Flux by Berkeley Scientific

STAFF

EDITOR’S NOTE

Editor-in-Chief Elettra Preosti Managing Editor Melanie Russo Features Editors Jonathan Hale Marley Ottman Interviews Editors Ananya Krishnapura Esther Lim Research and Blog Editors Nanda Nayak Rebecca Park

Elettra Preosti

Layout Editors Aarthi Muthukumar Stephanie Jue Publicity and Finance Chairs Afroze Khan Hosea Chen Copy Editors Leighton Pu Noah Bussell Senior Advisor Jonathan Kuo Layout Designers Daniel Cui Isaac Chang Features Writers Anisha Iyer Anna Castello Cassidy Biellak Leighton Pu Letian (Jane) Li Merve Ozdemir

Noah Bussell Pierre Letellier Rebecca Hebert Shreya Ramesh Varun Upadhyay

Interviews Team Alexandra Du Allisun Wiltshire Andrew Delaney Anjuli Niyogi Carolyn Qian Grace Guan

Gunay Kiran Laurentia Tjang Lexie Ewer Luke Lyons Michael Xiong

Research and Blog Team Ali Fazal Leighton Pu Bryan Kim Mark Ortega Corey Dodson Miriam Goodwin Elizabeth Chen Riya Bhatia Eunice Tsang Sinead de Cleir Javier Santillan Swathy Natarajan Katherine De Lange Xiaopei Chen

Berkeley Scientific Journal | SPRING 2022

Melanie Russo

Within this loud, energetic universe, even the things that are the most still are in a state of flux. Even rocks that have stood sturdy for billions of years are comprised of vibrating atoms whose subatomic particles are whizzing around at great speeds. These seemingly motionless rocks are planted on our planet’s surface, which in itself is constantly spinning around its axis, playing tug of war with the moon, orbiting around the sun, and whirling around the Milky Way galaxy. Everything from the micro to the macro lives in a state of dynamic equilibrium—a state of constant flux. Many astrophysicists have reason to believe that the net energy of the universe is zero, implying that all energy in the universe is a temporary quantum fluctuation out of nothingness. Furthermore, it is believed that while quantum fluctuations bring about energy, cosmic fluctuations in the Cosmic Microwave Background are the seed from which galactic superclusters are born, thereby shaping the structure of the entire universe. So, whether they are quantum or cosmic, we have fluctuations to thank for our entire existence. In this issue, our authors discuss the flux—the perpetual state of flow and change over time—that surrounds us, exploring the ever-changing dynamics within astronomy, mental health, and more. For instance, author Shreya Ramesh explains how the James Webb Telescope came to be while its images show us how dynamic our universe really is, even though it may not seem like it when staring at the night sky. Similarly, in a panel interview with Dr. Daniel Eisenberg and Dr. Igor Chirokov, we better understand how mental health has fluctuated as a result of the pandemic and what tools we can use to bring our minds to a steadier baseline. It is exciting to track how scientific research flows and changes with time, adapting to current events and converging to an optimized future. Through the fascinating scientific advancements discussed in this issue, one can see that the world works by ebbing and flowing, not by standing still. We hope the Berkeley Scientific Journal and the science we share helps you find your balance in this world of oscillations and universal Flux.

Melanie Russo Managing Editor

TABLE OF OF CONTENTS CONTENTS TABLE Features 4. 12. 21. 30. 40. 45. 54. 59. 70. 74.

Uncovering the New Mysteries of Our Universe: The Origins of the James Webb Space Telescope Shreya Ramesh Meditation and the Brain Anna Castello The Underappreciated Value of Awe Leighton Pu On the Sunny Side of Science Noah Bussell Using Deep Reinforcement Learning to Peer Into the Unconquerable Mind: How Do Animals Learn to Track Odor Trails? Anisha Iyer Antibiotic Resistance: A Silent Pandemic Merve Ozdemir Alzheimer’s Disease and the Neurobiology of Long-Term Memory Formation Varun Upadhyay What Determines Coffee Aroma and Flavor? Jane Li Do Memes Behave Like Viruses? Pierre Letellier Neoantigens: The Future of Personalized Cancer Cassidy Biellak

Interviews 8.

The Beautiful Biology of an Evolutionary Arms Race (Dr. Kimberly Seed)

16.

Water & the People: A Relationship in Flux (Dr. David Sedlak)

24.

Science and Society: A Novel Approach to Decision-Making

34.

Corrupted Clocks: How Parasites Utilize the Circadian Rhythm (Dr. Filipa Rijo-Ferreira)

49.

The Decline of Mental Health: An Overlooked Consequence of the Pandemic

64.

Reactivating Regeneration: The Power of Neural Crest Cells in Repairing Damaged Tissues (Dr. Megan Martik)

Andrew Delaney, Laurentia Tjang, Ananya Krishnapura Alexandra Du, Allisun Wiltshire, Ananya Krishnapura Gunay Kiran, Carolyn Qian, Ananya Krishnapura

Caroline Kim, Anjuli Niyogi, Michael Xiong, Esther Lim

Andrew Delaney, Michael Xiong, Ananya Krishnapura, Esther Lim

Lexie Ewer, Grace Guan, Luke Lyons, Esther Lim

Research 77.

Prognostic Potential of Extracellular Vesicles: Noninvasive Monitoring of Chemotherapeutic Resistance Development Jennifer C. Hall, Thomas R. Carey, Lydia L. Sohn

81.

Renewable Energy Economics: Understanding the Costs and Capacity of Green Energy in the United States

86.

Epidemiological Analysis of Acute Flaccid Paralysis (AFP) Surveillance in Conflict-Affected Syria

Megan J. Mehta, Abrar Rahman

Raneem Rayes, Rohini J. Haar, Naser AlMhawish

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UNCOVERING THE NEW MYSTERIES OF OUR UNIVERSE:

THE ORIGINS OF THE JAMES WEBB SPACE TELESCOPE BY SHREYA RAMESH

INTRODUCTION

ithin billions of galaxies in our uni verse, there are trillions of star sys tems, each with its own planets, moons, asteroids, and comets. Our planet exists in its own pocket of outer space, and it is easy to forget that ours is within uj st one solar system in the vast universe. We have barely begun to uncover and answer the mysteries of the cosmos and our very existence, and there are plenty of answers we do not yet have. e uH bble Telescope is one of the most well-known telescopes in modern histo ry, thanks to its pivotal role in helping us begin to visualize and understand the uni verse we call home. oH wever, despite its vital contribution to the advancement of astronomy, its dated technology has begun hindering us from answering the increas ingly complex questions we have about our universe. To address this issue, NASA recently launched the James Webb p S ace Telescope (JWST), named aer NAs’ SA second administrator, who is credited with

the success of the pA ollo missions. is telescope is the culmination of decades of cutting-edge research and technological advancements meant to provide us with unique, never-before-seen insights into the mysteries of the universe. ORIGINS nI the 1940s, almost a century be fore the James Webb p S ace Telescope was launched, uH bble was conceived as a thought experiment. 1 sA tronomers dreamed of having a telescope, positioned outside of the Earth’s atmosphere, that would be powerful enough to observe the universe. oF llowing years of planning, the project’s design and creation began in 197. nI 190, the telescope was then launched into orbit on the p S ace h S uttle Discovery mission. 1 vE erything was running smooth ly until scientists discovered an aberra tion in one of uH bble’s main mirrors a few months aer deployment, resulting in distorted images. e rst servicing mis sion to clean the lenses took place in 193,

Berkeley Scientific Journal | SPRING 2022

and aer this service, uH bble took what is arguably one of its most impactful images, the uH bble Deep Field, in 195. e uH b ble Deep Field focused on a single section of space and took 342 separate exposures over the course of 10 days. 1 nI the end, the combined images helped us visualize pre cisely how many galaxies and stars exist in a tiny patch of our sky. Now, the James Webb Telescope will oer us the chance to build on this knowledge and push our un derstanding even further.

“Now, the James Webb Telescope is here to dive deeper and continue answering hard-hitting questions about our universe.”

FEATURES

Figure 1: The James Webb Space Telescope. ENGINEERING AND DESIGN James Webb is an infrared telescope equipped with unique technology designed to capture signals from some of the farthest and never-before-seen reaches of space. The telescope is currently positioned at the second Lagrange point of the Earth-Sun system, a point in space such that the telescope maintains its displacement from the Earth as it revolves around the Sun.2 The telescope uses three primary features to make observations: the Optical Telescope Element, the Integrated Science Instrument Module, and the Spacecraft Element.3 The Optical Telescope Element contains one of the telescope’s iconic features: the foldable, hexagonal mirror. The unit consists of 18 gold-coated, hexagonal mirror segments, made of beryllium, that come together to form a 25 square-meter primary mirror—seven times bigger than Hubble’s.4 The large primary mirror maximizes the amount of light collected, which increases the sensitivity of the telescope and produces sharper results. Scientists designed the foldable mirror as a solution to the issue of fitting the telescope’s large structure on a space transport system. Beyond eased transport, the foldable design also allows for scientists to adjust the

FEATURES

hexagonal mirrors.4 The hexagonal shape also directs the incoming light to be concentrated and focused on key areas of the telescope’s detectors.5 The Integrated Science Instrument Module consists of several important devices such as a near-infrared camera and spectrograph. These instruments are meant to receive and interpret signals from far-away objects in space, including galaxies and other star systems.6 Infrared signals are valuable when the observed object is not very bright or has little energy compared to neighboring objects. Such signals also easily pass through dust and other sources of signal interference due to these signals’ longer wavelengths compared to visible light.7 Additionally, JWST’s sensors can help us see what galaxies and other objects looked like in the past. When light from far-away galaxies and objects travels through space, its wavelength gets longer since the rate of the universe’s expansion is faster than the speed of light.7 As a result of this Doppler shift phenomenon, the light that reaches the sensors is “stretched out” light from a source that is rapidly moving further away, and therefore also very old. James Webb can then use this data to gather information and make inferences about our universe and its history. Once the infrared signals from a

source have been gathered, the signals are diverted into different detectors depending on their wavelength. Shorter wavelengths (0.6-5 μm) are diverted through mercury-cadmium-telluride H2RG detectors while signals of slightly longer wavelengths (5-28 μm) are diverted through arsenic-doped silicon detectors.8 After the light travels through the detectors, the constituent photons pass through a thin layer of semiconductor absorber and are processed through a silicon readout integrated circuit (ROIC) that interprets the results.8 In order to ensure that JWST can continue its operations in the harsh conditions of outer space, NASA engineers created the Spacecraft Element of James Webb. The Spacecraft Element consists of the sun-shielding system and the support functions of the spacecraft, including the electrical power, communications, data handling, and propulsion systems, collectively known as the “Spacecraft Bus”. The sun-shielding system is critical to main operations as it ensures that the sensors onboard the telescope work properly by shielding them from the sun’s heat. The precise sensors usually require temperatures as low as 50 K (-370 Fahrenheit), and exposure to direct sunlight causes overheating and malfunction.9 To keep these sensitive components cool, the sunshield, which is coated with five layers of

Figure 2: The Hubble Space Telescope in orbit around the Earth.

SPRING 2022 | Berkeley Scientific Journal

physical and chemical properties of these planets.11 They are also seeking to use the telescope for projects related to understanding the evolution of stellar populations as the telescope’s infrared sensors enable astronomers to see through the dust in stellar nurseries. JWST allows us to see such systems with greater resolution and sensitivity than Hubble, such as for galaxies that are farther away.12 By better understanding what causes star formation and the processes involved in stellar death, we can gain a broader insight into the origins of our solar systems and our universe. UNDERSTANDING OUR PLACE

Figure 3: Integrated Science Instrument Module. WHAT COMES NEXT

“Over the course of its decades-long development, it marked a wave of groundbreaking optical technology focused on creating innovative solutions to complex

The telescope is currently slated to be used for a wide variety of projects such as imaging exoplanets and cosmological phenomena. Scientists plan on investigating other solar systems nearby by using cutting-edge spectroscopy to analyze the

James Webb is an incredible step forward in the fields of astronomy and astrophysics. Over the course of its decades-long development, it championed a wave of groundbreaking optical technology focused on creating innovative solutions to complex problems. This telescope is a new chapter in deepening our understanding of the universe by solving century-old problems, and in the process, we may uncover larger mysteries that govern the universe. James Webb will soon help us better understand our universe and its future, and consequently, more about humanity and our place in the cosmos.

problems.”

aluminum, doped-silicon, and Kapton—a lightweight material that helps to irradiate heat—acts as a giant shade.9 In addition, JWST has a cryocooler to keep the Mid-infrared Instrument (MIRI), a device that observes wavelengths from 5 to 28 microns, at less than 7 K (-447 Fahrenheit), MIRI’s optimal temperature. The cryocooler is a novel three-stage cooling system that uses liquid helium and has no moving parts, minimizing vibrations and allowing for clear images.

Figure 4: The specialized Sunshields on JWST.

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FEATURES

REFERENCES 1.

Garner, R. (2018, September 4). About - Hubble History Timeline [Text]. NASA. http://www.nasa.gov/ content/goddard/hubble-historytimeline 2. Orbit - Webb/NASA. (n.d.). Retrieved April 8, 2022, from https://webb.nasa. gov/content/about/orbit.html 3. Garner, R. (2015, February 20). The James Webb Space Telescope Observatory. NASA. http://www. nasa.gov/mission_pages/webb/ observatory/index.html 4. NASA - NASA’s Largest Space Telescope Mirror Will See Deeper Into Space. (n.d.). Retrieved April 4, 2022, from https://www.nasa.gov/ vision/universe/starsgalaxies/mirror_ size.html 5. Mirrors Webb/NASA. (n.d.). Retrieved April 6, 2022, from https:// webb.nasa.gov/content/observatory/ ote/mirrors/index.html 6. Garner, R. (2015, February 20). The James Webb Space Telescope Instruments [Text]. NASA. http:// www.nasa.gov/mission_pages/webb/ instruments/index.html 7. Infrared Astronomy. (n.d.). WebbTelescope.Org. Retrieved April 4, 2022, from https://webbtelescope. org/home/webb-science/theobservatory/infrared-astronomy 8. Infrared Detectors Webb/NASA. (n.d.). Retrieved April 4, 2022, from https://webb.nasa.gov/content/about/ innovations/infrared.html 9. The Sunshield Webb/NASA. (n.d.). Retrieved April 4, 2022, from https:// jwst.nasa.gov/content/observatory/ sunshield.html 10. Cryocooler Webb/NASA. (n.d.). Retrieved April 19, 2022, from https://webb.nasa.gov/content/about/ innovations/cryocooler.html 11. Astro2020-white-paper-BeichmanC. pdf. (n.d.). 12. Astro2020-white-paper-MeixnerM. pdf. (n.d.).

Retrieved July 12, 2022, from https://www.flickr.com/photos/ nasawebbtelescope/52211883534/in/ album-72177720300469752/. USA, N. G. S. F. C. / C. G. from G., MD. (2016). English: The primary mirror of NASA’s James Webb Space Telescope consisting of 18 hexagonal mirrors looks like a giant puzzle piece standing in the massive clean room of NASA’s Goddard Space Flight Center in Greenbelt, Maryland. Appropriately, combined with the rest of the observatory, the mirrors will help piece together puzzles scientists have been trying to solve throughout the cosmos. Flickr. https://commons. wikimedia.org/wiki/File:James_ Webb_Space_Telescope_Mirrors_ Will_Piece_Together_Cosmic_ Puzzles_(30108124923).jpg NASA. (2009). English: An STS125 crewmember aboard the Space Shuttle Atlantis captured this still image of the Hubble Space Telescope as the two spacecraft continue their relative separation on May 19, after having been linked together for the better part of a week. http:// spaceflight.nasa.gov/gallery/images/ shuttle/sts-125/html/s125e012033. html. https://commons.wikimedia. org/wiki/File:Hubble_2009_close-up. jpg Telescope, N. J. W. S. (2012). The Near Infrared Camera (NIRCam) [Photo]. https://www.flickr.com/photos/ nasawebbtelescope/8738663480/ Telescope, N. J. W. S. (2014). NASA’s Webb Sunshield Stacks Up to Test! [Photo]. https:// www.flickr.com/photos/ nasawebbtelescope/14753947223/

IMAGE REFERENCES 1.

“Cosmic Cliffs.” Flickr | Image Galleries Webb/NASA.

FEATURES

SPRING 2022 | Berkeley Scientific Journal

the

Beautiful Biology of an

Evolutionary Arms Race INTERVIEW WITH DR. KIMBERLEY SEED

Kimberley Seed, PhD, is a professor in the Plant and Microbial Biology Department at UC Berkeley. Dr. Seed received her PhD in Microbiology and Biotechnology from the University of Alberta and completed her postdoctoral training at Tufts University School of Medicine. Her research focuses on understanding the host-pathogen co-evolutionary arms race between bacterial immunity and phage genome dynamics. In this interview, we discussed the beautiful biology of mobile genetic elements and how the presence of these genes in bacteria can defend against phage transmission.

BSJ

: Your lab’s research focuses on immune strategies employed by the bacteria Vibrio cholerae as well as the viruses (i.e. “phages”) it is infected by. What are phages, and what motivated you to choose V. cholerae as your model organism?

: Phages are viruses that only infect bacteria. Viruses are fascinating for many reasons. They are master manipulators of the cells; their whole propagation depends on getting into a susceptible host cell to reproduce more and more viruses. I have been interested in phages since I was a graduate student. When I started my postdoc research, I started working with cholera. It is a good model organism because it is very well-studied and is genetically tractable (the organism’s genome can be manipulated). You can grow it in the lab, work with it fairly easily, and it is not super dangerous to work with. In addition, the literature has suggested that phages were really important in controlling Vibrio cholerae levels for over the last 15 years at least, if not over 100 years. The hypothesis is that if you have lots of phages killing the V. cholerae, then you get less cholera disease. This hypothesis has been put forward to explain why we see cholera disease in certain areas go up and down every year. We do not understand what triggers cholera outbreaks and what causes them to end, so people think phages might have something to do with the fluctuating cycle of V. cholerae levels. At the time I started my lab, people did not really understand these viruses and their interactions with cholera bacteria, so that is where my lab comes in.

BSJ

: In many of your papers, you refer to mobile genetic elements as sequences that can have significant impacts on the fitness of organisms. What are mobile genetic elements, and what are some examples of how they have affected the co-evolutionary arms race between V. cholerae and phages?

KS BY: ANDREW DELANEY, LAURENTIA TJANG, AND ANANYA KRISHNAPURA

Berkeley Scientific Journal | SPRING 2022

: Mobile genetic elements are segments of the genome that can move as a unit between cells. It is a way for a bacterium to acquire multiple genes or multiple traits at once, unlike drift or mutation. It is like a fast-forward form of evolution for an organism where it can acquire a whole new biosynthetic pathway or new defense system at once. Generally speaking, the field as a whole is starting to see a pattern where most phage defense systems are encoded on mobile genetic

INTERVIEWS

elements. Phages can quickly become resistant to these defense systems and counter adapt, so it is beneficial for bacteria to be able to rapidly respond and evolve via these mobile elements. The fact that they are mobile means that you can just swap them around and keep trying until something works. However, because of this, they usually have high fitness costs to the bacteria since it needs to acquire and maintain this big block of genes. Because of this cost, they can often end up lost from the bacterial genome. This is probably the main reason why the presence of mobile genetic elements tends to fluctuate in bacterial populations, especially for cholera. PLEs (phage-inducible chromosomal island-like elements) are one such example of mobile genetic elements that my lab studies. PLEs are a major driving force in phage resistance. I discovered PLEs when I was a postdoc; there was initially nothing known about them, but now my lab is responsible for analyzing and figuring out any molecular details about them. The other mobile genetic elements we study are called SXT ICEs (integrative and conjugative elements). They have been well known for a number of years in the literature because they confer multidrug resistance to antibiotics. They are five times bigger than PLEs and include many more genes.

BSJ KS

: How exactly do PLEs inhibit phages?

: PLEs function like parasites of phages. They live in the bacterial genome, and they divide and replicate along with the bacterium. In the absence of phage infections, they are pretty quiet. When a phage infects the cell though, they can sense the infection. In response, they excise from the bacterial chromosome and start replicating and stealing proteins from the phage. They are parasites of the phage because they hijack important aspects of the phage’s lifecycle. They have evolved ways of tinkering with the phage’s capacity to replicate and package its own genome such that at the end of the infection cycle, more PLEs are released encapsulated in phage proteins compared to the actual phage genome. They then spread their genome to neighboring bacterial cells, ultimately making use of phage particles to spread and replicate. In many ways, PLEs are really just a parasite of phages, but we think of them as a defense mechanism for Vibrio. By inhibiting a given phage from producing new progeny viruses, PLEs are protective to the population of bacteria.

BSJ

: Phages and CRISPR are two terms that have received a lot of concurrent attention recently. How is it that bacteriophages can take advantage of CRISPR to overcome PLEs?

: When I was a postdoc, I discovered this phage called ICP1 that normally encodes a fully functional CRISPR-Cas system. It was a huge surprise when I discovered it because CRISPR is supposed to be in bacteria, but I found it in a phage! It turns out that the phage’s CRISPR-Cas system functions in the same way that CRISPR-Cas works in bacteria. Its job is to use sequence-guided nucleases to chop up a target that is otherwise dangerous to the phage. Rather than a bacterium using the system against the virus, this virus now uses it against its parasites, PLEs, in order to regain the upper hand. The data suggest that CRISPR systems in phages are very rare. Because of that, I think the overall hypothesis is that they emerged

INTERVIEWS

Figure 1: Bacterial PLE defense system. PLEs are mobile elements that act as bacteriophage satellites (“viruses” of viruses) encoded in the V.cholerae genome. PLEs are activated by phage infection and block phage DNA replication during infection. This process prevents the transmission of phages. in bacteria on multiple independent occasions and then have been hijacked by some viruses for their own purposes in the same way that viruses will hijack and reuse other cellular components for their own purposes. Since CRISPR-Cas systems are usually quite big, for many viruses, it is too big of an investment to be able to encode that much DNA. But if the going gets tough enough, some viruses, like ICP1, will find a way to encode it.

BSJ KS

: What other pathways does ICP1 implement to combat PLEs?

: We found another nuclease called Odn which functions in a similar way as CRISPR-Cas systems for the phage. It targets PLEs to stop them from stealing proteins from the phage, but it does not have the adaptability of a CRISPR-Cas system. It can only target a single, predetermined target. The downfall for the phage is that PLEs can mutate. If a phage relies only on Odn, and if PLE ends up losing the site for Odn cleavage, PLE regains the upper hand. What we envision with ICP1 phages that have a CRISPR-Cas system is that this system was selected in evolutionary history because, at some point, the phage was not able to use Odn anymore to cleave the intended targets. Both CRISPR-Cas systems and Odn make use of nucleases, but we have discovered one other mechanism in the lab. It is a small protein in phage that can overcome some PLE variants. It does not look like a nuclease on its own, unlike CRISPR and Odn, but rather it interacts with a PLE protein that is a nuclease. Normally, that nuclease is very specific, and it only cuts certain segments. Our data collectively suggest that this phage protein turns that protein from a good, well-behaving nuclease into a very badly behaved nuclease

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Figure 2: Phage CRISPR system against bacterial element PLE. In this diagram, the bacterium uses PLE to interfere with phage replication, while the phage uses a CRISPR-Cas system to block PLE functioning.

that targets the PLE. In this way, the phage protein manipulates PLEs into turning on themselves.

BSJ

: Your lab has conducted much of its research on a second type of mobile genetic element found in γ-proteobacteria: SXT ICEs. How prevalent are these elements in these bacteria, and how do the phage defense systems encoded by these SXT ICEs compare to those from PLEs?

: Most epidemic strains of Vibrio cholerae encode SXT ICEs. They are very common, but they come in different “flavors,” encoding different antibiotic resistance or phage defense profiles. The major difference between how PLEs function and how these SXT ICE phage defense systems function is that SXT ICEs are constitutively on, essentially guarding the bacterium from diverse phages or other mobile elements that are trying to compromise the cell. SXT ICEs are much more promiscuous and less specific to a given phage. They operate by blocking DNA replication. Once the phage gets its DNA into the cell, SXT ICEs have ways of either recognizing the foreign DNA right away and cutting it or they stop the phage DNA from replicating. In this case, the individual cell survives the infection, whereas PLEs only protect the bacterial population as a whole rather than the initially infected cell.

BSJ teria?

: Could the phage resistance conferred by SXT ICEs have implications on the study of antibiotic resistance in bac-

: SXT ICEs in cholera can confer antibiotic resistance. In fact, they were discovered because of that phenotype. When antibiotics started being used against cholera, cholera strains with

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SXT ICEs were selected for. Now, it is at the point where basically all strains of epidemic cholera have these SXT ICEs. They are undoubtedly responsible for antibiotic-resistant pathogens. We mentioned in a recent Science paper that SXT ICEs can sense when a cell has been infected by a phage. So, in some cases, it might make more sense for SXT ICEs to leave their infected hosts and try to gain access to other cells. Similarly, a PLE excises from the genome, steals phage material to package itselfs, and then leaves the original cell. However, after phage infection, PLEs only transmit when the cell is dying or dead, whereas SXT ICEs can transmit while the original cell is still alive. This means that if you infect the bacteria with a phage, you can stimulate the transfer of an element that will confer antibiotic resistance. I think what is really important as people start to consider phages for therapeutic applications is that we think critically about the consequences of our actions. If we do not actually understand what those consequences are and fail to ask, “What are the risks if we want to replace antibiotics or use phages in conjunction with antibiotics? Could we actually be making the problem worse?” the consequences could be catastrophic. At this point, we may not necessarily have the answers to these questions because we have not studied these interactions in depth.

BSJ

: Since phages were discovered in the early 20th century, scientists have continuously proposed or discovered potential uses for these viruses in a multitude of fields. Were there any scientists in particular whose work inspired you to pursue the research you conduct today?

: I have to be honest: I am not inspired to do the research that I am doing because I want to use phages to treat bacterial infections. I am inspired by the fact that there is this dynamic

INTERVIEWS

mind is much more open when you are in that state of following biology rather than solving particular problems.

BSJ KS

Figure 3: SXT ICEs over time. SXT ICEs are responsible for phage resistance in V. cholerae. SXT ICEV1and SXT ICEV2 refer to bacteria containing these two variants of SXT ICEs, while SXT ICE(-) refers to bacteria where SXT ICEs are absent. The abundance of SXT ICEs in bacterial populations fluctuates with time for reasons that are still unknown. evolutionary back-and-forth between bacteria and their viruses or sometimes viruses and their viruses. This relationship is just so fascinating and reveals totally unexpected mechanisms, like the CRISPR-Cas systems in a virus. My research has some direct implications for people who are interested in phage therapy because my work can help us answer many relevant questions: “Will this organism be effectively killed by the phages being administered? How can we engineer or select phages that can overcome this phage defense system?” However, I personally am inspired by the plain beauty of these systems and the discovery of novel biology; I cannot say that I was primarily motivated by some scientists who studied phages for their application in other fields. I am much more inspired by simply discovering amazing biology and being willing to follow that biology and see where it takes me. My postdoc mentor was someone similar who was super excited about any kind of science, and his enthusiasm for all cool ideas and discoveries definitely has made a huge impact on my scientific career. Frankly, you never know where the next big discovery is going to be. CRISPR would not be something we all know and use in our labs and medicine unless people were just fundamentally interested in this back-and-forth war between phages and bacteria. For example, when scientists first studied CRISPR, it was not because they thought they were going to generate a genome editing tool that would change the field of biology and medicine. They were studying it because their bacterial cultures were dying of phage infection in a dairy industry setting. They were trying to make yogurt, and it was not working because phages kept killing all their hosts. They asked themselves, “How do we get these phages to stop killing our hosts? What are these spontaneous mutants that are resistant to viruses?” Well, they were CRISPR acquisition mutants, and that is what led to the discovery of CRISPR. In essence, you never know when the act of following beautiful biology will reveal something amazing. Your

INTERVIEWS

: Can you provide any insight on the future trajectory of your research?

: I think one of the things I really love about our research is that we rely on sampling that we do in collaboration, primarily in Bangladesh, with Dr. Munir Alam’s lab. We get stool samples from cholera patients every year, and it is like we go on a treasure hunt, except in stool. One of the things we found last year that was really surprising is that although it seems like CRISPR is much more effective than Odn and the phages we saw needed to have CRISPR because some variants of PLEs present were resistant to Odn, we actually found that CRISPR and PLEs disappeared and Odn was once again favored in the V. cholerae and phage populations. I was very nervous about that because my career is largely based on studying PLEs. However, we now have the next cohort of samples, and I am happy to say that a new PLE has emerged. This PLE is resistant to CRISPR, resistant to Odn, and resistant to this other protein mechanism that we have identified. Now, the battle seems to be back in PLEs’ favor, and we are very eager to see what phages are going to do in nature to circumvent this, because they will. I think just following this biology—this coevolutionary arms race—in almost real time is so amazing because you never know what you are going to find. We were not initially looking for the SXT ICEs. We did not have a hypothesis that SXT ICEs were responsible for this phage-resistant phenotype. We found it by looking in the stool and asking, “Well, why are you not infecting these bacteria anymore?” I truly enjoy looking into these genomes, seeing what gets selected for, and trying to reconstruct what could have happened that led to the current situation. REFERENCES 1. 2.

3. 4.

Headshot: [Photograph of Kimberley Seed]. Seed Lab. https://vcresearch.berkeley.edu/faculty/kimberley-seed. Image reprinted with permission. Figure 1: Barth, Z. K., Silvas, T. V., Angermeyer, A., & Seed, K. D. (2020). Genome replication dynamics of a bacteriophage and its satellite reveal strategies for parasitism and viral restriction. Nucleic aAcids Rresearch, 48(1), 249–263. https://doi.org/10.1093/nar/gkz1005 Figure 2: Research. Seed Lab. (n.d.). Retrieved March 31, 2022, from http://www.kimseedlab.com/research Figure 3: LeGault, K. N., Hays, S. G., Angermeyer, A., McKitterick, A. C., Johura, F.-tuz, Sultana, M., Ahmed, T., Alam, M., & Seed, K. D. (2021). Temporal shifts in antibiotic resistance elements govern phage-pathogen conflicts. Science, 373(6554). https://doi.org/10.1126/science. abg2166 Barth, Z. K., Nguyen, M. H. T., & Seed, K. D. (2021). A chimeric nuclease substitutes a phage CRISPR-cas system to provide sequence-specific immunity against subviral parasites. ELife, 10. https://doi.org/10.7554/elife.68339

SPRING 2022 | Berkeley Scientific Journal

MEDITATION AND THE BRAIN BY ANNA CASTELLO

rom claims that meditation can improve interpersonal relationships, enrich mental health, or turn practitioners into better people, it seems that there is nothing that a little sitting down in the quiet cannot fix. Before researching meditation for this article, I, like many others, was quite suspicious and frankly tired of having so-called ‘wellness gurus’ tell me how to live my life. But the research might suggest that these ‘wellness gurus’ might be on to something after all. Though sitting in a dark room with eyes closed for half an hour a day, or saying affirmations in the mirror is not for everyone, taking a brief moment to breathe and shift perspectives while driving, for example, is enough to reap wonderful benefits. MINDFULNESS MEDITATION In the West, “meditation” often refers to the practice of mindfulness medita-

tion, where one permits thoughts to pass through oneself in the present moment without interpretation or judgment. This is often done by redirecting attention to one’s breathing when a thought hijacks our stillness or awareness. But can this redirection of attention really change someone’s life? Since the early 2000s, studies have purported to show that meditation can change the structure of the brain. Unfortunately, many of these studies had flawed methodologies, as meditation and well-being can be two factors that are hard to test in controlled settings due to their innately subjective nature and lack of universal definitions.1 In more recent years, however, more well-designed research on mindfulness meditation has come to light. One meta-analysis found that the brains of regular meditators tend to be linked to being of larger size in parts of the insula and cingulate cortex, orbitofrontal cortex, and the prefrontal cortex, the re-

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gions involved in emotional self-awareness, self regulation, and attention respectively (Figure 1).2 A number of studies have highlighted the potential of meditation programs such as Mindfulness-Based Stress Reduction (MBSR), an eight-week long program developed at University of Massachusetts with the goal of reducing stress by teaching participants how to be present in one’s thoughts and body. MBSR has been linked to increases in the cortical thickness of the hippocampus, an area of the brain responsible for learning and memory retention, as well as a reported decrease in the volume of the amygdala: the brain region mediating fear, anxiety, and stress. This suggests that MBSR can help reduce stress in meditators.3,4 These changes demonstrate that not only does meditation shape our brain, but it does so in a manner where we actually feel better. Surprisingly, practitioners of MBSR seem to feel the effects of this eight-week

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Figure 1: Brain Anatomy. Highlighted in blues and green are some areas of the brain that demonstrate an increase in size in regular meditators. long course even years after the training. In a study designed to test the long-term effects of MBSR patients diagnosed with anxiety disorder, from a previous study where they underwent MBSR training, were analyzed again after three years. All 18 patients had maintained their improvements from the original study. Moreover, the majority of participants in the study continued regularly meditating even after the completion of the course.5 With a plethora of studies suggesting that mindfulness meditation can be beneficial for one’s mental well-being, how does it compare to other tried and tested methods such as exercise and antidepressants? A study looking precisely to compare MBSR to aerobic exercise, an alternative stress reduction intervention, used functional magnetic resonance imaging (fMRI), a tool that looks at brain activity, to investigate changes in the brain in areas associated with emotional regulation. Participants with diagnosed generalized social anxiety disorder were divided in two groups and had fMRI taken before and after their experiences. The first group underwent the eight-week long MBSR training course and the second had to adhere to an eight-week long aerobic exercise training course. The study demonstrated that participants who underwent MBSR were better suited to emotionally regulate their negative self-beliefs compared to those who underwent the eight-week aerobic exercise training, indicating the MBSR is a viable option to help with generalized social anxiety disorder (Figure 2).6 MBSR has also been compared

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to antidepressants. A meta-analysis done at Johns Hopkins University found meditation to be as effective as antidepressants at reducing depression, anxiety, and pain.7 Because mindfulness meditation is rooted in focusing one’s attention, it is no surprise that it has been found to help with concentration. Adhering to a twoweek mindfulness meditation program has been shown to help improve cognitive function. One study looked at the effects of meditation on concentration, working memory capacity, occurrence of distracting thoughts, and reading comprehension scores during the GRE. On average, the participants experienced a 16 percentile increase in the verbal reasoning section of the GRE.8 These results suggest that the effects of mindfulness meditation are tangible in an academic setting. Discoveries such as this have caused an increase in mindfulness meditation programs targeted towards schools with the goals of helping students concentrate and improve academically. A 2018 study looked at 16 classrooms from districts with two different socio-economic backgrounds exposed to daily audio-guided mindfulness training. The study found that those who participated in the training reported an increase in math, social studies, and overall GPA scores. The researchers did highlight, however, that there was a lot of pre-existing variation between the schools leading to a decrease in statistical significance of these results.9 This study does still greatly contribute to the conversation of implementing meditation programs across schools,

something that especially over Zoom has started to occur more widely. COMPASSION MEDITATION Mindfulness meditation seems to be linked to an abundance of benefits, but when it comes to altruism, compassion meditation is a fierce competitor.10 Compassion meditation cultivates compassion by encouraging practitioners to repeat mantras, think purposeful thoughts, and wishing everyone to be free from suffering. What exactly is compassion, however, and why is it something we want to actively foster?

