Feeding 8 Billion People: Engineering Crops for Climate Resiliency
By Shaina Eagle, Global Disease Biology ’24
Feeding the world’s 8 billion– and growing– people [2] is an Augean task that requires cooperation between farmers, scientists, government agencies, and industry stakeholders across the globe. Agriculture and climate are deeply intertwined and climate conditions play a critical role in determining agricultural productivity and have a significant impact on global food security. The climate crisis poses immense challenges to food security and the farmers whose livelihoods depend on crop production. As the consequences of the climate crisis increase and intensify, developing resilient agricultural systems is essential to ensuring that our food and those who grow it can adapt without further depleting carbon and water resources.
Climate-smart agriculture identifies technologies that can best respond to the impacts of climate change, such as increasing temperatures and heat waves, changing rainfall patterns, severe storms, drought, and wildfires that adversely affect crop yield and quality [1]. Agronomists, plant biologists, and farmers are working to develop crops that will increase sustainable production and better withstand a changing climate via various genetic techniques.
Clonal Seeds
A team including a UC Davis Assistant Professor of Plant Sciences, Imtiyaz Khanday, genetically engineered rice seeds that reproduce clonally, or without sexual reproduction, in order to maintain the desirable traits found in the F1 generation (Vernet et al. 2022). They developed a breeding technique that allows for high-frequency production– or the ability to produce a large quantity in a short amount of time in a cost-effective manner– of hybrid rice using synthetic apomixis. Apomixis, a type of asexual reproduction in plants, allows for the production of seeds without fertilization, which can be useful in hybrid breeding programs. The study used CRISPR/Cas9 gene editing to introduce mutations in the genes responsible for sexual reproduction in rice. These seeds were planted and produced the F1 generation of plants, which were genetically stable and had high yield potential. Subsequent generations were clonally propagated from the F1 plants. In agriculture, high-frequency production has the ability to produce a large number of crops or seeds using advanced breeding techniques. High-frequency production is important for meeting the increasing demand for food and other agricultural products, as well as for improving the efficiency and profitability of farming operations.
The study suggests that this technique could be a valuable tool for plant breeders to produce high-quality hybrid rice seeds with more efficient and cost-effective methods. Clonal propagation can help maintain desirable traits as the climate crisis threatens agriculture, such as disease resistance, yield potential, or drought tolerance that might otherwise be lost through sexual reproduction. It is a faster alternative to sexual reproduction methods such as cross-breeding, which can take several generations and require extensive testing to identify desirable traits.
De Novo Domestication
De novo is a Latin term that means “from the beginning” or “anew”. In the context of genetics and plant breeding, de novo refers to the creation of something new or the starting point for the development of a new organism or trait. De novo domestication, for example, refers to the process of identifying and selecting wild plants with desirable traits and developing them into new crops that are better adapted to agricultural use. This approach differs from traditional domestication, which involves selecting and breeding plants that have already been used by humans for thousands of years. Eckhardt et al. highlight the potential benefits of de novo domestication, including the creation of new crops that are better adapted to changing environmental conditions, and the conservation of genetic diversity by using previously unexploited wild species.
A study by Lemmon et al. (2021) aimed to create a domesticated tomato variety with desirable traits by introducing mutations into genes related to fruit size and shape via CRISPR-Cas9. While there are many tomato cultivars available, they often have limitations in terms of yield, quality, or other traits that are important for consumers and growers. Therefore, there is a need to develop new tomato varieties with improved characteristics, and the de novo domestication of a wild tomato variety using genome editing offers a potential solution to this challenge. The domesticated variety has several desirable traits, including larger fruit size, smoother fruit shape, reduced seed count, and prolonged fruit shelf life. Additionally, the domesticated tomato plants have increased branching and produced more fruit per plant compared to the wild-type tomato plants.
Kaul et al. (2022) conducted a de novo genome assembly of rice bean (Vigna umbellata), a nutritionally rich crop with potential for future domestication. The study revealed novel insights into the crop’s flowering potential, habit, and palatability, all of which are important traits for efficient domestication. Flowering potential refers to the crop’s ability to produce flowers, which is important for seed production and crop yield. Understanding the genetic basis of flowering potential can help breeders select plants that flower earlier or later, depending on their needs. Habit refers to the overall growth pattern of the plant, such as its height, branching, and leaf morphology. Understanding the genetic basis of habit can help breeders select for plants that are more suitable for specific growing conditions or cultivation methods. Palatability refers to the taste and nutritional value of the crop, which are important factors for its acceptance as a food source. Identifying genes involved in carbohydrate metabolism and stress response can help breeders develop crops with better nutritional value and resistance to environmental stressors. Overall, these traits are desirable because they can contribute to the development of a more productive, nutritious, and resilient crop. The researchers also identified genes involved in key pathways such as carbohydrate metabolism, plant growth and development, and stress response. Climate change is expected to have a significant impact on crop yields, water availability, and soil fertility. One NASA study found that maize yields may decrease by 24% by 2030 [3]. Understanding the genetic basis of stress response and carbohydrate metabolism can help breeders develop crops that are more resilient to environmental stressors, such as drought, heat, and pests. Furthermore, identifying genes involved in plant growth and development allows breeders to introduce desirable traits, such as earlier flowering or increased yield. This is important for domestication because it can help accelerate the process of crop improvement and make it easier to develop new varieties with desirable traits. Overall, the genes identified in the study provide a foundation for developing crops that are better adapted to changing environmental conditions and more suitable for cultivation, which is crucial for ensuring food security in the face of climate change.
Genetically enhancing common crops
Molero et al. (2023) identified exotic alleles (germplasm unadapted to the target environment) associated with heat tolerance in wheat through genomic analysis and conducted breeding experiments to develop new wheat with improved heat tolerance. The exotic alleles were obtained from wheat lines that originated from diverse regions around the world, including Africa, Asia, and South America. The identified alleles increased heat tolerance in wheat under field conditions, and the effect was consistent across multiple environments. The authors obtained these lines from the International Maize and Wheat Improvement Center (CIMMYT) and used genomic analysis to identify the specific exotic alleles associated with heat tolerance. These alleles were then incorporated into breeding programs to develop new wheat varieties with improved heat tolerance.
The authors used genomic analysis to identify these alleles, which had diverse functions, including regulating heat shock proteins, osmotic stress response, and photosynthesis. The study provides evidence that the use of multiple exotic alleles could lead to the development of wheat varieties with improved heat tolerance under field conditions. The authors crossed the heat-tolerant lines carrying the exotic alleles with local commercial varieties to develop new breeding populations. They then evaluated the heat tolerance of these populations under field conditions to identify the lines with improved heat tolerance. The selected lines were further evaluated in multiple environments to confirm their performance and stability. Heat tolerance was measured by exposing the plants to high temperatures under field conditions and evaluating their performance. Specifically, they conducted experiments in three different environments, including a dry and hot irrigated environment, a semi-arid rainfed environment, and a temperate irrigated environment, all of which are known to impose high-temperature stress on wheat. The authors evaluated multiple traits related to heat tolerance, including yield, plant height, spike length, and the number of spikes per plant.
They also measured physiological traits such as chlorophyll fluorescence, canopy temperature, and photosynthetic activity. By evaluating these traits, they were able to identify the wheat lines with improved heat tolerance. By combining both phenotypic and genomic analyses, they were able to identify the wheat lines and alleles with the greatest potential for improving heat tolerance in wheat under field conditions. This demonstrates the potential for the use of exotic alleles in plant breeding to improve crop performance and address the challenges of climate change.
Porch et al. (2020) report the release of a new tepary bean germplasm (seeds or plant parts that can be passed onto the next generation and are helpful in breeding efforts) called TARS-Tep 23, which exhibits broad abiotic stress tolerance, as well as resistance to rust and common bacterial blight. Tepary bean (Phaseolus acutifolius) is a drought-tolerant legume crop that is native to the southwestern United States and northern Mexico. Tepary beans are generally grown in arid and semi-arid regions of North America, including the Sonoran Desert, Chihuahuan Desert, and the Great Basin. They are also grown in parts of Central and South America. According to FAO statistics, the total world production of tepary beans in 2019 was around 4,000 metric tons. Rust and common bacterial blight are two diseases that can affect the growth and productivity of tepary beans. Rust is a fungal disease that causes orange or brown spots on the leaves and stems of plants, leading to reduced photosynthesis and yield loss. Common bacterial blight is a bacterial disease that can cause wilting, necrosis, and reduced yield in affected plants.
The researchers conducted field trials and laboratory experiments to evaluate the performance and traits of TARS-Tep 23 under different conditions. Laboratory experiments involved inoculating TARS-Tep 23 with rust and common bacterial blight pathogens, then comparing the performance and traits with other tepary beans under these conditions. Field trials were carried out under conditions such as normal rainfall, drought, and heat stress. The results showed that TARS-Tep 23 had higher yields and better growth under drought and heat stress compared to other tepary bean varieties. It also showed high resistance to rust and common bacterial blight. The release of TARS-Tep 23 provides a valuable resource for breeding programs and can contribute to enhancing the productivity and sustainability of tepary bean cultivation. Developing climate-resistant germplasm is a critical resource for crop improvement and biodiversity cultivation, and it is used by plant breeders and researchers to develop new varieties with desirable traits such as disease resistance, stress tolerance, and improved yield.
Conclusion
The urgent need to address the challenge of climate change and its impact on global food security cannot be overemphasized. The world is already experiencing food shortages due to the adverse effects of climate change, and this problem is likely to worsen in the future unless appropriate measures are taken. Significant strides are being made in the research and development of new agricultural and genetic technologies that can engineer crops for climate resiliency. These technologies offer hope for a more sustainable future by enhancing food production, increasing resilience to extreme weather conditions, and mitigating the impact of climate change. However, it is essential to recognize that research and development efforts should not only focus on genetic engineering but should also involve all levels of the food production process, including better management practices, more efficient use of resources, and improved supply chain management. Only by taking a comprehensive approach can we hope to achieve a sustainable and resilient food system that can withstand the challenges of climate change.
References
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[2] Frayer, Lauren. “Earth Welcomes Its 8 Billionth Baby. Is That Good or Bad News… or a Bit of Both?” NPR, November 15, 2022, sec. Goats and Soda. https://www.npr.org/sections/goatsandsoda/2022/11/15/1136745637/earth-welcomes-its-8-billionth-baby-is-that-good-or-bad-news-or-a-bit-of-both.
[3] Gray, Ellen. NASA’s Earth Science News. “Global Climate Change Impact on Crops Expected Within 10 Years, NASA Study Finds.” Climate Change: Vital Signs of the Planet. Accessed May 30, 2023. https://climate.nasa.gov/news/3124/global-climate-change-impact-on-crops-expected-within-10-years-nasa-study-finds.
[4] Jägermeyr, Jonas, Christoph Müller, Alex C. Ruane, Joshua Elliott, Juraj Balkovic, Oscar Castillo, Babacar Faye, et al. “Climate Impacts on Global Agriculture Emerge Earlier in New Generation of Climate and Crop Models.” Nature Food 2, no. 11 (November 1, 2021): 873–85. https://doi.org/10.1038/s43016-021-00400-y.
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Interview: John Davis
By Isabella Krzesniak.
INTRODUCTION
John Davis is a 5th year Ph.D. candidate in the Integrative Genetics and Genomics graduate group at UC Davis. He works in the Maloof Lab and uses bioinformatics to analyze genetic variation among native California wildflowers in the Streptanthus clade in different environments and uses data to create gene models.
The project he is working on has two main goals. First, he aims to create genomic resources for Streptanthus clade species through reference genomes and transcriptomes, which can be used to analyze differential gene expression in different individuals. Second, he aims to examine the germination niche of Streptanthus clade species, the conditions required for them to germinate and the gene networks expressed during this life stage.
