptsd symptoms and their mechanism within the disorder 7 . though the directional relationship between ptsd and in�ammation is unknown, accumulating evidence points to the adaptive and innate immune systems’ roles in the pathophysiology of ptsd. there are often variations in peripheral in�ammatory markers in those with ptsd including c-reactive protein, il-6, il-10, and tumor necrosis factor-alpha (tnf-α) 7 . there have also been several genomic studies that have identi�ed multiple genes related to the immune system that are altered in those with ptsd. more speci�cally, transcriptomic studies performed on us marines showed the upregulation of immune-related genes and the overexpression of genes related to the innate immune response. interferon signaling pre-deployment was able to predict post-deployment ptsd 7 . berkeley pharma tech journal of medicine | 51 ptsd is associated with variations in regions of the brain that regulate fear, anxiety, and threat detection. these regions mainly consist of the amygdala, hippocampus, and hypothalamus. upon stress, people with ptsd have heightened amygdala activation and increased pro-in�ammatory cytokine levels 7 . this is associated with depression, social disconnection, fatigue, and cognitive disturbance, which often come with ptsd. the hippocampus, which is involved in fear and memory processing, is often found in smaller volumes in those with ptsd. a smaller hippocampus is associated with increased in�ammation. in a rat model, it was found that in�ammation of the hippocampus suppresses neurogenesis and stimulates apoptosis of neuronal progenitor cells, indicating that in�ammation of the hippocampus may greatly contribute to cognitive and emotional issues that come with ptsd. several more peripheral structures are a�ected by in�ammation and ptsd, which a�ect neurotransmitter release and reuptake, again correlating the emotional issues of ptsd and in�ammation 7 . of the two fda-approved ssris, none target in�ammation. there are several proposed potential treatments in this review that target the in�ammation associated with ptsd, though none are fda approved for ptsd treatment. monoclonal antibodies are approved for the treatment of berkeley pharma tech journal of medicine | 52 autoimmune disorders and cancers 7 , and the elevated levels of il-1β, il-6, and tnf-α give reason to believe that blocking these pro-in�ammatory cytokines using monoclonal antibodies could better regulate gene expression and reduce in�ammation. though there are no studies on this intervention for ptsd, several tnf inhibitors have been reported to reduce anxiety and depression in people with psoriasis, indicating the potential application of tnf inhibitors for other diseases that cause in�ammation 7 . non-steroidal anti-in�ammatory drugs (nsaids) and cyclooxygenase 2 (cox-2) inhibitors can regulate the pro-in�ammatory cytokine production in the body, thereby directly reducing in�ammation 7 . celecoxib, a cox-2 inhibitor, was able to reduce depression symptoms in people with major depressive disorder, meaning it potentially reduced il-6 levels, one of the upregulated genes in people with ptsd. ibuprofen, an nsaid, reduced anxiety in a rat model of ptsd and decreased expression of il-1β and tnf-α while increasing bdnf expression in the hippocampus 7 . this indicates that the e�ect of ibuprofen on ptsd was moderated by decreased anti-in�ammatory activity and increased expression of bdnf in the brain. glucocorticoids are another class of drugs that can be employed to treat the symptoms of ptsd. glucocorticoids work to suppress the in�ammatory response following exposure to stress by promoting the production of anti-in�ammatory cytokines and suppressing the production of pro-in�ammatory cytokines. clinical trials have shown that glucocorticoid treatment combined with psychotherapy can improve ptsd symptoms. glucocorticoid administration after prolonged exposure, however, has been shown to not have signi�cant e�ects on eliminating ptsd symptoms alone. studies investigating the prophylactic e�ects of glucocorticoids following trauma exposure found that glucocorticoid treatment following an acute traumatic event was able to signi�cantly reduce stress symptoms and the incidence of ptsd 7 . in addition to these approaches, angiotensin-converting enzyme inhibitors, noradrenergic beta-receptor blockers, angiotensin receptor blockers, and cannabinoids also have bene�cial e�ects when used to treat ptsd. there are several mechanisms by which they do this through suppressing in�ammation 7 . there is a growing body of strong evidence that supports berkeley pharma tech journal of medicine | 53 the use of immunotherapy to reduce ptsd symptoms, including in�ammation. the drugs in this review are generally safe with proper oversight and are underutilized for ptsd. given that glucocorticoids and noradrenergic beta-receptor blockers can work prophylactically to reduce the incidence of ptsd following a traumatic event, further research and applications are warranted for trauma-related conditions. if there is further evidence that glucocorticoids can work prophylactically, they can be a supplement to other treatments, such as stellate ganglion blocks, for individuals who arrive at the hospital after an intensely traumatic event, possibly blocking the development of ptsd. the link between ptsd, the brain, and in�ammation produces the potential for anti-in�ammatory treatment as a prophylactic intervention after an acute traumatic event, but before the onset of ptsd. discussion this qualitative systematic review aims to highlight several novel therapies for ptsd as well as the limitations of their current studies. some common limitations in novel research for ptsd include low adherence rates, funding/participant bias at military hospitals funded by the department of defense, and low generalizability rates given the limited demographics of study participants. these issues, in combination with a disorder that is not yet fully understood, greatly hinder widespread access to groundbreaking treatments for ptsd. the identi�cation of pressure-sensitive and oxygen-sensitive genes through hyperbaric oxygen therapy can possibly be used in combination with gene therapy to upregulate these genes. this could potentially be used to enhance the e�ects of hyperbaric oxygen therapy under normal atmospheric conditions. hyperbaric oxygen therapy is commonly used to assist in neural regeneration after brain injuries or ischemic strokes, heal large wounds, and restore hearing after sudden deafness. hbot exhibits several di�erent mechanisms in which it has proven to be e�ective, but induced hyperoxia is ultimately the objective of the treatment. forcing extra oxygen to perfuse throughout the body has many e�ects, and the oxygen works directly to berkeley pharma tech journal of medicine | 54 �ght bacterial infections by releasing stem cells and various growth factors4. this in turn helps regulate the immune system and e�ectively reverse hippocampal tissue damage. immune disorders are very commonly comorbid with ptsd, indicated by elevated levels of in�ammatory markers in individuals with ptsd. these novel therapies have the potential to be administered in unison to maximize the e�ects of treatment 7 . stellate ganglion blocks take an interesting approach to ptsd in that they utilize local anesthetic to block signal transmission to the brain. stellate ganglion blocks are used for a wide variety of disorders, and the studies reviewed in this paper indicate that these injections can provide immediate relief to individuals with ptsd. these injections are safe when performed correctly, and more than one can be administered if one’s symptoms return after an initial injection. though there isn’t relevant longitudinal research, it has shown to be safe to intermittently administer more anesthetic, indicating potential long-term relief with the use of several injections. the main side e�ect of sgbs, horner’s syndrome 16 , is a temporary condition that causes minor neurological symptoms. stellate ganglion blocks are low-risk treatments that are supported by several in vivo studies and clinical trials. the available data warrants their use as therapy for ptsd to provide immediate relief of physical, cognitive, and emotional symptoms. topiramate is another low-risk treatment that aims to reduce alcoholism in individuals with ptsd 8 . topiramate is a widely accepted anticonvulsant that is now being used in other ways. two clinical trials have yielded statistically signi�cant results in di�erent areas [9,22] , indicating that topiramate can be an e�ective medication for alcohol use disorder comorbid with ptsd. some of these interventions have produced great results, assessed by a decrease in total symptom severity. aside from stem cell therapy, these treatments are safe and e�ective for many individuals with ptsd and other disorders, and the qualitative evidence supports the mainstreaming of these therapies when administered in concert with psychotherapy. berkeley pharma tech journal of medicine | 55 conclusion with the limitations seen in the discussed studies, further in-depth investigation of all methods would increase the strength of statistical evidence, expand on ptsd-speci�c e�ects of each treatment, and generalize the outcomes to more diverse groups of patients. o�ering di�erent and more feasible approaches to these treatments would also popularize the e�ective treatment of ptsd symptoms according to their speci�c mechanism and signi�cance in terms of the bene�ts, risks, and limitations they provide. berkeley pharma tech journal of medicine | 56 references 1. bailey cr, cordell e, sobin sm, neumeister a. recent progress in understanding the pathophysiology of post-traumatic stress disorder: implications for targeted pharmacological treatment. cns drugs . 2013;27(3):221-232. doi: 10.1007/s40263-013-0051-4 2. alexander w. pharmacotherapy for post-traumatic stress disorder in combat veterans: focus on antidepressants and atypical antipsychotic agents. p t . 2012;37(1):32-38. 3. doenyas-barak k, catalogna m, kutz i, et al. hyperbaric oxygen therapy improves symptoms, brain’s microstructure and functionality in veterans with treatment resistant post-traumatic stress disorder: a prospective, randomized, controlled trial. plos one . 2022;17(2). doi:10.1371/journal.pone.0264161 4. friedman mj. va.gov: veterans a�airs. history of ptsd in veterans: civil war to dsm-5. https://www.ptsd.va.gov/understand/what/history_pts d.asp. published august 17, 2018. accessed october 21, 2022. 5. lynch, jh. stellate ganglion block treats posttraumatic stress: an example of precision mental health. brain behav . 2020; 10:e01807. https://doi.org/10.1002/brb3.1807 6. liu q, zhang l, zhang j. induced pluripotent stem cell-derived neural progenitor cell transplantation promotes regeneration and functional recovery after post-traumatic stress disorder in rats. biomedicine & pharmacotherapy . 2021;133:110981. doi:10.1016/j.biopha.2020.110981 7. katrinli s, oliveira ncs, felger jc, michopoulos v, smith ak. the role of the immune system in posttraumatic stress disorder. transl psychiatry . 2022;12(1):313. published 2022 aug 4. doi: https://doi.org/10.1038/s41398-022-02094-7 8. cooper js, phuyal p, shah n. oxygen toxicity statpearls ncbi bookshelf. https://www.ncbi.nlm.nih.gov/books/nbk430743/. published january 2022. accessed october 22, 2022. 9. batki sl, pennington dl, lasher b, et al. topiramate treatment of alcohol use disorder in veterans with posttraumatic stress disorder: a randomized controlled pilot trial. alcohol clin exp res . 2014;38(8):2169-2177. doi: 10.1111/acer.12496 10. chater te, goda y. the role of ampa receptors in postsynaptic mechanisms of synaptic plasticity. frontiers . https://www.frontiersin.org/articles/10.3389/fncel.201 4.00401/full. doi: 10.3389/fncel.2014.00401 published january 1, 1ad. accessed october 22, 2022. 11. bhangoo sk, swanson gt. kainate receptor signaling in pain pathways. mol pharmacol . 2013;83(2):307-315. doi: 10.1124/mol.112.081398 12. va.gov: veterans a�airs. clinician-administered ptsd scale for dsm-5 (caps-5). https://www.ptsd.va.gov/professional/assessment/adult-i nt/caps.asp. published september 24, 2018. accessed october 22, 2022. 13. beck, a. t., steer, r. a., & brown, g. (1996). beck depression inventory–ii (bdi-ii) [database record]. apa psyctests . https://doi.org/10.1037/t00742-000 14. weaver lk, wilson sh, lindblad as, et al. hyperbaric oxygen for post-concussive symptoms in united states military service members: a randomized clinical trial. undersea hyperb med . 2018;45(2):129-156. 15. skipper ld, churchill s, wilson sh, deru k, labutta rj, hart bb. hyperbaric oxygen for persistent post-concussive symptoms: long-term follow-up. undersea hyperb med . 2016;43(5):601-613. berkeley pharma tech journal of medicine | 57 16. rae olmsted kl, bartoszek m, mulvaney s, et al. e�ect of stellate ganglion block treatment on posttraumatic stress disorder symptoms: a randomized clinical trial. jama psychiatry. 2020;77(2):130–138. doi: 10.1001/jamapsychiatry.2019.3474 17. scott hughey, md, mc, usn, je�rey schafer, md, mc, usn, jacob cole, md, mc, usn, gregory booth, md, mc, usn, ralph tuttle, do, mc, usn, eric stedje-larsen, md, mc, usn, ultrasound versus fluoroscopy for stellate ganglion block: a cadaveric study, pain medicine , volume 22, issue 10, october 2021, pages 2307–2310, https://doi.org/10.1093/pm/pnab182 18. james h. lynch, mc usa, sean w. mulvaney, mc usa, eugene h. kim, mc usa, jason b. de leeuw, ms usa, matthew j. schroeder, phd, shawn f. kane, mc usa, e�ect of stellate ganglion block on speci�c symptom clusters for treatment of post-traumatic stress disorder, military medicine , volume 181, issue 9, september 2016, pages 1135–1141, https://doi.org/10.7205/milmed-d-15-00518 19. dai d, zheng b, yu z, et al. right stellate ganglion block improves learning and memory dysfunction and hippocampal injury in rats with sleep deprivation. bmc anesthesio l. 2021;21(1):272. published 2021 nov 8. doi: 10.1186/s12871-021-01486-4 20. lynch jh, muench pd, okiishi jc, means ge, mulvaney sw. behavioral health clinicians endorse stellate ganglion block as a valuable intervention in the treatment of trauma-related disorders. j investig med . 2021;69(5):989-993. doi: 10.1136/jim-2020-001693 21. fariba ka, saadabadi a. topiramate. [updated 2022 may 2]. in: statpearls [internet]. treasure island (fl): statpearls publishing; 2022 jan-. available from: https://www.ncbi.nlm.nih.gov/books/nbk554530/ 22. yeh ms, mari jj, costa mc, andreoli sb, bressan ra, mello mf. a double-blind randomized controlled trial to study the e�cacy of topiramate in a civilian sample of ptsd. cns neurosci ther . 2011;17(5):305-310. doi: 10.1111/j.1755-5949.2010.00188.x 23. henig i, zuckerman t. hematopoietic stem cell transplantation-50 years of evolution and future perspectives. rambam maimonides med j . 2014;5(4):e0028. published 2014 oct 29. doi: 10.5041/rmmj.10162 berkeley pharma tech journal of medicine | 58 abstract schizophrenia is a neurological disorder that affects an individual’s perception of themselves and the world around them. in recent years, research on schizophrenia has plateaued, with much of the focus placed on post-onset treatments, such as the use of antipsychotic medications and psychotherapy. although the disorder has been extensively studied, the prodromal stage—preceding the onset of psychosis—remains relatively underexplored. we assert in this scientific review that the prodromal stage offers significant promise in the prevention or strong reduction of schizophrenic symptoms and has been overlooked by previous research. emerging studies suggest that both pharmacological and psychological interventions targeting epigenetic markers may offer promising new approaches for early-stage treatment. preventing schizophrenia: insights from epigenetic research by: cheyenne fischer-zimmermann, khushi shah, laney seligson and noah perazzo berkeley pharma tech journal of medicine correspondence: cheyeneathome@gmail.com keywords: epienetics schizophrenia prodrome psychosis published july 31, 2025 full open access creative commons attribution license 4.0 1. introduction schizophrenia is a highly debilitating neurodevelopmental disorder that significantly affects an individual's mental health, cognition, and behavior. affecting nearly one percent of the u.s. population, the disorder currently has no known cure. most research to date has focused on alleviating the severity of symptoms following the onset and formal diagnosis. common symptoms of schizophrenia include hallucinations, delusions, and disorganized thinking, speech, and movement.1 1.1 background on schizophrenia globally, schizophrenia affects approximately 0.32% of the population—about 1 in 300 individuals. the disorder also exhibits a strong hereditary component, with genetic studies suggesting up to a 90% likelihood of heritability from one generation to the next. schizophrenic symptoms are generally categorized into two broad groups: positive and negative symptoms. both types are detrimental to an individual’s functioning and tend to worsen over time.1 positive symptoms are any change in behavior or thought that was not originally present. these include delusions, hallucinations, confused thinking, and confused speech. these symptoms can be managed by antipsychotic medications. on the contrary, negative symptoms are classified as a withdrawal from the world. individuals with schizophrenia experiencing these symptoms take no interest in the things they used to care about and essentially appear emotionless. examples of these symptoms include apathy, little emotion, poor attention and concentration, and depression. unlike positive symptoms, negative symptoms are more resistant to pharmacological treatment and often emerge months or even years before positive symptoms, making early detection and intervention particularly challenging.1 1.2 clinical overview of schizophrenia schizophrenia is a chronic disorder that affects both men and women equally. as of 2022, it is estimated to impact around 1 in 222 adults globally.1 the typical age of onset falls within early adulthood, generally berkeley pharma tech journal of medicine | 143 between the late teens and early thirties, with men tending to exhibit symptoms earlier than women. while childhood-onset schizophrenia is rare, cases have been documented in individuals as young as thirteen.2 schizophrenic symptoms can range in severity from mild to disabling and are typically classified into three categories: positive, negative, and cognitive. positive symptoms reflect the presence of abnormal thoughts or behaviors, such as auditory or visual hallucinations, persistent delusions, disorganized thinking, and incoherent speech. negative symptoms represent a reduction or absence of normal emotional and behavioral functions. these include diminished motivation, anhedonia (lack of pleasure), flat affect, limited vocabulary and speech detail, and social withdrawal. cognitive symptoms involve deficits in attention, memory, and executive functioning, which can impair decision-making, judgment, and problem-solving abilities.2 according to the diagnostic and statistical manual of mental disorders, fifth edition (dsm-5), a diagnosis of schizophrenia requires the presence of at least two of the following symptoms for a significant duration: delusions, hallucinations, disorganized speech, grossly disorganized or catatonic behavior, and negative symptoms. crucially, at least one of the symptoms must be delusions, hallucinations, or disorganized speech. additionally, other psychotic or schizotypal disorders must be ruled out in the diagnostic process.2 if left untreated, patients with schizophrenia may suffer psychological distress, relationship difficulties, employment issues, struggle with substance abuse, and self-harm. schizophrenia is further associated with a variety of comorbidities, the most common of which being comorbid substance abuse and depression in about half of individuals with the condition.3 additional psychiatric comorbidities include ptsd, ocd, and panic disorder. the addition of these psychiatric conditions can greatly hinder the proper diagnosis, and treatment of schizophrenia. this can be due to the possibility of an individual’s symptoms having crossover from both conditions, meaning it can be hard to pinpoint whether the individual has both conditions at the same time. furthermore, there could be different medicinal treatment methods for schizophrenia and an additional condition that may have conflicts.3 berkeley pharma tech journal of medicine | 144 the timeline of schizophrenia development has been insufficiently studied, largely due to the challenges in accurately identifying and tracking the early progression of symptoms. while a subset of individuals experience an acute onset of the disorder, approximately three out of four cases are preceded by a prodromal phase.4 this phase varies in duration but typically lasts between one and five years. during the prodrome, negative symptoms and some cognitive impairments often begin to emerge. in some cases, a first-episode psychosis (fep) may occur later in the prodromal period, following the appearance of mild psychotic symptoms. during this phase, individuals might experience subtle changes in perception, thought patterns, or behavior that can precede the onset of more severe psychotic symptoms, such as delusions or hallucinations. recognizing and understanding these early signs can be crucial for timely intervention and treatment. this is particularly relevant for clinical high-risk (chr) individuals, who exhibit early signs or symptoms indicating a heightened likelihood of developing a psychotic disorder, such as schizophrenia, in the future. several screening tools have been developed to identify chr individuals, with varying degrees of success. the early detection, intervention, and prevention of psychosis program (edippp) employed the structured interview for prodromal syndromes (sips) in conjunction with the scale of prodromal symptoms (sops) to detect early signs of psychosis with notable accuracy.5 the positive and negative syndrome scale (panss), the basel screening instrument for psychosis (bsip)6, and the comprehensive assessment of the at-risk mental state (caarms)7 have all proven to have valid and reliable methods of screening for individuals at risk for psychosis. however, despite the efficacy of these tools in detecting prodromal symptoms, their ability to accurately predict progression to a full psychotic disorder remains limited. this raises ethical and clinical concerns, as individuals labeled as chr may never develop psychosis yet still face stigmatization due to their risk status. although research on the prodromal phase remains limited, emerging evidence suggests that early intervention during this stage can significantly reduce the likelihood of transition to active psychosis.8 berkeley pharma tech journal of medicine | 145 1.3 environmental risk factors environmental risk factors play a significant role in the development of schizophrenia, often interacting with underlying genetic predispositions. key environmental contributors include prenatal complications, urban living conditions, and socioeconomic adversity. prenatal exposure to maternal infections—particularly viral infections such as influenza, rubella, and toxoplasmosis—has been linked to an increased risk of schizophrenia in offspring. furthermore, birth complications, including hypoxia (oxygen deprivation) and elevated maternal stress, may disrupt neurodevelopment and increase susceptibility to the disorder. growing up in urban environments is also associated with a heightened risk of schizophrenia compared to rural areas. contributing factors may include social isolation, environmental stress, pollution, and limited community cohesion, all of which are more prevalent in densely populated settings.8 additionally, socioeconomic disadvantage—characterized by poverty, low educational attainment, unemployment, and social marginalization—has consistently been linked to increased schizophrenia risk. other relevant factors include exposure to chronic stress, inadequate access to healthcare, and systemic social inequalities, all of which may exacerbate vulnerability to psychosis.8 1.4 genetic risk factors schizophrenia is highly hereditary, as several genetic risk factors have been implicated. individually unique genomic variations, otherwise known as copy number variations, have been linked to an increased risk of schizophrenia. these variations can arise through genomic mechanisms such as deletions and duplications of chromosomal segments. additionally, single nucleotide polymorphisms (snps) are genetic variations that can also lead to mutations that are associated with a higher risk of schizophrenia. among these, variations in the neuregulin 1 (nrg1) gene have garnered attention due to the gene's role in neural development, synaptic plasticity, and signaling pathways related to schizophrenia. moreover, genes encoding proteins involved in dopaminergic neurotransmission have been extensively studied, supporting the dopamine hypothesis of schizophrenia. this theory berkeley pharma tech journal of medicine | 146 posits that dysregulation of dopamine pathways contributes to the positive symptoms of the disorder, such as hallucinations and delusions.8 1.4.1 dopamine hypothesis the dopamine hypothesis is a long-standing theory suggesting that dysregulated dopamine activity contributes significantly to the pathophysiology of schizophrenia. research has particularly implicated dopamine d2 receptors in the subcortical and limbic regions of the brain, where hyperactivity is thought to underlie positive symptoms such as hallucinations and delusions. additionally, dopamine d1 receptors have been associated not only with positive symptoms but also with negative and cognitive manifestations of the disorder.8 the origins of the dopamine hypothesis stem from pharmacological evidence: antipsychotic medications such as haloperidol and clozapine reduce dopamine activity—especially by antagonizing d2 receptors—leading to symptom relief. conversely, substances that elevate dopamine levels, such as amphetamines and cocaine, can induce psychotic-like behaviors in otherwise healthy individuals, further supporting the link between dopamine dysregulation and psychosis.8 despite its foundational role, the dopamine hypothesis does not fully account for the wide spectrum of symptoms observed in schizophrenia. current understanding acknowledges that the disorder likely results from complex interactions between genetic and environmental factors, potentially affecting early brain development and leading to broader neurochemical and structural abnormalities beyond dopamine alone.8 2. background on epigenetics epigenetics is the study of heritable changes in gene expression that occur without alterations to a dna sequence itself. these changes are mediated by chemical modifications to dna or histone proteins, as well as through non-coding rna molecules. epigenetic mechanisms play a crucial role in regulating gene activity in response to environmental cues, developmental stages, and cellular differentiation. they contribute to diverse processes that function throughout an organism’s lifetime. epigenetics provides insights berkeley pharma tech journal of medicine | 147 into how external factors influence genetic activity, offering profound implications for understanding disease susceptibility, evolution, and even personalized medicine. by unraveling the complexity of epigenetic marks, scientists are gaining new perspectives on human biology and the intricate ways in which our experiences leave lasting imprints on our genetic code.9 epigenetics overall is a relatively new concept that is being put more into practice each year. the term “epigenetics” was created in the human biology setting by the embryologist conrad waddington in 1942. the biggest advancements in epigenetics and the understanding of them have occurred in the 21st century. this includes but is not limited to, a better understanding of dna methylation, further development of crispr/cas9 skills, more knowledge of histone modifications, and the expansion of knowledge of epigenetics with disease treatment.9 2.1 epigenetic mechanisms epigenetic mechanisms refer to reversible modifications that regulate gene expression without altering the underlying dna sequence. these mechanisms include dna methylation, histone modifications, and the regulation of gene activity by non-coding rnas. epigenetic modifications play crucial roles in various biological processes, including development, where they regulate gene expression during cell differentiation; differentiation, by influencing how stem cells mature into specific cell types; and disease, as seen in conditions like cancer, where abnormal dna methylation patterns can lead to the silencing of tumor suppressor genes. these modifications can also affect brain function and contribute to neurodevelopmental disorders such as schizophrenia. dysregulation of epigenetic mechanisms has been implicated in numerous diseases, including cancer, neurodevelopmental disorders, and autoimmune diseases.9 2.2 dna methylation dna methylation involves the enzymatic addition of methyl groups (–ch₃) to the cytosine residues of dna, typically at cpg islands, which are regions rich in cytosine and guanine nucleotides. this epigenetic mark generally acts to suppress gene transcription by reducing the accessibility of dna to transcription factors and other regulatory proteins. when berkeley pharma tech journal of medicine | 148 methylation occurs in promoter regions of genes, it often leads to transcriptional silencing, effectively turning the gene "off." aberrant dna methylation patterns—either hypermethylation or hypomethylation—are implicated in a wide range of diseases, including schizophrenia, where they may alter the expression of genes critical for brain development and function.9 2.1.1 histone modification histone modifications are chemical changes to the histone proteins around which dna is wrapped to form chromatin. these modifications affect how tightly or loosely dna is packaged and thereby influence gene accessibility and expression. acetylation causes the chromatin to relax by neutralizing the positive charge on histone proteins, which lessens their attraction to negatively charged dna and, therefore can assist with gene activation. phosphorylation acts in a similar capacity to altering the histone charges, but instead, it can make it either relaxed or compact. methylation is most commonly used to promote gene silencing by enhancing the interaction between the dna and histones. ubiquitination can play a dual role and could both loosen or compact the chromatin. histones are proteins around which dna is wrapped to form chromatin. they play a fundamental role in packaging and organizing dna into a compact and orderly structure which is essential for the regulation of gene expression, among other processes.9 additionally, non-coding rnas—including micrornas (mirnas) and long non-coding rnas (lncrnas)—contribute to epigenetic regulation by interacting with chromatin remodeling complexes and transcription factors. these rna species can modulate gene expression post-transcriptionally or by guiding epigenetic modifiers to specific genomic loci, further increasing the complexity of epigenetic regulation in health and disease.9 2.1.2 chromatin chromatin is the complex of dna and proteins found within the nucleus of cells helping to organize and compact dna within the nucleus into a manageable structure. specifically, chromatins condense into visible chromosomes during cell division. it is important to note that its structure berkeley pharma tech journal of medicine | 149 is highly dynamic and can be altered by various epigenetic mechanisms such as dna methylation, histone modifications, and non-coding rna regulation. these epigenetic modifications regulate gene expression by controlling the accessibility of dna to the transcriptional machinery. essentially, chromatin structure and its modifications are central to the field of epigenetics, playing critical roles in the regulation of gene expression, cellular differentiation, development, and disease.9 2.2 prior utilization of epigenetics in medical research although there has been slow progress in the implementation of epigenetic pharmaceuticals due to their low specificity, there are several medicinal drugs currently on the market for clinical use focused on modulating the epigenome. controlling gene expression pre-transcriptionally is desirable in treating a variety of diseases. epigenetic drugs targeting tumor and cancer growth have been approved by the fda for oncological use.9 histone deacetylase (hdac) inhibitors and dna methyltransferase (dnmt) inhibitors have also been suggested as epigenetic drugs that may be relevant in combating schizophrenia. histone deacetylases are enzymes that remove acetyl groups on histones. this allows chromatin to wrap more tightly around histone proteins and thus serve as an obstacle to restrict transcription, eventually permitting the chromatin structure to be more relaxed and accessible for transcription factors. therefore, histone deacetylases can help restore the normal expression of genes which can assist in alleviating symptoms such as cognitive impairments and memory deficits which are common symptoms of schizophrenia. dnmt contributes to the abnormal methylation of regions of dna. additional second and third-generation epigenetic drugs have explored additional enzyme inhibitors, however, ensuring low toxicity and high specificity has thus far proved to be a major hurdle. the use of dnmt inhibitors in schizophrenia treatment would assist in reversing abnormal dna methylation patterns that may have contributed to the onset of schizophrenia.9 berkeley pharma tech journal of medicine | 150 figure 1: chromatin modifications mediated by methylation and acetylation. this figure illustrates the sequence of mechanisms that cause methylation–the addition of a methyl group on the dna. it also shows the different effects of methylation in activating or inhibiting a gene’s expression. 3. previous exclusion of epigenetics in schizophrenia research although schizophrenia is a disease with a known high heritability, and thus offers strong promise for early prevention through epigenetic treatment, there has been little to no high-quality research examining the potential for the use of drugs such as hdac or dnmt inhibitors in responding to early psychotic symptoms. while the broader medical field has embraced the principle that prevention is the most effective form of treatment, and epigenetics has been extensively applied in oncology and other areas for identifying trauma-related and disease-specific biomarkers, the field of schizophrenia research has largely overlooked the potential of epigenetic approaches. this neglect may stem, in part, from the complex and multifactorial etiology of schizophrenia, which continues to resist simple genetic or biochemical explanations.9 berkeley pharma tech journal of medicine | 151 nevertheless, numerous well-characterized environmental and genetic risk factors—including prenatal infection, early-life stress, and gene variants—could serve as promising starting points for epigenetic investigation. given that epigenetic modifications are reversible, unlike permanent gene-editing interventions, they offer a more flexible and potentially safer therapeutic avenue. this reversibility enables dynamic modulation of gene expression in response to treatment, positioning epigenetics as a compelling but underutilized frontier in schizophrenia research.9 4. current models of interventional treatment the current treatment models for schizophrenia continue to rely predominantly on reactive interventions, initiated after the emergence of clinical symptoms, rather than on preemptive or preventive strategies. 4.1 psychological intervention psychological interventions, particularly psychosocial treatments, play an essential role in the comprehensive management of schizophrenia. psychosocial treatment refers to a broad category of therapeutic approaches that involve not only the patient but also family members, caregivers, and support networks. these interventions may include individual or group therapy, family counseling, peer support groups, and community-based rehabilitation programs.10 psychoeducation helps patients and their loved ones understand the condition and available treatment options. additionally, psychosocial rehabilitation teaches patients skills to manage daily activities.10 although therapy is often incorporated into treatment plans after a formal diagnosis, emerging evidence supports its early integration. cognitive behavioral therapy (cbt), in particular, has demonstrated effectiveness in reducing symptom severity, improving medication adherence, and enhancing overall psychosocial functioning. when combined with pharmacological treatment, cbt can contribute to more favorable long-term outcomes for individuals living with schizophrenia.10 berkeley pharma tech journal of medicine | 152 4.2 pharmacological intervention pharmacological treatment for schizophrenia has been largely guided by the dopamine hypothesis, which attributes positive symptoms—such as hallucinations and delusions—to dopaminergic hyperactivity, particularly in the mesolimbic pathway. this model led to the development and widespread use of first-generation (typical) antipsychotics, such as chlorpromazine, which alleviate positive symptoms by blocking dopamine d2 receptors.11, 12 however, this narrow pharmacological focus has several limitations. while effective for positive symptoms, d2 antagonists provide limited benefit for negative symptoms (e.g., lack of motivation, social withdrawal) and cognitive deficits, which often appear earlier and more persistently impair daily functioning. moreover, excessive d2 receptor blockade is associated with significant side effects, including extrapyramidal symptoms and tardive dyskinesia, which may hinder long-term adherence. current pharmacological strategies, therefore, risk overlooking core domains of the disorder that are critical to long-term recovery, highlighting the need for more holistic and targeted treatments—potentially including non-dopaminergic and epigenetic approaches.12 4.3 limitations and complications current schizophrenia treatment models often overemphasize the dopamine hypothesis, primarily targeting positive symptoms while overlooking the full spectrum of the disorder. additionally, treatment typically begins after the onset of schizophrenia, despite evidence that early intervention during the prodromal phase may significantly reduce symptom severity or prevent full disease progression. unfortunately, the prodromal stage remains under-researched and underutilized in preventative treatment approaches. psychological interventions, though beneficial, are commonly introduced only after initial symptom onset. yet research indicates that earlier implementation may yield better outcomes. these interventions are often combined with pharmacological treatment, but their effectiveness depends heavily on patient engagement. a major barrier to treatment is anosognosia—a condition in which individuals are unaware of their own illness, often due to delusions, hallucinations, or disorganized thinking. berkeley pharma tech journal of medicine | 153 anosognosia complicates medication adherence and therapy participation, as patients may not recognize their need for treatment. furthermore, individuals with schizophrenia often score low on conscientiousness, a personality trait linked to routine and self-discipline. as a result, they may struggle to follow structured treatment regimens, such as attending therapy or taking medication consistently. when treatment is delayed until later stages of illness, these challenges can become even more pronounced. there is also the risk of the affected individual developing anosognosia if treatment isn’t sought out before the full onset of symptoms. anosognosia affects individuals who do not believe they have a disability and therefore do not seek out treatment. they often actively avoid any assistance offered. therefore, the current models of interventional treatment will not be effective due to needing participation from the individual. potentially, their symptoms could have been first eased by preventative treatment, so they lessen their chances of developing anosognosia by the time of the onset of symptoms.13 cultural and socioeconomic barriers also contribute to delayed or avoided treatment. in some contexts, individuals may fear discrimination, stigmatization, or the loss of legal and social rights following a psychiatric diagnosis. in many countries, limited access to affordable mental health care—including psychiatric evaluations and follow-up services—remains a significant obstacle, particularly in low-income or rural areas.13 berkeley pharma tech journal of medicine | 154 figure 2: the consequences of anosognosia in schizophrenic individuals13 berkeley pharma tech journal of medicine | 155 5. epigenetic regulation in preventative treatment 5.1 psychological potential therapy and broad psychological intervention are already an established part of schizophrenic intervention plans; however, the connection between psychological stress and its epigenetic impact on an individual’s risk for developing schizophrenia has not been greatly explored as a means of early prevention. awareness about the transgenerational effects of prenatal maternal stress and the promotion of cognitive behavioral therapy in at-risk individuals are promising as early responses to pre-psychosis.13 5.1.1 prenatal sensitivity to stress prenatal maternal stress (pnms) is defined by a pregnant woman experiencing stress during her pregnancy whether it is physical or psychological. the fetus can sense stress stimuli through elevated levels of maternal stress hormones, such as cortisol, which can cross the placenta. this exposure may trigger physiological adaptations, potentially altering gene expression. during development, the fetus takes cues from its environment to optimize its growth and function, but excessive stress exposure can lead to long-term changes in brain structure and function. this can alter the expression of the fetus’ genes because, during development, the fetus takes cues from its environment on how best to develop.14 it has been shown that pnms in combination with other factors such as genetic disposition and childhood trauma increases the risk of schizophrenia. it has been shown that individually, pnms does not have a statistically significant effect on the fetus in developing schizophrenia. however, it is important to acknowledge pnms’ role in combination with other factors that influence schizophrenia to help identify high-risk individuals and be able to create an early intervention plan. pnms has also been shown to affect sleep, diet, and inflammation which could also contribute to schizophrenia development. identifying pnms’ role in affecting other factors that could contribute to schizophrenia, is crucial in order for more research to be conducted on pnms’ indirect effect on the development of schizophrenia.14 berkeley pharma tech journal of medicine | 156 5.1.2 individual therapy and psychoeducation cognitive behavioral therapy (cbt) is a widely practiced, evidence-based psychotherapy that targets maladaptive thoughts, emotions, and behaviors to improve overall mental health. when applied during the prodromal phase of schizophrenia, cbt has been shown to reduce the risk of transition to active psychosis, making it a promising early intervention strategy15. additionally, enrolling at-risk individuals in psychoeducational programs may help prevent behaviors linked to schizophrenia through epigenetic pathways.15 one notable comorbidity of schizophrenia is substance abuse, particularly the high prevalence of tobacco use among individuals with the disorder. a leading theory suggests that tobacco may serve a self-medicating function, offering short-term cognitive benefits for patients by interacting with epigenetic mechanisms. animal studies have demonstrated that nicotine exposure reduces levels of dna methyltransferase 1 (dnmt1) in telencephalic gabaergic neurons in the frontal cortex—a brain region critical to cognition. dnmt1 is an enzyme responsible for methylating cpg islands, which suppresses gene transcription and plays a vital role in maintaining epigenomic stability. notably, elevated dnmt1 levels have been observed in the gabaergic neurons of patients with schizophrenia16. this suggests that nicotine’s ability to lower dnmt1 expression may temporarily improve cognitive function, potentially explaining the high rates of tobacco use in this population.16 however, despite these cognitive effects, the long-term consequences of tobacco use remain profoundly harmful. integrating psychoeducation into early intervention programs could help at-risk individuals understand the biological basis of their susceptibility to tobacco use and inform more tailored, health-conscious treatment plans. this approach may enhance both the prevention and management of schizophrenia by addressing underlying epigenetic vulnerabilities.16 5.2 pharmacological potential as the majority of schizophrenia treatments have focused on treating the dopaminergic pathway and the subsequent positive symptoms, the berkeley pharma tech journal of medicine | 157 pharmacological potential of epigenetically adjusting abnormal levels of other neurotransmitter pathways has been overlooked. in the prodrome, where negative and cognitive symptoms take precedence over positive symptoms, epigenetically regulating other neurotransmitter pathways that are imbalanced through pharmaceuticals could prove to be a powerful preventative measure against full schizophrenic onset.17 figure 3: a chart depicting the contributors to each kind of schizophrenic symptom category. figure 3 shows the relevant neurotransmitters and their connections to each of the three symptom categories of schizophrenia. certain neurotransmitters play little to no role in contributing to certain symptoms, so this serves as a means of narrowing what is worth researching for treatments for specific symptoms. berkeley pharma tech journal of medicine | 158 5.2.1 dna methylation focused pharmacologic treatment multiple post-mortem studies of patients with schizophrenia have identified hypermethylation in the promoter regions of genes in gabaergic neurons, leading to reduced gaba neurotransmission—a key inhibitory process in the brain.17 this epigenetic suppression results in the downregulation of mrna for glutamic acid decarboxylase 67 (gad67) and reelin (reln), both of which are crucial for normal synaptic function. gad67, encoded by the gad1 gene, catalyzes the conversion of glutamate to gaba, and its reduced expression in the prefrontal and temporal cortices has been associated with cognitive impairments, particularly deficits in working memory and executive function, seen in individuals during the prodromal phase of schizophrenia.18 another promising molecular target is the neural cell adhesion molecule (ncam), a glycoprotein involved in axon growth, synaptic plasticity, and neuronal migration. ncam is found to be overexpressed in the prefrontal cortex and hippocampus of individuals with schizophrenia. in animal studies, the presence of anti-ncam1 autoantibodies has induced schizophrenia-like behaviors, suggesting that such antibodies could serve as a biomarker for the disease.18 the elevated levels of dna methyltransferase 1 (dnmt1) found in patients with schizophrenia can potentially be normalized through the use of dnmt inhibitors or dna demethylation inducers. dnmt inhibitors prevent the addition of methyl groups to dna, while demethylating agents remove existing methyl marks. both strategies may restore gad67 and reln expression, thereby enhancing gabaergic signaling and potentially ameliorating cognitive and synaptic deficits associated with the disorder. as epigenetic regulators, these agents offer a promising route to reverse transcriptional repression of key genes implicated in schizophrenia.18 5.2.2 histone modification-focused pharmacologic treatment previous epigenetic research has found that schizophrenic-like symptoms can be replicated by administering mitotoxin (a cytotoxic molecule that causes cell death by interfering with protein or dna synthesis) on day 17 of the embryonic prenatal stage. the mitotoxin administration had several berkeley pharma tech journal of medicine | 159 effects on various epigenetic mechanisms, including decreased methylation in certain promoter zones of cannabinoid receptor 1.19-23 using mam-e17 has replicated schizophrenic symptoms and has also been used as a method to examine the impact on histones. tri-methylated histone h3 at lysine (k) (h3k4me3) facilitates gene transcription by opening chromatin. the opening of chromatin, allows transcriptional mechanisms such as rna polymerase and transcription factors to have better access to the dna, which means the genes are more likely to be activated. mam-e17 experimentation has found lower levels of h3k4me3 near gad1 promoters, thereby hindering the production of gaba23. adjusting the expression of the setd1a gene may be an epigenetic point of interest, as this gene regulates the downstream levels of h3k4me3.23 figure 4: a chart detailing the inhibitory or excitatory effects of various pharmaceuticals targeting histones. this figure shows the way in which epigenetic therapeutics target transcription, when dna gets copied into rna. when transcription is inhibited and inactive, proteins are not produced, and the gene is not expressed. 