“This study does still greatly contribute to the conversation of implementing meditation programs across schools, something that especially over Zoom has started to occur more widely.”

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Compassion is when one is sensitive to the emotional suffering of others, whilst empathy is when one feels the malaise of others. It is important to curate compassion over empathy in our daily lives as it helps people connect without causing personal suffering or burnout. When looking at a systematic review on the effects of meditation on empathy, compassion, and prosocial behavior (characterized by behavior through which people benefit others), 74% of the studies in this meta-analysis found significant self-reported improvements.11 A particular study conducted in Leipzig, Germany used fMRI to examine blood flow before and after eight-hour compassion trainings to see which areas of the brain were more active. The participants were divided into two groups, one exposed to compassion meditation training, involving a six hour class on extending kindness to oneself and others followed by three 45-minute sessions, and the other not. They were then shown videos of people suffering and were asked to empathize with them. The fMRI showed that people without the training had an active insula, a region of the brain related to personal pain. Those with the training, however, had regions of the brain related to love, like that of a parent for a child, light up, indicating that just eight hours of compassion meditation training has drastic effects on one’s outlook and can help alleviate personal pain in stressful situations. This suggests that compassion meditation can be a useful tool in preventing burnout and allowing for better social responses.12 Another fMRI study looked at highly experienced practitioners of compassion meditation and found that this practice triggers the brain’s motor centers which then allow the body to physically move. This study demonstrates that compassion meditation incentivises practitioners to help people in need even while they are still in the brain scanner.13 WHAT NOW? DO WE ALL START MEDITATING? Though there seems to be a lot of promise regarding both mindfulness and compassion meditation, there is still a lot more research that is needed to definitively show how they affect the brain and their

Figure 2: This bar graph indicates the % reduction of negative thoughts after completion of an eight week Mindfulness-Based Stress Reduction course compared to an eight week long aerobic exercise regimen. The star indicates a statistical significant difference from baseline levels of control of negative emotions when compared to after treatment. This graph demonstrates that individuals who practice MBSR are better suited to regulate negative emotions than those who implement an aerobic exercise routine. REFERENCES 1.

“This suggests that compassion meditation can be a useful tool in preventing burnout and allowing for better social responses.”

3. efficacy in daily life. In fact, a recent 2022 study from Brown University highlights that meditation-related adverse effects are not rare.14 This study highlights the importance of looking into how individual people can be affected by long-term meditation, indicating that this practice may not be for everyone. Many promises are made by hopeful practitioners that meditation can fix everything, and though it is linked to many wonderful improvements, it is important to listen to one’s body and mind first.

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Awasthi, B. (2013). Issues and perspectives in meditation research: in search for a definition. Frontiers in Psychology, 3, 1-9. https://doi. org/10.3389/fpsyg.2012.00613 Fox, K. C. R., Nijeboer, S., Dixon, M. L., Floman, J. L., Ellamil, M., Rumak, S. P., Sedlmeier, P., & Christoff, K. (2014). Is meditation associated with altered brain structure? A systematic review and meta-analysis of morphometric neuroimaging in meditation practitioners. Neuroscience and Biobehavioral Reviews, 43, 48–73. https://doi. org/10.1016/j.neubiorev.2014.03.016 Hölzel, B. K., Carmody, J., Vangel, M., Congleton, C., Yerramsetti, S. M., Gard, T., & Lazar, S. W. (2010). Mindfulness practice leads to increases in regional brain gray matter density. Psychiatry Research. Neuroimaging, 191(1), 36–43. https://doi.org/10.1016/j. pscychresns.2010.08.006 Singleton, O., Hölzel, B. K., Vangel, M., Brach, N., Carmody, J., & Lazar, S. W. (2014). Change in brainstem gray matter concentration following a mindfulness-based intervention is correlated with improvement

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in psychological well-being. Frontiers in Human Neuroscience, 8, 33–33. https://doi.org/10.3389/ fnhum.2014.00033 5. Miller, J. J., Fletcher, K., & KabatZinn, J. (1995). Three-year followup and clinical implications of a mindfulness meditation-based stress reduction intervention in the treatment of anxiety disorders. General Hospital Psychiatry, 17(3), 192–200. https://doi. org/10.1016/0163-8343(95)00025-M 6. Goldin, P., Ziv, M., Jazaieri, H., Hahn, K., & Gross, J. J. (2013). MBSR vs aerobic exercise in social anxiety: fMRI of emotion regulation of negative self-beliefs. Social Cognitive and Affective Neuroscience, 8(1), 65–72. https://doi.org/10.1093/scan/ nss054 7. Goyal, M., Singh, S., Sibinga, E. M. S., Gould, N. F., Rowland-Seymour, A., Sharma, R., Berger, Z., Sleicher, D., Maron, D. D., Shihab, H. M., Ranasinghe, P. D., Linn, S., Saha, S., Bass, E. B., & Haythornthwaite, J. A. (2014). Meditation programs for psychological stress and well-being: a systematic review and meta-analysis. JAMA Internal Medicine, 174(3), 357–368. https://doi.org/10.1001/ jamainternmed.2013.13018 8. Mrazek, M. D., Franklin, M. S., Phillips, D. T., Baird, B., & Schooler, J. W. (2013). Mindfulness training improves working memory capacity and gre performance while reducing mind wandering. Psychological science 24(5), 776–781. https://doi. org/10.1177/0956797612459659 9. Bakosh, L. S., Tobias Mortlock, J. M., Querstret, D., & Morison, L. (2018). Audio-guided mindfulness training in schools and its effect on academic attainment: Contributing to theory and practice. Learning and Instruction, 58, 34–41. https://doi.org/10.1016/j. learninstruc.2018.04.012 10. Bibeau, M., Dionne, F., & Leblanc, J. (2015). Can compassion meditation contribute to the development of psychotherapists’ empathy? A review. Mindfulness, 7(1), 255–263. https:// doi.org/10.1007/s12671-015-0439-y

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11. Luberto, C. M., Shinday, N., Song, R., Philpotts, L. L., Park, E. R., Fricchione, G. L., & Yeh, G. Y. (2017). Can compassion meditation contribute to the development of psychotherapists’ empathy? A reviewMindfulness, 9(3), 708–724. https://doi.org/10.1007/s12671-0170841-8 12. Klimecki, O. M., Leiberg, S., Lamm, C., & Singer, T. (2013). Functional neural plasticity and associated changes in positive affect after compassion training. Cerebral Cortex (New York, N.Y. 1991), 23(7), 1552–1561. https://doi.org/10.1093/ cercor/bhs142 13. Lutz, A., Brefczynski-Lewis, J., Johnstone, T., & Davidson, R. J. (2008). Regulation of the neural circuitry of emotion by compassion meditation: effects of meditative expertise. PLoS ONE, 3(3), e1897– e1897. https://doi.org/10.1371/ journal.pone.0001897 14. Goldberg, S. B., Lam, S. U., Britton, W. B., & Davidson, R. J. (2022). Prevalence of meditation-related adverse effects in a populationbased sample in the United States. Psychotherapy Research, 32(3), 291–305. https://doi.org/10.1080/105 03307.2021.1933646 IMAGE REFERENCES 1. 2. 3.

Banner Image: By Author Figure 1: By Author. Figure 2: Goldin, P., Ziv, M., Jazaieri, H., Hahn, K., & Gross, J. J. (2013). MBSR vs aerobic exercise in social anxiety: fMRI of emotion regulation of negative self-beliefs. Social Cognitive and Affective Neuroscience, 8(1), 65–72. https:// doi.org/10.1093/scan/nss054

SPRING 2022 | Berkeley Scientific Journal

Water & the People: A Relationship in Flux

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: I did not set out to study urban water infrastructure; I did not even know what it was when I was a college student. My undergraduate major was environmental science, and after graduating I was trained as an environmental chemist in the University of Wisconsin, where I was interested in the transport of chemicals and their effects on the environment. It was not until I arrived here in Berkeley that I became interested in water infrastructure, particularly urban water infrastructure, and started studying it more seriously. In part, that was because California and the rest of the West are quite different from the East and the Midwest. Whereas water is widely available in the East and Midwest, that is not the case for the West; here, we are more focused on where our water comes from and more tightly control where it goes after use. This means there are a lot of opportunities for us to use treatments to remove contaminants and directly minimize their impact on surface waters.

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INTERVIEW WITH DR. DAVID SEDLAK David L. Sedlak, PhD, is the Plato Malozemoff Chair Professor in UC Berkeley’s Civil & Environmental Engineering Department. He is currently the Co-Director of the Berkeley Water Center, the Deputy Director of the NSF Engineering Research Center for Reinventing the Nation’s Urban Water Infrastructure (ReNUWIt), and Chair of the Research Advisory Council in the National Alliance for Water Innovation (NAWI). In addition to his research and advisory policy work, Dr. Sedlak frequently engages the public through interviews, TED talks, books, and more. He is a leading expert on water reuse management and the author of Water 4.0: The Past, Present, and Future of the World’s Most Vital Resource. In this interview, Dr. Sedlak discusses potential water reuse systems and point-of-use devices along with their respective limitations.

BY: ALEXANDRA DU, ALLISUN WILTSHIRE, ANANYA KRISHNAPURA

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: What led you to study urban water infrastructure?

: What is the current “water revolution,” and why is it important?

: The water systems that we have today have essentially remained unchanged for the past 70 years or so, at least in most places within the wealthy parts of the world. They are what people refer to as “linear systems,” where water comes in one side from a reservoir or a series of groundwater wells, and we then treat it to make it safe for people to drink. People use it, we treat it again in sewage treatment plants, and then we release it into the environment. That system has worked pretty well for those 70 years, but now a lot of places are dealing with water scarcity. As water becomes more scarce, there is a benefit in using it multiple times. The revolution that is going on concerns this idea of recycling water. In other words, using it multiple times. There are other revolutions going on simultaneously—for example, transitioning away from a reliance on water that is imported over great distances and replacing it with locally-sourced water. That would mean capturing the rainwater that falls within a city and making it part of the water supply. We could also use shallow groundwater in cities, which oftentimes in the past had been considered too contaminated for use. Finally, there is another revolution that is taking place in many parts of the world, which is the use of desalination to make seawater and salty groundwater drinkable. All of these things are upending the current way that we provide water to cities.

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: Which cities have been indicated as possible potable water reuse adopters? Why those in particular?

: There are two parts of the world that are leading the drive toward potable water reuse. One is Southern California, and the other is Singapore. Southern California has

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been at it the longest, starting in the early 1960s as water stress and water shortage drove the need to find new water supplies. Utilities in Los Angeles and Orange County have adopted potable water reuse as a way of increasing their supply. They have pioneered transformative technologies, using advanced treatment combined with groundwater storage to make water reuse possible. The model developed in Orange County was picked up in the 1990s by Singapore. As a city state without its own water supply, it had historically been reliant upon Malaysia for water imports. Singapore considered their water supply a vulnerability, so gaining water independence became an issue of national security. When they saw what was happening in Orange County, they realized that this was a way to reduce their vulnerability to having their supply cut off. Southern California and Singapore are two main places that everyone looks to, but if you look a little closer, there are many other cities that have been quietly pursuing potable water reuse. For example, Atlanta, Georgia, has two sizable potable water reuse facilities that have been in operation for over 20 years. Northern Virginia, outside of Washington, D.C., has a large potable water reuse facility. Phoenix, Denver, and Perth, Australia, are all moving in the same direction as well.

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: What is reverse osmosis?

: In the 1960s the US government funded a program with the goal of making it possible to desalinate seawater. Up until then, the way in which seawater was desalinated was essentially distillation. You just boiled the water and captured the steam to turn it into freshwater, leaving the salts behind. However, that was a very energy intensive and expensive process, so the federal government funded a program to try to drop the cost. A team of scientists at UCLA then discovered a new way of taking salt out of water that involved thin plastic polymeric membrane. This was a polymer made out of cellulose acetate, the material that they used to use to make movie films. If you apply pressure to it, you can drive water molecules through the plastic membrane and leave the salts behind. This is

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called “reverse osmosis” because you are working against the osmotic pressure and the salt gradient to push the water through, but it just lets the water molecules go through. The basis for most modern seawater desalination and potable water recycling is now the reverse osmosis membrane, a University of California product designed at UCLA and improved upon gradually by the rest of the world.

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: How can each water-stressed location figure out a water reuse plan and/or reverse osmosis (RO) management style that is right for their location? Who should be responsible for designing a water reuse plan for each community?

: The decision to adopt water reuse depends upon local geography, local economics, and local politics. It is very much tailored to the specific city and their needs. As an example, in the Western US, one of our limitations is that our water supply often starts out with high levels of salt. When water is used in someone’s home, it picks up even more salt. It drives utilities toward employing reverse osmosis because this removes the salt as part of the treatment process. In the southeast places in Atlanta, Northern Virginia, and Texas, a lot of the cities that are interested in water reuse are inland. They prefer not to remove the salts from the water because their

“The basis for most modern seawater desalination and potable water recycling is now the reverse osmosis membrane, a University of California product designed at UCLA and improved upon gradually by the rest of the world. “

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sive. We have been trying to develop a less expensive approach, and in the process we have come up with a way of doing it that provides other societal benefits. In particular, our nature-based treatment systems are a low energy way of removing those residual contaminants from the concentrate. In the process, we create a wildlife habitat called a horizontal levee, which can support a wetland ecosystem and also offer coastal communities protection from storm surges.

BSJ DS Figure 1: Horizontal levee in Oro Loma created as part of the NSF’s ReNUWIt project is an example of nature-based water treatment. Scan the QR code for a video of the site’s construction and an overview of the coastal protection offered by the levee. water supply is already low in salt. If they were to remove the salts from their water using reverse osmosis, they would then have to find a place to put it, so they instead use other treatment technologies. Therefore, the decision of how exactly to implement potable water reuse depends upon whether you are on the coast or inland and whether the water supply starts with high levels of salt or not. The decision on how and when to implement these technologies, though, is really a community decision. The process is normally initiated by a water utility because they are the water suppliers, but quite frequently, there are one or more politicians who take it upon themselves to then become advocates and supporters for it.

BSJ

: Could you describe the benefits of a nature-based treatment of RO concentrate over the more conventional method of ozone treatment paired with biological activated carbon (O3/ BAC)?

: When recycling wastewater effluent from a sewage treatment plant, the reverse osmosis membranes do an excellent job removing not only the salts but also the microbes, chemicals, and metals that are left behind. The material they leave behind in the membrane is called “concentrate” because it has all the stuff that was in the wastewater effluent but concentrated by a factor of five or six. The early adopters of potable water reuse were coastal cities who put this material in a great big pipe and sent it out to the ocean, where it was diluted. When you just think in terms of the mass, the amount that was flowing to the ocean was no different than what was going there before they started doing reverse osmosis. However, over time, more and more cities have expressed interest in using reverse osmosis for recycling projects occurring inland. When that happens, we run into the problem that the concentrate might need to be treated before it is released to the environment. Traditionally engineered approaches for concentrate treatment, like ozonation followed by activated carbon, turned out to be quite expen-

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: Are there any limitations associated with constructed wetlands?

: Each time we embark on these projects is essentially like the “first time” we are building different aspects on such a large scale, so every day brings a new lesson. I can give you an example regarding the first system that we built. We used construction materials for the gravel layer underneath where the water flows; however, we think that because we used local gravel from the Bay Area that is rich in serpentine minerals, we ended up with levels of nickel that are higher than we would have liked. In the Bay Area, gravel and rock is often rich in nickel and chromium, whereas in most other parts of the world, the gravel is much more benign when it comes to those metals. Next time we build a demonstration or full-scale project, we are going to look a little more carefully to make sure we get low-nickel gravel. One of the interesting aspects of building something for the first time is you learn by making mistakes and keeping your eyes open.

“One of the interesting aspects of building something for the first time is you learn by making mistakes and keeping your eyes open.”

BSJ

: What has the data shown from pilot-scale projects in California of RO concentrate treatment in constructed wetlands?

: We have built two types of constructed wetlands in California. One type treats the effluent directly from sewage treatment plants, while the other treats reverse osmosis concentrate. We built some pretty large wetlands in Southern California to treat a river where the flow is mostly wastewater effluent, and these efforts have been pretty successful. The wetlands do a good job removing not only nitrate, which is one of the key pollutants, but also some of the pharmaceuticals and other chemicals that we find in wastewater. What we are really excited about now is the second type, the horizontal levee system, which treats the reverse osmosis concentrate from potable water reuse facilities. We are finding that it does an excellent job of removing the excess nutrients and most of the chemicals. There are a few chemicals that it is still not capable of removing, but it is

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supply through potable water recycling. Most chemicals are removed, especially if you have a reverse osmosis membrane which only really lets water molecules through. If a compound has a charge on it—and many, many chemicals in wastewater do—they are very easy to keep out. However, some of the uncharged compounds, especially those that are of low molecular weight, can work their way through the membrane in the same way as the water molecules do. We have been particularly interested in a chemical called NDMA or nitrosodimethylamine. This is a potent carcinogen at very low concentrations, but reverse osmosis membranes typically remove only half of it from wastewater. Luckily, for most of the water recycling projects here in California, there is an additional treatment step after reverse osmosis using an ultraviolet lamp. There, NDMA molecules absorb UV light and break down into benign byproducts.

BSJ DS Figure 2: Adsorption quality of Stacked MoS2 Membranes. MoS2 membranes are a POU device designed to remove lead from wastewater due to its high adsorption selectivity. a big improvement to the current practice, which is to release the concentrate to the environment without any treatment at all.

BSJ DS

: What are some common contaminants found in water?

: Traditionally, when people talked about wastewater and its possible effects on the environment, they were concerned with both nutrients—because these could cause excessive algal growth through eutrophication—and metals like copper, nickel, and mercury because they can be toxic to fish at low concentrations. More recently, over the last 20 years, people have become more interested in the trace amounts of organic chemicals that remain after the wastewater treatment process. When it comes to wildlife and aquatic ecosystems, I can give you two examples of chemicals that are particularly interesting to us. One is an antibiotic called sulfamethoxazole. It is a common antibiotic, and it partially survives the sewage treatment process. The levels of sulfamethoxazole in RO concentrate may be high enough to cause stress or damage to not only the fish but also the insects that live in aquatic environments. Another chemical that has been interesting to us in our studies has been fipronil, which is an active ingredient in flea shampoos and topical products used on dogs and cats. It is also sometimes used for controlling ants around the house. The levels of fipronil in RO concentrate are high enough to be of concern to wildlife. When it comes to wastewater and using it as a water supply, we are not only concerned about the effects of the concentrate on the environment but also about chemicals that might make it through the treatment process and find their way back into the drinking water

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: Would point-of-use (POU) devices be a potential solution for these contaminants that are difficult to remove?

: Yes, POU water treatment systems are another emerging way to think about water treatment. We are all somewhat familiar with the very simplest of POU treatment devices. Many people have household-scale reverse osmosis units, especially if you live in a place where the water is quite salty. People may also be familiar with water filters like the ones marketed by Brita. These examples demonstrate this idea that we can take water quality into our own hands and purify the water right before use, which is essentially what POU devices do. From a societal standpoint, it is probably better to treat everyone’s water and not get to a point where only people who are wealthy enough to afford treatment devices are protected. However, in many circumstances, people still feel the need to have a POU water treatment system. For example, folks who do not live in

“It would be incredibly unfortunate if people’s health depended upon whether they could properly operate one of these systems.” cities often have private wells. Those private wells typically have little or no water treatment, so if your home is supplied by a well, a POU treatment system may be a very effective way to protect yourself. In low- and middle-income countries or in parts of the US where the infrastructure is not well maintained, people often feel compelled to take matters into their own hands. For example, the “under-thesink” reverse osmosis market has taken off in places like China and India where people cannot rely on the safety of the water coming out of the tap.

BSJ

: Would you say POU devices have good long-term potential for general wastewater treatment rather than being used solely by individuals?

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: The big challenge with POU water treatment systems is that we need to find a way for them to get into everyone’s hands. One of the things that I like most about our existing centralized water supply system is that no matter your income or personal attitude toward water quality, every individual gets the same quality water. If we move to POU systems, some people may not have enough money or technical skill to operate these systems while others may not care enough to do so. It would be incredibly unfortunate if people’s health depended upon whether they could properly operate one of these systems. I am not saying that it is not possible to make an inexpensive, reliable POU device. When you think about our homes, we have gas furnaces in the basement that, if not properly operated, could set the house on fire or poison us with carbon monoxide gas. It seems like we figured out how to make a gas furnace that is reliable enough where you do not have to have a great deal of expertise or resources to operate it. Someday, I think we could see these POU devices as standard fittings on faucets and sinks in addition to a system for maintaining and replacing them that takes the user out of the equation. But for now, those who have been thinking about this are quite concerned about the need to make sure everyone’s water quality remains protected.

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: Although 2D (molybdenum disulfide) MoS2 is the most selective for lead, it is also highly selective for copper. Does this make MoS2 more or less ideal as a candidate for POU lead removal?

: I have been working with my colleague here in Civil & Environmental Engineering, Professor Baoxia Mi, on these molybdenum disulfide filters. What is really interesting about them and other types of novel materials is we are trying to go beyond reverse osmosis, which is generally an all-or-nothing kind of approach: either you take all the salts out or you take none of them out. Instead, we develop these tailored membrane materials that selectively remove contaminants of concern. The MoS2 membrane is very good at removing so-called “soft metals” that have lots of electrons in their valence shell. These metals, like lead and mercury, are often the ones that we are most concerned about in regards to human health, so MoS2 is an ideal candidate for use in POU devices. If it does prove to be something that can be manufactured inexpensively and robust enough to be left in a home water system, it might become the preferred way to selectively remove lead, which is one of the largest concerns as far as contaminants in drinking waters go.

BSJ DS

: How do you remain optimistic about climate solutions and the water revolution?

about the security of their water. In low- and middle-income countries, we are reaching a point where water security is going to get easier and easier. Even though there are some startling statistics about the number of people who lack access to clean water, the situation improves every year. With the UN Sustainable Development Goals, we are starting to talk about a day when everyone on the planet has access to safe water. Climate change is going to throw a lot of curveballs at us, and we are going to have to be able to adapt. But seeing the creativity of people, not just in technology but also in conservation and policy makes me optimistic that most seemingly permanent water crises stem from a lack of imagination and a lack of will and not an intractable problem. How is that for optimism?

BSJ

: Your book Water 4.0, published in 2014, details the past three “water revolutions” that have enabled the modern use of water. If you were to write a follow-up to this book today, what new information would you include?

: The book talks about the “fourth revolution” that is currently going on. Since I wrote it, in 2010 to 2013, a lot has changed. I am currently in the process of writing a sequel to that book. Water 4.0 was focused on my experience in wealthy countries. This new book is a little broader. It not only addresses wealthy cities but also discusses more generally how we get water to grow food, how we treat water to protect the environment, and how people in lower and middle-income countries are going to transition to secure water sources. In every case, I find reasons for concern but also lots of reasons for optimism, and I am really excited to share them with people. REFERENCES 1. 2. 3.

Headshot. [Photograph of David L. Sedlak]. Image reprinted with permission. Figure 1: Horizontal Levee Project. Oro Loma Sanitary District. (2021, August 30). Retrieved April 4, 2022, from https://oroloma.org/horizontal-levee-project/ Figure 2: Wang, Zhongying, et al. (2020). Superselective removal of lead from water by two-dimensional MoS2 nanosheets and layer-stacked membranes. Environ. Sci. Technol., 54(19). https://doi.org/10.1021/acs.est.0c02651 Scholes, Rachel, et al. (2021). Enabling water reuse by treatment of reverse osmosis concentrate: The promise of constructed wetlands. ACS Environ. Au, 1(1) 7-17. https://doi.org/10.1021/acsenvironau.1c00013

: My optimism for water systems is based upon seeing places like Los Angeles, Singapore, the Bay Area, Atlanta, and a host of other cities where there have never been serious concerns about running out of water. The places that are getting it right never make it into the news. It is only the places where people have not been paying enough attention and failed to make investments to forestall water crises that make the news. I think that in wealthy countries, there is no reason that people should ever have to worry

Berkeley Scientific Journal | SPRING 2022

INTERVIEWS

The Underappreciated Value of Awe BY LEIGHTON PU Figure 1: View of a sunset, which is a known source of awe.

n integral part of the human condition is the constant experience of emotions.1 Eating with friends induces happiness, while sadness ensues loss. A student’s anxiety is maintained at a baseline throughout a semester and spikes during midterm and finals season. Emotions like happiness, sadness, and anxiety are commonly recognized and discussed. Yet awe is not afforded the same attention, and research on its experiential impact only began in the last two decades.2 Even with its powerful ability to cause acute emotional responses, awe receives less attention than more familiar emotions.3 Psychologists still hold different opinions on if awe is an emotion. UCSF professor emeritus Paul Ekamn thinks of awe as an emotion like any other, whereas the pioneering emotional psychologist Richard Lazarus described awe as a psychological state consisting of emotional qualities.3 Though both ideas have their merits, a definition of awe proposed by UC Berkeley’s professor of psychology Dacher Keltner and American social psychologist Jonathan Haidt aligns more with Ekman’s idea. They define awe as an emotion felt when

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one experiences vastness and the need for accommodation.4 Vastness can occur in the presence of colorful sunsets, physically impressive mountains, and looming skyscrapers. It can result from considering the accomplishments of figures like Gandhi or Einstein, or from pondering profound concepts like the golden ratio.3 In other words, vastness is a “stimulus that challenges one’s accustomed frame of reference in some dimension.”4 The challenge vastness brings to an individual’s frame of reference necessitates accommodation—the other condition of awe.4 Accommodation occurs when one changes their mental structures to assimilate a new experience of vastness.4 For instance, accomodation follows observing an incredible shade of green for the first time, in a forest rich in color, or taking in the powerful force of air on a windy day. Awe must meet the conditions of vastness and accommodation, but can nonetheless be induced by simple experiences.5 So long as an individual experiences vastness and the need for accommodation, one experiences awe.4 Simply put, awe is the experience of being struck by something unfamiliar.

On average, people experience awe twice a week.5 The high frequency of awe may be due in part to the many sources of awe. Taking in the height of towering trees, discovering mutual connections with a stranger, or a nice arrangement of clouds can elicit awe. Feats big and small, from a friend facing adversity, but earning high marks, to looking at the Egyptian pyramids, can produce a collective of intense emotions connected to awe. Oftentimes,

“They define awe as an emotion felt when one experiences vastness and the need for accommodation.” researchers studying awe show participants striking images of nature or ask them to recall moments in which they experienced a deep sense of awe. Other elicitors

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Figure 2: Art is a common elicitor of awe. Pictured above is van Gogh’s The Starry Night. of awe include other people, music, art, and beauty.3 Deriving a sense of awe from these or other sources can be nice, but beyond simple pleasure, awe confers many benefits such as increased prosocial values.4 One study that established correlation between awe and stimulated prosociality induced awe in an experimental group by instructing participants to view tall trees. Afterwards, a researcher approached participants with a questionnaire and a box of pens. Each time the researcher approached, they intentionally dropped the box and recorded picking up pens as a prosocial act of helping. Significantly more participants in the experimental group helped pick up the pens than did those in a control group who were instructed to gaze at a large building and did not experience awe. This behavioral difference correlates awe with prosocial behavior. Feelings of a “small self ” leading to diminished self focus, caused by awe, are thought to be responsible for the observed increase in prosociality.6 Awe-prone individuals are less concerned about personal costs incurred when engaging in helping behavior. Whether it be bending down to pick up objects or going out of their way to help a senior citizen cross the street, those that more frequently experience awe seem to possess greater bandwidth to help others, due largely to decreased concern for

themselves. The link between awe and feelings of a small self were later solidified in a study that collected daily measurements of those feelings. Collective engagement was also observed and found to be the result of the small self. Unsurprisingly, the same feelings that increase prosociality also increase engagement with groups.7 Put another way, individuals more willing to help and more inclined to engage with a collective are more likely to be integrated into a social group. Belonging to a collective is important for its ability to act as a support network and source of social stimulation. A feeling of belonging is so important that it was found to be related to enhanced physical health.8 Like physical health, general well being increased in participants of a study that provided them an experience conducive to causing awe. One study brought military veterans and at risk youth white water rafting as a medium to induce awe. Participants were asked to rate certain emotions and social experiences in diary entries before and after rafting. Researchers observed an increase in general well-being after individuals had gone rafting, indicating that nature-induced awe was responsible for the reported increase. This was further established in an experiment studying col-

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lege students that reported how often they experienced nature in their everyday life, how often they experienced positive emotion, and daily life satisfaction. Just as with those that went white water rafting, college students experienced greater well-being on days during which they were more immersed in nature.9 All together, awe is responsible for a wide range of benefits. This singular emotion is so powerful that it has been suggested as a therapeutic tool to address mental health issues.10 As such, cultivating awe is a powerful strategy to live an improved life. But how exactly does one increase encounters with awe? Common methods of induction in the literature on awe suggest nature as a common source of awe. Nonetheless, anything from music and art, to the achievements of other people, or learning about a profound concept in class can all lead to feelings of vastness necessitating accommodation. It is first important to subject oneself to a wealth of experiences that have the potential to induce awe, whether it be looking to the sky on nature walks or attending orchestral concerts. Some awe inspiring experiences can be extraordinary, but many are quite ordinary, such as witnessing someone giving food to the homeless or noticing the changing colors of leaves in autumn.1 Equally as important is to be mindful of the emotions an awe-inspiring experience causes, such as admira-

“By practicing mindfulness and actively putting oneself in situations conducive to facilitating awe, one opens doors to boosting quality of life in multiple facets, from cultivating feelings of belonging to boosting general well-being.”

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Figure 3: Scenes at which experimental and control groups looked at in a study linking prosociality and awe. On the left are a grove of towering trees intended to elicit awe. On the right is a building that the control group was instructed to look at.