These models have many applications concerning adaptation in the wake of climate change; for instance, they can help ecologists make informed decisions such as whether a crop will function well in a given region as the climate warms. Davis’ work is part of a collaborative study between the Maloof, Gremer, Strauss, and Schmitt labs in the Department of Plant Biology.
What does your research consist of and what are its potential applications?
We’re looking at how plant populations persist in different environments. So even though it’s wildflowers that are closely related, you can also look at how they differ in terms of survival in different environments. If you have an environment that’s great for one crop, but it’s either getting wetter or hotter, the crop might not survive very well. But if you know which genes it has or how it functions, you can move it to a different location or potentially just bring in a different crop that will function well in that region. From an ecological standpoint, it’s a matter of which species will survive and which ones will die off. Underlying all of it are what genes the plant has.
What work are you doing with the project in particular?
The main thing I’m doing right now is building genomic references. We’re trying to do gene expression studies, but if we don’t know what the genes are, we can’t compare the differences in gene expression. So, one of the things I’m doing is building these reference genomes and transcriptomes to determine which genes are in the species. And then from there, I hope to build gene models, construct coexpression networks, and predict germination based on gene expression profiles. To analyze the data, I use Python, Linux, Excel, and R. Another thing I’m doing is building transcriptomes which are collections of just the genes that are expressed. Then, ideally, my goal would be to develop gene networks that would basically tell us which species have these genes that are needed to survive in these environments and which ones don’t.
Why are you studying Streptanthus in particular and what exactly are you doing as part of the study?
The Streptanthus clade has a well-documented phylogeny of closely-related species. Adding genomic resources will improve our ability to perform genetic analyses.
After seed collection, what steps do you take to analyze your data?
We took our seeds and sent them to a collaborating company where they extracted the RNA and then prepared RNA-seq libraries (where they extracted the RNA and then prepared the data), which were then sent to the UC Davis Genome Center where they were sequenced. and then the Genome Center sends us back the sequence reads. We have those reads, we use those to assemble transcripts and to also do gene expression analysis, where we start relating and making models to compare gene expression to different climate variables like precipitation, temperature, and elevation. They’re all correlated.
What kind of models do you employ for data analysis?
It’s just basic linear models and other types of models. You have your variable, which in our case would be germination proportion, and that is a function of gene expression. Gene expression is affected by temperature, genotype, and precipitation, so it’s just models on models.
Has the project been successful?
We did what we set out to accomplish with the funding. Right now, the final bit of sequencing data is coming in and then we’re actually starting to dive into it and produce actual results.
What are the difficulties of working with plants?
I love genetics and genomics stuff and I just fell into working with plants. Plants are the hardest compared to bacteria and humans. Plant genomes are ridiculous and weird things happen all the time. Humans are diploid–we’re boring. I finished working on a project with Brassica napus. It’s an allotetraploid (having four sets of chromosomes derived from different species), which is a hybrid of two different plants, Brassica rapa and Brassica oleracea, so it has two separate diploid genomes in itself. You have the two genomes that are crossing over with each other through homeologous exchange. So when you’re going try to assemble that genome, you don’t know if it came from the Rapa genome or the Oleracea genome. I think strawberries are up to eight copies of each chromosome, so it makes it a lot harder when you’re trying to find alleles. When you’re doing an experiment where you’re trying to knock out alleles of a genome, you have to knock out every copy in each chromosome. Whereas in humans, you only have to knock out two of them to make it homozygous. But in a strawberry, you have to knock out all six of those mutations. Plants just seem like the hardest of the group. And then you have pine trees where genomes have 22.5 gigabases (20 billion base pairs) and humans only have 3.2 gigabases.
How has extreme weather (wildfires, flooding) over the past years affected the study?
So one of the struggles of our project is that we’re looking at how the climate affects germination, but at the beginning of our project, there were droughts like crazy and wildfires, and that affects the genetics of the population and what survives and what doesn’t.
You’re trying to do all these environmental studies that look at the long-term effects compared to now, but when you’re a grad student on a grant, the grant only lasts four to five years. But, how do you take four to five years of data and project it out decades ahead without having data from decades prior? It just gets difficult when you only have four seasons that you can collect data from, and two of those are on fire and one of them is flooding. None of this seems like normal conditions historically. So it can make it a little bit tough to tease out what’s long-term variation in genetics in response to what’s happening in the environment right now.
What makes ecological, as opposed to transgenic research, difficult?
With our studies, we don’t knock out any genes or use any transgenics. Ours is all ecology. That’s the difficulty of our project. With Arabidopsis (a model organism in plant biology), the genes are pretty much homozygous and it’s a lot easier. In our case, all the seeds are collected in the wild, so they’re going to be heterozygous. We can try to make more of the seed by breeding the greenhouse to expand our seed stock, but we can only do so much since it takes up space to make more seed. The field is always going to be changing too. When you collect seeds from one year, the genetics could be completely different from the genetics of the next year.
Why don’t you use transgenics in your studies?
You don’t want to dive into transgenics (organisms whose genes have been altered) because there’s so much pushback against it. These are all natural California species and you don’t want to put something in the environment that can outcompete the natural population.
We’re trying not to affect the study environment that we’re looking at. When we do seed collections, we don’t take from at-risk populations of the certain species, and when we collect seeds, we only take a percentage of the seeds from each plant. We don’t want to affect the growth for the next season, so ultimately, we’re trying to do the minimum amount of disruption to the environment that we’re studying. We potentially hope to use our results for rehabilitation efforts. We’ll be able to tell which ones need more help to survive and which ones are fine.
Canine Cloning: History and Recent Trends
By Sara Su, Animal Science and English ’24
INTRODUCTION
In 1996, Dolly the sheep was the first mammal to be successfully cloned [1, 2]. Since then, 22 other animal species have been cloned, including rats, mice, cattle, goats, camels, cats, pigs, mules, and horses [3-12]. Among these, about 19 species have clones surviving to adulthood. In 2005, the first cloned dog to survive to adulthood was named Snuppy, who was derived from somatic cells from the ear-skin of a male Afghan hound. He was the 15th animal to be cloned and lived to the age of 10 [13]. He was cloned using Somatic Cell Nuclear Transfer (SCNT), a common method that involves removing the nucleus from an oocyte (egg cell) and replacing it with a nucleus from a somatic cell, typically a fibroblast [14]. Fibroblasts are a type of stromal cell found in connective tissues such as the skin and tendons – they are often used in cloning because they are relatively easy to culture. After nuclear transfer, this reconstructed oocyte, which is similar to a fertilized egg, is then activated and transferred into the oviduct of a surrogate female, usually in groups of 10-15 cells. After pregnancy is confirmed, viable offspring are born via C-Section [13-16]. Though SCNT is the most viable method of cloning so far, it remains very inefficient and the live birth ratio is extremely low [13, 17, 18]. Nearly 30 years after Dolly, little is understood about cloning, which presents unique challenges to different species; this review will discuss the relevance of the canine model in regards to human health, canine-specific challenges in using SCNT for cloning, as well as recent trends among successfully cloned dogs.
The Interest in Dogs as a Medical Model
Overall, dogs have become increasingly relevant as a medical model for human diseases in the 21st century. This is because many heritable canine diseases are orthological to human ones, which means they share similar traits and functions [19]. The dog genome is found to be closer to the human genome than the mouse genome – while mice are commonly used as medical models for humans, canine models have also proven to be useful for comparative studies due to their relatively long life, larger size, and similar tissue functions[20]. There are at least 350 shared genetic diseases between dogs and humans discovered so far, affecting a variety of systems such as the dermatological, lysosomal, hematological/immunological, and muscular/skeletal systems [21]. All of these contribute to the rising application of canine medical models to study disease mechanisms for well-known conditions such as Alzheimers and diabetes, while also being able to explore clinical therapies for rare genetic diseases that would otherwise be difficult to study. Other fields of study using canine colonies include but are not limited to: organ transplants, drug development, non-invasive biomarker generation, and psychological disorders [19-24].
Dog-Specific Challenges in Cloning
There are a few species-specific reasons why dog cloning remains inefficient. Cloning efficiency, defined as the ratio of live offspring coming from the number of transferred reconstructed oocytes, is usually not more than 3% across all species, regardless of the age or type of donor cell. Additionally, the average cloning efficiency between breeds does not differ significantly [21]. For canines, cloning efficiency is higher than many other reported species, at 2% [25]. However, this is still an extremely low number, and a specific challenge when it comes to dogs is the viable maturation of oocytes in vitro [13, 26]. It should be noted that the pregnancy rate can be increased by increasing the number of reconstructed oocytes injected into surrogates, but cloning efficiency itself is not changed. Another issue with canines is the vast number of breeds within the species – it is difficult to select for compatible nuclear/oocyte donors, in addition to adequate surrogate selection [21]. Finally, a widespread issue with cloned individuals is postnatal care – although survival is pretty much guaranteed for clones that are born healthy, cloned animals are just as vulnerable to disease and poor management as any other species [21]. Dolly the sheep died early from such an instance, rather than complications directly related to cloning. Overall, there is insufficient knowledge of the nuances of canine reproductive systems, including a lack of comprehensive protocols regarding oocyte maturation in culture and specific methods of post-natal clone care, leading to further difficulties in dog-specific cloning.
Snuppy and His Clones
As previously stated, the first dog to be successfully cloned and survive to adulthood was a male Afghan hound named Snuppy, short for Seoul National University Puppy. Snuppy was born in 2005, and was the only survivor out of 123 recipient surrogates. Snuppy was cloned from fibroblast cultures derived from the biopsy of the ear-skin of an Afghan hound named Tai. He was confirmed to be genetically identical to Tai through the use of canine-specific biomarkers [27]. For this experiment, 3 out of 123 surrogates resulted in pregnancies, 2 were carried to term, and 1 survived to adulthood – the other puppy died on day 22 due to aspiration pneumonia after experiencing neonatal respiratory distress. Although the efficiency of cloning is very low in the first place, this particular experiment had a cloning efficiency rate much lower than expected – 2 puppies were born to 123 surrogates, or 1.6% [13]. Snuppy ended up living to be 10 years old, while his donor Tai lived to be 12 years old – both individuals died of cancer-related causes, but were generally healthy until then. It should be noted that the median lifespan of Afghan hounds is reported to be 11.9 years, so their lifespans were not out of the norm [28].
In 2017, Snuppy was cloned. This time the cloning efficiency and success rates were much higher and resulted in 3 clones who are still alive today. Rather than using fibroblasts, this experiment used adipose-derived mesenchymal stem cells (ASCs). Then, ASCs were cultured with Dulbecco’s Modified Eagle Medium(MDEM), a technique that increases oocyte fusion rate in SCNT[29]. This experiment resulted in pregnancy and delivery rates of 42.9% (3 dogs out of 7 recipients) and 4.3% (4 clones out of 94 embryos). Compared to Snuppy’s 2.4% (3 out of 123) and 0.2% (2 from 1,095), these changes in technique correlated in a huge jump in overall efficiency [13, 28].
Other studies have also been published exploring the viability of cloned working dogs. For dogs, SCNT can be used regardless of sex, age, and breed [13, 30]. It was recently concluded that cloned dogs have similar behavior patterns to their cell donors, and can lead healthy lives with life spans comparable to naturally bred dogs [21, 29]. Overall, about 20% of dog breeds recognized by the American Kennel Club have been successfully cloned, which is highly successful compared to other mammals [21]. Though more research is needed to improve dog cloning efficiency, it has already been proven that clones of drug detection dogs[31] and cancer-sniffing dogs[33] outperform naturally bred dogs, scoring higher averages on qualification tests for these services [32-33].
