Bowdoin Science Journal

Biology

From Milk to Malignancy – Breast Cancer and its Metabolic Implications 

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The annual rise of cancer cases has created a high demand for new innovative treatments and has made cancer a prominent topic in the scientific community. According to the American Cancer Society (ACS), approximately 20 million new cancer cases were diagnosed worldwide in 2022, leading to 9.7 million deaths [1]. It is expected that by 2050, cancer cases will reach 35 million, largely due to population growth [1]. While significant advancements have been made in cancer research, the complexity of different cancer types presents challenges. 

One of the most prevalent forms is breast cancer, which, in 2022, was the second most common cancer in the U.S., with 2.3 million new cases, predominantly affecting women [2]. Unlike many cancers, breast cancer is not a single disease but a collection of subtypes characterized by distinct clinical, morphological, and molecular features. This heterogeneity makes it challenging to study and treat effectively. A recent study published in Nature Metabolism explores the metabolic differences between normal mammary cells and breast cancer cells [4]. Understanding these metabolic processes could pave the way for new, targeted therapies. Researchers have identified specific metabolic vulnerabilities in mammary epithelial cells, which line the breast tissue.

 

Figure 1. Non-tumorigenic Mammary Gland Components. A diagram of a non-tumorigenic mammary gland showing a cluster of alveoli containing luminal and basal cells. Luminal cells line the milk ducts and alveoli and are responsible for milk secretion during lactation. Basal cells are believed to play a role in transporting milk to the nipple during lactation. Source: Created in BioRender, [4], [10], [11].

In the normal mammary gland, various types of cells carry out specific functions, one of which is the progenitor cells. These progenitor cells generate distinct alveolar structures that continuously form in the adult breast, and their activity is crucial for maintaining normal mammary homeostasis [5]. Progenitor cells are located in the luminal compartment [6], which is also home to the luminal cells. The luminal cells play a key role in lactation by lining the milk ducts and alveoli, where they secrete milk (Figure 1)[7]. In contrast, basal cells are located around the luminal cells and are believed to function during lactation by helping to transport milk to the nipple (Figure 1)[7]. Although these mammalian epithelial cells (luminal and basal cells) are important to the function of normal mammary glands, these also serve as a tumour cell of origin [4].

In their study, Mahendralingam et al. used mass spectrometry to analyze the metabolic profiles of normal human mammary cells [8]. They discovered that luminal progenitor cells primarily rely on oxidative phosphorylation for energy, whereas basal cells depend more on glycolysis [4]. This distinction is crucial because oxidative phosphorylation is an efficient, oxygen-dependent process that generates substantial energy, while glycolysis, though faster, is less efficient and does not require oxygen — a pathway often favored by cancer cells to support rapid growth [9]. Targeting these distinct energy pathways could lead to more effective treatments for different breast cancer subtypes.

However, a new discovery was that breast cancer cells appear to adopt the metabolic programs of their cells of origin [4,9]. This complicates treatment since the cancer cells may still be vulnerable to metabolic pathways that are important for normal cell function. As a result, treatments designed to target specific metabolic pathways might not work as expected, since the cancer cells might behave similarly to the healthy cells from which they originated. 

The results from Mahendralingam et al. can form a basis for future metabolic studies that may lead to specific anti-tumoral drug therapies designed to treat specific breast cancer subtypes. This type of research lays a foundation for targeted approaches but further studies are needed to assess how findings, such as this one, can translate into clinical practice. As breast cancer continues to rise, understanding the complexity is more important than ever. 

 

Work Cited: 

  1. Global Cancer Facts & Figures. (n.d.). Retrieved October 27, 2024, from https://www.cancer.org/research/cancer-facts-statistics/global-cancer-facts-and-figures.html
  2. Global cancer burden growing, amidst mounting need for services. (n.d.). Retrieved October 27, 2024, from https://www.who.int/news/item/01-02-2024-global-cancer-burden-growing–amidst-mounting-need-for-services
  3. Sánchez López de Nava, A., & Raja, A. (2024). Physiology, Metabolism. In StatPearls. StatPearls Publishing. http://www.ncbi.nlm.nih.gov/books/NBK546690/
  4. Alfonso-Pérez, T., Baonza, G., & Martin-Belmonte, F. (2021). Breast cancer has a new metabolic Achilles’ heel. Nature Metabolism, 3(5), 590–592. https://doi.org/10.1038/s42255-021-00394-8
  5. Tharmapalan, P., Mahendralingam, M., Berman, H. K., & Khokha, R. (2019). Mammary stem cells and progenitors: Targeting the roots of breast cancer for prevention. The EMBO Journal, 38(14), e100852. https://doi.org/10.15252/embj.2018100852
  6. Tornillo, G., & Smalley, M. J. (2015). ERrrr…Where are the Progenitors? Hormone Receptors and Mammary Cell Heterogeneity. Journal of Mammary Gland Biology and Neoplasia, 20(1–2), 63–73. https://doi.org/10.1007/s10911-015-9336-1
  7. New Paradigm for Mammary Glands. (n.d.). Massachusetts General Hospital. Retrieved December 8, 2024, from https://www.massgeneral.org/cancer-center/clinician-resources/advances/new-paradigm-for-mammary-glands
  8. Mahendralingam, M. J., Kim, H., McCloskey, C. W., Aliar, K., Casey, A. E., Tharmapalan, P., Pellacani, D., Ignatchenko, V., Garcia-Valero, M., Palomero, L., Sinha, A., Cruickshank, J., Shetty, R., Vellanki, R. N., Koritzinsky, M., Stambolic, V., Alam, M., Schimmer, A. D., Berman, H. K., … Khokha, R. (2021). Mammary epithelial cells have lineage-rooted metabolic identities. Nature Metabolism, 3(5), 665–681. https://doi.org/10.1038/s42255-021-00388-6
  9. ZHENG, J. (2012). Energy metabolism of cancer: Glycolysis versus oxidative phosphorylation (Review). Oncology Letters, 4(6), 1151–1157. https://doi.org/10.3892/ol.2012.928
  10. Fig. 3 Stem cell in glandular and stratified epithelia. A A schematic… (n.d.). ResearchGate. Retrieved December 7, 2024, from https://www.researchgate.net/figure/Stem-cell-in-glandular-and-stratified-epithelia-A-A-schematic-model-depicting-the_fig3_374804603
  11. Model of normal mammary gland structure. This tissue is composed of… (n.d.). ResearchGate. Retrieved December 8, 2024, from https://www.researchgate.net/figure/Model-of-normal-mammary-gland-structure-This-tissue-is-composed-of-ducts-which-are_fig1_357239665

Genomics of severe and treatment-resistant obsessive-compulsive disorder treated with deep brain stimulation: a preliminary investigation

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Obsessive-compulsive disorder (OCD) can be severely disabling, and some patients do not respond to standard treatments like medication and therapy. Deep brain stimulation (DBS), an invasive neurosurgical intervention where thin electrodes are connected to a neuro-pacemaker and introduced into subcortical central structures of the brain to modulate pathological neuronal activity with electrical current, has shown promise for these treatment-resistant cases. However, responses to DBS vary widely, prompting a need to identify genetic factors that might predict which patients will benefit. Understanding these genetic markers may ultimately lead to more personalized, effective approaches for treatment-resistant OCD.

This study (Chen et al, 2023) conducted a preliminary genomic analysis on a small cohort of patients with severe, treatment-resistant OCD who received DBS. Researchers sequenced the patients’ DNA to examine specific genetic variants. These included instances where a single nucleotide in a genomic sequence was altered in a phenomenon known as single nucleotide variants and among other genetic markers previously associated with psychiatric disorders and traits related to treatment resistance. Statistical analysis was then applied to explore any associations between these genetic markers and the clinical outcomes of DBS in these patients.

