MORE THAN SCI FI: ASTROBIOLOGY’S SEARCH FOR ALIEN LIFE

FIONA MURPHY `23

Figure 1 A representation of planets and stars beyond Earth — the subjects of astrobiology’s search for extraterrestrial life.

Since humanity acquired the knowledge of an expansive universe beyond Earth, many have wondered about the possibility of life existing on other planets. The idea of alien existence captivates humans, excites our creative faculties, and even provokes our deepest fears. Humankind’s widespread fascination with extraterrestrial life is clearly exemplified in popular culture. One only has to look to iconic movies like E.T. the Extra-Terrestrial, beloved literature like The Hitchhiker’s Guide to the Galaxy, or even the growth of conspiracy theories around topics like Area 51 to understand the enamor and curiosity of humans with foreign life. For many, extraterrestrial life is an interesting possibility to consider, but a seemingly impossible reality to prove. Combining the limitless quality of the universe with humankind’s limited technological capabilities makes discovering unfamiliar life forms an extremely difficult task. However, there are scientists across diverse disciplines who study the potential for life outside of Earth and the methods by which this life can be confirmed. Through the possible discovery of extraterrestrial life and the conditions it requires, humanity can hopefully learn more about the origins of our own unique existence in addition to discovering future planets that may be hospitable for human life.

Habitable Zone

Because Earth has optimal conditions for supporting life, researchers reduced Earth’s qualities to basic criteria that other planets must meet in order to potentially host alien life. One foundational requirement a planet

must meet is the ability to sustain surface water, particularly in liquid form. In the presence of water, which is essential for metabolic processes, carbon-based organisms undergoing photosynthesis can survive and multiply enough to be detected. This requirement allowed researchers to establish the habitable zone (HZ), or the area around a Sun-like star where a planet typically must be located to maintain liquid water, and thus life (1). To provide a clearer definition of the inner limit of the HZ, Dr. James Kasting and his colleagues at Pennsylvania State University classified three main parameters — runaway greenhouse, moist greenhouse limit, and recent Venus limit — based on a planet’s ability to preserve liquid water (1). The inner limit of the HZ is restricted by a planet’s proximity to its star; if a planet is too close, extreme temperatures will cause the surface water to evaporate. It is highly likely that at temperatures above 647 K, the critical temperature of water (Tw), or the temperature at which there is no difference between liquid and gaseous water, any ocean will completely evaporate. With this knowledge, Kasting and his associates determined that temperatures above Tw indicate a runaway greenhouse climate, where a planet will be unable to host life indefinitely as its surface water will eventually evaporate into space. Additionally, they defined the moist greenhouse limit as a planet whose climate has temperatures below Tw, and yet is still too warm to prevent total evaporation. Kasting and his colleagues made this distinction by reasoning that on planets with atmospheric pressures comparable to Earth’s, these temperatures can cause the saturation mixing ratio of water, or air’s capacity to hold water before condensation, to increase to high levels. As a result, greater amounts of gaseous water are found in the stratosphere, an altitude at which water cannot condense (1). Finally, Kasting and his colleagues identified the recent Venus limit based on evidence suggesting that Venus once contained bodies of water that evaporated due to proximity to the Sun at least 1 billion years ago. With this information, the researchers postulated that an orbital path of 0.75 astronomical units (AU), 4% larger than Venus’s orbital path, is a likely minimum distance that a planet must be from its star to maintain surface water (1). The Kasting group also defined the outer limit of the HZ based on the assumption that a planet hosting life must also contain large amounts of carbon, mainly in the form of carbon dioxide (CO2). CO2, an important greenhouse gas, helps maintain the temperature of a planet by trapping energy from the neighboring star in the planet’s atmosphere. The maximum greenhouse limit defines the outer limits of the HZ. At this maximum distance away from a planet’s energy source, it is reasoned that atmospheric CO2 allows for the lowest intake of solar energy required to maintain an optimal 273 K temperature of the planet. Using previously established concepts, Kasting and his group understood that beyond this maximum distance there will be a high albedo due to Rayleigh scattering — the former defined as the amount of light that is reflected by a surface and the latter as the scattering of light by small particles such as CO2. With this principle in mind, the group was able to conclude that more light will be reflected by CO 2 than is trapped inside the planet, and thus planets outside of the outer limit will begin to cool to temperatures incapable of supporting life (1). To obtain a more accurate estimate of the number of life-supporting planets, Dr. Amri Wandel of Hebrew University of Jerusalem implemented the Drake equation and its parameters in his own calculations and statistical analyses. One important factor of the Drake equation remains unknown — Fb, or the probability of life developing on a planet in the HZ. Statistically, this factor can take on any value because Earth is currently the only confirmed planet to host life. However, Wandel suggests that this parameter could fall in the range of 10% to 100%, anticipating that current research endeavors searching for exoplanets containing life markers are successful (2). Coupled with the assumption that life can develop on planets orbiting red dwarf stars (smaller and more abundant than Sun-like stars), Wandel proposes that there may be between 109 and 1010 feasible life-supporting planets in the galaxy, indicating that the nearest one could be around 10 light-years away (2). To determine how many alien civilizations may exist, the Drake equation requires two more factors: the probability that life develops into a complex civilization capable of communication through radio signaling and the longevity of a civilization with such capabilities. The communicative property of radio signaling is necessary for determining the likelihood of detecting such a distant civilization. Based on the extremely low probabilities of these additional Drake equation parameters, Wandel posits that the nearest complex alien civilizations may be several thousand light-years away (2).