CONCLUSION
To conclude, both studies regarding the creation of Snuppy and the subsequent cloning of his cells demonstrate great potential for the common use of canine clones in the modern world. Multiple obstacles regarding canine cloning were recognized and overcome, though the cloning efficiency rate can be further improved by obtaining greater knowledge of the canine reproductive system. Additionally, it was proven that clones who are born healthy aren’t at a larger risk for diseases or a shortened lifespan – they are comparable to the average puppy. All of this contributes to the feasibility of cloning working dogs – studies are already exploring the possibility of using clones of dogs that perform drug- and disease- detection, knowing that the physical qualifications for such jobs are strongly linked to specific genetic traits.
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How does prenatal nicotine exposure increase the chance of a child developing asthma?
By Madhulika Appajodu, Cell Biology ’24
Author’s Note: My name is Madhulika Appajodu and I am a 3rd Year Cell Biology major at UC Davis. I am a pre-medical student and hope to go on to medical school. I chose Cell Biology as a major because I found the focus on cell organization and function to be very interesting. I am a volunteer at Shifa Community Clinic and a member of MEDLIFE, SEND4C, and H4H. I am also a BioLaunch Mentor and a Learning Assistant for the Physics Department. I wrote this piece to answer the question: “How does prenatal nicotine exposure increase the risk of asthma in offspring?” I wrote this for undergraduate students in the field of epigenetics/prenatal exposures and experts/professors in the field but also for the general public who have some knowledge in science. I chose this topic in particular because epigenetics interests me greatly. I find that environmental factors likely play a large part in the life outcomes of people who may be genetically similar but grew up in different environments. I hope that readers will understand how important environmental factors are in the grand scheme of physical, emotional, and mental health for not just the reader but their future families (if they choose to have them) health as well.
ABSTRACT:
Previous studies have studied prenatal nicotine exposure and its effects which follow offspring over the course of their lives. One of these effects is asthma. Asthma is a chronic respiratory condition characterized by the narrowing of one’s airways in response to an allergen or irritant. It is a widespread condition, affecting over 25 million people currently in the US alone. The mechanisms of asthma and its causes are currently being investigated. However, researchers agree that prenatal nicotine exposure increases the risk of asthma in offspring exponentially.
There is currently no cure for asthma, only methods to lessen the intensity of asthmatic episodes, such as through the use of an inhaler. This literature review details the mechanisms through which prenatal nicotine exposure increases the risk of asthma in offspring, according to current research. The three potential causes of this increased risk are placental damage, epigenetic alteration, and nicotine exposure alone. The mechanisms will be evaluated through a synthesis of experimental and survey data in mice and human models in studies done in the past seven years. Comparisons will be drawn between articles that cite the same mechanism as the cause of the increased risk of asthma. Once the mechanism(s) are identified, research can be done to identify a solution so asthma due to prenatal nicotine exposure can be prevented.
INTRODUCTION
In the United States, approximately 25 million people are currently diagnosed with asthma [1]. Asthma is a respiratory condition characterized by difficulty breathing due to narrowing airways, caused by inflammation and excess mucus production. This inflammatory response is often triggered by viruses or air-borne allergens. Researchers are currently investigating the underlying immune mechanisms that cause the intense inflammatory response, which is often more intense when someone has been subjected to risk factors such as prenatal nicotine exposure. Since there is currently no cure for asthma, research about the underlying mechanisms of the inflammatory response is vital so that asthma can be prevented rather than simply managed.
Researchers have studied prenatal nicotine exposure and its effects on offspring for decades, focusing on human subjects who smoked while pregnant. Over the past thirty years, there has been a shift toward using animal trials to investigate the mechanisms associated with the risk factors for asthma.
The primary model in asthma research in mice is the house dust mite (HDM) model. The HDM model involves exposing one group of pregnant mice to tobacco smoke-infused air and another group of pregnant mice to filtered air. The offspring of both groups are exposed to house dust mites– a common allergen– and their inflammatory immune response is examined. There are variations to the model, such as exposing the fathers to nicotine prior to mating or exposing the female mice to nicotine prior to or during pregnancy.
Current literature cites three main factors that contribute to an increased risk of asthma: nicotine smoke exposure alone, placental damage induced by nicotine, and epigenetic alterations induced by nicotine. Nicotine passes from the mother’s blood to the fetus through the umbilical cord during pregnancy. Nicotine can also damage the placenta through vasoconstriction of blood vessels and alter the fetus’ epigenetic markers through DNA methylation.
The purpose of this literature review is to examine precisely how prenatal nicotine exposure increases the risk of asthma, first in experimental data using the HDM model and then in experimental & survey data regarding humans.
Prenatal Nicotine Smoke Exposure
In 2015, Eyring et al proposed that nicotine use in pregnant women increased the risk of asthma in offspring through epigenetic alterations [2]. Eyring et al exposed one group of female mice to tobacco infused smoke (ETS) for five weeks and mated them to male mice and examined the offspring. The pregnant female mice were then exposed to ETS until they gave birth. There was also a control group of female mice exposed to filtered air and mated to male mice. The offspring of the ETS exposed group did display an increased inflammatory response when exposed to house dust mites compared to the control group. However, the level of DNA expression of both groups were not statistically different. Thus, Eyring et al. came to the conclusion that prenatal nicotine exposure can cause an increased risk of asthma in offspring, but was unable to identify the mechanism through which prenatal ETS causes an increased inflammatory response [2]. It is possible that the Bisulfite sequencing equipment at the time of Eyring et al.’s study was not sensitive enough to detect the difference in methylation that newer studies observed.
Figure 1. Expression levels of IL-5 (Th2 cytokine producing protein) are the same for the CS (ETS exposed group) and FA (filtered air group) mice when exposed to house dust mites (HDM). This indicated that the gene expression levels were not affected by ETS.
A three-generation survey study on human subjects found a correlation between maternal smoking and the increased risk of asthma in offspring, as well as a correlation between grandmothers smoking during pregnancy and their grandchildren having an increased risk of asthma, regardless of the intermediate generation’s smoking habits [3]. The researchers also found a correlation between paternal smoking and an increased risk of asthma in the offspring [3]. They have hypothesized that paternal smoking causes altered microRNA (miRNA) in the sperm. MiRNA is a nucleic acid that regulates expression of genes. During fertilization, this altered miRNA can change the gene expression of the progeny, increasing the risk of asthma in the offspring. The conclusion of this study is that maternal, paternal, and grandmaternal nicotine exposure is correlated with an increased risk of asthma in offspring. The researchers also proposed epigenetic alteration as the mechanism of increased asthma risk, but due to the nature of the study, they were unable to confirm this hypothesis [3].
Placental Damage
A survey of mothers who smoked and mothers who did not smoke by Zacharasiewicz et al. concluded that prenatal exposure to nicotine causes placental damage by decreasing nutrient delivery to the fetus [4]. Prenatal nicotine exposure decreases alveolar surface area, thereby decreasing the tidal volume of fetal lungs after birth [5]. Tidal volume is the amount of air that enters the lungs per breath. A decreased tidal volume results in less oxygen entering the body under standard conditions and a vastly reduced amount of oxygen entering the body when exposed to an allergen. Placental damage also results in the increased aging of the fetus’ lungs as pulmonary cells perform less glycogenolysis and glycolysis, causing cells to die prematurely [6]. The premature death of lung cells means the lungs are weaker, unable to exchange a normal amount of oxygen, and therefore more prone to intense allergic reactions.
Similarly, a study by Cahill et al. using the HDM mice model found that inhaling nicotine causes vasoconstriction– the narrowing of blood vessels– in the mother, resulting in less oxygen and nutrients delivered to the fetus [7]. They also found that placental HSD2 (a crucial enzyme in fetal development) is decreased when pregnant mothers are exposed to nicotine. Cahill et al also observed placental damage from nicotine use which resulted in decreased birth weights and lung size in fetuses [7]. Decreased lung size leads to intense asthmatic episodes because the airways are smaller and narrower than the airways of an individual not exposed to nicotine prenatally. Ultimately, Zacharasiewicz and Cahill came to the same conclusion that nicotine consumption or exposure in pregnant women increases the risk of asthma in their offspring by negatively affecting the offspring’s lungs [4,7].
Epigenetic Alteration
Researchers agree that DNA methylation is the one of the mechanisms leading to an increased asthma risk [8]. DNA methylation, the primary form of epigenetic alteration that occurs when a fetus is exposed to nicotine, is a chemical reaction where a methyl (-CH3) group is added to a cytosine base. This methyl group prevents transcription factors from binding to DNA and recruiting repression proteins, resulting in underexpressed genes, which in this case is a disproportionate inflammatory response. However, there is disagreement among researchers about which genes are being alternatively methylated. Christensen et al. conducted an HDM mouse study and found that methylation of genes which produce and regulate Th2 cytokines was decreased in the offspring of mothers exposed to ETS [9]. Cytokines are small proteins that regulate the immune response; Th2 cells produce cytokines that encourage inflammation. Thus the increased expression of Th2 intensifies the inflammatory response that occurs in response to the asthma trigger of house dust mites. Christensen et al. found that Th1 cytokine levels remained constant and methylation was unaffected [9].
Conversely, Singh et al. found that Th1 cytokine levels decreased due to hypermethylation [10]. Singh et al. did also find that Th2 cytokine levels increased due to hypomethylation, which concurs with the findings of Christensen et al [9-10].
Figure 2. Expression levels of IL-3 (Th2 producing gene) in the groups that were exposed to tobacco infused smoke (SS) or filtered air (FA). There is a statistically significant increase in expression in the SS group indicating a decrease in methylation.
Christensen et al. exposed pregnant female mice to either tobacco smoke-infused air or filtered air and then examined the offspring [9]. Singh et al. exposed both male and female mice to tobacco smoke-infused air or filtered air prior to mating and then examined the offspring [10]. This variation in experimental methods could contribute to the difference seen in the methylation of Th1 cytokine-producing genes. However, both researchers concluded that the nicotine-induced DNA methylation levels changed in genes that produced inflammatory responses to allergens [9-10].
Zakarya et al. found that DNA methylation levels were altered in genes associated with fetal growth and nicotine detoxification [11]. This review examined epigenome-wide association studies (EWAS) on patients suffering from asthma whose mothers smoked or vaped during pregnancy. These studies showed increased methylation in placental, whole blood, and fetal lung genes [12]. These results differed from the research done by Singh et al. and Christensen et al. both in the affected genes and the way that methylation was altered [9-10]. The difference in results can be attributed to the difference between mice and humans as well as the variation in experimental design. Christensen and Singh used the HDM model on mice and controlled the levels of nicotine the mice were exposed to [9-10]. Zakarya et al. used data from children of women who reported smoking during pregnancy [11]. The levels of nicotine that the subjects were exposed to was not controlled and varied greatly. These differences between the studied species and experimental design could explain the different conclusions that the researchers drew.
CONCLUSION
There is not a simple answer about the mechanism by which nicotine use during pregnancy increases the risk of asthma in offspring. However, both epigenetic alterations and placental damage due to nicotine exposure play a role in increased asthma risk.
Research citing nicotine-induced epigenetic alteration as the main cause of the increased risk of asthma identifies various genes being altered by DNA methylation. The HDM studies cited in this review conclude that genes producing cytokines had a decrease in methylation, while a study using human subjects concluded that genes involving fetal growth and nicotine detoxification had an increase in methylation. Further research should determine which altered genes are increasing the risk of asthma so that methylation can be induced or repressed in those genes as a preventive measure for asthma. Further research should also focus on which aspect of nicotine-induced placental damage is the biggest factor in the increased risk of asthma so that a solution can be found to address that aspect.
Future research studies should continue to investigate the two presented mechanisms and identify the factors that are increasing the risk of asthma so that nicotine-induced asthma can be prevented in future generations.
