STATIC Review of Innovation, Volume 3.

STATIC

ST ANDREWS TECHNOLOGICAL INNOVATION CLUB REVIEW ISSUE 3 | AUG 25

FEATURING: NANOCATALYSTS AND THEIR USE IN BIOFUEL PRODUCTION

Table of Contents Acid mine drainage……………………………………………………….………….Page 5 The Discovery of Neurotransmitters: From Frog Hearts to Modern Neuroscience…Page 12 The Use of Psilocybin and MDMA in the Treatment of PTSD……………………..Page 17 Advancing Biodiesel Production through various Nanomaterial Productions………Page 22 Colouring the Molecular World: How Cryo-EM is Redefining Biological Imaging..Page 26 Computation and the Classification of Finite Groups………………………………..Page 31 Developments Towards Practical Quantum Computing……………………………..Page 36 Could we use Artificial Intelligence to help prove whether P = NP?...........................Page 45 How the Misalignment of Incentives is Sabotaging Scientific Progress: is Decentralised Science the Solution?....................................................................................................Page 50 The Impact of Artificial Intelligence on Modern Software Development……………Page 57 Exploring the Efficacy of Focused Ultrasound Treatment in Patients with Tremor Dominant Parkinson’s Disease…………………………………………………………………..Page 61 Oncolytic Virotherapy for Glioblastoma; Improving Median Survival Time Through an Enhanced Immune Response…………………………………………………………Page 70 Extended Reality (XR) in Exposure Therapy for PTSD: A Rapid Review of Clinical Potential and Limitations………………………………………………………………………..Page 76 CRISPR-mediated Reactivation of Foetal Haemoglobin (HbF) as a Sustainable Therapeutic Solution for Beta-Thalassemia………………………………………………………...Page 82 PSMA – From Diagnosis to Treatment: The Key to Prostate Cancer Management? Page 86 The Use of AI and 3D Printing in Personalised Paediatric Cardiac Surgery………….Page 89 The Use of AI and 3D Printing in Personalised Paediatric Cardiac Surgery………….Page 93 Ozempic on the Brain: Investigating the Rise of Glucagon-Like Peptide-1 Receptor Agonists and Their Potential to Treat Neurodegenerative Diseases……………………………..Page 97 Cracking the Code of Chronic Pain: Molecular Gateways and Emerging Treatments.Page 109 Advancements and Implications of Robotic-Assisted Coronary Artery Bypass Grafting: A Minimally Invasive Revolution………………………………………………………Page 124

COMMITTEE

2024-2025

Founders Note

Leadership

___________________

Tom Burton Ideja Bajra Operations Bryn Carney Isobel Fenemore Kimy Pedersen Abigail AradiPosylkin Creative

When we launched STATIC three years ago, it was little more than an idea fuelled by curiosity and a shared belief that St Andrews deserved a platform to highlight fresh perspectives on STEM innovation. What began as a small-scale club has grown into a publication that has sparked conversation and brought together voices from across the University and beyond.

This third edition marks a special moment for us. Not only as the latest chapter in STATIC’s journey, but also as the point where we, the founders, hand over the reins. As we step into the next stage of our own careers, we do so with immense pride in what STATIC has become and with deep gratitude to the innovative writers, editors and readers who have shaped it along the way.

We are confident that STATIC will continue to evolve and inspire, carrying forward its mission within the St Andrews STEM community.

Watching its next chapter unfold will be one of our greatest joys and we can’t wait to see where the next generation will take it.

Violeta Vigo Victoria SchroederFinckh Constantin Mareschal Ailsa Bruce

With thanks and excitement, Ideja and Tom The Founders of STATIC.

Incoming Presidents Note

Sector Heads

On joining STATIC this year, we’ve been introduced to a different side of science in St Andrews. With talks from industry pioneers, events aimed at sharing research goals and opportunities to learn more about the scientific publishing process, STATIC offers students an amazing chance to take their work to a different level.

Through the spaces and events we organised to share innovative ideas, we also built meaningful connections that fostered both professional and personal relationships.

Pedro Lourenco

Hannah Betts Andrew Linz Sushmhitah Sandanatavan

Editing Leadership Ava Grönberg

As Tom and Ideja graduated they leave behind a strong network of professional connections that allow St Andrews students a glimpse into the reality of professional work. With the guidance of Ideja and Tom, we’re looking to take STATIC to an even wider audience, and further embracing all of the scientific excellence that St Andrews Students have to offer.

Tom Dunlop

We hope that current members join us on this journey in the next academic year!

Amelie Baveja

Bryn and Tom

Louisa Willan

Editing Alethea Stevenson Ghaith Albarakati Mackensie KawaguchiFabian Gayatri ChatterjiSharma

1

Acid Mine Drainage Violet Melcher Edited by Hannah Betts and Tom Burton

I. I NTRODUCTION

The mining industry is notorious for its impact on the environment with Acid Mine Drainage (AMD) lying at the forefront of issues. In the US alone, AMD has contaminated 22,000 kilometers of streams and 180,000 acres of freshwater reservoirs (Tuffnell, 2022). The term Acid Mine Drainage describes the movement of waters with high concentrations of heavy metals. As varying surface water sources including rainwater, snowmelt, or pond water chemically react with rocks that contain sulfur-bearing minerals in shallow subsurface water sulfuric acid is created (US EPA, 2015). This reaction causes mobilization of heavy metals and increasing acidity in the host water (US EPA, 2015).

The toxic contaminants found within these waters are dominated by high percentages of arsenic, lead, and mercury, ultimately depositing them into water bodies impacting communities and various natural landscapes (US EPA, 2015). Recent attention and activism have driven the development of sustainable AMD treatment technologies to reduce waste sludge and brine that result from these acidic, metalladen waters, prompting a shift towards more efficient and environmentally responsible remediation strategies (Naidu et al., 2019). While traditional AMD treatments have centered on neutralization and containment, advancements in biological and chemical technologies, alongside hybrid approaches have led to a more sustainable and effective path towards remediation. Based on the limitations of single-method approaches, a combination of innovative treatments, including sulfate-reducing bioreactors, constructed wetlands, and hybrid adsorption systems, offer promising solutions for AMD management, minimizing impact on the environment, prioritizing economic efficiency, and adapting to site-specific conditions.

II. E XPLANATION Acid Mine Drainage increases in severity when ore bodies are disturbed by mining practices. As water, associated with mineral exploration and harvesting, percolates through sulfides, oxidation can occur resulting in the formation of sulfuric acid (US EPA, 2015).

Pyrite is one of Earth’s most abundant sulfide minerals, commonly found in a vast range of geological settings, and is known to be a leading cause of AMD generation due to its commonality (Murphy and Strongin, 2009). Pyrite oxidation is the greatest contributor to AMD formation (Figure 1).

Figure 1: The generalised reactions in the oxidation of Pyrite The oxidation of sulfide to sulfate solubilizes the ferrous iron, which is then oxidized to ferric iron.

These reactions can occur instantaneously or can catalyze the microorganisms that gain energy from oxidation. The ferric irons that are produced can then oxidize an additional Pyrite. These reactions release hydrogen ions, which lowers the pH of the host water (Mitchell and Craddock, 2011). When sulfides are exposed to water, air, or microbial activity, a reaction can occur resulting in the development of sulfuric acid, creating and releasing hydrogen ions (Yuan et al., 2022).

Oxidation can continue to release acid and metals into surrounding water sources for decades after mining has stopped, making this process common in both active and abandoned mining sites (Moncur et al., 2005). This reaction occurs in waste rock

2

h!

Figure 2: AMD formation and contamination pathways (Yuan et al., 2022) piles, tailings, underground tunnels, leach pads, and open pits (Moncur et al., 2009). The acid created can dissolve metals and metalloids such as arsenic from the surrounding rock, accumulating a slurry of toxic components within the water body.

Uncontaminated stream water is infiltrated by toxic runoff produced in the pit waters of a mine following precipitation and oxidation, lowering the pH of the flow (Yuan et al., 2022). This runoff can spread into the terrestrial biosphere by way of plant roots, harming aquatic life through contaminated stream waters (Figure 2) (Yuan et al., 2022). The acidity of these waters has been shown to alter surrounding environments, killing fish, flora, and organisms in the water and soils (Malmström et al., 2008). This is exemplified in cases such as the pollution in the Odiel and Tinto Rivers in Spain, where pollution from AMD persists for more than 50 kilometers downstream, damaging estuarine sediments at the Rı́a de Huelva estuary and the Gulf of Cadiz (Alvarenga et al., 2021). These areas have been significantly degraded ecologically due to this prolonged contamination (Alvarenga et al., 2021).

Treating AMD is essential to mitigate its environmental impact. Treatment methods can broadly be categorized as passive methods or active methods.

Passive methods rely on natural processes to increase pH and remove contaminants, requiring minimal human intervention and few energy resources (Skousen et al., 2016). Passive treatment systems are commonly used in low-flow, low-contamination environments or in addition to other treatment methods (Skousen et al., 2016). Thus, they are often inefficient in cases of large-scale pollution (Fripp et al., 2000). A prime example of a passive AMD treatment is utilized in West Virginia sites, where

anoxic limestone drains intercept AMD. As the acidic water percolates through the limestone, the flow reacts with the calcium carbonate, increasing alkalinity and pH, and facilitating the precipitation of dissolved metals. This process reduces the acidity and metal concentrations in the flow without continuous chemical addition or management (Skousen et al., 2016). Comparatively, active methods often involve chemical additives to neutralize acidity and facilitate metal precipitation. An active approach requires continuous operation, energy input, and maintenance (Skousen et al., 2016). However, active methods are more applicable in widespread AMD pollution events.

Traditionally, AMD remediation focuses on neutralizing the acid and reducing material exposure to reduce the potential for oxidation. Neutralization can be used as both a passive and active treatment method (Taylor et al., 2005). When used as an active method, alkaline substances are added to polluted water bodies to increase the pH, neutralize the acid, and increase dissolved metal precipitation (Keane, 2025). Alkaline agents such as lime or limestone are prevalent in these neutralization-based methods (Iakovleva et al., 2015). As an active approach, alkaline chemicals must be continuously added to the water body creating an ongoing need for materials, equipment, and labor (Keane, 2025). This process also creates sludge, a metal laden waste product that can settle in waterways. As neutralization precipitates metals, substantial sludge volumes can be generated (Keane, 2025). This sludge requires disposal, contributing further to the labor-intensive process and exacerbating environmental denudation.

Additionally, while neutralization reduces acidity, it does not completely remove all dangerous contaminants, such as certain heavy metals which remain a source of pollution and further degrade the landscape (Sheoran and Sheoran, 2006).

The passive approach to neutralization relies on natural processes, creating an alkalinity source while following the same general process as the active treatment though not requiring continuous human intervention (Skousen et al., 2016). This method can be extremely cost and labor efficient. However, it is not a substantial source of AMD treatment as it rarely occurs on large scales (Skousen et al., 2016).

Containment is another traditional method for AMD remediation. Containment isolates mine waste that could produce AMD if given the opportunity to

3

oxidize (Keane, 2025). The specifics of containment practices can take multiple different forms. Containment often involves isolating mine waste from groundwater, relocating and covering waste with impermeable material, restricting water infiltration (Keane, 2025). Other methods of containing the waste include diverting water pathways away from waste storage locations, isolating the mine waste under water to prevent oxygen exposure, and filling abandoned mines to prevent AMD formation (Keane, 2025). These techniques have variations with differing benefits and drawbacks. Some popular critiques include the longevity of containment, covering waste does not always eliminate the ability for AMD to form but rather delays oxidation. Barriers can degrade and if containment is breached, due to erosion, extreme weather, or material failure, the generation of AMD can continue without restriction (Skousen et al., 2016). More broadly, containment does not remove or treat AMD but prevents it.

If waste is already oxidized or containment fails, additional treatment is necessary (Skousen et al., 2016).

Though neutralization and containment techniques have been used for over a century, recent studies are highlighting that no singular approach can aptly treat or remediate AMD (Rambabu, 2020). This has led to the development of not only countless innovations but also use of multiple overlapping methods.

Recent advancements in AMD treatment have emphasized adsorption techniques and sulfate reducing bacteria within hybrid approaches (Mosai et al., 2024). Adsorption employs materials including activated carbon or bio-sorbents to bind pollutants and increase water quality (Mosai et al., 2024). Sulfate reducing bacteria is a form of bioremediation, utilizing organic matter to remove dangerous elements from water streams impacted by AMD (Mosai et al., 2024).

Adsorption targets heavy metals, effectively removing them from a solution. When AMD encounters an adsorbent the dissolved metals in the contaminated flow attach to the surface of the solid adsorbent, removing the metals from the solution (Yang et al., 2024). Unlike techniques that attempt to target all contaminants, adsorption targets metal ions, separating them from sulfate ions (Yang et al., 2024).

A variety of innovative materials can be used as the adsorbent, from natural substances like zeolite,

sawdust and eggshells, to more advanced materials such as magnetic graphene oxide (Dehghani et al., 2023). This method provides promising innovations not only because of its relative simplicity but also because most absorbents can be cleaned and reused, reducing the overall waste produced from treatment (Dehghani et al., 2023). Adsorption has yet to be used on a large scale, with most research restricted to lab settings and small water samples. However, current studies focus on applying adsorption in large-scale treatment systems and suggest that this technique could become more widely used in future (Yang et al., 2024).

Sulfate reducing bacteria (SRB) is another active area of research in innovative AMD treatments. SRB can reduce sulfates to sulfides when provided with an organic carbon source such as lactate and glucose (Mosai et al., 2024). The sulfate ions in AMD act as electron acceptors during metabolism of organic matter and are converted to hydrogen sulfide resulting in highly stable metal sulfide precipitates, allowing for the removal of metals from the polluted water source (Mosai et al., 2024). This process is cost effective and leads to an increase in pH in drainage waters due to the generation of alkalinity from the metabolism of the bacteria.

These approaches can then be combined within hybrid frameworks to address the limitations of the traditional methods. The combination of both traditional and innovative techniques enhances the efficient removal of contaminants and optimizes treatment sustainability and cost (Nguegang et al., 2022).

III. D ISCUSSION The introduction of innovative treatment techniques for AMD has presented new avenues for AMD mitigation that can reduce waste, exemplified in the recyclability of many adsorption techniques.

However, due to ongoing research, these techniques promise advancements that can soon be applied on large-scale issues but currently do not have the ability to address real-world pollution events at scale.

A central example of these innovations can be seen within the newly developed CloSURE technologies pioneered by Mintek in South Africa (Mosai et al., 2024). CloSURE is an SRB technology of AMD treatment using sulfate content. Figure 3 simply illustrates the CloSURE process.

4

h!

Figure 3: Simplified Illustration of AMD Treatment with CloSURE (Mosai et al.,2024)

CloSURE consists of sulfate reduction through biological processes followed by sulfide oxidation and the production of biosulfur resulting in a clean water source (Mosai et al., 2024). Sulfate is reduced through dissimilation. The sulfide created in this process interacts with metals in the solution, forming metal sulfides which can be easily removed due to its low solubility and high stability (Mosai et al., 2024). If unchecked, a high concentration of sulfides can lead to corrosion. However, the biological removal of sulfides transforms toxic metals such as mercury leading to their easy removal from the water body (Mosai et al., 2024). The use of naturally occurring anaerobic prokaryotic microorganisms allows for this technology to be easily applied in hard-to-reach environments. . Additionally, significantly less solid waste is produced compared to traditional techniques. The key drawback to an approach comprised solely of innovative techniques is that methods such as CloSURE have only been used in a limited number of pilot tests. That said, when used, it has been shown to remove more than 85% of sulfate and sulfide (Mosai et al., 2024).

Technologies such as CloSURE present an exciting next step for AMD remediation strategies, but with testing still underway, this cannot be an end all solution.

As shown in Figure 3, these new treatment methods integrate traditional approaches such as neutralization. Similarly, many academic sources present the possibility of combining multiple methods, both innovative and traditional, to maximize productivity and minimize waste (Beauclair et al., 2023). Each method explained thus far has its own limitations, including amount of waste produced and applicability on large scales (Wibowo et al., 2024).

Adsorption for example, is a low-cost and rapid approach for AMD treatment, but even with adsorbent recycling, sludge can be generated and applicability at larger scale may be challenged by the need for ef-

ficient contact between the adsorbent and dissolved metals (An et al., 2024).

To improve efficiency of treatment and achieve more comprehensive treatment of polluted water bodies, the challenge of AMD remediation requires not only innovative approaches but also involvement of multiple methods to complement the restrictions of any singular treatment (Sahoo et al., 2013). A key consideration when combining approaches is that AMD in one environment can be different from AMD in another. In the Witwatersrand Basin in South Africa, the water contamination greatly differs from those in the Makum Coalfield in India (Wibowo et al., 2024). Goldmining executed at the South African site leaves water bodies strewn with high concentrations of arsenic and uranium while the site in India has high levels of iron leading to elevated levels of dissolved metals and higher rates of acidification (Wibowo et al., 2024). The unique characteristic of each site requires a different combination of treatments tailored specifically to the location and conditions (Figure 4) (Wibowo et al., 2024). A simplified process of the development

Figure 4: The site-specific capabilities of AMD treatment (Wibowo et al., 2024) of physical, chemical, and biological options for remediation can be designed specifically for the issue at hand. Wibowo et al. (2024) touches on the applicability of techniques such as biological treatment methods including SRB, adsorption, and neutralization emphasizing the advantage of hybrid technologies’ customization potential to site-specific conditions, contaminant composition and treatment aims. The adaptability of the multi-method approach centers a dynamic and ever-evolving approach to AMD treatment. A range of possible combinations of both traditional and innovative approaches exist,

5

approaches to minimize harmful waste products, and ensure treatment success. Promising modern research is being conducted to make impactful advances within AMD remediation and safeguard the natural environment from further degradation. Traditional treatments have centered on neutralization and containment for decades. Hybrid approaches integrate newly developed innovative technologies, presenting a more efficient and sustainable path towards reducing and restricting AMD. Due to the limitations of traditional methods, research is required to further develop innovative techniques tailored to site-specific conditions for effective AMD management.

Figure 5: Varied combinations of AMD treatment approaches emphasizing the possibility of sitespecific hybrid AMD techniques (Mosai et al., 2024)

V. R EFERENCES

Alseno Kagiso Mosai, Gebhu Ndlovu and Hlanganani Tutu (2024). Improving acid mine drainage treatment by combining treatment including the integration of CloSURE technology. technologies: A review. The Science of The However, these combinations are hypothetical, and Total Environment, 919, pp.170806–170806. have not been tested experimentally (Mosai et al., doi:https://doi.org/10.1016/j.scitotenv.2024.170806 2024). Wibowo et al (2024) presents a similar hesitation explaining that hybrid technologies for Alvarenga, P., Guerreiro, N., Simões, I., Imaginário, AMD remediation are still being developed, Figure M.J. and Palma, P. (2021). Assessment of the 5 has been calibrated from computational modelling Environmental Impact of Acid Mine Drainage rather than on-site applications. However, these ap- on Surface Water, Stream Sediments, and Macrophytes Using a Battery of Chemical proaches may have strong future potential.

Traditional methods have objective flaws, with in- and Ecotoxicological Indicators. Water, 13(10), novative techniques such as adsorption and SRB p.1436. doi:https://doi.org/10.3390/w13101436 presenting some possible solutions to address limitations. The most promising next step towards An, W., Liu, Y., Chen, H., Sun, X., Wang, efficient and sustainable AMD treatment is multi- Q., Hu, X. and Di, J. (2024). Adsorption properties method approaches. Still, further research must be of Pb(II) and Cd(II) in acid mine drainage by conducted to graduate from computational mod- oyster shell loaded lignite composite in different elling, developing instead real-world solutions to morphologies. Scientific Reports, [online] 14(1). doi:https://doi.org/10.1038/s41598-024-62506-0 AMD issues.

IV. C ONCLUSION

Advancements in AMD treatment technologies have greatly evolved from conventional methods to innovative approaches. While many innovative approaches offer significant improvements from traditional methods, they have yet to be applied on large scales, remaining pilot programs or lab experiments. Hybrid models provide the most promising step forward, building on a foundation of traditional techniques while integrating innovative site-specific

Beauclair, N., Vhahangwele, Masindi, Msagati, T.A.M. and Memory, T. (2023).

Acid Mine Drainage Treatment Technologies.

Hybridized Technologies for the Treatment of Mining Effluents, pp.245–285. doi:h t t p s : //doi.org/10.1002/9781119896920.ch8 Dehghani, M.H., Ahmadi, S., Ghosh, S., Othmani, A., Osagie, C., Meskini, M., AlKafaas, S.S., Malloum, A., Khanday, W.A., Jacob, A.O., Gökkuş, Ö., Oroke, A., Martins Chineme, O.,

6

Karri, R.R. and Lima, E.C. (2023). Recent advances on sustainable adsorbents for the remediation of noxious pollutants from water and wastewater: A critical review. Arabian Journal of Chemistry, [online] 16(12), p.105303. doi:https://doi.org/10.1016/j.arabjc.2023.105303

Moncur, M.C. (2013). Acid mine drainage: past, present...future? [online] Wat on Earth.

Available at: https://uwaterloo.ca/wat-on-earth/new s/acid-mine-drainage-past-presentfuture.

Fripp, J., Ziemkiewicz, P. and Charkavorki, H. (2000). Acid Mine Drainage Treatment. [online] Available at: https://apps.dtic.mil/sti/pdfs/ADA37 9475.pdf [Accessed 21 Mar. 2022].

Moncur, M.C., Jambor, J.L., Ptacek, C.J. and Blowes, D.W. (2009). Mine drainage from the weathering of sulfide minerals and magnetite.

Applied Geochemistry, 24(12), pp.2362–2373. doi:https://doi.org/10.1016/j.apgeochem.2009.09.01 3.

Gusti Wibowo, Y., Safitri, H., Khairurrijal, K., Taher, T., Ode Arham, L., Jasipto, A., Akbari Danasla, M., Fadhilah, R., Kharisma Army, E., Zul Hakim, H., Tawfiequrahman Yuliansyah, A. and Tri Bayu Murti Petrus, H. (2024). Recent advances in acid mine drainage treatment through hybrid technology: Comprehensive review of scientific literature. Environmental Nanotechnology, Monitoring & Management, [online] 21, p.100945. doi:https://doi.org/10.1016/j.enmm.2024.100945

Murphy, R. and Strongin, D.R. (2009). Surface reactivity of pyrite and related sulfides.

Surface Science Reports, 64(1), pp.1–45. doi:https://doi.org/10.1016/j.surfrep.2008.09.002.

Naidu, G., Ryu, S., Thiruvenkatachari, R., Choi, Y., Jeong, S. and Vigneswaran, S. (2019). A critical review on remediation, reuse, and resource recovery from acid mine drainage. Environmental Pollution, [online] 247, pp.1110–1124.

Iakovleva, E., Mäkilä, E., Salonen, J., Sitarz, doi:https://doi.org/10.1016/j.envpol.2019.01.085.

M., Wang, S. and Sillanpää, M. (2015). Acid mine drainage (AMD) treatment: Neutralization and toxic Nguegang, B., Masindi, V., Msagati Makudali, elements removal with unmodified and modified T.A. and Tekere, M. (2022). Effective treatment limestone. Ecological Engineering, 81, pp.30–40. of acid mine drainage using a combination of MgO-nanoparticles and a series of constructed doi:https://doi.org/10.1016/j.ecoleng.2015.04.046. wetlands planted with Vetiveria zizanioides: Keane, C. (2025). What can be done to prevent or A hybrid and stepwise approach. Journal of clean up acid mine drainage? [online] American Environmental Management, 310, p.114751.

Geosciences Institute. Available at: https://professi doi:https://doi.org/10.1016/j.jenvman.2022.114751. on.americangeosciences.org/society/intersections/f aq/what-can-be-done-prevent-or-clean-acid-mine-d Rambabu, K. (2020). Biological remediation of acid mine drainage: Review of past trends rainage/prevent-or-clean-acid-mine-drainage/. and current outlook. Environmental Science Malmström, M.E., Berglund, S. and Jarsjö, J. and Ecotechnology, [online] 2, p.100024.

(2008). Combined effects of spatially variable doi:https://doi.org/10.1016/j.ese.2020.100024. flow and mineralogy on the attenuation of acid mine drainage in groundwater. Applied Sahoo, P.K., Kim, K., Equeenuddin, Sk.Md. Geochemistry, 23(6), pp.1419–1436. doi:h t t p s : and Powell, M.A. (2013). Current Approaches for Mitigating Acid Mine Drainage. //doi.org/10.1016/j.apgeochem.2007.12.029.

Reviews of Environmental Contamination Toxicology Volume 226, pp.1–32.

Mitchell, G. and Craddock, T. (2011). An and Introduction to Acid Mine Drainage. [online] doi:https://doi.org/10.1007/978-1-4614-6898-1 1.

Available at: https://dep.wv.gov/WWE/getinvolved /sos/Documents/Presentations/AMD Introduction. Sheoran, A.S. and Sheoran, V. (2006). Heavy pdf?utm source=chatgpt.com [Accessed 28 Feb. metal removal mechanism of acid mine 2025]. drainage in wetlands: A critical review.

7

Minerals Engineering, 19(2), pp.105–116. doi:https://doi.org/10.1016/j.mineng.2005.08.006.

Skousen, J., Zipper, C.E., Rose, A., Ziemkiewicz, P.F., Nairn, R., McDonald, L.M. and Kleinmann, R.L. (2016). Review of Passive Systems for Acid Mine Drainage Treatment. Mine Water and the Environment, 36(1), pp.133– 153. doi:https://doi.org/10.1007/s10230-016-0417-1.

Taylor, J., Pape, S. and Murphy, N. (2005).

A Summary of Passive and Active Treatment Technologies for Acid and Metalliferous Drainage (AMD) EARTH SYSTEMS PTY LTD Environment -Water -Sustainability. [online] Available at: https://www.earthsystems.com.au/wp-content/uploa ds/2012/02/AMD Treatment Technologies 06.pdf.

Tuffnell, S. (2022). Acid drainage: the global environmental crisis you’ve never heard of. [online] www.history.ox.ac.uk. Available at: https://www.history.ox.ac.uk/article/acid-drainage-g lobal-environmental-crisis-youve-never-heard.

US EPA (2015).

Abandoned Mine Drainage. [online] US EPA. Available at: https://www.epa.gov/nps/abandoned-mine-drainage.

Yang, Y., Wang, Y., Xue, C., Lin, Y., Lee, J.-F., Yi, X. and Dang, Z. (2024). Efficient removal of heavy metals from acid mine drainage by ϵ-MnO2 adsorption. Journal of Cleaner Production, [online] 452, p.141936. doi:https://doi.org/10.1016/j.jclepro.2024.141936.

Yuan, J., Ding, Z., Bi, Y., Li, J., Wen, S. and Bai, S. (2022). Resource Utilization of Acid Mine Drainage (AMD): A Review. Water, 14(15), p.2385. doi:https://doi.org/10.3390/w14152385.

1

The Discovery of Neurotransmitters: From Frog Hearts to Modern Neuroscience Dariush Khodakhah Edited by Hannah Betts and MacKenzie Kawaguchi Fabian

I. A BSTRACT The understanding of neurotransmitters has advanced significantly in the past century, shaping modern neuroscience and expanding the knowledge of synaptic communication. This was propagated through clarification of chemical vs. electrical signaling by Otto Loewi and his experiment of frog hearts and Henry Dale’s work with muscarinic receptors, which led to the discovery of acetylcholine and the joint award of the 1936 Nobel Prize in Physiology or Medicine. Subsequent discoveries of dopamine, serotonin, and other neurotransmitters were based heavily on fluorescence, histochemistry, and electrophysiology. Through understanding the composition of these chemicals and the mechanism in the synaptic cleft, scientists have been able to further our neuroscientific understanding. As research continues, new insights into neurotransmitter dynamics are the basis for novel therapeutic approaches, advances in neurotechnology, and a greater understanding of neuroplasticity.

II. I NTRODUCTION Neurotransmitters are the chemical messengers in our brain that transmit the electrical signal from nerves through the synaptic cleft. Early research on how neuronal signals were propagated debated whether conduction was fully electric or potentially involved a chemical component. A pivotal discovery was made by Otto Loewi and Henry Dale, winning them both the Nobel Prize in 1936. One of Loewi’s most well-known experiments was his 1921 ’Dream experiment’, which, as the name suggests, came to him in his sleep.

The experiment began with two frog hearts placed in separate containers of Ringer’s solution, a substance that keeps organs alive outside of the body for extended periods of time. Frog hearts were chosen as they beat readily outside of the host in the correct

solution. Heart one had its vagus nerve intact, heart two had no neuronal connections, only chemical exposure through the fluid. Loewi then electrically stimulated the first heart’s vagus nerve. This caused a decrease in heart rate (bradycardia), which was consistent with the known parasympathetic effect of the vagus on the heart. Loewi took some of the Ringer’s solution from the first heart’s container and added it to the container with the second heart. The fluid transferred also caused heart two to slow down, indicating that it was a substance within the fluid from the vagus nerve that caused the parasympathetic substance from the vagus nerve, which he termed ’vagusstoff’ (Loewi, 1921). This experiment set the precedent that neuronal transmission was not solely through electrical conduction: the chemical component, vagusstoff, was later implicated in neuronal transmission.

Henry Dale’s research built upon Loewi’s findings, exploring the nature of vagusstoff. Dale had previously isolated acetylcholine in 1913 along with Arthur Ewins at the Wellcome Physiological Research Laboratories (Tansey, 2006). It was purified from a rare extract from a batch of ergot, and in a letter to a fellow physiologist he wrote: ”We got our of our... ergot extract... acetyl-choline and a most interesting substance. It is much more active than muscarine, so easily hydrolysed that its action, with exactly similar effects, ... like adrenaline.” (Dale, 1913).

The subsequent year, Dale published a paper on the pharmacology of choline derivatives, noting two main features of acetylcholine: its main effects could be reproduced through injections of acetylcholine or muscarine (Dale, 1914).

In 1926, Loewi further built upon his vagusstoff experiments, investigating why the effects of the substance were so short-lived. He believed that the heart produced a substance that degraded the vagusstoff. Loewi and his colleague Navratil then

2

extracted a vagusstoff-containing fluid and left different samples of it in different temperature solutions. The longer the fluid was left, the less effect it had on the heart. When left at 56°C the fluid prevented this loss suggesting that enzymes in the fluid that degraded vagusstoff had been denatured in the solution. They had discovered that vagusstoff must therefore be a choline-ester and the enzyme that degraded it a choline-ester (Loewi and Navratil, 1926). Acetycholine, however, had still not been discovered in vivo. In 1929 Dale and his colleague Dudley accidentally discovered it while looking for endogenous histamines. Fresh spleens from oxen and horses were collected and placed on ice to preserve biochemical integrity. The spleens were then homogenised in a buffer solution with filtering to leave the liquid. The presence of acetylcholine was tested by application of the filtrate to isolated frog hearts. Acetylcholine was known to have bradycardic effects, and on the application of the liquid this was seen. To confirm this response was from acetylcholine and not another substance, atropine, a muscarinic receptor antagonist, was added. Prevention of bradycardia would indicate competitive antagonism took place and that acetylcholine was responsible for the initial bardycardic effect. The results were consistent with this hypothesis, and thus acetylcholine was finally discovered to naturally exist in mammals (Dale and Dudley, 1929). The culmination of the work confirmed the electrochemical transmission across synapses and confirmed the identity of vagusstoff as acetylcholine. The research set the precedent for the discovery of many other neurotransmitters, with the next to be discovered being norepinephrine.

III. A P EAK IN I NTEREST Ulf von Euler, a Swedish physiologist, was interested in the activity of endogenous substances, having previously discovered prostaglandin, vesiglandin (v. Euler, 1935) and piperidine in 1942. In 1946 he discovered norepinephrine to be a substance distinctly different from adrenaline. The experiment required the extraction of sympathetic nervous tissues from a variety of animals, namely cats and rabbits. The tissues were then homogenised and treated with an acid solution to extract aminecontaining products. The sympathomimetic amines were then isolated through butanol-based extraction,

where butanol is added to the aqueous solution in a specific ratio before shaking vigorously to allow butanol to dissolve the target compounds.

The solution is then left to settle to separate into two layers, with the layers then separated using a separating funnel. The dissolved amines were tested using paper chromatography and the separated amines then compared using UV spectra.

The key substance in the sympathetic nerve terminals had an absorption profile distinct from that of epinephrine, a key finding. The physiological effects of the substance were tested by injection into cats and rabbits; acting as bioassays. Blood pressure responses, heart rate changes, and changes in vascular tone were observed. On application of the substance to isolated muscle, adrenergic nerve stimulation occurred, confirming that the substance was a neurotransmitter (v. Euler, 1946).

The discovery of noradrenaline not only demonstrated the existence of multiple catecholamines but also provided crucial evidence that neurotransmitters are selectively stored and released from nerve endings. This helped distinguish the chemical mediators of the sympathetic and parasympathetic nervous systems, solidifying the concept that different neurotransmitters regulate distinct autonomic functions.

The increase in interest surrounding catecholamines and their actions on the nervous system was furthered by the identification of dopamine in the human brain. This was by Montagu in 1957 (Montagu, 1957). The more commonly known researcher, Avid Carlsson, then used an assay approach in 1957 to prove that dopamine was not only an intermediate to catecholamine synthesis, but a neurotransmitter. Carlsonn used reserpine, a drug known to deplete monoamine neurotransmitters, and observed severe motor deficits in mice, such as akinesia, eyelid ptosis, and a depletion of brain serotonin. The mice were then either administered a precursor of serotonin or catecholamines, and observed to see if the akinesia ameliorated.

It was only in the catecholamine precursor 3,4dihydroxyphenylalanine (DOPA) that the akinesia was able to be improved. A further injection of MOA inhibitor significantly reduced the amount of DOPA required to save the mouse from the akinetic phenotype (Carlsson et al., 1957). The following year, Carlsson did some more work to confirm the presence and distribution of dopamine in the

3

brain. Homogenised brain was made to be a pH of around 5.3 using hydrochloric acid, as this allows for characteristic changes in the fluorometric properties of the catecholamines (Carlsson and Waldeck, 1958). This allowed for the structural differentiation of dopamine from adrenaline and norepinephrine distinguishing dopamine as a separate substance with properties alike neruotransmitters.

The discovery of dopamine by Carlsson was a catalyst of interest for scientists in the 1950s to further explore monoamines and neurotransmission. Serotonin, termed ’enteramine’ by Vittorio Erspamer in 1937, was identified as a vasoactive substance extracted from enterochromaffin cells (Erspamer and Vialli, 1937). It wasn’t until eleven years later that Rapport and colleagues were able to isolate and name serotonin. The isolation required five steps: ethanol precipitation of proteins, acetone precipitation of salts, chloroform extraction of inactive substances, butanol extraction of the active substance, and dilituric acid precipitation (Rapport et al., 1948a). The result was an impure crystal.

Following an acetone precipitation and a methanol extraction, the extracted substance was named serotonin, ’ser’ referring to the source of the extract, cow serum, and ’tonin’ referring to the observed vasoconstrictive effects (Rapport et al., 1948b). The following year, the structure of serotonin or 5hydroxytryptamine was discovered (Rapport, 1949).

Now that the structure of serotonin was discovered, the significance of serotonin in the body and neural transmission would require novel localization techniques to be employed. It was the FalckHillarp method that allowed this breakthrough. The technique involved the exposure of freeze-dried tissues to formaldehyde vapour. This would convert dopamine, noradrenaline and serotonin into fluorescent yellow-green molecules (Falck, 1962).

Dahlström and Fuxe employed this technique to localise serotonin to certain nerve terminals, establishing it as a neurotransmitter found in the vesicles of certain nerones, specifically those of the raphe nuclei (Dahlström and Fuxe, 1964). These molecules and mediators of the action potential were the basis to which modern neuroscience has sprouted – currently flourishing with a vast array of research areas.

IV. M ODERN N EUROSCIENCE Having understood the basic principles of neurotransmission and the sites at which certain neurotransmitters are localised, neuroscience has been able to expand significantly. Some novel research regarding neurotransmitters include the localisation of neurotransmitter receptors to certain parts of the brain (and hence the tracks of specific neurotransmitters localisation), and linked abnormalities in these receptor tracks to thirteen disorders (Hansen et al., 2022).

One hundred years after Loewi’s discoveries, connectomics, a field that aims to map the connections of neurons to each other, has emerged as a forefront of neuroscientific interest. This would allow a much better understanding of how different parts of the brain are interconnected to form functional networks. Being able to understand how different tracts run would allow for a better understanding of cognition in both humans and animals and the use of ’comparative connectomics’ to understand how changes in the connectome (the collection of connections in the brain) manifest as behavioural differences in individuals (Catherine Caruso, 2023).

An entire Drosophila melanogaster connectome has been mapped, so this relatively new field should expect a rapid expansion once imaging and databases for human brains become established (Dorkenwald et al., 2024).

Neuromodulation is the process by which neurones strengthen or weaken connections to other neurones. Understanding the mechanisms by which this works would allow scientists to understand neuroplasticity and therefore how learning works on a mechanistic level, hence also allowing for therapeutic use. From a paper in which the use of SSRIs were used for patients suffering from neuropsychiatric disorders, SSRIs were not found to help with cognitive decline, but a gap in the literature exists for the use of the drugs for treating post-TBI PTSD (Yue et al., 2017). Another example of future research is how specific serotonin receptors and their localisation link to neuromodulation and the broader behavioural modulation. To put it briefly, different receptors of serotonin convey different effects and are part of different tracks and therefore the specific functions of each should be further explored. The idea was explored by Salvan et al., proving the heterogenous distribution of the

4

receptors and thus precipitated this area of future research (Salvan et al., 2023).

It was only through an experiment involving two amphibians’ hearts procured in a German’s sleep that set off the explosion of the comprehension of neural communication. With new precedents being set, the translational effects should soon be innumerable - whether in the clinic, laboratory, or novel technologies. Concurrently, technological advancements in imaging modalities will also allow for future expansion and perhaps mastery of what makes us who we are, unweaving the intricate web of complexity that is neural science.

V. R EFERENCES A New Field of Neuroscience Aims to Map Connections in the Brian — Harvard Medical School [WWW Document], 2023.

URL https://hms.harvard.edu/news/new-fieldneuroscience-aims-map-connections-brain (accessed 3.31.25).

Carlsson, A., Lindqvist, M., Magnusson, T., 1957.

3,4-Dihydroxyphenylalanine and 5-Hydroxytryptophan as Reserpine Antagonists.

Nature 180, 1200-1200. https://doi.org/10.1038/1801200a0 Carlsson, A., Waldeck, B., 1958. A Fluorimetric Method for the Determination of Dopamine (3-Hydproxytryamine.). Acta Physiol. Scand.

44, 293-298. https://doi.org/10.1111/j.17481716.1958.tb01628.x Dahlström, A., Fuxe, K., 1964. Localization of monoamines in the lower brain stem. Experientia 20, 398-399. https://doi.org/10.1007/BF02147990 Dale, H.H., 1914. THE ACTION OF CERTAIN ESTERS AND ETHERS OF CHOLINE, AND THEIR RELATION TO MUSCARINE. J. Pharmacol. Exp. Ther. 6, 147-190. https://doi.org/10.1016/S0022-3565(25)08268-0 Dale, H.H., 1913. Dale to Elliott.

Dale, H.H., Dudley, H.w., 1929. The presence of histamine and acetylcholine in the spleen of the ox and the horse. J. Physiol. 68, 97-123.

Dorkenwald, S., Matsliah, A., Sterling, A.R., Schlegel, P., Yu, S., McKellar, C.E., Lin, A., Costa, M., Eichler, K., Yin, Y., Silversmith, W., SchneiderMizell, C., Jordan, C.S., Brittain, D., Halageri, A., Kuehner, K., Ogedengbe, O., Morey, R., Gager, J., Kruk, K., Perlman, E., Yang, R., Deutsch, D.,

Bland, D., Sorek, M., Lu, R., Macrina, T., Lee, K., Bae, J.A., Mu, S., Nehoran, B., Mitchell, E., Popovych, S., Wu, J., Jia, Z., Castro, M.a., Kemnitz, N., Ih, D., Bates, A.S., Eckstein, N., Funke, J., Collman, F., Bock, D.D., Jefferis, G.S.X.E., Seung, H.S., Murthy, M., 2024. Neuronal wiring diagram of an adult brain. Nature 634, 124-138. https://doi.org/10.1038/s41586-024-07558-y Erspamer, V., Vialli, M., 1937. Ricerche sul secreto delle cellule enterocromaffini. Boll Soc MedChir Pavia 51, 357-363.

Falck, B., 1962. Observations on the Possibilities of the Cellular Localization of Monoamines by a Fluorescence Method. Acta Physiol. Scand. Suppl.

197, 1–25.

Hansen, J.Y., Shafiei, G., Markello, R.D., Smart, K., Cox, S.M.L., Nørgaard, M., Beliveau, V., Wu, Y., Gallezot, J.-D., Aumont, É., Servaes, S., Scala, S.G., DuBois, J.M., Wainstein, G., Bezgin, G., Funck, T., Schmitz, T.W., Spreng, R.N., Galovic, M., Koepp, M.J., Duncan, J.S., Coles, J.P., Fryer, T.D., Aigbirhio, F.I., McGinnity, C.J., Hammers, A., Soucy, J.-P., Baillet, S., Guimond, S., Hietala, J., Bedard, M.-A., Leyton, M., Kobayashi, E., RosaNeto, P., Ganz, M., Knudsen, G.M., PalomeroGallagher, N., Shine, J.M., Carson, R.E., Tuominen, L., Dagher, A., Misic, B., 2022. Mapping neurotransmitter systems to the structural and functional organization of the human neocortex. Nat. Neurosci.

25, 1569–1581. https://doi.org/10.1038/s41593-02201186-3 Loewi, O., 1921. Über humorale übertragbarkeit der Herznervenwirkung. Pflüg. Arch. Für Gesamte Physiol. Menschen Tiere 189, 239–242. https://doi.org/10.1007/BF01738910 Loewi, O., Navratil, E., 1926. Über humorale Übertragbarkeit der Herznervenwirkung. Pflüg.

Arch. Für Gesamte Physiol. Menschen Tiere 214, 678–688. https://doi.org/10.1007/BF01741946 Montagu, K.A., 1957. Catechol Compounds in Rat Tissues and in Brains of Different Animals. Nature 180, 244–245. https://doi.org/10.1038/180244a0 Rapport, M.M., 1949. SERUM VASOCONSTRICTOR (SEROTONIN): V. THE PRESENCE OF CREATININE IN THE COMPLEX. A PROPOSED STRUCTURE OF THE VASOCONSTRICTOR PRINCIPLE. J. Biol. Chem. 180, 961–969. https://doi.org/10.1016/S0021-9258(19)51208-X

5

Rapport, M.M., Green, Arda.Alden., Page, I.H., 1948a.

PARTIAL PURIFICATION OF THE VASOCONSTRICTOR IN BEEF SERUM. J. Biol. Chem. 174, 735–741. https://doi.org/10.1016/S0021-9258(18)573555 Rapport, M.M., Green, Arda.Alden., Page, I.H., 1948b. SERUM VASOCONSTRICTOR (SEROTONIN): IV. ISOLATION AND CHARACTERIZATION. J. Biol. Chem. 176, 1243–1251. https://doi.org/10.1016/S00219258(18)57137-4 Salvan, P., Fonseca, M., Winkler, A.M., Beauchamp, A., Lerch, J.P., Johansen-Berg, H., 2023. Serotonin regulation of behavior via large-scale neuromodulation of serotonin receptor networks. Nat. Neurosci. 26, 53–63. https://doi.org/10.1038/s41593-022-01213-3 Tansey, E.M., 2006. Henry Dale and the discovery of acetylcholine. C. R. Biol. 329, 419–425. https://doi.org/10.1016/j.crvi.2006.03.012 v. Euler, U.S., 1935. Über die Spezifische Blutdrucksenkende Substanz des Menschlichen Prostataund Samenblasensekretes.

Klin.

Wochenschr.

14, 1182–1183. https://doi.org/10.1007/BF01778029 v. Euler, U.S., n.d. A Specific Sympathomimetic Ergone in Adrenergic Nerve Fibres (Sympathin) and its Relations to Adrenaline and Nor-Adrenaline - EULER - 1946 - Acta Physiologica Scandinavica - Wiley Online Library [WWW Document]. URL https://onlinelibrary.wiley.com/doi/10.1111/j.17481716.1946.tb00368.x (accessed 3.30.25).

Yue, J.K., Burke, J.F., Upadhyayula, P.S., Winkler, E.A., Deng, H., Robinson, C.K., Pirracchio, R., Suen, C.G., Sharma, S., Ferguson, A.R., Ngwenya, L.B., Stein, M.B., Manley, G.T., Tarapore, P.E., 2017. Selective Serotonin Reuptake Inhibitors for Treating Neurocognitive and Neuropsychiatric Disorders Following Traumatic Brain Injury: An Evaluation of Current Evidence. Brain Sci. 7, 93. https://doi.org/10.3390/brainsci7080093

1

The Use of Psilocybin and MDMA in the Treatment of PTSD Kaavya Chandrasekar Edited by Hannah Betts and Louisa Willan

I. A BSTRACT Post-Traumatic Stress Disorder (PTSD) is a wellknown mental health condition that, while typically associated with members of the armed forces, can affect anyone who has experienced trauma. Despite the availability of psychotherapy, the condition remains highly prevalent, underscoring the need for novel therapeutic approaches. One such approach currently under exploration is psychedelic-assisted therapy (PAT). Substances explored thus far include psilocybin, more colloquially termed ‘Magic Mushrooms’, and 3,4-methylenedioxymethamphetamine (MDMA). This report aims to summarise the effects of psychedelic assisted therapy on the human body, evaluate its application in the treatment of PTSD and finally compare the efficacy of psilocybin and MDMA in clinical use.

II. I NTRODUCTION Post-Traumatic Stress Disorder (PTSD) affects just over 2.5 million individuals in the UK. It is estimated that 1 in 10 individuals will experience it in their lives (PTSD UK, n.d.).

PTSD is a mental health condition triggered by traumatic experiences, typically resulting in severe psychological distress (Oxford Press, n.d.). It can result in flashbacks—vivid recall of the experience—as well as general negative changes in outlook and mood.

There is currently no universally standardised or consistently effective treatment for PTSD, but psychotherapy remains the most widely explored therapeutic approach. However, psychotherapy is usually applied in an evidence-based manner, relying on clinical expertise and individualised treatment planning, unlike pharmacological treatments, which are often developed and evaluated using standardised empirical trials (Corrigan & Hull, 2015).

The differences between psychotherapeutic and pharmacological approaches to PTSD have led to increased research in both areas, with the aim of integrating them into a more holistic and effective treatment strategy. Among emerging pharmacological approaches, psilocybin and 3,4methylenedioxymethamphetamine (MDMA) have received the most research attention.

III. T REATING PTSD In 2013, a review regarding the diagnostic process of PTSD was undertaken by the Diagnostic and Statistical Manual of Mental Disorders. As a result, significant changes were made to PTSD diagnosis, shifting PTSD from an anxiety disorder to a stress and trauma disorder. The number of symptom clusters for the disorder increased, with a novel inclusion of dissociation as a key symptom (Watkins, et al., 2018). In 2019, the World Health Organisation (WHO) introduced complex PTSD (cPTSD) as a separate disorder, characterising it through symptoms such as emotional dysregulation and difficulties with human interaction and communication.

As the diagnostic processes and symptomatic evidence of PTSD changed over time, treatment options were explored and modified. Modern treatments are split into two categories: trauma focused interventions and non-trauma focused interventions.

Trauma focused interventions are designed to directly address trauma, process memories, and manage coping with them. Non-trauma focused interventions address the symptoms of PTSD rather than the trigger that causes them, emphasising coping skills (Watkins et al., 2018).

Highly recommended treatments, by both the American Psychological Association and the department of Veteran Affairs, include Prolonged Exposure (PE), Cognitive Processing Therapy (CPT) and Cognitive Behavioural Therapy (CBT).

2

Prolonged Exposure involves in vivo exposures, in which individuals directly confront feared situations, imagination exposure, and breathing retraining. The approach encourages patients to systematically engage with fear-inducing stimuli, process the associated emotional content, and work on controlled breathing techniques to help modify dysfunctional fear structures (Watkins, et al., 2018).

The main intention of Cognitive Processing Therapy is to identify and promote a cognitive change to a patient’s maladaptive thoughts. It aims to challenge negative thinking, particularly self-blame, and replace those ideas with a more realistic perspective, via a process known as accommodation. Overall, it shifts the patient’s ideas of self, society, and the world around them (Watkins, et al., 2018).

Cognitive Behavioural Therapy draws from the models outlined in both PE and CPT. Prior to treatment, individuals with PTSD often develop maladaptive cognitive and behavioural responses, such as avoidance or thought suppression. These can block the brain from modifying the negative appraisal of traumatic memories (Ehlers, et al., 2005), contributing to a cycle of guilt and continued avoidance of trauma-related stimuli (Watkins, et al., 2018). CBT aims to disrupt this cycle by teaching the patient to identify trauma triggers and restructure their thinking in a more rational, adaptive way.

All these psychotherapies have proven effective in the treatment of PTSD. However, modern research is proving more can be done. The use of psychedelics as adjunctive therapies has become a rapidly growing area of research. Of particular interest is the focus on their potential to enhance neuroplasticity and induce altered states of consciousness (Morland & Woolley, 2025).

IV. P SILOCYBIN Psilocybin is a naturally occurring psychedelic compound found in over a hundred species of fungi worldwide. Typically referred to as ‘magic mushrooms,’ the effects of psilocybin include hallucinations, intense emotions, nausea, increased heart rate and a loss of sense of time and space (National Institute on Drug Abuse, 2024).

Its mechanism of action remains under debate as neuroimaging studies have produced conflicting results. However, evidence suggests that the brain’s Default Mode Network (DMN)—the active region

of the brain when at rest—plays a pivotal role, particularly involving the prefrontal cortex and hippocampus (Smausz, et al., 2022). These regions are responsible for the formation and retrieval of episodic memories, and work together to support normal learning and memory mechanisms (Preston & Eichenbaum, 2013).

Psilocybin was first used by the Aztecs and Mazatecs, for healing, divination, and spirituality, before the 16th century (Wasson, 1980).

There have been many efforts to explore past uses of psilocybin, however due to the decadeslong global arrest of research on psychedelic drugs, research has been limited (Nutt, et al., 2020). This was partly due to the 1960s Small Heroin Epidemic (Mold, 2007). To combat this, President Richard Nixon passed the Controlled Substances Act, which the United Nations similarly followed, placing a halt on the exploration of psychedelic drugs, including psilocybin. In the 1990s, funding and resources for clinical trials eventually re-emerged (Smausz, et al., 2022).

In 2001, the psychoactive effects were described in a study by Griffiths et al. where participants underwent a double-blind placebo trial. The participants who received psilocybin were significantly more likely to report experiencing mystical experiences compared to those in the placebo group.

Following a general increase in the personality trait of open mindedness, the use of psilocybin resulted in positive life impact in 80% of patients. (Griffiths, et al., 2006).

More recently, in 2021, research revealed that psilocybin interacts primarily with serotonin receptors—particularly the 5-HT2A receptor—rather than acting through general monoamine neurotransmission, thereby advancing our understanding of the relationship between psychedelics and neuroplasticity.

V. MDMA 3,4-Methylenedioxymethamphetamine (MDMA), commonly known as ecstasy, was manufactured in the early 20th century as a drug to control bleeding (Dunlap, et al., 2018). Its psychoactive effects were rediscovered in the late 1960s leading to growing recreational interest. By the 1970s, MDMA had been identified in street samples in more than 10 US states, particularly along the West Coast. It was

3

not originally placed in the Controlled Substances Act, and was only added in 1985 after it started to be widely synthesised as a ‘legal alternative’ to the popular 3,4-methylenedioxyamphetamine (MDA) (Passie & Bezenhofer, 2016).

