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Effects of Aerobic Exercise Training on Longevity in Aging Adults

April 1, 2022

By Hastings Lorman, Human Development

Author’s Note: This paper was written as a term paper for my HDE 117 class in which my professor, Dr. Carey, suggested that I submit this paper to the Aggie Transcript. Not only did I write this paper for my class, but I wrote it for myself as one of my goals while taking this class was to improve my writing skills. I chose to write on the effects of aerobic exercises on longevity because of my personal interest in successful aging and general health and well-being. The topic is similar to a previous term paper I wrote for HDE 100C which was The Effects of Aerobic Exercise on Executive Function. I earned a C+ on that paper and I saw this assignment as a redemption and a way to build my research and writing abilities. I hope that the reader is able to take away information on how to avoid or minimize the risk of chronic, degenerative diseases in adulthood. I also hope to introduce the reader to the concept of exercise as medicine.

 

ABSTRACT 

Physical health and cognition are determinants of mortality. These factors are also measurements to discern if aerobic exercise influences longevity. Aerobic exercise is a treatment that has been extensively studied and has been shown to have a positive effect on healthy aging and longevity, such as, lowering mortality in older age, improving VO2 uptake, and increasing lean body mass, which can facilitate greater physiological function in aging adults. Physical activity can ameliorate adverse symptoms resulting from cardiovascular diseases such as high blood pressure, diabetes, and stroke compounded by the presence of Alzheimer’s Disease. An active lifestyle has been linked to greater cognitive function and improved mood. Those who exercise have, on average, greater brain volume which can stall the deteriorating effects of neurological conditions to lengthen one’s life expectancy. The implementation of aerobic exercise and healthy lifestyle practices is a key factor contributing to overall successful aging and mitigating risk of morbidity [1].

Introduction 

Physical exercise is a highly effective and often prescribed treatment for a plethora of physical and mental health conditions. There is a strongly reviewed association between daily, moderate exercise and improved health, which becomes more imperative for successful aging. [2]. Healthy aging involves maintaining high function of physiological and cognitive abilities while resisting major diseases such as high blood pressure or Alzheimer’s disease [3]. One means of extending longevity in aging adults is a consistent aerobic exercise regimen such as brisk walking or jogging. By following a succinct, cardiovascular workout routine repeated multiple times a week, multimorbidity, the presence of two or more chronic illnesses, significantly improved. 

Procedure 

Table 1. The effectiveness of individualized aerobic exercise training combined with telephone-based motivational interviewing on physical activity amount based on mixed model analysis [4].

In a longitudinal study, designed to test an aerobic exercise treatment for aging adults, participants were found to have an increase of muscle protein synthesis leading to greater lean muscle and body mass. Physical exercise as a prophylactic was linked to combating frailty. Participants were selected for age and health condition [5]. Aging adults were defined as being 65 years or older and were most often suffering from pre-existing chronic conditions such as cardiovascular diseases and Alzheimer’s due to the subjects being residents of rehabilitation centers. Studies were also selected for individuals who did not already exercise on a regular schedule. 

 One-hundred fifty minutes of aerobic exercise a week is the recommended benchmark for healthy aging [6]. Physical exercises used as treatment were brisk walking and jogging, on a treadmill or outdoors, and cycle ergometer. Aerobic workouts were performed outdoors with weekly check-ins or completed in a lab or rehabilitation center. Aerobic exercise treatments were structured to be an hour long occurring 3-5 times a week for 12-18 weeks long. Physical and psychological measurements were taken before and after the training routine. 

Activities were recommended to either be intentional, such as exercises done with the purpose for gaining health benefits, or for leisure, such as walking to destress. Since lower muscle mass is associated with cancer-related disease, researchers paid attention to increases in muscle mass and VO2 uptake, which can determine brain function and metabolic health [7-8]. More vigorous exercises were allowed but subjects were encouraged to participate in moderate physical activities [9]. 

RESULTS 

Improved cardiovascular measures positively correlates with greater VO2 uptake, a leaner body mass (measured by BMI), and improved endurance in aerobic exercise [10]. Greater cognitive function is correlated to a decrease in depression after receiving an exercise treatment. Participants exhibited a decrease in high blood pressure, which is a predictor of chronic health conditions. Alzheimer’s patients after exercising on a weekly basis had greater brain volume. Aerobic exercise can help aging adults retain their autonomy in daily life by reducing the naturally occurring physiological stress that accompanies longevity.

Effects on Physical Health 

 Consistent aerobic exercise can improve different measures of health such as weight, energy and pre-existing conditions such as Type II diabetes and other cardiovascular diseases. Moderate aerobic exercise can have anti-inflammatory effects on the aging body and provide significant relief for chronic pain [11]. Exercises such as walking and jogging on a weekly basis contribute to an increase in peak oxygen consumption by 10 to 15 percent when targeting frailty, the physical and cognitive decline that develops with age [5]. Subjects who adhered to the exercise regimen were reported to have a lower BMI, a greater heart rate reserve, and a lower blood pressure reading after 12 weeks [4]. For people struggling with cardiovascular diseases such as Type II Diabetes, exercise can be used to help alleviate symptoms, produce healthier measures of fitness, and contribute to an increase in longevity [12].

Lack of physical exercise is linked to an increase in noncommunicable diseases such as diabetes and respiratory diseases. In addition, lack of physical exercise can increase hypertension and obesity. Twenty percent of deaths in the United States are due to obesity and Type II Diabetes [9]. The Copenhagen City Heart Study contributed additional evidence to the benefits that aerobic exercise can have on physical health and, in turn, life expectancy. The study concluded that those who are physically active have at least a 30 percent lower mortality risk when compared to inactive participants [13]. Non-joggers treated with an exercise regimen of light to moderate jogging had a significantly lower mortality rate than the sedentary control to prevent the accumulation of diseases that can contribute to high morbidity risk.

Aerobic activities can also improve the quality of life in aging adulthood. Capacity for movement and strength drastically decrease after 65 years, but the addition of aerobic activities in daily life can lead to better blood pressure and bone density [14]. This can help alleviate physical strain from activities of daily living [15]. Aerobic exercise is especially crucial for women, who, on average, experience a more drastic loss in bone density after twenty years. An active lifestyle should be prescribed as medicine and should be performed as a prophylactic and preservation of current ability. Along with reinforcing bone density in older age, aerobic exercise is also linked to retention of muscle mass [16]. As life becomes more sedentary for the aging population, health professionals urge aging populations to keep as mobile as possible. Short durations of exercise can help limit the physiological effects of aging and reduce the impact of chronic disease on activities of daily living [17].

Routine exercise can reduce the risk of many debilitating conditions that can become more prevalent at an older age, such as certain types of cancers and osteoporosis. Exercise benefits are directly linked to intensity and quantity of the workout. Aerobic exercise can also increase measures of fitness such as muscle tone, flexibility, and cardiorespiratory function. Oxygen carrying capacity decreases with age which correlates with a 5 to 10 percent decrease in physical ability. The risk of sarcopenia, a decline in muscle strength and volume, increases with age which is why physical fitness becomes more imperative for successful aging [18].

Effects on Cognition 

Aerobic exercise has led to improvements in memory processing and improvement in pre-existing neurological conditions such as Alzheimer’s Disease and dementia. The progression of Alzheimer’s Disease has been linked to a decrease in brain volume in the entorhinal cortex and hippocampus [19]. These brain regions are correlated with episodic memory which stores personal experiences of previous events [20]. Brisk walking on a regular basis significantly slows the growth of brain atrophy in Alzheimer’s, reducing the amount of nonfunctioning years of a patient’s lifespan.

Those with mild cognitive impairment are expected to live 3.5 years (male) and 4.1 years (female) after receiving a diagnosis. Aerobic exercise cannot stop the biological clock, an organism’s natural time and physiological cycle, nor can it reverse the effect of dementia. However, aerobic exercise can extend quality of life and extend the functioning years of those with memory diseases [27]. Biological age, the measurement of age based on biological health, is a more accurate predictor of mortality than chronological age [28]. Leukocyte telomere length, a biological age marker, appeared to be nine years healthier in active participants compared to sedentary ones [29]. Aerobic activities can compress the prevalence of symptoms in aging adults [18]. 

 Aerobic exercise is associated with activation of brain-derived neurotrophic factors (BDNF), promoted by the mild stress caused by physical activity. The exposure to mild stress with the purpose to build tolerance and improve cognitive function, known as the process of hormesis, is associated with greater neuroplasticity and thus successful aging [3]. Structural connectivity is not only correlated with better cognitive, overall operational processing, and executive function, working memory and self-regulation, but also with the prevention of Alzheimer’s and dementia. Exercise can help slow down the loss of gray matter and brain volume, contributing to greater longevity in those with degenerative conditions. 

As brain volume begins to decrease with age, moderate exercise has been shown to increase cortical thickness in preclinical Alzheimer patients. Decrease in brain volume is one of the most present symptoms of Alzheimer’s, and an active lifestyle could combat the progression of memory loss. Aerobic exercise specifically benefits homeostatic functions such as brain plasticity and neurogenesis [3]. Physical exercise can attenuate amyloid, an abnormal protein made in bone marrow, which can lead to greater cognition. The presence of amyloid beta proteins, an inactive part of the protein amyloid, is linked to memory diseases such as Alzheimer’s [22].

Risk of heart failure along with Alzheimer’s disease greatly increases with age due to rapid myocardial dysfunction and a higher rate of perfusion and inflammation. These risk factors can be worsened by cardiovascular diseases and can lead to cognitive impairment. The progression of Alzheimer’s is linked to a greater risk of stroke due to a buildup of amyloid beta proteins between brain cells. Cognitive decline is found in both conditions and is believed to be linked to the presence of amyloid beta protein build up to form plaque in the brain which affects myocardial function [23]. Likewise, heart disease is linked to the deposition of amyloid plaque into the heart which leads to swelling and stiffness of the soft tissue [24]. Exercise has been shown to improve some of the cognitive deficits brought upon by Alzheimer’s such as executive function. Cognitive test scores measuring memory, attention, and mood significantly improved after a physical exercise treatment consisting of cycling or psychomotor activities administered 2 to 6 times over the course of 7 weeks [25].

Exercise is significantly correlated to improvement in mood for people with memory conditions such as mild cognitive impairment and dementia. Adherence to an exercise regimen has decreased dependency on others for activities of daily living (ADL) along with increased mood and feelings of self-efficacy. Depression can cause both physiological and psychological stress leading to rapid degeneration due to worsening cognitive symptoms. Physical activity along with environmental enrichment can induce hippocampal neuro-genesis resulting in greater benefits than either physical activity or an enriched environment alone. Aerobic treatments stimulate neural pathways found in the hippocampus, an area of the brain that decreases in function in those with neurodegenerative diseases, leading to greater retention of cognitive operation [25].

Fig. 2. “Vicious circle” of inactivity and positive effects of regular physical activity [18].

Fig .3. The links between physical inactivity, abdominal adiposity, inflammation, and disease [11].

Fig. 4. Hypertension. A linear dose–response association exists between leisure-time physical activity (LTPA) and risk for hypertension [11].

Discussion 

Aerobic exercise should be prescribed to aging adults to preserve cognitive function with the purpose of extending longevity and improving quality of life. Exercises such as walking or jogging are non-invasive treatments that produce significant improvements in blood pressure, body mass, and cognition. Direct implementation of exercise is crucial for healthy aging and early intervention is associated with a lower risk of developing adult-onset diseases later in life [26]. Participants who have experienced many life stressors were able to find health benefits through routine exercise, especially when combined with other life pillars such as adequate nutrition, well-being, and sleep. Public health professionals and researchers encourage the public to use exercise as medicine [11]. There are currently no FDA-approved medications for functional decline [30]. Aerobic exercise is now being regarded as a lifestyle medicine to combat chronic diseases that can develop later in life.

The goal of aerobic exercise is compression morbidity which is reducing the number of years one is disabled and extending the ability to perform ADLs in older age. Successful aging consists of both biological and psychological measures. Causes of death have shifted from infectious diseases to age-related chronic diseases meaning that personal wellness and successful aging are becoming more salient with age. Absence of disease has appeared on surveys measuring feelings of content throughout aging and is a key factor in emotional wellness as well as physical [28].

Aerobic exercise should be done in consideration to the individual. Exercises performed should not negatively impact other physiological functions and can be uniquely designed for the individual’s ability. The CDC recommends 150 minutes per week such as 30 minutes of walking 5 times a week or 75 minutes of vigorous exercise per week such as running [31]. Aerobic exercise is prescribed as safe and doable for most individuals regardless of socioeconomic status or other sociological categories which makes it a more equitable treatment than pharmaceuticals or invasive surgeries. Aerobic exercises do have barriers that should be accounted for such as access to an appropriate location, lack of time, and disease-specific symptoms that infer with the ability to participate [4].

Conclusions 

The aging population of this generation is healthier than any previous cohort. To ensure the health of this generation and those to come, aerobic exercise must be incorporated into the weekly routine of the general population. It is important to consider exercise as medicine and as public health policy. With increasing longevity, older populations should place a greater focus on physical independence and the maintenance of well-being in old age. Social institutions, starting as early as primary school and as late as retirement home should encourage walking and light jogging for aging adults to maintain health and foster longevity. Education and encouragement to engage in practices that contribute to successful aging should be promoted to further ensure the health and longevity of future generations. Aerobic exercise is a crucial factor in healthy aging and elicits health benefits that decrease the likelihood of early morbidity.

 

References:

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mRNA Vaccines: A Safe and Effective Technology

March 18, 2022

By Elexia Butler, Human Biology, ’23

Author’s Note: This article was written to reveal how the COVID-19 vaccines are produced and how they are a safe technology used to help reduce the number of sick individuals. Throughout the article, I will discuss the safety and efficacy of mRNA vaccines as well as the limitations that scientists overcome. I chose this topic because mRNA vaccines are a “new” technology that many of us don’t understand and has led to a larger social debate. The controversy surrounding mRNA vaccines stems from people’s questions regarding the vaccines’ safety and necessity. After reading this article, I hope the reader is able to take away the fact that the mRNA vaccines are safe and effective. 

 

Abstract:

As the world begins to settle after the past year and a half of operating with the COVID-19 pandemic, we look to mRNA vaccines to help return to a sense of normalcy. With both Moderna and Pfizer leading the market of mRNA vaccines since April 2021, we have seen a large decline in cases [27]. However, many people across the country are still skeptical of this “new” mRNA vaccine technology [8] and remain hesitant about getting the vaccine. Additionally, the COVID-19 vaccine controversy has left many individuals wondering if the vaccine is truly a safe way to fight the spread of COVID-19 or not. Currently, 54.7-59% of Americans have been fully vaccinated, but based on a PBS poll 24% have chosen to not receive any dose of the vaccine [40-41]. The goal of this article is to demonstrate the safety of mRNA vaccines, their development, limitations, and potential for treating future diseases. 

Introduction:

Messenger RNA (mRNA) vaccines are not a new technology, in fact they have been researched for years. mRNA is a small genetic molecule that encodes specific proteins [33]. The discovery of mRNA in 1961 sparked an entire field of research related to gene regulation [1-5]. 