5.3 epigenetic biomarkers in identifying individuals at risk for schizophrenia, an accurate and reliable screening method is necessary. h3k4me3 deficits near gad1 promoters have been noted in people with schizophrenia23. brain-derived neurotrophic berkeley pharma tech journal of medicine | 160 factor (bdnf) levels were found to be frequently elevated along with c-reactive protein (crp) elevation24. these may both contribute to lowered cognitive function or dysregulation in patients suffering from schizophrenia. unfortunately, there are very few identified reliable biomarkers for the prodromal period of schizophrenia. this may be influenced by the difficulty of enrolling prodromal individuals into invasive trials to reliably determine prodromal biomarkers. however, biomarkers already associated with schizophrenia are suggested to be prodromal biomarkers as well. a deletion of a piece of chromosome 22 (22q11.2), noted in about 1% of all individuals with schizophrenia, is also theorized to be a genetic risk factor and subsequent biomarker of schizophrenia.25 certain biomarkers also suggest the role of prenatal stress in the potential for developing schizophrenia. dnmt1 and tet1 were found to be higher in prenatally stressed mice and are promising early biomarkers as well.26 5.4 methods for screening for epigenetic abnormalities and markers since biomarkers are relatively unreliable for early detection of prodromal schizophrenia on their own, additional screening measures should be used. individuals with a reported familial history of schizophrenia may be recommended to partake in regular annual screenings to catch early identifiers due to the high heritability of schizophrenia. the medical field already often suggests annual check-ups and screenings such as pap smears during certain years of life. this is less common regarding mental health and neurodevelopmental disorder prevention, likely contributed to by significant historical stigma regarding psychiatric disorders and mental illness. recently, similar propositions for recommended mental health check-ups have been suggested by psychologists and psychiatrists alike, although little progress has been made as of yet.27 there are a variety of symptom screening tests to identify early prodromal symptoms. the structured interview for the prodromal syndrome (sips), paired with the scale of prodromal symptoms (sops), has been reliable and used in many clinical trials to determine individuals at high risk of developing psychosis. in sips, a clinician performs a lengthy interview with the patient examining their psychological, social, and health history. using berkeley pharma tech journal of medicine | 161 sops, the patient’s responses are ranked on a four-item scale based on positive, negative, disorganized, or general symptoms. individuals may be classified as being already psychotic, at high risk for psychosis, or not at risk for psychosis.27 the third syndrome diagnosis is the least common, but highly relevant to those with schizophrenia, being the genetic risk and deterioration prodromal syndrome (grds)27. individuals with a general decline or deterioration in their well-being who have a first-degree relative with a history of a psychotic disorder or who may have a schizotypal personality disorder may be diagnosed with grds.27 the positive and negative syndrome scale (panss) also utilizes a clinician-based interview and has been used since the 1980s to measure the severity of symptoms in affected individuals. it has use in screening for early psychosis as well, although its interview questions are less focused on identifying prodromal individuals similar to the basel screening instrument for psychosis (bsip).27 the comprehensive assessment of the at-risk mental state (caarms), is often used alongside sips/sops to determine prodromal individuals for early psychosis. it is reliable in tracking the development of psychosis and the onset of the fep. caarms includes many subscales that measure the well-being of various aspects of the individual’s life and includes many interview questions across seven domains of interest in order to appropriately gauge the individual’s risk. questions may explore the individual’s psychopathology, positive and negative symptoms, changes in their cognition, emotions, behavior, and physiology. caarms uses the social and occupational functioning assessment scale (sofas), which rates individuals similarly to sops and determines if they meet the criteria for full onset psychosis, bips, or aps.27 a major obstacle with prodromal screening is that they often require highly trained clinicians who can offer appropriate interviews. the interviews themselves can also be quite lengthy and inaccessible to individuals who have significant life obligations.27 berkeley pharma tech journal of medicine | 162 6. discussion developing pharmaceuticals that target the epigenome has historically proven to be a challenging endeavor, which has discouraged widespread research into their therapeutic potential. compounding this difficulty are ethical concerns related to privacy and genetic data, which have further hindered funding and institutional support for epigenetics research. as a result, the application of epigenetic therapeutics in schizophrenia treatment remains limited, despite substantial evidence supporting its promise. currently available epigenetic drugs often suffer from issues such as low target specificity, off-target effects, and high toxicity, reducing their viability in clinical settings. furthermore, many research institutions prioritize areas with higher academic prestige or more secure funding, creating a significant opportunity cost for pursuing epigenetics-based research. these factors have collectively slowed progress in exploring how epigenetic interventions could be used to prevent schizophrenia and other neurodevelopmental disorders. nonetheless, recent advances in developmental psychology and genetics have contributed to the reframing of the nature vs. nurture debate, now widely accepted as an interactionist model. the consensus among psychologists is that both genetic predispositions and environmental exposures influence developmental outcomes, including disease susceptibility. this evolving perspective has opened the door for greater exploration of epigenetics in medicine, as it accounts for how environmental factors can modulate gene expression without altering the dna sequence. in the case of schizophrenia, while heritability remains high, growing evidence indicates a significant interplay between epigenetic modifications and environmental influences in shaping the onset and progression of the disorder. as discussed in section 4.2, pharmacological intervention — particularly the use of antipsychotic medications—remains the most common treatment model for schizophrenia. these medications have proven effective in reducing positive symptoms such as hallucinations, delusions, and disorganized thinking by modulating dopaminergic activity 21. however, research also shows that pharmacological treatments are most effective when combined with psychological therapies, such as cognitive behavioral berkeley pharma tech journal of medicine | 163 therapy (cbt), which equip patients with coping strategies and cognitive restructuring tools. despite the therapeutic utility of antipsychotics, there is a growing need to shift the focus toward prevention. early-stage, non-pharmacological interventions may help delay symptom onset, reduce severity, and lessen reliance on medication. preventative treatment is particularly important for individuals who cannot tolerate antipsychotic medications due to severe side effects or treatment-resistant schizophrenia22. given the unpredictability of medication efficacy and tolerability, alternative or complementary preventative strategies are critical to improving outcomes. another vital consideration is the social stigma historically associated with schizophrenia and other psychotic disorders. being labeled as “at risk” for psychosis can significantly impact a person’s self-perception, social standing, and mental health, even if symptoms never manifest. stigma may lead to ostracization, bullying, or discrimination, particularly if peers or family members are unfamiliar with the disorder. these social pressures may contribute to the development of comorbid conditions, such as depression, anxiety, or panic disorders. therefore, any preventative framework for schizophrenia must be implemented with cultural sensitivity and clinical precision. accurate screening tools are essential to avoid false positives and mislabeling, which may do more harm than good. furthermore, public education is a key component of reducing stigma. increasing awareness and understanding of mental disorders among the general population can foster a more supportive and inclusive environment for those identified as at risk. building such a community is vital—not only for the effectiveness of treatment but also for maintaining morale, social integration, and overall mental well-being. 7. conclusion the field of schizophrenia research has historically underemphasized prevention, despite mounting evidence supporting the efficacy of standardized preventative measures, including psychoeducation, therapy, early detection, and epigenetic interventions. as a result, many individuals berkeley pharma tech journal of medicine | 164 continue to suffer from lifelong, severe symptoms that might have been mitigated—or even prevented—through early response strategies. stigma surrounding schizophrenia further delays diagnosis and treatment, exacerbating the burden on affected individuals. emerging research suggests that epigenetics may offer a powerful avenue for early intervention by enabling reversible modifications to gene expression. these interventions have the potential to reduce symptom severity or prevent the onset of psychosis altogether. to fully realize this potential, greater investment is needed from pharmaceutical and biotechnology companies, as well as from clinical psychologists, to explore the interplay between epigenetic regulation and schizophrenia pathophysiology. though still a developing field, epigenetics presents transformative possibilities not only for schizophrenia but also for a broad spectrum of conditions, including neurological disorders, autoimmune diseases, metabolic syndromes, and cardiovascular conditions. continued exploration and application of epigenetic science stand to advance both preventive medicine and precision therapeutics, reshaping the future of healthcare. berkeley pharma tech journal of medicine | 165 references 1. schizophrenia. world health organization. accessed may 3, 2024. https://www.who.int/news-room/fact-sheets/d etail/schizophrenia 2. substance abuse and mental health services administration. dsm-iv to dsm-5 schizophrenia comparison. in: impact of the dsm-iv to dsm-5 changes on the national survey on drug use and health. accessed 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targets in schizophrenia development and therapy. brain sci. berkeley pharma tech journal of medicine | 167 https://pubmed.ncbi.nlm.nih.gov/31284882/ https://pubmed.ncbi.nlm.nih.gov/18474208/ 2023;13(3):426. https://www.ncbi.nlm.nih.gov/pmc/articles/pm c10046502/ 27. loewy rl, pearson r, vinogradov s, bearden ce, cannon td. psychosis risk screening with the prodromal questionnaire–brief version (pq-b). schizophr res. 2011;129(1):42–46. https://www.ncbi.nlm.nih.gov/pmc/articles/pm c3113633/ berkeley pharma tech journal of medicine | 168 https://www.ncbi.nlm.nih.gov/pmc/articles/pmc10046502/ https://www.ncbi.nlm.nih.gov/pmc/articles/pmc10046502/ 5_fischer-zimmermann final ver_article 5.pdf zimmerman fp copy of zimmerman body paragraph.docx.docx (1).pdf copy of zimmerman reference page.pdf chang references berkeley pharma tech journal of medicine correspondence: bchang092@gmail.com keywords: pharmaceutical non-pharmaceutical medicine pain nanotechnology published july 31, 2025 full open access creative commons attribution license 4.0 abstract pain medicine has demonstrated its significance from brief surgical procedures to managing chronic illnesses. historically, a range of analgesic and opioid agents have been utilized as treatment options, and although effective to a certain extent, traditional approaches exhibit limitations. restricted by the constraints of technology, current approaches fall short in specificity and unintended effects. this review article delves into the evolving landscape of pain management, focusing on emerging treatment modalities within and beyond the pharmaceutical setting. non-pharmaceutical treatment modalities, including myofascial release, transcutaneous electrical nerve stimulation (tens), photobiomodulation, and passive treatment, such as capsaicin patches, introduce promising, non-invasive treatment options for those experiencing chronic pain. the advent of nanotechnology has also introduced an exciting new avenue that may herald a new era in personalized medicine. with many promising treatment modalities currently under investigation, this review article aims to examine and highlight the advancements in pain medicine. it is imperative to understand the potential remedies these novel modalities may have in a multitude of neurodegenerative disorders, cancer, and other diseases. innovations in pain management: from traditional analgesics to novel therapeutic modalities by: brandon chang, danica hergenroeder, glory gage and nathan tan 1. introduction pain serves as the body's protective response to external stimuli that pose potential harm. while essential for signaling bodily distress, persistent or chronic pain can persist even after the underlying issues have been resolved, significantly hindering the quality of life for those affected. 1.1 pathways of pain pain can be classified as either chronic (long-term) or acute pain (short-term). the fundamental mechanisms causing this pain can be categorized into two main groups: nociceptive and neuropathic pain1. nociceptive pain is caused by the activation of nociceptors in response to surface-level tissue damage, representing the most immediate level of pain perception. examples of nociceptive pain include minor injuries such as bruising, scratches, and cuts, as well as some deeper tissue damage like fasciitis, tendonitis, and fractures. nociceptive pain normally subsides with time, although various physical therapies and pharmacological remedies offer immediate pain alleviation. neuropathic pain arises from dysfunction or damage to the nervous system, specifically nerves responsible for transmitting pain signals to the spinal cord or brain. consequently, pain is perceived even in the absence of external stimuli. whether caused by injury, disease, or other factors, damage to nerve fibers disrupt their ability to transmit accurate signals from the periphery to the central nervous system. neuropathic pain syndromes can include post-stroke pain, spinal cord injury, multiple sclerosis (ms), and they have also been associated with other pain groups. currently, the first line of treatment for neuropathic pain is using pharmaceuticals which include tricyclic antidepressants (tca), gabapentin, anticonvulsants, and antidepressants2. in addition, there have been recent studies to show botulinum toxin has helped by inhibiting proinflammatory mediators3. recognizing the interconnectedness of nociceptive and neuropathic pain is vital for developing comprehensive approaches to pain management. acute pain typically subsides when the primary stimulus is removed, whereas chronic pain persists even after the primary stimulus is no longer present. berkeley pharma tech journal of medicine | 2 classifying pain in two broad categories requires some more specificity to obtain effective characterization. the different conditions can be broken down into different categories in terms of chronic pain. these groups are inflammatory, musculoskeletal, and psychological pain4. inflammatory pain is characterized by heightened sensitivity of nociceptive pain receptors due to inflammation5. hallmark symptoms of this type of pain include increased pain perception, which is the result of the higher influx of pain mediators that localize with the increase in blood flow. this heightened sensitivity can manifest as allodynia, where pain occurs in response to stimuli that would not normally induce pain, or hyperalgesia, an exaggerated response to painful stimuli. common examples of conditions associated with inflammatory pain include arthritis and various infections. surgery and opioid use can also trigger hyperalgesia by increasing the concentration of calcium and upregulating calcium-dependent kinases, serving as a trigger for the release of neurotransmitters. the next classification of pain to consider is musculoskeletal pain. it is highly prevalent in the general population, affecting approximately 37% of the united states population, with an economic burden of $635 billion per year6. musculoskeletal pain is discomfort that arises in muscles, bones, ligaments, tendons, and various supportive tissues in the body, as a result of defects of certain receptors. the most common agitators for this type of pain include overuse, poor posture, and various underlying medical conditions, leading to other ailments including both osteoarthritis and rheumatoid arthritis7. it remains closely connected with inflammatory pain, as damaging the tissue as a result of bad posture or overuse, may cause inflammation. during inflammation or tissue injury, damaged cells and immune cells release a variety of substances known as inflammatory mediators, such as bradykinin, nerve growth factor (ngf), prostaglandin e2 (pge2), pro-inflammatory cytokines [e.g. interleukin (il)-1β, il-6, tumor necrosis factor-α (tnf-α)] and chemokines (e.g. chemokine ligand 2). these inflammatory mediators act both directly on peripheral nociceptors, eliciting sensitization, and indirectly by promoting inflammation and the release of prostaglandins8. berkeley pharma tech journal of medicine | 3 psychological pain is relatively a new term and is still being discussed as to what the full criteria are. it is generally being discussed as relating to either psychological disorders such as severe depression or relating to physical pain like migraines and headaches9. in addition, due to the developing research on this type of pain, there is no definite way to treat this type of pain and no concrete definition of this pain yet. across various pain classifications, a shared pathway of pain reception emerges, initiated by a-delta and c-fibers – a-delta fibers emit rapid and localized signals, while c-fibers transmit non-localized pain signals10. these primary afferent axons serve as modalities for sensory perception, transducing pain signals from chemical to electrical within neurons. the electrical signals are transmitted along neuronal pathways to the central nervous system. upon reaching synapses, axons convert electrical signals into chemical signals, which are then reconverted into electrical signals upon reception by dendritic cells. the presence of myelinated sheaths serves to expedite conduction speed along these pathways. notably, ions such as calcium, potassium, and sodium play crucial roles in the transmission of these neurotransmitters. at a broader level, stimulatory molecules, including various growth factors, prostaglandins, and proteases are released upon contact with noxious stimuli. these molecules then access various channels, which facilitates the release of more pain mediators from nerve terminals, such as glutamate and interleukins. the signals are then sent to the central nervous system, specifically the spinal cord or dorsal horn, where they are interpreted in the primary sensory cortex. understanding the intricacies of this pain perception pathway is essential for elucidating mechanisms behind pain perception and advancing targeted therapeutic modalities. 1.2 physiology of pain having understood the pain reception mechanisms and pathways, the next step in designing an effective pain medicine is identifying proper targets. some of the mediators previously discussed, like glutamate, have significant interactions with nmda pain receptors, primarily situated in the central nervous system. this interaction heightens sensitivity to pain while berkeley pharma tech journal of medicine | 4 simultaneously reducing the effectiveness of opioid receptor agents. therefore, n-methyl-d-aspartate (nmda) receptor-targeted modalities involve much of the research in pain medicine11. another receptor of interest, dopamine, is classified into different types, each with distinct roles in pain modulation. d1 dopamine receptors, for instance, trigger the production of higher levels of camp (cyclic-adenosine-monophosphate), which in turn enhances pka (protein kinase a) activity. in contrast, d2 dopamine receptors inhibit camp and pka activity, exerting an opposing effect on d1 receptors. d1 receptors play a role in pain development and maintenance, while d2 receptors act to block pain signals. shifting our focus to opioids; there are three main types of opioid receptors: delta-opioid peptide/receptor (dop), kappa-opioid peptide/receptor (kop), and mu-opioid peptide/receptor (mop), each with their subtypes. in addition, there is a fourth receptor named nociception receptor (nop) that is considered a non-opioid receptor of the opioid receptor family. nop works similarly to the opioid receptors but does not bind or become affected by naloxone, the common opioid antagonist. due to its lack of response to naloxone, it is frequently questioned if the classification of the opioid receptor is correct. activation of dop receptors may lead to spinal analgesia and reduced gastric motility12. kop receptor activation also induces spinal analgesia but may increase urination and raise the risk of depression. mop receptor activation provides analgesic effects but can lead to respiratory depression, sedation, cardiovascular complications, and nausea. though these interactions serve as promising pain remedies, it is important to understand the unintended effects they may cause. drugs that are capable of capitalizing on these reception functionalities will be reviewed later in the document. 1.3 comorbidities pain is most commonly encountered in conjunction with its comorbidities, which is where most of the dedicated research efforts have been focused. neurodegenerative disorders are normally associated with loss of coordination, memory, and motor movements. understandably, treatment for these neurodegenerative disorders is focused on either restoring or retarding the progression of the disorders. however, patients report great levels of pain; thirty-eight to seventy-five percent in alzheimer's disease and berkeley pharma tech journal of medicine | 5 other dementias, forty to eighty-six percent in parkinson's disease and related disorders, and nineteen to eighty-five percent in motor neuron diseases report a high prevalence of pain13. scientists are currently struggling to classify the type of pain experienced by patients, due to poor diagnostics, difficulties in patient self-reporting, and complex neuronal pathways. this article will be analyzing the ongoing research on the modalities of pain in parkinson’s disease (pd), the most rapidly growing neurodegenerative disorder, which can be applied to other neurodegenerative disorders. in a study conducted by cattaneo et al., a classification of pain at four levels was reported. the first level of this grouping isolates pain into pd-related and non-pd-related (level 1: relationship to pd). this idea stems from correlation, as many patients with pd are elderly. in the subsequent level, the two aforementioned types of pain are classified as nociceptive or neuropathic; if it is not clear which of the two classifications ought to be picked, pain is included in the "miscellaneous" classification (level 2: the broader categorical system). in the third level, the different types of pain are categorized according to the categories: musculoskeletal, visceral, cutaneous, peripheral, or central (tier 3: broad type). in the last (fourth) level, different aspects from a clinical, pathogenetic, and therapeutic point of view are specified (tier 4: specific structures and pathology). another important classification method that is frequently used is that of ford, which considers five categories: musculoskeletal, dystonic, neuropathic/radicular, central/primary, and akathisia14. a significant proportion of diabetes patients experience chronic pain. musculoskeletal pain and neuropathy are the common symptoms of pain, stemming from alterations in the structural matrix and mechanical properties of periarticular connective tissues, owing to an unusual deposition of collagen15. these defects further lead to rheumatic problems such as the reduced mobility of joints, stiff hand and carpal tunnel syndromes, shoulder capsulitis, and tenosynovitis16. these trends may reflect correlation rather than causation. but according to a study sampling populations with nondiabetics, diabetics, and prediabetics, the results showed that, compared to prediabetic and nondiabetic individuals, diabetic subjects have a higher prevalence of lower limb pain (11.1%), back pain (8.9%), abdominal pain (6.7%), and neck pain (4.4%). a chi-squared test confirmed that diabetic and prediabetic patients had a significantly higher berkeley pharma tech journal of medicine | 6 prevalence of chronic pain17. if this problem is not properly dealt with, chronic pain may spiral into larger health problems, causing a greater disruption in the lives of many. cancer-related chronic pain affects about 40-70% of patients with cancer diagnostics and 33-40% of long-term cancer survivors suffer from chronic pain18. there exist numerous sources of pain, including the tumor itself, chemotherapeutic agents, and surgical interventions. all of these factors can not only cause physical pain, but also significant psychological pain. delivery of therapeutics is currently a hot area of research in cancer research and will be covered in this article. 2. previous treatment modalities 2.1 nonopioid drugs popular pharmaceutical drugs fall into three primary categories: nonsteroidal anti-inflammatory drugs (nsaids), acetaminophen, and adjuvant analgesics. nsaids work primarily by inhibiting the activity of the cox-1 and cox-2 enzymes, which are subsets of the calcium-dependent kinases mentioned earlier19. this inhibition leads to reduced prostaglandin expression, resulting in analgesic effects and decreased inflammation. however, a significant drawback of nsaids is the increased risk of cardiovascular issues and gastrointestinal complications. nsaids are responsible for 30% of adverse drug reactions such as strokes, heart attacks, renal damage, and bleeding. common examples of nsaids include aspirin, ibuprofen, and naproxen. the other common drug, acetaminophen, commonly recognized as tylenol, operates similarly to nsaids. however, unlike nsaids, it does not interfere with the cox pathway in the peripheral nervous system; instead, its effects are limited to the central nervous system. as a result, tylenol effectively alleviates pain perception and reduces fever but lacks efficacy in diminishing inflammation. when used in appropriate doses, tylenol is generally considered safe. however, prolonged or excessive usage can lead to liver damage, occasionally fatal. adjuvant analgesics include most non-opioid drugs that are not classified as acetaminophen and nsaids. adjuvant analgesics refer to medicine that berkeley pharma tech journal of medicine | 7 was originally engineered for purposes other than pain management; popular agents include antidepressants and anticonvulsants. because of the nervous system’s interconnectedness, drugs that operate in the nervous system for conditions such as depression or convulsions affect pathways involved in pain reception. specifically, their effects on the spinal cord and dorsal horn overlap. antidepressants suppress neuropathic pain by enhancing noradrenaline influx in the spinal cord20. this neurotransmitter binds to α2-adrenergic receptors, initiating downstream signaling cascades that inhibit calcium channel activity in the dorsal horn and dampen neurotransmitter transmission, thereby inhibiting hyperalgesia and allodynia. additionally, antidepressants hold therapeutic effects on the locus coeruleus, a brain region rich in noradrenergic nerve cells. following injury, the locus coeruleus releases noradrenaline in response to noxious stimuli, contributing to nociceptive analgesia. however, with prolonged injury, this response diminishes, compromising the descending noradrenergic inhibitory system. antidepressants intervene by restoring locus coeruleus function and increasing noradrenaline levels21. moreover, they elevate levels of brain-derived neurotrophic factor (bdnf), crucial for ampa receptor-mediated activation in the locus coeruleus, further promoting analgesic effects. another common class of adjuvant analgesics, anticonvulsants, exert their analgesic effects by modulating neurotransmission to prevent seizure activity. the drug acts on the brain to serve its primary pharmaceutical purpose, which also inhibits neurotransmission in pain reception. though the listed adjuvant analgesics manage pain well, they do have side effects; headaches, decreased alertness, and bleeding are just some of the problems that can arise. 2.2 opioid drugs the most hazardous class of medications is opioids. among the commonly known opioids are morphine, oxycodone, codeine, dihydrocodeine, and loperamide, some of which are available over the counter without prescriptions. despite the wide array of opioid-based drugs available, they all share a common mechanism of action, targeting the mu, kappa, and delta receptors in the nervous system. in the context of pain medicine, the mu berkeley pharma tech journal of medicine | 8 receptor is of interest for its ability to elicit analgesic effects. opioids bind to the mu receptor, decreasing the excitability of nociceptors and the release of pro-inflammatory peptides22. however, this is not all the drug does. the kappa receptor induces the commonly known drug effects, like euphoria and depression of bodily function. the addictiveness of the euphoria makes this combination especially dangerous. as depressants, opioids effectively slow down bodily functions, including respiration, which can lead to respiratory depression and neuronal damage. the unreliability of this drug makes it undesirable as a pain medicine. nanoparticles, which will be discussed further on, hold promise for providing synergistic effects with opioids, making them safer to use. additionally, it is important to note that many of the commonly used opioids do not act on all of the opioid receptors. although the majority of them act mainly on the mu receptor, they do not act on the other two receptors. for example, oxycodone, codeine, and loperamide are all opioids that are agonists on the mu receptor. morphine affects each of the receptors but binds mainly to the mu receptor. when morphine binds with these receptors it leads to an activation of the descending inhibitory pathways of the central nervous system resulting in reduced nociceptive signal transmission23. although morphine has been shown to decrease blood pressure and heart rate, it has some unfavorable effects such as constipation, nausea, depression, and vomiting. pentazocine is not as commonly used at present but is a synthetic opioid used as an agonist on the kappa (κ) receptor24. pentazocine does have the chance of common adverse effects of opioids such as nausea, respiratory depression, vomiting, and constipation. the final opioid receptor, the delta receptor, is known to associate with the drug named buprenorphine. buprenorphine is also known to be a partial agonist to mu receptors and a weak antagonist at kappa receptors, but its effects on delta receptors are also weak . it is a synthetic opioid that is used for pain treatment and opioid use disorders (oud). it acts differently from normal opioids by exhibiting slow dissociation kinetics, allowing for fewer adverse effects. berkeley pharma tech journal of medicine | 9 though current drugs on the market have proven their use over the years, there are many avenues of innovation in the field of medicine that can elevate the current drugs on the market as well as provide alternate approaches to pain remedies. 3. pharmaceutical remedies advancements 3.1 nanoparticles in contrast to the pharmaceutical remedies mentioned earlier, nanoparticles demonstrate therapeutic effects stemming from the emerging field of nanotechnology. the wide attributes and modifications for nanoparticles enable treatment for a diverse spectrum of diseases, as well as an avenue to the next age of precision medicine. this review article will explore the various avenues and types of nanoparticles that are relevant in a pharmaceutical context. 3.2 zno & mgo np starting with zinc oxide, its wide range of capabilities places it at the forefront of scientific research. zinc, recognized as an essential trace element, plays a crucial role in pain perception modulation, proving its ability to analgesia through various pathways25. zinc ions have been shown to inhibit the release of glutamate, a previously mentioned neurotransmitter by producing more gaba, an inhibitory neurotransmitter. furthermore, zinc ions noncompetitively inhibit the nmda receptor, which diminishes the activation of the central nervous system, the perception pathway for pain. what differentiates the nanoparticle from the microparticle form is the penetration efficiency; nanoparticles are three orders of magnitude smaller, contributing to their greater motility through various tissues26. as a result, smaller doses of zinc oxide are required to induce saturation. furthermore, zinc oxide demonstrates photocatalytic capabilities, due to the high reactivity of surface oxygen atoms. it has proven to oxidize stimulants on peripheral receptors, and various tumors in cancer therapy. magnesium oxide serves a similar role, as it is another vital cation in the human body that offers promising avenues for pain relief. by preventing the calcium ions from entering cells through blocking nmda receptors, it serves as another berkeley pharma tech journal of medicine | 10 analgesic agent. one study27 was conducted to test this reaction on mice. varying doses of zinc oxide nanoparticles were injected into rats (0, 1, 5, 10, 20 mg/kg) and the latency in pain was measured. as shown in figure 1, the latency period was longest at about 10 mg/kg, meaning pain was delayed for about 90 minutes. just like zinc, magnesium reduces the need for relaxant drugs, intraoperative anesthetics, and morphine. figure 1. results from zinc oxide nanoparticles on rats 3.3 magnetite nanoparticles magnetic metal oxides represent a distinct class of nanoparticles. ferromagnetic compounds, including magnetite (fe3o4), have shown ferromagnetism, meaning that they readily interact with applied external magnetic fields. similar to zinc and magnesium oxide, magnetite has demonstrated potential in directly alleviating pain. therapeutic effects that the nanoparticle possesses were revealed by a study that concluded diminished macrophage activity and diminished expression of inflammatory biomarkers with the treatment of ferromagnetic particles. however, the most intriguing aspect of magnetite is unveiled by its name: magnetism. it has been widely used as a nanocarrier because of its capability to control the vesicle externally through magnetic fields. the strong interaction between the nanoparticle and the external magnetic fields enhances the targeting efficacy of this method, contributing to precise delivery. just like zinc oxide nanoparticles, the nanoparticles themselves hold therapeutic effects. berkeley pharma tech journal of medicine | 11 in a mice study28, iron oxide nanoparticles were injected, and the prevalence of biomarkers was checked. these biomarkers include reactive oxygen species, cd68, and mpo (a neutrophil marker). pain levels were also measured through von frey filaments, a band that measures mice’s paw withdrawal response, an indicator of pain perception. after the injection of treatment into inflamed tissue, there were signs of suppressed expression of the previously expressed proinflammatory biomarkers. further taking advantage of the magnetic properties of magnetite, they are currently being studied in the field of nanoparticle-induced magnetic hyperthermia. bioactive glass doped with magnetite nanoparticles can generate heat in the presence of an alternating magnetic field29. when the magnetic field alternates, the magnetism of the nanoparticle also alternates, but at a delay, a phenomenon called magnetic hysteresis. as a result, heat is capable of being generated in a highly specific area. 3.4 gold nanoparticles another metallic nanoparticle worth covering is gold nanoparticles. currently, at the forefront of cancer research, gold nanoparticles boast a myriad of conformations and high membrane customizability. one of its most notable features is the surface plasmon resonance (spr), a phenomenon that occurs when electron oscillatory frequencies align with the frequency of incoming light, allowing the generation of a magnetic field30. though showing great promise, its toxicity must be considered when evaluating its efficacy. gold nanoparticles can alter dna function and bioaccumulate. depending on the intended application, coatings added onto these nanoparticles, such as folic acid, polyacrylamide, polyvinylpyrrolidone, and polyacrylic acid can serve to either mask their cytotoxicity or exploit it when targeting cancer cells for treatment. for instance, in a study31 that tested the absorption of gold nanoparticles, antibody-modified gold nanoparticles demonstrated a 600% increased retention to tumor cells, allowing greater release of cytotoxicity into the cell. looking into the therapies driven by gold nanoparticles, photothermal and radiofrequency therapy stand as promising candidates. as previously mentioned, the ability of nanoparticles to absorb and scatter electromagnetic radiation has captured significant interest in the realm of berkeley pharma tech journal of medicine | 12 photothermal therapy. the application of this phenomenon has been studied in localized hyperthermia, as well as radiofrequency therapy, as its great specificity and morphology control have contributed to great levels of localization. its impact has also been seen in neurodegenerative disorders, such as parkinson’s disease, multiple sclerosis, and lou gehrig’s disease. the commonality amongst these painful illnesses includes the decline of the nad+/nadh ratio in the brain. reversing this energy deficit promises slower neurodegenerative decline and even partial recovery. in the phase 2 clinical trial conducted in 202432, gold nanoparticles demonstrated their capability to improve this nad+/nadh balance. 11 participants with relapsing multiple sclerosis and 13 with parkinson’s disease were the subjects of this clinical trial and took cnm-au8 (the gold nanoparticle-based therapeutic agent) for 12 weeks. the result included an average of 10.4% improvement in nad+/nadh ratios. further validated testing was performed to test the motor skills of these patients and improved motor skills were demonstrated by the patients. without diagnosing any adverse effects of this treatment, the gold nanoparticles have cemented themselves as a promising candidate for neurodegenerative disorders. the next class of nanoparticles this review article will be covering includes nanocarriers, one of the most active areas of research in nanotechnology. the capability to fine-tune the solubility and mimic human cells grants them easy entry into specific cells, while their ability to vary delivery mechanisms based on environmental factors ensures controlled and precise drug release. by doping the nanoparticles with various membranes or certain receptors, there are many ways to specify the targeted location of the cell. beyond their ability to precisely target cells, their modifications enhance drug stability and cellular compatibility. with high modification potential, nanocarriers hold immense promise for personalized medicine and theranostic applications. we will be taking a look at a few nanocarriers that are currently being researched in the field of nanotechnology33. 3.5 liposomes liposomes are mainly composed of cholesterols and phospholipids, boasting great biocompatibility, biodegradability, high loading capacity, and permeability. over the past decades, liposomes have gradually been berkeley pharma tech journal of medicine | 13 improved to localize more precisely, with improved responsiveness to ph and temperature as conditions, though they still encounter issues. some of its applications have been heavily explored in co-morbidities with chronic pain, including cancer. in a clinical study34 conducted in 2022 on the efficacy of liposomes in children’s anticancer therapy 74 trials were conducted with 70 being intervention trials and 4 being observational trials, with 28.6% being in phase 3 trials, 30.0% in phase 1 trials, and 24.3% in phase 2 trials. in total, 17 liposomal drugs for 123 types of cancer were investigated, consisting mainly of organic chemicals. of these cancers, the highest proportion was leukemia (15.4%), followed by lymphoma (9.8%) and ovarian cancer (8.9%). it is worth noting that accurate statistical masking is required to produce accurate results, and only 30% of the data collected was properly masked. ultimately, many challenges were found to be associated with this treatment modality. leakage, uncontrollable drug release, instability in storage, and difficulty in drug loading limited the use of liposomes in cancer therapy. however, they did demonstrate high loading capacity for hydrophobic substances. ultimately, liposomes are still an active area of research for scientists and possess many challenges that must be overcome. 