Genetic algorithms: An overview of how biological systems can be represented with optimization functions
By Aditi Goyal, Genetics & Genomics, Statistics ‘22
Author’s Note: As the field of computational biology grows, machine learning continues to have larger impacts in research, genomics research in particular. Genetic algorithms are an incredible example of how computer science and biology work hand in hand and can provide us with information that would otherwise take decades to obtain. I was inspired to write a review overviewing genetic algorithms and their impact on biology research after reading a news article about them. This paper is not intended to serve as a tutorial of any kind when it comes to writing a genetic algorithm. Rather, it serves to introduce this concept to someone with little to no background in computer science or bioinformatics.
Introduction
In 2008, Antoine Danchin wrote that “there is more than a crude metaphor behind the analogy between cells and computers.” [1] He also stated that the “genetic program is more than a metaphor and that cells, bacteria, in particular, are Turing machines.” [1] This is the fundamental theory that has been the basis of systems biology and has inspired the development of genetic algorithms. Genetic algorithms (GAs) provide a method to model evolution. They are based on Darwin’s theory of evolution, and computationally create the conditions of natural selection. Using genetic algorithms, one can track the progression of a certain gene or chromosome throughout multiple generations. In this paper, we discuss the components of a genetic algorithm, and how they can be modified and applied for various biological studies.
Background
GA’s are an example of a metaheuristic algorithm that is designed to find solutions to NP-hard problems [2, 3]. NP problems, aka Non-deterministic Polynomial-time problems, describe optimization problems that take a polynomial amount of time to solve via a brute force method. This is best understood through an example, the most classic one being the Traveling Salesman Problem [4]. If a salesman has to travel to five different locations, how should he pick the order of his destinations, in order to minimize the distance he travels? The solution to this problem is to calculate the total distance for each combination and pick the shortest route. At five destinations alone, there are 120 possible routes to consider. Naturally, as the number of ‘destinations’ increases, the number of possible routes will increase, as will the time it takes to calculate all options. In more complicated scenarios, such as an evolution prediction system, this problem becomes exponentially more difficult to solve, and therefore requires optimization.
GA’s are “problem independent” optimization algorithms [2, 3]. This means that the parameterization of the algorithm does not depend on any certain situation, and can be modified by the user depending on the situation. This class of optimization algorithms is often referred to as a metaheuristic algorithm. The key idea is that these types of optimization functions trade accuracy for efficiency. Essentially, they aim to approximate a solution using the least amount of time and computing power, as opposed to providing a high degree of accuracy that may take significantly more resources.
Components of a Genetic Algorithm
There are only two components essential to creating a GA: a population, and a fitness function [I*]. These two factors are sufficient to create the skeleton of a GA. Withal, most GA’s are modeled after Darwin’s theory of evolution [j*]. They use the components of fitness, inheritance, and natural variation through recombination and mutation to model how a genetic lineage will change and adapt over time [j*]. Therefore, these components must also be incorporated into the GA in order to more accurately mimic natural selection.
Population
A population is defined using the first generation, or F1. This can be a set of genes, chromosomes, or other units of interest [7]. This generation can be represented in several ways, but the most common technique is to use a bit array where different permutations of 0’s and 1’s represent different genes [7].
Selection & Fitness Functions
Now that a population has been initialized, it needs to undergo selection. During selection, the algorithm needs to select which individuals from the population will be continuing onto the next generation. This is done through the fitness function [3]. The fitness function aims to parameterize the survival of a certain individual within the population and provide a fitness score. This accounts for the fitness of each genetic trait and then computes the probability that the trait in question will continue onwards. The fitness score can be represented in different ways. A common method is using a binary system. For example, consider a chromosome being defined as a set of bits (see Figure 1). A neutral, or wild-type allele can be represented with a zero. A beneficial allele or one that confers some sort of advantage over the wild-type is represented using a 1. The fitness function would then be defined to calculate the fitness of each chromosome. In this example, the fitness is equivalent to the sum of the binary scores.
Chromosomes with a higher fitness score represent chromosomes that have more beneficial traits as compared to chromosomes with lower fitness scores. Therefore, chromosomes that maximize the fitness score will be preferred.
Inheritance & Genetic Variation
The fittest individuals are then propagated onwards to the “breeding” phase, while only a small proportion of the fewer fit individuals are carried forward. This is the step that mimics “natural selection”, as we are selecting for the more fit individuals, and only a small proportion of the fewer fit individuals are surviving due to chance.
Now that the survivors have been identified, we can use GA operators to create the next generation. GA operators are how genetic variation is modeled [7]. As such, the two most common operators in any GA are mutation rates and recombination patterns. The F2 generation is created by pairing two individuals from F1 at random and using our operators to create a unique F2.
Mutations are commonly represented using bit changes [3]. Because our original population was defined in binary, our mutation probability function represents the probability of a bit switch, i.e. the probability that a 0 would switch to a 1, or vice versa. These probabilities are usually quite low and have a minor impact on the genetic variation.
Recombination, or crossovers, is where the majority of new genetic variations arise. These are modeled by choosing a point of recombination, and essentially swapping bit strings at that point. A simple GA uses a single point crossover, where only one crossover occurs per chromosome. However, a GA can easily be adapted to have multiple crossover points [8, 9].
On average, via the mutation and crossover operators, the fitness level of F2 should be higher than F1. By carrying some of the fewer fit individuals, we allow for a larger gene pool and therefore allow for more possibilities for genetic combinations, but the gene pool should be predominated by favorable genes [3].
Termination
This three-step pattern of selection, variation, and propagation is repeated until a certain threshold is reached. This threshold can be a variety of factors, ranging anywhere from a preset number of generations to a certain average fitness level. Typically, termination occurs when population convergence occurs, meaning that the offspring generation is not significantly better than the generation before it [10].
Modifications to GA’s
As one can see, this is a rather simplistic approach to evolution. There are several biological factors that remain unaddressed in a three-step process. Consequently, there are many ways to expand a GA to more closely resemble the complexity of natural evolution. The following section shall briefly overview a few of the different techniques used in tandem with a GA to add further resolution to this prediction process.
Speciation
A GA can be combined with a speciation heuristic that discourages crossover pairs between two individuals that are very similar, allowing for more diverse offspring generations [11, 12]. Without this speciation parameter, early convergence is a likely possibility [12]. Early convergence describes the event that the ideal individual, i.e. the individual with the optimized fitness score, is reached in too few generations.
Elitism
Elitism is a commonly used approach to ensure that the fitness level will never decrease from one generation to the next [13]. Elitism describes the decision to carry on the highest-rated individuals from one generation to the next with no change [13, 14]. Elitism also ensures that genetic information is not lost. Since each offspring must be ‘equal or better’ than the generation before it, it is guaranteed that the parental genotypes will carry through generations, changing at a much slower rate than a pure GA would model [15].
Adaptive Genetic Algorithms
Adaptive Genetic Algorithms (AGA’s) are a burgeoning subfield of GA development. An AGA will continuously modify the mutation and crossover operators in order to maintain population diversity, while also keeping the convergence rate consistent [16]. This is computationally expensive but often produces more realistic results, especially when calculating the time it would take to reach the optimal fitness. The Mahmoodabadi et al team compared AGA’s to 3 other optimization functions and found that “AGA offers the highest accuracy and the best performance on most unimodal and multimodal test functions” [17].
Interactive Genetic Algorithms
As previously stated, the fitness function is critical to creating a GA. However, there arise several instances where a fitness function cannot be accurately defined. This is particularly true for species that have elaborate mating rituals, as that is a form of selection that would be computationally expensive to recreate. In these situations, one can use an interactive genetic algorithm (IGA). IGA’s operate in a similar fashion to GA’s, but they require user input at the fitness calculation point.
While this method does provide some way of modeling a population without having a predefined fitness function, it has glaring drawbacks. Primarily, this process is not feasible for large populations, as it puts the burden of calculating the fitness on the user, and it also leaves room for subjective bias from the user. However, this subjective component been viewed as an advantage in several fields, particularly the fashion industry [18]. Designers have been investigating IGA’s as a method to generate new styles, as the algorithm depends on user validation of what is considered to be a good design versus a bad one [18].
Applications
Genetic algorithms have a far-reaching effect on computational efforts in every field, especially in biology. As the name suggests, genetic algorithms have a huge impact on evolutionary biology, as they can assist with phylogeny construction for unrooted trees [19]. Oftentimes, evolutionary data sets are incomplete. This can result in billions of potential unrooted phylogenetic trees. As the Hill et al team describes, “for only 13 taxa, there are more than 13 billion possible unrooted phylogenetic trees,” [19].
Testing each of these combinations and determining the best fit is yet another example of an optimization problem– one which a GA can easily crack. Hill et al applied a GA to a set of amino acid sequences and built a phylogenetic tree comparing protein similarities [19]. They found that a program called Phanto, “infers the phylogeny of 92 taxa with 533 amino acids, including gaps in a little over half an hour using a regular desktop PC” [19].
Similarly, the Wong et al team tackled the infamous protein folding prediction problem using GA’s [20]. They used the HP Lattice model to simplify a protein structure and used the iterative nature of a GA to find a configuration that minimized the energy required to fold a protein into that shape. The HP Lattice model stands for Hydrophobic Polar Lattice and seeks to model the hydrophobicity interactions that occur between different amino acid residues in the secondary structure of a protein [20]. They found that a GA performed better than some of the newer protein folding predictive programs available today [20].
GA’s are an incredible tool for cancer research as well. The Mitra et al team used a GA to study bladder cancer [21]. They conducted quantitative PCR on tissue samples from 65 patients and identified 70 genes of interest. Of these 70 genes, three genes in particular, were identified in a novel pathway. They discovered that ICAM1 was up-regulated relative to MAP2K6, while MAP2K6 was up-regulated relative to KDR. This pathway was considered to be novel because individually, all three genes displayed no signs of significant changes in regulation. By applying a GA, the Mitra team was able to identify this pattern between all three genes. Uncoincidentally, “ICAM1 and MAP2K6 are both in the ICAM1 pathway, which has been reported as being associated with cancer progression, while KDR has been reported as being associated with the vascularization supporting tumors” [21, 22, 23].
Another groundbreaking discovery was made by applying GA’s to p53 research. P53 is an essential tumor suppressor [24]. Most cancerous tumors can be attributed, in part, to a mutation in the p53 gene, making it an excellent candidate for oncology research. The Millet et al team investigated a possible p53 gene signature for breast cancer, hoping to find an accurate prediction system for the severity of breast cancer [25]. They analyzed 251 transcriptomes from patient data and found a 32 gene signature that could serve as a predictor for breast cancer severity [23, 25]. They also found that “the p53 signature could significantly distinguish patients having more or less benefit from specific systemic adjuvant therapies and locoregional radiotherapy,” [25].
GA’s have also had a huge impact on immunology, vaccine development in particular. Licheng Jiao and Lei Wang developed a new type of GA called the Immunity Genetic Algorithm [26]. This system mimics a typical GA but adds a two-step ‘immunological’ parameter (Figure 3). Much like a GA, the fitness function is applied to a population, which then triggers mutation and crossover. However, after these steps, the program mimics ‘vaccination’ and ‘immune selection. These two steps are referred to as the “Immune Operator” [26]. They are designed to raise a genetic advantage in individuals who respond well to the vaccine and confer a disadvantage to those with a ‘weaker’ immune response. In essence, the vaccination step acts as a secondary mutation, as it is acting as an external change factor in each individual’s fitness. Similarly, the ‘immune selection’ step acts as a secondary fitness function, as it measures the immune response post-vaccine. If evolution is continuing as it should, each generation should have an improved immune response to the vaccine until convergence is reached.
Conclusion
GA’s have a broad reach in all fields of research, from fashion to immunology. Their success is due to three critical components underlying their programming: they are simple to write, easy to customize, and efficient to run. This flexibility and independence are what will allow programs like GA’s to become commonplace in research, across all disciplines. In particular, as biology research continues to merge with computer science and advanced modeling techniques, applications like GA’s have the potential to solve problems and raise questions about our world that we may have never imagined before.