Traditional vaccines work by introducing an antigen (a foreign substance that is recognized by the immune system) to elicit an immune response and cause the body to produce antibodies against that antigen [13]. For nucleic acid vaccines (DNA and RNA vaccines), rather than directly injecting the antigen, the instructions for producing the antigen are introduced into the cell [14]. The cell can then use these instructions to “make a protein—or even just a piece of a protein—that triggers an immune response inside our bodies” [16]. In the case of COVID-19, Pfizer and Moderna mRNA vaccines encode the instructions to make a viral spike protein from SARS-COV-2 (the virus that causes COVID-19). The spike protein won’t cause sickness on its own, it trains the immune system to defend against the real SARS-COV-2 virus [38]. While research has been conducted on both DNA and RNA based nucleic acid vaccines, it has been shown that RNA vaccines are able to elicit a stronger immune response and are likely safer [15]. The technology of mRNA vaccines became increasingly promising as scientists used the speed of production of the technology to develop a safe and effective mRNA vaccine to their advantage [50-51]. One of the many reasons the Moderna and Pfizer vaccines work is the way they modify the stability of the mRNA and establish a method for efficient delivery, allowing for a strong immune response when administered [17, 45-47]. Though hesitancy remains surrounding the COVID-19 vaccine, the Moderna and Pfizer vaccines are both effective and have significantly reduced the infection rate of COVID-19 [27]. This hesitancy has been fueled by reports of conspiracies as well as possible health effects, which all have been proven false and will be discussed later in larger detail.

Figure 1. This diagram demonstrates how the SARS-COV-19 vaccine was produced and how it elicits an immune response. Through the mRNA being introduced into the body, the cells gain instructions on how to produce the spike protein and forms antibodies. 

Proof of Principle:

The COVID-19 mRNA vaccine has brought hope to the medical field because they are effective and can continue to develop. With this technological advancement, it is important to maintain a certain standard of success to build confidence in the vaccines.  The Food and Drug Administration (FDA) has set a standard for success of “at least 50%” efficacy, or the prevention of the spread of infection due to the vaccine [18, 53]. The Moderna and Pfizer mRNA vaccine clinical trials exceeded this standard, granting them Emergency Use Authorization (EUA). The application of the mRNA vaccine demonstrated an effectiveness of “90% for full immunization and 80% for partial immunization” [10]. A study, conducted by the CDC in March of 2021, was used to assess the real world application and effectiveness of the vaccine in a potentially infectious setting. As reported by the CDC, the group of vaccinated first responders and essential health care workers were prevented from infection. This study showed that the Moderna and Pfizer vaccines are highly effective in the real world.

Along with this, there have been observational studies that show the vaccines have reduced the amount of transmission and need for hospitalization [9, 23]. Through a recent study by the Center of Disease Control and Prevention (CDC), it was concluded that the “SARS-CoV-2 vaccines significantly reduce the risk for COVID-19–associated hospitalization in older adults and, in turn, might lead to commensurate reductions in post-COVID conditions and deaths.” [9] 

The vaccines have created an opportunity for the world to return to a somewhat normal reality through the concept of herd immunity. Herd immunity is the idea that a “large portion of a community becomes immune to a disease … As a result, the whole community becomes protected—not just those who are immune” [30]. In other words, as more people get vaccinated, the transmissibility of SARS-COV-2 will be significantly reduced. Proof of this comes from the CDC as they discovered that in 1000 working days, infections among unvaccinated individuals (1.38 infections) were significantly higher than both fully vaccinated (0.04 infections) and partially vaccinated individuals (0.19 infections) [10]. To put it simply, the COVID-19 vaccine works. The vaccine has protected individuals throughout the past 6 months, and now that it is readily available we are seeing a massive decline in cases [27]. 

Figure 2. This diagram demonstrates how herd immunity functions in our society. As shown, the more people that are vaccinated, they are less likely to become infected. 

Versatility:

         Researchers have started studying possible applications of mRNA vaccines to diseases such as AIDS and other incurable diseases. It has been difficult to make regular vaccines due to the fact that there are so many mutations and strains, however the mRNA vaccine has been able to sidestep that by teaching the body to make antibodies and proteins. Before the modern advancements of mRNA vaccines that the COVID-19 vaccine brought forward, there was no efficient and effective way to deliver mRNA into the cell [31-32]. According to Mu et al. until these recent developments, there were major bottlenecks that hindered such research because mRNA is very unstable and can easily denature [31-32]. With new research, Moderna has begun trials on various mRNA vaccines, including one for HIV and AIDS [29]. 

Along with HIV, there has been research into using mRNA vaccines to treat cancer. Two types of vaccines have been proposed for cancer: preventative vaccines and treatment vaccines. Preventative vaccines attempt to protect the body from viruses that can potentially lead to cancer.  HPV and Hepatitis B are two infections where vaccines have been made in an effort to prevent these infections and stop the development of cancer [43]. In this method, the body “mount[s] an attack against cancer cells … Instead of preventing disease, they are meant to get the immune system to attack a disease that already exists” [43]. Treatment based vaccines, meanwhile, are more personalized to an individual’s genome [49]. To implement this, there must be an understanding of the individual’s specific cancer genome [49]. Scientists identify the mutated genes that are responsible for the tumor growth in the individual. They then encode and inject the mutant mRNA into the body, providing the individual’s immune system with instructions to create the mutated protein. This mRNA enables the body to identify and attack the cells with markers for the mutated gene, which are not present in non-cancerous cells. Moderna implemented a similar approach and found that the method reduced tumor size in 30% of human participants when combined with checkpoint inhibitors, a drug which activates proteins to regulate the immune system when attacking cancer cells  [49, 54]. Through the use of an mRNA vaccine, this allows the body to fight the tumors on its own rather than using harsh chemical mixtures, like chemotherapy, to stop the growth of the cancer. 

In regards to the multitude of other infectious diseases, much of the research around mRNA vaccines has already started and will continue. With the full approval of the Pfizer vaccine and current EUA of Moderna, the opportunity for future mRNA vaccines seems promising. As noted in previous research for mRNA vaccines targeting Zika and other diseases, there was a lack of knowledge regarding mRNA vaccines that impacted the ability to create a successful vaccine [19]. Due to the recent advancements, the opportunity to revisit these vaccines is possible.  

Limitations:

Several major hurdles continue to limit the broad application of mRNA vaccines which include cost, safety concerns, and instability of mRNA affecting storage.

Cost: 

Due to the severity of COVID-19, funding was readily available in an effort to mitigate the spread of this deadly virus. The federal government was one of the major financial suppliers as they “pledged to give nearly $500 million to Moderna alone for its COVID-19 vaccine”, and this was able to support one of the first COVID-19 vaccines brought forward [24]. Dr. Nathaniel Wang, chief executive of Replicate Bioscience developing RNA-based treatments for cancer, said “it’s pretty hard to talk people into taking bets on this type of technology for vaccines in infectious diseases” because it is seen as “new” technology [19]. This has been gravely apparent regarding RNA vaccines for diseases like Zika [19]. These financial constraints delayed progress and it made mRNA vaccines a nonviable strategy of treatment for Zika, COVID, and other diseases previously discussed. 

Safety: 

The safety concerns regarding the COVID-19 vaccine have been particularly contentious in the U.S. This fear is fueled by misinformation such as rumors of infertility caused by the vaccine and other false claims that have been reported in opinion pieces online. Many of the conspiracy theories and stories that damaged the image of the vaccine originated from social media[21]. A study polled that a majority of Americans believe there was “rushed approval for the COVID-19 vaccine without the assurances of safety and efficacy” causing people to believe that the vaccine bypassed all the regulatory steps [22]. The FDA defines that “for an EUA to be issued for a vaccine… FDA must determine that the known and potential benefits outweigh the known and potential risks of the vaccine” [39]. Through years of advanced research, the trials and production of the vaccine were able to run in parallel without compromising the safety of the vaccine [50]. While there are some valid concerns specific to the COVID-19 mRNA vaccines, including myocarditis, blood clots, and potential allergic reactions, the COVID-19 mRNA vaccines have been deemed as safe and effective by the CDC [26].

Side effects:

It is possible that individuals will experience certain side effects ranging from pain, swelling in the arm, nausea and fever, along with some more serious side effects, for example myocarditis and blood clots, reported by the CDC. It is important to note that if these less serious side effects even occur they are generally present for less than a week. A small price to pay for a vaccine that has been effective in preventing the spread of COVID-19 [23]. This was shown through mouse and hamster trials, as they noted that they had full immune system responses that protected against COVID-19, similar to that of humans [57]. In another study done with rats, they focused on the vaccine’s potential impact on pregnant rats to simulate that of a pregnant woman and found that there are potential side effects on that impact fetal development, female fertility, and early offspring development, but none were observed [58]. 

Through a variety of trials, scientists have determined that the body has been able to perform a timely immune response to the vaccine. A measurement of this has been the body’s reaction in the form of specific side effects [52]. Only a small number of cases include more serious reactions, such as anaphylaxis (2.5 per 1 million Moderna vaccines). Most cases will only have small reactions and no long-term side effects have been recorded [34, 35]. Though the majority of people only have minor reactions, these side effects show that the vaccine has gotten into the cell and the body has identified the viral mRNA [52]. 

Through the immense amount of data showing the vaccine’s efficacy, Pfizer has received FDA approval while Moderna has begun the FDA approval process [36, 37]. This milestone highlights the safety and efficacy of both mRNA vaccines. 

Storage: 

Due to the fact that both Moderna and Pfizer need lower temperatures for stability, they require the vaccines be kept below freezing around -20 to -80 degrees C for long term storage [25]. RNA needs to be stored at lower temperature as it will degrade due to alkaline hydrolysis, (breakdowns on its own in basic conditions) and RNAse activity (a nuclease that cleaves RNA). There have been cases of COVID-19 vaccines being discarded due to improper storage [55]. This limits packaging, shipment, and regions of the world allowed to have access to these vaccines because their storage will require specialized equipment and refrigeration. 

Conclusion:

The COVID-19 vaccines have paved a way for more mRNA vaccines to be brought to the medical field. If there is a steady increase in funding, researchers can begin to establish these kinds of vaccines for a variety of different diseases. By working through setbacks and finding a way to deliver vaccinations to the masses as well as bringing money to research, many of the limitations of mRNA vaccines can be mitigated in the future. The COVID-19 vaccine has proven to be quite efficacious and the recent FDA approvals are evident of this. These vaccines have been able to set a precedent of how mRNA vaccines can be used throughout health care as a protective measure.  mRNA vaccines are still considered a “new” technology and will continue to be researched and applied to a wide variety of fields in the future.

 

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Computational Strategies in the Treatment and Analysis of COVID-19

March 11, 2022

By Surya Vishnubhatt, Biomedical Engineering, ’22

Author’s Note: The devastating COVID-19 pandemic, having resulted in the death of millions of people worldwide, has spurred innovation in countless sectors of academia, namely in the field of bioinformatics and computational biology. By using computer science techniques, researchers have been able to rapidly identify treatments and further analyze the SARS-CoV-2 virus; the following review synthesizes computational advancements in COVID-19 research through immunoinformatics, docking servers, machine learning, and microRNA analysis. This review also incorporates current computational approaches in the treatment and analysis of COVID-19 viral variants. The use of bioinformatics and computational biology, in pursuit of analyzing and treating all forms of COVID-19, has yielded fast and effective therapeutic treatments in conjunction with crucial analytical findings. With much of the United States now opening up, and the virus likely to become a global endemic, the need for fast, computational analysis of the disease, regarding its progression and spread, is invaluable in ensuring public safety.

 

Introduction

The COVID-19 pandemic is a global public health emergency, with the fast spreading virus having engulfed the world within a few months. The respiratory disease, as of February 2022, has resulted in the death of 5.9 million people worldwide [1]. The viral pathogen behind COVID-19 is SARS-CoV-2 (severe acute respiratory syndrome coronavirus 2). This relentless virus has a mutation rate of 9.8 x 10-4  substitutions per site per year, which refers to the replacement of a specific DNA base pair with another by means of nucleotide substitution. Given the scale of the genome (the human genome is 3.2 billion base pairs long), the mutation rate of SARS-CoV-2 allows for substantial changes to the virus’ spike protein, allowing it to evade its host’s defenses and causing new viral variants to crop up around the world [2]. As the prevalence of these variants increases worldwide, the importance of effective computational analysis of COVID-19 protein and antibody dynamics cannot be overstated.

Bioinformatics and computational biology are highly similar, interdisciplinary areas of study that use the core tenets of computer science to analyze biological data. In general, bioinformatics and computational biology are crucial in understanding and analyzing protein dynamics, primarily in regards to sequence, structure, and evolution based analysis (which tracks changes in protein composition over time) [3].

A variety of computational approaches have been and are being investigated in the hopes of better understanding how COVID-19 operates in order to develop effective therapeutic treatments. One such approach concerns the field of immunoinformatics, a subset of bioinformatics and computational biology, which uses protein structures and genome sequences to develop vaccines [4]. Other areas of interest involve the use of docking servers (which predict and model protein-ligand binding interactions) and machine learning to identify and develop COVID-19 therapeutics [5]. Further research is also being conducted in analyzing micro RNA to better understand and exploit the cellular dynamics of COVID-19 [6]. 

This review will investigate established computational approaches and will also explore novel research regarding COVID-19 in the hopes of stimulating further research in COVID-19 variant analysis.

A Brief History of COVID-19 

The SARS-CoV-2 virus emerged in December of 2019, first reported in Wuhan, China. The unique virology of SARS-CoV-2 allowed for its rapid progression and spread. The virus itself is covered with spike proteins on its surface; each spike protein consists of three monomeric units which bind to human ACE2 receptor cells [7]. ACE2 receptors are present on the surface of human muscle cells, primarily in the lungs, and act to mediate vascular constriction and inflammation. During COVID-19 pathogenesis, the SARS-CoV-2 spike protein can bind to the human ACE2 receptor, invade the cell, and proliferate, leading to lung damage. The spike protein has an incredibly high affinity for the ACE2 receptor due to contact interactions which occur at the interface between the receptor binding domain of the spike protein and the ACE2 receptor, contributing to the widespread nature of the disease [8].

Figure 1: The SARS-CoV-2 infection mechanism. 

Currently, in the United States, vaccines have been created for the original viral strain, namely the double dose Pfizer and Moderna (mRNA based) vaccines and the single dose Johnson and Johnson (adenovirus based) vaccine [9]. These vaccines stimulate the host to synthesize a non-pathogenic version of the spike protein, which triggers an immune response and is then targeted by host-specific antibodies (generated in response to the host-mediated spike protein), rendering immunity to the major COVID-19 strain. However, with the rise of new variants, most notably, the omicron B.1.1.529 strain, the effectiveness of mRNA (e.g. Pfizer and Moderna) vaccines are diminishing from 95% efficacy to 35% efficacy, with booster shots required to increase efficacy to 75% [10, 11]. Similarly, the Johnson & Johnson adenovirus based vaccine declined from 94% to 85% efficacy in adenovirus based vaccine therapies in individuals who received booster shots [12, 13].

Applications of Bioinformatics and Computational Biology in COVID-19 Research

The field of bioinformatics and computational biology deals in the collection and analysis of biological data, namely genomic and proteomic data, in the hopes of better understanding disease pathogenesis [3]. 

3.1 Immunoinformatics and COVID-19 Vaccine Development

The field of immunoinformatics is a subset of the field of bioinformatics and computational biology. Specifically, it uses computational, analytical, and mathematical data tied in with computer science processing techniques, to formulate predictions about immunity and vaccine development [14]. In the immunoinformatics-based approach to COVID-19 vaccine development and drug discovery, it is important to note that only the receptor binding domain (RBD) of the SARS-CoV-2 spike protein is in contact with the human ACE2 receptor, making the RBD the major functional region of the virus. Accordingly, the major immunoinformatics-based approaches to COVID-19 antibody development target the RBD of the spike protein, preventing its attachment to the ACE2 receptor [15]. 

There are two major methodologies of vaccine discovery through immunoinformatics: reverse vaccinology and structural vaccinology [4]. Reverse vaccinology analyzes expressed genomic sequences in order to identify various antigens as potential vaccine targets, as these identified antigens are, ideally, to be synthesized and subsequently targeted by the host immune system. Meanwhile, structural vaccinology uses 3D protein models to engineer immunogenic conformations of antigens in the hopes of eliciting antibody responses against pathogenic attack [16, 17]. Structural vaccinology is not explicitly used in COVID-19 drug discovery, but is incorporated within modern reverse vaccinology techniques [4]. 