3.6 solid lipid carriers and nanostructured lipid carriers solid lipid nanocarriers have been around for decades and have been extensively researched. many versions of this nanoparticle have been produced. a solid lipid nanocarrier, at its core, is a solid lipid (lipid that is solid at room temperature) that is commonly composed of triglycerides, fatty acids, waxes, and phospholipids35. these lipids form a matrix of crystalline structure that encapsulates the drug to be delivered. surrounding this solid lipid nanocarrier core exists a surfactant layer. in chemistry, surfactants decrease the surface tension between adjacent surfaces and are amphiphiles. the hydrophilic portion of the surfactant faces outward toward the aqueous environment, while the lipophilic portion interacts with the lipid core, which acts as a stabilizer for the solid lipid core, preventing aggregation or coalescence. the first generation of this nanocarrier was used as a vesicle to deliver topical anti-inflammatory drugs. great success was found in the controlled release of the drug, allowing for berkeley pharma tech journal of medicine | 14 bioaccumulation in the stratum corneum, the outermost layer in the epidermis. it has also served as an effective nanocarrier for various pain drugs. epalrestat36, a treatment for streptozotocin-induced diabetic neuropathic pain, was delivered with solid lipid nanocarriers, and the effect was measured in a rat-modeled study. the study tested the efficacy of epalrestat encapsulated nanoparticles at varying concentrations (0.25, 0.50, 1, and 5 mg/kg) on streptozotocin-injected rats. the various parameters linked with peripheral neuropathy were subsequently measured. the results were promising. solid lipid nanoparticles demonstrated a large encapsulation efficacy at 88 ± 2%. analyzing the results, the tail-flick latency time and hot plate response time, improved linearly with greater doses of solid lipid nanoparticles. figure 2. the formation of solid lipid nanocarriers. the second generation of this nanocarrier saw the advent of nanostructured lipid carriers (nlcs). the main differentiating factor between these nanocarriers and solid lipid nanocarriers lies in the liquidized lipid core37. the oils in the core function by decreasing the crystallinity of the lipid core, preventing drug expulsion from the matrix, enhancing drug loading capacity, and stability, and ensuring long-term physical and chemical stability. as a result, its loading capacity saw an increase, and its encapsulation rate rose to 99.5%. now solving many of the problems that plagued the solid lipid nanocarrier, one of its applications was the delivery enhancement of topical therapeutic agents like butyl-substituted benzocaine analog butamben, a local anesthetic. it proved to decrease berkeley pharma tech journal of medicine | 15 toxicity and improve analgesic effects. building off of all the applications of solid lipid nanoparticles, it has been explored in the delivery of gene therapy, chemotherapy, and other treatments. 3.7 hydrogels hydrogels in their simplest form are networks of water-absorbing polymers. some commonly used polymers include polyethylene glycol (peg), poly(acrylic acid) (paa), polyvinyl alcohol (pva), hydroxypropyl methylcellulose (hpmc), alginate, collagen, and chitosan; some of these hydrophilic polymers are naturally forming polymers. in the world of anesthetics, the brief lifetime of the anesthetics and toxicity has been at the center of focus when attempting to engineer effective therapeutics. hydrogels have demonstrated great promise in combination therapy with traditional anesthetics in blocking peripheral nerve blocks. a study38 tested the logistics of polycaprolactone (pcl) hydrogels in delivering topical agents. the study determined that drugs enriched in a pcl core and chitosan shell demonstrated a steady release of drugs. they also tried determining the optimal ropivacaine (rpv) and dexamethasone (dem) nanoparticle composition and determined that rpv/dem ch-pcl nps-3 delivered the best effects, the mix with the largest nanoparticle size. hydrogels have also been extensively researched in the field of oncology, as a result of their impressive biodegradability and biocompatibility39. specifically, it has shown great promise in the field of immunotherapy. with difficulties arising in the delivery of monoclonal antibodies (immune checkpoint inhibitors), hydrogels serve as the perfect transport vesicle. (li et al., 2023)40 reported that the alginate hydrogel with protoporphyrin ix (ppix)-modified iron oxide (fe3o4) nanoparticles demonstrated high control over the performance of photodynamic and chemodynamic therapies41. another study conducted by li et al., 2021) demonstrated how a hybrid peptide hydrogel of melittin (rada-320, titanium, and doxorubicin (dox) controlled the release of therapeutics-that activated immune cells, depleting the m2-like tumor-associated macrophages, effectively reshaping the immunosuppressive tumor microenvironment42. altering the berkeley pharma tech journal of medicine | 16 microenvironment of melanoma gives promise to the capability to alter other tumor microenvironments present in other cancers. figure 3. hydrogel into a cell 3.8 micelles in its simplest form, micelles are aggregates of amphiphilic molecules that have self-assembled into a spherical geometry. they are formed by submerging common amphiphiles, like surfactants, in an aqueous solution, where the hydrophilic heads shield the hydrophobic tails from the polar aqueous environment. this structure mirrors that of the phospholipid bilayer of cells, as there exists a hydrophilic head with hydrophobic tails. when applied to the field of therapeutics development, micelles can encapsulate drugs in their inner compartment. what differentiates micelles from other nanocarriers is their minute size, being sub 50 nanometers in size. combined with their cell-like coatings, these particles have accessibility to deeper tissue locations, increasing the range of drug delivery distance. micelles are also easily manufactured, increasing their accessibility relative to the previously discussed nanocarriers. though there are many benefits to this nanocarrier, a major drawback includes instability in fluctuating environments. as a result, it may disintegrate in variable environments, like the bloodstream, thereby reducing therapeutic effectiveness of the nanocarrier. current research efforts are investigating the possibility of crosslinking various structures to prevent premature release of therapeutic agents43. like any other nanocarriers, micelles are currently being investigated for their role in delivering therapeutic agents to tumors. their small size allows them to permeate well through certain systems, like the kidney, and evade elimination by the liver or spleen. their minute size allows precise localization in certain tissues, improving therapeutics delivery. berkeley pharma tech journal of medicine | 17 by modifying the surface of micelles with ligands that can specifically recognize and bind to receptors overexpressed on the tumor cells, a targeting modality is established. this is evident in approved polymeric nano micelle-based drugs on the market for cancer treatment44. figure 4. loading of a micellar nanocarrier genexol-pm, a micelle-based drug, has been approved for the treatment of breast cancer, non-small-cell lung cancer, and ovarian cancer. it is also undergoing clinical trials for a variety of cancers. in a phase ii study of genexol-pm in patients with locally advanced or metastatic pancreatic cancer, common side effects were comparable to those of taxol in a dose of 300 mg/m2 every three weeks. the general reaction rate was 6.7%, with 1 patient in complete reaction and 2 patients in halfway reactions, and the infectious prevention rate was 60%. the median pfs was 2.8 months, and the median overall survival was 6.5 months. neutropenia (40.0%), fatigue (17.8%), infection (13.3%), dehydration (13.3%), neuropathy (13.3%), and abdominal pain (11.1%) were the most common grade 3 toxicities. genexol-pm had sufficient antitumor activity as second-line chemotherapy in patients with urothelial cancer after gemcitabine-platinum failure in a phase ii study in 37 patients with advanced urothelial cancer who had previously received gemcitabine and platinum combination chemotherapy45. of 34 evaluable patients, the general reaction rate was 21%, with 1 patient in complete reaction. the median pfs was 2.7 months, and the median overall survival was 6.5 months. grade 3/4 non-hematologic poison levels included neutropenia (14.7%) and contamination (5.9%). hematologic toxicities of grade 3/4 were observed in only one patient. the low rates of high-grade toxicities give promising results prospects for micelles in the field of oncology. 3.9 dendrimers berkeley pharma tech journal of medicine | 18 dendrimers are large branched molecules that are composed of generation zero (the core molecule) with branched polymer chains synthesized onto the central molecule. drugs can be integrated into the various branches of this molecule through reactions. their differentiating characteristics from other nanocarriers include hyperbranching, well-defined spherical structure, and high compatibility with biological systems. beyond operating as a nanocarrier, dendrimers can also be used to enhance drug solubility and as a stabilizing agent for various drugs. these two attributes are synergized when finding applications of dendrimers. the most well-known dendrimers incorporate poly(amidoamine) (pamam) dendrimers, polypropylene dendrimers, polyesters, and triazines. dendrimers can be conjugated to various molecules in addition to the functional groups that are found on their external surface. dendrimers are a productive solvency enhancer of nsaids, which is enhanced by weak hydrogen bonds provided by branch units, and electrostatic action from surface groups. koc et al. investigated the solubility of pamam dendritic macromolecules loaded with nsaids (ketoprofen, ibuprofen, and diflunisal) in a buffer solution46. the outcomes showed that the solvency improvement execution of hydrophobic medications in pamam dendritic atoms was several times greater than ethylenediamine-cored pamam dendritic macromolecules because of their original polypropylene oxide cores. as a result, dendrimer carrier optimization and application have significant potential for pain-induced inflammation treatment. 3.10 poly(lactic-co-glycolic acid) nps (plga) nanoparticles plga nanoparticles are synthesized from lactic and glycolic acids, with customizability deriving from the myriad of lactic and glycolic acid concentrations. the differentiating factor of this nanocarrier lies in its simple preparation, biodegradability, and high drug-loading capacity. it also demonstrates great control over drug release. a study conducted on the release of ketamine through plga nanoparticles was conducted by han et al; biocompatible and biodegradable ketamine-loaded polyethylene-glycol plga nanoparticles were tested in pain treatment in the context of pain medicine47. the results demonstrated a 41.8% drug loading, with preservation of the drug for up to 7 days and controlled release for up to 21 berkeley pharma tech journal of medicine | 19 days. furthermore, the decay of ketamine below the lower limit of quantitation rose to ~103 hours and ~80 hours with plga in comparison to only 24 hours when injected intravenously without a nanocarrier. further optimization of drug distribution in the polymer matrix as well as plga composition is being performed to better formulate drugs with broader and more effective applications. 3.11 other nanocarriers there remains a multitude of nanocarriers currently being explored for their therapeutic applications. to list a few, thermogels, emulgels, various films, and silicon nanoparticles are currently being researched. it is important to note the limitations when it comes to nanotechnology, including stability, cytotoxicity, and solubility, as they pose great barriers to delivering many drugs. 4. pharmaceutical remedies advancements 4.1 myofascial release myofascial release is an external method that refers to the manual application of a low-load, long-duration stretch of the myofascial complex. its objective is to restore the ideal length of the fascial tissue to decrease perceived pain and enhance mobility. this method targets fascia tissue and other associated soft tissues. fascia tissue is considered connective tissue and it is not just the muscular aspect but the connective tissue that also surrounds organs48. overall fascia tissue goes under a lot of stress from the body and may become increasingly rigid over time. in addition, fascia tissue can be elastic and go in and out of the normal shape, but over time the overuse may gradually deform the tissue, allowing the rigidness to take place49. tissue rigidity or deformity may contribute pain for an individual. the method of myofascial release is to target those tightened points of fascia tissue and either the individual or clinician will add pressure to the pain point and hold the pressure until there is no longer any resistance from the tissue50. when myofascial release is used by the individual, the tools commonly used are a foam roller and roller massager51. variety of motions and movements used with each of these tools target specific painful areas. berkeley pharma tech journal of medicine | 20 figure 5. fascia tissue normal vs abnormal shape a clinical, double-blind, parallel sham-controlled trial with a balanced randomization trial performed in 2021 investigated the magnitude of myofascial release’s relief on chronic lower back pain (clbp)52. in this trial, fifty-four participants between the ages of eighteen and sixty years old diagnosed with nonspecific clbp for at least three months were chosen. these participants were then divided into two groups: myofascial release (n=27) and sham (n=27) with a random number generator. patients’ pain was measured through the short form mcgill pain questionnaire (sf-mpq) and visual analog scale (vas) and disability was measured with the roland morris questionnaire. the trial showed a significant improvement in pain, as displayed with the sf-mpq compared to the sham group while showing no significant difference in vas. 4.2 tens transcutaneous electrical nerve stimulation (tens) is an external non-invasive treatment used to treat neuropathic and nociceptive pain53. tens, in practice, is an inexpensive mode of electrically stimulating targeted tissues that excite a neuronal complex. the most common form of tens can be found as a small battery-run device that can be self-administered, with the dosage of electrical currents being delivered through electrical pads that are attached to the skin. in addition, there are three different techniques of tens that are used with this type of device to evoke different nerve fibers to treat different types of pain. the three different techniques are conventional tens, acupuncture tens, and intense tens. berkeley pharma tech journal of medicine | 21 conventional tens is the most commonly used technique and is characterized by a low-intensity and high-frequency stimulation54. the overall purpose of this approach is to target afferents (a-beta fibers) that are connected to the central nervous system and are related to pain. conventional tens stimulation affects a broader neural area and can inhibit nociceptive transmission55. acupuncture tens (al-tens) is considered a high-intensity low frequency technique used for hyperstimulation. al-tens targets a smaller area and aims for higher threshold peripheral afferents (a-delta). during this technique, patients may have painless muscle twitches because of the placement of the electrode pads. intense tens is viewed as a high intensity high frequency technique that is used as a “counter-irritant” to stop nociceptive transmission in the peripheral nerves before it reaches the central nervous system. this technique is different compared to the other two because this method can only be used for a small amount of time compared to the conventional method, which can be used as long as the patient desires. the overall method of tens affects the surrounding area by reducing the transmission of nociceptive neurons. tens can also activate extra-segmental areas including the midbrain and the medulla. the activation in the midbrain and medulla complexes then triggers descending inhibitory systems which reduces hyperalgesia, an increased sensitivity to pain, or an extreme response to pain. although the main intent of tens is through electrical stimulation, some neurochemicals help further the effects of tens. low frequency stimulation has demonstrated the involvement of the mu opioid receptor and high frequency has displayed the use of the delta opioid receptors. tens is still being researched and garnering more information but has shown promising results in multiple clinical trials. berkeley pharma tech journal of medicine | 22 figure 6. common tens device a heat application studied in clinical trials garnered positive effects56. when applying tens and heat stimulation in a 2021 clinical trial, the results showed a significant improvement in average pain experienced by those affected by chronic lower back pain. the clinical trial was a randomized, double-blinded controlled trial to test the early intervention of tens for a spinal injury. a treatment of 30 minutes of tens therapy twice a week for 8 weeks or a sham tens therapy was supplied as a placebo (n=26). there were 4 pads applied, 2 located paraspinally to the spinal injury and the other 2 located ventrally within the dermatome of the injury. the overall end showed one patient from the tens treatment and 2 from the sham treatment having adverse reactions related to the study. with many clinical trials showing promising results, they have also shown some complications with interactions with pre-dispositioned illness. people with epilepsy, who are pregnant or have a pacemaker have been shown to have more risk of complications. in addition, there have been negative effects due to inappropriate positioning of the electrode pads. current common errors in placement of the pads include over the chest near the heart, eyes, and internally unless specified for internal use. overall the use of berkeley pharma tech journal of medicine | 23 tens has been shown to be safe with little to no side effects for most people. 4.3 photobiomodulation photobiomodulation is an external treatment that uses laser and led light to affect the action potentials of the pain pathway. the method uses low-intensity red or near-infrared light that is brought near the surface of the skin, relative to other energy densities in laser therapy used for ablation, cutting, and the thermal coagulation of tissue57. it interferes with the stimulation of action potentials within the mitochondria by initially interacting with the peripheral nerves and having the electrons join part of the photoreceptor on the skin. these photoreceptors stimulate membrane potential and cause extra absorption into the potassium and sodium channels. this increases the reabsorption power of sodium and potassium which keeps the action potential from crossing the threshold needed. the electron transfer by photons in the visible and near-infrared light spectrum through the modulation of cytochrome c-oxidase activity, increasing atp production, modulation of redox states, and inducing transcription factors58. due to limited understanding of its effects on the molecular, cellular, and tissular mechanisms, photobiomodulation has not been widely accepted in clinical settings. in recent years, the effects of photobiomodulation have been further studied59. figure 7. photobiomodulation mechanism of action a double-armed, randomized, sham-controlled, double-blinded clinical trial of photobiomodulation therapy with a 3:4 ratio (n=70) to treatment or berkeley pharma tech journal of medicine | 24 sham arms was conducive to less neuropathic pain in treating chemotherapy-associated peripheral neuropathy. each patient was given 18 treatments of 30-minute duration three times a week, which included laser exposure to areas of pain which included the legs, feet, cervical spine region, and lumbar spine region for anywhere between 3 to 30 minutes, based on the severity of the symptoms. the change in mean for the pain score between the time of randomization and by the end of the experiment for photobiomodulation therapy and sham treatment were -6.8 and 0.2 respectively. the higher magnitude in a change of mean for photobiomodulation therapy demonstrates its efficacy in attenuating neuropathic pain symptoms. heat is a limitation in receiving optimal results as the skin surface temperature increases, and the effects of photobiomodulation decrease. heat can be generated through the absorption of radiation by cells and tissue targeted by the laser. however, this energy transfer from the photons produced by the laser that the skin absorbs is allocated to non-targeted tissues that surround the targeted tissues, leading to the unwanted production of heat that reduces the efficacy of photobiomodulation. absorption of light is a characteristic that is attributed to any material and, thus, is impossible to completely eradicate. therefore, an external procedure that decreases the temperature of the surface of the targeted tissue must be performed to induce a substantial response toward photobiomodulation. other limiting factors include reflection and scattering60. reflection of light from the targeted tissue implies that the energy provided by the light is not being completely absorbed and that energy is erroneously expended towards non-targeted tissues. scattering on the other hand disperses light in different directions due to wave-particle interactions. this is significant to photobiomodulation efficiency because varying wavelengths of light pertain to different scattering properties when penetrating tissue, diffusing collimated beams into a cloud of photons. this dilutes the potency of the light because rather than focusing on a single area, surrounding non-target tissue receives unnecessary treatment. recent debates on using pulsed waves instead of continuous waves to maximize therapeutic efficiency have emerged because pulsed waves are less thermally strenuous to irradiated berkeley pharma tech journal of medicine | 25 tissues which may improve deep tissue penetration, but this is only conjecture since evidence for this hypothesis is scarce. photobiomodulation has also been studied in the context of its promising effects on the musculoskeletal system. due to its cellular activity enhancement capabilities, photobiomodulation is often used in recovering athletes affected by musculoskeletal-related issues. it has been found that when using an 810 nm laser on athletes to observe muscle performance and postexercise recovery, 50 j is the optimum dosage. the study only focused on three different doses at 10 j, 30 j, and 50 j, which implies that an upper limit is still unknown and that a higher dose may be conducive to more benefits. similarly, an 830 nm laser was used on another group of athletes before and after exercise divided into three groups: placebo, pre-fatigue laser, and post-fatigue laser. this wavelength of light reduced serum lactate and creatine kinase levels in both pre-fatigue laser and post-fatigue laser groups. however, a more significant reduction was observed in the post-fatigue laser group. it has also been found that the source of light is responsible for the resulting beneficial effects of photobiomodulation. in a study observing musculoskeletal performance and postexercise recovery in healthy males, a 30 j dose provided the best results by decreasing delayed-onset muscle soreness and improving biochemical markers related to musculoskeletal damage61. therefore, maximum musculoskeletal system recovery benefits stemming from photobiomodulation therapy are empirically attainable through optimizing wavelength, light source, and application during fatigue. 4.4 passive treatment capsaicin patch is an effective and accessible treatment for neuropathic pain. capsaicin is a compound that when used topically, attenuates cutaneous hypersensitivity to heat and mechanical stimuli through the degeneration of nerve fibers. trpv1, a cationic channel that is activated by capsaicin, stimulates the increased movement of sodium and calcium ions into sensory cells that depolarize nociceptive neurons and cause action potential firing. this creates an analgesic effect that desensitizes the nociceptive areas to which the patch or topical treatment is applied to. berkeley pharma tech journal of medicine | 26 figure 8. capsaicin mechanism of action unlike other topical alternatives, patches are unique in their method of drug delivery as they can provide extended release of the drug in regulated concentrations to prevent overdose and unwanted prolonged effects. neuropathic pain is a type of chronic pain. therefore, capsaicin effectively attenuates pain for longer periods depending on several factors that are being researched in recent developments. the duration of the analgesic effect is affected by the concentration of capsaicin, capsaicin-induced calpain-mediated ablation of axonal terminals, axonal mitochondrial dysfunction, and microtubule disorganization62. calpain is a protease activated by the influx of overbearing levels of calcium into the cell which cleaves cytoplasmic and nuclear substrates causing cells to end up undergoing apoptosis. while calpain enzymes are involved in processes such as cell division, differentiation, and migration, their apoptotic response allows for protection from other malignant cell lines including hepatocarcinoma, prostate cancer, human glioma, breast cancer cells, and human gastric cancer. therefore, even in different contexts regardless of the causation of said neuropathic pain, capsaicin appears to be a versatile remedy. a randomized clinical trial involving the application of ngx-4010, a high-concentration capsaicin patch, to treat painful hiv-associated neuropathy areas was conducive to a decrease in the average pain felt by participants (n=422)63. the study involved four groups: ngx-4010, 60 berkeley pharma tech journal of medicine | 27 minutes (60 min of high concentration capsaicin 640 mcg/cm^2), control group, 60 minutes (60 min of low concentration capsaicin 3.2 mcg/cm^2), ngx-4010, 30 minutes (30 min of high concentration capsaicin 650 mcg/cm^2), control group, 30 minutes (30 min of low concentration capsaicin 3.2 mcg/cm^2). the change in mean for the pain score between weeks 2-12 of the study period on a scale from 0-10 (0 = no pain, 10 = worst pain) for the above groups listed were -32.8, -30.0, -26.2, and -19.1 respectively. the “ngx-4010, 60 minutes” group exhibited the largest reduction in pain score. this suggests that a higher concentration of capsaicin and a longer duration of application is optimal. another common analgesia-induced patch used to treat neuropathic pain is through the local anesthetic lidocaine64. frequently juxtaposed with capsaicin, both drugs are mediators for neuropathy, but should not be used interchangeably. their mechanism of action, situational application, and analgesic effects differ. lidocaine works to block nerve signals in the area of application by inhibiting the action potentials occurring between nerves through interference with sodium channels. it is mainly used in medical procedures that require localized analgesia, resulting in a numbing sensation in the area due to the blockage of transmission signals. however, both lidocaine and capsaicin are effective neuropathic pain relievers that produce anti-inflammatory effects, it is believed that lidocaine is more effective short term like in cases of dental procedures or minor abrasions while capsaicin is more effective long term involving treatment of allodynia and hyperalgesia. a double-blinded, placebo-controlled, parallel-group study was conducted to observe the development of adverse effects in type 2 diabetics experiencing symptoms of peripheral neuropathy (pricking sensations, numbness, burning, and aching in feet) for more than 10 years. only males ages 40-60 were chosen as participants for this study (n=273) to avoid possible hormonal issues as variables. patients scored from 0-10 (0 = no pain, 10 = worst pain). the study groups are as follows: group ll (5% lidocaine patch 700 mg lidocaine for 60 min), group lp (placebo patch for 60 min), and group lc (8% capsaicin patch 179 mg for 60 min). after 24 weeks, group lc displayed significantly reduced pain scores compared to group lp. the average pain scores for the groups are as follows: ll: 6.4 to 4.9, lp: 6.1 to 5.7, lc: 6.7 to 3.6. berkeley pharma tech journal of medicine | 28 group average satisfaction scores (0 = poor, 4 = excellent) are as follows: ll: 2.1 to 3.2, lp: 2.2 to 2.5, lc: 2.0 to 4.2. showing higher and improved scores than group ll in both average pain score and satisfaction score, group lc demonstrates a more potent analgesic effect. 5. future direction/implications the emergence of nanotechnology, external modalities, and passive treatments has changed the future of the pain management industry. with the status of pain alleviation not curing or fully treating the problem, these new developing treatments show promising outcomes. they have shown fewer side effects and complications because of their external and non-invasive nature. for the side effects, they also showed no addiction, respiratory depression, and irregular bowel movements that are common in the current treatments such as opioids. in addition, the style of these procedures has been demonstrated to be more cost-effective when compared to surgical procedures and the repetitive prescriptions of ongoing pain medicine. even with these advantages of the treatments, there still needs to be consideration that these are developing and still relatively new with more research needing to be done before widespread usage. additionally, more research must be done to make sure that each method would address the root causes of pain. despite these treatments still being developed, there would be ethical considerations to address during their development. those ethical considerations include obtaining informed consent, ensuring patients understand both the benefits and risks, guaranteeing access to treatment without financial or insurance-related barriers, and addressing conflicts of interest among those promoting the treatments. these therapies have currently optimistic results and will change the future of pain treatment. 6. conclusion as the field of biotechnology continues to grow, the information gained here will help influence and promote numerous treatment modalities that will help address chronic pain in patients. these modalities would offer more options for the large population of individuals who suffer from pain. in addition, they would be able to treat chronic pain and relieve it greater berkeley pharma tech journal of medicine | 29 than the ongoing treatments. nanotechnology can effectively work with opioids but enhance their safety profile with their controlled delivery and high specificity. the non-pharmaceutical and external treatments such as myofascial release, tens, photobiomodulation, and the capsaicin patch are non-invasive and give little to no complications and chances of infections. although these treatments require further research, they have shown positive outcomes with a good impact on the future of pain management. furthermore, pain treatments will continue to advance and evolve, expanding the range of procedures available to patients, ultimately improving their wellbeing. berkeley pharma tech journal of medicine | 30 references 1. armstrong sa, herr mj. physiology, nociception. statpearls. treasure island (fl): statpearls 2. mitsikostas dd, moka e, orrillo e, et al. neuropathic pain in neurologic disorders: a narrative review. cureus. 2022;14(2). 3. park j, park h. botulinum toxin for the treatment of neuropathic pain. toxins. 2017;9(9):260. 4. dydyk am, conermann 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nerve stimulation. continuing education in anaesthesia critical care & pain. 2009;9(4):130-135. 54. teoli d, an j. transcutaneous electrical nerve stimulation (tens). nih.gov. published june 28, 2019. 55. johnson m. transcutaneous electrical nerve stimulation: mechanisms, clinical application and evidence. reviews in pain. 2007;1(1):7-11. 56. felix e, university of miami, national institute on disability, independent living, and rehabilitation research. randomized, double-blinded, controlled trial of early-intervention tens for the reduction of the prevalence and severity of chronic neuropathic pain during the first year after spinal cord injury. clinicaltrials.gov. 57. de oliveira mf, johnson ds, demchak t, tomazoni ss, leal-junior ec. low-intensity laser and led (photobiomodulation therapy) for pain control of the most common musculoskeletal conditions. european journal of physical and rehabilitation medicine. 2022;58(2). 58. friedmann h, lipovsky a, nitzan y, lubart r. combined magnetic and pulsed laser fields produce synergistic acceleration of cellular electron transfer. laser therapy. 2009;18(3):137-141. 59. de freitas lf, hamblin mr. proposed mechanisms of photobiomodulation or low-level light therapy. ieee journal of selected topics in quantum electronics. 2016;22(3):348-364. 60. toich s. photobiomodulation: light-tissue interaction, and the current and future states of pbm. www.lumitex.com. 61. gendron dj, hamblin mr. applications of photobiomodulation therapy to musculoskeletal disorders and osteoarthritis with particular relevance to canada. photobiomodulation, photomedicine, and laser surgery. 2019;37(7):408-420. 62. arora v, campbell jn, chung mk. fight fire with fire: neurobiology of capsaicin-induced analgesia for chronic pain. pharmacology & therapeutics. 2021;220:107743. 63. brown, s., simpson, d.m., moyle, g. et al. ngx-4010, a capsaicin 8% patch, for the treatment of painful hiv-associated distal berkeley pharma tech journal of medicine | 34 http://nih.gov sensory polyneuropathy: integrated analysis of two phase iii, randomized, controlled trials. aids res ther 10, 5 (2013). 64. hussain n, amira s.a. said, javaid fa, et al. the efficacy and safety profile of capsaicin 8% patch versus 5% lidocaine patch in patients with diabetic peripheral neuropathic pain: a randomized, placebo-controlled study of south asian male patients. journal of diabetes and metabolic disorders. 2021;20(1):271-278. berkeley pharma tech journal of medicine | 35 glioblastoma body.docx berkeley pharma tech journal of medicine correspondence: daniyalatiq7@gmail.com keywords: neoantigen glioblastoma monoclonal antibodies immune checkpoint inhibitors personalized therapy submitted: may 8, 2023 accepted: july 14, 2023 published: december 30, 2023 full open access creative commons attribution license 4.0 exploring novel neoantigen-based treatment methods for glioblastoma by: daniyal atiq, yacoub alokam, and suraj shah abstract glioblastoma, constituting almost half of all malignant brain tumors, presents a formidable challenge in treatment due to the blood-brain barrier's protective role against certain therapies. these tumors, characterized by rapid growth, tissue invasion, and diverse evolving cells, afflict individuals of all ages and resist conventional cancer treatments despite extensive biomedical research. an innovative approach to glioblastoma treatment involves leveraging neoantigens, specific to cancer cells and targetable by the immune system. clinical trials suggest that neoantigenbased treatments hold promise, offering more effective and personalized options for patients. exploring this avenue, including vaccines, immune checkpoint blockers, and adoptive cellular therapies, is crucial for improving outcomes. this paper reviews novel therapeutic options within neoantigen-based treatments, providing insights into potential advancements against glioblastoma. 1. introduction the aim of this paper is to explore and evaluate the e�cacy of neoantigen-based vaccines, immune checkpoint blockers, and adoptive cellular therapies as potential treatment methods for glioblastoma. by referencing scienti�c literature and clinical trials, this research seeks to accomplish the following objectives: 1.1. reviewing the history and background of glioblastoma treatment: conduct a comprehensive analysis of the historical context and background of glioblastoma treatment methods utilizing scienti�c databases such as pubmed, google scholar, cinahl, etcetera. highlight the limitations and challenges associated with conventional treatment approaches, such as surgery, radiation therapy, and chemotherapy. 1.2. investigating neoantigens as potential treatment options for glioblastoma: review scienti�c literature to examine the role of neoantigens in glioblastoma immunotherapy. explore the mechanisms through which neoantigens can elicit an immune response against glioblastoma tumor cells. evaluate the e�cacy and safety of neoantigen-based vaccines in preclinical and clinical settings. 1.3. assessing the e�cacy of immune checkpoint blockers in glioblastoma treatment: examine the role of immune checkpoint blockers, such as pd-1/pd-l1 inhibitors and ctla-4 inhibitors, in modulating the immune response against glioblastoma. analyze the outcomes of clinical trials and real-world evidence regarding the use of immune checkpoint blockers in glioblastoma patients. assess the potential of combination therapies involving immune checkpoint blockers and other treatment modalities. 1.4. exploring adoptive cellular therapies for glioblastoma: berkeley pharma tech journal of medicine | 30 investigate the use of adoptive cellular therapies, including chimeric antigen receptor (car) t-cell therapy and tumor-in�ltrating lymphocyte (til) therapy, in glioblastoma treatment. analyze the e�cacy and safety pro�les of adoptive cellular therapies in glioblastoma patients. evaluate the potential of enhancing the e�ectiveness of adoptive cellular therapies through genetic engineering and personalized medicine approaches. by addressing these objectives, this article aims to contribute to the advancement of glioblastoma treatment by shedding light onto the potential of neoantigen-based vaccines, immune checkpoint blockers, and adoptive cellular therapies as innovative and promising therapeutic strategies. 2. glioblastoma background every year, it’s estimated that approximately 10,000 people will be diagnosed with glioblastoma, which yields a 25% one-year survival rate as well as a 6.8% 5-year survival rate.1 on average, patients survive eight months before succumbing. glioblastoma is a grade iv brain tumor2—signifying that the cells are actively dividing, and the tumor has dead tissue as well as abnormal blood vessel growth—that stems from malfunctioning astrocytes, glial cells that provide structural support to neurons, modulate synaptic activity, and act as a major component in the blood-brain barrier.3 figure 1. glioblastoma statistics the age-adjusted incidence of glioblastoma has a positive correlation with age, being 0.15 per 100,000 in children to 15.03 per 100,000 in patients berkeley pharma tech journal of medicine | 31 between 75 and 84 years of age.4 glioblastoma develops most commonly in the frontal lobe, but has appeared in the temporal, parietal, and occipital lobes, and could even grow into surrounding brain tissues.5 common symptoms of glioblastoma include seizures, coordination issues, paralysis, fatigue, severe headaches, and cognitive impairment. major risk factors have yet to be identi�ed; however, it was recently discovered that exposure to high ionizing radiation is a major contributor towards glioblastoma development. moreover, exposure to vinyl chloride, pesticides, smoking, petroleum re�ning, and synthetic rubber have shown occasional positive association with glioblastoma emergence.6 currently, the main treatment methods used include surgical resection, alkylating chemotherapy, radiation therapy, and tumor treating fields.6 the majority of gliomas have been found to contain point mutations in isocitrate dehydrogenase 1 and 2. in glioblastoma, molecular alterations that have been discovered include mutations in genes regulating receptor tyrosine kinase (rtk), rat sarcoma (ras), phosphoinositide 3-kinase (pi3k), p53, and retinoblastoma protein (rb) signaling.7 current research shows mutations in egfr (57% of gbm patients), her2, pdgfra (13%), c-met (1.6%), fgfr (3.2%), pten (41%), and vegfr genes are therapeutic targets due to having shown ampli�cations or mutations from dysregulated cell signaling cascades in glioblastoma.7 kinase inhibitors haven’t proven to be e�ective in glioblastoma therapy due to their low e�cacy in penetrating the blood-brain barrier; however, a multitude of experimental treatments have emerged and shown consistent progress. azd3579 (egfr inhibitor) has shown e�ective blood-brain barrier penetration in vivo in rats and monkeys, however its safety and e�cacy in humans has yet to be tested. epitinib (egfr inhibitor) was reported to have optimal bbb penetration, is well-tolerated in patients, and is e�ective in treating brain metastases as well. wsd0922 (egfr inhibitor) is reported to have high bbb penetration, reasonable safety, and has shown antitumor properties in glioblastoma pdx models.7 tucatinib (her2 inhibitor) can cross the blood-brain barrier and create survival bene�ts in mice. for patients with breast cancer and brain berkeley pharma tech journal of medicine | 32 metastasis, tucatinib produced better progression-free survival and overall survival. neratinib (her2 inhibitor)had limited bbb penetration in mice and was not successful in a phase ii trial for patients that had her2-positive brain metastases.7 additionally, for newly diagnosed glioblastoma cases, clinical trials involving the drug temozolomide—a monofunctional dna alkylating agent—have shown signi�cant progress. adding the temozolomide regimen improved the overall survival and progression free survival in patients with gbm compared with radiotherapy alone. temozolomide is a lipophilic molecule that crosses the blood-brain barrier and is stable at the acidic ph of the stomach and is therefore administered orally.7 temozolomide is currently being tested, in a phase ii interventional clinical trial led by wen et al. (nct02977780) via combination therapy with neratinib, to study overall survival compared to standard treatment in glioblastoma patients.8 3. neoantigen background neoantigens are a class of proteins that arise in cancer cells with the mutation of tumor dna. the �rst discovery of neoantigens was made in 1988 by de plaen and his colleagues.9 by utilizing cdna library screening on a mouse tumor model, they observed a single nucleotide di�erence between the normal and tumor gene, resulting in a noticeable amino acid change. this novel �nding led to the coining of the term “neoantigen” which was used in further studies on human tumors including melanoma and renal cell carcinoma.9 neoantigens have proven e�cient immunogenic targets because of their localization to cancer patients. the mechanism of action by which they form explains the tumor-speci�city of neoantigens and their associated potency in developing cancer treatment. first, mutations in tumor dna cause rise of new and mutated proteins in tumor cells. after completion of function, these proteins are proteolyzed by the proteasome and the degraded peptides are sent to the endoplasmic reticulum via a transporter associated with antigen processing (tap)protein.10 the protein-peptide complex is then sent to the golgi apparatus and then exported to the plasma membrane with chaperone proteins. there, the major histocompatibility berkeley pharma tech journal of medicine | 33 complex (mhc) displays protein fragments (post-proteolysis) to the immune system to �ght infection by pathogens. neoantigens are a class of proteins that arise in cancer cells with the mutation of tumor dna. the �rst discovery of neoantigens was made in 1988 by de plaen and his colleagues.9 by utilizing cdna library screening on a mouse tumor model, they observed a single nucleotide di�erence between the normal and tumor gene, resulting in a noticeable amino acid change. this novel �nding led to the coining of the term “neoantigen” which was used in further studies on human tumors including melanoma and renal cell carcinoma.9 neoantigens have proven e�cient immunogenic targets because of their localization to cancer patients. the mechanism of action by which they form explains the tumor-speci�city of neoantigens and their associated potency in developing cancer treatment. first, mutations in tumor dna cause rise of new and mutated proteins in tumor cells. after completion of function, these proteins are proteolyzed by the proteasome and the degraded peptides are sent to the endoplasmic reticulum via a transporter associated with antigen processing (tap) protein.10 the protein-peptide complex is then sent to the golgi apparatus and then exported to the plasma membrane with chaperone proteins. there, the major histocompatibility complex (mhc) displays protein fragments (post-proteolysis) to the immune system to �ght infection by pathogens. figure 2. mechanism of action of neoantigens berkeley pharma tech journal of medicine | 34 in humans, the mhc is referred to as human leukocyte antigens (hla), which are encoded by genes on chromosome 6.11 hlas work with t cells to identify 'self' and 'foreign' cells and tissues in the body by displaying short polypeptides to t-cell receptors. these polypeptides can be recognized as ‘foreign’ by tumor-in�ltrating lymphocytes (tils) which results in the activation of strong tumor-speci�c immune responses.12 there are two main groups of mhc molecules, which bind to di�erent peptide sequences and are recognized by separate t-cell types. mhc-class i genes consist of hla-a, hla-b and hla-c, and their attached peptides are recognized by cd8+ t-cells.11 mhc-class ii genes, on the other hand, consist of hla-dr, hla-dq and hla-dp, and attached peptides are recognized by cd4+ t-cells.11 figure 3. role of the major histocompatibility complex (mhc) neoantigens are commonly confused with other tumor antigens that exist,of which there are three di�erent classi�cations that t-cells can target.11tumorassociated (taa) antigens are a group of proteins characterizedby overexpression in cancer cells compared to normal tissue. they consist of differentiation antigens, which are normal proteins that are increasingly expressed due to uncontrolled division of cells that have speci�c functions [eg. prostate-speci�c antigen (psa) in prostate cancer.13 the other class is overexpressed antigens, which are proteins that are greatly expressed due to mutated genes characteristic of cancer [eg. hepsin in prostate cancer].14 a complete library of tumor-associated antigens can be found in the cancer antigenic peptide database: https://caped.icp.ucl.ac.be/peptide/list. cancer testis (ct) antigens are a family of taa that are strictly found on the testis and placenta. due to their expression speci�city in the germ cells, ct antigens found in other regions of the body indicate oncogenic causes. berkeley pharma tech journal of medicine | 35 https://caped.icp.ucl.ac.be/peptide/list hence, ct antigens are considered promising therapeutic targets. there is also the fact that the testis do not express mhc-class i, therefore ct antigens are recognized as ‘non-self.’ a comprehensive list of ct antigens can be found in the ctdatabase: http://www.cta.lncc.br/. finally, viral antigens are a family of taa that are caused by a viral infection. examples include the human papillomavirus (hpv)which can cause cervical and several other cancers, as well as hepatitis c which may lead to liver cancer and non-hodgkin's lymphoma. neoantigens are unique to the aforementioned antigen types because they arise from the onset of cancer, thus it is the genomic mutations from the cancer that lead to the rise of new (neo-)antigens in the body.14 in exploring neoantigen-based treatment, there is the distinction to be made between public and private neoantigens. public (shared)neoantigens are common across cancer patients and typically occur in driver oncogenes and tumor suppressor genes. private (personalized)neoantigens are unique to each patient and therefore require individualized treatment. 