PENNSCIENCE JOURNAL OF UNDERGRADUATE RESEARCH
VOLUME 20 ISSUE 2 SPRING 2022
GENETIC EPILEPSY PAGE 24
FUELING A CLEANER FUTURE
mRNA VACCINES PAGE 20
PAGE 22
fuel
SCIENCE PIONEERS AND SCIENTIFIC ADVANCEMENTS
Spring 2022 | PENNSCIENCE JOURNAL
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PENNSCIENCE
PennScience is a peer-reviewed journal of undergraduate research and related content published by the Science and Technology Wing at The University of Pennsylvania and advised by a board of faculty members. PennScience presents relevant science features, interviews, and research articles from many disciplines, including the biological sciences, chemistry, physics, mathematics, geological science, and computer science. PennScience is funded by the Student Activities Council. For additional information about the journal including submission guidelines, visit www.pennscience.org or email pennscience@gmail.com.
EDITORS IN CHIEF MAGNOLIA WANG
BRIAN SONG
FACULTY ADVISORS DR. M. KRIMO BOKRETA
DR. JORGE SANTIAGO-AVILES
WRITING MANAGERS
EDITING MANAGERS
BUSINESS MANAGERS
DESIGN MANAGERS
DAHYEON CHOI SAGAR GUPTA
ANUSHKA DASGUPTA MEHEK DEDHIA
GLEN KAHAN
AMARA OKAFOR BIANCA VAMA
WRITING COMMITTEE
EDITING COMMITTEE
BUSINESS COMMITTEE
DESIGN COMMITTEE
ANYA JAYANTHI AVINASH SINGH BEATRICE HAN EASHWAR KANTEMNENI AVINASH SINGH BENJAMIN BEYER ELENA CRUZ-ADAMES CAITLYN PRABOWO COLLIN WANG LYNNE KIM BRIAN LEE NEHA SHETTY DANISH MAHMOOD PHUONG NGO CHINASA EMERIBE ESHA MISHRA CYNTHIA SCHNEIDER TECHNOLOGY SARASWATI SRIDHAR ISAIAH LEE EMILY NG COMMITTEE JESSICA LVOV GUYIN (CICE) CHEN KONSTANTINOS TSINGAS GRACE LEE ISABEL ENGEL SPENCER GIBBS JONATHAN TRAN SUKHMANI KAUR JUNLE (RICHARD) CHEN ZOE LU KEVIN GUO MICHELLE PAOLICELLI REBECCA NADLER SARAH PHAM
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PENNSCIENCE JOURNAL | Spring 2022
TABLE OF CONTENTS 06 10 12 15 18 20 22 25 27 29 31 33 35 38 42 How Environmental Factors Influence Antibiotic Resistance
Guyin (Cice) CHEN ....................................................................................................................................................
Engineering the Future with CRISPR/Cas9
Brian Lee...................................................................................................................................................................
Defense Against Resistant Diseases: Microbial Therapeutics and CanceR
Junle (Richard) Chen..............................................................................................................................................
The Power of Doodling: Impossible Symmetries and Diffraction Patterns; Roger Penrose
Michelle Paolicelli................................................................................................................................................
Expansion of Quantum Computing
Benjamin Beyer........................................................................................................................................................
mRNA vaccines: The Solution To the Return to Normal
Chinasa Emeribe.......................................................................................................................................................
Fueling a Cleaner Future
Jonathan Tran..........................................................................................................................................................
Genetic Epilepsy: Discovering Novel Mutations in the SCN3A Gene
sarah pham...............................................................................................................................................................
Single Cell Sequencing for Cancer Research
Rebecca Nadler.......................................................................................................................................................
Restoring the Mind: Stem Cell Therapy and Neurodegenerative Diseases
Beatrice Han..............................................................................................................................................................
Acres of Skin: The Cost of Scientific Innovation
Cynthia Schneider..................................................................................................................................................
Genetic Testing: There’s More at Stake Than Just Our Ancestry
Isabel Engel.............................................................................................................................................................
MODERN PIONEERING IN CORAL REEF RESTORATION: MICROFRAGMENTATION
Emily Ng....................................................................................................................................................................
The Various Clinical Applications of Nanomedicine
kevin guo....................................................................................................................................................................
RESEARCH FEATURE: The Memory Trace of Addiction
Raena E. Greenbaum, Steven J. Simmons, William R. Haury, Amelia J. Eisch .........................................
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To Our Readers, We are beyond thrilled to present our Spring 2022 and twenty-second issue of the PennScience Journal of Undergraduate Research. We are extremely grateful for our members at PennScience who worked diligently throughout the semester and have gone above and beyond to make this issue possible. We extend our thanks to the students who submitted their research findings for publication, and to the greater Penn community for actively engaging in our events and contributing to the scientific discourse on campus. This semester, in honor of Rare Disease Day, we are proud to have welcomed field experts Drs. Cynthia Powell of UNC School of Medicine and Susan Furth of CHOP at our annual PennScience Speaker Series to highlight the importance of raising awareness for the rare disease community. For many years, PennScience has explored an array of topics that span the areas of science, innovation, and technology. Here at Penn, we are proud to be the birthplace of scientific breakthroughs, and are pleased to feature Science Pioneers and Scientific Advancements as our publication theme this semester. We highly commend our Committees who have taken a multifaceted approach to manifest our theme in many different directions. In this issue, Cice Chen examines the influence of environmental factors on antibiotic resistance, and Brian Lee discusses engineering a future using CRISPR/Cas9 technology. Richard Chen explores the potential of microbial therapeutics in providing defense against resistant diseases and cancer, while Michelle Paolicelli highlights the oft-overlooked significance of doodling, which contributed to breakthrough discoveries in crystallography and diffraction patterns. Benjamin Beyer brings to light the current and future directions in quantum computing, and Chinasa Emeribe explains the power of mRNA vaccines in shifting our society to the post-pandemic normal. Jonathan Tran examines up-and-coming hydrogen fuel cells that pave the way for a cleaner future, and Sarah Pham analyzes novel mutations of SCN3A as a genetic basis for the onset of epilepsy. Rebecca Nadler taps into the power of single-cell RNA sequencing as a novel strategy to expose biomarkers and aid in the development of cancer treatment, while Beatrice Han conducts a thorough investigation into stem cell therapy in ameliorating neurodegenerative disease. Cynthia Schneider underscores the costs of scientific innovation implicated in the manipulation and abuse of minority groups, and Isabel Engel sheds light on the commercialization of genetic testing and future concerns for consumer privacy. Finally, Emily Ng delves deeper into micro fragmentation as an instrumental tool in coral reef restoration, while Kevin Guo elucidates the numerous clinical applications of nanomedicine. Along the vein of scientific discovery and advancements, we are pleased to showcase the original research of Penn alumna Raena Greenbaum on the impact of drug-linked memories on emotionality and drug-seeking behavior. PennScience is dedicated to featuring the breadth and depth of undergraduate contributions as well as promoting greater scientific literacy in the Penn community. We are incredibly thankful to our Writing, Editing, Design, Business, and Technology Committees for their continued diligence, as well as the Science and Technology Wing of King’s Court English College House and the Student Activities Council for their funding. Finally, we express our sincere appreciation to faculty advisor Dr. Krimo Bokreta for his constant mentorship and ardor for PennScience. Last but not least, we thank you! PennScience cherishes your support, and we hope you enjoy reading our issue as much as we enjoyed curating it. Sincerely, Magnolia Wang (C’23) and Brian Song (C’22) Co-Editors-in-Chief
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PENNSCIENCE JOURNAL | Spring 2022
Looking for a chance to
publish your research? Pennscience is accepting submissions for our Fall 2022 issue! Research in any scientific field will be Submit your independent considered, including but not limited to: study projects, senior design products, reviews Biochemistry and other original research projects to share Biological Sciences Biotechnology with fellow Chemistry undergraduates at Penn Computer Science and beyond. Email Engineering submissions and any Geology questions at Mathematics pennscience@gmail.com Physics Physiology
Spring 2022 | PENNSCIENCE JOURNAL
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FEATURES
How Environmental Factors Influence Antibiotic Resistance Written By Guyin (Cice) Chen Designed By Bianca Vama
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PENNSCIENCE JOURNAL | Spring 2022
FEATURES
D
ue to the abuse of antibiotics during this century, increased antibiotic resistance from bacteria has become a serious global problem; each year, more than 700,000 people are killed by antibioticresistant bacteria, and according to the World Health Organization (WHO), the figure is likely to reach ten million in 2050.1 The environment, being the largest reservoir for bacteria and microbes, plays a significant role in manipulating antibiotic resistant bacteria; specifically, soil, animals, and wastewater influence the spread of antibiotic resistance. Potential solutions for policymakers include creating global surveillance systems, regulating the use of antibiotics, and controlling the release of wastes and sewage into the environment.
The Emergence of Antibiotics and Antibiotic Resistance
through the soil, interaction with animals, and intake of contaminated food and water.4
Soil: Largest Storage of Resistant Genes Soil acts as one of the most important reservoirs for resistant genes. Through manure, sewage sludge, and fertilizers, the soil receives a large amount of excreted antibiotics. This includes improper disposal or handling of drug wastes, contamination of aquaculture and plants, and wastewater streams of some factories. Due to the close proximity of bacteria in the soil, when one strain develops resistance towards a certain antibiotic, the resistant genes can be easily transferred in the colony utilizing genetic exchange mechanisms such as HGT. Bacteria from the soil can be transferred to humans by opportunistic pathogens, which receive resistant genes from human-associated pathogens and then transfer the genes back to humans.4
The first antibiotic, penicillin, was discovered by Alexander Fleming in 1928. Penicillin and other antibiotics developed since then directly kill or inhibit the growth of bacteria by interfering with the bacterial cell walls and protein synthesis. They also supplement the immune system when there is an excessive amount of pathogens detected. Although antibiotics kill most bacteria, strains that are resistant to the antibiotics survive and reproduce via natural selection. Over time, the amount of antibiotic resistant strains increases, making it more difficult to eliminate them. These resistant strains can also increase in number through Horizontal Gene Transfer (HGT) in which bacteria transfer their resistant genes to neighboring bacteria, causing resistance to spread quickly and effectively.2
In a study in China, a total of 36 samples were collected in 2010 from three Chinese provinces in typical large-scale swine farms. Based on quantitative PCR arrays, there were more than 149 unique resistance genes among the samples, with 63 genes being enriched from 192 to 28,000 fold compared to antibiotic free manure and soil.7 Based on the study, there was an increased concentration of antibiotics in both manure and soil in the farm, and the number of resistant genes tripled from the control manure and soil due to resistant gene transfer. It was estimated that resistant genes could be found in one out of two bacteria in the field.1
However, bacterial infection has progressively worsened due to increases in antibiotic resistance. From 1998 to the present, the widespread dependency on antibiotics in various fields, including human health, animal husbandry, and veterinary medicine, have led to the spread of resistant bacteria and viruses. Antibiotic resistance has become an international issue, with WHO declaring it as one of the top ten global public health threats.3
Animals can serve as intermediate hosts for resistance genes which then transfer those genes to humans either directly or indirectly.4 Since many farms and companies regularly feed antibiotics to animals to boost their weight and health, a large amount of antibiotics accumulate inside animals’ bodies.
Environmental Factors Influencing Antibiotic Resistance The environment facilitates the spread of antibiotic resistance in several ways, including
Animals: Resistant Genes Transporters
Resistant bacteria then spread from animals to humans via direct consumption. For example, urinary tract infection caused by E. coli is usually transmitted from the food chain.5 Wild birds and animals can also harbor bacteria that carry resistant genes from the environment, including soil, which is Spring 2022 | PENNSCIENCE JOURNAL
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FEATURES
Figure 1: The exchange of antibiotic resistant genes between humans and environmental factors, including air, livestock, crops, antibiotics, feces. spread through their migration and flight patterns. When they encounter humans, the genes can be transferred through consumption or interaction.4 Nutrient-rich animal manure is also often used as fertilizer for crops. Since animal wastes do not undergo secondary treatments, such as disinfection, like human wastes do, resistant genes inside animals’ bodies can enter crop fields and farms through their wastes.5
Food and Water Contamination Contaminated food and water can also play a role in the development and spread of antibiotic resistance. Drinking water from surface water sources can contain antibiotic-resistant organisms.5 Exposure to water that is contaminated by fecal residues can lead to infections. Untreated hospital wastewater fosters the growth of multi-resistant E. coli under various situations. Inadequate infrastructure also
Figure 2: The antibiotic concentration of different wastes. 8
PENNSCIENCE JOURNAL | Spring 2022
FEATURES causes sanitary issues, including contaminants in wastewater.6
released into the environment.4 Also, pharmaceutical company employers could help limit the discharge of antibiotic production wastes into the environment through disinfection.6 Policymakers can plan policies Potential Solutions accordingly to minimize the threat of antibiotic Although there are many environmental resistance globally. mechanisms which increase the spread of antibiotic Advanced technologies also allow us to resistant bacteria, there is strong global concern for stopping and reducing the effects of antibiotic reduce the spread of antibiotic resistance. For example, researchers can increase the concentration resistance. of antibiotics through potentiation, which manipulates Policymakers can enact and enforce another part of the bacteria to make the organism more regulations to reduce antibiotic resistance. For sensitive to the antibiotics. Moreover, vaccines can example, global surveillance can be implemented to also act as preventative methods to counter antibiotic regulate the exchange of antibiotic resistance across resistance through preventing infections and decrease the globe. Currently, Global Antimicrobial Resistance the use of antibiotics. More effective vaccines can and Use Surveillance System, established by WHO in be developed through manipulating the bacteria’s 2015, works on incorporating data from surveillance outer membrane vesicles to make recombinant virusof antibiotic resistance in humans, food chains, like particles to eliminate infections. Glycoconjugate medicines, and environments. It sets standards for vaccines, which use protein carriers to improve collecting, analyzing and interpreting data between immune response to polysaccharide antigens, can also allow the development of vaccines of influenzae type B countries.6 and other targets.10 With the enhanced understanding Moreover, public health regulation for waste of antibiotic resistance and rapid development in and manure could be implemented and enforced technology, we could potentially combat this worldby veterinary and officials. Standards for mixed wide problem. farming and animal manure usage to crops can be developed as well.6 Important policies include use of exactly prescribed amounts of antibiotics and safe disposal of used antibiotics.8 With each mass-scale farm, limited use of antibiotics could be applied for treating poultry, and the waste from the farms can be treated with secondary disinfection before being
REFERENCES
Spring 2022 | PENNSCIENCE JOURNAL
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t FEATURES
Engineering the Future with CRISPR/Cas9 C
hinese scientist He Jiankui made international headlines when he claimed to have created the world’s first two genetically-modified human babies. He applied CRIPSR/Cas9 to change their DNA so that they would be immune to HIV, a disease which attacks the body’s own immune system. Naturally, his unsanctioned experiments have stirred much controversy: where do scientists draw the line with genetic editing? Are genetic modifications safe in the real world? Can they potentially save lives?