Exoplanet Discovery

To confirm theories of alien existence, researchers are currently seeking to discover life-supporting exoplanets in the HZ. NASA’s Kepler Mission, which ran from 2009 to 2013, made great advancements in the area of exoplanet discovery within the Milky Way galaxy. The Kepler spacecraft detected exoplanets within the HZ of their neighboring star via the “transit method,” monitoring for a decrease in the light intensity of a star, indicating that a planet is passing in front of it. By assessing the size of the planet’s orbital path, the size of the planet, and its temperature, NASA researchers determined whether the planet fell in the HZ and could host life (3). Over the course of the Kepler mission, 2,394 exoplanets were discovered and 391 of which were deemed either candidate or verified exoplanets in the HZ of their star (4).

Nearest Neighbors

Scientists determine the possible existence of nearby life-sustaining planets using the Drake equation — Nc = R*F s

Fp F e nhzFbF c

Lc. The Drake equation considers various factors like the rate of star formation in the Milky Way, the proportion of stars with planets, and the number of planets with environments capable of supporting life, among others, to loosely estimate the number of alien civilizations existing in our galaxy. Modified versions of the equation approximate the number of planets that host any form of life (2).

Figure 2 NASA’s Kepler telescope, which made breakthrough advancements in exoplanet discovery during its four-year run, identifying 2,394 exoplanets. Today, mechanisms such as TESS, or the Transiting Exoplanet Survey Satellite, continue the work begun by the Kepler mission. Initiated in 2018, TESS takes a similar approach to the Kepler spacecraft; it searches for a plunge in a star’s brightness to reveal an orbiting planet. TESS also

has technological features that make it more sensitive to red wavelengths, granting the satellite an improved ability to observe red dwarf stars and any orbiting exoplanets. The satellite is anticipated to identify upwards of one thousand exoplanets, some projected to be Earth-sized (5).

Figure 3 TESS, the Transiting Exoplanet Survey Satellite, orbiting in space, as it searches for exoplanets as they pass in front of distant stars.

Biomarker Detection

To further confirm the existence of extraterrestrial life, biomarkers, or the organic molecules found in and produced by living organisms, may be used. In the past, researchers identified biomarkers via pyrolysis-GC-MS, wherein molecules are heated at extreme temperatures and then detected through gas chromatography and mass spectrometry. However, scientists could only detect very small organic molecules that are also produced via abiotic processes, rendering any results as inconclusive. Additionally, the interactions that occurred during pyrolysis-GC-MS were highly volatile, causing the molecules to decompose, making detection difficult (6). In recent years, Spanish Astrobiology Center researcher Victor Parro and his colleagues devised new approaches to biomarker detection, such as the Signs of Life Detector (SOLID) chip, which utilizes immunoassay technology. Immunoassays use sensitive antibodies to identify molecules in low concentrations (7). In general, small lab-on-chips like SOLID are more effective as they do not cause volatile interactions, and can detect both small and large organic molecules, including cells. One molecule of interest for discovery is ATP, or adenosine triphosphate, which quickly deteriorates outside of a living organism and is thus a good indicator of the presence of life (6). When designing technologies to be used in space exploration, scientists must consider that resources are limited on a spacecraft. Instruments used to conduct biomarker detection must be small and energetically efficient. An issue with current chips is that their use of fluorescence detection methods requires external equipment to be operated, which takes up space and power. To solve these issues, Dr. Augusto Nascetti and his fellow researchers at Sapienza University of Rome have recently designed the Planetary Life Explorer with Integrated Analytical Detection and Embedded Sensors (PLEIADES). This chip utilizes chemiluminescence detection instead of fluorescence, such that detection is completed via small-scale chemical reactions as opposed to the use of an external photoexcitation mechanism. Thus, PLEIADES does not require supplementary machines and actually eliminates some of the side effects of fluorescence methods that impact the data (6).