The results identified several genetic markers such as missense variants in the gene KNCB1 that seemed to correlate with positive or negative DBS responses. However, because the study involved a small number of participants, these findings are considered preliminary. Certain genetic variants showed potential as predictors for treatment outcomes, but further research with a larger sample size is needed to validate these associations and understand the mechanisms by which they influence DBS response.

This study provides initial evidence that genetics may play a role in how patients with treatment-resistant OCD respond to DBS. If validated by larger studies, these findings could pave the way for genetically-informed approaches to selecting and optimizing DBS candidates, contributing to more precise, personalized treatment strategies for severe OCD cases.

References:

Long Long Chen, Matilda Naesström, Matthew Halvorsen, Anders Fytagoridis, David Mataix-Cols, Christian Rück, James J Crowley, Diana Pascal (2023) Genomics of severe and treatment-resistant obsessive-compulsive disorder treated with deep brain stimulation: a preliminary investigation, medRxiv , https://www.ncbi.nlm.nih.gov/pmc/articles/PMC10153313/

Unveiling the Potential of PGCCs in Future Cancer Treatments

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Poly-ADP ribose polymerases (PARPs) are proteins that participate in the process of repairing single strand DNA breaks (SSBs). PARPs carry out surveillance in the cell and when DNA damage occurs, PARPs bind to and recognize DNA damage, setting off DNA repair pathways (Morales et al., 2014). PARP inhibitors (PARPi) block the DNA damage repair pathway, leaving the SSBs unrepaired. Eventually, the SSBs will accumulate and form double stranded breaks (DSBs).

Normal ovarian cells use BRCA proteins to recruit repair complexes to the DSB region and help repair the DSBs using a method called homologous recombination (HR) (Dilmac and Ozpolat, 2023). Homologous recombination is a process in which identical and undamaged nucleotide sequences from homologous chromosomes are used to repair DSBs. In BRCA-mutant ovarian cancer, DSBs are unable to be repaired because BRCA proteins lose function and are unable to recruit repair complexes.

While PARPi is a viable treatment for BRCA-mutant ovarian cancer, 40-50% of BRCA-mutant patients do not respond to PARPi due to innate or acquired PARPi resistance (Dilman and Ozoplat, 2023). Acquired resistance happens because cancer cells reproduce and adapt quickly to overcome the PARPi lethality. In addition, more than 50% of HGSC patients are do not have BRCA mutations, meaning that PARPi treatment are not substantially beneficial to a large majority of patients (Zhang et al., 2023). This highlights the importance of finding a different mechanism by which we can potentiate the therapeutic effect of PARPi treatment. The solution may lie in polyploid giant cancer cells (PGCCs).

Fluorescence light micrograph of a Polyploid giant cancer cell (PGCC) from a triple-negative breast cancer. The other cellular components highlighted here are actin (red), mitochondria (green), and nuclear DNA (blue).
Source: University Of Pittsburgh Cancer Institute. “Polyploid Giant Cancer Cell From Breast.” Photograph. Science Photo Gallery.

PGCCs are large cancer cells that are usually thought to be senescent, meaning that the cells age but do not die or divide. However, scientists are not entirely certain on the formation or function of PGCCs (Zhou et al., 2022).

In Targeting polyploid giant cancer cells potentiates a therapeutic response and overcomes resistance to PARP inhibitors in ovarian cancer, Zhang et al. investigates the association of PGCCs with the PARPi (specifically, Olaparib) resistance in ovarian cancer. Their research looks at high-grade serous carcinoma (HGSC), the most common subtype of ovarian cancer. First, they found that PGCCs grow in Olaparib-resistant HGSC patient-derived xenografts (PDX) and that they were able to induce PGCC formation using Olaparib. PDX is a model of cancer where cancer tissues are collected from patients and planted into an immunodeficient mouse. Second, while PGCCs are usually thought to be senescent, they found that PGCCs are also able to escape senescence and produce daughter cells that are able to divide. A portion of these mitotic competent daughter cells also have therapeutic resistance, increasing PARPi resistance within the tumor. So far, Zhang et al. found that not only can PGCCs become resistant to PARPi treatment, but they can also produce more cancer cells that are also resistant to PARPi treatment. However, this isn’t all bad news.

From previous studies, we know that PGCCs dedifferentiate and mimic early embryonic development (Niu et al., 2017). Leveraging this knowledge, Zhang et al. reasoned that certain contraceptives, such as Mifepristone, may be able to block the life cycle of PGCCs. Indeed, they found that Mifepristone was able to suppress tumor growth in both Olaparib-naive and Olaparib-resistant HGSC cells, treating the original issue of PGCC and daughter cells being resistant to Olaparib. The results showed that PGCCs are most sensitive to a combination of Olaparib and Mifepristone treatment. In conclusion, Zhang et al. found a way to grow PGCCs in HGSC cells and then use the PGCCs to suppress cancer growth while also overcoming PARPi resistance.

Zhang et al. mentioned that PGCCs can be used to potentiate the therapeutic response of PARPi treatment and combat more than one HGSC subtype. Previously, PARPi treatment worked best with BRCA mutant patients. However, Zhang et al. suggest that PGCCs, rather than the BRCA status of tumor, are associated with acquired Olaparib resistance. This means that PGCCs can be used as an alternative treatment regime, broadening applications of PARPi to HGSC patients who do not have BRCA mutations.

 

References:

Dilmac, S., & Ozpolat, B. (2023). Mechanisms of PARP-Inhibitor-Resistance in BRCA-Mutated Breast Cancer and New Therapeutic Approaches. Cancers, 15(14), 3642. https://di.org/10.3390/cancers15143642

Morales, J. C., Li, L., Fattah, F. J., Dong, Y., Bey, E. A., Patel, M., Gao, J., & Boothman, D. A. (2014). Review of Poly (ADP-ribose) Polymerase (PARP) Mechanisms of Action and Rationale for Targeting in Cancer and Other Diseases. Critical Reviews in Eukaryotic Gene Expression, 24(1), 15–28.

Niu, N., Mercado-Uribe, I., & Liu, J. (2017). Dedifferentiation into blastomere-like cancer stem cells via formation of polyploid giant cancer cells. Oncogene, 36(34), 4887–4900. https://doi.org/10.1038/onc.2017.72

Zhang, X., Yao, J., Li, X., Niu, N., Liu, Y., Hajek, R. A., Peng, G., Westin, S., Sood, A. K., & Liu, J. (2023). Targeting polyploid giant cancer cells potentiates a therapeutic response and overcomes resistance to PARP inhibitors in ovarian cancer. Science Advances, 9(29), eadf7195. https://doi.org/10.1126/sciadv.adf7195

Zhou, X., Zhou, M., Zheng, M., Tian, S., Yang, X., Ning, Y., Li, Y., & Zhang, S. (2022). Polyploid giant cancer cells and cancer progression. Frontiers in Cell and Developmental Biology, 10, 1017588. https://doi.org/10.3389/fcell.2022.1017588

The Dark Side of Antibiotics

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Antibiotics are medications that fight infections caused by bacteria. But they can also lead to mental health issues, such as anxiety and depression, later in life. 

The discovery of antibiotics was one of the greatest medical advances of the 20th century. Antibiotics have significantly reduced mortality from infectious diseases and increased average life expectancy. However, they have a variety of side effects, including cognitive impairment and emotional disorders in adulthood (Adedji 2016). 

Antibiotics destroy bacteria in the gut, which can disrupt brain function. Gut microbiota are an important component of the gut-brain axis–the two-way line of communication between the gastrointestinal tract and the central nervous system. Gut microbiota produce neurotransmitters that regulate mood, such as dopamine, norepinephrine, and serotonin, which travel through the vagus nerve to the brain (Karakan et al 2021). Previous studies have found that antibiotic-induced gut microbiota depletion causes dysfunction of the gut-brain axis, increasing anxiety and depression-related behaviors (Mosaferi et al 2021).