A new study used reverse vaccinology programs as well as novel computation techniques such as the Molecular Mechanics Poisson-Boltzmann Surface Area calculation approach, to design a COVID-19 antibody protein that can provoke a wide array of host immune responses [18]. This immunological approach, in its emphasis on reverse vaccinology, has also been successfully implemented in the design of a multi-epitope subunit vaccine, triggering immunity in both humoral and cell-mediated contexts [19]. Using DNA/PCR visualization software, researchers observed that the multi-epitope vaccine has highly specified, targeted responses to pathogenic invasion via host-mediated immune response [19, 20]. 

3.2 COVID-19 Docking Analysis

The SARS-CoV-2 docking procedure binds the pathogen to the host’s ACE2 receptors. It is a key point of interest for many researchers who aim to disrupt this binding configuration to prevent COVID-19 infection [21]. Free energy simulators can be used to visualize the stability of various binding configurations of proteins to ligands with a lower binding free energy value indicating a more stable protein-ligand configuration. Using these computational free-energy simulators that bind ligands to the spike protein, potential antibodies can be developed to block or destabilize host-virus interactions [22]. A variety of preliminary studies have been able to identify potential therapeutic compounds from which drug development can progress.

Furthermore, COVID-19 binding can be simulated by docking servers which model how small molecules, peptides, and antibodies bind to potential targets on SARS-CoV-2. In 2020, a team from China created a free meta-server to predict COVID-19 target-ligand interactions to promote drug discovery [23]. This server has been used in a variety of studies. One study used the server to test docking scores of a variety of potential antiviral agents and found that scalarane-based sesterterpenes (a biochemical) showed promise in developing COVID-19 vaccines [24]. Another study, using the same server, identified teicoplanin, an antibiotic, as a potential source of drug design in combating SARS-CoV-2 infection [25]. 

Several other studies have used docking servers to analyze potential plant-based therapeutic targets; including the compounds of the Boerhavia diffusa, the phytochemicals of the Phyllanthus amarus and Andrographis paniculata, and hesperidin [26-28]. These compounds were initially chosen due to their therapeutic properties and have been previously used to treat a wide array of diseases. Upon further analysis, resulting simulations show that these chemicals can destabilize the ACE2-spike protein complex, thus rendering host immunity [28]. In addition, in silico molecular docking techniques were used in identifying the antiviral drugs Remdesivir and Mycophenolic acid acyl glucuronide as potential candidates to be repurposed towards COVID-19 treatment, due to their preferential binding to the main protease of SARS-CoV-2. This preferential binding can then be used to disrupt the binding of SARS-CoV-2 to human ACE2 receptor cells [29]. 

3.3 Machine Learning and COVID-19

The field of machine learning is a branch of computer science which trains an algorithm to “learn” through feeding it enough data such that it can make logical predictions about a variety of different sets of conditions [30]. 

Reverse vaccinology can be combined with machine learning practices to design COVID-19 vaccines. The machine learning tool, known as Vaxign-ML, incorporates biochemical and physicochemical characteristics into its reverse-vaccinology analysis [31]. This platform can then be incorporated with machine learning algorithms, to identify “cocktail” vaccines, consisting of structural and non structural proteins (a protein that is encoded but not part of the viral body), which stimulate an immune response to COVID-19 [32]. 

Another aspect of machine learning in COVID-19 research involves a more external approach to attacking the problem. Researchers from the Sri Ramaswamy Memorial Institute of Science and Technology were able to train a machine learning algorithm to analyze abnormal chest x-rays and CT scan data in patients exhibiting signs of COVID-19. The study yielded a 93% recall score of CT scan images and 88% precision in analyzing chest x-ray images [33]. Similar machine learning algorithms were used in a different study to analyze abnormal features in the CT scan data of patient lungs. This study yielded an accuracy of 91.94% in diagnosing COVID-19 infection [34]. 

3.4 MicroRNA (miRNA) and COVID-19 Analysis

MicroRNAs or miRNAs are pieces of RNA which act to regulate gene expression post-transcriptionally [35]. Researchers from Italy and Singapore found that various miRNAs are regulated by the spike protein of SARS-CoV-2 and the human ACE2 receptor in conjunction with the enzyme histone deacetylase (HDAC). Through computational analysis, using in silico methods and the query miRNet analytics platform, these researchers were able to identify that HDAC inhibitors limited interactions between the spike protein and the ACE2 receptor [36]. Further studies confirmed the effectiveness of HDAC inhibitors as a preventative drug to restrict SARS-CoV-2 entrance into the host, using a wide array of laboratory tests and culture analyses [37]. Using similar methodologies, another study constructed its own computational meta-analysis framework to identify how host miRNAs bind to SARS-CoV-2 RNA and suggests the repurposing of anti hepatitis C, RNA based, drugs in the treatment of COVID-19, due to its substantial binding affinity [38]. 

Current Computational Efforts in COVID-19 Variant Analysis 

COVID-19 variants are formed when the virus’ spike protein mutates, making it harder for the established antibodies in vaccinated people to recognize and bind to the pathogen. In some cases, the established antibodies may be able to bind well enough against the variant molecule, while in other cases, a breakthrough infection may occur and the virus is able to override the host’s defense systems [39, 40]. Currently the four major variants of COVID-19 in the United States are: Alpha (B.1.1.7), Beta (B.1.351), Gamma (P.1), Delta (B.1.617.2), and Omicron (B.1.1.529) [41]. 

Figure 2: A phylogenetic tree depicting the dominant COVID-19 variants as of December 2021.

By using machine learning algorithms, and comparing genome sequences, researchers were able to obtain an overall picture of the spread of variants throughout all continents [42]. Similarly, other researchers were able to track the global progression of coronavirus variants by aggregating data, concerning the worldwide evolution of COVID-19 nucleotide-substitutions, and building an open source web application known as COVID CG to reflect their findings [43]. Other population-orientated studies investigate the genetic, topological, and evolutionary progression of SARS-CoV-2 in order to understand its emergence on the global scale and how to homogeneously apply vaccines to heterogeneous populations, in the hopes of preventing the spread of COVID-19 and its variants in the future [44-46].

Figure 3: The COVID CG tool, from the end user perspective.

Other studies use computational modeling mechanisms to determine how variants interact with ACE2 receptor cells. One such study modeled a wide array of mutations to the spike protein in order to determine variant transmissibility, which can aid in establishing future safety precautions [47]. Another study was able to model the transmission dynamics of COVID-19 by computationally comparing it with dengue infection (as dengue fever and COVID-19 have similar symptoms in the earlier stages of infection) to obtain alternate insights into COVID-19 disease progression [48].

Conclusion

The field of bioinformatics and computational biology is expansive in its coverage; it can be narrowed down to analyze specific protein-to-protein interactions on the molecular scale or expanded to examine the global progression of disease. With much of the United States reopening its borders, and students returning to in-person classes, the rapid computational analysis of COVID-19 disease progression on both a micro and macro scale is invaluable in ensuring public safety. 

As of January 2022, actions to curb the spread of variants have been taken in the form of booster shots and the Pfizer pill. Booster shots reintroduce the same material as the previously mentioned vaccinations to “boost” or reinforce host immunity by increasing the count of memory B and T cells [49]. Additionally, the FDA approved Pfizer COVID-19 Oral Antiviral Treatment, or Paxlovid, consists of nirmatrelvir and ritonavir, with nirmatrelivr acting to prevent viral replication while ritonavir reduces the breakdown of nirmatrelvir. Furthermore, Paxlovid has been proven to be effective against COVID-19 variants in in vitro studies [50-52]. Ultimately, due to the virus’ rapid evolution, most experts have reached a common consensus, that COVID-19 is likely to continually circulate as an endemic, with yearly vaccines needing to be developed and administered, much like the flu [53]. Like the flu, we must continually stay “ahead” of the virus and its variants. Thus, the need for fast, effective computational analysis of the disease and its mutations is essential in mitigating its potentially detrimental effects. 

 

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Investigating Anthelmintics for Vector Control

February 11, 2022

Investigating the use of anthelmintic drugs in the context of disease vectoring arthropod control

By Anna Cutshall, Animal Biology, Global Disease Biology minor ’21

Author’s Note: When considering the topic of my literature review and analysis, I wanted to select work that I could continue research on in graduate school. As I entered academia, my career and life experiences had prepared me well for the unique intersection of veterinary medicine, ecology, and epidemiology. I have been on a pre-veterinary track for many years and have worked professionally in the veterinary field for more than three years. As an Animal Biology major and Global Disease Biology minor, my coursework largely centered around the emerging threat of zoonotic and vector-borne diseases. These experiences considered, my primary research interests lie in how we may integrate veterinary medicine into One Health practices to better combat emerging disease threats. In this literature review, I investigate the viability of anthelmintic drugs against arthropod vectors of disease. The use of anthelmintics against arthropods is fairly new, and the pool of current literature is limited but promising. This review was written for those, like myself, who are interested in new approaches to the control of tropical diseases, especially through the lens One Health. I hope to leave readers with a clear picture of what is next for this field, what gaps in the data should be filled, and how we can use information gained in responsible, sustainable ways to combat both emerging and established vector-borne diseases.

 

Abstract

This literature review analyzes the efficacy of currently available anthelmintic drugs against key disease vectoring arthropods. When comparing effective dosages between different drugs and vector genera, we found that relatively low concentrations are effective against most vectors, but there is evidence to suggest that ivermectin resistance has been established in some species (Aedes spp). The avermectin drug class also displayed limited efficacy over time, as the drugs degrade in vertebrate species faster than the isoxazoline drug class or fipronil. We determined that the current findings related to this method of vector control are promising. However, further research must be conducted before we implement anthelmintics for mass drug administration as a part of integrated vector management.

Keywords: anthelmintics, insecticides, vector, disease vector, mosquito, sandfly, One Health, integrated vector management, mass drug administration

 

Introduction

Vector-borne diseases threaten the well-being of hundreds of millions of people globally. This is predicted to increase as climate change and human activity facilitate the spread of vector species to previously unoccupied locations. In a press release by the Sacramento-Yolo Mosquito & Vector Control District, it was reported that multiple invasive mosquito species, including Aedes aegypti, had been identified in northern California [1]. Recent literature suggests that these habitual expansions may be due, in part, to climate change as these species are able to adapt to broader regions that are of similar climate to their native regions [2]. The continued spread of these species leaves unprepared countries at risk for outbreaks of the diseases vectored by invading species. Moreover, most vector-borne diseases remain uncontrolled in endemic regions. The most direct way to mitigate the threat of globalizing tropical vector-borne diseases is to control the species that are vectoring them. Unfortunately, traditional insecticide-based methods of vector control have become ineffective due to the emergence of insecticide resistance. In 2012, the World Health Organization identified the status of insecticide resistance as “widespread”, as most of the globe reported resistance in at least one major malaria vector [3]. Traditional spray and topical insecticides have been compromised by such resistance. Therefore, it is essential that new methods of vector control,without acquired resistance, be discovered, evaluated, and implemented.

There are many new methods of vector control currently under evaluation. These include genetically modifying vectors to render them sterile, the use of entomopathogenic fungi and viruses, trapping, repellents, and environmental modification [4]. As we continue to evaluate each method for its efficacy, the Integrated Vector Management (IVM) method may be our best option for the elimination of many tropical diseases. Through IVM, we take careful and integrated approaches to vector control via intersectional communication between Public Health officials, Governments, Non-Governmental Organizations, and communities in which we hope to implement our strategies [5]. IVM calls for multiple vector control strategies, and increasing control efficacy via synergy between control efforts. Unfortunately, the primary tool utilized for the control of adult mosquitoes, insecticides, has lost efficacy over time. This is a result of vector insect populations developing resistance to common insecticides, such as pyrethroids and organophosphates, that are used to control adult mosquito populations. However, there is a reservoir of insecticides that have not been utilized against human disease-vectors, which therefore have minimal acquired resistance . This class is oral insecticides, or insecticides ingested by vertebrates that act when a vector is exposed via blood meal from a treated animal. The use of oral insecticides has been standard in veterinary medicine for years, in the form of flea and tick prevention. Common classes of oral insecticides include avermectins, isoxazolines, and phenylpyrazoles. These compounds have been standard in human and/or veterinary medicine as ectoparasiticides, demonstrating their safety for use in vertebrates. Avermectins, isoxazolines, and phenylpyrazoles have similar modes of action as neurotoxins, with both interrupting the function of GABA-gated chloride ion channels, resulting in insect paralysis [6, 7]. Importantly, there is still diversity within these classes as tools against vector species, as they bind to different sites on the GABA receptors [7, 8]. Investigating the efficacy of these drugs for use as insecticides, against key vectors of diseases such as malaria, zika, west nile virus, leishmaniasis, and African Trypanosomiasis, could be part of the solution to the increasingly urgent problem of insecticide resistance.

Research is currently underway, across the globe, to investigate the efficacy of ectoparasiticides against disease vectors. The question still stands, however, if the approach of oral insecticides is any more effective than the traditional insecticides available. To answer this question, we assessed the current literature regarding the testing of ectoparasiticides against disease vectors, and developed a database of studies testing the efficacy of these compounds against vector insects. This analysis aims to determine the relative efficacy of these compounds to determine if these drug classes are worth consideration for use in vector control and management.

 

Materials and methodology

To establish a database of the relevant literature, we first mined the scientific literature via the UC Davis Library. Using access granted to undergraduate students, the search terms utilized were input as follows: title/abstract contain “vector” AND “veterinary” AND “control” AND “arthropod” in the key word function. Papers were then selected for further analysis. These articles were input into an AI-based literature analysis tool, “Research Rabbit”, to identify additional relevant studies [9]. In addition, studies were selected from the works cited of previously selected works. Papers not testing the efficacy of oral insecticides on adult disease vectors were excluded from the study. Additionally, papers without comparable data (did not supply direct mortality or density data) were also excluded.

Each paper was analyzed to extract relevant data on the efficacy of oral insecticides against disease vectors. The data was collated into a Microsoft Excel spreadsheet. Categories selected for further evaluation included: drug type, the concentrations used, associated concentration resulting in 50% mortality (LC50 values), time to mortality, the reduction of vectors present in field study by visual count (resting density), and drug effects on vector fecundity. However, for the purposes of this study, we focused on LC50 and temporal values. Other categories were not consistent across publications.

When creating data visualizations for comparison of different drug types, R’s “ggplot2” package, “dplyr” package and “esquisse” package were used [10-13]. The categories determined to be best for visual comparison were “Temporal Data” and “LC50” data. After initial visualization was made in R, figures were exported to Adobe Illustrator to edit aesthetically, which was limited to modification of font types and caption content [14]. When creating visualizations for LC50 data, both sandfly and mosquito vectors were compared on the same figure, to compare the efficacy of not only drugs in relation to each other, but also drugs in relation to their efficacy against different disease vectors. When creating the data visualization for this comparison, the drugs “Moxidectin” and “NTBC” were excluded. Moxidectin’s LC50 value was too high to allow for reasonable comparison to other drugs, and NTBC only had a value for tsetse flies (Glossina spp), which were not represented in any other drug. Additionally, the Lutzomyia spp displayed LC50 values too high to be effectively compared to other disease vectors. When visualizing temporal data, only Anopheles spp and Phebotomus spp had enough supporting information in the literature for effective comparisons. There were 5 studies that supplied data for Anopheles spp temporal data and 2 studies that supplied data for Phlebotomus spp temporal data. These temporal data were plotted as Day of Feeding against Mortality, faceted by drug type, and grouped by dose. 2 Figures were created, one for Phlebotomus spp and another for Anopheles spp.

 

Results

Database Creation
From the initial search in the UC Davis Library system, 15 studies were selected. Then, based on the output from Research Rabbit, an additional 5 studies were selected. Finally, 3 additional papers were identified and integrated into the analysis from the references of the 20 studies. These 23 studies were then evaluated individually from January, 2021 through March, 2021.