MDMA releases monoamines, such as serotonin and dopamine by acting on two receptors.

It functions as an antagonist at the Trace AmineAssociated Receptor 1 (TAAR1), triggering cascades of intracellular signals that promote neurotransmitter release. It also inhibits the Vesicular Monoamine Transporter 2 (VMAT2), reducing neurotransmitter storage within vesicles and promoting their release (Dunlap, et al., 2018).

Despite the lack of experimental evidence, theoretical evidence suggests that MDMA produces positive therapeutic effects by increasing the availability of serotonin (Patel & Titheradge, 2015). Thus, the psychological responses elicited by MDMA support its potential use in trauma-related psychotherapy (Sessa, et al., 2019).

VI. T HE C OMPARISON Both psilocybin and MDMA show great potential for use in psychedelic-assisted therapy. There is also the possibility of combining treatments sequentially, which may be explored in future research (Fonseka & Woo, 2023).

Currently, more research has been conducted on the use of MDMA for PTSD treatment, with studies finding that it is well tolerated and lacks significant abuse potential. However, adverse reactions such as hyperthermia and rhabdomyolysis have been reported in cases of recreational use, especially when combined with alcohol consumption. Clinically administered MDMA does not produce the same adverse effects, demonstrating that the context of administration can influence both its safety and efficacy in treating PTSD. Ethical concerns remain, including how MDMA’s therapeutic use may influence public perception of PTSD and whether its approval for therapeutic purposes could lead to increased recreational use (Hartstein & Menon, 2024).

On the other hand, while psilocybin is still in an earlier stage of exploration as a psychedelic treatment option for PTSD, current evidence suggests it poses no major health risks. The most significant concern is the risk of a bad trip (NYU

Grossman School of Medicine, 2025). This is said to involve frightening feelings, including anxiety, fear, and a sense of losing oneself (Gashi, et al., 2021). Although the risks are relatively low, some effects, such as flashbacks and muscle issues, remain a concern. From a utilitarian perspective, while the use of psilocybin offers substantial benefits, the potential for individuals to experience harmful side effects, particularly in public settings, raises important moral and ethical concerns regarding its broader societal use.

Modern clinical research has significantly elevated our understanding of both psilocybin and MDMA in the treatment of PTSD. In 2023, following a phase III trial conducted by the Multidisciplinary Association for Psychedelic Studies for MDMA-assisted therapy, it was reported that 67% of participants no longer met the diagnostic criteria for PTSD after treatment and 88% had a significant reduction in clinical symptoms (Mitchell, et al., 2023).

Psilocybin is currently in phase II trials. In 2022, a double-blind phase II trial studied the combination of 25mg of psilocybin with psychotherapy for individuals with treatment-resistant depression (TRD).

This resulted in a 29% reduction in depression scores after three weeks (Goodwin, et al., 2022).

Nevertheless, despite the expanding evidence base, limitations such as narrow participant criteria, small sample sizes and overarching regulatory constraints continue to restrict the progress of current research.

Future large scale and diverse clinical trials are essential to comprehensively evaluate the safety and efficacy of psychedelic-assisted therapies.

VII. C ONCLUSION In conclusion, both MDMA and psilocybin demonstrate significant therapeutic potential for PTSD treatment. Clinical research shows that both drugs facilitate a reduced fear response and promote emotional processing and psychological healing.

These therapeutics used in conjunction with psychotherapy, enhance feelings of trust and emotional openness, making it easier for patients to confront traumatic memories. Psilocybin, in particular, has been associated with profound psychological insights and increased neural plasticity, which may contribute to long-term improvements in mental health. While both drugs have shown promising

4

results in experimental settings, further research and clinical trials are necessary to fully understand their efficacy, safety, and long-term effects. Nonetheless, the narrative of growing evidence certainly suggests the transformative promise of these psychedelics in advancing PTSD treatment.

VIII. R EFERENCES Corrigan, F. & Hull, A., 2015. Neglect of the complex: why psychotherapy for post-traumatic clinical presentations is often ineffective. BJPsych bulletin, 39(2), pp. 86-89.

Dunlap, L., Andrews, A. & Olson, D., 2018.

Dark Classics in Chemical Neuroscience: 3,4Methylenedioxymethamphetamine (MDMA). ACS chemical neuroscience, 9(10), p. 2408–2427.

Ehlers, A. et al., 2005. Cognitive therapy for posttraumatic stress disorder: development and evaluation. Behaviour research and therapy, 43(4), pp.

413-431.

Fonseka, L. & Woo, B., 2023.

Therapeutic role of psilocybin and 3,4methylenedioxymethamphetamine in trauma: A literature review.. World J Psychiatry, 13(5), pp.

182-190.

Gashi, L., Sandbery, S. & Pederson, W., 2021. Making “bad trips” good: How users of psychedelics narratively transform challenging trips into valuable experiences. International Journal of Drug Policy, Volume 87.

Goodwin, G. M., Aaronson, S. T., Alvarez, O. & Arden, P. C., 2022. Single-Dose Psilocybin for a Treatment-Resistant Episode of Major Depression..

The New England Journal of Medicine, 387(18), p.

1637–1648.

Griffiths, R., Richards, W., McCann, U. & Jesse, R., 2006. Psilocybin can occasion mystical-type experiences having substantial and sustained personal meaning and spiritual significance. Psychopharmacology, 187(3), pp. 268-283.

Hartstein, G. L. & Menon, S. N., 2024. New Territory: Ethical Challenges Associated With MDMAAssisted Psychotherapy for PTSD’. American Journal of Psychiatry Residents’ Journal, 20(1), pp. 1922.

Mitchell, J. M. et al., 2023. MDMA-assisted therapy for moderate to severe PTSD: a randomized, placebo-controlled phase 3 trial.. Nature medicine, 29(10), p. 2473–2480.

Mold, A., 2007. Illicit drugs and the rise of epidemiology during the 1960s. Journal of epidemiology and community health, 61(4), pp. 278-281.

Morland, L. & Woolley, J., 2025. PsychedelicAssisted Therapy for PTSD. [Online] Available at: https://www.ptsd.va.gov/professional/treat/ txessentials/psychedelics assisted therapy.asp#four [Accessed 1 April 2025].

National Institute on Drug Abuse, 2024.

Psilocybin (Magic Mushrooms).

[Online] Available at: https://nida.nih.gov/research-topics/ psilocybin-magic-mushrooms#what-is-psilocybin [Accessed 11 March 2025].

Nutt, D., Erritzoe, D. & Carhard-Harris, R., 2020.

Psychedelic Psychiatry’s Brave New World. Cell, 181(1), pp. 24-28.

NYU Grossman School of Medicine, 2025.

Post-Traumatic Stress Disorder Treatment with Psychedelic Drugs.

[Online] Available at: https://med.nyu.edu/ departments-institutes/population-health/ divisions-sections-centers/medical-ethics/ education/high-school-bioethics-project/ learning-scenarios/ptsd-treatment-psychedelics#: ∼:text=Also%2C%20while%20it%20has%20not, and%20vomiting%2C%20and% [Accessed 3 April 2025].

Oxford Press, n.d. The Oxford English Dictionary. Oxford: Oxford University Press.

Palamar, J. et al., 2023. Trends in illicit ketamine seizures in the US From 2017 to 2022. AMA Psychiatry, 80(7), pp. 750-751.

Passie, T. & Bezenhofer, U., 2016. The History of MDMA as an Underground Drug in the United States, 1960-1979. Journal of psychoactive drugs, 48(2), pp. 67-75.

Patel, R. & Titheradge, D., 2015. MDMA for the treatment of mood disorder: all talk no substance?.

Ther Adv Psychopharmacol, 5(3), pp. 179-188.

Preson, A. & Eichenbaum, H., 2013. Interplay of hippocampus and prefrontal cortex in memory.

Current biology : CB, 23(17), pp. R764-R773.

PTSD UK, n.d. Post Traumatic Stress Disorder stats and figures. [Online] Available at: https://www.ptsduk.org/ptsd-stats/#: ∼:text=4%20in%20100%20people%20in, population%20in%20this%20age%20range [Accessed 8 Februry 2025].

Sessa, B., Higbed, L. & Nutt, D., 2019. A Review of 3,4-methylenedioxymethamphetamine (MDMA)-

5

Assisted Psychotherapy. Frontiers in psychiatry, 10(138).

Smausz, R., Neill, J. & Gigg, J., 2022. Neural mechanisms underlying psilocybin’s therapeutic potential – the need for preclinical in vivo electrophysiology. Journal of psychopharmacology, 36(7), pp.

781-793.

Wasson, R. G., 1980. The Wondrous Mushroom: Mycolatry in Mesoamerica. 17th Edition ed. New york: McGraw-Hill.

Watkins, E., Sprang, K. & Rothbaum, B., 2018.

Treating PTSD: A Review of Evidence-Based Psychotherapy Interventions. Frontiers in behavioral neuroscience, 12(258).

1

Advancing Biodiesel Production through various Nanomaterial Productions Hannah Orchison Edited by Hannah Betts and Tom Dunlop

Abstract - The use of nanocatalysts in biodiesel production represents a significant advancement in renewable energy technology. Biodiesel is an alternative fuel, derived from biomass through the process of transesterification. Biodiesel is a promising alternative to fossil fuel due to its high energy density and lower environmental impact.

Nanocatalysts (predominantly metal oxides) offer distinct advantages over conventional catalysts, including higher surface area-to-volume ratios and greater recyclability when used in biodiesel production [2]. This report examines the potential of various nanocatalysts that use organicallyderived molecules, including calcium oxide derived from waste snail shells and lipase extracted from fungi, in enhancing biodiesel production efficiency. The findings indicate that nanocatalysts significantly improve biodiesel yield while reducing production costs and environmental impact. With the global demand for sustainable energy production, using nanotechnology in biofuel production could reduce greenhouse gas emissions and mitigate climate change.

I. I NTRODUCTION Climate change is a global issue at the heart of the 21st century. Our planet can only produce a finite amount of natural resources and withstand minimal temperature fluctuation before the complex balance of our global environment is irrevocably disrupted with catastrophic consequences. Over 75% of climate-causing greenhouse gases are produced when fossil fuels, such as gas, oil, and coal, are burnt to generate energy for transport, residential use, industrial processes or (most commonly) electricity production [1]. As concerns over climate change continue to grow, the search for more sustainable energy alternatives with fewer adverse environmental effects has become increasingly urgent.

Biofuels, particularly biodiesel, have emerged as a promising solution due to their renewable nature, lower emissions, and compatibility with existing fuel infrastructure.

II. W HAT IS B IODIESEL ?

Biofuel is a type of fuel produced from living matter (biomass). This includes food waste, agricultural by-products, and plants [5]. Biodiesel is a common biofuel produced via transesterification, a process where vegetable oils/animal fats (biomass) react with methanol in the presence of a catalyst.

Compared to petroleum-based fuels, biodiesel is biodegradable, non-toxic, and emits fewer greenhouse gases, making it a viable option for reducing the environmental impact of fuel production [2].

A key challenge in biodiesel production is the efficiency and cost of catalysis. Traditional catalysts have many drawbacks—enzyme (biological) catalysts can be damaged by acidic methanol and are extremely expensive [6]. In contrast, acid catalysts can corrode equipment and lead to slow reaction rates. Nanocatalysts have emerged in recent years as cutting-edge innovations within the field of biodiesel production, as they have fewer limitations than regular catalysts.

III. W HAT ARE NANOMATERIALS ?

Nanomaterials (also known as nanoparticles) are particles which are approximately 1–100 nm (10−9 to 10−8 m) in diameter, much smaller than the particle size of bulk materials, which are usually in ranges of microns (10−6 m). When a particular nanomaterial is used as a catalyst, it is referred to as a nanocatalyst. Nanoparticles of many different materials make suitable catalysts because they exhibit unique physiochemical properties compared to ‘normal-sized’ molecules, enabling efficient and sustainable biodiesel production.

These properties include a high surface area-tovolume ratio, which allows more of the catalyst’s surface to participate in reactions; lower activation energy, meaning the transesterification process can occur more readily and at lower temperatures; and enhanced reactivity due to their high surface energy and small size, which increases interaction with reactant molecules and boosts overall yield.

Several types of nanocatalysts are under investigation for use in biodiesel synthesis. Metal oxide nanocatalysts, such as calcium oxide, have shown promise due to their strong basicity, ease of recovery, and cost-effectiveness. Magnetic nanocatalysts, which incorporate magnetic properties for easier separation and reuse, offer additional advantages in industrial applications. Other emerging nanomaterials, including zeolite and carbon-based catalysts, are also being explored for their potential to enhance biodiesel yields and reduce waste generation.

This study examines nanocatalysts which use organically derived molecules (i.e. from living organisms). These are considered some of the most sustainable nanocatalysts because they already exist in nature, so don’t require expensive, energy-intensive synthesis, and they use renewable materials [3].

from other products. After seven reaction cycles, the immobilised lipase retained 25.79% of its initial activity, with near-complete retention over the first four cycles [4]. This reusability reduces operational costs, making the process economically viable and more sustainable.

Additionally, the immobilised lipase exhibited enhanced thermal and pH stability, performing optimally at 40°C and pH 8 but tolerating a wide range of ph. In comparison, free lipase (without the nanocatalyst) performed optimally at 37°C and pH 7 [4]. However, its relative activity at higher pH levels drops more rapidly than that the immobilised lipase. This broader operational range is crucial for industrial-scale biodiesel production, where reaction conditions may fluctuate and a catalyst that is not significantly impeded by minor changes in pH is useful.

IV. F UNGI -D ERIVED NANOBIOCATALYSTS Nanobiocatalysts combine nanoparticles and their desirable properties, including high surface area and stability, with biological catalysts to enhance biodiesel production. A study by T. Touqeer et al.

(2019) successfully developed a high-performance nanobiocatalyst through the immobilisation of Aspergillus terreus (a type of fungi) lipase (a type of enzyme) onto polydopamine (PDA)-coated Fe3 O4 nanoparticles. The PDA coating helped securely attach the enzyme onto the nanoparticles whilst maintaining 97.27% of the lipase’s native activity (17.82 U/mg/min) [4].

The catalyst showed 97% efficiency in converting waste cooking oil into biodiesel, with a high yield of 92% under optimised conditions (37°C, 30 hours) [4]. Unlike traditional chemical catalysts, this nanobiocatalyst worked efficiently with low-quality oils and did not produce harmful byproducts.

The magnetic nature of Fe3 O4 nanoparticles allowed easy separation and reuse of the nanobiocatalyst using an external magnet, a critical advantage over conventional catalysts, which are difficult to remove

Figure 1: Effect of pH on the activities of free and immobilised lipase (T. Touqeer et al., 2019) V. E GGSHELL -D ERIVED M ETAL OXIDE PARTICLES A metal oxide nanocatalyst comprises metal and oxygen atoms (e.g., CaO, TiO2 , ZrO2 , Fe3 O4 ) on the nanoscale. These combine the catalytic properties of metal oxides with nanoscale advantages. One such example would be calcium oxide nanocatalysts, which can be synthesised from eggshells and used to catalyse the transesterification of soybean oil to produce biodiesel [7].

Mansir et al. (2018) demonstrated that calcined eggshell nanoparticles form a highly efficient bifunctional catalyst when modified with transition metals like tungsten oxide (WO3 ). The CaO component facilitates transesterification of triglycerides, 2

while the acidic WO3 sites simultaneously esterify free fatty acids (FFAs). As a catalyst, CaO can enable greater than 90% biodiesel yield from waste cooking oil, under moderate conditions (65°C, 2–6 hours).

Unlike conventional homogeneous catalysts (e.g., NaOH), these nanocatalysts avoid soap formation, eliminating the need for costly pre-treatment steps.

Their solid-phase nature also simplifies separation; centrifugation or filtration can recover the catalyst for reuse, reducing waste and operational costs. After seven cycles, the catalyst retained 8̃0% activity, with minimal leaching of active sites [7].

VII. C OMPARISON The three nanocatalysts each offer unique advantages for biodiesel production. The fungi-derived nanobiocatalyst excels in sustainability and biocompatibility, achieving high yield with minimal byproducts and exceptional pH and thermal stability, though it shows lower reusability after multiple cycles. Eggshell-derived CaO-WO3 nanocatalysts provide a strong balance of efficiency and durability, with high yield and excellent reusability, thanks to their bifunctional ability to handle both triglycerides and free fatty acids. Snail-shell-derived CaO-Fe3 O4 nanocomposites outperform the others in biodiesel yield and thermal resilience, combining strong catalytic activity with easy magnetic recovery. Overall, while all three are eco-friendly and cost-effective, the choice of catalyst may depend on specific feedstock quality, desired process conditions, and scalability needs.

VI. S NAIL S HELL D ERIVED M ETAL OXIDE NANOPARTICLES A 2024 study by Ao et al. developed a metal oxide nanocatalyst by transforming waste snail shells into calcium oxide-magnetite (CaO-Fe3 O4 ) nanocomposites. This catalyst harnesses the strong basicity of CaO for transesterification, and the magnetic properties of Fe3 O4 for easy recovery.

The nanocatalyst demonstrated remarkable performance, achieving 97.7% biodiesel yield from soybean oil under optimised conditions (24:1 methanolto-oil ratio, 8.6 wt% catalyst loading, 66°C for 62 minutes). This significantly outperformed conventional CaO catalysts due to its dual functionality.

The CaO component efficiently converted triglycerides while the Fe3 O4 interface sites esterified free fatty acids, preventing soap formation common with low-quality feedstocks [8].

Like the nanobiocatalyst, a key advantage of the snail-shell derived nanocatalysts was magnetic recoverability, which allows simple separation with an external magnet following reactions. The catalyst maintained 68.1% activity after seven reuse cycles, a significant improvement over conventional CaO catalysts which typically suffer from rapid deactivation.

The system showed excellent thermal stability, withstanding temperatures up to 80°C without degradation, making it suitable for industrial-scale processes [8]. Post-reaction characterisation revealed that the nanocatalyst retained its core structure even after multiple cycles, though some magnetisation loss did occur (from 7.2 to 4.2 emu/g) [8].

Source Nanocatalyst MateType rial

Key Components

Biodiesel Yield

FungiDerived Nanobiocatalyst

Lipase Fungi+ PDAAspergillus coated Terrus Fe3 O4

EggshellDerived Metal Oxide

Waste eggshells

CaO modified with WO3

> 90%

Snail-Shell Derived Nanocatalyst

Waste snail shells

CaO Fe3 O4 nanocomposite

98%

92%

Reusability Retains ∼ 26% activity after 7 cycles Retains ∼ 80% activity after 7 cycles Retains ∼ 68.1% activity after 7 cycles

Table I: Comparison of key properties of three nanocatalysts

VIII. C ONCLUSION This study highlights the significant potential of organically derived nanocatalysts, in advancing sustainable biodiesel production. By leveraging the unique physiochemical properties of nanomaterials, these catalysts offer a compelling alternative to conventional chemicals for biofuel production.

Fungi-derived nanobiocatalysts and metal oxide nanocatalysts synthesised from eggshells and snail shells demonstrate not only high biodiesel yields (over 90%) but also impressive stability, 3

reusability, and environmental compatibility.

These materials reduce reliance on non-renewable resources, lower production costs, and simplify post-reaction separation processes. Incorporating magnetic materials further enhances catalyst recovery, making these approaches highly attractive for industrial applications.

As the global demand for clean energy intensifies, nanotechnology presents a scalable and environmentally-focused solution to improve biodiesel efficiency while addressing waste management and reducing greenhouse gas emissions. Future research should focus on optimising synthesis techniques, improving longterm catalyst durability, and evaluating large-scale implementation to fully realise the transformative potential of nanocatalysts in renewable fuel production.

1147-1157 https://doi.org/10.1016/j.renene.2018.08.

108 [6] Norjannah, B., Chyuan Ong, H., Masajuki, H.H, Juan, J.C., Chong W.T. (2016) ‘Enzymatic transesterification for biodiesel production: a comprehensive review’ RSC Advances, 6, pp. 6003460055 https://doi.org/10.1039/C6RA08062F [7] Mansir, N., Teo ,S. H., Rashid, U., Saiman, M.I., Tan, Y. P., Alsultan, G.A., Taufiq-Yap, Y.H. (2018) ‘Modified waste egg shell derived bifunctional catalyst for biodiesel production from high FFA waste cooking oil. A review’, Renewable and Sustainable Energy Reviews, 82(3), 3645-3655 https://doi.org/10.1016/j.rser.2017.10.098 [8] Ao, S., Greer, H.F, Alghamdi, L.A., Rashid, U., Halder, G., Wheatley, A.E.H., Rokhum, S.L. (2024) ‘Snail shell derived magnetic nanocatalysts for biodiesel production: Process optimization through response surface methodology, kinetics, and thermodynamic studies’ Biomass and IX. R EFERENCES Bioenergy, 191, 107442 https://doi.org/10.1016/j.

[1] SEI, IISD, ODI, Climate Analytics, CICERO, biombioe.2024.107442 and UNEP. (2019). The Production Gap: The discrepancy between countries’ planned fossil fuel production and global production levels consistent with limiting warming to 1.5°C or 2°C. link: http: //productiongap.org/ [last accessed 01/04/2025] [2] Narasimhan, M., Chandrasekaran, M., Govindasamy, S., Aravamudhan, A. (2021) ‘Heterogeneous nanocatalysts for sustainable biodiesel production: A review’, Journal of Environmental Chemical Engineering, 9(1), 104876 https://doi.org/ 10.1016/j.jece.2020.104876 [3] Changmai, B., Vanlalveni, C., Ingle, A.P., Bhagat, R., Rokhum, S.L. (2020) ‘Widely used catalysts in biodiesel production: a review’, RSC Advances, 10, pp. 41625-41679 https://doi.org/10.

1039/D0RA07931F [4] Touqeer, T., Mumtaz, M.W., Mukhtar, H., Irfan, A., Akram, S., Shabbir, A., Rashid, U., Nehdi, I.A., Choong, T.S.Y. (2019) ‘Fe3O4-PDA-Lipase as Surface Functionalized Nano Biocatalyst for the Production of Biodiesel Using Waste Cooking Oil as Feedstock: Characterization and Process Optimization’, Energies, 13(1), 177 https://doi.org/10.3390/ en13010177 [5] Silva César, A., Conejero, M.A., , Barros Ribeiro, E.C., Batalha, M.O., (2019) ‘Competitiveness analysis of “social soybeans” in biodiesel production in Brazil’, Renewable Energy, 133, pp.

4

1

Colouring the Molecular World: How Cryo-EM is Redefining Biological Imaging Ananya Darshan Edited by Andrew Linz and Alethea Stevenson

Abstract—Cryogenic-electron microscopy (cryo-EM) has revolutionized structural biology by enabling highresolution imaging of biological molecules in their nearnative states. Unlike conventional electron microscopy, cryo-EM overcomes key limitations such as radiation damage and sample dehydration, allowing researchers to visualize protein structures, molecular interactions, and misfolded proteins linked to disease. Its applications are broad, ranging from fundamental biological research to pharmaceutical studies - including drug-binding site analysis and investigations into neurodegenerative diseases. A significant recent development is REEL cryo-EM, which integrates electron energy loss spectroscopy (EELS) with scanning transmission electron microscopy (STEM). This approach produces color-coded, element-specific maps of biological structures, enabling precise identification of atomic composition and localization. Such capabilities provide critical insights into metal toxicity and metaldependent enzymatic processes. As cryo-EM technology continues to advance, it is redefining our understanding of cellular architecture and molecular function, solidifying its role as an indispensable tool in both research and drug discovery.

I. I NTRODUCTION The development of structural biology techniques such as x-ray crystallography and cryogenicelectron microscopy (cryo-EM) has revolutionized the field of molecular biology and biochemistry, providing unprecedented insights into the complex structures that form the foundation of life (Calloway, 2020). Although the existence of certain molecular interactions has already been acknowledged, the mechanisms behind them remain unclear; microscopic imaging techniques have since enabled scientists to observe these processes in action, driving rapid advances across biological disciplines.

Today’s advanced microscopy techniques allow researchers to visualize complex biological structures at high resolution, and the recent introduction of colorization (Stahlberg, 2024) to these images marks a crucial step forward by enhancing the inter-

pretability of molecular data. This development, particularly impactful in visualizing protein-metal complexes, demonstrates that the field continues to hold considerable potential for further innovation (Stahlberg, 2024). These capabilities have made microscopy an indispensable tool in modern biological research, driving innovations in medicine, genetics, and our fundamental understanding of life at its most basic level. This article explores cryo-EM, its key advancements, current applications, and future developments.

II. W HAT IS E LECTRON M ICROSCOPY ?

Microscopy, at a fundamental level, harnesses the interactions between radiation and physical matter to generate images that would otherwise be invisible to the naked human eye. Light is a form of electromagnetic radiation, existing on a spectrum that includes radio waves, microwaves, infrared, ultraviolet, X-rays, and gamma rays. The light microscope uses visible light as the source of radiation, and a series of carefully aligned lenses and apertures help magnify the resulting image allowing us to view cells and multicellular structures. The term radiation, however, is not limited to the electromagnetic spectrum. Both electromagnetic waves and particle beams, such as accelerated electrons, can function as radiation sources for microscopy since they exhibit wavelike properties that interact with specimens.

The scale of what can be imaged depends directly upon the source of radiation and the relative size of its wavelength to the sample. Visible light alone has a small enough wavelength to image the fundamental structures of biological matter; however, a highly accelerated beam of electrons has a wavelength roughly 100 times smaller than visible light (Milne et al., 2012) allowing for imaging at much higher resolutions.

2

III. L IMITATIONS OF E LECTRON M ICROSCOPY Electron microscopy has proven to be a highly promising piece of technology; however, it is not without its limitations, the most apparent being the susceptibility of organic matter to high doses of electron radiation. High energy electron irradiation leads to changes within the electronic configuration of molecules, resulting in the breakdown of chemical bonds and subsequent losses to the shape, position and structure of the sample (Egerton et al., 2004). The various degradation processes are characterised by their critical dose – the amount of electron radiation a sample can tolerate before being destroyed (Egerton et al., 2004). An imaging process which leads to the destruction of the very sample being imaged is less than ideal. Lower energy electron beams can be used to minimise the damage incurred by the sample. However, since the energy of the beam is indirectly proportional to the electron wavelength, lower energy beams result in lower resolution images (Stahlberg, 2024).

Another issue arises due to the operating environment of an electron microscope, as the electrons must be accelerated in a vacuum in order to prevent the beam being deflected off course by air particles. Since biological matter contains a high percentage of water, which vaporises in a vacuum, untreated samples are prone to structural collapse and deformation as their internal moisture rapidly evaporates during imaging (Oikonomou and Jensen, 2017). Flash-freezing samples before imaging offers a solution to both problems (Cressey and Calloway, 2017). This technique, perfected by an international team of scientists who won the 2017 Nobel Prize in Chemistry, preserves the specimen’s hydration and natural biological structure through rapid immersion in cryogenic liquids – most commonly liquid ethane (Bock and Grubmüller, 2022). The development of cryo-electron microscopy (cryo-EM) combined this freezing approach with specialized image processing software, and for larger samples the addition of cryoprotectants to assist with freezing, to facilitate the imaging process (Hurbain and Sachse, 2012).

IV. A DVANCING PROTEIN STRUCTURE ANALYSIS Cryo-electron microscopy has emerged as a transformative analytical platform, opening unprecedented windows into the molecular architecture that underlies cellular processes. The technique’s

diverse applications have fundamentally reshaped how researchers explore and interpret the intricate structural landscape of biological systems. One key application of cryo-EM is enabling scientists to identify previously unknown atomic compositions within complex protein structures, revealing crucial molecular details that remained hidden with other techniques. Amino acids form the basic building blocks of proteins, whose structure determines their ability to function properly (Alberts et al., 2015).

This powerful method captures the intricate arrangements of amino acids and the folding patterns that determine protein structure, illuminating how proteins achieve their functional three-dimensional shapes and operate in molecular interactions. A compelling example comes from the characterization of amyloid- fibrils, fibre like assemblies of misfolded proteins extracted directly from Alzheimer’s disease brain tissue (Kollmer et al., 2019). Cryo-EM analysis uncovered multiple distinct morphologies, including a novel C-shaped peptide fold (a peptide being a short chain of amino acids that can function as building blocks for proteins or perform specialized roles in biological systems (Alberts et al., 2015)) and an unexpected right-hand twist structural features absent in laboratory-grown fibrils (Kollmer et al., 2019). This discovery highlights the critical importance of studying disease-relevant structures in patient-derived materials rather than relying exclusively on artificial models. Moreover, cryo-EM provides unprecedented insights into protein misfolding diseases such as Alzheimer’s and Parkinson’s, where specific misfolded conformations drive disease development (Negi, Khurana, and Duggal, 2024). By visualizing these diseasespecific fibril structures at near-atomic resolution, researchers can now decipher the structural mechanisms of neurodegeneration and design targeted therapeutics that precisely interact with damaging or disease-causing protein structures.

V. P HARMACEUTICAL RESEARCH Pharmaceutical research has seen significant advancements with the advent of cryo-electron microscopy, benefiting in three key areas. First, cryoEM offers detailed structural insights into drugbinding sites, enabling researchers to visualize how therapeutic compounds interact with their molecular targets. A drug-binding site is a specific region on

3

proteins such as enzymes, receptors, or transporters where a drug can attach (Chufan, Sim, and Ambudkar 2015). The interaction between the drug and the binding site can profoundly influence the protein’s function. Understanding these interactions is essential in drug discovery, as it allows researchers to design more effective compounds. The ability to observe binding sites at such high structural resolution is vital for understanding binding mechanisms at the atomic level. Leading pharmaceutical companies, including Pfizer, have incorporated cryo-EM into their structural biology workflows, utilizing it to advance the discovery and optimization of drug targets (Ilitchev 2019). Additionally, the high-resolution structural data obtained through cryo-EM enables the design of more selective and potent drugs. By providing a clearer understanding of the three-dimensional shapes of proteins and complexes, cryo-EM allows researchers to develop drugs that more precisely interact with their targets, improving their effectiveness. These improvements in drug design and target discovery contribute to the overall acceleration of drug development. By providing precise molecular-level information, cryoEM streamlines the drug development process, reducing timelines and increasing the likelihood of successful therapeutic outcomes.

Gardiner et al., 2024). Scanning transmission electron microscopy (STEM) focuses a fine electron beam that scans across samples in a precise pattern, distinguishing it from traditional transmission electron microscopy (TEM) which uses a broad beam (Williams and Carter, 2009). As electrons pass through the sample, their interactions generate signals that reveal internal structure at atomic-scale resolution (Williams and Carter, 2009). The integration of STEM with EELS creates a powerful analytical approach: STEM provides high-resolution structural imaging while EELS simultaneously delivers elemental composition data from the same sample regions, significantly advancing our understanding of complex biological systems (Pfeil-Gardiner et al., 2024). A groundbreaking feature of REEL cryoEM is its ability to introduce colour to cryo-EM imaging (Pfeil-Gardiner et al., 2024). By assigning specific colours to different atomic elements, scientists can directly visualize elemental distribution across complex structures. The energy loss data collected by the EELS detector generates a threedimensional, color-coded map where each colour represents a different element, providing detailed spatial distribution of atoms throughout the sample.

This innovative combination of techniques enables researchers to examine biological materials with unprecedented detail and chemical context. REEL cryo-EM is setting the stage for deeper insights into VI. F UTURE DEVELOPMENTS : COLOUR biological macromolecules, their interactions, and Recent advancements in cryo-EM have intro- their roles in health and disease, representing one duced Reconstructed Electron Energy Loss Spec- of the most significant recent developments in cryotroscopy (REELS), an innovative technique that electron microscopy. combines electron energy loss spectroscopy (EELS) with scanning transmission electron microscopy VII. K EY S CIENTIFIC ADVANCEMENTS (STEM) to analyse the atomic composition of T OXICITY biological and macromolecular structures at unprecedented resolution (Pfeil-Gardiner et al., 2024).

Recent breakthroughs in cryo-electron miEELS measures energy lost by electrons as they croscopy, particularly through REEL analysis, have interact with atoms in a sample. When an electron transformed our ability to pinpoint heavy atoms beam encounters material, some energy transfers within intricate protein structures12. This advanceto atoms, with the amount lost depending on the ment represents a watershed moment for metal specific elements and their chemical bonds. By detection in biological samples, illuminating critical analysing this energy loss pattern, researchers can aspects of metal toxicity mechanisms and metalidentify the atomic types present and understand dependent enzymatic functions (Pfeil-Gardiner et their chemical environment and bonding states. This al., 2024) (Stahlberg, 2024). By enabling the idencapability makes EELS an effective tool for map- tification of specific atoms with near-atomic preping the elemental composition of complex materi- cision, these innovations address one of structural als at the atomic level, crucial for comprehending biology’s most persistent challenges - accurately structural properties of biological systems (Pfeil- determining atomic composition within complex

4

macromolecular assemblies (Pfeil-Gardiner et al., 2024). The implications for toxicology research are profound. Cryo-EM now allows scientists to visualize with remarkable clarity how toxic metals accumulate within specific cellular compartments (PfeilGardiner et al., 2024), providing crucial spatial context that could transform our understanding of heavy metal poisoning at the cellular level. This precision extends to neurodegenerative research, where cryo-EM offers unprecedented insights into diseases linked to metal dysregulation (Pfeil-Gardiner et al., 2024). Scientists can now directly observe the relationship between copper or iron accumulation and conditions like Alzheimer’s disease, revealing in striking detail how metal imbalances compromise cellular architecture and function.

VIII. C ONCLUSION The evolution of electron microscopy, particularly cryo-EM technology, represents one of the most transformative developments in modern molecular biology. From addressing fundamental limitations of conventional electron microscopy through flashfreezing techniques to the groundbreaking REEL cryo-EM that introduces colour visualization at atomic resolution, these advancements have revolutionized our ability to explore the molecular foundations of life. The applications of cryo-EM span critical areas of scientific inquiry, including detailed protein structure analysis of diseaserelevant proteins and protein complexes, structureguided drug discovery for large proteins that do not crystallize, and pharmaceutical research that accelerates drug development by enabling precise visualization of binding sites. The technology’s capacity to identify specific atomic compositions within complex macromolecular structures has filled a longstanding gap in structural biology, allowing unprecedented insights into protein folding, misfolding diseases, and metal-dependent biological processes. Furthermore, the development of colour in cryo-EM through the integration of EELS and STEM techniques marks a new frontier in molecular visualization. By allowing for the collection of element-specific visual information, coloured cryoEM images enable researchers to simultaneously analyse both structural architecture and chemical composition of biological samples. As this technology continues to advance, its implications for

understanding fundamental biological mechanisms, neurodegenerative diseases, and metal toxicity will likely expand even further. The journey from early microscopy to today’s sophisticated cryo-EM techniques illustrates how methodological innovations drive scientific discovery, ultimately deepening our understanding of the intricate molecular processes that underpin life itself. Cryo-EM stands as a testament to how technological breakthroughs can transform theoretical concepts into visible reality, continually redefining the boundaries of what scientists can observe, analyse, and ultimately comprehend about the microscopic world.

IX. R EFERENCES Works Cited: Alberts, B., Johnson, A., Lewis, J., Raff, M., Roberts, K., Walter, P. (2015).

Molecular Biology of the Cell (6th ed.).

Garland Science. Alberts, B., Johnson, A., Lewis, J., Raff, M., Roberts, K., Walter, P. (2015). Molecular Biology of the Cell (6th ed.).

Garland Science Bock, L.V. and Grubmüller, H. (2022). Effects of cryo-EM cooling on structural ensembles. Nature Communications, 13(1). doi:https://doi.org/10.1038/s41467-02229332-2. Callaway, E. (2020). Revolutionary cryo-EM is taking over structural biology.

Nature, [online] 578(7794), pp.201–201. doi:https://doi.org/10.1038/d41586-02000341-9. Chufan, E.E., Sim, H.-M. and Ambudkar, S.V. (2015). Molecular Basis of the Polyspecificity of P-Glycoprotein (ABCB1).

ABC Transporters and Cancer, [online] pp.71–96. doi:https://doi.org/10.1016/bs.acr.2014.10.003.

Cressey, D. and Callaway, E. (2017).

Cryo-electron microscopy wins chemistry Nobel.

Nature, 550(7675), pp.167–167. doi:https://doi.org/10.1038/nature.2017.22738.

Egerton, R.F., Li, P. and Malac, M. (2004). Radiation damage in the TEM and SEM. Micron, [online] 35(6), pp.399–409. doi:https://doi.org/10.1016/j.micron.2004.02.003.

Hurbain, I. and Sachse, M. (2011). The future is cold: cryo-preparation methods for transmission electron microscopy of cells.

Biology of the Cell, 103(9), pp.405–420. doi:https://doi.org/10.1042/bc20110015.

Ilitchev, A. (2019). How Pfizer is Driving Drug Discovery with Cryo-EM. [online]

5

Life in Atomic Resolution. Available at: https://www.thermofisher.com/blog/atomicresolution/how-pfizer-is-driving-drug-discoverywith-cryo-em [Accessed 4 Jun. 2025]. Kollmer, M., Close, W., Funk, L., Rasmussen, J., Bsoul, A., Schierhorn, A., Schmidt, M., Sigurdson, C.J., Jucker, M. and Fändrich, M. (2019).

Cryo-EM structure and polymorphism of A amyloid fibrils purified from Alzheimer’s brain tissue. Nature Communications, 10(1). doi:https://doi.org/10.1038/s41467-019-12683-8.

Milne, J.L.S., Borgnia, M.J., Bartesaghi, A., Tran, E.E.H., Earl, L.A., Schauder, D.M., Lengyel, J., Pierson, J., Patwardhan, A. and Subramaniam, S. (2012). Cryo-electron microscopy - a primer for the non-microscopist. FEBS Journal, [online] 280(1), pp.28–45. doi:https://doi.org/10.1111/febs.12078.

Negi, S., Khurana, N. and Duggal, N. (2024). The Misfolding Mystery: -syn and the Pathogenesis of Parkinson’s Disease.

Neurochemistry International, 177, pp.105760–105760. doi:https://doi.org/10.1016/j.neuint.2024.105760.

Oikonomou, C.M. and Jensen, G.J. (2017). The development of cryo-EM and how it has advanced microbiology. Nature Microbiology, [online] 2(12), pp.1577–1579. doi:https://doi.org/10.1038/s41564017-0073-7. Pfeil-Gardiner, O., Higor, R., Riedel, D., Chen, Y.S., Lörks, D., Kükelhan, P., Linck, M., Müller, H., Petegem, V. and Murphy, B.J. (2024). Elemental mapping in single-particle reconstructions by reconstructed electron energyloss analysis. Nature Methods, [online] 21(12), pp.2299–2306. doi:https://doi.org/10.1038/s41592024-02482-5. Stahlberg, H. (2024). Cryo-electron microscopy in color. Nature Methods, 21(12), pp.2233–2234. doi:https://doi.org/10.1038/s41592024-02427-y. Williams, D. B., Carter, C. B. (2009).

Transmission Electron Microscopy: A Textbook for Materials Science (2nd ed.). Springer.

1

Computation and the Classification of Finite Groups Sarah Aoudia Edited by Andrew Linz and Gayatri Chatterji Sharma

Abstract - This article explores the intersection of computational tools and abstract group theory. After introducing fundamental group theoretic concepts, such as the dihedral and symmetric groups, it examines the long-standing challenge of classifying all finite groups - a task made difficult by their infinite variety. The article highlights historical classification efforts by mathematicians like Cayley and Hölder, before turning to recent breakthroughs made possible by the advent of computational group theory (CGT). Central to this progress are software systems like GAP (Groups, Algorithms, Programming), which enables efficient storage, exploration, and isomorphism checking for vast numbers of groups. While full classification remains elusive, GAP’s capabilities continue to grow, opening new pathways in understanding group structure. The article concludes with a discussion of ongoing challenges and open questions in the finite classification problem.

The advent of computational tools continues to have a profound impact on modern mathematics. From everyday items like calculators to groundbreaking uses of artificial intelligence to solve complex problems, postulate new conjectures and even produce rigorous proofs of longstanding mathematical statements, the bounds of what we can do are stretching. Within number theory, mathematicians use powerful computers to search for huge prime numbers - numbers only divisible by themselves and 1 - that humans could never check by hand.

A striking example came in October 2024, when a computer discovered the current record-holder: 2136279841 − 1, a prime consisting of over 41 million digits (Mersenne.org, 2024). Within the realm of computational group theory, a programming system known as GAP (Groups, Algorithms, Programming) stands as a cornerstone (GAP, 2024). This article will first give some background on group theory and the open question of classifying finite groups, before examining the significance of GAP in progress thus

far and as a potential tool to further classification in future I. BACKGROUND The abstract definition of a group was first introduced in 1854 by Arthur Cayley (Cayley, 1854). A group (G, ∗) consists of a collection of objects (G) together with a binary operation (∗) that satisfies the following four rules for all objects g,h and k in G: 1) Closure: g ∗ h is in G When two objects in G are combined using the operation ∗, the result is also in G 2) Associativity: (g ∗ h) ∗ k = g ∗ (h ∗ k).

Combining the three objects can be done in either order above.

3) Identity: There always exists an object in G, namely e, such that e ∗ g = g ∗ e = g.

This object does not change the result when combined with any object in G in any order.

4) Inverse: For each g, there exists g −1 in G such that g ∗ g −1 = g −1 ∗ g = e.

Each object has a unique ‘inverse’, such that they cancel each other out when combined to give the identity, e.

This formal definition of a group allows for structures with very different appearances and properties. We now explore two such examples, beginning with the dihedral group of size 6, D3 , which consists of all the symmetries of an equilateral triangle.

D3 = {p, p2 , p3 , r, s, t} is a group of 3 rotations and 3 reflections (see Figure 1).

We can easily show the set D3 is a group by checking the group axioms (see Figure 1), where we

2

Figure 1: A diagram showing the elements of D3 Figure 2: A visual representation of the elements visually as rotations and reflections of a triangle and of S3 , showing where each element A,B and C a table explaining transformations of each element. maps. Left to right, we will label these elements f1 , f2 , . . . , f6 for ease of notation in discussion Rotations p: A rotation 120 degrees clockwise, sending A → B,B → C,and C → A. p2 : A rotation 240 degrees clockwise, sending A → C,B → A,and C → B. p3 : A rotation 360 degrees clockwise, sending A → A,B → B,and C → C.

Reflections r: A reflection on the axis through A, swapping B and C. s: A reflection on the axis through B, swapping A and C. t: A reflection on the axis through C, swapping A and B.

Table I: D3 rotations and reflections define the operation (∗) so that a ∗ b means: perform transformation a first, followed by b. For example, performing p ∗ s one would rotate the triangle 120 degrees clockwise, then reflect across the s axis.

With a little thought, one can clearly see this is the same transformation as simply performing t , so p ∗ s = t.

Closure

Associativity

Identity

Inverse

Any combination of rotations and reflections will always be equivalent to either a rotation or a reflection (i.e. another element of D3 ).

Performing ‘(a then b) then c’ is equivalent to ‘a then (b then c)’. p3 , rotating 360 degrees, brings each vertex back to its original position hence composing p3 with any element does not change the output. p3 ,r,s and t are all their own inverses. p and p2 are inverses of each other, so all elements have a (unique) inverse element.

Table II: Showing D3 satisfies the group axioms.

The second group we will discuss is the symmetric group on three points, S3 , which is the collection of

all possible one-to-one functions on 3 points. Essentially, these are all the possible ways of ‘pairing up’ the three points (of which there are 6 options).

We will use the points A,B and C (see Figure 2 for the elements of S3 ).

The group operation here is ‘composition of functions’, similarly to D3 . For example, performing f3 ∗ f6 means send A to C and then C to B, so overall we send A to B. Similarly, we send B to A and C to C, giving us f4 , so f3 ∗ f6 = f4 .

Although D3 and S3 may appear to describe very different groups, they are in fact structurally identical in how their elements combine. In group theory, such a relationship is called an isomorphism, denoted D3 ∼ = S3 . This means there exists a one-toone correspondence between the elements of the two groups that preserves the group operation. Using a Cayley table to show how all elements of each group combine with one another (see Figure 3), we can see how these two tables are identical, up to relabelling the names of the elements.

To a group theorist, saying that D3 and S3 are isomorphic is as good as saying they are ”the same” group in terms of their algebraic structure. Every group element in D3 can be matched to a unique element in S3 such that their respective Cayley tables are identical up to relabelling. The key insight here is that group isomorphism abstracts away the specific interpretation (rotations and reflections vs. matching up elements) and focuses purely on the behaviour of the group operation.

3

introducing errors such as Cayley’s initial classification of groups of size 6 (Cayley archive, 2021).

This corpus already covers a substantial number of finite groups, laying much of the groundwork on which modern, computer-aided classifications now build.

Figure 3: A side-by-side comparison of the Cayley tables of D3 and S3 , showing their similarity.

III. E NTER C OMPUTATIONAL G ROUP T HEORY Specifically, performing the relabelling: p3 → f1 , r → f 2 , s → f 3 , t → p → f 5 , p2 → f 6 , r → f 2 , In 2000, a team led by Besche, Eick, and O’Brien s → f3 , t → f4 produces two identical Cayley used computers to push classification to all sizes tables, proving D3 ∼ = S3 . ≤ 2000 (except size 1024) (Besche et al., 2001), discovering 49,910,529,483 distinct, non-isomorphic groups. The sheer scale of this number shows why Clearly, isomorphic groups must have the same size brute-force approaches were no longer viable be(number of elements) to match the elements up oneyond size 215. to-one. This provides a first criterion for isomorThis was one of the first feats of Computational phism. If we could classify exactly when any two Group Theory (CGT) - an interdisciplinary field groups of the same finite size are isomorphic, we blending abstract group theory with the practical would achieve a complete classification of all finite power of computer science to study groups using groups - a fundamental goal in abstract algebra. algorithms and software. Its importance lies in its ability to handle groups of such immense size II. E ARLY D EVELOPMENTS IN C LASSIFYING and complexity that manual calculations become F INITE G ROUPS intractable or impossible. One prominent tool is Unsurprisingly, there is a huge difficulty in the GAP (Groups, Algorithms, Programming), a system classification of all finite groups. Although we only developed collaboratively over several decades and consider groups of a finite size, there are infinitely significantly supported by researchers at institutions many such groups in total as there is at least one like the University of St Andrews, UK, and Vrije for every positive integer size. As such, a full clas- Universiteit Brussel, Belgium. GAP enables resification will likely never be achieved. However, searchers to perform otherwise impossible calculatremendous progress has been made in classifying tions: generating group Cayley tables, checking for large, meaningful subsets of them. For example, isomorphisms, finding identity elements, and much for each prime number p, there is only one group more. For example, the SmallGroups library in GAP of size p up to isomorphism, so sizes 2, 3, 5, 7, contains complete data on all groups of size up to 11... are easily classified (Cayley, 1854). Hölder 2000 (except 1024), groups whose size factorises in gave complete classifications for sizes p3 ,p2 q,pqr at most three primes and groups without a factor p2 andp4 for any distinct primes p,q and r (Hölder, for any prime p (Eick, 2024). These are stored in ef1893), marking the first systematic treatment of ficient coded forms and users can call up any group mixed prime powers. At the turn of the twentieth from the library with a simple command. Extending century, Bagnera extended the catalogue to size our work on D3 and S3 from earlier, we can seek p5 (Bagnera, 1898), Western settled the p3 q case out these groups in the SmallGroups library, and (Western, 1899), and Le Vavasseur determined all even build a basic function called AreIsomorphic groups of size p2 q 2 (La Vavasseur, 1899) (Eick, (see Figure 4) to perform a simple isomorphism 2024). Enumerating all groups of size at most 215 check in GAP (Colazzo, 2025). As seen in Figure by hand was a remarkable feat by Lunn & Senior 4, it now takes very few lines of code to achieve (Lunn & Senior, 1934-1935), with the exception of the same result as all the work we achieved in our sizes 128 and 192. All of these were undertaken background section. This is a very simple example by hand and required pages and pages of hand- of what can now be achieved for groups of size computation – unsurprisingly becoming tedious and several thousand!

4

Open problems in achieving a full classification of finite groups often arise from issues in developing algorithms that can navigate these three challenges and produce accurate outputs in a sensible amount of time with our current computational power. We will list three open problems brought to attention

by Prof. Bettina Eick, which demonstrate some Figure 4: A screenshot of some GAP coding, where remaining gaps in classification (Eick, 2024). red is user input and black is output. We seek 1. All groups of size 1024: The number of the groups D3 and S3 stored in GAP (as defined groups of size 1024 (= 210 ) is enormous. We in Background) and use a simple function built have a complete enumeration of these groups using inbuilt GAP functions for isomorphism to test ∼ (Besche et al., 2001), however the list has whether D3 = S3 , which we have previously seen never been fully written out or stored in full to be true. due to its sheer size. Meaningful progress now hinges on new computational tools or IV. C HALLENGES AND O PEN Q UESTIONS applying existing algorithms using massive computing power.

Despite these powerful tools, the classification problem remains far from solved. GAP’s capabilities are expanding - the SmallGroups library is constantly being extended, and new GAP packages are frequently being added to deal with increasingly complex groups - but several deep challenges persist. We will briefly consider three such issues.

1. Algorithm Efficiency: In the background of the simple inbuilt GAP functions are many complex algorithms, many of which grow exponentially with group size, making them slow or impractical for large inputs.

Developing algorithms that are efficient both in theory and practice remains critical.

2. Representation Issues: Groups can be represented in many ways, such as permutations (S3 ), matrices, or more abstractly as the symmetries of a polygon (D3 ). Different representations vary greatly in computational efficiency and lead to vastly different algorithmic possibilities.

Even for small groups, choosing the ‘right’ representation is often a non-trivial task.

2. Groups containing five prime factors: These are groups with size of the form pqrst, where the primes may or may not be distinct. So far, we have group classification of size up to four primes.

3. Mixed prime powers of the form p5 q and p6 q, where p and q are distinct. Groups of size pn q for n ≤ 5 have been enumerated by Eick and Moede (Eick & Moede, 2018), but they have not been listed as in Problem 1. It is believed by Prof. Bettina Eick (Eick, 2024) that proofs in Eick & Moedes’ 2018 paper could be made ‘constructive’ as to translate into an explicit listing of groups with sizes of this form rather than a proof of how many exist. On full completion of p5 q, a next logical step would be considering p6 q and subsequent powers of p.

It is important to note that solving these problems is a practical CGT problem, as theoretically we can compare finite-sized Cayley tables and perform straightforward isomorphism checks - the novelty comes in developing practical algorithms to solve these problems. Classifying the groups of these 3. Isomorphism Problem: For large groups, it’s forms would create richer databases of finite groups impractical to compare full Cayley tables that mathematicians can use in their research. While (which have size n × n for group size n), so the full classification of finite groups may never more sophisticated tests are required to find be reached, the pursuit of the goal has already isomorphisms between two groups - or, even transformed our understanding of algebra and symmore difficult, to prove there is none! metry. From the hand-drawn Cayley tables of the

5

19th century to today’s cloud-based repositories hosting billions of groups, the field has evolved dramatically. This leap was driven by computational group theory, powered by systems like GAP and sustained by an enthusiastic international community of mathematicians and computer scientists.

V. R EFERENCES

9. Senior, J.K. and Lunn, A.C. (1934) ‘Determination of the groups of orders 101–161, omitting order 128’, American Journal of Mathematics, 56, pp. 328–338.

10. Senior, J.K. and Lunn, A.C. (1935) ‘Determination of the groups of orders 162–215, omitting order 192’, American Journal of Mathematics, 57, pp. 254–260.

1. Mersenne.org. Mersenne Prime Discovery - 2136279841 − 1 is Prime! Available at: h t t p s : 11.

The collected mathematical papers //www.mersenne.org/primes/?press=M136279841 of Arthur Cayley: Cayley, Arthur, 1821[Accessed 9 June 2025].

1895: Internet Archive.

Available at: https://archive.org/details/collectedmathema 2.