Aging plays an important role in the development of both gut microbiota and the brain. Microbiota first appear at birth and rapidly colonize the intestinal tract. The composition and diversity of gut microbiota resembles adult level by 2 years of age and remains stable throughout adulthood before decreasing in old age. The brain develops until the mid-to-late 20s and starts to decline in middle age. It has been shown that antibiotic-induced gut microbiota depletion has negative effects on the brain (Li et al 2022). However, no prior research has been done on this relationship during the different stages of life. This study aimed to determine the connection between gut microbiota and cognitive and emotional function during the different life stages. 

In this experiment, the researchers used mice as models for human subjects, randomly assigning 75 mice to five groups. One group served as the control (Veh group) and was given distilled water from birth to death. The other four groups were given an antibiotic cocktail at different life stages: birth to death (Abx group), birth to postnatal day 21 (Abx infant group), postnatal day 21 to 56 (Abx adolescence group), and postnatal day 57 to 84 (Abx adulthood group).  

At postnatal day 85, the researchers randomly selected thirteen mice from each group for testing. They measured the cognitive function and emotion of the mice by using four traditional behavioral tests: open-field test (OFT), passive avoidance test (PAT), morris water maze test (MWM), and tail suspension test (TST). The OFT measured anxiety level by observing how long the mice moved in an open field for 5 minutes. The PAT measured short-term memory, which was defined as the difference in latency–the time it took mice to reenter a room that delivered an electric shock–between day 1 and day 2 (Jahn-Eimermacher et al 2011). The MWM measured spatial memory, which was defined as incubation time, or how long it took mice to find a submerged platform in a pool after a 5-day training period. The TST measured depression state by observing the duration of quiescence (motionless state) of the mice while they were suspended upside down for 4 minutes.

Figure 1 | Results of OFT, PAT, MVM, and TST tests. Researchers conducted four traditional behavioral tests on mice given water, as well as mice given antibiotics at different stages of life: birth to death, infancy, adolescence, and adulthood. They found that exposure to antibiotics from birth to death and in infancy led to the most severe cognitive and emotional dysfunction, followed by exposure in adolescence and adulthood (Li et al 2022).

The results of this study suggested that life cycle stages influence the relationship between gut microbiota and cognitive and emotional function. For the OFT test, the total movement time of the Abx, Abx infant, and Abx adolescent groups was significantly lower than the Veh group, indicating they were more anxious than the Veh group (Figure 1). In other words, exposure to antibiotics from birth to death, in infancy, and in adolescence caused anxiety-related behaviors. For the PAT test, the difference in latency for every Abx group was significantly lower than the Veh group, meaning all Abx groups reentered the room with the electric shock more quickly after training. These results signaled that short-term memory loss was greater in the Abx groups than the Veh group; exposure to antibiotics at any stage of life caused short-term memory loss. The MWM test found that the incubation time after the 5-day training period was significantly higher for the Abx and Abx infant groups than the Veh group, so they experienced more spatial memory loss than the Veh group; exposure to antibiotics from birth to death and in infancy caused spatial memory loss. The TST test found that the duration of quiescence in the Abx and Abx infant groups was significantly higher than the Veh group, implying they were more depressed than the Veh group. In other words, exposure to antibiotics from birth to death and in infancy caused depression-related behaviors.

The researchers’ findings align with previous work showing that depletion of gut microbiota causes cognitive impairment and emotional problems (Lach et al 2020). Furthermore, the researchers demonstrated that life cycle stages are an important factor in the relationship between gut microbiota and cognitive and emotional function. In particular, their findings strengthened the idea that infancy is a crucial stage of development of gut microbiota and the brain (Hunter et al 2023). Gut microbiota lost in infancy recovers over time; however, this depletion has lasting cognitive effects. In this study, mice given antibiotics in infancy exhibited similar behaviors in adulthood as mice given antibiotics from birth to death: anxiety, depression, memory loss, and learning ability decline. Exposure to antibiotics in infancy and in the long term led to the most severe cognitive and emotional dysfunction, followed by exposure in adolescence and adulthood.

The researchers’ findings also have implications for the treatment of mental health illnesses. Previous studies have shown that probiotics replace depleted gut microbiota, alleviating symptoms of anxiety and depression. Antidepressants and anxiolytics–current medications for anxiety and depression–cause side effects such as nausea, weight gain, insomnia, constipation, dizziness, agitation, and restlessness. Probiotics are associated with milder side effects, including gas and bloating (Bistas et al 2023). Probiotics are unlikely to treat severe depression and anxiety, but they are promising treatments for people with milder conditions. The next step is to identify and manufacture effective probiotics, which would revolutionize the field of psychiatry and improve the lives of people around the world.

 

References

Adedji, W.A. 2016. THE TREASURE CALLED ANTIBIOTICS. Annals of Ibadan Postgraduate Medicine. 14(2):56-57. https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5354621/.

Bistas KG, Tabet JP. 2023. The Benefits of Prebiotics and Probiotics on Mental Health. Cureus Journal of Medical Science. 15(8):e43217. doi:10.7759/cureus.43217.

Hunter S, Flaten E, Petersen C, Gervain J, Werker JF, Trainor LJ, Finlay BB. 2023. Babies, bugs and brains: How the early microbiome associates with infant brain and behavior development. PLOS One. 18(8):e0288689. doi:10.1371/journal.pone.0288689.

Jahn-Eimermacher A, Lasarzik I, Raber J. 2011. Statistical analysis of latency outcomes in behavioral experiments. Behavioural Brain Research. 221(1):271-275. doi:10.1016/j.bbr.2011.03.007.

Karakan T, Ozkul C, Akkol EK, Bilici S, Sobarzo-Sánchez E, Capasso R. 2021. Gut-Brain Microbiota Axis: Antibiotics and Functional Gastrointestinal Disorders. Nutrients. 13(2):389. doi:10.3390/nu13020389.

Lach G, Fülling C, Bastiaanssen TFS, Fouhy F, O’Donovan AN, Ventura-Silva AP, Stanton C, Dinan TG, Cryan JF. 2020. Translational Psychiatry. 10(1):382. doi:10.1038/s41398-020-01073-0.

Li J, Pu F, Peng C, Wang Y, Zhang Y, Wu S, Wang S, Shen X, Li Y, Cheng R, He F. 2022. Antibiotic cocktail-induced gut microbiota depletion in different stages could cause host cognitive impairment and emotional disorders in adulthood in different manners. Neurobiology of Disease. 170:105757. doi:10.1016/j.nbd.2022.105757.

Mosaferi B, Jand Y, Salari AA. 2021. Gut microbiota depletion from early adolescence alters anxiety and depression-related behaviors in male mice with Alzheimer-like disease. Scientific Reports. 11:22941. doi:10.1038/s41598-021-02231-0.

Saffron Can Reduce Cognitive Impairment and Biomarkers of Alzheimer’s Disease

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Saffron is a popular food condiment known to have a positive effect against neurodegenerative diseases such as dementia, Alzheimer’s, and Parkinson’s. Saffron could potentially alleviate some of the biological hallmarks of Alzheimer’s disease, including the accumulation of Aβ plaques outside of cells and neurofibrillary tangles (NFTs) inside of cells. It could also target the neurotransmitter system that involves acetylcholine (ACh), which when disrupted can lead to the progression of dementia. This system, called the cholinergic system, is essential for attention, learning, and memory. ACh plays a role in memory acquisition, encoding, and consolidation (Patel et al. 2024). 

A recent study looked at how saffron extract (CSE) supplementation affects behavioral activity, memory, Aβ plaques, and NFTs in rats. The study also investigated how saffron affects a disrupted cholinergic system. When the system breaks down, there is an increase in Aβ plaques and NFTs. There is also an increase in AChE, which is the protein that breaks down ACh. All of these factors can ultimately contribute to Alzheimer’s. The current study investigated how CSE could reduce these effects and ultimately prevent progression of dementia-related diseases (Patel et al. 2024).  

