PCR - Fall 2021

PC R. Pingry Community Research

Fall 2021 Showcasing the next generation of scientific researchers.

Contents.

Novel Research A Structural Basis for the Catalytic Activity and Unique Functions of Dnase1 Family Endonucleases 7-19 Interleukin-33 Expression is Upregulated in Colon Stem Cells After Irradiation 19 Examining Star Formation in the Evolution of Elliptical Galaxies 20-22 The Post-Transcriptional Role of Igf2bp3 and its Interactions with NF-кB in Cancer 23-26 The Role of Narrative-based Advocacy in Advancing the Talanoa on Climate 26-37 Understanding Protein Interactions with BACE1 38-40

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Reporter Articles 23.46 Cents: Exploring the Development of 12 TET Through History and Understanding the Cosmic Flaw at the Center of All Tuning Systems 41-44 Crime in the Era of COVID-19 45 Maggot Debridement Therapy 46-47 The Incredible Mozart Effect 47 The Perseverance Rover’s Scientific Instruments 48-50 Possible Dinosaur DNA Discovered in Remarkably Preserved Hypacrosaurus stebingeri Cartilage 51-54 Temozolomide in the Treatment of Gliomas 54-55 Oncolytic Viruses in Cancer Treatment 56-57 Transcriptomic Analysis of COVID-19 Patients 58-60 Trisomy Formation Within Cancers 61 Treatment of Metastatic Cancer with Statins 62

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Editor’s Note.

Welcome to the Fall 2021 issue of the Pingry Community Research (PCR)

Journal. We are delighted to showcase Pingry’s top scientific talent, both in terms of research skills and knowledge of scientific concepts and discoveries.

The PCR journal provides students the opportunity to publish novel research. Through a written medium, students demonstrate their in-depth understanding of complex, collegiate-level scientific topics, and their applications in research at Pingry.

The fall edition of PCR highlights work in two categories: reporter articles, which are written by students on a scientific topic of their choosing, and novel research articles, which communicate the findings of novel research conducted by Pingry students outside of school in a myriad of fields.

Through the PCR journal, we hope to spark intellectual curiosity and promote scientific inquiry amongst the next generation of Pingry researchers.

Read, learn, and inquire. Dive into the wonders of Pingry Research through this issue of PCR: Pingry’s foremost journal of scientific research.

Kristin Osika (VI), Editor-in-Chief Caitlin Schwarz (VI), Head Copy Editor Christine Guo (VI), Head Layout Editor

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Editorial Staff.

Editor-in-Chief: Kristin Osika (VI)

Head Layout Editor: Christine Guo (VI)

Head Copy Editor: Caitlin Schwarz (VI)

Layout Editors: Sarah Gu (IV) Leon Zhou (IV) Sriya Tallapragada (III)

Copy Editors: Brian Li (VI) Aanya Patel (VI) Ashleigh Provoost (VI) Max Watzky (V) Mirika Jambudi (V) Hansen Zhang (IV) Ali Santana (IV) Annabelle Shilling (IV) Sophia Odunsi (IV) Sarina Lalin (IV) Gabrielle Marques (IV)

Art Editors:

Allen Wu (VI) Connor Chen (VI) Lleyton Lance (VI) Kelly Cao (IV) Ava Khan (III) Faculty Advisor: Mr. Maxwell

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Novel Research.

A Structural Basis for the Catalytic Activity and Unique Functions of Dnase1 Family Endonucleases

by Kristin Osika (VI), Jon McCord2, Dr. Peter Keyel1 Department of Biology, Texas Tech University, Lubbock, TX Health Sciences Center, Texas Tech University, Lubbock, TX

1 2

Abstract DNA degradation is a key post-apoptotic process which is facilitated by the Dnase1 family of endonucleases. By cleaving DNA, these enzymes mitigate the aggregation of highly inflammatory chromatin microparticles; thus, they play a critical role in prevention of multiple human autoimmune diseases, such as lupus erythematosus and parakeratosis, which result from DNA accumulation. While Dnase1 has been extensively investigated, structural questions remain surrounding the other Dnase1 family enzymes, Dnase1L1, Dnase1L2, and Dnase1L3. All of these endonucleases degrade DNA, but each is distinctly localized to specific parts of the human body, and each exhibits unique structural characteristics; these structural features have not been thoroughly examined in silico to date, but they could provide unique insight into the enzymatic activity of Dnase1 family members and their crucial role in human health.

We computationally predicted structures for Dnase1L1, Dnase1L2, and animal homologs of Dnase1L3; to attain high accuracy, we utilized a three-track neural network approach, comparative modelling against Dnase1 and the newly-crystallized human Dnase1L3, and Rosetta-based energy minimization. Based on these models, here we uniquely illustrate the structural basis for Dnase1L2 catalysis and highlight the stabilizing impact of Dnase1L2’s distinct proline rich domain. We also elucidate the structural implications of catalytic residue discrepancies between human Dnase1L3 animal models, which provide renewed support for alternative catalytic mechanisms across Dnase1 family members. Finally, we identify contact residues facilitating Dnase1L1-EtpE binding, a process which mediates Ehrlichia chaffeensis endocytosis, and thus pathogenic entry as a whole; our molecular Dynamics (MD) simulations isolated Dnase1L1-EtpE binding and Dnase1L1 activity to within 45Å of the cell membrane.