What is genome editing? What is CRISPR/Cas9? Genome editing involves tools that allow scientists to change DNA inside a cell. DNA is a large polymer made out of individual units called nucleotides. The cell has internal machinery which “reads through” the sequence of nucleotides in DNA to make proteins. DNA is the instruction set for the cell, dictating all of its functions. Genome editing 10 PENNSCIENCE JOURNAL | Spring 2022
Written By Brian Lee Designed By Caitlyn Prabowo
is used to understand the functions of genes or to engineer a new diagnostic or therapeutic. Editing technologies are capable of removing or adding genes at very specific locations in the genome. CRISPR/Cas9, short for Clustered Regularly Interspaced Short Palindromic Repeats and CRISPRassociated protein 9, is the most recent advancement in genome editing.1 It won the 2020 Nobel Prize in Chemistry for being a more accurate and more efficient tool than a majority of other gene-editing methods available to scientists.2
Where did CRISPR/Cas9 come from? How does it work? CRISPR/Cas9 actually comes from nature (with some modifications in the lab). Bacteria use CRISPR complexes to defend themselves very effectively from viral infections.1, 3
FEATURES Since viruses are not ‘alive,’ they must rely on a host (i.e. cells in our bodies) to replicate the proteins they need for replication. Figure 1 summarizes this process. Just like our cells, viruses rely on DNA to encode the instructions to build their components; therefore, if a bacterial cell had a method to destroy a virus’s DNA, it would stop the viral infection in its tracks. This is where the genius of CRISPR/Cas9 comes in.
engineer a CRISPR/Cas9 complex that can be used to specifically turn off that portion of a transcript within a model organism (i.e. bacteria), allowing for very precise alterations in model organism genomes.1
How
can
CRISPR/Cas9 medicine?
transform
Given its ability to specifically alter unique genes, CRISPR/Cas9 has the potential to cure After a viral infection, the bacteria will a range of conditions with a significant genetic/ capture segments of the virus’s DNA to create a heritable component, such as genetic/congenital “spacer” CRISPR sequence. When the bacterium is neurodegenerative diseases, blood disorders, cancers, invaded again, this spacer sequence is the key to the or ocular disorders. bacterium’s self-defense: it can be used to search out viral DNA in the cell. If a match is found, the Cas9 CRISPR/Cas9 technology is already being enzyme would bind to the identified viral DNA and used to treat genetic diseases. For example, Sickle cell then (physically) cut it, effectively disabling the virus disease is caused by a mutation in the HBB gene that by halting replication.3 This process is depicted below leads to malformations in hemoglobin, the essential in Figure 2. oxygen-carrying protein in red blood cells. These CRISPR/Cas9 is revolutionary because of this ability to shut off specific portions of the genome: potentially, if a scientist has a specific stretch of DNA in mind, they can use its sequence to find a unique spacer sequence. Then, they can proceed to
malformations may lead to red blood cells collapsing into a dysfunctional ‘sickle’ shape, giving the disease its name. Since sickle cell disease currently has no cure, scientists have investigated using CRISPR/ Cas9 to undo the genetic mutation in HBB. Using CRISPR/Cas9, scientists targeted enhancers key in
Figure 1: Replication of Viruses.1 Spring 2022 | PENNSCIENCE JOURNAL 11
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FEATURES sickle cell disease, allowing for life-saving increases in COVID-19 virus’s genetic material (denoted Cas12b), fetal hemoglobin, transfusion independence, and the these tests can accurately detect COVID-19 in under an hour without the use of complex equipment such elimination of vaso-occlusive episodes.4 as expensive RT-qPCR machines for regular nasal Scientists have also investigated CRISPR/Cas9 swab testing.6, 7 as a potential treatment for transthyretin amyloidosis, Several kits have additionally been granted a terminal cardiovascular disease characterized by the accumulation of misfolded transthyretin protein an Emergency Use Authorization from the FDA and in the nerves and heart. The TTR gene is responsible therefore may be in use today.8 They have sensitivity for the faulty folding of transthyretin that causes the similar to RT-qPCR, but can be done in under an disorder, so scientists have attempted to target this hour with minimal equipment, allowing for testing gene via CRISPR/Cas9 treatments. In a specific study, without a sophisticated scientific lab. CRISPR/Cas9 was directly injected into a patient’s The discovery of this bacterial DNA-finding blood as treatment for this disease, which successfully modified the TTR gene and led to an 87% decrease and gene-editing enzyme complex has undoubtedly after only one infusion. This proves the viability and reshaped both the lab and the clinic. From treating safety of CRISPR to lead to advancements in patient life-threatening genetic diseases to quickly detecting COVID-19 genetic material, scientists and clinicians care.5 are constantly using CRISPR/Cas9 technology to push Amazingly, CRISPR/Cas9 can also be used to the envelope of molecular biology/genetics laboratory diagnose viral diseases such as COVID-19. Multiple techniques. With its revolutionary precision, we can kits (such as the SHERLOCK, STOPCovid, and only wonder how this tool will be continually used to DETECTR kits) have been developed. Using a read and edit the genetic material that serves as the modified CRISPR complex that automatically cleaves language of life. a fluorescent (biotin) marker upon recognition of the
REFERENCES (1) Virus invades bacterial cell
(2) New spacer is derived from virus and integrated into CRISPR sequence
Targeting
(4) CRISPR RNA guides molecular machinery to target and destroy viral genome
Figure 2: Mechanism of CRISPR/Cas9 in bacteria. 2 12 PENNSCIENCE JOURNAL | Spring 2022
(3) CRISPR RNA is formed
Adaptation
Production of CRISPR RNA
FEATURES
Defense Against Resistive Diseases Microbial Therapeutics and Cancer Written By Junle (Richard) Chen Designed By Avi Singh
I
Introduction
n concurrent fields of modern medicine, breakthroughs across genetics, molecular biology, and biochemistry have led to the development of novel treatments and cures for a wide spectrum of diseases, significantly prolonging the average human life expectancy. However, despite these interdisciplinary advancements and improvements, many diseases related to cellular proliferation are still deemed “incurable” or have posed challenges to modern medicine. One such disease is cancer, being resistant to most chemotherapeutic methods and maintaining a high mortality rate. With conventional therapies failing to cure cancer due to its rapid mutation and evolution against treatments, microbe-based drugs and therapeutics emerge as potential solutions.
History of Antibiotics The medical community has long used microbe-based therapeutics, specifically antibiotics, for the welfare of humankind. Such application can be seen in the discovery and development of penicillins and arsphenamine (the first modern antimicrobial agent)1. However, the connection between medical application of bacteria and cancer is rare. The first
clinical, bacterial application in cancer appeared in the early 1900s, when William Coley’s bacteria anticancer treatment was nullified due to the lack of antibiotic knowledge at the time2. However, recently, with advances in cancer biology, interests in bacterial cancer treatments have resurged.
Basis of Microbe-based Therapeutics Before delving into how specific bacterial functions and physiology grant them advantages in treating cancer, it is important to understand the general novel treatment techniques that microbes bring in tackling diseases challenging to current clinical practices.
Multi-Faceted Mechanisms Previous research have demonstrated that combination treatments, namely the multi-faceted attacks implemented by drug cocktails comprising both traditional drugs and therapeutic microbes, are effective against diseases and infections.3,4 Such treatment originates from microbes’ multiprolonged survival mechanism, where they encompass immune and defense characteristics against extreme environments.5 Thus, when countering treatment resisting diseases, microbes provide a comprehensive arsenal of mechanisms that allow pharmacologists to combine them with vaccines and other drugs. Spring 2022 | PENNSCIENCE JOURNAL 13
FEATURES Rapid Evolution
pathogen within a site, making microbial therapeutics customizable to individual patients.8
Microbes employ rapid evolution to acquire advantageous traits that optimize survivability, allowing them to effectively and efficiently adapt to their environments.6 Through modifying this characteristic via directed evolution, microbes can be engineered specifically to target pathogens and become self-adjusting therapeutics.7 Extraction of such microbial agents can also reveal information about disease pathology and drug efficacy, both of which can guide clinical designs.
Quick Replication The quick replication rate of microbes make them customizable therapeutics against different diseases. Microbes can replicate at the site of infection, through which they amplify the drug dosage precisely in desired locations within the environment without delivering negative effects elsewhere. The mechanism also allows microbes to adjust their dosage according to the amount of
Immune System Stimulation Research has shown that with hosts being colonies of microorganisms, microbes can stimulate the hosts’ innate immune system to attack pathogens: after triggering the adaptive immune system (sub-immune-system responsible for diminishing pathogens), microbes activate the hosts’ antimicrobial defense to recognize specific antigens. Through this mechanism, microbes can not only indirectly enhance other microbial mechanisms against pathogens but also coordinate different immune systems in producing long-term immunity.9
Genetic Engineering Microbes can be genetically engineered via molecular biological and biochemical methods to achieve specific goals. Most of the genetic
Attack on Vascular Structure
Tumor Degradation
Microbial Therapeutics Against Cancer
Macrophage/Immune Activation
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FEATURES engineering methods modify the genetic makeup and sequences of microorganisms.10
Specified Microbial Therapeutic Against Cancer Regardless of their species, all microbial anti-cancer mechanisms can be classified into three categories: direct antitumor mechanism, vasculature destruction, and immune activation. Related treatments can also be enhanced by genetic engineering.
Direct Antitumor Mechanisms Microbes can eliminate tumor tissues by infecting and killing host cells through virulent mechanisms that often lead to cell lysis, the breaking of cell membranes. Some microbes infect tumor cells internally, while many others attack extracellularly. For instance, toxin-bearing microbes produce peptides and virulent elements that can damage tumor tissues and suppress vital functions like immune systems, which is more effective than chemotherapy, as the latter targets all tissues and can cause fatal side effects.11 Microbes can be engineered to transform molecules with little pharmacological activity into toxins or active chemotherapy reactants within target tissues.12 Notably, microbes can also embed radioisotopes-coupled antibodies into their membranes to deliver lethal radiation to tumors.13
Attacks on Vascular Structures Many scholarships have demonstrated that cancerous tumors rely on vasculature, namely blood vessels, to provide oxygen and nutrients for their high metabolism.14 Current chemotherapies, although effective at inhibiting the growth of new blood vessels, are impotent against treating existing vessels that support tumor growths. However, through genetic expressions that allow the development of specific pathway-binding protein structures, microbes can not only suppress tissue growth with higher success rate than traditional treatments but also trigger apoptosis (programmed cell death) in malignant and blood vessel cells.15,16,17 The destruction of harmful vasculature would facilitate immune cells’ targeting on cancer cells and support therapeutic bacterial infections.
Immune Activation Tumors have developed mechanisms that allow them to inhibit and suppress immune cells within tumors.18 However, through both genetic engineered and intrinsic properties, microbes emerge as a useful tool to activate immune systems and attract immune cells. For instance, through infecting tumors, microbes can trigger inflammatory responses of the host’s innate immune system to destroy both cancerous tissues and related vasculatures.19 Specifically, microbes can be engineered to produce cytokine proteins to attract immune cells like macrophages (cells that can filter through tumor membranes, infuse therapeutic microbes, and trigger antitumor inflammations)20 and neutrophils (white blood cell that can stimulate non-specific inflammation).21 Additionally, microbial vaccines can deliver tumortargeting antigens like tumor necrosis factor alpha (TNF-�) to activate adaptive immune responses independent of tumor’s infection status, which allow the implementation of multifaceted attacks.22 Eventually, by inducing immune memory, microbes could help to establish a surveillance system that identifies and removes metastatic cells before they create further dysregulation.23
Concerns One risk of applying microbe-based therapeutics is induced septic shock—a body-wide bacterial infection. When a microbial vaccine activates immune systems, some bacterias might be lysed during the release process of macrophages and excrete toxins that can lead to fatal septic shock. Thus, it is essential for future treatments to minimize that risk. A potential solution is to engineer phage to delete genes corresponding to lysing. Another concern surrounds immune system clearance, which can rapidly clear therapeutic phages and significantly reduce their efficacy. One potential solution is couple directed evolution that can increase phages’ lifetime with genetic engineering that boosts their immunity against the hosts’ immune system.24 REFERENCES
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FEATURES
THE POWER OF DOODLING: IMPOSSIBLE SYMMETRIES AND DIFFRACTION PATTERNS Written By Michelle Paolicelli Designed By Saraswati Sridhar
T
he mark of a great scientist is someone who is constantly questioning. Questioning the world around them, the way things work, and why things are the way they are. But beyond this, a scientist with the potential to pioneer will question established truths. The things that others take for granted and believe to be unequivocally true. Many people exhibit a passion for science, but what sets pioneers of science apart is their hunger to explore a subject further than anyone else before them. Sometimes their discoveries may not be the most flashy or exciting, but their impacts are more profound than anyone could have imagined. Roger Penrose did just that, opening the door to the discovery of an entirely new form of matter, and it all started with a handful of shapes. Penrose, a contemporary of Stephen Hawking, is a British mathematician and relativist. His work on black holes earned him the 2020 Nobel Prize for Physics. Alongside Hawking, Penrose proved that
all matter within a black hole reduces to a point of infinite density and zero volume, also known as a singularity. In addition to this, Penrose created a way to model the effects of a black hole upon an object as it approaches a black hole. Although Penrose’s achievements in Physics are extraordinary on their own, his love for mathematics and doodling led to perhaps his most interesting contribution to science in the field of crystallography. 1 In crystallography, tiling is defined as the filling of all space with a repeating geometric pattern, called a motif, without gaps or overlaps. As a general rule, only motifs that result in global 1, 2, 3, 4, and 6-fold rotational symmetry are able to do this. This was a widely accepted truth. However, Roger Penrose discovered a way to tile while creating 5-fold symmetry using a motif of only four shapes. This was revolutionary as it was thought to be impossible. Shortly after this initial discovery, two other types of Penrose tiling were discovered using different motifs. As Penrose tiling was further explored, scientists soon realized that infinite variations of Penrose tiling exist in which a pattern with five-fold rotational symmetry filled all space.2 This redefined an absolute truth within crystallography, and Penrose tiling has been found to naturally occur in quasicrystals and used in architecture and art. What makes Penrose tiling so revolutionary is that its novelty goes beyond its forbidden 5-fold symmetry. Other scientists studied Penrose tiling at length, coming up with their own variations, identifying new patterns and features, and translating the 2D pattern to a 3D representation of matter.3 Apioneer himself, Penrose’s work inspired others to push further and search for greater meaning in Penrose tiling. One person of note who extended the meaning of Penrose tiling was Robert Ammann. Ammann’s contribution to Penrose tiling is unique in that he was an amateur mathematician. He did not complete a college degree but rather spent his time developing
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FEATURES his genius and a knack for pattern recognition. He was first introduced to Penrose tiling through Martin Gardner’s Mathematical Games column in Scientific American. Ammann discovered his own aperiodic sets of tiles, some more simplistic than the kite and dart version Penrose himself proposed. He also developed a way to help form new tilings by placing lines on a plane in a semi-regular pattern. These lines are known as Ammann bars and show that all edges of an aperiodic tiling connect via straight lines. More intriguing is that the spacing of these lines follows Fibonnacci’s sequence, also known as the golden ratio.1 As an amateur mathematician, but surely a genius, Ammann’s contributions to crystallography and the understanding of Penrose tiling are not to be overlooked. Another great scientific discovery that was enabled by Penrose tiling was the idea of quasicrystals. Quasicrystals are just what their name sounds like– crystal-like. The classic definition of a crystal is an ordered, periodic structure with predictable makeup. Quasicrystals modify this description as they are ordered structures, but they are aperiodic in nature. This means that although a quasicrystal pattern can fill all space, it lacks the translational symmetry of traditional, periodic crystals. In addition, crystals follow the symmetry restrictions of 2, 3, 4, and 6-fold symmetry while quasicrystals possess the forbidden symmetrys.3 It is this connection to Penrose tiling that led from one discovery to the other. Prior to the publishing of Penrose tiling, it was widely accepted in the field of crystallography that the symmetry rules were not broken under any circumstances. With this understanding came the acceptance of the idea that 2, 3, 4, and 6-fold symmetries were the only to occur in nature.4 However, Penrose tiling cast doubt upon this tenet of crystallography and scientists began to look deeper. Prior to the introduction
of Penrose tiling, diffraction patterns of many different crystals had been made. Most often, they followed the established rules of symmetry. However, when they did not, scientists found some other explanation for the appearance of other symmetries. One explanation was that the crystals were twinning, or overlapping, causing the diffraction pattern to appear to possess a forbidden symmetry. In some instances, this was the case, but in others the blind trust in established theories led to a missed opportunity for discovery.1 Paul Steinhardt, formerly of the University of Pennsylvania, refused to accept the notion that quasi crystals did not exist in nature. His work with Penrose tiling led him to the theory of naturally occuring forbidden symmetries and after years he finally discovered icosahedrite as the first known naturally occurring quasicrystal.1 This discovery was a culmination of Steinhardt’s entire career of work and was enabled entirely by his tendency to question and discover things on his own. Today, quasicrystals are better understood. From two-dimensional Penrose tiling to threedimensional diffraction patterns observed in nature, quasicrystals are the result of the collaboration of manypioneering minds. Applications of quasicrystals include serving as steel-hardening agents, non-stick pan coatings, solar absorbers, and biomedical uses among others.5 Penrose’s legacy stretches forward decades and reaches back generations to explain previously mysterious phenomena and behaviors. His original tiling patterns have formed the basis of other scientists’ careers as they work to uncover all their hidden meanings, all because he liked to doodle.