In order to study the effects of CSE, the researchers used the drug scopolamine to disrupt the cholinergic system in rats. Scopolamine mimics the memory loss observed in dementia, and it is often used to study the cholinergic system in animal models of dementia. It primarily impairs the acquisition and encoding of memories (Paul 2019). Scopolamine increases the level of AChE in the brain, which means that more ACh is broken down. With less ACh available, the cholinergic system is disrupted, causing cognitive impairments (Figure 1). The study observed the potential of CSE to reduce this disturbance. They used behavioral tests to observe memory and spatial acquisition. Blood samples and brain samples were collected for examination (Patel et al. 2024). 

Figure 1. The role of acetylcholinesterase (AChE). AChE breaks down acetylcholine (ACh) as it moves from one neuron to another. An increase in AChE leads to a decrease in ACh. (Adapted from What is the role of acetylcholinesterase at a synapse?)

The researchers found that CSE supplementation significantly improved behavioral activity and memory in rats. CSE improved performance on the reversal task, which requires the ability to switch between learned and new responses, a reflection of cognitive flexibility. 

Importantly, the study offers insight into the biological parameters that lead to the improved cognitive abilities. CSE lowered the levels of AChE, meaning that less acetylcholine was broken down. The dose of 20 mg/kg of CSE lowered the amount of AChE in the scopolamine-treated rats as significantly as Rivastigmine did (Figure 2). Rivastigmine is a pharmaceutical drug currently used to treat symptoms of Alzheimer’s by inhibiting AChE (Emre et al. 2010). An increase in the level of acetylcholine can improve memory, as demonstrated by the improved performance of the rats on the memory tests. ACh has been shown to promote nerve conduction related to memory and learning ability, helping memories get encoded and consolidated in the brain. 

Figure 2. CSE supplementation lowered the levels of AChE and inhibited its effects in rats who received scopolamine administration. Thus, less acetylcholine was broken down and disruption of the cholinergic system was attenuated. This can lead to an improvement in memory (Adapted from Patel et al, 2024). 

CSE supplementation also significantly reduced the formation of Aβ plaques and NFTs, the two classical pathological hallmarks of Alzheimer’s in the hippocampus in the brain. The 20 mg/kg dose of CSE produced the most significant reduction. CSE reduced the number of Aβ plaques and NFTs more than Rivastigmine did. In fact, the Rivastigmine group still had significant levels of NFTs and Aβ plaques (Figure 3). Accumulation of Aβ plaques and NFTs lead to neuronal dysfunction and ultimately dementia. Moreover, AChE and Aβ can bind together to form complexes that exaggerate neurodegeneration even more. Thus, if CSE can attenuate their formation, neuronal function may be preserved, preventing the progression of dementia.  

Figure 3. CSE supplementation lowered the density of Aβ plaques in rats who received scopolamine administration. This can lead to an improvement in memory (Adapted from Patel et al, 2024). 

Figure 4. Summary of the article. Scopolamine administration led to a disruption of the cholinergic system (a part of the nervous system involving acetylcholine that helps with memory and learning), neuroinflammation, and accumulation of NFT and Aβ plaques. When CSE (saffron extract) was administered, the rats’ memory improved and the biomarkers of Alzheimer’s were reduced (Adapted from Patel et al, 2024). 

Because CSE supplementation reduces the formation of Aβ plaque and NFTs in the hippocampus, inhibits the activity of the enzyme that breaks down acetylcholine, and counters neuroinflammation, a saffron-based botanical supplement could be used to help manage dementia. Currently, manufactured drugs such as donepezil and memantine have been used to treat symptoms of Alzheimer’s, but saffron could potentially offer a safer alternative that is just as effective (D’Onofrio et al. 2021). The current study tested the effects of CSE against Rivastigmine, another drug that is approved to treat symptoms of Alzheimer’s. CSE produced the same improvements as Rivastigmine. Rivastigmine, along with other chemical drugs that inhibit AChE in order to treat Alzheimer’s, has been shown to cause gastrointestinal side effects and does not have great patient compliance (Emre et al. 2010). Saffron supplementation could offer an effective treatment without such side effects, increasing quality of life. The research offers promise that saffron can improve the cognitive impairments associated with Alzheimer’s and dementia. 

 

Literature Cited

Emre M, Bernabei R, Blesa R, Bullock R, Cunha L, Daniëls H, Dziadulewicz E, Förstl H, Frölich L, Gabryelewicz T, et al. 2010. Drug Profile: Transdermal Rivastigmine Patch in the Treatment of Alzheimer Disease. CNS Neurosci Ther. 16(4):246–253. 

D’Onofrio G, Nabavi SM, Sancarlo D, Greco A, Pieretti S. 2021. Crocus Sativus L. (Saffron) in Alzheimer’s Disease Treatment: Bioactive Effects on Cognitive Impairment. Curr Neuropharmacol. 19(9):1606–1616. 

Patel KS, Dharamsi A, Priya M, Jain S, Mandal V, Girme A, Modi SJ, Hingorani L. 2024a. Saffron (Crocus sativus L.) extract attenuates chronic scopolamine-induced cognitive impairment, amyloid beta, and neurofibrillary tangles accumulation in rats. Journal of Ethnopharmacology. 326:117898. 

Paul J. 2019. Chapter 5 – Experimental Medicine Approaches in CNS Drug Development. In: Nomikos GG, Feltner DE, editors. Handbook of Behavioral Neuroscience. Vol. 29. Elsevier. (Translational Medicine in CNS Drug Development). p. 63–80. [accessed 2024 Feb 25]. 

What is the role of acetylcholinesterase at a synapse? | Socratic. Socratic.org. [accessed 2024 Mar 21]. 

Microplastic burden in marine benthic invertebrates depends on feeding strategies

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Microplastic pollution is a global issue effectively impacting all aquatic systems from the poles to tropical reefs. Current emission patterns project to around 35 – 98 metric tons of annual microplastic emission by 2030. Yet, this may only be an underestimation, as our current understanding of microplastic concentrations based on traditional sampling practices overlooks smaller debris (Lindeque et al. 2020, Borrelle et al. 2020). With this scale of rapid increase in concentrations, the implications of microplastic accumulation in marine systems have become an increasing concern. In response to this global concern, Adam Porter and his team looked towards the ocean’s floor to better understand how microplastics interact with dynamic ecosystems.

Microplastics emitted into the marine environment can adversely impact a wide range of processes from cellular metabolism to digestive functions, fertility, locomotion, and growth (Foley et al. 2018; Bour et al. 2018). Furthermore, bioaccumulation, or trophic transfer when contaminated prey is consumed by predators, magnifies microplastic burdens in organisms higher in the food chain. These above properties, in conjunction to the rapidly increasing environmental concentrations, highlight the pressing need to quantify how much microplastics marine organisms are ingesting.

Historically, our understanding of individual microplastic burdens has often assumed that levels of environmental contamination directly map onto their uptake by marine organisms. However, studies have found that this isn’t always the case. Other factors, such as feeding strategies and community composition, also impact a species’ uptake rate (Pagter et al. 2021; Bour at al. 2018). 

To bridge the mismatch of environmental concentration and individual burden, Porter et al. reviewed 412 studies on marine invertebrates from around the globe to investigate how different species traits could influence microplastic uptake. First, they gathered data from each study and assigned a geographic sector to each sampling site. Next, they evaluated each observation for a variety of variables, including feeding mode, position within the sediment, and wet weight (mass) of the individual. Then, Porter’s team used statistical tests to examine the potential influence each parameter had on plastic uptake with statistical analyses tests and visualized their findings. 

Geographically, the Pacific Northwest, Yellow Sea and Japan Trench, had the highest mean individual microplastic burden. In terms of animal class, the highest mean burden occurred in the Malacostraca class. Malacostraca encompasses common commercial species such as crabs and lobsters, which could have commercial implications on industries like lobster fishing and aquaculture. 