Our findings reveal distinct structural mechanisms underlying the unique functions of

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multiple Dnase1 family endonucleases, and in the future, our work could provide the basis for future endonuclease-based therapeutics and interventions beneficial to human health as a whole.

cations Mg2+ and Ca2+, is inhibited by Zn2+, and cleaves DNA into 3’-OH 5’-P oligonucleosomal fragments (1). Furthermore, Dnase1 family members share common structural characteristics, such as a N terminal signal sequence and two histidines which are vital for catalytic activity (2); however, in vitro and in vivo studies have also identified unique functions, domains, and characteristics specific to each Dnase1 endonuclease. Their respective absences are responsible for the onset or progression of inflammatory human autoimmune

Introduction DNA degradation precludes the aggregation of highly inflammatory nucleic acids after cell death. The Dnase1 family comprises four distinct endonucleases, Dnase1, Dnase1L1, Dnase1L2, and Dnase1L3, which all degrade DNA. Each Dnase1 family member requires divalent

Figure 1: Dnase1L1 and Dnase1L2 homology models illustrate folding and distinct protein-specific domains a. PyMOD model of Dnase1L1 b. PyMOD model of Dnase1L2 c. Robetta model of Dnase1L1 d. Robetta model of Dnase1L2

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Figure 2: Comparison of Dnase1L2 and Dnase1L2 ΔPRD structures Dnase1L2 is in pink. Dnase1L2 ΔPRD is in teal.

diseases specific to locations within the human body. Dnase1 is highly expressed in the digestive tract and blood (3) while Dnase1L3 can be found in myeloid cells in the blood, liver, and spleen (4) (5) (6); both are linked to Systemic Lupus Erythematosus (SLE) (3). Dnase1L1 is found in skeletal and cardiac muscle (3) and has been tentatively associated with Pompe’s disease (7) and muscular health (8), while Dnase1L2 is largely present in keratinocytes (9) and is associated with parakeratosis (10). Structural insight into the Dnase1 family has been limited. Of the Dnase1 family, only Dnase1 has publicly available crystallized structures. Dnase1L3 was recently crystallized by the Sutton lab at Texas Tech University Health Sciences Center; their structures are awaiting publication. In contrast, the structures of Dnase1L1 and Dnase1L2 have not been solved, leaving questions about the structural basis of their catalytic activity and unique functions unanswered.

rich domain (PRD) and optimal catalytic activity at an acidic pH. While the PRD has been observed to have no effect on catalytic activity (11), the function and stability of the PRD, along with the potentially correlated structural basis for Dnase1L2’s low pH optimum, have not been identified. Since Dnase1L2 is active at an acidic pH rather than a neutral pH, it also may employ an alternative catalytic mechanism. There have been two proposed mechanisms for Dnase1 family endonuclease catalytic activity which may apply: one suggests that two histidines play a role in acid-base catalysis, while the other implicates D168 as the catalytic base in Dnase1 (12). Previous mutagenesis indicates that both histidines and D168 are catalytically required; however, D168 could also be a divalent cation coordinating residue.

Similarly, the catalytic mechanism may be different for atypical Dnase1L3 variants expressed by other organisms. To compare Dnase1L3 catalytic sites across species, we first generated a

Dnase1L2 is distinguished by both its proline

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Table 1: Energy Scores of Dnase1L2 and Dnase1L2 ΔPRD. All scores are rounded to three decimal places.

Relaxed energy scores are the average of 30 trials.

multiple sequence alignment (MSA), intending to predict the catalytic residues of six specific animal homologs which displayed distinct sequence variation from human Dnase1L3 (Figure 4). The MSA showed that all DNA binding contacts and cation contacts in Dnase1L3 were conserved by sequence location across species, but residues at the same sequential position of human Dnase1L3 catalytic histidines varied dramatically. Of particular interest are the owl monkey and box turtle variants, which are each missing nearly the same stretch of residues, which includes the first catalytic histidine, a residue present in all other homologs (Figure 4). Homology modeling based on the recent Dnase1L3 crystal structure could provide insight into the catalytic potential of these homologs. Understanding each variant’s catalytic potential could elucidate the molecular evolution of Dnase1L3 and the Dnase1 family in general.

Unlike other Dnases, Dnase1L1 has a glycosylphosphatidylinositol (GPI) anchor on its C-terminus which permits membrane binding (13).

In macrophages, Dnase1L1 uniquely facilitates the endocytosis of Ehrlichia chaffeensis, a tickborne rickettsial pathogen. It binds to EtpE, an Erlichia receptor protein, potentially via a “zipper mechanism” (14); however, the structural basis for this protein-protein interaction, and thus for pathogenic endocytosis of Ehrlichia as a whole, has yet to be identified. (15).

Using computational analysis, including homology modeling, molecular dynamics, and Rosetta-based protein energy minimization, our study investigates the unique structural

features of the Dnase1 family. It concurrently details these features’ proposed implications on Dnase1 enzyme catalytic activity and protein-specific functions. We hypothesized that the Dnase1L2 PRD located off of its core nuclease domain would prove energetically (and thus structurally) unfavorable, but we found that this domain actually enhances molecular stability. Though we proposed that catalytic site differences between Dnase1 and Dnase1L2 would account for Dnase1L2’s acidic pH, key catalytic and DNA binding residues are nearly identical among the two. Similarly, we comparatively investigated catalytic sites of Dnase1L3 animal homologs. We illustrate the active site conformation of key catalytic residues in these variants, and specifically note the surprising folding pattern and catalytic residues of the box turtle variant. We additionally confirmed our hypothesis that Dnase1L1 would function within near-membrane proximity, as it was simulated to remain within 45 Å of the plasma membrane. Finally, binding contacts between Dnase1L1 and Ehrlichia protein EtpE were identified. Overall, our results illustrate distinct Dnase1 family structural features in silico and their functional implications, and they may have significant applications in human health.

Results Homology modeling predicts structures for Dnase1L1 and Dnase1L2 Recent advances in computational technology permit the prediction of unsolved protein structures using homology modeling. Here, two preeminent approaches, PyMOD and Robetta, were used to generate putative structures for

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Table 2: Residues near the DNA binding site in Dnase1 and Dnase1L2. Text colors are based on the residue colors in Figure 3.