We obtained multiple data categories for comparison between the selected papers. Figure 1 displays the summary of the resulting database. Due to the recent nature of this research, resources from which to draw for our database were limited. Table 1 shows a summary of the data types and the number of papers each data type was collected from. Within each paper, some investigated efficacy against multiple vector genuses while others investigated only one. Based on the data we were able to collect, we will be moving forward directly comparing temporal data as well as LC50 data.

Comparing Effectiveness of Dosages Between Drugs
We sought to compare the concentrations of drugs required to be effective against the disease vectoring arthropods studied. Figure 2 displays the LC50 values chosen for comparison as a “lollipop” plot. Within the plot, each “dot” represents a single datapoint taken from a study, and 7 studies were compiled to create the plot. In this figure, we are able to compare 2 major drug classes: avermectins and isoxazolines. The isoxazoline drug class had more available data across insect families, and it is clear that the LC50 value is variable between genuses. Sandflies have more resistance to isoxazolines (especially fluralaner) than mosquito species. Amongst the avermectins, Anopheles spp. Display the most consistent, and relatively low LC50 values. However, Aedes spp. displays higher resistance to ivermectin compared to Anopheles spp.

 

Comparing Temporal Data
Temporal data involving different insecticides were first to be compared. In temporal data, the “Days Post Feeding (Day of Blood Meal)” represents the number of days after the initial dosing of the vertebrate animals (for example, “Day 3” indicates mosquitos that fed on an animal 3 days after it was given the drug). We were able to create 2 figures for comparing the efficacy of drugs over time at various doses. Doses were represented as variance in color in the figures. Figure 3 displays the efficacy of oral dosing to vertebrates of eprinomectin and ivermectin against Anopheles spp. over time. Both of the drugs in this comparison were of the avermectin class, and neither displayed robust effects on mortality past the 15 day mark after single-dosing of vertebrates with the drugs. Additionally, we observe great variance in the efficacy of ivermectin, even within dosages (that is, mortality varied within dosages between different studies). Unfortunately, mosquito genuses outside of Anopheles did not provide enough data to compare efficacy over time.

Created in a similar fashion, Figure 4 displays the efficacy of oral dosing of vertebrates with fipronil and fluralaner. Here, two separate drug classes were tested for efficacy. While fluralaner (a member of the isoxazoline drug class) acts in a similar mode of action to avermectins, it maintains efficacy over time in Sandflies. Because mosquitos displayed more sensitivity to isoxazolines than sandflies, (Figure 2) one may predict similar, if not more deadly, effects when isoxazolines are tested over time for mosquitos. The drug fipronil displays varying efficacy between doses. Unfortunately, the study involving fipronil did not collect data past 21 days of administration, but it is possible some of the dosages would have remained effective from visual interpretation of the figure. Due to the limited amount of studies investigating the efficacy of oral insecticides, we were not able to compare the efficacy of all drugs over time, as other studies used different methods of efficacy measurements.

 

Discussion

Based on the findings of our review of currently available literature, oral insecticides certainly show promise as a method of Disease Vector management. As displayed in Figure 2, we are able to determine effective dosages for each drug as a concentration in blood. However, there was significant variance in the data between taxa and between drugs. The highest resistance was observed in Aedes spp. against ivermectin, which could be evidence of acquired resistance due to the common use of ivermectin in humans as an anthelmintic [15]. Another significant variance observed was the relative resistance of sandflies to isoxazolines, requiring approximately twice the concentration or more compared to mosquito species [16]. It is unclear if this effect carries over to other drugs, as there is no available data.

There were also studies analyzed that were not included in the visual data analyses performed. These included studies that investigated the efficacy of isoxazolines against the kissing bug, of nitisinone against the tsetse fly, a field study, and data from otherwise integrated studies measuring the effect of the drugs on fecundity of arthropods [16-22]. The investigation by Loza et al. regarding the efficacy of isoxazolines against the kissing bug showed similar temporal data results to papers investigating isoxazolines against Sandflies, which was visualized in Figure 4 [17]. The isoxazoline drug class, then, has been shown to be effective against 3 major disease vector families. Another drug class also shows promise. A study by Sterkel et al proposes the use of nitisinone (traditionally used in the treatment of hereditary tyrosinemia type 1, a genetic disorder) as an insecticide dispensed to vertebrates, and investigates its efficacy against the African Trypanosomiasis vector, the tsetse fly. This study highlights the importance of looking for alternative methods to vector control, and manipulates a characteristic of a drug originally developed to aid in human disease against disease vectors. The 2021 study found that concentrations above 0.5 micrograms per milliliter in blood impacted survival of feeding tsetse flies significantly, while also studying pharmacokinetics when ingested by mouse models [18]. Pharmacokinetic data supplied by the mouse models in this study may assist in any later calculations for human dosage. No evidence is available on the effectiveness of nitisinone on other disease vectors.

Three studies supplied data involving sublethal effects on adult arthropods, including fecundity. These studies found that there were significant effects on Anopheles spp. fecundity, regardless of vertebrate being dosed and observed across multiple doses [20-22]. There is no currently available data involving the effects of isoxazolines or phenylpyrazoles. Should they be provided, however, they show additional promise as vector management tools. When an insecticide is able to exhibit both lethal and sublethal effects, particularly regarding fecundity, insects that survive the initial exposure produce less offspring than their unexposed peers.

In order for these methods to be effective in Disease Vector management, there would need to be a considerable number of individuals in the population participating to make a significant impact on the burden of vector borne diseases [16, 19]. Mass Drug Administration (MDA) is expensive, and cost is a limiting factor in many of the areas we hope to lower disease burden in. Due to this, and issues related to the accessibility of MDA, it is important that drugs remain effective for an extended period of time. Fortunately, we found this to be the case. As evident in Figure 4, both isoxazoline drugs display extended efficacy on mortality of sandflies over 40 days after initial vertebrate dosage. Additionally, it may be that fipronil displays a similar effect in higher tested dosages, following the trajectory of the available data. Unfortunately, there is limited literature on this subject, so we are unable to say with absolute certainty that these effects would carry over to mosquito species. Figure 3 suggests that the avermectin drug class does not have the same long-term effect on arthropod mortality. For both ivermectin and eprinomectin, mortality dropped below 50% overall after just 14 days from initial dosage. For this reason, the isoxazoline and phenylpyrazole drug classes may be more effective for MDA, although their testing for safety in humans is less extensive (than ivermectin).
Additionally, there is the question of if MDA should be dispensed to humans or livestock. The field study by Poche et al. applied previous findings to the field, dosing cattle in several tribes in Africa and visually measuring the effects on the density of mosquitos found in nearby homes. They observed that the dosage of livestock with fipronil reduced the “resting density” of mosquito species known to feed on both cattle and humans, but did not significantly affect the resting density of particularly anthropophilic species [19]. This study highlights the importance of catering an MDA to the specific species you want to impact by ensuring dosage to vertebrates that it is likely to take a blood meal from.

When considering a drug for use in MDA, the safety of the drug must be copiously studied, and current findings are promising. At the dosages used in the study that were effective against adult arthropods, vertebrates suffered no severe adverse effects attributed to the dosages in all of the studies analyzed. This strongly suggests that these drugs are safe for use in IVM. Additionally, when considering MDA, taking a “One Health” approach will also be key to success. Too often, non-Governmental Organizations have gone into regions with targeted endemic diseases, and neglected to listen to native perspectives on previously used methods of disease control and basic needs. While investigating the efficacy of these drugs is important to protecting communities against vector-borne disease, giving aid to impoverished communities must first address the baseline health of individuals at risk. Only then can we hope to earn the trust of native populations, and continue to help them in sustainable ways. Continuing to thoroughly investigate the efficacy and safety of this sector of vector management before beginning any large implementation will also be essential.

Overall, it can be inferred from the amount of studies performed that there needs to be an enormous amount of research performed before we integrate oral insecticides, especially in humans, into IVM. What we do know, though, gives promise in the face of the insurmountable resistance to traditional pesticides.

 

References:

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  17. Loza, A. et al. Systemic insecticide treatment of the canine reservoir of Trypanosoma cruzi induces high levels of lethality in Triatoma infestans, a principal vector of Chagas disease. Parasit Vectors 10, 344 (2017).
  18. Sterkel, M. et al. Repurposing the orphan drug nitisinone to control the transmission of African trypanosomiasis. PLOS Biology 19, e3000796 (2021).
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  37. Smit, M. R. et al. Pharmacokinetics-Pharmacodynamics of High-Dose Ivermectin with Dihydroartemisinin-Piperaquine on Mosquitocidal Activity and QT-Prolongation (IVERMAL). Clin Pharmacol Ther 105, 388–401 (2019).

Potential Methods of Life Detection on Ocean Worlds

August 29, 2020

By Ana Menchaca, Biochemistry and Molecular Biology ‘20

Author’s Note: As a biochemistry major who is interested in pursuing astrobiology research, I initially wrote this literature review for an assignment in my Writing in Biology course. Methods of life detection and what we know about life is a field in which we still have much to discover and explore, given Earth as our only example, and I hope to be involved in this exploration myself in the future.

 

Abstract

Ocean worlds, such as Enceladus, Saturn’s largest moon, provide intriguing environments and the potential for life as we continue to explore the Solar System. Organic compounds have been discovered in plumes erupting from the moon during flybys and point towards the presence of amino acids and other precursors of life. The data collected from these flybys, in turn, has been used to calculate the theoretical amounts of amino acids present in the oceans of Enceladus. While this data is intriguing, it relies on a limited definition of life, based on organisms and macromolecules that have only been observed on Earth. Other methods, including using nucleic acids or nanopores for detection, have been proposed. Nucleic acids utilize binding to identify a broad spectrum of compounds, while nanopores utilize the measurement of ionic flow. These alternative methods allow for a broader spectrum of compound detection than terran-based methods, creating the potential to detect unfamiliar kinds of life. Research into more holistic detection should continue.

Keywords: astrobiology, life detection, planetary exploration, biosignatures

 

Introduction

The search for life elsewhere in the Solar System is becoming increasingly relevant, and more importantly, feasible. Icy moons, such as Europa, Titan, and Enceladus, have been identified as holding the greatest potential for extraterrestrial life within the Solar System [1]. Europa and Enceladus, with seas below icy crusts, have geysers with unidentified fluctuations, along with evidence of tidal warming and geologic activity [2]. The Cassini spacecraft identified these geysers on Enceladus during flyby in 2006, spouting from four specific fractures on the surface of the moon [3]. Analysis of the vapors produced show that they mainly consist of water, along with CO2, N2, CO, CH4, salts, other organic compounds, and silica particulates [3, 4]. This points towards evidence of hydrothermal activity, the movement of heated water, which has the potential to provide necessary energy for life [4]. Additionally, the discovery of volatile aliphatic hydrocarbons in these plumes potentially indicate some degree of organic evolution within the seas of Enceladus [4].

However, there is no consensus yet on how to detect and identify life [1, 2]. Some scientists propose looking for life based on the shared ancestry hypothesis, which proposes all life shares the same genetic ancestry [2]. Others propose there is a potential for extraterrestrial life to present variations from terran life that we may neither be able to recognize nor detect with our current methods of biochemical detection [5]. Experimentally, the potential for nucleic acids based on different backbones has already been identified [5]. Here, we examine the range of proposed methods for identifying extraterrestrial life. 

 

Proposed theories and methods based on current knowledge of life

Collection of amino acids

Current data collected from Enceladus’ plumes presents organic compounds that provide potential evidence of amino acid synthesis taking place in the oceans of the moon. Steel et al. used the thermal flux at the moon’s South Polar Terrain (SPT) to predict the hydrogen produced by hydrothermal activity. The predicted rates of production ranged between 0.63 and 33.8 mol/s of H2, and from there, amino acid production rates were estimated to be between 8.4 and 449.4 mmol/s [4]. Annual biomass production was also modelled in these calculations and estimated at 4 · 104  to 2 · 106 kg/year, compared to 1014 kg/year on Earth. These estimates, however, are dependent on the environment being an abiotic, steady state ocean; the actual production rates could be different if there is life present in Enceladus’ ocean [4]. 

While this limits our predictions of Enceladus’ true environment, it still provides a basis that can be extrapolated for use in the design of modules to be sent out. One such module that has been proposed is the Enceladus Organic Analyzer, which is designed to analyze amino acids through chain length variations [3]. To properly collect and analyze the amino acids proposed to be in Enceladus’ oceans, there are several requirements. The sample must be collected from the subsurface ocean with minimal degradation, isomerization, racimerization, and contamination of biological molecules and amino acids [3]. A collection chamber made of aluminum has been modeled, designed to reduce the thermal heating caused by collection of samples, in order to best preserve them. If the moon contains bacteria as postulated, this design will lyse and kill collected cells through either heat or shock but release their more stable chemical components for analysis [3]. This depends highly upon current knowledge as a starting place, focusing with a limited scope on amino acid and cell identification. Another such method using cell identification is digital holographic microscopy. 

Digital holographic microscopy

The development and improvements of microscopy, while beneficial, depend heavily on the assumption that life in the same form as terran cells will be found. Investigators propose digital holographic microscopy (DHM) as a more efficient alternative over traditional light microscopy [1]. This technology produces a 100-fold improvement in the depth of field and is able to monitor both intensity and phase of images. However, even with the increase in resolution, differentiation of cells and cell-shaped structures is difficult, even before taking into account potential differences in extraterrestrial life. Refraction, an emerging field, was able to differentiate experimentally between crystalline structures and cells in the study’s Arctic samples. While the technology can be miniaturized and discriminate between cells and minerals, it depends highly on actual capture of a sufficient number of cells from plumes. This experimental data was obtained using dye-less techniques, which still function in the context of organisms without DNA or RNA, and refraction with the potential to differentiate structures [1]. DHM is both useful for detection of cells based on collected data and for the potential discovery of organisms without nucleic acids as we know them. 

 

Expanding outside the current knowledge of life

Detection using nucleic acids
Other experiments and proposals, while not explicitly targeting life outside the current perceptions, propose a more holistic collection of data. This carries the potential of identifying life outside our current scope, as opposed to focusing directly on known amino acids and cells. Using a broader concept of nucleic acids as a means of detection and identification is one such method. 

Oligonucleotides, through forming secondary and tertiary structures, have specificity and affinity to a wide variety of molecules, both organic and inorganic [2]. Even at a length of only 15 base pairs and within complex mixtures, these molecules can bind to what is being analyzed, or the analytes. Systematic evolution of ligands by exponential enrichment (SELEX) is a process that can identify oligonucleotides that bind very specifically to analytes. However, this method proposes the use of low affinity and low specificity nucleic acids that are typically discarded in this process. Unlike antibodies, this method requires no prior knowledge of the surface attributes or the three-dimensional structure of the molecule that is being bound. Through accumulating a wide range of binding sequences and statistical analysis, a vast number of compounds can be collected and environmental variations identified. Additionally, this method posits that the optimal means of capturing sequences is through proximity ligation assay (PLA), a technique currently used in scientific fields. PLA purifies the binding species based on ligation and amplification, producing a lower background than sieving, which separates based on size. It is also capable of capturing a vast range of sequences and structures, including inorganic, organic, or polymeric molecules [2], and thus is more capable of providing holistic results.