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Human Cryopreservation: An Opportunity for Rejuvenation
By Barry Nguyen, Biochemistry & Molecular Biology ‘23
Author’s Note: I became interested in ways to bypass built-in lifespans after taking HDE 117, a longevity class with Dr. James Carey. During the course of the class, I was exposed to many different ways to extend the human lifespan. However, I was most interested in cryogenics and its prospects of human rejuvenation, prompting me to explore the possibilities of human cryopreservation.
Summary
This paper is focused on exploring the prospects of human cryopreservation. The first section discusses cryogenics and its relevance in the discussion of human cryopreservation. The following section utilizes empirical modeling to support the relationship between temperature and reaction rates. Next, the paper discusses the cryopreservation procedure itself and explores how the definition of death can be reimagined. We then transition to discussing cryopreservation’s possibility for rejuvenation. Specifically, we redefine the definition of aging itself and discuss aging phenomena on the molecular scale and use both of these as a basis for the discussion of immortality. The succeeding section is concerned with the limitations of human cryopreservation. Finally, the paper concludes with a brief discussion of the possible future of cryogenic technology.
Cryogenics
Cryogenics is a field of study focused on material behaviors at very low temperatures, ranging from -150°C to -273°C. At these extremely low temperatures, chemical properties are altered, and molecular interactions are halted [1]. By halted, it is not correct to say that all molecular interactions have been stopped. Rather, the molecular interactions have come as close to theoretically possible to ceasing and are at the lowest possible energy state. At these temperatures, chemical properties are also altered and unique phenomena emerge, allowing for extensive applications, most notably human cryopreservation. Because heat is related to the motion of particles, at these temperatures the biochemical activities within living systems are effectively reduced [9]. The prospects for preserving an individual at extremely cold temperatures have been increasing throughout the years as research within the field continues to develop. As of now, human cryopreservation seems more of a speculation than reality. Freezing an individual is one thing, but there is no guarantee that the individual will wake up from such an extensive period of suspension. Although extremely low temperatures serve as an appropriate basis for human cryopreservation, many more factors must be considered to avoid consequences that may occur during the procedure and after revival.
Empirical Modeling
The rates of biochemical processes at extremely low temperatures can be modeled mathematically [4]. The Arrhenius equation, proposed by Arrhenius in 1889, establishes a relationship between temperature and reaction rates. In figure 1, K is the reaction rate, Ea is the activation energy, A is the frequency factor (related to the orientations of molecules necessary to produce a favorable reaction), R is the universal gas constant, and T is the temperature. Manipulating the equation, we produce a form that directly shows the relationship between the reaction rate and temperature, as depicted in Figure 2. We will use the enzyme lactate dehydrogenase to illustrate the relationship between K and T [4]. With its activation energy defined as 54, 810 J/mol, we can explore the enzyme’s reaction rate at a 10°C difference. With T1 and T2 at 40°C and 30°C respectively, we get a reaction rate ratio of 2.004. This tells us that a 10°C difference is enough to cut the reaction rate of the enzyme exactly in half.
The relationship between reaction rates and temperature, as expressed by the Arrhenius equation, lends weight to the viability of cryopreservation. If a 10°C difference is enough to cut a reaction rate in half, imagine how much the reaction rate would be reduced within cryonically preserved individuals at extremely low temperatures. Furthermore, the biochemical processes that are occurring in the body at these levels are paused—not in the sense of being physically stopped, but rather the time needed for the processes to go to completion is relatively infinite.
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| Figure 1. The Arrhenius equation | Figure 2. Manipulation of the Arrhenius equation to compare reaction rates at two different temperatures |
Human Cryopreservation
By understanding that at these extremely low temperatures, biochemical reaction rates are suppressed, the practice of cryogenically preserving a whole individual became a reality [14]. For this process to begin, the individual must be induced in the death state. Once an individual enters the initial stages of death, the human body initiates its decomposition phase. The body’s cell walls begin to break down and in turn, release digestive enzymes that process the tissues in the body [11]. Because the body begins to break down at such a rapid pace, it is imperative that the patient, once induced in the death stage, be worked on immediately.
The process of chilling the human body to extremely low temperatures is a delicate and slow process and is very important in the initial steps of the cryopreservation procedure. Once the patient arrives in the death state, the circulation and respiration of the cryonic subject is restored and they are ready to be cooled [4]. First, the subject’s blood is replaced with 10% cryoprotectants to prevent ice formation. A small percentage of cryoprotectants are added initially to avoid an elevated osmotic shrinking response. Once the intracellular and extracellular cryoprotectant volume reaches equilibrium, the cells are ready for cooling which is done at a very slow pace (1°C/min) [5].
The cryoprotectant used typically consists of nutritional salts, buffers, osmogens, and apoptosis inhibitors, ingredients necessary in the maintenance of isotonic concentrations of the cell [5]. In doing so, cells within the human body can avoid swelling and shrinking. Additionally, another key formulation of cryoprotectant mixture is non-penetrating cryoprotectants which are typically large molecular polymers. These play a large part in the inhibition of ice growth and prevention of injury due to being subjected to the extreme cold [5].
To understand the prospects of human cryopreservation, it is helpful to redirect ourselves back to the definition of death. In 1988, the scientific community reviewed and redefined the definition of death from being in cardio-respiratory arrest to brain death [8]. In cryonically preserved patients, the extremely cold temperatures are thought to preserve the neural structures, which store long-term memory and the identity of the person. In this way, utilizing extremely low temperatures to preserve neural structures and prevent them from being compromised is a prospect worth noting. Individuals who are cryonically preserved should not be viewed as being dead or alive, but rather be viewed as being temporarily suspended in time [8]. The normal cycles of biological processes such as growth and decay are paused, providing an opportunity for resuscitation and reanimation in the future [10]. To give a new perspective, cryopreservation can be viewed similarly to frozen embryos: just as embryos preserved in extremely cold temperatures gain life once implanted in a uterus, the cryopreserved patient may reenter the living state through the process of human reanimation.
Prospects for Immortality
The process of human cryopreservation aims to allow individuals to escape imminent death by first being induced into a transient death state [8]. Essentially, individuals are given the opportunity to bypass human mortality. Dr. James Hiram Bedford, a former psychology professor at UC Berkeley had his life threatened by renal cancer. He decided to undergo the cryopreservation process and became the first human to be cryonically preserved in 1967 [13]. By agreeing to enter this process, he hoped that, in the future, technology would be advanced enough to revive him and cure his illness. Ever since interests in cryopreservation have increased substantially, and as of 2014, about 250 corpses have been cryogenically preserved in the US [13].
Shifting Views on Aging
Aging is a degradative process that entails a whole array of pathologies. If we were to view aging as a disease itself that can be treated, cryopreservation opens a wide range of possibilities. Specifically, the process of cryopreservation allows an individual to avoid the effects of aging pathologies by having the opportunity to be treated once technology has advanced enough. This provides hope to bypass the mechanically built-in lifespans of humans, and essentially, provides prospects for immortality.
On a larger scale, as we age, the probability of dying increases significantly [7]. To put it simply, as we age, there are more health factors in place to compete for our lives and the chance of survival through older ages decreases. In such cases, aging can be correlated with functional decline. Similarly, on the molecular scale, aging can be seen as a direct consequence of telomere shortening [6]. Telomeres are nucleoprotein structures that exist at the ends of chromosomes and are essential to the integrity of our DNA. During the process of DNA replication, telomeres protect the ends of chromosomes and prevent loss of genetic information [16]. However, as we age, and as our body continues to undergo DNA replication, the telomeres shorten leading to the joining of ends of various chromosomes, pathological cell division, genomic instability and apoptosis.
In short, the health consequences that come with aging are inevitable but human cryopreservation can be seen to offset these inevitable aging phenomena. The process allows an individual who is suffering from a presently incurable disease to be temporarily frozen in time. In this way, they may be revived when society is advanced enough to deal with the disease successfully. In essence, the human cryopreservation process can be seen to bypass inevitable health consequences, providing rejuvenating possibilities for any individual.
Technological Limitations
Although successfully preserving an individual through extreme temperatures is certainly an exciting prospect, little evidence exists to indicate that successful preservation and remanimation is possible [15]. At present, there are many challenges that need to be overcome to even support the viability of such an extensive process. According to Professor Armitage, the director of tissue banking at the University of Bristol, preserving the whole human body is an entirely new challenge [15]. Society is not even at the stage of cryopreserving organs. Organs, alone, are very complex, containing different types of cells and blood vessels that all need to be preserved. Similarly, Barry Fuller, another professor at the University of College London, has stated that before exploring the prospects of human cryopreservation, society must be able to demonstrate that human organs can be cryopreserved for transplantation [15]. Hence, as of current, there is close to zero evidence that a whole human body can survive cryopreservation.
In the previous section, we discussed the arrhenius equation which derived the relationship between temperature and metabolic rates. However, the equation itself does not explore the consequences of raising the temperature of the human body during reanimation. While thawing, the frozen tissues and cells can experience physical disruptions which can damage them [3]. To a greater extent, an individual’s epigenetic markers can even be affected, causing epigenetic reprogramming, which can change the expression of certain genes. However, the biggest hurdle is the successful preservation of the brain. The human brain is arguably one of the most important organs in the body, and cryopreservation must be successful in preserving the integrity of the neural structures. Prospects of successfully cryopreserving whole human brains are slim due to minimal research. Moreover, experiments with frozen whole animals’ brains have not been reported since the 1970s [3]. Obviously, research on this matter is severely limited.
Discussion
Despite the overwhelming uncertainties surrounding human cryopreservation and society’s current limits, the prospects of being able to defy death or possibly avoiding it in the future are becoming a topic of increasing interest. When an individual is brought to the brink of death, the uncertainties around the cryopreservation procedure, specifically its unproven track record of success, seem inconsequential in the long run. If society were to overlook the field of preservation based purely on unsubstantiated results and the unlikelihood of success, advancements would never occur. All in all, the increase in technological advancements and research within cryogenics is making the prospects of reviving a frozen individual in the future ever so likely.
References:
- Britannica, T. Editors of Encyclopaedia. “Cryogenics.” Encyclopedia Britannica, May 26, 2017. https://www.britannica.com/science/cryogenics.
- “What Is Cryogenics? “Gaslab.com. Accessed May 2, 2021. https://gaslab.com/blogs/articles/what-is-cryogenics.
- Stolzing, Alexandra . “Will We Ever Be Able to Bring Cryogenically Frozen Corpses Back to Life? A Cryobiologist Explains.” The Conversation, March 26, 2019. https://theconversation.com/will-we-ever-be-able-to-bring-cryogenically-frozen-corpses-back-to-life-a-cryobiologist-explains-69500.
- Best, Benjamin P. “Scientific Justification of Cryonics Practice.” Rejuvenation Research 11, no. 2 (2008): 493–503. https://doi.org/10.1089/rej.2008.0661.
- Bhattacharya, Sankha. “Cryoprotectants and Their Usage in Cryopreservation Process.” Cryopreservation Biotechnology in Biomedical and Biological Sciences, 2018. https://doi.org/10.5772/intechopen.80477.
- Blasco, M. A. “Telomere length, Stem Cells and Aging.” Nature Chemical Biology, 3, no.10 (September 2007): 640–649. doi:10.1038/nchembio.2007.38
- Carey, J.R. 2020, June 13. Limits of morbidity compression. Longevity (HDE/ENT 117) lecture notes, UC Davis.
- Cohen, C. “Bioethicists Must Rethink the Concept of Death: the Idea of Brain Death Is Not Appropriate for Cryopreservation.” Clinics 67, no. 2 (2012): 93–94. https://doi.org/10.6061/clinics/2012(02)01.