Dnase1L1 and Dnase1L2 (Figure 1). Though Robetta uses RoseTTA fold, a fairly comprehensive, three-track neural network approach (16), PyMOD-based (17) models had the advantage of additionally including Dnase1L3 structures recently crystallized by the Sutton Lab, which are not yet available online. PyMOD modeling based on the Dnase1 and Dnase1L3 structures generated a more compactly folded structure for Dnase1L1, whose C-terminus adhered tightly to the rest of the structure (Figure 1a), and Dnase1L2, whose proline rich domain (PRD) also folded closer to the protein (Figure 1b).

In contrast, the Robetta models depict an extended C-terminus loop in Dnase1L1 (Figure 1c) and a protruding PRD in Dnase1L2 (Figure 1d). These homology models serve as the basis for our further structural experimentation.

Dnase1L2 proline rich domain enhances protein stability In order to compare the structural stability of native Dnase1L2 with Dnase1L2 lacking its Proline rich domain (Dnase1L2 ΔPRD), we used PyMOD homology modeling to determine a structure for Dnase1L2 ΔPRD, having cleaved the proline rich domain (PRD) from G140 to A160 (Figure 2). Both Dnase1L2 and Dnase1L2 ΔPRD were scored using the Rosetta all-atom energy function and relaxed to identify the most

energetically favored conformation (18). Excepting the removal of the PRD, Dnase1L2 ΔPRD remains otherwise unchanged in comparison to Dnase1L2, as evidenced by the seamless overlay of the respective beta sheets (Figure 2).

Surprisingly, Dnase1L2 demonstrated a lower energy score than Dnase1L2 ΔPRD, and despite its large, protruding PRD loop, this lower score indicates it is the more structurally stable of the two enzymes (Table 1). Dnase1L2’s enhanced stability could explain its generally high expression, specifically in keratinocytes (9), since the PRD does not affect Dnase1L2 catalytic activity (11).

The structural basis for Dnase1L2 catalytic activity and intermolecular contact The structural basis for Dnase1 DNA binding and catalysis has been illustrated (19), but it could benefit from further investigation. Aligning DNA bound Dnase1 (PDB ID: 1dnk) with the homology model of Dnase1L2 (Figure 3) illustrated that all catalytic residues, critical DNA contacts, and cation contacts were positionally conserved in the structure of Dnase1L2 (Table 2) (20) (21).

Though we anticipated structural differences between the Dnase1 and Dnase1L2 catalytic sites, they appear to be nearly identical, so it is unlikely that Dnase1L2’s DNA binding

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domain or catalytic mechanism is responsible for its optimal activity at an acidic pH. Atypical Dnase1L3 animal homologs display varied catalytic residues To extend our understanding from primary to tertiary structure, we homology modeled and relaxed (18) each of the six atypical Dnase1L3 animal homologs, using the newly-crystallized Dnase1L3 structures from the Sutton Lab as our basis. In general, human Dnase1L3 and animal homolog structures aligned closely, while the Rosetta structural relax function further enhanced energetic favorability (Table 3). Aligning with the sequence prediction, the owl monkey variant was missing one beta sheet and alpha helix, corresponding to A144-N182 in human Dnase1L3 (Figure 5a). Of note, the absence of this region did not significantly alter the rest of the folded variant, or the residues located at its catalytic site, because though the owl monkey homolog is missing one histidine, its remaining catalytic residues and critical DNA contacts remain structurally conserved (Figure 6).

In contrast, though the box turtle Dnase1L3 sequence resembled that of the owl monkey, its 3D structure exhibited a surprising secondary structure pattern and catalytic residue positioning. The box turtle variant’s missing region, which sequentially includes residues from D126 to W159 in human Dnase1L3 (Figure 4), does not structurally result in a missing helix or sheet. Instead, the box turtle residues corresponding to K160 onwards still fold in an astonishingly similar pattern to Dnase1L3, despite having a sequence shifted by 33 residues; its relaxed energy score and R.M.S.D are weaker than most of the other variants (Table 3).

As a result, although all box turtle catalytic residues downstream of this shift sequentially align to human Dnase1L3 (Figure 4), they do not structurally align. Therefore, similarity appears to be isolated to a primary structural level, as the hypothesized catalytic residues are not located near the catalytic site and therefore cannot participate in catalysis.

Furthermore, the catalytic histidines structurally align to M150 and L267 respectively.

M150 and L267 are both uncharged and hy drophobic side chains and therefore cannot give or receive a proton in acid base catalysis.

All variants excepting the box turtle displayed catalytic residues consistent with our sequence prediction; these may still be catalytically active (Figure 6). For example, in the chimp homolog, the first catalytic histidine is conserved, and the residue in place of the second catalytic histidine, serine, could still hydrogen bond to other catalytic residues, and participate in catalysis (Figure 6b). The Chinese alligator variant also possesses the first catalytic histidine, but in contrast, its second histidine is substituted for a valine; though the valine is unlikely to be involved in catalysis, depending on the exact biochemical catalytic mechanism in Dnase1s, the variant could still remain active (12). Of particular interest is the fact that all variants possess glutamate and aspartate cation contacts; each one also possesses an aspartate catalytic site residue, and all but

Figure 3: Dnase1L2- DNA Binding. Dnase1 is in lime green. Dnase1L2 is in pale blue. Cation contacts are in purple. Critical DNA binding contacts are in red. Catalytic residues are in blue. Dnase1 residues are darker in color than those of Dnase1L2.

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Figure 4: Multiple sequence alignment of seven different species isoforms of Dnase1L3 Residues are highlighted based on their structural position. Cation contacts are in purple. Critical DNA binding contacts are in red.