Conclusion

Many researchers suggest that there is a very real possibility that extraterrestrial life does exist, although it has yet to be detected. Each day, small advancements are being made in the realm of astrobiology that bring humanity closer to confirming whether there is truly life beyond planet Earth. If we are able to conclude that alien life exists, there is potential to learn more about how the universe developed, and whether humans may be able to thrive on other planets in the future. However, what we do know for sure is that the mystery of extraterrestrial existence is one that is not likely to be resolved any time soon.

References

1. J. Kasting, et. al., Remote life-detection criteria, habitable zone boundaries, and the frequency of Earth-like planets around M and late K stars. Proceedings of the National Academy of Sciences of the United States of America 111, 12641-12646 (2014). doi: 10.1073/pnas.1309107110. 2. A. Wandel, How far are extraterrestrial life and intelligence after Kepler?. Acta Astronautica 137, 498-503 (2017). doi: 10.1016/j.actaastro.2016.12.008. 3. B. Dunbar, Mission overview. NASA, (2018). 4. A. Laity, et. al., Exoplanet and candidate statistics. NASA Exoplanet Archive, (2020). 5. A. Krishnamurthy, et. al., Precision characterization of the TESS CCD detectors: Quantum efficiency, charge blooming and undershoot effects. Acta Astronautica 160, 46-55 (2019). doi: 10.1016/j.actaastro.2019.04.016 6. A. Nascetti, et. al., Integrated chemiluminescence-based lab-on-chip for detection of life markers in extraterrestrial environments. Biosensors and Bioelectronics 123, 195-203 (2019). doi: 10.1016/j.bios.2018.08.056 7. V. Parro, et. al., SOLID3: A multiplex antibody microarray-based optical sensor instrument for in situ life detection in planetary exploration. Astrobiology 11, 15-28 (2011). doi: 10.1089/ ast.2010.0501

Images retrieved from: 1. https://cdn.pixabay.com/photo/2020/09/12/00/03/planets-5564482_1280.jpg 2. https://upload.wikimedia.org/wikipedia/commons/thumb/1/16/Kepler_Telescope_in_sapce. jpg/1600px-Kepler_Telescope_in_sapce.jpg 3. https://upload.wikimedia.org/wikipedia/commons/0/0d/Tess_Satellite.jpg 4. https://live.staticflickr.com/1880/44799056732_f4735d89c7_b.jpg

Figure 4 The first image captured by TESS, featuring many stars as well as solar systems that are known to host exoplanets.

Antibiotic Resistance:

a modern problem with ancient solutions

JESSICA GEORGE `24

Figure 1 Bacterial biofilms serve as major protective mechanisms against antibiotics.