 

Nanopore-based sensing

Nanopore-based sensing, presented as an alternative to current methods, detects and analyzes genetic information carriers in watery systems without making assumptions about its chemical composition [5]. This system relies upon the restrictions placed on these sorts of compounds within watery systems, as the repeating charge of backbones keeps polymer strands from folding and favors solubility in water. A nanopore is a hole with a diameter of a few nanometers, surrounded by an insulating membrane within two chambers containing an electrolyte solution. Due to its diameter, only single-stranded DNA can pass through the nanopore, allowing for slow movement and characteristic signals that produce data clearly distinguishable from other molecular data. This method can detect and analyze molecules by measuring the ionic flow across the membrane. While biological nanopores are able to detect and resolve individual terran bases, nonbiological, solid-state nanopores provide the same function, avoiding the limitations of detecting terran molecules that may be present in biological nanopores. Graphene, with its crystalline form, can have its membrane adjusted to only accommodate one nucleotide at a time or can be sculpted to produce varying sizes of nanopores. This could allow for the detection of other polymers with chemical and sterical properties that vary from currently known polymers. This approach has the potential to analyze a broad range of molecules without any assumptions regarding the external structure’s outside charge and linearity. Few identified nonbiological polymers are structured this way, so any data picked up by nanopores would be significant [5].

There are, however, limitations to this approach. Nucleic acids have high electroporation speeds, making it necessary to find methods of slowing these speeds down for accuracy [5]. Electroporation uses an electrical charge to make the cell membrane more permeable. Potential methods include control through physical factors, such as temperature, salinity, and viscosity. Conditions of collection on other planets also pose the problem of extreme dilution of the target molecules, which depends on a large number of variables [5]. 

 

Conclusion

Radiation and stability are major concerns in moving forward with any sort of data collection from extraterrestrial worlds. Mechanisms and samples are potentially open to the detrimental effects of extreme vacuum and solar radiation [5]. These problems should be addressed in conjunction with the technology actually being used for analysis to produce the most beneficial results. Some of these issues have been addressed to some extent, such as using microfluidics for collection because they are unaffected by the vacuum of space due to their own internal surface tension [3]. However, these problems need to be explored further in all cases to ensure that each method can function in uncontrolled or non-terran environments.

The presented data indicates potential for the existence of amino acids in these environments. Even though prediction and detection of these amino acids seems a logical step forward, the development of further, broader technology for life detection should also be pursued. Current knowledge is limited by the qualities of terran life; while that is a well-supported starting point, methods that leave open the potential of deviation from this point may allow for the detection of otherwise overlooked forms of life. Moving forward, it seems only logical to combine these methods that can detect both the known and the unknown, allowing scientists to gather the widest possible array of data in future missions, especially on promising worlds like Enceladus. 

 

References

  1. Bedrossian M, Lindensmith C, Nadeau JL. 2016. Digital Holographic Microscopy, a Method for Detection of Microorganisms in Plume Samples from Enceladus and Other Icy Worlds. Astrobiology 17(9):913–925.
  2. Johnson SS, Anslyn EV, Graham HV, Mahaffy PR, Ellington AD. 2018. Fingerprinting Non-Terran Biosignatures. Astrobiology 18(7):915–922.
  3. Mathies RA, Razu ME, Kim J, Stockton AM, Turin P, Butterworth A. 2016. Feasibility of Detecting Bioorganic Compounds in Enceladus Plumes with the Enceladus Organic Analyzer. Astrobiology 17(9):902–912.
  4. Steel EL, Davila A, Mckay CP. 2017. Abiotic and Biotic Formation of Amino Acids in the Enceladus Ocean. Astrobiology 17(9):862–875. 
  5. Rezzonico F. 2014. Nanopore-Based Instruments as Biosensors for Future Planetary Missions. Astrobiology 14(4):344–351.

Gene editing invasive species out of New Zealand

March 6, 2020

By Jessie Lau, Biochemistry and Molecular Biology ‘20

Authors Note: Since the advent of Clustered Regularly Interspaced Short Palindromic Repeats-CRISPR Associated Protein 9 (CRISPR/Cas9) discovery and biotechnological breakthroughs thereafter, this revolutionary application has been primarily focused on human health, particularly fostering solutions to numerous debilitating ailments. However, the general public has offered little attention towards the use of this engineering feat in a broader ecological system. Upon watching the new Netflix original Unnatural Selection, discussion of considering the use of CRISPR/Cas9 in New Zealand’s effort to completely eradicate invasive species piqued my interest. The following article is an exploration of CRISPR/Cas9 prospect into New Zealand’s bold environmental pursuit and its potential ecological impact.

 

Abstract

Since it has become feasible to cross oceans to reach unforeseeable land masses, invasive alien species (IAS) are an increasing threat to international biodiversity. Moreover, no other region faces as great of a peril as New Zealand (NZ), which holds the record as the nation with the highest survival rate of threatened avifauna (birds of a particular region) species [5]. In 2012, New Zealand physicist Sir Paul Callaghan introduced a large-scale eradication program to permanently remove eight invasive mammalian predators (rodents: Rattus rattus, Rattus norvegicus, Rattus exulans, Mus musculus; mustelids: Mustela furo, Mustela erminea, Mustela nivalis; and the common brushtail possum: Trichosurus vulpecula) [5]. Four years after this grand proposal, the NZ government committed to a national challenge titled “Predator Free 2050” (PF 2050) to pursue this audacious goal. 

 

Introduction

Approximately 85 million years ago, NZ was one of the first landmasses to split from the supercontinent Gondwana and as it shifted away, it did not carry mammals until bats flew and aquatic species swam to this island. [7]. With the introduction of rodent species initially through Polynesian settlement some 750 years ago and thereafter European seafaring ventures, the endemic species of NZ have been left vulnerable to novel predators. Consequently, at least 51 native bird species that have evolved adaptive skills of remaining close to the ground have been unsuccessful at surviving alongside these rodent predator species [4]. These invasive omnivorous rodents prey on birds, eggs, seeds, snails, lizards, and fruits. As a result, their varied diets prompt competition with the native fauna, further placing pressure on their vulnerable survival [7]. Despite CRISPR/Cas9 modifications offering the greatest potential in gene drive to eliminate these unwanted predators, several techniques implemented to control these invasive populations,  such as pesticides, trapping innovations, and biological factors have given favorable results.  

 

Past Successes and Future Endeavors 

For decades, the NZ government has pursued eradication initiatives to permanently eliminate foreign invasive species on small local islands. Through concerted efforts, several of these projects have proven successful in the restoration of the natural biodiversity this island nation boasts. 

In June of 2009, the NZ Department of Conservation (DOC) undertook a multi-action plan to simultaneously eradicate rodent, rabbit, stout, hedgehog, and cat species in Rangitoto and Motutapu islands. After three years of aerial dispersion of anticoagulant Pestoff 20RTM combined with trapping and indicator dogs, the island witnessed total elimination of the islands’ stoats and four rodent species; declination of rabbit and hedgehog population by 96%; and over 50% reduction in cats [7]. 

Despite these successful feats, the invasive species fecundity and ability to adapt to these challenges still present an overwhelming challenge to reach the goal of complete eradication. Recently, more direct approaches delving into novel genetic inheritance techniques have been explored to serve as a potential permanent solution. Termed the“Trojan Female Technique” (TFT), the method makes use of the correlation of sperm fitness dependence on the abundance of mitochondrial DNA (mtDNA) [2]. Healthy sperm is dependent on sufficient mitochondrial level for energy production to manage motility and fertilization. For example, experiments conducted on Drosophila melanogaster supports the causation of reduced spermatogenesis and sperm maturation due to induced mutations in cytochrome mitochondrial gene [9]. Contrasting female eggs, the asymmetric greater dependence of sperm on mtDNA for normal functionality results in only male populations to be the sole source of target. Induction of mitochondrial mutations in females to compromise total sperm viability in future male progenies will serve as an effective control to population growth. 

Although seemingly promising, TFT is not guaranteed to completely eradicate propagation for several reasons. For starters, males with impaired fertility can still provide sufficient sperm count to fertilize eggs on a population-based scale. Furthermore, in circumstances where females do receive nonviable sperm count, they can still seek adequate functioning sperm through matings with other males. On a larger scale, should the mutation pervade, selection pressures could still inadvertently choose for nuclear modifications to make up for the mitochondrial defects [2]. These flaws raise the need for more pervasive and permanent resolutions.

 

Daisy Chain CRISPR Gene Drive

The recent biochemical breakthrough underpinning the ability to effectively and precisely modify genes with CRISPR/Cas9 has allowed for potential biotechnology to boom in the realm of ecology. The simple generation of a short RNA sequence into a virus or bacterium to serve as a vector, guides the cutting mechanism of Cas9 to specific regions in the genome to be excised, prompting for these double stranded breaks to be fixed through DNA repair mechanisms. While these fixtures can potentially repair the gene, it can also raise the possibility for the gene to be disabled, introduce a new function, or create an unforeseen mutation. Given the right specific targeting in the germ line, this approach houses the innovation for exterminating entire species through gene drive [6].

The mechanism behind gene drive overthrows the traditional Mendelian sexual reproduction concept of proportional contribution from both the male and female parent. The power comes from the ability of one genetically modified (GM) contributor encoding for the ‘gene drive’ to cut the other set of chromosomes lacking these genes and replace this excision with a self-replicating copy. In effect, this divisive modification would push for an otherwise heterozygous offspring from a wild-type mating with a GM partner to become homozygous for certain genes to be carried on and propagated by future generations. Given that these genetic alterations do not affect the fitness of the organism, dissemination of 1% of the population with CRISPR modified genes can lead to 99% of the local population carrying the genetic indicator in as little as nine generations (The use of gene editing to create gene drives for pest control in New Zealand).

Such a unilateral approach poses political and ethical challenges amongst neighboring nations with diverging ecological approaches to confront pest control. Should this pervasive gene drive program reach beyond its intended border, great difficulty would arise in maintaining this ecological enclosure. For example, possum is on the list of invasive species in NZ while just 2,500 miles west, its neighbor Australia keeps this species of marsupials under protection. As such, scientists have devised a simple model to localize gene alterations, coined The Daisy Chain.

Unlike the original gene drive method in which all components necessary for transformation (CRISPR, edited DNA, and guide RNAs) are provided on the same chromosome, Daisy Chain provides a self-exhaustive means of guaranteeing genetic edits. This tool is designed so that  each component required for genetic alteration is dependent on the presence of a different element upstream on the gene found on the same locus to be activated [8]. The most downstream portion of this chain contains the “load” of engineered dominant lethal genes preventing reproduction, which will be promoted to higher frequency in the population within several generations. For instance, should an engineered allele contain three elements A, B, and C, element C would render element B to drive, which will in turn cause element A carrying the final load to drive. The initial element (C in this case) does not actually drive, thus is restricted by the number of altered individuals released into the wild and will be lost via natural selection over time. During initial implementation, the presence of C will increase B in abundance, but B will eventually decline and finally disappear as C is lost in the population. The rapid rise in abundance of B will also cause A to increase in frequency within the local population; however, with the decrease of B, A would not be driven and will ultimately vanish as well [1]. Using MIT Professor Esvelt’s analogy, “… the elements of a daisy drive system are similar to booster stages of a genetic rocket: those at the bottom of the base of the daisy-chain help life the payload until they run out of fuel and are successively lost.”

 

Challenges with Daisy Chain CRISPR Gene Drive

CRISPR/Cas9 technology’s ability to potentially alter these invasive species’ fecundity provides an avenue of pursuing NZ’s goal of PF 2050. Despite the developed understanding of how to carry out this plan, scientists in NZ are still working to piece together the genomes of stoats and possums in order to understand where to properly facilitate the engineered RNA sequence. Other barriers that must be acknowledged are the unprecedented approach to genetically modify marsupials and the difficulty of implanting hundreds to thousands of oocytes to be dispersed amongst their population. 

Beyond these known difficulties, scientists still tread in unknown terrains pertaining to whether these mutations can have pernicious effects in the survival, health, and reproductive success in propagating these mutations within their populations. Further exploration into the development of these modifications, and the potential impacts they can have on these animals, must be investigated on model organisms prior to widespread use. 

Of the eight listed mammalian species vied to be permanently eradicated from NZ, Mus musculus holds the most promise, given the extensive knowledge of the Mus musculus genome. With the help of scientists outside of New Zealand, joining in on the efforts to identify which germline gene to focus on, this project has received international attention.

Although the daisy drive provides promising potential, research collaborators at MIT and Harvard have identified a possible risk of, “… DNA encoding a drive component from one element to another, thereby creating a ‘daisy necklace’ capable of a global drive” [1]. Due to this rare recombinatory event arising from the similarity of DNA sequences, these investigators have looked into circumventing the problem by creating numerous alternatives to CRISPR components and selecting the model with the greatest diversity. 

 

Conclusion

From their renowned aviary to reptilian species, New Zealand’s islandic geographical region houses some of the most biodiverse fauna known to man. The arrival of human settlement has introduced predatory species, causing endemic species to experience extinction at concerning rates [4]. With the purpose of preserving their unique remaining diversity, New Zealand has committed to concerted efforts of varying methods to eradicate these invasive vertebrate pests. Investigation into genetic modifications can provide for more expansive and thorough techniques to eliminate these human introduced pests and allow for these endangered species to thrive once again. By further exploring daisy chain CRISPR/Cas9, this effort can be genetically inherited by offspring, allowing for nature to carry out this effort. As opposed to continued efforts of targeting each individual one by one, conservation ecologists can borrow from molecular biologist’s toolkit to revolutionize the means of pursuing pest control and perhaps even pave the road for future endeavors with similar pursuits. 

 

References

  1. Esvelt, Kevin M. “Daisy Drives.” Sculpting Evolution, www.sculptingevolution.org/daisydrives.
  2. Gemmell, Neil J., et al. “The Trojan Female Technique: a Novel, Effective and Humane Approach for Pest Population Control.” Proceedings of the Royal Society B: Biological Sciences, vol. 280, no. 1773, 2013, pp. 1–6., doi:10.1098/rspb.2013.2549.
  3. Griffiths, Richard, et al. “Successful Eradication of Invasive Vertebrates on Rangitoto and Motutapu Islands, New Zealand.” Biological Invasions, vol. 17, no. 5, 2014, pp. 1355–1369., doi:10.1007/s10530-014-0798-7.
  4. Owens, Brian. “The Big Cull: Can New Zealand Pull off an Audacious Plan to Get Rid of Invasive Predators by 2050?” Nature, vol. 541, 12 Jan. 2017, pp. 148–150.
  5. Russell, James C., John G. Innes, Philip H. Brown, and Andrea E. Byrom. “Predator-Free New Zealand: Conservation Country.” BioScience 65, no. 5 (October 2015): 520–25. https://doi.org/10.1093/biosci/biv012.
  6. Saey, Tina Hesman. “Explainer: How CRISPR Works.” Science News for Students, 4 Dec. 2017, www.sciencenewsforstudents.org/article/explainer-how-crispr-works.
  7. “Why Predator Free 2050?” Department of Conservation. Accessed November 18, 2019. http://www.doc.govt.nz/nature/pests-and-threats/predator-free-2050/why-predator-free-2050/.
  8. Dearden, Peter K., et al. “The Potential for the Use of Gene Drives for Pest Control in New Zealand: a Perspective.” Journal of the Royal Society of New Zealand, vol. 48, no. 4, 2017, pp. 225–244., doi:10.1080/03036758.2017.1385030.
  9. Wolff, Jonci N., et al. “Mitonuclear Interactions, MtDNA-Mediated Thermal Plasticity and Implications for the Trojan Female Technique for Pest Control.” Scientific Reports, vol. 6, no. 1, 2016, doi:10.1038/srep30016.
  10. Min, John, Jason Olejarz, Joanna Buchthal, Alejandro Chavez, Andrea L. Smidler, Erika A. DeBenedictis, George M. Church, Martin A. Nowak, Kevin M. Esvelt, and Charleston Noble. “Daisy-Chain Gene Drives for the Alteration of Local Populations.” PNAS. National Academy of Sciences, April 23, 2019. https://www.pnas.org/content/116/17/8275.

A History of Vaccines and How they Combat Disease

February 28, 2020

By Vishwanath Prathikanti, Political Science ‘23

Author’s note: The anti-vaccination movement has recently gained traction with many families across the nation and I wanted to tackle the idea of anti-vaccination and where it came from. I also wanted to see if there was any credit due to the anti-vaccinators and see if there was any truth to the idea that more vaccinations might be bad.