Catalytic residues are in blue.

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the box turtle possess a glutamate catalytic residue.Whether these structural facets support the single divalent carboxylic acid mechanism or the principal importance of divalent cation coordination in the active site is not yet clear, but our results indicate the relative importance of these site residues, given their high conservation.

Dnase1L1 membrane binding dictates the range of its catalytic activity and facilitates Ehrlichia chaffeensis pathogenic entry Dnase1L1 binds to the cell membrane via a Glycosylphosphatidylinositol (GPI) anchor. To identify the impact of membrane localization on the range of Dnase1L1 catalytic activity, we conducted a NAMD-based CHARMM36 force field molecular dynamics simulation in explicit solvent in the presence of the physiologically relevant membrane, with Dnase1L1 attached to a GPI anchor embedded in the membrane.

Dnase1L1’s simulated location was consistently within around 45 Å of the membrane, indicating its catalytic activity is isolated within that region as well (Figure 7) (Supplementary Video 1).

In order to predict the structure of EtpE, which has been shown to bind to Dnase1L3 but has not been widely investigated, we ran

a BLAST search to identify sequence homologs. We discovered that a cAMP-dependent protein kinase (PDB ID: 4HPT) exhibited the highest sequence similarity. We then homology modeled EtpE against 4HPT and relaxed its structure. We found that polar contacts between Dnase1L1 and EtpE included hydrogen bonds between Val97 and Gly369 and Tyr452 and Thr66, and therefore we predict that these residues are critical to the EtpE-Dnase1L1 binding interface (Figure 8). Identification of the key binding residues is critical to targeted disruption of EtpE-Dnase1L1 affinity, which could prove critical to therapeutic intervention.

Discussion Here we demonstrate in silico the structural characteristics of Dnase1 family nucleases which contribute to their crucial catalytic activity and specialized cellular functions. We employed homology modeling as the basis for our investigation, generating novel structures for Dnase1L1, Dnase1L2, and six animal homologs of Dnase1L3. We found that the Dnase1L2 proline rich domain stabilized the molecular structure. We also identified the structural mechanism for Dnase1L2 catalytic activity, and its similarity to Dnase1. We further investigated

Figure 5: Comparison of Animal Homologs to Human Dnase1L3. The specific human Dnase1L3 region missing from the owl monkey (T122-W159) and box turtle (D126-W159) homolog sequences is highlighted in red. a. Owl monkey Dnase1L3 (grey) and human Dnase1L3 (black). The owl monkey Dnase1L3 magenta loop consists of four residues to either side of the missing region, for illustrative purposes. b. Box turtle Dnase1L3 (brown) and human Dnase1L3 (black).

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Table 3: Dnase1L3 Animal Homolog R.M.S.D. and Energy Scores. Energy scores are rounded to three decimal places.

catalysis by modeling Dnase1L3 animal homologs, in which we observed the structural implications of catalytic residue discrepancies between human Dnase1L3 animal models. Most prominently, the Box turtle variant was missing a stretch of residues which included its first catalytic histidine, and this variant’s tertiary structure folded differently than our primary structure-based prediction. Finally, we propose contact residues between Dnase1L1 and EtpE that mediate Ehrlichia chaffeensis endocytosis.

Proteins with PRDs are known to be difficult to crystallize; often, the PRD must be cleaved to successfully execute the crystallization process.

Homology modeling demonstrated the positive impact of the PRD on energetic favorability and therefore structural stability, indicating it likely exists for a specific function. Though the PRD does not impact on the catalytic activity of Dnase1L2, its purpose has yet to be identified (11). We hypothesize the PRD may be an SH3 binding domain (22) and hope to investigate this possibility as a course for further study.

Our Dnase1L3 animal homolog homology models also had the unique advantage of utilizing crystallized human Dnase1L3 structures as their basis. These structures reveal unique folding patterns which were not recognizable

simply by sequence analysis, and which could have broader significance in understanding Dnase1 family molecular evolution. Notably, the box turtle Dnase1L3 was of particular interest due to its unique folding mechanism, which resulted in distinctive catalytic residues unlike those of any other variant. These results raise new questions as to Dnase function and folding. The varied residues substituted for the second catalytic histidine in many of the homologs, coinciding with the conservation of aspartate and glutamate cation binding residues, and conservation of separate aspartate and glutamate catalytic residues, could support an alternative biochemical basis for Dnase catalytic mechanism as well. Here we also illustrated that the residues likely involved in Dnase1L2 DNA binding and catalysis are nearly identical to those in Dnase1. Understanding specific structural facets driving DNA-degrading activity can open new doors for therapeutics or have alternative applications in human health.

Our simulation of membrane-bound Dnase1L1 activity demonstrated the membrane proximity which is likely necessary for DNA degradation. This specific membrane localization is a distinguishing feature to Dnase1L1, though further studies are needed for confirma-

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tion. Furthermore, homology modeling EtpE and docking it to Dnase1L1 uniquely illustrated key residues involved in the interaction.

Because Dnase1L1-EtpE interaction is absolutely critical for the pathogenic endocytosis of Ehrlichia chaffeensis, the residues we illustrated could serve as therapeutic targets to prevent the human disease Ehrlichiosis.

One shortcoming of homology modeling is its failure to perfectly account for unique do-

mains. For example, many of the animal homologs, most obviously the owl monkey variant, possess lengthy domains (often at the N- and C-terminus) which are not conserved in human Dnase1L3; though none interfere with catalytic activity, these may not be modeled in exactly the correct position. Due to this shortcoming and the generally putative nature of in silico research, further in vitro studies are necessary to validate and expand upon our notable structure-based

Figure 6: Active sites of Dnase1L3 animal homologs Human Dnase1L3 catalytic residues are in blue. Animal homolog residues are in cyan. The human Dnase1L3 backbone is black. Each specified animal homolog has a colored backbone.