Figure 4: Visualization of positive emotions reported in diary entries of participants that went white water rafting. Awe exhibited a strong association with white water rafting, indicating a link between awe and nature. tion, respect, wonder, curiosity, confusion, and love. By practicing mindfulness and actively putting oneself in situations conducive to facilitating awe, one opens doors to boosting quality of life in multiple facets, from cultivating feelings of belonging to boosting general well-being. REFERENCES 1.

van Kleef, G. A., Cheshin, A., Fischer, A. H., & Schneider, I. K. (2016). Editorial: The social nature

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of emotions. Frontiers in Psychology, 7. https://www.frontiersin.org/ article/10.3389/fpsyg.2016.00896 Allen, S. (2016, September 28). Eight reasons why awe makes your life better. Greater Good Magazine. https://greatergood.berkeley.edu/ article/item/eight_reasons_why_awe_ makes_your_life_better Shiota, M. N., Keltner, D., & Mossman, A. (2007). The nature of awe: Elicitors, appraisals, and effects on self-concept. Cognition and

Emotion, 21(5), 944–963. https://doi. org/10.1080/02699930600923668 4. Keltner, D., & Haidt, J. (2003). Approaching awe, a moral, spiritual, and aesthetic emotion. Cognition and Emotion, 17(2), 297–314. https://doi. org/10.1080/02699930302297 5. Keltner, D. (2016, May 10). Why do we feel awe? Greater Good Magazine. https://greatergood.berkeley.edu/ article/item/why_do_we_feel_awe 6. Piff, P. K., Dietze, P., Feinberg, M., Stancato, D. M., & Keltner, D. (2015). Awe, the small self, and prosocial behavior. Journal of Personality and Social Psychology, 108(6), 883–899. https://doi.org/10.1037/pspi0000018 7. Bai, Y., L. Maruskin, S. Chen, A. Gordon, J. Stellar, G. McNeil, K. Peng, and D. Keltner. (2017). Awe, the diminished self, and collective engagement: Universals and cultural variations in the small self. Journal of Personality and Social Psychology. 113(2), 185–209. https://doi. org/10.1037/pspa0000087. 8. Hale, C. J., Hannum, J. W., & Espelage, D. L. (2005). Social support and physical health: The importance of belonging. Journal of American College Health, 53(6), 276–284. https://doi.org/10.3200/ JACH.53.6.276-284 9. Anderson, C. L., Monroy, M., & Keltner, D. (2018). Awe in nature heals: Evidence from military veterans, at-risk youth, and college students. Emotion, 18(8), 1195–1202. https://doi.org/10.1037/emo0000442 10. Chirico, A., & Gaggioli, A. (2021). The potential role of awe for depression: Reassembling the puzzle. Frontiers in Psychology, 12. https:// www.frontiersin.org/article/10.3389/ fpsyg.2021.617715 11. Gibson, K. (2019). The hunt for the best Berkeley sunset, [Image]. University of California, Berkeley, CA, United States. https://beartalk. berkeley.edu/2019/03/25/the-huntfor-the-best-berkeley-sunset/ 12. van Gogh, V. (1889). The starry night [Painting]. Museum of Modern Art, New York City, NY, United States. https://www.moma.org/collection/ works/79802

SPRING 2022 | Berkeley Scientific Journal

Science and Society A Novel Approach to

INTRODUCTION

Science uses various tools to achieve rational deliberation and productive discussion. These tools are neither taught in schools nor written in books; however, they can prove to be exceedingly helpful and relevant for daily decision-making on personal, social, and political matters. In this interview, we talk with Prof. Saul Perlmutter, Prof. Alison Gopnik, and Prof. Johann Frick about their course, “Sense, Sensibility, and Science” (LS 22), where they attempt to convey these scientific and critical thinking tools to their students in order to help them effectively tackle some of the major controversies we find in the world today.

BY GUNAY KIRAN, CAROLYN QIAN, AND ANANYA KRISHNAPURA

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INTERVIEWS

Saul Perlmutter is a professor of physics and the holder of the Franklin W. and Karen Weber Dabby Chair at the University of California, Berkeley. He shares the 2011 Nobel Prize for his discovery of the accelerating expansion of the universe. Dr. Perlmutter is the leader of the International Supernova Cosmology Project as well as a current advisor on the President’s Council of Advisors on Science and Technology. He has served as an instructor for LS 22 since its creation.

Alison Gopnik is a professor of psychology and affiliate professor of philosophy at the University of California, Berkeley. Her current research focuses on children’s ability to discover the world through their powerful, causal learning mechanisms. Dr. Gopnik is the author of many bestselling books, such as The Philosophical Baby and The Scientist In The Crib. She has written for various media outlets, including the New York Times, Science, and the Wall Street Journal’s “Mind and Matter” science column.

Johann Frick is an associate professor of philosophy at the University of California, Berkeley. His research focuses on topics in moral and political philosophy, practical reason, and applied ethics. Dr. Frick received his Ph.D. in philosophy from Harvard University, and he previously served as an associate professor in the Department of Philosophy and the Center for Human Values at Princeton University. This is his first semester serving as a professor for LS 22.

INTERVIEWS

SPRING 2022 | Berkeley Scientific Journal

BSJ SP

: What motivated you to create and serve as an instructor for LS 22 (Sense, Sensibility, and Science)?

: Almost 10 years ago, we were looking at our society as it was, trying to make decisions about what seemed like fairly practical topics such as the debt ceiling, and we realized that these practical issues were being treated as if they were religious debates. We were not getting any kind of rational deliberation. In general, it felt like many of the discussions in our society could benefit from the tools and styles of deliberative thinking that scientists were taking for granted and using on a daily basis in their discussions. We thought that there should be a way to articulate these techniques that we had unconsciously learned via “osmosis” by way of simply being within a community of scientists. I thought, “We should be able to teach these things explicitly.” I realized that it was not good enough for me alone to tackle these topics from a physics background. Some of the questions in the course would need perspectives from social psychology, philosophy, or public policy, so I found faculty and graduate students from these other departments to develop this course. We would meet on Friday in the afternoon, and everybody would stick around for a couple of hours. This went on for nine months. Eventually, we came up with 23 ideas, and we tried to figure out methods to experientially teach them so that people could apply these concepts to other parts of their lives. We created LS 22 in 2014, and since then, we have always taught it in the spring with three faculty instructors—one from the social sciences, one from humanities, and one from the natural sciences. We are now starting to have the course taught at other universities. Both last year and this year, it was taught at Harvard, and this quarter it was taught at UC Irvine. We are continuing to receive calls from other schools that would also like to launch this course.

: Before I started teaching LS 22, Saul and I were friends, and I used to hear him talk about this fascinating class. As a psychologist, I study how people figure out the world around them. Psychologists examine how people learn and how they come to form different kinds of beliefs. In my career, over the course of many years, my biggest argument has been that children are similar to little scientists in what they do and how they figure out the world around them. That gave me a different kind of perspective; instead of seeing science as this niche field for the highly educated and brilliant, I realized that it is really something that is within all of us. The real question for us to ask is, “How can we all use these inherent capabilities to deal with the world around us, and why or when do we choose not to do so?” This is the kind of question I address in the course.

: Unlike Alison and especially Saul, who both have taught this class multiple times, this is my first time teaching this course. When Saul asked me about co-teaching this class last fall, I was immediately captivated by the idea of the course. I thought to myself, “This is the kind of course that I would have loved to take myself as an undergraduate at university.” I think there are two reasons why I was keen to get involved on the teaching side. First, over the last few centuries, science has assembled a set of incredibly powerful concepts that have allowed us to make unprecedented progress in our understanding of the natural and social world around us. I thought that having the opportunity to get a crash introduction to some

Berkeley Scientific Journal | SPRING 2022

“We thought that there should be a way to articulate these techniques that we had unconsciously learned via ‘osmosis’ by way of simply being within a community of scientists.” - SP of these ideas from two preeminent scientists like Saul and Alison sounded well worth the price of admission in itself. The second reason is that I was gripped by the central credo of this class, which is that familiarity with some of the basic tools and methods of modern science can stand us in really good stead outside of the lab. It just seems to me that in our everyday lives as private individuals, we often have to face decisions that require us to reason well, but decisions are made in the face of uncertainty or in situations where it is incredibly easy to fool yourself or to fall into error. For example, these can be situations where you have to sift through messy data to find the signal amidst the noise or where there are various cognitive biases at work that are liable to lead us astray. It seems to me that the tools that modern science has developed for trying to reason well and avoid cognitive errors can be incredibly helpful in an everyday context. Personally, unlike Saul and Alison, I am not a scientist by training. I am a philosopher; I work in moral and political philosophy, which is the part of philosophy that concerns itself with questions of value. Despite not being a scientist myself, a discipline like philosophy has an important contribution to make to a class like this. A sensitivity to questions of values and norms can help us understand the role of science in society and do science “better.”

Taking “Sense, Sensibility, and Science” has refined my understanding of how we know what we know. I feel much more equipped to engage with and understand new information. -Brian Delahunty

BSJ

: As you have mentioned, the fields of science depend upon collaborative deliberation and discussion. As such, do you believe that there is a shared, mind-independent reality whose collective existence helps guide scientists to similar conclusions?

: In the course, we discuss how science has progressed dramatically by the acknowledgment that the world out there is independent of each of us, and we all are trying to get access to more information about this same world. If scientists had each gone off to their own corners, assuming that, “Well, I guess my world just looks

INTERVIEWS

different from everybody else’s,” the field of science never would have progressed as far as it did. In some sense, we take a bit of a pragmatic view on this incredibly deep philosophical question. Though, I should pass the ball back to our philosopher.

: I am a realist. I believe that there is a physical world out there that exists independently of what humans might think and how they perceive it. In that sense, we all inhabit a common reality. However, there are a couple of important caveats. Holding such a broadly realist view about the world is completely compatible with acknowledging that we all have various perspectives on the world and diverse ideas about it. What I would reject is the notion that an

After taking LS 22, I feel more aware of my surroundings and have a motivation to question information I receive on a daily basis. -Anna Benzel

individual’s personal viewpoints define their own world. I think we all have different perspectives about what is real, but it is not true that we each live in our own separate reality. A second caveat is that not all aspects of reality are mind-independent. There are many aspects of our reality that reflect people’s moral beliefs and cultural customs. In one of our plenaries, we talked about funeral practices in different cultures, and I made the point that what is considered a respectful way of disposing of a dead body is often culturally relative. Therefore, the discussion of what is a respectful way of disposing of a dead body is not mind-independent. However, I do think there are many questions that do not fall into this category. For instance, consider the questions, “Does the sun revolve around the earth?” or “Is planet Earth more than 5000 years old?” There are objectively true answers to these questions. People might hold different views and different beliefs on these questions, but ultimately, these beliefs are accountable to a reality that is out there, and that exists independently of what we say or think about it.

: The pragmatic idea that Saul mentioned is incredibly significant. From the perspective of a psychologist, if you want to know how human understanding of the world evolved in the first place, the idea that we are tracking something that is real about the world is a very good way of explaining how it is that we could be existing, surviving, and doing things in the world that actually end up having particular, predictable consequences. We can send a rocket up and we have some idea of where it will go. We can make predictions about the consequences of our actions. However, I would also echo what Johann said, which is that it is interesting that for humans there are all these social and cultural phenomena that are “real.” An example is the concept of marriage. It is real and it is a true fact that my husband is upstairs, and yet marriage is something that

INTERVIEWS

we constitute as part of our social world rather than something that is physically out there the way that the sun or the moon is.

BSJ

: During the LS 22 discussions of whether a country should be governed by a democracy or epistocracy (i.e. rule by experts), a majority of students stated that they would prefer an epistocracy. However, they later learned that experts actually tended to be overconfident in their statements, which could make them more inclined to make mistakes. What are your opinions on the subject, and how do you self-calibrate against overconfidence in your own career?

: As scientists, we are always being self-reflective. We contend with this constant process of change and revision that differs from what is experienced in other areas of human life. On a normal day, you might not want to be thinking all the time about what it is that you are doing and whether it is right. However, under a scientific way of thinking, those questions are critical; they are the means by which we figure out whether or not we are actually being overconfident. This relates back to what we mentioned earlier regarding how a scientific way of thinking can help inform reasonable decision-making in other contexts. Most of the time, it is not so much about an individual person being able to make these considerations, but rather about an individual person putting themselves in a social group or within a set of institutions that enables us to do this.

My greatest takeaway from this course is learning how to think critically about things that are perceived as facts and learning how to look at scientific information with more uncertainty. -Julia Bates

: I was struck by the results of this student poll. A majority of students seem to think perhaps democracy has had its day, and it is time to turn practical decisions over to the experts. There are extremely seductive arguments in favor of epistocracy. If you really care about the stakes of practical decisions and if it really matters to you that you get the right answer, one could ask why you would give the uninformed votes. Why would you not just consult the experts? However, it seems to me that there is a powerful ethical case to be made for democracy as well: People should have a say in decisions that directly concern them. This is the fundamental ethical principle that underlies the case for democracy. For instance, suppose you want to invest money. There are a number of options that you have: You could put it in the stock market, you could invest in real

SPRING 2022 | Berkeley Scientific Journal

estate, or you could put it in a savings account. If you want to make a rational decision about how to spend or invest your money, it would be a very good idea to consult an expert like a financial advisor. However, ultimately, the decision of what to do with your money is still yours to make. After all, you will be the one directly impacted by that decision. The financial advisor can not just invest it in a way that seems most prudent to them without your consent. I think this

“Every approach we have needs to be constantly reexamined since it is inevitable that we will find flaws in almost anything we are doing given enough time.” - AG provides quite a powerful argument in support of democratic input. Yet, in a democracy, we are never just deciding for ourselves; our vote also has an impact on others. This places an ethical duty on us to try to be well-informed when we vote. Therefore, I think there is a very important role for scientific experts to play: advising our political decision-makers, and informing the population at large, so that they can make these widely impactful political decisions. We should not see these two systems as diametrically opposed. The input of experts and decision-making by the population at large should be combined.

BSJ AG

: What is the best way to hold scientists accountable to publishing results that meet a certain rigor or criteria?

: The peer review approach is what everyone depends on now. One of the themes we discuss in the course, though, is that every approach we have needs to be constantly reexamined since it is inevitable that we will find flaws in almost anything we are doing given enough time. Just in the past ten years, many of the procedures that we have adopted in our field have forced us to be more robust and reliable in our work. For example, take pre-registration: Before beginning my research, I will document what my predictions are, how many kids I am going to test, and the kind of statistical analysis I am going to perform. I only just started doing it, but I believe it has greatly improved the science that goes on in my lab. And for all those years, I thought I was a good scientist!

: A similar concept to pre-registration is blind analysis. Unlike the typical blind experiments done in medicine, blind analysis involves a new, extra wrinkle of blinding yourself in the analysis so that you do not know the consequences of your analysis choices until you have committed to them. It is used particularly in certain areas of physics and cosmology, but it is becoming something that people in other areas are starting to look at as well.

Berkeley Scientific Journal | SPRING 2022

BSJ

: As a researcher, how do you make the distinction between when to continue pursuing a line of inquiry and when to consider alternate explanations? How do you distinguish between scientific optimism and naive optimism?

: Let us consider the trade-offs between false positives and false negatives. When deciding whether to continue pursuing an avenue of research or give up and try something else, you have to strike the right kind of balance between these two kinds of errors. The first is a type I error, in which you choose to persist with your line of inquiry when the solution to the problem is beyond your reach or your hypothesis is simply incorrect. On the other hand, there is also the possibility of committing a type II error, in which you give up when the solution to your problem was within your grasp, and had you persisted, perhaps you would have found it. In deciding whether to give up or to persevere, you need to weigh the relative costs of these two kinds of possible errors. You should ask yourself how important of a scientific advance would your discovery be if your research yielded fruitful results. With all else being equal, the more important a result, the longer you should persist. Of course, you should also ask yourself how high the opportunity costs are to persist with that research question if it turns out to be a blind alley. How valuable are alternative uses of your time as a researcher? Another factor that should incline us toward greater persistence is that even a failed hypothesis or research program can still have value to the scientific community. Science is a collaborative activity that is pursued by a whole community of researchers, not in isolation.

The strategies that I learned from LS 22 changed the way I approach a problem in other science classes and even in daily decision-making. It taught me how to think when I am making decisions. -Rohit Jha

Scientific endeavors have value since other scientists can learn from your mistakes. They will already know that a certain possibility has been tried exhaustively in the past, which can save them time and allow them to investigate other possibilities. That is why the costs of persisting with an idea that turns out to be a failure are often not as high as we might think in the beginning. When Newton said he saw so far because he was standing on the shoulders of giants, we need not interpret that remark as, “I saw so far because I was standing on the shoulders of the positive results that previous scientists had established.” The errors of his predecessors are part of what allowed him to see further.

INTERVIEWS

“When Newton says he saw so far because he was standing on the shoulders of giants ... [t]he errors of his predecessors are part of what allowed him to see further.” - JF

BSJ

: Did you ever observe an association between two variables and predicted causation, but you could not perform the necessary experiment(s) to test this hypothesis? If so, did you try to find an alternate way to conclude causation?

: It is often the case that we have a relationship between two things, and we are trying to determine what the causal connection is between them. In astrophysics, there are some surprising connections right now between the mass of galaxies and the behavior of certain small events in these galaxies. One example is that right now, it looks like there is a relationship between the mass of a galaxy and the brightness of a supernova that we use as a distance indicator. The question is, “Can we get away with adjusting an empirical correlation and not understanding the causal connections, or do we feel that it is important for us to test and potentially uncover a causal connection?” If we believe that testing for causation is important, we try to invent new tests based on similar principles to those in Hill’s criteria.1

: There is an interesting contrast between the approaches used in physics and psychology. It is difficult to make concrete observations in the physics that Professor Perlmutter is talking about. You will potentially have one or two observations, and you are trying to discern what other factors are responsible. In psychology, our problem is usually that we observe too much. Every phenomenon we see is correlated with another, so the question becomes, “How do we sort out which factors are causal and which ones are not?” For example, we might say that kids who are better at one activity are also better at another, different activity, but then it turns out that kids that are better at the first activity are older than those who are not doing as well, which is clearly going to affect performance. We combat this issue by using controlled studies. We measure the kids in different circumstances and then see whether the results are different. For example, to test the correlation between a child’s theory of mind and their executive function, we study the executive function while controlling for other variables. Other tools like regression, statistical analysis, or control conditions help when we cannot do these experiments.

BSJ

: Have you ever communicated or encountered a false positive in your research? How did you react when you realized this?

INTERVIEWS

: We thought that we had detected a very dramatic event, which was the creation of a pulsar, and even more importantly, it would have been the very first example of a planet orbiting a star outside of our own solar system; it turned out to be a false positive. It was certainly embarrassing for the group at the time because we had to retract a paper that made an appearance in one of the most visible journals, Nature. It is amazing how random events can sometimes look like a very well-structured signal. Even though, at the time, it seemed like the natural world was treating us a bit unfairly, as scientists we know that random events can happen.

: We conducted the “broccoli and crackers” experiment in the 1990s in which we discovered that 18-month-olds could figure out what somebody else’s desire was, and this got a lot of attention. Many people replicated the effect with 24-month-olds, but not 18-month-olds. Now, when I talk about this finding, I say that children have this capacity somewhere in their second year. We were not sure if the kids that we were looking at were very advanced or if we did the experiment in a slightly different way. It gives a sense of how tricky it can be to be doing developmental psychology work. Part of the problem with kids, for example, is that it is easy to get a false negative. There are a million different factors to look out for; most of the time, when you test kids, you get random noise. Finding any signal is difficult, and it can depend on a factor as unexpected as the lighting in a room. For example, a fun activity is to take a distinguished philosopher, put them in a chair opposite a child, and get them to try and conduct the experiment. I can tell you that the experiments do not work as well when conducted by a nervous philosopher than by a friendly undergraduate research assistant. FOOTNOTES 1.

The Bradford Hill Criteria, known as Hill’s Criteria, are nine standards that can help establish a causal relationship between two factors, especially when randomized control trials cannot be used. An example is the consistency criterion, which states that if findings can be reproduced by multiple individuals in different places and times, this increases the probability that the phenomenon is not simply a result of location. Other criteria include strength, biological gradient, specificity, and temporality. IMAGE REFERENCES

Nobel Prize 2011 Press Conference. (n.d.). Wikimedia. Retrieved April 11, 2022, from https://upload.wikimedia.org/ wikipedia/commons/5/5b/Nobel_Prize_2011-Press_Conference_KVA-DSC_7744.jpg. Alison Gopnik SkeptiCal. (n.d.). Wikimedia. Retrieved April 11, 2022, from https://upload.wikimedia.org/wikipedia/commons/thumb/c/c7/Alison_Gopnik_SkeptiCal. jpg/440px-Alison_Gopnik_SkeptiCal.jpg. Philosophy at Berkeley. (n.d.). Johann Frick. Retrieved April 11, 2022, from https://philosophy.berkeley.edu/person/photo/584/medium/JohannFrick18_0007.jpg.

SPRING 2022 | Berkeley Scientific Journal

On the Sunny Side of Science BY NOAH BUSSELL

hile the lamps along the Charles Bridge and the vibrant colors of the aurora borealis exemplify an ethereal yet romantic quality of light, to a scientist, light is also immensely practical. From photosynthesis to drug design, interactions between matter and light are responsible for the progression of many organic processes, our ability to elucidate the structures involved, and these structures’ significant contributions to the mechanisms of chemical action at work. The relationship between light, structure, and energy is one of the most fundamental and widely utilized concepts in science—and science fiction for that matter. In Jean Luc Goddard’s dystopian film Alphaville, where science and logic are the law of the land, the “laws’’ are E=mc2 and E=hν. The latter serves as the scientific statement that quantifies light’s energy as well as the fundamental equation underlying spectroscopy. While current developments in light-matter research are highly unlikely to plunge us into the technocratic society of Goddard’s film, absent of emotion and free thought, they instead have immense potential to (and currently are working to) usher us into a healthier and more sustainable future. Even historically, advances in our abil-

ity to harness and manipulate light have been linked to the advancement of science. Due to the strong relationship between the structure and function of organic systems, obtaining detailed information about the structure of biomolecules by manipulating and measuring light allows for advances in

Figure 1: Scene from Jean Luc Goddard’s Alphaville portraying the “laws” of the dystopian technocracy, Source: Boake1. fundamental research and health. In 19521953, the English biochemists James Watson and Francis Crick were attempting to uncover the structure of DNA via model building while similar efforts coupled with the chemical analysis of Nobel Laureate Linus Pauling—who inaccurately proposed a triple helix structure—did much of the

Berkeley Scientific Journal | SPRING 2022

same. Nonetheless, it was an image procured by Rosalind Franklin, dubbed Photograph 51, captured via the interaction of X-rays with DNA molecules that led to the fundamental breakthrough. Light provided the answer. Watson and Crick’s postulate in their seminal 1953 Nature paper that “the specific pairing we have postulated immediately suggests a possible copying mechanism for the genetic material” was precinct, and the discovery has revolutionized the field of biochemistry. Their specific contributions to the project were perhaps exaggerated, however. This was in accordance with the sexist environments of the Cavendish and King’s College laboratories where Watson and Crick and Rosalind Franklin were respectively conducting their research.2 As our knowledge of light-matter interactions, and how to manipulate them, continues to advance, scientists are progressing in their ability to make more precise measurements and study increasingly complex systems. Notably, in 2020, a scientific group spread across several European technical institutions published their research on the morphology and energetics of a protein that plays a critical role in energy transport within living systems. Their PNAS paper outlines the mechanism

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Figure 2: (a) Chemical structure of cytochrome c; (b) Structural changes of ferric cytochrome c via excitation and relaxation through spin states, Source: Bacellar, et al.3

“In the case of cytochrome c, the researchers discovered that the protein takes up a domed configuration very soon after excitation by light. As cytochrome c undergoes a change in energy it accordingly shifts to this new conformation.” by which this protein, known as ferric cytochrome c, “relaxes” and simultaneously shifts in shape after light excitation.3 “Ferric” simply refers to the fact that the iron atom in the protein has +3 charge as opposed to the +2 charge of ferrous iron, a subtle yet important distinction in this study. Cytochrome c is an integral component in a collection of proteins that compose the electron transport chain (ETC), a biochemical network essential to life. Cytochrome c specifically channels electrons and their energy to proteins downstream

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in the chain and towards the production of adenosine triphosphate (ATP), commonly referred to as an energetic “currency” in biochemistry.4 Alongside this role in the ETC, cytochrome c can also increase in energy by absorbing light and then subsequently relax in energy. This relaxation occurs through “spin states” in the cytochrome c system; electrons can be either in a spin-up or spindown state, and collectively, these spins can arrange parallel or antiparallel. This alignment in turn affects the energy of the cytochrome c molecule. In certain spin states, molecules have high energy, affecting molecular bonds and geometry. To think of it one way, atoms are attracted to some particles in their environment and repulsed by others. Naturally, they try to move to locations where they are most attracted—when the atoms find a sweet spot where there are a lot of attractive interactions and few repulsive ones, the molecule is in a state of low energy. By exciting cytochrome c proteins with light, electrons are pushed to higher energy states. Then, to compensate for the simultaneous change in bond lengths as atoms move around, the molecule needs to change geometry. In the case of cytochrome c, the researchers discovered that the protein takes up a domed configuration very soon after light excitation. As cytochrome c undergoes a change in energy, it accordingly shifts to this new conformation. While doming had been shown to play a key role

in the respiratory function of ferrous iron proteins, this finding challenges a prior hypothesis that ferric cytochrome proteins (such as the cytochrome c protein in this study) did not undergo doming. Thus, this study provokes inquiries into the biochemical relevance of doming and future studies of ferric cytochrome proteins. To probe the precise structure of these molecules, researchers had to use ultrafast spectroscopy techniques, physical techniques that measure the absorption and emission of light to garner information about chemical systems on miniscule time scales. Ultrafast spectroscopy is an especially useful experimental approach as it can distinguish the various shapes and siz-

“Ultrafast spectroscopy is an especially useful experimental approach as it can distinguish the various shapes and sizes of molecules on the femtosecond time scale (1 millionth of 1 billionth of a second).”

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es of molecules on the femtosecond time scale (1 millionth of 1 billionth of a second). One type of ultrafast spectroscopy used in this study is known as X-ray emission spectroscopy (XES). By “pumping” cytochrome c systems with high energy X-ray light, electrons that are held very closely to atomic nuclei can be ripped away, leaving behind a desirable, low-energy vacancy for another electron to fill.5 This process outputs a spectroscopic signal known as a “Kβ-line,” which is a stream of light that marks this vacancy filling and details where the electrons that filled these gaps came from. While many of these signatures exist, Kβ-lines are specifically significant in the cytochrome c complex. These Kβ-lines are a signature of electrons (here, referred to as 3p electrons) coming from regions where there exists interaction with other electrons that are responsible for most of the molecule’s spin behavior (3d electrons in the cytochrome c system). As such, Kβlines are effectively a marker of both 3p and 3d electrons. In the context of this study, in a low spin state (more antiparallel alignment), it is easier for 3p electrons to produce Kβlines. This results from the enhanced ability of 3d electrons in low spin states to push 3p electrons away towards the low-energy

vacancies. Thus, the Kβ signals that chemists measure are stronger in low-spin states. Using this principle, the group discovered that ferric cytochrome c relaxes to low energy states after light excitation by progressing through these spin states, as opposed to other thermal mechanisms that were previously postulated. To validate their findings, the group used an array of other techniques as well. In fact, there is currently an abundance of research into light-matter interactions, using a whole array of these spectroscopic techniques, with applications ranging from fundamental scientific inquiry to developments in health and sustainable energy. For instance, Professor Naomi Ginsberg’s laboratory at the University of California, Berkeley in collaboration with scientists from Lawrence Berkeley National Laboratory and the Kavli Energy NanoSciences Institute have shown how proteins can serve as a scaffold for the arrangement of chromophore (color-related) complexes. This was accomplished in part through the use of ultrafast spectroscopic techniques. These protein-chromophore complexes mimic the photosynthetic process for light harvesting, an active area of research relevant to the reduction of atmospheric carbon dioxide concentrations. By modifying the lengths and configurations of organic

“By modifying the lengths and configurations of organic “linker” molecules, the proximity and interaction of chromophores, proteins, and their solvent environments are altered with the potential for the development of more efficient light harvesting systems.” “linker” molecules, the proximity and interaction of chromophores, proteins, and their solvent environments are altered with the potential for the development of more efficient light harvesting systems.7 This means that just as plants harvest sunlight

Figure 3: Photograph 51 with analysis, Source: Ho & Carter6.

Berkeley Scientific Journal | SPRING 2022

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to synthesize chemicals that are useful for them, scientists are tweaking the process so that we can channel the sun’s energy to the production of renewable energy. Today, spectroscopy techniques are rapidly advancing, and physical chemists are pushing the field further forward. Dynamic information can be collected on a range of systems on the femtosecond time scale, allowing researchers to not only visualize the structures of molecules, but also the ways in which charge, heat, and energy flow through. Light is even being used to activate drugs, enabling highly targeted, and consequently safer, cancer therapies.8 In addition to its plentiful practicality and the fundamental role it plays in nature, there is something more to light that equations and spectroscopy can’t quite capture. The physicist Erwin Schrödinger once wrote in his book, Mind and Matter, that “the sensation of colour cannot be accounted for by the physicist’s objective picture of light-waves.”9 Regardless, as scientists and philosophers excite organic systems with light to unearth their structure and artists excite our emotions with it, light’s practical and sentimental nature continues to advance and enrich the world around us.

REFERENCES 1.

2. 3.

Boake, T. M. (2008). Alphaville discussion questions. Arch 443/646: Architecture and Film Fall 2008. http://www.tboake.com/443_ alphaville_f08.html Maddox, B. (2003). Rosalind Franklin: The dark lady of DNA. HarperCollins. Bacellar, C., Kinschel, D., Mancini, G. F., Ingle, R. A., Rouxel, J., Cannelli, O., Cirelli, C., Knopp, G., Szlachetko, J., Lima, F. A., Menzi, S., Pamfilidis, G., Kubicek, K., Khakhulin, D., Gawelda, W., Rodriguez-Fernandez, A., Biednov, M., Bressler, C., Arrell, C. A., … Chergui, M. (2020). Spin cascade and doming in ferric hemes: Femtosecond X-ray absorption and X-ray emission studies. Proceedings of the National Academy of Sciences, 117(36), 21914–21920. https://doi. org/10.1073/pnas.2009490117 Rosamond, R., Keeler, A., & Diaz, J. (n.d.). The role of cytochrome c in the

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electron transport chain. Chemistry Texas A&M University. https://www. chem.tamu.edu/rgroup/marcetta/ chem362/HW/2019%20Student%20 Posters/The%20Role%20of%20 Cytochrome%20c%20in%20the%20 Electron%20Transport%20Chain.pdf Bergmann, U., & Glatzel, P. (2009). X-ray emission spectroscopy. Photosynthesis Research, 102(2), 255. https://doi.org/10.1007/s11120-0099483-6 Ho, P. S., & Carter, M. (2011). DNA structure: Alphabet soup for the cellular soul. In H. Seligmann (Ed.), DNA replication. IntechOpen. https:// doi.org/10.5772/18536 Delor, M., Dai, J., Roberts, T. D., Rogers, J. R., Hamed, S. M., Neaton, J. B., Geissler, P. L., Francis, M. B., & Ginsberg, N. S. (2018). Exploiting chromophore–protein interactions through linker engineering to tune photoinduced dynamics in a biomimetic light-harvesting platform. Journal of the American Chemical Society, 140(20), 6278–6287. https:// doi.org/10.1021/jacs.7b13598 Liu, J., Chen, H., Ma, L., He, Z., Wang, D., Liu, Y., Lin, Q., Zhang, T., Gray, N., Kaniskan, H. Ü., Jin, J., & Wei, W. (n.d.). Light-induced control of protein destruction by opto-PROTAC. Science Advances, 6(8). https://doi.org/10.1126/sciadv. aay5154 Schrödinger, E. (1958). Mind and matter. University Press.

SPRING 2022 | Berkeley Scientific Journal

Corrupted Clocks:

How Parasites Utilize the Circadian Rhythm

INTERVIEW WITH DR. FILIPA RIJO-FERREIRA BY: CAROLINE KIM, ANJULI NIYOGI, MICHAEL XIONG, ESTHER LIM

Berkeley Scientific Journal | SPRING 2022

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Dr. Filipa Rijo-Ferreira is an assistant professor of Infectious Diseases and Vaccinology at Berkeley Public Health. Much of her research focuses on the relationship between circadian rhythms—the cycles by which organisms keep track of time—and parasitic infections. Some of her groundbreaking work includes the discovery of circadian rhythms within parasites, such as those responsible for malaria and sleeping sickness, as well as the ability of parasites to influence the circadian rhythms of their hosts. She hopes that these innovations can help inspire ways for scientists to advance medication and treatment of infectious diseases.