4. potential treatment methods 4.1 neoantigen-based vaccines the process of creating a neoantigen vaccine typically involves sequencing the patient's tumor cells to identify the unique neoantigens present. once these neoantigens are identi�ed, they can be used to create a personalized vaccine that is tailored to the patient's speci�c cancer. the goal of the vaccine is to prime the immune system to recognize and attack the cancer cells that display these neoantigens. this approach has the potential to be more e�ective than traditional cancer treatments because it targets the cancer cells directly while sparing normal cells. berkeley pharma tech journal of medicine | 36 http://www.cta.lncc.br/ figure 4. mechanism of action for protein-peptide vaccine 4.2 protein-peptide vaccines protein peptide vaccines contain speci�c antigens which are immunogenic and unique to the patient's tumor, with potential immunomodulating and antineoplastic activities.15 upon vaccination, with the neoantigen peptide vaccine, the peptides stimulate the host’s immune system to elicit a speci�c cytotoxic t-lymphocyte (ctl)response against the tumor cells that are expressing the targeted neoantigens, thereby resulting in tumor cell lysis. according to the fda, with respect to the mechanism of action, neoantigen-based protein-peptide vaccines use short peptide fragments to induce highly targeted immune responses while simultaneously avoiding allergic responses. peptide epitopes can bind antibodies in three conformations: alpha-helical, beta-strand/extended, or loop. since the peptide epitope’s spatial conformation among the antigen-antibody complex is signi�cant, vaccines must be engineered in a conformationally correct way to produce optimal results. to constrain peptide epitopes when epitope conformation is important, some methods utilized include covalent side chain-side chain cross linking and integration into a larger sca�old. emulsions, which act as delivery systems for various peptides, form a depot at the site of injection that attracts immune cells.16 the presence of antigen depots at organs releasing low-level antigens induces a strong immune response and promotes tolerance. the stability of these emulsions as delivery systems plays a key role in vaccine safety and e�cacy.16 berkeley pharma tech journal of medicine | 37 the process of developing a neoantigen-based peptide vaccine starts with extracting a sample of peripheral blood.17 peripheral blood mononuclear cells (pbmcs) are then isolated from the blood sample, and subsequently are di�erentiated into dendritic cells—the antigen presenting cells. simultaneously, the peripheral blood sample and a tumor sample are run through sequencing and computational neoantigen identi�cation: whole-genome sequencing (wgs), whole-exome sequencing (wes), and next-generation sequencing (ngs).18 the identi�ed neoantigens are validated in vitro via elispot, mhc tetramer, and tumor organoids, which have shown in studies to retain the neoantigen features of parental tumors. then, the selected neoantigens are loaded onto dendritic cells and integrated into peptide vaccines for treatment.17 wgs, one of the previous sequencing identi�cation methods, determines the order of the nucleotides in the individual’s dna, however its production of complex datasets that require bioinformatics expertise makes it ine�cient timewise. wes utilizes probes and hybridization to analyze the exome, which despite only constituting 1-5% of the genome, contains approximately 85% of disease-related variants. wgs is more time-e�cient and allows for greater sequencing capabilities. ngs, re�ective of wgs, is used to determine nucleotide sequences in targeted regions of dna and is capable of sequencing an entire human genome within one day.19 it’s evident that with these three sequencing methods, identifying neoantigens and somatic mutations would be carried out e�ciently with regards to time and costs. in two clinical trials reported by hilf et al. (30568303) and keskin et al. (30568305) in 2019, neoantigen-peptide vaccines produced somewhat successful therapeutic results for glioblastoma.20,21 in hilf et al.’s trial, �fteen patients, all of whom were recently diagnosed with glioblastoma, were found to possess the human leukocyte antigen (hla)-a*-2:01 or hla-a*24:02. in apvac1 (actively personalized vaccine 1), patients received a vaccine that targeted unmutated antigens, and in apvac2, the vaccine targeted neoantigens. compared to classic treatment, patients who received apvac1 and apvac2 had an increase in median overall survival of 29 months, and an increase in median progression-free survival of 14.2 berkeley pharma tech journal of medicine | 38 months. in the tumor, the vaccine injection induced t-cell expansion; however, at the vaccine’s injection site, nearly all patients experienced some form of disease.20 in a trial led by keskin et al., ten patients with newly diagnosed glioblastoma were treated with the personalized neoantigen-based vaccine after being treated surgically and with radiotherapy. though some experienced an increase in cd8+ t cells in the environment of recurrent tumors, as well as a small increase of pd-1+ tumor in�ltrating cd8+ t cells, all of the patients ended up dying as a result of disease progression, with an average survival of 16.8 months and progression-free survival of 7.6 months.21 reardon et al. (nct02287428) is conducting an ongoing phase i interventional clinical trial studying the neoantigen-speci�c peptide vaccine neovax, alongside the monoclonal antibody pembrolizumab, radiation therapy, and temozolomide in subjects with glioblastoma.22 the clinical trial involves �ve cohorts, each receiving either separate individual or combinations of treatments. the �rst cohort receives neovax and radiation therapy, the second starts pembrolizumab within two weeks of the start of radiation therapy and continues triweekly for two years, the third starts pembrolizumab about three weeks after completion of neovax priming and continues triweekly for two years, the fourth receives a single dose of pembrolizumab two weeks after the start of radiation therapy and restarts concurrently three weeks after completing neovax priming, and the �fth enrolls patients with tumors for which the mgmt status is (partially) methylated and receives standard temozolomide with radiation and adjuvantly for six cycles after radiation therapy. the primary outcomes are the number of participants with adverse events to determine the tolerability and safety (all cohorts), the number of participants with at least ten actionable peptides to measure the feasibility of the study (cohort 1), and the number of participants able to initiate vaccine therapy after radiation therapy within 12 weeks of the date of surgery (cohort 1). the secondary outcomes are the number of participants who experience ifn-γ t-cell responses at week 16 (all cohorts), the number of participants who are alive and don’t demonstrate glioblastoma progression eight months after resective surgery (cohorts 1, 1a, 1b, 1c), and the number of participants who are alive without having glioblastoma progression eleven months after berkeley pharma tech journal of medicine | 39 resective surgery (cohort 1d).22 as the study is ongoing, �nal results and data have not been collected yet. 4.3 nucleic acid (dna and mrna vaccines) nucleic acid vaccines are another category of treatment methods available for cancer patients, of which there are two types: deoxyribonucleic acid (dna) and messenger ribonucleic acid (mrna) vaccines.23 for dna vaccines, a bacterial plasmid is used as a vector to introduce a gene encoding antigens. the plasmids then replicate, and by using antibiotics, the vectors exhibiting antibiotic resistance can be separated. dna vaccines are able to activate both humoral and cellular immune responses, and they can be delivered intramuscularly (im), intradermally (id), mucosally, and/or transdermally. the process by which genomic alterations occur include internalization of the dna vaccine into the cell, transcription of the genomic information at the nucleus, and translation of that info within the cytoplasm.24 this ultimately leads to expression of proteins in vaccinated hosts, however there are three distinct mechanisms by which these proteins reach the t-cells for recognition and targeting. one way includes the use of mhc i complexes in somatic cells, which presents proteins to cd8+ t cells. another method uses professional antigen presenting cells (apcs), like dendritic cells (dcs),that are transfected with plasmid dna and then present antigens to t cells through mhc i or ii complexes. a third way involves the phagocytosis of plasmid-transfected somatic cells by apcs, resulting in the cross priming and presentation of antigens to both cd4+ and cd8+ t cells.23 berkeley pharma tech journal of medicine | 40 figure 5. molecular mechanism of dna vaccines there are several current clinical trials experimenting with the use of dna vaccines in patients with glioblastoma. in johanns et al, the initiative is to evaluate the safety, feasibility, and immunogenicity of a personalized neoantigen-based vaccine in subjects with newly diagnosed, unmethylated glioblastoma.25 in this trial, patients will each receive standard radiation therapy as well as administered neoantigen dna vaccines using the cellectra®2000 ep device. the vaccine used is a combination of gnos-pv01 + ino-9012, dna vaccines which have previously shown immunogenicity in newly diagnosed gbm patients when administered alongside libtayo, radiation, and temozolomide. safety of the vaccine will be determined by dose-limiting toxicities, as per the common terminology criteria for adverse events version 5.0 (ctcae v5) published by the u.s. department of health and human services (usdhhs). alongside safety, feasibility of this dna vaccine is also being considered, for which there are three necessary conditions: e�ciency of vaccine manufacturing, administration, and ability of the vaccine to identify neoantigens in the patient. additionally, the immunogenicity of the vaccine will be determined by percentage of neoantigens that elicit a neoantigen-speci�c t cell response, as well as the associated cd8 t-cell response, and overall survival rate.25 berkeley pharma tech journal of medicine | 41 some clinical trials are testing the use of dna vaccines in combination with other immunotherapies to achieve a higher magnitude and breadth of neoantigen-speci�c t cell responses. in another trial by johanns et al, a personalized neoantigen vaccine is being used concurrently with retifanlimab pd-1 blockade therapy for patients who are newly diagnosed with glioblastoma.26 this work extends to other cancer types as well. in gillanders et al, patients with triple negative breast cancer (tnbc) are being actively recruited to conduct a study on the e�cacy of adjuvant therapies after administering neoantigen-based dna vaccines. in this trial, both groups will be given standard therapy as needed (ie. chemotherapy, surgery, radiation therapy, etc.), however one group of patients will be given the dna vaccine alone. the other group will be given the vaccine along with durvalumab to test for increased immunogenicity. 27 the purpose of these clinical trials, by both johanns and gillanders et al, is to investigate the e�ects of using certain dna vaccines in combination with standard therapies. the �ndings from these trials will lead to future advancements in the treatments used for diagnosed gbm patients. the other type of nucleic acid vaccines, mrna vaccines, currently holds two widely acknowledged forms: non-amplifying mrna and self-amplifying mrna, and each bears its mechanistic di�erences.23 the main di�erence between the two is in the additional length of self-amplifying mrna, which is attributed to the nonstructural proteins that extend the duration and amplitude of gene of interest (goi) expression.23 in contrast to dna vaccines, mrna vaccines do not have to be transcribed by the host. instead, the antigen-encoding genetic information is directly delivered to antigen-presenting cells (apcs). after reaching the host cell, the mrna can be released and translated into proteins which are then proteolyzed into peptide epitopes. then, the epitopes are sent to the golgi apparatus, after which they are transported to the plasma membrane and combined with mhc class i complexes via a cross-presentation pathway.23 an immune response arises when cd8+ t cells are activated as a result of the peptides reaching the cell surface of apcs. berkeley pharma tech journal of medicine | 42 figure 6. molecular mechanism of mrna vaccines currently, there are studies being done assessing the potency of mrna vaccines in patients with glioblastoma (gbm). in sayour et al, the objective is to �rst assess the manufacturing feasibility and safety of a rna-lipid particle (rna-lp) vaccine and then evaluate the maximum tolerated dose in gbm patients.28 participants will take part in a dose-escalation study using the bayesian optimal interval (boin) design with an initial embedded accelerated titration design (atd).28 in other studies, such as desjardins et al, mrna is currently being combined with dendritic cell vaccines and then evaluated for feasibility, potential adverse e�ects, and survival rates.29 similar to clinical trials using dna vaccines, the purpose of the experiments led by sayour and desjardins et al. is to advance the therapies available for gbm patients. as their approach uses less-studied mrna vaccines, the current initiative is to evaluate the potency of these vaccines in patients. 4.4 autologous dendritic cells (dc) vaccines autologous dc vaccines are composed of autologous, immature dendritic cells (dcs) with potential immunostimulating and antineoplastic abilities. upon leukapheresis, immature dendritic cells are isolated and re-administered intratumorally. the immature dcs internalize and process berkeley pharma tech journal of medicine | 43 the neoantigens or tumor-associated antigens (taas), migrate to the lymphatic system, and expose the immune system to the taas, thereby inducing a speci�c cytotoxic t-lymphocyte (ctl) response against the cancer cells, leading to tumor cell lysis. the process of vaccination has the potential to either reinforce the reaction against taas or induce a new reaction. the mechanism of action for dc vaccines involves the mhc-ii molecules on the surface of the dcs, which make them professional antigen-presenting cells (apcs). the dcs move between lymphoid and nonlymphoid tissues to regulate chemokine gradients and cytokine gradients and active t-killers. to create a dc vaccine, immature dendritic cells are isolated from human blood, then utilize a cytokine cocktail and autologous tumor antigens to promote maturation, then readminister the autologous dcs back into the human body via the dc vaccine.30 figure 7. an overview of the mechanism of action of autologous dc vaccines the e�cacy of the dc vaccine is dependent on the quantity of neoantigens present in the tumor. the tumor mutational burden (tmb), representing the frequency of neoantigen-associated mutations per megabase, is about 10 mutations/1.4 mb.31 for patients undergoing tmz chemotherapy, the tmb typically experiences an increase, thus making neoantigen discovery more feasible. berkeley pharma tech journal of medicine | 44 the autologous neoantigen-based dc vaccine neo-modc was tested in a patient with advanced metastatic gastric cancer (35661819). the patient had initially undergone laparoscopic-assisted d2 radical distal gastrectomy. multiplex immunohistochemistry (ihc) revealed low expression of pd-1 and high expression of pd-l1. once the patient chose to alter their treatment to receive the aforementioned trial’s treatment methods, neo-modc vaccines were created using autologous dendritic cells generated from monocytes in peripheral blood mononuclear cells.14 neo-modc was administered on its own initially, however after two months, due to continued progression of the tumor, it was administered alongside combination therapy with nivolumab—a monoclonal antibody used to treat di�erent forms of cancer. the neo-modc vaccine elicited a healthy immunogenic response via an increase in neoantigen-speci�c cd4+ and cd8+ t cell activation, as well as an increase in neoantigen-speci�c t cell clones in peripheral blood. upon the onset of combination therapy, the tumor volume experienced a rapid decrease, and continued application of the neo-modc vaccine resulted in complete regression for 25 months until the present. to study t cell clone activation due to the neo-modc vaccine, primary tumor tissue and blood samples at di�erent intervals of vaccination were collected. activated peripheral blood lymphocytes (pbls) containing mutant peptides, tcrb clonotypes, and cdr3-regions of the tcrb chain were also analyzed. approximately 35.3% to 86.5% of tcrb clones in pbls were found in the tumor tissues, and the frequency of tumor-enriched tcrb clones in pbls increased from 0% to 16.8% after four doses of neo-modc.14 evidently, neo-modc induced immunogenicity, however it required concurrent administration of nivolumab to produce signi�cant results in tumor regression. 4.5 immune checkpoint blockade therapy many signaling pathways associated with the development of cancer. but there are also key regulatory checkpoints termed immune checkpoints that can negatively regulate these same signaling pathways. immune checkpoint blockade therapy takes advantage of these checkpoints and suppresses them, thereby limiting tumor progression. some known signaling molecules berkeley pharma tech journal of medicine | 45 associated with pathways that lead to cancer development include pd-1, pd-l1, and ctla-4.32,33 figure 8. mechanisms of immune checkpoint inhibitors (icis). icis act on certain key proteins involved in the signaling pathways that activate t cells. (a) t cells, via their cd28 protein, interact with antigen-presenting cells (acps) and their cd80 or cd86 proteins. this interaction can lead to subsequent activation of the t-cell. another signaling protein on the same t-cell, ctla-4, has more a�nity for the cd80 and cd86 proteins and can lead to subsequent inactivation of the t-cell. anti-ctla-4 inhibitors prevent the inactivation of the respective t-cell by disrupting the function of the ctla-4 protein. (b) cytotoxic t cells cd8+ interact via their pd-1 protein with tumor cells via their berkeley pharma tech journal of medicine | 46 pd-l1 protein. the interaction between these signaling proteins can lead to negative regulation of the anti-tumor t cell response. anti-pd-1 and anti-pd-l1 inhibitors prevent the binding of pd-1 and pd-l1 and thus prevents inactivation of t cell anti-tumor responses. figure 9. a comparison of normal t-cell receptor binding vs. t-cell receptorbinding after the addition of anti-pd-l1 ligand and pd-1 blockade.(a) t-cell binds to pdl1 on tumor cells via pd-1 receptor; anti-tumor killing response is inhibited. (b) tcell’s pd-1 receptor binds toanti pd-1 and pd-l1 binds to anti-pd-l1; subsequent activation of theanti-tumor killing response is observed. more speci�cally, monoclonal antibodies are being used to target these signaling molecules. when understanding the mechanisms by which monoclonal antibodies function, we focus on a key immune cell, the b cell. b cells are a very important part of the immune system since they have antibodies on their surface membrane. moreover, each b cell has antibodies that are speci�c to one antigen and are useless against other antigens. this can be advantageous or disadvantageous depending on the type of antibody and respective antigen. when a b cell and its antibody interact with a berkeley pharma tech journal of medicine | 47 speci�c antigen, the b cell becomes activated and di�erentiates into either a plasma cell or memory b cell. plasma cells produce a vast amount of their speci�c antibody and memory b cells remain in the host and serve as a part of immune memory so that if they are needed again, they can be activated.32,34 looking further into antibodies or immunoglobulins, they are made up of two heavy chains and two light chains and are arranged in a y-shape. the lower portion of the antibody contains an fc portion, which remains the same amongst all antibodies and is then used to bind to cells of the immune system. cells of the immune system have an fc receptor that binds to the fc portion of the antibody. the upper portion of the antibody contains the variable region, which has many con�gurations and is designed to attach only a single type of antigen. there are many ways by which antibodies can stimulate the immune response which include by activating the classical complement system, by attaching themselves to antigens in order to neutralize toxins, by attaching themselves to certain receptors to disrupt function of that receptor, by attaching themselves to a certain pathogen or disease causing agent and agglutinate, or by acting as opsonins, which involves antibodies attaching themselves to pathogens or disease causing agents and making it convenient for the phagocytes to recognize and destroy those pathogens that would have otherwise not been able to recognize them because of primitive recognition structures associated with certain immune cells.32,33,35 another very important role they can play is simulating antibody-dependent cell mediated cytotoxicity, in which antibodies attach themselves to pathogens or abnormal cells and then they help immune cells to recognize the pathogen and destroy it.32,35 utilizing the information that antibodies provide an e�ective way of targeting certain disease-causing agents and are very speci�c as to what antigen they target, this can be translated over to mechanisms by which monoclonal antibodies function. when treating a patient, it is possible to translate this key process of recognition and subsequent action to stopping a disease-causing pathway. certain proteins or antigens that are associated with harmful processes can be identi�ed and targeted by monoclonal antibodies. monoclonal antibodies are a single type of antibody [mp1] that target a speci�c protein. there are many types of monoclonal antibodies on the market today that have their own speci�c target protein.33,34 many berkeley pharma tech journal of medicine | 48 monoclonal antibodies have been designed to target previously mentioned key signaling molecules to inhibit immunosuppression and restore antitumor responses of the immune system. these antibodies include nivolumab, ipilimumab, avelumab, atezolizumab, retifanlimab, and many more that have proven to be e�cacious against many cancer types, including glioblastoma.32 multiple clinical studies have been undertaken to understand the e�cacy of monoclonal antibodies against cancer. figure 10. an overview of the mechanism of action for a monoclonalantibody berkeley pharma tech journal of medicine | 49 figure 11. monoclonal antibodies (mab) against tumor cells. monoclonal antibodies can be found in many di�erent forms and thus based in many di�erent ways. mabs can be organized into three categories: (1) naked-mab, (2) immunoconjugates, (3) multistep targeting. all these categories represent recombinant or “chimeric” mabs that are capable of mediating antibody dependent cellular toxicity. one such phase i clinical trial, observed treatment with neo-modc (vaccine) and nivolumab, which is a monoclonal antibody currently being studied for glioblastoma therapy (fda). pd-l1 and pd-l2 are ligands that typically bind to pd-1 receptors in t-cells, which subsequently inhibit t-cell proliferation and cytokine production (fda).14 nivolumab competitively inhibits pd-1 by binding to the pd-1 receptor and preventing pd-l1 and pd-l2 from binding, thus allowing enhanced t-cell function as well as a resulting anti-tumor response. the recommended dosage for nivolumab, as tested in unresectable and metastatic melanoma, metastatic non-small cell lung cancer, advanced renal cell carcinoma, and classic hodgkin lymphoma is typically 240 mg per 2 weeks or 480 mg 4 weeks, as well as 3 mg/kg every 2 weeks for pediatric patients under 40kg. some possible adverse reactions include fatigue, rashes, musculoskeletal pain, pruritus, diarrhea, nausea, asthenia, dyspnea, arthralgia, urinary tract infection, and upper respiratory tract infection. although its safety has not been assessed in humans yet, there were no notable e�ects caused by berkeley pharma tech journal of medicine | 50 nivolumab in the male and female reproductive organs in monkeys, however in animal models inhibition of pd-1 signaling led to an increase in the severity of some diseases, such as tuberculosis in mice.14 phase ii clinical trial nct03718767 was an interventional study of neoadjuvant nivolumab in patients with glioblastoma. the patients were due to receive 3mg of nivolumab every 2 weeks (anti-pd-l1 and pd-l2). its primary outcome measures were changes in the level of expression of pd-l1 by tumor cells and lymphocytes, measured from the baseline until the end of nivolumab treatment. the secondary outcome measures were e�cacy, with the response rate assessed by response assessment in neuro-oncology (rano) criteria, and safety, with toxicity being assessed by common toxicity criteria (ctc). the study revealed that neoadjuvant nivolumab resulted in enhanced expression of chemokine transcripts, higher immune cell in�ltration and augmented tcr clonal diversity among tumor-in�ltrating t lymphocytes, supporting a local immunomodulatory e�ect of treatment.36 as such, the use of neoadjuvant nivolumab can be used a method of early termination for developing tumor. some key things to mention are that this study involved the use of patients who have idh1 or idh2 mutated gliomas with hyper mutator phenotypes (hmp). these patients had a high number of mutations in their tumors and the study revolved around understanding if neoadjuvant nivolumab can stop or reduce tumor size, showing characteristics of controlled tumor growth. one of the drawbacks of this study was their secondary outcome measure of improvements in quality of life, which was self-reported alongside symptom severity and interference with daily activities. self-reporting may lead to biases or false speculation in the reports from the patients’ end, which in turn would produce skewed secondary results. an additional combined monoclonal antibody treatment for glioblastoma (nct03367715) that is currently being studied involves nivolumab and ipilimumab (fda). for the mechanism of action, ipilimumab binds to ctla-4 to block its interaction with the ligands cd80 and cd86—which negatively regulate t-cells, therefore resulting in tumor penetrating t-e�ector cell activation and proliferation (fda). inhibiting ctla-4 signaling reduces t-regulatory cell function, contributing to an increase in berkeley pharma tech journal of medicine | 51 https://clinicaltrials.gov/ct2/show/nct03718767 https://clinicaltrials.gov/ct2/show/nct03367715?term=nivolumab%2c+ipilimumab&cond=glioblastoma&draw=2&rank=1 t cell responsiveness. when ipilimumab and nivolumab are used concurrently, pd-1 and ctla-4 dual inhibition results in enhanced t-cell function that is more e�ective than either antibody on its own, and they’ve shown to produce more signi�cant antitumor responses.38 currently, there are many trials being conducted to test the e�cacy of single-monoclonal antibody treatments as well as combined monoclonal antibody treatments. the aforementioned trials are just some of those few. 4.6 adoptive cell therapies (act) adoptive cell therapies (act) involve the use of in vitro ampli�cation of key immune cells, which include dendritic cells (dc), tumor-in�ltrating lymphocytes (til) and cytokine-induced killer cells. these cells are extracted from a patient, are ampli�ed, and then injected back into the patient to enhance the tumor-killing response. more speci�cally til based cell therapies have proven to be e�cacious in recognizing antigenic epitopes on the surface of tumor cells and killing them.39,40,41 act with tils has been proven to be e�cacious against metastatic melanoma.42 neoantigen speci�c t cells were detected in the tils of patients with metastatic melanoma and neoantigens were identi�ed that increased the response of t lymphocytes in the peripheral blood post-til infusion.33,43 however, the process that goes into amplifying t cells is complex and it is di�cult to obtain high a�nity tcr+ t cells and t cells produced in vitro do not last for a prolonged period of time following infusion.33,40,42 in addition, the tissue from which these t cells are extracted and then ampli�ed can lead to variation amongst the types of antigens produced, making it less likely for a shared/public treatment method.41,42 berkeley pharma tech journal of medicine | 52 figure 12. a sample work�ow of t-cell therapy in a phase iii clinical trial nct05685004 a combination of tvi-brain-1 immunotherapy and standard therapy compared to standard therapy alone as a treatment for newly diagnosed mgmt unmethylated glioblastoma patients. the general procedures include the collection and testing of cancer tissue samples after surgery and chemoradiation therapy (radiation and temozolomide). for the patients randomized into the investigational study treatment group, they will also receive two vaccinations created from their own cancer cells, undergo leukapheresis to collect immune t-cells from their blood, and transfer of those activated e�ector t-cells after chemoradiation therapy. all patients are followed with mris at follow-up visits. this study acknowledged mgmt promoter methylation changes and investigated whether these changes should be considered in the treatment decision.44,45 there is a related therapy called the chimeric antigen receptor (car) t cell therapy which utilizes an antibody and t cell receptor complex. car can be used to modulate host t cells in order to enhance the cancer killing response of the immune system.41 car t-cell therapy is a type of immunotherapy that uses a patient's own immune cells, called t-cells, to �ght cancer. the therapy involves genetically modifying a patient's t-cells in the laboratory to produce chimeric antigen receptors (cars) on their berkeley pharma tech journal of medicine | 53 https://clinicaltrials.gov/ct2/show/nct05685004?term=nct05685004&draw=2&rank=1 surface.40,41,46 these receptors can then recognize and bind to speci�c proteins on cancer cells, which helps the t-cells to identify and attack the cancer cells. however, the use of car t-cell therapy for the treatment of solid tumors is still being studied in the early stages of clinical testing. one of the challenges in treating solid tumors with car t-cell therapy is that the car t-cells need to be able to penetrate the tumor microenvironment and e�ectively target the cancer cells, which can be di�cult to achieve.41,46 figure 13. a simple work�ow of car-t cell therapy in a phase i clinical trial nct04003649, the e�cacy of il13r alpha 2-car t cells when given alone or together with nivolumab and ipilimumab and their e�ects on treating patients with glioblastoma that has come back (recurrent) or does not respond to treatment (refractory). biological therapies, such as il13r alpha 2-car t cells, use substances made from living organisms that may attack speci�c glioma cells and stop them from growing or kill them. immunotherapy with monoclonal antibodies, such as nivolumab and ipilimumab, may help the body's immune system attack the cancer, and may interfere with the ability of tumor cells to grow and spread.47 it was found that il13r alpha 2-car t berkeley pharma tech journal of medicine | 54 https://clinicaltrials.gov/ct2/show/record/nct04003649?term=car+t&recrs=ad&cond=glioblastoma&draw=4&rank=5 cells enhance anti-tumor activity and t-cell persistence and showed evidence of bioactivity in patients. in addition, it was found that local intracranial delivery of car t cells increased anti-tumor e�cacy as compared to intravenous administration.40,47 overall, this study de�ned parameters for the clinical translation of car t cell therapy for the treatment of brain tumors. 4.7 combination therapies with neoantigen-based vaccines the use of tumor vaccines, monoclonal antibodies, and adoptive cell therapies are potential and e�cacious treatment methods for glioblastoma. speci�cally looking at tumor vaccines, they have extreme potential when developing personalized treatment methods for patients. anti-tumor vaccines, such as the proposed neoantigen vaccines, activate certain pathways in the host immune system that can create a susceptible environment for concurrent or subsequent treatment methods.48,49 this is an important aspect to note when considering neoantigen-based vaccines as a treatment method for glioblastoma. as shown in previous clinical trials and studies, cancer vaccines are a very innovative way to approach cancer treatment but they have very low e�cacy, with an objective clinical response rate of only >7% and an overall rate of clinical bene�t of only ~20%.49,50 there is no question that cancer vaccines stimulate and modulate the immune system, and this can be taken advantage of when treating patients. utilizing neoantigen-based vaccines can prime the host immune system to be more responsive to subsequent therapies that can enhance the tumor-killing response of the host immune system.48 in a phase ii clinical trial nct03047928, the programmed death 1 (pd-1) regulatory antibody nivolumab and a peptide vaccine consisting of programmed death ligand 1 (pd-l1) and indoleamine 2,3-dioxygenase (ido) peptides were tested in patients with metastatic melanoma.51 the primary endpoints for this study was feasibility and safety of the combined treatment. the secondary endpoints were e�cacy and immunogenicity. overall, the study revealed that the vaccine-reactive t cells comprised cd4+ and cd8+ t cells with activity against idoand pd-l1-expressing cancer and immune cells, thus increasing the immunomodulatory e�ect of the combination therapy.51,52 berkeley pharma tech journal of medicine | 55 https://clinicaltrials.gov/ct2/show/nct03047928 there are many other clinical trials currently being conducted to understand the use of combination therapies as a treatment method for di�erent types of cancers including glioblastoma. overall, while further research is needed to fully understand the potential of neoantigen vaccine combination therapies in glioblastoma, early results suggest that these approaches may hold promise as a way to improve outcomes for patients with gbm. 5. practical considerations glioblastoma is a type of brain cancer that is di�cult to treat. novel neoantigen-based treatment methods are being explored as a potential solution to this problem. neoantigens are unique proteins that are expressed on the surface of cancer cells, but not on healthy cells, making them a promising target for cancer treatment.53 here are some practical considerations when exploring novel neoantigen-based treatment methods for glioblastoma: identi�cation of neoantigens: the �rst step is to identify the neoantigens that are present in the glioblastoma cells. this can be done through genomic analysis, transcriptomic analysis, and/or proteomic analysis. the identi�cation of neoantigens is critical for developing a personalized treatment plan. personalized treatment plans: because each patient's glioblastoma is unique, a personalized treatment plan is necessary. this involves identifying the patient's speci�c neoantigens and developing a treatment plan that targets those speci�c neoantigens. delivery methods: one of the challenges of neoantigen-based treatment methods is delivering the treatment to the brain. traditional delivery methods, such as intravenous injections, are not e�ective for brain tumors. therefore, new delivery methods, such as direct injection into the brain, are being explored. immunogenicity: it is important to ensure that the neoantigen-based treatment method is immunogenic, meaning that berkeley pharma tech journal of medicine | 56 it stimulates an immune response. this is critical for the treatment to be e�ective. combination therapies: neoantigen-based treatment methods may be more e�ective when used in combination with other therapies, such as chemotherapy or radiation therapy. therefore, the potential for combination therapies should be considered. clinical trials: clinical trials are necessary to test the safety and e�cacy of neoantigen-based treatment methods. these trials should be designed to address the speci�c challenges associated with treating glioblastoma, such as delivery to the brain and the development of personalized treatment plans. overall, exploring novel neoantigen-based treatment methods for glioblastoma requires careful consideration of several practical factors, including identi�cation of neoantigens, personalized treatment plans, delivery methods, immunogenicity, combination therapies, and clinical trials. 6. conclusion glioblastoma remains a formidable challenge in the �eld of oncology due to its aggressive nature and limited treatment options. however, advancements in immunotherapeutic approaches, such as neoantigen-based vaccines, immune checkpoint blockers, and adoptive cellular therapies, have demonstrated promising e�cacy in repressing glioblastoma tumor cells. our comprehensive review of scienti�c literature and clinical trials has shed light on the potential of these treatment methods and identi�ed areas for improvement to further enhance their e�ectiveness. neoantigen-based vaccines have shown promise in priming the immune system to recognize and attack cancer cells displaying unique neoantigens. protein-peptide vaccines have been utilized to stimulate a speci�c cytotoxic t-lymphocyte (ctl) response against tumor cells expressing targeted neoantigens. the clinical trials conducted by hilf et al. and keskin et al. demonstrated increased overall survival and progression-free survival in berkeley pharma tech journal of medicine | 57 patients who received neoantigen-based vaccines, albeit with some side e�ects. ongoing trials, such as the reardon et al. trial, are investigating the combination of neoantigen-based vaccines with other therapies to assess their synergistic e�ects. nucleic acid vaccines, including dna and mrna vaccines, have also emerged as potential treatment methods. dna vaccines introduce a gene encoding antigens using bacterial plasmids as vectors, while mrna vaccines deliver the antigen-encoding genetic information directly to antigen-presenting cells. clinical trials led by johanns et al. and sayour et al. are evaluating the safety, feasibility, and immunogenicity of personalized neoantigen-based dna and mrna vaccines in glioblastoma patients, respectively. combination therapies with dna vaccines, such as the trial by johanns et al., show promise in enhancing immunogenicity and improving patient outcomes. autologous dendritic cell (dc) vaccines aim to stimulate a speci�c ctl response against cancer cells by presenting neoantigens or tumor-associated antigens to the immune system. the neo-modc vaccine demonstrated immunogenicity and induced tumor regression in a patient with advanced metastatic gastric cancer when administered alongside nivolumab. the �ndings suggest the potential of autologous dc vaccines in treating glioblastoma, but further research is needed to optimize their e�cacy and explore combination strategies. in addition to vaccine methods, immune checkpoint blockade therapy, particularly through the use of monoclonal antibodies, has shown promising results in the treatment of glioblastoma and other cancers. by targeting key signaling molecules such as pd-1, pd-l1, and ctla-4, immune checkpoint inhibitors can suppress immunosuppression and restore anti-tumor responses, leading to improved outcomes for patients. monoclonal antibodies provide a highly speci�c and e�ective way to target disease-causing proteins or antigens. by utilizing their ability to recognize and bind to speci�c targets, monoclonal antibodies can disrupt harmful pathways and stimulate the immune response against cancer cells. various monoclonal antibodies, including nivolumab, ipilimumab, avelumab, and berkeley pharma tech journal of medicine | 58 atezolizumab, have shown e�cacy in glioblastoma and other cancer types. combination therapies, such as the concurrent use of nivolumab and ipilimumab, have demonstrated enhanced t-cell function and signi�cant antitumor responses. these combinations target multiple checkpoints simultaneously, leading to more e�ective immune responses against cancer cells. adoptive cell therapies, such as til-based cell therapies and car t-cell therapy, o�er another avenue for glioblastoma treatment. til-based cell therapies can recognize and kill tumor cells, while car t-cell therapy genetically modi�es a patient's t-cells to enhance their ability to target and attack cancer cells. however, further research is needed to optimize these therapies for solid tumors like glioblastoma. to further advance glioblastoma treatment, several areas of improvement should be considered. first, optimization of sequencing methods, such as whole-genome sequencing (wgs), whole-exome sequencing (wes), and next-generation sequencing (ngs), can enhance the identi�cation of neoantigens and somatic mutations e�ciently in terms of time and cost. additionally, strategies to overcome immune evasion mechanisms employed by glioblastoma, such as upregulation of immune checkpoint molecules, should be explored. combination therapies involving neoantigen-based vaccines, immune checkpoint blockers, and adoptive cellular therapies hold promise in overcoming resistance and enhancing treatment responses. in conclusion, the development and utilization of neoantigen-based vaccines, immune checkpoint blockers, and adoptive cellular therapies have opened new avenues for glioblastoma treatment. while these treatment methods have demonstrated e�cacy, ongoing research and improvements in sequencing techniques, combination therapies, and immunomodulatory strategies are crucial for achieving further breakthroughs. the integration of personalized medicine approaches and a deeper understanding of the complex interplay between the tumor microenvironment and the immune system will contribute to the development of more e�ective and targeted therapies for glioblastoma patients, ultimately improving their prognosis and quality of life. berkeley pharma tech journal of medicine | 59 references 1. wirsching hg, galanis e, weller m. glioblastoma.handb clin neurol. 2016;134:381-397. doi:10.1016/b978-0-12-802997-8.00023-2 2. glioblastoma (gbm) american brain tumor association | learnmore. published august 22, 2022. accessed february 16, 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https://doi.org/10.1038/nm1100 https://doi.org/10.1038/s41591-021-01544-x metastatic melanoma. clinicaltrials.gov; 2022. accessedmay 5, 2023. https://clinicaltrials.gov/ct2/show/nct03047928 53. chen i, chenmy, goedegebuure sp, gillanders we. challenges targeting cancer neoantigens in 2021: a systematic literature review. expert rev vaccines. 2021;20(7):827-837. doi:10.1080/14760584.2021.1935248 berkeley pharma tech journal of medicine | 64 https://clinicaltrials.gov/ct2/show/nct03047928 https://clinicaltrials.gov/ct2/show/nct03047928 https://doi.org/10.1080/14760584.2021.1935248 pappas et al. reference page template berkeley pharma tech journal of medicine correspondence: isabelle.pappas02@gmail.com keywords: ptsd brain development stress trauma adolescents neurodevelopment neuroimaging youth published july 31, 2025 full open access creative commons attribution license 4.0 abstract the developing adolescent brain is particularly susceptible to the effects of stress and trauma which can result in long-lasting morphological and psychological effects. this review paper aims to highlight the important changes caused by childhood stress and trauma, outline current treatments, and provide insight into future research directions. studies suggest that exposure to stressors and traumatic events at a young age increases overall volume in the amygdala while decreasing volume in the hippocampus and frontocortical regions. however, due to a discrepancy in results, there is a need for future studies to control for age-based and trauma differences. additionally, analysis of commonly used treatments reveals that a combination of therapeutic approaches, hormonal treatments, and lifestyle changes is generally most effective to address both underlying mechanisms and psychological effects. ultimately, studies show that adolescent stress and trauma leads to significant hormonal and morphological changes in the brain that result in psychological changes such as anxiety and depressive symptoms. adolescent brain development under stress and trauma: evidence-based treatments by: isabelle pappas, tzung yu, olivia liu and nainika srinivasan 1. introduction trauma is defined as an experience that triggers intense physical and psychological stress reactions, including heightened arousal, intrusive thoughts, and emotional numbness. the stress induced by trauma releases hormones like cortisol which can disrupt bodily systems and is associated with increased risk of anxiety, depression, and post-traumatic stress disorder (ptsd).1 researchers often model trauma in test subjects using chronic variable or restraint stress, enabling the study of these impacts.2 a severe outcome of trauma is ptsd, characterized by abnormalities in frontolimbic circuitry that result in increased threat sensitivity and reduced emotional regulation.3 ptsd is linked to poor academic performance, higher rates of depression, suicide attempts, and substance abuse.3 in adults, ptsd manifests through symptoms like flashbacks, avoidance, hyperarousal, and mood changes.4 adolescent development is a time of significant psychological and physiological vulnerabilities. for instance, there are significant volumetric increases in the hippocampus and amygdala in the early stages of puberty.2 furthermore, there are periods of cortical thinning in the frontal and temporal cortical volumes during adolescence following the initial increase in volume during childhood.2 these are regions intimately involved in emotional and cognitive processes, and it can be inferred that alterations to the developmental process of these brain regions would affect the emotional and cognitive abilities of the individual; however, the precise nature of this structure-function relationship in the adolescent brain is unclear. 