Antibiotic resistance: A modern problem with ancient solutions

Since the early 1940s, antibiotics have diminished mortality associated with bacterial infections, contributing to a 29.9 year increase in life expectancy, earning the reputation of being one of the most revolutionary achievements in the history of medicine (1). However, inappropriate and excessive use of antibiotics has given rise to a new deadly era of antibiotic-resistant bacteria. In the U.S. alone, antibiotic resistance is responsible for over 2.8 million infections and 35,000 deaths annually (2). Current solutions are unsustainable as they are limited to modifying previously used antibiotics. In recent years, botanical species, such as blackberry root and Brazillian peppertree berry, and clay minerals have caught the attention of researchers as potential options in understanding and eradicating antibiotic-resistant bacteria. Years of experimentation with natural remedies provides researchers with a foundational understanding of where to focus their studies. Additionally, natural substances are less toxic and offer decreased chances of engendering resistance in bacteria due to increased amounts of active principles involved (3). Though the issue of antibiotic resistance is ceaseless, natural remedies could be a significant component of the multi-faceted approach to provisionally preventing antibiotic resistance in bacteria. Antibiotics are designed to be lethal to their bacterial target. However, some microbes naturally accumulate random mutations, becoming resistant to the antibiotic’s effects by means of natural selection. The surviving bacteria then proliferate and pass on their antibiotic-resistant genes to the next generation, in time producing a generation of antibiotic-resistant bacteria. Additionally, antibiotics may also kill bacteria that are vital in protecting the body from infection, further exacerbating the effects of antibiotic-resistant bacteria. Bacteria are also equipped with defense strategies that help them actively defend against antibiotics. These mechanisms include utilizing bacterial plasmids that house the necessary genetic information for the bacteria to develop resistance. A unique property of plasmids includes the ability to share this valuable DNA from one microbe to another, leading to a chain of resistant strains of bacteria (4).

Botanical cures to MRSA:

Researchers have been exploring botanical species as a potential solution to methicillin resistance, the leading cause of hospital and community associated infections around the world, and the blackberry plant appears to be a promising candidate. Ethnobotanist researcher Cassandra Quave discovered the use of blackberry pomace extract (BPE) in ancient treatments of abscesses during her visit to southern Italy. When sent to her lab in Atlanta, researchers discovered that when molecules of phenolic acids of BPE were tested in vitro with cultured MRSA (methicillin-resistant Staphylococcus aureus), the acids inhibited the formation of biofilms, an adhesive entanglement of bacteria that easily coheres to tissue and inhibits the ability of medicine to penetrate the target bacteria (5). Phenolic acids of BPE act to disrupt the membrane and slime layer of biofilms, while their delocalized electron system destabilizes the cytoplasmic membrane, leading to the collapse of proton motive force. Through confocal imaging, Qauves’ group demonstrated that 220D-F2 (a BPE extract from the root that contains high levels of gallic acid) inhibited the formation of biofilms when incubated with methicillin resistant strains of bacteria. Wild-type strains that were not treated with 220D-F2 formed biofilms that were 88–92 µm thick while the strains exposed to 220D-F2 developed patchy biofilms with only a small number of isolated clumps of adherent cells (6). Additionally, when 220D-F2 was combined with antibiotics clindamycin and oxacillin and used to treat methicillin-resistant strains, the anti-biofilm effects were enhanced. After seven days, a 2.5 log decrease in biofilm colony count in comparison to the antibiotic alone was observed (6). The significant decrease in biofilm formation exhibits the effectiveness of 220D-F2 in impeding a biological