BSJ FRF

: What led you to pursue research in infectious diseases, specifically regarding the circadian rhythms of parasites?

: When I was in college, I started becoming very interested in the world of microbiology and how much the immune system of the host and pathogens, viruses, bacteria, or parasites have co-evolved and learned to be together. Parasites, for example, are able to maintain long chronic infections; they do not ‘want’ to kill the host, whilst the host is trying to get rid of the parasite. This really intrigued me. Then I started being interested in neuroscience. I worked in a lab studying a parasite-related condition called sleeping sickness, but there was so little known about the neuroscience side. Sleeping sickness parasites lead to the disruption of sleep, and in particular, patients are not sleeping more but rather, are sleeping at the wrong time of the day, causing increased daytime instead of nighttime sleep. When I went to graduate school, I could not let go of this question that I had been so intrigued about. I wanted to do a project combining both parasitology and circadian rhythms, and that is how it all started.

BSJ

: What is sleeping sickness and what are the symptoms associated with it? Which areas of the world does it predominantly affect?

FRF

: Sleeping sickness is caused by a parasite called Trypanosoma brucei. The fly that transmits the infection is endemic in Africa, especially sub-Saharan Africa. The disease itself has an early blood-stage and is then able to cross the blood-brain barrier and reach the brain. That is when, traditionally, most of the complicated sleep disruption occurs. But there are many different disruptions beyond sleep; there is also dysregulation of rhythmic temperature. Usually, temperature profiles get disrupted, hormone

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secretion gets disrupted, and some very complex neuropsychological disorders come with it. It creates inflammation in the brain and eventually leads to a coma. It is almost 100% lethal if left untreated and unfortunately, the treatments that we have available are also quite toxic. It is believed that about 5% of people that have taken the treatment die of the treatment itself. However, the treatments are still worth taking because the other route is very devastating. There is a lot of further research that needs to be done on the pharmaceutical side, but also on understanding how the disease happens.

BSJ

: One of the most important observations made was a phase advance in the circadian rhythms of the mice. What is a phase advance, and what are its consequences?

FRF

: A rhythm usually has a period, a phase, and an amplitude. It is an oscillation: the phase is the peak of that rhythm, and advance means that it is happening earlier. If we think about sleep having its phase in the night, if there is now a phase advance, that means that sleep is happening earlier. That is most likely what is causing the traditional disruption in humans, in which people experience daytime sleepiness and nighttime insomnia.

BSJ

: It is known that body temperature and circadian rhythms go hand in hand. How did you measure altered body temperature regulation, and what did you discover?

FRF

: When you are active, your temperature rises. Temperature is higher during the day for us and lowers at night when we sleep. It is the opposite for mice that are nocturnal. We can implant a telemetry device in the mouse abdomen that is recording the temperature fluctuation into a receiver in real-time. We were able to find that in the first few days upon infection, there was a high

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Could you describe the SCN and its relationship to circadian rhythms?

FRF

Figure 1: Trypanosoma brucei shortens the period of cells. Infection by T. brucei reduced the length of daily rhythms in mouse tissue samples from a number of different organs. A bioluminescent reporter of the circadian cycle (PERIOD2::LUCIFERASE) showed that circadian rhythms of cells in the presence of parasites drifted away from those of noninfected cells over the course of several days. On average, the period of infected fibroblast cells was shorter than that of healthy fibroblasts. increase in temperature like a fever. Then, the body of a mouse that is infected is able to re-adjust the temperature rhythms, becoming normal again. As they get sick, they will have a hypothermia-like phenotype, so the temperature also drops. There is dysregulation in both the rhythmicity and even on the absolute levels of the temperature.

BSJ

: You measured the locomotor activity of infected mice, which is driven by the suprachiasmatic nucleus (SCN).

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: The SCN is a very small nucleus in the hypothalamus of mice and humans. You have direct connections with the eyes through the optic chiasm. That is how our circadian rhythms are linked to environmental changes from light to dark. We can get our rhythms synchronized with the environment directly because the eye is connected to the SCN. This nucleus is made of 20,000 neurons and they are all coupled to each other; they keep a rhythm because we have a molecular clock inside these neurons and virtually all cells of our body. We have some genes that are transcription factors and transcribe many other genes, among them their own repressors. This creates a loop: the activators transcribe the repressors, which then repress the activators from initiating transcription of them and other genes. This is what is going on in the SCN. It is keeping time, receiving information from light, and then sending out this information through the body to keep other organs and cells synchronized with one another. That is through direct connections like neuronal connections, and also humoral connections like hormones.

BSJ T. brucei?

: What is the function of the Suramin drug? How did you use it in your study to analyze the mechanism of action of

FRF

: Suramin is one drug that is used clinically to treat sleeping sickness. It acts only on the blood stage of the parasite, because this drug in particular is not able to cross the bloodbrain barrier. As I mentioned, there are two phases. If you are able to detect the infection early on, you can give this drug. It kills the parasite. It is believed to act on the metabolic pathways of the parasite. For this study, we took advantage of the drug not crossing the bloodbrain barrier but being able to save mice from dying early on, to better study and mimic human infection. In the mouse model, mice do not have a long lasting chronic infection, possibly because they

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did not co-evolve with this parasite. Once we sub-cure the mouse by killing parasites and decreasing the inflammation in the periphery, the mouse survives for much longer. Only the few parasites that have already crossed to the brain are going to remain. So we are able to mimic low parasitemia (low levels of parasites in the blood) and the long-term behavior changes that we see later on in humans.

BSJ

: Your study establishes that the T. brucei parasite causes disruptions in the host’s circadian rhythm and sleep patterns. How do the findings of this research change the way sleep sickness and sleep disorders are viewed? What do you think are the next steps for this research?

FRF

: Before, we were aware of sleep disorders and circadian disorders, but most of them were commonly associated with genetic variations or mutations from the host itself. Now, this is a new perspective of a parasite or pathogen that is somehow able to modulate and disrupt the clock. This is new information that we did not have before because now it is not just about natural variation in the human population, but specific pathogens that are able to modulate the clock. In regards to what comes next, that would be trying to find out how the parasite is doing all of that, as it is important to understanding sleeping sickness. One of the things we found is that the parasite appears to be secreting a molecule, but we do not know what it is. If we are able to identify this molecule, it could be useful. If it is not pathogenic, we could use the molecule itself to modulate the clock of humans. Imagine you are going through jet lag, or if you have a phase delay disorder or delayed sleep pattern, and you want to bring your phase earlier, you may benefit from using that molecule. Figure 3: Image of red blood cells of mice infected with Plasmodium chabaudi, a parasite of the Plasmodium genus responsible for causing malaria. Small purple-dyed dots within the red blood cells refer to the parasite; large dark purple circles refer to immune cells.

BSJ

: In one of your studies, you infected mice with the parasite Plasmodium chabaudi, one of the many species in the Plasmodium genus that is responsible for causing malaria. What were the main goals going into this study, and how were they shaped by your previous research with T. brucei?

FRF Figure 2: The life-cycle of the malaria parasite Plasmodium chabaudi. Within the bloodstream, P. chabaudi reproduces by infecting red blood cells. The parasite begins as an immature trophozoite within the red blood cell, appearing as a ring. Next, the parasite matures into a schizont, which replicates and bursts from the cell, ready to begin another round of infection.

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: Once we became aware that parasites and circadian clocks can interact with one another, and learned that parasites themselves like T. brucei may have their own clock, we came to this question about malaria. We were very intrigued about the rhythmic fevers that malaria is known for. Even before we knew it was caused by a parasite, we already knew about these fevers that would come and go at certain times of the day. This periodicity actually has a multiple of 24 hours, which is a circadian characteristic, as you can imagine. It was very difficult to distinguish if the fevers in malaria were a consequence of the host circadian rhythm, or if it was more complex than that, and actually had a contribution from both the host and the parasite. And that was the main goal: Can we test if there is a contribution beyond the circadian rhythm of the host—is

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there also a parasitic contribution to it?

BSJ

: You discuss how the intrinsic clock of the malarial parasite is the underlying mechanism for malarial rhythmic fevers. What cellular processes occurred during these fevers?

FRF

: There is this amazing phenomenon. These unicellular parasites invade red blood cells, but they do it coordinately. All the other parasites are also invading red blood cells of their own. They are multiplying and replicating inside the red blood cell, and then they burst the red blood cell. When they do this, they burst all millions of red blood cells at once, exposing newly formed parasites and all the metabolic waste that the parasite generated while living in these red blood cells. That triggers a massive immune response which causes fevers.

BSJ FRF

: How is the cell cycle and gene expression of a parasite linked to its intrinsic clock?

: That is actually a very difficult question. We know from other organisms, like mammalian cells, that the circadian clock and the cell cycle are usually independent, but they do regulate one another. So they may also be linked to the parasite. But we also know from the mammalian system that cells that have grown enough to cover an entire petri dish, called confluent cells, do not divide anymore. There is no more space to create new ones, so they

do not divide anymore, but they still have circadian rhythms. One does not drive the other, even though they might interact with one another. In the malaria research that we did, we were looking into blood, where there is definitely also replication of the parasite. We are now going to have to dissociate these two, and try to understand which genes are cell cycle-related, and which genes were circadian rhythm-related. Most likely, we need to go to a stage of the parasite that has no division.

BSJ

: In the process of proving that the malarial parasite has rhythms of its own, you had to control for several confounding factors, like food and light exposure. How could these factors have influenced the circadian rhythms you observed?

FRF

: The way to prove that there is an intrinsic clock is through removal of all of the possible confounders. For example, light is known to be an important biological cue that many organisms respond to, and our team was quite worried as to whether or not this was also important for malaria parasites. We tested this by eliminating any light in the parasite’s environment, and instead, having complete darkness to ensure that the parasite’s rhythms were generated on its own. After we established light was not a required cue, we also thought of considering food, because there have been studies showing that parasites sense when the food is being consumed and align the timing at which they burst red blood cells. If you gave the food at the wrong time of the day, then the parasites would burst at an abnormal time. What we did to solve this was to introduce automated feeders to the mice to abolish a normal feeding rhythm. We gave food evenly distributed throughout the day so that the mice were eating small portions of food every hour and a half, which is very different from what they normally do, which is eating a lot in 12 hours and not eating at all in the other 12 hours. Even after removing the food confounder, we still observed a consistent rhythm in the parasite, and so we were able to conclude that food is a potential factor that the parasites sense, but is not completely necessary for their synchronization.

BSJ

: The models that you developed led you to the conclusion that malaria parasites use host activity to synchronize their internal clocks. Why might the malaria parasite benefit from having its own circadian rhythm rather than simply taking cues from its host?

FRF Figure 4: Graph depicts the distribution of gene expression of the Plasmodium chabaudi parasite throughout a 24-hour cycle. The green distribution represents the parasites of mice that received food spread out during the day, whereas the blue distribution represents parasites that followed a regular feeding schedule (control). The distribution concludes how host cues, such as feeding patterns, did not significantly influence gene expression of the parasite.

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: This is really the big “why” question: Why would parasites go through the effort of having an intrinsic clock if they can just rely on the host? I think the most important aspect to consider in answering this question is understanding the difference between responding and anticipating. When we think of plants, for example, there is a very obvious need for sunlight to conduct photosynthesis. If plants had a circadian clock that allowed them to anticipate when light was coming, they could be more prepared to photosynthesize by making all of the relevant proteins earlier, even halfway through the night because, in a few hours, the sun will come out. If they had just waited for the sun, on the other hand, they wouldn’t be prepared to photosynthesize as effectively until much later in the day. We can think about this reasoning in the context

INTERVIEWS

of parasites. By predicting when nutrients are going to be available, they could time when to replicate. Our immune system is also highly rhythmic—the number of lymphocytes in circulation is very different from night and day. Sometimes they are in the lymph nodes and other times, they are in the blood. If the parasites can predict these and other changes in their environment, they might be at an advantage.

BSJ FRF

: How do the results of your study give us more insight into how we can best treat these parasitic infections?

: This new research on circadian rhythms and parasitic diseases may open doors for further research on when the best time to treat these infections is. When we need to take medication, we are often not informed of when it is the best time to take it because we have not studied it. Regarding the sleeping sickness parasite, our team found that the parasite is more susceptible to the suramin drug at specific times of the day. Just from this information, we can improve the outcome of the disease by administering the drug when the parasite is much more vulnerable. There is a window of opportunity to kill parasites more effectively, and this is something that is important to consider in treating parasitic infections.

BSJ FRF

: What is the most rewarding aspect of your work? What do you love most about being a scientist?

: For me, the most rewarding part is the teamwork that goes into trying to discover what is unknown. And this kind of work cannot be done alone—being able to work with students, postdocs, and other faculty members has made my work very fun and rewarding. In this particular malaria paper that we have just discussed, I had a very strong collaboration with a mathematician who helped us model it. And that is just one example of how interdisciplinary science can be and how fun it is. REFERENCES 1. 2.

Headshot: [Photograph of Filipa Rijo-Ferreira]. Image reprinted with permission. Figure 1: Rijo-Ferreira, F., Carvalho, T., Afonso, C., Sanches-Vaz, M., Costa, R. M., Figueiredo, L. M., & Takahashi, J. S. (2018). Sleeping sickness is a circadian disorder. Nature Communications, 9(1). https://doi.org/10.1038/s41467-017-02484-2 Figures 2 and 4: Rijo-Ferreira, F., Acosta-Rodriguez, V. A., Abel, J. H., Kornblum, I., Bento, I., Kilaru, G., Klerman, E. B., Mota, M. M., & Takahashi, J. S. (2020). The malaria parasite has an intrinsic clock. Science, 368(6492), 746–753. https://doi. org/10.1126/science.aba2658 Figure 3: [Microscope image]. Rijo-Ferreira Lab. Image reprinted with permission.

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SPRING 2022 | Berkeley Scientific Journal

Using Deep Reinforcement Learning to Peer Into the Unconquerable Mind: How Do Animals Learn to Track Odor Trails? BY ANISHA IYER

early all pet owners and park frequenters have witnessed an animal searching furiously along a scent trail. Dogs and other olfaction-dependent animals are expert trail trackers, often relying on odor trails to perform life-sustaining tasks like foraging for food or navigating complex environments. However, odor trails in nature are laden with gaps, intersecting routes, and sporadic or incomplete odor cues which interfere with an animal’s ability to develop a clear picture of a trail. Such constraints provide much to overcome for olfaction-dependent animals who rely almost solely on olfaction for vital behaviors. With such a computationally intensive task, one must wonder: What takes place inside an animal’s brain to evaluate and optimize such a complex set of dynamic variables in real-time? Olfaction-based trail tracking is a complex and precise behavior, which makes it a challenging investigative endeavor for theoretical neuroscientists and researchers in related fields. Scientists can simulate odor trail tracking in laboratory settings and have described experimentally observed tracking strategies using statistics and geometry. However, questions regarding the animal’s cognition still remain unanswered, largely due to the difficulty of representing such far-reaching questions in a testable manner. As a result, there is a broader goal in

asking how scientists might reach conclusions about the interconnected systems of the infinitely complex brain. The quest to find ways to represent cognitive processes spans further than this specific research question and is complicated by a scientist’s need for control in scientific experiments. If scientists require an observable version of the system in question and a controlled way to manipulate its variables, how might they find an equivalent for the vast and unconquerable brain? QUESTIONS IN SYSTEMS NEUROSCIENCE Despite centuries of neuroscience research, the precise workings of the brain’s olfactory and navigational systems remain relatively unclear. Neuroscience is studied at a cellular and anatomical level, but processes like odor trail tracking require a robust theoretical framework for scientists to develop a system-wide understanding. While scientists conceptualize odor trail tracking as searching consecutive trail sectors using complex mathematical techniques, it seems unreasonable to credit an animal’s impressive tracking abilities to conscious and deliberate statistical approximations. Rather, an animal following a scent must develop some kind of navigational intuition to deduce probabilities without calculating them.

Berkeley Scientific Journal | SPRING 2022

Figure 1: Dog tracking odor trail To understand these biological systems, scientists combine experimental and computational research to acquire a system-wide understanding, which is the basis of a field called systems biology. By simulating a subset of the system’s components, scientists use models and theoretical exploration to glean insights into the behavior of widely expansive and intricately complex biological systems. Systems biology relies on data engineering and machine learning techniques to obtain

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Figure 2: Art depicting neuronal migration. The heterogeneous makeup of this image, in both artistic and symbolic cellular contexts, represents the complex task of trying to capture or recreate elements of neuroscience that we do not fully understand. a system-wide understanding of biological problems. For odor trail tracking, scientists have used machine learning techniques to recreate an animal’s learning process as it tracks a trail by modeling the way an animal learns through trial and error in a simulated environment.1 MACHINE LEARNING One of today’s most rapidly growing technical fields, machine learning (ML), focuses broadly on constructing self-improving computer systems and understanding the statistical, computational, and theoretical laws that govern all learning systems on a fundamental level. Across a number of disciplines, techniques, and applications, ML models make inferences based on trends in existing data. Furthermore, these inferences, which operate by using machine-based computation, actually reflect physical neuroscientific learning processes that occur in animal life. Throughout computer science and a

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wide range of other industries, ML is revolutionizing technology: In commercial sectors, ML optimizes consumer services and logistic chains, and in academia, ML serves laboratory curiosity. Accordingly, various empirical sciences have recruited ML methods to analyze high-throughput data in new ways.2 ML can be distilled into three branches: supervised learning, unsupervised learning, and reinforcement learning. Supervised learning (SL) is the most straightforward branch of ML, in which models make inferences by training on labeled and categorized data. SL requires a knowledgeable, external supervisor to label the training data and subsequently simplify the task for the machine. For instance, if an SL model were trained on a dataset of labeled images of cats and dogs, the model could learn to predict whether a new image is a cat or a dog, performing as a classifier. Unsupervised learning (UL) is similar to SL in that it also requires previously obtained data, but it uses chaotic and un-

“Furthermore, these inferences, which operate by using machine-based computation, actually reftect physical neuroscientific learning processes that occur in animal life.” filtered data that has not been labeled or categorized. ML models for unsupervised learning would recognize trends and patterns in chaotic data and use these observations to make inferences and predictions about new data. Beyond SL and UL, reinforcement learning employs supervised, unsupervised, and novel learning techniques to learn optimal strategies for success in a goal-oriented environment.

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Figure 3: A schematic of an artificial neural network (ANN), modeled after a biological neuron with input-receiving dendrites, a cell body that takes in electrochemical information from dendrites, an axon hillock which conditionally triggers an electrical action potential if the voltage surpasses a threshold, an axon down which the action potential propagates, and a synaptic terminal where the output is sent to the next neuron in the circuit. Here, the ANN takes in several numerical inputs, which undergo a linear transformation by the transfer function, or net input function, to integrate biases and tunable weights representing the mathematically-identified importance of data from that node. Transformed inputs are sent into an activation function which conditionally activates the neuron depending on whether the output surpasses a threshold. Deep neural networks have more complicated architectures than ANNs, with several hidden layers and more sophisticated mathematical processing. REINFORCEMENT LEARNING Reinforcement learning models concern an agent as it fine-tunes its strategy to seek reward in an environment, solely through trial and error. Rather than identifying whether an image’s features match more to a dog or a cat, RL problems typically center around an active agent trying to learn an optimal strategy, or policy, in a dynamic environment, such as a winning strategy for a game of chess. Broadly, RL agents learn to interact with their unknown and dynamic environment with no prior knowledge, where the in-progress policy is responsible for selecting present and potential future actions that affect each progressive state of the environment. While there are certain elements that must be controlled or simplified by scientists when setting up an RL model, these models are arguably the most accurate representations of policy-based human learning developed thus far, where policy-based learning refers to the fine-tuning of strategy that someone like a chess player would use to win a chess game.2 In every state of the environment, there are pathways through which the agent can achieve reward, which are closely dependent on the agent’s own actions. With an RL agent’s capacity to affect the

state of the environment, the agent’s actions play a large role in opening or closing pathways to reward. In the short term, an agent takes actions that change small aspects of the state and alter the potential for expected reward. However, if the agent takes a suboptimal action, it could limit future avenues for reward, metaphorically closing a door to a particular outcome. Building upon its short-term goal to optimize statistical descriptions of reward

in the environment, the agent pursues its ultimate goal of traversing the maximal reward pathway through complicated and highly unintuitive “black box” algorithms. Such algorithms are termed “black box” algorithms because it is too complex to try to obtain a comprehensive understanding of their inner-workings. Resultantly, scientists ignore the question of how “black box” algorithms work, when working with them, focusing only on which inputs to

Figure 4: Reinforcement learning; depicts agent-environment interaction and relationship.

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“The foundational principle of using reward to reinforce behaviors connects directly to neuroscience, rendering RL algorithms intentional microcosms of the adaptable brain.”

feed in and what outputs to expect. As a result of this architectural composition, RL has great potential to aptly represent situations where a conscious being crafts an optimal policy through trial and error. RL AS IT RELATES TO NEUROSCIENCE RL algorithms allow us to recreate and optimize models of complex tasks, like playing a game of chess or tracking a surface-born odor trail. For the latter, an RL model can recapitulate the behavior of an animal searching for the source of an odor, a task that requires complex computation and statistical optimization that exceeds any animal’s conscious computational capacity. Through this simulation, scientists have access to a model whose variables can be manipulated to understand the extent to which conditions like environmental, spatial, and physical constraints affect or limit the agent’s behavior. By testing the agent’s ability to overcome simulated constraints, RL algorithms can lend insight into the extents of an animal’s tracking ability. Foundationally, RL theory is based on psychological and neuroscientific principles of learning and reward. Much like how an infant is born with no prior knowledge and only a sensorimotor connection to its environment, RL agents begin with no background and only a means to take actions to affect the state of the environment. From feedback, an infant modifies its behaviors to develop a more powerful understanding of how to optimally behave

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in its environment. In the same way, a reinforcement learning agent, which would begin naive, builds upon a nonexistent understanding of its environment through corrective feedback to reach an optimal strategy. The foundational principle of using reward to reinforce behaviors connects directly to neuroscience, rendering RL algorithms intentional microcosms of the adaptable brain. Methodologically, the broader goal of RL algorithms to achieve maximal reward is emblematic of the brain’s limbic system and neurochemical reinforcement during the formation of neuronal connections. Animals learn from neurochemical reinforcement signals that are naturally embedded in the process of trial and error.3 As a result, the parallel between the neurochemical learning process of an animal and the RL training of an agent is a curious one that enables multidirectional biomimicry. Roughly speaking, RL learning mimics the formation of synaptic connections from neurochemical reinforcement by upticking the model’s quantification of reward to reinforce smart actions. Deepening the connection to neuroscientific learning, RL algorithms function in this manner to reinforce actions with simulated neurochemical reinforcement, once again maintaining a strong, but general, connection to the growth and reinforcement of neural synapses on a cellular and molecular level. Moreover, using RL to represent a system of animalian learning is particularly significant because the method of RL learning is based on principles of neuroscientific learning. An agent’s encounters with positive reinforcement, for the computational purposes of training, are indicative of the positive neurochemical reinforcement an animal receives while it learns a task in nature. This proposes a conceptual translation of modeled neurochemical reinforcement, via numeric upticking of reward, into actual neurochemical reinforcement, via excitatory neurotransmitters and other biophysical potentiation mechanisms during the training of the real animal. As a result, RL is highly applicable to cognitive neuroscience, with strong quantitative components. Through the direct application of RL for simulation in neuroscience, as well as foundationally and methodologically, RL maintains inten-

tional, direct, and symbolic connections to neuroscience. CONCLUSION Questions in cognitive neuroscience span the vast inner-workings of the brain and its interconnected systems, augmenting the need to develop scientific approaches which can answer them. A common barrier in systems biology is the task of creating an accurate representation of the system in question, which would offer insight into the complex process of interest, such as odor trail tracking. RL enables scientists to establish a model of the neurological systems needed to create this level of precision and optimization in an animal incapable of consciously calculating high-level statistical computations. For odor trail tracking, RL algorithms allow scientists to obtain further insight into the requirements for cognitive neuroscience’s abilities and the extents of neuroscientific systems. With RL’s ability to recreate cognitive neuroscience problems and to represent an agent’s development through models, the field of RL opens up possibilities to further understand cognitive neuroscience systems and peer into the unconquerable mind. As science continues to deepen its interest in the use of RL for neuroscience, the field holds a promising future for how scientists can use RL to shed light on cognitive neuroscience’s complex processes and systems.

“An agent’s encounters with positive reinforcement, for the computational purposes of training, are indicative of the positive neurochemical reinforcement an animal receives while it learns a task in nature.”

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REFERENCES 1. 2.

Kitano, H. (2002). Computational Systems Biology. Nature, 420(6912), 206–210. Jordan, M. I., &amp; Mitchell, T. M. (2015). Machine learning: Trends, Perspectives, and prospects. Science, 349(6245), 255–260. https://doi. org/10.1126/science.aaa8415 Schultz, W., Dayan, P., & Montague, P. R. (1997). A neural substrate of prediction and reward. Science, 275(5306), 1593–1599. https://doi. org/10.1126/science.275.5306.1593 Vergassola, M., Villermaux, E., & Shraiman, B. I. (2007). ‘Infotaxis’ as a strategy for searching without gradients. Nature, 445(7126), 406–409. https://doi.org/10.1038/ nature05464 Lillicrap, T. P., Hunt, J. J., Pritzel, A., Heess, N., Erez, T., Tassa, Y., Silver, D., & Wierstra, D. (2019). Continuous control with deep reinforcement learning (arXiv:1509.02971). arXiv. http://arxiv.org/abs/1509.02971 Jinn, J., Connor, E. G., & Jacobs, L. F. (2020). How Ambient Environment Influences Olfactory Orientation in Search and Rescue Dogs. Chemical Senses, 45(8), 625–634. https://doi. org/10.1093/chemse/bjaa060 Khan, A. G., Sarangi, M., & Bhalla, U. S. (2012). Rats track odour trails accurately using a multi-layered strategy with near-optimal sampling. Nature Communications, 3(1), 703. https://doi.org/10.1038/ncomms1712 Mnih, V., Kavukcuoglu, K., Silver, D., Rusu, A. A., Veness, J., Bellemare, M. G., Graves, A., Riedmiller, M., Fidjeland, A. K., Ostrovski, G., Petersen, S., Beattie, C., Sadik, A., Antonoglou, I., King, H., Kumaran, D., Wierstra, D., Legg, S., & Hassabis, D. (2015). Human-level control through deep reinforcement learning. Nature, 518(7540), 529–533. https:// doi.org/10.1038/nature14236 Reddy, G., Murthy, V. N., & Vergassola, M. (2022). Olfactory Sensing and Navigation in Turbulent Environments. Annual Review of Condensed Matter Physics, 13(1), 191–213. https://doi.org/10.1146/

annurev-conmatphys-031720-032754 10. Reddy, G., Shraiman, B., & Vergassola, M. (n.d.). Sector search strategies for odor trail tracking. 28. 11. Silver, D., Huang, A., Maddison, C. J., Guez, A., Sifre, L., van den Driessche, G., Schrittwieser, J., Antonoglou, I., Panneershelvam, V., Lanctot, M., Dieleman, S., Grewe, D., Nham, J., Kalchbrenner, N., Sutskever, I., Lillicrap, T., Leach, M., Kavukcuoglu, K., Graepel, T., & Hassabis, D. (2016). Mastering the game of Go with deep neural networks and tree search. Nature, 529(7587), 484–489. https:// doi.org/10.1038/nature16961 12. Sutton, R. S., & Barto, A. G. (1998). Reinforcement learning an introduction. A Bradford Book.

from https://commons.wikimedia. org/wiki/File:Rl_agent.png.

IMAGE REFERENCES 1.

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Greg Bradshaw. (2011, April). New Guinea Singing Dog sniffing the ground [Photograph].Wikimedia Commons. https://commons. wikimedia.org/wiki/File:New_ Guinea_Singing_Dog_sniffing_the_ ground.jpg Harris, P. B. (n.d.). Neuronal migration is an artwork depicting many very young neurons that have been produced in the neuroepithelium migrating to their appropriate destinations in the brain. This image highlights the future of neuroscience showing different classes of cells colour coded. There is no available technique to do this now, but it is not far off considering the advances that have been made with brainbow mice. The brainbow technique allows for different cell types to be tagged with fluorescent proteins to track their development and connections with other cells. Wellcome Collection. Retrieved from https://wellcomecollection.org/works/ u2mrc7w5. Saini, G. (2017). Artificial Neural Network.png. Wikimedia Commons. Retrieved from https://commons. wikimedia.org/wiki/File:Artificial_ neural_network.png. Notfruit. (2017). Rl agent.png. Wikimedia Commons. Retrieved

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Antibiotic Resistance:

A Silent Pandemic BY MERVE OZDEMIR

n 1928, in a London laboratory, Scottish bacteriologist Alexander Fleming looked into one of his culture plates, on which he was growing colonies of Staphylococcus, a genus of Gram positive bacteria that grows in grape-like clusters. To his surprise, he noticed a colony of contaminated mold around which the bacteria could not grow (Figure 1).1 Fleming had accidentally discovered a substance produced by the fungus Penicillium chrysogenum that was able to inhibit bacterial growth. The mystery substance had contaminated Fleming’s sample, killing off the bacteria and producing a clear, bacteria-free zone in the dish. Though Fleming could neither identify nor purify the substance at the time, he named it “penicillin.”2 It was only a decade later that scientists at Oxford University, Howard Florey, Ernst Chain, and Norman Heatley, succeeded in purifying penicillin and proposing it as a drug.3 In 1945, Florey and Chain were awarded the Nobel Prize together

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with Fleming. Shortly after, mass production of the antibiotic began. While penicillin was the first antibiotic to be discovered, it certainly was not the last. Between 1940 and 1960, the period often called the “Golden Age of Discovery,” scientists discovered many more antibiotics, including those still commonly used like streptomycin, erythromycin and chloramphenicol.2 Today, antibiotics are one of the most commonly used medicines, prescribed to treat a variety of bacterial infections. They work by disrupting essential processes in the bacteria, which results in either their immediate death or arrest in replication. But modern healthcare is facing a serious threat. After decades of treating patients with antibiotics, bacteria are learning to fight back, and our drugs are no longer the almighty killers they used to be. There are a number of bacterial strains that have developed resistance to one or more antibiotics, and resistance is spreading at a pace

faster than new drug developments can keep up. Currently, drug resistant infections kill around 700,000 people every year, and the United Nations estimates that the annual death toll could go up to 10 million by 2050, which would make drug resistant pathogens deadlier than cancer.4 “In essence,” proclaimed a 2013 editorial in Na-

“After decades of treating patients with antibiotics, bacteria are learning to fight back, and our drugs are no longer the almighty killers they used to be.”

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Figure 1. Fleming’s photograph of a Staphylococcus culture plate with bacterial clearing around the penicillium colony.

“The data points to a concerning possibility, where simple infections that are easily treated today could become, in the not-so-distant future, a death sentence.” ture, “we are engaged in an arms race with pathogenic bacteria — and we are losing.”5 The data points to a concerning possibility, where simple infections that are easily treated today could become, in the not-so-distant future, a death sentence. HOW DID WE GET HERE? When Fleming won the Nobel Prize in 1945, he warned the public of a concerning possibility. In his Nobel lecture, he explained, “The time may come when penicillin can be bought by anyone in the

shops. Then, there is the danger that the ignorant man may easily underdose himself, and by exposing his microbes to non-lethal quantities of the drug, make them resistant.”6 Fleming’s fear of resistance has certainly become a reality today, but it is important to ask nevertheless: how did we get here? Contrary to popular belief, antibiotic resistance did not start with us. In the natural world, where bacteria compete with each other for limited resources, antibiotics have been bacteria’s biggest weapons against other bacteria. Thus, antibiotics have existed in nature even before humans started using them clinically, evolving slowly through natural processes. But the current rate at which resistance is evolving is much faster than natural evolution, making it almost impossible for us to catch up.7 What is causing resistance to evolve so quickly? In a way, humans are. Millions of pounds of antibiotics are used each year, but the usage of these drugs is poorly regulated. In 2020, the CDC reported that healthcare providers have prescribed 201.9 million antibiotic prescriptions.8 According to another report, at least 28% of annual prescriptions in outpatient settings were completely unnecessary.9

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Studies have also shown that around 3060% of antibiotics prescribed in hospital intensive care units (ICUs) are “unnecessary,” “inappropriate,” or “suboptimal.”10 But extensive antibiotic prescriptions to patients is only part of the problem. Around 80% of antibiotics sold in the United States are used in livestock to prevent infections, improve the health of animals, and increase yields.11 The resistant bacteria that thrive in livestock under antibiotic pressure are then transferred to humans upon ingestion, where they can cause infection. Furthermore, the excretion of antibiotics from livestock via urine and stool cause the drugs to get mixed in soil and disperse through fertilizer and groundwater.12 So while humans did not create the biological phenomenon of antibiotic resistance, we certainly played a massive role in turning resistance into the large-scale threat it is today. The excessive use of antibiotics has created an evolutionary stress on bacterial populations, driving the development of resistance through positive selection for resistance genes. HORIZONTAL GENE TRANSFER

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infection.13 Conjugation, unlike transformation and transduction, requires physical contact. It occurs through a conjugation “pilus,” a bridge-like structure through which genetic material is transferred from the donor to the recipient (Figure 2).14 WHAT’S NEXT?