5 in animal models, particularly mouse models, adolescence is also marked by increased hippocampal volume and vulnerability to stress, making this period of development particularly vulnerable to the morphological changes that result from stress and trauma. mouse models are particularly useful because of their genetic similarity to humans and the ability to control their environmental factors. exposure to stressful stimuli in adolescent mice (between 5 and 7 weeks old) results in short term as well as long term morphological changes in their brains, suggesting similar results for berkeley pharma tech journal of medicine | 37 https://www.zotero.org/google-docs/?9diot8 https://www.zotero.org/google-docs/?6tsmlw https://www.zotero.org/google-docs/?qpoue5 https://www.zotero.org/google-docs/?ojzbq4 https://www.zotero.org/google-docs/?b18z0q https://www.zotero.org/google-docs/?sckoog https://www.zotero.org/google-docs/?c5u2in https://www.zotero.org/google-docs/?js7v5k adolescent humans.6 behaviorally, mice exhibit impaired cognitive functions and social withdrawal, which mirror the psychological effects of stress in adolescent humans. thus, mouse models can allow researchers to identify biological indicators of disorders caused by stress. additionally, the mouse models can aid physician scientists in developing the most effective treatment plans to mitigate the adverse effects of stress and trauma. the impact of trauma is most pronounced during adolescence, affecting critical brain regions at specific ages: the hippocampus at 14, the amygdala between 10 and 11, and the prefrontal cortex between 14 and 16.3 adolescents exhibit higher stress reactivity, with stress hormones like acth and corticosterone taking longer to normalize post-stress compared to adults.2 this necessitates study and prevention of abuse and trauma for adolescents due to the vulnerable nature of that time period and the long term effects these changes can have on the psychology of these individuals. figure 1. physical changes characteristic of adolescence in humans. berkeley pharma tech journal of medicine | 38 https://www.zotero.org/google-docs/?ylo0ox https://www.zotero.org/google-docs/?kyo530 https://www.zotero.org/google-docs/?zyug2f figure 2. physical characteristics of mice (left) vs. human (right) brains. mice models are commonly used for the studies reviewed in this paper. 2. morphological and psychological effects 2.1 amygdala the amygdala, critical for emotional reactivity and cognitive processes such as memory, possesses a high concentration of glucocorticoid receptors that causes it to be susceptible to stress.7,8 during postnatal development, the amygdala undergoes a period of rapid growth which peaks during ages 9-11 and diminishes.9 these changes highlight the importance of the amygdala in studies regarding adolescent stress and trauma. childhood trauma and adversity have been found to be correlated with heightened amygdala reactivity to negative stimuli in functional magnetic resonance imaging (fmri) studies of both healthy and psychiatric youth.10–12 in addition, amygdala hyperactivation in youth with ptsd compared to healthy youth has also been reported, similar to the hyperactivation in adult ptsd.11,13–15 on the other hand, many studies also berkeley pharma tech journal of medicine | 39 https://www.zotero.org/google-docs/?cum479 https://www.zotero.org/google-docs/?d6xv1j https://www.zotero.org/google-docs/?z1q2ad https://www.zotero.org/google-docs/?oewurs report no difference in amygdala hyperactivation.16–18 this contrast in findings may be due to a delayed expression of amygdala hyperactivation until adulthood, as shown when analyzing amygdala activation using age-related differences demonstrates that typically developing youth show decreased activation with age whereas youth with ptsd have increased activation with age.19 the same authors established that amygdala activation in youth with ptsd was lower at ages below 15 years compared to healthy youth, possibly due to younger children compensating for stress by downregulating amygdala activity, but this becomes less effective as they age. amygdala hypertrophy caused by childhood trauma is expressed in adulthood. in a longitudinal sample, adult subjects exposed to maltreatment had higher right and left amygdala volumes than healthy controls.20 furthermore, by using the maltreatment and abuse chronology of exposure scale, it was revealed that the right amygdala undergoes a sensitive period during 10-11 years, becoming prone to enlargement from exposure to maltreatment.21 interestingly, even small amounts of maltreatment in healthy controls triggered hypertrophy during this sensitive period, despite not meeting the threshold for moderate exposure. left amygdala volume was also shown to be correlated to attachment disruption at 18 months.1 this underscores the susceptibility of the amygdala to stress and trauma during youth development and highlights the need for further understanding in this topic. in rats, early life stress exposure led to increased amygdala volume and reactivity for prolonged periods of time.22–24 developing rats exposed to restraint stress exhibited an increase in dendrite complexity and frequent, spontaneous neuron firing in the basolateral amygdala which resulted in amygdala hypertrophy.25,26 further studies found that exposure to psychological stress or stress hormone lead to increased excitability and spine formation on pyramidal cells, a basis for exhibiting anxiety-like behaviors.27–29 additionally, studies have shown that development of threat learning occurred earlier in rodents exposed to early life stress compared to the controls.30 this evidence supports previous trials indicating an association with early life stress and enhanced threat bias in youth.23 these findings suggest that hyperreactive amygdala in adolescents exposed to berkeley pharma tech journal of medicine | 40 https://www.zotero.org/google-docs/?knrdja https://www.zotero.org/google-docs/?kg4g2o https://www.zotero.org/google-docs/?hdnant https://www.zotero.org/google-docs/?hnqqb4 https://www.zotero.org/google-docs/?ztrsw0 https://www.zotero.org/google-docs/?dnoghg https://www.zotero.org/google-docs/?dmbdpl https://www.zotero.org/google-docs/?zvhxyc https://www.zotero.org/google-docs/?2yrzq5 https://www.zotero.org/google-docs/?omwmta trauma and stress may be of evolutionary advantage to detect threat. similarly, chronic restraint stress during adolescence also resulted in short-term difficulties unlearning fear responses, such as freezing, which disappeared in the long term.26,31 however, when adolescent and adult rats were exposed to corticosterone as a chronic stressor, only adolescents had impaired extinction retention, showing the importance of sensitive periods during development.32 2.2 hippocampus the hippocampus, which plays key roles in memory, learning, and emotion, grows rapidly during adolescence.33 additionally, this region is densely populated with glucocorticoid receptors, causing it to be highly susceptible to the increased levels of glucocorticoids due to childhood maltreatment.34 studies have shown that high exposure to glucocorticoids can lead to changes in hippocampus regions, such as reversible atrophy of dendritic processes in the cornu ammonis and suppression of neurogenesis in the dentate gyrus.35 due to the rapid development and sensitivity to stress hormones of the hippocampus, this region is important in our understanding of the effects of childhood trauma and stress. current literature proposes that childhood stress and trauma are associated with lower volume of the hippocampus, aligning with the reduced hippocampal volume present in adult ptsd.36 one study on patients with childhood ptsd concluded a significant 12% reduction in left hippocampal volume compared with typically developing controls.37 another paper analyzing structural mri images determined that childhood trauma was associated with less gray matter in the hippocampus.38 however, some report that hippocampal volume is not altered between youth with ptsd and typically developing youth. for example, multiple studies have found no significant reduction in hippocampal volume in children with ptsd.39–41 one possible explanation for the discrepancy between these studies is that, similar to the amygdala, the hippocampus has delayed onset of reduced volume. for instance, analyses show that subjects in studies reporting no significant reduction had a mean age of 11.26 years whereas studies reporting significant hippocampus reduction had a mean age of berkeley pharma tech journal of medicine | 41 https://www.zotero.org/google-docs/?9kwozy https://www.zotero.org/google-docs/?5ysh3i https://www.zotero.org/google-docs/?sqeefo https://www.zotero.org/google-docs/?jxbspw https://www.zotero.org/google-docs/?dr6usj https://www.zotero.org/google-docs/?ozh8jn https://www.zotero.org/google-docs/?feulwx https://www.zotero.org/google-docs/?s6hotc https://www.zotero.org/google-docs/?4ufr4c 12.65 years, suggesting that there is a silent period between maltreatment and neurobiological effects.42 additionally, when analyzing differences using age-related differences, hippocampal volume increased in typically developing youth but decreased in ptsd youth with age.3 rodent studies have also shown impaired hippocampus growth. one study reported that chronic restraint stress on both male and female adolescent rats resulted in lowered dendrite complexity of pyramidal neurons in the hippocampus compared to controls.25 rats exposed to stress also exhibited depressive behaviors. in another study, male rats that were exposed to chronic stress during adolescence exhibited short-term increase in hippocampal growth in the ca1 region only.43 however, a later examination revealed a significant long-term decrease in ca1 and dg regions and ca3 growth arrested. these structural changes correlated with impaired abilities in navigation which demonstrates that adolescent stress can significantly change brain structure and function in the long-term. 2.3 frontocortical regions 2.3.1 prefrontal cortex the prefrontal cortex (pfc) is a region of the brain that controls complex processes such as emotion, thought, and actions. studies have analyzed mri images and found that gray matter in the frontal lobe increased during adolescence.44 growth peaked at 12.1 years for males and 11.0 years for females then decreased during post-adolescence. additionally, past literature has stated that the pfc is particularly vulnerable to stress-induced morphological changes which makes it important to understand changes in the pfc from stress and trauma in developing youth.45 youth with ptsd have shown a decreased volume in the pfc compared to typically developing youth. for example, multiple articles found decreased gray matter volume in the right ventromedial prefrontal cortex (vmpfc), bilateral ventrolateral prefrontal cortex (vlpfc), dorsomedial prefrontal cortex (dmpfc), and dorsolateral prefrontal cortex (dlpfc)11,46,47 while other studies report increased gray matter in the vmpfc, vlpfc, dmpfc, and dlpfc in individuals with childhood trauma.16,17,19,38,48 one possible explanation for this disparity could be differences in sample ptsd severity, berkeley pharma tech journal of medicine | 42 https://www.zotero.org/google-docs/?cukehs https://www.zotero.org/google-docs/?kem4kh https://www.zotero.org/google-docs/?xlwbtd https://www.zotero.org/google-docs/?r7gqaj https://www.zotero.org/google-docs/?yskbla https://www.zotero.org/google-docs/?i1ssyy https://www.zotero.org/google-docs/?roxxpl https://www.zotero.org/google-docs/?thneg4 trauma-related factors, or age-related differences. studies that report a decrease in gray volume also note that lower left ventral and left inferior prefrontal matter are associated with higher cortisol levels.49 additionally, childhood ptsd was found to be associated with loss of neuronal integrity in the pfc.47 these results demonstrate the need to further investigate the underlying mechanisms and contributing factors to better understand the impact of ptsd on brain development and to develop targeted intervention strategies. studies using rodent models have achieved similar results. when chronic restraint stress is applied to adolescent rats, those exposed to stress had reduced pfc dendrite complexity in pyramidal neurons.25 the same protocol combined with early weaning caused rats to show decreased pfc neuronal activity in stress-inducing environments.50 furthermore, social isolation rearing led to decreased spine density, dendritic branching in pyramidal neurons, and volume in the pfc.51–54 exposure to isolation stress also resulted in lasting lower concentrations of proteins spinophilin, synaptophysin, and myelin basic protein in the pfc of adolescent rats.55 post-adolescent rats that were exposed to isolation stress in childhood exhibited down-regulation of immediate early genes and genes that regulate differentiation and apoptosis in the medial pfc.56 these genetic changes were associated with the degree of hyperlocomotion and social isolation in stressed rats which offers a molecular basis for anxiety-like behaviors expressed in individuals exposed to stress and trauma. 2.3.2 anterior cingulate cortex the anterior cingulate cortex (acc) regulates emotions, motivation, cognition, and motor abilities during conflict. due to the importance of the acc in body processes, it is crucial for us to further understand the effects of stress and trauma on its development during adolescence. previous studies have found that the dorsal acc becomes hyperactivated when exposed to threat and emotional pictures in youth with ptsd.18,19 however, this contrasts with dorsal acc becoming hypoactivated in adults with ptsd which could be due to overcompensating for emotional regulation during adolescence which fades with age.57 moreover, reduced gray matter in the acc has been associated with maltreatment during berkeley pharma tech journal of medicine | 43 https://www.zotero.org/google-docs/?3mj8yq https://www.zotero.org/google-docs/?rivk8b https://www.zotero.org/google-docs/?chj3sj https://www.zotero.org/google-docs/?xykbxu https://www.zotero.org/google-docs/?yumjbu https://www.zotero.org/google-docs/?kk1wbn https://www.zotero.org/google-docs/?nhcqad https://www.zotero.org/google-docs/?ciqukf https://www.zotero.org/google-docs/?1xxupi adolescence.58 since acc volume has been correlated with depressive symptoms, there is an urgent need for more understanding of trauma effects on this region of the brain.59 2.4 corpus callosum the corpus callosum connects the left and right brain hemispheres and allows them to communicate and coordinate actions. studies show that children who have ptsd or suffered abuse have a smaller corpus callosum than controls.4 for example, multiple studies have reported an association between maltreatment and a significant reduction in corpus callosum volume, specifically higher in adolescent male samples than females.39,60–63 this may be due to males having a larger sensitive period lasting throughout infancy and early childhood. notably, one study reported that at corpus callosum segments ii and iii, adolescents with low risk of mental disorders had the greatest fractional anisotropy followed by controls then adolescents with high risk.64 these results demonstrate the possibility that corpus callosum size may indicate vulnerability to mental disorders. however, analysis on the bucharest early intervention project demonstrated that significant reductions in the corpus callosum were mitigated when orphans were placed in foster care compared to those who remained in institutions.65 these findings are particularly impactful since they demonstrate the potential irreversibility of the damage to the corpus callosum caused by stress and trauma. 2.5 larger scale effects 2.5.1 hypothalamic-pituitary-adrenal (hpa) axis during adolescence, the hypothalamic-pituitary-adrenal (hpa) axis undergoes significant growth and developmental changes. the hpa axis is intricately intertwined with the hypothalamic-pituitary-gonadal (hpg) axis which releases more gonadal steroids, triggering the onset of puberty. hpa activity is influenced by the hpg axis, resulting in heightened activity along with an increase in mineralocorticoid and glucocorticoid receptors in the hippocampus during this particular period of development.66 basal activity of the hpa axis, characterized as the resting rate of hormones associated with this axis, increases during adolescence. this increase in basal berkeley pharma tech journal of medicine | 44 https://www.zotero.org/google-docs/?ayuqqq https://www.zotero.org/google-docs/?agyyka https://www.zotero.org/google-docs/?r8hq8t https://www.zotero.org/google-docs/?gx6bdx https://www.zotero.org/google-docs/?rbctxl https://www.zotero.org/google-docs/?hdar84 https://www.zotero.org/google-docs/?1taylz activity results in hypersensitivity and reactivity to stimuli, which can make adolescents particularly vulnerable to traumatic experiences.67 in rodent models, stress results in higher levels of adrenocorticotropic hormone (acth) and cortisone which last longer during periods of adolescence than during adulthood. studies have revealed sex-specific changes in the hpa axis due to stress in adolescent rodent models. for example, “males that have been subjected to adolescent isolation have lower corticosterone responses to restraint stress compared with controls, whereas females develop greater restraint-induced corticosterone responses.”67 additionally, rats that are exposed to repeated stressful environments, such as prolonged isolation, exhibit an increase in hormonal reactivity after being paired with a foreign cagemate. prolonged exposure to stress can also lead to habituation or desensitization of the hpa axis in rodent models, which suggests that the same can occur in adolescent humans under conditions of chronic stress and trauma. maladaptive changes, such as hyperactivity or subdued activity of the hpa axis, can make adolescents more vulnerable to mental health disorders, such as anxiety and depression. 2.5.2 the dopaminergic system the dopaminergic system is involved in the regulation of motor, cognitive, and motivational brain regions.68 furthermore, dopamine (da) modulates emotional processing at multiple levels, which affects the amygdala, medial temporal lobe, and prefrontal cortex.69 the dopaminergic system has three main dopaminergic pathways, affecting regions of the brain spanning the prefrontal cortex and midbrain.70 in rats, the development of this system has several maturation steps. firstly, the activity and levels of da and 3,4-dihydroxyphenylacetic acid rises in the nucleus accumbens and striatum at postnatal day (pd) 28, which remains constant until adulthood 71. da synthesis and turnover increases in the pfc at approximately pd 30 but decreases after several days.72–75 da fibers mature earlier in the striatum and nucleus accumbens (~pd 35) compared to projections in the pfc.76–78 d1 and d2 receptor densities peak at pd 28 in the nucleus accumbens and striatum, and peak in the pfc at around pd 40-60.78–83 berkeley pharma tech journal of medicine | 45 https://www.zotero.org/google-docs/?qkuna2 https://www.zotero.org/google-docs/?mkpy8s https://www.zotero.org/google-docs/?oqspw6 https://www.zotero.org/google-docs/?ruhk9t https://www.zotero.org/google-docs/?pnz8lo https://www.zotero.org/google-docs/?imvuc5 https://www.zotero.org/google-docs/?uwy2rd https://www.zotero.org/google-docs/?wzdlmf https://www.zotero.org/google-docs/?zkwtpd there are changes to the da system during adolescence that make it particularly sensitive to the effects of stress.67 for example, extracellular da levels in the dorsal and ventral striatum are lower during adolescence, while da activity in the pfc peaks during adolescence.84 this can also be seen when rats were subjected to social isolation rearing, where several effects on the da system were observed. there was an increase in the in vivo firing rate and burst-like activity of putative da neurons in the ventral tegmental area (pd 25-90),85 da release and da transporter activity was increased in the nucleus accumbens and dorsomedial striatum (pd 28-77),86 and da innervation and turnover decreased in the mpfc (starting from pd 21-28).76,87,88 furthermore, during adolescent social isolation rearing, da activity and turnover increased in the nucleus accumbens.76,77,89,90 da levels declined in the pfc in adulthood after repeated prolonged social isolation episodes in periadolescent mice (pd 15-21).91 the predator odor model in adolescent mice also showed decreased levels of d2 receptors in the mpfc.92 there was also lower basal tissue and extracellular da levels and upregulated da transporters in the pfc as a result of social defeat stress.93–96 restraint stress and footshock increased responsivity of vta da neurons in adults when adolescent rats (pd 31-40) were exposed to these stressors.97 lastly, da levels in the mpfc were elevated in adulthood as a result of chronic variable physical stressors during adolescence (pd 27-33).98 2.5.3 default mode, salience, and central executive networks recent research has highlighted the significant impact of larger brain networks in ptsd, extending the frontolimbic model to include key networks such as the default mode network (dmn) for self-referential thought, the salience network (sn) for detecting relevant cues, and the central executive network (cen) for goal-directed behavior and emotion regulation.99 typically, the dmn and cen function in opposition, switching between internal processing and external tasks. in adult ptsd, studies show increased sn activity, decreased dmn and cen activity, and poor regulation between the cen and dmn, which may explain symptoms like hypervigilance and poor emotion regulation.100 berkeley pharma tech journal of medicine | 46 https://www.zotero.org/google-docs/?dsicvh https://www.zotero.org/google-docs/?kibqsd https://www.zotero.org/google-docs/?6m1ipr https://www.zotero.org/google-docs/?epsxuv https://www.zotero.org/google-docs/?apohwq https://www.zotero.org/google-docs/?ula038 https://www.zotero.org/google-docs/?kzpdrg https://www.zotero.org/google-docs/?wdnvpb https://www.zotero.org/google-docs/?6oec9o https://www.zotero.org/google-docs/?quqaw1 https://www.zotero.org/google-docs/?agzyni https://www.zotero.org/google-docs/?epgjw0 https://www.zotero.org/google-docs/?7p9und pediatric ptsd research suggests a hyperactive sn and decreased cen engagement as children age, though large-scale network function studies in this group are limited. initial research in youth with ptsd indicates increased dmn connectivity and greater anti-correlation between the dmn and cen/sn compared to adults.101 stronger cen/sn connectivity correlates with fewer re-experiencing symptoms, suggesting compensatory use of executive control systems, which might also contribute to dissociative symptoms. however, additional research is required to understand network function in pediatric ptsd and its development over time. during acute stress, connectivity increases between dmn and cen regions while decreasing between the sn and both dmn and cen.102 greater polyvictimization is associated with reduced connectivity between the dmn and the left insula of the sn, potentially disrupting emotion regulation. further studies are necessary to clarify these dynamics and their impact on ptsd symptoms in youth. 2.6 brain region connectivity 2.6.1 changes and importance of neuroplasticity neuroplasticity is the ability for the brain to form and reorganize synaptic connections following learning, experience, or injury. this is particularly important during development since stress strongly impacts social interactions and behaviors in developing adolescents, magnifying negative effects on neuroplasticity due to social isolation.103 studies have found stress to impact the ability to develop neurons during adolescence. in two experiments, female rats exposed to chronic restraint stress or social instability during adolescence exhibited lower levels of neurogenesis in contrast to another study that reported increased neurogenesis in males.104–106 interestingly, levels of brain-derived neurotrophic factor as well as neurogenesis was reduced in mildly-stressed adult animals but increased in adolescents.106 due to this discrepancy, more studies are needed to determine the short and long-term effects of stress and trauma on neuroplasticity in male and female adolescents. 2.6.2 alterations in brain region connectivity berkeley pharma tech journal of medicine | 47 https://www.zotero.org/google-docs/?hgxncw https://www.zotero.org/google-docs/?0g8ck7 https://www.zotero.org/google-docs/?9b1cbs https://www.zotero.org/google-docs/?vsktr6 https://www.zotero.org/google-docs/?ddblqm previous studies have found changes in connectivity between various brain regions in adolescents exposed to stress and trauma. for example, less coupling occurred between the amygdala and the acc/dmpfc in youth with ptsd, which is also inversely associated with the severity of ptsd.18,19,101 these results are of interest because adolescents exposed to adversity that downregulate amygdala and acc/dmpfc coupling exhibit depressive and anxiety symptoms during late adolescence.57 furthermore, coupling between the amygdala and the vmpfc decreased in youth with ptsd in contrast to increases shown in typically developing youth.18 however, similar to discrepancies discussed previously, age-related differences can also be seen. compared to typically developing youth, younger adolescents with ptsd show greater connectivity between the amygdala and vmpfc which reverses as they age.18 overall, when accounting for age differences, amygdala and acc/pfc connectivity has been shown to decrease in adolescents exposed to stress and trauma. additionally, studies have shown that adolescent ptsd patients have decreased connectivity between the vmpfc to amygdala and vlpfc to hippocampus during development compared to healthy youth.47 in the posterior cingulate cortex (pcc) of healthy individuals, activity was correlated with activity in regions of the default network such as the mpfc, precuneus, lateral parietal cortices, inferior and middle temporal cortices, thalamus, and cerebellum.107 however, patients with early life trauma expressed only correlation between the pcc and right superior frontal gyrus and left ventrolateral thalamus. additionally, connectivity between the pcc and precuneus, right amygdala, right hippocampus, mpfc, right insula, and bilateral lateral parietal cortex was higher in healthy controls than those with early life trauma. these results suggest that early life trauma may strongly impact development of the right hemisphere and the default network. berkeley pharma tech journal of medicine | 48 https://www.zotero.org/google-docs/?llwsl8 https://www.zotero.org/google-docs/?8m5bjl https://www.zotero.org/google-docs/?li3fkn https://www.zotero.org/google-docs/?uqxcxj https://www.zotero.org/google-docs/?jwlxhh https://www.zotero.org/google-docs/?r9pad5 (a) (b) figure 3. morphological effects of stress and trauma on the adolescent (a) outer brain regions and (b) inner brain regions. berkeley pharma tech journal of medicine | 49 figure 4. physiological effects of stress and trauma on adolescent brain connections. 3. discussion 3.1 overall effects adolescence is a period of significant volumetric increases in the hippocampus and amygdala, cortical thinning in the frontal and temporal lobe, as well as heightened sensitivity to stress. this is mirrored by the time periods in which different parts of the brain are the most vulnerable to change due to abuse as mentioned above, exacerbating the importance of the study, prevention, and treatment of trauma and ptsd of adolescents. the physiological changes to the brain caused by trauma present differently depending on parts of the brain, but they largely affect neural networks that are related to emotional processing and memory. it is worth noting that many of the studies investigating this topic have been unable to differentiate the effects of different kinds of trauma on the developing brain, as it is often the case that youth with ptsd are subject to multiple and repeated traumas instead.3 furthermore, many symptoms common to adults with ptsd like reduced hippocampal volume and hyperactivity of the amygdala and insula do not consistently present themselves in adolescents with ptsd. this is due to the increased stress sensitivity of the developing neural system, as well as delayed developmental effects.3 berkeley pharma tech journal of medicine | 50 https://www.zotero.org/google-docs/?awbd4i https://www.zotero.org/google-docs/?ueuxkd ptsd in youth exhibits changes to the frontolimbic circuits that contribute to the psychological changes like increased threat reactivity and reduced emotion regulation capacity as they age, which can persist well into adulthood.2,3 it has commonly been speculated that these changes to the brain caused by trauma and ptsd are adaptations to aversive and threatening environments, as the neural circuits affected by trauma in adolescents affect the brain’s capacity to detect threat, threat extinction, and threat reactivity.3,47 in particular, the increased volume and activity of the amygdala, as well as the abnormal connectivity between the amygdala and different parts of the prefrontal cortex are indicative of early maturation of these processes, at the potential cost of sustained hypervigilance and an inability inhibiting threat responses. this ultimately leads to an improved automatic detection of threat, increased responsivity of the hypothalamic-pituitary axis stress response, impaired threat regulation, etc.3 there are many parts of the pfc that get affected as a result of early life stress and trauma, including the dorsolateral pfc, ventrolateral pfc, ventromedial pfc, precentral gyrus, and dorsomedial pfc. as stated above, there are discrepancies in how trauma affects gray matter volume in these areas, which seem to be due to differences in age-related factors and trauma/ptsd severity. the abnormal development of the pfc also showed evidence of decreased intrinsic connectivity with the amygdala and hippocampus over time. furthermore, there has been mixed evidence on the activation of these parts as a result of ptsd, including the ventrolateral pfc, ventromedialpfc/rostral acc, and dorsomedial pfc. there are many possible reasons for this, including task differences, trauma-related factors, sex differences, and age differences in the studies. a particularly important region of the pfc for emotional processing is the ventromedial pfc. the ventromedial pfc has been associated with the top down modulation of the amygdala responses and threat response inhibition. abnormal development of the ventromedial pfc and reduced ventromedial pfc-amygdala coupling we’ve seen in both youth and adults with ptsd contributes to the reduced threat extinction and negative emotional processing capabilities of these youth.3,47 berkeley pharma tech journal of medicine | 51 https://www.zotero.org/google-docs/?cx10ht https://www.zotero.org/google-docs/?hhxlm6 https://www.zotero.org/google-docs/?mqapip https://www.zotero.org/google-docs/?3jjyv6 adult ptsd is characterized in the hippocampus with reduced volumes. however, this is not consistently found in youth with ptsd.47 a possible reason for this is the use of selective serotonin reuptake inhibitors (ssris) for the treatment of ptsd, which has been shown to increase hippocampal neurogenesis and increase hippocampal gray matter volume in adults with ptsd.47 this suggests that the use of ssris can counteract the effects of trauma on the hippocampus by preventing the reduction of hippocampal volume. another reason is the delayed developmental effect mentioned above, where the physiological effects of trauma take time to present themselves. both the ventrolateral pfc and dorsolateral pfc have shown decreased connectivity with the anterior hippocampus in adolescents with ptsd. this is significant as the anterior hippocampus has many projections into the amygdala, and is involved in unconditioned threat responses. both of these pfc regions are heavily involved in emotional processes; the ventrolateral pfc is associated with selection and inhibition of cognitive appraisals, while the dorsolateral pfc is associated with explicit emotion regulation through cognitive reappraisal.47 it can be hypothesized that this reduction in connectivity between the two parts of the pfc and the anterior hippocampus can result in a loss of inhibitory control of unconditioned threat responses, increased threat acquisition, and impaired threat extinction. however, the study by heyn et al. was unable to confirm this, and further study including threat learning and emotion regulation is required.47 the physiological changes in the hippocampus as a result of stress doesn’t just affect the emotional and threat responses of the individual, but spatial memory and navigation as well. this is evident by the reduced spatial navigation capabilities in rats tested using the morris water maze, which coincides with reduced volumes of the cornu ammonis 1, 3, and dentate gyrus.2 these reductions in volume in different parts of the hippocampus as a result of stressors shows that early life trauma does not just affect the emotional and threat responses of individuals, but other parts of mental capacity as well, such as spatial navigation and memory. berkeley pharma tech journal of medicine | 52 https://www.zotero.org/google-docs/?ogcuq6 https://www.zotero.org/google-docs/?slb9lj https://www.zotero.org/google-docs/?8hehfb https://www.zotero.org/google-docs/?vgz6vu https://www.zotero.org/google-docs/?bvcgym overall, the physiological changes in adolescent brains caused by trauma contribute to the symptoms of ptsd that persist through adulthood, such as improved threat detection, impaired threat extinction, and reduced negative emotion regulation. these changes include abnormal gray matter volumes and connectivities throughout different parts of the pfc, amygdala, and hippocampus. these changes do not immediately present themselves, however, and can take many years to appear. furthermore, these changes affect not only the emotional and threat processing circuits, but other brain regions responsible for thinking and memory as well. these effects of these physiological changes on the brain and emotional capabilities on the individuals highlight the importance of studying, treating, and prevention of trauma in adolescents. 3.2 therapeutic treatments the most effective treatments for adolescents who have experienced stress and trauma include a combination of therapeutic, hormonal, and lifestyle changes. medication alone may not be effective in treating a patient with severe psychological illness, but medication combined with therapy may be. cognitive behavioral therapy (cbt) is one of the most effective methods for helping individuals cope with trauma. cbt focuses on the interplay between thoughts, feelings, and behaviors. this therapy helps individuals challenge negative thought patterns and develop problem-solving skills. cbt is particularly desirable for adolescents because it equips them with the skills to manage distressing behaviors and emotional reactions. trauma-focused cognitive behavior therapy (tf-cbt) is a subset of cbt designed to help those who have experienced significant trauma in their lives. this form of therapy often focuses on controlled exposure to emotions associated with the trauma, the creation of a trauma narrative, and relaxation techniques.108 tf-cbt is a short-term treatment, lasting up to 16 sessions of this type of therapy, and its main focus is to address post traumatic stress. tf-cbt relies on the individual’s ability to master the therapeutic techniques learned during the session in order to practice them routinely after the sessions have ended.109 a meta-analysis of published and berkeley pharma tech journal of medicine | 53 https://www.zotero.org/google-docs/?uifpvf https://www.zotero.org/google-docs/?uqrft5 unpublished data regarding the efficacy of cbt for ptsd in adolescents found that “participants who received cbts-tf had lower mean post-traumatic stress symptoms after treatment than those who received the control conditions, after adjusting for post-traumatic stress systems before treatment.”110 according to an article by evgenia gkintoni et al. assessing the efficacy of different therapeutic interventions for ptsd in adolescents: “among the array of therapeutic interventions analyzed, cbt and specifically tf-cbt emerge as the most effective and extensively employed methodologies for addressing ptsd in children and adolescents.”111 eye movement desensitization and reprocessing (emdr) is another popular form of therapy used to treat post traumatic stress. emdr is based on the adaptive information process (aip) model of storing memories, which suggests that some traumatic events can remain “unprocessed” by the brain and stay “stuck” in the body, resulting in negative emotions and adverse responses to similar situations as the traumatic event. by having the individual “focus on external stimuli” while recalling these traumatic events, emdr aims to desensitize the individual to their own traumatic experiences.112 emdr helps to mitigate the effects of stress and trauma on the brain by using bilateral stimulation, in the form of guided eye movements, while the brain recalls traumatic events. the eye movements are thought to stimulate the information processing region of the brain, which enables the reprocessing of traumatic memories into less distressing ones. bilateral stimulation allows the participant to reconstruct a more coherent traumatic narrative, reducing the emotional impact of the event itself.113 significant research suggests that emdr is beneficial for minimizing the effects of ptsd by desensitizing the participant, allowing the individual to think about their traumatic event without inciting an intense emotional response.114 while emdr was originally meant to be used on adults, the therapy has been relatively recently extended to help children of all ages. emdr would be beneficial for adolescents who have experienced any sort of trauma because it is a holistic approach to healing and provides them with the tools to manage distressing emotions that may arise in their future. 3.3 hormonal treatments berkeley pharma tech journal of medicine | 54 https://www.zotero.org/google-docs/?7yucuy https://www.zotero.org/google-docs/?evifqw https://www.zotero.org/google-docs/?sspp8r https://www.zotero.org/google-docs/?ggqwsk https://www.zotero.org/google-docs/?yvm46q in addition to employing a myriad of therapeutic treatments, adolescent trauma survivors can also take medication to alleviate the psychological symptoms of ptsd. the hormonal treatments focus on addressing the dysregulation of the hpa axis which is often disrupted in adolescents who have experienced stress and trauma. most recently, glucocorticoid-therapy based treatments have become popular for treating ptsd in individuals of all ages.115 the glucocorticoids are used to normalize levels of cortisol, commonly known as the stress hormone, in order to mitigate the physiological and psychological effects of stress on the individual. glucocorticoids are thought to prevent the overactivation of the hpa axis which is commonly seen in individuals suffering from ptsd.116 glucocorticoids impair memory consolidation and impairs the retrieval of adverse memories. glucocorticoids have been used both as a treatment and preventative method for individuals that may be more susceptible to developing ptsd.115 although the use of glucocorticoids presents promising results, there are some negative side effects that should be taken into consideration when choosing the most effective treatment plan for the patient. some side effects of glucocorticoid use are insomnia, edema, and hypomania. these side effects, coupled with the daily struggles of adolescence, may make this relatively new form of therapy not entirely desirable for this age group. it may be a few years before biochemists and physician-scientists refine the treatment approach for this therapeutic drug, but the potential for glucocorticoids to prevent and treat ptsd is there.117 psychedelic drugs, such as ketamine and mdma, are being used as alternative hormonal therapies to treat ptsd and other stress-related disorders. ketamine is a dissociative anesthetic which acts on the brain’s n-methyl-d-aspartate (nmda) receptors involved in mood regulation and synaptic plasticity. ketamine infusions are intravenous and have been most widely used to treat major depressive and anxiety disorders, specifically in previously treatment-resistant patients.118 there is a current clinical trial entitled “ketamine-assisted psychotherapy for adolescents ptsd (kap)” that aims to investigate the efficacy of ketamine infusions for adolescents diagnosed with ptsd. the study is set to commence in august 2024 and conclude a year later. participants will receive three intravenous ketamine administrations before receiving psychotherapy sessions over a six week berkeley pharma tech journal of medicine | 55 https://www.zotero.org/google-docs/?fam0b4 https://www.zotero.org/google-docs/?lilunf https://www.zotero.org/google-docs/?syilw6 https://www.zotero.org/google-docs/?att3dp https://www.zotero.org/google-docs/?wu2quo course. then, participants’ sleep will be monitored using smartsleep eeg recording headset for seven consecutive days. the researchers hypothesize that, following the ketamine infusion therapy, the participants’ ptsd symptom severity will decrease.119 as opposed to psychedelics, selective serotonin reuptake inhibitors (ssris) are more widely accepted drugs that have been used since the late 1980s to treat major depressive disorders and anxiety, which can result from complex trauma and stress in adolescents. ssris function by increasing the amount of serotonin in the brain, a neurotransmitter responsible for feelings of happiness and mood regulation. ssris can often mitigate the symptoms of depression and anxiety by reducing the intensity of emotions and emotional responses.120 some ssris include sertraline (zoloft), paroxetine (paxil), and fluoxetine, but only sertraline and paroxetine are currently fda approved as treatments for ptsd.121,122 ssris are most effective when used alongside other therapeutic treatment methods, like cbt or emdr. additionally, this hormonal treatment is appealing to adolescents suffering from ptsd because it stabilizes mood and regulates complex emotions that arise from traumatic experiences. that being said, ssris can also have adverse side effects, such as significant weight gain and changes in appetite which can be particularly harmful to adolescents who already struggle with body dysmorphia and/ or disordered eating as a result of the stress and trauma. finally, the use of ssris can result in dependency, which makes tapering off them an added challenge for adolescents who may not want to take them long term.123 in addition to ssris, alpha-adrenergic blockers (prazosin) is also an effective hormonal treatment for ptsd in adolescents. alpha-adrenergic blockers, also known as alpha-1 adrenergic receptor antagonists, block alpha-receptors on cells within the body, which can be used to treat high blood pressure and stress-related disorders like ptsd. within the scope of this article, alpha-adrenergic blockers limit adrenergic activity which reduces the body’s stress response. research suggests that alpha-adrenergic blockers are effective at minimizing hyperarousal symptoms associated with ptsd which can manifest in adolescents as extreme moodiness and heightened anxiety. by blocking the alpha-1 adrenergic receptor, the body decreases its berkeley pharma tech journal of medicine | 56 https://www.zotero.org/google-docs/?tyvobe https://www.zotero.org/google-docs/?mml1n0 https://www.zotero.org/google-docs/?j61v8q https://www.zotero.org/google-docs/?aj2ezl parasympathetic and sympathetic responses, which are responsible for activating the “fight or flight” feelings.124 finally, ptsd can disrupt particular stages of the sleep cycle, like the rapid eye movement (rem) stage, and these alpha-adrenergic blockers are known to restore this sleep stage. while alpha-adrenergic blockers have been most suitable for treating ptsd in war veterans due to the high incidence of sleep disturbances and night terrors in this population, studies suggest that it may also be used for treating ptsd and other stress-related disorders in other groups as well.125 maintaining proper sleep quality is essential for adolescents in particular as sleep regulates emotions and determines cognitive functioning during the day. the negative side effects of prazosin include dizziness and headaches due to slight hypotension.125 just as with other hormonal treatments, alpha-adrenergic blockers are most effective when used in combination with other therapeutic treatments discussed above. an ongoing clinical trial entitled “sleep and emotion processing in adolescent post traumatic stress disorder” explores the role of sleep in emotional processing and reactivity in adolescents with ptsd. subject ages from 12 to 17 years old, and all have been previously diagnosed with ptsd. the researchers plan to use electroencephalogram (eeg) tests to determine the quality of sleep in their subjects. using sleep enhancement algorithms, researchers hope to investigate if the deepest sleep, non-rapid-eye-movement (nrem), can be clinically enhanced in adolescents suffering from sleep disturbances due to ptsd.126 this study could potentially lead to targeted interventions for improving sleep and mitigating the effects of ptsd in adolescents. 