mechanism of antibiotic resistant bacteria. Additionally, separate tests comparing the colony count of the methicillin-resistant bacteria biofilm in catheters with the antibiotics (daptomycin, clindamycin, and oxacillin) and catheters with both antibiotic and 200 µg/mL of 220D-F2 were conducted. The catheters that were exposed to only the antibiotics yielded a colony count of 1.01x107 while the catheters exposed to a combination of antibiotic and 220D-F2 yielded a significantly smaller colony count of 3.3x103 (6). The data observed suggests that antibiotics do not need to be abandoned but rather natural remedies can be used along with pre-existing antibiotics to create an enhanced effect. Quave’s discovery was especially momentous as the phenolic acid molecules did not prove fatal to the MRSA microbes like most antibiotics do. Instead, the plant molecules were able to hinder the bacteria’s defense mechanism, possibly impeding the development of resistance. Researchers at the University of Maryland also explored the abilities of the blackberry plant with regards to gene expression. Methicillin efficiency is restored by suppressing expression of genes involved in antibiotic resistance, such as gene mecA, and others like norA associated with efflux pumps that expel antibiotics from within the cell (5). Researchers prepared BPE by combining blackberry and blueberry (1:1 ratio) and placed it with MRSA strains. Through utilization of quantitative RT-PCR assay, researchers examined the differential expression levels of genes in the methicillin bacteria, discovering that BPE significantly (p < 0.05) down-regulated the aforementioned gene products by inactivating antibiotic binding proteins that are utilized by efflux pumps to identify which substance to expel (5). By obstructing the defense mechanisms of bacteria without killing them, BPE is able to effectively inhibit the negative effects of the microbes without engendering resistance through evolution. Additionally, the phenolic acids of BPE were found to inhibit the growth of MRSA. Testing via antibiogram with broth microdilution exhibited that BPE and gallic acid decreased the minimum inhibitory concentration (MIC) of MRSA from 512 μg/mL to 4 μg/mL (5). By reducing the lowest concentration at which a substance is able to hinder the growth of bacteria, gallic acid of BPE successfully increases the effort it takes for MRSA to proliferate. Quave’s team observed that another plant, Schinus terebinthifolia (Brazillian peppertree), also displayed virulence inhibiting properties. 430D-f5 (a flavone-rich extract of brazillian peppertree berry) extracts from the leaf of this species were used to treat cutaneous skin lesions in rats and extract from the bark was used to treat beef cattle for 17 days, causing improved wound healing, improved clotting, and formation of a fibrin net, a fibrous protein mesh that aids in clotting, at the site of the wound for both animals (7). In the mice, the size of the lesion remained under 0.1cm2 throughout the 14 days after a single 50μm dose of 430D-F5 . The control group on the other hand, showed a significant spike up to a lesion size of 1.0cm2 and then a decrease to 0.25cm2 by day 14 (7). Prevention of lesions is a significant property as open wounds allow for more bacteria and other microbes to infect the individual. 430D-f5 inhibited the expression of accessory gene regulators, which control many of the virulent mechanisms of bacteria including initial infection of cells, evasion of an immune system response, quorum signalling, and the destruction of tissues (7). 430D-f5 was able to shrink bacterial MRSA biofilms and prevent the formation of skin lesions. The incredible facility of these botanical species to disrupt bacterial function without off-target physiological toxicities builds the foundation for future combat with resistant bacterial infections.

Application of clay minerals in the treatment of bacterial ulcers:

Kisameet clay (KC), a type of natural glacial clay, has its origins in northern British Columbia and has been documented to have a history of therapeutic applications, particularly in remedying skin infections due to its anti-inflammatory and antiseptic properties. Recent research with kisameet clay conducted by Shekooh Behroozian and his team at the University of British Columbia has shown that, in vitro, it demonstrates antibacterial properties against ESKAPE (Enterococcus faecium, Staphylococcus aureus, Klebsiella pneumoniae, Acinetobacter baumannii, Pseudomonas aeruginosa, and Enterobacter) pathogens (8). ESKAPE pathogens are the leading cause of nosocomial infections, or those originating in a hospital, and earn their name as a result of their ability to “escape” the mechanisms of antibiotics. Behroozian and his team tested the KC leachate (water percolated through KC) with P. aeruginosa and S. aureus in biofilm microplate assays to determine the antimicrobial effects of the minerals in the clay. Quantification of P. aeruginosa biofilms displayed that the clay leachate reduced biofilm formation to approximately 30% of the initial material in 10 hours (8). In S. aureus, the clay leachate also inhibited biofilm formation to 30% of the initial material in approximately 24 hours, reaching 25% of the initial material by 48 hours (8). The ability of KC to significantly reduce one of the bacteria’s most useful defense mechanisms is of high importance as it is essential that new solutions be found where the microbes are not completely killed. Furthermore, KC’s capacity to obstruct the formation of biofilm in an accelerated manner is of great value as bacteria rapidly proliferate and a fast-working solution is necessary to combat this. KC has demonstrated success in eradicating ulcer-causing bacteria due to its ability to partially or completely inhibit the growth of the microbes. To gain understanding into this process, researchers Shelley E. Haydel, Christine M. Remenih, and Lynda B. Williams at Arizona State University investigated the mineral components of the clay. One clay mineral, CsAg02, displayed bactericidal properties against a variety of bacteria. To assess the effects of this mineral, researchers placed CsAg02 with bacterial cultures of E. coli, ESBL E. coli, S. enterica serovar Typhimurium, and P. aeruginosa. The results of the susceptibility testing demonstrated that the clay mineral completely killed the antibiotic-sensitive bacteria (9). Further

Figure 2 Botanical species such as Rubus ulmifolius (blackberry) have been discovered to possess antibacterial prop erties.