Figure 2. Conjugation pilus between donor and recipient bacteria. One way that bacteria speed up the evolution of drug resistance is through a process called horizontal gene transfer (HGT), the exchange of genetic material between non-genetically related bacteria. Similar to how humans pass on genes to their offspring (vertical gene transfer), bacteria can exchange segments of DNA between each other without needing to have a parent-offspring relationship. Genes conferring resistance can spread among different bacterial species through HGT. In the presence of antibiotics, the spread of resistance is so favorable for survival that some bacteria even actively kill their neighbors to speed up the process of horizontal gene transfer.7 Genes can be transferred horizontally through three mechanisms: conjugation, transformation, and transduction. Transformation is the process in which free, “naked” DNA from the environment is incorporated into the genome of a bacteria. Transduction is similar but requires mediation by a bacteriophage, a special type of virus that infects bacteria. Genes from the host bacterium are carried in the genome of the bacteriophage, then incorporated into the recipient bacterium upon phage

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Unfortunately, little progress is being made to overcome the antibiotic resistance crisis. The development of new, effective antibiotics is incredibly slow, mostly due to economic barriers. Developing an antibiotic is costly, and the revenue from selling the drug is relatively low. For pharmaceutical companies, investing in this area of research is simply not profitable. Many companies have left the search for new drugs and have instead moved on to more profitable areas like cancer therapeutics.4 Healthcare workers, policy makers, governments, and even economists have been slow to act. Despite the enormity of the threat, its gradual development over time compared to more sudden crises like the COVID-19 pandemic has led people to underestimate the urgency of the problem. But hope remains. Hospital-based antibiotic use can be improved with stewardship programs, and patients can be better educated about the importance of taking antibiotics in the correct dose and duration throughout their treatment. Similarly, public education about when and why antibiotics are necessary is essential. For example, antibiotics are useless against viral infections, because viruses have a significantly different structure than bacteria, rendering them resistant to the action mechanism of antibiotics. Preventative measures like hygienic practices can also be taken by individuals to limit the risk of infections in the first place. Antibiotic usage in agriculture and livestock must be better regulated, taking into account the long-term effects of these drugs on the environment and human health.15 CONCLUSION The problem of antibiotic resistance seems to be growing at an alarming rate. Though the numbers are certainly cause for alarm, we can come back from this. Bacteria are diverse and sophisticated, yet

they only want two simple things: to survive and to replicate. It is our actions that disrupted the balance and forced them to wage war on us. It is not too late to reestablish peace. Public education, better policies, collective action and a detailed understanding of the mechanisms behind resistance is our biggest chance. This is just the beginning. REFERENCES 1.

Fleming, A. (1929). On the antibacterial action of cultures of a penicillium, with special reference to their use in the isolation of B. influenzae. British journal of experimental pathology, 10(3), 226. 2. Gaynes R. (2017). The Discoveryof penicillin—new insights after more than 75 years of clinical use. Emerging Infectious Diseases, 23(5), 849–853. https://doi.org/10.3201/ eid2305.161556 3. Chain, E., Florey, H. W., Gardner, A. D., Heatley, N. G., Jennings, M. A., Orr-Ewing, J., & Sanders, A. G. (1940). Penicillin as a chemotherapeutic agent. The lancet, 236(6104), 226-228. 4. Plackett, B. (2020). Nature, 586, 5053. 5. The antibiotic alarm. (2013). Nature,495(7440), 141–141. https:// doi.org/10.1038/495141a 6. Fleming, A. (1942). Nobel Lecture, December 11, 1945. Nobel Lectures, Physiology or Medicine, 1962, 83–93. 7. Cooper, R. M., Tsimring, L., & Hasty, J. (n.d.). Inter-species population dynamics enhance microbial horizontal gene transfer and spread of antibiotic resistance. ELife, 6, e25950. https://doi.org/10.7554/eLife.25950 8. Center for Disease Control & Prevention (CDC). Outpatient Antibiotic Prescriptions—United States, 2020. (n.d.). 5. 9. Center for Disease Control & Prevention (CDC). Measuring Outpatient Antibiotic Prescribing. (2021, October 12). https://www.cdc. gov/antibiotic-use/data/outpatientprescribing/index.html 10. Luyt, C.-E., Bréchot, N., Trouillet, J.-L., & Chastre, J. (2014). Antibiotic

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11.

12. 13.

14.

15.

stewardship in the intensive care unit. Critical Care, 18(5), 480. https://doi. org/10.1186/s13054-014-0480-6 Spellberg, B., & Gilbert, D. N. (2014). The future of antibiotics and resistance: a tribute to a career of leadership by John Bartlett. Clinical Infectious Diseases: An Official Publication of the Infectious Diseases Society of America, 59(Suppl 2), S71–S75. https://doi.org/10.1093/cid/ ciu392 Ventola, C. L. (2015). The antibiotic resistance crisis. Pharmacy and Therapeutics, 40(4), 277–283. Soucy, S. M., Huang, J., & Gogarten, J. P. (2015). Horizontal gene transfer: building the web of life. Nature Reviews Genetics, 16(8), 472–482. https://doi.org/10.1038/nrg3962 Brinton, C. C. (1971). The properties of sex pili, the viral nature of “conjugal” genetic transfer systems, and some possible approaches to the control of bacterial drug resistance. CRC Critical Reviews in Microbiology, 1(1), 105–160. https:// doi.org/10.3109/10408417109104479 Bartlett, J. G., Gilbert, D. N., & Spellberg, B. (2013). Seven ways to preserve the miracle of antibiotics. Clinical Infectious Diseases, 56(10), 1445–1450. https://doi.org/10.1093/ cid/cit070 IMAGE REFERENCES

Figure 1: Fleming, A. (1929). On the antibacterial action of cultures of a penicillium, with special reference to their use in the isolation of B. influenzae. British journal of experimental pathology, 10(3), 226 Figure 2: Brinton, C. C. (1971). The properties of sex pili, the viral nature of “conjugal” genetic transfer systems, and some possible approaches to the control of bacterial drug resistance. CRC Critical Reviews in Microbiology, 1(1), 105–160. Banner Image: Pixabay. (2016, August 26). Pills. Wikimedia. https:// commons.wikimedia.org/wiki/ File:Pill_1.jpg

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The Decline of Mental Health An Overlooked Consequence of the Pandemic Due to the challenges brought about by the pandemic, it is no surprise that so many young adults have struggled with their mental health over the past few years. As a result, mental health has risen to the forefront of conversation in both science and public policy as individuals have increasingly shared their recent experiences. In this issue, the Berkeley Scientific Journal investigates the mental health struggles that are plaguing young adults. We interviewed two accomplished professionals in the fields of public health and psychology to discuss the effects of the pandemic, the stigma around seeking mental health services, and the future of mental health among young adults.

BY: ANDREW DELANEY, MICHAEL XIONG, ANANYA KRISHNAPURA, AND ESTHER LIM

Daniel Eisenberg, PhD, is a Professor of Health Policy and Management at UCLA’s Fielding School of Public Health. His research is aimed at understanding how to effectively invest in the mental health of young people. Eisenberg’s expertise in young adult mental health disorders has been sought by the New York Times, the Los Angeles Times, and The Nobel Conference.

Igor Chirikov, PhD is Director of the Student Experience in the Research University (SERU) Consortium. He conducts research focused on improving the learning outcomes of students in higher education. At the start of the pandemic, he coordinated a survey on the impact of COVID-19 on the mental health of students.

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INTERVIEWS

BSJ

: Given that you research mental health, some individuals may be surprised to know that your educational background is in economics. Can you provide insight into why you study mental health and the relationship between mental health and economics?

: I initially studied economics because I have always been interested in issues whose consequences affect entire communities and societies, and economics provides a good set of tools to study issues that are broad in scale, like social and policy issues. Similarly, mental health is a public health issue that is relevant to nearly everybody in a population or a community. Thus, the two fields share this common perspective, and that intersection is what motivated me since I want to do research that would have an impact on a large number of people. In economics we try to understand how best to use limited resources. One of the best ways we could use resources would be to address mental health early in life; therefore, much of my research is specifically aimed at quantifying the benefits of investments in mental health in early ages and understanding which kinds of investment strategies would be most impactful.

BSJ

: What does your role as Director of the Student Experience in the Research University (SERU) Consortium entail, and what inspired you to write your report on student mental health?1

: The Student Experience in the Research University, or SERU, Consortium is a non-profit international community of research universities. A major goal of the Consortium is to systematically collect data on student experience over the years and then share that data to better understand who our students are, what their characteristics and experiences are, and how they have changed compared to previous years. It is primarily used to support decision-making on various campus initiatives. When the pandemic hit, we focused on the aforementioned areas and published a series of policy briefs on different topics, one of which centered on mental health and well-being. For this policy brief in particular, we conducted one of the first large-scale multi-institutional surveys whose results clearly highlighted that there was a mental health crisis in universities during the pandemic; students were struggling not only in terms of learning, but also in terms of their mental health. This is what drove us to write the report. Previously, there were rumors going around campuses and some university counselors had anecdotally noted that student mental health was declining, but this was conclusive data to support these observations. Effects of the Pandemic

BSJ

: During the COVID-19 pandemic, we have seen the rates of mental health illness among teenagers and young adults rise. From your research, can you describe some of the factors that have contributed to this rise in mental illness?

: First, it is important to recognize that mental health problems were on the rise before the pandemic, especially for adolescents and young adults. So, it is not entirely clear whether the pandemic has changed the overall trajectory of mental illness to a

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significant degree. I do not mean to downplay the impact that the pandemic has had on many peoples’ mental health because we can all see in our lives that the pandemic has been extremely difficult in many ways. The pandemic has perhaps accelerated the increase in mental illness, but the overall rates of depression, anxiety, and suicide risk for young people were already rising before the pandemic. As we emerge from this pandemic in the coming months and years, we must realize that mental health struggles are not going away, and they probably will not even diminish very much, if at all. Undoubtedly, the pandemic has introduced new challenges and issues, but fundamentally we are still in the same place we were before the pandemic–mental health is perhaps the most important public health issue for young people today.

BSJ

: Your 2020 report “Undergraduate and Graduate Students’ Mental Health During the COVID-19 Pandemic” found an alarming increase in major depressive and generalized anxiety disorders following the pandemic. How was this data collected, and how were you able to draw these conclusions?

: Nine large public research universities participated in this survey. We used two screeners that are widely used in the literature for data collection. One is the patient health questionnaire or PHQ-2. It is a very simple two-item scale that screens for both major depressive disorder and generalized anxiety disorder. The important thing to note is that these screens are not actual diagnoses. We cannot determine for sure whether these students suffer from anxiety or from major depressive disorder. However, for our purposes and for many other research studies, these serve as rough indicators of the existence of the problem. Due to our large sample size, we can then disaggregate by student characteristics, such as gender, race and ethnicity, sexual orientation, socioeconomic status, or caregiving status. This allows us to highlight which groups are most impacted by the conditions we screened for.

BSJ IC

: What factors contributed to this rise in mental health disorders, and which groups were most at risk?

: In terms of factors, I do not think our data can answer that question specifically. We can only speculate that COVID contributed to a significant extent. Anecdotal evidence and evidence from other surveys also support this claim. It was not necessarily COVID itself that directly contributed to this rise but rather the fact that campuses were closed, which led to many students reporting loneliness or living in less safe environments. This is especially true for LGBTQ+ students who went back to their hometowns, where their families or friends were not as accepting of their sexual orientation or identity. In terms of the kind of general trend that we see for undergraduates, about a third of students had symptoms of a major depressive disorder and more than a third–39%–had symptoms of generalized anxiety disorder, which is much higher compared to levels in pre-pandemic surveys. Additionally, low-income students and working-class students have a higher percentage who screen positive for depression and anxiety. African American, Latino, Asian, female, and LGBTQ+ students were at higher risk of exhibiting symptoms of both major depressive disorder and generalized anxiety disorder.

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These same student subpopulations were less academically engaged and experienced more food and housing insecurity. We cannot disaggregate which specific factors led to this higher rate, but I think it is a cumulative effect. For example, some of these students had parents who lost their jobs, and they needed to help them make money and could not spend as much time on their studies. In this survey design, you cannot distinguish the individual effects on mental health each factor had. In more detailed studies, maybe we will be able to see what contributed the most, whether that be employment conditions, location, or other factors. For this particular report, it was important to alert people that declining mental health was a reality and universities need to pay more attention to vulnerable groups. Stigma Surrounding Mental Health

BSJ

: Many people still believe that there is a stigma surrounding seeking mental health services. How do you think mental health professionals and society as a whole can work to combat this stigma?

: It is certainly true that there is still some form of stigma, but I think it has changed over time. In the past, say, twenty or thirty years, the stigma was quite discriminatory or overtly negative. There were negative attitudes about having a diagnosis of a mental health condition or seeking treatment for mental health. Today, I think it is not as blatant. Now, for most people, especially younger people, the stigma is more in the form of not assigning enough priority to mental health. Particularly, one of the main reasons why young adult students do not seek help even when they would benefit from seeking help is because they do not recognize that their problems require treatment. Many are aware that they are stressed but think that their stress is pretty normal because they see it all around them. Or, some students feel that they do not have time to take the steps to seek care, even though they might want to do so. I still think that mental health is not a high enough priority for many individuals and communities. When we sit down and think about it, most of us acknowledge that mental health is fundamental to our well-being and success. Yet, we do not always act in a way that prioritizes our mental health, and I think that is the form of stigma that is most prevalent now. I think the solution is for professors and the administration in college communities to emphasize the importance of mental health and the fact that mental health requires effort and attention. We must realize that mental health is not something that we can easily address and then move on from. Similar to learning and succeeding in college, your mental health requires hard work every day, week after week. Mental health requires persistent attention and effort, whether that entails therapy, medication, or just being mindful of spending enough time with friends and other supportive people. The Future of Mental Health

BSJ

: In your report, you made several recommendations as to how best we can approach the mental health crisis. These included allocating more resources to and expanding mental health programs alongside encouraging faculty to promote such services. Have you seen any of these changes implemented, and if so, what

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impact have they made?

: We probably cannot talk about the impact because we are still trying to analyze the follow-up data. The surveys are run every year, so we currently have data from 2021. However, anecdotally, I have seen and heard of numerous efforts in this area. The pandemic led to a nationwide focus on the mental health crisis, and as a result, other publications came out that supported the same results that we had. For example, there was a survey of university presidents done by Inside Higher Ed where they asked students about the major challenges they faced. At the beginning of the pandemic, students cited budget concerns, but as time went on, student mental health and well-being were consistently brought up. So, this truly became a priority for many universities. I would say all of them increased resources allocated to counseling services. Remote counseling also became increasingly available at many institutions; however, the problem with that is students often find themselves in places where they cannot speak openly about their experiences. Imagine they live in a house with a person that abuses them. They cannot talk with the counselor about this via remote counseling from their home because their abuser could be nearby and hear everything, though I know that some universities had creative solutions to address that. Personally, I think what was harder to track was the faculty side of the issue because I know that some faculty were very supportive of students while others were not. Some provided encouragement, mental health breaks, and even brought food to students. When we were doing the survey, we got a lot of positive feedback about what the faculty did for their students. But at the same time, it varies because some faculty were not accommodating at all; therefore, it is harder to meaningfully track the effects of faculty support among different universities. In summary, we saw that there was significant institutional effort to support students, but it was less coordinated at the level of departments and at the level of faculty. This is important because prevention is easier and cheaper than treatment. Before, universities were dealing with consequences more than they were trying to prevent the problems from happening in the first place. Prevention is hard because it seems easier to just hire more counselors and put more resources into treatment. But ultimately, the more important task going forward is to redesign and create an environment to encourage students’ well-being. That requires a more concerted effort from administration, faculty, students, and staff. During the pandemic, everyone was in emergency mode and it was hard to strategize. Hopefully, we are now approaching the light at the end of the tunnel. Many of the institutions we work with are very competitive and challenging in terms of coursework. Sometimes that is good, but they need to understand the impact it has on students. They need to do more in providing the support that is needed for students to not feel alienated, depressed, or anxious. My hope is that there will be no further major outbreaks of COVID-19–or that we will at least be better prepared for them. However, universities still need to redesign their learning environments to address the concerns that have come to light as a result of the pandemic. You cannot say that well-being is important during the pandemic, but that it no longer is after the pandemic ends. Universities need to be more strategic and work with faculty, staff, and students to create an environment that is supportive.

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: Now that we are over two years deep in this pandemic, what hopes or concerns do you have about the current situation and the future of student mental health?

: Even though there has been a rise in distress over time, I think there have been a lot of positive developments. Even before the pandemic, there was a rise in the use and awareness of mental health services, and overall, individuals were becoming more inclined to prioritize mental health. Also, during the pandemic, there was an acceleration of the use of digital resources for mental health. Not just tele-therapy, but a variety of self-guided tools like meditation apps, mindfulness apps, and self-guided therapy came into widespread use, aiding the general public in improving their mental health. In short, there are various digital resources to support mental health that are now able to reach a lot more people as a result of the pandemic. Thus, going forward, one of the challenges will be how to merge digital resources with traditional treatment. Digital resources are so low-cost and convenient, so there should be a place for them even after the pandemic. I think campus communities are in a really good position to figure out that optimal combination. These communities can provide in-person support, help connect students to digital resources, and help them sort through the many options to figure out which resources would be most helpful for each individual. REFERENCES 1.

2. 3.

Chirikov, I., Soria, K. M, Horgos, B., & Jones-White, D. (2020). Undergraduate and Graduate Students’ Mental Health During the COVID-19 Pandemic. UC Berkeley: Center for Studies in Higher Education. https://escholarship.org/uc/item/80k5d5hw Headshot: [Photograph of Daniel Eisenberg]. Eisenberg Lab. https://ph.ucla.edu/faculty/eisenberg. Image reprinted with permission. Headshot: [Photograph of Igor Chirikov]. Center for Studies in Higher Education. https://cshe.berkeley.edu/ people/igor-chirikov. Image reprinted with permission.

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Alzheimer’s Disease and the Neurobiology of Long-Term Memory Formation BY VARUN UPADHYAY THE PHENOMENON OF MEMORY

hink back on a happy memory. While you may not be able to recall the details vividly, you will probably be able to remember what made you happy, as well as a general recollection of the event’s context. Now try to think back to what you ate for lunch two weeks ago—odds are you will not remember. The concept of longterm memory formation is an incredible phenomena. It is the biological process of storing and retrieving information that, although seemingly trivial and second-nature, functions as the foundation for shaping one’s identity and informing one’s decision-making. However, the mechanisms and neurobiology behind this remarkable ability remain far from understood. How is it possible for the assembly of atoms and molecules that comprises the human brain to have the capacity to “remember?” Novel research in this branch of neuroscience may provide a glimpse into exactly how a collection of neurons across the brain’s neural network work in tandem to make this process possible.

be definitively proven. However, an abundance of cutting-edge research supports the current dominating theory on longterm memory formation, which revolves around a revolutionary concept termed “synaptic plasticity.” Synaptic plasticity refers to the ability for neurons to strengthen or weaken their connections with one another in response to an increase or decrease in their activity. Essentially, if neuron 1 consistently succeeds in activating neuron 2, the connection between the two becomes stronger. Likewise, if neuron 1 fails in activating

neuron 2, the connection becomes weaker. As these connections are developed, memories become encoded within the brain’s neural network. These lasting increases or decreases in synaptic strength are called “long-term potentiation” (LTP) and “longterm depression” (LTD) respectively and are influenced by a variety of neurobiological factors.1 One such factor recently discovered to play an important role in long-term memory formation is the neuronal PAS domain protein 4 (Npas4). Within the hippocampal region of the brain—the region

HOW DO MEMORIES FORM? While scientific research on long-term memory formation has been underway for decades, the underlying mechanisms by which this process occurs have yet to

Figure 1: An overview of a neural cell with brief descriptions of important regions. At the cell’s dendrites, messages are received from other neurons, and at the terminal branches, the nerve impulses are transmitted to another cell.

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Figure 2: A diagram showing the process that occurs at the synapse — the junction between neurons where the impulse is transmitted via neurotransmitters. The signal travels from the terminal branches of the axon from the presynaptic neuron to the dendrites of the postsynaptic neuron.

“Although Staufen2 and Npas4 are both examples of proteins that play a pivotal role in the process of memory formation, it is important to note they are but a small part of the molecular machinery involved with memory. ” primarily responsible for learning, memory formation, and storage—Npas4 was found to be directly responsible for synaptic maintenance. Npas4 plays an important role in memory formation through its regulation of plk2, a gene responsible for controlling the shrinking of the postsynaptic

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Figure 3: An overview of various functions associated with specific regions of the brain. The hippocampus is the primary region involved with memory formation and storage.

structures of neurons. As Npas4 activates plk2, synaptic size and strength decreases. This suggests that without Npas4, synapses would become excessively strong, inhibiting their capacity to encode memories through further strengthening.2 Due to its remarkable ability to control the strength of connections between neurons, Npas4 plays an integral role in synaptic plasticity and the complex process of long-term memory formation. An additional neurobiological factor with important ties to memory formation is the protein Staufen2. While studying this protein, researchers were able to pinpoint its impact on the efficiency of signal transmission across synapses in the hippocampus. It was found that synthetically reduced levels of Staufen2 within mice enhanced LTP and impaired LTD, which had a negative impact on memory. This implies that the deficiency of Staufen2 makes synapses more responsive than normal. Researchers hypothesize that as synapses become highly responsive, not enough become suppressed during the process of memory consolidation in which experiences are encoded into long-term memory. These findings illustrate that the absence of

Staufen2 and the consequent imbalance in LTP and LTD may lead to the destabilization of long-term memory formation.3 Although Staufen2 and Npas4 are both examples of proteins that play a pivotal role in the process of memory formation, it is important to note they are but a small part of the molecular machinery involved with memory. The complex process of long-term memory formation relies on an incredible array of intricate interactions between neurobiological processes that work in tandem to strengthen connections across the neural network and encode a person’s experiences into long-term memories. Understanding how synaptic plasticity works may provide the foundation for not only understanding human memory but also the neurodegenerative disorders that can result from a fault in these complex processes. WHERE CAN MEMORY FORMATION AND RETENTION GO WRONG? Alzheimer’s disease (AD) is one of the most common forms of a neural network malfunction in which brain cells degenerate and die. This neurodegenerative

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Figure 4: A graphic representation depicting the two different pathways of APP cleavage. On the left side, illustrating the non-amyloidogenic pathway, APP is cleaved and becomes p3 and CTFγ. On the right side, illustrating the amyloidogenic pathway, APP is cleaved and becomes CTFγ and the toxic protein Aβ. disease may cause symptoms such as severe memory loss, the inability to form new memories, and erratic changes in behavior. Moreover, Alzheimer’s disease is the cause of 60%-80% of all dementia cases and typically leads to a fatal prognosis after 10 years of onset.4 The hallmarks and neurobiology of AD are centered around the development of two toxic proteins: amyloid-beta proteins and tau proteins. These result in the problematic growth of amyloid-beta plaques and neurofibrillary tangles respectively. Although it is not clear to what extent each of these pathologies— plaques or tangles—causes AD, both are suspected to contribute to the deterioration of cognitive functions, including the ability to form and recall memories. In amyloid-beta plaque formation, the gradual buildup of plaque from small molecules of amyloid-beta (Aβ) proteins covers large portions of the brain. This serves as a marker for the progression of AD and possibly leads to neural cell death as well. Aβ proteins are formed when a large protein involved in neural growth and neurorepair, amyloid-beta precursor protein (APP), is broken down via two distinct pathways.5 In the first pathway—generally regarded as “non-amyloidogenic” for its hypothesized non-toxic products— APP is cleaved in two by the enzyme α-secretase. This results in the formation of the protein fragments C83 and sAPPα,

the latter of which is hypothesized to have neuroprotective effects. C83 is again cleaved by γ-secretase, resulting in a new protein called p3. Although largely regarded as benign, research is starting to shed light on the specific effects of the accumulation of p3, as it may actually have toxic, amyloidogenic properties. Previous studies have found that this protein may be linked to the formation of aggregates and amyloid fibrils, and thus may not be nonamyloidogenic as previously thought.6

“ The hallmarks and neurobiology of AD are centered around the development of two toxic proteins: amyloidbeta proteins and tau proteins.” In the second pathway—which may be associated with genetic abnormalities— APP is instead cleaved by a different enzyme, β-secretase, forming sAPPβ and a protein fragment called C99.7 Next, C99 is cleaved by γ-secretase, resulting in another protein fragment and the 42-amino acid Aβ peptide. As the function of a protein is determined by its specific amino acid sequence and how these amino acids

organize, Aβ is considered toxic due to its resulting insolubility and its tendency to form long fibrils that create dense plaques on neural cells, which interfere with synaptic function.8 This gradual accumulation of plaque in the brain may result in decreased cognitive function and ultimately lead to neural cell death.9 An additional characteristic of Alzheimer’s disease is the presence of neurofibrillary tangles (NFTs), which are the result of the aggregation of tau proteins within neurons. The normal function of tau proteins is to form the cytoskeleton, or the framework, of a neuron. Tau proteins carry out this function by promoting the development and stabilization of microtubules—structures which provide support for a neuron and facilitate transportation of substances, such as neurotransmitters, across the cell. However, as tau proteins become impaired during the progression of AD, its function changes and its capacity to bind to and support microtubules becomes hindered. As it falls off the microtubule, it then begins to aggregate, forming a series of tangles which negatively affect neuronal function.10 Although the neurobiological mechanisms facilitating the progression of tau pathology are still unclear, evidence suggests the presence of abnormal tau proteins with extra phosphate groups attached (termed hyperphosphorylated) may be a contributing factor.11 In fact, following studies by Ksiezak-Reding et

Figure 5: When tau proteins aggregate, they form loosely intertwined paired helical filaments (PHFs) and tightly wrapped straight filaments (SFs). The result is a deviation from normal microtubule structures (as shown on the leftmost picture) and the formation of neurofibrillary tangles (NFTs).

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guidance throughout the writing process. REFERENCES 1.

4. Figure 6: A graphic showing the differences between a healthy neuron and a diseased neuron. al., 6-8 mol of phosphate per molecule of tau were found in the autopsied brain of an individual suffering from Alzheimer’s, as opposed to 2-3 mol in a healthy brain.12 Because tau phosphorylation is regulated by the activities of cellular enzymes and protein kinases—the molecules which facilitate the attachment of phosphate groups—an imbalance could potentially result in the neurotoxic hyperphosphorylation of tau proteins, and the subsequent impairment of cognitive function.13 CONCLUSION The mechanism by which memories are encoded via the synaptic plasticity of the brain’s neural network is an incredible process—it brings to light various questions revolving around the molecular machinery which makes this function of memory possible. Moreover, by studying the intertwining nature of synaptic plasticity and neurodegenerative diseases, promising possibilities exist in developing therapies for common pathologies such as Alzheimer’s disease. Although no cure has been developed for AD, a treatment does exist that helps slow its progression. The aducanumab medication, the only current-

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ly approved treatment, is a form of immunotherapy which targets the Aβ protein to reduce the formation of amyloid plaques in the brain.14 Although it may seem diminutive to reduce one’s most defining memories to mere electrical impulses and interactions of proteins, it is fascinating to realize that these neurobiological phenomena serve as the foundational mechanisms which govern one’s identity and thinking. Uncovering the complexities of what allows us to remember is an essential stepping stone; it provides the key towards unlocking not only a greater hope for effectively treating those with neurodegenerative diseases, but also towards providing a greater understanding of human thinking and the basis of what makes us who we are.

ACKNOWLEDGEMENTS I would like to thank and acknowledge Dr. William Jagust, professor of public health and neuroscience at UC Berkeley, for his helpful feedback and expertise in Alzheimer’s research, which helped greatly in the writing process. I’d also like to thank my editors Jonny Hale and Marley Ottman, of the Berkeley Scientific Journal Features Department, for their thorough edits and

Queensland Brain Institute. (2016, December 2). How are memories formed? The Brain. https://qbi. uq.edu.au/brain-basics/memory/howare-memories-formed Trafton, A. (2018, February 8). Study reveals molecular mechanisms of memory formation. MIT News. https://news.mit.edu/2018/studyreveals-molecular-mechanismsmemory-formation-0208 Ludwig-Maximilians-Universitaet Muenchen. (n.d.). Neurobiology: The chemistry of memory. ScienceDaily. Retrieved March 14, 2022, from https://www.sciencedaily.com/ releases/2017/11/171123095409.htm American Brain Foundation. (n.d.). Alzheimer’s disease. Brain Diseases. Retrieved March 14, 2022, from https://www. americanbrainfoundation.org/ diseases/alzheimers-disease/ Dawkins, E., & Small, D. H. (2014). Insights into the physiological function of the β-amyloid precursor protein: Beyond Alzheimer’s disease. Journal of Neurochemistry, 129(5), 756–769. https://doi.org/10.1111/ jnc.12675 Kuhn, A. J., Abrams, B. S., Knowlton, S., & Raskatov, J. A. (2020). The Alzheimer’s disease “non-amyloidogenic” p3 peptide revisited: A case for amyloid-α. ACS Chemical Neuroscience, 11(11), 1539–1544. https://doi.org/10.1021/ acschemneuro.0c00160 Robertson, S. (2014, January 17). What are amyloid plaques? News Medical Life Sciences. https://www. news-medical.net/health/What-areAmyloid-Plaques.aspx Goodsell, D. (2006, July). Molecule of the month: Amyloid-beta precursor protein. RCSB PDB-101. http:// pdb101.rcsb.org/motm/79 Zhang, X., & Song, W. (2013). The role of APP and BACE1 trafficking in APP processing and amyloid-β generation. Alzheimer’s Research & Therapy, 5(5), 46. https://doi.

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org/10.1186/alzrt211 10. Iqbal, K., Liu, F., Gong, C.-X., & Grundke-Iqbal, I. (2010). Tau in Alzheimer disease and related tauopathies. Current Alzheimer Research, 7(8), 656–664. https://doi. org/10.2174%2F156720510793611592 11. Alonso, A. D., Cohen, L. S., Corbo, C., Morozova, V., ElIdrissi, A., Phillips, G., & Kleiman, F. E. (2018). Hyperphosphorylation of tau associates with changes in its function beyond microtubule stability. Frontiers in Cellular Neuroscience, 12. https://www.frontiersin.org/ article/10.3389/fncel.2018.00338 12. Chong, F. P., Ng, K. Y., Koh, R. Y., & Chye, S. M. (2018). Tau proteins and tauopathies in Alzheimer’s disease. Cellular and Molecular Neurobiology, 38(5), 965–980. https://doi. org/10.1007/s10571-017-0574-1 13. Wang, J.-Z., Grundke-Iqbal, I., & Iqbal, K. (2007). Kinases and phosphatases and tau sites involved in Alzheimer neurofibrillary degeneration. European Journal of Neuroscience, 25(1), 59–68. https://doi.org/10.1111/j.14609568.2006.05226.x 14. National Institute on Aging. (n.d.). How is Alzheimer’s disease treated? Health Information. Retrieved May 19, 2022, from https://www.nia.nih. gov/health/how-alzheimers-diseasetreated

Research & Therapy, 5(5), 46. https:// doi.org/10.1186/alzrt211 Figure 5: Jie, C., Treyer, V., Schibli, R., & Mu, L. (2021). TauvidTM: The first

IMAGE REFERENCES 1. 2.