Of all the outlined parameters, feeding strategies had the greatest impact on microplastic uptake. Omnivores were shown to have the highest rate of uptake, followed by predators, herbivores, grazers, suspension feeders, deposit feeders, and lastly scavengers. These findings support the bioaccumulation theory, one of several hypotheses concerning microplastic uptake patterns (Wang 2014). According to the bioaccumulation theory, microplastics enter the food web through primary consumers like suspension feeders, grazers, and filter feeders. The plastic they retain in their systems will then be ingested by higher trophic levels like secondary and tertiary consumers that are omnivores,predators, and scavengers. Accordingly, the microplastic burdens would be highest in predators and omnivores, which matches the study’s findings.

In addition to the quantity of microplastics retained, feeding patterns were also found to influence the size and type of microplastics consumed were also different across groups. The most reported shape was fibers. The mean sizes of these fragments ranged from 0.2 micrometers to 17 centimeters, and herbivores in general retained the largest particles, but the precise mechanisms driving these patterns remain unclear.  

These findings precisely highlight our gap in knowledge of microplastic distribution amongst marine communities. As Porter et al. highlights, a holistic consideration of subtle processes related to feeding patterns is essential in fine tuning our understanding of how our world is changing. Thus, although the study describes general trends on a global scale, future research focusing on regional subtleties is important. Subsequently, applying these findings as policy is crucial, as many marine organisms are frequently consumed commercial species. Being major consumers of seafood, the microplastic accumulation in marine animals can directly impact humans. This is particularly concerning in context of our status as the apex predator, and therefore the final stop in the chain of bioaccumulation. As the microplastic burden in marine organisms is rising at an alarming pace, the need for action is more urgent than ever.

 

Works Cited

Borrelle, S. B., Ringma, J., Law, K. L., Monnahan, C. C., Lebreton, L., McGivern, A., Murphy, E., Jambeck, J., Leonard, G. H., Hilleary, M. A., Eriksen, M., Possingham, H. P., De Frond, H., Gerber, L. R., Polidoro, B., Tahir, A., Bernard, M., Mallos, N., Barnes, M., & Rochman, C. M. (2020). Predicted growth in plastic waste exceeds efforts to mitigate plastic pollution. Science, 369(6510), 1515–1518. https://doi.org/10.1126/science.aba3656 

Bour, Agathe, Carlo Giacomo Avio, Stefania Gorbi, Francesco Regoli, and Ketil Hylland. “Presence of Microplastics in Benthic and Epibenthic Organisms: Influence of Habitat, Feeding Mode and Trophic Level.” Environmental Pollution (Barking, Essex: 1987) 243, no. Pt B (December 2018): 1217–25. https://doi.org/10.1016/j.envpol.2018.09.115.   

Foley, Carolyn J., Zachary S. Feiner, Timothy D. Malinich, and Tomas O. Höök. “A Meta-Analysis of the Effects of Exposure to Microplastics on Fish and Aquatic Invertebrates.” The Science of the Total Environment 631–632 (August 1, 2018): 550–59. https://doi.org/10.1016/j.scitotenv.2018.03.046.   

Lindeque, P. K., Cole, M., Coppock, R. L., Lewis, C. N., Miller, R. Z., Watts, A. J. R., Wilson- McNeal, A., Wright, S. L., & Galloway, T. S. (2020). Arewe underestimating microplastic abundance in the marine environment? A comparison of microplastic capture with nets of different mesh-size. Environmental Pollution, 265, 114721. https://doi.org/10.1016/j.envpol.2020.114721

Pagter, Elena, Róisín Nash, João Frias, and Fiona Kavanagh. “Assessing Microplastic Distribution within Infaunal Benthic Communities in a Coastal Embayment.” Science of The Total Environment 791 (October 15, 2021): 148278. https://doi.org/10.1016/j.scitotenv.2021.148278. 

​​Porter, A., Godbold, J. A., Lewis, C. N., Savage, G., Solan, M., & Galloway, T. S. (2023). Microplastic burden in marine benthic invertebrates depends on species traits and feeding ecology within biogeographical provinces. Nature Communications, 14(1), 8023. https://doi.org/10.1038/s41467-023-43788-w 

Wang, W. -X. “Chapter 4 – Bioaccumulation and Biomonitoring.” In Marine Ecotoxicology, edited by Julián Blasco, Peter M. Chapman, Olivia Campana, and Miriam Hampel, 99–119. Academic Press, 2016. https://doi.org/10.1016/B978-0-12-803371-5.00004-7.

Engineered Nanoparticles Enable Selective Gene Therapy in Brain Tumors

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Inborn protective mechanisms present challenges for therapies targeting cancers of the brain. Engineered nanoparticles permit the selective delivery of CRISPR-Cas9 to glioblastoma tumors.

Glioblastoma accounts for almost half of all cancerous tumors originating in the brain.1 Even with maximum safe treatment, the median survival period for patients is less than 1.5 years.2 However, survival varies widely by age, from a 0.9% 5-year survival rate for patients over 75 years old to an 18.2% 5-year survival rate for patient 0-19 years old.3 Nevertheless, the overall 5-year survival rate remains low at 5%2 and novel therapies are urgently needed for glioblastoma treatment. Zou et al. present a highly specific CRISPR-Cas9-based gene therapy for glioblastoma.4

Gene therapies offer an attractive treatment for cancers. The goal of gene therapy is to mutate or remove deleterious DNA sequences such that they are unable to be transcribed and translated into functioning proteins. In the most prevalent technique, CRISPR-Cas9, single guide RNA (sgRNA) identifies and binds to the target DNA sequence. It then recruits Cas9 proteins to excise parts of the target DNA sequence. Mutations are generated as the cell tries to repair the damaged DNA.5

While a powerful tool, researchers have struggled to effectively deliver CRISPR-Cas9 to their cellular target. Delivery is particularly complex in brain tumors such as glioblastoma. Traditionally medications are trafficked through the body and delivered to their target through the bloodstream. However, the brain is a more complex and protected system. Primarily, selective delivery of therapeutics to tumor cells is necessary to protect neuronal function. Moreover, the brain is separated from the blood stream by a thin layer of cells termed the blood-brain barrier (BBB). The BBB allows for selective permeation of compounds into the brain, shielding neurons from toxins while permitting the passage of essential nutrients.6 Previous studies have sought to transport CRISPR-Cas9 therapies across the BBB using viruses as delivery capsules7 or circumvent the BBB altogether via intercranial injection of therapeutics.8 Yet these methods carry risk, either an immune response to the viral vector or complications from the invasive injection.

Zou et al. sought to resolve the issues of specific cell targeting and BBB permeability in CRISPR-Cas9 delivery by encapsulating the gene editing complex within a nanoparticle. The researchers chose to target Polo-like kinase 1 (PLK1) using CRISPR-Cas9 gene therapy. PLK1 is an attractive for selective gene therapy due to its higher overexpression by glioblastoma cells and by the proliferative glioblastoma subtype in particular.9 Moreover, inhibition of PLK1 has been shown to reduce tumor growth and induce cell death.9

The researchers encapsulated Cas9 and sgPLK1 in a neutrally charged nanoparticle for delivery. The small size of nanoparticles, which are measured in nanometers, allow for easy transport through the bloodstream and uptake by cells. Taking advantage of the high expression of lipoprotein receptor-related protein-1 (LRP-1) on both BBB endothelial cells and glioblastoma tumor cells, they decorated the nanocapsule surface with LRP-1 ligand angiopep-2 peptide to facilitate selective uptake by BBB and glioblastoma cells (Figure 1). Zou and her colleagues bound the nanoparticle together with disulfide bonds as an added layer of selectivity for glioblastoma cell delivery. In the high glutathione environment of a glioblastoma cell, the nanoparticle dissolves, releasing its contents. However, glutathione concentrations are lower in BBB endothelial cells and healthy neurons, reducing the dissolution of the nanoparticles and leaving healthy DNA alone.