testing with M. smegmatis and M. marinum resulted in the complete inhibition of growth for M. marinum and a 1,000-fold reduction in growth of M. smegmatis in comparison to the bacterial cultures grown without CsAg02 (9). In regards to the mineral’s mechanisms of action, researchers hypothesize that CsAg02 eradicates bacteria indirectly by engendering an unfavorable environment for the antibiotic resistant bacteria. Researchers found that the iron ions (Fe2+) in clay minerals saturate the membranes of bacteria, causing the oxidation of the inner cell (10). This oxidation process causes the production of hydroxyl radicals which are deadly to the microbes. Additionally, the minerals in clay get their bactericidal properties from their ability to either deprive bacteria of their essential nutrients (like iron) and disrupt the homeostatic balance bacteria require for metabolic processes. CsAg02 was found to buffer both pH and oxidation state, creating conditions that were not ideal for the microbe to cause harm. The data exhibits CsAg02’s broad-spectrum antibacterial properties which is useful in expanding the application of this mineral to various other bacterial infections. Eradication of the resistant bacteria by targeting internal environment stability through the use of CsAg02 presents a successful alternative to the use of antibiotics, which have more toxic components than the natural clay.

Conclusion

Presently, treatments with natural remedies are not widely implemented with patients in clinical settings. Traditional remedies using natural substances are often overlooked as pseudoscientific homeopathy. However, dismissing all natural materials as a potential solution to modern issues eliminates the possibility of implementing an innocuous and sustainable treatment to bacterial conditions. As modern research suggests, natural medicine offers many promising candidates worthy of clinical investigation. A paradigm shift in how medicine is developed and operates is imperative in advancing modern medicine. As evident from the extensive deaths due to antibiotic-resistance, current solutions to bacterial infections have proved unsustainable, calling for novel approaches where bacteria are understood instead of completely eliminated. The studies into blackberry root, Brazillian peppertree berry, and clay minerals present several forerunners as potential solutions to the antibiotic resistance crisis but further study into the remedies of the natural world is essential. Delving deeper into ancient remedies may give rise to a new age of medicine that could work towards resolving some of the most pressing threats to public health.

References

1. Achievements in public health, 1900-1999: Control of Infectious Diseases. CDC, (1999). 2. Antibiotic resistance threats in the United States 2019. CDC, (2019). 3. P.D. Gupta and J. B. Tannaz, Development of 3 botanicals to combat antibiotic resistance. Journal of Ayurveda and Integrative Medicine 8, 266-275 (2017). doi: 10.1016/j.jaim.2017.05.004. 4. How antibiotic resistance happens. CDC, (2020). 5. S. Salaheen, et al., Eradication and sensitization of methicillin resistant Staphylococcus aureus to methicillin with bioactive extracts of berry pomace. Frontiers in Microbiology 8, (2017). 6. C.L. Quave, et al., Ellagic acid derivatives from Rubus ulmifolius inhibit Staphylococcus aureus biofilm formation and improve response to antibiotics. Plos One, (2012). 7. A. Muhs, et al., Virulence inhibitors from Brazillian peppertree block quorum sensing and abate dermonecrosis in skin infection models. Sci Rep, (2017). 8. S. Behroozian, et al., Broad-spectrum antimicrobial and antibiofilm activity of a natural clay mineral from British Columbia, Canada. mBio, (2020). 9. S. Haydel, C. Remenih, and L. Williams, Broad-spectrum in vitro antibacterial activities of clay minerals against antibiotic-susceptible and antibiotic-resistant bacterial pathogens. J Antimicrob Chemother, (2008). 10. L. Williams, et al., What makes a natural clay antibacterial? Environ Sci Technol, (2011).

Images retrieved from: 1. https://upload.wikimedia.org/wikipedia/commons/2/28/Mixed-culture_biofilm.jpg 2. https://upload.wikimedia.org/wikipedia/commons/c/c5/Illustration_from_Medical_Botany%2C_digitally_enhanced_from_rawpixel%27s_own_original_plates_88.jpg

Botanical species such as Rubus ulmifolius (blackberry) have been discovered to possess antibacterial prop-

The incredible facility

of these botanical species to disrupt bacterial function without off-target physiological toxicities builds the foundation for future combat with resistant bacterial infections...