Figure 1: AP Psychology Community. (2022, March 16). https:// appsychology.com/ Figure 2: Lumen. (n.d.). Anatomy and physiology - communication between neurons. https://courses. lumenlearning.com/ap1/chapter/ communication-between-neurons/ Figure 3: Sinha Dutta, S. (2010, May 4). Hippocampus functions. News Medical Life Sciences. https:// www.news-medical.net/health/ Hippocampus-Functions.aspx Figure 4: Zhang, X., & Song, W. (2013). The role of APP and BACE1 trafficking in APP processing and amyloid-β generation. Alzheimer’s

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any people start their day with a cup of coffee. As one of the few natural sources of caffeine—the most widely consumed psychoactive drug—coffee has become an essential part of modern daily life.1 Although some people choose coffee as their first beverage of the day, due to its stimulating effects, others drink coffee for leisure and enjoy its odor or taste. Indeed, the price, quality, and uniqueness of coffee depend on the aroma obtained after processing raw beans; therefore, coffee aroma is of great commercial and consumer interest.2 But how does coffee obtain its different flavors and aromas? CHEMICALS —VOLATILE AND NONVOLATILE COMPOUNDS OF COFFEE A cup of coffee consists of over

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1,000 chemicals which can produce various aromas and flavors.3 Volatile organic compounds—compounds with a high vapor pressure at room temperature—and non-volatile organic compounds are produced in multiple phases of coffee production, from green (raw) beans to the brewed coffee we consume. Studies have shown that some volatile compounds determine the aroma while non-volatile compounds make up the taste or flavors.3 Some key non-volatile compounds include alkaloids (caffeine and trigonelline), chlorogenic acid (CGA), carbohydrates (sucrose), and lipids.4,5 Both caffeine and CGA contribute to the bitter flavor, but CGA, which degrades rapidly and forms phenolic compounds,also produces astringent and acidic flavors.5 Trigonelline, on the other hand, leads to an overall ar-

omatic perception and has a weak, bitter taste, but it also degrades during roasting, producing volatile compounds such as pyridines or pyrroles. Lipids contribute to the texture and mouthfeel of coffee, while carbohydrates act as an aroma precursor and degrade quickly, leading to other volatile and non-volatile compounds that contribute to crucial flavors such as sweetness and acidity.6 Some key volatile compounds that influence aroma include pyrazines, pyrroles, furans, aldehydes, ketones, and phenolic compounds.5 Pyrazines and pyrroles often lead to roasted, nutty, and burnt aromas. Furans contribute to malty and sweet roasted flavors. Aldehydes usually exhibit fruity notes, while ketones are associated with buttery flavor notes. Phenolic compounds often contribute to spicy aromas.3,5,12,13

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Figure 1. Non-volatile compounds: A) caffeine, B) chlorogenic acid, C) trigonelline

“Some of the most known cultivars of Arabica—all of which have unique characteristics, flavors, and aromas—include Typica, Blue Mountain, Bourbon, and Yunnan Xiaoli.” As chemicals determine coffee’s aroma, it is important to consider that a variety of variables influence the chemical composition of your morning brew. COFFEE SPECIES AND CULTIVAR One of the most apparent factors contributing to the wide variety of different

chemicals is the species of coffee plant, a distinction dating back to coffee’s Middle Eastern roots. Several stories about the origin of coffee exist, but possibly the most well known version tells of a goat-herder named Kaldi. Around 850 CE, he noticed that his goats became more alert at night after eating the berries from bushes near the Red Sed. The beans, it seemed, had stimulating properties. Kaldi knew he had found something important and set about proclaiming his discovery to the world. Historically, the wild coffee plant is indigenous to Ethiopia and was cultivated in the Arabian colony of Harar; thus, the earliest grown species is known as Arabica coffee (C. Arabica).8 Arabica, along with another species Robusta (C.Robusta, also known as C. Canephora), are cultivated widely and compose most of the worldwide coffee market. In the coffee industry, the words “variety”, “cultivar”, and “hybrid” are used interchangeably to describe different types of coffee beans. However, there are some

differences. According to the Speciality Coffee Association of America (SCAA), a cultivar is a cultivated variety not generally found in natural populations.9 Arabica coffee plants have been grown in different areas and this has resulted in many cultivars. Some of the most known cultivars of Arabica—all of which have unique characteristics, flavors, and aromas—include Typica, Blue Mountain, Bourbon, and Yunnan Xiaoli.10 On the other hand, Robusta is more noted for its resistance to diseases in the natural environment than its cultivars, varieties, or hybrids. Furthermore, Robusta, generally, is less vulnerable to adverse weather conditions than Arabica and is thus easier to grow and produces fruit more quickly. Robusta green beans are hard and have lower sucrose levels than Arabica green beans, which confers a stronger and harsher taste as well as a less acidic flavor after roasting. Since acidity is a crucial feature of high-quality beans, Arabica coffee is considered by coffee enthusiasts to possess superior flavor.11

Figure 2. Volatile compounds: A) pyrazine, B) furan, C) aldehyde, D) ketone, E) phenol.

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“The most common three ways of processing coffee are natural processing (dry processing), washed processing (wet processing), and honey processing.”

NATURAL IMPACTS Environmental elements such as elevation and light exposure could also influence chemicals in coffee beans. Some studies have shown that altitude is correlated with glucose content in coffee beans. Coffee trees growing at higher altitudes typically have higher glucose content, thus improving the coffee’s sensory attributes.6 One of the most famous cultivars of coffee, known for its premium flavor, is Blue Mountain coffee, which is grown at an elevation of up to 2,350 metres above sea level and with regular rainfall and volcanic soil. Blue Mountain will not exhibit as good of a taste if it is not grown in its preferred mountainous environment.12 More often, bigger coffee beans are considered more flavorful, and consisten-

cy in size leads to a more even degree of roasting.11 Shading, or avoiding direct sunshine, results in coffee beans with greater and more unified bean size and with higher levels of lipid content.6 Therefore, shadegrown coffee beans have higher market prices.11 In addition, high temperatures could cause faster ripening of the coffee cherries and Figure 4: Coffee berry anatomy. thus immature, green coffee beans with higher sucrose, trigonelline, and chlorogenic acid concen- cherries in the sun, allowing them to fertrations, leading to more bitter and astrin- ment. In this process, all the layers usually remain intact, leading to a deeper-tasting gent tastes.11 Environmental factors have a signif- coffee with fruity and syrupy notes. Wet icant effect on coffee’s final flavor profile. processed coffee requires depulpers to reFarmers thus grow specific cultivars in pre- move the skin, pulp, and mucilage from the ferred environments that could potentially seeds before drying. Once this is done, the seeds are washed in water and then finalcultivate higher quality coffee. ly dried out in the sun. These coffee beans are typically more acidic and cleaner. This PROCESSING AND ROASTING process is efficient but usually considered IMPACTS environmentally unfriendly due to the Once coffee cherries are harvested, the amount of wastewater produced as a byseeds (which we call beans) are fermented product. Honey processed coffee combines and dried via one of the many processing wet and dry methods, producing coffee methods that influence the aromas and with flavors similar to both of the previflavors of coffee. The most common three ously described methods, but that is sweetways of processing coffee are natural pro- er and more complex. The mucilage—a cessing (dry processing), washed process- layer of sugary substance surrounding the ing (wet processing), and honey process- seed—is what the “honey” refers to. After the depulper removes the seed from the ing. Dry processing coffee is the most tra- cherry, the mucilage stays on the seed as it 13 ditional process and involves drying coffee dries in the sun.

Figure 3: Coffee processing process.

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tastes and mouthfeels of coffee.11 THE COFFEE TASTER’S FLAVOR WHEEL

Figure 5. Wet and Dry coffee processing. These processes get us from coffee cherries to green coffee beans. However, green bean coffees are typically odorless. It is not until roasting triggers certain chemical processes that coffee develops its distinctive aromas. Generally speaking, lightly roasted coffee contains more of the characteristics of green beans and generates more acidic and fruity flavors. When roasting time is increased and coffee beans are roasted more darkly, we obtain coffee

with an oilier surface and less acidity, emphasizing a bolder and deeper aroma that is chocolaty and nutty.11 Other factors, such as level of grinding, water to coffee ratio, brewing method, temperature, and extraction process, could likely influence coffee’s aroma as well. Many coffee shops also blend coffee beans from multiple origins to obtain a more balanced flavor. These factors could affect consumer preference and cause different

Given a cup of coffee, people might perceive its taste and aroma differently, and a number of factors can explain this difference in perception and preference including genetics and environment. Studies have shown an association between taste receptor variants and bitter taste preference.14 Genome-wide association studies (GWAS) have also indicated that genetic differences in the stimulating effects of caffeine result in different preferences for caffeine.15 Environmental and social factors such as parents, peers, and food access may also affect individual choice. In order to address these differences, the coffee taster’s flavor wheel, initially published in 1995 and updated in 2016 as a collaboration between Specialty Coffee Association (SCA) and World Coffee Research (WCR), can help people characterize their cup of coffee. The wheel encompasses all the tastes and flavors of coffee and has served as the industry standard since it was published. Coffee culture is growing while many people are starting to pursue unique flavoring. With some understanding of what contributes to the aromas and flavors of coffee, we can better choose our cup of coffee when entering a coffee shop. A cup of coffee is simple yet complex, with so many processes and factors influencing its taste, so we should appreciate every cup of coffee we drink. REFERENCES 1.

Figure 6. The coffee taster’s flavor wheel: Taste descriptors near the center are the most broad and get more specific towards the outside.

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Nehlig, A. (1999). Are we dependent upon coffee and caffeine? A review on human and animal data. Neuroscience & Biobehavioral Reviews, 23(4), 563–576. https://doi. org/10.1016/S0149-7634(98)00050-5 Caporaso, N., Whitworth, M. B., & Fisk, I. D. (2022). Prediction of coffee aroma from single roasted coffee beans by hyperspectral imaging. Food Chemistry, 371, 131159. https://doi. org/10.1016/j.foodchem.2021.131159 Wood, J. (2019). Determination and correlation of volatile and nonvolatile

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compounds with coffee quality . Southern Illinois University Honors Theses. https://opensiuc.lib.siu.edu/ uhp_theses/456 4. Angeloni, S., Mustafa, A. M., Abouelenein, D., Alessandroni, L., Acquaticci, L., Nzekoue, F. K., Petrelli, R., Sagratini, G., Vittori, S., Torregiani, E., & Caprioli, G. (2021). Characterization of the aroma profile and main key odorants of espresso coffee. Molecules, 26(13), 3856. https://doi.org/10.3390/ molecules26133856 5. Heo, J., Adhikari, K., Choi, K. S., & Lee, J. (2020). Analysis of caffeine, chlorogenic acid, trigonelline, and volatile cin crew coffee using highperformance liquid chromatography and solid-phase microextraction—gas chromatography-mass spectrometry. Foods, 9(12), 1746. https://doi. org/10.3390/foods9121746 6. Cheng, B., Furtado, A., Smyth, H. E., & Henry, R. J. (2016). Influence of genotype and environment on coffee quality. Trends in Food Science & Technology, 57, 20–30. https://doi. org/10.1016/j.tifs.2016.09.003 7. Caporaso, N., Whitworth, M. B., Grebby, S., & Fisk, I. D. (2018). Non-destructive analysis of sucrose, caffeine and trigonelline on single green coffee beans by hyperspectral imaging. Food Research International, 106, 193–203. https:// doi.org/10.1016/j.foodres.2017.12.031 8. Smith, R. F. (1985). A history of coffee. In M. N. Clifford & K. C. Willson (Eds.), Coffee: Botany, Biochemistry and Production of Beans and Beverage (pp. 1–12). Springer US. https://doi. org/10.1007/978-1-4615-6657-1_1 9. Specialty Coffee Association of America. (n.d.). A botanist’s guide to specialty coffee. Resources. Retrieved March 13, 2022, from http://scaa.org/index. php?goto=&page=resources&d=abotanists-guide-to-specialty-coffee 10. Gibson, M., & Newsham, P. (2018). Chapter 18—Tea and Coffee. In M. Gibson & P. Newsham (Eds.), Food Science and the Culinary Arts (pp. 353–372). Academic Press. https://

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11.

12.

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15.

1. 2. 3.

doi.org/10.1016/B978-0-12-8118160.00018-X Seninde, D. R., & Chambers, E. (2020). Coffeef: A review. Beverages, 6(3), 44. https://doi.org/10.3390/ beverages6030044 Jamaica Blue Mountain Coffee. (n.d.). Location. Blue Mountain Coffee Group Ltd. Retrieved April 13, 2022, from https://www. bluemountaincoffeejamaica.com/en/ location Bean & Bean. (n.d.). Coffee processing methods. Blogs. Retrieved March 23, 2022, from https://beannbeancoffee.com/blogs/ beansider/coffee-processing-methods Diószegi, J., Llanaj, E., & Ádány, R. (2019). Genetic background of taste perception, taste preferences, and its nutritional implications: A systematic review. Frontiers in Genetics, 10, 1272. https://doi.org/10.3389/ fgene.2019.01272 Cornelis, M. C., & van Dam, R. M. (2021). Genetic determinants of liking and intake of coffee and other bitter foods and beverages. Scientific Reports, 11(1), 23845. https://doi. org/10.1038/s41598-021-03153-7

Policy, 22. https://doi.org/10.1007/ s10098-020-01841-y Figure 6: Specialty Coffee Association of America. (n.d.). A botanist’s guide to specialty coffee. Resources. Retrieved March 13, 2022, from http://scaa.org/index. php?goto=&page=resources&d=abotanists-guide-to-specialty-coffee

IMAGE REFERENCES Cover image: Designed by artist Yue Wu Figures 1, 2: Created by author Figure 3: Bean & Bean. (n.d.). Coffee processing methods. Blogs. Retrieved March 23, 2022, from https://beannbeancoffee.com/blogs/ beansider/coffee-processing-methods Figure 4: Lagrasta, F. P., Pontrandolfo, P., & Scozzi, B. (2021). Circular economy business models for the Tanzanian coffee sector: A teaching case study. Sustainability, 13(24), 13931. https://doi.org/10.3390/ su132413931 Figure 5: Sengupta, B., Priyadarshinee, R., Roy, A., Banerjee, A., Malaviya, A., Singha, S., Mandal, T., & Kumar, A. (2020). Toward sustainable and eco-friendly production of coffee: Abatement of wastewater and evaluation of its potential valorization. Clean Technologies and Environmental

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e r activating regeneration The Power of Neural Crest Cells in Repairing Damaged Tissues INTERVIEW WITH DR. MEGAN MARTIK Megan Martik, PhD, joined UC Berkeley in July 2021 as an assistant professor of genetics, genomics, and development in the department of Molecular and Cell Biology. The Martik lab studies the neural crest–a multipotent and migratory cell population that differentiates into various tissues. They use developmental biology approaches to understand how gene regulation impacts neural crest differentiation, disease, evolution, and adult regeneration. Some of her current projects include studying how neural crest cells contribute to adult tissue regeneration and vertebrate evolution, as well as studying the dysregulation of neural crest gene regulatory networks in neural crest-derived cancers. Her continued work in these areas has implications for human disease treatment and regenerative medicine.

BY LEXIE EWER, GRACE GUAN, LUKE LYONS, AND ESTHER LIM

BSJ

: Your research centers around elucidating the role of neural crest cells in developing embryos. What drew you to research this cell population in particular?

: The neural crest is a stem cell population that is important during embryonic development. The neural crest is fascinating because it can make many different derivatives in the adult body plan—ranging from the craniofacial skeleton to cardiovascular derivatives, pigmentation in the skin, and many aspects of the peripheral nervous system. I became interested in this population, in particular, because we can unravel the different regulatory programs that control cell state transitions from multipotency to differentiated cell types, then use those programs as tools to understand other complex biological problems. For example, we discover how tissues can regenerate, how the cell types facilitate vertebrate evolution, and how organs become dysregulated to give rise to diseases or cancers.

BSJ

: You recently published a paper which revealed that neural crest cells aid in the development of cardiomyocytes in the ventricles of the heart in birds and mammals. What is the significance of your findings?

: Before our paper, it had been shown that the neural crest could give rise to cardiomyocytes in zebrafish hearts. Those previous papers showed that this contribution existed, but also that when you ablate this population, it would give rise to a poorly functioning adult heart. The adult zebrafish without a neural crest component suffered from severe hypertrophic cardiomyopathy, and would not survive when exposed to stress tests. Due to limitations in tools, there was never a description of this population of cells to amniote (like mammals and chickens) hearts. We were the first

INTERVIEWS

“We were the first to show, using new techniques, that neural crest cells were actually contributing a similar proportion of cells to cardiomyocytes in the heart.” to show, using new techniques, that neural crest cells were actually contributing a similar proportion of cells to cardiomyocytes in the heart. This is important because not only do we show that they represent a significant population of cardiomyocytes in the adult heart, but we also show that this population of cells is very important for cardiac regeneration. Knowing that chickens and mammals could have this population of cells in their heart got us one step closer to understanding how we can tinker with these programs that are controlling regeneration.

BSJ

: The primary technique that you employed to determine neural crest cell involvement with cardiomyocytes was retroviral lineage analysis. What advantage does lineage tracing have over other developmental and molecular biology approaches?

: I should talk a little bit about the history of how people have lineage-traced using chicken embryos before. Classical embryology experiments, including those that are foundational to understanding neural crest contributions to different lineages, use two different approaches. One approach is to make chimeric embryos by tissue grafting, for example, from quail to chick

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Figure 1: Retrovirally-labeled chick cardiac neural crest cells reveal novel derivatives. A replication incompetent avian retrovirus encoding H2B-YFP, a fusion protein that fluorescently labels cell nuclei, was injected into the lumen of the hindbrain—the origin of cardiac neural crest cells in a chick embryo. By embryonic day 6, the cardiac crest cells (labeled in green in the above panels), migrated to the heart and settled in the pharyngeal arch arteries (F), aorticopulmonary septum (G), and the outflow tract and ventricles (H). Panel H also displays that cardiac neural crest cells express the myocardial marker Trop T (labeled in magenta), indicating that the neural crest contributes to cardiomyocytes.

or chick to chick, then trace where the donor graft migrates in the host embryo. That is a beautiful, elegant technique, but it comes with many problems because you have a graft that has been put into a host environment that needs to heal. You lose a lot of information due to cells that die around the incision or cells that are not receptive to taking new cells on. The second approach involves lineage tracing done with a lipophilic dye called DiI that is used to trace cells over developmental time. However, as cells proliferate, this dye will dilute over time. This results in a loss of ability to do long-term analysis. In developmental biology, some research organisms such as mice and fish can be genetically manipulated to make transgenic lines to permanently lineage trace cells from early in development into adulthood. With chickens, we cannot make transgenic lines easily due to various restrictions, such as housing the chickens. In our paper, we use retroviral lineage analysis, which uses viruses to permanently label different cell populations. In chickens, we are able to specifically inject these retroviruses into the cells that give rise to the neural crest. Then, we track over time what cells they give rise to. This is similar to the dye injections, but now with a permanent label because the virus will integrate a fluorophore lineage tracer directly into the host genome. Each replication does not dilute the tracer over time allowing for long-term analysis which had not been used before. That is why people had not seen this contribution of cardiomyocytes previously.

BSJ

: Why is it important that the cells involved in regeneration are embryonic-derived? What is known about the mechanism zebrafish use to reactivate neural crest cell genes?

: There are two different lineages to cardiomyocytes in the adult heart and both are embryonic-derived—the mesoderm and the neural crest. It is interesting because they activate their own gene regulatory programs during development. Yet, they both give rise to physiologically indistinguishable cardiomyocytes in the adult, until injury. After an injury, the neural crest-derived

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cardiomyocytes will reactivate these developmental programs, and we are not sure how that works yet. Figuring out what that trigger is to facilitate this reactivation is crucial to understanding how we can hijack this information for any type of therapeutic approach in the future.

BSJ

: Given the contribution of neural crest cells in cardiomyocyte regeneration, you mention the possibility of new therapeutic approaches these results indicate. What information is necessary to translate this research into a human therapeutic?

: The first thing is understanding how nature has already figured this out. How does it work in something that can regenerate, like zebrafish? Once we have the information about the programs that are being reactivated, we need to know how the programs are being reactivated. Given this contribution of cells exists in mammalian hearts, this implies whatever regeneration is happening in zebrafish that is not happening in mice, or possibly humans, can be switched on. It is just a program, a regulatory logic that needs to be driven, and not a cell population that does not exist. In terms of therapeutics, we have to understand how it works, figure out what that trigger is, and go into mammalian systems to determine how we can reactivate this, either by activating or suppressing the trigger in order to drive a regenerative process. In terms of actual therapeutics, if it is a genetic switch, we can use CRISPR-based therapeutics to facilitate some kind of reactivation after injury.

BSJ MM

: Are neural crest cells likely to be involved in regeneration of any other organ systems?

: This is something I think so much about, because neural crest cells are multipotent, and they give rise to so many differentiated derivatives in the adult body plan. You can imagine the neural crest as a special population in the adult

INTERVIEWS

Figure 2: Cardiac neural crest contributes to zebrafish heart regeneration. A transgenic line of zebrafish is used to fluorescently turn cells green upon transcription of Sox10, a gene which is expressed in migrating cardiac neural crest cells but is down-regulated once these cells reach the heart. In an uninjured zebrafish heart, there is very little expression of Sox10. However, 7 days post amputation (dpa) of approximately 20% of the zebrafish heart, Sox10 is reactivated, shown by the presence of green cells in the middle panel. After 21dpa, the heart regenerated and green cells are seen surrounding the site of injury, indicating that cardiac neural crest cells reactivate transcriptional programs to contribute to heart regeneration. heart. For instance, it holds some kind of “stemness” to be able to dedifferentiate, proliferate, and tell other cells around them that it is time to respond to injury. That same process could be happening in other organ systems that the neural crest contributes to, like the craniofacial skeleton. In terms of differentiated derivatives of the neural crest, I do think that they hold a special ability to regenerate different organs, and that is something we definitely want to look at in the future.

BSJ

: Since the publication of this article, what new information has been discovered concerning the connection between neural crest cells and cardiomyocytes?

: In the lab, we have made a lot of headway on what programs are controlling multipotency to differentiate cardiomyocytes. Since differentiation during development and regeneration are very similar programs, learning more about these differentiation programs allows us to learn more about how they control regeneration, and vice versa. Also, in terms of regeneration, we have seen that if you use genetic tools to ablate these cells after injury, the hearts are not able to regenerate. We assumed this was going to be the case, but it is nice to see that whenever you do not have neural crest cells after injury, the heart cannot regenerate. Now, we are starting to look at what the cells are doing besides reactivating genes. What are they doing in order to drive this regeneration process, and how are they responding to an injury?

BSJ

: In another one of your papers, you discussed the evolution of the “new head” and its relation to neural crest circuits. What can researching the neural crest tell us about vertebrate evolution?

: The neural crest as a whole population is a vertebrate innovation, meaning they are exclusive to vertebrates.

INTERVIEWS

However, sea lampreys, a basal vertebrate, lack the full complement of neural crest derivatives. They do not have a jaw and do not have a lot of other derivatives, like organized sympathetic ganglia. The neural crest is really interesting to study in terms of evolution because, throughout vertebrate evolution, we see the increasing complexity of these different derivatives and their regulatory networks.

“[T]he neural crest is thought to be intimately linked with vertebrate evolution.”

BSJ MM

: Could you briefly explain to our readers what the new head hypothesis is?

: About 40 years ago, the new head hypothesis proposed that, with the advent of the neural crest and the cranial placodes, the craniofacial skeleton and highly complex sensory system arose, which allowed the elaboration and expansion of the vertebrate brain. With these complexities, invertebrate chordates, which were predominantly filter feeding animals, became active predators. That is crucial for the success of vertebrates as a lineage. Because this is a neural crest-derived feature, the neural crest is thought to be intimately linked with vertebrate evolution.

BSJ

: You posit an alteration to the new head hypothesis, stating that the neural crest portion of the new head arose via continued regulatory modifications rather than all-at-once at the base of the vertebrate lineage. What implications do these findings have for the study of neural crest evolution?

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Figure 3: Evidence of neural-crest derived cells in mouse myocardium. Figure 3A is an overview of a mouse heart showing locations of images B, C, D, E (cell nuclei labeled with DAPI-blue). Two transgenic mice lines were used: a Wnt1-ZsGreen line (B, C) and an improved Wnt1-mtmg line (D, E). Figures 3B and 3D compare the outflow tracts of the myocardium, while Figure 3C and 3E focus on the ventricles. Cells shown in green represent cardiac neural crest cells, indicating that both mice lines contain neural crest cells in the heart. Dashed box C’ and E’ shows enhanced magnification to highlight dual-staining between myocardial marker Trop T (in gray) and green neural crest cells.

: The paper is expanding upon what we know about this new head hypothesis. Instead of the neural crest arising as a whole set of derivatives at the base of vertebrates, we are essentially saying they arose from a homogenous, very rudimentary population. The complexity we now see arose via continued evolution and regulatory modifications. It is not really the case to think of all vertebrates having this new head or full set of derivatives. What happens is that throughout vertebrate evolution, we get more complexity.

BSJ

: Can you elaborate further on what mechanisms you believe are responsible for these neural crest regulatory modifications within jawed vertebrates?

: The short of it is no, but I can speculate. With these modifications, it is on the cis-regulatory level, meaning it is modifications in enhancer sequences and elements that control the activation of gene expression. Looking deeper at this regulatory DNA compared to gnathostomes (jawed vertebrates), we can get an understanding of which features have been acquired with the regulatory genome. This helps us to understand more about how novel gene expression and regulatory circuits are being acquired to give rise to these complex derivatives. But I do not have an answer.

BSJ

: Your lab is also interested in researching the gene regulatory circuitry underlying the formation of neuroblastomas. Can you describe some of the projects you have planned to research this?

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: We want to know how this multipotent stem cell becomes a differentiated sympathetic nervous system derivative, as the sympathetic nervous system is where neuroblastomas arise. More often than not, it is because of a dysregulation of the events culminating in these differentiated cell types. We want to understand the trajectory of neural crest cells to sympathetic neurons, chromaffin cells, and other derivatives of the sympathetic nervous system. With that information, we can then understand how those programs that are controlling differentiation become dysregulated to give rise to neuroblastomas. While we are doing that kind of research, neuroblastoma formation requires us to develop better animal models to recapitulate this tumorigenesis phenotype. We use zebrafish in the lab as our main model organism for understanding tumorigenesis. Hopefully, all of this will culminate in the ability to make therapeutics, maybe patient-specific therapeutics, by modeling tumor formation in zebrafish.

BSJ

: How do you see the field of regenerative medicine evolving in the near future, and how will your lab be a part of the effort to translate regeneration from one animal to another?

: The first thing is figuring out how this works in something that can regenerate. That is what we are really focused on now. Our immediate next steps are understanding what is not being reactivated and what is not happening in humans and mammals that is happening in zebrafish. Using the mouse as a model system, as well as human-derived cell culture and organoids, we can

INTERVIEWS

“I think we are on the precipice of developing translational strategies because the tools and technologies that we have now allow us to uncover these things rather quickly.”

model these things in human-derived tissues and see, using genome wide screens, what is not being reactivated after injury. Following this, we can use CRISPR-based approaches to reactivate these regenerative genes in the mammalian system. As we unveil candidates that are necessary for this reactivation, it becomes very easy to stimulate them with tools that already exist. I think we are on the precipice of developing translational strategies because the tools and technologies that we have now allow us to uncover these things rather quickly. REFERENCES 1. 2.

Headshot: [Photograph of Megan Martik]. Image reprinted with permission. Figures 1, 2, and 3: Tang, W., Martik, M. L., Li, Y., & Bronner, M. E. (2019). Cardiac neural crest contributes to cardiomyocytes in amniotes and heart regeneration in zebrafish. ELife, 8. https://doi.org/10.7554/elife.47929 Figure 4: Martik, M. L., Gandhi, S., Uy, B. R., Gillis, J. A., Green, S. A., Simoes-Costa, M., & Bronner, M. E. (2019). Evolution of the new head by gradual acquisition of neural crest regulatory circuits. Nature, 574(7780), 675–678. https://doi.org/10.1038/s41586-019-1691-4

INTERVIEWS

Figure 4: Evolution of neural crest subpopulations. Figure 4a illustrates the evolution of neural crest (NC) subpopulations, and the similarities between vertebrate NC and amniote trunk subpopulations. Early vertebrate evolution led to the division of NC into cranial and trunk groups. Figure 4b shows the molecular differences between lamprey and gnathostome NC, namely that lamprey lack differentiation between cranial and trunk NC.

SPRING 2022 | Berkeley Scientific Journal

Do Memes Behave Like Viruses? BY PIERRE LETELLIER

any internet memes go “viral.” Indeed, the action of memes spreading rapidly across the web evokes the concept of a spreading virus. In The Selftsh Gene, Richard Dawkins deftnes me mes as “evolutionary machines”. 1 Material or not, these machines satisfy three crite ria to stay alive: “variation,” “heredity,” and “selection.”

Genetics can be considered a meme: random mutations create variations with in the gene pool, heredity ensures genes spread throughout a population, and the external environment acts as a Thlter by selecting advantageous characteristics. What about internet memes? One can see a meme, download it, and modify it. When the meme is reuploaded, there is a slight

chance for it to “go viral.” Not every meme has success: when it is too speciThc or peo ple are not persuaded to share it, it dies oft and stays forgotten. fiis bold statement that anything from genes to internet memes can behave in a biological way was recently support ed by researchers exploring the “virality” of memes on the internet. fiey argue that

Figure 1: “Instagram,” ”Halloween,” and “Nyan Cat” on Google Trend, a big data tool which provides the number of times requests were entered into the google search bar across time.

Berkeley Scientific Journal | SPRING 2022

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there are two principles of internet user attention motivating the spread of popular internet content. The first is that internet users focus on what has already received attention. Contradictorily, the second, is that they are interested in what is new. These principles result in “a typical form of web success, where attention on certain elements concentrates, then disperses, and moves to new elements”.2 In 2011, researchers attempted a new method of modeling the growth of internet memes — an approach still being improved today.3,4 They were motivated by the idea that treating memes as viruses presents the opportunity to apply pre-existing epidemiological solutions to internet issues such as education, advertising, or the mitigation of fake news. Using Google Trend — a big data tool that allows people to explore how the interests of internet users on certain topics evolve across time — researchers noticed how the curve of the Nyan Cat meme (see Figure 1) seemed familiar. It looked exactly like a pandemic infection over time. First, the researchers stored the Google Trend data carefully. Then, they got off their computers to brainstorm a way to

Figure 2: Schema of the logic behind the differential equations of the SIR model. Here, the Infected (“I”) are the individuals who were “contaminated” by the meme; they google the topic, and have a chance “α” during their online interactions to share the meme and infect someone Susceptible (“S”). Out of boredom, a fraction “γ” of the infected people Recover (R) from the meme across time. However, there is still a chance “β” for them to get re-infected, if their interest is piqued again.

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Figure 3: Optimization of the model (line function) on the data (scatter plot). All of the curves are fit to as many of the dots extracted from Google Trend as possible. verify the virality of memes. In order to mathematically account for the evolution of an epidemic, researchers used a computational model called the “SIR model.” It is part of a branch of compartmental models in epidemiology. It’s called “compartmental” because it divides the population into several categories. In the case of a pandemic, the categories are “susceptible,” “infected,” and “recovered.” There is also a way for people to move from one category to another, over time. This is what differential equations do: they compute the difference between one state and another. Now that they have a theoretical model and data, researchers can test their model with a bit of code. The searchers did not share the source code of their optimization method. However, a way to replicate the approach is to try many different combinations of these parameters α, γ, β, and keep the epidemiologic model which is the closest to the Google Trend data. The code loops every combination of α, γ, β and computes each model. Then, it estimates the correlation between the Google Trend data and the model and saves the model with the highest correlation. This process is a “brute-force” algorithm, since it relies on sheer computing power and trying every possibility rather than more efficient algorithms. After a bit of waiting and a few iterations, this method found that the epidemiologic model provided excellent fits for the memes “Gangnam Style” and “Grumpy Cat”. However, it also found that the Goo-

“Blackmore and Dawkins invite us to follow the point of view of the meme. To memes, humans are nothing more than ‘copying machines’.” gle search “Math” does not provide a good fit for the model. Therefore, “Math” does not behave like a virus, even if the model does its best to fit (p-value > 0.05). The “Gangnam Style” meme has a higher infection factor “α” than Grumpy Cat (5 > 4), indicating that the song spreads with greater efficiency. However, the recovery factor “γ” is also more significant for Gangnam style than for Grumpy Cat (0.75 > 0.55). This means that people got tired of Gangnam Style quicker! Now is the time for some analysis. What does all of this mean? Susan Blackmore’s book The Meme Machine serves as a surprising introduction to “memetics.”5 This proposed field of research explores the question of evolution in a broader perspective. It applies evolutionary theory to the spread of culture just like it was applied to the spread of ideas, rumors, and ideology.6,7,8 Under

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this paradigm, the vast majority of cultural concepts come from memes: all music, history, language are principles vary by mutation, transmit by heredity, and are selected by the environment. In fact, religion is arguably one of the first memes in history.9 For Dawkins, the meme is “selfish” because it lives through us, at our expense. Blackmore and Dawkins invite us to follow the point of view of the meme. To memes, humans are nothing more than “copying machines.’’ For instance, some physically harmful memes can live at our expense. Many dangerous internet challenges re-

quire us to perform something and then to nominate other people to do it in turn. For example, the “Ice Bucket Challenge”: once nominated, people have to pour a bucket of ice over their heads, and then donate in the next 24 hours to the Motor Neurone Disease Association. Even if at least one death has been linked to the challenge, the scale of this meme was so enormous that President Obama himself had to speak on the subject.10 He refused to participate despite his nomination — and still donated! This confirms how the virality of ideas can be strategically used to serve a purpose, and

Berkeley Scientific Journal | SPRING 2022

“For Dawkins, the meme is ‘selfish’ because it lives through us, at our expense.”