 

Figure 1. Nanoparticles enable permeation of the blood brain barrier (BBB) and selective delivery of the CRISPR/Cas9 system to glioblastoma (GBM) cells. Angiopep-2 peptides, which decorate the nanoparticle’s surface, bind with lipoprotein receptor-related protein-1 (LRP-1) overexpressed on BBB and GBM cells. Following uptake into GBM cells, the nanoparticles dissolve in the high glutathione environment, releasing the Cas9 nuclease and single guide RNA (sgRNA) targeting Polo-like kinase 1 (PLK-1) genes. Zou et al. demonstrated the selective mutation of PLK-1 induced apoptosis in GBM cells with minimal off-target effects.

 

Through Cas9/sgPLK1 delivery by nanoparticle, Zou et al. demonstrated a 53% reduction in expression of the targeted gene in vitro. PLK1 gene editing was cell selective, with negligible genetic mutation in the healthy surrounding brain tissue. While nanoparticles with and without disulfide cross-linking were capable of gene editing, the disulfide cross-linked nanoparticle induced almost 400% more mutations. Glioblastoma cells treated with disulfide cross-linked nanoparticles were also over 3 times more likely to undergo apoptosis cell death. Mice grafted with patient glioblastoma tumors treated with Cas9/sgPLK1 nanocapsules experienced an almost 3-fold extension in life expectancy, suggesting this treatment as a viable anti-glioblastoma therapy.5

Yet in order to be effectively applied as a cancer therapy, the efficiency of this nanoparticle delivery system must be increased. In mouse glioblastoma models, Zou et al. only achieved a maximum accumulation of 12% and effected a 38% knockdown of PLK1.5 Despite their low magnitude, these values are far greater than similar gene therapy treatments currently studied, suggesting nanoparticles present an innovation in the delivery of CRISPR-Cas9 to difficult to access tumors.

In addition to the improved efficacy over existing systems, the work of Zou et al. opens the door for less invasive administration of treatment. In contrast to previous gene therapies administered directly to the tumor through intercranial injection, the BBB penetration and tumor-specific accumulation of the nanoparticles may permit systemic administration. Zou et al. injected the nanoparticles intravenously, but treatment may even be given as a pill taken orally, eliminating any surgical intervention. Moreover, due to their modular design, the engineered nanoparticles may be adapted as targeted treatments for other tumors. Exchanging the angiopep-2 peptides for another ligand would facilitate uptake by cells expressing the corresponding receptor. The load carried within the nanoparticle could be altered to contain a different sgRNA targeting a new gene or another therapeutic entirely as a complementary treatment. Nanoparticle delivery systems like that studied by Zou et al. contains many layers of selectivity, offering hope for effective delivery of treatment to previously inaccessible tumors.

 

Works Cited:

  1. Wirsching, H.-G. & Weller, M. Glioblastoma. in Malignant Brain Tumors : State-of-the-Art Treatment (eds. Moliterno Gunel, J., Piepmeier, J. M. & Baehring, J. M.) 265–288 (Springer International Publishing, Cham, 2017). doi:10.1007/978-3-319-49864-5_18.
  2. Delgado-López, P. D. & Corrales-García, E. M. Survival in glioblastoma: a review on the impact of treatment modalities. Clin Transl Oncol 18, 1062–1071 (2016).
  3. Ostrom, Q. T. et al. CBTRUS Statistical Report: Primary Brain and Central Nervous System Tumors Diagnosed in the United States in 2007–2011. Neuro Oncol 16, iv1–iv63 (2014).
  4. Lino, C. A., Harper, J. C., Carney, J. P. & Timlin, J. A. Delivering CRISPR: a review of the challenges and approaches. Drug Delivery 25, 1234–1257 (2018).
  5. Zou, Y. et al. Blood-brain barrier–penetrating single CRISPR-Cas9 nanocapsules for effective and safe glioblastoma gene therapy. Sci Adv 8, eabm8011.
  6. Dotiwala, A. K., McCausland, C. & Samra, N. S. Anatomy, Head and Neck: Blood Brain Barrier. in StatPearls (StatPearls Publishing, Treasure Island (FL), 2024).
  7. Song, R. et al. Selection of rAAV vectors that cross the human blood-brain barrier and target the central nervous system using a transwell model. Molecular Therapy Methods & Clinical Development 27, 73–88 (2022).
  8. Lee, B. et al. Nanoparticle delivery of CRISPR into the brain rescues a mouse model of fragile X syndrome from exaggerated repetitive behaviours. Nat Biomed Eng 2, 497–507 (2018).
  9. Lee, C. et al. Polo-Like Kinase 1 Inhibition Kills Glioblastoma Multiforme Brain Tumor Cells in Part Through Loss of SOX2 and Delays Tumor Progression in Mice. Stem Cells 30, 1064–1075 (2012).

Invasive Species: Ecological Shapeshifters?

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Watershed reeds of midcoast Maine provide a deeper look into the field of epigenetics

Forests, grasslands, and marshes are ecological battlegrounds. In the fight to hold territory, maintain access to resources, and reproduce, many organisms compete directly to occupy the same niche– the role played by a specific organism in an ecosystem. An organism’s ability to carry out these roles is dictated by its “fitness” or capacity to survive and contribute its genes to the next generation. Naturally, relative reproductive success is incredibly environmentally dependent. Most organisms are tailor-made to thrive within their native habitats via natural selection. However, this biological narrative is challenged by the proliferation of invasive species in competition with their native counterparts. In their 2016 study, Spens and Douhovnikoff argue that epigenetics may be key to understanding ecological invasiveness and that the common reed (Phragmites australis) is “an ideal model species” (Spens & Douhovnikoff, 2016) for studying this rapidly expanding subfield of genetics.

Among other things, greater phenotypic plasticity, or “the ability of individual genotypes to produce different phenotypes when exposed to different environmental conditions” (Fusco & Minelli, 2010), increases an organism’s potential to adjust to its surroundings and occupy a vast variety of niches. This becomes possible through epigenetics.  Epigenetic modifications alter gene expression without changing the underlying DNA sequence (Weinhold, 2006). Methylation, the process by which methyl groups are added to DNA, is the key turning genes “on” and “off” (Menezo et al., 2020). The addition of methyl groups prevents DNA-transcribing proteins from accessing the DNA strand, stopping the gene’s expression as a protein. This has the potential to create significant differences in structural and even cellular function among individuals that are otherwise genetically identical.

Clonal plants provide a unique opportunity to study environmental pressures on epigenetics, as these individuals can act as their own genetic control. Reeds are an excellent example of this: as facultatively clonal plants, they can utilize both sexual and asexual reproduction. Exploiting this integral feature, and the existence of multiple subspecies of reed in midcoast Maine, researchers studied the genomes of both native and invasive reeds in two separate locations, Libby and Webhannet. They addressed two questions: Do introduced subspecies exhibit greater epigenetic variation (indicating that epigenetics plays a role in the success of an invasive species)? And will the variation between subspecies genotypes be lesser than the variation within a single genotype’s epigenetic markers (suggesting that epigenetic variation can be used to adapt to an incredibly variable environment)?

Researchers sought answers by studying clusters of reeds called ramets. Since all the reeds within a ramet were genetically identical, they could selectively measure epigenetic variation. These clones were grown within heterogeneous microhabitats that contain varying combinations of nutrients and conditions. Extracted DNA fragments were compared based on the level of methylation among subspecies, genotype, and ramet.

In both sites, the invasive reed demonstrated greater epigenetic diversity than the native reed (Figure 1). Up to 71% of epigenetic variation at the Webhannet site is attributed to differences among genotypes. These results suggest that clones adjust to the demands of their environments via epigenetics, rather than genotypic adaptation. Flexibility of this kind allows for rapid specialization in response to the hyper-individualized environmental conditions of each ramet. Additionally, each site developed an epigenetic “signature” with both subspecies exhibiting distinct, location specific, morphological characteristics. The significant differences in epigenetic markers between sites hint at the potential for large scale shifts due to epigenetics, should genotype not be a factor in these differences. The distinct characteristics displayed by each species demonstrate the vast alterations necessary to survive in an environment with subtle differences.