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sualized in PyMOL; homology modeling of DNase1L2 ΔPRD was based on the same aforementioned Dnase1 and Dnase1L3 structures.

Dnase1L3 Animal Homologs Sequence and Structure Assessment: We used Clustal Omega to generate the multiple sequence alignment of DNase1L3 animal homologs (23). Structures were generated using PyMOD homology modeling and were based on four crystallized Dnase1L3 chains (unpublished).

Within PyMOD, SALIGN was used to generate the sequence alignment, while MODELLER was used to predict the target 3D structures.

Figure 7: Dnase1L1 is bound to the cell membrane via a GPI anchor Full-atom molecular dynamics in explicit solvent modeled on a phospholipid membrane with lipid components: 74 cholesterol, 72 POPE, and 67 PSM in the upper leaflet and 73 cholesterol, 75 POPE, and 67 PSM in the lower leaflet. The MD setup was performed with CHARMM-GUI, and ran with NAMD in the CHARMM36m force-field. The centroid of the Dnase1L1 core domain is roughly 45 Å from the surface of the cell membrane. For the full simulation, please view Supplementary Video 1.

results. In the future, further understanding the structural facets of Dnase1 family proteins will create new pathways for preventing, treating, and mitigating human autoimmune diseases.

Materials and Methods Homology Modeling Dnase1, Dnase1L2, and Dnase1L2 ΔPRD: Homology models for uncrystallized proteins were generated using PyMOD, a PyMOL plugin (17), unless otherwise specified. Dnase1L1 and Dnase1L2 structures were based on four crystallized Dnase1 structures (PDB IDs: 4awn, 3dni, 1dnk, 1atn, 2a40) and four crystallized Dnase1L3 chains (unpublished).

SALIGN was used to generate the sequence alignment, while MODELLER was used to predict the target’s 3D structure. Cleavage of the PRD in Dnase1L2 was conducted and vi-

Rosetta structural relax: Before testing each of our homology modeling-based hypotheses, we employed Rosetta’s FastRelax and all-atom energy function in order to achieve, assess, and compare energetically favorable conformations of each of our homology models (18).

Dnase1L1 GPI anchor attachment and membrane simulation: We used CHARMM-GUI membrane builder to attach a GPI anchor at the C-terminus to our homology model of Dnase1L1. The GPI anchor was embedded in the membrane using the orientation of proteins in membrane prediction and the Molecular Dynamics input files were generated for NAMD. The simulation was run for 100 ns on the High Performance Computing Center (HPCC) at TTU. Dnase1L1 EtpE binding: A BLASTp search (24) identified sequence homologs for EtpE. We noted the highest sequence identity match with a crystallized structure, which was a cAMP-dependent protein kinase (PDB ID: 4HPT). We used PyMOD to homology model EtpE against 4HPT. SALIGN was used to generate the sequence alignment, while MODELLER was used to predict the target’s 3D structure. We modeled EtpE docking to Dnase1L1 on ZDock (25) The ZDock output was relaxed with Rosetta (see Rosetta structural relax) to account for any residue clashes.

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Figure 8: EtpE-Dnase1L1 binding EtpE is shown in marine blue. Dnase1L1 is shown in cyan. Critical binding contacts are highlighted in red.

Supplementary Video 1: Membrane-bound Dnase1L1 activity https://drive.google.com/file/d/1qcnMBTdJGrHZbqBH36b2NVx0AOkNscDQ/view?usp=sharing Works Cited 1. Shiokawa, D. and S. Tanuma, Characterization of human DNase I family endonucleases and activation of DNase gamma during apoptosis. Biochemistry, 2001. 40(1): p. 143-52.

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Interleukin-33 Expression is Upregulated in Colon Stem Cells After Irradiation

by Mirika Jambudi (V), Ya-Yuan Fu PhD1 Memorial Sloan Kettering Cancer Center, New York, NY

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Abstract Interleukin-33 (IL-33), a pleiotropic cytokine, plays critical roles in intestinal immunity, contributing to tissue homeostasis and responses to infection and inflammation. Extracellular IL-33 can be sequestered by its decoy receptor, soluble ST2 (sST2). While sST2 has been identified as a predictive biomarker of intestinal injury, including graft-versus-host disease and inflammatory bowel disease, the role of IL-33 in intestinal stem cell (ISC) compartment remains unclear. In the colon, the ISC compartment consists of colonic stem cells necessary for ep-

Muscle layer of colon tissue, stained with DiD and DAPI

ithelial regeneration upon damage and niche cells providing growth factors to the stem cells.

Given the inability of traditional two-dimensional (2-D) imaging to precisely evaluate the IL-33-producing cell localization and its relationship to the ISC compartment, we sought to develop an approach using three-dimensional (3-D) microscopy of intact colonic tissue from IL33-GFP reporter mice following total body irradiation (TBI) to analyze the specific locations of IL-33-producing cells within colon after damage. Using this approach, we found that IL33-GFP+ cells were predominantly located at the submucosal layer while others were located at the crypt base during homeostasis.

IL33-GFP+ epithelial cell number significantly increased in the colonic crypt base after TBI. To phenotype IL33-GFP+ epithelial cells in the colonic stem cell compartment at crypt base, Lgr5-GFP ISC reporter mice were used to establish an approach for evaluating the colonic stem cells and niche cells. Using Lgr5-GFP mice with whole-mount immunofluorescence staining, cKit immunostaining labeled Lgr5-GFPcolonic niche cells and Smoc2 immunostaining labeled Lgr5-GFP+ colonic stem cells in the stem cell compartment. Assessment of IL33GFP+ epithelial cells in the stem cell compartment with immunostaining of cKit and Smoc2 demonstrated upregulation of IL-33 expression in Smoc2+ colonic stem cells after radiation injury. These findings suggest that IL-33 plays a role in colonic stem cells after intestinal damage.