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can also be harmful to humans. All of this said, Blackmore’s memetics field is not recognized by most researchers in the social sciences. It is considered a failed paradigm. Rather, scientists now use the concept of cultural evolution, which offers a similar set of ideas.11 Everything in our culture — from language to our most complex scientific concepts — is the result of evolution.12 These principles, or memes, have been modified over many generations to provide us today with extremely powerful and refined conceptual tools. What makes humans so intelligent when compared to other animals is our collective intelligence and our cumulative and evolving culture!13 Individually, nobody is omniscient; we always rely on past knowledge to build new ones. Memes are a good example of this principle. Understanding the evolution of memes is like understanding the deep logic of our culture.14 REFERENCES 1.

5. 6.

Thomas Beauvisage, Jean-Samuel Beuscart, Thomas Couronné and Kevin Mellet (2011). Is success on the internet based on contagion? An analysis of research on virality, Tracés. DOI: 10.4000/traces.5194 Wang, L., Wood, B. C. (2011). An epidemiological approach to model the viral propagation of memes. Applied Mathematical Modelling, 35, 5442–5447. https://doi.org/10.1016/j. apm.2011.04.035 Adam Lonnberg, Pengcheng Xiao, Kathryn Wolfinger, (2020). The growth, spread, and mutation of internet phenomena: A study of memes, Results in Applied Mathematics, Volume 6, https://doi. org/10.1016/j.rinam.2020.100092. Blackmore, Susan J. (1999). The meme machine. Oxford [England]: Oxford University Press, ISBN 0-19850365-2 R. Dawkins. (1976). The Selfish Gene, Oxford University Press. ISBN: 9780198788607 L.M.A. Bettencourt, A. CintrónAias, D.I. Kaiser, C. Castillo-Chávez, (2006). The power of a good idea: quantitative modeling of the spread

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of ideas from epidemiological models, Physica A 364 513–536. DOI: 10.1016/j.physa.2005.08.083 7. K. Kawachi, (2008). Deterministic models for rumor transmission, Nonlinear Anal.: Real World Appl. 9 1989–2028. DOI: 10.1016/j. nonrwa.2007.06.004 8. F.J. Santonjaa, A.C. Tarazonaa, R.J. Villanueva, (2008). A mathematical model of the pressure of an extreme ideology on a society, Comput. Math. Appl. 56. Volume 56, issue 3, pages 836–846. ISSN 0898-1221, DOI: 10.1016/j.camwa.2008.01.001. 9. Radim Chvaja (2020). Why did memetics fail? Comparative case study. Perspectives on Science. 28 (4): 542–570. doi:10.1162/posc_a_00350. 10. Alex Mesoudi. (2011). Cultural evolution: how darwinian theory can explain human culture and synthesize the social, Broché, 1 septembre 2011. ISBN: 9780226520452 11. Gayon, Jean. (2015). Cultural evolution, theories and models, Jean-François Dortier éd., Révolution dans nos origines. Éditions Sciences Humaines, pp. 352-365. ISBN : 9782361063245 12. Durham, W. (1991). Coevolution: genes, culture and human diversity. Stanford: Stanford University Press. Chapter 5. ISBN: 9780804715379. IMAGE REFERENCES 1. 2. 3. 4. 5.

6. 7.

Instagram. Instagram Logo, 2016. Toby Ord. Jack o’ Lantern, 2003. Christopher Torres, prguitarman, Nyan Cat screenshot, 2012. The Brave Fighter of Sun Fighbird, screenshot, 1991. Park Jai-Sang, Yoo Gun-Hyung, youtube, Gangnam Style 2012. https://www.youtube.com/ watch?v=9bZkp7q19f0. Bryan Bundesen, Grumpy Cat, 2012. Designed by Freepik, https://www. freepik.com/free-photos-vectors/ background.

Reviewed by Abi Rajan (UC Berkeley Mathematics Department)

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Neoantigens: The Future of Personalized Cancer Treatment BY CASSIDY BIELLAK

ver the last century, there have been numerous advances in oncological research, each one getting us closer and closer to treating one of the most fatal diseases of our generation: cancer. Presently, many treatments may only be effective for a short amount of time and finding long lasting cancer treatments has been a struggle for many patients.8 Chemotherapy–the use of chemicals to eradicate cancer cells–can be effective yet it kills many healthy cells in the process, leaving patients with painful side effects. Likewise, the most common forms of radiation therapy can also cause damage to healthy tissue, prolonging treatment.11 Naturally, neither of these two options sound particularly appealing, and while many people do not have the luxury of choice when handed a diagnosis, there are less destructive treatments on the horizon. Immunotherapy, the term for using one’s own immune system to fight cancer, is different from other types of treatments as it only attacks tumor cells. Although the immune system’s job is to attack foreign cells, tumor cells start out as normal, healthy cells until they proliferate out of control. As such, the immune system does

not recognize them as foreign cells. It is for this reason that current immunotherapy treatments are only viable for a short amount of time, with patients having to continually switch treatments to ensure that tumors are not growing. However, recent advancements pertaining to cancer immunology have begun to widen the efficacy and length of treatments. In recent years, there has been a promising solution to the shortcomings of modern cancer treatment: personalized cancer treatment tailored to the individual. Tailored treatment could provide a solution to some of the issues that come with mainstream cancer therapy. This specificity is achieved by creating fewer side effects and directly targeting only cancer cells.1 As put by the Journal of Molecular Biomarkers and Diagnosis, “the suitability of this ‘onesize-fits-all’ approach to cancer therapy is called into question. Precision medicine, the proposed future for the treatment of disease, is based on a tailored approach for selecting therapy at the individual patient level.”2 The human body possesses a complex immune system with many types of cells.

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Before speaking on some of the new personalized immunotherapy treatments, a short background discussion about how our immune systems work is needed. There are two main types of white blood cells present in the immune system: T-cells and B-cells. T-cells, which originate in the lymph nodes, recognize foreign intruders and threatening cells within the body and will attack them to reduce harm to the body. B-cells interact with antigens, which are specific foreign molecules that will elicit an immune response. B-cells will recognize certain antigens as foreign and will then produce antibodies (proteins) that will attach to foreign materials and act as a flag down for the immune system to locate and destroy them.10 A major characteristic of immune cells is their ability to recognize molecules on the surface of cells, which is the mechanism that personalized immunotherapy treatments take advantage of by using neoantigens. A neoantigen is a new protein that forms on the surface of cancer cells when certain mutations occur in tumor DNA. Neoantigens play an important role in helping the body build an

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immune response against cancer cells.3 Neoantigen-based cancer treatments are revolutionary, as the immune system will be wired to only destroy cells that display neoantigens (cancer cells), and all other cells in the body will be unaffected–a feat that chemotherapy and radiation therapy treatments have not been able to achieve. According to initial studies of neoantigen-based vaccines, scientists have generated a multitude of evidence surrounding “antitumor activity in patients with melanoma”, with neoantigens playing a key role in the T-cell immune response against tumor cells.4 A recent paper published in 2021 completed a study on a group of 729 breast cancer patients, and patients who were reported to have a “high level of neoantigen expression” demonstrated improved survival. Furthermore, neoantigen-based vaccines have been shown to be effective in mouse models for cancers such as skin, colon, and bone cancers.5 Neoantigens have also been shown to be effective in the clinical realm, as a recent clinical trial completed by the Center for Cancer Research utilized neoantigens in a breast cancer study; in total, they treated six women with metastatic breast cancer using Tumor Infiltrating Lymphocytes (T-Cells that have neoantigens on their surfaces) that were personalized to each patient, and found that “tumors shrank in three of the six women”, one of which became completely cancer free after the treatment.6 How can medical researchers train an immune system into attacking cancer

“A neoantigen is a new protein that forms on the surface of cancer cells when certain mutations occur in tumor DNA.”

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Figure 1: B cells binding to antigens and releasing antibodies.

cells? The first step is to sequence the patient’s normal genomic DNA as well as the tumor’s DNA. The two sequences are then compared to find mutations within the tumor DNA; these mutations will eventually be the “targets” for the immune system to aid in recognizing cancer cells. In order for a mutation to be an effective target for the creation of neoantigens, the mutation must encode for a protein that is expressed on the surface of the tumor.9 This protein must also be able to be recognized by the immune system, so an immune response can be generated. Once a mutation that meets these requirements is found, there are a couple of different ways to induce the neoantigen treatment into the patient, including vaccines and T-cell therapy.7 In neoantigen based vaccines, the neoantigens identified during mutation analysis are injected into the body and will elicit an immune response from T-cells and B-cells. These immune cells will recognize the neoantigen vaccine as foreign and are programmed to attack any material within the body presenting the specific neoantigen. In the process, they will also attack neoantigen-presenting tumor cells. In T-cell therapy, neoantigens are added to the patient’s isolated T-cells in the lab and are grown until the T-cells reach a high enough concentration. T-cells have specific receptors that are programmed to recognize the specific neoantigen sequence that was previously isolated. Once the cells reach this concentration, they will be injected back into the patient and the T-cells will start attacking any foreign materials expressing that specific neoantigen.7

CONCLUSION While investigators have made great strides in personalized neoantigen cancer treatments, if widespread use of these treatments is to take place, more research is required on how to produce them on a large scale for many patients. An issue with the current neoantigen treatment research is that it is difficult to find a mutated sequence that can be targeted and expressed as a neoantigen. Because of this, it can take a long time to find a suitable sequence for one patient, and most cancer patients do not have this time to wait.2 Furthermore, not all patients have tumors that are easily accessible, making it difficult to sequence the tumor DNA, which is essential for neoantigen treatments. Clinical and preclinical trials are currently being completed and are aiming to target these issues. Despite these challenges, the promising research surrounding neoantigens provides a foundation for a new generation of cancer treatments, focused around diminishing harmful side effects and creating more tumor specificity. These treatments will be free of toxic chemicals or harmful radiation, and consequently, cancer patients will eventually have a higher quality of life that is largely pain-free. REFERENCES 1.

Jain K. K. (2021). Personalized immuno-oncology. Medical Principles and Practice: International Journal of the Kuwait University, 30(1), 1–16. https://doi.

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Figure 2: Personalized cancer therapy procedure.

“T-cells have specific receptors that will be programmed to recognize the specific neoantigen sequence that was previously isolated.”

org/10.1159/000511107 Maciejko, L., Smalley, M., & Goldman, A. (2017). Cancer immunotherapy and personalized medicine: Emerging technologies and biomarker-based approaches. Journal of Molecular Biomarkers & Diagnosis, 8(5), 350. https://doi. org/10.4172/2155-9929.1000350 Understanding Cancer Immunotherapy Research. (2020). What is neoantigen-based therapy? Therapies. https://www.ucir.org/ therapies/neoantigen-based-therapy Blass, E., Ott, P.A. (2021). Advances in the development of personalized neoantigen-based therapeutic cancer vaccines. National Review of Clinical Oncology 18(4), 215–229. https://doi. org/10.1038/s41571-020-00460-2 Jiang, T., Shi, T., Zhang, H., Hu, J.,

Song, Y., Wei, J., Ren, S., & Zhou, C. (2019). Tumor neoantigens: From basic research to clinical applications. Journal of Hematology & Oncology, 12(1), 93. https://doi.org/10.1186/ s13045-019-0787-5 6. Li, W., Amei, A., Bui, F., Norouzifar, S., Lu, L., & Wang, Z. (2021). Impact of neoantigen expression and T-cell activation on breast cancer survival. Cancers, 13(12), 2879. https://doi. org/10.3390/cancers13122879 7. Zhang, Z., Lu, M., Qin, Y., Gao, W., Tao, L., Su, W., & Zhong, J. (2021). Neoantigen: A new breakthrough in tumor immunotherapy. Frontiers in Immunology, 12. https://www. frontiersin.org/articles/10.3389/ fimmu.2021.672356 8. Zhang, X., Sharma, P. K., Peter Goedegebuure, S., & Gillanders, W. E. (2017). Personalized cancer vaccines: Targeting the cancer mutanome. Vaccine, 35(7), 1094–1100. https:// doi.org/10.1016/j.vaccine.2016.05.073 9. Borden, E. S., Buetow, K. H., Wilson, M. A., & Hastings, K. T. (2022). Cancer neoantigens: Challenges and future directions for prediction, prioritization, and validation. Frontiers in Oncology, 12. https:// www.frontiersin.org/articles/10.3389/ fonc.2022.836821 10. Aldous, A. R., & Dong, J. Z. (2018). Personalized neoantigen vaccines: A new approach to cancer immunotherapy. Bioorganic & Medicinal Chemistry, 26(10), 2842–2849. https://doi.org/10.1016/j. bmc.2017.10.021

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11. Majeed, H., & Gupta, V. (2022). Adverse effects of radiation therapy. In StatPearls. StatPearls Publishing. http://www.ncbi.nlm.nih.gov/books/ NBK563259/ IMAGE REFERENCES 12. Figure 1: B cells binding to antigens and releasing antibodies | Arizona Science Center; https://askabiologist. asu.edu/b-cell | Arizona State University. (2011, February 16). B-cells. Ask a Biologist. https:// askabiologist.asu.edu/b-cell 13. Figure 2: Personalized cancer therapy procedure | Jeyang259 | Jeyang259. (2020, February 27). Personalized cancer therapy. Wikimedia Commons. https:// commons.wikimedia.org/wiki/ File:Personalized_Cancer_Therapy. png

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Prognostic Potential of Extracellular Vesicles: Noninvasive Monitoring of Chemotherapeutic Resistance Development Jennifer C. Hall, 2Thomas R. Carey, 3Lydia L. Sohn Research Sponsor: 3Lydia L. Sohn

ABSTRACT Chemotherapy remains the most common modality of cancer treatment, used both independently and in combination with other systemic or localized therapies. It has been shown that patient response to chemotherapeutics is a potent predictor of prognosis and over 90% of cancer patient mortalities are related to drug resistance. Resistance to chemotherapy can develop during the course of treatment when tumor cells become less sensitive to therapy and is particularly dangerous to patient survival due to the difficulty of detection. As such, it has become essential to develop efficient methods of monitoring changes in patients’ response to treatment. Our research demonstrates the potential of extracellular vesicles (EVs) in rapid, noninvasive monitoring of tumor response to chemotherapeutics. Through analysis of EV mRNA cargo, trends in gene expression observed in cells are shown to be conserved in their derived EVs. To determine tumor response to treatment, we devised a model system to mimic interactions between tumor cells, chemotherapeutic(s), and gene expression alterations that confer resistance. We isolated EVs from MCF7/wt and MCF7/ADR cell culture supernatant to model EVs derived from doxorubicin-sensitive and resistant tumors, respectively. We then extracted mRNA from EVs and quantified the expression of top2a. We observed downregulation of top2a, which confers resistance to doxorubicin, in MCF7/ADR (doxorubicin resistant) EVs relative to MCF7/ wt EVs. Our findings establish the feasibility of using mRNA in tumor-derived EVs to assess drug sensitivity of tumors via liquid biopsy. Major, Year, Departmental: 1Class of 2021, Molecular and Cellular Biology Neurobiology Emphasis, Department of Molecular and Cellular Biology, University of California, Berkeley; 2UC Berkeley–UC San Francisco Graduate Program in Bioengineering, University of California, Berkeley; 3Department of Mechanical Engineering, University of California, Berkeley, 5118 Etcheverry Hall, Berkeley, CA 94720, US

INTRODUCTION Cancer treatment often employs an array of modalities utilized independently, in sequence, or concurrently. Treatment modalities include surgery, radiation, and systemic therapies such as chemotherapy or immunotherapy. Chemotherapy remains the most common form of cancer treatment in the US (Figure 1A).1 Patient response to administered chemotherapeutics is a strong predictor of prognosis, and over 90% of cancer patient mortalities are related to drug resistance.2,3 Chemotherapeutic resistance is categorized as either intrinsic or developed. Intrinsic resistance inhibits a drug’s mechanism of action and exists in tumor cells prior to exposure. Developed resistance occurs when tumors evolve decreased sensitivity after initial or prolonged exposure. In conjunction with the difficulty of detection, this makes developed resistance particularly threatening

to positive patient outcomes. At present, determination of tumor response to chemotherapy treatment typically requires invasive tumor biopsies. This demonstrates the need for more efficient, noninvasive mechanisms for monitoring tumor response to treatment frequently throughout the treatment course. As a result of the frequency of chemotherapy use and the implications of losses in sensitivity to treatment, much research has gone into elucidation of the mechanisms and gene expression alterations conferring chemotherapeutic resistance (Figure 1B). Similarly, pathways and gene targets of many chemotherapeutics are well understood. For example, the chemotherapy drug doxorubicin acts by inhibiting topoisomerase-IIα (TOP2A) function within the nucleus of tumor cells (Figure 1C), and decreases in TOP2A gene expression are indicative of doxorubicin resistance. Broadly, altered gene expression within drug-resistant tumor cells provides clear ev-

Figure 1. Chemotherapeutics remain the most prescribed cancer treatment and accordingly, mechanisms of drug action and drug resistance are well-characterized for many drugs. Chemotherapy utilized in breast cancer treatment (A) demonstrates its prevalence, particularly in late-stage cases. This prevalence has led to research into the different mechanisms of drug resistance (B). Likewise, much research has further illuminated the different mechanisms of action of given drugs. In a specific example (C), doxorubicin is shown entering the cell and nucleus to inhibit the TOP2A enzyme.

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Table 1. A model system was devised to measure gene expression alterations associated with developed chemotherapeutic resistance. MCF7/wt and MCF7/ADR cell lines were used to model doxorubicin-sensitive and resistant tumors, respectively. TOP2A is an enzyme that acts in DNA repair and has been shown to be downregulated in doxorubicin resistant cells.

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RESULTS Using RT-qPCR, we measured top2a expression in both cells and EVs derived from the MCF7/ADR and MCF7/wt cell lines. In MCF7/ADR cells, we observed a 3.58-fold downregulation relative to MCF7/wt cells; in MCF7/ADR EVs, we observed a 24.29-fold downregulation relative to MCF7/wt EVs (Figure 2). We normalized top2a expression in cells and EVs derived from both cell lines to baseline expression of the glyceraldehyde-3-phosphate dehydrogenase (gapdh) housekeeping gene. These data strongly support the notion that gene expression alterations in cells can be detected through quantification of the

A. 10

ΔCt values of MCF7/wt and MCF7/ADR cells

Fold-down regulation of TOP2A for MCF7/ADR cells and EVs

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7 6 5

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MCF7/ADR cells

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Fold-down regulation

8 7

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idence of resistance when compared to that of drug-sensitive tumor cells.4 However, this method is limited in its clinical potential because probing for altered gene expression in tumor cells fails to eliminate the need for invasive biopsy procedures. To address this limitation, we put forth a method for the efficient and noninvasive monitoring of altered gene expression through the examination of mRNA cargo within extracellular vesicles (EVs) derived from tumor cells. EVs are lipid bilayer-delimited nanoparticles secreted by cells and heralded as a primary mechanism for cell-tocell communication. As a result of their formation via exocytosis or membrane budding, EVs display protein surface markers and contain mRNA cargo conserved from their cell of origin. In addition, it has been shown that EVs travel throughout the body within biofluids.5 These characteristics of EVs highlight their potential application in a method for noninvasively monitoring tumor cell sensitivity to cancer treatment. For our research, a model system was devised to mimic the development of chemotherapeutic resistance in tumor cells (Table 1). The system was comprised of a chemotherapy drug, sensitive and resistant tumor cells, and a target gene known to be differentially expressed in association with drug resistance. We utilized the common anthracycline antibiotic, doxorubicin, as the model chemotherapeutic. We used the MCF7 (MCF7/wt) breast cancer cell line to model drug-sensitive tumor cells, and we used the MCF7/ ADR cell line—known to be multi-drug resistant—to model tumor cells with developed drug-resistance. We then isolated EVs from the cell culture supernatant of the drug-sensitive and drug-resistant cell lines. Finally, we selected top2a as the target gene because it is highly downregulated in doxorubicin-resistant MCF7 cells, top2a codes for TOP2A, an enzyme involved in DNA repair, and is a primary target in the doxorubicin mechanism.4 With this model, we demonstrated mRNA within tumor-derived EVs reflects the gene expression alterations observed in tumor cells associated with developed resistance to doxorubicin. This finding indicates the highly impactful clinical motivation and potential of our research.

5 4 3

2 1 0

MCF7/wt EVs

MCF7/ADR EVs

Cells

EVs

Figure 2. Relative expression of top2a in doxorubicin-resistant tumor cells and their derived EVs compared to MCF7/wt cells and EVs, respectively. Comparison of the ΔCt values (normalized to gapdh) of (A) cells and (B) EVs reveals decreased expression of top2a in both MCF7/ADR cells and EVs. (C) A 3.58-fold downregulation was observed in MCF7/ADR cells compared to MCF7/wt cells. A 24.29fold downregulation was observed in MCF7/ADR EVs compared to MCF7/wt EVs.

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mRNA cargo of their derived EVs. In particular, decreased top2a expression in MCF7/ADR cells agrees with established trends observed in MCF7 cells with developed doxorubicin resistance and further underscores the potential of using EVs to noninvasively monitor tumor resistance development.4 DISCUSSION Through our research, we have presented a novel method for noninvasively monitoring gene expression alterations in tumor cells through quantification of a target gene in the mRNA cargo of their derived EVs. Observation of differential expression of the target gene, top2a, demonstrates the conservation of trends in gene expression from cells to EVs (Figure 3). Our findings illuminate clear next steps for the implementation of this method into a clinical setting. In addition to tumor-derived EVs, biofluids contain abundant healthy-cell-derived EVs, as well as cells, proteins, and free oligonucleotides. Accordingly, future work should improve upon the current EV isolation protocols to more effectively address complex biofluid samples. Coupled with the properties of EVs, including their ability to travel great distances from the site of their genesis and their conserved biomarkers derived from their cell of origin, it is possible to select for tumor-derived EVs among samples comprised of EVs secreted from a diverse range of cell types.5,6 A potential mechanism for this selection is immuno-capture where antibody-functionalized microbeads bind EVs based on surface markers differentially expressed on tumor cells. This is possible because these surface markers are inherited by EVs secreted from tumor cells and may be used to differentiate them from other EV populations. Implementation of a two-step immuno-capture procedure has previously been shown to increase selectivity.6 The primary step aims to diminish background and consists of negatively selecting for EVs from non-tumor cell types with a first round of microbeads coated with surface markers known to be generally expressed. This is then followed by a secondary step which utilizes functionalized microbead interactions with known tumor surface markers yielding positive selection of tumor-derived EVs. Captured EVs can be lysed

on the bead to release their cargo, enabling subsequent isolation of their mRNA and quantification of target genes. The addition of tumor-derived EV selection furthers the clinical potential of utilizing EVs in liquid biopsy applications. Additional future directions include developing a streamlined protocol incorporating isolation/selection of tumor-derived EVs, extraction of their mRNA cargo, and quantification of target genes that confer drug resistance. All together, this would fulfill the need for an efficient, noninvasive method of determining patient prognosis through the rapid evaluation of response to chemotherapy. As long as drug therapies remain a prominent tool for limiting disease progression, there will likewise remain the need to verify tumor sensitivity to prescribed drugs. To assess this, we have presented a novel method to noninvasively monitor tumor response to chemotherapeutics through examination of tumor-derived EV mRNA. Our findings highlight the potential use of EVs in liquid biopsy applications that may be performed at frequent timepoints throughout a cancer treatment course to support improved patient outcomes. METHODS Cell culture and growth conditions The MCF7/wt and MCF7/ADR cell lines were obtained from the UCB Cell Culture Facility supported by the University of California, Berkeley. Both cell lines were cultured in an attached monolayer in DMEM media (Thermo Fisher Scientific, USA) supplemented with 10% exosome depleted fetal bovine serum (Thermo Fisher Scientific, USA) and 1% penicillin-streptomycin (Roche Molecular Systems, USA). Additionally, cells were grown in either T25, T75, or T175 attached type, filter-cap culture flasks (Thermo Fisher Scientific, USA). Cells were incubated in a 37 °C humidified atmosphere with 5% CO2. EV isolation EVs were isolated from cell culture supernatant using a membrane-affinity-based commercial isolation method (ExoEasy Maxi, Qiagen, Germany). Briefly, cell culture supernatant was clarified by centrifugation at 3000 rcf for 15 minutes, and the resulting supernatant was mixed with a binding buffer and added to the spin column.

Figure 3. Conservation of gene expression from cells to EVs. Decreased top2a expression was observed in EVs derived from cell types in which top2a expression was similarly decreased. This demonstrates the conservation of trends in gene expression from cells to their derived EVs.

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EVs were captured on the column, washed, and lysed using QiaZOL. RNA isolation RNA from both EVs and cells was isolated using a phenol-chloroform method with the RNeasy® Mini Kit (Qiagen, Germany) according to the manufacturer’s protocol. An additional RNase-free DNase step was performed according to manufacturer’s protocol and utilizing an RNase-free DNase kit (Qiagen, Germany). This step was carried out to ensure total elimination of genomic DNA. Finally, to ensure purity of RNA, optical density was measured using a NanoDrop® spectrophotometer at 260 and 280 nm and 260/280 ratios were compared to published qualifications of purity (Thermo Fisher Scientific, USA). Gene quantification The top2a gene was quantified in the isolated RNA of both cells and EVs using a 48-well RT qPCR assay. Each well containing samples also contained the components necessary for the PCR reaction (New England BioLabs, USA) as well as a primer for either top2a or gapdh. RT-qPCR was performed following the manufacturer’s protocols. The gapdh housekeeping gene was used to normalize top2a expression. top2a and gapdh primers (Integrated DNA Technologies, USA) were used at an in-well concentration of 40 nM.7 This primer concentration was found to minimize primer dimer formation observed in initial testing. Initial testing also produced an empirical top2a primer efficiency value of 99.43% which translates to an amplification value of 1.99 used in gene expression fold-change calculations. Data analysis Data analysis was performed using the delta delta cycle threshold method (ΔΔCt) and an amplification factor of 1.99. Fold-increases or -decreases in gene expression were calculated using the following equation: fold-increase or decrease = (amplification value)- ΔΔCt 1. 2.

REFERENCES American Cancer Society. (2019). Cancer treatment & survivorship facts & figures. https://www.cancer.org/research/ cancer-facts-statistics/survivor-facts-figures.html Pierga, J. Y., Robain, M., Jouve, M., Asselain, B., Diéras, V., Beuzeboc, P., Palangié, T., Dorval, T., Extra, J. M., Scholl, S., & Pouillart, P. (2001). Response to chemotherapy is a major parameter-influencing long-term survival of metastatic breast cancer patients. Annals of Oncology,, 12(2), 231–237. https://doi.org/10.1023/a:1008330527188 Bukowski, K., Kciuk, M., & Kontek, R. (2020). Mechanisms of multidrug resistance in cancer chemotherapy. International Journal of Molecular Sciences, 21(9), 3233. https:// doi.org/10.3390/ijms21093233 AbuHammad, S., & Zihlif, M. (2013). Gene expression alterations in doxorubicin resistant MCF7 breast cancer cell line. Genomics, 101(4), 213–220. https://doi.org/10.1016/j. ygeno.2012.11.009 Zhang, J., Nguyen, L. T. H., Hickey, R., Walters, N., Wang, X., Kwak, K. J., Lee, L. J., Palmer, A. F., & Reátegui, E.

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10.

11. 12.

13.

14.

15.

(2021) Immunomagnetic sequential ultrafiltration (iSUF) platform for enrichment and purification of extracellular vesicles from biofluids. Scientific Reports, 11. https://doi. org/10.1038/s41598-021-86910-y Ko, J., Hemphill, M., Gabrieli, D., Wu, L, Yelleswarapu, V., Lawrence, G., Pennycooke, W., Singh, A., Meaney, D. F., & Issadore, D. (2016).Smartphone-enabled optofluidic exosome diagnostic for concussion recovery. Scientific Reports, 6, https://doi.org/10.1038/srep31215 Szlachta, K., Manykyan, A., Raimer, H. M., Singh, S., Salamon, A., Guo, W., Lobachev, K. S., & Wang, Y. (2020). Topoisomerase II contributes to DNA secondary structure-mediated double-stranded breaks. Nucleic Acids Research, 4812), 6654–6671, https://doi.org/10.1093/nar/ gkaa483 Burgess, D. J., Doles, J., Zender, L., Xue, W., Ma, B., McCombie, W. R., Hannon, G. J., Lowe, S. W., & Hemann, M. T. (2008). Topoisomerase levels determine chemotherapy response in vitro and in vivo. Proceedings of the National Academy of Sciences, 105(26), 9053–9058. https://doi. org/10.1073/pnas.0803513105 Thorn, C. F., Oshiro, C., Marsh, S., Hernandez-Boussard, T., McLeod, H., Klein, T. E., & Altman, R. B. (2011). Doxorubicin pathways: pharmacodynamics and adverse effects. Pharmacogenetics and Genomics, 21(7), 440–446. https:// doi.org/10.1097/FPC.0b013e32833ffb56 An, X., Xu, F., Luo, R., Zheng, Q., Lu, J., Yang, Y., Qin, T., Yuan, Z., Shi, Y., Jiang, W., & Wang, S. (2018). The prognostic significance of topoisomerase II alpha protein in early stage luminal breast cancer. BMC Cancer, 18(1). https://doi. org/10.1186/s12885-018-4170-7 Stevic, I., Buescher, G., & Ricklefs, F. L. (2020).Monitoring therapy efficiency in cancer through extracellular vesicles. Cells, 9(1), 130. https://doi.org/10.3390/cells9010130 Kosaka, N., Yoshioka, Y., Fujita, Y., & Ochiya, T. (2016). Versatile roles of extracellular vesicles in cancer. The Journal of Clinical Investigation, 126(4), 1163–1172. https:// doi.org/10.1172/JCI81130 Choi, D. S., Lee, J., Go, G., Kim, Y., & Gho, Y. S.. (2013). Circulating extracellular vesicles in cancer diagnosis and monitoring. Molecular Diagnosis & Therapy, 17, 265–271. https://doi.org/10.1007/s40291-013-0042-7 Redzic, J. S., Ung, T. H., & Graner, M. W. (2014). Glioblastoma extracellular vesicles: reservoirs of potential biomarkers. Pharmacogenomics and Personalized Medicine, 7, 65–77. https://doi.org/10.2147/PGPM.S39768 Zhou, S., Yang, Y., Wu, Y., & Liu, S. (2021). Review: Multiplexed profiling of biomarkers in extracellular vesicles for cancer diagnosis and therapy monitoring. Analytica Chimica Acta, 1175. https://doi.org/10.1016/j.aca.2021.338633

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Renewable Energy Economics: Understanding the Costs and Capacity of Green Energy in the United States Megan J. Mehta Research Sponsor (PI): Abrar Rahma ABSTRACT Although the cost of renewable energy has dramatically decreased in the United States over the past 10 years, many energy experts raise concerns regarding the efficiency of production and capacity to meet demand. This paper evaluates how demand for renewable energy in various sectors has changed over time in relation to cost, and if renewable energy has the capacity to meet growing demand in the future. Despite substantial efforts to maximize the generation of renewable energy, form government contracts with renewable energy companies, and incite investments in research and development for producing clean energy faster, production of renewables will likely not meet its exponentially growing demand. Furthermore, renewable energy is harder to implement in industrial and commercial sectors than traditional energy sources, due to infrastructure constraints. Although demand for energy will continue to increase, the use of natural gas is expected to remain constant, and it is essential that the United States continues expanding its capacity for renewable energy. Major, Year, Departmental: Computer Science, Environmental Economics & Policy, 1st Year, Data Science Department University of California, Berkeley

INTRODUCTION As we continue to battle the climate crisis, discussions about the harms of burning fossil fuels and regarding partially or completely switching to renewable energy—energy from sources that are “inexhaustible in duration but limited in the amount of energy that is available per unit of time” —have increased in prevalence. Unlike fossil fuels, renewable energy sources can be accessed an infinite number of times.1 However, the amount of energy available for each use is limited. As such, voting for policies that encourage the government to fund research and development into renewable energy efficiency, as well as motivating corporations and individuals to utilize green energy, is essential for reducing the long-term consequences of burning fossil fuels and its impact on climate change. According to Figure 1 from the U.S. Energy Information Administration, we still primarily burn fossil fuels (petroleum and natural gas) to generate energy. But currently, renewable energy consumption has surpassed that of coal, indicating that we are moving towards a more sustainable future.1 To better understand how

projections of clean energy production relates to the overall demand for energy in various sectors, this paper weighs the effectiveness of solar, hydroelectric, and wind energy sources in meeting growing energy demand.