Figure 1. Epigenetic markers clustered by species (native, introduced) and location (Libby, Webhannet). Differences within a single genotype were greater than variation between genotypes, particularly for the introduced species. Figure adapted from Spens and Douhovnikoff

While this study was small scale, it supports the position that epigenotype variation provides a strong competitive advantage in the natural world. It also suggests that further study would provide more valuable information about the relevance of epigenetics in ecology. In our rapidly changing environment, due to climate change and other human influences, these native genotypes are in danger of being displaced from their niches. Despite a species’ history with its habitat, subtle alterations can have vast impact on individuals that demonstrate low plasticity or tolerance for change. Introduced organisms who demonstrate more flexible epigenotypes have the potential to outcompete their neighbors, eroding local ecosystems beyond repair. This reality drives ecological research in the direction of epigenetics, not only for the sake of discovery, but also in hopes of protecting species who cannot adapt as quickly as we disrupt.

 

Works Cited

Fusco, G., & Minelli, A. (2010). Phenotypic plasticity in development and evolution: Facts and concepts. Philosophical Transactions of the Royal Society B: Biological Sciences, 365(1540), 547–556. https://doi.org/10.1098/rstb.2009.0267

Menezo, Y., Clement, P., Clement, A., & Elder, K. (2020). Methylation: An Ineluctable Biochemical and Physiological Process Essential to the Transmission of Life. International Journal of Molecular Sciences, 21(23), 9311. https://doi.org/10.3390/ijms21239311

Spens, A., & Douhovnikoff, V. (2016). Epigenetic variation within Phragmites australis among lineages, genotypes, and ramets. Springer International Publishing. https://link.springer.com/article/10.1007/s10530-016-1223

Weinhold, B. (2006). Epigenetics: The Science of Change. Environmental Health Perspectives, 114(3), A160–A167.

Smoke Signals: The Unexpected Long Term Effects of Smoking on the Immune System

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Image Source: “Smoking has a Lasting Impact on the Immune System, 2024”

When we get sick, our bodies’ immune systems work to fight off infections by invading pathogens, or organisms like bacteria and viruses that cause disease. However, many factors such as lack of sleep and poor nutrition weaken our immune system, meaning that we are less able to stay healthy. It has been known that smoking is another one of these factors that weaken our immune systems, but a recent study from a group at the Institut Pasteur in France looking at the effects of a variety of factors on the immune system showed that the extent to which smoking plays a role is much higher than many would think. But to understand the results of this study, it is important to first understand the mechanisms the immune system uses to fight infection. 

The immune system has many different moving components, including two distinct branches. The first is the faster, more general innate immune system which has a similar response to all infections. The second is the adaptive immune system which is slower, memory-based, and is involved in pathogen specific response. Although the innate immune system involves general molecules that interact with all cells and the adaptive immune system has specialized molecules that interact with pathogens based on memory of past infection, they share one important class of signaling molecules. These molecules are called cytokines and their role is to coordinate both of these types of immune response. Cytokines are small molecules that are released by immune cells to communicate with other parts of the body and each other. This signaling results in deployment of a response by other immune cells against invading pathogens. However, levels of cytokine production exist in a very fine balance. In order to get the desired immune response, you need the exact right level of cytokines present. If levels are too high or too low, they could cause abnormalities including overactive immune response and inflammation or impaired immune responses. 

To investigate the effects of a variety of different factors on the immune system and cytokine responses of healthy individuals, a project called the Milieu Intérieur put together a cohort of 1000 healthy participants and has been studying variability in the immune system between these individuals (“The Milieu Intérieur Project”). In an investigation of this data, the group from Institut Pasteur, Saint André et al, analyzed 136 variables measured in the Milieu Intérieur Project that could be causing differences in cytokine secretion and immune response (Luo and Stent 2024). These variables included everything from demographics, to diet, to health habits like smoking, to social and environmental characteristics (Saint-André et al. 2024).

After they performed their initial statistical analysis, Saint André et al measured production of 13 disease relevant cytokines as a quantitative measure of immune response in populations with different demographics, health habits, and other characteristics. In the lab, they exposed blood samples from their sample population to 12 different molecules meant to serve as stimulants for the immune system (these molecules included things like viral and bacterial proteins). After this exposure, the authors tested cytokine production in both innate and adaptive immune cells, and once they had that data, they took their results one step further. The group also used epigenetics, or the study of changes in gene expression rather than the DNA code that makes up the genome to investigate possible reasons for variability in immune responses associated with factors tested. Their epigenetic evaluation consisted of analyzing the extent to which one epigenetic process, DNA methylation, occurred at specific regulators of signaling and metabolism (Saint-André et al. 2024) to assess changes associated with smoking. 

As previously stated, one of the authors’ main findings from the initial statistical analysis was that smoking had a large effect on cytokine response. In fact it had the same effect as age, sex, and genetics, three things many would consider much more directly impactful to the immune system than smoking. In their in vitro simulations, they found that smoking had a temporary effect on the ability of the innate immune system to function properly. This result is a relatively intuitive one. If you do something that is considered bad for you, it makes sense that you would get sick more easily. 

However, more surprisingly, they also found that smoking leaves a lasting effect on memory based adaptive immune responses even after cessation of smoking, meaning that even after people quit smoking, their immune systems still are impacted. They found that in samples from individuals who smoked there were higher levels of cytokine expression, especially of an inflammatory cytokine called CXCL5 that is secreted in response to bacterial infection. Secretion of this cytokine is associated with the presence of an inflammatory protein called CEACAM6 in the blood. Consistent upregulation of levels of this protein has been found to have links with multiple cancers such as colon cancer (Wu et al. 2024). In Saint André et al’s epigenetic investigation of this association, they found that DNA methylation, which results in a downregulation of gene expression and in this case an increase in cytokine production, is linked to smoking’s lasting effect on the immune system (Greenberg and Bourc’his 2019). DNA methylation was decreased at many of the sites they tested which are involved in regulation of signaling genes and metabolism. Decreased DNA methylation was likely impacting levels of cytokines in response to detection of pathogens. In these populations, smoking caused lasting changes in gene expression which resulted in long term changes in addition to the expected short term effects on the immune system. 

This study demonstrates that smoking can have lasting negative impacts on your health which are not limited to just lung damage. It is also associated with pro-inflammatory cancer pathways and epigenetic markers that cause increased cytokine production. This overproduction of cytokines can confuse cells and also cause increased inflammation. Over time the extra inflammation can damage tissues and lead to developments of other conditions, like the cancers previously mentioned and complications associated with overproduction of cytokines (“What are Cytokines”). These recent findings emphasize that it is important to consider the possible implications of smoking and all things that we expose ourselves to, and to keep in mind that new data is still being discovered.

Works Cited

The Milieu Intérieur Project Institut Pasteur. Luo,Y. and Stent,S. (2024) Smoking’s lasting effect on the immune system. Nature, 626,  724–725.

Saint-André,V., Charbit,B., Biton,A., Rouilly,V., Possémé,C., Bertrand,A., Rotival,M., Bergstedt,J., Patin,E., Albert,M.L., et al. (2024) Smoking changes adaptive immunity with persistent effects. Nature, 626, 827–835.

Wu,G., Wang,D., Xiong,F., Wang,Q., Liu,W., Chen,J. and Chen,Y. (2024) The emerging roles of CEACAM6 in human cancer (Review). International Journal of Oncology, 64, 1–15.

Greenberg,M.V.C. and Bourc’his,D. (2019) The diverse roles of DNA methylation in mammalian development and disease. Nat Rev Mol Cell Biol, 20, 590–607.

What are Cytokines? Types and Function Cleveland Clinic.

Smoking has a lasting impact on the immune system, a new study finds (2024) Euronews.