GAP Groups, Algorithms and 02cayluoft/page/122/mode/1up?view=theater Programming, Version 4.14.0. Available at: [Accessed: 9 June 2025]. https://www.gap-system.org/ [Accessed: 9 June 2025].

12. Besche, H.U., Eick, B. and O’Brien, E.A. (2001) ‘The groups of order at most 2000’, 3. Cayley, A. (1854) ‘On the theory of groups, Electronic Research Announcements of the as depending on the symbolic equation n = 1’, American Mathematical Society, 7(1), pp. 1–4.

Philosophical Magazine 7(42), pp. 40–47. Reprinted doi:10.1090/S1079-6762-01-00087-7. in: Cayley, A. (1889) The Collected Mathematical Papers of Arthur Cayley. Vol. 2. Cambridge: 13. Ilaria Colazzo (2025). [online] Available Cambridge University Press, pp. 123–130. at: https://www.ilariacolazzo.info/gap/slides/lectur e2.html#34 [Accessed 9 Jun. 2025].

4. Hölder, O. (1893) ‘Die Gruppen der Ordnungen p3 , p q 2 , p q r, p4 ’, Mathematische Annalen, 43, 14. Eick, B. and Moede, T. (2018) ‘The enumeration pp. 301–412. of groups of order pn q for n ≤ 5’, Journal of Algebra, 507, pp. 571–591.

5. Bagnera, G. (1898) ‘La composizione dei gruppi finiti il cui grado è la quinta potenza di un numero primo’, Annali di Matematica Pura ed Applicata, Serie III, 1, pp. 137–228.

6. Western, A. (1899) ‘Groups of order p3qp3 qp3q’, Proceedings of the London Mathematical Society, 30, pp. 209–263.

7. Le Vavasseur, R. (1899) ‘Les groupes d’ordre p2 q 2 , p étant un nombre premier plus grand que le nombre premier q’, Comptes Rendus de l’Académie des Sciences de Paris, 128, pp. 1152–1153.

8. Eick, B. (2024) ‘Classification of finite groups: recent developments and open problems’, Foundations of Computational Mathematics. Available at: https://doi.org/10.1007/s10208-024-09688-1 (Accessed: 9 June 2025).

1

Developments Towards Practical Quantum Computing Malachi Johns Edited by Andrew Linz and Tom Burton

Abstract - Efforts towards quantum computing have gained increasing attention and attracted increasing amounts of investment in recent years, with excitement over new developments comparable to the hype around new artificial intelligence products. However, it is still unclear whether quantum computers will become a commercially viable option, and if so, when we can expect their widespread use. This review explores the strengths and weaknesses of current research developments to examine the future viability of quantum computers.

I. I NTRODUCTION As demand increases for higher computational power and solutions to increasingly complex problems, even the most advanced supercomputers can be insufficient. For many applications, such as quantum chemical simulations, logistical problems, cryptography, and more, classical computing is surprisingly inefficient – which is why more and more governments are investing in the development of quantum computers (QCs) (Seatter, 2025, Duranton, 2024, Griffith, 2024).

One of the most important applications would be in cryptography: the Rivest-Shamir-Adleman (RSA) encryption scheme, which protects sensitive information on the internet, is based on the difficulty that classical computers have in factoring large numbers into primes (Shafique, 2024). While a classical computer can factorise large numbers, it tends to take an extremely long time – it took two years and a great deal of computational power for one computer to factorise a 768-bit RSA key, while most RSA keys are 1024 or 2048 bits long (Bhatt, 2024).

A quantum computer would, in theory, be able to factorise these numbers in much shorter timeframes, which has raised concerns about internet security –

though the largest number factorised by a quantum computer so far was 56,153, which is only 16 bits long (Dattani, 2014).

The initial idea of a QC is often attributed to Richard Feynman’s 1981 paper “Simulating Physics with Computers” (Feynman, 1982), in which he noted the advantages that a QC would have over a classical computer for simulating quantum systems: classical computers require incredible amounts of processing power to model quantum systems, but if a computer already has a fundamentally quantum nature, it won’t be so difficult for it to mimic the similar quantum nature of the molecules that make up the universe. A QC will also have a dramatically increased processing power compared to a classical computer with the same number of bits: while the power of a classical computer increases linearly with the number of transistors (hence the exponential growth in transistors in a computer in the past few decades (Auth, 2023)), a QC’s ability to access quantum mechanical superpositions means that its power increases as 2N, where N is the number of qubits in the processor (Gill, 2025).

While the potential benefits are significant, functional QCs have been difficult to implement. Current state-of-the-art QCs are still unreliable and have little processing power (Dargam, 2023), and it is still unclear how long it will take to create an efficient QC that definitively out-performs classical computers (McKenzie, 2023). Nonetheless, there have been some impressive recent developments in the most challenging areas of development, such as noise suppression (Singh, 2023, Williams, 2023), error correction (Google Quantum AI and Collaborators, 2025), and scalability (Main, 2025, Oxford, 2025). This article aims to analyse a key recent developments in each of these areas to consider the probable future of QCs.

2

II. BACKGROUND A quantum computer has many of the same functioning parts as a classical computer: bits that carry information, gates which perform operations on the bits to carry out calculations, and memory (Wong, 2022). The difference lies in the nature of the bits themselves. Whilst a classical computer uses binary bits that can be in one of two distinct states 0 or 1, the quantum bits or “qubits” in a QC can exist in superposition between these states (Zygelman, 2018, LaPierre, 2021, Wong, 2022). This means they can occupy both states simultaneously and to different extents. A qubit can be half in state 0 and half in state 1, or two-thirds in state 0 and onethird in state 1; but this superposition will collapse when the qubit is measured so that the measured value will be either 0 or 1. After the measurement, the qubit will be entirely in the measured state (LaPierre, 2021). Superposition turns out to be an incredibly useful attribute for QCs because it allows a single processor effectively to trace out multiple computational pathways simultaneously by being in multiple states simultaneously (LaPierre, 2021).

The other difference between qubits and classical bits is that qubits can be connected through entanglement (Zygelman, 2018, Wong, 2022). Classical bits can only interact with each other via electrical signals, which can travel only as fast as the speed of light. An entangled pair of qubits, on the other hand, can transmit information instantly between arbitrary distances. Entanglement means that the state of each individual particle is unknown, but the state of both particles together is known (LaPierre, 2021): for instance, you could have a pair of particles such that one is in state 0 and the other in state 1.

This says nothing about the state of each individual particle: there is a fifty percent chance of the first particle being in state 0, and an equal chance of it being in state 1, but we still know the state of the entire system. This allows us to infer the state of the second particle only after we measure the state of the first: if the first particle’s state is 0, we know the second’s is 1. Practically, this can’t be used to transport information faster than light, as the quantum information stored in the entangled particles must be accompanied by classical information transported in classical communication channels (LaPierre, 2021), but allowing qubits to “measure” each other through entanglement allows

for simpler and thus more fault-tolerant processors.

The continuing development of QCs is mostly focused around maintaining superposition and entanglement of the qubits and protecting qubits from decoherence, the collapse of these quantum states.

The state of a qubit is extremely sensitive, and even the smallest disruptions or “noise” from the environment or even from other qubits can cause decoherence. The problems this presents lie in the persistence of environmental noise that disrupts the extremely sensitive qubits, the difficulty of correcting errors in the quantum circuits, the issue of scaling these systems to meet requirements for use in industry, and making some way of unifying developments in each of these areas so that the quantum computing world has some level of standardisation.

This review will discuss recent developments in each of these areas to assess how viable quantum computers are for commercial use.

III. D ISCUSSION A. Noise Noise is often considered the most significant challenge to the development of QCs. It introduces uncertainty and errors in quantum systems by disrupting the constituent qubits. Vibrations in the environment, impurities in any functional components, or even cosmic waves can cause decoherence; by interacting with the qubits, they effectively measure the qubits’ state – because the qubits’ reaction to the environmental noise will depend on their states, so a qubit’s state can be inferred from the effect it has on the noise – which collapses any superpositions.

Most QCs operate at extremely low temperatures of a few millikelvin to decrease disorder in the surrounding system and are thoroughly cushioned from external vibrations, but these systems remain vulnerable to other influences from qubits close to each other or controlled by the same hardware.

Recent developments have implemented the idea of directly cancelling the effects of noise on the qubits in a quantum chip rather than trying to shield the chip (Song, 2023) (Gupta, 2020) (Majumder, 2020) (Williams, 2023) (Singh, 2023). This method allows for the suppression of correlated errors by measuring and cancelling the noise from the system itself as well as the environment. One design (Singh, 2023) uses an array of spectator qubits interspersed with data qubits; the data qubits are the

3

ones that perform calculations, while the spectator qubits measure the noise in the system. A classical control system takes those measurements of the environment around the data qubits and sends a corrective feedforward signal to negate the effect of the noise on the system.

The array contains data qubits made of trapped rubidium atoms and spectator qubits of trapped caesium atoms (Singh, 2022, Singh, 2023). Neutral atom qubits are atoms that have been cooled to near-absolute zero and arranged into a processing chip (NIST, 2025); the data is represented in the atomic energy levels (Ebadi, 2021, Scholl, 2021).

Because the spectator qubits are made of a different element from the data qubits, they have a different electronic excitation spectrum and can absorb different wavelengths of light. When the spectator qubits scatter light from a measuring laser, the emitted wavelengths then cannot disrupt the data qubits, but the dispersed system of spectator qubits can still measure a spatially varying noise environment. Dur-

be observed. As shown in Fig. 2, with the feedforward from the spectator qubits, the coherence of the data qubits was recovered in each repetition of the experiment, and for field strengths less than 11 mG the coherence of the data qubits was stable. At higher field strengths, the spectator qubits could no longer accurately detect phase errors and coherence was once again lost.

The spectator qubits also influenced the way the

Figure 2: Data qubits remain coherent longer with the feed-forward from the spectator qubits (green) than without the feed-forward signal (blue). Taken from (Singh, 2023).

Figure 1: A schematic of the method of using midcircuit readouts for noise correction, taken from (Singh, 2023). The spectator qubits provide midcircuit readout measurements, shown in yellow, which are sent to a classical processor (grey) that then sends a feedforward signal to the data qubits for real-time correction. ing a calculation, the spectator qubit measurements are used to infer the noise around the data qubits via a classical algorithm in real time, as shown schematically in Fig. 1. To demonstrate the efficacy of this method, the researchers added artificial noise around the processor with comparable attributes to those found in laboratory environments. The qubits in the processor showed correlated errors that were then suppressed using a feedforward signal that updated the phase of the data qubits.

In the first test, the noise was adjusted so that without the spectator qubits it would cause complete decoherence of the data qubits and no pattern would

data qubits reacted to different frequencies in the surrounding noise: while the data qubits would experience high decoherence rates near resonant frequencies, the spectator qubits were able to achieve a net gain in the measured coherence in these regions. This effect is shown graphically in Fig. 3. There were additional effects due to the finite amount of time it takes to take and analyse the measurements of the spectator qubits, so further experimentation will be necessary to reduce this time. This method is applicable to many quantum computing platforms, which will assist in efforts towards scalability as greater numbers of interacting qubits generally become more error-prone The procedure is also convenient because it does not require multiple-qubit gates that would necessitate more complex hardware. It may also be used to measure spatially varied noise environments, though this was not measured in the study. The results of this study support further developments in error correction and long-range qubit entanglement, which is important for scalability as well.

4

does not disrupt the entire calculation, but this only works if the physical error rate is below a certain threshold. If the physical error rate is below this threshold, the error rate of logical qubits decreases approximately exponentially with increasing code distance, a measure of how many physical qubits are used in each logical qubit (Google Quantum AI and Collaborators, 2025)

(d+1)/2 p ϵd ∝ pt hr Figure 3: Qubits are protected from decoherence at resonant frequencies by the noise cancelling effect of the spectator qubits. Taken from (Singh, 2023).

With the feed-forward signal off (blue), the data qubits show high decoherence rates near the first and third harmonic, whereas with the feed-forward signal on (green), the data qubits are much more stable with resonant frequencies of noise.

B. Error Correction For QCs to be useful in most applications, it is necessary to achieve error rates under 10−10 , much lower than the current lowest error rates of 10-3 (Google Quantum AI and Collaborators, 2025). In classical computers, errors occur in the form of bit flips, where a bit erroneously switches between the 0 and 1 states (Microsoft, 2025), resulting in data corruption and device malfunctions (Baraniuk, 2022).

If every one in a thousand bits in a computation flipped unpredictably, the result would be unusable.

A popular method to reduce errors in quantum devices is to use quantum error correction, constructing logical qubits from the combination of multiple physical qubits so that the state of the logical qubit is more stable (Google Quantum AI and Collaborators, 2025, Microsoft, 2024, Roffe, 2019). A logical qubit is the bit that carries information to carry out calculations, while the physical qubits in the logical qubit are the individual components such as trapped ions or neutral atoms. Calculations are performed on the logical qubits in much the same way as classical calculations are performed on classical bits, but the logical qubit still has the desirable quantum properties from its constituent physical qubits. The individual physical qubits can effectively check each other so that one mismatch in one physical qubit

where ϵd is the logical error rate, p is the physical error rate, pthr is the threshold error rate, and d is the code distance. There are 2d2 − 1 physical qubits in each logical qubit, for reasons that will be clear later. We can also define an error suppression factor (Google Quantum AI and Collaborators, 2025) which represents the decrease in logical error rate when the code distance is increased by two. Λ = ϵd /ϵd+2 ≈ pthr /p The physical error rates for most quantum processors haven’t been definitively below this threshold, which makes quantum error correction difficult to implement usefully. However, as of December 2024, Google’s Quantum AI team have shown definitive below-threshold error rates in two of their Willow processors as well as making significant progress in other error mitigation techniques such as system stability, and decoding in real time (Google Quantum AI and Collaborators, 2025). A schematic of the 105-qubit processor is shown in Fig. 4, with superconducting data qubits in yellow and measure qubits interspersed around them in blue, just like the spectator qubits discussed previously. The array has d data qubits on each side – thus there are d2 data qubits and d2 − 1 measure qubits, totalling 2d2 − 1 physical qubits in the processor, with the side length of the array becoming the code distance.

The generated measurements are then decoded with one of two offline decoders, one in real time and one after the fact.

To establish that the code operates below the threshold error rate, it is obviously necessary to measure the error rates at different distances. The state of the logical qubit is measured, decoded, and compared with the initial logical state to determine whether an error has occurred. Physical error rates are measured using the measure qubits: an error is detected if the state of the measure qubit is different from expected

5

Figure 4: A schematic of the layout of the data and measure qubits, taken from (Google Quantum AI and Collaborators, 2025). The data qubits are shown in yellow, forming a grid interspersed with measure qubits in blue. Leakage removal qubits are shown in green. from a noiseless environment. The physical error rate tends to increase as the code distance increases because of interference between qubits, but this effect will likely reach a limit at larger processor sizes. Using these measurements, it is established that the code operates below the error threshold in distance-3, 5 and 7 subgrids of the 105-qubit processor: the increasing distance results in significantly lower error rates per computational cycle. Higherdistance logical qubits are simulated using a noise model based on component error rates measured experimentally, accounting as well for leakage and qubit interference, and this data is shown alongside the empirical results. Importantly, the distance-7 logical qubit had a lifetime of over twice that of its longest-lived constituent physical qubit and three times the median lifetime of the physical qubits.

This suggests that using more stable logical qubits made of many less stable physical qubits is a viable pathway to more fault-tolerant QCs. C. Scalability The most obvious remaining problem is that these quantum processors have troublingly few logical qubits. A functional QC would need millions of qubits to be definitively more useful than classical

Figure 5: Error probabilities over time of codes of various distances, taken from (Google Quantum AI and Collaborators, 2025). Higher distance codes (teal and blue) tend to have lower logical error probability than even the best physical qubit, represented by the dotted green line.

computers in many contexts – but as established previously, processors with higher qubit counts tend to have greater physical error rates due to interference between the constituent qubits. Even if those errors saturate at higher processor sizes or could be suppressed, the processor itself would be prohibitively big; so the problem of scalability becomes one of connecting multiple processors so that logical operations can be performed across multiple modules.

This approach is called distributed quantum computing (DQC), where individual processing modules are linked together, allowing for greater total power without sacrificing the overall performance of the device. While DQC seems like it should be feasible enough, a scalable DQC architecture was only found in February of 2025 (Main, 2025, Oxford, 2025).

The study, published in Nature (Main, 2025), uses two distinct processing modules, each with their own network and circuit qubits, connected with classical and quantum information channels. Qubits from each module are entangled with those of the other module so that the data qubits on each module are effectively connected and can run calculations in concert with each other. Then information can

6

be transferred between the modules using quantum teleportation, so the processing is deterministic and repeatable even though the entanglement process is probabilistic.

The qubits from each module are entangled using interference of photons between the modules, which is especially beneficial for scaling QCs because photons can carry quantum information over long distances without the need for cryogenics or vacuums, so there is no need to cool the optical cables to a few millikelvin like the processor itself. The use of quantum teleportation also avoids the possibility of information loss that would occur if quantum information were transferred directly between modules; quantum gate teleportation (QGT) relies only on a simple pair of entangled qubits and the exchange of two classical bits between them to transfer complete information between the modules.

Because the quantum information channel is used exclusively for generating entangled pairs, channel losses can be overcome by repeating the process, and the distance between modules can be increased arbitrarily by adding quantum repeaters between the modules. The resulting teleportation protocols after entanglement is established allow for the continuous and deterministic operation of the QC, which is crucial for scalability: if the information transfer between modules is probabilistic, then a large-scale computer will produce an exponentially large set of undesirable outcomes along with one singular outcome, because every instance of information transfer has a finite probability of the information being changed. This would necessitate a protocol for finding the one desirable outcome out of the larger set of undesirable outcomes, which would be a problem in itself.

D. Standardisation Perhaps the most obvious challenge that will come with implementing the ideas described above is that each proof of concept is run on a unique quantum device by researchers who know how to handle that device specifically (Donne, 2025) – unlike today’s computers, which almost anyone can run with ease, even without knowing anything about the individual computer they’re using, quantum devices are diverse and unique. If quantum computers are to become an integral part of industry, there will need to be some level of standardisation

between processor designs, a way of running any programme on any quantum computer regardless of its design parameters and of having quantum computers communicating with each other to form a “quantum internet”.

Designing an operating system (OS) for quantum computers is not a simple task – the first design of an OS for quantum networks (QIA, 2025) was presented in Nature in March (Donne, 2025). The operating system is called QNodeOS, an abbreviation of quantum node operating system, and it can do many of the fundamental tasks that a classical computer’s OS can do: converting programmes into tasks for the quantum processors, managing multiple processors of different design, and multitasking (Donne, 2025).

First, it is assumed that the programmes comprise classical and quantum blocks of code, organised by a compiler or by a programmer (Donne, 2025). The classical code blocks are largely what you would expect from a classical computer, local or networked operations carried out on a classical processor or using network devices. The quantum code blocks contain the operations that a quantum processor might run, as well as networked quantum operations that would be run through multiple processors in concert, e.g. entanglement. The quantum blocks can also have classical control logic, for example in the mid-circuit readout procedure in a noisecancelling programme. The OS architecture is correspondingly divided into three parts: the classical network processing unit (CNPU) that executes classical code blocks, the quantum network processing unit (QNPU) that manages the execution of quantum code blocks, and the QDevice, the quantum processor that runs operations on qubits.

One of the design challenges that the team faced was that each part of their proposed system would operate on wildly different timescales (Donne, 2025): classical operations generally take milliseconds to run, while the execution time of a quantum operation can be on the order of nanoseconds, a million times shorter – and the classical control algorithms paired to a quantum programme will correspondingly need precision on the nanosecond scale. In addition, the OS would need to be able to facilitate the generation of entangled pairs of qubits on different processors (Donne, 2025), which also necessitates precise management of when each processor generates entanglement. The entangle-

7

ment schedule is further complicated by its interdependence with the classical network schedule, as entanglement generation cannot be carried out simultaneously with the local operations (Pompili, 2022, Krutyanskiy, 2019) in any currently functioning processors. The solution here comes from the separation of the architecture into three logically separate parts which can be implemented at different timescales as necessary (Donne, 2025).

A broad-strokes description of how QNodeOS works sounds incredibly intuitive (Fig. 6): a process is created on the CNPU and registered on the QNPU, which makes its own QNPU process.

Physical instructions are sent to the QDevice in response to QNPU and classical network processes, and the device returns the result of the operations back to the QNPU. The only part of the architecture that depends on the design of the quantum chips is the QDriver, which translates quantum operations written in the language of the OS into physical instructions that the individual processor (Donne, 2025). QDrivers for a few specific systems (Teller, 2021, Pompili, 2022, Hermans, 2022, Teller, 2023) have already been presented. The team demonstrated the validity of their system by executing an arbitrary quantum network process on a twonode system of one qubit per node. Their results presented high reliability, showing that QNodeOS could run programmes using entanglement, classical communication, and millisecon-level memory lifetimes (Donne, 2025). They also demonstrated the capability of QNodeOS to multitask by running multiple quantum applications simultaneously. All applications were started in the CNPU concurrently, so the QNPU was sent instructions for all of the programmes simultaneously and continuously that it then scheduled separately for the client node to run (Donne, 2025). The results of each application were equal to what would have been expected if it were run without multitasking, but the fraction of runtime during which the quantum chip was actively executing physical instructions increased with increasing number of simultaneous processes to run – eliminating the inefficiency caused by a quantum processor waiting for classical computations to run before executing its next step by instead running part of another application.

This progress suggests the possibility of many new areas of research both in quantum information science and in computer science research, as well as

Figure 6: The logical design of QNodeOS. The system is split into three modules: the CNPU shown in grey, the QNPU in green, and the QDevice in pink. The CNPU and most elements of the QNPU are hardware-independent, while only the QDriver and the QDevice are hardware-dependent. Figure taken from (Donne, 2025). inspiring a very positive outlook on the development of classical-quantum hybrid devices or a quantum internet.

IV. C ONCLUSION As quantum technologies progress and become more important for many industrial applications, it is still unclear whether QCs will soon be as useful as theoretical predictions suggested. There are already functional QCs, but the difficulty of producing and maintaining one far outweighs the benefits so far. However, these recent developments in noise cancellation, distributed quantum computing, error correction, and standardisation provide an inspiring

8

outlook.

Given the developments described here, it seems likely that quantum computers will become commercially viable within the forseeable future – it’s only a question of when and who does it first.

Continued investment will be imperative for future technologies, but the benefits of QCs to the search for better batteries pharmaceuticals, artificial intelligence, cybersecurity, and modelling make the difficulty worthwhile.

V. R EFERENCES AUTH, C. 2023. Evolution of Transistors: Humble Beginnings to the Ubiquitous Present.

IEEE Solid-State Circuits, 15. BARANIUK, C. 2022. The Computer Errors from Outer Space.

BBC Future [Online].

Available: https://www.bbc.co.uk/future/article/2022101 1- how- space- weather- causes- computer- errors [Accessed 20 May 2025].

FEYNMAN, R. P. 1982. Simulating Physics with Computers. International Journal of Theoretical Physics, 21.

GILL, S. S. 2025. Quantum Computing: Vision and Challenges. Quantum Computing: Principles Paradigms. Elsevier.

GRIFFITH, A. 2024. Unlocking the potential of quantum: £45 million investment to drive breakthroughs in brain scanners, navigation systems, and quantum computing.

GUPTA, R. S. 2020. Integration of spectator qubits into quantum computer architectures for hardware tune-up and calibration. Phys. Rev. A, 102.

HERMANS, S. L. N. 2022. Qubit teleportation between non-neighbouring nodes in a quantum network. Nature, 605.

BHATT, H. 2024. What is RSA? How does an RSA work? Available: https://www.encrypti KRUTYANSKIY, V. 2019.

Light-matter onconsulting.com/education-center/what-is-rsa/ entanglement over 50km of optical fibre. npj [Accessed 20 May 2025].

Quantum Information, 5.

COLLABORATORS, G. Q. A. A. 2025. Quantum LAPIERRE, R. 2021. Introduction to Quantum error correction below the surface code threshold. Computing, Springer Cham.

Nature, 638.

MAIN, D. 2025. Distributed quantum computing DARGAM, J. 2023. What Is NISQ Quantum across an optical network link. Nature, 638.

Computing? Available: https://thequantuminsider. com/2023/03/13/what-is-nisq-quantum-computing/. MAJUMDER, S. 2020. Real-time calibration with spectator qubits. npj Quantum Information, 6.

DATTANI, N. S. 2014. Quantum Factorization of 56153 with only 4 qubits.

MCKENZIE, J. 2023. When will quantum computers finally break into the market? Available: DONNE, C. D. 2025. An operating system https://physicsworld.com/a/when-will-quantum-c for executing applications on quantum network omputers-finally-break-into-the-market/ [Accessed nodes. Nature, 639.

20 May 2025] DURANTON, S. 2024. Quantum Computing MICROSOFT. 2024. Introduction to Quantum Error Takes Off With $55 Billion In Global Investments. Correction. Available: https://learn.microsoft.com/ Forbes [Online]. Available: https://www.forbes.com en-us/azure/quantum/concepts-error-correction. /sites/sylvainduranton/2024/06/26/quantum-now/.

MICROSOFT. 2025. Quantum Error Correction.

EBADI, S. 2021. Quantum phases of matter Available: https://quantum.microsoft.com/en-us/insi on a 256-atom programmable quantum simulator. ghts/education/concepts/quantum-error-correction Nature, 595.

[Accessed 20 May 2025].

9

TELLER, M. 2021. Heating of a Trapper Ion NIST. 2025. Quantum Computing Explained. Induced by Dielectric Materials. Phys. Rev. Lett., Available: https://www.nist.gov/quantum- infor 126. mation-science/quantum-computing-explained [Accessed 20 May 2025].

TELLER, M. 2023. Integrating a fiber cavity into a wheel trap for strong ion–cavity coupling.

OXFORD. 2025. First distributed quantum AVS Quantum Sci., 5. algorithm brings quantum supercomputers closer.

Available: https://www.ox.ac.uk/news/2025-02-06-f WILLIAMS, S. C. P. 2023. ‘Noise-cancelling’ irst-distributed-quantum-algorithm-brings-quantum qubits developed at UChicago to minimize errors -supercomputers-closer [Accessed 20 May 2025]. in quantum computers. Available: https://pme.uchi cago.edu/news/noise-cancelling-qubits-developed-u POMPILI, M. 2022. Experimental demonstration chicago-minimize-errors-quantum-computers. of entanglement delivery using a quantum network stack. npj Quantum Information, 8.

WONG, H. Y. 2022. Introduction to Quantum Computing: From a Layperson to a Programmer QIA. 2025. QIA researchers create first Operating in 30 Steps, San José, CA, USA, San José State System for Quantum Networks. Available: University. https://quantuminternetalliance.org/2025/03/12/qia -researchers-create-first-operating-system-for-qua ZYGELMAN, B. 2018. A First Introduction ntum-networks/[Accessed 20 May 2025]. to Quantum Computing and Information, Springer Charm.

ROFFE, J. 2019. Quantum Error Correction: An Introductory Guide.

SCHOLL, P. 2021. Quantum simulation of 2D antiferromagnets with hundreds of Rydberg atoms. Nature, 595.

SEATTER, M. 2025. How to Invest in Quantum Computing Companies. Investing News Network [Online]. Available: https://www.nasdaq.com/artic les/how-invest-quantum-computing-companies.

SHAFIQUE, M. A. 2024. Quantum Computing: Circuits, Algorithms, and Applications. IEEE Access, 12.

SINGH, K. 2022. Dual-Element, Two-Dimensional Atom Array with Continuous-Mode Operation.

Phys. Rev. X, 12.

SINGH, K. 2023. Mid-circuit correction of correlated phase errors using an array of spectator qubits. Science, 380.

SONG, H. 2023. Optimized mitigation of randomtelegraph-noise dephasing by spectator-qubit sensing and control. Phys. Rev. A, 107.

1

Could we use Artificial Intelligence to help prove whether P = NP? Thomas Burton BSc. (Hons) Edited by Pedro Lourenco and Tom Dunlop

Abstract - This article explores one of the most important open questions in mathematics and computer science: the P vs NP problem.

It begins by explaining the meaning of the two complexity classes in accessible terms, then reviews the strategies researchers have used to try and resolve the question, including direct proofs, relativisation, natural proofs, and circuit complexity. The article then shifts focus to the emerging role of artificial intelligence (AI), particularly large language models (LLMs), as tools to aid in hypothesis generation, formal reasoning, and proof construction. Finally, it considers the transformative implications that solving P vs NP would have across science, technology, security and society. The review argues that while AI may not yet be the key to a solution, it is reshaping the way we approach one of the most enduring problems of our time.

I. I NTRODUCTION Every day, whether we realise it or not, we rely on algorithms to help us solve problems—finding the fastest route to work, sorting our emails, recommending songs we might like. Many of these tasks are straightforward for computers to handle.

But lurking beneath the surface of these seemingly simple solutions is one of the biggest unanswered questions in mathematics and theoretical computer science: Is every problem whose solution can be quickly checked also one that can be quickly solved? This is the essence of the P vs NP problem.

P, which stands for “polynomial time,” refers to the class of problems that computers can solve efficiently. That is, given an input, there’s an algorithm that can find a solution relatively quickly, even as the input gets larger. A classic example is sorting a list of names alphabetically. Whether the list has ten names or ten thousand, modern computers can do

it fast, due to well-understood sorting algorithms.

Another example might be finding the shortest path between two points on a map; your phone does this every day using algorithms designed to keep calculations quick.

NP, on the other hand, stands for “nondeterministic polynomial time.” These are problems where, even if we don’t know how to solve them quickly, we can at least check a solution quickly if someone hands it to us. In mathematics, we call this a polynomial time verifier. Think of a Sudoku puzzle. Solving one from scratch can take a long time. But if someone gives you a completed grid and claims it’s a valid solution, it’s relatively easy to check that all the rules are followed; each number appears once per row, column, and box. This is an NP problem: difficult to solve, easy to verify.

So, the big question is: Are these two types of problems the same? Is every problem that can be verified quickly also solvable quickly? That’s what mathematicians mean when we ask whether P = NP. This is more than just an intellectual puzzle. The answer has enormous implications. If P = NP, we could potentially revolutionise fields like medicine, logistics and beyond. If not, it would confirm that some problems will always be fundamentally hard, or impossible to solve in polynomial time, no matter how clever our algorithms become.

Even if P = NP, it doesn’t automatically imply practical solvability. Many polynomial-time algorithms may feature extremely large constant factors that render them inefficient in practice. This incidental inefficiency is precisely why tasks like cryptographic encryption remain secure: although they may be solvable in theory, their real world runtimes are still prohibitive. The following rapid review article will look at recent developments in the field, particularly the intriguing possibility that AI, especially LLM’s could help us explore or even solve this problem. Finally, I will explore what it

2

would mean for the world if we did, one day, finally Relativisation settle the question of whether P equals NP. One method, popular in the 1970s, was to see what happens to P vs NP when computers II. C AN I T B E P ROVEN ? A L OOK AT THE are given extra ”powers”—like a magic box that S TRATEGIES B EHIND P VS NP can instantly answer specific types of questions.

For decades, some of the sharpest minds in Surprisingly, when researchers tried this, they mathematics and computer science have been found examples where P = NP under one setup, trying to prove whether P equals NP or not. But and P ̸= NP under another. This told us something despite all the brainpower, research and even a important: this approach alone couldn’t settle the million-dollar prize for whoever solves it, the question. It’s like trying to solve a riddle, but question remains stubbornly unanswered. Why? depending on what tools you’re allowed to use, you Because proving something like this is incredibly get completely different answers. As one expert hard, especially when we don’t even know which in complexity theory explained, “the P vs NP side of the equation is true. Let’s start with what a question has contradictory relativizations,” which proof means in this context. effectively shows that “the P vs NP question It’s not about running a million simulations or cannot be solved by any proof techniques [that testing every puzzle and seeing how fast we can rely on relativization]” [1]. In other words, if solve it. That would take an infinite amount of two different versions of the same problem yield time. What mathematicians are looking for is a opposite outcomes depending on the ‘rules of the logical argument; a step-by-step explanation that game’, then those rules are not enough to give us shows why P must be equal to NP, or why it isn’t. the real answer.

It must work for every single problem in the class, not just some. Over the years, researchers have Natural Proofs tried many different angles to get there. Most fall into a few big categories: Another major idea was to try to prove things using methods that seem “natural” or intuitive Direct Proofs and Contradictions methods that capture common patterns across many problems. But researchers discovered that if Some have tried to directly prove that P equals you rely too much on these types of proofs, you NP or that it doesn’t, by using traditional end up running into walls. In fact, in the 1990s, mathematical techniques. Often, this involves proof two computer scientists showed that if P ̸= NP by contradiction, trying to assume the opposite is true, then many of our natural ways of trying of what you want to show (for example, assume to prove it might be doomed to fail. That was a P = NP), and then finding a logical inconsistency sobering moment for the field; it meant that even that proves it can’t be true. The challenge? You are our best ideas might be blind to the answer. This dealing with abstract concepts that stretch across insight was summarised in a foundational paper hundreds of different types of problems. It’s like which argued that “if you define a natural proof trying to prove that every key in the world fits as one that relies on a natural property. . . then it into a particular type of lock; it’s not enough to follows that no natural proof can separate P from check a few, you must account for all possible NP.” [2] The implication was clear: conventional combinations. As supported through an article mathematical intuition might simply be too limited published by Princeton University, “the simplest to break open this puzzle. version of a proof that P = NP would be a proof that an NP−complete problem can be solved in Circuit Complexity polynomial time” [1] which captures just how broadly such a proof would need to apply; it would One promising direction is to think of problems in have to work for every problem in the class, not terms of circuits—sort of like simplified blueprints just one or two. for a computer. If you can show that certain problems require massive circuits that no small

3

or efficient design could ever handle, that would prove they can’t be solved quickly. This would be a win for the “P ̸= NP” side. But building these arguments is extremely tricky, and progress has been slow. It’s like trying to prove that no matter how you design a machine, it will always need 10, 000 parts—you must eliminate every possible clever shortcut. In fact, researchers studying this approach found that “even a very rough approximation of the maximum clique of a graph requires super polynomial size monotone circuits,” [3] highlighting just how demanding some problems are when translated into circuit form.

These results offer some of the strongest evidence we have, though they still fall short of settling the general case.

Despite these challenges, the effort has not been wasted. Each attempt has taught us more about the limits of computation, and where we might find the cracks in the wall. Still, most experts today believe that P is not equal to NP, but proving it formally is still out of reach. So, the question now becomes: could a new kind of thinker bring something new to the table? The following section will explore what artificial intelligence, especially today’s most powerful models, might bring to the search for a solution.

III. H OW AI C OULD H ELP P ROVE P VS NP AI is already transforming how we communicate, code and create. In recent years, researchers have begun exploring whether LLM’s could be used to approach, assist with, or even generate steps toward a proof of whether P equals NP. While we are far from seeing a computer independently produce a rigorous proof, there are growing signs that AI could become a powerful ally in the journey.

One of the earliest and most promising directions is using AI as a hypothesis generator, not to find final answers, but to help map out potential arguments.

In a 2023 experiment, researchers prompted GPT 4 with a series of structured questions about P vs NP, leading the model through nearly 100 steps of what they called Socratic reasoning. Remarkably, the model generated a consistent framework and even proposed a structured outline of a proof sketch. The researchers noted that “GPT 4 successfully produces a proof schema and engages in rigorous reasoning

. . . concluding ‘P ̸= NP’” [4]. Although this outline does not meet the standards of a formal proof, it demonstrates AI’s growing ability to explore complex logical terrain, sometimes highlighting paths that human researchers may overlook.

Beyond brainstorming, AI is also making strides in formal mathematics; the step-by-step language that professional mathematicians use to validate theorems. New tools like Goedel Prover, an opensource language model fine-tuned for mathematical proof writing, are outperforming earlier benchmarks in formal environments such as Lean. These systems can generate precise mathematical arguments using logical syntax. Similarly, a tool called Logic LM blends language models with symbolic reasoning: it translates natural-language math statements into formal logic, checks them with symbolic solvers, and refines the output. The result, as its creators reported, was a “39% improvement in accuracy over LLMs alone,” [4] demonstrating how combining AI’s language fluency with exact mathematical checking produces more reliable results.

Perhaps even more exciting is AI’s ability to critique and improve its own reasoning with structured feedback. In a recent study, researchers demonstrated that LLMs, when paired with external verifiers, could iteratively refine complex logic chains. One paper explained that integrating large models with soundproof assistants made it possible to “repair flawed proof attempts more reliably,” [5] opening the door to a process of continuous machine-guided revision. This model mirrors how mathematicians often work, drafting an argument, testing it, finding holes and revising until it holds together. These developments are being pulled together into what some call collaborative proof pipelines. A recent system called APOLLO takes a generated proof from an LLM, sends it to Lean (a formal verifier) and then prompts the LLM to revise it based on Lean’s feedback. According to researchers, this system reduced the number of invalid attempts and improved the speed at which valid proofs were constructed. In practice, it’s as if the AI is becoming not just a clever guesser, but an apprentice mathematician; learning to write, revise and formally defend ideas.

Yet while these advances are impressive, recent research reminds us not to mistake polished output for true understanding. A study released in June 2025, “The Illusion of Thinking” [6], found that

4

even the most advanced language models struggle significantly when problem complexity increases.

Their accuracy often collapses; even when given correct instructions, suggesting that what appears to be structured reasoning may in fact be surface-level pattern-matching. In certain puzzles, the models often failed despite having all the steps available.

This highlights the ongoing gap between generating a fluent explanation and performing deep logical reasoning, an important caution as we explore AI’s role in tackling major open problems like P vs NP. So, what does this mean for P vs NP? While AI is still far from independently solving the problem, its ability to generate ideas, construct structured arguments, and refine them through formal logic is changing the landscape. AI may not give us a final answer on its own, but it might soon help researchers reach insights faster, test more possibilities and structure clearer arguments than ever before.

As tools become more sophisticated and collaborative models improve, the question is no longer can AI help, but how much it will accelerate progress, and who will guide it?

IV. W HAT W OULD H APPEN I F W E S OLVED P VS NP? For a problem as abstract as P vs NP, its resolution would carry enormous real-world consequences that could ripple across science, technology, industry and everyday life. Whether it turns out that P equals NP or not, finally settling this question would reshape how we understand the power and limits of computation. But if it turns out that P = NP, and we are also able to find practical algorithms to match, the implications would be transformative.

At the heart of this lies the idea that many of today’s most time-consuming problems, like scheduling airline routes, predicting protein folding, cracking codes or optimising delivery logistics, fall into the NP category. We can check good solutions when we see them, but we can’t always find them efficiently (in polynomial time). If someone could prove not only that P = NP but also show how to make those efficient solutions work in real life, it would effectively unlock the ability to solve every problem we can currently only verify by inputting a solution.

The impact on medicine could be revolutionary.

Imagine being able to compute the optimal structure

for a new drug compound in seconds or quickly simulate and test countless climate models to find the most likely outcomes. Fields that rely on massive trial-and-error like genetics, physics, chemistry and materials science would have shortcuts to insight.

Complex simulations, currently too costly to run at scale, could become routine. In technology and industry, optimisation problems that currently require vast computing power could be handled with ease.

Companies could optimise and resource allocation models in near real time. Financial markets could run hyper-efficient risk calculations. Even basic tasks like automated design and software testing could be made exponentially faster. And then there’s AI itself. If AI systems could access solutions to hard problems efficiently, their planning, decisionmaking, and learning capabilities would skyrocket.

Systems that today struggle with combinatorial logic could suddenly operate at levels of performance we can’t yet imagine. Entire new categories of AI applications could emerge.

Of course, the implications would not be purely positive. Modern cryptography, which protects nearly everything we do online, from banking and messaging to national security, is built on the assumption that certain problems (like factoring large numbers) are computationally hard. If P = NP and more efficient methods were found, it could render most existing encryption breakable overnight. We would need to rapidly rethink how to secure digital systems in a world where nothing is computationally out of reach. But even a proof that P ̸= NP would still have enormous value. It would finally confirm what most researchers suspect: that some problems are just inherently hard and unsolvable in polynomial time, meaning there are no shortcuts. This would redirect effort toward better approximation algorithms and even entirely new paradigms like quantum computing, methods that accept these limits and work around them creatively.

Perhaps the most exciting implication is this: if AI can assist in solving P vs NP, even by guiding humans, organising ideas or exploring patterns, it would show that we are entering a new era of collaborative mathematical discovery. Then the question would be, how are we splitting the million-dollar prize?

5

V. C ONCLUSION The P vs NP problem sits at the heart of theoretical computer science, yet its implications reach far beyond academia. From logistics and medicine to cybersecurity AI, the question of whether problems that are easy to check are also easy to solve could define the limits of what technology can achieve.

After decades of effort, traditional approaches have fallen short of delivering a definitive answer. Now, AI is entering the conversation as a powerful collaborator. LLM’s and formal reasoning tools are showing early promise in structuring arguments, checking logical consistency and guiding the creative process of proof exploration. While AI has not yet delivered a breakthrough, it may well become the accelerant that propels researchers toward one. Whether P is equal to NP or not, the process of solving it will continue to deepen our understanding of complexity, proof and the evolving relationship between human and machine intelligence.

VI. R EFERENCES [1] Arora, S. and Barak, B. (2007). Computational Complexity: A Modern Approach. [online] Available at: https://theory.cs.princeton.edu/ complexity/book.pdf.

[2] Katz, J. (2008). Lecture on Relativization. [online] Available at: https://www.cs.umd.edu/ ∼jkatz/complexity/f05/relativization.pdf.

[3] Stanford University (2019). The Natural Proofs Barrier and P =? NP. [online] Available at: https://theory.stanford.edu/∼liyang/teaching/ projects/natural-proofs-barrier-and-P-NP.pdf [4] Dong, Q., Dong, L., Xu, K., Zhou, G., Hao, Y., Sui, Z. and Wei, F. (2023). Large Language Model for Science: A Study on P vs. NP. [online] arXiv.org. doi: https://doi.org/10.48550/arXiv.2309.

05689 [5] Github.io. (2025). Goedel-Prover. [online] Available at: https://goedel-lm.github.io/ [6] Apple Machine Learning Research. (2025).

The Illusion of Thinking: Understanding the Strengths and Limitations of Reasoning Models via the Lens of Problem Complexity. [online] Available at: https://machinelearning.apple.com/research/ illusion-of-thinking

1

How the Misalignment of Incentives is Sabotaging Scientific Progress: is Decentralised Science the Solution?

Hugo Okada and Osian Guthrie Edited by Pedro Lourenco and Ava Grönberg

Abstract - Systemic incentive misalignments within traditional science inhibit progress across research funding, knowledge dissemination, and the translation of discoveries, causing inefficiencies, biases, and restricted access. This review critically analyses these issues and evaluates Decentralised Science (DeSci) as a transformative solution. DeSci proposes to realign these incentives through blockchain technology [1], Decentralised Autonomous Organisations [2] (DAOs), and token economics, offering novel models for democratised funding (e.g. VitaDAO), open-access publishing (e.g. ResearchHub), and streamlined intellectual property [3] management via IP-NFTs[4]. While presenting promising frameworks to address long-standing challenges, DeSci faces significant hurdles. These include robust DAO governance, crypto-asset volatility, regulatory uncertainties, and adoption barriers. The potential of DeSci, therefore, depends on effectively addressing these pitfalls by ensuring DeSci’s incentives align with the greater goal of scientific progress.

I. I NTRODUCTION From the discovery of DNA to the harnessing of electricity, science has been the engine of human progress; yet paradoxically, the very structures designed to accelerate it now inhibit advancement.

Misaligned incentives undermine the efficacy of research funding, knowledge dissemination, and the translation of scientific discoveries into mainstream use.

This misalignment pervades research funding; rather than direct scientific output, the system incentivises peripheral activities. For example, researchers dedicate substantial time to administrative tasks, particularly grant writing, because grant[5] success is a

primary currency for academic prestige, institutional standing, and continued laboratory operation (Herbert et al., 2013). This focus can also lead to biased allocation, with funding bodies sometimes favouring established researchers or institutions, or less risky, incremental projects over potentially transformative novel ideas (Bol et al., 2018).

Similarly, in scholarly publishing, incentives are skewed toward publication in high-impact, often paywalled journals such as Nature. This dynamic has created an oligopolistic market dominated by a few large publishers, restricting access to publicly funded research (Larivière et al., 2015). The pressure to publish contributes to detrimental outcomes such as the replication crisis and, in extreme cases, research fraud, because researchers are incentivised to prioritise novelty and positive results over reproducibility.

Furthermore, the translation of basic scientific discoveries into practical applications is frequently stymied by the ”valley of death.” This gap persists, in part, because academic reward systems - driven by metrics like publication counts in high-impact journals and grant acquisition for basic research - often undervalue the complex, time-consuming, and sometimes less-cited work of translational science (Butler, 2008; Seyhan, 2019; Qing Ke, 2019).

This effectively disincentivises the crucial bridgebuilding between laboratory findings and real-world solutions.

In response to these deeply rooted incentive misalignments, Decentralised Science (DeSci) has emerged. Utilising blockchain technology, Decentralised Autonomous Organisations (DAOs), and token economics. DeSci is not merely a collection of novel tools, but rather a concerted attempt to fundamentally realign the incentives within the scientific ecosystem. It proposes alternative models for

2

funding, collaboration, publication, and intellectual property management, aiming to foster a more efficient, open, and impactful scientific enterprise. This report critically evaluates incentive misalignments within traditional science and analyses DeSci’s potential in realigning these incentives by examining their potential to address long-standing challenges and the risks associated with these nascent approaches.

II. F UNDING Funding is the cornerstone of all scientific progress and experimentation. Without funding, trials would not take place, and scientists would not be paid. Yet, the incentive structures embedded within traditional science frequently cultivate a culture of inefficiency and bias, diverting resources and researchers’ attention away from the science itself. These misalignments start from the very beginning of the funding process.

Researchers dedicate a disproportionate amount of time to administrative tasks, particularly writing grants, rather than to direct discovery. A report from Australia’s National Health and Medical Research Council found that the preparation of proposals for a single grant round consumed an estimated 550 working years, translating into approximately $41 million in salary costs with a success rate of only 21% for these arduously crafted proposals (Herbert et al., 2013). This administrative load persists because the system itself incentivises such bureaucracy: grant acquisition is a primary metric for academic success, institutional prestige, and thereby the continued operational viability of laboratories. Consequently, a time-consuming application process is normalised, leading to extensive delays and resource diversions that directly undermine the capacity for rapid scientific progress.

Beyond sheer inefficiency, the current funding ecosystem is frequently criticised for being biased, which has further knock-on effects on the misdirection of resources and the stifling of innovation. As mentioned, the skewed incentive structure often favours those with prestige (researchers, institutions), creating a Matthew effect whereby those who already possess resources and recognition are more likely to receive further support (Bol et al., 2018). This systemically

perpetuated preference inadvertently penalises novel, high-risk-high-reward research, which may struggle to gain traction against more incremental proposals emanating from well-established figures in familiar fields (Azoulay et al., 2011). A famous example is the discovery of CRISPR in the 1990s by researchers at the University of Alicante, which, due to the novelty (among other biological reasons), was repeatedly rejected by journals until it was finally published in 2005. Researchers from the University of Alicante finally gained recognition in 2013 when scientists from MIT and Harvard first successfully applied CRISPR-gene editing on human cells. This highlights how biases towards prestigious institutions can hinder scientific progress - imagine what progress may have been made if the CRISPR breakthrough had been published in the 90s?

In response to these issues, DeSci utilises several mechanisms aimed at recalibrating funding incentives to create a more agile, transparent and meritocratic system for resource allocation.

Key among these are Decentralised Autonomous Organisations (DAOs), exemplified by platforms such as VitaDAO, which funds longevity research, and Athena DAO, which advances women’s health research. DAOs such as these usually have a treasury from which funding is drawn. They facilitate community-driven voting on funding proposals, aiming to reduce institutional bias and expedite decisions by shifting power from overly centralised committees. VitaDAO is the largest DeSci DAO; it has a community of approximately 10,000 members, with 2,000 individuals holding the governance token with which votes are cast (Lukacs et al., 2023). To date, VitaDAO has successfully raised in excess of $10 million, a portion of which originates from Pfizer Ventures, a traditional pharmaceutical sector entity. Although this may call into question the supposed decentralisation within these systems, DAOs have taken steps to address issues such as plutocracy (ibid).

To address potential plutocracy in voting, whereby wealth dictates influence in voting, DAOs use mechanisms such as quadratic voting, which make it harder for single, heavily invested entities to dominate governance (Lustenburger et al. 2025).

In other words, the cost of voting increases quadratically with the number of votes. This means

3

about funding it. Lastly, the volatility in prices of governance tokens such as the aforementioned $RSC makes long-term budgeting for research projects highly uncertain for scientists.

III. R EALIGNING K NOWLEDGE D ISSEMINATION I NCENTIVES

Figure 1: Illustrates the democratic and communityfocused dynamic of a DAO (GREED Academy, 2025).

it becomes increasingly expensive for one person to dominate a decision, giving more balanced power across the community. This alleviates the risk of vote-buying or off-chain collusion by influential stakeholders, which could sway funding decisions away from pure scientific merit (Caravello 2025).

In terms of meritocracy, DeSci platforms offer onchain reputation systems, such as ResearchHub’s Reputation (REP) score. These systems reward diverse scientific contributions, which create verifiable track records independent of institutional affiliation and thus incentivise valuable community participation. Complementing this, direct tokenised incentives, like ResearchHub’s ResearchCoin ($RSC), offer a monetary reward for specific actions such as peer-reviewing or sharing reproducible research, contributions which are often undervalued and under-incentivised in traditional models.

Although promising, DeSci comes with its challenges and, by existing on the blockchain, it presents potential risks absent in traditional models.

Within DAOs themselves, low participation and voter apathy are persistent concerns across the broader DAO ecosystem, and DeSci is not immune (Caravello, 2025). Many DAOs struggle with active voter participation rates that can be as low as 5-10% of token holders for important protocols (Lukacs et al. 2023) can severely undermine the credibility of funding rounds, while the voting mechanisms, although decentralised, can be slow.

Moreover, DAO members voting on funding may not possess the specific scientific expertise needed to accurately assess complex proposals, albeit those disinterested in longevity would likely care

Traditional scientific publishing suffers from systemic incentive misalignments that limit research accessibility, inflate costs, and exacerbate the reproducibility crisis. This issue stems not from the aforementioned pitfalls of prestige-based funding but rather from prestigious publishing houses themselves. The career progression of an academic disproportionately rewards publication in highprestige, frequently paywalled journals, enabling large publishers to form an oligopoly. Deutsche Bank describes it as a ”triple payment” system where public funds support the initial research, compensate researchers and peer reviewers, and then public institutions and individuals pay again to access the published findings. Nearly two decades ago, major academic publishers in the science, technical, and medical sectors were already reporting substantial annual revenues of around $20 billion - a figure that would be significantly higher today. Profit margins for some publishers like Elsevier are reaching nearly 40%, exceeding Microsoft, Google, and Coca-Cola (Becerril-Garcı́a, 2024). Journals charge exorbitant fees, with Nature requiring $11,000 (Oransky and Marcus, 2021) and The Lancet Global Health charging up to $7,000 per paper published (Lancet, no date), beyond any subscription costs.

The competitive pressure to secure publications in highly selective journals can inadvertently compromise scientific integrity. This system fosters bias toward publishing exclusively positive or ostensibly novel results, leading to the underreporting of null or negative results, despite their importance to scientific process. In more severe instances, these pressures have been implicated in research fraud, where photoshopped dementia results led to $34 billion in funding being misdirected away from discovering a treatment for over two decades (OHA, 2025). Yet, this should not be surprising, considering that 70% of scientists cannot reproduce another researcher’s results, highlighting the corruption in

4

science (Baker, 2016).

courages sharing all research outputs regardless of their perceived ”publishability” in conventional journals. Publications on these platforms can evolve dynamically, allowing updates with new data and corrections.