Figure 1: Wind and hydroelectric energy dominate renewable energy consumption, but petrol and natural gas dominate the overall consumption market.

Figure 2: Global renewable energy generation complared to total energy consumption.

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Global Energy Demand and Renewable Energy Capacity It’s possible that nations that consume more energy per person (such as the U.S. when compared to countries with denser populations like China or India) have more energy draining residential and commercial sectors. This is visualized in Figure 2.2 We also have to account for each nation’s state of development, which influences how many people have access to electricity. That being said, the U.S also has a greater annual change in renewable energy generation (larger capacity to generate more energy through greener sources).3 This, however, should not undermine the enormous capacity for implementing renewable energy infrastructure in other nations—there is great potential to do so around the globe. In fact, many countries (especially in the European Union)

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Figure 3: U.S. Energy supply and demand compared with source of renewable energy generation.

have contracts with corporations to expand renewable energy capacity and the “market for corporate power-purchase agreements—longterm deals to supply electricity—[boomed] in 2020.”4 Renewables are expected to “make up to 35% of electricity generation by 2030,” mostly from wind and solar sources.5 Renewable Energy in the United States U.S. renewable electricity generation has doubled since 2008, indicating improvements in renewable energy technology and increases in efficiency. The largest improvements are from solar and wind energy sources (Figure 3).6 Figure 4 further highlights the fact that the electric power sector is the largest consumer of wind, hydropower, and solar power. We can conclude that the sale of renewable energy is more lucrative than that of fossil fuels because the infrastructure for green energy production costs less than fossil fuel extraction, in most cases, especially when depleting fossil fuel reservoirs force the relocation of mines or drills or presents added challenges to worker safety, labor equipment costs, and liability.7 If the costs of implementing green energy continue to decrease and the electric power sector continues obtaining most of its energy

from these sources, we can conclude that the consumption of fossil fuels will dramatically decrease. Oil consumption fell 9.1 million barrels/day (9.3%), a record low since 2011. Most of the demand originated from the U.S. and decreases in demand for oil led to a production decrease of 6.6 million barrels/day. Refinery utilization fell to 74.1%—the lowest level since 1985.8 Despite this trend, demand for natural gas drastically increased as prices fell to $1.99/mmBtu in the U.S. and the share of gas in primary energy rose to 24.7%.8 As stated before, solar and wind capacity increased; solar electricity rose by 20%. China, the U.S., and Europe spearheaded the largest renewable growth change, largely due to policies that support investments in renewable energy research and infrastructure.8 Some examples of such policies include the European Green New Deal, Germany’s proposal to reach 50% renewable energy by 2030 and 80% by 2050, and 15 EU nations that reached 18% renewable energy in 2020.16,17,18 An increase in efficiency and quantity of energy generated from renewable energy, along with government funded implementation and research and development subsidies, lead to a significant decrease in cost, fueling a positive cycle to better implement these technologies and move further away from burn-

Figure 4: U.S. Energy supply and demand compared with source of renewable energy generation.

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ing fossil fuels. Renewable energy continues to cost less and will be a significant part of our future. My research project aims to analyze how energy demand projections compare with renewable energy production and consumption within each sector, and how this demand may change if costs of developing and using renewable energy continue to decrease. This will help us better understand if renewable energy has the capacity to meet projected demands for energy and if it will continue to be a significant part of energy economics. METHODOLOGY Sources All my datasets come from the United States Energy Information Administration (US EIA), within the U.S. Department of Energy and I mainly studied data from the Energy Consumption by Sector and Type of Energy Consumed by Each Sector. I also used the interactive model feature on their websites for datasets that were too large or had too many sub-files to download, and referenced various articles and data visualizations provided by these government websites or other reliable sources. Technology I mainly used the Python libraries Pandas, NumPy, Seaborn, and Matplotlib for my analyses and visualizations after downloading the data and logging it into a Jupyter Notebook. For datasets that were in an Excel file format, I opened the file in Excel and made visualizations using the built-in software. RESULTS Demand for Energy With the data from the Energy Consumption by Sector dataset, I created the Total Energy Consumption by Sector within figure 4 which visualizes how much the demand for energy from various sources has changed from 1949 to 2020. All measurements are in Trillion btus (British Thermal Unit of energy measurement). I would like to mention that we can attribute the dramatic decrease in energy consumption in 2020, especially in the industrial and transportation sector, to the COVID-19 pandemic because of the consequent shutdown of countless offices, businesses, and factories. Over the past 70 years, demand for energy has exponentially increased in each sector—and it will continue to do so before plateauing at a high consumption amount. Since we primarily rely on natural gas and petroleum for electricity, if we fail to generate enough energy to meet demand from renewable sources, we will be forced to continue burning these “dirty” fuels and harming our planet. The limited supply and increasing costs of procuring “dirty” fuels also puts our economy at risk. Even minor disturbances in this supply chain could result in disastrous energy crises and recessions, since the health and stability of many industries relies on steady energy costs and production. DISCUSSION Costs of Energy According to Our World in Data, “wind was 22% [and] solar [was] 223% more expensive than coal.” Figure 5 best visualizes the changes in costs of various sources of energy from 2009 to 2019. The most dramatic decrease is in solar photovoltaic power by 89% followed by onshore wind by 70%.9 Although the cost of renewable energy has de-

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Figure 5: Visualizations of changes in costs for various energy sources. creased, the overall costs of energy have increased between 20102020 by 1.61 cents/kilowatt hour for residential consumers, 0.4 cents/kilowatt-hour for commercial consumers, yet decreased by 0.1 cents/kilowatt-hour for industrial consumers, and 0.66 cents/ kilowatt-hour for transportation consumers, as shown in figure 6.10 The overall staggering decrease in fossil fuel consumption for electricity generation, as shown in figure 5 could also influence the changes in price for each of these sectors. Implementations of Renewable Energy A 100% shift to renewable energy can be an unrealistic goal because lots of existing infrastructure, especially in the commercial and industry sector, was built to consume fossil fuels. For example, New York’s ambitious goal to reach 100% Clean Energy by 2040 is facing challenges as their “grid must grow to supply 75% more power” and “has just nine years to more than double the share of electricity it uses that is generated from wind, sun, and water to 70 percent, from less than 30 percent today”.11 We can hope for more dramatic shifts in the residential sector because it is easier to build new greener homes and remodel older ones, especially when there is an incentive to do so—if it increases

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Figure 6: Consumption of fossil fuels, average price of electricity per kilowatt-hour, savings from increased energy efficiency. property value. Figure 6 further supports this notion because it shows the total decrease in energy used in newer homes; the primary energy source is not natural gas.12 Since homes can more easily adapt to renewable energy, it is possible that the slight increase in costs resulted from the initial costs of incorporating renewable energy infrastructure at the beginning of the decade. Again, costs for doing so have now dramatically decreased, making it easier for future homeowners to make the switch. Although it does not specify what sources create the “Electricity” category, we can eliminate one of the largest dirty fuel sources, natural gas, because Figure 7 Average cost of gas in the US per week since 1990 (adjusted for inflation) it is in a different category. Additionally, governments have subsidized the costs of installing renewable energy infrastructure in the residential sector to encourage movements towards a greener economy.13 We can also hope for larger shifts in the transportation sector; companies like Tesla, Ford, Toyota, and Chevrolet have brought electric vehicles to the forefront, and existing strict emission regulation policies have incentivized the sales of such vehicles for consumers and sellers through emission subsidy programs. Since the number of consumers is projected to increase, as shown in figure 6, any progress towards renewable energy implementation in any sector is better than nothing.10 Despite these efforts, the average price of gas per gallon per week since January 1st, 1990 has dramatically decreased, as visualized in figure 7. Maintaining low gas prices is essential for a healthy economy as it allows consumers to have more “disposable income [which would not] weigh on discretionary spending,” Such low prices, however, could reduce incentives for consumers to switch to electric or hybrid vehicles.14 Although this may motivate electric power companies and clean energy suppliers to double-down on research and development

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investments to further reduce the costs of electricity—countering the low gas prices to remain a player in an extremely market with exponential demand—the overall convenience for consumers’ continued reliance on gas may slow progress towards a greener future. Again, the energy industry responds as governments and car manufacturers offer even more EV savings and emphasize the fact that low maintenance and operations cost could save “consumers up to an extra $7,000” of which “$800-$1,000 are from fuel costs alone”.15 CONCLUSION Demand for energy is projected to increase in every sector, most notably in the total electric industry. As efficiency for renewable energy increases, most of the generated energy will be allocated to this sector. Again, costs for sustainable energy must continue to decrease and remain significantly cheaper than fossil fuels to provide the total electric industry sector an incentive to invest in this energy. We can conclude that the use of natural gas will largely remain constant while the use of renewable energy will increase, especially in the residential sector. The use of coal is projected to decrease; since we are still burning natural gas for most of our energy, we will still be damaging our environment. As such, renewable energy has tremendous potential and will decrease our use of certain fossil fuels, but it may fail to diminish the burning of natural gas by a notable amount. To ensure that we can develop renewable energy technology that can meet the ever-increasing demand and prevent dependency on natural gas, we must continue to fund research in the materials sciences, energy sector, and energy economics.

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Figure 7: Average cost of gas in the US per week since 1990 (adjusted for inflation). ACKNOWLEDGEMENTS Thank you to those that helped organize the Fall 2021 Berkeley EECS and Research Symposium (BEARS), where I presented my research. I would also like to acknowledge the U.S. Department of Energy and U.S. Energy Information Administration for compiling data on renewable energy consumption and demand in the United States and making it extremely accessible. Finally, big thank you to those that are advocating for renewable energy, sustainability, and energy independence across the globe and conducting more research into energy efficiency and renewable energy economics. 1.

REFERENCES U.S. Energy Information Administration. (2022, January). Renewable energy explained. Retrieved from U.S. Energy Information Administration - Independent Statistics & Analysis: https://www.eia.gov/energyexplained/renewable-sources/ Our World in Data. (2019, December). Primary Energy Consumption. Retrieved from Our World in Data - Energy Data Explorer : https://ourworldindata.org/explorers/energy?facet=none&country=USA~GBR~CHN~O WID_WRL~IND~BRA~ZAF&Total+or+Breakdown=Total&Energy+or+Electricity=Pri mary+energy&Metric=Annual+consumption. Our World in Data. (2019, December). Primary Energy Consumption. Retrieved from Our World in Data - Energy Data Explorer : https://ourworldindata.org/explorers/energy?facet=none&country=USA~GBR~CHN~O WID_WRL~IND~BRA~ZAF&Total+or+Breakdown=Total&Energy+or+Electricity=Pri mary+energy&Metric=Annual+consumption. The Economist Group. (2021). The use of renewable energy is accelerating. Retrieved from The Economist: https:// www.economist.com/graphic-detail/2021/05/11/the-use-

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of-renewable energy-is-accelerating Center for Climate and Energy Solutions. (2022). Renewable Energy. Retrieved from Center for Climate and Energy Solutions - Technology Solutions: https://www.c2es. org/content/renewableenergy/#:~:text=Renewables%20 made%20up%2019.8%20percent,come%20from%20wi nd%20and%20solar 6. U.S. Energy Information Administration. (2019, March 19). U.S. renewable electricity generation has doubled since 2008. Retrieved from U.S. Energy Information Administration - Independent Statistics & Analysis: https://www. eia.gov/todayinenergy/detail.php?id=38752 7. U.S. Energy Information Administration. (2020, December). U.S. renewable energy consumption by source and sector. Retrieved from Independent Statistics & Analysis: https://www.eia.gov/totalenergy/data/monthly/pdf/flow/ renewable_energy_2020.pdf British Petroleum. (2021). Statistical Review of World Energy. Retrieved from BP Energy Economics: https://www.bp.com/en/global/corporate/energy-economics/statistical review-of-world-energy.html 8. Our World in Data. (2020, December 1). Why did renewables become so cheap so fast? Retrieved from Our World in Data: https://ourworldindata.org/cheap-renewables-growth U.S. Energy Information Administration. (2020). Summary Statistics for the United States 20102020. Retrieved from U.S. Energy Information Administration - Data and Figures: https://www.eia.gov/electricity/ annual/html/epa_01_02.html 9. Anne Barnard, G. A. (2021, November 21). New York: Can New York Really Get to 100% Clean Energy by 2040. Retrieved from The New York Times: https://www.nytimes. com/2021/11/29/nyregion/hochul-electrical-grid-climate change.html 10. U.S. Energy Information Administration. (2018, May). 5.

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11.

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14. 15.

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Residential Energy Consumption Survey (RECS). Retrieved from U.S. Energy Information Administration Independent Statistics & Analysis: https://www.eia.gov/ consumption/residential/data/2015/hc/php/hc1.3.php California Distributed Generation Statistics. (2022). California Solar Initiative. Retrieved from California Distributed Generation Statistics - Statistics and Charts: https:// www.californiadgstats.ca.gov/charts/ Emma Goldberg, C. M. (2021, November 20). Family Unvisited, Travel a No-Go: The Hard Costs of High Gas Prices. Retrieved from The New York Times: https://www. nytimes.com/2021/11/20/business/high-gas-prices.html Preston, B. (2020, October 8). EVs Offer Big Savings Over Traditional Gas-Powered Cars. Retrieved from Consumer Reports: https://www.consumerreports.org/hybrids-evs/ evs offer-big-savings-over-traditional-gas-powered-cars/ International Energy Agency (IEA) (2020), Germany 2020, IEA, Paris https://www.iea.org/reports/germany-2020 International Trade Administration (ITA) (2020, October 10) Italy - Country Commercial Guide. Retrieved from https://www.trade.gov/country-commercial-guides/italy-renewable-energy#:~:text=Italy%20ranks%20third%20 in%20Europe,versus%20a%2017%25%20target). European Commission (2022) A European Green New Deal. Retrieved from https://ec.europa.eu/info/strategy/ priorities-2019-2024/european-green-deal_en

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Epidemiological Analysis of Acute Flaccid Paralysis (AFP) Surveillance in Conflict-Affected Syria Raneem Rayes; Rohini J. Haar, MD, MPH; Naser AlMhawish, MD ABSTRACT Syria has been polio-free since 1999; however, due to factors including mass displacement, insecurity, and the decreasing functionality of public health infrastructure, poliomyelitis (polio) reemerged in 2013-2014 and 2017. The aims of this study were to describe trends in the incidence and surveillance of the clinical diagnosis of polio, acute flaccid paralysis (AFP), in northern Syria over a three-year period during the ongoing conflict. We conducted a retrospective analysis of reported AFP incidence and surveillance among AFP cases in people under the age of 15 years from January 2018 to December 2020. We utilized data collected from the Early Warning Alert and Response Network (EWARN) operated by the Assistance Coordination Unit (ACU). A total of 1124 cases of AFP were reported in northern Syria during the study period. More than half of the children were under five years of age (n= 837, 68.9%) and 57.7% (n= 701) were male. All cases were classified as non-polio cases based on serology, with one case classified as polio compatible. Surveillance indicators were constantly above the minimum targets on a national level, with a significant increase in the adequacy of stool specimens and rank of reporter over the period studied. AFP surveillance data are the final measure of a country’s progress towards polio eradication. While the ACU has strengthened the sensitivity and quality of the AFP surveillance system in northern Syria over time, additional efforts are needed to strengthen subnational sensitivity. Major, Year, Departmental: School of Public Health, University of California, Berkeley; School of Public Health, University of California, Berkeley, Division of Epidemiology; Assistance Coordination Unit, Turkey

INTRODUCTION Since the Syrian civil war began in 2011, the country’s health care system has suffered due to destruction of health facilities, an exodus of health professionals, shortages of medical supplies, disruption of preventative services, and mass displacement.1-5 In the first year of conflict, disease surveillance broke down. It was not until 18 months after the beginning of the crisis that this deficit began to be addressed.6 The conflict has led to a drop in vaccination rates and the re-emergence of poliomyelitis (polio) in mid-2013.7 The spread of infectious diseases is attributable, among many other factors, to uncoordinated and delayed response efforts caused by mass displacement. Nationwide acute flaccid paralysis (AFP) surveillance is the gold standard for detecting cases of polio, and only one in two hundred non-immune infected individuals develop the condition.14,17,18 Therefore, the great majority of those infected with poliovirus (90- 95%) are asymptomatic and do not exhibit paralysis.1,11 This underscores the need for early warning and intervention, effective management of infectious disease, and timely information systems.12 In this study, we use a disease surveillance dataset focused on case reporting for AFP, the clinical case description for suspected polio, and describe the characteristics of cases in conflict affected northern Syria based on epidemiological surveillance indicators. Although many studies have been conducted on the causes of AFP and its surveillance in different countries, few aim to describe the incidence, distribution, and surveillance performance of AFP in Syria.1,10,13–15 We hope that this data will contribute to a broader understanding of outbreak vulnerabilities in regions affected by conflict and highlight the need for robust early warning surveillance systems in complex emergencies. METHODS Study Design and Period We conducted a retrospective time-series analysis of AFP surveillance data in conflict affected northern Syria between January

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1st, 2018, and December 31st, 2020. This data was collected by the Early Warning Alert and Response Network (EWARN) and operated by the Assistance Coordination Unit (ACU), a national Syrian non-governmental and non-profit organization. We describe EWARN data and AFP surveillance indicators and then review the statistical analysis. EWARN Surveillance The re-emergence of polio in 2013 takes place in the context of an increased incidence in AFP documented by both the EWARN operated by the ACU; and the Early Warning and Response System (EWARS) operated by the World Health Organization (WHO). These two surveillance systems operate independently within Syria but are seen as complementary, providing a full profile of epidemic-prone disease burden.19,21,22 The immediate reporting and weekly reporting components of these systems emphasize effectiveness and proactiveness–an essential method for the early detection of polio where clinical illness (AFP) is documented only in a minority of infected individuals.6 Study Setting For this study, the complex landscape was split into two categories of authority within Syria: areas under control of the Syrian government (government-controlled areas), and those not in control of the Syrian government (non-state-controlled areas).18 This retrospective analysis focused on data routinely collected by the EWARN in non-state-controlled areas of northern Syria. The EWARS is a similar system but focuses on government-controlled areas and is jointly administered by the WHO and the Syrian Ministry of Health.6 Areas that are phased out of the coverage region after the Syrian government regained control are reported as having missing case reports. We do not report on data from EWARS. As of 2020, Syria’s population is estimated at about 17 million people according to UN data.19 The country’s administration is divided by 14 regional governorates (muhafazat), which are further

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divided into 61 districts (manatiq) which in turn are subdivided into subdistricts (nawahi).20 The EWARN collects data on 13 infectious diseases from more than 480 health facilities and covers a population of about 12 million.21 AFP Surveillance Indicators The ACU defines an AFP case as a child less than 15 years of age presenting with recent or sudden onset of floppy paralysis or muscle weakness due to any cause, or any person of any age with paralytic illness if polio is suspected by a clinician. The alert threshold is one suspected case, while the outbreak threshold is one confirmed case (including laboratory confirmation) of polio.6,22,23 The WHO has established surveillance indicators that ensure the quality and sensitivity of the AFP surveillance system is achieved and maintained. For this analysis, we focused on the following indicators: non-polio acute flaccid paralysis rate (NP-AFP rate), early detection, rank of reporter, and stool adequacy. Non-Polio Acute Flaccid Paralysis (NP-AFP) rate The sensitivity of the AFP surveillance system is reflected by the annualized non-polio AFP rate.9,15 The NP-AFP rate is the incidence of AFP cases due to conditions other than polio. It measures the number of non-polio AFP cases in a population aged under 15 years that should be detected over one year. An NP-AFP of at least 2 per

100,000 aged under 15 is considered sensitive in endemic regions and is the minimum target.9 Early Detection Timely reporting is crucial. Within seven days of the onset of paralysis, the case should be reported to the health system, which classifies it as early detection. The minimum target of early detection is 80% of all AFP cases.9 Rank of Reporter Another characteristic of detection is the rank of the reporter–the order of the individual who notified the suspected case to EWARN. A ranking of “1” is considered best in terms of early detection, implying that the first person who observed the AFP case notified EWARN; a ranking of “2” signifies that the second person who observed the case reported to EWARN (signifying that one person prior detected the case and did not report), and so on. The rank was coded as “1, 2, and 3+,” with the “3+” category being from “3” to “5.” Stool Adequacy Stool adequacy is the collection of adequate specimens collected from AFP patients and helps determine the quality of the sample submission process to the lab and resulting confidence in lab results. Adequate stools are defined as two stool specimens collected from an AFP pattern person 24 to 48 hours apart, within 14 days of onset of symptoms, and in good condition. A stool specimen is said to have arrived in such condition if ice packs in stool carrier still have frozen water, specimen is of adequate quantity (8-10 g), presents no leakage or desiccation, and with complete documentation.14 At least 80% of all AFP cases should have adequate stool specimens.9 Furthermore, an AFP case is classified as polio compatible if the stool specimens were not adequate to rule out poliovirus and the case had developed residual paralysis after 60 days follow up, died within 60 days or was last to follow. These cases were presented to an independent Expert Review Committee (ERC) to provide the classification of whether the specimens were compatible or discarded.14 Data Analysis

Table 1: Reported AFP cases and population under 15 by district and year, northern Syria, January 2018 to December 2020. *POPL15 = Population under 15 years of age within the area by year.

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Figure 2: Surveillance indicators, northern Syria, January 2018 to December 2020.

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Figure 3: Rank of reporter, northern Syria, January 2018 to December 2020. Descriptive analysis was performed to describe the epidemiology of reported AFP cases in conflict-affected Syria, and statistics based on the WHO recommended surveillance indicators for AFP surveillance were generated. Mapping was performed to visualize the distribution of AFP cases by location and year. We conducted a statistical analysis of surveillance data to report on the study population characteristics, which included variables for age, sex, and geographic district. The study aimed to analyze the rank of reporter and adequacy over time to examine whether there were significant improvements of these variables over the threeyear study period. Linear regression analysis was used with the year being considered the explanatory variable, with a reference year of 2018 and p-value of <0.05 chosen as the threshold for significance. . RESULTS Descriptive Analysis of Reported AFP Epidemiology From January 2018 to December 2020, EWARN identified a total of 1214 cases of AFP from children under 15 years of age reported in conflict-affected northern Syria. Table 1 shows the governorate of Idleb (n= 397, 32.7% of cases) had the highest numbers of total AFP cases, followed by Aleppo (n= 336, 27.7% of cases) and Deirez-Zor (n= 202, 16.6% of cases) (Figure 1). None of the reported cases classified as polio; however, the ERC classified one AFP case as polio compatible in 2018 (located in the district of Menbij, Aleppo). The study population included a total of 1214 individuals between the ages of 0-15. Within the coverage region, the average age of children was 4.24 years (±3.23). Children under five years of age accounted for 68.9% (n= 837) of the total population; the average age of children in this age group (≤ 5 years) was 2.39 years (±1.18). There was no significant difference between males and females un-

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der the age of 15 years (p-value = 0.845): males accounted for 701 of the 1214 total cases (57.7%) and females accounted for 513 (42.3%). The mean NP-AFP rate during the study period was 12 per 100,000 children under 15 years: 10.8/ 100,000 in 2018, 14.9/ 100,000 in 2019 and 10.2/ 100,000 in 2020. These rates consistently exceeded the recommended benchmark of 2/ 100,000. The proportion of AFP cases with two stool specimens collected within 14 days after paralysis onset, 24-48 hours apart, and in “good condition” (adequacy) remained stable from 2018 to 2019 (91%) and increased to 96% in 2020. Lastly, the proportion of AFP cases notified to the health system within 7 days after paralysis onset (early detection), was 89.7% in 2018, 88.1% in 2019 and 91.5% in 2020. Overall, reporters ranked “1” for 85% of total cases (n= 1037),

Figure 4: Simple Linear Regression comparing rank of reporter between each year.

Figure 5: Simple Linear Regression comparing stool adequacy (%) between each year.

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“2” for 11% of cases (n= 130) and “3+” for 4% of cases (n= 47). Doctors reported the most cases (n= 963, 79.3%), followed by nurses (n= 90, 7.4%) and pharmacists (n= 41, 3.4%). In 2018, 82% (n= 327) of reporters ranked as “1”; in 2019, 84% (n= 359); and in 2020, 91% (n= 351) (Figure 3). Linear regression was done to compare each year to 2018, and the analysis had 1211 degrees of freedom and yielded a p-value of 0.003. There was a significant difference between 2018 and 2020 in terms of rank of reporter–the order of the individual who notified the suspected case to EWARN (p-value = 0.004). The year 2019 was not statistically different from 2018. The proportion of AFP cases with two stool specimens collected within 14 days after paralysis onset, 24-48 hours apart, and in “good condition” between the years was compared using simple linear regression analysis. The analysis had 1211 degrees of freedom and yielded a p-value of 0.011. In 2018 and 2019, adequacy was 91% and increased to 96% in 2020. The year 2019 was not statistically different from 2018. However, 2020 was statistically different from 2018 (p value = 0.019). DISCUSSION The present study focused on descriptive and statistical analyses of reported AFP based on surveillance indicators in northern Syria over a three-year study period using data obtained from EWARN. The Syrian humanitarian crisis has left much of the population vulnerable to disease outbreaks. Throughout the first three years of conflict (2011-2013), a decrease in vaccination coverage occurred, along with disruptions in previously well-functioning disease surveillance systems and poor sanitation.24 Vaccination coverage dropped across the country from 90% before the crisis to 50% in December 2013.24 Throughout the years 2014 to 2017, health services suffered, civilian violence exponentially increased, and disease surveillance was low. Among children born during the war, the estimated proportion of unvaccinated children with non-polio AFP (NP-AFP) increased from 3% in 2015 to 6% by the end of 2016.25 Wild poliovirus, which had been eliminated in Syria since 1999, had a resurgence in 2013-2014, occurring against the backdrop of declining routine vaccination coverage and in one of the most contested areas of the country.25,26 Surveillance systems were established and multiple rounds of supplementary immunization activities (SIAs) were implemented in response to the outbreak in 2013-2014. However, the frequency and quality of SIAs lessened after the outbreak was declared over in early 2014.25 The disease resurged in 2017 partly due to a lack of treatment and control, with the first cases reported in the governorate of Deir-ez-Zor. An 18-month long intensive vaccination campaign successfully halted this outbreak, despite large-scale population movement and accessibility issues.26 Following the outbreak, a strengthened response during 2018 and onwards has contributed to an increase in reporting and surveillance performance, even amidst the 2020 COVID-19 pandemic. Nonetheless, the viral strain responsible for the outbreak in 2017 was circulating for approximately a year before the isolation of cVDPV. This delay in reporting, along with gaps in AFP surveillance performance and the ongoing conflict, contributed to the inability to earlier detect of the outbreak.25 Non-governmental organizations continually strengthen data collection methods and efforts to en-

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hance surveillance for polioviruses and immunization programs.26,27 Out of the 1214 total AFP cases, no cases of polio were reported from 2018 through 2020, with one case classified as polio compatible by the Expert Review Committee. The high numbers of reported AFP cases in the governorates of Idleb (n= 397, 32.7% of total cases) and Aleppo (n= 336, 27.7% of total cases) can be explained by the high population of children under 15 years of age in these areas. As for Deir-ez-Zor (n= 202, 16.6% of total cases), experiencing two outbreaks within five years may have resulted in high sensitivity among healthcare providers and led to high reporting. The EWARN coverage region phased out of the governorates of Dar’a, Homs, Rural Damascus, and Quneitra once the Syrian government regained control, therefore there were little to none reported cases due to the lack of reporting. Our findings showed that 68.9% of AFP cases occurred in children under five old, confirming that young age was a risk factor, as shown in previous literature.10,14,15,28 Although the differences in reported AFP by sex was not statistically significant, our findings indicate that the majority (57.7%) of AFP cases involved males. This is also consistent with previous literature and may be explained by sex differences in the susceptibility to infectious agents.10,14,15,28 The minimum NP-AFP, early detection, and stool adequacy targets set by the WHO were surpassed at the national level in all three years analyzed. When looking at the mean NP-AFP by governorates, all met the recommended NP-AFP goal of 2/ 100,000 and all governorates exceeded the early detection target of 80%. The success of an AFP surveillance system not only depends on the detection of AFP cases, but also on the investigation and reporting of the cases. The mean proportion of AFP cases with adequate specimens collected within 14 days of onset of paralysis exceeded the target of 80% throughout the study period. Results showed disparities in adequacy performance among governorates, as the governorate of Homs (50%) did not meet the minimum target. This may have been accounted for by the changing coverage region; as the Syrian government regained control of the area, the EWARN lost half of collected specimens. While surveillance indicators increased over the three-year study period, we found significant increases in the rank of reporter and adequacy in 2020 compared to the reference year of 2018. The rank of reporter signifies the order of the individual who notified the suspected case to EWARN. Our findings indicate a gradual increase of the rank of reporter being “1” throughout the study period, signifying a statistical increase in reporting methods. Additionally, adequacy significantly increased in 2020, signifying increased quality of AFP surveillance. Nonetheless, challenges still exist in terms of AFP surveillance activities, especially throughout the COVID-19 pandemic. These challenges include: the partial closures of a considerable number of private physicians’ practices; the inability to conduct supplementary AFP surveillance activities such as Area Coverage Survey and outof-household contact sampling; delay in lab results; lack of PPEs with the field staff; and inability to conduct in-person Expert Review Committee sessions as a precaution measure against COVID-19. Mitigation efforts should be made to address such challenges. These efforts include communicating with surveillance focal points on a regular basis to ensure immediate notifications of AFP cases, maintaining communication channels with the reference laboratory to

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ensure timely feedback, and looking into alternative laboratories to run needed tests.33,34 Active surveillance for AFP depends, above all, on early detection and timely action. Staying ahead of the virus requires a well-managed system for detecting AFP cases that entails immediate investigation. EWARN has helped detect epidemic-prone diseases in Syria, where the risk of disease outbreaks is greatly increased by cross-border movements of highly mobile populations.24 This paper is unique in analyzing the rank of reporter as a surveillance indicator over time, along with assessing the WHO recommended surveillance indicators in conflict affected Syria. The most recent polio outbreak occurred in 2017. Although Syria maintained a three year period (2018-2020) without any confirmed polio cases throughout the country, the resurgence of poliovirus remains a potential threat; therefore, it is essential to maintain high quality AFP surveillance, supplemented by mitigation measures.29 The recent outbreaks in Syria emphasize the significance of increasing financial and logistical support for immunization efforts, especially in complex emergencies where epidemic-prone diseases can reemerge. Furthermore, the gradual disintegration of the Syrian public health system has led to a greater reliance on NGOs.24 Although Syrian NGOs provide 75% of the support in Syria currently, these NGOs receive less than 1% of the international funding.30 It is critical to resolve the Syrian conflict, continue the surveillance of infectious diseases, and further support NGOs.

3. 4. 5.

7. 8.

Limitations Limitations include the changing coverage region based on the political situation–territories starting off as non-state-controlled areas that later fall out of the coverage region after becoming government-controlled areas were reported as having missing case reports and therefore excluded from this analysis. Additionally, analysis was done by aggregating for the entire coverage region by year. National performance can obscure the subnational performance and prevent early detection of AFP cases, which can occur at the sub-county level.15 Therefore, surveillance indicators should be analyzed at the governorate and district level to uncover underperformance that might be concealed by country level analyses.

ACKNOWLEDGEMENTS I would like to thank the healthcare workers working in Syria along with the staff of the ACU for their support to the people of Syria. I would also like to thank Dr. Rohini Haar and Dr. Naser AlMhawish for their continuous guidance and making this research possible. Lastly, I would like to thank Dr. Kristine Madsen and Dr. Lisa Barcellos for their advice and support as well as the D-Lab at UC Berkeley for their technical guidance.

12.

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RESEARCH

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Flux

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Epidemiological Analysis of Acute Flaccid Paralysis (AFP) Surveillance in Conflict-Affected Syria

Renewable Energy Economics: Understanding the Costs and Capacity of Green Energy in the United States

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