REFERENCES
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FEATURES
EXPANSION OF
QUANTUM
COMP U TI N G Written By Benjamin Beyer Designed By Bianca Vama
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hroughout history, people have used computers for a variety of purposes and they have become ubiquitous in our society. Research into computers is ongoing and will continue as they are useful tools that can enhance every aspect of our daily lives. Around 1982, Richard Feynman formulated an early iteration of quantum algorithm to complete quantum calculations, and this was fleshed out in 1983 by David Albert. The basis of quantum computing is called a qubit, and this was realized in 1998 with the first qubit registers. While normal computers use binary to store and transmit data, quantum computers use qubits to store data which have the power to store data much more concisely than binary. This is because of the quantum mechanical principle of superposition which allows the qubit to be in either a “0” state, “1” state, or a combination of these two states, and this is the reason that quantum computers can more efficiently store and complete calculations. Throughout the 2000s, this technology has been scaled up and complicated quantum algorithms have been developed which have proven to be faster than their classical counterparts due to quantum principles such as superposition.
As these computers are scaled up (they are about the size of a fridge) and their size scales down, 18 PENNSCIENCE JOURNAL | Spring 2022
we can start to think about the commercial advantages of quantum computing and the expansion of quantum computing into the consumer world. For example, electric car companies are investing in this idea to see if they can enhance the capabilities of electric car batteries. They are doing research into the chemistry of electric car batteries with the help of quantum computing’s vast power and calculating capabilities. As quantum computers continue to improve, their capabilities will continue to challenge and surpass the capabilities of classical computers which could help further expand the electric car industry. The extent to which quantum computing can revolutionize some industries is not yet known. While this power is used in electric car battery development, quantum computing is also forecasted to be able to be used in drug discovery. This is one of many very new developments in the quantum computing industry. While often people consider wet labs to be at the forefront of drug development, the forefront of drug development has been pivoting into computational realms. For many of the purposes that drug development requires, classical computing suffices. Companies hope that quantum computing will be able to predict certain qualities of molecules like reactivity, structure, and behavior of large and
FEATURES complicated drug molecules. It would also increase the precision and usefulness of our models such that fewer materials would be needed to test and perfect the drugs themselves. While this is not currently being used, companies are exploring the possibilities of harnessing quantum computing power in pharmaceutical development. As this technology improves and becomes better at helping in drug synthesis, this would be able to revolutionize drug development and testing. While the power of quantum computing will be able to be harnessed in the future of many consumer fields, quantum computers are also being used to increase our knowledge of theoretical physics.
The CERN Supercollider is one such instance of these computers being used to progress our knowledge of particle physics, namely the Standard Model and the Higgs Boson. This elusive particle is one that is hypothesized to be the origin of mass in the universe, but it’s elusivity has been a topic of contention for years. At CERN in Switzerland, classical computers process immense amounts of data using the most stateof-the-art classical systems, but as quantum computers improve, CERN will begin using these computers to more efficiently and more precisely sift through the data it receives to resolve the mystery of the Higgs Boson. In the future, more aspects and quirks of quantum mechanics will be harnessed by these computers and they will only become even more advantageous to use compared to their classical counterparts, especially as they become more commercially available. One of the most promising ways that innovation is being done in quantum computing is the use of dual-atom spreads, which—simply put—will allow for even more data to be stored within the qubit system. This will only improve quantum computers as this technology becomes more understood and available. While classical computers have become such a large industry, there is undeniable promise in their quantum counterparts and these quantum computers may one day be as ubiquitous as the classical computers we use daily.
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mRNA Vaccines: The Solution To the Return to Normal Written by Chinasa Emeribe Designed by Amara Okafor
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he COVID-19 pandemic has been one of the most impactful pandemics in modern history. The virus, SARS-CoV-2, was highly contagious Over the course of two years, the CDC reported that up to 80 million Americans were infected, with 978,254 people passing away from COVIDrelated deaths. American life changed drastically overnight, with the population entering heavy lockdown periods. With the arrival of a newer, albeit not completely new type of vaccine, messenger RNA (mRNA) vaccines, America was finally able to begin administering vaccines.
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FEATURES Pharmaceutical vaccine such as Pfizer-BioNTech’s and Moderna’s Messenger RNA vaccines work differently than conventional vaccines. In order to generate the body’s immune response, many vaccines put a weakened live virus or an inactivated virus into the body for antibodies to be generated. Alternatively, mRNA vaccines use mRNA synthesized in a laboratory, which introduce an mRNA sequence to code for the creation of a certain antigen protein. The body will then create antibodies for the antigen, which will build up immunization for exposure to the disease.
in early clinical stage trials. Currently, there are studies actively recruiting for mRNA vaccine trials in multiple countries at universities, medical centers, and hospitals. These new trials include both for testing of new COVID-19 vaccines and boosters, as well as for other viruses and illnesses, such as liver cancer, multiple sclerosis, and Respiratory Syncytial Virus. The safety of the mRNA vaccine has been verified as safe for people to take. Dr. Weissman, a infectious disease expert at Penn Medicine, has been researching RNA for use in vaccines, and co-developed the mRNA technology for the Modera and Pfizer vaccines. He asserts that there is the clinical risk/ Several types of mRNA vaccines exist benefit ratio, which measures the risk of the vaccine currently. These include non-replicating mRNA, in to the risk of the disease, and that the vaccine is safe to vivo-self-replicating mRNA, and In vitro dendritic take. As documented in mRNA vaccine clinical trials, cell non-replicating mRNA vaccine. Non-replicating RNA molecules are broken down easily, which help mRNA is by far the simplest type of RNA vaccine prevent drug toxicity. There is also the lipids used with a single mRNA strand. In vivo-self-replicating in the mRNA vaccine that are natural are therefore mRNA packages the main pathogen-mRNA strand less toxic to the body. While some may experience additional RNA strands, which allows for more moderate side flu-like side effects, these side effects antigen production for a smaller amount of vaccine. are both not dangerous and quickly reside. In vitro dendritic cell non-replicating mRNA vaccines use Dendritic cells, immune cells that can present Currently, Penn Medicine has started a new antigens on their cell surface to cause an immune project on the use of mRNA vaccines for Human response. Dendritic cells are transfected with the Immunodeficiency Virus, or HIV. The HIV/AIDS mRNA vaccine to stimulate a stronger immune Prevention Research Division will be hosting trials in reaction. a study know as HVTN 302, which is scheduled to be completed by July 2023, with results published by the There are multiple medical and financial following October. This study will be one of the first benefits to using mRNA vaccines. Since production studies to attempt HIV-infected DNA from the body of RNA vaccines is able to be quickly replicated in in the hopes of neutralizing the deadly disease. laboratories from DNA in a standardized manner, the production of the vaccine is less expensive and lengthy. This differs from conventional vaccines, which often use mammalian or chicken cells instead to grow the virus inside of, which then have to be isolated and purified. Efficacy is very good in mRNA vaccines, as one RNA molecule can make 1,000 to 100,000 proteins, which amplifies the immune response. The REFERENCES REFERENCES safety of mRNA vaccines is safer than traditional vaccines, as they do not use any inactive infectious elements,with mammalian cells, often chicken cells being used instead to grow the virus inside of. Although COVID vaccines were the first type of mRNA vaccine to be produced and used on a population on a mass scale, they have been researched for decades, researching immunization efficacy and success with diseases such as influenza, ebola, Zika, rabies, HIV cytomegalovirus, and even cancer. However, there are still other mRNA vaccines Spring 2022 | PENNSCIENCE JOURNAL 21
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merica consumes twenty percent of all oil produced in the world despite being the home of less than five percent of the world’s population1. Our dependence on oil creates economic and political pressures, as we have seen with recently inflated gas prices2. When these concerns are compounded by the damaging environmental effects of oil extraction, transport, and use, it is easy to understand why the U.S. is turning toward alternative fuel sources. Hydrogen fuel cells are one of the most promising alternatives that has been researched over the last few decades.
These advantages have motivated researchers and engineers to explore and refine the processes and technologies that are essential to hydrogen fuel cells. Many different types of hydrogen fuel cells have been developed, including alkali fuel cells, molten carbonate fuel cells (MCFC), and proton exchange membrane fuel cells (PEMFCs)4. These are all variations of the same fundamental process of reacting hydrogen and oxygen to generate power. To better understand how these fuel cells work, one can study PEMFCs, which many scientists believe to be a promising candidate for use in homes and vehicles.
These fuel cells have several advantages over conventional fossil fuels3. One such advantage is the ability to generate power without combustion or carbon emissions. Even if one were to account for emissions due to production, when measured against gasoline vehicles, vehicles that utilize fuel cells are able to reduce their total carbon dioxide by half if the hydrogen is produced by natural gas and by 90% if the hydrogen is produced by renewable sources. Other upsides include the accessibility of hydrogen and oxygen in our atmosphere and the ability to be electrical grid-independent, which is especially important for critical load functions such as hospitals and military applications.
There are four main components to a PEMFC: the anode, the cathode, the electrolyte, and the catalyst5. Respectively, the anode and cathode are negative and positive electrodes (conductors that connect nonmetallic materials to a circuit). That is, both the anode and cathode are the mechanisms with which electrons are directed throughout the fuel cell. They also have channels which are responsible for distributing gases evenly across the catalyst6. Next, the electrolyte, commonly referred to as the proton exchange membrane, functions by conducting positive charges. Lastly, the catalyst is where the hydrogen and oxygen reactions occur. It is engineered from carbon paper or cloth coated with platinum nanoparticles.
Fueling A e r u t u F r e N Clean han Tra by Jonat ng Ngo Written by Phuo d e n ig s e D
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HYDROGEN STATION
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FEATURES of researchers led by Mengxiao Li at Chongqing University have explored numerous experiments and simulations to model ways in which this temperature change can be minimized. To approach this problem, they first determined the parameters that affect the final temperature of the gas, which include but are not limited to the initial temperature of the hydrogen, the initial pressure, and the flow rate. By employing mathematical models that relate gas parameters, Li and her team have been able to predict the effects of different refueling methods.