Despite compelling advantages, established journals have oligopolised the academic incentive structures, presenting adoption barriers for DeSci platforms.

While platforms like ResearchHub demonstrate potential with 850 weekly contributors, achieving comparable credibility remains challenging. Additionally, DeSci platforms must navigate evolving regulatory landscapes while avoiding new biases that might favour research popular in online communities over complex fundamental science or inadvertently exclude researchers less familiar with the requisite technology.

IV. R EALIGNING T RANSLATION & I MPACT I NCENTIVES

Figure 2: Shows the “file drawer” phenomenon, where scientists abandon research which is less likely to be published (Franco et al., 2014).

DeSci offers mechanisms designed to fundamentally realign knowledge dissemination incentives, cultivating a research ecosystem that is inherently more open, transparent, and collaborative. DeSci initiatives champion Open Access Platforms and Decentralised Repositories, exemplified by projects like ResearchHub, coined the ”GitHub for science.”

These platforms aim to dismantle paywalls and circumvent profit-extracting intermediaries, such as Nature and Science, by facilitating direct sharing of research results, methodologies, datasets, preliminary findings, and preprints, making them accessible to the global scientific community.

Complementing this infrastructural shift, DeSci promotes incentivised collaboration and openness throughout the research lifecycle, rather than confining peer interaction to pre-publication review. Scientific contributions often overlooked in academia, such as null results, replication studies, and the creation of analytical tools, can be directly rewarded on DeSci platforms (e.g. ResearchHub) through platform-specific tokens. This model en-

The journey from laboratory discovery to commercial application faces a ”valley of death” in translational research. For context, 90% of drug candidates entering clinical trials fail FDA approval (Millar, Hill and Whelan, 2022). Developing a single drug takes approximately 10-15 years and $2.6 billion, a 145% increase from early 2000s estimates (Le et al., 2020 & Waltman and Larivière, 2021) The valley of death stems from misaligned academic incentives, rewarding publications, citations rather than the translation of discoveries into applications.

Seyhan (2019) argues that the lack of appropriate incentives in academia to pursue the translation of discoveries into clinical innovations contributes to extremely low success rates. Empirical research

by Qing Ke (2019) demonstrates that clinical research papers consistently receive significantly fewer citations compared to basic science[6] publications. - a proxy for the academic rewards that drive researchers’ careers and recognition. This citation disparity creates powerful disincentives for pursuing translational work despite its huge societal importance.

DeSci utilises the technology of blockchain to provide transparent pathways from discovery to application. IP-NFTs (Intellectual Property Non-Fungible Tokens) serve as immutable digital

5

Figure 3: The translation gap, or “Valley of Death”, where the majority of drug discoveries fail.

certificates of ownership on-chain, creating verifiable IP records manageable through smart contracts. Smart contracts self-executing pieces of code that live on the blockchain; once certain pre-agreed conditions are met - such as a product reaching a sales milestone - the smart contract automatically carries out the defined terms of an agreement without needing a traditional intermediary. This model allows for fractional ownership, allowing researchers to raise capital without ceding complete control of their research (Shilina, 2023). In addition, smart contracts can streamline licensing agreements by automatically connecting researchers with commercial partners and removing bureaucracy in the drug-approval process (Boicova-Wynants, 2022). Most importantly, embedded smart contracts can distribute royalties to researchers and contributors when commercial milestones are achieved, such as bringing a product to market or when a certain sales goal is reached, creating incentives aligned with translation outcomes rather than just academic metrics (Białas, 2025).

DeSci DAOs, such as HairDAO for hair loss, VitaDAO for longevity, or even PsyDAO for medical psychedelic research, offer a promising solution to the translational ”valley of death”. These DAOs have mission-driven research communities that target specific medical challenges or disease areas (Shilina, 2023). Unlike traditional research institutions with broad mandates, these specialised DAOs concentrate resources, expertise, and funding directly on a single issue (Lee, 2023). This targeted approach breaks down silos between academic researchers, clinicians, patients, and industry partners by uniting them within a governance structure that aligns incentives toward translational outcomes. By combining domain-specific focus

with transparent funding allocation and milestonebased rewards embedded in smart contracts, DeSci DAOs can strategically direct collective resources toward bridging critical gaps in the translational pathway that traditional institutions often neglect due to commercial uncertainty or misaligned academic incentives (Lopez, 2025).

Despite these promising frameworks, DeSci’s approach faces implementation challenges. IPNFTs exist in uncertain regulatory territory, raising questions about their legal status across jurisdictions (Białas, 2025). Tokenised IP also risks introducing commercialisation challenges if governance structures around it are inefficient, potentially stalling R&D (Lopez, 2025). Additionally, blockchain-based IP management faces adoption barriers as many researchers and institutions lack the technical expertise to navigate on-chain systems and smart contracts, making it difficult to integrate practically into the existing academic infrastructure (Białas, 2025).

While DeSci addresses legitimate pain points in translational research, its effectiveness depends on thoughtful implementation that preserves scientific integrity, ensures equitable access, and maintains support for the full spectrum of research from fundamental inquiry to practical application.

V. C ONCLUSION The misaligned incentives entrenched within the system of traditional science demonstrably impede progress across research funding, knowledge dissemination, and the translation of discoveries into tangible, real-world impact. Current frameworks often prioritise administrative burdens over efficient resource allocation, foster paywalled publishing oligopolies detrimental to open access, and perpetuate the valley of death, thereby fostering inefficiencies and biases. DeSci emerges as a novel paradigm proposing the recalibration of these incentives through blockchain-based mechanisms such as DAOs, tokenised rewards, and IP-NFTs. These tools aim to cultivate a more democratic, transparent, and efficient scientific enterprise.

However, while offering compelling solutions to long-standing issues, DeSci confronts significant implementation challenges. Concerns regarding robust DAO governance, including voter apathy and

6

plutocracy, the volatility of crypto-assets, the uncertain legal and regulatory landscape for tokenised IP, and barriers to widespread adoption within the established scientific community must be addressed.

The ultimate success of DeSci in transforming scientific practice hinges on its capacity to rigorously navigate these complexities. Further research and careful, iterative development are crucial to ascertain whether DeSci can mature into a superior model for advancing scientific discovery and application.

Network Blog, 21 December. Available at: https://blog.shutter.network/dao-voting-confidence -is-in-decline-how-to-restore-it/ (Accessed: 5 June 2025).

Greed Academy (2023) Governance. Medium, 12 March. Available at: https://medium.com/@Gree dAcademy/governance-623612c5707d (Accessed: 5 June 2025).

Herbert, D.L., Barnett, A.G., Clarke, P. and Graves, N. (2013) ‘On the time spent preparing grant VI. R EFERENCES proposals: an observational study of Australian Al-Shahi Salman, R., Dennis, M.S. researchers’, BMJ Open, 3(5), e002800. and Sandercock, P.A.G. (2014) ‘Research misconduct: time for a united approach’, Ke, Q., Ahn, Y.-Y. and Sugimoto, C.R. (2019) ‘A Nature, 533(7603), pp. 452–453. Available at: systematic identification of scientific disciplines’ h t t p s : / / w w w. n a t u r e . c o m / a r t i c l e s / 5 3 3 4 5 2 a orientation toward basic or applied research’, (Accessed: 5 June 2025).

Journal of Informetrics, 13(1), pp. 383–394.

Azoulay, P., Graff Zivin, J.S. and Manso, G. Larivière, V., Haustein, S. and Mongeon, P. (2011) ‘Incentives and creativity: evidence from (2015) ‘The Oligopoly of Academic Publishers in the academic life sciences’, The RAND Journal of the Digital Era’, PLoS ONE, 10(6), e0127502.

Economics, 42(3), pp. 527–554.

Lee, J.Y. (2023) ‘Decentralized Autonomous Białas, P. (2023) How Decentralized Organizations (DAOs) Unchained: A New Form Science (DeSci) Is Revolutionizing Research. of Corporate Governance?’, University of Illinois Ulam Blog, 21 February. Available at: Law Review, 2023(4), pp. 1293–1348. Available https://www.ulam.io/blog/how- decentralized at: https://illinoislawreview.org/wp-content/upl -science-is-revolutionizing-research (Accessed: 5 oads/2023/08/Lee.pdf (Accessed: 5 June 2025).

June 2025).

López-López, A.V., Navas-Martı́n, M.Á. and Larrú-Rupérez, L. (2024) ‘Decentralized Science Boicova-Wynants, A. (2022) Research (DeSci): An Exploratory Study of Its Possibilities project: NFTs in IP Strategy of a Company. in Open Science’, Laws, 13(4), p. 131. Available at: IP Business Academy.

Available at: https://doi.org/10.3390/laws13040131 (Accessed: 5 https://ipbusinessacademy.org/research- proje June 2025). Lukacs, M., Pinter, N., Rožon, R. and ct-nfts-in-ip-strategy-of-a-company (Accessed: 5 Pólvora, A. (2023) Opportunities and Limitations of June 2025).

Decentralization in Decentralized Science. Preprint.

Available at: https://www.researchgate.net/publica Bol, T., de Vaan, M. and van de Rijt, A. tion/373824086 Opportunities and Limitations of Decentralization in Decentralized Science (2018) ‘The Matthew effect in science funding’, Proceedings of the National Academy of Sciences, (Accessed: 5 June 2025).

115(19), pp. 4887–4890.

Lustenburger, et al. (2025) [Article on quadratic Butler, D. (2008) ‘Translational research: Crossing voting in DAOs]. Supposedly Applied Sciences, the valley of death’, Nature, 453(7197), pp. 15(7), Article 3491. OHA Editorial Team (2025) The Alzheimer’s Research Scandal. Occupational 840–842.

Health Assessment, 10 February. Available at: Caravello, A. (2023) DAO Voting Confidence https://occupationalhealthassessment.com/2025/02 is in Decline. How to Restore it? Shutter /10/the-alzheimers-research-scandal (Accessed: 5

7

June 2025).

Seyhan, A.A. (2019) ’Lost in translation: the valley of death across preclinical and clinical divide – identification of problems and overcoming obstacles’, Translational Medicine Communications, 4, article number 18. doi: 10.1186/s41231-019-0050-7.

Shilina, S. (2023) Intellectual property (in science): The potential & advantages of NFTs. Preprint.

Available at: https://www.researchgate.net/publica tion/369952984 Intellectual property in science The potential advantages of NFTs (Accessed: 5 June 2025).

Wager, E. and Kleinert, S. (2014) ‘Why science needs standards of evidence’, Science, 345(6200), pp. 1210–1211. Available at: https://www.science. org/doi/10.1126/science.1255484(Accessed: 5 June 2025).

[1] A distributed ledger with growing lists of records (blocks) that are securely linked together via cryptographic hashes.

[2] A blockchain-based system that enables people to coordinate and govern themselves mediated by a set of self-executing rules deployed on a public blockchain, and whose governance is decentralised (i.e. independent from central control).

[3] Intangible property that is the result of creativity and innovation and which can be owned in a similar way to physical property.

[4] Provide a secure and immutable proof of ownership for intellectual property on-chain in the form of an NFT (non-fungible token), meaning that each is 1:1 and cannot be replicated.

[5] Enables a user to request specific individuals or groups to address a research challenge or complete a task.

1

The Impact of Artificial Intelligence on Modern Software Development Nifemi Awotorebo Edited by Pedro Lourenco and Ava Grönberg

Abstract - The rise of artificial intelligence and generative AI, such as ChatGPT and Claude, has had massive impacts on almost every industry.

Particularly within software development and testing. AI tools like GitHub Copilot are now integrated into most IDEs, while completely AIpowered integrated development environments (IDEs) like Cursor are emerging. As these tools develop, they raise questions about what impact this will have on the way developers work, the extent to which certain tasks can be automated, and the risks behind integrating AI into the workspace. A 2023 survey by GitHub found that 92% of software developers are using AI professionally and personally [6]. However, new OpenAI research has also shown that AI models like Claude still fall short in real-world software engineering tasks compared to human developers [2]. This contrast highlights both the potential and limitations of AI within software development. This paper explores how AI is transforming Software development and the risks associated with its integration.

I. I NTRODUCTION : A T URNING P OINT IN D EVELOPMENT

process for them.

Meta CEO Mark Zuckerberg recently suggested that AI will be capable of replacing mid level engineers[8], accelerating the automation of routine coding tasks. This statement reflects a broader industry debate: What role will human developers play as AI generated code becomes more efficient and better at solving complex problems? Whilst AI can lead to increased productivity, it also introduces risks such as unreliable, insecure code and overreliance on automation.

This report explores how AI is shaping software development, the challenges it presents and some of its potential future use cases.

II. K EY T ECHNOLOGICAL D EVELOPMENTS Before diving into its impact. It is important to give a brief overview regarding the technical advancements that have allowed for this shift to be possible.

- Transformer Architectures: The foundation of modern generative models such as ChatGPT and Claude.

Software engineering and technology has always - Larger Context Windows: Models such been synonymous with being fast paced and innoas GPT-4 turbo now support > 100K vative, with new technologies constantly arriving. tokens[10] which allows better understanding Requiring developers and companies alike to either of complex text and codebases allowing for a adapt or be left behind. From the rise of Integrated more appropriate and context driven response Development Environments (IDEs) that helped imto any queries. prove code development to collaboration tools like Git that revolutionised and improved how teams - Retrieval Augmented Generation (RAG): work together. Tools are constantly being developed This allows AI models to pull in relevant to make development more efficient. However, the external information to improve responses. rise of AI represents a fundamental shift, where tools are not being used to aid developers but These advancements have fueled the development of are taking on significant parts of the development modern AI tools leading to more capable systems.

2

III. T HE C HANGING ROLE OF D EVELOPERS

of one [2]. These findings suggest that AI cannot As models have grown more capable the role yet replace human developers but can augment them of the software engineer is changing in response. effectively.

Tools like GitHub Copilot and Cursor now assist in everything from boiler plate code generation to suggesting implementation and design decisions.

Google CEO Sundar Pichai recently revealed that AI is generating over 25% of their code, and it is now the engineer’s job to either accept or reject the code[5]. Airbnb cut a multi month code migration project down to 6 weeks using Large Language Models[11]. This showcases a major strength of AI: automating traditionally time-consuming tasks.

Allowing companies and developers alike to focus on more high-level tasks rather than routine maintenance.

But these tools don’t eliminate the need for engineers, instead they shift responsibilities. Their job now includes verifying that AI generated code is secure, efficient, and reliable. This evolving collab- Figure 1: Model performance on SWE-Lancer oration brings new challenges, especially when AI- ($1M earnings pool). Claude 3.5 Sonnet earned generated code sometimes does not meet real world the most, capturing 40.3% of available earnings, standards. followed by o1 (38.0%) and GPT-4o (30.4%).

Source: https://www.bensbites.com/p/swe-lancercan-ai-make-it-as-a-freelance software-engineer.

IV. B ENCHMARKS V ERSUS R EALITY AI tools often perform well in standard coding benchmarks, but real-world development is much harder to quantify. Many benchmarks are often “gamed”[7] meaning models are fine tuned to perform well on them which inflates their perceived competence.

A recent OpenAI study found that household models such as Claude struggle with real world problemsolving tasks compared to human developers. The best model only completed a mere 26.2% of tasks correctly [2]. This is a stark reminder that AI models still fall short of human reasoning and problemsolving abilities. The study highlighted the fact that the models fell short in key areas crucial to being an industry standard software engineer such as: understanding project wide context, problem solving and handling edge cases and writing tests.

However, the study also highlighted some key strengths. AI models outperformed humans in navigating large code bases, locating function definitions and evaluating existing code. Success rates also significantly improved when models were given more than 1 attempt. OpenAI’s o1 model nearly tripled it’s accuracy when given seven tries instead

V. T HE D ECLINE OF C OMMUNITY VALIDATION Historically, developers relied heavily on peer reviewed sites such as Stack Overflow. As of late 2022 the site had over 100 million monthly visitors.

However within six months of ChatGPTs release, traffic fell by over 25% as seen in Figure 2.[11] This swift decline can be attributed to the fact that LLMs such as ChatGPT can generate responses almost instantaneously, and more specific follow up questions can be asked and the end user can receive tailored responses, unlike more traditional forum websites. However, a key upside of Stack Overflow was the ability to upvote and downvote responses based on their correctness. Yet with AI generated code there is no mechanism of peer validation.

This can quickly become an issue as AI models tend to sound completely correct even when they get things wrong. An issue so common it has its own name, “AI hallucinations”. This can lead to developers of all skill levels simply copying and pasting correct looking code without realising that it is completely incorrect, potentially introducing

3

VII. H UMAN -AI C OLLABORATION Given these challenges, the path forward is not to abandon AI, but to use it wisely. The most promising results come when humans and AI collaborate with the model serving more as an assistant than an authoritative figure. As touched on before, AI models excel at tasks such as refactoring code, navigating large file structures and locating relevant functions quickly. But they consistently fail in areas where human developers thrive in such as writing secure and robust tests, reasoning through ambiguity and managing edge cases. The future of software Figure 2: tack Overflow traffic drop development is not AI versus humans. It is AI with after ChatGPT’s release.

Source: humans. Developers who know how to critically ashttps://techxplore.com/news/2024-09-chatgptsess AI output, understand its limitations and know linked-decline-knowledge-online.html how to integrate these tools into their workflow will be the ones who thrive most in the future. security risks and bugs into systems. This loss of VIII. E MERGENCE OF AI C ODING S YSTEMS communal knowledge sharing also reduces collaboAI in software engineering is evolving from much rative learning and knowledge validation. Making it more of a passive tool to an active agent capable of harder for developers to understand why a solution reasoning and executing tasks freely. Unlike tools works and does what it does. such as GitHub Copilot which suggests code line by line, newer systems such as OpenAI’s Codex can operate more autonomously. Codex can read, test and edit real codebases in isolated environVI. S ECURITY AND P RIVACY R ISKS ments. Developers can guide its behaviour by using The risks on relying on AI without proper over- configuration files, enabling it to follow project sight extend beyond correctness and into privacy conventions. These agentic systems mark a new and security. A Stanford study found that developers phase in the software life cycle. Where AI doesn’t who utilised OpenAI’s codex model were more just assist developers but also collaborates with likely to introduce security vulnerabilities [3]. This them. As highlighted throughout this paper, this aligns with a broader trend that AI may speed up transformation carries both opportunity and risk. On development but can also spread insecure or faulty the one hand, we have faster development stages code if used incorrectly. Major companies have and iteration, but on the other hand, there are still already taken steps to reduce the risk of AI-induced concerns about reliability and security. security issues. Samsung made headlines in 2023 for banning the use of ChatGPT after the accidental leak of internal code to the openAI model [4].

The company reportedly feared that the data shared with the bot could be stored on servers owned by companies such as Google, OpenAI and Microsoft.

There is also, the fear that sensitive data shared with ChatGPT could be shared with end users. The combination of the lack of transparency, hallucinated content and potentially unsecure outputs makes it clear that current AI tools if not used appropriately can become more of a burden to major organisations than aids.

IX. C ONCLUSION While the pace of technological change is nothing new, the rise of generative AI marks a shift not just in tools but also responsibilities. Developers are no longer just limited to building and testing applications; they are involved in reviewing and overseeing code written by machines, demanding new skills from the future and current engineers alike. Though current models still fall short in real world tasks, their rapid improvement suggests that the gap will continue to narrow. The challenge ahead will not be about resisting or trying to stop

4

this improvement but rather learning to guide it and ensure that AI enhances human creativity and problem-solving abilities rather than replacing them.

The future of software development will not be AI trying to outdo humans or vice versa. Humans will work with AI to enhance software and make developers more efficient than ever. Similar to the introduction of the first major IDE in Microsoft Visual Basic, in 1991.

X. R EFERENCES [1] SignHouse. (2024). Stack Overflow Growth and Usage Statistics (2024) SignHouse. [online] Available at: https://usesignhouse.com/blog/stack-overflow-stats/ [Accessed 20 Mar. 2025].

[2] Smith, T. (2025). AI Coding: New Research Shows Even the Best Models Struggle With Real-World Software Engineering - DevOps.com. [online] DevOps.com. Available at: AI Coding: New Research Shows Even the Best Models Struggle With Real-World Software Engineering DevOps.com

[3] University, S. (n.d.). Dan Boneh and team find relying on AI is more likely to make your code buggier — Stanford Electrical Engineering. [online] ee.stanford.edu. Available at: https://ee.stanford.edu/dan-boneh-and-team-find-rel ying-ai-more-likely-make-your-code-buggier.

[4] Ray, S. (2023). Samsung Bans ChatGPT among Employees after Sensitive Code Leak. [online] Forbes.

Available at: https://www.forbes.com/sites/siladityaray/20 23/05/02/samsung-bans-chatgpt-and-other-chatbot s-for-employees-after-sensitive-code-leak/ [5] Kelly, J. (2024). AI Writes Over 25Look Like For Software Engineers?

Forbes. [online] 1 Nov. Available at: https://www.forbes.com/sites/jackkelly/2024/11/01/ ai-code-and-the-future-of-software-engineers/ [6] Hurler, K. Finds 92[online]

(2023). GitHub Survey Gizmodo. Available at:

https://gizmodo.com/github- survey- 92- perce nt-programmers-are-using-ai-tools-1850534570 [7] Syed, T. (2024a). Teaching to the Test: How Benchmark Gaming Could Influence AI Progress. [online] Boxcars.ai. Available at: https://blog.boxcars.ai/p/teaching-to-the-test-how -benchmark?utm source=chatgpt.com [Accessed 16May 2025].

[8] Marks, G. (2025). Business Tech News: Zuckerberg Says AI Will Replace Mid Level Engineers Soon. Forbes. [online] 26 Jan. Available at: https://www.forbes.com/sites/quickerbettertech/2 025/01/2/business-technews-zuckerberg-says-ai-wil l-replace-mid-levelengineers-soon/.

[9] OpenAI (2024). New models and developer products announced at DevDay. [online] Openai.com. Available at: h t t p s : / / o p e n a i . c o m /index/new-models-and-developer-products-annou nced-at-devday/?utm source=chatgpt.com.

[10] Simone, S.D. (2025). How Airbnb Used LLMs to Accelerate Test Migration. [online] InfoQ. Available at: https://www.infoq.com/news/2025/ 03/airbnb-llm-test-migration/ [Accessed 16 May 2025].

[11] Hub, S. (2024). ChatGPT’s rise linked to decline in public knowledge sharing on online Q&A platforms. [online] Techxplore.com. Available at: https://techxplore.com/news/2024-09-chatgpt-l inked-decline-knowledge-online.html [Accessed 20 Mar 2025].

1

Exploring the Efficacy of Focused Ultrasound Treatment in Patients with Tremor Dominant Parkinson’s Disease Katherine Mellor Edited by Sushmhitah Sandanatavan and Alethea Stevenson

Abstract—Tremor Dominant Parkinson’s Disease (TDPD) presents a significant therapeutic challenge, as pharmacological interventions are insufficient or poorly tolerated. MRI-guided focused ultrasound (MRgFUS) has surfaced as a novel, incisionless, neurosurgical technique, that can provide precise and targeted ablation of the brain regions associated with tremor generation. This study aims to evaluate the efficacy and safety of MRgFUS in the treatment of TD-PD, with a focus on motor symptom control, the safety profile and it’s outcomes comparatively to those from deep brain stimulation (DBS).

I. I NTRODUCTION Parkinson’s disease (PD) is the second most common neurodegenerative disorder globally, after Alzheimer’s disease, it poses a significant burden on healthcare and patients’ quality of life (Aarsland et al., 2021). With an ageing global population, the incidence and prevalence of PD are increasing, making it a public health concern. According to the Global Burden of Disease study (Feigin et al., 2020), more than 8.5 million individuals were living with PD in 2019; this is expected to double by 2040 (Dorsey et al., 2018).PD is characterised by motor symptoms including: resting tremor, bradykinesia, rigidity and postural instability, that progressively impair the individual’s mobility and independence. Additionally there are non-motor symptoms such as, mood disorders, depression, cognitive dysfunction, hallucinosis and complex behavioural disorders (Poewe, 2008). The rising prevalence of this diease coupled with the extensive life-limiting symptoms highlights the need for innovative, effective and minimally invasive treatments. PD arises from the progressive loss of dopaminergic (DA) neuromelanin-containing neurons in the substantia nigra (SN) and noradrenergic neurons in the locus coeruleus (Dickson, 2012). The SN is divided into

two regions: pars reticulata (SNpr) and pars compacta (SNpc). Where SNpr contains GABAergic neurons and acts as an output nucleus of the basal ganglia, and the latter contains dopamine producing neurons that project into the striatum (Partanen and Achim, 2022). DA projections, referred to as the nigrostriatal pathway, are essential in the regulation of voluntary movement (Chan et al., 2010). Dopamine released from the SNpc terminals modulates the activity of striatal neurons in two circuits, the direct and indirect pathways. Where the direct pathway facilitates movement and the indirect inhibits it (Young CB, 2023). In PD, degeneration of SNpc leads to a dopamine deficit, thus reducing the activation of the direct pathway and inhibition of the indirect. This prompts excessive inhibitory output from globus pallidus internus (GPi) to the thalamus which reduces its ability to excite the motor cortex (McGregor and Nelson, 2019). Consequently, thalamocortical motor drive is diminished, manifesting as bradykinesia, motor rigidity and resting tremor.

In the presence of motor dysfunction, approximately 50 Another pathological hallmark of PD is the presence of Lewy Bodies (LB), primarily composed of misfolded -synuclein protein. The presence of a presynaptic neuronal protein, -synuclein, is involved in synaptic vesicle trafficking and neurotransmitter release (Mahul-Mellier et al., 2020). In PD, -synuclein protein undergoes misfolding and aggregation into -sheet-rich fibrils which accumulate and become neurotoxic. They contribute to neuronal dysfunction through impairment of synaptic function, mitochondrial damage, lysosomal and proteasomal dysfunction and activation of inflammatory responses (Calabresi et al., 2023). Braak staging denotes the distribution of LB, beginning in the dorsal motor nucleus of the vagus nerve and the

2

olfactory bulb (stages 1-2), progressing to SNpc and other midbrain nuclei (stage 3), and cortical regions (stages 5-6)(Braak et al., 2004). This correlates with substantial non-motor dysfunction including sleep disturbances, anosmia and GI disturbances, that often precede motor symptoms (Radad et al., 2023).

DA neurons in the SNpc are vulnerable to mitochondrial dysfunction due to high metabolic demand and reliance on calcium buffering, thus demonstrating reduced activity of mitochondrial complex I in SNpc of PD patients (Vos, 2022). This leads to increased generation of reactive oxygen species (ROS) which are responsible for oxidative damage to lipids, proteins, and DNA and also contribute to neuronal injury and cell death. The selective susceptibility of DA neurons may come from dopamine itself; dopamine may auto-oxidise to form quinones and ROS, amplifying oxidative stress in an already compromised environment (Zhou et al., 2023).

Chronic neuroinflammation assists the progression of PD as residential immune cells of the brain (microglia) are activated in response to neuronal injury and the presence of aggregated -synuclein protein (Ferreira and Romero-Ramos, 2018). Activated microglia cause an excessive release of proinflammatory cytokines such as TNF-, IL-1, IL-6, ROS and nitric oxide which creates a toxic environment that exacerbates neuronal damage (Çınar et al., 2022). PD is associated with dysfunction of two major intracellular protein degradation systems: ubiquitin-proteasome (UPS) and autophagylysosomal pathway (ALP) (Pan et al., 2008). Impairment of UPS is linked to the accumulation of ubiquitinated proteins in LB and mutations in several genes involved in protein degradation, Parkin (E3 ubiquitin ligase) and UCHL1, both associated with familial PD (Doss-Pepe et al., 2005). ALP is crucial in the degradation of large protein aggregates and damaged organelles. Mutations in genes, LRRK2, GBA and ATP13A1, that affect lysosomal function are linked to PD (Pang et al., 2022). Additionally, impaired autophagy promotes -synuclein accumulation and increased neuronal vulnerability.

Whilst PD is generally considered a dopaminergic disorder, the motor function of the striatum is based on the equilibrium between dopamine and acetylcholine, DA activity within the SNpc is modulated by glutamatergic and GABAergic innervations (Aosaki et al., 2010). PD displays a multisystem

pattern of degeneration, whereby neurodegeneration of noradrenergic neurons in the locus coeruleus and serotonergic neurons in raphe nuclei are associated with mood disorders and autonomic dysfunction.

Whilst neurodegeneration of cholinergic neurons in the pedunculopontine nucleus is linked to gait abnormalities (Barone, 2010).

Pharmacological therapy remains the first-line treatment for PD, focusing on symptoms management rather than a cure. Levodopa is the gold standard for treating motor symptoms, and is often combined with adjunctive therapies such as dopamine agonists, MAO-B inhibitors and COMT inhibitors to enhance dopaminergic tone and delay the levodopa initiation (Regensburger et al., 2023). However, chronic levodopa therapy is associated with motor complications like dyskinesia and the “on-off” phenomenon, where patients experience dramatic fluctuations in mobility (Lees, 1989).

When pharmacological interventions become inadequate in advanced PD, surgical interventions such as deep brain stimulation (DBS) are considered.

DBS targets the subthalamic nucleus (STN) and GPi to modulate neuronal activity through continuous electrical stimulation. Whilst multiple studies have exhibited the efficacy of DBS, it remains an invasive procedure with risks such as haemorrhage, infection and hardware discomfort that limit its applicability (Fenoy and Simpson, 2014).

MRI-guided focused ultrasound (MRgFUS) is an emerging, non-invasive therapy that utilises highintensity ultrasound waves to ablate specific regions of the brain in order to treat the motor symptoms of PD. Preprocedural planning includes imaging of the target structures such as the ventral intermediate nucleus (VIM) of the thalamus, GPi and STN, to determine the suitability of the patient for treatment (Ghanouni et al., 2015)t. MRgFUS integrates three key components: a focused ultrasound transducer array, MRI scanner and a thermal dose controller (Jung and Chang, 2018). The transducer arrays emit ultrasound rays, known as sonication, that converge at a defined local point where the energy is then absorbed by the brain tissue. This results in disruption of abnormal neuronal activity by heating to 5560°C. MRI thermometry allows for real-time monitoring of temperature, using temperature sensitive MR parameters, to ensure the safety and efficacy of the treatment (Rieke and Butts Pauly, 2008).

Prior to permanent ablation, low energy sonication

3

is applied to the target area to observe for symptom relief and side effects, this temporary ‘reversible lesioning’ helps fine tune the final target which is observed in three planes (sagittal, axial and coronal) (Schlesinger et al., 2017). Once confirmed, ultrasound of increasing intensities is used to raise the temperature to 46-50°C, up to a maximum temperature of 60°C, until an improvement in the tremor is accomplished (Lu et al., 2023). To achieve ablation a gradual increase in total energy is required, either by increasing intensity or exposure time to sonication, which is then stopped when control of the tremor is achieved. Targeting the STN reduces pathological inhibition via the indirect pathway to alleviate symptoms of bradykinesia and rigidity, which is also achieved in ablation of the GPi by decreasing overactive basal ganglia output. VIM, a key component of the cerebellothalamic circuit, exhibits abnormal oscillatory activity when relaying motor signals to the cortex in PD. Targeting the VIM via DBS or MRgFUS has been proven to be effective in reducing tremor symptoms (Schlesinger et al., 2017).

The non-invasive nature of MRgFUS exhibits exciting advantages such as absence of pre-, intra- and post-surgical complications as well as the absence of implants which eliminates the need for devicerelated maintenance. With its immediate symptom relief and ability to be an outpatient procedure, this innovation proves to be an exciting advancement in the care of PD patients. This review aims to explore and evaluate the use and efficacy of MRgFUS in the treatment of PD. It will compare MRgFUS to existing pharmacological and surgical treatments, through examination of its advantages, limitations and clincal outcomes. Finally, it will discuss the prospects of MRgFUS, including bilateral applications, neuromodulation and drug delivery.

II. M ETHODS Studies were collated by manually searching PubMed, Medline, Embase, Cochrane Library and Google Scholar. The search utilised the following key words: “Parkinson’s Disease”, “PD”, “focused ultrasound”, “MRgFUS”, “motor symptoms”, “safety” and “efficacy”. Additional papers were selected to provide adequate background information regarding PD and MRgFUS. Inclusion Criteria Randomised clinicals trials (RCT), meta-analysis and systematic reviews that

were published in English were included. Studies were screened by both title and abstract to determine their applicability for this review. Literature that examined the efficacy and challenges of the use of MRgFUS in PD were kept.

Exclusion Criteria Non-human studies, narrative reviews and conference abstracts were excluded.

Any studies that could not be accessed and those involving atypical or secondary parkinsonism were excluded.

III. R ESULTS A total of eleven studies examining tremordominant PD treated with MRgFUS were analysed, including observational cohorts, RCTs, prospective studies, reviews and meta-analyses. The majority targeted the VIM, whilst a subset explored pallidothalamic tract (PTT), STN and GPi. IV. M OTOR S YMPTOM I MPROVEMENT Unified Parkinson’s Disease Rating Scale (UPDRS-III) Seven of the analysed studies utilised the MDS-UPDRS-III to determine motor scores following MRgFUS in both the “on-medication” and “off-medication” states, however the “onmedication” state was more widely reported (Tian et al., 2023, Cesarano et al., 2024, Monteiro et al., 2024, Armengou-Garcia et al., 2024, Ge et al., 2021, Xu et al., 2021, Liang et al., 2025). Tian et al. (2023) reported results in more frequent intervals than the other studies, reporting on the “on-medication” and “off-medication” state, at one, three, six and twelve months (Tian et al., 2023).

Across 258 participants in the “on medication” state the mean UPDRS-III score was 12.2 (95 These results were corroborated for the “on” state by (Cesarano et al., 2024) indicating a 37 Tremor Scores (CRST) The Clinical Rating Scale for Tremor (CRST) score was used by five of the papers (Tian et al., 2023; Shiramba et al., 2025; Monteiro et al., 2024; Agrawal et al., 2021)(Xu et al., 2021). The papers determined varying extents of efficacy, with research conducted by Tian et al. (2023) unable to determine the efficacy due to insufficient data and Shiramba et al. (2025) displaying significant reduction across one, three, six and twelve months post operation (Tian et al., 2023, Shiramba et al., 2025).

4

V. Q UALITY OF L IFE (Q O L) AND NON - MOTOR SYMPTOMS

Six studies applied varied measures including QUEST scores, PD-39, depression and cognitive (MoCA) assessments to determine the impact that MRgFUS has on QoL and non-motor symptoms (Shiramba et al., 2025, Agrawal et al., 2021, Monteiro et al., 2024, Ge et al., 2021, Xu et al., 2021).

Across the six studies a varied effect was observed, with Ge at al. (2021) and Xu et al. (2021) reporting no significant improvement. Whilst Shiramba et al.

(2025) displayed three studies with QUEST scores of -1.5 (95% CI: -2.3; -0.7) and -1.5 (95% CI: -2.1; 0.8) at one and three months respectively (Shiramba et al., 2025). Agrawal et al. (2021) reinforces a reduction with a QUEST score of -1.5 (95% CI: -2.93; -0.04) illustrating an inconclusive result (Agrawal et al., 2021).

VI. L ONG - TERM EFFICACY Both Monteiro et al. (2024) and Tian et al. (2023) showed diminishing efficacy at the twelve-month mark, with little research going beyond this time frame (Monteiro et al., 2024, Tian et al., 2023).

Agrawal et al. (2021) identified a RCT that followed patients for 3 years which found that these patients experienced a 63% reduction in disability score and a 50% improvement in QoL (Agrawal et al., 2021).

Tian et al. (2023) noted in four studies that eight patients, out of fourty-nine, experienced a relapse of tremor symptoms at various points including: one week, one month, three and six months (Tian et al., 2023). Shiramba et al. 2025 revealed a statistically significant decrease in hand tremor reduction between one month and five years post MRgFUS (Shiramba et al., 2025).

VII. S AFETY AND A DVERSE E FFECTS Nine studies noted adverse events (AEs) which can be grouped into: AEs associated with MRgFUS procedure and AEs associated with ablation (Armengou-Garcia et al., 2024, Agrawal et al., 2021, Balduino de Souza et al., 2025a, Chua et al., 2023, Ge et al., 2021, Monteiro et al., 2024, Shiramba et al., 2025, Tian et al., 2023, Xu et al., 2021). Balduino de Souza et al. (2025) reported an incidence rate (IR) of 2.63 AEs per patient whereas the control demonstrated an IR of 1.05 AEs. The most common AEs associated with MRgFUS were

dizziness and headaches, with an odds ratio (OR) of 4.35 (95% CI: 1.23-14.64) and 0.88 (95% CI: 0.194.16), this is supported by all nine papers (Balduino de Souza et al., 2025a). Other notable symptoms included vertigo, nausea and vomiting, sensory disturbances and pin-site complications. However, all symptoms reported were transient and mild, resolving within hours to days. Ablation AEs comprised of gait disturbances, dysarthria, orofacial paraesthesia, ataxia and hemiparesis. Gait disturbances were the most frequent AE associated with ablation, with an OR of 4.53 (955% CI: 0.79-26.07) (Balduino de Souza et al., 2025a). In a large prospective cohort, gait disturbances, as with other ablationrelated AEs, continued to three to four months post operation. Chua et al. (2023) displayed an IR for: gait imbalance (38%), weakness (17%), dysarthria (6%) and by one year most side effects were mild (Chua et al., 2023). Balduino de Souza et al. (2025) also suggests that there was no statistical difference in life-threatening complications between the treatment and control groups (Balduino de Souza et al., 2025a).

VIII. C OMPARISON WITH DBS Liang et al. (2025) examined numerous trials with over 3,000 patients involved, comparing the efficacy between DBS and MRgFUS in both the “on” and “off” medication state across different treatment modalities. In the “off” state, DBS focusing on the STN significantly improved the UPDRS-III result.

In the “on” state, DBS targeting the STN and GPi and MRgFUS addressing the STN significantly improved UPDRS-III score, with a SMD of -0.83, 0.80 and -1.83 respectively. QoL was also assessed, with the use of STN-DBS and GPi-DBS showing significant improvement in QoL (Liang et al., 2025).

IX. D ISCUSSION This discussion evaluates the use of MRgFUS for tremor-dominant PD patients with a focus on its clinical outcomes and potential AEs. A comparison is drawn between DBS and MRgFUS to determine its suitability compared to the current gold standard treatment of DBS. X. S UMMARY OF MAIN FINDINGS MRgFUS offers an exciting non-invasive treatment alternative to current pharmacological and

5

surgical interventions, providing consistent and substantial tremor and motor symptom relief. A reduction in UPDRS-III scores of 30-40% across 1-12 months post-treatment, was observed with patients reporting moderate to substantial overall improvement in the “on” state (Tian et al., 2023, Cesarano et al., 2024). As time post-intervention increased, scores rose highlighting a potentially diminishing effect (Agrawal et al., 2021, Monteiro et al., 2024, Shiramba et al., 2025, Tian et al., 2023). Whilst in the “off” state, results were largely inconclusive with a variety of clinical outcomes reported. Nonmotor functions remained mostly unchanged and medication dependence was stable. Some studies demonstrated an increase in QoL and decrease in disability scores demonstrating the effectiveness of MRgFUS (Agrawal et al., 2021, Shiramba et al., 2025, Liang et al., 2025). Headaches, dizziness and sensory disturbance were common complications associated with MRgFUS. These AEs were transient during the treatment process and were resolved within days at most. Complications associated with ablation brought a different set of AEs including gait disturbances, ataxia, dysarthria, orofacial paraesthesia and hemiparesis. In a minority of patients, AEs related to ablation persisted for months, however they were all classed as moderate AEs. (Balduino de Souza et al., 2025b, Chua et al., 2023). In the “on” state, both DBS and MRgFUS significantly improved motor symptoms compared to the baseline, although STN-DBS ranked first overall (Liang et al., 2025).

XI. L IMITATIONS AND B IASES Despite the promising therapeutic potential of MRgFUS in managing tremor-dominant PD, the existing literature has notable limitations and sources of bias. These limitations originate from study design, sample selection, measurement tools, generalisability, which must be further assessed to evaluate the strength of evidence and navigate future research.

One of the most significant limitations is the narrow availability of large RCTS. Systematic reviews and meta-analyses relied heavily on single-arm, small prospective cohort series, due to there only being a few blinded RCTs in existance. The lack of robust RCT data means our ability to establish relationships as well as control and identify confounding variables is diminished. Additionally, the lack

of blinding gives rise to potential performance and expectation bias, whereby patients and clinicians unintentionally influence outcomes or reporting of symptoms. Most studies involved a small cohort, of only around 20 participants, and as a result of this statistical power of the study was reduced, the subgroup analyses were limited and effect sizes exaggerated. For a complex and multifactorial disorder such as PD larger sample sizes are necessary to understand the spectrum of disease presentations and treatment responses that can occur. Selection bias is evident in most MRgFUS studies, as participants are frequently older adults with tremordominant PD with mild-moderate disease severity and few comorbidities. Thereby generalisability is limited to the broader PD population including early onset, severe cognitive impairment or advanced multisystem involvement. Most participants were screened for favourable skull-density ratios (SDRs), a parameter crucial for eligibility for successful ultrasound transmission. As a result, patients with low SDRs were excluded, further emphasising the selection bias at play (Kyle et al., 2024). Another limitation originates from the variability in target areas across studies resulting in heterogeneity. Most studies ablate the VIM, some explore PTT, STN and GPi and combinations thereof (Campins-Romeu et al., 2024). Each of these targets different motor circuits and may display distinct variability in clinical effect (Liang et al., 2025). Differences in lesion targeting, energy delivery protocols and intraoperative monitoring may therefore influence efficacy and AEs. This variability complicates the interpretation of results and without standardised protocols it becomes challenging to compare outcomes, especially when coupled with different patient phenotypes and symptom focuses. Technical parameters employed in MRgFUS including targeting coordinates, thermal thresholds and sonic duration, are not standardised due to MRgFUS being an emerging treatment (Pan et al., 2024). Some centres employ higher peak temperatures whilst others may maintain lower thresholds to minimise AEs (Campins-Romeu et al., 2024). Further variability is introduced via SDRs as patients with low SDRs required an adjusted protocol or were excluded, leading to inconsistency in eligibility criteria. There was significant variation in how treatment outcome was measured, with some focusing on tremor reduction using CRST scores whilst others used subscores of UPDR. Whilst

6

appropriate, these scales neglect other symptoms like gait instability and non-motor manifestations.

Few trials consider QoL, cognitive and behavioural changes or sleep, which results in a narrow focus that overlooks the greater impact that MRgFUS can have on patient autonomy and well-being. The majority of published MRgFUS studies report followups from 3 to 12 months, with few lengthening to 5 years. As PD is a progressive neurodegenerative disorder, short follow-ups may not reflect the sustained effects or efficacy of MRgFUS on patients long-term.

XII. F UTURE D IRECTIONS Current MRgFUS treatment is performed unilaterally due to concerns about bilateral lesion induced AEs like cognitive changes and balance issues (Bruno et al., 2020). Future directions should include the development of safe bilateral treatment protocols through staged procedures and altering dosing. Advancements in real-time thermal monitoring and intraoperative imaging may help to mitigate risks by ensuring greater precision and development of larger lesion volumes and eventual bilateral treatments. Research should examine and determine outcomes based on targets beyond VIM, such as STN and PTT, which may broaden MRgFUS use beyond tremor suppression based on location dependent outcomes. Current evidence for MRgFUS is limited to relatively short-term outcomes, longitudinal studies with follow-ups extending further than five years would be beneficial to determine the durability of clinical outcomes, emerging AEs and any tremor recurrence. Establishing standardised treatment regimens and developing large blinded RCTs will help facilitate a better understanding of the clinical outcomes and how they vary by target and population. Such data could also contribute to cost-effectiveness analyses for determining policy and funding decisions.

As neuromodulation therapies continue to evolve, future research comparing MRgFUS and DBS must evolve beyond efficacy comparisons and address a wider array of implications. These two interventions are opposite in their invasiveness but can be used synergistically in personalised PD management. To date, few RCTs have compared MRgFUS and DBS with small sample sizes and short-follow up durations. Future trials should be stratified by motor phenotype i.e. tremor-dominant vs akinetic-rigid and

include standardised outcome measurements such as UPDRS. It is vital that long-term outcomes are assessed to determine motor symptom progression, medication dependence and any need for repeated treatment. Additionally, they should explore bilateral intervention, a current limitation of MRgFUS, as bilateral DBS is used routinely. A comparison in cost-effectiveness between MRgFUS and DBS should include direct procedural costs as wells as long term maintenance, battery replacements and programming for DBS and potential tremor recurrence with MRgFUS. MRgFUS has the potential to go beyond its ablation applications by leveraging low-intensity focused ultrasound (LIFU) for neuromodulation and blood-brain barrier (BBB) modulation (Baek et al., 2017). Reversible neuromodulation with LIFU can be used to modulate neuronal activity without creating a permanent lesion by influencing membrane permeability, ion channel activity and neuronal excitability (Baek et al., 2017). Thus, there is an opportunity to temporarily suppress pathological oscillatory activity associated with tremors and also hopefully offer a reduced range of AEs that are associated with irreversible ablation or surgical electrode placement. Another promising avenue of MRgFUS is its ability to reversibly open the BBB when combined with intravenous injected microbubbles (Luo et al., 2022). This technique temporarily disrupts the tight junction of the endothelial cells in cerebral vessels allowing the passage of large molecules into basal ganglia (Li et al., 2023). This would overcome the limited ability of drugs to penetrate the CNS, one of the major barriers to PD treatment, which in turn would reduce the need for high systemic doses that increase peripheral AEs. In this model, MRgFUS would become a multifunctional therapeutic tool supporting precision neurology adapting protocol to disease stage and symptom presentation.

XIII. E THICAL I MPLICATIONS As MRgFUS are irreversible, unlike DBS, concerns surrounding the finality of the decision are raised which becomes particularly relevant when dealing with younger patients or those in early stages of the disease who may wish to preserve future options. Due to the lack of longitudinal studies, long-term outcomes of MRgFUS are under investigation. Patients undergoing irreversible

7

treatments in the absence of research into long-term RODRIGUEZ-OROZ, M. C. 2024. Unilateral Magefficacy may encounter unexpected complications. netic Resonance-Guided Focused Ultrasound Lesion of the Subthalamic Nucleus in Parkinson’s Disease: A Prospective Study. Movement disorders : XIV. C ONCLUSION official journal of the Movement Disorder Society, MRgFUS is an emerging, non-invasive treatment 39, 2230-2241. BAEK, H., PAHK, K. J. KIM, H. alternative for tremor dominant PD, particularly in 2017. A review of low-intensity focused ultrasound patients who are unsuitable or reluctant to undergo for neuromodulation. Biomed Eng Lett, 7, 135invasive procedures such as DBS. A growing body 142. BALDUINO DE SOUZA, A. L., MENDES, of evidence displays the sustained benefit in tremor T. P., ROCHA DOS SANTOS, M. A., MORO, P., severity and enhancement of motor function by NIRTA, L. DE LIMA XAVIER, L. 2025a. Safety targeting key structures in the basal ganglia thala- of Focused Ultrasound Ablation in Parkinson’s Dismocortical circuit. The minimally invasive nature ease: A Meta-Analysis of Randomized Controlled of the procedure combined with real-time MRI Trials. Journal of clinical neuroscience : official guidance and the absence of implanted hardware journal of the Neurosurgical Society of Australaaugments its favourable risk profile by lowering sia, 135, 111159. BALDUINO DE SOUZA, A. infection risk and hardware related complications. L., MENDES, T. P., ROCHA DOS SANTOS, M. Regardless of its advantages, MRgFUS still has A., MORO, P., NIRTA, L. DE LIMA XAVIER, limitations. As MRgFUS is approved for unilat- L. 2025b. Safety of Focused Ultrasound Ablation eral use only its utility in patients with bilateral in Parkinsonx2019;s Disease: A Meta-Analysis of symptoms is limited. Whilst tremor response is Randomized Controlled Trials. Journal of Clinical excellent, MRgFUS shows inconclusive efficacy on Neuroscience, 135. BARONE, P. 2010. Neurotransaddressing other symptoms such as bradykinesia or mission in Parkinson’s disease: beyond dopamine. rigidity, which remain better managed using DBS. Eur J Neurol, 17, 364-76. BRAAK, H., GHEOngoing research into bilateral treatment, patient BREMEDHIN, E., RüB, U., BRATZKE, H. DEL selection criteria and long-term outcomes is critical TREDICI, K. 2004. Stages in the development of to optimising MRgFUS and its integration in PD Parkinson’s disease-related pathology. Cell Tissue management.

Res, 318, 121-34. BRUNO, F., CATALUCCI, A., VARRASSI, M., ARRIGONI, F., GAGLIARDI, A., SUCAPANE, P., CERONE, D., PISTOIA, F., XV. R EFERENCES RICCI, A., MARINI, C. MASCIOCCHI, C. 2020.

AARSLAND, D., BATZU, L., HALLIDAY, G. Bilateral MRgFUS thalamotomy for tremor: A safe M., GEURTSEN, G. J., BALLARD, C., RAY solution? Case report and review of current inCHAUDHURI, K. WEINTRAUB, D. 2021. Parkin- sights. Clinical Neurology and Neurosurgery, 197, son disease-associated cognitive impairment. Na- 106164. CALABRESI, P., MECHELLI, A., NAture Reviews Disease Primers, 7, 47. AGRAWAL, TALE, G., VOLPICELLI-DALEY, L., DI LAZM., GARG, K., SAMALA, R., RAJAN, R., ZARO, G. GHIGLIERI, V. 2023. Alpha-synuclein NAIK, V. SINGH, M. 2021. Outcome and in Parkinson’s disease and other synucleinopathies: Complications of MR Guided Focused Ultra- from overt neurodegeneration back to early synapsound for Essential Tremor: A Systematic Re- tic dysfunction. Cell Death Disease, 14, 176. view and Meta-Analysis. Frontiers in Neurology, CAMPINS-ROMEU, M., CONDE-SARDON, R., Volume 12 - 2021. AOSAKI, T., MIURA, M., SASTRE-BATALLER, I., MORATA-MARTINEZ, SUZUKI, T., NISHIMURA, K. MASUDA, M. C., LOSADA-LOPEZ, M., LEON-GUIJARRO, J. 2010. Acetylcholine-dopamine balance hypothesis L., RAGA-RODRIGUEZ, L., PEREZ-GARCIA, in the striatum: an update. Geriatr Gerontol Int, J., GUTIERREZ-MARTIN, A., LOZANO, A. M., 10 Suppl 1, S148-57. ARMENGOU-GARCIA, L., BAVIERA-MUNOZ, R. MARTINEZ-TORRES, I. SANCHEZ-CATASUS, C. A., AVILES-OLMOS, 2024. MRgFUS subthalamotomy in Parkinson’s disI., JIMENEZ-HUETE, A., MONTOYA-MURILLO, ease: an approach aimed at minimizing Lesion G., GOROSPE, A., MARTIN-BASTIDA, A., Volume. npj Parkinson’s Disease, 10, 230. CEGONZALEZ-QUARANTE, L. H., GURIDI, J. SARANO, S., SAPORITO, G., SUCAPANE, P.,

8

BRUNO, F., CATALUCCI, A., PISTOIA, M. L., SPLENDIANI, A., RICCI, A., DI CESARE, E., TOTARO, R. PISTOIA, F. 2024. Staged magnetic resonance-guided focused ultrasound thalamotomy for the treatment of bilateral essential tremor and Parkinson’s disease related tremor: a systematic review and critical appraisal of current knowledge. Front Neurol, 15, 1409727. CHAN, C. S., GERTLER, T. S. SURMEIER, D. J. 2010. A molecular basis for the increased vulnerability of substantia nigra dopamine neurons in aging and Parkinson’s disease. Movement Disorders, 25, S63S70. CHENG, H.-C., ULANE, C. M. BURKE, R. E. 2010. Clinical progression in Parkinson disease and the neurobiology of axons. Annals of Neurology, 67, 715-725. CHUA, M. M. J., BLITZ, S. E., NG, P. R., SEGAR, D. J., MCDANNOLD, N. J., WHITE, P. J., CHRISTIE, S., HAYES, M. T., ROLSTON, J. D. COSGROVE, G. R. 2023. Focused Ultrasound Thalamotomy for Tremor in Parkinson’s Disease: Outcomes in a Large, Prospective Cohort.