Novel Gene Drive Demonstrates Promise in Reducing Transmission of Malaria

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Recent progress has been made in malaria elimination using insecticides, bed nets, and clinical management (CDC). However, efficient mosquitos carrying deadly species of malaria parasites paired with favorable climate in regions where the disease is prevalent, weak infrastructure, and high intervention costs create difficult barriers to malaria elimination (CDC). Similarly, new threats are arising from drug- and insecticide-resistant parasites that pose a risk of expansion to densely populated areas with no prior exposure to the parasites and an unprepared public health system (Garrood et al., 2022). Amidst malaria control efforts, many scientists are investigating a promising new method of inhibiting mosquitos’ ability to transmit malaria: gene drives.

Gene drives, or “selfish” genes, are transmitted at a frequency greater than 50%, above what is predicted by Mendelian genetics. These genes occur naturally in populations; however, we can also engineer gene drives using CRISPR/Cas9 technology to insert a desired gene into a population (Bier, 2022, Hillary et al., 2020). The CRISPR/Cas9 system targets a certain DNA sequence and cuts it, after which the DNA will repair itself either randomly (end-joining, or EJ) or following a provided template (homology-directed repair, or HDR) (Adolfi et al., 2020, Hillary et al., 2020). In the case of gene drives, researchers create a drive, or set of genes, containing the desired genes to be inserted as well as genes encoding necessary CRISPR/Cas9 elements. The presence of CRISPR/Cas9 elements ensures that offspring can make the CRISPR/Cas9 proteins necessary to insert the desired gene into their genome. The desired inserted genes can serve a variety of purposes; in the case of malaria, scientists are inserting antimalarial effector genes into gene drives (Gantz et al, 2015, Isaacs et al., 2012).

Prior research demonstrates use of a gene drive, nRec, to insert antimalarial effector genes that inhibit parasite transmission into a mosquito population (Gantz et al, 2015, Isaacs et al., 2012). The drive targets the essential kynurenine hydroxylase (kh) gene, causing a loss-of-function mutation that reduces female survival and reproduction (Gantz et al., 2015). This type of drive, although effective in preventing malaria transmission, ultimately proves unsustainable given that the loss-of-function results in poor fitness and selection against the drive. A recent study proposes a solution to this problem via a gene drive rescue system that restores the function of the kh gene (Adolfi et al., 2020). The restoration of kh gene function ensures sustainable propagation of the drive and alleviates the fitness cost of the drive.

In a study led by Adriana Adolfi at University of California, Irvine, researchers aimed to target the same essential kh gene while also providing a rescue sequence that maintains the function of the gene (Adolfi et al. 2015). Although their drive does not include the antimalarial effector genes used in prior drives, they do not anticipate any challenges to incorporating the genes. Their approach addresses two problems: it minimizes the impact of drive-resistant alleles and reduces the fitness cost of the drive. By targeting the essential kh gene, drive-resistant mosquitoes are selected out because of the fitness cost that they take on while those that are not resistant do not suffer a fitness cost. The EJ repair that occurs in drive-resistant mosquitoes results in a loss-of-function mutation to the kh gene that makes the mosquitoes less fit for survival than their nonresistant counterparts who repair the Cas9 cleavage with HDR. HDR restores the original sequence of the kh gene by repairing the DNA according to a provided template, thus preventing the loss-of-function mutation, the fitness cost, and ultimately a population crash. In contrast, if the researchers had chosen to target a non-essential gene, this undesirable EJ event would not confer a fitness cost and would then be maintained while preventing transmission of the drive. Ultimately, the drive acts by replacing the previously targeted portion of the kh gene with the original kh sequence to alleviate prior fitness costs of the drive and minimize survival of drive-resistant individuals.

Reckh mosquitos, those that acquired the drive, demonstrated fitness equal to wild-type (WT) mosquitos, while mosquitos with a loss-of-function mutation in the kh gene demonstrated decreased fitness and were eliminated from the population. The drive was released into caged mosquito populations with varied ratios of Reckh males to WT males (1:1, 1:3, and 1:9). In all cages except for one 1:3 cage, the drive was present in more than 95% of mosquitoes by the eleventh generation. Notably, the population did not crash, and the drive was not selected out of the population. In prior research with the nRec drive, few cages reached greater than 95% introduction of the drive before the population crashed or the drive was selected out. The consistent spread of the Reckh drive demonstrates its ability to combat previous challenges of drive sustainability. 

 

Fig. 3

Figure 1: The drive reached >95% introduction by the eleventh generation in populations seeded with ratios of Reckh:WT males of 1:1, 1:3, and 1:9 while drive-resistant individuals were eliminated from the population. Green represents individuals with the drive, gray represents those without the drive, and orange represents drive-resistant individuals. (Adapted from Adolfi et al., 2020)

Furthermore, analysis of mosquitos with non-functional kh– alleles revealed that they were selected out of the population at a rate higher than expected. The researchers propose that lethal/sterile mosaicism factors into elimination of individuals with non-functional kh– alleles. During reproduction, offspring inherit one kh allele from their mother and one kh allele from their father. In the proposed model, CRISPR/Cas9 elements inherited from a mother with a non-functional kh– allele target and cut the WT kh+ paternal allele. This results in EJ mutations in the WT paternal allele that render the offspring less fit and accelerates its elimination from the population. The recoded kh gene sequence is not present in the mothers because they have a non-functional kh– allele, thus the mosquitoes cannot use HDR to repair the cut paternal allele and must instead use EJ. This lethal/sterile mosaicism ensures that non-functional kh– alleles act dominantly to eliminate drive-resistant individuals from the population.

The proposed gene drive shows promise towards sustainability of antimalarial gene drives in caged trials. To further explore the potential of this gene drive, the researchers propose further experimentation with the antimalarial effector genes inserted into their drive. Based on prior research, they do not anticipate any fitness disadvantage to the insertion of the antimalarial genes nor any challenges due to the large size of the drive. Although much research is required before this gene drive can be implemented in wild populations, it introduces a sustainable and effective method of malaria control.

 

References

  1. Adolfi, A., Gantz, V. M., Jasinskiene, N., Lee, H.-F., Hwang, K., Terradas, G., Bulger, E. A., Ramaiah, A., Bennett, J. B., Emerson, J. J., Marshall, J. M., Bier, E., & James, A. A. (2020). Efficient population modification gene-drive rescue system in the malaria mosquito Anopheles stephensi. Nature Communications, 11(1), 5553. https://doi.org/10.1038/s41467-020-19426-0
  2. Bier, E. (2022). Gene drives gaining speed. Nature Reviews Genetics, 23(1), 5–22. https://doi.org/10.1038/s41576-021-00386-0
  3. Gantz, V. M., Jasinskiene, N., Tatarenkova, O., Fazekas, A., Macias, V. M., Bier, E., & James, A. (2015). Highly efficient Cas9-mediated gene drive for population modification of the malaria vector mosquito Anopheles stephensi. Proceedings of the National Academy of Sciences of the United States of America, 112(49), E6736–E6743. https://doi.org/10.1073/pnas.1521077112
  4. Garrood, W. T., Cuber, P., Willis, K., Bernardini, F., Page, N. M., & Haghighat-Khah, R. E. (2022). Driving down malaria transmission with engineered gene drives. Frontiers in Genetics, 13, 891218. https://doi.org/10.3389/fgene.2022.891218
  5. Hillary, V. E., Ceasar, S. A., & Ignacimuthu, S. (2020). Chapter 18 – Genome engineering in insects: Focus on the CRISPR/Cas9 system. In V. Singh & P. K. Dhar (Eds.), Genome Engineering via CRISPR-Cas9 System (pp. 219–249). Academic Press. https://doi.org/10.1016/B978-0-12-818140-9.00018-0
  6. How Can Malaria Cases and Deaths Be Reduced? (2019, January 28). Centers for Disease Control and Prevention. https://www.cdc.gov/malaria/malaria_worldwide/reduction/index.html
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