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One of Li’s most significant findings was in the exploration of the different tank materials used for hydrogen storage. To date, there are two types of units considered most suitable for hydrogen storage—the difference between the two largely being in the inner The actual process of producing energy layer of the tanks. One is made of aluminum, and the begins with high pressure hydrogen gas entering the other is constructed from high density polyethylene, anode side5. This pressure forces the gas through the a plastic. By modeling the thermal conductivity of catalyst, which breaks the hydrogen into two protons both tanks, Li reports a lower final temperature for and two electrons. These electrons flow through the the aluminum tank because of its higher thermal anode, into an external circuit, and finally to the diffusivity. The plastic absorbs heat slower which cathode. It is in the external circuit that the fuel cell is ultimately results in less heat dissipated during the able to produce work from the electron flow. refueling process. Because the plastic tank is worse at counteracting the increase in hydrogen temperature, On the cathode side, a similar process is simultaneously the aluminum tank is superior in this regard. Li and occurring, as oxygen gas is forced through the catalyst5. her team to date continue to research such factors The oxygen is broken into two oxygen anions, which in hydrogen refueling in hopes of optimizing this then react with incoming hydrogen cations to form complex process. one of the products of this reaction—water. As the world continues to grapple with the This process generates very little voltage from effects of climate change and heavy dependence a single reaction (about seven-tenths of a volt, which on fossil fuels, hydrogen fuel cells are becoming an is about half of what a typical AA battery provides); increasingly important and appealing alternative. however, when fuel cells are connected using bipolar The government and the private sector are making plates, large amounts of voltage can be efficiently enormous investments in this rapidly improving produced by what is known as a fuel-cell stack. technology. Currently, states across the U.S. are vying for almost $10 billion in federal funding for hydrogen Scientists are currently conducting extensive fuel cell projects8. As these investments continue to research into optimizing the efficiency and practicality drive innovation and production, hydrogen fuel cells of hydrogen fuel cells. One prominent example are becoming ever closer to drastically changing the is the study of refueling technologies, which is way we think about energy in the U.S. and around the especially important for hydrogen fuel cell vehicles. world. As previously explained, these fuel cells require high pressure hydrogen gas as an input to the anode. However, engineers have found that the transfer of hydrogen at high pressures can cause a sudden increase in the temperature of storage systems7. This can decrease the fuel effectiveness, damage the fuel tanks, and create safety concerns. In response, a group
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Written By Sarah Pham Designed By Caitlyn Prabowo
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FEATURES Introduction
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pilepsy is one of the most common neurological conditions that arise from a combination of acquired and genetic factors. Seizure syndromes are representative of different genetic mechanisms in epilepsy that may include issues with electrical signaling, proper positioning of neurons within the layers of the brain’s cortex, and developmental abnormalities among others.1 Describing an even more distinct subset of this neurological disorder, genetic epilepsy arises from single-gene mutations or structural changes in chromosomal DNA sequence. Specifically, mutations in genes encoding voltage-gated sodium channels contribute widely to a variety of genetic epilepsy syndromes. The scientific community has identified a vast majority of these mutations existing in a gene called “SCN1A.” Mutations in SCN1A lead to an expression of overlapping phenotypes such as in: Dravet syndrome, a severe myoclonic epilepsy of infancy also known as SMEI, intractable childhood epilepsy with generalized tonic clonic seizures (ICEGTC), and generalized epilepsy with febrile seizures plus (GEFS+).1 If even one copy of the SCN1A gene is passed down from parent to offspring, these genetic epilepsy phenotypes will appear. For this reason, the SCN1A gene affirms its mark as an area of major genetic epilepsy research. However, some members of the scientific community have branched off to identify other possible pathogenic, or causative, variants. Novel research pinpoints mutations in the “SCN3A” gene
that give rise to an intriguing overlap of features in SCN3A patients including epilepsy, brain (cortical) malformation, and intellectual or developmental disorder.
The SCN3A Gene The SCN3A gene is highly expressed in the embryonic brain during the fetal gestational weeks (WKSG) and gradually decrease postnatally (after birth).4 As you can imagine, elucidating the pathogenic mechanisms of this gene would require meticulous study of the developing neuron due to the narrow time-span of SCN3A’s peak expression levels. De novo mutations are new genetic changes (not passed down from parents) caused by mutagenesis during the formation of male and female gametes. De novo mutations represent the most extreme form of rare genetic variation as they tend to have more deleterious effects compared to inherited variants and can be passed down to offspring as inherited mutations.3 Current research establishes De novo pathogenic variants in SCN3A as the cause of developmental and epileptic encephalopathy.5 Three unique aspects of SCN3A that make it worthwhile to study include: gain-of-function in sodium (Na+) channels, cortical malformations, and the unique spectrum of disease. Sodium (Na+) channels are primarily found in the brain and control the flow of Na+ ions into neurons. These voltage-gated Na+ channels mediate transfer of electrical signals between and among neurons called action potentials and serve as critical
Figure 1: Development of babies via germination, embryonic, and fetal stages. Spring 2022 | PENNSCIENCE JOURNAL 25
FEATURES regulators of electrical excitability. SCN1A and SCN3A differ in the types of voltage-gated Na+ channel � subunits Nav 1.1 and Nav 1.3, respectively.4 Nearly all patients with mutations in the SCN3A gene develop a “gain-of-function (GoF)” in Na+ channels that emerges in developing neurons and lead to an expression of over-excited neurons.4 These particular GoF properties describe mechanisms in which the Na+ channel opens too soon, closes too slowly, and leaves a trail of excess Na+ current that remains from the initial influx of Na+ that depolarizes (increase in membrane potential during an action potential) of the neurons.
segments 4 to 6 of domains II to IV.5 These variants occur in regions of the Na+ channel called the “poreforming” regions that are crucial for the dynamic structure changes during opening and closing. As you can imagine, issues with the influx and efflux of Na+ ions would disrupt the electrical and chemical communication of neurons, exacerbating uncontrolled excitability.
Conclusion
SCN3A-related developmental disorders continue to be extensively studied as a novel area of genetic epilepsy research. The distinct trait phenotypes linked to SCN3A gene mutation prompts Malformations in the cortical region of the researchers to delve into this particular subfield of brain may be considered one of the more surprising study surrounded by obscurity. In elucidating the phenotypes in patients with the SCN3A variant. In particular mechanisms of the SCN3A gene mutation, fact, over 75% of patients (15/19 individuals) developed the scientific community may soon generate innovative a type of brain malformation called perisylvian therapies for epilepsy and other developmental polymicrogyria (PMG).5 PMG refers to a condition disorders. characterized by abnormal development of the brain before birth in which there are excessive small References folds.2 Common symptoms include seizures, learning disabilities, behavioral concerns, motor delay, and more.2 Differing from the other genes encoding ion-channels, SCN3A is the only ion channel with a phenotype of brain malformation in a majority of individuals with the SCN3A variant.5 In SCN3A-related developmental disorder, pathogenic variants cluster in transmembrane
Figure 2: Sodium Channel with linker segments implicated in epilepsy. 26 PENNSCIENCE JOURNAL | Spring 2022
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Single Cell Sequencing for Cancer Research
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he first study utilizing scRNA-seq examined the whole transcriptome of single cells of a four-cell stage murine embryo. A study of single cell resolution from the University of Cambridge accelerated the general understanding of gene expression and regulation under this new innovation. They were able to detect the expression of 5,720 more genes than microarray, the previous standard method for transcriptomics analysis.1 This represents a 75% increase in unique transcripts detected by the microarray technique, which clearly provides more in-depth information about the cellular states.
Tang et al. ultimately showed cell types can be clustered by tracking transcriptomic changes through scRNA-seq. While their technique was successful, there were many challenges that slowed development of the technology. The scRNA-seq libraries were generated manually in individual tubes after isolating single cells, which is a time and labor-intensive process.2 This inability to increase the
Written By Rebecca Nadler Designed By Saraswati Sridhar scale of scRNA-seq limited potential applications for several years. Researchers at Harvard Medical School innovated a method to utilize microfluidics, whereby a single cell and functional bead are contained within a droplet in an oil emulsion in order to compartmentalize cell lysing, barcoding, and reverse transcription. This procedure allowed researchers to build up large-scale sequencing libraries and is regularly used today. There is a standard methodological protocol generally used to conduct scRNA-seq.3 Arguably, the most important step of scRNA-seq is the primary isolation of viable, single cells from the cell population of interest. Establishing an unbiased, representative sample of cells is a critical step for ensuring validity of downstream data analysis. The cells are then lysed to capture RNA molecules, which are converted to complementary DNA (cDNA) via reverse transcription. In doing so, adaptor sequences are added to the ends of the cDNA molecules to serve as unique molecular barcoding tags for analysis, allowing researchers to analyze multiple samples together without ambiguity. However, reverse transcription only provides a minimal amount of cDNA, so it is amplified exponentially by polymerase chain reactions (PCR). The resulting cDNA is pooled and sequenced using Next Generation Sequencing techniques, and bioinformatic methods are utilized
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FEATURES to interpret the vast amount of scRNA-seq data generated. With the spread of scRNA-seq technology, increasingly accessible and affordable commercial kits and reagents are now available for researchers to carry out each step of the protocol.3
this technology will facilitate the discovery of tissue microenvironment pressures on cellular infiltration in a tumor setting and may enable profiling of an entire organ. With a greater understanding of the hallmark cellular phenotypes that dominate certain cancer types across organs, researchers will be better able to Much information can be obtained from design targeted therapies for patients, as they will be scRNA-seq analysis to bolster our understanding able to assess the barriers and vulnerabilities within of tumor progression. Cancer develops through the tumor. the random accumulation of somatic mutations — those in non-germ line cells — creating gene Today, scRNA-seq has already provided key mutation heterogeneity and genomic instability. Both insights to accelerate the theoretical understanding of genetic phenomena create tremendous intra-tumor tumor development and progression. Gu et al. also heterogeneity, particularly influencing the expression demonstrate how it may provide clinical benefits in the of genes that contribute to cancer development. context of cervical cancer. Across five cervical biopsy scRNA-seq technology can be employed in the study samples, they identified differential gene expression of tumor heterogeneity for analysis of the characteristic and activation of various signaling pathways that differ cell state of malignant cells, while also elucidating between chemoresistant and chemosensitive cervical the influence of genetic factors and epigenetic cancer patients.7 In examining the immune infiltrate, modifications.4 In addition to the assessment of they found that immune checkpoint molecules intratumoral heterogeneity, scRNA-seq can be were highly expressed in a manner that inhibits utilized to distinguish between tumoral cells and the cytotoxicity, indicating that immunotherapy could be tumor microenvironment. Immune cells, fibroblasts, useful in cervical cancer. Furthermore, inhibition of and endothelial cells typically comprise the tumor certain components of the pathways upregulated in microenvironment, all of which are implicated in chemoresistant samples has the potential to overcome tumor development.5 Supplementary investigation resistance. Ultimately, this information provides novel of these cell types may reveal mechanisms of insights into human cervical cancer and has the potential to advance both diagnostic and treatment immunosuppression and cancer growth. efforts. More recently, researchers are pairing Thus far, the scientific journal Nature Methods scRNA-seq technology with other assays in order to understand individual cell transcriptomics with greater named spatially resolved transcriptomics the 2020 depth. Researchers at the University of California San Method of the Year.8 It has the potential to expose Francisco recently developed the XYZeq workflow novel biomarkers and therapeutic targets to aid in as a means of incorporating information about the cancer treatment. scRNA-seq may also guide clinical spatial organization of cells with scRNA-seq libraries. decision-making throughout treatment, as it can be This enables scientists to simultaneously cluster cells used to track drug resistance and tumor evolution. present in sample tissue by physical location and gene As personalized medicine becomes more prevalent in expression. Using this approach, Lee et al. identified the clinical setting, scRNA-seq will prove incredibly spatially variable patterns of gene expression beneficial for identifying candidate treatment options and heterogeneity within lung cancer-associated for patients according to their specific disease mesenchymal stem cells, specified by proximity presentation. REFERENCES to the tumor core.6 The authors hypothesize that
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estoring MIND the
Stem Cell Therapy and Neurodegenerative Diseases
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ew diseases are as terrifying as neurodegenerative diseases. Classified as disorders that target the central or peripheral nervous system, neurodegenerative diseases are amongst the most difficult disorders for patients, doctors, and researchers.1 Without long term symptomatic relief from existing treatments, these diseases place the healthcare system under considerable strain.2 Of all the conditions encompassed by the broad umbrella of neurodegenerative disease, Huntington’s disease (HD) and Parkinson’s disease (PD) are two common disorders that affect seven million people worldwide.2 Each disease has unique molecular mechanisms that present challenges to existing modes of treatment. Huntington’s disease is an inherited disease linked to a dominant genetic mutation that triggers the production of toxic proteins that kill medium spiny neurons, a type of neuron that performs the crucial function of receiving dopamine signals.3 Unlike Huntington’s disease, which induces damage to receptor cells, Parkinson’s disease is characterized by a loss of dopamine-supplying (DA) neurons due to an accumulation of misfolded proteins. This may alter the cellular environment enough to damage DNA and compromise neuron survival.3 Huntington’s disease patients generally suffer neuropsychiatric symptoms, while PD patients are affected by both motor and psychiatric symptoms. Both diseases are considered “incurable,” in that current treatments are only stop-gap measures, toothless against the inevitable breakdown of the brain. Even symptomatic-relief care for PD sometimes fails, as the strategy of supplying
Written By Beatrice Han Designed By Avi Singh
pharmaceutical agents that convert to dopamine in the brain suffers from declining efficacy over time. Worse, it triggers adverse side effects, including worsening motor control and impulse control disorders.4 Fortunately, stem cell therapies offer an alternative option. Stem cells differentiate into many other cell lines and have the capacity to restore lost neurons and repair damaged cellular environments. For HD especially, marked by the loss of a specific cell line, stem cell therapy is an especially attractive option. With no natural consistent source of neurogenesis in the central nervous system, where most neurodegenerative diseases are localized, the need for stem cell therapies is especially urgent.5 Mesenchymal stem cells (MSCs) provide a promising solution for neurodegenerative disease treatment with their ability to differentiate into non-mesoderm derived cells like neurons. Unlike embryonic stem cells–the prototypical stem cell with almost unlimited differentiation capacity– MSCs are adult stem cells taken from mesodermal tissue, including bone, cartilage, and muscle.5 MSCs sidestep the thorny ethical controversies that plague ESCs, which face pushback because they are sourced from fetal tissue.2 Because MSCs display the unique capacity to “home” in on injured tissues, they are considered promising targets for future treatments.7 This selectivity is especially important because MSCs may be able to cross the blood brain barrier–which Spring 2022 | PENNSCIENCE JOURNAL 29
FEATURES usually prohibits therapeutic agents from entering the Regulation of the unpredictable immune brain –allowing them to localize to injury sites and responses to grafts of stem cells poses another threat to exert a full therapeutic effect.2 stem cell transplantation. Triggered by both the act of grafting itself–which is inherently surgically intrusive– Once in the brain, MSCs address and the presence of foreign cells in a host body, these neurodegeneration through a two-fold response. immune responses may further inflame the nervous First, MSCs can differentiate into neuron-like cells system or reject the transplanted stem cell altogether, once exposed to specific environmental conditions, negating what therapeutic benefit the treatment may allowing them to directly replace lost neurons. have been achieved.3,8 Other challenges include Indeed, when rats displaying the motor symptoms ensuring the differentiated neuron properly interacts of PD were implanted with a meshwork of MSCs, with the brain environment. To restore a damaged motor function improved because the transplanted pathway, it is not enough to implant a cell without MSCs differentiated into DA neurons, targeting the triggering an adverse response, as the cell must survive root of the dopamine deficiency in PD.7 Second, and regrow its axon to the targeted region. Because beyond directly replacing DA neurons, MSCs can of the difficulties associated with ensuring the correct exert an immunomodulatory and neurotrophic connections are made, many clinical and animal trials effect; they secrete trophic factors, which are have yielded inconsistent functional recovery post chemicals that promote the growth of neurons and stem-cell transplant.5 protect existing neurons from degeneration. Trials in rodents manipulated to express the symptoms of Even the ability of stem cells to give rise to HD have even indicated MSCs can be genetically multiple cell lines can become a double-edged sword. modified to secrete more of these trophic factors, As stem cells often mimic the characteristics of cancer creating a favorable extracellular environment to cells–including changes in tumor-suppressor genes stave off degeneration.3 Many of these studies, which and a long life span, they may induce tumorigenesis, engineered MSCs to secrete excess trophic factor, saw or cancer growth.9 ESC-derived treatments, which the new treatment prolong their subject’s lives and have been found to trigger tumor growth in animal reduce behavioral abnormalities associated with HD.4 subjects, have risk of turning cancerous because of their origin in fetal tissue, high differentiation capacity, Although many of the trials involving MSCs and ability to proliferate indefinitely.10,11 Though have taken place in animals, clinical trials in humans MSC therapies have never been found to trigger have been conducted, with varying outcomes. cancer growth in human patients, they should not Some trials have concluded MSCs yielded positive be exempt from scrutiny; some MSC therapies have outcomes with enrichment in DA-neuron tissue with uncovered stem cells with unbalanced chromosomal 24 year survival, improvement of motor symptoms, arrangements and other tumor-adjacent traits.12,13 and integration of MSCS into surrounding neural circuits.5 However, because of an inconsistent supply Like any novel therapy, stem cell-derived of MSCs and the heterogeneous nature of many treatments should be approached cautiously. At the transplants, which are derived from multiple donors, same time, their potential to achieve the impossible– clinical results have been inconsistent.5 Indeed, reversing the progress of diseases once characterized deriving non-heterogeneous cell lines for grafts and by their inevitability–must also be recognized. As transplants remains a pressing challenge, as pooling research on stem cells progresses, a door to the future cells from multiple donors for transplants makes of neurodegenerative disease treatment may open. it difficult to source enough tissue for widespread REFERENCES clinical applications while rendering results extremely inconsistent.