- Jang, Tae Hoon, Sung Choel Park, Ji Hyun Yang, Jung Yoon Kim, Jae Hong Seok, Ui Seo Park, Chang Won Choi, Sung Ryul Lee, and Jin Han. “Cryopreservation and Its Clinical Applications.” Integrative Medicine Research 6, no. 1 (2017): 12–18. https://doi.org/10.1016/j.imr.2016.12.001.
- Lemke, Thomas.“Beyond Life and Death. Investigating Cryopreservation Practices in Contemporary Societies,” Soziologie, 48. No. 4 (April 2019):450-466.
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- Luke Davis. “The Difference between Cryonics and Cryogenics,” August 10, 2020. https://logicface.co.uk/difference-between-cryonics-and-cryogenics/.
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- Purtill, Corinne. “Fifty Years Frozen: The World’s First Cryonically Preserved Human’s Disturbing Journey to Immortality.” Quartz. Quartz. Accessed May 2, 2021. https://qz.com/883524/fifty-years-frozen-the-worlds-first-cryonically-preserved-humans-disturbing-journey-to-immortality/.
- Roxby, Philippa. “What Does Cryopreservation Do to Human Bodies?” BBC News. BBC, November 18, 2016. https://www.bbc.com/news/health-38019392.
- Trybek, Tomasz, Artur Kowalik, Stanisław Góźdź, and Aldona Kowalska. “Telomeres and Telomerase in Oncogenesis (Review).” Oncology Letters 20, no. 2 (2020): 1015–27. https://doi.org/10.3892/ol.2020.11659.
Epigenetics as a Tool for Personalized and Targeted Care
By Parmida Pajouhesh, Neurobiology, Physiology & Behavior ‘23
Author’s Note: For as long as I can remember I wanted to attend medical school and become a pediatrician. More recently, I have been exposed to the study of epigenetics, which has unveiled the importance of prioritizing prevention of disease and furthered my interest in the field of medicine. In hopes of practicing internal medicine in the future, I wanted to investigate how providing care and support to the patient on an individualized level is crucial for effective treatment in the long term.
Put simply, epigenetics means “on top of” genetics, derived from the Greek prefix “epi.” Epigenetics describes the relationship between one’s gene activity and their environment, whereby the activity of the genes is altered but the DNA sequence is not directly modified. Genes can be turned “on” or “off” and changes to our epigenome are fortunately reversible. Researchers studying this phenomenon have been trying to answer the following questions: how does our knowledge of epigenetics help to further advance the study of illness and disorders, such as cancer and schizophrenia? How can understanding this phenomenon help scientists, doctors and researchers provide personalized care for patients?
Epigenetic changes, which impact our phenotype without directly affecting our genotype, have been previously tied to aging, our environment and even lifestyle [1]. Prenatal and early postnatal environmental factors have influenced an offspring’s risk and vulnerability to developing a health condition [2]. For instance, in the 1940s, children who were born during a period of increasing famine had exceedingly greater rates of coronary heart disease after maternal exposure to famine, as opposed to those not exposed. This was linked to a decrease in DNA methylation—an epigenetic alteration—of the gene responsible for insulin growth [2].
These epigenetic modifications continue to take place throughout an individual’s lifespan. Exposure to pollution can alter methyl tags on DNA and make an individual even more susceptible to neurodegenerative diseases [3]. The foods we consume can have an impact on our epigenome; one study has shown that a high-fat, low-carb diet could open up chromatin and thus improve mental ability [1].
A closer look into our epigenome
Common and widely studied epigenetic alterations include histone modifications and DNA methylation. These mechanisms regulate the expression of genes as well as “cellular and biological functions related to homeostasis, allostasis and disease” [4]. DNA methylation adds a methyl group (-CH3) to cytosine at a promoter region containing repetitive sequences of CpG (cytosine–phosphate–guanine) dinucleotides. Proteins then bind to the methylated CpG islands, which correlates with transcriptional repression and affects gene expression [4]. This form of methylation has been previously linked to cancer [5].
As for the modification of histones, ubiquitin—a molecule which is attached to a protein destined for degradation—has been linked to neurological disorders including Parkinson’s disease and Angelman syndrome [5]. Likewise, histone methylation has been linked to several biological processes: DNA repair, stress responses, development, differentiation and aging [4]. If any one of these processes is altered, whereby histone methylation is either activated or inhibited, this can result in the development and progression of disease. For example, H3K4me2, a post-translational dimethylation at the lysine 4 residue of the histone H3 protein, located at the promoter of active genes, is downregulated in cases of lung, kidney, prostate and pancreatic cancers [6]. Histone modification contributes to the cell cycle, growth, DNA replication and other processes. Therefore, abnormal histone modifications can lead to the development and progression of tumors [6].
Personalized medicine and targeted care
Inevitably, individuals are prone to changes in their epigenome, which makes providing targeted care an even more challenging task. These changes occur not only between individuals but also within a single individual over time. Therefore, genomic approaches that include identifying specific variations in DNA and RNA sequences can help to bridge the gap between epigenetics and personalized care. Health care professionals have shifted their attention towards diagnostic tests that use genomic data to more accurately assess the extent of a patient’s risk for disease or illness, to determine appropriate dosage amounts and to make conclusions about the benefits of a specific drug or treatment. According to a study by Mahmood Rasool that highlights epigenetics as a contributing factor to personalized and individualized care, “various factors such as nutrition, age, body weight, sex, genetic behavior, infections, co-medications and organ function are important considerations that are unavoidable during the course of treatment for a disease” [4].
Combatting epigenetic changes. What’s next?
Changes due to our environment are unpredictable; therefore, we must take precautions as early as possible. Being wary of how our lifestyle can impact the activation of our genes is crucial to our health and development. We must take preventative measures early in our life and determine which lifestyle changes will benefit us in the long term. Exposure to hazards in our environment is not fully noticeable until years and sometimes decades later. If we place emphasis on the prevention of disease early on, we are much less likely to encounter abrupt and irreversible effects to our well-being. While epigenetic biomarkers are being evaluated for use in environmental risk assessment, more immediate lifestyle changes include reducing exposure to harmful air pollutants, implementing specific dietary changes and altering medication use that will provide long-term benefits, as opposed to only short-term relief [4]. Our diet can result in profound changes in our epigenome, leading to human disease. For instance, lacking essential amino acids in your diet can result in colon cancer, which “impairs biosynthesis of the active precursor for DNA methylation.” Similarly, exposure to nicotine and other toxins can cause epigenetic changes in smokers, “affecting the genes involved in normal pulmonary function.” Exercise can also have important effects on the skeletal-muscle epigenome [7].
With this being said, we must recognize the importance of integrative medicine in primary care. Physicians, and even specialists, consider the patient as a whole person and are cognizant of their lifestyle, diet, genetic background and even mental health. This holistic approach to medical care provides patients with a greater sense of what they need to accomplish to keep their body and mind healthy. This not only strengthens the connection between practitioner and patient, but it demonstrates the importance of taking preventative measures prior to development of the illness. Understanding epigenetics can increase our awareness of how physical space alters our well-being and reinforce that providing holistic and preventative care reduces the negative impacts of epigenetic changes.
References (online)
- Baccarelli, Andrea, and Valentina Bollati. “Epigenetics and environmental chemicals.” Current opinion in pediatrics vol. 21,2 (2009): 243-51. doi:10.1097/mop.0b013e32832925cc
- Heerboth, Sarah et al. “Use of epigenetic drugs in disease: an overview.” Genetics & epigenetics vol. 6 9-19. 27 May. 2014, doi:10.4137/GEG.S12270
- Moosavi, Azam, and Ali Motevalizadeh Ardekani. “Role of Epigenetics in Biology and Human Diseases.” Iranian biomedical journal vol. 20,5 (2016): 246-58. doi:10.22045/ibj.2016.01
- Rasool, Mahmood et al. “The role of epigenetics in personalized medicine: challenges and opportunities.” BMC medical genomics vol. 8 Suppl 1,Suppl 1 (2015): S5. doi:10.1186/1755-8794-8-S1-S5
- Tollefsbol, Trygve O. “Dietary epigenetics in cancer and aging.” Cancer treatment and research vol. 159 (2014): 257-67. doi:10.1007/978-3-642-38007-5_15
- Li, Simin, et al. “Association between H3K4 Methylation and Cancer Prognosis: A Meta-Analysis.” Thoracic Cancer, vol. 9, no. 7, 2018, pp. 794–99. Crossref, doi:10.1111/1759-7714.12647.
- Feinberg, Andrew P. “The Key Role of Epigenetics in Human Disease Prevention and Mitigation.” New England Journal of Medicine, edited by Dan L. Longo, vol. 378, no. 14, 2018, pp. 1323–34. Crossref, doi:10.1056/nejmra1402513.
References (print)
- Baccarelli and Bollati. Current opinion in pediatrics vol. 21,2 (2009): 243-51.
- Heerboth, et al. Genetics & epigenetics vol. 6 9-19. 27 May. 2014.
- Moosavi and Ardekani. Iranian biomedical journal vol. 20,5 (2016): 246-58.
- Rasool, et al. BMC medical genomics vol. 8 Suppl 1,Suppl 1 (2015): S5.
- Tollefsbol. Cancer treatment and research vol. 159 (2014): 257-67.
- Li, et al. Thoracic Cancer, vol. 9, no. 7, 2018, pp. 794–99.
- Feinberg. New England Journal of Medicine, edited by Dan L. Longo, vol. 378, no. 14, 2018, pp. 1323–34.
Strimvelis: An Application of Personalized Medicine
By Aditi Goyal, Genetics & Genomics, Statistics, ‘22
Author’s Note: I heard about this therapy during a freshman seminar, and I presented on this during that class. This article is an adaptation of that presentation.
ADA-SCID is a rare, autosomal recessive disease that cripples one’s immune system. ADA SCID stands for Severe Combined Immunodeficiency due to Adenosine Deaminase Deficiency, which occurs due to a mutation in the ADA gene [1]. This gene normally assists in the production and regulation of lymphocytes, also known as white blood cells [1]. Specifically, ADA (Adenosine Deaminase) breaks down deoxyadenosine, which is toxic to lymphocytes [2]. In the absence of a working ADA gene, this deoxyadenosine collects in the body and continues to degrade lymphocytes. Eventually, the lack of functioning lymphocytes leads to severe combined immunodeficiency (SCID) [2].
ADA-SCID is typically screened for at birth and has a variety of treatment options. The most common treatment is a bone marrow transplant from a sibling. In this process, stem cells are taken from someone with a matching blood type, and transplanted into the patient, with the hope that these cells will proliferate and produce healthy lymphocytes [3]. While this approach is effective approximately 70% of the time, the real challenge is in matching a patient to a donor. Because the patient’s immune system is already so impacted, there is a high possibility of rejection of the transplant. Additionally, for patients who do not have a sibling or someone in the family who is able to donate, finding a match can be incredibly difficult.
For patients unable to have a transplant, enzyme therapy is also a possible form of treatment [3]. Enzyme Replacement Therapy, ERT, is simply providing the patient with a working copy of an enzyme, ADA in this case [4]. The drawback to this form of treatment is that it requires a patient to be dependent on a hospital for their entire lives. They cannot travel too far away from a hospital for too long, because if they miss a delivery of the enzyme, there can be drastic consequences [5]. Additionally, ERT can lose effectiveness over the years [4].
The third, and still experimental, treatment option is a gene therapy known as Strimvelis [6]. Strimvelis is one of the first gene therapy products to be used anywhere in the world. While it has yet to be approved by the FDA in the United States, it marks a milestone in the development of personalized medicine.