 

In April 2019, public health officials declared a measles outbreak in Los Angeles. To many, this sounded almost absurd; measles was eradicated in the United States in 2000 [4]. The outbreak highlighted the severity of a movement that many had declared irrelevant: the anti-vaccination movement. In light of this event, many had to question: what is the anti-vaccination movement? When did it begin? Is there any truth to the movement?

To understand the anti-vaccination movement, one must first understand vaccines and their history. Centers for Disease Control and Prevention (CDC) defines a vaccination as, “a product that stimulates a person’s immune system to produce immunity to a specific disease, protecting the person from that disease.” [1]. The human immune system uses white blood cells to fight infections in the body; specifically, there are three types of white blood cells that work together to fight infections: macrophages, B-lymphocytes and T-lymphocytes [2]. When a cell becomes infected or dies, it releases a chemical that attracts macrophages, which will engulf and degrade the cell. If the cell was damaged or died due to a virus or bacteria, the macrophage will leave behind antigens, which are recognized by the immune system as harmful [10]. When the immune system recognizes the antigens, B-lymphocytes will produce antibodies to attack the antigens and T-lymphocytes will attack cells in the body that have been infected by the identified antigen. After the infection is dealt with, the immune system will create memory cells that act immediately if the body encounters the same germ again. Vaccines work by imitating an infection; they do not cause illness but they will stimulate the production of T-lymphocytes, B-lymphocytes and memory cells to fight the disease in the future. Most vaccines require multiple doses to ensure full immunity, and how frequent these dosages are required depends on the vaccine [2]. 

Our knowledge of vaccines has not always been as vast as it is today. Evidence suggests that the earliest form of inoculation was in China during the late 1600s when emperor K’ang Hsi had his children inoculated after surviving smallpox (the process involved grinding smallpox scabs and inhaling them) [5]. The practice of vaccination has grown considerably since then, becoming vastly popular in the West by the 17th century. In 1853, Britain passed a law that made it mandatory for citizens to receive a smallpox vaccination and in 1855, Massachusetts passed the first U.S. law mandating vaccination for smallpox, allowing vaccinations to grow and develop. 

In the late 20th century, research on the negative effects of vaccines started to emerge. A 1995 study published in The Lancet linked the measles-mumps-rubella (MMR) vaccine with bowel disease. Wakefield, a gastroenterologist and researcher in the study, went on to further speculate that persistent infection with the vaccine caused disruption of the intestinal tissue that could lead to autism. This led to the study that would capture the attention of parents for decades to come. In 1998, Wakefield and his colleagues published a case series study in which, out of 12 children who had recently been administered their MMR vaccine, eight had the measles virus in their digestive system and were demonstrating symptoms for autism. Wakefield then went on to claim that the combined vaccination led to this, and advocated instead to adopt single-antigen vaccinations as opposed to combined MMR vaccines [3]. He did not, however, list how he came to this conclusion, saying “the combined measles, mumps, and rubella vaccine (rather than monovalent measles vaccine) has been implicated” [3].

The link between autism and the MMR vaccination was studied intensively over the next few years, and no reputable study ever found a similar link. Additionally, a study published in The Journal of Pediatrics, while acknowledging a slightly lower than average antibody count when the combined vaccination was employed, stated that there was no significant reason why single antigen vaccinations should be favored over combined vaccinations. The lower antibody count was deemed irrelevant in light of the fact that failure of the vaccine was extremely rare in fully immunized children [7]. In 2010, The Lancet formally retracted the paper, and three months later, Britain’s General Medical Council banned Wakefield from practicing medicine in Britain. Finally, in 2011, it was revealed that Wakefield had falsified most of his data; in his study, he reported eight children developed symptoms of autism when in reality, there were at most two cases. In addition, two of the children had developmental delays that were not mentioned in the final published work [3].

Despite the study being completely discredited by the scientific community, the damage to society had been done; after the Wakefield paper was published, vaccination rates dropped below 50 percent in some parts of London. Luckily, immunization rates drastically rose since then, with over 90 percent in the UK vaccinated in 2013, with BBC declaring a “universal recovery” [8]. Although vaccination rates are high, the US still faces about 60 cases of the measles every year, caused by international travelers who carry the disease [9]. While the spread of misinformation due to the Wakefield paper has mostly subsided, its legacy continues keeping a minority of children in the US unvaccinated and susceptible to antiquated and preventable diseases.

 

References

  1. Centers for Disease Control and Prevention “Immunization: the basics” https://www.cdc.gov/vaccines/vac-gen/imz-basics.htm
  2. Centers for Disease Control and Prevention “Understanding how vaccines work” https://www.cdc.gov/vaccines/hcp/conversations/downloads/vacsafe-understand-color-office.pdf
  3. History of Vaccines “Do vaccines cause autism?” https://www.historyofvaccines.org/content/articles/do-vaccines-cause-autism
  4. Centers for Disease Control and Prevention “History of measles”  https://www.cdc.gov/measles/about/history.html
  5. History of Vaccines “All timelines overview” https://www.historyofvaccines.org/timeline#EVT_1 
  6. Wakefield A, et al. RETRACTED:—Ileal-lymphoid-nodular hyperplasia, non-specific colitis, and pervasive developmental disorder in children. Lancet. 1998; 351(9103): 637-641. https://www.thelancet.com/action/showPdf?pii=S0140-6736%2897%2911096-0
  7. Heinz J. Schmitt, et al. “Primary vaccination of infants with diphtheria-tetanus-acellular pertussis–hepatitis B virus– inactivated polio virus and Haemophilus influenzae type b vaccines given as either separate or mixed injections.” The Journal of Pediatrics. 1999. https://www.sciencedirect.com/science/article/pii/S0022347600260885
  8. BBC “Measles outbreak in maps and graphics” 2013. https://www.bbc.com/news/health-22277186
  9. NPR “Fifteen Years After A Vaccine Scare, A Measles Epidemic” 2013. https://www.npr.org/sections/health-shots/2013/05/21/185801259/fifteen-years-after-a-vaccine-scare-a-measles-epidemic
  10. Arizona State University “Macrophages” https://askabiologist.asu.edu/macrophage

How Are California Bears Doing?

February 14, 2020

By Timur Katsnelson, Neurobiology, Physiology, and Behavior ‘19

Conservation biology has always been an interesting field to me. After having previously submitted two neuroscience-related articles to the Aggie Transcript, I decided to explore a new topic. The bear-sighting on campus last spring was on my mind, mainly because I began to wonder about the status of bears in our state. Given their symbolic status in California, I imagined that their conservation would be well-documented. This article serves as a brief report of the black bear’s current status in California, and the population genetics methods used by researchers keeping track of the animal.

 

Shortly before 6 a.m. on June 4, 2019, a very rare occurrence captured the attention of the entire Aggie community. A young, male black bear was spotted wandering near the UC Davis Arboretum’s Redwood Grove [4]. The campus police and fire departments, in conjunction with California’s Department of Fish and Wildlife (CDFW), worked to tranquilize the straggler and release him to the nearest habitat west of the city of Davis. This moment of excitement on campus sparked the curiosity of many who care about wildlife and conservation. According to CDFW, black bear populations have been on the rise over the past quarter-century, so what are the challenges the species face and how do biologists keep track of populations? It is also important to evaluate the evolutionary track of the species to understand if it can withstand changing environments, so what is the genetic diversity of the state’s population of black bears?

California’s history with bears is complicated. Many sports teams in the state are fondly named after them and, most notably, a prominent grizzly roams a patch of grass on the state’s flag. While it is a cherished symbol of the state, the grizzly faced a savage end to its reign as apex predator. The Spanish threw the bears in fights to the death against bulls and dogs, and later American settlers hunted them into oblivion. The last California grizzly was seen in 1924 and has since been extinct [2]. Nearly one hundred years later, environmentalists are aspiring to reintroduce the grizzly bear through back-breeding, cloning, or genetic engineering [2]. Many might consider these to be aspirational long-term goals, and in the meantime have focused on the current population of bears in the state. What is known for sure is that the absence of the grizzly in the state has opened up more room for a different population to flourish [5]. 

The real challenge in the many decades since the last grizzly has been observing and managing the population of California black bears. Unlike their phylogenetic cousins, black bear populations still exist in the state and are relatively stable. The department estimates that between 25,000 and 30,000 black bears occupy 52,000 square miles in California. There are three subpopulations of the bears which are recognized as the North Coast/Cascade, Sierra, and Central Western/Southwestern regions. Unsurprisingly, about half of the state-wide population of black bears resides in the North Coast/Cascade region [1]. 

In 2016, a population genetics study of California Black Bears was published by the CDFW in conjunction with the Wildlife Population Health and Genetics Laboratory at the UC Davis School of Veterinary Medicine. The study analyzed the Central Western subpopulation, specifically in Monterey and San Luis Obisbo counties and compared genetic samples to bears in Mono County, which is between Yosemite National Park and the border with Nevada. Research of this sort evaluates abundance of the species and the genetic diversity of small populations to predict migrating patterns, check for genetic bottlenecking and inbreeding, and to examine the overall strength of the genetic pool [3].

One way researchers acquired genetic material was through a hair sampling technique. Two rungs of barbed wire were tied around a circle of trees. At the center of this sample area was fish bait and a sweet scent bait made of honey and berries. As bears approached the bait, their hair would get caught in the wires. The spacing of each sample station was strategically determined from a grid design that considered habitable ranges for the bears and safe distances from human-related dangers such as roads and watch points. DNA extraction and further genotyping was used to identify unique individual bears. From there, most of the work came from computer-programmed statistical tests such as Bayesian genetic clustering algorithms to evaluate the population structure for each data range [3]. 

Researchers have come to believe that the central coastal population of bears has been in the region for about 50 years and are descendants of the population in the Sierra region that have migrated towards the coast. The report concluded that there are very few bears populating in Monterey County because there has not been enough time for them to disperse adequately within the coastal region. A potential reason for their slow migration towards Monterey County could be urbanization and the construction of highways that provide real physical barriers [3]. 

A 2009 study (Brown et al.) by researchers at the UC Davis School of Veterinary Medicine also analyzed the population genetics of California’s black bears, this time on a statewide scale. Using historical documents to track translocations of bears by humans, but also the analysis of microsatellite DNA from subpopulations of the bears, the researchers came to similar conclusions as the aforementioned CDFW report. It is believed that the extinction of the California grizzly, which roamed a significant portion of the state’s central coast, made room for the black bear to begin to inhabit regions such as Monterey and San Luis Opisbo [5]. Brown et al. used genetic samples from 540 bears, which were collected between 1990 and 2004 throughout the state. To determine the genetic structure of the California populations, the group used a computer program that “groups individuals into clusters based on genotype without consideration of sampling geography.” Following this, the determined clusters were tested for Hardy-Weinberg equilibrium, one of a handful of tests for genetic populations. This is an evaluation method that assumes no evolutionary changes are taking place in the genetic pool. Doing this makes it possible to simply analyze the allelic frequencies within the population without accounting for potential changes. 

Wildlife management is an increasingly important and difficult operation for any organization. Urbanization and global climate change will certainly become more prominent issues in black bear conservation. This is not even considering the laws on hunting or other policy-related challenges that may arise. The CDFW, in conjunction with researchers from around the state, has made a concerted effort to observe this population. If we are to learn a lesson from the past, it would be to not take for granted the abundance of the bear population in California. There has been a lot of excitement about the black bear’s proliferation, but our state will need to be attentive if we are to keep the health of our wildlife in balance. 

 

References

    1. California Department of Fish and Wildlife “Black Bear Biology” https://www.wildlife.ca.gov/Conservation/Mammals/Black-Bear/Biology
    2. Los Angeles Times “Column: Will the California Grizzly Make a Comeback?” https://www.latimes.com/opinion/op-ed/la-oe-arellano-grizzlies-20180718-story.html
    3. Sherman et al., “Population Genetics Study of California’s Black Bears” https://lpfw.org/wp-content/uploads/2017/02/Sherman-Ernest-CDFW-Final-Report-Population-Genetics-Study-of-California%E2%80%99s-Black-Bears.pdf
    4. UC Davis “Bear Caught in the Morning, Freed 5 Hours Later” https://www.ucdavis.edu/news/bear-caught-morning-freed-5-hours-later/ 
    5. Brown et al., “Black Bear Population Genetics in California: Signatures of Population Structure, Competitive Release, and Historical Translocation” Journal of Mammalogy https://doi.org/10.1644/08-MAMM-A-193.1

Genetically Engineered Crops: A Food Security Solution?

February 7, 2020

By Roxanna Pignolet, Biochemistry and Molecular Biology 20’

Author’s Note: Since I started working on plant metabolites as an undergraduate researcher in the Shih Lab, I’ve developed a great appreciation for the power of plant genetic engineering to address a wide variety of problems. A uniquely global and increasingly relevant concern is how to continue to feed the world’s growing population in the face of climate change. I decided to write this paper to provide a snapshot of the current research being done to innovate crop species that will survive in the face of climate change. As part of this review. I also wanted to address ongoing concerns about the safety and impact of GMOs on consumers and the environment, and whether these genetic engineering strategies have the potential to make a positive impact on food security.

 

Introduction

As the world population continues to rise, climate change is also having an increasingly large impact on agriculture in the form of rising temperatures and intensified weather variations. Population growth is challenging researchers and farmers to find new ways to increase crop yields without access to more land or freshwater. Population is expected to increase from the current 7.7 billion to 9 billion by 2050 (1,2). However, it was found in 2000 that about 70% of the available freshwater was already in use. Meanwhile, climate change is introducing new challenges to crop productivity and stability. By 2050, the global crop demand may increase as much as 110%, which emphasizes the need for new, powerful strategies for crop improvement.

Genetically engineered crops have been used in agriculture since the mid-1990s, and have been instrumental in overcoming serious agricultural challenges such as disease outbreaks and overuse of toxic insecticides (3). In contrast to traditional breeding, genetic engineering allows for a direct transfer of one or more genes of interest from either closely or distantly related organisms. In some cases, a plant is modified solely by turning on or off one of its own genes (4). These methods allow for fast and precise changes that target a specific trait. Since their introduction, numerous studies have measured their potential for health and environmental risks, as well as their benefits. This review will discuss the impacts of genetically engineered crops from an environmental and health perspective. Additionally, I will look at how genetically engineered crops are currently being applied to address food security concerns in the face of climate change.

 

What is the Impact of Genetically Engineered Crops?

Environment

As genetically engineered crops have now been used in the field for many years, the environmental impacts can be assessed. The most abundant type of genetically engineered crops are insect resistant crops, specifically Bacillus thuringiensis (Bt) resistant corn and cotton. Bt is a soil bacterium which produces proteins that are toxic to certain insects (5). Bt crops have been modified to produce Bt genes as protection against specific pests (3). These crops have been grown commercially since 1996 (2), which has allowed long term environmental studies to be conducted. In a two-year field trial on the impact of transgenic maize on soil fauna, Fan et al. found that there was no impact on biodiversity, abundance or composition of the soil fauna. They compared samples taken in varying conditions from either transgenic maize or non-transgenic maize controls. The researchers found that the insecticide transgene did not affect the soil ecosystem, while factors such as time of year, pH, sampling time, and root-biomass all had significant effects (6). In a 2003 review on Bt crops, Mendelsohn et al. also found that there were no negative impacts observed on species of endangered insects, earthworms, or non-target insects. However, one negative that applies to all insecticides is that pests will eventually gain resistance. Engineering crop varieties to have several different resistance genes has been shown to slow this process (2).