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Acres of Skin: The Cost of Scientifc Innovation Written By Cynthia Schneider Designed By Phuong Ngo
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ome of the greatest scientific advancements of the last century have been at the expense of ethics. The famous HeLa cells were extracted from an unconsenting African-American woman with cancer, but now polio is almost entirely eradicated. Even a former University of Pennsylvania dermatologist, researcher, and scientific pioneer in dermatology, Albert Kligman, performed unethical experiments on African-American prisoners for years. However, these experiments propagated a revolutionary antiaging/acne prescription known as Retin-A. Retin-A is a commonly used topical prescription medication within a group of chemicals made of vitamin A known as retinoids, including Retin-A and retinol (a slower-acting retinoid for skincare treatment)1. The skincare and the anti-aging industry is a multi-billion dollar industry that relies on the wonders of these products like Retin-A that was discovered by Albert Kligman. Kligman’s 1986 study titled “Topical tretinoin for photoaged skin” detailed how 0.05% topical tretinoin, which is Retin-A, heals sun-damaged and acne-prone skin2. Kligman’s work determined that tretinoin could be used to aid those with damaged skin because of its properties of repairing skin, removing any atypical tissues, forming collagen, increasing newly formed small vessels in the dermis, and reducing precancerous sites2. Kligman mentions multiple times throughout the manuscript the difference in the skin between white and black subjects, which
we now understand was the black prisoners used in many of his studies in the 20th century. His work gave rise to dermatology becoming a more respected and researched scientific field. He published over a hundred papers detailing other retinoic acid effects such as acne progression, herpes simplex, the human hair cycle, or simply testing the effects of different products on the skin (shampoos, deodorants, drugs, etc.). This wealth of data Kligman acquired during his time as a researcher transformed the field3. He invented Retin-A, which is still widely regarded as an effective prescription for those with acne, wrinkly skin, or damaged skin (sundamaged, precancerous, etc.). He conducted most of his infamous experiments at Holmesburg Prison in Philadelphia in what is now known as “The Holmesburg Prison Experiments”. While his contributions to the skincare industry cannot go unnoticed, his data was from prisoners who did not give informed consent, a concept in medical ethics where test subjects are required to have sufficient information to make a
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FEATURES decision about participating in a study. Following Kligman and others’ studies, prisoners are now considered a vulnerable and protected group in medical law. Many of his experiments inflicted immense pain on the prisoners. He tested the effects of materials like shampoo, deodorant, drugs, and sometimes radioactive material on the skin of prisoners4. Kligman’s studies irrevocably damaged the prisoners’ skin, leaving them with permanent cysts, lesions, and even resulted in death for some3.
“Penn Medicine acknowledges that the work done by Dr. Kligman was terribly disrespectful of individuals – many of whom were imprisoned Black men – denying them the autonomy and informed consent which the medical community now considers to be foundational underpinnings for conducting ethical research. Legality, of itself, does not excuse these activities, which are not now, and never were, morally acceptable, even if Dr. Kligman and his contemporaries believed them to be”5.
His dangerous studies were mainly performed on unconsenting African American prisoners. An example of this in his work are his findings on tretinoin’s effect on melanocytes. Kligman did not care for the medical ethics laws of the late 20th century, even going on to say that “things were simpler then. Informed consent was unheard of. No one asked me what I was doing. It was a wonderful time”3. His incarcerated “volunteers” in his studies were subjects not people to him. When Kligman details his time conducting research in the prison, he said the infamous quote “all I saw before me were acres of skin”3.
The abuse of minorities, specifically black and African American people, for scientific advancement is common in modern medical history. Henrietta Lacks and Tuskegee syphilis experiments are examples of how researchers, especially in the 20th century, choose science over ethics. Despite the lasting contribution of Kligman’s findings, ethical consideration is essential for the improvement of research—working towards revolutionary change but not at the expense of ethics.
In August of 2021, Penn made a statement about Kligman, removing his name from the prominent Penn Medicine lectureships/professorships due to the dark history of his actions. The university still acknowledges the importance of his work as a clinician and researcher but has worked since 2019 to formulate new action in regard to Kligman’s legacy at Penn Medicine5. A quote from Penn’s statement acknowledges the ethical implications of Kligman’s discoveries:
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Genetic Testing: There’s More at Stake Than Just Our Ancestry Written By Isabel Engel Designed By Bianca Vama Introduction
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magine sequencing your genome from the comfort of your living room couch. What was once a far-off concept limited to science fiction movies has now become a reality. With the mainstream commercialization of at-home genetic testing, people can better understand their DNA, genetic biomarkers, and ancestral history. Companies like 23andMe market their services as “real science, real data and genetic insights that can help make it easier for you to take action on your health”.1 But beyond flashy branding and consumer appeal, what is really at stake when it comes to at-home genetic testing? It’s time to take a look.
Genetic Counselors (NSGC), genetic counseling is defined as “the process of helping people understand and adapt to the medical, psychological and familial implications of genetic contributions to disease.”2 At-home genetic testing ranges in type, from Y-chromosome testing (designed to follow male ancestry) to mitochondrial DNA testing (that identifies genetic variations through mitochondrial DNA) to single nucleotide polymorphism testing (used to determine ethnic backgrounds).3 Through human saliva, all at-home genetic tests aim to look at regions of human DNA attributed to specific traits. 23andMe, for example, uses the HumanOmniExpress BeadChip Kit by Illumina as a means of inputting genetic material and scanning for variants.
They describe the genotyping technology and personalized reports as information “based on The Technology of well-established scientific and medical research.”4 Though the technology is relatively straightforward Genetic Testing and offers the general public streamlined access As outlined by The Genetic Counseling to their own genetic information, not all within Definition Task Force of the National Society of the science community are sold on this growing technology. While the technology elucidates genetic
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FEATURES variations, ancestry, and potential diseases, critics through all 30 million letters on your 23 pairs of chro argue that much more is at stake. mosomes, these tests look for a handful of common specific markers that vary between people, called single nucleotide polymorphisms (SNPs, pronounced The Concerns ‘snips’). But one SNP on its own is not enough to have American author and journalist Charles Seife sweeping effects on what10food you should be eating, of Scientific American warns that, despite positive, or what wine you’ll like.” medically-oriented branding, 23andMe’s “Personal Genome Service isn’t primarily intended to be a medical device. It is a mechanism meant to be a front end for a massive information-gathering operation against an unwitting public.”5 While the public may not realize it, in collecting the data of millions of consumers, 23andMe becomes “the Google of personalized health care.”6 While some consumer data go to important genetic and medical research, it is also used in sales. Seife writes that “23andMe reserves the right to use your personal information— including your genome—to inform you about events and to try to sell you products and services.” Knowing about individuals’ specific genetic concerns may prompt insurance companies to target the individual with medicines or sponsored products. What’s more, if insurance companies get their hands on the data, they may be able to sell (or deny) coverage to users. Specifically, genetic testing may impact life insurance. Though the Genetic Information Nondiscrimination Act (GINA) ensures that employers and insurance providers cannot obtain access to genetic testing nor discriminate on the basis of genetic results, not all are protected by GINA (including those in the US military and Veterans Administration).7 GINA also does not protect life insurance, long-term care, or disability insurance – meaning individuals’ premiums may change if their insurance companies get hold of genetic testing information. Thus, 23andMe, a technology with a branded low “consumer barrier to entry” may ultimately raise medical expenses in the long run.8 Another concern for critics of genetic testing is the weight placed on genetic results. The National Society of Genetic Counselors emphasizes that the results of genetic testing should not be regarded in isolation. Rather, they should be considered alongside family history, personal education about genetics and disease, and medical counseling.9 In her WIRED article, Amit Katwala writes of the concerns with single nucleotide polymorphism technology – and its inability to be all-encompassing: “People may think they’re getting their entire genome sequenced, but that’s not usually the case for most of the cheaper products. Instead of crunching 34 PENNSCIENCE JOURNAL | Spring 2022
Individuals cannot extrapolate medical diagnoses or guidance from the limited results of genetic testing. Without additional medical consultation, the results of genetic testing can be misleading. Critics warn that commercialized genetic testing is a strong start to a more informed public, but should not be treated as an end-all-be-all medical solution not be used at the expense of consumer’s privacy.
Looking to the Future All of this is not to say that at-home genetic testing should be outright abolished. It certainly allows for unprecedented mainstream access to genetic information. Regardless of socioeconomic status, geographic location, gender, or race, people can be armed with more information about their ancestral history and genetic variations. By removing cost barriers to genetic information, individuals can use genetic knowledge to consult medical professionals in hopes of leading healthier and longer lives. The testing, though, should be taken with a grain of salt. Technologies like 23andMe are not a catch-all solution. As critics suggest, American consumers should be cautious and informed before they willingly spit into a vial.
references
FEATURES
MODERN PIONEERING IN CORAL REEF RESTORATION: MICROFRAGMENTATION
C
Introduction
oral reefs are vital components of nature’s aquatic ecosystems. Over 1 million aquatic species depend on coral reefs to serve as feeding, habitation, and nursing grounds. Furthermore, approximately 25% of ecosystems depend on the existence of these reefs for habitation. Coral reefs also serve as a form of natural coastal protection, preserving life against destructive phenomena like tsunamis and storms. However, climate change has led to the endangerment of coral reefs in recent decades. At the time of its enactment in 2014, the Endangered Species Act stated that 22 coral species were considered a s
Written By Emily Ng Designed By Lynne Kim threatened, and 2 are endangered. Since then, the number of endangered coral species have increased rapidly, which also places the lives of various aquatic organisms in jeopardy. A specific consequence of climate change includes mass bleaching, which remains as one of the prime drivers of coral reef deterioration. The rise in intensity of mass coral bleaching is due to the increase in global temperatures. Bleaching mortality is rapidly escalating in various regions in the world—even the Great Barrier Reef, the largest coral reef in the world, is facing increased levels of bleaching. Public interest has been gradually pushing for international efforts to preserve and recover coral reefs. Microfragmentation, an extensive process in which coral is regenerated from its smaller counterparts, is a prominent example of modern solutions. Several groups have been advocating for more attention on this subject, such as Mote Marine Laboratory in Florida, an independent nonprofit that is currently undertaking microfragmentation as a recent initiative. Such efforts, however, have also been countered by the need for more funding and governmental cooperation within global intervention.
Mass Bleaching Derived from climate change, mass bleaching is a key driver of coral reef deterioration. The survival of coral depends on their symbiotic relationship with algae. Algae produces sugars using sunlight, and most are passed into the coral. In exchange, coral gives carbon and nitrogen to algae from their waste. Coral has the tendency to bleach when heat stress disrupts their symbiotic relationship with algae. One proposal suggests that bleaching may be due to damage toward algae’s photosynthetic processes from high temperatures. Struggles to process light properly produce reactive oxygen and nitrogen species (e.g., hydrogen peroxide) as a harmful byproduct. Such Spring 2022 | PENNSCIENCE JOURNAL 35
FEATURES molecules can damage proteins, so the coral host cell rejects these defective algae, which halts their symbiotic relationship and leads to bleaching. Furthermore, a 2018 study on parasitism in a coral reef ecosystem suggests that algae may become hostile in elevated water temperatures. Kim Ritchie, microbiologist in the Mote Marine Laboratory in Sarasota, recently discovered that higher temperatures can eliminate probiotic bacteria in coral reefs. These antibioticproducing bacteria fight off other pathogenic strains. This has led to an extensive and expanding search for finding bacteria that would grant coral immunity against such pathogens. In recent years, studies have determined that some coral reefs may possess thermal adaptations in response to prolonged high temperatures. Nevertheless, regions such as Micronesia and western Polynesia are still extremely vulnerable to climate change, and only rapid, monumental efforts in suppressing climate change would eliminate mass bleaching in these regions.
Microfragmentation Former restoration efforts have revolved around the usage of large fragments of coral. In this process, these fragments grow larger in size using in situ coral nurseries located at the original location of the corals. Once fully grown, they are often planted onto degraded reefs, and their development and survival are recorded. However, the need for larger pieces in this process requires the rapid growth of large coral reef species. Most massive corals usually possess relatively slow growth rates, which are unable to keep up with the immediate need for recovery.