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Examining Star Formation in the Evolution of Elliptical Galaxies

by Max Watzky (V), Milenka Men (V), Inimai Subramanian1, Juliana Karp1 1

Astronomy Camp, University of Arizona, Tucson, AZ

Abstract The predominant theory of galaxy evolution holds that elliptical galaxies are formed when two or more spiral galaxies merge with one another. We hypothesized that if elliptical galaxies do indeed form in this way, they would exhibit a low star formation rate (SFR) compared to other types of galaxies, as they had exhausted their reserves of star-forming gas in violent collisions. In order to test this hypothesis, we used a hydrogen-alpha (H-alpha) filter, which isolates light from the ionized hydrogen found in stellar nurseries, to image star-forming regions in multiple galaxies at different stages in the galaxy merger process. Although we are still in the process of drawing quantitative measurements from the data, we speculate that star formation in the sample elliptical galaxy is less pronounced than in the sample spiral and merging galaxies, indicating that it may have been formed in a galaxy merger.

Introduction According to Hubble’s Galaxy Classification System, there are four major types of galaxies: spiral, barred spiral, elliptical, and irregular. Spiral and barred spiral galaxies like the Milky Way were formed by collapsed clouds of gas that clumped together into larger hierarchical structures. Many spiral galaxies exist in structures known as galaxy clusters, which consist of several galaxies gravitationally bound to one another. Within such clusters, spiral galaxies frequently collide and merge with one another. As galaxy mergers take place, high-density regions of interstellar gas collide, creating an environment of enhanced star formation. In these violent events, the merging galaxies burn through their reserves of star-forming gas, meaning that the resulting galaxies would then exhibit relatively little active star formation. Elliptical galaxies, which have well-established

Figure 1: M106 in all visible light

Figure 2: M106 in H-alpha

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Figure 3: Stephan’s Quintet in all visible light

Figure 4: Stephan’s Quintet in H-alpha

low SFRs, match this description, leading some to believe that they are formed in galaxy mergers. Alternative hypotheses attribute ellipticals’ low SFR to ram pressure stripping and thermal evaporation, processes by which spiral galaxies in galaxy clusters and groups lose star-forming gas through interactions with the surrounding medium. The merger theory of elliptical evolution is supported by the fact that relatively few ellipticals are spotted billions of light years away, indicating that they had not yet had time to form via collision in the early universe. In an effort to confirm or refute this theory, we decided to compare the SFRs of spiral and merging galaxies to that of ellipticals, a proven technique which has been used by several prior studies to investigate elliptical evolution. Our comparison was performed by imaging samples of the different types of galaxies using a filter that isolates light in the H-alpha wavelength.

Hydrogen-alpha is a spectral line emitted when electrons in hydrogen atoms fall from the third energy level to the second. This energy level jump is indicative of hydrogen ionization, which sometimes occurs when hydrogen is exposed to newly-formed stars. Thus, by imaging the galaxies with a H-alpha filter, we were able to detect star formation. Using this kind of filter, we took data from the spiral galaxy M106, a group of merging galaxies in Stephan’s Quintet, and the elliptical galaxy NGC 5813. In order to take baseline measurements of the galaxies’ luminosity, we also imaged M106, Stephan’s Quin-

tet, and NGC 5813 with filters that allowed all light from the visible spectrum to pass through.

Results In the full visible spectrum image of the spiral galaxy M106, a central nucleus, an inner disc containing two prominent spiral arms, and two faint outer arms are visible (Fig. 1). By comparison, only the central nucleus and two inner spiral arms are discernible in the H-alpha image (Fig. 2). In both the H-alpha image and the full visible spectrum image of Stephan’s Quintet (Fig. 3, 4), two galaxies with spiral arms, NGC 7318a and NGC 7318b, can be seen undergoing collision at the center. However, the spiral arms of another galaxy above and to the left of the colliding pair, NGC 7319, are only clearly visible in the full spectrum image. A fourth galaxy, NGC 7320, is visible below NGC 7319, but this galaxy is actually hundreds of millions of light-years away from the others, and not involved in the merger. In the full visible spectrum image of the elliptical galaxy NGC 5813, a bright core surrounded by a faint yellow disc can be seen (Fig 5.), but these features are considerably dimmer in the H-alpha image (Fig 6.).

Discussion ​​Due to the use of various telescopes and instruments in this project, our images are of differing quality, meaning that reliably interpreting the data has been difficult, and the effort to produce quantitative measurements of

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star formation is still ongoing. Nonetheless, a few aspects of the results stand out to us. Only some of the features detected in the full visible spectrum image of the spiral galaxy M106 are defined in the H-alpha data (Fig. 1, 2), indicating that star formation is localized to certain regions of the galaxy. By contrast, the features of the colliding galaxies in Stephan’s Quintet, NGC 7318a and NGC 7318b, are well defined in both images (Fig. 3, 4), suggesting a higher SFR in the merging pair than in M106. The fuzzy disc and central core of the elliptical galaxy NGC 5813 are much dimmer in the H-alpha image than in the full visible spectrum image, which we speculate reflects a low SFR compared to the spiral and merging galaxies (Fig. 5,6). If correct, this observation indicates that NGC 5813 may be the result of a galaxy merger. However, because NGC 5813 is located in a small galaxy group, ram pressure stripping or thermal evaporation may have played a role in its low SFR, although the extent of these influences is unclear. All things considered, our results’ qualitative nature, the plausibility of alternative explanations for the data, and the small scope of our project make it difficult to draw a meaningful conclusion. We hope that future quantitative analysis of our data and the conduction of broader surveys will offer greater insight into the evolution of elliptical galaxies.