Another class of genetically modified crops that are currently in use are herbicide-tolerant crops. Herbicide-tolerant crops are designed to be tolerant to broad-spectrum herbicides that can be used to control surrounding weeds. Use of herbicide-tolerant corn and soybeans has been shown to decrease the use of highly toxic herbicide sprays in favor of an amino-acid derived, non-toxic alternative (Roundup), and has also encouraged low-till farming practices which have been correlated to significant reductions in greenhouse gasses (2). Weed resistance is a concern with herbicide-resistant crops, especially when a single herbicide gene is overused. In some cases, high selection pressures caused by overuse of a single broad-spectrum herbicide have led to resistant weeds. If unchecked, these resistant weeds can spread across farms and negatively impact crop growth (7). New varieties of crops resistant to multiple types of herbicides should help mitigate this problem by allowing farmers to rotate several types of herbicides. A widespread adaptation of these new varieties and consistent practice of sustainable herbicide application will be important to avoiding negative outcomes of herbicide-tolerant crop use.

Implementing these genetically engineered crops has contributed to overall decreases in the amount of toxic insecticide and herbicide sprayed. Just as with chemical pesticide and herbicide sprays, proper steps must be taken with insect-resistant or herbicide-resistant crops to delay resistance in the affected insect or weed. These steps include rotating planting of herbicide-resistant crops and using weed control tactics with different modes of action to avoid putting high selection pressure on one type of resistance.

Health

The consensus from long term studies carried out to address biosafety concerns of genetically modified crops, is that they are just as safe as their natural counterparts. Genetically engineered crops are subjected to a variety of tests on a case by case basis before they are implemented, and now long term data shows that there have been no side effects from possible unintended chemical compositions of crops, making them just as safe as those derived from traditional breeding. There are, however, concerns about next generation genetic engineering, which targets regulator genes instead of a single functional gene. Targeting regulator genes could allow scientists to target plant stress response pathways, and engineer plants to have multiple desirable traits (8). Additional research must be conducted to assess the plant-wide changes caused by affecting a player in a signaling cascade.

New Approaches to Crop Improvement

While the current genetically engineered crops have been found to have a positive effect on crop yields, the increases are not enough to keep up with projected population growth. Additionally, climate change is predicted to cause stressors to crops such as drought, rising temperatures, and weather variations among other things (2). Therefore, scientists are looking for new and creative genetic engineering techniques to create robust and high-yielding crops for our future.

One of the main targets for genetically engineered crops is adaptions to grow and produce quality yields under higher temperatures. In a study investigating the genes responsible for creating lower quality, chalky rice grains under high temperature conditions, Nakata et al. looked at the role of a starch metabolizing enzyme, known as amylase, in the packing of starch into rice grains. Their team used transgenic rice modified with a reporter gene attached to each isotype of the amylase gene. By comparing the activity of plants overexpressing each variety, they were able to identify specific amylase genes as targets for genetic modification. Rice variants with these modifications would remain higher quality, with tightly packed starch, even if grown under non-optimal higher temperatures (9). Another study tested the responses of a previously created transgenic rice line called HOSUT under high amounts of carbon dioxide (CO2), a heat wave, and nitrogen enriched conditions. They found that the transgenic line, which has enhanced sucrose transport, has a superior yield than the control line (Certo), and that increased CO2 conditions resulted in higher yields in Certo with only minimal increases for HOSUT. They concluded that the minimal response of HOSUT to the increased CO2 was indicative of HOSUT already being saturated due to its optimized transport capabilities. The HOSUT line is already optimized for translocation of carbon, which they were able to show by increases in starch in the grains in HOSUT only. HOSUT also produced more yield in response to increased nitrogen, making it a good option for producing high rice yields under variable climate change conditions (10) The HOSUT line is a great example of how genetic engineering can be used to fortify and optimize crops to both survive under atypical conditions and produce enough yield to keep up with demand.

Another problem that researchers are addressing through genetic engineering, is drought. Selvaraj et al., developed and field tested two drought tolerant rice lines, created by introducing an Arabidopsis stress response gene (galactinol synthase) with a maize promoter. Galactinol synthase produces galactinol, a sugar that functions as an osmoprotectant, keeping water from leaving the cells. These galactinol synthase genes were introduced into two commercially available rice lines and tested in the field under drought and well-watered conditions. Under drought conditions, the collection of galactinol resulted in higher grain yields, while under well-watered conditions no significant yield increase was observed. Galactinol is a sugar that functions as an osmoprotectant, keeping water from leaving the cells. The results of these field trials show that these rice lines are ready to be integrated into ongoing breeding programs (11). Wang et al. also tackled the problem of drought stress caused by global warming on fruit such as apple trees. They transgenically expressed an aquaporin gene found in Fuji apples that has increased expression during fruit growth in tomato. The transgenic plants did have an increased drought tolerance, observed as an increased sensitivity of their stomata to water loss, and a larger fruit size when compared to wild type. This research will be continued in apples next with the goal of producing plants with larger fruits when well-watered, which will also be more tolerant to drought due to increased water transport efficiency (12).

A third target for genetic engineering solutions is circadian rhythms. Understanding and controlling circadian rhythms in crop plants has the potential to adapt plants to radically different environments. One group at the Guru Jambheshwar University of Science and Technology is tackling this challenge in rice. This group expressed an Arabidopsis transcription factor known as Circadian Clock Associated1 (CCA1) under the Timing Of Cab Expression 1 (TOC1) promoter, which are both part of the circadian clock machinery in Arabidopsis. They found that overexpression of the CCA1 in rice had negative results, while repressing it caused positive changes to plant morphology. The researchers used RNAi, which is a biological process where small fragments of RNA are used by the cell to target complementary mRNA for destruction, thus silencing expression of the encoded protein. By comparing RNAi constructs based off of three different parts of the CCA1 gene for silencing the gene expression, they found that the RNAi derived from the 3’-terminal end of the CCA1 gene had the best impact on plant morphology (13). This study is an important first step towards unlocking the power of using circadian clock genes to breed plants better adapted to a changing environment.

One new strategy being considered is a CRISPR/Cas9 genome editing method that could be used to quickly develop improved crop varieties without transgenes. CRISPR/Cas9 can introduce specific changes into a plant genome without being limited by existing variation. Applying this method, scientists will be able to stack multiple edits into a plant within a single generation, resulting in transgene-free progeny. One benefit of this method is that it may allow for more complex changes to polygenetic traits or signaling pathways. For example, this could be helpful for targeting complex plant stress response pathways. This technology is currently limited by the availability of annotated reference genome sequences for plants other than Arabidopsis. Scheben et al. suggest that taking a genomics-based approach would allow for a comparison of species-wide genome diversity, making differences in copy-number visible and thus available for editing. While the authors suggest that this method creates plants that are indistinguishable from those created through natural breeding and random mutations, bans against genetically modified crops may target methodologies rather than the final result (14).

 

Conclusion

Currently implemented genetically engineered crops, have been shown, through years of testing and trials to be at least as safe, both towards the environment and in terms of human health, as naturally bred varieties. While new transgenic lines must be screened and tested on a case-by-case basis, the overall benefits of this technology make it an important tool that may be necessary to confront upcoming challenges to agriculture. Climate change and population growth are putting steep demands on crops to survive in more hostile environments while also producing higher yields. Current efforts are focusing on vital crops, such as rice, corn, wheat, and fruits, to create drought-tolerant, heat-tolerant, and yield-optimized plants.

 

References

  1. “World Population Clock: 7.7 Billion People (2019) – Worldometers.” n.d. Accessed November 18, 2019. https://www.worldometers.info/world-population/.
  2. Ronald, Pamela. 2011. “Plant Genetics, Sustainable Agriculture and Global Food Security.” Genetics; Bethesda 188 (1): 11–20.
  3. Mendelsohn, Mike, John Kough, Zigfridais Vaituzis, and Keith Matthews. 2003. “Are Bt Crops Safe?” Nature Biotechnology 21 (9): 1003–9. https://doi.org/10.1038/nbt0903-1003.
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  5. “Bacillus Thuringiensis (Bt).” n.d. Accessed November 18, 2019. http://npic.orst.edu/ingred/bt.html.
  6. Fan, Chunmiao, Fengci Wu, Jinye Dong, Baifeng Wang, Junqi Yin, and Xinyuan Song. 2019. “No Impact of Transgenic Cry1Ie Maize on the Diversity, Abundance and Composition of Soil Fauna in a 2-Year Field Trial.” Scientific Reports 9 (1): 1–9. https://doi.org/10.1038/s41598-019-46851-z.
  7. Resources, University of California, Division of Agriculture and Natural. n.d. “Herbicide Tolerance.” Accessed November 18, 2019. http://sbc.ucdavis.edu/Biotech_for_Sustain_pages/Herbicide_Tolerance.
  8. Ortiz, R., Andy Jarvis, P. Fox, Pramod K. Aggarwal, and Bruce M. Campbell. 2014. “Plant Genetic Engineering, Climate Change and Food Security.” Working Paper. CGIAR Research Program on Climate Change, Agriculture and Food Security (CCAFS). https://cgspace.cgiar.org/handle/10568/41934.
  9. Nakata, Masaru, Yosuke Fukamatsu, Tomomi Miyashita, Makoto Hakata, Rieko Kimura, Yuriko Nakata, Masaharu Kuroda, Takeshi Yamaguchi, and Hiromoto Yamakawa. 2017. “High Temperature-Induced Expression of Rice α-Amylases in Developing Endosperm Produces Chalky Grains.” Frontiers in Plant Science 8. https://doi.org/10.3389/fpls.2017.02089.
  10. Weichert, Heiko, Petra Högy, Isabel Mora-Ramirez, Jörg Fuchs, Kai Eggert, Peter Koehler, Winfriede Weschke, Andreas Fangmeier, and Hans Weber. 2017. “Grain Yield and Quality Responses of Wheat Expressing a Barley Sucrose Transporter to Combined Climate Change Factors.” Journal of Experimental Botany 68 (20): 5511–25. https://doi.org/10.1093/jxb/erx366.
  11. Selvaraj, Michael Gomez, et al. “Overexpression of an Arabidopsis Thaliana Galactinol Synthase Gene Improves Drought Tolerance in Transgenic Rice and Increased Grain Yield in the Field.” Plant Biotechnology Journal, vol. 15, no. 11, Nov. 2017, pp. 1465–77. PubMed, doi:10.1111/pbi.12731.
  12. Wang, Lin, Qing-Tian Li, Qiong Lei, Chao Feng, Xiaodong Zheng, Fangfang Zhou, Lingzi Li, Xuan Liu, Zhi Wang, and Jin Kong. “Ectopically Expressing MdPIP1;3, an Aquaporin Gene, Increased Fruit Size and Enhanced Drought Tolerance of Transgenic Tomatoes.” BMC Plant Biology 17, no. 1 (December 19, 2017): 246. https://doi.org/10.1186/s12870-017-1212-2.
  13. Chaudhury, Ashok, Anita Devi Dalal, and Nayan Tara Sheoran. 2019. “Isolation, Cloning and Expression of CCA1 Gene in Transgenic Progeny Plants of Japonica Rice Exhibiting Altered Morphological Traits.” PLOS ONE 14 (8): e0220140. https://doi.org/10.1371/journal.pone.0220140.
  14. Scheben, Armin, Felix Wolter, Jacqueline Batley, Holger Puchta, and David Edwards. 2017. “Towards CRISPR/Cas Crops – Bringing Together Genomics and Genome Editing.” New Phytologist 216 (3): 682–98. https://doi.org/10.1111/nph.14702.

Idiopathic Pulmonary Fibrosis (IPF): PHMG-P and Other Disinfectant-associated Chemicals as Potential Causes, the Mechanism, and Potential Treatments

January 11, 2020

By Téa Schepper, Biological Sciences ‘19

Author’s Note
I would like to give special thanks to Professor Katherine Gossett (UC Davis) for encouraging me to write this paper and Dr. Angela Haczku (UC Davis Health) for her expertise in pulmonary diseases. Last fall, I decided to research idiopathic pulmonary fibrosis after my grandfather was hospitalized and diagnosed with it over the previous summer. I quickly discovered that there wasn’t much research on the disease itself or how to treat it due to its rarity. The purpose of this literature review is to inform others about idiopathic pulmonary fibrosis and to encourage further research on the subject. With time, this research could be vital in saving lives just like that of my grandfather.

Abstract
Idiopathic pulmonary fibrosis (IPF) is an irreversible and fatal disease of the lungs. Although it has been associated with genetic predisposition, cigarette smoking, environmental factors (e.g. occupational exposure to gases, smoke, chemicals, or dusts) and other conditions such as gastroesophageal reflux disease (GERD), the mechanism and causes of IPF are not yet fully understood by researchers. However, recent studies have provided evidence that IPF may be caused by the generation of reactive oxygen species (ROS) due to the inhalation of chemicals commonly found in household disinfectants. These chemicals have been identified as polymethylene guanidine phosphate (PHMG-P), didecyldimethylammonium chloride (DDAC), polyhexamethylene biguanide (PHMB), oligo(2-(2-ethoxy)-ethoxyethyl) guanidinium chloride (PGH), and a mixture of chloromethylisothiazolinone (CMIT) and methylisothiazolinone (MIT). It has been suggested that the generation of ROS by these chemicals is responsible for damaging the cellular structures of the lungs and triggering the development of IPF through the activation of the transforming growth factor β (TGF-β) signaling pathway. Studies have also shown microRNAs to be key regulators of the TGF-β pathway and the development of the disease. Several promising future treatments of IPF involve the inhibition of the TGF-ꞵ signaling pathway either through the administration of drugs containing sesquiterpene lactones, matrine, or oridonin compounds; or through the replenishment or inhibition of certain miRNAs. The studies detailed here highlight the importance of further research on IPF.
Keywords: idiopathic pulmonary fibrosis | PHMG-P | TGFβ | miRNA

Introduction
Pulmonary fibrosis is an irreversible, fatal disease that results in scarring of the lung tissue and decreased function of the lungs. Idiopathic pulmonary fibrosis simply means that the cause is unknown. Patients with IPF typically experience difficulty breathing, with death caused by either respiratory failure or incurrent pneumonia. [1] The disease is characterized by marked collagen deposition and other alterations to the extracellular matrix (ECM), a network of macromolecules that provide structural support to the lungs. [1] These alterations to the ECM remodel and stiffen the lung’s airspaces and tissues. [1] It is also characterized by diffuse interstitial inflammation and respiratory dysfunction. [2] Although its cause remains unknown, it is believed that the main steps in the pathogenesis of IPF are initiated by the transforming growth factor β (TGF-ꞵ) signaling pathway and involves the migration, proliferation, and activation of lung fibroblasts and their differentiation into myofibroblasts. [3] Fibroblasts are cells that have a high ability to proliferate and to produce ECM and fibrogenic cytokines. [3] Fibrogenic cytokines are multifunctional immunoregulatory proteins that contribute to the inflammatory cell recruitment and activation needed to promote the development of fibrosis. [4] These cytokines can activate myofibroblasts, which are primarily responsible for the synthesis and excessive accumulation of ECM components, collagen and fibronectin, during the repair process that leads to fibrosis. [5], [6]

A 2011 outbreak of pulmonary fibrosis in South Korea prompted an onslaught of research as to how IPF may be caused and treated. [7] Specifically, this research has provided evidence that certain chemicals commonly found in household disinfectants can cause IPF through the generation of reactive oxygen species (ROS). ROS have a powerful oxidizing capability that can induce the destruction of cellular and subcellular structures in the lung, including DNA, proteins, lipids, cell membranes, and mitochondria. [8] This damage caused by ROS has been found to promote the activation of the TGF-ꞵ signaling pathway and the development of numerous characteristics associated with IPF. [4] This research has been invaluable for the discovery of new potential treatments for patients with IPF.