3.4 lifestyle changes in addition to biochemical and therapeutic treatments, adolescents can also make lifestyle changes that may mitigate the effects of stress and trauma on the body and the brain. perhaps the most important lifestyle change for treating ptsd is to engage in physical activity. exercising releases endorphins, which reduce symptoms of anxiety and depression and is known to stabilize mood. research suggests that “aerobic exercise, which improves cardiorespiratory fitness, is an effective treatment for depression, berkeley pharma tech journal of medicine | 57 https://www.zotero.org/google-docs/?sziluh https://www.zotero.org/google-docs/?7vxkp3 https://www.zotero.org/google-docs/?wkvbeh https://www.zotero.org/google-docs/?jyglqp anxiety, and schizophrenia through both physiological and psychological mechanisms, and may be comparable or superior to other common treatments, such as psychotherapy and pharmacology.”127 additionally, a balanced diet plays an important role in maintaining emotional wellbeing. a diet rich in whole grains, vitamin d and b12, and lean proteins can minimize physiological and psychological effects of stress on the brain as well as regulate mood and emotions. in an article recently published by harvard t.h. chan school of public health, a study suggests that a mediterranean diet may help to mitigate the symptoms of ptsd.128 additionally, caffeine in chocolate, coffee and other fountain drinks act as a stimulant, enhancing the body’s stress response and ultimately exacerbating ptsd symptoms. this is particularly important for this age group, as coffee and energy drinks are popular beverages among middle and high school students. figure 5. infographic for effective treatments for ptsd in adolescents some effective therapeutic treatments for ptsd in adolescents include cbt, tf-cbt, and emdr. additionally, hormonal treatments, such as glucocorticoids and ssris can be used to mitigate symptoms from stress-related disorders. finally, lifestyle changes like maintaining a well-balanced diet and a routine sleep schedule can help to normalize cortisol levels in the body.129–136 3.5 future research future research on the effects of stress and trauma on adolescent brain development should focus on several key areas to enhance our understanding and ultimately improve health outcomes. for one, berkeley pharma tech journal of medicine | 58 https://www.zotero.org/google-docs/?dnxhwx https://www.zotero.org/google-docs/?zfp7bq https://www.zotero.org/google-docs/?hgtkcc physician-scientists could conduct long-term research that follows subjects from childhood, through adolescence, and into adulthood to investigate the long term effects of trauma on the mind and body. additionally, these studies could investigate which period of development interventions are most effective. it would also be beneficial to explore resilience factors that may affect how trauma manifests itself in the body as well as the healing process. factors like social support, coping mechanisms, and genetic predispositions all play significant roles in the body’s response to stress and trauma. since this article is geared towards adolescents, it would be important to investigate how the use of social media might reduce or exacerbate the effects of stress and trauma on the brain. additionally, there needs to be more research done on the socio-economic and cultural factors that might influence how adolescents process trauma and ultimately heal from it as well. access to mental health resources may mitigate the effects of trauma on the adolescent brain, so this would be an important area to explore when determining health outcomes. finally, it would be interesting to investigate how collective traumas like cultural diasporas and natural disasters affect the brain, morphologically and psychologically. this information could provide a point of comparison to how individual traumas, like experiences in childhood, affect the adolescent brain. 4. conclusion 4.1 summary of main ideas adolescent trauma has been shown to induce severe physiological and psychological stress, including disorders such as ptsd, anxiety, and depression. some regions in the adolescent brain have also been shown to be in a sensitive period during that time and are particularly vulnerable to stress and trauma. studies in both animal and human models reflect this sensitivity since exposure to stressors during adolescence results in lasting brain changes and impaired cognitive function. this underscores the need for more research and treatment efforts to address stress and trauma in adolescents. berkeley pharma tech journal of medicine | 59 clinical trials as well as rodent studies have mapped the effects of stress and trauma on adolescent brain development. most notably, individuals with exposure to stress or trauma exhibited altered volume in brain regions with increased volume in the amygdala, reduced volume in the hippocampus, and various changes in pfc matter according to specific location. additionally, studies have shown reduction in corpus callosum volume, decreased brain connectivity, and changes in the hpa axis, da levels, and larger brain networks. it is important to mention that many studies reported conflicting results; however, many of these discrepancies were eliminated when analyzing based on age-related effects. the most effective treatments for adolescent ptsd often combine hormonal, therapeutic, and lifestyle treatments. cbt, specifically tf-cbt, addresses negative thought patterns associated with ptsd and is one of the most common treatments for adolescents. another therapeutic option, emdr, helps those with ptsd process memories. therapeutic treatments, which mitigate psychological symptoms, are often combined with hormonal treatments, including glucocorticoids, ketamine, ssris, and alpha-adrenergic blockers which target underlying biological processes. additionally, lifestyle changes such as exercise, a balanced diet, and limited caffeine intake are commonly recommended. 4.2 practical applications this research article has several practical applications that may impact mental health treatment and public health policies. understanding how stress and trauma affect the brain as well as how these morphological changes manifest themselves as emotional and behavioral changes is important for early detection of ptsd and other stress-related disorders. early identification is necessary for timely intervention, which significantly improves health outcomes. therefore, this article can be used as an informational guide for those who care for and work closely with adolescents, such as parents, pediatricians, and educators. in school settings, teachers can be trained to recognize the signs of trauma in their students, which would help early detection and prevention. along with teachers, students could attend required educational sessions on trauma so that they, themselves, are aware of the effects of trauma and can spot warning signs berkeley pharma tech journal of medicine | 60 well in advance of symptoms worsening. finally, this review article could be used for the development of new and effective mental health treatments and policy advocacy. public health policies could leverage the insights gained from this article to advocate for community-based initiatives that address several systemic causes of stress and trauma such as poverty and familial violence. 4.3 acknowledgements we would like to thank ms. vanloan nguyen for her guidance and support throughout the writing process of this paper. we would also like to thank hayden loc-jun wong, aditi shankar and luke wang for feedback on our 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05ffd3aa-fa04-4657-8f75-d033f5886ee6.pdf pappas et al. reference page template.pdf the role of the gut microbiome in neuromodulation therapies as a potential treatment adjunct for multiple sclerosis berkeley pharma tech journal of medicine correspondence: gabybentolila5@gmail.com keywords: cellular senescence neurodegenerative diseases neuroinflammation sasps (senescence-associated secretory phenotypes) senolytics senomorphics tau protein submitted july 19, 2024 accepted august 19, 2024 published december 27, 2024 full open access creative commons attribution license 4.0 abstract there is strong evidence that cellular senescence is involved in the pathogenesis of neurodegenerative diseases such as alzheimer’s and parkinson’s disease. presence of abnormal tau protein in mouse models is implicated in senescence phenotypes leading to cognitive decline, while the significance of pro-inflammatory molecules called senescence-associated secretory phenotypes (sasps) in inflammation and degeneration highlights its potential importance in disease treatment. understanding the involvement of senescence in disease progression is vital to develop effective treatment strategies. clearance of such cells can be performed through senolytics drugs that disturb non-apoptosis pathways. alternatively, senomorphics involve the suppression of senescence burden, rather than elimination through apoptosis, through the intervention of sasp factors that elicit neuroinflammation and tau toxicity. other therapeutic approaches involve targeting mitochondrial dysfunction to attenuate neuroinflammation and senescence stressors, thereby preventing the formation of a feedback loop. this review offers insights into the mechanisms of neuro-degeneration driven by cellular senescence in the brain and outlines novel therapeutic strategies to reduce the senescence burden and slow disease progression. exploring the role of senescent cells in neurodegenerative pathology: a window into promising therapeutic avenues by: gabriela bentolila, niharika agrawal, julie phung, linh van, and gatik trivedi 1. introduction cellular senescence is a state of irreversible growth arrest in cells that have aged and ceased their ability to multiply, yet they avoid undergoing apoptosis. this process is triggered in response to various stressors such as dna damage, telomere shortening, mitochondrial dysfunction, tumor suppression, oxidative stress, and ionizing radiation (fig. 1a). while senescence plays a protective role in development, aiding in wound healing and limiting tumor progression, its accumulation also leads to harmful effects associated with aging and age-related diseases. the harmful impact of senescence is mainly attributed to the release of sasps. research comparing biomarkers of senescence in young and aged mouse brains has shown that senescent cells increase with age, along with a rise in sasp factor genes.1 consequently, this contributes to decreased tissue regeneration and increased neuroinflammation, paving the way for neurodegenerative diseases like alzheimer's disease (ad), parkinson's, and multiple sclerosis. furthermore, gene ontology analysis has revealed a strong connection between senescence-related genes, mild cognitive impairment, and neurodegenerative diseases.2 treating senescence has shown promise in improving cognitive function among patients with neurodegenerative diseases. glial cells are the primary cell type in the brain undergoing senescence, contributing to neurodegenerative diseases.3 glial cells play a supportive role in maintaining neuronal functions, and when compromised through senescence, they trigger sasp production, neurotoxicity, and neuroinflammation—common characteristics of neurodegenerative diseases.4 astrocytes, which are abundant in the brain and regulate synaptic signaling, neurotrophic support, and glutamate metabolism, have been found to be affected by senescence. likewise, microglia, essential for cns homeostasis, experience senescence during aging and are associated with neuroinflammatory phenotypes and neural impairment when increased in number.1 moreover, oligodendrocytes (olg), derived from oligodendrocyte progenitor cells (opcs), are involved in responding to neuronal injury and demyelination. in areas of the brain of ad patients, where there is elevated aβ plaque and neural degeneration and berkeley pharma tech journal of medicine | 44 inflammation, there is a notable correlation with the presence of senescent opc populations.5 considering the role of senescent glial cells in aging-related neurodegenerative diseases, targeting these cells has emerged as a potential therapeutic approach to tackle such conditions effectively. we may develop strategies to promote healthier aging and reduce the burden of neurodegenerative diseases. 2. mechanisms of senescence-associated neurodegeneration in the realm of neurodegeneration, the intricate interplay between cellular senescence and the emergence of pathological hallmarks such as sasps and tau pathology has garnered significant attention. these mechanisms not only shed light on the complex processes underlying aging-related neurological disorders but also offer insights into potential avenues for therapeutic interventions. 2.1 sasp it was previously believed that senescent cells lack function; however, in reality they simply have altered important morphological functions.6 many altered functions fall under sasp, which is a core feature of most senescent cells.7 sasp is mainly characterized by the production and secretion of factors like cytokines, chemokines, and other molecules that create an inflammatory microenvironment.6 the main function of sasp is to recruit immune cells for tissue damage repair; however, an abundance of senescent cells has a negative effect on tissue restoration and leads to a chronic, low-grade inflammation called “inflammaging”.8 activation of inflammaging results in decreased clearance of senescent cells, establishing a positive feedback mechanism that ultimately further fuels inflammaging. in an autocrine or paracrine manner, senescent cells can secrete sasp factors that induce neighboring cells to senescence, furthering the accumulation of senescent cells (fig. 1b). when this process occurs in the brain, it leads to the loss of neurons, resulting in the initiation, severity, and progression of neurodegeneration.9 berkeley pharma tech journal of medicine | 45 figure 1: mechanisms triggering cellular senescence. (a) cellular senescence is triggered by stressors such as dna damage, telomere shortening, mitochondrial dysfunction, tumor suppression, oxidative stress, ionizing radiation, and many other abnormal factors. (b) through the release of sasp factors, senescent cells can use autocrine or paracrine signaling to induce senescence in neighboring cells. early sasp is often induced by transforming growth factor-β (tgf-β), secreted by senescent cells. tgf-β can also help maintain the secretory phenotype if its target cell is already senescent. if early sasp is upregulated for an extended period of time, it transforms into late sasp, which is regulated mainly through transcription factors ccaat/enhancer-binding protein β (c/ebp β) and nuclear factor (nf)-κb. c/ebp β regulates the expression of various sasp factors, such as interleukin (il)-1β, il-6, and il-8. late sasp expression is induced when c/ebp β levels increase, suggesting c/ebp β is a key regulator of the early to late sasp transition.7 however, nf-κb signaling is the main signaling pathway in late sasp induction. nf-κb activation induces expression of inflammatory mediators il-6, il-8, and il-1α. in a positive feedback loop, il-1α contributes to sasp maintenance by regulating nf-κb and c/ebp β dna binding activities to induce further il-6 and il-8 transcription. this feedback loop is regulated by rapamycin-mtor signaling.8 nf-κb signaling is also provoked by dna damage through signaling pathways that include p38 mitogen-activated protein kinase (p38 mapk) and retinoic acid inducible gene-1 (rig-1).10 p38 mapk acts to stimulate nf-κb through the activation of mapk-activated protein kinase 2 (mk2),8 and p38 mapk berkeley pharma tech journal of medicine | 46 signaling itself can be induced by environmental stress or chronic inflammation, such as inflammaging (fig. 2).10 persistent nf-κb activation is partially maintained by tumor necrosis factor (tnf)-α. upon tnf-α activation, ak kinase is phosphorylated, which subsequently phosphorylates signal transducers and activators of transcription 3 (stat3). these phosphorylated stat3 molecules then migrate to the nucleus, contributing to prolonged activation of nf-κb and increased expression of sasp factors.8 interestingly, tnf-α itself is a common cytokine secreted by sasp.4 thus, its ability to induce sasp through nf-κb activation presents another positive feedback loop that serves to maintain sasp. persistent sasp expression through nf-κb, as well as other contributing factors, induces proinflammation, which promotes tissue dysfunction, cellular aging, and other aging-associated issues (fig. 2).8 figure 2: sasp regulation and neuroinflammation pathways. several different molecules act through the nf-κb pathway to activate and regulate sasp. upon acquisition of sasp, the secreted sasp factors promote autonomous neurotoxicity and disrupt normal function in the brain. in fact, it was found that treatment of senescent astrocytes with an antibody that neutralizes il-6, a sasp-associated cytokine, alleviates neuronal cell death.4 in vitro studies showed that il-6 production was found to have the most dramatic increase in senescent astrocytes, matching in vivo findings of il-6 berkeley pharma tech journal of medicine | 47 at elevated levels in the cns in ad patients.6 moreover, in a study with primary cortical neurons from senescence accelerated mouse prone 8 (samp8) mice, sasp factor plasminogen activator inhibitor 1 (pai-1) was found to promote neuron apoptosis; however, the exact mechanisms by which pai-1 carries out this activity is still unknown.11 several sasp factors have been linked to axonal and myelin injury. activation of sasp in neurons through the lymphotoxin-nf-κb pathway was found to trigger expression of chemokine cxcl13, which recruited cd8+ t cells. cd8+ t cells then activated caspase 3 in injured dorsal root ganglia (drg), which was found to hinder signaling and lead to the repression of the neuron’s axonal regeneration capabilities. however, the same study showed that regenerative failure in drg could be restored by the neutralization of chemokine cxcl13 with monoclonal antibodies.12 similarly, myelin injury and regenerative failure can occur if sasp is activated in olgs.13 a study with mice showed that persistent nf-κb signaling in olgs led to chronic neuroinflammation and post-mitotic senescence. nf-κb signaling also promoted white matter degeneration (wmd) in the brain, a well-known process of aging. wmd often manifests in myelin loss, which was confirmed by ultrastructural analysis of the mice that revealed impaired myelin protein expression. additionally, the mice exhibited neurological deficits within three weeks following treatment.14 olgs have also been found to be affected by secretion factor high-mobility group box-1 (hmgb1) in vivo. when extracellular hmgb1 was released by senescent progenitor cells, differentiation of opcs to myelinating olgs was prevented, ultimately inhibiting cns remyelination.15 moreover, hmgb1 prevailed at higher levels in the brains of patients with primary progressive multiple sclerosis (ppms) compared to healthy controls. more specifically, hmgb1 was found at high concentrations in white matter lesions of the brain, and progenitor cells were identified to be the source.16 this highlights the contributing role of sasp factor hmgb1 in the limited remyelination found in ppms, as well as potentially other neurodegenerative diseases. examples of other neurotoxic sasp factors include il-8, il-1, matrix metalloproteinases, insulin-like growth factors (igfs), endothelial growth berkeley pharma tech journal of medicine | 48 factor (egf), epidermal growth factor receptor (egfr), specific mirnas, and many other molecules.7 2.2 biomarkers understanding the relationship between cellular senescence and neurodegenerative conditions is crucial for developing effective therapeutic strategies. to this end, researchers have focused on identifying biomarkers that link cellular senescence to neurodegeneration. several enzymes, cytokines, chemokines, genes, and other biomarkers have been implicated in senescence-related processes in the brain. these biomarkers provide valuable insights into the mechanisms underlying neurodegenerative diseases and offer potential targets for therapeutic interventions. enzymes ● senescence-associated beta-galactosidase (sa-β-gal): sa-β-gal is one of the earliest markers identified for the detection of senescent cells in situ within tissues.7 this hydrolytic enzyme becomes active in senescent cells and is commonly used as a hallmark to distinguish senescent cells from non-senescent cells. ● caspase 3: caspase 3 is an enzyme involved in apoptosis. it suggests a potential link between senescence and cell death pathways.18 cytokines cytokines are signaling molecules that play critical roles in inflammation and immune responses. certain cytokines have been found to be associated with the development of neurodegeneration. ● il27: this cytokine is involved in the activation of natural killer cells in the dentate gyrus, which leads to the elimination of neuroblasts and subsequently contributes to neurodegeneration and cognitive decline.19 ● il-6, il-8, il-1β: il-6, il-8, and il-1β are among the cytokines that mediate tumor suppressor functions. studies conducted by bussian et al. andmusi et al. have identified these cytokines in the brains of mouse models with tauopathy. they were found to be part of the sasps observed in these models.20,21 berkeley pharma tech journal of medicine | 49 chemokines chemokines are small signaling proteins involved in cellular migration and immune responses. one chemokine that has been linked to senescence-associated neurodegeneration is ccl2.22 ● ccl2: studies have implicated ccl2 in the development of neurodegenerative processes. it may contribute to the recruitment of immune cells, such as microglia, to the site of neurodegeneration, further promoting inflammation and tissue damage.23 apart from its role in immune cell recruitment, ccl2 can also directly influence neuronal function. it has been suggested that ccl2 might affect synaptic plasticity and neurotransmission, potentially contributing to cognitive and functional impairments seen in neurodegenerative diseases.22 genes several genes have been identified as crucial regulators of cellular senescence, and their dysregulation may contribute to neurodegeneration. ● p16, p21, p53: these genes encode cell cycle inhibitors and repressors and are essential components of the senescence program. exposure to tau has been shown to increase the expression of senescence-associated markers, including p16ink4a and p21waf1, in microglia, potentially contributing to neurodegeneration.24 ● cdkn2a and cdkn2d/p19: neurons expressing cdkn2d/p19, a gene related to cdkn2a, were found to be more prone to containing tau aggregates and displaying signs of neurodegeneration, suggesting a potential role for senescent neurons in the development of tauopathy.25 additionally, nfts (neurofibrillary tangles) have been directly linked to cdkn2a upregulation.21 other biomarkers other biomarkers associated with senescent cells and neurodegeneration have been identified. berkeley pharma tech journal of medicine | 50 ● b2m: b2m is an extracellular epitope of senescent cells and serves as a membrane marker for mhc class i molecules. its expression may contribute to immune responses and inflammation in the context of senescence.26 ● pai-1: plasminogen activator inhibitor-1 is a serine protease inhibitor that increases with aging and has been found to contribute to brain cell senescence. however, the specific mechanisms linking pai-1 to neurodegeneration are still being investigated.11 ● hmgb1: high mobility group box 1 serves as a crucial biomarker. this multifunctional protein plays significant roles within the cell, particularly in dna repair and autophagy regulation. studies conducted by gaikwad et al. and rouilliard et al. have brought to light its potential implications in the advancement of neurodegenerative diseases. hmgb1's involvement as part of the sasp is particularly noteworthy, as it contributes to neuroinflammation and impedes the growth of myelin, which is essential for nerve fiber insulation. 15,27 ● lamin b1: lamin b1 is a protein that plays a crucial role in maintaining the structural integrity of the cell nucleus. it is a component of the nuclear lamina, a network of proteins that provides support to the nuclear envelope. lamin b1 has been implicated in the aging process as its expression levels decline with age in various tissues.28 reduced levels of lamin b1 have been observed in certain senescent cells, and its loss has been linked to nuclear envelope disorganization and altered gene expression, contributing to cellular dysfunction and aging-related changes.28 ● igfbp5: insulin-like growth factor binding protein 5 is a member of the insulin-like growth factor-binding protein family and plays a role in regulating the bioavailability and activity of insulin-like growth factors (igfs). igfs are essential for cell growth, proliferation, and survival. igfbp5 modulates the actions of igfs by binding to them and regulating their interactions with cell surface receptors.29 additionally, igfbp5 has been found to be involved in processes related to brain development and function, and its dysregulation may influence neuronal survival and synaptic plasticity, potentially contributing to neurodegeneration.29 berkeley pharma tech journal of medicine | 51 2.3. tau pathology the tau protein, predominantly located within neurons, plays a vital role in maintaining the structural integrity of nerve cells by stabilizing microtubules and facilitating efficient axonal transport.4 in addition to its structural functions, tau protein is involved in essential cellular processes such as cell signaling, synaptic plasticity, and the preservation of genomic stability, and in olgs cells, it also plays the role of myelination.30,31 when tau becomes abnormal, it can contribute to the development of neurodegenerative diseases. excessive phosphorylation, genetic mutations, and mis-splicing contribute to the abnormal aggregation of tau protein, resulting in the formation of either tau oligomers or neurofibrillary tangles (nfts).32 tau oligomers are small aggregates of abnormal tau protein that form prior to the development of nfts, which are larger insoluble clumps of hyperphosphorylated tau protein. both tau oligomers and nfts contribute to pathogenesis of neurodegenerative diseases. abnormal tau can be secreted by neurons into extracellular space, which are taken up by healthy neighboring neuronal and glial cells through endocytosis, pinocytosis, or phagocytosis means, thereby contributing to the propagation of tau pathology across interconnected brain regions.27 in addition, extracellular tau was found to induce human astrocyte senescence, inflammation, and sasps. the inflammatory state can cause induction of senescence in nearby cells in a paracrine-like manner, further propagating neurodegeneration, which is why overall extracellular tau may be more neurotoxic than intracellular tau.4 one pathological change caused by abnormal tau accumulation is the disruption of cell function and the blood brain barrier (bbb), which is responsible for protecting the cns from pathogens and maintaining homeostasis in the microenvironment of the brain.31 other tau pathologies include chronic neuroinflammation, dna damage, and oxidative stress, which are known stressors of senescence. these tau-related pathologies contribute to the establishment of a vicious cycle, where tau pathology triggers senescence, leading to the release of toxic sasp factors that, in turn, exacerbate the formation of abnormal tau (fig. 3). berkeley pharma tech journal of medicine | 52 neurons senescent cells are present in both ad and non-ad brains. however, it has been determined that the number of senescent cells was significantly higher in ad brains. additionally, a subset of senescent cells in ad brains were neurons that expressed the gene cdkn2d/p19. these neurons also showed signs of tau neuropathology.25 moreover, an examination of the transcriptomic profile of cortical neurons containing neurofibrillary tangles (nfts) in ad brains revealed that those neurons displayed a senescence-like phenotype. this means that the gene expression patterns observed in these neurons resemble those typically seen in senescent cells, suggesting that ad neurons undergo a cellular aging process akin to senescence.21 astrocytes tau can be transmitted from neurons to astrocytes, causing the astrocytes to senesce and produce sasps. studies have shown that astrocyte senescence is detrimental to dendritic and synaptic structure and density. this suggests that pathogenic soluble tau-induced astrocyte senescence may contribute to synaptic dysfunction and loss in ad.33 recent research has highlighted that tau oligomers play a role in promoting the release of hmgb1 from astrocytes during inflammation. notably, the release of hmgb1 not only leads to inflammation but also drives paracrine senescence in neighboring cells.27 moreover, tau oligomers have been specifically implicated in promoting the aggregation of p53 in ad patients. in the study, this interaction between tau oligomers and p53 was observed exclusively in ad brains, compared to control, resulting in the sequestration or aggregation of p53 outside the nucleus. consequently, this phenomenon hinders the ability of p53 to carry out dna repair processes, leading to dna damage and ultimately triggering senescence. the accumulation of senescent cells further exacerbates the progression of ad.34 one study found that astrocytes and microglia are the two major types of senescent cells that accumulate in the dentate gyrus of the hippocampus in ad.20 this was supported by the laboratory of human carcinogenesis, where they found that astrocytes are the primary type of senescent cells present in patients with ad, amyotrophic lateral sclerosis (als), aged individuals and those receiving cranial radiation.4 flow cytometry analysis of ps19 mouse models, which overexpress human mutant tau, also validated these findings by berkeley pharma tech journal of medicine | 53 revealing increased expression of senescence-associated genes, including p16ink4a, in isolated astrocytes and microglia, while olgs and neurons did not exhibit the same upregulation. furthermore, the study reported that senescent astrocytes and microglia exhibit inflammatory reactions to extracellular tau, leading to damaged neurons.20 in the context of neuronal damage, it has been identified that astrocyte senescence is associated with the downregulation of glutamate and potassium transporter genes. consequently, this impairment of glutamate clearance increased susceptibility of neurons to glutamate-induced toxicity and ultimately neuronal cell death (fig. 3).35 microglial in the aged human brain, many microglial cells showmorphological features indicative of senescence or degeneration rather than activation. this suggests that microglial senescence and associated loss of neuroprotection could be involved in aging-related neurodegenerative diseases. in fact, streit et al. reports that dystrophic microglial cells are implicated in the development of ad, associated with severe tau pathology. investigation of microglial dysfunction by examining their morphology in the vicinity of tau-positive structures revealed that microglial degeneration may precede the onset of nft pathology, meaning that the loss of microglial structural integrity contributes to neurodegeneration.36 karabag et al. conducted a study demonstrating that exposure to tau can induce microglial senescence. specifically, they found that an exposure to 15 nm concentration of tau resulted in increased levels of cell cycle arrest and a dna damage marker. it also led to the loss of nuclear envelope protein lamin b1 and the histone marker h3k9me3, thereby affecting the clearance of tau and impairing cell migration (fig.3). additionally, this exposure caused alterations in cell morphology and triggered the formation of a sasp.24 oligodendrocytes tauopathies involving olgs have been identified in specific dementias, such as progressive supranuclear palsy (psp), corticobasal degeneration, and frontotemporal dementia, which includes pick's disease and certain cases of ad.37 because pathological tau has the ability to propagate from neurons to other cell types both trans-cellularly and trans-synaptically, it can contribute berkeley pharma tech journal of medicine | 54 to the spread of neurodegenerative disease.38 however, studies now propose that olgs can contribute to the spread of tau without passing through intra-neuronal transport in a glial-to-glial fashion, playing a further role in the spreading of tauopathies.39 the spread of tau pathology to olgs is closely linked to the loss of high-firing pyramidal neurons, a crucial neuron type essential for cognitive function. the depletion of these neurons can lead to cognitive decline, as assessed through various behavioral tests in mice. in one study, mice injected with human tau aggregates displayed prolonged search times in locating the platform in the morris water maze, reduced visits to the correct arm in the y -maze, and decreased exploration time of the novel object compared to control mice.37 the aggregation of tau in olgs may be a contributing factor to the development of gait abnormalities and myelin loss in neurodegenerative diseases (fig. 3).30 in fact, luo et al., found that olg lineage cells from old donors had lower myelination potential than olg lineage cells from younger donors.40 figure 3: cellular morphological changes in senescence. senescent cells undergo morphological changes, such as altered cell size and structure, that lead to neurodegeneration. these cellular changes make up a vicious cycle as they contribute to the propagation of other senescent cells. berkeley pharma tech journal of medicine | 55 3. therapeutic approaches 3.1. senolytics senolytics eliminate senescent cells by inducing apoptosis. this is done by targeting senescent cell anti-apoptotic pathways (scaps) such as p53 and bcl-2. through removing senescence, senolytics have the potential to reduce neuroinflammation, enhance tissue regeneration, and improve cognitive function. the effects of senolytics are currently mainly tested through mouse models and cell populations characterized with an age-related disease. dasatinib and quercetin the combination of dasatinib and quercetin (d+q) is one of the most extensively studied senolytics, shown to be effective in numerous age-related diseases by selectively targeting senescent cells while maintaining the viability of non-senescent cells. dasatinib is a tyrosine kinase inhibitor that was initially discovered for use in cancer treatments to prevent cancer cell growth. by targeting pro-survival pathways, dasatinib induces apoptosis to senescent cells. quercetin is a natural flavonoid and enhances the effectiveness of dasatinib with its anti-inflammatory and pro-apoptotic properties. when used together, their effectiveness as a senolytic increases, as they are able to target a larger variety of senescent cell types. beyond targeting scaps, quercetin has also been found to improve cognitive function in ad patients through activating amp-activated protein kinase leading to a reduction in aβ-induced mitochondrial dysfunction, a main characteristic in early ad pathogenesis.41 through clearance of senescent cells by targeting bcl-2 and other anti-apoptotic proteins, d+q has shown to be effective in numerous age-related conditions. by decreasing senescence, d+q relieves tissue degeneration, inflammation, and other sasp factors.42 d+q acts in a dosage-dependent manner positively correlating with increased senescent cell death in mice with ad, specifically in olgs. these mice were also found with reduced levels of aβ plaque associated proinflammatory cytokines such as il-1β5. nfts are also reduced by d+q in tau transgenic mice with effects of improved cerebral blood flow and berkeley pharma tech journal of medicine | 56 decreased neurodegeneration.21 intermittent treatment with d+q on mice showed improved cognitive function by reducing senescent microglia. y oung and aged mice were put through phenotypic, memory, and cognitive assessments. assessment results of young mice were unchanged while aged mice improved significantly in a stone t-maze following treatment of d+q suggesting high effectiveness of d+q during aging.43 a current clinical trial of d+q involving early-stage older ad patients suggests promising results. the patients were treated intermittently with d+q for 12 weeks while their cognitive function and progression of ad were measured. the only statistically significant changes in safety parameters of d+q observed was an increase in total cholesterol, yet this higher total cholesterol remained in a normal range, and one possibly related adverse event, yet this event was resolved between 1 to 16 days. several tests were conducted to test changes in cognitive function including the montreal cognitive assessment (moca) and the clinical dementia rating sum of boxes (cdr sob). there was no significant change in the results from these cognitive assessments from the baseline to post-treatment. mris also showed no significant change from the baseline to post-treatment, suggesting a stable brain morphology over the treatment period. plasma and cerebrospinal fluid (csf) levels revealed that d was able to penetrate through the bbb while q was not able to. overall, the study’s results are promising in the safety and potential of d+q as a senolytic, yet further studies are needed to examine long-term efficacy.44 fisetin with studies of quercetin, a natural flavonoid, being an effective senolytic, other flavonoids were explored for their senolytic abilities such as fisetin. fisetin, present in various fruits and vegetables, was found to possess great seno-therapeutic qualities.45 it is assumed that with similar chemical structures, fisetin and quercetin hold similar therapeutic and anti-inflammatory effects.46 with a fisetin diet, abundance of p16 was reduced in aged mice and levels of p16 and sasp factors continued to remain in significantly low levels following treatment. senescence was reduced in multiple organs of the mice along with markers of inflammation and oxidative stress.45 through blocking mitogen activated protein kinase (mapk) and nf-κb signaling pathways, fisetin is able to reduce berkeley pharma tech journal of medicine | 57 inflammation.46 specifically, fisetin targets senescence through the scap networks of bcl-xl and hif-α. fisetin has also shown to be able to extend median and maximal lifespan, suggesting promising results for age-related treatments.47 fisetin also attacks the p13k/akt pathway, which promotes cell survival, to induce apoptosis.48 human adipose tissue explants treated with fisetin had a decrease in sa-β-gal positive cells and sasp expression of il-6, il-8, and mcp-1. similar to d+q, fisetin was effective in reducing senescence without affecting proliferating cells.45 currently, fisetin has proven to be successful as a senotherapeutic agent, yet further testing is needed to study its safety for age-related and neurodegenerative treatments.47 navitoclax navitoclax, also known as abt263, is another discovered senolytic. initially used as an anti-cancer drug, navitoclax works by inducing apoptosis in cancer cells. this mechanism is likewise in its role as a senolytic drug, disrupting bcl-2 and pro-death proteins to induce apoptosis in senescent cells.49 this disruption is caused by navitoclax binding to bh3 of bcl-2, resulting in the displacement of a pro-apoptotic protein, bim, that causes cell apoptosis (fig. 4).50 aged mice treated with navitoclax enhanced neurovascular coupling, regulating cerebral blood flow that is essential for cognitive function. examination through a radial arms water maze, navitoclax improved on the learning capabilities and memory retention in aged mice.51 hippocampal neurogenesis reduced by aging can be restored by navitoclax. by targeting senescent cells that impair neural progenitor cells (npcs), hippocampal neurons and hippocampus-dependent spatial memory function was increased following navitoclax treatment.52 trails of navitoclax were tested on p16 transgenic mice in biweekly cycles. apoptosis of senescent cells increased in these mice, yet the possibility of thrombocytopenia and neutropenia as side effects rose.48 berkeley pharma tech journal of medicine | 58 figure 4: navitoclax mechanism of action in senescence. the figure demonstrates navitoclax targeting the bcl-2 pathway to release caspase 3 and cause apoptosis in senescence. ap-20187 ap-20187 (ap) is a drug that targets senescence by disrupting scaps and activating a pro-apoptosis protein.53 a study on young and old ink-a ttac mice revealed significant effectiveness in clearing senescence during aging following intermittent treatment of ap. cognitive impairment was reduced without affecting the physicality of the mice. specifically, aging-induced senescent microglia was reduced in aged mice with ap treatment.43 in another study, ap increased neuron density and decreased tau aggregation in ps19 mice, which express models of tauopathy, with twice-weekly administrations. with treatment, expression of senescent genes were measured at levels compared to that of control mice. treatment of ap was able to mitigate short-term memory loss in novel-scent discrimination assessments.20 berkeley pharma tech journal of medicine | 59 limitations senolytics is an emerging field and therefore there remains a lack of information on the exact mechanisms of senolytics in neurodegenerative diseases and long-term effects. future studies are necessary to test the safety and efficacy of senolytics long-term before being able to be used beyond clinical trials.54 while senolytics have shown to improve lifespan, cognitive functions, and anti-inflammation, senolytics have the risk of eliminating benefits of senescence such as wound healing and tissue development. since senescent cells are not replaced when removed by senolytics, fibrosis can occur, resulting in scarring and thickening of tissues.55 as there are other cell types that are non-senescent yet can have a high p16 expression, some senolytics such as navitoclax and ap can have off-target effects and eliminate non-senescent cells.43,56 3.2. senomorphics senomorphic medications intervene with sasp factors without inducing apoptosis. unlike senolytics, the benefit of senomorphic drugs is that they do not kill functioning senescent cells, which are still important for anti-tumor cell signaling (fig. 5). senomorphic medication mainly targets signaling pathways: nf-κb, mtor, il-1α, p38 mapk, etc. there are currently multiple senomorphic drugs being tested to varying levels of success that will be covered in the following sections. berkeley pharma tech journal of medicine | 60 figure 5: comparison of senolytic and senomorphic mechanism of action and effect. senolytics eliminate senescent cells, while senomorphics modulate sasp factors to mitigate inflammation without inducing apoptosis. metformin metformin has a neuroprotective effect in animal models, and is shown to reduce inflammation and oxidative stress.57 metformin is a drug that was originally approved for the treatment of type 2 diabetes, but its therapeutic effects have expanded into age-related disorders and neurodegenerative diseases. it is effective in suppressing cellular senescence and sasps; for example, metformin reduced cellular sa-β-gal activity and down-regulated the expression of senescence and sasp factors in human diploid fibroblasts—cells commonly found in the perivascular spaces, meninges, and choroid plexus of the brain—among other cells.58,59 metformin also increases the lifespan of different model organisms, including mice.60 despite the fact that metformin has been tested in detail, its exact mechanism of action remains unknown, and is an area for further experimentation and testing. metformin has been proven to reduce senescence via transcriptional up-regulation of nrf2-medicated gpx7 in human diploid fibroblasts, but it also decreases cellular senescence and sasps via the microrna processing protein dicer1.61,62 metformin has been found to influence all hallmarks of aging, which is why understanding its mechanism of action is so complicated. its impact has been found in nutrient signaling pathways, insulin signaling, repairing oxidative damage, and inhibiting protein synthesis, among others.63 currently, there is an ongoing clinical trial (nct04098666) for alzheimer’s prevention using metformin. at baseline and after study visit at 18 months a brain mri, physical exams, plasma tau and amyloid beta levels are being measured to assess prevention of ad, along with clinical interviews, physical exams, and brain mris. results are inconclusive as of yet.57 resveratrol resveratrol is an sirt1 activator that plays an important role in promoting anti-inflammatory and antioxidant properties in the body, and has an effect on cellular senescence.8 it also mitigates neuroinflammation by promoting microglia