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Microfragmentation introduces smaller pieces of fragments that have the potential to improve the rate of coral reef growth. Prior to outplanting, such pieces are cut to around 1 cm2 or less and grown to about 6 cm2. This is extremely small in comparison to larger pieces that have been used in stimulating regrowth of coral reefs (16-64 cm2). Even so, early success in research trials have presumed microfragmentation to be more high-yielding. A 2013 study by Mote Marine Laboratory on the outplanting of Orbicella faveolata and Montastrea cavernosa fragments on reefs in the Florida Keys demonstrated major findings. Similar survival rates were found between 6 microfragments and 1 larger fragment, and all of these fragments were derived from the same material. Positive results like these continue to drive efforts to refine microfragmentation. Rather than just serving as a means of effective and efficient recovery, microfragmentation can also be used as an experimental tool. Microfragmentation could determine areas in which growth of certain corals are the most fit as well as causes of death. The method can also be used to identify which coral species are especially vulnerable to bleaching as well as which are resistant. Notably however, microfragments are subject to predators, such as parrotfish, at a higher rate than larger fragments of coral. In reference to Mote’s 2013 study, it suggests that the microfragments of the coral reef species O. faveolata can produce around 10 times more tissue than its larger counterparts. This deduction, however, only took into account arrays of microfragmentation exhibiting less than 40%
FEATURES predation relative to other arrays. Thus, predation is a necessary factor to consider when refining the process of microfragmentation. The need to protect microfragments throughout their stages of growth suggests that rapid recovery of coral reefs is only possible with increased investment. Thus, funding is crucial in implementing microfragmentation on a global scale. Furthermore, it is still unclear if successful reproduction of coral is dependent on size, not just age.
The Need For Funding Facilitating microfragmentation on a global scale requires various equipment and cooperation. For instance, Frank Mars’s Coral Reef Rehabilitation Project has installed over 8,600 hexagonal steel structures (“spiders”) to plant microfragments onto the ocean floor nearby existing coral reefs. However, the potential of these
positive advancements are limited without sufficient funding. In the case of this particular rehabilitation project, much of the funding comes directly from philanthropists, not from federal agencies. No two coral reefs are the same, rendering experimentation in regrowth a primary concern. Mandated funding for coral research is crucial to providing such necessary flexibility. Furthermore, funding for coral reef restoration is often limited in duration and in quantity. The short cycles of a majority of restoration projects inhibits precision and accuracy across all data collected from various sources. Such funding also motivates many researchers to limit the sample size of their experiments, which hinders the ability to create effective conclusions regarding the success of microfragmentation. The sources of increasing necessary funding remain unknown. At the moment, there is still not enough global concern to push for support behind microfragmentation and on coral reef recovery efforts on the whole. Some experts believe that such efforts would be relatively cheap, but it is up to others to be willing to take this leap that will lead to change. Dave Vaughan, who is spearheading efforts in microfragmentation at Mote Marine Laboratory, predicted that only $1-2 million is needed to save elk horn coral populations in Florida. Similar efforts across coral reef restoration are also cheaper than anticipated, and their enactment ultimately comes down to the willingness from international powers to collaborate with scientific intervention. As research continues to search for more sustainable methodology, the need for recovery becomes more and more pressing.
REFERENCES
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FEATURES
The Various Clinical Applications of Nanomedicine Written By Kevin Guo Designed By Amara Okafor
Overview When we first think about the term “nano-”, we immediately think of small. But how small is nano really? Nano means on a scale of 10-9, meaning one over one billion! For comparison, a sheet of paper and a strand of hair are about 100,000 nanometers thick, and a strand of DNA is 2.5 nanometers thick. Nanotechnology is technology often dealing with the scale of less than 100 nanometers.
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FEATURES
T
he idea of nanotechnology was first conceived by quantum physicist Richard Feynman in a presentation at the California Institute of Technology. He raised the question, “Why can’t we write the entire 24 volumes of the Encyclopedia Britannica on the head of a pin?” With these ideas, he jump-started and pioneered nanotechnology as we know it today. Thanks to nanotechnology, we have evolved our understanding of several fields ranging from information technology to medicine and food safety. One emerging branch of the technology is nanomedicine, which applies the knowledge and tools of nanotechnology to prevent and treat diseases. The first specific instance of nanomedicine was introduced by nanotechnology researchers Drexler, Peterson, and Pergamit in a 1991 book titled “Unbounding the Future: the Nanotechnology Revolution” to describe how nanobots or biocompatible particles could be used in the clinical setting to treat various illnesses. Since then, interdisciplinary research involving biology, chemistry, and physics has built upon the foundations set by Feynman and Drexler, Peterson, and Pergamit.3
The most common applications of nanotechnology include using nanoparticles and smart sensing nanobot probes for various purposes ranging from the diagnosis of certain diseases to drug delivery. 1, 2can interact with cells much easier and bypass barriers traditional medications alone have difficulty overcoming.
alleviate the symptoms of diseases such as syphilis, diarrhea, and mental illnesses. Furthermore, nanoparticles that are artificially synthesized biological structures can serve as delivery systems for drug pathways. Many different types of nanostructures, ranging from phospholipid-bilayer liposomes to branched symmetrical dendrimers are utilized often in conjunction with the same metal nanoparticles to optimize the specificity of drug delivery.The size, shape, and biochemical interactions in the environments within and outside the cell determine the efficacy of the drug delivery system. In order to synthesize these drug delivery systems, nanomaterials used such as polyvinyl alcohol and polyethylene glycol must have high biocompatibility, which is being able to interact with humans with little toxicity, and biodegradability (see Figure 1). 6 Certain models of drug delivery may have low efficacy and may require an increased dosage of the drug in order to achieve the necessary therapeutic effect. 8, 9
Imaging Diagnostic imaging often incorporates these nanoparticles for drug delivery to determine whether a patient may have a disease. For example, X-ray fluorescence analysis (XRF) is used in tandem with nanoparticles in recent years to track cells for nanoparticle uptake or drug delivery studies. This same principle could be applied to the clinical setting
Clinical Applications in Drug Delivery
100 nm
1 nm
One of the most important applications of nanomedicine is efficient drug delivery to eliminate disease or improve the overall health of the human body. One of the most common methods of treatment is using metal nanoparticles to enhance radiation therapy through exposure to radiation and producing heat that can kill the cancer cells without affecting healthy cells. Many of these metals including gold and ruthenium had already been well documented for their ability to Figure 1: various structures mimicking cellular structures such as solid-lipid particles resembling cellular membranes and protein-drug conjugates in antibodies.
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FEATURES in the form of magnetic resonance imaging (MRI), computerized tomography (CT), ultrasound (US), and positron emission tomography (PET). These scanning techniques involve the injection of nanoparticle-based contrast agents into the patient and exposure of the patient to radiation in order to determine sites of abnormal tissue. Without diagnostic imaging, we would not be able to easily detect and prevent the effects of diseases at an early stage like cancer.5, 6 Despite the relatively novel approach to imaging, there may be multiple factors and variables for wholebody imaging using nanoparticles that may lead to improper conclusions. Although fluorescencebased imaging is effective in -vitro, in -vivo imaging is much more difficult to achieve as the increased tissue thickness may make it more difficult to distinguish disease image diagnoses from control backgrounds, decreasing the sensitivity of imaging. 8,9
Cancer Therapy As opposed to correcting any issues with unhealthy human cells, nanoparticles can also be used to kill cancer cells. If nanoparticles can be internalized in cancer cells without affecting normal tissues, they can exhibit inherently high cytotoxicity (i.e., toxic to cancer cells) and lead to high mortality rates of cancer cells. This effect is then further enhanced by radiation therapy to heat the metal nanoparticles and induce cancer cell-killing without significant effects on surrounding cells. Another interesting application is being able to elicit immune responses in the checkpoint blockade of immunologically “cold” tumors. In one study, a bismuth metal-based nanoscale metal-organic framework was created to manipulate the tumor microenvironment for enhanced radiotherapy (see Figure 2). When injected in combination with checkpoint blockade therapy, a form of immunotherapy that turns off signals that deactivate the immune system, the model was able to stimulate the immune system’s T-cells into tumors and properly target primary and distant tumors.7 Anti-cancer nanomedicines often have physiological transport barriers and thus have limited clinical outcomes. In cancer therapy, when directing nanoparticles to tumors, less than 1% of intravenously injected nanoparticles normally reach the tumor. This lack of specificity is often caused for concern as metal nanoparticles may exhibit some cytotoxicity (toxicity to normal 40 PENNSCIENCE JOURNAL | Spring 2022
cells) that could cause further damage to the human body.
Figure 2: bismuth forming a lattice matrix with organic molecules to rearrange the environment containing tumors.
Conclusion It is important to highlight how nanomedicine has contributed to medicine as a whole and could potentially influence future approaches to treating diseases. With possibly new diseases emerging that we may have never seen before, it’s critical that we learn all the methods and fundamental knowledge that we can in a field such as nanomedicine to adapt and respond to these future scenarios. Nanomedicine along with other rising trends such as AI machine learning and the increased digitalization of healthcare may soon predominate medicine, so it is crucial to understand its tenets.
References
AS THE OPIOID EPIDEMIC CONTINUES TO WORSEN, REFERENCES
“More than 107,000 deaths were reported in the United States between December 2020 to December 2021”
“Overdose deaths hit a historic high in 2020.”
“The epidemic affects every state”
“Kansas saw 54 percent increase in drug overdoses during 1st half of ’21.“
“SAN FRANCISCO opioid epidemic: Fewer people died of overdoses in 2021 than 2020, but crisis still unprecedented.”
ADDICTION RESEARCH IS MORE IMPORTANT THAN EVER.
GREENBAUM, SIMMONS, HAURY, AND EISCH, STUDY How Drug-linked Memories Impact Emotionality and Drug-seeking BEHAVIORS IN an Experimental Model of Opioid Abuse Spring 2022 | PENNSCIENCE JOURNAL 41
RESEARCH
THE MEMORY TRACE OF ADDICTION: HOW DRUGLINKED MEMORIES IMPACT EMOTIONALITY AND DRUG-SEEKING IN AN EXPERIMENTAL MODEL OF OPIOID ABUSE Raena E. Greenbaum, Steven J. Simmons, William R. Haury, Amelia J. Eisch
Children’s Hospital of Philadelphia Research Institute; Abramson Pediatric Research Center; 3615 Civic Center Boulevard; Philadelphia, PA, USA.
ABSTRACT While efforts have been made to interpret the emotional effects of drug-taking and drug-seeking in humans, there is currently a gap in research on these affective changes in animal models for opioid use disorders (OUDs). Indeed, one way to measure emotional changes in a model of OUD is to study ultrasonic vocalizations (USVs) from the drug-taking rat, as two main frequency ranges are interpreted to reflect positive (50-kHz) and negative (22-kHz) affect. While used in studies on other drugs of abuse like cocaine, this study is the first to use USVs as a measure in the self-administration of a prescription opioid and during recall of drug-linked memories. We put rats (n=66) through intravenous self-administration of the prescription opioid oxycodone by training them to press a lever for intravenous drug infusions during behavioral sessions. This was followed by an extinction of the drug-taking behavior in either the drug-linked context or a novel context. This paper examines the results of two studies. The first was an analysis of emotionality over the course of the acquisition phase, which involved recording drug infusions earned, lever presses made, and USVs emitted by self-administering rats to characterize affective states associated with the development of addiction. We found that rats will experience greater negative affect early in acquisition, and greater positive affect in later acquisition sessions, and that a positive anticipatory affect will be experienced when the rats are exposed to the drug-paired context before the start of the session commences. The second study involved recording USVs emitted by rats during one single extinction probe test with different levels of drug-memory linkage to assess whether context and new action-outcome learning impact emotionality. The analysis of this extinction data is in progress, results of which hold important insights for understanding the neurobiology of drug-linked memory retrieval.
INTRODUCTION
experiences typified in opioid addiction, preventing such dependence has proven to be a serious clinical There has been a sharp increase in the prescription challenge and is an important research focus in the rate of opioids due to their unmatched capability to face of the current epidemic. relieve pain. However, their highly addictive qualities have led to severe consequences that have proven When patients were given a list of prescription opidifficult to address. Roughly 21-29% of patients pre- oids and asked to identify the one most desirable to scribed opioids for pain have reported misusing them themselves, the one most desirable among drug-using (SAMHSA 2017), and 75% of heroin users reported communities, and the one they deemed most addicthat their first opioid was a prescription drug (Cicero tive, oxycodone was ranked most highly in all catet al. 2014). Each day over 130 people in the Unit- egories (Remillard et al. 2019). Due to its immense ed States die due to overdose on opioids (SAMHSA “likability” in users, oxycodone has one of the highest 2017) and as of October 16, 2017, the United States potentials for abuse and dependence compared with government declared the opioid epidemic a public other prescription opioids. Subjective effects of the health emergency. high of a drug like oxycodone may contribute to the extent to which the drug is sought after initial use and It is clear that people do not take prescription opioids thus informs measures such as proclivity to opioid with the intention to become dependent; rather, the abuse as well as relapse propensity. Because relapse effect of the drugs includes intense emotional experi- is one of the main challenges in combating addiction, ences that are craved after the high wanes. Because an important next step in research is to understand much is still unknown about the dynamic subjective the subjective effects of prescription opioid use. 42 PENNSCIENCE JOURNAL | Spring 2022
RESEARCH Experimentally, opioid abuse disorder (OUD) can be modeled in rats by training them to “self-administer” intravenous oxycodone infusions following a behavior such as lever pressing as an operant response. We know that when given the opportunity, rats willingly and continually self-administer oxycodone (Bossert et al. 2018, Mavrikaki et al. 2017), which is known as the acquisition phase. Once drug is no longer available, as rats learn that a lever press will not lead to an infusion, a behavioral process described as extinction will occur. This refers to the elimination of the target response when its reinforcing consequence, in our case the oxycodone infusion, is no longer presented (Rey et al. 2020). The extinction occurs as a result of new action-outcome learning, which is the realization and learning that pressing the active lever will no longer lead to an infusion of oxycodone. Animal models are vital in the development of novel therapies to manage OUDs, but while efforts have been made to interpret the emotional effects of drug-taking in humans (Remillard et al. 2019), there is currently a gap in research on these affective changes in animal models for opioid use disorders (OUDs). One way to measure emotional changes in animals is to analyze the animals’ ultrasonic vocalizations (USVs) as has been performed in prior work (Burgdorf et al. 2000). In rats, USVs are emitted in two frequency ranges that are interpreted to reflect positive (50-kHz; e.g., “tickling” [Panksepp and Burgdorf 2000], electrical stimulation of brain reward structures [Burgdorf et al. 2000]) and negative (22-kHz; e.g., electrical footshock [Tonoue et al. 1986], sight of predator [Blanchard et al. 1991]) mood states. USVs have been used in several ways to study substance abuse, including the response to the presentation of drug-related cues, experimenter- and self-administered drug, drug withdrawal, and relapse tests (Barker et al. 2015). Emotionality in response to other drugs of abuse such as heroin, cocaine has been studied through the use of USVs (Avvisati et al. 2016, Simmons et al. 2018, Mahler et al. 2013), and other studies have demonstrated that rats become dependent on oxycodone in a self administration model of abuse (Bossert et al. 2018, Mavrikaki et al. 2017). Yet, oxycodone has never been a focus of acquisition and extinction USV studies, which is an essential gap in knowledge given oxycodone’s essential contribution to the opioid crisis in the US (CDC, 2017). Our study uniquely contributes an understanding of the emotionality behind oxycodone dependence which further drives opioid-taking behavior. We performed two studies to address these knowledge gaps in the
field by recording and analyzing USVs during drug acquisition and an extinction probe trial. Study 1 will add important information by characterizing the subjective effects of acquisition of oxycodone dependence in a self-administration animal model of abuse. Through Study 1, we had four main goals. The first was to compare positive and negative affect in early acquisition (day 1), the second was to compare positive and negative affect later in acquisition (days 7 and 13), the third was to examined a positive anticipatory affect before later sessions (days 7 and 13) commenced, and the fourth was to check for the presence of a correlation between the number of 50kHz calls and total number of infusions. Study 1 holds important insights for creating effective preventative screening methods for OUD as well as clinical treatments for diminishing opioid dependence and protecting against relapse. Study 2 will test how drug linked memories impact emotionality during the extinction of self-administration behavior by recording and analyzing USVs during a single-session extinction probe test, where drug is not available. Through Study 2, we had two main goals. The first was to examine the emotional experience of undergoing extinction in the drugpaired context compared to a novel context. The second was to examine the emotional effect of new action-outcome learning, represented by the pressing of the previously active lever not leading to a drug infusion. This study holds important implications for understanding the neurobiology of drug-linked memory retrieval in order to develop mechanisms targeted to mitigate the salience of relapse-promoting druglinked memories.