Potential inducers of idiopathic pulmonary fibrosis
After the 2011 outbreak in South Korea, researchers were able to find a connection between IPF and exposure to chemicals commonly found in household disinfectants, such as those found in humidifiers and pools. They have suspected that these chemicals can cause pulmonary fibrosis by infiltrating the respiratory system as aerosol particles to induce cellular damage. The chemicals polymethylene guanidine phosphate (PHMG-P), didecyldimethylammonium chloride (DDAC), polyhexamethylene biguanide (PHMB), oligo (2-(2-ethoxy) ethoxyethyl guanidinium chloride (PGH), and the mixture of chloromethylisothiazolinone (CMIT) and methylisothiazolinone (MIT) attracted particular interest.

In a study evaluating registered lung disease cases in South Korea, it was revealed that 70 percent of registered patients that suffered from IPF or other forms of household humidifier disinfectant-associated lung injury had used humidifier disinfectants containing the chemicals PHMG, PGH, or a mix of CMIT and MIT prior to their development of the disease [7] It was determined that the aerosol water droplets emitted by the humidifiers may have acted as carriers to deliver these chemicals into the lower part of the respiratory system, causing humidifier disinfectant-associated lung injury. [7] It was also revealed that most of the affected patients in the study had used humidifier disinfectant containing the chemical PHMG. [7]

Another study detailed that even slight exposure to PHMG could cause cell death triggered by the generation of reactive oxygen species (ROS). [8] Injury by ROS is typically followed by a fibrotic repair process involving increases in TGF-ꞵ expression, increased fibronectin, collagen synthesis, and a marked increase in the deposition of the ECM, all key characteristics of IPF. [4]

One way that ROS promote ECM deposition and IPF is by activating transcription factors like nuclear factor kappa B (NF-κB). [4] NF-κB is a regulator of proinflammatory cytokines that is typically bound to a cytoplasmic inhibitor. [9] One study found that exposure to the biocide (substance that destroys/prevents growth in organisms) and preservative PHMB was able to generate significant ROS levels and activate the NF-κB signaling pathway through the degradation of its inhibitor. [10] This is significant because the activation of proinflammatory cytokines is necessary for the recruitment and activation of myofibroblasts responsible for the increased ECM deposition that is characteristic of IPF patients. PHMB is also a cationic chemical and there is evidence that it can bind to negatively charged mucins, located within the mucous membranes of various organs. This can cause organs located in the respiratory tract to acquire increased susceptibility to PHMB and, in effect, a higher likelihood for the development of IPF. [10] Although the study did not match the exposure conditions of PHMB in humans, it has illuminated another way that individuals may develop IPF. [10]

In a study investigating the role of DDAC—one of the aerosols— in causing pulmonary fibrosis, mice exposed to DDAC exhibited fibrotic lesions that increased in severity over time. [11] Exposure to the chemical DDAC increased TGF-β signaling and appeared to maintain the differentiation of myofibroblasts. [11] This was complemented by the high expression of genes responsible for the production of collagen in fibrogenic lungs. [11] Overall, the form of pulmonary fibrosis that was induced by DDAC was mild, and so more research must be conducted before it can be concluded that the chemical DDAC is responsible for irreversible, severe pulmonary fibrosis. [11] It is also possible that some of the patients affected with humidifier disinfectant-associated lung injury may have experienced synergistic or additive effects from using multiple humidifier disinfectants, but this can be difficult to determine. [7] However, this study does indicate that exposure to DDAC can result in the development of several characteristics typically associated with IPF.

PHMG-P as a potential causative of IPF
Of the chemicals listed in this literature review, PHMG-P has received the most attention by researchers. PHMG-P is a biocide that exhibits its antibacterial effect by disrupting the cell wall and inner membrane of bacteria, causing cellular leakage. [12] In a similar manner, PHMG-P can infiltrate the lungs in the form of aerosol particles and may cause IPF in individuals through the generation of ROS and the disruption of the ECM’s alveolar basement membrane. [4]

Disruption of the basement membrane occurs through increased expression of matrix metalloproteinases (MMPs), enzymes that degrade various components of connective tissue matrices. [6] Metalloproteinase MMP2, in particular, destroys the basement membrane by solubilizing ECM elastin, fibronectin, and collagen, helping immune cells and fibroblasts migrate to alveolar spaces. [12] This can lead to severe damage of the lung architecture and aberrant ECM deposition typical of IPF. [4]

In a study using an air-liquid interface (ALI) co-culture model to study the pathogenesis of fibrosis, PHMG-P was shown to trigger ROS generation, airway barrier injury, and inflammatory response. [4] Recall that exposure to other chemicals suspected of being potential inducers of IPF had similar effects. Therefore, it can be concluded that PHMG-P infiltrates the lungs in the form of aerosol particles and induces airway barrier injury by ROS. [4] This would result in the release of fibrotic inflammatory cytokines and trigger a wound-healing response that would eventually lead to pulmonary fibrosis. [4]

In an animal study, mice exposed to PHMG-P experienced difficulty breathing and exhibited pathological lesions similar to the pathological features observed in humans affected with IPF. [12] A time course of 10 weeks was even established for PHMG-P-induced pulmonary fibrosis. [12] Throughout this period, it was found that a single instillation of PHMG-P contributed to an increase in proinflammatory cytokine levels and elicited an influx of inflammatory cells into lung tissue. [12] This recruitment of inflammatory cells contributes to the deposition of ECM components in the lungs and, as a result, the development of IPF. The instillation of PHMG-P was also suspected of blocking T cell development and impairing its function in the immune system. [6] This would result in an insufficient resolution of inflammation caused by the increased levels of proinflammatory cytokines and result in stacked fibrotic changes and the progression of IPF. [6]

Another study claimed that PHMG-P could cause pulmonary fibrosis through the activation of the NF-κB signaling pathway. [9] Recall that the NF-κB signaling pathway is responsible for the production of proinflammatory cytokines associated with the development of IPF. According to the study, mice exposed to PHMG-P generated a large amount of ROS and produced significant levels of the cytokines IL-1β, IL-6, and IL-8 in a dose-dependent manner. [9] These cytokines produced by the NF- κB signaling pathway are known to activate the TGF-β signaling pathway, increase collagen production, and promote wound-healing and tissue remodeling responses. [4] As these responses are characteristic of IPF and the cytokines exhibited in this study are known to be produced through the activation of the NF-κB signaling pathway, there is strong evidence that PHMG-P can induce IPF through the NF-κB signaling pathway.

The Mechanism of IPF
TGF-β’s importance in the mechanism
Various studies of IPF have indicated that transforming growth factor β (TGF-β), one of the most significant fibrotic cytokines, plays a key role in the mechanism that induces IPF. TGF-β1 is credited with inducing the differentiation of fibroblasts to myofibroblasts and upregulating the secretion of ECM proteins (like collagen) in IPF. [13]

Specifically, growth factor TGF-β1 binds directly to the TGFβ receptor II (TGFβRII), triggering the recruitment and activation of receptor TGFβRI by TGFβRII. [14] This step leads to the increased production of collagen through the activation of a collection of proteins called the Smad 2/3 complex. [13] The activated Smad 2/3 complex accomplishes this by entering the nucleus to enhance the transcription of profibrotic genes such as those that produce collagen. [13] This idea has been heavily supported by experimental evidence. Exposure to the chemical DDAC was found to increase cellular mRNA levels of TGF-β1 by two-fold. [11] This increase contributed to the activation of the Smad 2/3 complex [11] and induced the differentiation of fibroblasts to myofibroblasts. [15] Overall, this led to the development of pulmonary fibrosis-causing fibrotic lesions in mice. [11]

In another study, TGF-β was found to promote the development of IPF by inhibiting the expression of the microRNA let-7d, driving epithelial-mesenchymal transition (EMT) and increased collagen deposition. [1] Typically, epithelial cells are important to maintaining lung functionality by acting as a barrier against pathogens and other harmful compounds and secreting protective substances. [4] During EMT, however, these cells increase in cellular motility [16] and are transformed into myofibroblasts, resulting in the acceleration of IPF. [4] Additionally, epithelial cells during EMT promote the recruitment of fibroblasts, while simultaneously inhibiting collagen degradation and elevating the levels of the tissue inhibitor of metalloproteinase 1 (TIMP-1). [4] TIMP-1 binds to metalloproteinase MMP2 to promote the growth of fibroblasts and myofibroblasts, accelerating ECM deposition while preventing its degradation. [12] This corroborates the claim that the TGF-β signaling pathway is a crucial component in the mechanism of IPF.

MicroRNA’s role in TGF-β regulation and pulmonary fibrosis
MicroRNAs are mRNA sequences that bind to complementary mRNA of proteins to prevent their translation and expression. They are also involved in multiple steps of fibrosis, such as cell proliferation, apoptosis, and differentiation. [16] During the progression of IPF, miRNAs are known to regulate the process in which epithelial cells transition into myofibroblasts (EMT) to promote fibrosis. [16] Since each miRNA is specific to a particular mRNA sequence, miRNAs may function as either promoters or inhibitors of IPF. One study found that the miRNA, miR-433, can act as a promoter of IPF by upregulating receptor TGFβRI and growth factor TGF-β1 to amplify TGF-β signaling. [13] In a separate study, it was confirmed that miR-30c-1-3p may act as a negative regulator of pulmonary fibrosis through targeting the mRNA and preventing the expression of receptor TGFβRII. [15]

In a study headed by the Department of Pathology at the University of Michigan Medical School, it was concluded that the development and pace of progression of IPF may be due to abnormal miRNA generation and processing. [1] It was found that in rapidly progressing IPF biopsies, five miRNAs significantly increased and one decreased when compared to slowly progressive biopsies. [1] This indicates that miRNAs have a significant influence on the mechanism of IPF. Additionally, members of the miR-30c and let-7d family significantly decreased in both forms of IPF when compared with unaffected individuals. [1] As stated previously, certain members of the miR-30c family are believed to be negative regulators of IPF and members of the let-7d family are inhibitors of EMT. All of the stated evidence signifies that miRNAs, in addition to the TGF-β signaling pathway, play important roles in the development of IPF.

Other factors to consider in the mechanism
The NALP3 inflammasome is another important factor to consider in the mechanism of IPF. The NALP3 inflammasome is an innate immune system receptor suspected of being the main cause of persistent inflammatory response and exacerbation of fibrotic changes. [12] According to a study focused on researching PHMG-P-induced fibrosis in mice, the activation of the NALP3 inflammasome appeared to contribute to fibroblast proliferation and the progression of IPF due to the production of the cytokine IL-1β. [12] IL-1β is known to increase the production of ROS needed to induce lung tissue damage by upregulating the expression of the cytokine chemokine (C-X-C motif) ligand 1 (CXCL1). [6] This upregulation of CXCL1 and resulting tissue damage was exhibited in the study, reinforcing the claim that the NALP3 inflammasome is a central component in the IPF mechanism. [12]

Secretory immunoglobulin A (sIgA), an antibody that has an important role in the immune system, also may have a role in the mechanism of pulmonary fibrosis. In a study supported by the Japan Society for the Promotion of Science, immunoglobulin A, the most abundant human immunoglobulin, was compared with TGF-β in its role in inducing pulmonary fibrosis and inflammation. [3] In this study, sIgA enhanced collagen production and induced responses in cytokines IL-6 and IL-8, and monocyte chemoattractant protein 1 (MCP-1). [3] MCP-1, similar to IL-6 and IL-8, is responsible for stimulating collagen synthesis and TGF-β production in fibroblasts. [6] It was concluded that under IPF, sIgA may make contact with lung fibroblasts and result in exacerbating airway inflammation and fibrosis through enhancing the production of inflammatory cytokines and ECM collagen. [3]

Potential therapeutic approaches and alternative methods of treatment
According to recent studies, only two drugs, pirfenidone and nintedanib, have been approved by the FDA for IPF treatment, and they have still failed to be significantly effective in treating the disease. [13] However, current studies on therapeutics that inhibit the TGF-β signaling pathway appear promising. Two particular drugs of interest are oridonin and matrine, along with their derivatives.

Oridonin, a major compound found in the herb Rabdosia rubescens, has been used in traditional Chinese medicine to treat inflammation and cancer for hundreds of years. [2] In a study focused on testing its effectiveness in treating IPF, it was found that exposure to oridonin significantly decreased the levels of three major biomarkers of fibrosis—hydroxyproline (HYP), beta silicomolibdic acid (β-SMA), and collagen, type 1, alpha 1 (COL1A1)—in a dose-dependent manner. [2] Additionally, oridonin attenuated pathological changes such as alveolar space collapse and infiltration of inflammatory cells. [2] Oridonin was able to achieve this through significantly inhibiting the upregulation of collagen production and the activation of Smad 2/3 in lung tissues, an important step in the progression of IPF through the TGF-β signaling pathway. [2] This presents a strong case for the use of oridonin as a treatment for IPF.

Matrine, similar to oridonin, also has roots in traditional Chinese medicine. Matrine has been shown in several studies to exhibit significant antifibrotic effects through the inhibition of the TGF-β pathway. In one study, matrine was shown to have an inhibitory effect against liver fibrosis by reducing the expression of TGF-β1 and instead increasing the expression of hepatocyte growth factor (HGF). [13] Through the inhibition of the TGF-β/Smad pathway, matrine was also shown to exhibit antifibrotic activities on cardiac fibrosis. [13] These antifibrotic effects are not just held by matrine, but their derivatives as well. The matrine derivative MASM was also shown to exhibit potent antifibrotic effects. [13] As the TGF-β signaling pathway is a central component in the mechanism of IPF, matrine and their derivatives present themselves as strong candidates for anti-IPF therapeutics.

Other drug candidates for the treatment of IPF are sesquiterpene lactones. Sesquiterpene lactones are naturally occurring compounds that are known to harbor extensive connections with the TGF-β1 signaling pathway. [5] This makes these compounds and their analogues strong drug candidates for IPF treatment. In one study, two out of 44 semi-synthetic analogues of sesquiterpene lactones were found to highly inhibit the TGF-β1 signaling pathway, ECM production, and the formation of fibroblasts. [5] This inhibition of ECM production and the formation of fibroblasts corroborates the claim that administering sesquiterpene lactones is an effective treatment for IPF.

As mentioned earlier, studies have shown microRNAs to be negative regulators of IPF. One study suggests that the replenishment of miR-30c may be a promising treatment. [15] Increased levels of miR-30c would promote the negative regulation of the TGF-β signaling pathway, suppressing the differentiation of myofibroblasts and preventing excessive collagen accumulation. In this manner, the replenishment of miR-30c would attenuate IPF symptoms. The inhibition of certain miRNAs, such as miR-34a, has also been shown to be an effective treatment. The inhibition of miR-34a by treatment with the caveolin-1 scaffolding domain peptide (CSP) was found to prevent pulmonary fibrosis by preventing the overgrowth of fibroblasts. [17] Although the manipulation of miRNA expression has been shown to have a large impact on the development of IPF, there is one issue with this method of treatment. A single miRNA can target thousands of mRNAs, making the function miRNAs have in pathophysiological events involved in IPF unclear. [17]

Conclusion
Although there is limited research on the etiology of IPF, this should only serve to motivate researchers to study its causes, mechanism, and potential treatments further. Thus far, the chemicals that have been shown to be potential inducers of IPF are PHMG-P, DDAC, PHMB, PGH, and the mixture of CMIT and MIT. However, out of all the chemicals, only PHMG-P has been heavily researched, and so additional studies are needed to confirm the other chemicals’ involvement in inducing IPF. Additionally, this research could be expanded upon through the study of the effects of other household disinfectants on the human body to determine whether they are also factors in inducing IPF. Besides the discovery of potential causatives, studies have also further illuminated details about the mechanism. Specifically attracting interest is the TGF-β signaling pathway in addition to miRNAs and their involvement in the regulation of IPF. Furthermore, the manipulation of TGF-β and miRNA levels with oridonin, matirne, and sesquiterpene lactones has been linked to favorable outcomes in the treatment of IPF. With further research, these treatments could become common practice and improve the quality of life for patients suffering from IPF.

 

References

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