polarization towards the m2 phenotype it activates, pgc-1α: a transcription factor involved in the suppression of sasp.64 m2microglia are berkeley pharma tech journal of medicine | 61 involved in anti-inflammatory responses; they promote tissue repair and protect neurons from damage; thus, promoting the m2 phenotype will help reduce neuroinflammation and protect neurons from further damage. in trials, long term resveratrol treatment prevented age-dependent decline in cellular viability and various cell parameters—metabolic, oxidative, inflammatory, and senescent—in hypothalamic astrocytes from aged rats. it also increased the expression of genes involved in cellular homeostasis, such as: nrf2, ho-1, sirt1, and pgc-1α.65,66 however, resveratrol has a biphasic effect. at low concentrations (below 10 μm), resveratrol acts as a senomorphic and prevents cellular senescence and suppresses sasps. for example, resveratrol prevented cellular senescence by activating the telomerase of endothelial progenitor cells via the p13k-akt pathway.67 the drug has also been shown to suppress sasp factors by inhibiting nf-κb and upregulating nrf2 pathways in the vascular smooth muscle cells of rhesus monkeys. however, at higher concentrations (over 25 μm), resveratrol triggers growth arrest and induces senescence of apoptotic death in cell lines. as such, controlling the dosage of resveratrol is very important for using it as a senomorphic therapy. rapamycin rapamycin is also known as sirolimus. while it was originally used as an immunosuppressive drug for the prevention of organ rejection in kidney transplantation, studies have shown that rapamycin could reduce cellular senescence and suppress sasp markers in a variety of mouse and human cell lines. it is an incredibly well-established senomorphic drug. in multiple invertebrate models, including yeast, flies, and worms, rapamycin has increased lifespan, and late-life administration has extended lifespan in male and female mice. in vivo studies have demonstrated that rapamycin alleviates age-related dysfunctions, decreases aging rate, and increases life-span. further, rapamycin treatment has reversed the cellular senescence phenotype in ppms npcs, as evidenced by the reduced expression of senescence markers and increased cellular proliferation. it further enhanced the ppms npc support for olg maturation, as seen in the increased expression of olg differentiation markers and decreased hmgb1 secretion.16 decreased hmgb1 secretion is important; it is seen that patients with pms exhibit higher levels of hmgb1, a senescence marker, berkeley pharma tech journal of medicine | 62 which inhibits the differentiation of progenitor cells into olgs. this is incredibly important for neurodegenerative disease progression, as olgs are what produce myelin to protect our neurons.16 rapamycin has side-effects, however: metabolic dysregulation and impaired wound healing, among others. rapamycin works by inhibiting torc1 activity in association with the intracellular protein fkbp12. torc1, or target of rapamycin complex 1, is a eukaryotic protein complex related to cell proliferation.68 the senomorphic and longevity effects of rapamycin relate to its inhibition of mtor signaling by reducing the phosphorylation of s6k and 4e-bp. other potential or secondary mechanisms by which rapamycin may regulate senescence and lifespan are being explored—for example, activating the nrf2 pathway and decreasing nf-κb activity to reduce il-1α production. it is also to be determined whether other mtor pathway inhibitors and rapalogues such as everolimus, temsirolimus, deforolimus, ridaforolimus and zotarolimus may exhibit senomorphic activities.63 aspirin aspirin has been found to partially prevent aβ-induced neuronal senescence and dna damage by upregulating sirtuin-1 (sirt1) in a dosage-dependent manner.23 it also delayed the onset of senescence in endothelial cells by increasing nitric oxide synthesis and decreasing oxidative stress, subsequently upregulating telomerase activity.69 however, other experimental studies indicate that the effects of aspirin on senescence vary with context, stress type, and dosage.63 ethyl pyruvate and glycyrrhizic acid ethyl pyruvate and glycyrrhizic acid inhibit the release of hmgb1, and can also inhibit p53 aggregation and prevent tau phosphorylation.27,34this indicates that the two drugs could have potential benefits in treating alzheimer’s. other areas that are only just being explored include nf-κb inhibition, p38 mapk inhibition, the janus kinase/signal transducer signaling pathway, mutated ataxia telangiectasia inhibitors, and statins.63 overall, senomorphic medications are a very promising area of research for treatment of neurodegenerative diseases, but there is still a lot of work to be done and discoveries to be made. berkeley pharma tech journal of medicine | 63 2.3. mitochondria-based therapies mitochondria are the dominant sources of reactive oxygen species (ros), which is produced as a by-product of the electron transport chain in mitochondria. under normal conditions, ros is central for organismal homeostasis, able to promote necessary signal transduction by mediating redox modifications of specific molecules; however, damaged mitochondria often result in an overproduction of this superoxide, leading to oxidative stress, another leading factor of cellular senescence. imbalances between ros production and detoxification of these reactive species, or antioxidant activity, is toxic to cells. it activates p53, which induces the cyclin-dependent kinase (cdk) inhibitor p21, causing cell cycle arrest and senescence. in addition, elevated ros levels have also been found to be strong modulators of inflammatory pathways that can accelerate cellular senescence and further aggravate inflammaging. as a result, dysfunctional mitochondria have become a hallmark of and contributor to aging and aging-related neurodegeneration. while the role of dysfunctional mitochondria in aging and cellular senescence is still being explored, many studies show that therapies targeting these defects have been effective in alleviating senescence-associated symptoms.7 nad+ supplementation nad+ supplementation has rapidly become a popular research avenue as a potential therapy for neurodegenerative diseases with underlying mitochondrial dysfunction-induced senescence causes. studies have shown that nadh improves motor symptoms in parkinson's disease and nad+ supplementation can prevent sting-induced senescence in a-t cells and mice, while promoting mitophagy to remove damaged mitochondria and prevent senescence and neuroinflammation.70,71 additionally, the cgas-sting pathway has been implicated in attenuating neuroinflammation, suggesting that boosting nad+ with nicotinamide riboside (nr) treatment could serve as a therapeutic approach to mitigate neurodegeneration.72 these findings highlight the potential of nad+ as a therapeutic strategy for addressing senescence-related issues in neurodegenerative diseases. berkeley pharma tech journal of medicine | 64 limitations studies have shown that increased nad+ levels could lead to the accumulation of potential toxic metabolites and may even contribute to tumorigenesis.70 additionally, excessive nad+ supplementation in young healthy mice, which already have sufficient nad+ levels, has been linked to elevated levels of pro-inflammatory cytokines such as il1β and il6, suggesting unintended consequences on inflammatory pathways.71 these findings highlight the need for cautious consideration of nad+ supplementation as a therapeutic approach, with attention to dosage and potential age-related variations to avoid adverse effects. further research is necessary to fully understand the risks and benefits of nad+ supplementation as a therapy for senescence and neurodegenerative diseases. mapks mitogen-activated protein kinase 15 (mapk15) is another potential target of mitochondria-based therapies. mapk15, an atypical mitogen-activated protein (map) kinase, has been found to control the mitophagic process by stimulating phosphorylation of unc-51 like autophagy activating kinase 1 (ulk1)-dependent parkin rbr e3 ubiquitin protein ligase (prkn) ser108, which induces recruitment of damaged mitochondria to lysosomal compartments for disposal. as such, by effectively getting rid of defective mitochondria, mapk15 helps prevent oxidative stress and dna damage accumulation, both of which are effective in inducing senescence. in a study conducted with human airway epithelial cells, it was found that the downregulation of mapk15 resulted in reduced cell proliferation, increased p21 levels, increased sa-β-gal activity, and increased expression of sasp cytokines, all of which are associated with senescence.73 the usefulness of other members of the map kinase family is still being investigated as they have a duality that both helps prevent and promote the negative effects of senescence. the activation of p38 mapk has been observed in senescent cells indicating an involvement in the cellular senescence process. in an α-synuclein mouse model of parkinson’s disease it has been shown to promote mitochondrial fission. chemical inhibition of p38 map with molecule sb203580 was shown to protect cells from mitochondrial dysfunction and cell death.74 additionally, p38mapk plays berkeley pharma tech journal of medicine | 65 a crucial role in the secretion of sasp factors. interestingly, p38mapk can induce sasp independently of the dna damage response (ddr), suggesting its significance as a separate pathway for sasp activation. thus, when p38 mapk is inhibited, it has been found to effectively suppress the sasp , potentially offering a promising target for therapeutic interventions aimed at mitigating the harmful effects associated with cellular senescence.75 furthermore, it is interesting to note that mapks and nf-κb have been found to regulate the expression of the other in a feedback mechanism, suggesting inhibition could lead to regulation of sasps.76,77 further research on p38 mapk's intricate role in senescence and its modulation may provide valuable insights into aging-related processes and age-associated diseases. while specific therapies that target mapks have not yet been thoroughly developed, these findings point to the great potential of mapk-based treatments. limitations excessive and constitutive mitophagy may contribute notably to the progression of neurodegenerative disease. in addition, in vitro studies on chronic obstructive pulmonary disease (cpod) epithelial cells found that cigarette smoke activated mapk15 signaling and led to oxidative stress, rather than the opposite, as what has been more commonly discovered in other studies.78 hence, patients with neurodegenerative diseases that smoke may encounter additional barriers to treatment with mapk15-targeted therapies.the p38 mapk has also been linked to oxidative stress, playing a role in the induction of senescence. thus caution of these therapies is essential in order to avoid producing the opposite effect. 4. future directions it is crucial to explore the heterogeneity of senescent cells and their distinct contributions to neurodegeneration. understanding the specific factors and signaling pathways that drive the detrimental effects of different senescent cells in neurodegenerative diseases will help guide the development of targeted therapies. one area that could be further explored is the use of targeted therapies. some research has been done but needs to be expanded upon for applications in neurodegenerative diseases. one of those is berkeley pharma tech journal of medicine | 66 second-generation senolytics: they offer a specific approach to enhance the effectiveness of existing senolytic drugs while minimizing the side effects. an example is b2m adc, an antibody conjugation that increases efficacy and selectivity when it comes to using senolytics to clear senescent cells.79 future research in combining strategies, like using senolytic drugs and immunotherapy simultaneously promises potential benefits as well. similar to how car t cell therapy has been used to treat cancers, this therapy can be applied and engineered to target senescent biomarkers for neurodegenerative diseases. so far very limited research has been done in this area but amor et al. successfully created a senolytic car t therapy that effectively eliminated senescent cells in mouse models and improved outcomes in liver fibrosis. however, this technique comes with its limitations. a condition called cytokine release syndrome may arise, in which an intense storm of t cells causes fever and impacts breathing and blood pressure.80 in addition, antibodies made to neutralize sasps are another promising avenue to take in the path of immunotherapy treatments. particularly, intervention of cytokine il27 as well as the upregulation of mhc class-i-related molecules, such as rae1 can be applied to alleviate senescence burden and interaction between nk cells and receptor nkg2d, which influences age-related decline in neurogenesis and cognition.19 another area of research is in leveraging the lymphatic system, which plays an imperative role in clearing senescent cells and therefore preventing neurodegenerative diseases. such diseases are known to cause chronic inflammatory cascades that damage hippocampal micro vessels and therefore lymphatic drainage. this in turn creates a dangerous cycle where the disease causes lymphatic blockage of senescent cell clearance and senescent cells clearance accelerates cognitive decline that further damages hippocampus. a study determined that such drainage is dependent on the vegf-c/ccl21 pathway. leveraging these pathways to improve lymphatic drainage could prove extremely beneficial in combination with senolytics like d+q. one such idea is dispersing astrocyte vegf-c into the csf promoting lymphangiogenesis, and thus enhancing drainage of parenchymal waste to the dural lymphatics. given that studies have demonstrated the aggravation of pathogenic tau accumulation in ad berkeley pharma tech journal of medicine | 67 mouse models when aqp4 water channels are pharmacologically inhibited, there is a need for further research strategies for mitigating the downregulation of aqp4 water channels in senescent astrocytes. these channels play a crucial role in facilitating cerebrospinal fluid and interstitial fluid exchange, thereby regulating glymphatic transport and helping clear senescent cells.23,35 overall, further research into second generation-senolytics, immunotherapy, and lymphatic drainage can pave the way for improved therapies to alleviate senescence burden and neurodegenerative disease onset and progression. 5. conclusion the field of neurodegenerative diseases is only just beginning to be explored; the brain is a complicated area of study, and cognitive decline even moreso. in recent years, many potential avenues have been explored for possible treatments, including senolytics, senomorphics, and mitochondrial-targeted therapies. senescence is a vital part of neurodegenerative diseases, and understanding its role proposes a powerful avenue of treatment. constant stimuli, such as nf-κb signaling, contributes to sasp , reinforcing inflammaging by its characteristic overproduction of proinflammatory cytokines and chemokines.8 in addition, it is clear that many factors function to induce and maintain sasp in a positive feedback manner, increasing senescent cell accumulation and exacerbating chronic inflammation. these factors contribute to a neurotoxic environment in which neuronal cell death occurs, advancing neurodegeneration in affected patients. extracellular tau can further cause oxidative stress and dna damage, which are known to be associated with induction of senescence.4 in addition, therapeutic strategies targeting senescence offer promising avenues for addressing neurodegenerative diseases. senolytics have shown potential in reducing neuroinflammation and improving cognitive function, while senomorphics provide alternatives by modulating sasp factors without inducing cell death. similarly, mitochondria-based therapies hold promise in mitigating oxidative stress and enhancing cellular health. however, challenges remain in understanding mechanisms, potential side effects, and disease-specific applications. collaborative research across disciplines is vital berkeley pharma tech journal of medicine | 68 to fully harness the potential of these approaches and reshape the 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keywords: anorexia nervosa gut-brain axis probiotics vitamins ketone supplements cognitive behavioral therapy dietary supplements published july 31, 2025 full open access creative commons attribution license 4.0 abstract anorexia nervosa (an), one of the most prevalent eating disorders in the us, is characterized by abnormally low body weight due to an obsessive fear of gaining weight and a distorted perception of one’s own body image and weight. this review aims to provide critical insights into the impact of improving patients’ gut microbiome diversity, and the use of probiotics, vitamins, and other supplements as potential therapeutic targets for an. given the intricate relationship between gut health and psychological well-being, researchers have proposed enhancing gut microbiome diversity through dietary supplements as a potential adjunctive treatment. the proposed thesis asserts that dietary supplements such as vitamins, minerals, and probiotics can be a potential neurobiological treatment method for an, based on their known ability to modulate gutmicrobiome diversity and address alterations in the gut-brain axis that are present in an. current treatments, such as cognitive behavioral therapy and family-based therapy, focus primarily on psychological and behavioral aspects. however, they may benefit from a more integrative approach that includes neurobiological treatments targeting the gutbrain axis. this review explores the pathophysiology of an, the gut-brain axis, and the potential for dietary supplements to modulate gut microbiome composition and improve therapeutic outcomes. additionally, it discusses the implications of such treatments in combination with existing therapies and explains why further research is needed to establish personalized treatment plans for an patients based on their unique microbiome profiles. promoting gut microbiome diversity in anorexic patients: a neurobiological review by: aditi shankar, hayden wong, izabel kwe and luke wang 1. introduction anorexia nervosa (an) is a psychiatric disorder characterized by restrictive eating behaviors, an intense fear of weight gain, and a distorted body image, leading to significantly low body weight. patients with an maintain an abnormally low body weight through restrictive eating behaviors, excessive exercise, and/or other compensatory mechanisms.1 it is a common and deadly disorder across the world, having the highest mortality of any mental illness.2 an patients typically show symptoms of fatigue, low blood pressure, dietary restriction, purging, malnutrition, and excessive exercise. these symptoms can lead to complications like heart failure, kidney problems, and may even alter brain structure. comorbid psychiatric conditions, including depression and anxiety, are frequently associated with an and can exacerbate the disorder’s severity.3 given adolescents' frequent use of social media, their perception of their bodies is constantly being shaped by others on the internet. this pressure causes more people to develop an, hence pushing research into more treatments for an. currently, cognitive behavioral therapy (cbt) is the primary psychotherapeutic approach for an, focusing on modifying maladaptive thoughts and behaviors related to eating and body image. this helps reduce anxiety surrounding food and promote healthier thought patterns. however, this only helps with the cultural and psychological causes of an, and is still limited in its effectiveness. another treatment is the family-based treatment (fbt), where the family of the patient takes over their diet.3 fbt has shown promise and patients have shown comparable remission rates to those receiving adolescent-focused therapy.4 while there is no fda-approved medication, some off-label pharmacotherapies (e.g., olanzapine) are used.5 this review will look into the relationship between the digestive system and an to propose a new treatment to combine the mental and physical aspects of an therapy. the enteric nervous system (ens) is a gastrointestinal tract neural network that controls digestive functions, while the vagus nerve serves as a link between the ens and brain to convey information and berkeley pharma tech journal of medicine | 74 mediate reflexes based on gut conditions, and the microbes present in the gut.6 containing the ens and the vagus nerve, the gut-brain axis (gba) is a complex network of nerves spanning from the gut to the brain, responsible for monitoring and designing the elaborate interactions between central and autonomic mechanisms regulating the emotional and cognitive parts of the brain along with peripheral intestinal functions such as entero-endocrine signaling, immune activation, intestinal permeability, and enteric reflex.7 due to its intricate connection between ens and the brain, the gba can play a role in influencing a person’s craving for food, feeling of hunger along with food intolerances, digestion, metabolism, and other cognitive functions.8 based on previous studies, there is growing evidence that gut microbiota can help in the regulation of the gba and physiologic homeostasis through the means of vagal transmission, gut hormones, the immune system, and the hypothalamic-pituitary-adrenal axis.8 this allows the microbiome-gut-brain axis to effectively communicate between the central nervous system and the gut. the gut microbiome is an ecosystem made up of the trillions of microbes that reside in the intestine and the environment surrounding it. these microbes can be categorized into bacteria, viruses, fungi and parasites. diet and external exposures largely dictate the makeup of the gut microbiome. maintaining its homeostasis is important as the gut microbiome interacts with different parts of our body such as the digestive, immune, nervous and endocrine system. bile acids, primarily synthesized in the liver, undergo microbial metabolism in the gut, influencing lipid digestion and signaling pathways.9 short chain fatty acids (scfa) produced by microbes in their metabolism regulates immune response by initiating anti-inflammatory cytokine production. these scfa are also responsible for releasing neurotransmitters such as serotonin which can affect memory and the learning process. finally, microbes interact with enteroendocrine cells to release gut hormones and control appetite, nutrition absorption, and digestion.10 given how gut microbiomes maintain proper metabolic and neurological functions, the symbiosis of host and microbes is crucial. such a relationship is regulated through a feedback berkeley pharma tech journal of medicine | 75 system and the improper balance or dysbiosis of the gut microbiome is linked with anxiety, depression, autism-spectrum disorder and eating disorders. the proper balance of a gut microbiome is measured by its diversity, abundance and integrity. metrics include alpha-diversity, which reflects the abundance of each taxon, and beta-diversity, which reflects differences in species composition. in gut dysbiosis, an increase in originally commensal species can act as pathogenic and the intestinal barrier integrity can be corrupted.10 although traditional treatments such as cognitive behavioral therapy (cbt) and family-based therapy (fbt) offer some relief, they often fall short in addressing the underlying neurobiological disruptions associated with the disorder. recent studies suggest that alterations in gut microbiome diversity and the gut-brain axis play a crucial role in the mechanisms of an. this review paper aims to explore the potential of dietary supplements, including vitamins, minerals, and probiotics, as therapeutic agents for an by restoring gut microbiome diversity and perturbations in the gut-brain axis. by integrating gut microbiome-targeted interventions with traditional psychotherapeutic approaches, a more holistic treatment model for an may emerge. this paper will investigate the gut-brain interactions associated with an, compiling and analyzing scientific literature to identify effective treatment strategies. by examining research on signaling pathways and neurobiological aspects linked to an, the review aims to highlight how substances affecting these pathways can serve as potential therapeutic targets. the primary objective is to provide insights into the impact of improving gut microbiome diversity and utilizing supplements like probiotics, vitamins, and other additives proposing a comprehensive treatment approach that integrates these with conventional therapies. given their ability to restore gut-microbiome diversity and modify the gut-brain axis, dietary supplements can be a viable treatment method for an if combined with traditional approaches, ultimately offering a more holistic approach to managing the disorder. berkeley pharma tech journal of medicine | 76 2. pathophysiology of anorexia 2.1 serotonin pathway serotonin, a monoamine neurotransmitter, is most prominently known for its role in the pathophysiology of many neuropsychiatric disorders. serotonin receptors, also known as 5-hydroxytryptamine (5-ht) receptors, are activated by the serotonin neurotransmitter and these receptors are found in central and peripheral nervous systems.10 these neurotransmitters participate in regulating essential functions in the body such as motor control, cognition, motivation, and the reward system pathway. for example, in the cns, serotonin plays a role in the regulation of mood and interpersonal perception. divided into 7 groups of g-protein-coupled receptors, each receptor activates a different intracellular secondary messenger cascade resulting in an inhibitory or excitatory effect. serotonin is synthesized through a two-step metabolic pathway that involves first hydroxylating tryptophan (trp) into 5-hydroxytryptophan by tryptophan hydroxylase. the second step involves the decarboxylation of 5-hydroxytryptophan by an aromatic l-acid decarboxylase that finally results in the end-product of 5-ht.11 5-ht receptors are responsible for regulating substrates including food consumption, anxiety control, learning, memory, locomotion, as well as prevention of depressive conditions. an is often observed when changes in connectivity and neurotransmission or dysregulation is present in serotonergic systems.12 past studies have shown that animals experience hypophagia or reduced ingestion of food when 5-ht1b and 5-ht2c receptors are stimulated. on the other hand, hyperphagia or the increased ingestion of food, is triggered through the activation of 5-ht1a and 5-ht2b receptors.13 as mentioned previously, serotonin is synthesized from trp, in the two-step metabolic pathway, however, only a fraction of the total trp available enters this metabolic pathway.14 many aspects of an research focus on this pathway of serotonergic systems since trp depletion caused by external factors such as stress and inflammation lowers berkeley pharma tech journal of medicine | 77 peripheral and central serotonin levels.14 some suggest that there are correlations between obsessive or anxious behaviors and serotonin and suggest that due to this correlation, starvation is a common outlet used by patients with an to reduce emotional discomfort, known as dysphoria. starvation however, negatively affects serotonergic transmission as it reduces the effectiveness of serotonin in the brain.14 not only does this lead to various psychological issues such as anxiety and depression, but also aggravates the course of an and any course of treatment. although these aspects of an research are of interest and are often of focus, many instances show that experimental data and clinical data are often conflicting in their results of the serotonergic system and how it is affected. therefore, it is uncertain whether the changes that occur from symptoms of an are causative or symptomatic. 2.2 dopamine pathway dopamine is a regulatory hormone within the mammalian nervous system. it is involved with regulating emotions and the motivation and reward system. the mesolimbic dopaminergic system is specifically associated with the food reward system. food consumption activates the mesolimbic dopaminergic pathway, leading to dopamine release in regions such as the nacc. this reinforces feeding behavior by associating food intake with pleasurable sensations, thereby motivating future consumption.15 dieting and excessive exercise activate the hypothalamic-pituitary-adrenal (hpa) axis, leading to cortisol release. this chronic stress response influences dopaminergic activity, contributing to heightened reward sensitivity associated with an behaviors. sustaining this release of dopamine eventually leads to the symptoms becoming addictive, causing subjects to display the symptoms shown in typical an patients. studies show that it becomes more and more rewarding to eat less and exercise more.16 this reward-driven behavior can become compulsive, reinforcing restrictive eating. berkeley pharma tech journal of medicine | 78 https://www.nature.com/articles/s12276-020-00532-4#%3a~%3atext%3dstress%20affects%20dopamine%20levels%20and%2cresponses%20to%20various%20environmental%20stimuli research has been done to investigate how dopaminergic neurotransmission is specifically affected by gut microbiota change.17 /gut microbiota can influence dopamine secretion through the vagus nerve by stimulating the nerve with metabolites, which impacts the dopamine level in the brain. through many other ways such as the immune system and the hypothalamus-pituitary-adrenal axis, microbiota are tied with dopamine levels.17 research shows that changes in these gut microbes target the dopaminergic transmission in the prefrontal cortex, striatum, hippocampus, nucleus accumbens (nacc), and the amygdala in the brain. these regions are all directly involved with cognitive functions and emotional control, with the nacc targeting motivation.18 meanwhile, intestinal microbe composition also affects the mesocorticolimbic circuit in the brain, which has dopamine as its main neurotransmitter. although the precise mechanisms remain under investigation, increasing evidence suggests a significant link between gut microbiota and dopaminergic regulation.19 numerous treatments have been tested to see how dopamine may be used to alleviate an symptoms. most studies theorize that altering the diet to normal levels would lower or normalize the dopamine levels.18 many trials have shown promising true remission in its patients. by using non selective dopamine antagonists, specifically cis-flupenthixol and olanzapine, an patients show reduced symptoms. meanwhile, other studies proposed to increase the dopamine turnover to bring about a normalized dopamine level when eating food.19 studies have demonstrated that tyrosine supplementation may help normalize dopamine levels disrupted in an, though further research is needed in human populations. fish oil has shown a similar effect, since it can normalize the expression of genes and neurotransmitters in the hippocampus and hypothalamus. more importantly, it increased the dopamine level in the hypothalamus.19 berkeley pharma tech journal of medicine | 79 2.3 appetite regulating pathway 2.3.1 orexigenic pathway figure 1. appetite regulating pathways of an. describes the orexigenic and anorexigenic signaling pathways that modulates appetite in an inter-dependent fashion. orexigenic: npy projects to lh and inhibits three anorexigenic neurons in pvn: oxt, th, and trh, to increase appetite and fat storage20; agrp acts as a competitive antagonist to α-msh for the melanocortin 4 receptor (mc4r) in pvn; orexin-a increases intracellular ca2+ concentrations to promote npy expression.21 on the other hand, orexin decreases intracellular ca2+ concentrations in pomc-expressing neurons21; ghrelin travels through the vagus nerve to promote npy/agrp neurons and inhibits pomc neurons through paracrine signaling22-23; anorexigenic: pyy3-36 depolarized pomc neurons and increased its action potential by 93%.24 pyy3-36 also stimulates mesolimbic dopamine pathways by acting as a selective y2 receptor agonist25; crh works through the glp-1 signaling pathway, which involves histamine and unknown feeding-relating neurons, to reduce food intake.26-27 ;leptin is responsible for lowering npy secretion by altering its voltage-gated k+ channels and decreasing its excitability.28 it also upregulates pomc through jak-stat3 phosphorylation pathway.29 in addition, leptin increases crh levels in the hypothalamus30;pomc cleavage produces a-msh which acts on mc4r in pvn to inhibit food intake. berkeley pharma tech journal of medicine | 80 orexigenic pathways are those that promote food intake and increase appetite. ingestive behaviors can be categorized into two types: appetite and consummatory. ingestive behaviors can be divided into two phases: appetitive behaviors, which include food seeking, foraging, and hoarding; and consummatory behaviors, which involve food intake and digestion. for the detailed description of how these appetite-regulating pathways are connected refer to figure 1. the primary orexigenic pathway involves neuropeptide y (npy), a 36-amino acid peptide predominantly expressed in the central nervous system, particularly within the hypothalamic arcuate nucleus (arcn), where it plays a key role in appetite regulation. the npy neurons are found in the arcuate nucleus (arcn). npy can detect hormones from stomach and fat adipocytes in promotion of food intake and fat tissue storage in adipose tissue cells. specifically, npy neurons in the arcn project to lateral hypothalamus and feeding-regulating neurons in pvn to enact orexigenic pathways.31 in the npy neurons, the orexigenic effects are mainly mediated through the y1 and y5 g protein coupled receptors.32 the y5 receptor is suggested to contribute more to the consummatory effects of food regulation as the y5 agonist, increased food intake by 225-800% in foraging hamsters.33 although studies remain inconclusive regarding the direct relationship between plasma npy levels and an, research indicates that reduced gut microbiome diversity, as observed in antibiotic-treated murine models, correlates with lower npy levels.34 agouti-related peptide (agrp), co-expressed with npy in the arcuate nucleus, serves as a key regulator of energy homeostasis by responding to metabolic signals, such as leptin (satiety) and ghrelin (hunger).35 agrp neurons are stimulated by the hunger hormone ghrelin and inhibited by the satiety hormone leptin, thereby modulating feeding behavior. mc4r signaling has been implicated as an inhibitory factor in food intake as administration of mc4r agonists into pvn reduces food intake by 48% four hours post-injection.36 agrp neurons also express gaba neurotransmitters which are capable of directly berkeley pharma tech journal of medicine | 81 inhibiting pomc anorexigenic neurons. due to the nature of agrp as an indicator of energy homeostasis, an patients are concluded to have abnormally higher agrp levels, as studies with acute and weight-restored an showed that the plasma agrp levels are elevated, and are correlated with a lower bmi score.35 the appetite-regulating pathway is interconnected and involves multiple parts of the brain. in fact, orexin are orexigenic hormones synthesized in the lateral hypothalamus (lh) nucleus and promote npy-positive neurons while inhibiting pomc-positive neurons in the arcn. orexin offers insight into how the physiological and psychological properties of an are connected. food aversion increases the expression of orexin in the lh, resulting in an increase in dopamine release in the accumbens nucleus.37 such a connection to the brain reward circuit contributes to aversion to food consumption and encourages anorexic behaviors. moreover, studies show that an patients have abnormally high neural responses to both pleasant and aversive food stimuli, suggesting a dysfunction in pathways leading up to the neural reward response.38 such evidence demonstrates the need to expand current treatment options for an to address the physiological issues. such interconnectedness is an example of the gut-brain relationship as ghrelin is a 28-amino acid peptide produced in the stomach that promotes npy/agrp expression. acylated ghrelin is orexigenic, while non acylated ghrelin is involved in synthesizing anorexigenic mediators like urocortin in the hypothalamus.39 ghrelin heavily relies on the vagus nerve to carry out its signaling functions.40 its secretion is up-regulated with negative energy balance such as fasting and an, and down-regulated with positive energy balance such as feeding and obesity. furthermore, ghrelin acts on the reward circuit through the dopaminergic pathway.41 paradoxically, ghrelin levels are elevated in berkeley pharma tech journal of medicine | 82 an patients, likely as a compensatory response to chronic energy deficit, despite persistent appetite suppression.42 2.3.2 anorexigenic pathways anorexigenic pathways are those that reduce food intake, suppress appetite, and are interconnected with regulation of orexigenic pathways. one of the key anorexigenic pathways involves peptide yy (pyy), a 36-amino acid peptide produced by enteroendocrine l cells in the ileum and colon.43 circulating pyy levels increase postprandially, peaking approximately 1–2 hours after feeding. pyy1-36 and pyy3-36, belong to the same family as npy and consist of five g protein-coupled receptors (y1, y2, y4, y5, y6). pyy is able to cross the blood-brain barrier and inhibit npy neurons through transmembrane diffusion from the circulation.44 anorexigenic actions are carried out through the y2 inhibitory receptor, which is abundantly expressed on npy neurons in arcn, since these effects were not observed in y2 receptor knockout mice.45 on the other hand, pyy3-36 in arcn promotes pomc neurons.45 pyy3-36 also stimulates mesolimbic dopamine pathways by acting as a selective y2 receptor agonist.46 this finding offers insights to evidence suggesting the dysfunction of the mesolimbic dopaminergic pathway in an patients.47 although results are inconsistent with regards to the pyy levels in an patients, increased pyy levels reduces food intake and fasting pyy levels are higher in all an presentations.48 although its specific anorexigenic action requires further research, corticotropin-releasing hormone (crh) acts as a hypothalamic neurotransmitter to reduce appetite.49 one of the ways crh exerts its anorexigenic effects is through the glucagon-like peptide-1 (glp-1) signaling.50 another study suggested that npy mediates the anorexigenic effects of crh, as impaired activity of crh neurons led to neuropeptide y (npy)-induced hyperphagia.51 within the hpa axis, adrenocorticotropic hormone (acth), a downstream product of crh, has been implicated in appetite suppression, as acth signaling precedes pomc activation, which exerts anorexigenic effects.52 crh levels are elevated in an patients, potentially as a berkeley pharma tech journal of medicine | 83 compensatory response to cortisol resistance, a phenomenon frequently observed in an.53-54 mirroring the role of orexigenic ghrelin, leptin acts as a “satiety hormone” and is responsible for regulating multiple feeding-regulating neurons including npy, pomc, crh.55-58 it is secreted from adipocytes, which are fat cells that store energy and its abundance is a key indicator of available energy due to its role as a peripheral hormone. in an patients, reduced plasma circulating leptin levels were observed while a significant increase in the npy inversely correlates with bmi scores.59 these studies hint at the dysregulation of neuropeptides and results in abnormal eating behaviors observed in an. finally, proopiomelanocortin (pomc) functions similarly npy/agrp and produces anorexigenic effects by producing a-msh to act on the pvn and decreases food intake. due to the permeability of arcn, pomc is heavily regulated by leptin and gaba. stress has been linked to be a marker for an, and under such conditions, pomc mrna expression increases and activation of its neurons results in anorexigenic effects.59 as pomc neurons can express a long isoform of leptin receptors, leptin can activate the stat3 pathway and increase the mrna expression of pomc. leptin modulates neighboring npy-gaba neurons by reducing gabaergic inhibition onto pomc neurons, thereby enhancing anorexigenic signaling.60 3. current treatments for an cognitive behavioral therapy (cbt) is among the most effective treatments for eating disorders, demonstrating relatively low relapse rates and significant symptom reduction. cbt begins by identifying sources of emotional distress that contribute to dysphoria. using that knowledge, the therapist can understand the behavior and motivation of the patients and develop their problem-solving skills to cope with their situations. each cbt strategy is tailored specifically between therapist and patient to help the patient eventually be able to practice berkeley pharma tech journal of medicine | 84 self-regulation.61 for patients with phobia, cbt has shown to have reduced hyperactivity in the insula and anterior cingulate cortex, regions known to have increased activity when processing phobic threats. in patients with ocd, cbt improved the hyperactivation of circuits that are associated with ocd, including the orbitofrontal cortex, anterior cingulate gyrus, and basal ganglia. in anxiety cases, patients had lower glucose uptake after cbt particularly in the right hippocampus, medial prefrontal cortex, and left ventral cingulate cortex, restoring the normal circulation.62 cbt facilitates cognitive restructuring, leading to neurobiological changes in brain regions involved in reward processing and emotional regulation. studies report a remission rate of 33% for an, with 77.3% of adult patients experiencing a significant reduction in disordered eating behaviors after a year of treatment.63-64 family based treatment (fbt) also focuses on restructuring the patient’s self-perception and any cognitive distortion on food intake. the major difference is that fbt seeks to include families as a resource to the child’s aid, as shown in figure 2 below. in phase 1 of fbt, parents assume full responsibility for meal planning and supervision, ensuring adequate nutritional intake while reducing conflict surrounding food. once the patient reaches a medically stable weight and eating-related distress decreases, phase 2 gradually returns control over eating to the adolescent under parental guidance. phase three revolves around resolving the still-lingering symptoms and any developmental issues disrupted by the condition, such as identity formation and self-image. the first randomly controlled trial (rct) of fbt on an patients showed that fbt was more efficient for 18-year-olds and younger children in preventing relapse and weight restoration. in other rcts, fbt has been proven to be effective in the long term for weight restoration and maintaining gains even five years after treatment. studies show that merely 10 sessions of fbt course is just as effective as 20 sessions of individual oriented course, in terms of weight maintenance.65 at six and twelve months after trials, fbt was shown to be statistically superior to adolescent-focused therapy, differing by over 20 percentage points in remission rates.66 berkeley pharma tech journal of medicine | 85 figure 2. family based therapy process flow for an. as aforementioned, fbt has multiple stages and one of its advantages is the gradual improvement the patient will experience as it goes through each phase. through creating a unity amongst the patient’s loved ones, the patient can feel more encouraged to correct their diet compared to cbt. berkeley pharma tech journal of medicine | 86 4. the relationship between the gut microbiome & gut brain axis figure 3. bi-directional gut-brain linkage. summary of examples of the bi-directional communications between the gut microbiota and the brain. this linkage suggests the important role gut microbiota play in mental health disorders. as mentioned previously and depicted in figure 3, the gba is a bi-directional linkage between intestinal processes and the central nervous system. the central nervous system mainly communicates through vagus and pelvic nerves to intestinal targets such as the enteric nervous system. the hypothalamic–pituitary–adrenal axis, endocrine pathways and bacterial metabolites via blood circulation are all major connections between the gba and gut microbiome.67 the gut microbiome consists of trillions of microbes that reside in the intestine and spans up to 500 species.68-69 the gut transmits signaling molecules through the enteric nervous system and vagus nerve, modulates immune responses and gut permeability, and maintains barrier integrity.8,67 the role of gut microbiota in neuropsychiatric disorders was first recognized in hepatic encephalopathy, where oral antibiotic treatment improved neurological symptoms.8 more recent studies link gut microbiome dysbiosis to an and other psychiatric conditions.68 more recently, the gut microbiota dysbiosis, referred to as the imbalance of microbial communities, has been linked closely with berkeley pharma tech journal of medicine | 87 anxiety, depression, autism-spectrum disorder and eating disorders.8,70 administration of lactobacillus and bifidobacterium strains can reduce cortisol release, which has been associated with reduced depression, lower pro-inflammatory cytokine levels and a healthy response to stress.71 the gut microbiome can communicate with the brain through their bacterial metabolites. gut microbes produce short chain fatty acids (scfa) such as butyric acid, propionic acid, and acetic acid during metabolism.67 scfas activate the sympathetic “fight or flight” nervous system, influences memory process, and modulates food intake. for example, butyric acid, the product of sugar fermentation by obligate anaerobic bacteria, is responsible for inhibiting the release of anorexigenic peptide, crh, levels and influencing appetite.72 these bacterial metabolites can also signal and regulate the synthesis of neurotransmitters in intestinal epithelial cells that act on the brain through the vagus nerve.67 one of the other ways the gut microbiome communicates with the brain is through neurotransmitters. the gut microbiota produces metabolites that include neurotransmitters or their precursors. as shown in figure 3, certain neurotransmitters and precursors produced by gut microbiota can travel through the blood-brain-barrier (bbb) for signaling or further synthesis of neurotransmitters.67 dopamine, serotonin and gaba are three neurotransmitters closely correlated to the pathology of an. as mentioned in earlier sections, hypophagia is present when 5-ht1b and 5-ht2c receptors are stimulated. staphylococcus in the gut can decarboxylate aromatic amino acid, dihydroxyphenylalanine and 5-hydroxytryptophan into dopamine and serotonin. dopamine can then be transported via the blood-brain-barrier and reach the mesolimbic dopaminergic system usually associated with food reward. on the other hand, serotonin is mainly synthesized through the metabolites, scfas, secreted by bacteria strains that signal enterochromaffin cells to produce tryptophan hydroxylase.73 in fact, 90-95% of serotonin are mainly present in the epithelial enterochromaffin cells of gi tract.74 the berkeley pharma tech journal of medicine | 88 serotonergic and 5-htergic system plays a role in mediating the onset of an. gaba production is influenced by gut microbiota, with dietary factors such as a ketogenic diet increasing gabaergic activity. as an inhibitory neurotransmitter, gaba plays a role in appetite suppression, which may contribute to an pathophysiology.75 on the other hand, since this gut-brain-axis is a bi-directional linkage, the brain also affects the make-up of gut microbiota. the brain primarily communicates with the gut microbiota through secretion of signaling molecules from neuron, immune, and enterochromaffin cells. these signaling molecules depend on the neurotransmitter receptors present on bacteria. for example, e. coli o157:h7 contains a receptor for host epinephrine and norepinephrine.76 the brain can also alter microbiota composition through influencing the intestinal permeability. stress increased colonic paracellular permeability by increasing the production of interferon-g and decreasing the expression of zo-2 and occludin.77 the crh receptors participate in colonic barrier dysfunction under mild stress in adult rats.78 stress in the brain also induces the production of an antimicrobial protein, a-defensin, influencing the existing microbiota composition. 