METHODS Animals The subjects were male Long-Evans rats (N=66) (Charles River Laboratories; Wilmington, MA, USA) aged 6-8 weeks upon arrival. Rats were kept on a 12h:12h light cycle (lights on at 7:00AM), with humidity and temperature controlled, and access to food and water ad libitum. Rats were initially pair-housed and given 5-7 days to acclimate before receiving jugular vein catheterization surgeries. After surgery, rats were switched to single housing. Two groups of rats were used for these studies, Study 1 rats (n=14; Eisch Lab designation M2.1&3.1) were used for Acquisition data, Study 2 rats (n=52, Eisch Lab designation M3.1, 3.2, 4.1, 4.2, 8.1) were used for Extinction probe trial data. All procedures were approved by the InstituSpring 2022 | PENNSCIENCE JOURNAL 43
RESEARCH tional Animal Care and Use Committee of The Chil- mL) was infused through the catheter of each rat to dren’s Hospital of Philadelphia. maintain catheter patency. All rats acquired self-administration behavior for 3 hours sessions, 6 days per Surgery week for 18 days, similar to a prior study (Bossert et A catheter (SAI Infusion Technologies) was surgically al. 2018). Catheter exit ports attached by PE-50 tubimplanted into each rat’s right external ing to the oxycodone-dispensing syringe. Each active jugular vein. Each catheter was connected to a dorsal lever (retractable) press would result in a dose of oxystainless steel exit port. The rats were initially anes- codone (0.10 mg/kg/inf during acquisition days 1-12, thetized with isoflurane gas anesthesia (5% induction, 0.05 mg/kg/inf during acquisition days 13-18), deliv1-3% maintenance) per the Anesthesia and ered over 3 s, paired with a ~7-kHz auditory tone. DiAnalgesia guidelines of Institutional Animal Care and rectly after the infusion the active lever would retract Use Committee (IACUC) at a flow rate of 1.2 – 1.5 L/ for 20 s to prevent overdose. Presses on the inactive min mixed with O2. Meloxicam (2 mg/kg, 10 mL/kg, lever resulted in no drug infusion but were recorded. s.c.) was used as a preoperative analgesic. The rat’s dorsal neck area and underarm were shaved and ster- Extinction Probe Test ilized using alcohol and iodine wipes. Then, with the The extinction probe test used in Study 2 was one sinrat lying on its ventral surface, an incision was made gle hour-long session, occurring in the same operant on the rat’s mid-scapular region using stainless steel chambers as acquisition. A new cohort of rats (Cosurgical scissors. Blunted scissors were used to sepa- hort 2) was used that had also underwent the same rate connective tissue. Then, the rat was flipped onto self-administration procedure. Two different contexts its dorsal surface, and an incision was made along (A and B) were used, half the rats acquired drug in the underarm area. The catheter tubing was pushed each. Context A included stainless steel bar floorunder the skin from the exit port at the dorsal insin- ing, a house light, fan on, and start time was 9:00am. cision until reaching the jugular vein, where a needle Context B included solid plexiglass floors, above-le(22-gauge) was used to puncture the vein and the end ver lights, fan off, and start was time 2:00pm. Half of the catheter was inserted. Wounds were closed us- the rats extinction probe test in the same context in ing surgical staples (9-mm, Braintree Scientific), and which they acquired drug (context sequence of AA rats were given 8-10 days to recover before beginning or BB), and half experienced the test in the novel conbehavioral sessions. text (context sequence of AB or BA). Rats assigned to the drug-context group had their catheters tethered to Self-administration the original tubing, whereas rats assigned to the novAll rats (both Studies 1&2) were taught to self admin- el-context group did not. ister oxycodone. Intravenous oxycodone self-administration sessions were conducted in operant chambers Ultrasonic Vocalizations (USVs) (MED Associates) consisting of a plexiglas enclosure Condenser microphones (UltraMic200K; Dodotron(29.53 H cm x 24.84 W cm x 18.67 H cm) with met- ic; Italy) were placed on top of the plexiglass, facing al bars as flooring. Inside, there are two levers (one down into the operant chambers. USVs were recordretractable, one stationary), a house light, cue lights ed at a sampling rate of 192-kHz (twice the maximum above the levers, a fan, an automated syringe pump, frequency detectable by the microphone). During and an auditory tone generator (Sonalert). Outside, acquisition, USVs of Cohort 1 were recorded of the syringes containing oxycodone solution are connect- entire 3 hour session once during week 1 (day 1 or 2 ed to tubing run through a metal spring leash for pro- referred to as “day 1”), once during week 2 tection against chewing, which attach to the catheter (day 7 or 8 - referred to as “day 7”), and once during exit ports on the back of the rats. The retractable lever week 3 (day 13, 14, or 15 - referred to as “day 13”). was used as the active lever and the stationary lever USV recording began 5-10 min prior to the start of was used as the inactive lever. All elements are con- the self-administration session to constitute an “anticitrolled by MED-IV software (Med Associates; Fairfax, pation” time epoch. During the extinction probe trial, VT, USA) on a desktop computer next to the cham- USVs of Cohort 2 were recorded during the duration bers. The physical context of the chamber used for of the one hour session. acquisition was metal bar flooring, house light on, fan on, and a start time of 9:00AM. USVs were analyzed using RavenPro (Cornell LabAt the start and end of each session, a saline flushing oratory of Ornithology) sound analysis software prosolution containing heparin and enrofloxacin (~0.1 gram which creates spectrograms of audio recordings 44 PENNSCIENCE JOURNAL | Spring 2022
RESEARCH for manual scoring of USVs and provides details such as frequency range, duration, and power of each manually scored USV. USVs greater than 15 ms in duration were included in analysis. USVs with a mean frequency from 18- to 32-kHz were considered “22-kHz calls, while USVs with a mean frequency of 38 kHz or greater were considered “50-kHz calls”. “50-kHz calls” that took place within the first 10 minutes of each session were considered “anticipatory calls”.
divided into 4 quartiles, each composed of 45 minute time bins of infusions to characterize drug taking over time within individual acquisition sessions which had not been shown in this model before. We found a main effect of quartile on infusions earned on all 3 days we looked at: day 1 [F(2.250, 27.00) = 4.330, p < 0.0200], day 7 [F(1.738, 20.85) = 2.998, p < 0.0778], and day 13 [F(2.263, 29.42) = 4.904, p < 0.0118]. On day 1, post hoc pairwise comparisons showed a statistical difference in infusions earned in quartile 1 and Statistics quartile 3 (p < 0.05), [t(12) > 2.480, p > 0.0171]. On All data were analyzed using Prism 8 software. Be- day 7, post hoc pairwise comparisons showed a statishavioral data (infusions, active presses, inactive press- tical difference in infusions earned in quartile 1 and es) were initially analyzed using a repeated-measure quartile 3, (p < 0.05), [t(12) > 0.7482, p < 0.0319] and mixed effects linear model to test for a main effect quartile 2 and quartile 3, (p < 0.05), [t(12) > 4.464, p of “Session”, reported in F values. Study 1 (Acquisi- < 0.0361]. On day 13, post hoc pairwise comparisons tion) dependent variables were: Time (Self Adminis- showed a statistical difference in infusions earned in tration, D1-D18); Time within Session (Self Adminis- quartile 1 and quartile 2, (p < 0.05), [t(13) > 4.896, p < tration, D1, 7, 13); Time (USVs, D1, 7, 13). If a main 0.0193] and quartile 2 and quartile 3 (p < 0.05), [t(13) effect was detected, Sidak-adjusted post-hoc pairwise > 4.734, p < 0.0237] (Figure 2B). comparisons proceeded against the respective Day 1 value, reported in t values. Quartile analyses of with- STUDY 1: USV data at 3 timepoints during in-session infusion data compared four 45 minute acquisition time bins on session days 1, 7, and 13. USV data were When examining the effect of session number on non-parametric, violating the assumption of normali- number and type of USVs produced, there was a main ty. Thus, we used repeated-measures Friedman tests, effect observed of session on 50-kHz USVs [�2(24) = reported in �2 values, followed by post hoc pairwise 8.000, p = 0.0183] (Figure 3A), and of session on 22comparisons, reported in Z values. Familywise alpha kHz USVs [�2(24) = 13.000, p = 0.0015] (Figure 3B). (Type I error) was established at 0.05 for all analyses. Post hoc pairwise comparison showed that there were Study 2 (Extinction) dependent variables were: Time significantly more 50-kHz USVs detected on day 7 (Self Administration, D1-D18), Context (USVs, Drug- compared to day 1 [Z =2.746, p = 0.0181] (Figure 3A), Cx Vs. NovelCx), and Lever Availability (USVs, Lvr and significantly more 22-kHz USVs detected on day Vs.NoLvr). Statistic analysis of Study 2 (the extinction 7 compared to day 1 [Z = 0.1890, p = >0.9999] and day 13 compared to day 1 [Z = 3.213, p = 0.0039] (Figure data) is in progress. 3B). The mean value for 50-kHz USVs across all rats for day 1 was 186.2±64.35, for day 7 was 461.8±115.4 RESULTS with a standard deviation of 416.2, and for day 13 was STUDY 1: Infusion and lever press data over the 399.6±106.2 (Figure 3A). The mean value for 22-kHz USVs across all rats for day 1 was 57.25±9.850 with a course of acquisition As seen in past studies (Bossert et al. 2018, Mavrikaki standard deviation of 36.86, for day 7 was 65.79±12.75 et al. 2017), we observed a main effect on all three with a standard deviation of 47.71, and for day 13 was measures of oxycodone self administration: infusions 21.69±6.018 with a standard deviation of 22.52 (Figure [F(2.359, 28.450) = 13.18, p < 0.0001], active lever 3B). presses [F(2.520, 30.38) = 11.21, p < 0.0001], and inactive lever presses [F(4.154, 49.84) = 2.868, p < 0.0309] There was also a phenomenon of anticipatory USVs earned during the 18 days of acquisition. The aver- seen beginning after the first day of acquisition, charage number of infusions ranged from lowest 10.64 ± acterized by 50-kHz calls made during the first 5 min1.868 (95% CI: 6.607, 14.86) on Day 2 to highest 42.45 utes the rats are placed in the chambers but before the ± 4.862 (95% CI: 31.62, 53.29) on Day 16. Post hoc lever extends and drug becomes available. On day 1, pairwise comparisons showed a statistical difference we see very few anticipatory USVs, with a mean of in infusions earned on day 15-18 compared with day 1 only 31.00±17.03 with a standard deviation of 35.04. On day 7, this increases to a mean of 242.1±51.36 with (p < 0.05). [all t(12) > 2.50, p < 0.05] (Figure 2A). The 3 hour session of each acquisition day was then a standard deviation of 39.5. On day 13, the mean Spring 2022 | PENNSCIENCE JOURNAL 45
RESEARCH number of anticipatory calls remains similar to day 7 at 202.0±73.73 with a standard deviation of 195.1. When looking for overall effect, we see that session number does have a main effect on number of anticipatory USVs [�2(24) = 10.29, p = 0.0036]. Post hoc pairwise comparison showed that there were significantly more anticipatory USVs detected on day 7 compared to day 1 [Z = 3.207, p = 0.0040] (Figure 3C). STUDY 1: Correlation between USVs and number of infusions From a scatter plot of number of infusions vs. number of USVs, no significant effect was observed from the linear regression for either 50-kHz calls (P = 0.2572, r = 0.3390, 95% CI -0.2607, 0.7499)(Figure 4A) or 22-kHz calls (P = 0.1771, r = 0.3825, 95% CI -0.1858, 0.7590) (Figure 4B). STUDY 2: Extinction probe test 50 kHz and 22 kHz USV data Analysis of extinction probe test data remains in progress (Figure 7A&B).