Movement Disorders, 38, 1962 EP - 1967. ÇıNAR, E., TEL, B. C. ŞAHIN, G. 2022. Neuroinflammation in Parkinson’s Disease and its Treatment Opportunities. Balkan Med J, 39, 318-333. DICKSON, D. W. 2012. Parkinson’s disease and parkinsonism: neuropathology. Cold Spring Harb Perspect Med, 2. DORSEY, E. R., ELBAZ, A., NICHOLS, E., ABBASI, N., ABD-ALLAH, F., ABDELALIM, A., ADSUAR, J. C., ANSHA, M. G., BRAYNE, C., CHOI, J.-Y. J., COLLADO-MATEO, D., DAHODWALA, N., DO, H. P., EDESSA, D., ENDRES, M., FERESHTEHNEJAD, S.-M., FOREMAN, K. J., GANKPE, F. G., GUPTA, R., HAMIDI, S., HANKEY, G. J., HAY, S. I., HEGAZY, M. I., HIBSTU, D. T., KASAEIAN, A., KHADER, Y., KHALIL, I., KHANG, Y.-H., KIM, Y. J., KOKUBO, Y., LOGROSCINO, G., MASSANO, J., MOHAMED IBRAHIM, N., MOHAMMED, M. A., MOHAMMADI, A., MORADI-LAKEH, M., NAGHAVI, M., NGUYEN, B. T., NIRAYO, Y. L., OGBO, F. A., OWOLABI, M. O., PEREIRA, D. M., POSTMA, M. J., QORBANI, M., RAHMAN, M. A., ROBA, K. T., SAFARI, H., SAFIRI, S., SATPATHY, M., SAWHNEY, M., SHAFIEESABET, A., SHIFERAW, M. S., SMITH, M., SZOEKE, C. E. I., TABARéS-SEISDEDOS, R., TRUONG, N. T., UKWAJA, K. N., VENKETASUBRAMANIAN, N., VILLAFAINA, S., WELDEGWERGS, K. G., WESTERMAN, R., WIJERATNE, T., WINKLER,

A. S., XUAN, B. T., YONEMOTO, N., FEIGIN, V. L., VOS, T. MURRAY, C. J. L. 2018. Global, regional, and national burden of Parkinson’s disease, 1990x2013;2016: a systematic analysis for the Global Burden of Disease Study 2016. The Lancet Neurology, 17, 939-953. DOSS-PEPE, E. W., CHEN, L. MADURA, K. 2005. -Synuclein and Parkin Contribute to the Assembly of Ubiquitin Lysine 63-linked Multiubiquitin Chains*. Journal of Biological Chemistry, 280, 16619-16624. FEIGIN, V. L., VOS, T., NICHOLS, E., OWOLABI, M. O., CARROLL, W. M., DICHGANS, M., DEUSCHL, G., PARMAR, P., BRAININ, M. MURRAY, C. 2020. The global burden of neurological disorders: translating evidence into policy. Lancet Neurol, 19, 255-265. FENOY, A. J. SIMPSON, R. K., JR. 2014.

Risks of common complications in deep brain stimulation surgery: management and avoidance. J Neurosurg, 120, 132-9. FERREIRA, S. A. ROMERORAMOS, M. 2018. Microglia Response During Parkinson’s Disease: Alpha-Synuclein Intervention.

Front Cell Neurosci, 12, 247. GE, Y., GU, F., YANG, X., CHEN, Z., DONG, W. WANG, Z. 2021.

Clinical application of magnetic resonance-guided focused ultrasound in Parkinson’s disease: a metaanalysis of randomized clinical trials. Neurological Sciences, 42, 3595 EP - 3604. GHANOUNI, P., PAULY, K. B., ELIAS, W. J., HENDERSON, J., SHEEHAN, J., MONTEITH, S. WINTERMARK, M. 2015. Transcranial MRI-Guided Focused Ultrasound: A Review of the Technologic and Neurologic Applications. AJR Am J Roentgenol, 205, 1509. JUNG, N. Y. CHANG, J. W. 2018. Magnetic Resonance-Guided Focused Ultrasound in Neurosurgery: Taking Lessons from the Past to Inform the Future. J Korean Med Sci, 33, e279. KYLE, K., MAAMARY, J., JONKER, B., PETERS, J., BARNETT, Y., BARNETT, M., D’SOUZA, A., MALLER, J., WANG, C. TISCH, S. 2024. Consistency is key: influence of skull density ratio distribution on the formation of clinically effective lesions and long-term tremor suppression following treatment with MR-guided focused ultrasound. J Neurosurg, 140, 648-656. LEES, A. J. 1989. The on-off phenomenon. J Neurol Neurosurg Psychiatry, Suppl, 29-37. LI, Q., TANG, Z., ZHANG, Y., YUAN, T., YUAN, B., DU, L. JIN, Y. 2023. Application of low-intensity ultrasound by opening blood–brain barrier for enhanced braintargeted drug delivery. International Journal of Phar-

9

maceutics, 642, 123191. LIANG, M., HOU, L., LIANG, J. BAO, S. 2025. Ameliorating motor performance and quality of life in Parkinson’s disease: a comparison of deep brain stimulation and focused ultrasound surgery. Front Neurol, 16, 1449973. LU, H., WANG, X. LOU, X. 2023.

Current applications for magnetic resonance-guided focused ultrasound in the treatment of Parkinson’s disease. Chin Med J (Engl), 136, 780-787. LUO, T., BAI, L., ZHANG, Y., HUANG, L., LI, H., GAO, S., DONG, X., LI, N. LIU, Z. 2022. Optimal treatment occasion for ultrasound stimulated microbubbles in promoting gemcitabine delivery to VX2 tumors. Drug Deliv, 29, 2796-2804. MAHULMELLIER, A.-L., BURTSCHER, J., MAHARJAN, N., WEERENS, L., CROISIER, M., KUTTLER, F., LELEU, M., KNOTT, G. W. LASHUEL, H. A. 2020. The process of Lewy body formation, rather than simply -synuclein fibrillization, is one of the major drivers of neurodegeneration. Proceedings of the National Academy of Sciences, 117, 49714982. MCGREGOR, M. M. NELSON, A. B. 2019. Circuit Mechanisms of Parkinson’s Disease.

Neuron, 101, 1042-1056. MONTEIRO, J. D. S., BB, E. S., DE OLIVEIRA, R. R., BORGES, P., PEREIRA, M., COSTA, K. A., NUNES, A. L. S., TELLES, J. P. M. VALENçA, M. M. 2024.

Magnetic resonance-guided focused ultrasound ventral intermediate thalamotomy for Tremor-Dominant Parkinson’s disease: a systematic review and metaanalysis. Neurosurg Rev, 47, 701. PAN, L., XIONG, Y., LI, Y., SUN, B., LI, X., ZHOU, S. LOU, X. 2024. Consensus for clinical applications of transcranial magnetic resonance-guided focused ultrasound. Chin Med J (Engl), 137, 2684-2686. PAN, T., KONDO, S., LE, W. JANKOVIC, J. 2008. The role of autophagy-lysosome pathway in neurodegeneration associated with Parkinson’s disease. Brain, 131, 1969-78. PANG, S. Y.-Y., LO, R. C. N., HO, P. W.-L., LIU, H.-F., CHANG, E. E. S., LEUNG, C.-T., MALKI, Y., CHOI, Z. Y.-K., WONG, W. Y., KUNG, M. H.-W., RAMSDEN, D. B. HO, S.-L. 2022. LRRK2, GBA and their interaction in the regulation of autophagy: implications on therapeutics in Parkinson’s disease. Translational Neurodegeneration, 11, 5. PARTANEN, J. ACHIM, K. 2022. Neurons gating behavior-developmental, molecular and functional features of neurons in the Substantia Nigra pars reticulata. Front Neurosci, 16, 976209. POEWE, W. 2008. Non-motor symptoms

in Parkinson’s disease. Eur J Neurol, 15 Suppl 1, 14-20. RADAD, K., MOLDZIO, R., KREWENKA, C., KRANNER, B. RAUSCH, W.-D. 2023. Pathophysiology of non-motor signs in Parkinson’s disease: some recent updating with brief presentation. Exploration of Neuroprotective Therapy, 3, 24-46. REGENSBURGER, M., IP, C. W., KOHL, Z., SCHRADER, C., URBAN, P. P., KASSUBEK, J. JOST, W. H. 2023. Clinical benefit of MAOB and COMT inhibition in Parkinson’s disease: practical considerations. J Neural Transm (Vienna), 130, 847-861. RIEKE, V. BUTTS PAULY, K. 2008. MR thermometry. J Magn Reson Imaging, 27, 376-90. SCHLESINGER, I., SINAI, A. ZAAROOR, M. 2017. MRI-Guided Focused Ultrasound in Parkinson’s Disease: A Review. Parkinson’s Disease, 2017, 8124624. SHIRAMBA, A., LANE, S., RAY, N., GILBERTSON, T., SRINIVASAIAH, R., PANICKER, J., RADON, M., OSMAN-FARAH, J. MACEROLLO, A. 2025. Efficacy and Safety of Magnetic Resonance-Guided Focused Ultrasound Thalamotomy in Essential Tremor: A Systematic Review and Metanalysis. Movement Disorders.

TIAN, X., HU, R., HE, P. YE, J. 2023. Efficacy and safety of magnetic resonance-guided focused ultrasound for Parkinson’s disease: a systematic review and meta-analysis. Frontiers in Neurology, 14, 1301240. VOS, M. 2022. Mitochondrial Complex I deficiency: guilty in Parkinson’s disease. Signal Transduction and Targeted Therapy, 7, 136. XU, Y., HE, Q., WANG, M., GAO, Y., LIU, X., LI, D., XIONG, B. WANG, W. 2021. Safety and efficacy of magnetic resonance imaging-guided focused ultrasound neurosurgery for Parkinson’s disease: a systematic review. Neurosurg Rev, 44, 115-127.

YOUNG CB, R. V., SONNE J 2023. Neuroanatomy, Basal Ganglia. StatPearls [Internet];lk. ZHOU, Z. D., YI, L. X., WANG, D. Q., LIM, T. M. TAN, E. K. 2023. Role of dopamine in the pathophysiology of Parkinson’s disease. Translational Neurodegeneration, 12, 44.

1

Oncolytic Virotherapy for Glioblastoma; Improving Median Survival Time Through an Enhanced Immune Response Kaitlyn Grosh Edited by Sushmhitah Sandanatavan and Alethea Stevenson

Abstract—Oncolytic virotherapy shows great promise in extending the lives of those diagnosed with glioblastoma (GBM). Standard treatment regimens include chemotherapy and radiation, but these modalities have been shown to have a median survival time of only 12-15 months.

By increasing the immunogenicity of the tumour microenvironment, these cold tumours can be turned “hot”, thus allowing for the body’s natural anti-tumour defence to be strengthened. Both the Oncolytic Herpes Simplex viruses (oHSV) and the Measles virus (MV) have been engineered for therapeutic use and so allow for infection of only the cancerous cells. Administration of an oncolytic virus enhances the immune response in the tumour by inducing viral replication, which facilitates destruction of the tumour. One trial has shown that oHSV increases median survival time to 30.5 months, compared to the average 12-15 months with a standard regimen. MV has similarly increased the one-year survival rate to 45.5%, which is significantly higher than what is seen with current treatments (17.7%). Additionally, the delivery method of oncolytic viruses can influence patient outcome. Intratumoral injection allows for the blood brain barrier to be bypassed, whilst also offering the ability to deliver a highly potent virus with minimal off-target effects. As a result of this, intratumoral injection may be recommended for integration into clinical trials in order to maximize effects.

The use of oHSV and MV for GBM treatment have shown significantly improved patient outcomes, however, further trials investigating safety and efficacy are needed prior to integrating these treatments into a new standard of care.

I. U NDERSTANDING G LIOBLASTOMA E TIOLOGY Glioblastoma (GBM) is a fast-growing, highly aggressive brain tumour that has a median survival time of only 12-15 months and a 5-year survival rate of less than 5% (Xu et al., 2024) ;(Gunasegaran et al., 2024). It is classified as a grade 4 tumour, which is the most severe grade a tumour can have, and typically develops in the frontal or temporal lobe.

Despite being the most common type of adult malignant brain tumour, treatment options are limited and

typically success rates are low. Conventional treatment begins with surgical resection of the tumour to the fullest extent, followed by a combination of radiation and temozolomide (TMZ) chemotherapy (Rose, 2022). Due to the low survival rate associated with traditional treatment, patients diagnosed with GBM may be encouraged to seek out clinical trials investigating new potential therapies such as laser interstitial thermal therapy, immunotherapy, and oncolytic viral therapy. While most patients do not express any identifiable risk factors, there have been some links highlighting potential environmental and genetic influences. Exposure to high-dose ionising radiation is the only confirmed risk factor for GBM that is not genetic, as a result certain military veterans are at higher risk of developing this tumour development due to their previous radiation exposure. (Gunasegaran et al., 2024). Some GBM patients have been previously diagnosed with neurofibromatosis, von Hippel-Lindau syndrome, and Li-Fraumeni syndrome, which could suggest a link between these genetic disorders and GBM formation (Bondy et al., 2008). Additionally, small genetic mutations in the genome can affect a person’s response to treatment. Isocitrate dehydrogenase (IDH) enzyme mutations are found in over 80% of GBM cases and are thought to drastically improve median survival (31 months vs 15 months) (Han et al., 2020). These enzymes are important as they catalyse isocitrate to alpha-ketoglutarate in the Krebs cycle.

It has also been found that the O6-methylguanineDNA methyltransferase (MGMT) gene plays a key role in conferring chemoresistance, as a result of this patients with a mutation in this gene are associated with poorer outcomes. (Kitange et al., 2009).

2

II. D ELIVERY OF O NCOLYTIC V IRUSES Oncolytic viruses can be administered in several ways, and currently there is no gold? standard delivery method yet determined. It is crucial to consider the role of the BBB(have you established this acronym?) when delivering virotherapy, especially for GBM. The immunogenicity of the virus should also be considered, as highly immunogenic viruses cannot sit in the bloodstream for long periods of time without causing adverse effects (Romanishin et al., 2024). Intratumoral injection is the most common method of administration, and involves the virus being injected directly into the tumour itself meaning the BBB can be bypassed and thus ensures the virus is delivered in full. This method is also beneficial for administering a highly immunogenic virus, which confers the largest immune response (Romanishin et al., 2024). However, this delivery method is invasive in GBM patients, as the tumour needs to be somewhat exposed to receive the injection. Convection enhanced delivery (CED) is a technique (+to help combat this issue)? that involves placing a catheter in the brain, directly into the tumour, that can be accessed through the skull, like a shunt. The virus is then delivered through the catheter allowing it to flow directly into the tumour (Romanishin et al., 2024). This helps bypass the BBB and also allows for re-dosing if needed without needing to perform a second surgery. It also helps protect surrounding healthy brain tissue from potential toxic effects of the virus (Romanishin et al., 2024). However, the catheter placement needs to be incredibly precise and this method of delivery may not be favoured by the patient. There are numerous systemic delivery options that are less invasive, such as intravenous and intraarterial delivery, but as a result of being less invasive they are typically not as effective intratumoral delivery(Romanishin et al., 2024). Intravenous delivery allows for convenient repeated dosing, yet does not circumvent the BBB and may yield off-target effects from highly immunogenic viruses (Romanishin et al., 2024). Intraarterial delivery is more direct to the brain than intravenous, but also does not ensure the virus will cross the BBB. Oncolytic virotherapy is often used on its own and does not always involve tumour resection and chemotherapy. There is potential to make oncolytic virotherapy less invasive, but this depends on the method of delivery. The most

successful mechanism is intratumoral delivery, as it allows the BBB to be bypassed and limits the off-target effects from highly immunogenic viruses.

Although the catheter placement for CED and intravenous delivery are still invasive, it limits the risk of damaging surrounding healthy tissues, which is often a concern with tumour resection.

III. C HALLENGES WITH C URRENT T REATMENT A PPROACHES There are numerous properties unique to GBM which may contribute to the poor prognosis. Current treatment begins with the removal of the tumour, but depending on size and location, it often cannot be removed in full without damaging surrounding tissues (Rose, 2022). Following surgery, patients will then undergo radiation and chemotherapy with TMZ. A mutation in the MGMT gene by hypermethylation occurs in over 40% of GBM patients (Kitange et al., 2009). This acts as a DNA repairing enzyme, but after chemotherapy treatment, apoptosis and cytotoxicity can occur due to chemotherapy-induced alkylation (Kitange et al., 2009). As MGMT is typically overexpressed in tumour cells, this can lead to challenges with standard treatments such as TMZ. Additionally, many drugs are large and cannot cross the blood-brain barrier (BBB), making the tumour even more difficult to reach with drugs (Rose, 2022). This resistance can lead to regrowth and an overall decreased median survival time. GBM are inherently resistant and often grow quickly meaning treatment can often be rendered ineffective. The tumour itself has a unique microenvironment with a poorly functioning immune response. The immune system naturally has mechanisms to recognise and kill tumour cells by employing natural killer cells, T cells, and B cells, however, with GBM, this process is interrupted (Razavi et al., 2016). Immunosuppressive cytokines are produced, which promote tumour growth and inhibit tumour necrosis factor-alpha (TNF- ) (Razavi et al., 2016). T cell proliferation is reduced and T cells become more vulnerable to apoptosis(, resulting in less in circulation)?. Tissue hypoxia, as well as abnormal blood flow, can occur due to the redirection of oxygen into proliferating tumour cells (Razavi et al., 2016). As a result of this several immunosuppressive pathways are activated, reducing the body’s natural defence against the tumour. This, in turn, creates a need for interventions

3

such as surgery and chemotherapy. However, these interventions are often not effective as the median survival time with treatment is still relatively low (+stat?) (Xu et al., 2024). GBM patients are in dire need of a new therapy to help prolong their lifespan and enhance their quality of life.

IV. O NCOLYTIC V IROTHERAPY: H OT AND C OLD T UMOURS Numerous clinical trials are in progress to study potential therapies that may improve median survival time, with one of the most promising techniques being oncolytic virotherapy. This involves the injection of a specific virus or viral vector into the tumour with the aim of enhancing the body’s anti-tumour response (Gunasegaran et al., 2024). Injection induces apoptosis and necrosis of the tumour, it causes the release of cytokines and cytotoxic lymphocytes which target the tumour, thus killing the cancer cells. This allows for only the tumour to be targeted, meaning healthy tissue can be left unaffected and the likelihood of the side effects that are often seen with chemotherapy is decreased. GBM are referred to as “cold” tumours due to their limited immune response in the tumour microenvironment. Cold tumours are skilled at evading anti-tumour immune responses, as they lack the necessary T cells and are immunosuppressive (Wu et al., 2024). “Hot” tumours, in contrast, have active immune systems and so are typically associated with better patient outcomes. This is because they are more responsive to a variety of therapies, such as immunotherapy, due to the enriched T cell microenvironment of the tumour and their favourable response to immune checkpoint inhibitors (Wu et al., 2024). Oncolytic virotherapy has the potential to turn a cold tumour into a hot tumour by enhancing the tumour microenvironment so it is immunosupportive, which is theorised to aid in improving the median survival time for patients with GBM (Wu et al., 2024). When an oncolytic virus enters the cancer cell, an immune response is triggered in the host.

Antigen-presenting cells (APCs) are then stimulated to release several signalling molecules which initiate the immune response (Romanishin et al., 2024).

Tumour-associated antigens (TAAs) are located on the surface of cancerous tumours, and they signal to the immune system to trigger a signalling cascade (Romanishin et al., 2024). Viral pathogenassociated molecular patterns (PAMPs) are part of

the oncolytic virus and they enhance the immune response by signalling to immune cells to respond to the tumour (Romanishin et al., 2024). The oncolytic virus can also cause the release of chemokines in response to cancer cell lysis, which is part of the destructive process. By introducing an oncolytic virus, an immune response is triggered through a signalling cascade, thereby recruiting necessary immune cells, such as T cells and cytokines, to aid in apoptosis of the cancer cells, thus influencing degradation of the tumour. This process also converts the tumour from cold to hot, making it more responsive to subsequent immunotherapy (Wu et al., 2024).

V. H ERPES S IMPLEX V IRUS -1 Over 30 different viruses have been studied in clinical trials thus far and have had varying success.

Most viruses used are either retroviruses or adenoviruses engineered to infect only cancer cells and incite viral replication and therefore kill the cancer cells. A phase I/II clinical trial of a triple-mutated oncolytic herpes simplex virus (oHSV), G47, was shown to safely increase median survival time (Todo et al., 2022). The study was conducted in adults with recurrent GBM following typical radiation and TMZ treatment. G47 was administered intratumorally, and the median overall survival from diagnosis was 30.5 months. The 1-year survival rate was 38.5%, which is slightly higher than the average (Todo et al., 2022). This was likely due to the induced lymphocyte infiltration of the tumour cells and viral replication, which caused the tumour to destruct. Although it has been deemed safe, G47 caused side effects in 12/13 patients, most commonly causing headache, fever, and vomiting (Todo et al., 2022). Another engineered HSV-1 strain, G207, has demonstrated some success in a phase I trial (Markert et al., 2014). It has been altered to not include the gene for neurovirulence in order to minimise off-target effects, whilst still being effective in fighting the tumour. G207 is infused into the tumour and then followed with radiation the next day to maximise efficacy. This regimen was deemed safe as patients experienced mild to moderate adverse effects, such as headache, seizures, and fever (Markert et al., 2014). The median survival time after injection was 7.5 months, with most injections taking place 18 months after diagnosis, and with many patients having at least a partial response to treatment (Figure

4

Fig. 1. Patient Response Pre and Post G207 Inoculation This patient shows a partial response to the G207 and radiation regimen, illustrated by the reduced tumour volume from before treatment (a) and one month following treatment (b).

1). The median progression free survival time was 2.5 months (Markert et al., 2014).

VI. M EASLES V IRUS Measles virus (MV) strains have also been derived as potential promising oncolytic virus therapies. In a phase I trial, a measles derivative, measles virus carcinoembryonic antigen (MV-CEA), was administered in patients with recurrent GBM either intratumorally or in the resection cavity (Galanis et al., 2024). The treatment was well tolerated, and the median overall survival was 11.6 months following treatment (Galanis et al., 2024). Additionally, the one-year survival was 45.5%, which is respectably high for a grade 4 tumour (Galanis et al., 2024).

No immunosuppression caused by treatment was observed. Biopsies revealed lymphocyte infiltration, as well as an increase in the pro inflammatory cytokines and chemokines that are needed to create an immunosupportive environment. A combined approach of MV and anti-programmed cell death protein 1 (anti-PD-1) has also been investigated to maximise the efficacy of the anti-tumour response (Hardcastle et al., 2016). This has only been done in vitro, but the results were promising. This combined approach caused human GBM cells to upregulate programmed cell death ligand 1, thus creating a pro inflammatory response (Hardcastle et al., 2016).

Although this still needs to be tested in vivo, it shows a promising combination approach that may improve survival odds.

VII. C ONCLUSION Whilst oncolytic virotherapy is still in early stages of development, much of the data shows promise that it could extend the median survival time for

patients diagnosed with this fatal tumour. With the current combination of chemotherapy and radiation, the median survival time is 12-15 months, and the one-year survival is approximately 17.7% (Ohgaki and Kleihues, 2005). Untreated GBM has a median survival time of only 3-6 months (Mazarakis et al., 2024). The oHSV derivative G47 was shown to extend the median survival time to 30.5 months after diagnosis in a phase I trial, essentially doubling the median survival time (Todo et al., 2022). Although most patients experienced side effects, they were all deemed mild (Todo et al., 2022). The average progression free survival (PFS) was only 8 days from the last G47 injection on average, however, one patient experienced 382 days without progression (Todo et al., 2022). G207 is another oHSV derivative which showed success in early trials (Markert et al., 2014). The median survival time from diagnosis was 25.5 months, and the average PFS was 2.5 months, which is much higher than G47 (Markert et al., 2014). Both derivatives have similar overall survival and side effects, so it is difficult to determine which one may work best for a patient. The longer PFS, however, may allow patients to feel fewer side effects of the tumour even if they do not live longer, thus enhancing the quality of life. Oncolytic MV have not been studied in as much depth, but they still show potential to enhance immunogenicity in GBM. MV-CEA demonstrated a median survival time of 11.6 months after treatment and a one-year survival rate of 45.5% (Galanis et al., 2024). The median time before treatment was not stated, which makes the data difficult to compare to oHSV trials. MV-CEA did increase the one-year survival rate significantly, to a higher rate than what was seen with oHSV administration (Galanis et al., 2024). Both oHSV and MV demonstrated safe and relatively effective treatments that enhanced GBM patient outcomes. Additionally, it can be concluded that intratumoral delivery appears to be the best form of administration. It allows for highly immunogenic viruses to be used, as the offtarget effects are minimized by targeted injection (Romanishin et al., 2024). It is more invasive than other methods such as intravenous and intraarterial delivery, but no more so than the current standard of care requiring tumour excision. Future research in the field is still needed to help further increase the efficacy of the treatment. Combination therapy using oncolytic viruses combined with other molecules,

5

such as anti-PD-1, may enhance the immunogenic effects even more (Hardcastle et al., 2016). This combination therapy involves the use of two different anti-tumour strategies, which may optimize patient outcomes. Current oncolytic virotherapy trials have shown promise in extending the median survival time for patients with GBM, particularly with the use of HSV1 and MV, highlighting its potential to revolutionize the standard of care for GBM. VIII. R EFERENCES Bondy, M.L., Scheurer, M.E., Malmer, B., Barnholtz-Sloan, J.S., Davis, F.G., Il’yasova, D., Kruchko, C., McCarthy, B.J., Rajaraman, P., Schwartzbaum, J.A., Sadetzki, S., Schlehofer, B., Tihan, T., Wiemels, J.L., Wrensch, M., Buffler, P.A. and Brain Tumor Epidemiology Consortium (2008). Brain tumor epidemiology: consensus from the Brain Tumor Epidemiology Consortium. Cancer, 113(7 Suppl), pp.1953–68. doi:https://doi.org 10.1002/cncr.23741.

Galanis, E., Dooley, K.E., Keith Anderson, S., Kurokawa, C.B., Carrero, X.W., Uhm, J.H., Federspiel, M.J., Leontovich, A.A., Aderca, I., Viker, K.B., Hammack, J.E., Marks, R.S., Robinson, S.I., Johnson, D.R., Kaufmann, T.J., Buckner, J.C., Lachance, D.H., Burns, T.C., Giannini, C. and Raghunathan, A. (2024). Carcinoembryonic antigen-expressing oncolytic measles virus derivative in recurrent glioblastoma: a phase 1 trial. Nature Communications, 15(1), p.493. doi:https://doi.org/10.1038/s41467-023-43076-7.

Gunasegaran, B., Ashley, C.L., Marsh-Wakefield, F., Guillemin, G.J. and Heng, B. (2024). Viruses in glioblastoma: an update on evidence and clinical trials. BJC Reports, 2(1), pp.1–21. doi:https://doi.org/10.1038/s44276-024-00051-z.

Han, S., Liu, Y., Cai, S.J., Qian, M., Ding, J., Larion, M., Gilbert, M.R. and Yang, C. (2020). IDH mutation in glioma: molecular mechanisms and potential therapeutic targets.

British Journal of Cancer, 122(11), pp.1580–1589. doi:https://doi.org/10.1038s41416-020-0814-x.

Hardcastle, J., Mills, L., Malo, C.S., Jin, F., Kurokawa, C., Geekiyanage, H., Schroeder, M., Sarkaria, J., Johnson, A.J. and Galanis, E. (2016). Immunovirotherapy with measles virus strains in combination with anti–PD-1 antibody

blockade enhances antitumor activity in glioblastoma treatment. Neuro-Oncology, 19(4), p.now179. doi:https://doi.org/10.1093 neuonc/now179.

Kitange, G.J., Carlson, B.L., Schroeder, M.A., Grogan, P.T., Lamont, J.D., Decker, P.A., Wu, W., James, C.D. and Sarkaria, J.N. (2009).

Induction of MGMT expression is associated with temozolomide resistance in glioblastoma xenografts. Neuro-Oncology, 11(3), pp.281 291. doi:https://doi.org/10.1215/15228517-2008-090.

Markert, J.M., Razdan, S.N., Kuo, H.-C., Cantor, A., Knoll, A., Karrasch, M., Nabors, L.B., Markiewicz, M., Agee, B.S., Coleman, J.M., Lakeman, A.D., Palmer, C.A., Parker, J.N., Whitley, R.J., Weichselbaum, R.R., Fiveash, J.B. and Gillespie, G.Y. (2014). A Phase 1 Trial of Oncolytic HSV-1, G207, Given in Combination With Radiation for Recurrent GBM Demonstrates Safety and Radiographic Responses.

Molecular Therapy, 22(5), pp.1048–1055. doi:https://doi.org/10.1038/mt.2014.22.

Mazarakis, N.K., Robinson, S.D., Sinha, P., Christos Koutsarnakis, Spyridon Komaitis, Stranjalis, G., Short, S.C., Chumas, P. and Georgios Giamas (2024). Management of glioblastoma in elderly patients: A review of the literature. Clinical and Translational Radiation Oncology, 46, pp.100761–100761. doi:https://doi.org/10.1016 j.ctro.2024.100761.

Mohammed, S., Dinesan, M. and Ajayakumar, T. (2022). Survival and quality of life analysis in glioblastoma multiforme with adjuvant chemoradiotherapy: a retrospective study. Reports of Practical Oncology and Radiotherapy, 27(6), pp.1026–1036. doi:https://doi.org10.5603/rpor.a2022.0113.

Razavi, S.-M., Lee, K.E., Jin, B.E., Aujla, P.S., Gholamin, S. and Li, G. (2016). Immune Evasion Strategies of Glioblastoma. Frontiers in Surgery, 3. doi:https://doi.org/10.3389 fsurg.2016.00011.

Romanishin, A., Vasilev, A., Khasanshin, E., Evtekhov, A., Pusynin, E., Rubina, K., Kakotkin, V., Agapov, M. and Semina, E. (2024). Oncolytic viral therapy for gliomas: Advances in the mechanisms and approaches to delivery. Virology, 593, pp.110033–110033.doi:https:/ doi.org/10.1016/j.virol.2024.110033.

Rose, L. (2022). QA: Understanding glioblastoma (GBM). MD Anderson Cancer Center. Available at: https://www.mdanderson.org/cancerwise/understanding glioblastoma.h00-158828856.html.

6

Todo, T., Ino, Y., Ohtsu, H., Shibahara, J. and Tanaka, M. (2022). A phase I/II study of triple mutated oncolytic herpes virus G47 in patients with progressive glioblastoma. Nature Communications, 13(1). doi:https://doi.org/10.1038/s41467022-31262-y.

Wu, B., Zhang, B., Li, B., Wu, H. and Jiang, M. (2024). Cold and hot tumors: from molecular mechanisms to targeted therapy. Signal Transduction and Targeted Therapy, 9(1). doi:https://doi.org/10.1038/s41392-024-01979-x.

Xu, J., Liu, X., Ding, J., Zhang, H., Yao, T., Li, S., Yang, R., Yu, N., Yue, Q., Zhan, C. and Gao, X. (2024). Nanosynergist-engineered oncolytic adenovirus enhancing immune virotherapy efficacy for glioblastoma via interrupting antiviral responses. Nano Today, 57, p.102328. doi:https://doi.org/10.1016/j.nantod.2024.102328.

1

Extended Reality (XR) in Exposure Therapy for PTSD: A Rapid Review of Clinical Potential and Limitations Jimin Lim Edited by Sushmhitah Sandanatavan and Alethea Stevenson

Abstract - Post-Traumatic Stress Disorder (PTSD) affects approximately 3.9% of the global population, with significantly higher rates among populations that have been exposed to war or interpersonal violence. While trauma-focused therapies such as exposure therapy are effective, limitations such as poor patient engagement and high dropout rates still persist. Extended Reality (XR), particularly Virtual Reality (VR), has emerged as a novel modality to enhance exposure therapy through provision of immersive, controlled environments for trauma reprocessing.

This rapid review aimsed to evaluate recent literature on XR-based exposure therapy for adults with clinically diagnosed or probable PTSD, assessing both clinical potential and current limitations. A structured search was conducted using OVID MEDLINE in accordance with PRISMA guidelines. Studies were included if they used XR as an intervention for exposure therapy, applied validated PTSD outcome measures, and were published from 2022 onwards. Four studies met the inclusion criteria. All studies utilised VR as the XR modality. VRET was found to be generally feasible, well-tolerated, and associated with short-term reductions in PTSD symptoms.

Studies reported strong patient engagement, particularly through multisensory input. However, several limitations were common: small sample sizes, high dropout rates, lack of control groups, non-standardised protocols, and absence of longterm follow-up. XR-enhanced exposure therapy, especially VRET, demonstrates clinical promise as a feasible and engaging intervention for PTSD. Nonetheless, further research is needed to establish its comparative efficacy, long-term durability, and optimal implementation formats across diverse trauma populations.

I. I NTRODUCTION Post-Traumatic Stress Disorder, otherwise known as PTSD, is defined by ICD-10 as a disorder displayed as a delayed response after a stressful event or situation of varying duration likely to cause significant distress to the majority of people (WHO, 2019). According to the DSM-5 criteria for diagnosis, someone can suffer from PTSD after an experience of direct exposure to a seriously traumatic event or witnessing an event as it happened to others or someone near them, such as family members or friends (APA, 2013). In these cases, individuals will show signs of dissociative reactions, otherwise known as flashbacks, when they feel as if they are experiencing a recurrence of the traumatic event (APA, 2013). Another sign of PTSD can be a continuous avoidance of related factors, such as avoiding talking about the thoughts and feelings felt during the event, and negative reactions such as irritable behaviour or anger (APA, 2013).

An estimated 3.9% of people in the world have suffered from PTSD at a particular stage in their lives (Koenen et al., 2017), and specific populations, such as people who are exposed to violent conflict or war, have three times the amount of people who are diagnosed with PTSD (Charlson et al., 2019).

There are various treatments for PTSD and they can be mainly divided into trauma-focused therapy and symptom-focused therapy (Schrader and Ross, 2021). While symptom-focused therapy is mostly through pharmacotherapy only, traumafocused therapy includes various methods such as cognitive behavioural therapy (CBT), prolonged exposure, written or narrative exposure therapy, and eye movement desensitisation and restructuring (EMDR) (Schrader and Ross, 2021). Among these various treatment options, is the method of exposure

2

therapy which typically involves two key concepts: imaginal exposure, which involves gradually revisiting the event through therapy sessions, and in vivo exposure, which involves real-life scenarios and objects related to the traumatic event (Norr et al., 2019).

However, since extended reality (XR), a rapidly evolving field, was introduced to the field of exposure therapy, the potential for a new treatment option was created. Out of the three terms included in XR, which are virtual reality (VR), augmented reality (AR), and mixed reality (MR) (Becker and Freitas, 2023), VR is seen to be the most commonly used, usually enabled through a head-mounted display (Difede et al., 2022). With this new technology, exposure therapy (ET) seeks to treat PTSD by visualising the actual event that the patients are suffering from, which is a much stronger effect of the imaginal exposure element (Difede et al., 2022). Preliminary studies suggest XR-enhanced exposure therapy may improve patient engagement and reduce avoidance, but the current evidence base remains limited.

This rapid review aims to explore the current literature on XR-based exposure therapy for PTSD by evaluating the design, outcomes, and limitations of existing literature and determining the clinical potential of XR-based exposure therapy and its prospects. To ensure that the current trends are examined, papers published for the past three years only will be selected in this rapid review, and the population will aim to include only patients clinically diagnosed with PTSD to ensure that the effect of VRET can be viewed as objectively as possible.

Inclusion Criteria All types of study methods including: - Pilot studies - Case studies - Case reports - Cohort studies - Longitudinal Studies

Studies that include a specific intervention of XR-based exposure therapy Studies published after 2022

Exclusion Criteria Publication types including: - Meta-analysis - Systematic review - Conference letters - Editorials - Critical reviews - Dissertation thesis - Editorial material - Commentary - Bibliometric analysis Studies that do not use XR for application to exposure therapy, such as for diagnosis purposes only Studies that include exposure therapy as an intervention without XR

Studies that include patients clinically diagnosed with PTSD

Table I: Inclusion and Exclusion Criteria to exposure therapy or set a combination of XR and other treatments for PTSD as their intervention were excluded.

The search string used for the literature search was (PTSD) AND (exposure therapy) AND (virtual reality OR extended reality OR augmented reality OR mixed reality OR xr OR vr OR ar OR mr). The details of the PEO framework used for this review can be seen in Table 2.

Population Adults who are 18 years or older who were clinically diagnosed with PTSD

Exposure XR-based exposure therapy

Outcome Validated PTSD measures e.g. CAPS-5, PCL-5 Secondary outcome only considered if symptom data present

Table II: Selection Criteria according to PEO framework

II. M ETHODS A search was conducted with the PRISMA guidelines using the OVID MEDLINE database, which will be available in the results section of this review.

The inclusion and exclusion criteria for the literature search can be seen in Table 1.

Publication types such as meta-analysis, systematic reviews, and bibliometric analysis were excluded as secondary research papers were not eligible for this rapid review. All primary research types were included in this review, including case studies, case reports, and cohort studies. Furthermore, studies that used XR for purposes other than application

To view the objective effect of the treatment as much as possible, the population was limited to adults who are 18 years or older with a clinical diagnosis of PTSD such as the criteria listed in DSM-5. The outcome was aimed to include the papers that used validated PTSD measures, and the secondary outcome was only considered if data related to PTSD symptoms were present.

III. R ESULTS After input of the search string mentioned above, MEDLINE showed 38 papers published over the

3

past three years. Four studies were selected in total for this rapid review, and the PRISMA flow diagram for this paper can be seen in Figure 1 (Page et al., 2021). The summarized results from

Figure 1: PRISMA Flow Diagram the selected studies can be seen in Table 3 (Found at end of article).

As seen from Table 3 above, all the papers explored virtual reality (VR) as the XR modality applied to exposure therapy in patients with a clinical or probable diagnosis of PTSD. Sample sizes ranged from 9 to 44, with studies based in Denmark, the UK, the USA and Netherlands. Populations consisted of military veterans with combat or MST-related trauma and adults with trauma from childhood sexual abuse. All studies used validated clinical outcome measures such as CAPS-5, PCL, and BDI. IV. D ISCUSSION All four studies show VRET being generally feasible, immersive, and well-tolerated, it can be

deduced from this that XR-based exposure therapy shows potential for clinical application in PTSD management. Furthermore, an overall theme that was seen in all four papers was the positive therapeutic effect of VRET. In Hannigan et al. (2023), some individuals within the dramatic improvement group reported immense emotional relief and improvements in CAPS-5 scores. Loucks et al. (2025) also showed this by showing the dramatic symptom reduction in the case of patients with MST-related PTSD with a drop in PCL-5 points by 32. This showed the potential of VRET for MST-related PTSD, a case where traditional imaginal exposure application is difficult. Although VR was embedded within a broader therapeutic context in this paper, this suggests potential for future use of VRET and for the treatment progression of MST-related PTSD. However, dropout rates were notable in Folke et al.

(2023) and van Meggelen et al. (2022), some of which were suggestive of improvements needed in the VRET delivery method. This highlights the need for variety in the delivery method of VRET, such as fully guided, semi-automated, or hybrid models.

Another area that is highlighted is the need to consider the degree of involvement of the therapist for support during these sessions since they also contribute to a change in the degree of involvement.

The technological details of XR based exposure therapy in all the studies also must be taken into consideration. Surprisingly, in Folke et al. (2023) patients reported that the VR scenario that was created did not properly map the context of their traumatic experience but did not impact their immersion in the scenario. However, in Folke et al. (2023) patients reported that the tactile and auditory stimuli enabled them to engage deeply in the treatment process. Furthermore, in Loucks, Rizzo and Rothbaum (2025), the paper reported that technological glitches occurred which impacted the patients’ VRET therapy sessions and thus became a limitation of their study. This shows that while the details of the VR scenario that patients create with their therapist do not have to be considered in depth, the technological smoothness of VRET needs to be taken into account in future studies to ensure a completely immersive experience. Another factor that needs to be taken into consideration is that VR was the only modality used for exposure therapy in all four papers, future research on whether other types of XR would be effective for exposure therapy

4

should to be undertaken.

Most of the studies also faced some common limitations. None of the studies used blinding designs, three papers lacked control groups, and most of the studies had a small participant sample. Another issue that arose was that the papers had non-standardised protocols, which limited the assessment of the clinical advantage and potential consideration of VRET as an alternative delivery method. Finally, most of the papers did not include long-term follow-ups in their study designs, which raises questions and potential concerns about the long-term effects of VRET and whether the improvements experienced made are temporary or longer term.

Despite these limitations, VRET is feasible and clinically promising in the aspect that it leads to symptomatic development and improvement in coping. In terms of structure, future research should aim towards devising research with a larger participant group that includes a control group, long-term follow-up, and better experimental circumstances such as blinding should be used. Regarding the evolution of VRET, there should be more measures put in to prevent technological glitches that would prevent the patients from complete immersion into the VRET sessions from occurring. Further studies need to prioritise larger, controlled trials while sticking to standardised clinical outcome measures such as PCL-5 and CAPS-5. Finally, considering that most of the VRET trials were undertaken on patients with military-related trauma, future studies need to consider the potential of VRET across patients with different trauma types and consider the suitability of VRET on treatment-resistant PTSD or those with difficulty accessing conventional therapy.

symptoms of their PTSD and in facing in vivo factors related to the traumatic event. However, it has to be remembered that most of these papers did not have a comparator group, a proper blinding process or a long- term follow-up, which should be prioritised in future studies. Furthermore, standardised methods for testing the effectiveness of XR-based exposure therapy alongside clinically validated measurements for outcomes should be the gold standard for future research in this area.

Although more exploration needs to be done in utilizing other XR modalities for exposure therapy, VRET currently suggests a promising future for the treatment of PTSD by providing an immersive experience which the patient’s highlight as a benefit potentially because it is something that imaginary exposure therapy lacks the ability to do. Improving access to XR technologies to help create a basis for future research, utilising other XR modalities for exposure therapy, and evaluation of the long-term durability of treatment effects will be essential in determining the future role of XR-based exposure therapy in PTSD treatment.

VI. R EFERENCES American Psychiatric Association (APA) (2013) Diagnostic and statistical manual of mental disorders. 5th edition. Arlington, VA. Becker, A. and Freitas, C. (2023) ‘Evaluation of XR Applications: A Tertiary Review’, Association for Computing Machinery, 56 (5). doi:10.1145/3626517.

Charlson, F., et al (2019) ’New WHO prevalence estimates of mental disorders in conflict settings: a systematic review and meta-analysis’, Lancet, V. C ONCLUSION 394(10194), pp. 240-248. DOI: 10.1016/s0140This rapid review aimed to evaluate the effec- 6736(19)30934-1. tiveness of XR-based exposure therapy as a treatment for adults with clinically diagnosed or proba- Folke, S., Roitmann, N., Poulsen, S. and Andersen, ble PTSD by doing a literature search using one S. B. (2023) ’Feasibility of Virtual Reality Exposure database following the PRISMA guidelines. Four Therapy in the Treatment of Danish Veterans with studies were selected for review after the literature Post-Traumatic Stress Disorder: A Mixed Method screening process, which all used VR as their main Pilot Study’, Cyberpsychol Behav Soc Netw, 26(6), modality for exposure therapy (VRET). Across all pp. 425-431. DOI: 10.1089/cyber.2022.0236. the studies, VRET proved to be highly engaging for the patients by providing an immersive experience, Hannigan, B., van Deursen, R., Barawi, K., and lead to noticeable improvements in both the Kitchiner, N. and Bisson, J. I. (2023) ’Factors

5

associated with the outcomes of a novel virtual reality therapy for military veterans with PTSD: Theory development using a mixed methods analysis’, PLoS One, 18(5), pp. e0285763. DOI: 10.1371/journal.pone.0285763.

Koenen, K. C., et al (2017) ’Posttraumatic stress disorder in the World Mental Health Surveys’, Psychol Med, 47(13), pp. 2260-2274.

DOI: 10.1017/s0033291717000708.

Loucks, L., Rizzo, A. and Rothbaum, B. O. (2025) ’Virtual Reality Exposure for Treating PTSD Due to Military Sexual Trauma’, Journal of Clinical Psychology, 81(2), pp. 81-92. DOI: https://doi.org/10.1002/jclp.23750.

Page, MJ. et al (2021) ‘PRISMA Flow Diagram’, BMJ, 372. DOI: 10.1136/bmj.n71. van Meggelen, M., et al (2022) ’A randomized controlled trial to pilot the efficacy of a computerbased intervention with elements of virtual reality and limited therapist assistance for the treatment of post-traumatic stress disorder’, Front Digit Health, 4, pp. 974668. DOI: 10.3389/fdgth.2022.974668.

World Health Organisation (WHO) (2019).

Internation statistical classification of diseases and related health problems. 11th edition. Available at: https://icd.who.int/browse10/2019/en#/F43.1

6

Author (Year) Country Folke et al.

(2023) Denmark

Hannigan et al.

(2023) UK

Loucks, Rizzo and Rothbaum (2025) USA

van Meggelen et al.

(2022) Netherland

XR Modality

VR Exposure

VR Exposure

VR Exposure

VR + limited therapistguided program

Sample Size (N)

Population

Measures

Main Outcome

Key Limitation

9 (dropout rate 33%)

Danish military veterans, probable diagnosis of PTSD

CAPS-5, PCL, BDI, WHO-5

Tactile and audio stimuli, confrontational/ engaging nature of VRET and reduced symptoms highlighted

Pilot study, small group, no medication accounted for, no control group, no follow up.

42

UK military veterans with treatmentresistant PTSD

CAPS-5, LEC-5, PHQ-9, GAD-7, MSPSS, WSAS, EQ-5D-5L

Improvement in coping, symptom relief, and emotional regulation (dramatic improvement, moderate improvement and minimal improvement groups)

Mixed methods; lacked long-term outcomes.

PCL-5

Reduced PTSD symptoms (PCL-5 decreased from 49-17), decrease of distress during trauma-related in vivo situations

No comparator group: non-randomized, technological glitches occurred, VR was part of broader therapy.

PCL-5, DES, BDIII, OQ-45-2, MINI

Decreased PTSD and depression symptoms in both 3MR and TAU groups (also in follow up), no significant differences in symptom changes remission rates

No strong control; digital-only limitations, no blinding, high dropout in 3MR group (45%)

20, focus on one patient case

44 (12 in 3MR intervention, 18 in TAU comparator)

US military veteran with PTSD due to military sexual trauma (MST) Adults clinically diagnosed with PTSD from childhood sexual abuse (CSA) and war veterans

Table III: Data Extraction Table: CAPS-5: clinician-administered PTSD scale for DSM-5, MINI: Mini-International Neuropsychiatric Interview, PCL-C: PTSD Checklist-Civilian Version, BDI-II: Beck Depression Inventory-II, WHO-5: WHO-Five Well-being Index, LEC-5: Life Events Checklist for DSM-5, PHQ-9: Patient Health Questionnaire, GAD-7: Generalised Anxiety Disorder, MSPSS: Multidimensional Scale for Perceived Social Support, WSAS: Work and Social Adjustment Scale, EQ-5D-5L: EuroQol five-dimensional descriptive system, OQ-45-2: Outcome Questionnaire wellbeing, DES: Dissociative Experience Scale

1

CRISPR-mediated Reactivation of Foetal Haemoglobin (HbF) as a Sustainable Therapeutic Solution for Beta-Thalassemia Ishani Badala Edited by Sushmhitah Sandanatavan and Alethea Stevenson

Abstract - Beta-thalassemia is a hereditary hemoglobinopathy caused by mutations in the β-globin (HBB) gene, that leads to impaired hemoglobin synthesis, chronic anaemia, and transfusion dependence. Reactivating foetal haemoglobin (HbF; α2 γ2 ) presents a mutationindependent strategy to help compensate for βglobin deficiency. This paper explores the use of CRISPR-Cas9 genome editing to induce HbF expression via two principal approaches: disruption of the erythroid-specific BCL11A enhancer and editing of γ-globin (HBG1/2) promoters to mimic hereditary persistence of foetal haemoglobin (HPFH). Both strategies target hematopoietic stem and progenitor cells (HSPCs) ex vivo, enabling durable HbF production posttransplantation. These therapies are especially valuable for patients with compound heterozygous mutations, as they offer a genotype-agnostic alternative to gene correction. While both approaches are safe and effective, some weaknesses limit current accessibility, particularly in lowresource settings. Future directions include base and prime editing, in vivo delivery, and nongenotoxic conditioning to improve precision, reduce risk, and expand access. CRISPR-mediated HbF reactivation thus represents a potentially curative strategy for β-thalassemia.

I. I NTRODUCTION Beta-thalassemia is an inherited blood disorder which is caused by mutations in the Haemoglobin subunit beta (HBB) gene that codes for the beta(β)-globin protein in β chains. These chains are a main component of adult haemoglobin (HbA) so as a result of this disorder, there is an imbalance between α and β-globin chains

(medlineplus.gov, 2020). This imbalance leads to defective red blood cell (RBC) production, chronic haemolytic anaemia, and other clinical symptoms depending on the genotype of the individual. It is common in the Mediterranean, South Asia, and Middle East, it is a problem in low-and middle-income countries (LMICs), where exposure to treatment is limited. Standard treatments like blood transfusions followed by iron chelation therapy, although effective in alleviating symptoms, are not a sustainable cure, due to risks of organ damage from iron toxicity and alloimmunization (Linder and Chou, 2021). Hematopoietic stem cell transplantation (HSCT)- is the only established curative approach but has limitations such as high costs, donor availability, and the risk of graft-versus-host disease.

Haemoglobin (Hb) primarily functions as an oxygen carrier molecule. It is a quaternary protein composed of four globin subunits that each contain a heme group respectively with an iron (Fe2+) ion at its centre, bound to a porphyrin ring. and primarily functions as an oxygen carrier molecule.

As a foetus Hb is expressed as foetal haemoglobin and contains two alpha and two gamma chainsHbF; α2 γ2 . However, after birth the gamma globin gene is silenced by transcriptional repressors, and beta-globin (HBB) expression is upregulated to produce adult Hb- HbA α2 β2 . In individuals affected by beta-thalassemia, HBB mutations lead to defective or absent β-globin, causing anaemia (Wu et al., 2017). Therefore the β-globin deficiency could be compensated for by reactivating HbF. Some patients with beta-thalassemia have compound heterozygous mutations, meaning they inherit two different allelic mutations in the HBB gene, and so production of beta-globin chains of haemoglobin is affected to varying degrees and

2

thus so is the severity of the beta-thalassemia.

Beta-thalassemia mutations are of two typesβ 0 (beta-zero) and β + (beta-plus) mutations. β 0 completely stops beta-globin production whereas β + mutations reduce beta-globin production. When both inherited alleles are severely affected, for example, β 0 /β 0 , the individual is diagnosed with thalassemia major which requires ring lifelong blood transfusions. When one mutation is milder (β 0 /β + ), it is less severe- thalassemia intermedia.

Lastly, individuals may inherit one normal and one defective HBB gene (β/β + or β/β 0 ), resulting in thalassemia minor, which causes only mild anaemia.