5. targeting anorexia by increasing gut microbiome diversity anorexia has been correlated with an altered gut microbiome due to the altered expression of orexigenic and anorexigenic peptides which are affected by a subpar gut microbiome shown in previous studies with anorexic mouse models.79 studies have shown that alterations in the normal microbial composition, also known as gut dysbiosis, can be linked to an patients, as well as the development of other eating disorders. in general, an patients have a lower microbial diversity than healthy controls.80 during different phases of an anorexic patient’s disorder, there are significant shifts in gut microbiome composition.81 more specifically, alpha diversity drastically decreases during the phase of weight loss.81 berkeley pharma tech journal of medicine | 89 morita et al. reported that an patients exhibit reduced colonization of butyrate-producing bacteria, including clostridium coccoides, clostridium leptum, and bacteroides fragilis, which may disrupt appetite-regulating signals.81 a decrease in butyrate-producing species and an increase in mucin-degrading species are also hallmarks of the gut microbiota in an patients.83 in addition, during the phase of weight loss in an patients, a review on the role of microbiota in the pathogenesis of eating disorders mentions that there is a reduction in firmicutes and scfas in the gut microbiome, while there is an increase in bacteroides, actinobacteria, enterobacteriaceae, and methanobrevibacter smithii.81 the review, consisting of 16 different studies, concluded that microbial richness increased in anorexic patients after weight regain through stool transplantation. they also concluded that scfa levels increased and the firmicutes/bacteroides (f/b) ratio normalized when there was weight gain and renourishment.81 as mentioned in the pathophysiology section, neuropeptide dysregulation is found in an and the gut microbiota plays a key role in maintaining npy, agrp, and pyy levels.84 an alteration in the gut microbiota affects neurotransmitters which is similar to the case borgo et al. reported. in the trial, an increase of enterobacteriaceae, an antigen, which produces bacterial peptide caseinolytic protease b, mimics a-msh and activates anorexigenic neurons.85 these studies associating an and gut-microbiome diversity, show that a restored gut microbiome may be a potential treatment option for eating disorders, and more specifically an. an patients’ altered gut microbiome conditions have been proven to influence gut permeability and inflammation, appetite changes, weight loss and gain, gastrointestinal symptoms, among other elements that impact the pathogenesis of an. understanding the relationship between a patient’s unique gut microbiome composition and their psychological condition can also be a key factor of focus for researchers and healthcare providers in order to determine the development of an. berkeley pharma tech journal of medicine | 90 6. dietary supplements as treatment method for anorexia nervosa 6.1 probiotics as mentioned in previous sections, an patients are often observed with gut dysbiosis, characterized with abnormal levels of pathogenic bacterial species, a disrupted intestinal barrier integrity, and lowered diversity. to study whether such gut dysbiosis contributes to an symptoms of weight loss and neuropsychiatric disturbances, fan et al. reported that germ-free mice colonized with an-derived microbiota resulted in reduced weight gain and increased anxiety-associated activities.86 another study by morisaki et al. conducted a study analyzing the gut microbiota of an patients using polymerase chain reaction (pcr), and reported that patients exhibited preserving gut dysbiosis during inpatient nutritional therapy, cbt, and supportive psychotherapy, despite improved weight gain and psychological functions.87 this highlights that gut dysbiosis may not be restored only by weight gain and indicates the importance of targeting the gut microbiota in combination with traditional nutritional intervention therapies. therefore, probiotics, including commercially available lactobacillus supplements, have been investigated as potential adjunctive treatments for an, with the goal of restoring gut microbiome balance. 6.1.1 lactobacillus lactobacillus species have been utilized in the clinical setting for improving gastrointestinal health.88 more recently, it has been linked with modulation of stress as studies of the gba matures. administration of lactobacillus bacterial strains can reduce the release of cortisol which leads to reduced depression, lower pro-inflammatory cytokine levels, and a healthy response to stress.89 schwarzer et al. recently discovered, lactobacillus plantarum probiotics supplementation attenuated weight loss and growth impairment in malnutritioned mice, suggesting the possible role of probiotic intervention for an.90 berkeley pharma tech journal of medicine | 91 lactobacillus rhamnosus gg (lgg) is one of the probiotic strains being studied for treating an due to its protective effect on the gut microbiota.91 it mostly resides in the colon mucosae and vagina and is key to maintaining genito-urinary health in preventing urinary tract infections. lgg has been identified as a key probiotic for an treatment due to its role in maintaining intestinal barrier integrity by upregulating tight junction proteins, such as zonula occludens-1, thereby reducing gut permeability. moreover, lgg modulates the gut-brain-axis by regulating the 5-htergic intestinal system, increasing gaba receptors in the brian, increasing serotonin receptor expression and, overall, relieving anxiety-like behaviors.91 bai et al. conducted a mouse model study on deoxynivalenol (don)-induced anorexia, demonstrating that lgg supplementation alleviated anorexic symptoms by reducing intestinal inflammation and modulating appetite-related signaling pathways. coinciding with information presented in previous sections, don-induced anorexia mainly releases 5-ht, which acts as a neurotransmitter and leads to anorexia.92 in the study’s experimental design, antibiotic-treated mice were separated into 4 study groups: control, don, don+lgg, lgg and after 4 weeks of treatment, researchers found that lgg prevented don-induced anorexia by increasing crypt depth of the jejunum and ileum, strengthening the barrier integrity, and promoting nutritional uptake. lgg also remodeled the tlr4-myd88-nf-κb signaling pathway and since tlr4-myd88-nf-κb is responsible for lipopolysaccharide (lps)-induced anorexia, lgg is able to alleviate anorexia mediated by pro-inflammatory cytokines. lgg also maintained butyric acid levels comparable to controls, whereas the don group had a significant drop. this affirms previous research done that shows an patients with lower butyric acid levels and demonstrates how lgg promotes food intake as butyric acid inversely correlates with anorexigenic crh by influencing cortisol receptor in the intestine.93,72 in addition, lgg’s regulation of appetite regulating pathways (npy, agrp, pyy) allows for upregulation of food intake and remodels the gut microbiota. more importantly, lgg remodeled berkeley pharma tech journal of medicine | 92 the gut microbiota to ensure long-term benefits as 11/16 metabolites were increased by don compared with control while they were decreased by lgg + don. moreover, bai et al. conducted fecal microbiota transplantation (fmt) to antibiotic-treated mice and reported that fmt-lgg+don prevented weight loss and low food intake present in fmt-don. further research will be needed to examine its efficacy on humans but its promising results can hopefully present a possible therapy in conjunction with nutritional and behavioral interventions for an . another treatment of interest is lactobacillus acidophilus (l. acidophilus) and has been associated with modulation of weight in multiple clinical studies. l. acidophilus’s main function is to produce lactic acid from lactose and has gained popularity for its digestive health benefits with the production of “acidophilus milk” when prevalence for obesity is significant in the us.94 although direct studies on l. acidophilus in an patients are lacking, its ability to restore gut microbiome diversity, regulate inflammatory responses, and improve intestinal permeability suggests its potential as an adjunctive treatment. first of such studies dates back to 1952, in which 124 bottle-fed infants, added with 500,000,000 viable l. acidophilus organisms per quart into daily formula, were compared with 123 healthy controls. the results showed significantly larger weight gain during the first month than did the controls.95 another 8-week randomized parallel study of the effects of probiotic drink on ldl-cholesterol and fibrinogen factors revealed that the group intaking yogurt with l. acidophilus showed weight gain.96 in a more recent study, researchers conducted a genomics study on seven lactobacillus species associated with weight gain and have been able to classify l. acidophilus and l. reuteri, among others, as weight gain-associated strains.97 although other studies indicate l. acidophilus’ capability in ameliorating obesity and reduce weight gain, such effects can be explained by l. acidophilus’ ability to restore gut microbiome diversity as this same study revealed its reversal of a obesity-induced gut dysbiosis. overall, l. acidophilus increased alpha-diversity and restored alterations in beta-diversity which is key to the treatment of an.98 berkeley pharma tech journal of medicine | 93 gut dysbiosis is characterized by a disrupted intestinal permeability leading to an outgrowth of gram-negative lps producing proteobacteria and circulating lps levels.98 the nutrient deficient environment of an disrupts the intestinal barrier integrity, as kleppe et al. reported zonulin family peptides, serum markers for integrity, are significantly lower than healthy controls.99 a disrupted barrier promotes lps release resulting in low grade inflammation which exacerbates an.100 in fact, lps plays a crucial role in the pathophysiology of an as it reduces food intake via a mechanism dependent on myeloid differentiation primary response 88 (myd88) signaling, an inflammatory signaling pathway, within endothelial cells lining brain capillaries and neurons cells.101 one of the ways l. acidophilus restores gut microbiome homeostasis is through improving intestinal integrity and lowering gut permeability. specifically, l. acidophilus strengthens the barrier by increasing the expression of epithelial mucosal proteins, intectin, and the tight junction proteins, occludin.98 a strengthened intestinal barrier results in decrease in circulating lps, as a clinical trial conducted on 71 obese women over the period of 12 weeks reported a decrease in lps levels by 20.14% after administering l. acidophilus.102 although these study subjects were not an patients, they were all diagnosed with low-grade systemic inflammation caused by a gut barrier permeability dysfunction, which is characteristic of anorexia. in culmination, l. acidophilus’ unique ability to restore gut microbiome homeostasis through modulating the intestinal barrier integrity and lps levels suggests its importance in treating an. currently, an ongoing phase 2 randomized controlled study conducted by the medical university of vienna is examining the effects of daily multistrain probiotics doses including l. acidophilus w22 in 30 adolescent an patients. this is the first study studying the administration of probiotics on adolescent an patients and can be conclusive due to a double-blind randomized longitudinal design, the large sample size and the comparison with sex and age-matched healthy controls. berkeley pharma tech journal of medicine | 94 a final bacterial strain of interest is lactobacillus reuteri (l. reuteri) and similar to l. acidophilus, studies have concluded that this strain is associated with weight gain.97 l. reuteri mainly produces organic acids, ethanol, which aids in inhibiting the colonization of pathogenic microorganisms. its usual tendency to colonize in the gi tract is the reason it is considered an optimal candidate for probiotic use.103 with regards to an, l. reuteri is capable of strengthening the intestinal barrier by preventing microbial translocation from the gut lumen to the tissues, restore microbiome homeostasis through inhibiting pathogenic microorganism colonization, and reduce the production of pro-inflammatory cytokines.104 a recent 3-year clinical trial in croatia investigated the effects of l. reuteri dsm17938 on 31 adolescent an patients with comorbid constipation. after six months, 93% of the l. reuteri group achieved weight normalization, compared to 63% in the placebo group.105 constipation is often frequently accompanied with an weight loss and can further discourage food intake.106 this study reported that after 3 months, constipation relief was higher in the l. reuteri than in the placebo group (87% vs. 63%), and such relief can pave the way for nutritional recovery interventions. overall, l. reuteri is a potential safe and effective way of mitigating an and constipation in adolescents, due to its positive effects on weight normalization, patient compliance, constipation relief, which can lead to more effective nutritional recovery. 6.2 vitamins vitamins are organic compounds that are essential to the human body for organs to perform various essential physiological processes, including metabolism, immune function, and cellular homeostasis. previous research has shown that there is significant association between vitamins and improving gut microbiome diversity, specifically with vitamin d, a, and e.108,115,121 most of these vitamins need to be consumed through one’s diet from sources such as meats, fruits, and vegetables. an patients are especially at risk of malnutrition can lead to gut complications like having deteriorating intestinal barriers, consequently leading to inflammation and general discomfort from berkeley pharma tech journal of medicine | 95 eating. vitamin d, a, and e have all been found to assist with preserving gut barrier function and promoting microbiome health. 6.2.1 vitamin d vitamin d is an important nutrient in development and especially in the regulation of the microbiome. with an patients consuming less food, patients are at high risk of vitamin d deficiency which could create a positive feedback loop of worsening symptoms. vitamin d influences the microbial composition through vitamin d receptor genes (vdr), which regulates immune responses and microbial homeostasis. vitamin d regulates the expression of antimicrobial peptides, such as cathelicidins and defensins, which help maintain gut mucosal integrity by preventing dysbiosis and promoting epithelial repair.108 through this, vitamin d controls the health of the gut by protecting against inflammation with the expression of vdr genes.109 this is shown in one particular study, in which the fecal microbiome of 50 adolescent women were tested after receiving vitamin d supplements. results showed an increase in firmicutes and bifidobacteria, with a reduction in bacteroidetes, a marker of inflammatory response. also, a reduction of vdr gene expression can increase the proteobacteria population, causing an increase in inflammation. this is also supported by a study in which vdr genes in mice were removed and subsequently the subjects showed severe gut inflammation, shown with increased amounts of bacteroidetes.110 with adequate supplementation of vitamin d, the vdr genes are able to prevent the pathogenic microbes from outcompeting the beneficial bacteria, the leading cause of gut dysbiosis.109 vitamin d can help alleviate many other organ complications arising from an. vitamin d deficiency is often associated with liver and kidney diseases, and with the inherent malnutrition experienced by an patients, they are more prone to such diseases. singh et. al tested the effect of vitamin d supplementation on subjects and discovered improved liver function along with decreasing the ratio of aspartate aminotransferase (ast)/alanine aminotransferase (alt). kidney function also improved as the ratio of serum blood-urea-nitrogen berkeley pharma tech journal of medicine | 96 (bun)/creatinine decreased. in the same study, the relative abundance of bifidobacterium and akkermansia increased, as did the firmicutes and healthy bacteroidetes probiotic phylums, while on the other hand, the prevotella phylum decreased. in addition, the study also showed that the vitamin d supplementation led to a statistically significant greater diversity of microbiota.111 although more research is still needed, scientists still speculate that an is related to the dysbiosis of the gut microbiota.112 if an association between gut dysbiosis and an is confirmed, vitamin d supplementation could serve as a potential adjunct therapy for improving gut health and alleviating gastrointestinal symptoms. an ongoing trial for the past two years aims to further study the effect of vitamin d on gut microbiota and intestinal barriers, especially in patients with irritable bowel syndrome (ibs). the researchers have currently found that when the concentration of vitamin d is reduced, symptoms seem to be connected to the concentration. supplementation of vitamin d has shown alleviation of ibs symptoms and researchers are investigating the mechanisms of dissecting fecal microbiome samples through 16s rrna gene sequencing and detecting the expressions of z0-1, occluding in intestinal mucosa. if vitamin d can indeed help with alleviating any organ complications arising from an, then vitamin d can be a good supplement to start helping alleviate symptoms of an. through feeding the patients their proper amount of vitamin d, they can prevent gut complications arising from an. as aforementioned, vitamin d supplements can be important in restoring the nutritional balance to maintain a healthy digestive system. vitamin d supplements will aid an patients who suffer from gastrointestinal issues in immune function and enhance nutrient absorption, a step in the overall recovery process. through alleviating the general discomfort and the organ complications arising from the disorder, patients can eventually feel comfortable again to eat healthily. berkeley pharma tech journal of medicine | 97 6.2.2 vitamin a vitamin a is an essential nutrient involved in various physiological functions, including the maintenance of gastrointestinal homeostasis.113 vitamin a is derived from two primary dietary sources: retinol, found in animal products such as meat and fish, and provitamin a carotenoids, obtained from fruits and vegetables. not only is vitamin a essential in homeostasis, but it also maintains a protective effect on the intestinal mucosal barrier and gut mucosal integrity.114 patients who suffer from an tend to not get the required nutrients and supplements they need to maintain their health and gut microbiome. without the consumption of foods like meat, fish, fruits, and vegetables, anorexic patients drastically lack vitamin a intake. a research trial conducted by pattanakitsakul et. al. studied the nature of vitamin a deficient rats and discovered that additional vitamin a supplementation protects the intestinal mucosal barrier even after chemotherapy treatment, which is known to drastically affect gut microbiome composition.115 this protective role of vitamin a can be useful in implementing and preserving a healthy gut microbiome composition that is required in anorexic patients. another study conducted in chongqing medical university located in china studied the gut microbiome composition of vitamin a-deficient and vitamin a-sufficient groups amongst patients experiencing diarrhea.116 they observed that gut microbiota richness was not different between the two groups, but that there was significantly lower microbiome diversity in patients with deficient vitamin a according to the shannon and simpson index, a traditional method used to measure species diversity.117 this means that patients with sufficient vitamin a had ample quantity of gut microbiota species, and in specific the study found that escherichia-shigella and clostridia phylotypes were found more prominently found in the vitamin a sufficient group, while the deficient group was largely composed of enterococcaceae, including the enteric pathogen enterococcus faecalis. enteric pathogens, such as enterococcus faecalis, are typically harmless in the intestinal environment of healthy individuals, but the species’ overabundance in the gut can quickly result in infections that spread throughout the berkeley pharma tech journal of medicine | 98 body and even promote tumor formation.118 in addition, people with underlying health problems, in this instance, patients with an, with a weakened immune system, are more likely to face additional health issues due to their diet lacking vitamin a amongst other nutrients. other studies have also shown that intake of retinol, a dietary supplement derived from vitamin a and as mentioned previously to be found in meat and fish, can increase or even re-establish ratios between proteobacteria and actinobacteria as well as proteobacteria and firmicutes.119 this would be useful in anorexic patients since there are significant changes in the quality, quantity, composition of gut microbiota during the phase of weight loss. these studies suggest that vitamin a may play a role in maintaining gut microbiome balance and mucosal integrity, which could be particularly beneficial for an patients experiencing intestinal dysbiosis. 6.2.3 vitamin e a fat soluble antioxidant, vitamin e functions as a fat-soluble antioxidant that supports plasma membrane repair and maintains intestinal barrier integrity by reducing oxidative stress and inflammation. vitamin e also helps protect the gut microbiota from pathogenic invasion. there are eight different types of vitamin e, but the most common form and most present and active in humans is a-tocopherol.120 significant vitamin e uptake shows a positive association with the firmicutes phylum and negative association with bacteroidetes. firmicutes are important gut bacteria for metabolism and bacteroidetes, while sometimes beneficial, overabundance of this species can be damaging for gut barriers as it degrades the mucus barriers of intestines.121 in addition, a study on lactating women found that those with higher vitamin e intake were associated with a decrease in proteobacteria, known to contain many pathogens and cause inflammation.119 this supports the idea that vitamin e deficiency can increase inflammatory response. due to anorexic patients’ lack of nutrient intake, vitamin e supplementation, along with other vitamins, could drastically improve the state of a patient’s gut microbiome and protect them from further health complications that can arise from pathogens and inflammation of the gut. berkeley pharma tech journal of medicine | 99 while research in determining the exact quantities of vitamins to administer in order to obtain the best results for the gut microbiome are lacking, specifically in regards to vitamin e supplementation, a recent study from 2020 shows that a lower-level consumption of vitamin e changes the composition of the gut microbiota while also increasing spleen and body weight. the study assigned mice with similar body weight to three varying groups, a control, low-level vitamin e (0.06 mg per 20 g of body weight), and high-level vitamin e (0.18 mg per 20 g of body weight). comparing the body weights of the mice before and after vitamin e supplementation showed that while all groups increased in weight (including control), the low vitamin e group had the most weight gain as well as the highest ratio of firmicutes to bacteroidetes in the gut microbiome.120 since the ratio of f/b is drastically altered in anorexic patients, implementation of lower quantities of vitamin e based on a patient’s body weight can promote weight gain and renourishment, while also enhancing gut microbiome composition. meanwhile, another study was conducted where iron and vitamin e were taken together as supplements. the test group was shown to have a differing microbiome composition with a decrease in bacteroidetes and an increase in firmicutes. there was also an increase in roseburia, a producer of butyrate, a short-chain fatty acid that can help with gut mucosal barrier. the study concluded that ultimately the combination of iron and vitamin e benefited microbiome composition by assisting the growth of butyrate-producing bacteria.122 6.3 fatty acids and ketone supplements ketones are metabolic intermediates formed as byproducts of fat metabolism. when people starve, their bodies enter ketosis, a state where the brain shifts from using glucose to using ketones for energy. this state can reduce anxiety and provide a sense of reward, which might explain why some people with an find starvation soothing. a ketogenic diet (kgd) can replicate the state of ketosis without actual starvation.123 the diet is high in fat from foods like avocados and nuts, moderate in lean protein like fish, eggs, and lean pork/beef, and is very berkeley pharma tech journal of medicine | 100 low in amounts of carbohydrates from leafy greens and non-starchy vegetables.124-125 it has been used effectively to reduce seizures in epilepsy and is being studied for other neurological conditions. this shift affects various brain pathways and can have significant effects on neurological health, similar to the effects seen in epilepsy treatment. while no published studies have evaluated kgd specifically for an, preliminary evidence suggests potential benefits in addressing energy deficits and gut microbiome alterations. however, further research is needed to determine its clinical applicability. 6.3.1 ketone body metabolism overview ketone body metabolism involves coordinated processes across multiple organs, including the liver, muscles, and brain, to regulate energy homeostasis. ketone bodies are vital energy sources for the nervous system, especially when glucose is scarce. in healthy adults, ketone levels fluctuate daily, contributing to about 5% of energy expenditure in the fed state and up to 20% during fasting or starvation.126 these levels rise significantly during fasting, exercise, neonatal periods, or late pregnancy.126 other tissues such as skeletal muscle, heart, and kidneys also metabolize ketone bodies. this turnover includes production from fatty acids in liver hepatocytes and disposal by extrahepatic tissues.127 fatty acids, released from adipose tissue, enter hepatocytes and undergo β-oxidation in the mitochondrial matrix, leading to ketone body production, primarily from acetyl-coa.127 ketogenesis is regulated by hepatic glucose availability, mitochondrial redox state, β-oxidation efficiency, tca cycle flux, and the hormonal balance between glucagon and insulin, which collectively determine ketone body production.126 overall, ketone bodies are crucial energy sources for various processes ranging across all bodily systems and mechanisms and changing levels in a person’s body highly affects how energy is utilized and the balance of hormones, and other substances, in the body. therefore, reinforcing the ketone body prevalence in a patient’s body may have a significantly positive impact on reestablishing homeostasis and energy efficiency throughout recovery from an. berkeley pharma tech journal of medicine | 101 6.3.2 ketones and short-chain fatty acids (scfas) ketone bodies serve as an alternative energy source during periods of low carbohydrate intake, supporting various organs, including the brain and muscles. they help modulate the redox state within cells, potentially reducing oxidative stress and supporting cellular health.126 they are precursors for lipid synthesis, playing a role in lipid storage and metabolism.126 ketone bodies can also act as signaling molecules that influence pathways involved in inflammation, cell growth, and differentiation.126 the production of ketone bodies in the gut is regulated by the microbiome and dietary factors, suggesting an intricate relationship between ketones and gut health. given the severe caloric deficits in an patients, ketone bodies could provide a critical energy source, helping to restore overall energy balance.128 an is often associated with gut dysbiosis which is essentially the imbalance of the microorganisms that reside in the body, especially in the gut or skin.129 due to their multifaceted role in the body, ketone bodies could help restore microbial diversity, which is crucial for gut health and nutrient absorption. they may reduce inflammation in the gut, a common issue in an, thereby promoting a healthier gut environment.126 the ability of ketone bodies to influence cell proliferation and differentiation in the gut lining could also help maintain or restore gut mucosal integrity, which is essential for proper nutrient absorption.127 the ambiguous roles of ketone bodies suggest they could be used as part of a comprehensive treatment strategy for an. their benefits in energy provision, gut health, and inflammation modulation make them a promising adjunct to traditional treatments like cbt and fbt which address the disease from a more neurological basis without also assessing the physical imbalances caused by an. according to other studies, ketone bodies, such as beta-hydroxybutyrate (bhb), significantly impact cerebral fuel metabolism.128 they exhibit a glucose-sparing effect by substituting for glucose in the brain, particularly noticeable during fasting or ketogenic diets.128 this metabolic adaptation not only supports brain function but also suggests potential therapeutic benefits for conditions like an, berkeley pharma tech journal of medicine | 102 where compromised glucose metabolism can occur.128 ketone bodies enhance cerebral energy metabolism without altering atp levels, indicating their efficiency as a neuroprotective substrate.128 additionally, ketone bodies possess antioxidant and anti-inflammatory properties, potentially mitigating oxidative stress and neuroinflammation, common in neurodegenerative diseases.128 these insights highlight the therapeutic potential of elevating ketone bodies in managing disorders characterized by impaired glucose metabolism, including those affecting brain health like an. there are a few ongoing clinical trials that are investigating the impact of short-chain fatty acids and ketone supplements on eating behavior and overall nutritional states. one current trial led by dr. elske vrieze in belgium is specifically testing the psychobiological effects of scfas in patients suffering from an.130 the study consists of administering scfas or a placebo through colon-delivery capsules over six weeks, in addition to standard psychotherapeutic treatment regimens, to evaluate changes in stress responses, eating habits, body mass index (bmi), gut microbiota, gastrointestinal symptoms, and related psychological conditions.130 these scientists aim to understand how metabolites in the gut microbiota can impact human stress systems and eating behaviors to facilitate potential therapeutic targets for anorexia and other psychiatric disorders.130 another ongoing clinical trial being done at uc san diego is specifically testing the effect of bhb as a nutritional supplement for anorexic and bulimic patients, and how bhb ingestion impacts brain function.131 researchers will compare the effects of ketone supplementation versus placebo on brain activity using eeg measurements, with participants taking a ketone drink twice daily for two weeks.131 overall, the growing number of studies surrounding the role of ketone bodies in human health and disease emphasizes the importance of understanding their role as potential therapeutic targets. while there are still limited studies on how ketone bodies directly correlate to an, there has already been substantial research regarding how it influences other psychiatric and chronic diseases like alzheimer’s, parkinson's disease, fatty liver disease, heart failure, among others.128 ketone berkeley pharma tech journal of medicine | 103 bodies play vital roles in energy metabolism, redox balance, lipid synthesis, and cellular signaling. for individuals with an, these properties suggest that ketone bodies might help address some of the condition's critical aspects, such as energy deficiency and gut health. they may serve as crucial mediators of a patient’s microbiome. while promising, these potential applications require further research to confirm their efficacy and safety in clinical settings, ensuring that interventions are tailored to the unique needs of an patients. 7. practical applications these findings highlight the potential role of dietary supplements, including vitamins, minerals, and probiotics, in treating an. integrating vitamin supplementation, dietary supplements, ketogenic diets with current therapies will overall provide a more comprehensive approach to treating an. this review paper may also encourage ongoing and future clinical trials to evaluate the effectiveness of these supplements and find ways to develop more personalized treatment plans based on individual gut microbiome profiles. further research on these substances may enable physicians to assess gut microbiome composition and optimize nutritional interventions tailored to individual patient needs. 7.1 clinical and nutritional rehabilitation treatment plans patients with an often suffer from severe nutritional deficiencies and altered gut microbiome, which contribute to both physical and psychological symptoms. effective nutritional rehabilitation is crucial for the recovery of an patients. traditional refeeding programs may benefit from the inclusion of dietary supplements to address specific deficiencies and support gut health. integrating specific dietary supplements and tailoring them to patient-specific needs introduces a way to provide a more personalized treatment plan for an patients. since the makeup of everyone’s gut microbiome is unique, understanding the impact of specific substances and how varying amounts of them affects each patient is key to targeting the distinct berkeley pharma tech journal of medicine | 104 ways the disorder is influencing them. dietitians can also create individualized supplement plans based on the specific needs and deficiencies of each patient. potential outcomes of individualized supplement-based interventions may include improved gut microbiome diversity, enhanced nutrient absorption, and potential benefits for mental well-being through gut-brain axis modulation. however, these effects require further clinical validation. 7.2 psychiatric support strategies an is also often accompanied by psychiatric symptoms such as anxiety, depression, and obsessive-compulsive behaviors, which can be influenced by gut health. incorporating dietary supplements into psychiatric support strategies can provide a holistic approach to treatment. psychiatrists and therapists can work with dietitians to ensure that patients receive supplements that support both mental and physical health. this integrated strategy aims to reduce psychiatric symptoms, improve adherence to treatment plans, and increase the likelihood of sustained recovery. the integration of dietary supplements into treatment strategies for an offers a promising approach to addressing the complex nutritional and psychiatric needs of patients. by restoring gut-microbiome diversity and supporting the gut-brain axis, these supplements can enhance both physical and mental health outcomes. continued research and careful implementation will be key to maximizing the benefits of this approach. 8. limitations/implications while dietary supplements hold promise as a potential treatment for an by restoring gut-microbiome diversity and addressing alterations in the gut-brain axis, significant limitations exist. addressing these limitations through rigorous research, personalized treatment approaches, and enhanced regulatory oversight is crucial to fully realize the potential benefits of dietary supplements in the treatment of an. berkeley pharma tech journal of medicine | 105 as of now, there is an extremely limited scope of research regarding incorporating these supplements into an treatment regimens. research on dietary supplements in an treatment is still in its early stages, with most studies being limited by small sample sizes, short follow-up periods, and a lack of randomized controlled trials. the limited scope of resources prevents us from fully examining variability in individual responses and the heterogeneity in patient responses necessitates personalized treatment approaches, complicating the standardization of supplement-based interventions. there is also lack of research on the potential risks and side effects of dietary supplements in relation to an, how they interact with other an treatments, and regulatory and quality control issues. over-supplementation of certain vitamins and minerals can lead to toxicity and therefore correct quantities of supplements to administer needs to be determined. probiotics may cause gastrointestinal disturbances in some individuals. additionally, the purity and quality of supplements can vary widely. the interactions between dietary supplements and other treatments are not well-understood, and there is a risk of adverse interactions or diminished efficacy when combined with certain medications or therapeutic modalities. variability in supplement quality, dosing, and labeling accuracy can also pose challenges for ensuring consistent and safe administration of supplements. another limitation to this treatment approach to consider is patients’ compliance and acceptance of the treatment regimen. individuals with an often exhibit resistance to treatment, including dietary interventions.132 fear of weight gain, mistrust of new treatments, and the psychological complexity of the disorder can lead to poor compliance with prescribed supplement regimens. ensuring patient compliance and acceptance of dietary supplements requires extensive education, support, and possibly behavioral interventions, which can be resource-intensive.132 overall, the limited scope of current research restricts our ability to draw definitive conclusions about the efficacy and safety of dietary supplements for treating an. berkeley pharma tech journal of medicine | 106 9. future directions while the components in this review show advantages for using dietary supplements as a treatment for improving gut microbiome diversity, there is limited research on its effectiveness in relation to an specifically. it is important to consider areas of future research that can maximize the potential of dietary supplements and improve the effectiveness of current treatment methods. as mentioned before, utilizing dietary supplements for an along with conventional therapies (i.e. cbt), can propose a comprehensive treatment approach that offers a holistic approach to managing and treating the disorder. studying this combination of traditional treatment methods with additional supplementation, scientists can compare the effectiveness of the combined approach versus solely traditional methods. future research should focus on comparative studies evaluating the efficacy of various dietary supplements, including probiotics, vitamins, fatty acids, and ketone supplements. examining their effects both individually and in combination may help identify the most effective strategies for improving gut microbiota diversity in an patients. in addition, although time consuming, conducting long-term studies would help assess the effects of dietary supplements in improving gut diversity in specifically anorexic patients as well as examine the longer term effect and side effects of using these supplements. these long-term studies can also be closely connected to evaluating the impacts of weight restoration in an patients while monitoring a patient’s psychological well-being. personalized medicine, including microbiome-based interventions and genetic screening, may improve treatment outcomes by tailoring dietary supplement regimens to individual an patients. however, further research is needed to develop standardized implementation strategies. since each individual’s gut microbiome is unique in composition and is developed fully approximately one to two years after birth, these personalized treatment plans targeting specific areas in a patient’s gut profile can improve, in this circumstance for example, microbiome imbalances in patients with an.133 these personalized berkeley pharma tech journal of medicine | 107 approaches can also take into account genetic predispositions which can be beneficial in tailoring dietary supplement intake and regimens to target specific deficiencies that are present in a particular patient. 10. conclusion patients with an suffer from both nutritional deficiencies and a disrupted gut microbiome, and traditionally nutritional intervention strategies and behavioral therapies are employed to restore weight gain and improve psychological functions. however, this leaves the gut microbiome unaddressed, as studies increasingly reported the presence of gut dysbiosis after traditional nutritional therapy, cbt, and supportive psychotherapy. dietary supplements can act as a promising add-on in meeting the unaddressed areas in an treatment. probiotic strains from the lactobacillus genus, such as l. acidophilus, l. reuteri, and l. rhamnosus gg, have proven to improve weight gain, relieve constipation (a symptom often preventing an patients from receiving nutritional interventions), positively modulate the appetite-regulating pathways, and strengthen the intestinal barrier integrity. while probiotics are the most extensively studied dietary intervention for gut microbiome modulation in an, further research is needed to determine the specific effects of different probiotic strains in human clinical trials. vitamin d plays an integral role in microbial composition through its receptor genes, regulation of gut mucosa, and is shown to positively remodel the gut microbial makeup of an patients. vitamin a maintains the intestinal barrier integrity and is positively correlated with microbiome diversity and vitamin e, similar to vitamin a and d, strengthens barrier integrity, protects against pathogenic microbes, and reduces intestinal inflammation. while some vitamins, such as vitamin d, have shown potential in microbiome modulation, direct clinical evidence supporting their role in an treatment remains limited, warranting further investigation. ketone bodies offer an alternative intervention acting as a critical energy source, restoring cerebral fuel metabolism, and in repairing gut mucosal integrity due to its role in influencing cell proliferation in the gut lining. fatty acids play a role in ketone body production, and berkeley pharma tech journal of medicine | 108 scfas modulate appetite-regulating pathways. while there are no published studies on the effects of ketogenic diet for an, ongoing clinical trials investigating scfas and ketone supplements as potential anorexic treatment are promising. by combining traditional therapies with dietary supplements, dietitians can tailor-make treatments to address the unique nutritional and gut microbiome deficiencies in each patient. however, more comprehensive and diverse clinical trials are needed to determine the efficacy of both individual dietary supplements and its combination with traditional therapies in the treatment for an. berkeley pharma tech journal of medicine | 109 references 1. neale j, hudson ld. anorexia nervosa in adolescents. british journal of hospital 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