Strimvelis treatment has three steps, starting with harvesting hematopoietic stem cells (HCS’s) from the patient. These cells carry the mutated ADA gene and are ineffective at supporting catalyzing deoxyadenosine. Once extracted, the corrected ADA gene is delivered to the HCSs in an ex vivo environment using a gammaretrovirus [7]. Once the cells have been transformed, they are delivered back to the patient using an IV drip, and take hold in the body, subsequent to a dose of Busulfan or Melphalan [8]. These two chemotherapy drugs are intended to kill any remaining damaged HCS’s in the body, allowing for the corrected cells to grow without interference. Once injected, the corrected cells will continue to proliferate, producing a healthy amount of ADA. The reason this therapy works well is that the patient’s own HCS’s are used, so there is little to no risk of rejection by the body’s immune system. Another key advantage to using Strimvelis is that it is a single treatment. Once the corrected HCS’s are delivered to the body, the patient is considered “cured” and is no longer reliant on any medical procedures to maintain their immunity.
The results of Strimvelis trials have been incredibly promising. A clinical trial conducted by the European Medicines Agency (EMA) found Strimvelis to have a 100% success rate, leading to its approval by the European Commission about one month later [9]. However, there have been rare cases of Strimvelis leading to patients developing T-cell leukemia [10]. These cases have led to the parent company of Strimvelis, Orchard Therapeutics, halting all administration of Strimvelis until an investigation on the possibly cancerous effects of Strimvelis has been completed [11].
Another primary drawback of Strimvelis is its cost. Strimvelis costs 594,000 euros per patient, which is equivalent to approximately 650,000 dollars [12]. While Strimvelis is not the most expensive gene therapy on the market, the cost is still incredibly restrictive, as the average middle-class family would not be able to afford this treatment.
The reason the cost for this treatment is so high is that ADA-SCID is considered an orphan disease. Orphan diseases are conditions that affect under 200,000 people worldwide [13], which means that from the perspective of a pharmaceutical company, it is not cost-effective to develop a treatment. ADA-SCID only affects around 350 people worldwide [2]. Therefore, the cost per patient is high, since there are not that many people affected by this disorder and because the therapy cannot be mass-produced.
Strimvelis is not perfect, by any means. There are still thousands of unknowns surrounding gene editing, and the side effects are dramatic. Even with Strimvelis on the market, it is not the number one treatment option for most ADA-SCID patients. Nevertheless, it is a step forward. In a world where we learn more about our genetics every day, Strimvelis is a milestone in the development of personalized medicine.
References
- Adenosine deaminase DEFICIENCY: MedlinePlus Genetics. (2020, August 18). Retrieved March 10, 2021, from https://medlineplus.gov/genetics/condition/adenosine-deaminase-deficiency/
- Hershfield M. Adenosine Deaminase Deficiency. 2006 Oct 3 [Updated 2017 Mar 16]. In: Adam MP, Ardinger HH, Pagon RA, et al., editors. GeneReviews® [Internet]. Seattle (WA): University of Washington, Seattle; 1993-2021. Available from: https://www.ncbi.nlm.nih.gov/books/NBK1483/
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- Adenosine deaminase deficiency: Treatment and prognosis. (n.d.). Retrieved March 10, 2021, from https://www.uptodate.com/contents/adenosine-deaminase-deficiency-treatment-and-prognosis#H2288540670
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- Aiuti, Alessandro et al. “Gene therapy for ADA-SCID, the first marketing approval of an ex vivo gene therapy in Europe: paving the road for the next generation of advanced therapy medicinal products.” EMBO molecular medicine vol. 9,6 (2017): 737-740. doi:10.15252/emmm.201707573
- Candotti F (April 2014). “Gene transfer into hematopoietic stem cells as treatment for primary immunodeficiency diseases”. International Journal of Hematology. 99 (4): 383–92. doi:10.1007/s12185-014-1524-z. PMID 24488786. S2CID 8356487.
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- Orchard statement on Strimvelis®, a Gammaretroviral Vector-Based gene therapy For ADA-SCID. (n.d.). Retrieved March 10, 2021, from https://ir.orchard-tx.com/index.php/news-releases/news-release-details/orchard-statement-strimvelisr-gammaretroviral-vector-based-gene
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Combating Malaria: Genetically Modified Mosquitoes Projected to Prevail Over Traditional Methods
By Marian Warner, Biotechnology ‘21
Author’s Note: I chose the subject of gene drives for UWP 104E (writing in science class) because I found it personally interesting and wanted to learn more about its controversy. The more I did research on the subject throughout the quarter the more I realized how much is unknown by the general public. The mechanism is a bit complex, so there are not many sources that attempt to explain it to a generalized audience. I hope this paper does a good job of helping those unfamiliar with gene drives become interested and gain a grasp on the scientific backing behind it. I also hope it aids them in forming an informed opinion on the subject or gives them an idea of what further questions they may want to ask.
Malaria, a mosquito-borne disease, has been a stubborn, unrelenting issue despite the established traditional methods used to combat it. Because the female mosquito in the Anopheles genus is the intermediary host transmitting the infectious agent, Plasmodium parasites, from an infected to a non-infected person, current preventative efforts mainly target the mosquitoes by dispersing insecticide-treated bed nets, insecticide for indoor use, and occasional environmental control by destroying larval habitats [1]. For infected individuals, there are treatment options such as antimalarial drugs that can suppress infections, but these target the Plasmodium parasites instead. Although these efforts have helped slow the spread of malaria, it still affected 229 million people and killed an estimated 409,000 in 2019 [2]. Fortunately, scientists such as Andrea Beaghton from Imperial College London have developed a promising strategy using genetically modified mosquitoes to rapidly spread the male-biased sex determination genes in order to exponentially decrease the amount of breeding in a population, which has proven to be effective in completely wiping out populations of malaria-carrying mosquitos in as little as 30 generations in a laboratory setting [3]. The complete collapse of malaria-carrying mosquito populations due to using this strategy in the wild would efficiently lead to the eradication of malaria.
The Role of Selfish Genetic Elements
The strategy works by utilizing a gene drive, a technique that spreads a gene throughout a population at an abnormally fast rate. Usually, any given copy of a gene has a 50 percent chance of being passed down to one’s offspring. This occurs because each diploid organism, such as a human or an insect, carries two different alleles, or variations, of a given gene. One allele is inherited from each of that organism’s parents.
When it comes to a gene drive, however, the allele in question is almost always inherited. Scientists use a naturally occurring selfish genetic element to propel gene drives. Selfish genetic elements, or selfish genes, are alleles that convert the other inherited allele into a copy of itself. In this scenario, the gene no longer has a 50 percent chance of being passed down, but instead nearly a 100 percent chance [Figure 1].
Figure 1
The Mechanism Behind the Selfish Genetic Element
To convert the other allele inherited into a replica of the selfish gene, the selfish gene exploits a natural process the genome uses to repair itself, known as homology driven repair, allowing harmful genes to bypass the rules of natural selection. The mechanism works by encoding a pair of biological scissors, an endonuclease, that cut DNA at the position of the second allele. Once the endonuclease slices open a segment of DNA, the DNA is more unstable and gets chewed back, resulting in the allele being lost. The cell registers the damage and repairs the region by copying and pasting the DNA sequence of the selfish genetic element into the spot where the naturally occurring allele was previously, resulting in both alleles being that of the selfish gene [4]. The only reason why the mechanism doesn’t work 100 percent of the time in practice is because of occasional issues the endonuclease has with recognizing the target allele [5].
The Anopheles Gambiae Gene Drive
With new powerful genome editing technologies, scientists can choose any gene to be a selfish genetic element. Many have focused on trying to identify and genetically modify an allele that could wipe out the Anopheles gambiae population, the most common mosquito species involved with the spread of malaria. However, this technique is not as simple as it seems at first glance. Trying to spread a deadly allele would not work, because the mosquitoes need to be alive and reproducing to spread it. Spreading an allele that makes mosquitoes weaker would not work either. For example, trying to eradicate their ability to fly could help suppress a population briefly, but the chances of mosquitoes forming resistance to the mechanism of this gene drive due to natural selection is high.
Instead, scientists have decided to target genes that are involved with sex determination, to make the population spread genes that reduce female survivorship. Under this gene drive, females are born with intersex mouth parts that do not allow them to feed, and therefore die relatively quickly. Targeting females is beneficial because they are the only ones capable of transmitting malaria. Additionally, as the population becomes increasingly more male-dominated, chances of reproduction become slimmer, and fewer mosquitos are born. The specific sex-determining genes for this are unlikely to invoke evolved resistance, because any changes to a pathway as specific as the sex determination pathway will most likely result in detrimental effects on the mosquito, preventing the spread of the mutations by natural selection [3]. As the gene drive rapidly spreads throughout the population, the number of malaria cases would rapidly decrease. Compared to traditional methods, the strategy would be incredibly efficient and a low cost, but there is a potential for unknown side effects, leading many to believe that using traditional methods is the best option for now.
Potential Consequences of Traditional Methods and Gene Drives
Despite the efficiency gene drives are thought to have, sceptics often argue that the current methods used to control malaria have a much lower risk of adverse side effects. Experts have approved pyrroles and pyrethroids as insecticides for mosquito bed nets because of their relatively low consequences on human health. However, mosquitos are now evolving resistance to pyrethroids. To reduce the odds of mosquitoes becoming resistant, many bed nets include multiple insecticides. However, there is not yet any evidence that these nets work in regions that already have high levels of pyrethroid resistance [6]. Similarly, Plasmodium parasites have also been found to harbor drug resistance to antimalarial drugs. Partial resistance to the drug artemisinin has already been detected in over five percent of some Plasmodium populations, and several other types of drug resistance have been detected as well. As of now, insecticides and antimalarial drugs can continue to be used effectively, but resistance to them must be closely monitored by collecting data on malarial drug treatment cases and looking for molecular markers of resistance in natural populations to prevent these methods from becoming useless in the future [1].
Many scientists agree that gene drives should be further studied as they currently have potential for more concerning side effects than traditional methods. One example of a significant concern is the unknown effect on the food chain from eliminating the A. gambiae populations [7]. So far, studies show that few animals rely solely on A. gambiae as a food source, so many experts believe the chance of negative environmental impacts are slim although there may always be the potential for side effects that were not studied in a specific sub-population or environmental niche [8]. Another concern is the potential for unethical uses with this new technology [7]. If, for example, someone releases a gene drive before enough research has been done and before it has been approved by a regulatory agency, serious environmental consequences could take place. Therefore, laws should be put in place to prevent such a thing from happening. Jim Thomas, a member of the Action Group on Erosion, Technology and Concentration says, “So far, all the proposals around gene drives are things like voluntary ethics codes and agreements between funders. They’re not binding in any way, so to what extent they can be enforced and who would be liable in the event of a problem — there’s none of that” [7]. Kevin Esvelt, one of the researchers who helped engineer the first gene drive, agrees that gene drive technology development could lead to consequences. “This isn’t just going to be about malaria,” Esvelt said. “This is potentially going to be something any individual who can make a transgenic fruit fly could build to edit all the fruit flies” [9].
The Costs of Traditional Methods and Gene Drives
Despite potential ethical and environmental concerns about the technology, the cost of gene drive research and implementation is overall lower than the cost of traditional methods and would save money in the long run. Comparatively, the cost of producing and dispersing nets, insecticides, and drugs each year is more expensive than developing and releasing a successful gene drive. The World Health Organization estimated that about $6.8 billion in resources for malaria prevention was needed in 2020, and that the cost will continue rising each year by an estimated additional $720 million [2]. As long as there is no complete way to prevent malaria, it is unlikely for the annual cost of these developments to disappear any time soon.
Unlike traditional methods, gene drive research has the potential to eradicate malaria completely and thereby curb all expenses involved in malaria research and malaria equipment dispersal. The Bill and Melinda Gates Foundation contributes a majority of grants going into gene drive research, donating about $7.4 million in grants in 2020. This funding has been going towards furthering promising research on gene drives and studies on the environmental effects of gene drives [10]. Ongoing research in future years may continue to require similar amounts of funding to the amount from current grants. However, this price is relatively small considering how much funding goes into malaria prevention and control annually, as well as the potential for a gene drive to completely curb the need for future funding of any kind. The cost of real life implementation is thought to be negligibly small, due to the process involving the release of only a small population of mosquitoes into the wild.