Patients with compound heterozygous mutations are interesting from a therapeutic perspective because they do not require regular transfusions however they still experience significant clinical complications. Due to the disorder’s ranging genotypes, an ideal solution would focus on something aside from the mutated gene. One such solution is reactivation of foetal haemoglobin (HbF) induced by CRISPR-Cas9. HbF forms functional haemoglobin tetramers with alpha chains thereby compensating for the deficient β-globin chains CRISPR-Cas9 disrupts erythroid-specific enhancer of BCL11A- a key transcriptional repressor of HbFand introduces Hereditary persistence of foetal haemoglobin (HPFH) mutations into the γ-globin gene promoters to reactivate foetal haemoglobin expression (Han et al., 2022). Since this does not directly correct the mutation, it could help treat compound heterozygotes and patients with variable genotypes.

Through this report, I aim to explore the potential of CRISPR-mediated induction of HbF via targeted disruption of BCL11A and direct editing of -globin promoters.

II. H OW DOES CRISPR W ORK ?

CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats) is derived from an adaptive immune response found in bacteria where Cas9 protein activity protects the cell from external pathogens upon infection with phages (bacteriophages) from viruses. When Cas9 protein is associated with CRISPR, it functions as an endonucleasean enzyme that cuts DNA.

If a bacterial cell survives phage infection, it cuts a portion of the invading pathogen’s DNA and inserts it into its own genome at the CRISPR locus region. This region is composed of repeated sequences integrated with inserts of viral sequences called ‘spacers’. If the bacterial cell is infected with the same phage again, it can transcribes the CRISPR region into an RNA strand which is further processed to form smaller guide RNAs. Guide RNAs (gRNA) are synthetic RNA molecules that match specific DNA sequences in the target genome. They comprise of a crRNA which binds to the DNA target, and a tracrRNA which binds to Cas9. gRNAs form complementary base pairs with the viral DNA sequence to induce Cas protein’s nuclease activity, thus allowing it to cut the foreign DNA and resolve the infection before replication occurs. Cas9 enzyme binds to the gRNA and cuts both strands of the viral DNA sequence at complementary base pairs to the target sequence, adjacent to the PAM (Protospacer Adjacent Motif) which is a 3nucleotide motif. Following the cutting of DNA, the cell repairs the break using either Non-Homologous End Joining (NHEJ) which ligates the DNA ends directly, introducing deletions or insertions (InDels) and disrupting gene function; or through HomologyDirected Repair (HDR) which uses a homologous DNA template to repair the break.

III. M ECHANISM OF H B F R EPRESSION AND R EACTIVATION A. THe Role of BCL11A and its Erythroid Enhancer BCL11A (B-cell lymphoma/leukaemia 11A) is a zinc-finger transcription factor that binds directly to promoter regions of γ-globin genes and recruits co-repressors (NuRD- nucleosome remodelling and deacetylase complex) to repress HbF transcription through histone deacetylation, condensation, and DNA methylation in adult erythrocytes. BCL11A expression in RBC precursors is regulated by the erythroid enhancer in intron 2 of the BCL11A gene. The enhancer is bound by transcription factors (GATA1, TAL1, KLF1) which activate it only in erythroid cells; and disrupting this enhancer via CRISPR Cas9 therefore stops BCL11A expression in RBCs, but preserves its function in other tissues, thus making it an ideal target in gene therapy.

3

B. HbF reactivation via BCL11A Erythroid En- γ-globin transcription and subsequently HbF levhancer Disruption- The CTX001 Trial els, replicating the HPFH phenotype (Khosravi et This CRISPR approach is used in the CTX001 al., 2019). CRISPR is also introduced into patient Trial conducted by CRISPR therapeutics and Vertex HSPCs by ex-vivo editing as described in the prePharmaceuticals to treat patients with transfusion- vious section. dependent Beta-thalassemia and sickle cell disease (Frangoul et al., 2020). CRISPR-Cas9 was used to disrupt the erythroid-specific enhancer in intron 2 of BCL11A in autologous CD34+ hematopoietic stem and progenitor cells (HSPCs) (Frangoul et al., 2020).

Patients’ HSPCs are collected by apheresis and edited ex-vivo. In this editing, a CRISPR-cAS9 ribonucleoprotein (RNP) complex was introduced into cells via electroporation, a technique that uses a short electrical pulse to open pores in the plasma membrane to allow the RNP complex to enter. The RNP complex is made up of the Cas9 protein and a gRNA. The double strand break (DSB) introduced

by Cas9 in the BCL11A erythroid enhancer in intron 2 disrupts the enhancer, downregulating BCL11A in RBCs, thus reactivating HbF (Frangoul et al., 2020). After completion of editing, myeloablative conditioning is done and the patient is administered a chemotherapy drug- busulfan, that wipes out bone marrow cells creating space for edited cells to engraft. The patient is then reinfused intravenously with edited CD34+ cells which start producing RBCs with high levels of HbF in the bone marrow.

C. HbF Reactivation via Editing the GammaGlobin Promoter, Point Mutations Mimicking HPFH The second approach of CRISPR-Cas9 involves editing the gamma-globin promoter. Hereditary Persistence of Foetal Haemoglobin (HPFH) is a benign condition caused by point mutations in the γ globin promoter (region 200 to 115) disrupting binding sites for BCL11A, thus preventing repression of γglobin, causing HbF levels to be elevated throughout life (Khosravi et al., 2019).

CRISPR introduces these mutations into the HBG1 and HBG2 promoter regions using Cas9, a repair template (HDR- homology-directed repair) and base editors which cause point mutations, i.e., change a single nucleotide without introducing any double strand breaks (Khosravi et al., 2019). These mutations remove or weaken the repressor binding sites as in HPFH, enabling transcriptional activators (GATA1, NF-γ) to bind to the promoter and increase

IV. C OMPARING THE E FFICACY OF BCL11A E NHANCER D ISRUPTION VERSUS γ- G LOBIN P ROMOTER E DITING IN THE C ONTEXT OF H B F I NDUCTION Both CRISPR approaches aim to increase expression of foetal haemoglobin (HbF) to compensate for the β-globin deficiency in beta-thalassemia.

However, they differ in terms of their molecular targets, editing mechanism, efficiency in HSPCs, and translational capacity.

BCL11A disrupts the erythroid enhancer which drives BCL11A expression in RBC precursors via interactions with transcription factors such as GATA1 and KLF1. Cas9 disrupts this enhancer using non-homologous end joining (NHEJ) and thus relieves repression of the γ-globin genes HBG1/2 by reducing BCL11A expression in RBCs. Results from the CTX001 trial referenced above showed an increase in HbF levels by greater than 40% and transfusion independence in the majority of treated patients (Wang, Siwko and Li, 2023) (Khosravi et al., 2019).

Contrastingly, γ-globin promoter editing induces HPFH point mutations directly into the HBG1/1 promoter regions (Benjamin, 2023). These edits disrupt the binding motifs for repressors like BCL11A, enhancing γ-globin transcription. To minimise genotoxicity, editing is done using base or prime editors thus avoiding DSBs. However, this method is not often used clinically due to the challenges associated with editing HSPCs such as how they are largely quiescent and resistant to homology-directed repair (HDR), additionally base editing efficiency in these cells is yet to be tested in humans.

BCL11A downregulates a master repressor causing epigenetic changes across the β-globin locus and activation of both HBG1 and HBG2. This causes erythroid cells to shift to a foetal gene expression profile. The γ-globin promoter however is locus specific and relies on the availability of existing chromatin and transcription factors at the promoter.

This leads to variability in expression in patients (Khosravi et al., 2019) (Benjamin, 2023).

4

V. R ELEVANCE TO C OMPOUND H ETEROZYGOTES

indicating that gene-edited autologous grafts can effectively supplant donor transplants from a clinical standpoint (www.aabb.org, 2022). Notably, because the patient’s own cells are used, the risk of graft rejection and chronic immunosuppression is essentially removed. Despite this, it is important to acknowledge that the CRISPR approach still requires myeloablation and transplantation of stem cells, which carry similar upfront risks and resource demands alike to those of BMT. Moreover, due to the aforementioned cost and infrastructure issues, allogeneic BMT (when a suitable donor exists) currently remains the more accessible option in many settings. Thus, in high-resource settings, CRISPR-based HbF induction is poised to gradually replace BMT for those without donors or those at high risk of transplant complications. But in low-resource settings, traditional transplantation (and palliative care) will likely continue until gene editing therapies become more affordable and simplified. In summary, while CRISPR-mediated HbF reactivation has clinically proven it can play the same curative role as a bone marrow transplant, its real-world replacement of BMT will depend on overcoming significant economic and logistical problems.

Patients with compound heterozygous beta thalassemia inherit two mutations in the HBB gene, and as a result they often have little or no adult β-globin production. This leads to a severe α-globin chain excess, these unpaired α chains precipitate in erythroid precursors and cause their premature death (Papizan et al., 2021). Elevating foetal haemoglobin (HbF, α2 γ2 ) can compensate for the β-globin shortfall by providing γ-globin chains that pair with α chains, forming functional HbF tetramers. This reduces the toxic α-globin surplus and ameliorates ineffective erythropoiesis (Rahimmanesh et al., 2022). Notably, reactivating HbF benefits patients regardless of the specific HBB mutations present , because it bypasses the need for any functional β-globin. CRISPR targets a common regulatory element rather than each patient’s unique mutation, making it broadly applicable. For example, BCL11A enhancer disruption focuses on an erythroid-specific consensus motif, so one gRNA can be used as a universal treatment for diverse β-thalassemia genotypes (Papizan et al., 2021).

Similarly, creating small deletions or HPFH-like point mutations in the HBG promoters prevents repressors like BCL11A or ZBTB7A from binding, VI. P OINT OF E XPLORATION AND F UTURE thus leading to HbF re-expression (Rahimmanesh D IRECTIONS et al., 2022). The induced γ-globin compensates for To make CRISPR cures more effective, accessidefective β-chains on both alleles. This approach also addresses α-globin chain imbalance by ble, and equitable the following could be considproviding alternate binding partners for excess α ered: chains,[I don’t think this next bit is clear] much as 1. In vivo genome editing: One transformative co-inheritance of α-thalassemia is known to reduce goal is to deliver gene editing tools directly free α-globin and improve beta-thalassemia balance into the patient’s body, which would allow for (VOON, WARDAN and VADOLAS, 2007). editing of blood stem cells in situ. In vivo A critical question is whether CRISPR therapies editing (for example, via injectable nanoparcan replace allogeneic bone marrow transplants as ticles or viral vectors) could reduce the need the standard curative approach for β-thalassemia. for bone marrow harvest and transplantation Allogeneic HSPC transplant is limited by donor (Molteni, 2023a). availability and risks such as graft-vs-host disease 2. Base editors: As explained earlier in gamma(Everette et al., 2023). In practice, fewer than 20% globin promoter editing, there are genome of patients have an HLA-matched sibling donor editors being developed that are able to make available (O’Hanlon Cohrt, 2020). CRISPR-based more precise DNA changes without creating therapy offers an autologous alternative – the double-strand breaks. Base editors (such as patient becomes their own donor, avoiding immune adenine or cytosine base editors) can conincompatibility. In trials, the efficacy of exa-cel in vert one nucleotide to another in a targeted eliminating transfusion needs and disease symptoms manner. One application for this is to introhas matched or exceeded what is seen with BMT, duce benign hereditary persistence of foetal

5

haemoglobin (HPFH) mutations: for example, Beam Therapeutics’ BEAM-101 uses a base editor to alter a single base in the γglobin promoters, preventing BCL11A binding and thus elevating HbF, all without cutting the DNA (Base editing boosts hemoglobin in SCD, 2024). Early clinical data for base editing in sickle cell disease is encouraging – in an ongoing Phase 1/2 trial, BEAM101 treated patients have shown ¿60% HbF in their blood and no vaso-occlusive crises, even at just 1–6 months post-treatment (Base editing boosts hemoglobin in SCD, 2024).

This suggests base editors can achieve at least comparable (potentially even higher) foetal haemoglobin induction with possibly fewer off-target concerns than CRISPR nuclease approaches.

3. Safer conditioning methods: An active area of research is finding alternatives to toxic chemotherapy (busulfan) for conditioning.

The current CRISPR therapy still relies on chemo-based myeloablation to allow edited cells to engraft, which can cause significant side effects (one patient in a base editing trial died from complications related to conditioning, not the gene edit itself (Base editing boosts hemoglobin in SCD, 2024)). Future regimens may look to use gentler nongenotoxic conditioning – for example, monoclonal antibodies targeting stem cell niches (such as anti-CD117) are being tested to clear bone marrow in a more selective way (Base editing boosts hemoglobin in SCD, 2024). In one approach, HSCs are edited to be resistant to an anti-CD117 antibody, so that administering the antibody selectively depletes only the patient’s unedited cells, making space for edited ones (Base editing boosts hemoglobin in SCD, 2024).

4. Ethical and regulatory considerations: One concern is long-term safety and monitoring – since CRISPR-edited cells could persist for life, patients must be followed for years to detect any late-occurring effects (such as clonal proliferation or unanticipated mutations). Patients need to understand the potential benefits and uncertainties associated with this; for example they should consider the potential unknown long-term effects or the

fertility implications of chemotherapy). There is also a broader ethical boundary to maintain as, while these therapies edit somatic cells, public concern remains high regarding any genetic manipulation.

5. HSPC Editing Considerations: In practice, patient CD34+ hematopoietic stem and progenitor cells (HSPCs) are collected, CRISPRedited ex vivo, and then reinfused after myeloablative conditioning (Papizan et al., 2021). A key consideration is maintaining HSPC potency and multi-lineage capacity after editing in other lineages. BCL11A, for instance, is critical in B-lymphoid and neural development, and germline BCL11A knockout is lethal with HSC and neurologic defects (Rahimmanesh et al., 2022). Thus, rather than silencing BCL11A throughout the body, CRISPR targets an enhancer active only in erythroid cells, leaving BCL11A intact elsewhere (Rahimmanesh et al., 2022). Edited HSPCs can then differentiate normally.

VII. C ONCLUSION CRISPR-induced reactivation of foetal haemoglobin is emerging as a potentially curative strategy for β-thalassemia and related disorders.

By upregulating the body’s own foetal-stage haemoglobin, this approach bypasses genetic defects in the β-globin gene – an especially important advantage for compound heterozygous patients, who carry diverse mutations that are difficult to correct with a single conventional therapy (Molteni, 2023). The significance for compound heterozygotes (and indeed, most β-thalassemia genotypes) is huge as it not only improves patients’ quality of life but also addresses the underlying disease in a sustainable way, potentially reducing the burden on healthcare systems in the long run. In summary, CRISPR-mediated HbF reactivation’s success in β-thalassemia showcases how precise gene editing can achieve outcomes unattainable with drugs alone. While challenges of cost, accessibility, and long-term consequences remain, the progress thus far confers great potential and it will likely serve as a model for future genetic therapies.

6

VIII. R EFERENCES

horizon, sickle cell patients ask hard questions about who can access them. [online] STAT. Han, Y., Tan, X., Jin, T., Zhao, S., Hu, L., Available at: https://www.statnews.com/2023/03/0 Zhang, W., Kurita, R., Nakamura, Y., Liu, 7/crispr-sickle-cell-access/.

J., Li, D., Zhang, Z., Fang, X. and Huang, S. (2022).

CRISPR/Cas9-based multiplex Papizan, J.B., Porter, S.N., Sharma, A. genome editing of BCL11A and HBG efficiently and Pruett-Miller, S.M. (2021). Therapeutic induces fetal hemoglobin expression. European gene editing strategies using CRISPR-Cas9 Journal of Pharmacology, 918, p.174788. for the β-hemoglobinopathies. Journal of doi:https://doi.org/10.1016/j.ejphar.2022.174788.

Biomedical Research, [online] 35(2), pp.115–134. doi:https://doi.org/10.7555/JBR.34.20200096.

Linder, G.E. and Chou, S.T. (2021). Red cell transfusion and alloimmunization in Base editing boosts hemoglobin in sickle cell sickle cell disease. Haematologica, 106(7). disease. (2024). Nature Biotechnology, 42(12), doi:https://doi.org/10.3324/haematol.2020.270546. pp.1759–1759. doi:https://doi.org/10.1038/s41587 -024-02517-4. medlineplus.gov. (2020). Beta thalassemia: MedlinePlus Genetics. [online] Available at: www.aabb.org. (2022). Gene Therapy Prevents https://medlineplus.gov/genetics/condition/beta-tha Vaso-Occlusive Crises in Patients With SCD. lassemia/#description. [online] Available at: https://www.aabb.org/news-r esources/news/article/2024/05/14/gene-therapy-pre Wu, L., Peng, Z., Lu, S., Tan, M., Rong, Y., vents-vaso-occlusive-crises-in-patients-with-scd.

Tian, R., Yang, Y., Chen, Y. and Chen, J. (2017). β-thalassemia caused by compound Khosravi, M.A., Abbasalipour, M., Concordet, heterozygous mutations and cured by bone J.-P., Berg, J.V., Zeinali, S., Arashkia, A., marrow transplantation: A case report. Molecular Azadmanesh, K., Buch, T. and Karimipoor, Medicine Reports, 16(5), pp.6552–6557. M. (2019). Targeted deletion of BCL11A gene doi:https://doi.org/10.3892/mmr.2017.7476.

by CRISPR-Cas9 system for fetal hemoglobin reactivation: A promising approach for gene Frangoul, H., Altshuler, D., Cappellini, M.D., therapy of beta thalassemia disease. European Chen, Y.-S., Domm, J., Eustace, B.K., Foell, J., journal of pharmacology, 854, pp.398–405. de la Fuente, J., Grupp, S., Handgretinger, R., doi:https://doi.org/10.1016/j.ejphar.2019.04.042.

Ho, T.W., Kattamis, A., Kernytsky, A., LekstromHimes, J., Li, A.M., Locatelli, F., Mapara, M.Y., Wang, L., Siwko, S. and Li, D. (2023). Waking up de Montalembert, M., Rondelli, D. and Sharma, the silenced beauty: CRISPR/Cas9 mediated reacA. (2020). CRISPR-Cas9 Gene Editing for tivation of fetal hemoglobin genes to treat severe Sickle Cell Disease and β-Thalassemia. New beta-thalassemia in young patients. Life Medicine.

England Journal of Medicine, [online] 384(3). doi:https://doi.org/10.1093/lifemedi/lnad009. doi:https://doi.org/10.1056/nejmoa2031054.

Benjamin, H. (2023). CRISPR-Cas9 promoting Rahimmanesh, I., Boshtam, M., Kouhpayeh, fetal hemoglobin production improves severe sickle S., Khanahmad, H., Dabiri, A., Ahangarzadeh, cell disease. [online] 2 Minute Medicine. Available S., Esmaeili, Y., Bidram, E., Vaseghi, G., at: https://www.2minutemedicine.com/crispr-cas9-p Haghjooy Javanmard, S., Shariati, L., Zarrabi, romoting-fetal-hemoglobin-production-improves-s A. and Varma, R.S. (2022). Gene Editing- evere-sickle-cell-disease.

Based Technologies for Beta-hemoglobinopathies Treatment. Biology, [online] 11(6), p.862. VOON, H., WARDAN, H. and VADOLAS, J. doi:https://doi.org/10.3390/biology11060862.

(2007). Co-inheritance of α- and β-thalassaemia in mice ameliorates thalassaemic phenotype. Blood Molteni, M. (2023). With CRISPR cures on Cells, Molecules, and Diseases, 39(2), pp.184–188.

7

doi:https://doi.org/10.1016/j.bcmd.2007.01.006.

Vertex Pharmaceuticals Newsroom. (2024).

Vertex Presents Positive Long-Term Data On CASGEVYTM (exagamglogene autotemcel) at the American Society of Hematology (ASH) Annual Meeting and Exposition and Provides Program Update — Vertex Pharmaceuticals Newsroom. [online] Available at: https://news.vrtx.com/news-r eleases/news-release-details/vertex-presents-positiv e-long-term-data-casgevytm-0.

BSH. (2024). Trial results for gene therapy for sickle cell and thalassaemia published. [online] Available at: https://b-s-h.org.uk/about-us/news/tri al-results-for-gene-therapy-for-sickle-cell-and-tha lassaemia-published . Everette, K.A., Newby, G.A., Levine, R.M., Mayberry, K., Jang, Y., Mayuranathan, T., Nimmagadda, N., Dempsey, E., Li, Y., Bhoopalan, S.V., Liu, X., Davis, J.R., Nelson, A.T., Chen, P.J., Sousa, A.A., Cheng, Y., Tisdale, J.F., Weiss, M.J., Yen, J.S. and Liu, D.R. (2023). Ex vivo prime editing of patient haematopoietic stem cells rescues sickle-cell disease phenotypes after engraftment in mice.

Nature Biomedical Engineering, [online] pp.1–13. doi:https://doi.org/10.1038/s41551-023-01026-0

1

PSMA – From Diagnosis to Treatment: The Key to Prostate Cancer Management?

Ridhima Ranade Edited by Sushmhitah Sandanatavan and Louisa Willan

I. I NTRODUCTION Cancer is currently the second leading cause of death globally, with incidence continuing to rise. It places an imposing burden on healthcare infrastructures and creates profound psychosocial and economic challenges for patients and their families (1).

In the UK, prostate cancer (PCa) is the most commonly diagnosed cancer in men, with approximately 55,000 cases diagnosed annually (2). This is of increasing concern, as incidence rates are projected to rise by 15% over the next 15 years (2). PCa presents with considerable heterogeneity, including differences across ethnic groups, phenotypic traits, grading and clinical progression. Therefore, it is imperative that robust diagnostic techniques are used after screening to confirm the presence of disease and determine its clinical significance. Prostatespecific membrane antigen (PSMA) positron emission tomography (PET) is a promising imaging technique with greater specificity and sensitivity in diagnosis than conventional imaging methods. Its use can help avoid unnecessary biopsies or potential overdiagnosis (3). This rapid review explores how PSMA PET can enhance PCa management by supporting diagnosis, staging, detection of recurrence, and therapeutic response, while also highlighting areas where further improvement is needed.

Currently, there is no single definitive test or screening method for the diagnosis of PCa. While the prostate-specific antigen (PSA) test may be useful in flagging up early PCa cases, its results are often inconclusive. For instance, elevated PSA levels can also be indicative of benign prostatic enlargement, a common and non-threatening condition. Therefore, PSA testing alone is not recommended as a screening tool. After a patient presents with symptoms, typically of a genitourinary nature, PSA blood tests, digital rectal examinations, MRI scans and biopsies can all be used to investigate the

presence of PCa (4).

To avoid overtreatment, PCa management is approached through a prognosis-focused lens that prioritises symptom control and quality of life over curative treatment, particularly in patients with localised cancer (5). However, where progressive disease is suspected, active surveillance through PSA monitoring and repeat prostatic biopsy is required. Treatment options, when indicated, include chemotherapy, radiotherapy, hormonal therapy, and radical methods such as prostatectomy, external beam radiotherapy, and brachytherapy (6).

An imaging technique gaining clinical adoption in nuclear medicine is PSMA PET, which relies on visualisation through radiotracers binding to PSMA expressed on PCa cells.

II. PSMA’ S VIABILITY AS A TARGET Prostate-specific membrane antigen (PSMA) is a membrane glycoprotein that is expressed 1001000 times more abundantly in prostatic carcinoma cells than in normal cells (7). Its exact function in prostatic epithelium is unknown; however, the protein is known to carry out various enzymatic functions, particularly associated with an internalisation signal (8). A study by Bostwick et al. examining PSMA immunohistochemical expression in 184 samples found a positive correlation with cancer severity. PSMA expression increased progressively from benign prostatic hyperplasia to high-grade prostatic intraepithelial neoplasia and was highest in prostatic adenocarcinoma, with staining rates rising from 69.5% in benign cases to 80.2% in the most aggressive cancers (9). Overexpression occurs in 95% of prostate cancer cells, making PSMA an attractive target for radiotracers which can then be detected by PET. In the UK, for diagnostic purposes, Gallium-68 and Fluorine-18 PSMA radiotracers are available through NHS England for men with highrisk primary prostate cancer when the results from

2

standard imaging techniques are inconclusive or when biochemical recurrence occurs after radical therapy (10). Biochemical recurrence is defined as an increase in prostate-specific antigen, occurring months or even years before recurrent disease becomes visible on standard imaging modalities (10, 11).

III. T HE USE OF PET/CT When compared with standard imaging techniques in isolation such as MRI, CT and bone scintigraphy, PSMA PET provides greater sensitivity and specificity, even when disease burden is low (3). It is particularly useful for staging high-risk primary PCa and for identifying spread to lymph nodes or bone, as these patients are at increased risk of metastasis. Detection of these ‘occult metastases’ is crucial for timely intervention and can improve patient outcomes. PSMA PET can also guide biopsies, which is especially valuable in biopsy-naı̈ve patients with clinical suspicion of disease, and provides additional benefit in repeatbiopsies following initial negative or inconclusive results (3, 11).

The first step in generating a PSMA PET image is the injection of a radiotracer. This binds to its target, PSMA, and emits positrons, which generate photons upon reacting with electrons in the body.

The resulting photon pairs, generated through a process termed annihilation, are detected by the PET scanner. Scanning proceeds from mid-thigh to the base of the skull, generating an image that shows radiotracer distribution throughout the body.

Fusion of this modality with an anatomical imaging technique, such as computed tomography (CT) allows detailed visualisation of radiotracer activity (12). Areas where radiotracers are highly concentrated, known as ‘hot spots’, indicate greater uptake due to abundant biomolecular targets or elevated metabolism and may reflect pathological activity (12).

Among patients who have undergone radical prostatectomy (i.e., curative treatment), approximately 5-20% continue to exhibit PSA levels above the critical threshold four to eight weeks after surgery (PSA ≥ 0.1 ng/mL) (3, 11). This may indicate incomplete tumour removal or previously undetected metastatic disease (3, 11). Even at low PSA levels (< 0.2 ng/mL), PSMA PET can detect

tumours in 33% of cases. This high sensitivity is particularly valuable for patients with biochemical recurrence, as it can help guide more precise treatment (3, 11).

IV. T HERANOSTICS PSMA is not only a useful target for imaging but can also play a therapeutic role. Theranostics is an innovative approach to PCa that aims to both diagnose and locate the tumour, as well as deliver targeted radiation using radioisotopes. A radioligand is selected to target PSMA and is first labelled with a diagnostic radioisotope, followed by a therapeutic radioisotope. Locating the tumour relies on the detection of positron emissions via PET scanning, whereas precision radiotherapy relies on alpha- or beta-emitting radionuclides. These emissions differ in their cytotoxic properties, enabling the selection of a radionuclide tailored to the tumour’s size and distribution. Alpha particles induce double-stranded DNA breaks and have a higher linear energy transfer (LET) over a shorter range (50–80 µm), making them effective at selectively irradiating target tissue while minimising damage to surrounding healthy tissue (13). Beta particles, by contrast, induce single-stranded DNA breaks and have a lower LET over a longer range (0.2–12 mm), making them more suitable for larger tumours (13).

Although radioligand therapy is not yet widely available in the UK, Lutetium-177, a β-emitting radioisotope, has shown promise in treating metastatic castration-resistant prostate cancer (mCRPC) and gained FDA approval in 2022. Ongoing trials continue to investigate its efficacy both as a standalone treatment and in combination with other therapies (14).

V. L IMITATIONS The PET/CT provides critical, life-saving diagnostic information; however, it is not without limitations. Each scan exposes patients to ∼25 mSv of radiation, equivalent to eight years of natural (background) radiation. Greater radiation exposure increases the risk of developing cancer, so thorough clinical justification is essential (15).

PSMA, the primary target in diagnostics and therapeutics, is not exclusive to prostatic epithelium.

PSMA overexpression is also observed in tumours

3

in other parts of the body, such as renal cell carcinomas, gliomas, and hepatocellular carcinomas, raising the risk of potential overdiagnosis (16). Some prostate cancers may not express PSMA and may therefore remain undetected by this imaging modality (17). Additionally, the lack of a global, standardised protocol for reporting represents another limitation. Theranostic agents targeting PSMA have been shown to reduce pain and extend progressionfree survival. However, due to PSMA’s expression in non-prostate tissues, off-target uptake in the salivary glands remains a significant concern with this approach (14).

VI. C ONCLUSION PSMA PET imaging and theranostic treatments have greatly improved the diagnosis and management of prostate cancer by enabling more accurate detection and personalised therapy. However, challenges such as radiation risks, off-target effects, and inconsistent reporting must still be addressed before these approaches can be implemented routinely. As treatments such as Lutetium-177 become more widely available, further research and the development of clear guidelines will be essential to ensure these advances benefit patients safely and effectively.

VII. R EFERENCES 1. Organization WH. Cancer 2025 [Available from: https://www.who.int/health-topics/cancer# tab=tab 1.

2. UK CR. Prostate cancer statistics 2024 [Available from: https://www.cancerresearchuk. org/health-professional/cancer-statistics/ statistics-by-cancer-type/prostate-cancer#prostate stats0.

3. von Stauffenberg F, Poyet C, Beintner-Skawran S, Maurer A, Schmid FA. Current Clinical Applications of PSMA-PET for Prostate Cancer Diagnosis, Staging, and Treatment. Cancers (Basel) [Internet].

2024 [cited; 16(24). Available from: 10.3390/cancers16244263.

4. NHS. Prostate cancer 2024 [Available from: https://www.nhs.uk/conditions/prostate-cancer/#: ∼:text=Tests%20for%20prostate%20cancer,a% 20biopsy 5. (NHS) NHS. Prostate cancer – Treatment 2021 [Available from: 18 October 2021.

6. (NICE) NIfHaCE. Prostate cancer – Management 2024 [updated 04-2025. Available from: https://cks.nice.org.uk/topics/prostate-cancer/ management/management/.

7. Ristau BT, O’Keefe DS, Bacich DJ. The prostate-specific membrane antigen: Lessons and current clinical implications from 20 years of research. Urologic Oncology: Seminars and Original Investigations [Internet]. 2014 [cited; 32(3):2729. Available from: https://doi.org/10.1016/j.urolonc.

2013.09.003.

8. Bařinka C, Rojas C, Slusher B, Pomper M. Glutamate carboxypeptidase II in diagnosis and treatment of neurologic disorders and prostate cancer. Curr Med Chem [Internet]. 2012 [cited; 19(6):856-70. Available from: 10.2174/092986712799034888.

9. Bostwick DG, Pacelli A, Blute M, Roche P, Murphy GP. Prostate specific membrane antigen expression in prostatic intraepithelial neoplasia and adenocarcinoma: a study of 184 cases.

Cancer [Internet]. 1998 [cited; 82(11):225661.

Available from: 10.1002/(sici)10970142(19980601)82:11¡2256::aid-cncr22¿3.0.co;2-s.

10. England N. Commissioning Policy: PSMA PET/CT Imaging for Individuals with High-Risk Primary or Recurrent Prostate Cancer. NHS England; 2025 [cited 30-05-2025].

11. Palot Manzil FF, Kaur H, Szabados L. Gallium-68 Prostate-Specific Membrane Antigen Positron Emission Tomography: A Practical Guide for Radiologists and Clinicians. Cureus [Internet]. 2022 [cited; 14(3):e22917. Available from: 10.7759/cureus.22917.

12. Imaging SoNMaM. Molecular Imaging and Prostate Cancer [Available from: https://snmmi.org/Patients/Patients/Fact-Sheets/ Molecular-Imaging-and-Prostate-Cancer.aspx.

13. Bauckneht M, Ciccarese C, Laudicella R, Mosillo C, D’Amico F, Anghelone A, et al. Theranostics revolution in prostate cancer: Basics, clinical applications, open issues and future perspectives. Cancer Treatment Reviews [Internet]. 2024 [cited; 124:102698. Available from: https://doi.org/ 10.1016/j.ctrv.2024.102698.

14. AlSadi R, Bouhali O, Dewji S, Djekidel M. 177Lu-PSMA Therapy for Metastatic Castration-Resistant Prostate Cancer: A MiniReview of State-of-the-Art. Oncologist [Internet].

4

2022 [cited; 27(12):e957-e66. Available from: 10.1093/oncolo/oyac216.

15. Society AC. Understanding Radiation Risk from Imaging Tests. 2018 [cited 30-062025]. Available from: https://www.cancer. org/cancer/diagnosis-staging/tests/imaging-tests/ understanding-radiation-risk-from-imaging-tests. html.

16. Van de Wiele C, Sathekge M, de Spiegeleer B, de Jonghe PJ, Beels L, Maes A. PSMA-Targeting Positron Emission Agents for Imaging Solid Tumors Other Than Non-Prostate Carcinoma: A Systematic Review. Int J Mol Sci [Internet]. 2019 [cited; 20(19).

Available from: 10.3390/ijms20194886.

17. Hope TA AR, Chee B, et al. PSMA PET in Prostate Cancer — From Approval to Appropriate Use. JAMA Oncology [Internet]. 2023 [cited; 9(5):637-8. Available from: 10.1001/jamaoncol.2023.0191.

1

The Use of AI and 3D Printing in Personalised Paediatric Cardiac Surgery Samuth De Silva Edited by Sushmhitah Sandanatavan and Louisa Willan

I. A BSTRACT Congenital heart defects (CHD) are the most common birth defect, appearing around 0.9% to 1.2% worldwide. These cases are often unique and complex due to anatomical variations between each infant’s heart and are often associated with lifelong comorbidities that reduce the quality of life. Recently, the rise of personalised paediatric cardiac surgery has been transformative in the management of congenital heart defects. Advancements in artificial inteligence (AI) and 3D printing have enhanced planning and accuracy of surgeries, resulting in improved patient outcomes.

AI facilitates diagnosis, predictive risk evaluation and decision-making within multidisciplinary teams. Meanwhile, 3D printing can be used to create patient-specific cardiac models to visualise, plan and prepare for surgeries. Despite growing interest, evidence supporting the use of these technologies to personalise care for individuals remains limited.

Current literature focuses on the accuracy and utility of 3D-printed models, along with emerging preliminary data on the use of AI in surgical planning. A deeper understanding of the quantitative benefits of these technologies has the potential to transform the management of congenital heart defects, improve surgical precision and enhance patient outcomes.

II. I NTRODUCTION 3.12 million babies were born with congenital heart conditions in 2019, with a total of 13.3 million individuals living with the condition (1). In the field of congenital heart disease, the use of AI has facilitated segmentation of cardiac structures by analysing echocardiograms, MRI and CT, thereby improving diagnostic accuracy (2). AI predictive models have demonstrated superior performance compared to traditional risk stratification

tools which rely on expert opinion, and are inherently subjective (3). By incorporating complex, high-dimensional data, AI models can generate evidence-based predictions for individual patients with high accuracy (3).

The emergence of 3D printing enables the use of common imaging modalities to provide clinicians with a spatial understanding of the complex cardiac anatomy in infants affected by CHD (4).

This increased spatial resolution can help with more difficult cases that involve more delicate structures such as valves and the atrial septum (4). This study aims to evaluate the clinical impact of AI-driven segmentation, AI predictive models and 3D printing in paediatric cardiac surgery.

III. C LINICAL NEED : CHD OVERVIEW AND SURGICAL CHALLENGES

Congenital heart defects represent a broad spectrum of structural abnormalities involving the heart and great vessels. Defects of the atrial and ventricular septa are the most common (5). Diagnosis relies on multimodality imaging, often beginning prenatally. Early detection and planning enable appropriate management of delivery, early interventions, and, in some cases, informed decisions regarding termination of pregnancy (5). The primary diagnostic tool is echocardiography, which is used in 90% of CHD cases. It provides a detailed view of the anatomy of the heart across various imaging planes.

This includes axial, sagittal, and long-axis views, needed for diagnosis, monitoring, and follow-up (6).

Prenatal diagnosis has been shown to significantly improve outcomes for infants requiring immediate post-birth care, leading to better blood pH, oxygen levels and a lower risk of severe organ damage (6). Despite advancements in imaging and surgical techniques, the management of CHD remains challenging due to the intricate and highly variable

2

anatomy of the paediatric heart, which requires a surgical approach tailored to each patient (7).

These surgeries have their own peri-operative risks associated with alterations in intracardiac shunting and vascular physiology, which can increase the risk of premature death (8, 9). Addressing these surgical challenges is important for reducing mortality and improving the long-term outcomes of children with congenital heart defects. The implementation of AI and 3D printing can help optimise surgical planning and address many of these challenges with greater precision and accuracy.

IV. AI AND 3D PRINTING APPLICATIONS IN CHD A. Image segmentation and predictive models AI has been shown to analyse fetal echocardiograms more effectively than the typical community clinician. In a model by Arnaout et al., AI detected tetralogy of fallot (TOF) with 75% sensitivity and 76% specificity, and hypoplastic left heart syndrome (HLHS) with 100% sensitivity and 90% specificity.

In comparison, clinicians detected TOF only 3050% of the time, and this trend was similar for HLHS (9).

Furthermore, deep learning models using graphbased matching to segment sections of the heart provide increased anatomical accuracy, with an average improvement of 11.9% over traditional techniques (11).

Figure 1 shows results from a systematic review that looked over 35 recent studies on AI models for predicting post-surgical outcomes. These outcomes included mortality, length of stay in hospital, and ICU stay and found that AI models outperformed traditional prediction tools, with some even achieving an area under the curve (AUC) of 0.997, indicating almost perfect predictive accuracy (3).

B. 3D printed patient-specific heart models 3D printing can provide accurate, personalised heart models that enhance three-dimensional understanding of the complex anatomy in congenital heart disease (12). The first step in this process is segmenting CT or MRI data to create an accurate digital model ready for printing.

In a study by Valverde et al., 3D models influenced 47.5% of surgical decisions in over 40

Fig. 1. A comparison between AI-generated segmentation and expertlabelled images, showing the anatomical precision achieved by deep learning methods (10) .

Fig. 2. An overview of the fabrication process for 3D printed heart models used in congenital heart disease (12).

patients (13). Other studies have reported significantly shorter ICU stays and reduced mechanical ventilation times when 3D printed models were used (p¡0.05) (14).

Additionally, 3D models improve doctor-patient and doctor-parent communication. A parental study

by Biglino et al. showed that 3D models increased satisfaction and understanding among 92 parents, with consultations lengthening from 16 to 21 minutes (15). Similar effects were observed in adolescent patients, who showed significant improvements in understanding (p¡0.005) and communication confidence (p¡0.001). However, 30% of patients also experienced increased anxiety, highlighting a potential psychological impact of 3D printed models (15).

3

V. L IMITATIONS AND CHALLENGES Despite the promising intergration of AI and 3D printing in personalised paediatric cardiac surgery, several limitations prevent widespread clinical implementation. Firstly, the majority of AI studies included in this review focused on small patient groups or homogenous data sets, limiting the generalisability of their findings to the broader population of individuals with CHD (3). Decisions made using AI models often lack explanation and transparency, which can reduce trust and limit their use in high-stakes environments such as paediatric cardiac surgery (2). Additionally, use of AI models involves the transfer of sensitive child health data, which is vulnerable to cyber-attacks and may compromise patient confidentiality (16).

One of the main limitations of 3D printing is the time and cost required to create models, making them less reliable and practical in emergency surgical cases (12). Additionally, the accuracy of printed models is heavily dependant on the quality of imaging and segmentation algorithms, which vary between institutions (4).

VI. F UTURE DIRECTIONS As technology advances, the future of paediatric cardiology is becoming increasingly intergrated with AI and 3D printing, enabling treatment that is safer, faster and more personalised. However, as previously discussed, several limitations prevent these technologies from becoming standard in the management of CHD. One proposed solution to address the lack of transparancy in AI models is the development of explainable AI (XAI), which can explain its reasoning and thereby reduce the risk of misdiagnosis (17).

Federated learning also offers a promising approach to data protection by keeping patient data locally at hospitals and sharing only the learning outcomes with the AI model, ensuring that no sensitive patient information is transferred (17).

Future research should focus on conducting larger, multicentre studies to further improve the accuracy, reliability and generalisability of AI models and 3D printing in paediatric cardiology.

VII. C ONCLUSION The integration of AI and 3D printing in paediatric cardiac surgery has led to significant advancements in the management of congenital heart

defects. AI-driven image segmentation and predictive models have demonstrated higher diagnostic accuracy and improved pre-operative planning.

Meanwhile, 3D printing has enhanced spatial understanding of CHD through printed patient-specific heart models, supporting surgical planning and communication with patients and their families.

Despite limitations such as lack of transparency, data protection concerns and data heterogeneity, technological innovations have been proposed to tackle this, including explainable AI models and federated learning. Moving forward, there is a need for large scale, multicentre studies to validate the accuracy and effectiveness of these technologies for widespread clinical implantation. Ultimately, AI and 3D printing have the potential to transform paediatric cardiac surgery and improve long-term outcomes for children with congenital heart defects.

VIII. R EFERENCES 1. Roth GA, Mensah GA, Johnson CO, Addolorato G, Ammirati E, Baddour LM, et al. Global Burden of Cardiovascular Diseases and Risk Factors, 1990–2019: Update From the GBD 2019 Study. J Am Coll Cardiol. 2020;76(25):2982-3021.

2. Sethi Y, Patel N, Kaka N, Desai A, Kaiwan O, Sheth M, et al. Artificial Intelligence in Pediatric Cardiology: A Scoping Review. J Clin Med. 2022;11(23).

3. Mohammadi I, Rajai Firouzabadi S, Hosseinpour M, Akhlaghpasand M, Hajikarimloo B, Zeraatian-Nejad S, et al. Using artificial intelligence to predict post-operative outcomes in congenital heart surgeries: a systematic review. BMC Cardiovasc Disord. 2024;24(1):718.

4. Capellini K, Ait-Ali L, Pak V, Cantinotti M, Murzi M, Vignali E, et al. Three-dimensional printed models as an effective tool for the management of complex congenital heart disease. Front Bioeng Biotechnol. 2024;12:1369514.

5. Caro-Domı́nguez P, Secinaro A, Valverde I, Fouilloux V. Imaging and surgical management of congenital heart diseases. Pediatr Radiol.

2023;53(4):677-94.

6. Haxel CS, Johnson JN, Hintz S, Renno MS, Ruano R, Zyblewski SC, et al. Care of the Fetus With Congenital Cardiovascular Disease: From Diagnosis to Delivery. Pediatrics. 2022;150(Suppl 2).

7. Holst KA, Said SM, Nelson TJ, Cannon BC, Dearani JA. Current Interventional and Surgical

4

Management of Congenital Heart Disease: Specific Focus on Valvular Disease and Cardiac Arrhythmias. Circ Res. 2017;120(6):1027-44.

8. Lui GK, Saidi A, Bhatt AB, Burchill LJ, Deen JF, Earing MG, et al. Diagnosis and Management of Noncardiac Complications in Adults With Congenital Heart Disease: A Scientific Statement From the American Heart Association. Circulation.

2017;136(20):e348-e92.

9. Raissadati A, Haukka J, Pätilä T, Nieminen H, Jokinen E. Chronic Disease Burden After Congenital Heart Surgery: A 47-Year Population-Based Study With 99% Follow-Up. J Am Heart Assoc.

2020;9(9):e015354. 10. Rima Arnaout MD LCMB, Erin Chinn MS, Yili Zhao PhD RDCS, Anita MoonGrady MD. Deep learning for improved prenatal diagnosis of congenital heart disease using fetal echocardiograms. 2018.

11. Xiaowei Xu TW, Dewen Zeng, Yiyu Shi, Qianjun Jia, Haiyun Yuan, Meiping Huang, Jian Zhuang. Accurate Congenital Heart Disease Model Generation for 3D Printing. arxiv. 2019.

12. Sun Z, Lau I, Wong YH, Yeong CH. Personalized Three-Dimensional Printed Models in Congenital Heart Disease. Journal of Clinical Medicine.

2019;8(4):522.

13. Sun Z. Patient-Specific 3D-Printed Models in Pediatric Congenital Heart Disease. Children (Basel). 2023;10(2).

14. Zhao L, Zhou S, Fan T, Li B, Liang W, Dong H. Three-dimensional printing enhances preparation for repair of double outlet right ventricular surgery.

J Card Surg. 2018;33(1):24-7.

15. Biglino G, Koniordou D, Gasparini M, Capelli C, Leaver LK, Khambadkone S, et al.

Piloting the Use of Patient-Specific Cardiac Models as a Novel Tool to Facilitate Communication During Cinical Consultations. Pediatr Cardiol.

2017;38(4):813-8.

16. Sen S, Ramakrishnan S. Artificial intelligence in pediatric cardiology: Where do we stand in 2024?

Annals of Pediatric Cardiology. 2024;17(2).

17. Mohamed Yaseen Jabarullaa TU, Thomas Jackb, Philipp Beerbaumb, Steffen Oeltze-Jafraa.

Artificial Intelligence in Pediatric Echocardiography: Exploring Challenges, Opportunities, and Clinical Applications with Explainable AI and Federated Learning. Arxiv.

1

Ozempic on the Brain: Investigating the Rise of Glucagon-Like Peptide-1 Receptor Agonists and Their Potential to Treat Neurodegenerative Diseases Hannah Betts Edited by Sushmhitah Sandanatavan and Gayatri Chatterji-Sharma

Abstract - Neurodegenerative diseases are life-altering and debilitating, and are defining a worsening health crisis affecting millions of people. Today, novel therapeutics are receiving approval to alleviate symptoms and delay some neurodegenerative diseases, but a cure remains out of reach. Research is investigating innovative approaches to unravel the complex pathology of neurodegenerative diseases, focusing on the brain’s relationships across the body and evaluating existing therapeutics for potential solutions to a class of currently incureable diseases. Glucagon-like peptide-1 receptor agonists are a therapeutic class with a fascinating history, and have recently gained attention as a breakthrough treatment for obesity. Today, glucagon-like peptide-1 receptor agonists are demonstrating neuroprotective effects, mitigating key aspects of Alzheimer’s and Parkinson’s disease pathology and delaying symptom onset. This evidence suggests that glucagon-like peptide-1 receptor agonists could offer novel insights into neurodegenerative disease pathology, and aid in the discovery and design of novel treatments to combat these complex diseases.

million people suffered from dementia in 2020, a figure predicted to increase to 139 million by 2050 (Alzheimer’s Disease International, 2025).

Worldwide, dementia costs totalled 818 billion USD in 2015, by 2030, this will rise to 2.8 trillion (Alzheimer’s Disease International, 2025). AD is an increasingly dire health crisis, with an urgent necessity for effective and safe therapeutics to combat this disease.

II. I NTRODUCTION

Many therapeutics have demonstrated potential to temporarily alleviate or stabilise symptoms of AD, but long-term efficacy and safety is unconfirmed due to variable off-target effects. The FDA has approved therapeutics that present potential disease-modifying effects, but these therapeutics demand stronger validation (Zhang et al., 2024).

As the mission for safer and more effective AD therapeutics persists, drug-repurposing has gained traction, utilising insights from pre-existing therapeutics and novel discoveries for application in treating AD. One class of therapeutic that has demonstrated unexpected promise to be repurposed, are glucagon-like peptide-1 receptor agonists (GLP1 RAs), or as they are more colloquially know, weight loss drugs, such as Ozempic. This proposes I. R ELEVANCE S TATEMENT that therapeutics used to treat type-2-diabetes (T2D) or obesity, could be applied to resolve an Alzheimer’s Disease (AD) is an aggressive entirely new problem, neurodegenerative diseases. and debilitating neurodegenerative disease, with life-altering symptoms. Nearly one third of AD cases worldwide are linked to modifiable risk III. AD PATHOLOGY AND A ETIOLOGY factors (Zhang et al., 2024). In the United States, one in three Americans die from dementia — a The current understanding of AD’s pathology higher death rate than breast and prostate cancer is a kaleidoscope of overlapping hypotheses.

(Alzheimer’s Association, 2025). Globally, over 55 Hallmark features of AD pathology include the

2

accumulation of Aβ plaques, neurofibrillary tangles (NFTS), neuroinflammation, and subsequent neuronal cell death. Many hypotheses exist, such as the cholinergic, amyloid, tau protein, inflammatory, oxidative stress, and microbiotagut-brain axis hypotheses (Fig. 1). Each proposes a different mechanism by which AD pathology and symptom onset occurs (Zhang et al., 2024).

For example, the microbiota-gut–brain axis hypothesis proposes that alterations in the gut microbiome may increase the permeability of the gut barrier and lead to an immune response, systematic inflammation, and impairment of the blood-brain-barrier, causing neuroinflammation and neurodegeneration (Kowalski and Mulak, 2019). However, these hypotheses lack conclusive evidence (Zhang et al., 2024).

AD symptoms can include cognitive impairments, severe memory loss, neuropsychiatric symptoms, and changes in behavioural and emotional presentation. There is currently no cure for AD, and with disease onset occurring decades before clinical symptoms present.Research has increasingly focused on targeting these preclinical stages of AD to identify therapeutic potential, before onset becomes unstoppable (Zhang et al., 2024).

IV. T ODAY ’ S A LZHEIMER ’ S D ISEASE T HERAPEUTICS Since 2020, three therapeutics have received FDA approval as treatments for AD: aducanumab (Aduhelm), donanemab (Kisunla), and lecanemab (Leqembi). Aducanumab (Aduhelm), manufactured

by Biogen, received accelerated FDA approval in 2021, but has since been discontinued. It was the first monoclonal antibody to be approved for AD treatment. The therapy removed Aβ plaques, reducing cognitive and functional decline in patients with early onset AD (Alzheimer’s Association, 2025). Aduhelm’s approval was a landmark event for AD research and treatment, and further therapeutics emerged soon after. Biogen cited Aduhelm’s discontinuance as a business decision to reprioritize resources focusing on AD and that it was unrelated to the therapeutics safety or efficacy (Alzheimer’s Association, 2025).

Figure 1: Diagram of Diversity of AD Pathology. This outlines the mechanisms underlying key hypotheses of AD pathology including the cholinergic hypothesis, the glutamatergic hypothesis, the amyloid hypothesis, the tau protein hypothesis, the inflammatory hypothesis, the microbiota-gut-brain axis hypothesis, the oxidative stress hypothesis, the metal ion hypothesis, and the abnormal autophagy hypothesis (Zhang et al., 2024).

Lecanemab (Leqembi), also manufactured by Biogen, received FDA approval in 2023 (Biogen, 2024). It is a monoclonal antibody delivered via intravenous fusion, primarily targeting and removing Aβ plaque formation in the brain.

This reduces cognitive and functional decline in patients with early onset AD, as well as mild cognitive impairment or mild dementia due to AD. Leqembi’s ability to slow disease progression allows patients to gain a better quality of life.

Leqembi has an annual cost of $26,500 USD for patients (Alzheimer’s Association, 2025).

Donanemab (Kisunla), manufactured by Eli Lilly, received FDA and MHRA approval in 2024 (Alzheimer’s Disease International, 2024; Hartnell, 2024). It is an anti-amyloid monoclonal antibody, administered intravenously every four weeks.

Donanemab trains our immune cells to recognize and remove amyloid protein. It demonstrated efficacy in early onset AD patients and patients suffering from mild cognitive impairment or mild dementia. In the clinical trial TRAILBLAZER-

3

ALZ2, three-quarters of patients taking Kisunla had amyloid successfully cleared from their brains (Hartnell, 2024). Additionally, after one year, half of the patients showed no decline in memory and cognitive skills. While Kisunla is an exciting therapeutic for AD treatment, it also presents key limitations. Firstly, 91.5% of patients in the clinical trials identified as white, highlighting the necessity for more diverse clinical trials to mitigate potential long-term side effects or off target effects (Hartnell, 2024). Additionally, side effects such as headaches, reactions to the intravenous drip, swelling, or microbleeds related to amyloid were observed (Hartnell, 2024). Donanemab has an annual cost of $32,000 USD for patients (Alzheimer’s Association, 2025).

As of today, research is continuing to discover a therapeutic that effectively and safely alleviates the debilitating symptoms and irreversible onset of neurodegenerative diseases such as AD. A therapeutic like this would not only change millions of people’s lives, but potentially, prevent memory loss in millions more. This is a discovery whose path will be paved by investigating the kaleidoscope of theories behind neurodegeneration, and delivering a clarified understanding fed by research spanning beyond the field of neuroscience.

So, when a novel therapeutic revolution, arrived on the doorstep of the pharmaceutical industry, neuroscience quickly took notice.