Methods In order to produce the hydrogen-alpha images of M106, Stephan’s Quintet, and NGC 5813, we took between 10 and 20 two-minute exposures of each target using the 24” Mt. Lemmon SkyCenter telescope, a 16 megapixel CCD camera, and a narrowband H-alpha filter. The images of each galaxy were then stacked together in the Maxim DL astronomy image processing software. We also took images of NGC 5813 and M106 using the 32” Schulman telescope, a 16.78 megapixel CCD, and red, green, and blue filters, which we stacked together to create full color images of the galaxies, which contain light from the entire visible spectrum. The same effect was achieved for Stephan’s Quintet by taking an image using a clear filter, which also allows light from the entire visible spectrum to pass through.

Figure 5: NGC 5813 in all visible light

Figure 6: NGC 5813 in H-alpha

Acknowledgements Thank you to the University of Arizona’s Mt. Lemmon SkyCenter Observatory for access to the telescopes, as well as Don McCarthy and all camp counselors at the University of Arizona Advanced Astronomy Camp 2021 for their guidance.

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The Post-Transcriptional Role of Igf2bp3 and its Interactions with NF-кB in Cancer

by Anuska Agarwal ‘21 Insulin-like growth factor 2 mRNA binding protein 3 (Igf2bp3) is part of a family of RNA-binding proteins that mediate post-transcriptional gene regulation. Insulin-like growth factor 2 mRNA binding proteins (Igf2bps) form stable complexes with target mRNA strands, effectively shielding them from microRNA (miRNA) degradation. They can bind a wide range of mRNAs and therefore have effects on various cellular pathways. Igf2bp3 expression in normal adult tissues is rare. Conversely, its expression in cancerous tissues is implicated in cell proliferation, adhesion, invasion, and migration (1).

I researched Igf2bp3 in the context of investigating genes of interest from RNA-sequencing (RNA-seq) data from mouse models with a RelA Thr505 mutation. RelA is an NF-кB subunit and transcription factor that can promote tumor formation. RelA Thr505 phosphorylation is linked to pro-apoptotic effects in response to replication stress; a RelA T505A mutation prevents Thr505 phosphorylation and therefore promotes tumor cell viability and proliferation. RNA-seq data of the RelA T505A cells revealed a log2(FC) increase of 3.5 for Igf2bp3 mRNA, and this was the focus of my research.

High Igf2bp3 expression has also been correlated with lower median survival rates in patients with multiple tumor types. Figure 1 displays Kaplan-Meier plots of patients with high and low Igf2bp3 expression in renal clear cell carcinoma, renal papillary cell carcinoma, lung adenocarcinoma, and pancreatic ductal adenocarcinoma.

Igf2bp3 expression has been identified in an estimated 80% of tumor types, and its overexpression is frequently associated with aggressive tumors and a higher risk of death (3). Expression varies by tumor type, but it is consistently detected in a majority of cases of lung cancer, germ cell cancer, colon cancer, pancreatic cancer, gastric cancer, liver cancer, and kidney cancer (3,4,5). In contrast, expression is rarely detected in gastrointestinal stromal cancer, leiomyoma, and bladder cancer (3,6). The cause of these variations in Igf2bp3 expression among tumor types is unclear. However, in cancers where it is commonly expressed, overexpression has been suggested as a biomarker for advanced grade tumors. Figure 1 demonstrates how high expression correlates with lower chances of survival in four sample tumor types.

Igf2bp3 binds RNA via four K homology (KH) domains, conserved sequences that are essential for target strand recognition (9). Mutations in Igf2bp3 KH domains are uncommon, but generally inhibit its ability to bind mRNA. Somatic mutations are present in less than 1% of cancer patients (10). Amplification is present in less than 2% of all cancer patients but occurs at a much higher rate in some cancer types (10). Copy number variation (CNV) gain occurs in over 30% of the tumors in autonomic ganglia tissues and pancreatic tissues (8).

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Igf2bp3’s ability to promote the translation of a range of target transcripts means that it can affect a range of cellular pathways. Its role in promoting cell proliferation and migration varies by tumor type. For example, its regulation of the oncogene Myc has been noted in mixed lineage leukemia, B-acute lymphoblastic leukemia, and pancreatic cancer (1,9). It has also been shown to promote the translation of several other common oncogenes, including Ras and Igf1r, and cell cycle factors such as cyclin D1 and cyclin D2 (1).

In the interest of further investigating the up-

regulation of Igf2bp3 in RelA T505A cells, I researched the relationship between Igf2bp3 and NF-кB. Igf2bp3 has been implicated in a regulatory role of RelA expression in glioblastoma, thereby promoting glioma cell migration (11). In renal clear cell carcinoma, Igf2bp3 has been shown to directly interact with the mRNA of all NF-кB subunits and promote their translation. Its overexpression also promotes NF-кB activity through increased phosphorylation of IкBa, an inhibitor of p50 and RelA (12). In addition, Igf2bp3 has been identified as a tran-

Figure 1: Kaplan-Meier plots of high and low expression of IGF2BP3 in various tumor types: a) renal clear cell carcinoma, 1% false discovery rate (FDR); b) renal papillary cell carcinoma, 1% FDR; c) lung adenocarcinoma, 5% FDR; d) pancreatic ductal adenocarcinoma, 1% FDR (2).

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scriptional target of RelA, providing evidence of a positive feedback loop between the two proteins (1,11). Expression of Igf2bp3 has been suggested as a marker for a greater risk of metastasis and lower survival rate in both glioma and renal clear cell carcinoma (13,14). One study suggests that Igf2bp3 expression in renal cell carcinoma correlates with a 42% increase in risk of death and nearly a five times greater risk of developing tumor metastases (15).