Efficiency of Traditional Methods and Gene Drives
Although cost is a big factor, the main reason for the huge support of gene drive research is the evidence pointing to a gene drive being a much more effective method than current traditional strategies. Current strategies such as insecticide use and drug use have not led and will unlikely lead to the elimination of malaria. Data suggests an overall trend towards fewer malaria cases likely due to traditional methods currently in place [1]. However, a full eradication of malaria around the world is the ultimate goal. Despite prevalent insecticide use and mosquito population control, there is still always a chance of a deadly mosquito bite in areas hard hit by malaria. Deadliness is especially the case when the issue of drug resistance pertains in Plasmodium. Mutations involved in partial drug resistance have already been detected in Plasmodium, and the more drugs continue to be used, the more likely resistance will continue to develop. Overall, insecticides can only be somewhat effective and cases of treatment failure are on the rise [1].
Gene drives, on the other hand, have been incredibly promising when it comes to efficiency. In the study by Beaghton, when the gene drive allele was released in a caged population and only made up 2.5 percent of the population, the entire population was predicted to crash in at least 30 generations [3]. Some models predict that even less mosquitos would need to be released into the wild in a real life scenario. One predicted that the release of just 500 gene drive mosquitoes could result in the complete collapse of a targeted mosquito species population in the timeframe of eight years [11]. Further mathematical models may be used in the future to calculate the optimal percent of the genetically modified mosquitoes that could be released to wipe out the population in the shortest timeframe feasible. From there, more gene drives targeting the other species of malaria-carrying mosquitos could be released, which would lead to a complete eradication of malaria. For now, scientists must continue to study the safety of gene drives by studying them in the lab, computationally, and perhaps in small contained real life settings. Additionally, further laws and policies will hopefully continue to be developed over gene drives in order to regulate the powerful technology. With enough time and research, affected communities and authorities may approve the gene drive strategy and begin implementing it in the near future.
Bibliography
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- Oberhofer, G., Ivy, T., & Hay, B. A. (2018). Behavior of homing endonuclease gene drives targeting genes required for viability or female fertility with multiplexed guide RNAs. Proceedings of the National Academy of Sciences of the United States of America, 115(40), E9343–E9352. https://doi.org/10.1073/pnas.1805278115
- Centers for Disease Control and Prevention. Insecticide-Treated Bed Nets. Accessed July 15, 2020. Available from: www.cdc.gov/malaria/malaria_worldwide/reduction/itn.html.
- Kahn, Jennifer. 2020. The Gene Drive Dilemma: We Can Alter Entire Species, but Should We? The New York Times Magazine.
- Collins, C. M., Bonds, J., Quinlan, M. M., & Mumford, J. D. (2019). Effects of the removal or reduction in density of the malaria mosquito, Anopheles gambiae s.l., on interacting predators and competitors in local ecosystems. Medical and veterinary entomology, 33(1), 1–15. https://doi.org/10.1111/mve.12327
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- Bill and Melinda Gates Foundation. Awarded Grants. Accessed 15 July 2020. Available from: www.gatesfoundation.org/How-We-Work/Quick-Links/Grants-Database#q/k=gene%20drive
- Eckhoff PA, Wenger EA, Godfray HC, Burt A. 2017. Impact of mosquito gene drive on malaria elimination in a computational model with explicit spatial and temporal dynamics. Proc Natl Acad Sci U S A [Internet]. 114(2):E255-E264. doi: 10.1073/pnas.1611064114.
Use of Transgenic Fish and Morpholinos for Analysis of the Development of the Hematopoietic System
By Colleen Mcvay, Biotechnology, 2021
Author’s Note: I wrote this essay to review the methods of utilizing Zebrafish as a model for understanding the mechanisms underlying the development of blood (hematopoietic) stem cells, for my Molecular Genetics Class. I would love for readers to better understand how the use of transgenic zebrafish and morpholinos have advanced our knowledge on the embryonic origin, genetic regulation and migration of HSCs during early embryonic development.
Introduction
Hematopoietic stem cells, or the immature cells found in the peripheral blood and bone marrow, develop during embryogenesis and are responsible for the constant renewal of blood throughout an organism. Hematopoietic development in the vertebrate embryo arises in consecutive, overlapping waves, described as primitive and definitive waves. These waves are distinguished based on the type of specialized blood cells that are generated and each occurs in distinct anatomical locations (7). In order to visualize and manipulate these embryonic developmental processes, a genetically tractable model must be used. Although many transgenic animals provide adequate models for hematopoiesis and disease study, the zebrafish (Danio rerio) proves to be far superior because of their easily visualized and manipulated embryonic developmental processes (6). Through the use of diagrams and analysis, this discussion will expand upon the mechanisms of the development of hematopoietic stem cells and explain how this knowledge is enriched through the use of transgenic animals and morpholinos, such as the zebrafish.
The Zebrafish Model
The zebrafish (Danio rerio) model has proven to be a powerful tool in the study of hematopoiesis and offers clear advantages over other vertebrate models, such as the mouse (Mus musculus). These advantages include the conserved developmental and molecular mechanisms with higher vertebrates, the optical transparency of its embryo and larvae, the genetic and experimental convenience of the fish, its external fertilization allowing for in vivo visualization of embryogenesis, and its sequential waves of hematopoiesis (9). Additionally, zebrafish allow for clear visualization of the phenotypic changes that occur during the transition from the embryonic to adult stages. This is beneficial in understanding and visualizing the hematopoiesis sequential-wave mechanism, as explained below (8). Mouse models on the other hand are embryonic lethal for many hematopoietic transcription factors, meaning the cells die in the embryo, therefore inhibiting that same visualization (12).
An Overview of Hematopoietic Development
The development of blood in all vertebrates involves two waves of hematopoiesis: the primitive wave and the definitive wave (4). Primitive hematopoiesis, involving an erythroid progenitor (or a cell that gives rise to megakaryocytes and erythrocytes), happens during early embryonic development and is responsible for producing erythroid and myeloid cell populations (5). The primitive wave is transitory, and its main purpose is to produce red blood cells to assist tissue oxygenation. These erythroid progenitor cells first appear in blood islands in the extra-embryonic yolk sac, however, they are neither pluripotent nor do they have renewal ability (11). Later in development (varying points in development for different species), definitive hematopoiesis produces hematopoietic stem and progenitor cells (HSPCs), that generate multipotent blood lineages of the adult organism (7). The HSC’s originate in the aorta-gonad-mesonephros (AGM) region of the developing embryo, where they then migrate to the fetal liver and bone marrow [Figure 1.]
Figure 1: Stages of Embryonic Hematopoiesis
This figure shows the establishment of primitive and definitive hematopoietic stem cells (HSC) during embryonic development. The first HSC’s appear in the blood islands in the extraembryonic yolk sac. The primitive wave is transient, and the successive definitive wave starts intraembryonically in the aorta-gonad-mesonephros (AGM) region. The definitive HSC’s are multipotent and migrate to the fetal liver where they proliferate and seed bone marrow. There is a systematic circulation of the embryonic hematopoiesis.
Hematopoietic Development in the Zebrafish Model:
Like all vertebrates, zebrafish have sequential waves of hematopoiesis. However, hematopoiesis in zebrafish occurs in a distinct manner compared to other vertebrate models, with its primitive HSC’s being generated intra-embryonically, in the ventral mesoderm tissue called the intermediate cell mass (ICM) (2). Throughout this primitive wave, the anterior part of the embryo creates myeloid cells while the posterior creates mostly erythrocytes, both of which circulate throughout the embryo from 24 hours post-fertilization (10). The next step involves hematopoiesis occurring in the aorta-gonad mesonephros (AGM) region, which is followed by the emergence of the HSC’s from the ventral wall of the dorsal aorta. The HSC’s then migrate to the posterior region in the tail called the caudal hematopoietic tissue (CHT). Finally, from 4 days post-fertilization, lymphopoiesis initiates in the thymus and HSC’s move to the kidney marrow (functionally equivalent to bone marrow in mammals) (11) (10)[Figure 2]. Although the anatomical sites of hematopoiesis are different in zebrafish and mammals, the molecular mechanisms and genetic regulation are highly conserved, permitting translation to mammals in many different ways. First, because zebrafish embryos can survive without red blood cells for a long time by passive diffusion, they are ideal for the identification of mutations that would be embryonic lethal in mice (9). These zebrafish mutants have been able to reveal genes that are critical components of human blood diseases and allow for the recognition of toxicity and embryonic lethality at an early stage of drug development. Additionally, the zebrafish model is amenable to forward genetic screens that are infeasible in any other vertebrate model simply due to cost and space requirements. Finally, zebrafish embryos are permeable to water-soluble chemicals, making them ideal for high-throughput screenings of novel bioactive compounds.
A:
B:
Figure 2: Hematopoiesis Development in the Zebrafish Model
A.) In embryonic zebrafish development, the sequential sites of hematopoiesis. Development first occurs in the intermediate cell mass (ICM), next in the aorta-gonad-mesonephros (AGM), and then in the causal hematopoietic tissue (CHT). Later hematopoietic cells are expressed in the thymus and kidney (Modified from Orkin and Zon, 2008).
B.) Timeline for the developmental windows for hematopoietic sites in the zebrafish (Modified from Orkin and Zon, 2008).
Transgenic Zebrafish & Morpholinos to Understand Genetic HSC Regulation and Migration
Transgenic zebrafish and morpholinos are easily manipulated and visualized through microinjection, chemical screening, and mutagenesis, all of which aid in identifying hematopoietic gene mutations and understanding gene regulation and migration in a vertebrate model. Epigenetic analysis of these mutations (through RNA sequencing, CHIP, microarray, and selective inhibition of a gene) have identified critical components to blood development that describe both the functions of these genes within hematopoiesis, and also describe phenotypes associated with defective development (1). Morpholinos target sequences at the transcriptional start site and allow for the selective inhibition of a targeted gene and analysis of the regulatory sequences in this mutant (Martin et al., 2011). Large-scale screening techniques (chemical suppressor screens, etc) of these mutations have identified many small molecules capable of rescuing hematopoietic defects and stopping disease, along with identifying new pathways of regulation (9). Although transgenic organisms have different origin sites and migratory patterns than mammalian hematopoiesis, the genetic regulation of HSC development and lineage specification is conserved, allowing for insights into the pathophysiology of disease.
Conclusion
The zebrafish is an invaluable vertebrate model for studies of hematopoiesis because of its amenability to genetic manipulations and its easily viewed embryonic developmental processes. This organism has become increasingly important in understanding the genetic and epigenetic mechanisms of blood cell development and the information produced is vital for the translation into regenerative medicine applications. Although more research is needed into specifics of HSC differentiation and self-renewal, zebrafish sufficiently allow for newly identified mutations and translocations of human hematopoietic diseases and cancers to be visualized and analyzed, unlike any other model organism. With this analysis, a more complete understanding of the molecular mechanisms of certain hematopoietic diseases can be made, thus aiding in the process of new treatments.
References
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- Galloway J. L., Zon L. I. (2003). Ontogeny of hematopoiesis: examining the emergence of hematopoietic cells in the vertebrate embryo. Curr. Top. Dev. Biol. 53, 139-158
- Kumar, Akhilesh et al. “Understanding the Journey of Human Hematopoietic Stem Cell Development.” Stem cells international vol. 2019 2141475. 6 May. 2019, doi:10.1155/2019/2141475
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- Jagannathan-Bogdan, Madhumita, and Leonard I Zon. “Hematopoiesis.” Development (Cambridge, England) vol. 140,12 (2013): 2463-7. doi:10.1242/dev.083147
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