V. GLP-1 R ECEPTOR AGONISTS AND THE W EIGHT L OSS T HERAPEUTIC R EVOLUTION It’s not often that a novel therapeutic becomes a household-name. But by 2025, Ozempic had become ever-present, not just in the pharmaceutical industry, but across pop-culture. As this novel therapeutic made its way onto the market, it simultaneously slipped into song lyrics, TikToks, and everyday conversations. With its rapid rise to fame came the question: where did this drug come from, and why is it generating such excitement? Ozempic, also known as semaglutide is a GLP-1 RA, a class of therapeutics whose histories are just as fascinating as their modern implications, rising from a story of accidental discovery, emotional-bias, and giant lizards (Kolata, 2023).

Obesity affects nearly 42% of Americans (Kolata,

2023). However, as research to find a treatment hit one dead end after another, and societal opinion erroneously grew to consider obesity a moral failing more than a chronic illness, the pharmaceutical industry’s interest in curing obesity waned if not completely plateaued (Kolata, 2023). Attention shifted instead to diabetes, a disease characterised by high blood sugar levels which is treated by the injection of insulin. Insulin is a hormone secreted by the pancreas that helps cells store sugar, decreasing blood sugar levels. However, research into diabetes revealed a serendipitous solution for obesity when the brain was exposed to high levels of a natural hormone, GLP-1. It was soon evident that the solution to one problem may emerge in the pursuit of solving another (Kolata, 2023).

GLP-1 is a hormone naturally produced in our gut.

GLP-1 RAs are synthetic mimics of this hormone, regulating blood sugar and acting on insulin to stimulate the pancreas when blood sugar rises too high. Targeting these mechanisms quickly became an appealing treatment for diabetes. While the initial discovery of GLP-1 RA was exciting, it came with a key limitation: the synthetic agonist didn’t last long. GLP-1 RAs degraded and disappeared before reaching the pancreas. Thus, research set out to find a longer-lasting solution and found its answer in a highly unexpected place. Gila monsters are giant lizards that use a GLP-1 variant to maintain blood sugar level stability when they lack a source of nutrition. These lizards go long-spans of time without food thus their GLP-1 variant lasts significantly longer than its human counterpart. In 2005, this longer-lasting GLP-1 RAs synthetic analog, exenatide, was patented and went on the market as Byetta (European Medicines Agency, 2024). However, Byetta required two injections daily, making it inconveniently time and cost consuming for both its patients and producers. It was clear, further innovation was needed to establish a more effective diabetes therapeutic (Kolata, 2023).

Novo Nordisk took a key first step, attaching GLP1 RA to a fatty acid, producing the GLP-1 RA liraglutide which binds blood proteins stabilizing the hormone and allowing it to circulate for up to 24 hours before degradation. This innovation led to the development of the therapeutic Victoza, which received FDA approval to treat T2D in 2010 (U.S. Food & Drug Administration, 2010). In the wake of its success, researchers noted a surprising side effect

4

to the diabetes therapeutic: patients experienced a slight loss in weight. This observation lit a spark in research to explore other naturally occurring hormones’ potential effects on weight loss. One such hormone, leptin, demonstrated promise. When genetically modified, leptin was found to affect the weight of mice. Mice with leptin were able to maintain their body weight, while mice with a leptin-knockout ate uncontrollably. Amgen soon bought the rights to leptin to begin human trials, but the weight loss effects did not translate from animal model to patient (Kolata, 2023). Novo Nordisk continued to interrogate GLP-1 RAs and research discovered that patients with an intravenous drip of GLP-1 RAs consumed nearly 12% less food than those with a placebo. A new therapeutic, Saxenda was approved in 2014 at a dose near twice that of Victoza (Caffrey, 2015). Patients taking Saxenda lost 5% of their body weight. The first GLP-1 RA weight-loss therapeutic had hit the market, but the primary goal remained: GLP-1 RAs needed to last longer (Kolata, 2023).

In 2017, came Ozempic, a breakthrough in diabetes and obesity treatment alike. Ozempic is an injectable therapeutic that was developed as an adjunct to exercise and diet, treating adults with T2D. It is administered subcutaneously using prefilled multidose injection pens. The drug is composed of the active substrate GLP-1 RA semaglutide, and has a 94% structural homology with human GLP-1 (Fig. 2) (Kommu and Whitfield, 2024).

pancreasreleases in response to food, aiding in the control of blood glucose levels. It further slows gastric emptying, increasing pancreatic β-cell proliferation and reducing glucagon release, subsequently reducing appetite.

Through activation of GLP-1 receptors in the gastrointestinal tract, pancreas, and brain Ozempic lowers levels of blood glucose between 1.2-1.8% over a period of 10-13 months. Other diabetes treatments achieve merely half that reduction (European Medicines Agency, 2025). Results from a 12-week single-centre, randomized, double-blind, placebo controlled parallel-group trial found that adults with T2D taking Ozempic for 12 weeks achieved an insulin response resembling healthy people (Fig. 3) (NovoMEDLINK, 2025). Additionally, Ozempic significantly reduces occurrence of serious health complications associated with diabetes, such as heart attack and stroke (European Medicines Agency, 2025; Kommu and Whitfield, 2024).

Figure 3: Ozempic’s Effectiveness Overtime. Results from a 12-week single-centre, randomized, double-blind, placebo controlled parallel-group trial demonstrate that adults with T2D taking Ozempic for 12 weeks achieved an insulin response resembling healthy people (NovoMEDLINK, 2025).

Additionally, patients taking Ozempic demonstrated significant weight loss, achieving a 15% loss in body weight, three times greater than that seen with Saxenda. However, Ozempic was designed to treat T2D, and was approved solely for this purpose.

Figure 2: The Ozempic Molecule. The modificaIt wasn’t until 2021 that it received approval to treat tions made to the native human GLP-1 hormone to weight loss under a new name: Wegovy. produce Ozempic are indicated (NovoMEDLINK, Ozempic was the first drug of its kind, a life 2025). changing therapeutic, and the world took notice.

US advertisements ran across screens promoting Ozempic increases the amount of insulin the the novel diabetes therapeutic’s miraculous weight

5

loss effects. Doctors began prescribing Ozempic off label for weight loss and social media fascination frenzied (Kolata, 2023). Fortunately, Wegovy caught up, and the Ozempic obsession has largely subsided.

Nonetheless, the fascination with weight loss therapeutics has only grown. Weight loss therapeutics like Wegovy cross the blood-brain barrier (BBB) and don’t act solely in the brain regions they’re intended to. They have been detected in areas of the brain unrelated to appetite regulation, slipping into places that GLP-1 shouldn’t exist, a phenomenon research has set out to investigate as it remains poorly understood (Kolata, 2023).

In 2022, GLP-1 RA tirzepatide, or Mounjaro, developed by Eli Lilly received FDA approval as a diabetes therapeutic. It too came with a side-effect of weight loss (Lilly, 2022). Mounjaro was approved to treat T2D mellitus in combination with diet and exercise. In 2023, it received approval in the US as an injectable weight loss therapeutic under the name Zepbound. Zepbound activates receptors for GLP1 and glucose-dependent insulinotropic polypeptide (GIP) in the intestine. It reduces appetite and food intake, and is distinct from Ozempic in that it additionally acts on GIP, a second incretin thought to work together with GLP-1 (FDA, 2023). This made Zepbound the first and only dual-GIP and GLP-1 RA approved for weight loss (Billingsley and Aungst, 2025). Studies have demonstrated that Zepbound results in greater weight loss than Wegovy, achieving an average weight loss of 25% per year, compared to Wegovy’s 16%. This is likely partially achieved by Zepbound higher weekly dosage, though the two therapeutics have yet to be directly compared. However, in a randomized controlled trial with over 1,800 participants, Zepbound was associated with more serious adverse effects and a higher discontinuation rate than Ozempic. Both medications are priced similarly (Puddick, 2025).

The most recent T2D therapeutic with weight loss therapeutic potential comes just this April of 2025, as Eli Lilly announced the success of Phase 3 clinical trials for its oral GLP-1 RA, Orforglipron.

It is the first oral small-molecule GLP-1 RA to successfully complete a Phase 3 clinical trial, setting it apart from its injectable counterparts. Further, Orforglipron can be taken at any time of day with no restrictions on food or water intake. Recently, Eli Lilly announced positive results from their Phase 3 clinical trial, ACHIEVE-1, a 40-week, randomized,

double-blind, placebo-controlled trial evaluating the safety and efficacy of Orforglipron compared to a placebo in adult patients with T2D. If Orforglipron receives FDA approval for T2D, it projected to impact an estimated 760 million adults by 2050.

ACHIEVE-1 is just one of seven trials examining the safety and efficacy of Orforglipron in individuals with diabetes and obesity. This trial demonstrated statistically significant results, and determined that Orforglipron’s safety profile is consistent with other therapeutics in the GLP-1 RA class. Adverse effects include gastrointestinal-effects, nausea, constipation, or vomiting. Eli Lilly expects to submit Orforglipron for regulatory approval for weight loss by the end of 2025 (Table 1) (Lilly, 2025).

Medication

Active Ingredient

Byetta

Exenatide

Victoza

Liraglutide

Saxenda

Liraglutide

Ozempic

Semaglutide

Wegovy

Semaglutide

Mounjaro

Tirzepatide

Zepbound

Tirzepatide

Orforglipron Orforglipron

Drug Class GLP-1 receptor agonist GLP-1 receptor agonist GLP-1 receptor agonist GLP-1 receptor agonist GLP-1 receptor agonist GLP-1 and GIP receptor agonist GLP-1 and GIP receptor agonist GLP-1 receptor agonist

FDA Approved Apusage(s) proval Type 2 dia2005 betes Type 2 dia2010 betes Overweight 2014 / Obesity Type 2 diabetes 2017 / heart disease prevention Overweight 2021 / Obesity 2022

Type 2 diabetes

2023

Overweight / Obesity

N/A

N/A

Table I: T2D Therapeutics with Weight Loss Therapeutics Counterparts

VI. GLP-1 RA’ S P OTENTIAL TO T REAT N EURODEGENERATIVE D ISEASES Weight loss therapeutics have captured the fascination of the pharmaceutical industry and the public. Now, they’re repositioning towards a novel breakthrough, demonstrating unexpected potential to treat neurodegenerative diseases such as Alzheimer’s Disease (AD) (McKenzie, 2024).

The jump from weight loss therapeutics to neurodegenerative diseases didn’t come completely

6

out of the left field. There is a long-dated history of interest in the intersection of diabetes and AD, with several studies suggesting a connection between insulin resistance and AD. Thus, curiosity rose regarding whether drugs for diabetes, targeting metabolic diseases and endocrine disease pathways may also be affective against AD. Meta analysis confirmed this curiosity, linking T2D to an increased risk of developing AD (McKenzie, 2024). Further, database reviews revealed similar implications: patients prescribed GLP-1 RA therapeutics like semaglutide were 10% less likely to develop dementia compared to those taking other diabetes therapeutics (Meca et al, 2024).

Semaglutide, the GLP-1 RA seen in Ozempic and Wegovy, has shown potential to reduce the risk of diabetes related cognitive decline and mitigate AD pathology by presenting anti-inflammatory and neuroprotective properties. Chronic inflammation is a well-established hallmark of neurodegenerative diseases, and semaglutide may counteract this by reducing levels of pro-inflammatory cytokines, inhibiting inflammatory pathways, reducing brain inflammation, and decreasing neuronal degeneration. Further, semaglutide may provide direct neuroprotective effects. These include enhancing cellular repair mechanisms, upregulating neurotrophic factors, reducing reactive oxygen species (ROS), and increasing the activity of antioxidant enzymes. These mechanisms support neuronal survival and promote neural growth (Allder, 2024).

Further studies have investigated the role of GLP-1 RAs in slowing the progression of neurodegeneration. Studies in rodents demonstrated diabetes and obesity therapeutics improved hallmarks of AD pathology (Smith and Blum 2025).

In AD models, semaglutide reduced Aβ plaque deposition, mitigated neuroinflammation, ameliorated behavioural deficits, and improved spatial learning and memory in maze navigation tests and novel object recognition assays (Fig. 4). These findings indicate multiple mechanisms of action and suggest semaglutide’s primary mechanism involves inhibition of acetylcholinesterase, increasing the availability of acetylcholine and enhancing the cholinergic system by supporting growth and maintenance of cholinergic synapses. It predominantly mitigates

Aβ and tau pathology in the CA3 hippocampal region, key areas implicated in AD pathology.

Further, in AD mouse models, the water navigation task was used to measure the effects of GLP-1 RA on memory and liraglutide, the GLP-1 RA in Victoza and Saxenda, demonstrated efficacy in preventing neuronal apoptosis (Meca et al, 2024).

In PD models, semaglutide elicited neuroprotective effects, modulated neuroinflammation, enhanced mitochondrial function, promoted neurogenesis, improved motor function, and ameliorated dopaminergic neural loss. Semaglutide demonstrates strong potential as a disease-modifying treatment strategy, specifically due to its involvement in mechanisms of impaired cognitive function associated with insulin resistance (Fig. 4).

Neuronal metabolism and synaptic efficacy suffer from desensitisation of insulin signalling pathways and insulin is key to activating growth factors that stimulates brain receptors, regulating mitochondrial functionality, cellular energy utilization, cell survival, and synaptic processing. Impaired insulin signalling can reduce or delay neuronal repair, increasing the risk of developing neurodegenerative pathologies. GLP-1 RAs like semaglutide may aid in compensating for deficiencies in insulin signalling, activating the same growth factor cascades seen in insulin. In PD, T2D and desensitized insulin signalling are linked to disease onset. Two further, GLP-1 RAs, liraglutide and lixisenatide, demonstrated efficacy in maintaining enzymes crucial to dopamine production. A trial of lixisenatide in early PD, demonstrated the drug presented neuroprotective properties, slowing the progression of motor dysfunction at 12 months in a phase 2 trial. However, gastrointestinal side effects were additionally noted (Meissner et al., 2024; Meca et al, 2024). In mouse models exenatide, the GLP-1 RA in Byetta, restored motor functionality and semaglutide prevented loss of dopaminergic neurons. Overall, semaglutide exhibited superior efficacy in restoring mitochondrial function and antioxidant balance by enhancing autophagy (Meca et al, 2024). Another study demonstrated exenatide improved motor function in patients with PD compared to placebo. Exenatide is currently being investigated in a larger trial population of patients with PD (Lenharo, 2024).

GLP-1 RAs have been implicated in multiple signalling pathways associated with

7

Figure 4: Semaglutide’s Mechanism of Action in AD and PD. This figure highlights semaglutide’s effect on neurodegenerative disease mechanisms (Meca et al., 2024).

neuroprotection. In incretin signalling chronic administration of GLP-1 RAs in mouse models of diabetes demonstrated that following binding and activation of GLP-1 receptors, cAMP levels increased rapidly, activating pathways involved in protein and mitochondrial biogenesis while inhibiting neuronal apoptosis. In the P13K/Akt signalling pathway, semaglutide was found to be experimentally correlated with neuroprotective properties, enhancing mitochondrial function, reducing neuro-apoptotic reactions, and inhibiting proinflammatory and neurodegenerative serine/threnonine kinase. Semaglutide treatment also reduced impairment of autophagic and lysosomal pathways. Overall, across in vivo and in vitro studies, the effects of GLP-1 RAs demonstrate immense promise as potential treatments for neurodegenerative diseases such as AD and PD, but the additional side effects and mechanisms that are not fully understood require further research to validate these findings (Meca et al, 2024).

Some GLP-1 RA therapeutics being investigated for repositioning towards neurodegenerative disease have even progressed to clinical trial. A Phase II trial of 200 AD patients was conducted across 24 clinics in the UK. Half of the patients were given an injection of up to 1.8 mg liraglutide while the other half received a placebo injection. Prior to the injection with liraglutide, MRIs of brain structure, PET scans of glucose metabolism, and detailed memory testing were conducted and evaluated

(Rey, 2024). The liraglutide cohort demonstrated an 18% slower decline in cognitive function compared to patients receiving a placebo (McKenzie, 2024). Further, patients who had taken liraglutide demonstrated a significant 50% reduction in brain shrinkage compared to placebo patients, specifically in the frontal, temporal, parietal lobes and total grey matter —, areas known to be involved in memory, learning, language, and decision making (Rey, 2024). These findings suggest liraglutide may be neuroprotective though research remains uncertain of the mechanisms by which it elicits these effects. It is believed liraglutide influences multiple processes in the brain including inflammation, tau protein aggregation, insulin resistance, and Aβ accumulation, slowing brain volume shrinkage (Rey, 2024). Following this, another study found that T2D patients taking semaglutide had a 40-70% reduced AD risk (McKenzie, 2024). These results further support the neuroprotective potential of GLP-1 RAs. There are many theories exploring the mechanisms by which weight loss therapeutics affect neurodegenerative diseases. Diabetes and obesity are risk factors for dementia. Consequently, weight loss therapeutics may simply reduce the risk of developing AD by improving metabolic health.

Further, neurodegenerative diseases demonstrate increased inflammation. The anti-inflammatory properties of GLP-1 RAs could also be responsible for improving AD pathology. Healthy insulin function is also a key aspect of memory formation.

Weight loss therapeutics curb insulin resistance and studies in animal models suggest that GLP-1 RA therapeutics improve insulin signalling in the brain. Finally, weight loss therapeutics may target meaningful regions of the brain associated with learning and memory. As previously addressed, liraglutide accessed regions of the brain beyond appetite control, including the hippocampus, an area associated with early damage in AD. Semaglutide demonstrated more mixed results, suggesting this therapeutic may struggle to pass through the blood-brain-barrier (Smith and Blum 2025).

Today, Kariya pharmaceuticals is developing KP045, a dual action therapeutic of GLP-1 RA and GIP receptor agonist. This aims to remove add-ons to GLP-1 RAs such as fatty acids that limit the therapeutics ability to reach the brain. Removing fatty acids allows for cell membrane receptors to

8

recognize the GLP-1 RA therapeutic, enabling it to reach the brain faster. This also aids in avoiding off-target effects and improving peripheral effects of the drug (McKenzie, 2024).

The investigation of GLP-1 RAs to treat neurodegenerative disease treatment has become an ongoing race of research, but even more so, a waiting game. Novo Nordisk, a long existing giant in the research of GLP-1 RAs is expecting readouts from two large clinical trials: EVOKE and EVOKE plus, testing semaglutide in patients with AD. These trials are expected to be completed in September 2025 (McKenzie, 2024). Other industry giants, such as Eli Lilly are also investigating GLP-1 RAs potential implications in treating neurodegenerative diseases. Results from these clinical trials could reveal imperative conclusions in the consideration of GLP-1 RAs as potential therapeutics to be repositioned towards neurodegenerative diseases.

However, it is key that as research continues to explore these therapeutic’s exciting potential, it does not do so without heavy consideration of the risk factors and patient safety.

VII. E THICS , L IMITATIONS AND C ONSIDERATIONS The celebration of the serendipitous discovery of GLP-1 RAs and consequently weight loss therapeutics must be coupled with an equivalent evaluation of associated risk factors. Large-scale analysis of data from nearly 2 million users of weight loss drugs revealed a prevalence of off-target effects.

Studies have found GLP-1 RAs put patients at a 146% higher risk of pancreatitis, a life-threatening disease (Guglielmi, 2025). Further adverse side effects include hypoglycaemia, thyroid c-cell tumours, diabetic retinopathy, anaphylaxis, and gall bladder disorders (Kommu and Whitfield, 2024).

According to Novo Nordisk’s MEDLINK resource, semaglutide induced dose-dependent and duration dependent thyroid c-cell tumours at clinically relevant exposure. These findings however note that it remains unclear whether Ozempic causes thyroid c-cell tumours in humans. Novo Nordisk advises patients receive further evaluation if serum calcitonin levels are found elevated or if thyroid nodules are found in physical examinations or neck imaging (NovoMEDLINK, 2025). The FDA states similar findings regarding Zepbound, warning that

Zepbound should not be used in patients with a personal or family history of medullary thyroid cancer or in patients with multiple endocrine neoplasia syndrome type 2 (FDA, 2023). These health risks should be carefully considered both in the current use of weight loss therapeutics and future research expanding the applications of GLP-1 RAs. The rising popularity of Ozempic additionally brought with it an increased prevalence of compounded and counterfeit preparation of semaglutide.

Patients should always seek prescription from a medical professional to ensure they are prescribed an approved and safe therapeutic (Kommu and Whitfield, 2024).

Alongside these risk factors, there are further impressions we should take away from the discovery and history of weight loss therapeutics. The weight loss therapeutic revolution rises from a history ingrained with emotional bias — something worth considering as novel technologies and innovation push the pharmaceutical industry into a new era of discoveries. A chief scientific officer at Novo Nordisk, a company which can be deeply highly credited for the modern success of GLP-1 RAs, once cited having to spend almost half a year convincing his CEO that obesity was not a moral failing or caused by a lack of willpower (Kolata, 2023). A belief once upheld and still lingers, yet one so poorly misinformed. It is essential that emotional bias does not impede scientific discovery and progress. It is interesting to reflect ten years ago, when GLP-1 RAs first emerged as weight loss therapeutics. In an era of Weight Watchers, diet fads, and media-driven body image ideals: how much sooner might life-changing therapeutics such as Wegovy and Zepbound have arrived had research not been hindered by judgment and bias?

The possibility of weight loss therapeutics being repurposed for neurodegenerative diseases is undeniably exciting, but these findings demand further evaluation and evidence to procure effective and safe therapeutics for patients. Preclinical studies demonstrating the benefits of GLP-1 RAs targeting core pathology of AD and PD have limited data.

Stronger results with a lower incidence of disease progression and exhibiting better safety could be instrumental in boosting the reputation of GLP1 RAs as treatments for neurodegeneration. More research must demonstrate safety and efficacy in chronic use of these therapeutics (McKenzie, 2024).

9

VIII. I NNOVATION AND F UTURE R ESEARCH GLP-1 RAs are a class of drugs with extraordinary promise not only as weight loss therapeutics, but across drug discovery. A risk assessment of GLP-1 RAs, including semaglutide, found broader implications for these therapeutics. The study, conducted over three years, followed more than 200,000 individuals with diabetes who were on GLP-1 RA therapeutics, and 1.7 million people with diabetes using blood-sugar-lowering medications. It tracked effects on 175 health conditions and found patients taking GLP-1 RA therapeutics had a lower risk of developing conditions such as heart disease, strokes, and kidney disease. Further, GLP-1 RAs seemed to lower the risk of psychotic disorders by 18% and addiction disorders by 13% compared to diabetes medication. This effect stands out as GLP-1 RAs are known to act on brain regions involved in reward and impulse control, lending it potential to curb cravings for tobacco, alcohol, cannabis, or opioids, and reduce dependence on substances involved in the current addiction epidemic (Guglielmi, 2025a; NCDAS, 2025).

As of 2025, weight-loss therapeutics have begun emerging across the pharmaceutical industry. A few weight-loss therapeutics currently being tested include: AMG133, Retatrutide, Orforglipron, MariTide, CagriSema, Amycretin, and Enobosarm. These therapeutics exhibit strong potential for weight loss, and mitigating limitations of present therapeutics. Future research and innovation will only aid in the evolution of GLP-1 RA therapeutics and the insights they may provide to areas of drug discovery beyond obesity and weight loss (Guglielmi, 2025b; (Kolata, 2023).

IX. C ONCLUSION Neurodegenerative diseases like Alzheimer’s disease are a worsening public health crisis. These diseases are particularly detrimental to patients, with widespread life-altering hallmark symptoms such as cognitive impairments, severe memory loss, and neuropsychiatric symptoms. However, one of the most powerful complexities of AD, is that disease onset can predate these symptoms by decades. It is a disease which has perfected the ability to hide, progressively affecting mechanisms and systems throughout the body with symptoms appearing long after disease mechanisms onset. Therapeutics

for AD aim to target the aetiology and cellular mechanisms responsible for early disease onset and progression. Therapeutics such as donanemab and lecanemab are the only existing FDA approved therapeutics to treat AD. These therapeutics, target key elements of AD aetiology such as Aβ plaques formation, and are effective in delaying disease progression and providing a better quality of life.

However, these therapeutics are intended for patients suffering from early onset AD. Further, these therapeutics are coupled with off target effects and are not a cure. Innovation and continued research are crucial to developing AD therapeutics that demonstrate efficacy and safety, an effort that has grown to include investigation of existing therapeutics for potential novel applications.

Many therapeutics have demonstrated promise for repositioning towards treating neurodegenerative diseases, but recently a more novel therapeutic class has stepped into the spotlight. GLP-1 RAs, a synthetic mimic of naturally occurring hormone GLP-1, have a fascinating therapeutic history, arising from serendipitous discovery, innovation, and repositing for a novel therapeutic purpose, obesity, despite bias. So-called weight loss therapeutics, such as Saxenda, Wegovy, and Zepbound arose from T2D therapeutics. Today, research suggests potential for these therapeutics to treat neurodegenerative diseases. In AD, GLP-1 RAs demonstrated a reduced risk of diabetes related cognitive decline and mitigated key AD pathology reducing brain inflammation, Aβ plaque deposition, and neuronal degeneration. These effects improved spatial learning, memory, and behavioural deficits in an animal model.

In PD, GLP-1 RAs were also neuroprotective, mitigating neuroinflammation, enhancing mitochondrial function, and promoting neurogenesis by improving insulin signalling along the insulin signalling pathway. These effects improved motor function and ameliorated dopaminergic neuron loss. GLP-1 RAs have demonstrated widespread neuroprotective effects and efficacy against disease mechanisms in a range of studies including in vitro experiments, in vivo models, and clinical trials. However, off-target effects are a considerable limitation of these therapeutics, and more data and concrete results from clinical trials are necessary to determine therapeutic safety and efficacy.

Research and clinical trials testing the potential of GLP-1 RAs to treat neurodegenerative diseases are

10

ongoing, but the results thus far are undeniably exciting. Finding safe and effective treatments to aid the growing number of people suffering the debilitating effects of neurodegenerative diseases demands innovation and novel discoveries. GLP-1 RAs may not be a cure, but the insights we can gain from these therapeutics and being attentive of modern drug discoveries is what pushes efforts to treat and cure neurodegenerative diseases forward.

The story of GLP-1 RAs, from their discovery and repositioning to public reception and emerging results in trials of neurodegenerative diseases highlights both the strengths and limitations of drug discovery and serves as a compelling example likely inspire continued progress across the pharmaceutical industry. As drug discovery advances and innovates, towards treating classed incurable diseases like neurodegenerative diseases, the story of GLP1 RAs invites us to wonder:, might the answers to the incurable already be within our reach, in our laboratories, our pharmacies, or even the shelves of our own homes?

NovoMEDLINK (2025) Ozempic semaglutide injection. Available at: https://www.novomedlink. com/diabetes/products/treatments/ozempic/about/m echanism-of-action.html Alzheimer’s Association (2025) Aducanumab Discontinued as an Alzheimer’ss Treatment.

Available at: https://www.alz.org/alzheimers-demen tia/treatments/aducanumab Kolata, G. (2023). We Know Where Weight Loss Drugs Came From, but Not Why They Work. The New York Times. Available at: https://www.nytimes.com/2023/08/17/health/weight -loss-drugs-obesity-ozempic-wegovy.html Alzheimer’s Association (2025) Donanemab Approved for Treatment of Early Alzheimer’s Disease. Available at: https://www.alz.org/alzheime rs-dementia/treatments/donanemab

Biogen (2024) Leqembi® (lecanemab) Authorized for Early Alzheimer’s Disease in Great Britain.

X. R EFERENCES Available at: https://investors.biogen.com/news-rel Alzheimer’s Association (2025) Alzheimer’s eases/news-release-details/leqembir-lecanemab-aut Disease Fact and Figures. Available at: h t t p s : horized-early-alzheimers-disease-great //www.alz.org/alzheimers-dementia/facts-figures#: ∼:text=in%20each%20state-,Quick%20facts,Amer Biogen (2023) FDA Grants Traditional Approval icans%20are%20living%20with%20Alzheimer’s for LEQEMBI® (lecanemab-irmb) for the Treatment of Alzheimer’s Disease. Available Alzheimer’ss Disease International (2025) Dementia at: https://investors.biogen.com/news-releases/news Statistics. Available at: https://www.alzint.org/abo -release-details/fda-grants-traditional-approval-leq ut/dementia-facts-figures/dementia-statistics/ embir-lecanemab-irmb Zhang, J., Zhang, Y., Wang, J. et al. Recent advances in Alzheimer’s disease: mechanisms, clinical trials and new drug development strategies.

Sig Transduct Target Ther 9, 211 (2024). doi: 10.1038/s41392-024-01911-3

Alzheimer’s Association (2025) Lecanemab Approved for Treatment of Early Alzheimer’s Disease. Available at: https://www.alz.org/alzheime rs-dementia/treatments/lecanemab-leqembi

Hartnell, I. (2024). What is donanemab?

European Medicines Agency (2025) Ozempic. Alzheimer’s Society. Available at: h t t p s : Available at: https://www.ema.europa.eu/en/medic //www.alzheimers.org.uk/blog/what- is- donan ines/human/EPAR/ozempic emab-alzheimers-drug Kommu S, Whitfield P. Semaglutide.

[Updated 2024 Feb 11]. In: StatPearls [Internet]. Treasure Island (FL): StatPearls Publishing; 2025 Jan-.

Available from: https://www.ncbi.nlm.nih.gov/books/NBK603723/

Guglielmi, G. (2025a) Obesity drugs: huge study identifies new health risks. Nature 637, 1034. doi: 10.1038/d41586-025-00173-5 Guglielmi, G. (2025b) The weight-loss drugs

11

being tested in 2025: will they beat Ozempic?

Nature 638, 591-592. doi: 10.1038/d41586-025- FDA. (2023). FDA Approves New Medication 00376-w for Chronic Weight Management. Available at: https://www.fda.gov/news-events/press-announcem Allder, S. (2024). The Potential of Ozempic ents/fda-approves-new-medication-chronic-weigh in Treating Alzheimer’s and Depression: A t-management#:∼:text=For%20Immediate%20Rel Promising Horizon. Re: Cognition Health Brian ease:%20November%2008,Espa%C3%B1ol and Mind Experts. Available at: https://recognitio nhealth.com/the-potential-of-ozempic-in-treating-a Billingsley, A and Aungst, C. (2025) Zepbound lzheimers-and-depression-a-promising-horizon/ vs. Ozempic: 5 Differences Between These Weekly Injections. GoodRx. Available at: Meca, A. D., Boboc, I. K. S., Mititelu-Tartau, https://www.goodrx.com/conditions/weight- l L., & Bogdan, M. (2024). Unlocking the oss/zepbound-vs-ozempic Potential: Semaglutide’s Impact on Alzheimer’s and Parkinson’s Disease in Animal Models. Current Puddick, R. and Lead, S. (2025). Zepbound vs issues in molecular biology, 46(6), 5929–5949. Wegovy. Second Nature. Available at: https://www. https://doi.org/10.3390/cimb46060354 secondnature.io/us/guides/lifestyle/glp1s/zepbound -vs-wegovy?srsltid=AfmBOopL1MeueDyLR6ng McKenzie, H. (2024) Evidence Mounts YC5VwoSQYrtkMSGPyEJfGz Eg886XiqeUiMV for Potential of GLP-1s in Alzheimer’s Disease. Bio Space. Available at: h t t p s : Lilly INVESTORS. (2025) Lilly’s oral GLP-1, //www.biospace.com/drug-development/evidence-m orforglipron, demonstrated statistically significant ounts-for-potential-of-glp-1s-in-alzheimers-disease efficacy results and a safety profile consistent with injectable GLP-1 medicines in successful Phase 3 Rey, S. (2024) Weight-loss drug may slow trial. Available at: https://investor.lilly.com/news-r Alzheimer’s decline. Imperial. Available at: eleases/news-release-details/lillys-oral-glp-1-orfor https://www.imperial.ac.uk/news/255214/weight-los glipron-demonstrated-statistically s-drug-slow-alzheimers-decline/ Meissner, W. G., Remy, P., Giordana, C., Smith, D.G. and Blum, D. (2025) To Protect Maltête, D., Derkinderen, P., Houéto, J. L., Against Alzheimer’s Researchers Look to Weight Anheim, M., Benatru, I., Boraud, T., BrefelLoss Drugs. The New York Times. Available at: Courbon, C., Carrière, N., Catala, H., Colin, https://www.nytimes.com/2025/01/31/well/mind/o O., Corvol, J. C., Damier, P., Dellapina, E., Devos, D., Drapier, S., Fabbri, M., Ferrier, zempic-alzheimers.html V., . . . LIXIPARK Study Group (2024). Trial of Lenharo, M. (2024) Obesity drugs have another Lixisenatide in Early Parkinson’s Disease. The New superpower: taming inflammation. Nature 626, 246. England journal of medicine, 390(13), 1176–1185. https://doi.org/10.1056/NEJMoa2312323 Doi: 10.1038/d41586-024-00118-4 Lilly INVESTORS. (2022) FDA approves Lilly’s Mounjaro™ (tirzepatide) injection, the first and only GIP and GLP-1 receptor agonist for the treatment of adults with type 2 diabetes. Available at: https://investor.lilly.com/news- releases/ news- release- details/fda- approves- lillys- m ounjarotm- tirzepatide- injection- first- and#: ∼ : text=News%20Release-,FDA%20approves%20Lil ly’s%20Mounjaro%E2%84%A2%20(tirzepatide) %20injection%2C%20the%20first,with%20type%2 01%20diabetes%20mellitus

European Medicines Agency. (2024) Byetta.

Available at: https://www.ema.europa.eu/en/medic ines/human/EPAR/byetta Kowalski, K., & Mulak, A. (2019). BrainGut-Microbiota Axis in Alzheimer’s Disease.

Journal of neurogastroenterology and motility, 25(1), 48–60. https://doi.org/10.5056/jnm18087 NCDAS National Center for Drug Abuse Statistics.

(2025) Drug Abuse Statistics. Available at:

12

https://drugabusestatistics.org/ US Food & Drug Administration. (2010) Drug Approval Package.

Available at: https://www.accessdata.fda.gov/drugsatfda d ocs/nda/2010/022341s000TOC.cfm Caffrey, M. (2015) Liraglutide Approved Under New Name to Treat Obesity. AJMC, 21(SP2).

Available at: https://www.ajmc.com/view/liraglutid e-approved-under-new-name-to-treat-obesity

1

Cracking the Code of Chronic Pain: Molecular Gateways and Emerging Treatments Leen Shanwar Edited by Sushmhitah Sandanatavan and Thomas Burton

Abstract - The prevalence of chronic pain reaches 20% of adults worldwide while it stands as a primary cause of disability. The standard pain management approaches including opioids and NSAIDs provide restricted pain relief benefits while exposing patients to major safety risks. The interest in targeted molecular and circuit-based therapies has increased because of this situation. This review synthesises findings from peer- reviewed systematic reviews, meta-analyses, and clinical trials identified via PubMed, Cochrane, and Google Scholar. Studies focused on four emerging intervention classes: neuromodulation (SCS, TMS, VNS), TRP channel blockers, cannabinoid-based therapies, and monoclonal/ion-channel inhibitors. Non-peer- reviewed, non-English, and animal-only studies were excluded. These therapies consistently deliver both pain relief and functional improvement to patients with neuropathic and back pain conditions. The pain relief from non- invasive neuromodulation techniques such as TMS and tVNS remains limited to small effects.

The preclinical development of TRP channel antagonists (TRPV1, TRPA1) faces challenges because of safety issues and trial-related inefficacy. Cannabinoids produce minimal but measurable pain relief although they cause noticeable cognitive side effects. The anti-NGF antibody treatment produces significant pain relief for osteoarthritis and low back pain patients, yet safety concerns exist regarding joint health. The clinical development of Nav1.7 sodium-channel blockers has not succeeded despite their strong genetic foundation. SCS and anti-NGF therapies demonstrate the most promising clinical benefits among current treatment options. The field requires additional research on long-term safety outcomes together with better biomarker-based patient selection methods and well-designed tri-

als that include diverse populations. The advancement of precise pain management depends on understanding the current developments in treatment approaches.

I. I NTRODUCTION Chronic pain affects 20% of adults globally, making it a major public health problem (Goldberg and McGee, 2011) and a leading factor in contributing to the global burden of disease, primarily through the burden of disability (Mills et al., 2019). Chronic pain is defined as “pain that persists or recurs for more than 3 months” (Treede et al., 2019) where pain is conceptualised as “an unpleasant sensory and emotional experience associated with, or resembling that associated with, actual or potential tissue damage” according to the International Association of the Study of Pain (IASP)(Pain, 2020).

The economic burden is significant, underscoring its urgent public health importance: in the US alone, annual costs range from $560 to $635 billion in 2010 dollars, surpassing those for costs for heart disease ($309 billion), diabetes ($188 billion) and cancer ($243 billion) (Gaskin and Richard, 2012).

Traditional pain treatments, particularly opioids, have significant shortcomings. While effective in the short-term pain relief, when used for an extended period of time, they can lead to dependence, physical and mental health problems, and even death from accidental overdoses or heart disorders caused by adverse effects (England, 2023).

The opioid crisis has become a major public health emergency because it resulted in more than 80,000 opioid-related deaths throughout the United States during 2022 while causing 76% of total overdose deaths (Spencer MR, 2024). The overdose death numbers decreased by 27% to approximately

2

80,000 in 2024 compared to 110,037 in 2023, yet these statistics demonstrate persistent weaknesses in the system (Statistics, 2025).

Traditional painkillers such as NSAIDs and anticonvulsants provide limited pain relief by reducing pain by 30 − 50% but their long-term use is restricted by adverse side effects (Finnerup et al., 2015).

The evident realities reveal the urgent need for therapeutics beyond the opioids. Recent studies therefore emphasise the need to look beyond these traditional modalities (Jayathilake et al., 2025, Johnson et al., 2025). In this context, four emerging categories of interventions have drawn particular interest as potential chronic pain therapies: neuromodulation therapies, TRP channel blockers, cannabinoid-based treatments, and monoclonal antibody therapies.

The to:

primary

objective

of

this

review

is

1. To describe the fundamental molecular processes that cause chronic pain.

2. To assess the effectiveness and safety of new therapeutic targets and their mechanisms of action in preclinical and clinical studies.

3. To identify gaps in current knowledge and promising areas for future research in chronic pain therapeutics.

4. To provide evidence-based recommendations for translating molecular discoveries into clinical practice.

The literature shows growing interest in these four emerging therapeutic strategies because of recent developments. These approaches demonstrate mechanism-based targeted strategies for chronic pain management which move beyond traditional systemic therapies.

II. M OLECULAR PATHOPHYSIOLOGY OF CHRONIC PAIN

The complex nature of chronic pain requires more than symptom analysis to understand it properly. Instead, a molecular-level framework has become essential to dissect the pathophysiology of different pain types: nociceptive, inflammatory, and neuropathic, each of which arises from distinct mechanisms and therefore demands tailored

therapeutic strategies (Finnerup et al., 2021, Vardeh et al., 2016).

Key molecular pathway in chronic pain includes ion channel dysregulation, neuroinflammation and immune-mediated signalling. TRPV1 functions as a key nociceptive signalling molecule which responds to heat and capsaicin stimuli while also participating in peripheral sensitisation processes (Julius and Basbaum, 2001). The voltage-gated sodium channels Nav1.7, Nav1.8 and Nav1.9 become overexpressed in chronic pain conditions to produce spontaneous neuronal activity and hyperexcitability (Dib-Hajj et al., 2013). The proinflammatory cytokines TNF-α, IL-1β and IL-6 which immune and glial cells produce enhance nociceptor excitability and contribute to central sensitisation (Miller et al., 2009). Inflammatory pain is also mediated by COX-2 and prostaglandin E2 (PGE2), which lower pain activation thresholds via EP receptors (Ricciotti and FitzGerald, 2011) , while activation of the complement system (particularly through C3a and C5a) further sensitises pain pathways via complement receptors on sensory neurons (Ren and Dubner, 2010).

Neuroinflammation functions as a fundamental pain driver which affects both inflammation and neuropathic chronic pain subtypes. Activated glial cells including microglia (Iba1+ , CD11b+ ) and astrocytes (GFAP+ , S100β + ) produce proinflammatory cytokines and chemokines and neurotrophic factors which disrupt synaptic homeostasis and cause neuronal hyperexcitability and central sensitisation (Ji et al., 2013). The persistent activation of glial cells has evolved into a recognised characteristic of maladaptive pain processing. The dysregulation of neurotrophic factors NGF and BDNF affects receptor sensitivity and synaptic strength through mechanisms that include NGF enhancement of TRPV1 activity and substance P and CGRP release in sensory afferents which strengthens pain signalling (Pezet and McMahon, 2006).

Sensitisation occurs at both peripheral and central levels. Peripherally, inflammatory mediators including prostaglandins and cytokines increase the activation threshold of nociceptors by modifying ion channels such as TRPV1 and sodium channels which results in increased pain sensitivity (Woolf and Ma, 2007). The spinal circuits develop longterm synaptic plasticity because of continuous

3

nociceptive input which results in changes to NMDA and AMPA receptor activity and plasticity-related transcription factors c-Fos and CaMKII expression leading to chronic pain states (Latremoliere and Woolf, 2009).

Pain classification has received additional refinement through recent genetic and molecular research. The PLAC8, ROMO1 and A3GALT2 gene trio demonstrates high accuracy (AUROC = 0.919) in distinguishing between neuropathic and nociceptive pain (Young et al., 2023). The expression of genes in chronic pain has been linked by epigenetic modifications which include histone methylation marks H3K27me3 and H3K9ac (Denk and McMahon, 2012). Similarly, microRNAs like miR-155 in inflammatory pain, miR-203 in neuropathic pain, and the let-7 family in nociceptive pain, are now understood to fine- tune molecular cascades including inflammation, ion-channel expression, and synaptic remodelling (Sakai and Suzuki, 2014).

The molecular differentiation of chronic pain types marks a fundamental shift in how we understand and classify pain. These distinct molecular mechanisms provide the foundation for developing targeted therapeutics and precision medicine approaches in chronic pain management which is essential for advancing beyond traditional analgesic approaches toward more effective, personalised pain treatment which will be discussed in the coming section. Understanding these pathophysiological mechanisms is critical for evaluating whether the emerging therapies truly target the underlying causes of chronic pain. The following sections assess four such interventions.

The following search terms were used, individually and in combination, to optimise comprehensives: “Chronic pain”, “Neuromodulation therapy”, “TRP channel blockers”, “Cannabinoid-based analgesia”, “Monoclonal antibodies pain”, “Molecular pain inhibitors”, “Nociception modulation”, “Molecular targets in pain” and “Peripheral and central sensitisation”. Additional studies were identified through reference mining of key articles to ensure conceptual depth.

Inclusion Criteria Studies were screened and compared manually by the author against predefined inclusion criteria. The literature review focused on four distinct treatment categories which included neuromodulation therapies, TRP channel blockers, cannabinoid-based treatments and monoclonal antibodies/molecular inhibitors. The selection of these modalities for review was based on previous studies and initial research findings and the existing scientific evidence supporting their potential applications. These four classes were selected based on promising clinical evidence and sufficient existing literature to allow meaningful evaluation within the review scope No restriction was placed on population demographics and there was no restriction on publication date.

Exclusion Criteria The research excluded case reports and editorials and non-peer-reviewed publications. The studies were excluded if they focused only on acute pain or non-targeted interventions and if they were not published in English.

III. M ETHODS Studies were manually identified by the author through targeted searches in Google Scholar, PubMed, ScienceDirect, ResearchGate and Embase. Clinical and therapeutic guidelines were additionally sourced from databases including the Cochrane library, NICE and NIH/NHS public repositories. The selection prioritised systematic reviews and clinical trials, followed by metaanalyses, randomised controlled trials, preclinical studies, and observational cohort studies relevant to molecular pain mechanisms and interventions.

IV. R ESULTS The initial search included systematic reviews and meta-analyses and clinical trials from PubMed, Cochrane, and Google Scholar databases which were narrowed down to four intervention classes: neuromodulation, TRP channel modulators, cannabinoid-based therapies, and monoclonal antibodies. The primary outcomes measured were changes in pain intensity (VAS/NRS, WOMAC), functional improvement, and safety.

4

Intervention Class

Neuromodulation

TRP Channel modulators

CannabinoidBased Therapies

Monoclonal Antibodies

Key Included Studies Huygen 2024 (SCS vs medical management, network meta-analysis); West 2023 (SCS lead migration, metaanalysis); Hoelzer 2017 (SCS infection rates); Jiang 2022 (rTMS, meta-analysis); Attal 2016 (rTMS vs sham/tDCS RCT); Lapa 2025 (rTMS in fibromyalgia); Costa 2024 (tVNS, meta-analysis); Farmer 2021 (tVNS consensus guidelines); Lange 2011 (fibromyalgia, implantable VNS trial); Duff 2024 (auricular VNS, metaanalysis).

Arendt-Nielsen 2016 (TRPV1 antagonist V116517, human pain model); Iftinca 2021 (TRPV1-targeted drugs, review); Koivisto 2024 (TRP channel therapies, review); Koivisto 2018 (TRPA1 antagonists, review); Mellado Lagarde 2024 (TRPA1 antagonist LY3526318, 3 RCTs).

Johal 2020 (cannabinoids in chronic non-cancer pain, meta-analysis); Wang 2021 (medical cannabis for pain, metaanalysis); Huggins 2012 (FAAH inhibitor PF- 04457845, OA knee pain RCT); Kaur 2016 (BIA 10-2474 safety review); Rezende 2023 (ECS review); Cecilio 2023 (cannabis vs neuromodulators review).

Schnitzer 2015 (anti-NGF antibodies, OA meta-analysis); Jayabalan 2017 (tanezumab in musculoskeletal conditions, review); Siebenga 2020 (Nav1.7 inhibitor PF-05089771, human pain models); Yang 2025 (Nav1.7 inhibitor translational gap, review); Wermeling 2005 (ziconotide, review).

V. E MERGING TREATMENTS IN CHRONIC PAIN MANAGEMENT

Recent advances in chronic pain research have highlighted four promising strategies that target pain at the molecular or circuit level.

First, neuromodulation therapies (e.g. spinal cord stimulation (SCS), transcranial magnetic stimulation (TMS), and vagus nerve stimulation (VNS)) directly alter neural pain circuits. Second, TRP channel blockers (antagonists of TRPV1, TRPA1, etc.) aim to silence overactive nociceptor channels. Third, cannabinoid-based approaches modulate the endocannabinoid system using THC/CBD or enzyme inhibitors (FAAH inhibitors).

Fourth, molecular inhibitors (such as anti–nerve growth factor antibodies and selective ion- channel blockers like Nav1.7 inhibitors) target key pain mediators. Each of these emerging therapies

has shown efficacy in some conditions but also important risks or limitations.

A. Neuromodulation therapies Neuromodulation uses electrical or magnetic stimulation to alter pain signalling in the nervous system. The paradigmatic example is Spinal Cord Stimulation (SCS), in which implanted electrodes deliver patterned pulses to the dorsal columns. By activating large-diameter afferents and inhibitory interneurons in the spinal cord (“gate control”) and engaging descending modulatory pathways, SCS can markedly reduce chronic neuropathic pain (e.g. failed back surgery syndrome, complex regional pain syndrome) (Huygen et al., 2024). In a 2024 systematic review and network meta- analysis (13 trials, 1,561 patients), both conventional SCS and newer high- frequency/paraesthesia-free SCS produced significantly greater pain relief than medical therapy alone for back and leg pain (Huygen et al., 2024). For example, novel SCS paradigms (e.g. high-frequency or differential target multiplexed SCS) were associated with much higher odds of ≥ 50% pain reduction (OR ≈ 9) than conventional medical management (Huygen et al., 2024). These studies also found modest functional improvements with SCS versus control (e.g. mean 7-point drop on WOMAC function) (Huygen et al., 2024). Notably, closed-loop (feedback-controlled) SCS systems are now in trial: the 36-month EVOKE RCT showed that adaptive closed-loop SCS (which adjusts stimulation in real time to maintain paraesthesia) produced more durable relief than traditional open-loop SCS, with higher rates of ≥ 50% pain reduction (77.6% vs.

49.3% at 36 months).

Despite efficacy, SCS carries device-related risks. A recent meta-analysis (53 studies, 2932 patients) estimated lead migration in about 10% of implants (West et al., 2023). Most migrations required revision surgery. Other complications include hardware failure, epidural hematoma, and infection. Infection rates in large series have ranged 2 − 6% (Hoelzer et al., 2017). These device issues, lead displacement, battery replacements, wound complications, limit SCS use to carefully selected patients. In addition, high costs and the need for

5

surgical implantation are important barriers.

1) Transcranial Magnetic Stimulation (TMS): This refers to non-invasive neuromodulation that uses focused magnetic pulses to excite or inhibit cortical regions involved in pain. Repetitive TMS (rTMS) over the motor cortex or dorsolateral prefrontal cortex can engage descending inhibitory pathways and alter maladaptive cortical plasticity.

Meta-analyses of randomized trials show that high frequency rTMS yields modest pain relief in neuropathic pain conditions. For example, Jiang et al. (2022) pooled 38 trials of rTMS for chronic neuropathic pain and found a significant analgesic effect versus sham (effect size ≈ −0.66, favouring active treatment) (Jiang et al., 2022).

Efficacy appeared to depend on parameters such as stimulation site, frequency, treatment course. In a head-to-head trial in patients with lumbosacral radiculopathy neuropathic pain, 10 Hz rTMS produced significantly greater pain reduction than sham or anodal tDCS (p=0.023) (Attal et al., 2016).

These studies show that rTMS is safe and can improve chronic neuropathic pain scores over short follow- up. However, results have been mixed in conditions like fibromyalgia, partly due to variable protocols (Lapa et al., 2025). Overall, evidence supports use of rTMS as an adjunct in refractory neuropathic pain, but its role is limited by modest effects, short duration of benefit, and heterogeneity of response.

2) Vagus Nerve Stimulation (VNS): This particularly non-invasive transcutaneous VNS (tVNS) has demonstrated analgesic potential in chronic pain by modulating brainstem nuclei such as the nucleus tractus solitarius and locus coeruleus.

A recent meta-analysis of 15 randomized controlled trials (auricular or cervical tVNS) published through October 2023 reported a moderate pooled effect size of Cohen’s d = 0.41 (95 % CI 0.17–0.66) favouring active stimulation over controls, with moderate heterogeneity (I2 ≈ 54%) (Costa et al., 2024) . In trials using non-active (sham or no-stimulation) comparators (n = 3), the effect size was larger at d ≈ 0.79 (95 % CI 0.25–1.33). Subgroup analyses further revealed similar moderate effects for both auricular tVNS (d = 0.42) and cervical tVNS (d = 0.36) (Costa et al., 2024) . Safety profiles were reassuring stimulation

was mild, mostly causing transient skin irritation or discomfort, with no serious adverse events reported across studies (Farmer et al., 2021). Early pilot trials have also indicated possible benefit in fibromyalgia, with implantable VNS showing preliminary improvement in tender points and tolerability in an open-label Phase I/II cohort of 14 patients (Lange et al., 2011) and in chronic knee osteoarthritis and pelvic pain, showing analgesic trends though limited by small sample sizes and preliminary designs (Duff et al., 2024). However, most studies remain small and short-term, and optimal patient selection criteria and stimulation parameters are not yet established.

Overall, neuromodulation shows promise for chronic pain by altering neural circuits. SCS has the strongest evidence for chronic back and neuropathic pain (Huygen et al., 2024), and innovations like closed-loop control can enhance outcomes. TMS provides a non-invasive option with moderate benefits in neuropathic pain (Jiang et al., 2022, Attal et al., 2016). VNS/tVNS is emerging with some positive trials but needs further validation. All device-based therapies carry limitations: surgical risks, device malfunction, and costs limit patient eligibility. The durability of effects (especially for TMS and VNS) is also a concern, and long-term benefit beyond months is not well established.