DISCUSSION STUDY 1: Lever Presses and Infusions These data indicate that there is an increase in drug seeking-behavior in rats over the course of oxycodone self-administration acquisition, as the rats are clearly differentiating between the active and inactive levers as one being drug-linked and the other not. The significant difference between infusions earned on the first day compared with later days indicates that the acquisition was successful and that the rats are taking substantially more drug after 3 weeks of self-administration. These findings are consistent with prior work with IV self administration of oxycodone (Mavrikaki et al. 2017, Bossert et al. 2019). Additionally, we found that there is a distinct pattern of drug taking within the individual sessions (Figure 2B-E). The significantly higher number infusions are earned in quartile 1 compared to later quartiles indicates the presence of a “load up” period, where the rats take a large number of infusions, and then are more satiated and have a lower, more consistent level of drug seeking throughout the session - just enough to “maintain” the high. This pattern has been consistently observed with cocaine self-administration in rodents (Ettenberg et al. 1982, Wise et al. 1995), and is characterized by “an initial burst of drug intake (loading) followed by more stable infusion rates (maintenance)” (Angarita et al. 2009). This phenomenon was not observed for cocaine use in humans (Angarita et al. 2009), and future 46 PENNSCIENCE JOURNAL | Spring 2022
work may test whether or not it is observed for opioid use in humans. STUDY 1: Ultrasonic Vocalizations (USVs) To my knowledge, this is the first study to assess and characterize the subjective effects of acquisition of oxycodone self-administration by recording and analyzing USVs. Other studies have been done examining USVs with other drugs of abuse, such as heroin (Avvisati et al. 2016), cocaine (Avvisati et al. 2016, Simmons et al. 2018), methamphetamine (Mahler et al. 2013), and bath salts (Simmons et al. 2018). Our study adds to this subset of knowledge by examining USVs with oxycodone addiction, which is an essential gap in knowledge given that oxycodone is one of the most commonly prescribed opioids in the U.S. and one of the most common drugs involved in prescription opioid overdose deaths (CDC, 2017). Through Study 1, we aimed to test four main hypotheses. First, we expected that rats to experience greater negative affect early in acquisition, when first exposed to the experience of oxycodone. Second, we expected to see increased positive affect over the course of acquisition as the operant self-administration behavior is developed and learned. Third, we expected to see an even further positive anticipatory affect when exposed to the drug-paired context before the start of the session commences. More specifically, we hypothesized that predominantly 22-kHz USVs will be observed during day 1, followed by predominantly 50-kHz USVs during the rest of intravenous oxycodone infusions, and that anticipatory 50-kHz USVs will be observed in week 2 and most robustly week 3. Fourth, we expected to see a correlation between the number of 50-kHz calls and total number of infusions, which would inform questions about the correlation between the initial affective experience of taking opioids and proclivity for abuse. Our data support our first two hypotheses that rats will experience greater negative affect early in acquisition, and greater positive affect as the acquisition sessions go on. The main effect observed of session on USVs and session-session comparisons indicates that negative affect, represented by 22-kHz USVs is most prevalent on day 1 and day 7 and decreases in the later sessions, while positive affect, represented by 50-kHz USVs, is low on day 1 and increases after the initial session. When first exposed to the experience of oxycodone, the rats are more likely to have an adverse reaction compared to when they are familiar with the effects and the operant self-administration behavior is developed and solidified. This is an inter-
RESEARCH esting result, as typically 22-kHz USVs are known to emerge during drug withdrawal in models of self-administration (Barker et al. 2015), but are not thought of as being an integral part of acquisition as well. A similar result of some initial negative affect early in acquisition followed by increasing positive affect has been shown in the self-administration of methamphetamine in rodents (Mahler et al. 2013). Our data also support our third hypothesis, that a positive anticipatory affect will be experienced when the rats are exposed to the drug-paired context before the start of the session commences. On day 1 there are almost no anticipatory 50-kHz calls, while there is a high number of anticipatory calls in later acquisition sessions due to the context serving as a conditioned stimulus to elicit the positive affect before drug is even presented. These findings show that anticipation of oxycodone taking elicits positive affective reactions due to learned associations with drug-paired contexts, which is different from what was found when a similar study was done with cocaine, in which both positive and negative affective anticipatory responses were observed when exposed to the drug-paired chamber (Coffey et al. 2013).
oxycodone, and were not compared to a saline-taking control group. However, studies have already shown that cocaine self-administration induces more 50kHz USVs than saline self administering (Barker et al. 2015), and we know that rats self-administering cocaine show a similar acquisition curve to that of oxycodone. Because of this, we decided to focus on a more novel concept: the patterns of emotionality over time throughout the acquisition period. Future studies comparing the USVs of oxycodone-self administering rats and saline self-administering rats are welcomed and needed to address this gap. STUDY 2: Lever Presses and Infusions Incorporating lever presses and infusion data in Study 2 aimed to demonstrate and confirm that rats that underwent the extinction probe trial had acquired self administration behavior as shown in Study 1 and in past studies with Oxycodone (Bossert et al. 2018, Mavrikaki et al. 2017). These data once again indicate that there is an increase in drug seeking-behavior in rats over the course of oxycodone self-administration acquisition, as the rats are clearly differentiating between the active and inactive levers as one being drug-linked and the other not. This is important for the new action-outcome learning we sought to examine through comparing USV data in the presence vs. absence of the active lever. Our findings indicate that the acquisition was successful and that the rats were taking substantially more drug after 3 weeks of self-administration, once again consistent with prior work (Mavrikaki et al. 2017, Bossert et al. 2019).
In contrast, our data did not support our last hypothesis, that we expected to see a correlation between number of 50 kHz calls and total number of infusions. This indicates that whether an individual animal is a particularly high caller or low caller does not necessarily have a relationship with the amount of drug they take. It remains unclear whether this is due to individual differences in call rate or individual differ- STUDY 2: Ultrasonic Vocalizations (USVs) ences in affective experience. While the extinction USV data analyses are still in progress, it is instructive to think about what the posGoing forward, it would be interesting to use similar sible outcomes mean for our study. Through Study data to compare USVs/emotional state during drug 2, we aimed to test two main hypotheses. Our first use relative to USVs emitted during tests of relapse, hypothesis was that we expected that extinction in as such studies hold important insights for creating the drug-paired context will also have an increased clinical treatments for diminishing opioid dependence emotional experience compared to extinction in a and protecting against relapse. Additionally, it would novel context, and expected to see a higher number be interesting to look further at what the difference in of both 50 kHz and 22 kHz USVs. It is clear that the call rate between animals means, and whether differ- physiological effects of drugs of abuse can become ent levels of sensitization to the drug can be represent- paired with contextual information, contributing to ed by USV rates, and if so, how different levels of sen- continued future drug-seeking behavior (Le Foll et sitization impact acquisition, extinction, and relapse al. 2006, Tropea et al. 2008, Kutlu and Gould 2016). rates. I believe that such findings may hold important In humans, we know that relapse to non-medical use insights for creating preventative screening methods of prescription opioids often occurs after exposure to for OUD, as well as clinical treatments for diminishing places previously associated with drug use and it is opioid dependence and protecting against relapse more challenging to wean off an addiction when conA reasonable criticism of Study 1 is that it does not stantly exposed to those places (Wikler 1973, O’Brien address the effect of the drug, given that all rats took et al. 1992). In rats, many studies have demonstrated Spring 2022 | PENNSCIENCE JOURNAL 47
RESEARCH context-induced relapse using a contextual renewal paradigm which involves a reinstatement probe trial in the acquisition context after extinction in a druglinked or alternate context (ABA vs AAA contextual sequence) (Bouton and Bolles 1979, Crombag and Shaham 2002, Crombag et al. 2008). Thus, investigating the emotional effect of undergoing extinction in the drug-paired context compared to a novel context holds important insights for understanding the neurobiology behind this behavior. Behaviors measured during drug self-administration paradigms such as lever presses have been shown to depend on interactions between several interconnected brain regions. Given that the hippocampus is the primary brain region associated with learning and memory, the perforant path from the entorhinal cortex to the dentate gyrus has garnered a growing body of evidence implicating these areas in coding information related to reward and generating contextual memories of drug-induced reward (Guo et al. 2016, Hernández-Rabaza et al. 2008). Our preliminary data (Figure 7A) suggests that DrugCx rats may produce more 50k kHz calls (positive affect) compared to NovelCx rats. Our interpretation of this result so far is that drug-linked contextual memory retrieval engages the dentate gyrus and associated brain areas to drive a positive anticipatory subjective state and promote drug-seeking behavior. Future research will explore how biochemical and molecular adaptations in these brain regions might play important roles in the behavioral manifestations of emotional affect and drug taking. Our second hypothesis was that that new action-outcome learning, represented by the pressing of the previously active lever not leading to a drug infusion, would have negative emotional affect. We predicted that the presence of the active lever would thus lead to diminished 50 kHz USVs and increased 22kHz USVs, compared to when the previously active lever is not present at all and the new action-outcome learning never occurs. In humans, studies on the role of action-outcome learning on affect indicate that positive events following a specific response are represented as desired outcomes by showing that anticipating positive events triggers the associated response. On the other hand, when that learned association is challenged, negative affect is elicited. We know that rodents, when given the opportunity, will be able to learn and form new action-outcome relationships, although the behavioral affect of such learning is yet to be explored (Laurent and Balleine 2015). 48 PENNSCIENCE JOURNAL | Spring 2022
Several brain structures have been implicated in the memory encoding of action–outcome learning, and studies have shown that the perforant path once again has ties to such behavior as part of an extended circuitry critical for maintaining habitual responding and responding to new stimuli (Corbit et al. 2002, Lex and Hauber 2010). Our preliminary data suggests that new action-outcome learning may lead to increased negative emotional affect, as seen by a possible increase in 22 kHz USVs with the lever present (Figure 7B). We suspect that the extinguishing of the memory association of drug-linked lever and the reward engages the dentate gyrus to drive a negative affective state, which would in turn cause a decline in drug-seeking behavior in response to the new learning. Future research would involve examining the explicit role of the perforant path in promoting this negative effect in order to understand the precise neurological mechanisms behind emotionality in drug-seeking behavior and how such pathways can be modified to diminish these tendencies and protect against relapse.
CONCLUSION Study 1 data collection, analysis, figure creation, and drafting components of the manuscript were completed during BIBB 399. Study 2 data collection, figure creation, discussions, and completion and finalization of the manuscript were completed during BIBB 499. Recommendations for future work on the Study 2 extinction USV dataset (Figures 7A&B) involves the statistical analysis of this data to confirm or deny my given hypotheses. This includes a normality and lognormality tests followed by two-way ANOVA without a repeated measure (given that data are normal) due to the presence of 2 variables (Context and Lever). Then, a post-hoc analysis is required consisting of either a Bonferroni or Tukey test. If significance is found in the interaction, the uncorrected Fisher test should be as a post-hoc analysis. If data are not normal in any test, a non-parametric test such as a rankbased test should be used.
ACKNOWLEDGEMENTS I would like to thank everyone in the Eisch lab for making BBB499 a great experience. Special thanks to Dr. Steven Simmons, my mentor, and Dr. Amelia Eisch, my PI, for all of their help and support in bringing this project to fruition. I am so appreciative for all that I am learning, and am grateful to be a part of the research being done in the lab.
RESEARCH STUDY 1 (Acquisition USV) FIGURES
Figure 1. Study 1: experimental timeline. After arrival and catheterization surgery, oxycodone self-administration training consisted of 18 3-hour sessions, 6 days per week for 3 weeks. During the first 2 weeks (12 sessions), lever pressing at the active lever under a fixed ratio 1 (FR-1) of response was reinforced by 0.10 mg/ kg/infusion of oxycodone. Then, for the remaining 6 sessions, FR-1 was reinforced by 0.10 mg/kg/infusion.
Figure 2. Study 1: Infusion and lever press data over the course of acquisition. (A) Number of infusions (black triangles), active lever presses (black circles), and inactive lever presses (open circles) over 18 days of acquisition of intravenous oxycodone self-administration in male rats. Black solid arrows indicate days of ultrasonic vocalization (USV) recordings. Gray dashed arrows indicate oxycodone concentration change. Plotted data are means. N=14. (B) Average number of infusions divided into 4 time bins (0-45 min, 45-90 min, 90-135 min, and 135-180 min) for days 1, 7 and 13 of acquisition. (C-E) Time of each oxycodone infusion over the 180 minute session on days 1, 7, and 13 for each rat in Mischief 2 and 3. Name of individual rat represented by mischief number, cohort number, and box number. Spring 2022 | PENNSCIENCE JOURNAL 49
RESEARCH
Figure 3. Study 1: USV data at 3 timepoints during acquisition. (A) Number of 50 kHz (N=14), (B) 22 kHz (N=14), and (C) anticipatory 50 kHz (N=7) USVs emitted by individual rats during acquisition day 1 (circles), 7 (squares) and 13 (triangles). Means ± standard error are shown. ** indicates a significant difference (p ≤ 0.05), * indicates a significant difference (p ≤ 0.0015) in a One-Way ANOVA Friedman test.
Figure 4. Study 1: Correlation between 50 kHz calls and infusion #. Scatterplots of number of infusions vs. number of (A) 50-kHz calls and (B) 22-kHz calls on day 1 of acquisition. Each filled circle represents an individual animal (n=14).
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RESEARCH STUDY 2 (Extinction Probe Trial USV) FIGURES
Figure 5. Study 2: experimental timeline and groups. (A) After arrival and catheterization surgery, oxycodone self-administration training consisted of 18 3-hour sessions, 6 days per week for 3 weeks. During the first 2 weeks (12 sessions), lever pressing at the active lever under a fixed ratio 1 (FR-1) of response was reinforced by 0.10 mg/kg/infusion of oxycodone. Then, for the remaining 6 sessions, FR-1 was reinforced by 0.10 mg/kg/infusion. Directly following the 18 days of acquisition, the extinction probe trial consisted of a 1 hour single-session event during which no infusions were given. (B) During the extinction probe test, the active lever was either available or unavailable (Lvr Vs. NoLvr), and rats were either placed in the same context in which they acquired drug or a novel context (DrugCx Vs. NovelCx). In total, there were 4 experimental groups: DrugCx-Lvr, DrugCx-NoLvr, NovelCx-Lvr, NovelCx-NoLvr. (C) To sufficiently differentiate between the two contexts, context A included stainless steel bar flooring, a house light, fan on, and start time was 9:00am. Context B included solid plexiglass floors, above-lever lights, fan off, and start was time 2:00pm. (D) Rats were divided up into two groups and acquired self-administration in either context A or context B. Rats in the DrugCx group extinguished in the same context that they acquired in (context sequence of AA or BB), rats in the Novel Cx group extinguished in the alternate context (context sequence of AB or BA).
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Figure 6. Study 2: Infusion and lever press data over the course of acquisition. Number of infusions (open triangles), active lever presses (black circles), and inactive lever presses (open circles) over 18 days of acquisition of intravenous oxycodone self-administration in male rats. Gray arrows indicate oxycodone concentration change. Plotted data are means. N=66.
Figure 7. Study 2: Extinction probe test 50 kHz and 22 kHz USV data (A) Number of 50 kHz (N=66), (B) 22 kHz (N=66) USVs emitted by individual rats during the extinction probe test (equivalent to extinction day 1) in each context sequence with the active lever available (Open Circles) or unavailable (black squares). Means ± standard error are shown.
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caine-conditioned place preference behavior.” J Neurochem 106: 1780–1790. 39. Wikler, A, Dynamics of drug dependence. 1973. “Implications of a conditioning theory for research and Treatment. Arch Gen Psychiatry”. 28(5):611-6. 40. Wise RA, Newton P, Leeb K, Burnette B, Pocock D, and Justice JB. 1995. “Fluctuations in 41. Nucleus Accumbens Dopamine Concentration during Intravenous Cocaine Self-Administration in Rats.” Psychopharmacology 120 (1): 10–20. Contributions: Study conception and methodology: Simmons, Eisch. Investigation: Greenbaum, Simmons. Data acquisition: Greenbaum, Simmons, Haury. Data visualization/presentation: Greenbaum. Writing of manuscript: Greenbaum. Supervision and critical review: Simmons, Eisch. Keywords: Opioid, Oxycodone, Self-administration, Ultrasonic Vocalizations, Affect, Acquisition, Extinction, Memory, Context, Action-Outcome.
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