B. TRP Channel Blockers Transient Receptor Potential (TRP) channels on nociceptor nerve endings transduce thermal and chemical stimuli into pain signals. Among these, TRPV1 (the capsaicin/heat receptor) and TRPA1 (the irritant/cold receptor) have been targeted for analgesia. TRPV1 antagonists aim to block noxious heat signalling and inflammatory sensitisation, while TRPA1 blockers aim to prevent amplification of pain by endogenous irritants. In principle, blocking these channels should reduce chronic pain signals at the periphery.

Several TRPV1 antagonists have been developed and tested in trials. These agents (e.g. V116517, SB705498, ABT-102) reliably raise heat pain thresholds in humans, confirming on-target effects. In a human capsaicin model, the TRPV1 antagonist V116517 significantly increased heat pain thresholds and suppressed capsaicin induced hyperalgesia

6

(Arendt-Nielsen et al., 2016). Notably, V116517 did not induce systemic hyperthermia in that trial (Arendt-Nielsen et al., 2016), unlike some earlier TRPV1 antagonists. However, an inherent issue is that blocking TRPV1 also blunts normal heat sensation. The V116517 study warned of ‘heat analgesia’ as a safety concern if patients cannot feel dangerous heat, they risk burns. More generally, many TRPV1 blockers in past trials caused hyperthermia (e.g. fever) or reduced the ability to sense high heat, limiting their clinical use (Iftinca et al., 2021, Arendt-Nielsen et al., 2016). Thus, despite proof-ofconcept analgesia in experimental pain, no TRPV1 antagonist has yet reached the market for chronic pain. By contrast, agonists of TRPV1 (capsaicin) are in clinical use (e.g. high-dose capsaicin patch) to desensitise nociceptors. Ongoing trials are exploring injected capsaicin or resiniferatoxin for arthritic and cancer pain (Koivisto et al., 2024), which sidesteps the hyperthermia issue by using agonism-induced defunctionalisation rather than blockade.

TRPA1 antagonists are also under investigation.

TRPA1 mediates pain from cold, oxidative stress and inflammatory mediators. Animal models show that blocking TRPA1 profoundly reduces mechanical and cold hyperalgesia with minimal side effects (Koivisto et al., 2018). A novel TRPA1 antagonist, LY3526318, was tested in three placebo-controlled trials of knee osteoarthritis, chronic low back pain, and diabetic neuropathy (the “Chronic Pain Master Protocol”) (Mellado Lagarde et al., 2024). Unfortunately, LY3526318 did not significantly reduce pain versus placebo at 4 weeks in any of these conditions.

There was a non-significant trend toward improvement in low back pain, but not in OA or diabetic neuropathy. Moreover, a concerning finding was that LY3526318 caused liver enzyme elevations in some patients (a potential hepatotoxic effect) (Mellado Lagarde et al., 2024). Thus, although TRPA1 is an attractive target biologically, clinical development faces challenges.

In summary, TRP channel blockade remains experimental. TRPV1 antagonists can relieve pain in models, but systemic hyperthermia and loss of heat sensation have stymied progress (Iftinca et al., 2021, Arendt-Nielsen et al., 2016). TRPA1 antagonists have shown efficacy in animals but thus far have failed to demonstrate clear benefit in human trials and may carry organ toxicity (Mellado Lagarde et al., 2024). Common adverse effects of TRP

blockers include altered thermosensation (elevated body temperature or inability to sense heat (Iftinca et al., 2021)) and potential organ-specific toxicity. In practice, translating TRP modulators into therapies is hindered by safety issues and the redundancy of pain pathways. Future directions may focus on localised delivery (e.g. intra-articular TRPV1 agonist injections) or combination therapies that mitigate thermoregulatory risk (Koivisto et al., 2024).

C. Cannabinoid-Based Therapies The endocannabinoid system (ECS) enables cannabinoids to control pain through CB1 and CB2 receptors that exist mainly in the central nervous system and immune cells and through natural ligands anandamide and 2arachidonoylglycerol (2-AG) (Rezende et al., 2023). Exogenous cannabinoids such as THC and CBD interact with these pathways through THC which functions as a partial CB1 agonist to produce analgesia and euphoria and CBD which lacks psychoactivity yet influences receptor activity (e.g., glycine, GABAA ) and enzymatic processes and inflammatory mediators (Cecilio and Júnior, 2023).

The therapeutic approach focuses on elevating endocannabinoid levels through fatty acid amide hydrolase (FAAH) enzyme modulation to provide pain relief without CB1 activation (Schlosburg et al., 2009).

Systematic reviews of clinical trials show that cannabinoids produce modest analgesia in chronic noncancer pain. For example, Johal et al. (2020) pooled 36 trials (4,006 patients) of smoked, oromucosal (spray), or oral cannabinoids. Compared to placebo, cannabinoids significantly reduced pain scores by ∼0.68 points on a 0–10 scale at 2–8 weeks (Johal et al., 2020). This reduction is small but statistically significant, and it was somewhat larger for oral cannabinoids (e.g. dronabinol, nabilone) than for inhaled/spray formulations.

Another meta-analysis (BMJ 2021) similarly found a small increase (∼10% more patients) achieving minimal pain relief with medical cannabis versus placebo (Wang et al., 2021). Thus, cannabinoids can improve pain and sleep/function to a small degree (e.g. ∼1–2 point change on quality-of-life scales) (Wang et al., 2021). However, these benefits are generally considered small in magnitude.

The evidence certainty is moderate for short-term

7

effects; longer-term benefits remain uncertain (Johal et al., 2020, Wang et al., 2021). Adverse effects and risks are important drawbacks. Meta-analyses show cannabinoids significantly increase non-serious side effects (nausea, dizziness, sedation, cognitive changes) compared to placebo (Johal et al., 2020, Wang et al., 2021). In the BMJ review, oral cannabinoids were associated with small absolute risks of dizziness (9–28%), cognitive impairment (∼2 6%), nausea, vomiting and drowsiness (Wang et al., 2021). Serious adverse events were rare and comparable to placebo in modern trials (Johal et al., 2020), but psychoactive THC can cause acute intoxication symptoms or, rarely, psychosis in vulnerable individuals. Thus, psychoactive risk is a clear concern, especially with high-THC products.

FAAH inhibitors (which block endocannabinoid breakdown) have been tested to avoid psychoactivity. The FAAH inhibitor PF-04457845 increased anandamide levels but failed to produce analgesia in osteoarthritis knee pain (Huggins et al., 2012). The drug was well tolerated, indicating that mere elevation of endocannabinoids may not suffice for pain relief. A first-in-human FAAH inhibitor (BIA 10-2474) tragically caused brain haemorrhages in a 2016 trial that has a total of 128 participants (Kaur et al., 2016), highlighting that off-target toxicity can doom this approach.

Dosing and formulation variability also limit cannabinoids. Plant-derived products vary widely in THC/CBD content, bioavailability and metabolism.

Clinical trials use regulated pharmaceutical formulations (dronabinol, nabiximols (THC+CBD spray), nabilone) with fixed dosing, but patients in practice may use inconsistent potencies. Thus, translating trial results to real-world dosing is difficult. Legal and regulatory contexts are critical.

Medical cannabis is legal for chronic pain in Canada and parts of the US while pharmaceutical cannabinoids (nabilone, dronabinol) are approved for other indications (e.g. chemotherapy nausea).

However, cannabinoids remain Schedule I (illegal) under US federal law, which complicates research and prescribing. Some agencies (e.g. the American College of Physicians) recommend considering cannabinoids only after conventional therapies fail, given the modest benefit (Wang et al., 2021). In summary, cannabinoids show limited analgesic benefit in chronic pain (Johal et al., 2020, Wang et al., 2021), tempered by psychoactive and cognitive

side effects. Their place in therapy is still being defined by ongoing trials and changing regulations.

D. Monoclonal antibodies and molecular inhibitors This category includes biologic and small molecule drugs targeting specific pain mediators.

The prototype is anti–nerve growth factor (NGF) antibodies (e.g. tanezumab, fulranumab, fasinumab).

NGF sensitises nociceptors in inflammation; blocking it can potently reduce pain. Multiple randomized trials in osteoarthritis (knee/hip) have shown that anti-NGF antibodies provide large analgesic effects. In one meta analysis of 13 trials, highdose tanezumab produced a standardized effect size of ∼0.7 on WOMAC pain (versus placebo) (Schnitzer and Marks, 2015). Even in later phase III, tanezumab significantly outperformed NSAIDs, yielding better pain and function (Schnitzer and Marks, 2015). Other anti-NGF agents (fulranumab, fasinumab) showed similar trends.

However, early trials revealed potential joint related risks such as rapidly progressive osteoarthritis, which prompted temporary regulatory halts.

Most reviews note that safety risks (requiring joint replacements) increase at higher antibody doses (Schnitzer and Marks, 2015). For example, Schnitzer et al. found that withdrawals due to adverse events were more frequent at higher anti-NGF doses, though low doses appeared safe. Current development is focused on lower doses and careful patient selection. A 2021 JAMA trial showed that low dose tanezumab (2.5 mg) still improved OA pain significantly, but with some cases of arthropathy (Schnitzer and Marks, 2015). Tanezumab development was halted in 2021 after the FDA advisory committee rejected approval over safety concerns.

Anti-NGF antibodies remain investigational, with approval dependent on optimising risk–benefit and ensuring long-term safety (Pfizer et al., 2021).

The Nav1.7 sodium channel serves as another molecular target because it is encoded by SCN9A. The SCN9A gene contains loss-of function mutations which result in congenital insensitivity to pain, thus making Nav1.7 an attractive analgesic target.

Numerous selective Nav1.7 blockers have been developed. Unfortunately, clinical trials have been disappointing so far. For example, the selective Nav1.7 inhibitor PF-05089771 was tested in a battery of

8

human pain models, alone and with pregabalin. It failed to produce any significant analgesic effect versus placebo (Siebenga et al., 2020). Previous diabetic neuropathy trials showed only modest, nonsignificant pain reduction. In healthy volunteers, PF05089771 did not differ from placebo on evoked pain measures (Siebenga et al., 2020). In fact, study authors concluded that Nav1.7 inhibition had no discernible analgesia in their test battery. Several similar compounds have been tested (for example in post-herpetic neuralgia, trigeminal neuralgia, etc.) with no robust success. It appears that selectively blocking Nav1.7 alone may be insufficient, possibly because of redundant pain pathways or inadequate peripheral bioavailability. Thus, Nav1.7 blockers remain promising in theory but unproven in practice (Siebenga et al., 2020). Other molecular inhibitors are in development (e.g. bradykinin antagonists, TRK receptor inhibitors, etc.), but evidence in chronic pain is limited. One example is ziconotide, a peptide N type calcium-channel blocker (delivered intrathecally) that provides potent analgesia for refractory pain, but its complexity (intrathecal pump, strict titration) limits use (Wermeling, 2005).

In summary, monoclonal antibodies like anti-NGF have demonstrated strong analgesia in osteoarthritis trials (Schnitzer and Marks, 2015) but carry safety issues of joint deterioration. Selective ion-channel blockers such as Nav1.7 antagonists remain experimental, with trials failing to show clear benefit (Siebenga et al., 2020). The use of NGF-related arthropathy and CNS effects of channel blockers as safety concerns limits the level of enthusiasm. The future directions include the refinement of dosing and regimen (e.g. intermittent NGF blockade) and the combination of targets, but at present these therapies are not part of standard chronic pain management.

VI. D ISCUSSION A. Comparative efficacy and clinical viability Emerging molecularly targeted therapies show varied efficacy relative to conventional chronic pain treatments. Neuromodulation has robust evidence of benefit. Spinal cord stimulation (SCS), especially with newer waveforms (high frequency, burst, etc.), consistently outperforms conventional medical management (CMM) in chronic neuropathic back and limb pain. As shown in the Huygen 2024

network meta analysis, both traditional and novel SCS approaches outperform conventional medical management, confirming its role in difficult-to-treat neuropathic pain ((Huygen et al., 2024). Repetitive TMS (rTMS) also yields statistically significant pain reduction. A 2021 systematic review noted that high-frequency stimulation over motor cortex (M1) produced the best outcomes, although protocols vary widely (Attia et al., 2021). Transcutaneous vagus nerve stimulation (tVNS) is newer but promising: a recent meta-analysis found a moderate pooled effect (Cohen’s d≈0.41) in favour of tVNS versus control for overall pain reduction (Costa et al., 2024). Notably, auricular versus cervical tVNS had similarly small-to moderate benefits.

By contrast, TRP channel blockers have mixed results. Topical capsaicin (a TRPV1 agonist) is in wide clinical use for neuropathic pain, often providing sustained relief with good tolerability (Koivisto et al., 2024, Zhou et al., 2023). A recent meta-analysis in arthralgia found capsaicin and other TRPV1-targeted preparations significantly reduced chronic joint pain (standardised mean difference ≈–0.53 versus placebo/NSAIDs) (Zhou et al., 2023). Both TRPV1 and TRPA1 antagonists remain experimental, with translation hindered by safety issues and limited efficacy in trials (Koivisto et al., 2024, Jain et al., 2022).

Cannabinoid-based therapies yield only modest benefit. The results of extensive reviews indicate THC/CBD produces small but measurable positive effects on chronic pain management. Meta-analyses show cannabinoids provide only small improvements in pain, with typical psychoactive and cognitive side effects (Bilbao and Spanagel, 2022). The overall therapeutic advantage is minimal because patients need to consider typical side effects which include cognitive and mood alterations and sedation.

FAAH inhibitors (which raise endocannabinoid levels) have so far failed to show clinical analgesia.

A Phase 2 osteoarthritis trial of the FAAH inhibitor PF-04457845 robustly inactivated FAAH yet showed no pain relief compared to placebo (Huggins et al., 2012), leading to early study termination for futility. This discordance between target engagement and pain outcomes highlights limitations.

Among monoclonal antibodies and molecular inhibitors, anti–nerve growth factor (NGF) antibodies have shown striking pain reductions. Multiple RCTs in osteoarthritis and low back pain report that anti-

9

NGF mAbs (e.g. tanezumab, fasinumab) achieve “clinically meaningful pain relief” (Jayabalan and Schnitzer, 2017) superior to placebo or NSAIDs, with effects that can persist in longer-term followups. These drugs tend to outperform traditional analgesics on intensity and function outcomes. In contrast, Nav1.7 sodium-channel inhibitors have not met expectations. The genetic evidence supporting Nav1.7 blockers (Nav1.7 mutations cause insensitivity to pain) has not translated into clinical pain relief in trials (Yang et al., 2025). The efficacy of anti-NGF therapy exceeds conventional analgesics for specific conditions but Nav1.7 inhibitors have not proven effective in clinical practice.

B. Promising Therapies and Precision Pain Management Given the evidence, anti-NGF antibodies and advanced neuromodulation currently seem most promising. Anti-NGF mAbs stand out for both strong analgesia and some evidence of sustained effect (Jayabalan and Schnitzer, 2017). Recent trials, especially at optimised lower doses, show that these biologics can meaningfully reduce osteoarthritis and chronic low back pain when traditional drugs fail. Likewise, SCS (particularly with new high-frequency or burst waveforms) is repeatedly shown to provide superior pain relief and functional gains compared to medications (Huygen et al., 2024, Head et al., 2019). Non invasive neuromodulation like TMS and tVNS may be useful adjuncts: they produce smaller effects but can be deployed without surgery and might benefit patients averse to drugs.

This body of evidence suggests a move toward precision pain medicine. For example, patient selection can be guided by mechanistic phenotyping. The ISC17536 trial illustrates that a TRPA1 antagonist could be effective in a defined subgroup (“pain phenotype”) (Jain et al., 2022). Genetic testing for SCN9A mutations will potentially identify patients who would benefit from Nav1.7 blockers when more potent or combination drugs become available.

Neuroimaging and sensory testing could predict TMS responders through motor cortex excitability patterns, and psychological profiling could determine who tolerates cannabinoid side effects. The integration of biomarkers (imaging, genetics, quantitative sensory tests) with these emerging therapies will enable personalised treatment by providing the appropriate therapy to the appropriate patient.

C. Research gaps and translational challenges The field has made significant progress, but major gaps continue to exist. The translation of numerous promising targets into clinical practice has failed because preclinical models have proven insufficient.

For instance, Nav1.7 blockers exemplify a “discordance” problem: most animal studies used young male rodents and measured evoked pain after a single dose, whereas human trials involved diverse adults with chronic neuropathy and repeated dosing (Yang et al., 2025). This mismatch likely contributed to clinical failure. Similarly, many neuromodulation trials lack long-term data: while 6- or 12-month results exist, we need more evidence on multi-year durability and optimal device programming.

Major research needs include better trial designs and endpoints. Chronic pain trials must contend with high placebo response and subjective outcomes.

The use of innovative designs such as crossover ‘withdrawal’ designs in some cannabis trials and objective biomarkers, including neuroimaging and nociceptive reflexes could enhance the rigor of the study. The heterogeneity of the sample population is a challenge because RCTs often combine different pain conditions that may have different responses (e.g. nociceptive vs neuropathic). Future trials should either stratify by mechanism or use adaptive designs that are guided by interim biomarker signals.

The current research lacks information about safety optimisation and dosing methods. The doseresponse relationships of TRP modulators and cannabinoids are intricate because higher doses lead to toxicity without providing significant additional pain relief.

The process of developing pain therapies from molecular targets needs to match preclinical models to human disease conditions while identifying patient benefits and collecting strong long-term outcome data.

D. Ethical and regulatory considerations The development of these new therapies brings multiple ethical and regulatory matters into play.

Neuromodulation devices present surgical risks together with high costs. The reversible nature of SCS implantation does not eliminate its invasive nature

10

which creates challenges regarding healthcare resource distribution and patient eligibility criteria.

The non-invasive methods TMS and tVNS provide safer alternatives but they remain costly and need specialised equipment and training. The disclosure of potential conflicts of interest stands as a fundamental requirement because tVNS studies frequently involve device manufacturers (Costa et al., 2024) thus requiring independent trials with strict protocols. The development of anti-NGF antibodies encountered regulatory challenges because early clinical trials showed patients developed rapidly progressive osteoarthritis. The FDA implemented strict risk mitigation strategies (e.g. dosing caps, exclusions) before reconsidering approval (Jayabalan and Schnitzer, 2017). The ethical requirement to monitor joint safety will become essential for these drugs to be used on a large scale.

Cannabinoid therapies create multiple legal and social problems. Medical cannabis and cannabinoids have received approval in certain jurisdictions, yet multiple regions maintain their prohibition status. Medical practitioners need to handle different regulatory frameworks while patients can access untested preparations whose strength remains unknown. The use of cannabinoids requires careful consideration because they can cause negative effects on the mind and behaviour of people who are young or have substance abuse problems. The fatal Phase I accident involving BIA 10-2474 has led to strict regulations for novel drugs including FAAH inhibitors while safety monitoring and ethical oversight of early trials continue to be essential priorities.

The last concern involves equal access and cost. The high cost of these therapies (implantable devices, biologic antibodies) may restrict their use to wellinsured patients and create health equity issues. We need to guarantee that pain management progress does not create more health disparities.

The emerging molecular treatments for chronic pain demonstrate promising results but they also present certain boundaries. The controlled trials demonstrate that these treatments perform better than standard medications (e.g. SCS and anti NGF antibodies (Huygen et al., 2024, Jayabalan and Schnitzer, 2017)), but their effectiveness does not extend to all patients. The variable duration of treatment effects can be explained through mechanistic understanding of channel function and neural

plasticity. The optimisation of treatment approaches through individual patient biological characteristics (e.g. sensory phenotypes, genetics) will lead to better results. The path to full integration of these new approaches with traditional therapies requires substantial translational work that includes better trial design and safety and ethical resolution.

VII. L IMITATIONS The review contains multiple essential limitations. The review only included English-language systematic reviews and meta-analyses which could have excluded unpublished data and non English trials while creating language/publication bias. The heterogeneity of trial designs and populations limits comparability and generalisability. The research duration was brief, and the participant numbers were limited which prevented scientists from fully understanding long-term effectiveness and safety outcomes. The study cannot confirm the presence of publication and reporting biases. The clinical heterogeneity of chronic pain makes it difficult to apply findings from one condition or mechanism to other conditions. Our conclusions face limitations because of the restricted scope and heterogeneity and bias and limited data availability.

VIII. C ONCLUSION AND F UTURE D IRECTIONS Our comprehensive review shows that spinal cord stimulation (SCS) and anti-nerve growth factor (anti-NGF) antibodies are the most effective emerging chronic pain treatments. Other interventions have produced either minimal or inconsistent therapeutic outcomes. Cannabinoids offer modest relief with notable psychoactive and cognitive side effects, limiting broader adoption (Banerjee and McCormack, 2019). TRP channel modulators exist in experimental stages because initial TRPV1 antagonist trials failed to deliver results and highconcentration agonists produce brief pain episodes (Koivisto and Szallasi, 2023). NaV1.7-selective inhibitors failed to demonstrate analgesic effects in clinical research despite their genetic potential.

Non-invasive neuromodulation techniques such as repetitive TMS produce temporary pain relief, but the diverse nature of protocols and results makes it impossible to draw definitive conclusions (Hamid et al., 2019). The most effective treatments for chronic pain appear to be SCS and anti-NGF therapies while

11

other modalities show promise but lack sufficient evidence.

The future of medical practice will depend on mechanistic phenotyping and biomarker-guided precision medicine. The biological diversity of chronic pain requires patients to receive treatment based on their specific underlying mechanisms which include sensory profiles and inflammatory markers and genetic signatures. The discovery of objective molecular biomarkers that predict pain state has created opportunities for “precision” trials (Niculescu et al., 2019). The integration of diagnostic tests into clinical research allows doctors to select treatments that will provide the most benefit to their patients.

Future research needs to conduct extended investigations about spinal cord stimulation durability and safety together with real-world data collection for patient retention rates and adverse event monitoring.

The use of adaptive and platform trial designs will speed up the testing process for different patient populations. The safety monitoring process needs to remain strict because of the anti-NGF therapy issues and potential cognitive side effects from cannabinoids. The inclusion of diverse and underserved populations in trials remains vital to establish precision pain management as an equitable treatment option for all patients.

Translating molecular insights into targeted therapies will be essential to achieving safe, effective, and equitable pain relief for millions living with chronic pain.

IX. R EFERENCES

sham controlled comparative study. Pain, 157, 12241231.

ATTIA, M., MCCARTHY, D. & ABDELGHANI, M. 2021. Repetitive Transcranial Magnetic Stimulation for Treating Chronic Neuropathic Pain: a Systematic Review. Curr Pain Headache Rep, 25, 48.

BANERJEE, S. & MCCORMACK, S. 2019.

CADTH Rapid Response Reports. Medical Cannabis for the Treatment of Chronic Pain: A Review of Clinical E ectiveness and Guidelines.

Ottawa (ON): Canadian Agency for Drugs and Technologies in Health Copyright © 2019 Canadian Agency for Drugs and Technologies in Health.

BILBAO, A. & SPANAGEL, R. 2022. Medical cannabinoids: a pharmacology-based systematic review and meta-analysis for all relevant medical indications. BMC Medicine, 20, 259.

CECILIO, S. & JÚNIOR, J. 2023. Cannabis versus neuromodulators in chronic pain. Brazilian Journal Of Pain, 6, 146-152.

COSTA, V., GIANLORENçO, A. C., ANDRADE, M. F., CAMARGO, L., MENACHO, M., ARIAS AVILA, M., PACHECO-BARRIOS, K., CHOI, H., SONG, J. J. & FREGNI, F. 2024. Transcutaneous vagus nerve stimulation e ects on chronic pain: systematic review and meta-analysis. Pain Rep, 9, e1171.

ARENDT-NIELSEN, L., HARRIS, S., WHITESIDE, G. T., HUMMEL, M., KNAPPENBERGER, T., O’KEEFE, S., KAPIL, R. & KYLE, D. 2016. A randomized, doubleblind, positive-controlled, 3-way cross-over human experimental pain study of a TRPV1 antagonist (V116517) in healthy volunteers and comparison with preclinical profile. Pain, 157, 2057-2067.

DENK, F. & MCMAHON, S. B. 2012. Chronic pain: emerging evidence for the involvement of epigenetics. Neuron, 73, 435-44.

ATTAL, N., AYACHE, S. S., CIAMPI DE ANDRADE, D., MHALLA, A., BAUDIC, S., JAZAT, F., AHDAB, R., NEVES, D. O., SOREL, M., LEFAUCHEUR, J. P. & BOUHASSIRA, D. 2016. Repetitive transcranial magnetic stimulation and transcranial direct-current stimulation in neuropathic pain due to radiculopathy: a randomized

DUFF, I. T., LIKAR, R., PERRUCHOUD, C., KAMPUSCH, S., KöSTENBERGER, M., SATOR, S., STREMNITZER, C., WOLF, A., NEUWERSCH-SOMMEREGGER, S. & ABDELSAYED, A. 2024. Clinical E icacy of Auricular Vagus Nerve Stimulation in the Treatment of Chronic and Acute Pain: A Systematic Review and

DIB-HAJJ, S. D., YANG, Y., BLACK, J. A. & WAXMAN, S. G. 2013. The Na(V)1.7 sodium channel: from molecule to man. Nat Rev Neurosci, 14, 49-62.

12

Meta analysis. Pain and Therapy, 13, 1407 1427.

adults: a systematic review and meta analysis.

Lancet Neurol, 14, 162-73.

ENGLAND, N. 2023. Reducing long-term opioid use [Online]. Available: https://www.england. FINNERUP, N. B., KUNER, R. & JENSEN, nhs.uk/longread/reducing-long-term-opioid-use/ T. S. 2021. Neuropathic Pain: From Mechanisms [Accessed 14 June 2025]. to Treatment. Physiol Rev, 101, 259-301.

FARMER, A. D., STRZELCZYK, A., FINISGUERRA, A., GOURINE, A. V., GHARABAGHI, A., HASAN, A., BURGER, A. M., JARAMILLO, A. M., MERTENS, A., MAJID, A., VERKUIL, B., BADRAN, B. W., VENTURA-BORT, C., GAUL, C., BESTE, C., WARREN, C. M., QUINTANA, D. S., HäMMERER, D., FRERI, E., FRANGOS, E., TOBALDINI, E., KANIUSAS, E., ROSENOW, F., CAPONE, F., PANETSOS, F., ACKLAND, G. L., KAITHWAS, G., O’LEARY, G. H., GENHEIMER, H., JACOBS, H. I. L., VAN DIEST, I., SCHOENEN, J., REDGRAVE, J., FANG, J., DEUCHARS, J., SZéLES, J. C., THAYER, J. F., MORE, K., VONCK, K., STEENBERGEN, L., VIANNA, L. C., MCTEAGUE, L. M., LUDWIG, M., VELDHUIZEN, M. G., DE COUCK, M., CASAZZA, M., KEUTE, M., BIKSON, M., ANDREATTA, M., D’AGOSTINI, M., WEYMAR, M., BETTS, M., PRIGGE, M., KAESS, M., RODEN, M., THAI, M., SCHUSTER, N. M., MONTANO, N., HANSEN, N., KROEMER, N. B., RONG, P., FISCHER, R., HOWLAND, R. H., SCLOCCO, R., SELLARO, R., GARCIA, R. G., BAUER, S., GANCHEVA, S., STAVRAKIS, S., KAMPUSCH, S., DEUCHARS, S. A., WEHNER, S., LABORDE, S., USICHENKO, T., POLAK, T., ZAEHLE, T., BORGES, U., TECKENTRUP, V., JANDACKOVA, V. K., NAPADOW, V. & KOENIG, J. 2021. International Consensus Based Review and Recommendations for Minimum Reporting Standards in Research on Transcutaneous Vagus Nerve Stimulation (Version 2020). Frontiers in Human Neuroscience, Volume 14 - 2020.

FINNERUP, N. B., ATTAL, N., HAROUTOUNIAN, S., MCNICOL, E., BARON, R., DWORKIN, R. H., GILRON, I., HAANPää, M., HANSSON, P., JENSEN, T. S., KAMERMAN, P. R., LUND, K., MOORE, A., RAJA, S. N., RICE, A. S., ROWBOTHAM, M., SENA, E., SIDDALL, P., SMITH, B. H. & WALLACE, M. 2015. Pharmacotherapy for neuropathic pain in

GASKIN, D. J. & RICHARD, P. 2012. The Economic Costs of Pain in the United States. The Journal of Pain, 13, 715-724.

GOLDBERG, D. S. & MCGEE, S. J. 2011.

Pain as a global public health priority. BMC Public Health, 11, 770.

HAMID, P., MALIK, B. H. & HUSSAIN, M. L. 2019. Noninvasive Transcranial Magnetic Stimulation (TMS) in Chronic Refractory Pain: A Systematic Review. Cureus, 11, e6019.

HEAD, J., MAZZA, J., SABOURIN, V., TURPIN, J., HOELSCHER, C., WU, C. & SHARAN, A. 2019. Waves of Pain Relief: A Systematic Review of Clinical Trials in Spinal Cord Stimulation Waveforms for the Treatment of Chronic Neuropathic Low Back and Leg Pain.

World Neurosurg, 131, 264-274.e3.

HOELZER, B. C., BENDEL, M. A., DEER, T. R., ELDRIGE, J. S., WALEGA, D. R., WANG, Z., COSTANDI, S., AZER, G., QU, W., FALOWSKI, S. M., NEUMAN, S. A., MOESCHLER, S. M., WASSEF, C., KIM, C., NIAZI, T., SAIFULLAH, T., YEE, B., KIM, C., ORYHAN, C. L., ROSENOW, J. M., WARREN, D. T., LERMAN, I., MORA, R., HAYEK, S. M., HANES, M., SIMOPOULOS, T., SHARMA, S., GILLIGAN, C., GRACE, W., ADE, T., MEKHAIL, N. A., HUNTER, J. P., CHOI, D. & CHOI, D. Y. 2017. Spinal Cord Stimulator Implant Infection Rates and Risk Factors: A Multicenter Retrospective Study. Neuromodulation, 20, 558-562.

HUGGINS, J. P., SMART, T. S., LANGMAN, S., TAYLOR, L. & YOUNG, T. 2012. An efficient randomised, placebo controlled clinical trial with the irreversible fatty acid amide hydrolase1 inhibitor PF-04457845, which modulates endocannabinoids but fails to induce e ective analgesia in patients with pain due to osteoarthritis

13

of the knee. Pain, 153, 1837-1846.

1179544120906461.

HUYGEN, F., SOULANIS, K., RTVELADZE, K., KAMRA, S. & SCHLUETER, M. 2024. Spinal Cord Stimulation vs Medical Management for Chronic Back and Leg Pain: A Systematic Review and Network Meta-Analysis. JAMA Netw Open, 7, e2444608.

JOHNSON, B. W., STRAND, N. H., RAYNAK, J. C., JARA, C., HABTEGIORGIS, K., HAND, B. A., HONG, S. & MALONEY, J. A. 2025.

Cannabinoids in Chronic Pain Management: A Review of the History, E icacy, Applications, and Risks. Biomedicines, 13.

IFTINCA, M., DEFAYE, M. & ALTIER, C. JULIUS, D. & BASBAUM, A. I. 2001. Molecular 2021. TRPV1-Targeted Drugs in Development for mechanisms of nociception. Nature, 413, 203-10.

Human Pain Conditions. Drugs, 81, 7-27.

KAUR, R., SIDHU, P. & SINGH, S. 2016.

JAIN, S. M., BALAMURUGAN, R., TANDON, What failed BIA 10-2474 Phase I clinical trial?

M., MOZAFFARIAN, N., GUDI, G., SALHI, Global speculations and recommendations for Y., HOLLAND, R., FREEMAN, R. & BARON, future Phase I trials. J Pharmacol Pharmacother, 7, R. 2022. Randomized, double-blind, placebo- 120-6. controlled trial of ISC 17536, an oral inhibitor of transient receptor potential ankyrin 1, in patients KOIVISTO, A., JALAVA, N., BRATTY, R. & with painful diabetic peripheral neuropathy: impact PERTOVAARA, A. 2018. TRPA1 Antagonists for of preserved small nerve fiber function. Pain, 163, Pain Relief. Pharmaceuticals (Basel), 11. e738-e747.

KOIVISTO, A. P. & SZALLASI, A. 2023.

JAYABALAN, P. & SCHNITZER, T. J. Targeting TRP Channels for Pain, Itch and 2017. Tanezumab in the treatment of chronic Neurogenic Inflammation. Int J Mol Sci, 25. musculoskeletal conditions. Expert Opin Biol Ther, 17, 245-254.

KOIVISTO, A. P., VOETS, T., IADAROLA, M. J. & SZALLASI, A. 2024. Targeting TRP JAYATHILAKE, N. J., PHAN, T. T., KIM, J., channels for pain relief: A review of current LEE, K. P. & PARK, J. M. 2025. Modulating evidence from bench to bedside. Curr Opin neuroplasticity for chronic pain relief: noninvasive Pharmacol, 75, 102447. neuromodulation as a promising approach.

Experimental & Molecular Medicine, 57, 501-514. LANGE, G., JANAL, M., MD, A., APN, J., FOBLER, M., COOK, D. & NATELSON, B. 2011.

JI, R.-R., BERTA, T. & NEDERGAARD, M. Safety and Efficacy of Vagus Nerve Stimulation in 2013. Glia and pain: Is chronic pain a gliopathy? Fibromyalgia: A Phase I/II Proof of Concept Trial.

PAIN, 154.

Pain Medicine, 12, 1406-1413 JIANG, X., YAN, W., WAN, R., LIN, Y., ZHU, X., SONG, G., ZHENG, K., WANG, Y. & WANG, X. 2022. E ects of repetitive transcranial magnetic stimulation on neuropathic pain: A systematic review and meta-analysis. Neurosci Biobehav Rev, 132, 130-141.

LAPA, J., DA SILVA, V. A. & CIAMPI DE ANDRADE, D. 2025. Repetitive transcranial magnetic stimulation for fibromyalgia: are we there yet? Pain Rep, 10, e1221.

LATREMOLIERE, A. & WOOLF, C. J. 2009.

Central Sensitization: A Generator of Pain JOHAL, H., DEVJI, T., CHANG, Y., SIMONE, Hypersensitivity by Central Neural Plasticity. The J., VANNABOUATHONG, C. & BHANDARI, M. Journal of Pain, 10, 895 926.

2020. Cannabinoids in Chronic Non Cancer Pain: A Systematic Review and Meta-Analysis. Clin MELLADO LAGARDE, M. M., WILBRAHAM, Med Insights Arthritis Musculoskelet Disord, 13, D., MARTINS, R. F., ZHAO, H. S., JACKSON,

14

K., JOHNSON, K. W., KNOPP, K. L., DIBENEDETTO, D. & BROAD, L. M. 2024.

Clinical proof-of-concept results with a novel TRPA1 antagonist (LY3526318) in 3 chronic pain states. Pain, 166, 1497 1518.

RICCIOTTI, E. & FITZGERALD, G. A. 2011.

Prostaglandins and Inflammation. Arteriosclerosis, Thrombosis, and Vascular Biology, 31, 986-1000.

MILLS, S. E. E., NICOLSON, K. P. & SMITH, B. H. 2019. Chronic pain: a review of its epidemiology and associated factors in population-based studies.

British Journal of Anaesthesia, 123, e273-e283.

SCHNITZER, T. J. & MARKS, J. A. 2015.

A systematic review of the e icacy and general safety of antibodies to NGF in the treatment of OA of the hip or knee. Osteoarthritis Cartilage, 23 Suppl 1, S8 17.

SAKAI, A. & SUZUKI, H. 2014. Emerging roles of microRNAs in chronic pain. Neurochem MILLER, R. J., JUNG, H., BHANGOO, S. Int, 77, 58-67. SCHLOSBURG, J. E., KINSEY, K. & WHITE, F. A. 2009. Cytokine and chemokine S. G. & LICHTMAN, A. H. 2009. Targeting fatty regulation of sensory neuron function. Handb Exp acid amide hydrolase (FAAH) to treat pain and Pharmacol, 417 49. inflammation. Aaps j, 11, 39 44.

NICULESCU, A. B., LE-NICULESCU, H., LEVEY, D. F., ROSEBERRY, K., SOE, K. C., ROGERS, J., KHAN, F., JONES, T., JUDD, S., MCCORMICK, M. A., WESSEL, A. R., WILLIAMS, A., KURIAN, S. M. & WHITE, F. A. 2019. Towards precision medicine for pain: diagnostic biomarkers and repurposed drugs.

Molecular Psychiatry, 24, 501-522.

SIEBENGA, P., VAN AMERONGEN, G., HAY, J. L., MCDONNELL, A., GORMAN, D., BUTT, R. & GROENEVELD, G. J. 2020. Lack of Detection of the Analgesic Properties of PF-05089771, a Selective Na(v) 1.7 Inhibitor, Using a Battery of Pain Models in Healthy Subjects. Clin Transl Sci, 13, 318-324.

PAIN, I. A. F. T. S. O. 2020. Definitions of Chronic Pain Syndromes [Online].

Available: https://www.iasppain.org/advocacy/ definitions-ofchronicpainsyndromes/ [Accessed 14 June 2025 2025].

SPENCER MR, G. M., MINIñO AM 2024.

Drug Overdose Deaths in the United States, 2002–2022 . NCHS Data Brief . Hyattsville, MD : National Center for Health Statistics PEZET, S. & MCMAHON, S. B. 2006. .

NEUROTROPHINS: Mediators and Modulators of Pain. Annual Review of Neuroscience, 29, 507-538. STATISTICS, N. C. F. H. 2025. U.S. Overdose Deaths Decrease Almost 27% in PFIZER, I., ELI, L. & COMPANY 2021. Pfizer 2024 [Online]. Hyattsville, MD: National and Eli Lilly and Company Announce Outcome Center for Health Statistics.

Available: of FDA Joint Advisory Committee Meeting for https://www.cdc.gov/nchs/pressroom/nchs press releases/2025/20250514.htm#print [Accessed].

Tanezumab.

REN, K. & DUBNER, R. 2010. Interactions TREEDE, R.-D., RIEF, W., BARKE, A., AZIZ, between the immune and nervous systems in pain. Q., BENNETT, M. I., BENOLIEL, R., COHEN, M., EVERS, S., FINNERUP, N. B., FIRST, M. Nature Medicine, 16, 1267-1276.

B., GIAMBERARDINO, M. A., KAASA, S., REZENDE, B., ALENCAR, A. K. N., DE KORWISI, B., KOSEK, E., LAVAND’HOMME, BEM, G. F., FONTES-DANTAS, F. L. & P., NICHOLAS, M., PERROT, S., SCHOLZ, MONTES, G. C. 2023. Endocannabinoid System: J., SCHUG, S., SMITH, B. H., SVENSSON, Chemical Characteristics and Biological Activity. P., VLAEYEN, J. W. S. & WANG, S.-J. 2019.

Chronic pain as a symptom or a disease: the IASP Pharmaceuticals, 16, 148.

Classification of Chronic Pain for the International

15

Classification of Diseases (ICD-11). PAIN, 160.

VARDEH, D., MANNION, R. J. & WOOLF, C. J. 2016. Toward a Mechanism-Based Approach to Pain Diagnosis. J Pain, 17, T50-69.

WANG, L., HONG, P. J., MAY, C., REHMAN, Y., OPARIN, Y., HONG, C. J., HONG, B. Y., AMINILARI, M., GALLO, L., KAUSHAL, A., CRAIGIE, S., COUBAN, R. J., KUM, E., SHANTHANNA, H., PRICE, I., UPADHYE, S., WARE, M. A., CAMPBELL, F., BUCHBINDER, R., AGORITSAS, T. & BUSSE, J. W. 2021.

Medical cannabis or cannabinoids for chronic non-cancer and cancer related pain: a systematic review and meta-analysis of randomised clinical trials. Bmj, 374, n1034.

WERMELING, D. P. 2005. Ziconotide, an intrathecally administered N-type calcium channel antagonist for the treatment of chronic pain.

Pharmacotherapy, 25, 1084-94.

WEST, T., ELSABAN, M., HUSSAIN, N., SCHAPPELL, J., ROGERS, K., ORHURHU, V., PROKOP, L. J. & D’SOUZA, R. S. 2023.

Incidence of Lead Migration With Loss of E icacy or Paresthesia Coverage After Spinal Cord Stimulator Implantation: Systematic Review and Proportional Meta-Analysis of Prospective Studies and Randomized Clinical Trials. Neuromodulation, 26, 917-927.

WOOLF, C. J. & MA, Q. 2007. Nociceptors— Noxious Stimulus Detectors. Neuron, 55, 353-364.

YANG, J., XIE, Y. F., SMITH, R., RATTé, S. & PRESCOTT, S. A. 2025. Discordance between preclinical and clinical testing of Na V 1.7-selective inhibitors for pain. Pain, 166, 481-501.

YOUNG, B., STEPHENSON, J., ISLAM, B., BURKE, N. N., JENNINGS, E. M., FINN, D. P. & MCHUGH, P. C. 2023. The Identification of Human Translational Biomarkers of Neuropathic Pain and Cross-Species Validation Using an Animal Model. Mol Neurobiol, 60, 1179 1194.

ZHOU, X., LI, Z. & XING, B. 2023. E icacy and safety of TRPV1-related preparations in the

treatment of inflammatory arthralgia. Medicine (Baltimore), 102, e36268.

1

Advancements and Implications of Robotic-Assisted Coronary Artery Bypass Grafting: A Minimally Invasive Revolution Pritja Wahi Edited by Sushmhitah Sandanatavan and Thomas Burton

Abstract - This paper discusses the promise of robotic-assisted CABG to revolutionise therapy of coronary artery disease, highlighting technological advancement as well as the major realworld issues that limit its widespread use. These technologies, supported by platforms like the da Vinci system, have reduced incision sizes, minimised surgical trauma, and improved precision in grafting procedures. This literature review examines the benefits of robotic-assisted CABG, including reduced recovery time, reduced infection risk, and reduced risk of stroke in relation to the traditional on-pump sternotomy CABG. While obstacles such as expense and technical learning curve still exist, robotic-assisted CABG presents optimistic outcomes, including reduced hospital stay and reduced postoperative complications. Future prospects of robotically assisted CABG will be discussed, with continued research further attempting to expand its application and further improving operating results in patients.

symptoms in patients [1]. This traditional approach involves a median sternotomy followed by removal of the great saphenous vein to use as a graft. The remainder of the procedure can be performed either with or without the usage of a cardiopulmonary bypass (CPB). In on-pump CABG, the heart-lung machine temporarily replaces the functions of the heart. This allows the heart to be stopped during surgery. In off-pump CABG, specialised instruments are used to stabilise the area of the heart where grafts are placed all while it continues to beat [1]. This approach comes with its own drawbacks such as large incisions, prolonged recovery times, and increased risk of infection. Robotic-assisted surgery has emerged as a transformative innovation in cardiac surgery, offering the promise of enhanced precision and reduced recovery times. The following literature review will closely examine the technological revolution to approaching a CABG procedure.

Further, it will explore the benefits provided by robotic-assisted surgery whilst weighing the implications of these perceived benefits. To conclude, challenges proposed by robotic assisted surgery and I. I NTRODUCTION how patient presentations are ultimately the final The integration of technology in healthcare is determinant in choosing the best course of treatment fundamental to the intent of driving efficiency and will be explored through a discussion of clinical improving patient-outcomes. The key question is indications and comparative outcomes whether newer technologies always offer better outcomes or if traditional methods should be mainII. A DVANCEMENTS IN ROBOTIC T ECHNOLOGY tained due to their long-standing success. The sigFOR CABG OVER THE YEARS nificance of this can be seen in the treatment of coronary artery disease. As the most commonly Minimally invasive approaches to CABG were performed surgical procedure [1], a Coronary Artery first introduced in 1994 [4], marking a significant Bypass Graft is a pioneering example of the treat- shift towards less invasive surgery. There are two ment procedure of overcoming atheromatous block- established types of robotic CABG techniques, the ages in coronary vessels and restoring blood flow to Robotic Assisted Minimally Invasive Direct CABG the ischemic myocardium. It restores function and (RA-MIDCAB) and Totally Endoscopic CABG viability of the myocardium while relieving anginal (TECAB) [2]. In RA-MIDCAB, robotic assistance

2

is utilised to harvest the internal mammary artery (IMA) whilst the anastomosis is performed under direct visualisation through a small anterior incision and is typically performed using an off-pump method. TECAB can be done either on-pump, offpump or on-pump with a beating heart and the entire operation is performed totally endoscopically [2].

Both have been shown to have considerable effects on lowered postoperative morbidity and mortality, and generally enhanced recovery [2]. This results in reduced surgical trauma, shorter recovery time and earlier return to normal activities [2]. Since its introduction, robotic CABG has evolved significantly, notably through the development of the da Vinci system, a key platform that revolutionised surgical capabilities. A particularly important historic step in developing the robotic CABG procedure was the release of the second and third generation da Vinci systems [4]. When compared to previous generations, the third generation enabled better vision, included a fourth arm, procedure specific instruments, and better instrument reach [3].

Advanced robotic systems now provide enhanced high-definition three-dimensional (3D) visualisation together with improved control, dexterity, and precision from four robotic arms [2]. For a MIDCAB procedure, robotic assistance provides 3D telemanipulation that further minimises access trauma of the LIMA harvest by avoiding larger incisions and wider rib spreading [2]. When compared with the “gold standard” on-pump sternotomy CABG, this method lends a much smaller incision needed [2].

With the evolution of robotic platforms, the potential benefits for patients have become clearer, especially in reducing surgical trauma and improving recovery times.

III. B ENEFITS OF M INIMALLY I NVASIVE ROBOTIC CABG Upon analysing the development of minimally invasive CABG techniques, it is evident that there are considerable patient benefits. The most appealing aspect of the technique is that the incision length and thus overall surgical trauma are significantly decreased [5]. When comparing the sternotomy and minimally invasive CABG techniques, it is important to consider that most patients receive a single vessel bypass graft, and the number of multivessel bypasses via a mini-thoracotomy has only recently

increased, thus, it is not always a direct comparison. Nevertheless, an important consideration when analysing benefits of minimally invasive treatment of coronary artery disease is the risk of stroke in the patient. A study conducted showing the intraoperative and postoperative performance of all methods currently practiced found a 1.3% stroke rate in a series of 234 patients undergoing sternotomy CABG for single vessel disease [5]. On the other hand, minimally invasive procedures demonstrate a stroke rate of 0.4% and 1.0% in robotically assisted MIDCAB and TECAB respectively in 25 years of minimally invasive CABG [5]. This is therefore very satisfactory and may indicate that less invasive procedures lead to relatively lower stroke rates.

Further, it is also important to consider infection risk. In larger CABG series we see rates of sternal wound infection in the range of 2.4% to 4.4% [5].

On the other hand, an infection rate of 0.7% and 1.0% in robotically assisted MIDCAB and TECAB respectively is demonstrated [5]. Therefore, it can reasonably be concluded that minimally invasive procedures maintained the levels of intraoperative and postoperative survival that would be expected of low complexity conventional CABG through sternotomy.

IV. C HALLENGES AND L IMITATIONS Patient outcomes are another important aspect to consider when deciding the viability of a treatment modality. To analyse this, a study comparing five studies with a mixed robotic CABG cohort reporting on mid-to-long term outcomes with a mean followup time of 54.5 months was consulted [2]. According to this, patients still required six-to-seven postoperative days in the hospital, which appears modest when compared to conventional CABG. However, this is not consistent with findings in highvolume centres, with patients staying an average of four days [2]. This is likely a result of the presence of a technical and learning curve required in a TECAB, and can thus be classified as a significant barrier to the wider utilisation of minimally invasive and robotic CABG in healthcare today. In more recent and larger propensity-matched analyses of the US National Inpatient Sample from 2012 to 2017, robotic CABG was found to be associated with lower in-hospital mortality, rate of acute kidney injury, transfusion, post-operative bleeding and hospital length of stay compared to conventional CABG

3

[2] In order to showcase this better, the Shanghai Jiao Tong University of China compared a MIDCAB and RACAB procedure. They discovered that the incision length was significantly shorter and the patient number with intercostal pain after surgery was significantly lower in the RACAB group. These results suggest there is less surgical trauma. The postoperative stay was also shorter in the RACAB group (9.2±3.8 vs. 7.8±3.0 days, P = 0.024) [6].

V. T HE FUTURE OF ROBOTIC CABG Although sternotomy is still widely considered the gold-standard of surgical intervention in coronary artery disease, sternotomy-free approaches are moving closer into the spotlight where they are applicable. This context-dependent approach has proven extremely effective in specific cases. Thus far, these have included: “Tricuspid valve: surgery without sternotomy, as a redo without pericardial dissection, with or without cross-clamping, Mitral valve: surgery without sternotomy, as a redo (specifically with patent mammary) with or without pericardial dissection, with or without cross-clamping, beating heart/fibrillating heart, Redo cases with previous sternal wound infection (specifically those with loss of sternal bone), Cases with morbid obesity, Frail patients with or without significant osteoporosis, Patients with large breast implants” [7].

Ongoing research is being conducted in terms of expanding this further into a more generalisable treatment plan. Addressing these barriers is ultimately the key to further advancement of the MIDCAB approach, particularly the RA-MIDCAB. According to Dr. Shahzad Raja’s article outlining the ‘New Clinical Advances in Minimally Invasive Coronary Surgery’ published in May 2025, ‘Ongoing research is essential to develop more advanced and userfriendly technologies, such as robotic systems with enhanced capabilities, improved imaging tools, and tactile feedback features.’ It can thus be concluded that the future of MIDCAB lies in the reservation of its application for cases where it provides a clear surgical benefit. Through research, this margin can be expanded in the future without compromising surgical completeness.

artery disease. The field has developed greatly in reducing trauma during surgery, enhancing accuracy, and improving recovery among patients. Techniques like RA-MIDCAB and TECAB have proven benefits, including minimal wounds, lower infection rates, and less risk of stroke, with similar, longterm outcomes to traditional CABG. Withstanding difficulties posed by a learning curve, cost, and the limited scope of its use, robotic CABG has unprecedented potential for application in specific clinical conditions. As studies continue advancing towards being more advanced and sophisticated in its approach, robot-assisted CABG will increasingly be at the forefront of the treatment of coronary artery disease, offering personalised, less-invasive, and cost-effective surgical options for patients.

VII. R EFERENCES

[1] Bachar, B., Manna, B. (2023) National Center for Biotechnology Information (NCBI) (2020) Coronary Artery Bypass Graft. Available at: https: //www.ncbi.nlm.nih.gov/books/NBK507836/.

[2] Hwang, B., et al. (2024) ’ Systematic review and meta-analysis of two decades of reported outcomes for robotic coronary artery bypass grafting’, Annals of Cardiothoracic Surgery. Available at: https://www.annalscts.com/article/view/17126/html.

[3] Canale, L. S., et al. (2013) ’ Robotically assisted totally endoscopic coronary artery bypass surgery’, Journal of Thoracic Disease, 25(2), 5 Suppl 6(Suppl 6), S641–S649. Available at: https://pmc.ncbi.nlm. nih.gov/articles/PMC3831835/.

[4] Bonatti, J. (2024) ’ Historical landmarks in the development of robotic coronary bypass grafting’, Annals of Cardiothoracic Surgery. Available at: https://www.annalscts.com/article/view/17127/html.

[5] Bonatti, J., et al. (2021) ’ Minimally invasive and robotic coronary artery bypass grafting—a 25-year review’, Journal of Thoracic Disease.

Available at: https://pmc.ncbi.nlm.nih.gov/articles/ PMC8024818/.

[6] Gong, W., et al. (2016) ’ Robot-assisted coronary artery bypass grafting improves short-term outcomes compared with minimally invasive direct coronary artery bypass grafting’, Journal of Thoracic Disease. Available at: https://pmc.ncbi.nlm.

VI. C ONCLUSION nih.gov/articles/PMC4805807/.

In conclusion, robotic CABG is an unprece- [7] Faerber, G., et al. (2023) ’ Is There a Fudented technology in the treatment of coronary ture for Minimal Access and Robots in Cardiac

4

Surgery?’, Journal of Cardiovascular Development and Disease. Available at: https://pmc.ncbi.nlm.nih. gov/articles/PMC10531980/.