These interactions between Igf2bp3 and NFкB are a possible explanation for the significant overexpression of Igf2bp3 in the RelA T505A cells that I initially observed. The upregulation of RelA may contribute to increased expression of Igf2bp3, thus promoting the translation of oncogenic transcripts and cell proliferation.

Interactions between Igf2bp3 and NF-кB have been noted in multiple cancers where Igf2bp3 serves as a prognostic for advanced grade tumors, metastases, and lower survival rates. Further research is required to elucidate the nature of these interactions, their contribution to tumor formation and metastases, and whether similar interactions exist within other tumor types.

Works Cited 1. Mancarella, C., & Scotlandi, K. (2020). IGF2BP3 From Physiology to Cancer: Novel Discoveries, Unsolved Issues, and Future Perspectives. Frontiers in cell and developmental biology, 7, 363. https://doi.org/10.3389/fcell.2019.00363 2. Nagy A, Lánczky A, Menyhárt O, Győrffy B. Validation of miRNA prognostic power in hepatocellular carcinoma using expression data of independent datasets, Scientific Reports, 2018;8:9227 3. Burdelski, C., Jakani-Karimi, N., Jacobsen, F., MöllerKoop, C., Minner, S., Simon, R., Sauter, G., Steurer, S., Clauditz, T. S., & Wilczak, W. (2018). IMP3 overexpression occurs in various important cancer types and is linked to aggressive tumor features: A tissue microarray study on 8,877 human cancers and normal tissues. Oncology reports, 39(1), 3–12. https://doi.org/10.3892/or.2017.6072 4. Tang, H., Wei, Q., Ge, J., Jian, W., Liu, J., Zhong, L., Fu, B., & Zhao, T. (2013). IMP3 as a supplemental diagnostic marker for Hodgkin lymphoma. Human pathology, 44(10), 2167–2172. https://doi.org/10.1016/j.humpath.2013.04.011 5. Pryor, J. G., Simon, R. A., Bourne, P. A., Spaulding, B. O., Scott, G. A., & Xu, H. (2009). Merkel cell carcinoma expresses K homology domain-containing protein overexpressed in cancer similar to other high-grade neuroendocrine carcinomas. Human pathology, 40(2), 238–243. https://

doi.org/10.1016/j.humpath.2008.07.009 6. Yamamoto, H., Arakaki, K., Morimatsu, K., Zaitsu, Y., Fujita, A., Kohashi, K., Hirahashi, M., Motoshita, J., Oshiro, Y., & Oda, Y. (2014). Insulin-like growth factor II messenger RNA-binding protein 3 expression in gastrointestinal mesenchymal tumors. Human pathology, 45(3), 481–487. https:// doi.org/10.1016/j.humpath.2013.10.010 7. Xu, W., Sheng, Y., Guo, Y., Huang, Z., Huang, Y., Wen, D., Liu, C. Y., Cui, L., Yang, Y., & Du, P. (2019). Increased IGF2BP3 expression promotes the aggressive phenotypes of colorectal cancer cells in vitro and vivo. Journal of cellular physiology, 234(10), 18466–18479. https://doi.org/10.1002/ jcp.28483 8. Tate, J. G., Bamford, S., Jubb, H. C., Sondka, Z., Beare, D. M., Bindal, N., Boutselakis, H., Cole, C. G., Creatore, C., Dawson, E., Fish, P., Harsha, B., Hathaway, C., Jupe, S. C., Kok, C. Y., Noble, K., Ponting, L., Ramshaw, C. C., Rye, C. E., Speedy, H. E., … Forbes, S. A. (2019). COSMIC: The Catalogue Of Somatic Mutations In Cancer. Nucleic acids research, 47(D1), D941–D947. https://doi.org/10.1093/nar/ gky1015 9. Wächter, K., Köhn, M., Stöhr, N., & Hüttelmaier, S. (2013).

Subcellular localization and RNP formation of IGF2BPs (IGF2 mRNA-binding proteins) is modulated by distinct RNA-binding domains. Biological chemistry, 394(8), 1077– 1090. https://doi.org/10.1515/hsz-2013-0111 10. Gao, J., Aksoy, B. A., Dogrusoz, U., Dresdner, G., Gross, B., Sumer, S. O., Sun, Y., Jacobsen, A., Sinha, R., Larsson, E., Cerami, E., Sander, C., & Schultz, N. (2013). Integrative analysis of complex cancer genomics and clinical profiles using the cBioPortal. Science signaling, 6(269), pl1. https:// doi.org/10.1126/scisignal.2004088 11. Bhargava, S., Visvanathan, A., Patil, V., Kumar, A., Kesari, S., Das, S., Hegde, A. S., Arivazhagan, A., Santosh, V., & Somasundaram, K. (2017). IGF2 mRNA binding protein 3 (IMP3) promotes glioma cell migration by enhancing the translation of RELA/p65. Oncotarget, 8(25), 40469–40485. https://doi.org/10.18632/oncotarget.17118 12. Pei, X., Li, M., Zhan, J., Yu, Y., Wei, X., Guan, L., Aydin, H., Elson, P., Zhou, M., He, H., & Zhang, H. (2015). Enhanced IMP3 Expression Activates NF-кB Pathway and Promotes Renal Cell Carcinoma Progression. PloS one, 10(4), e0124338. https://doi.org/10.1371/journal.pone.0124338 13. Gobbo, A. D., Vaira, V., Ferrari, L., Patriarca, C., Cristofori, A. D., Ricca, D., . . . Ferrero, S. (2015). The Oncofetal Protein IMP3: A Novel Grading Tool and Predictor of Poor Clinical Outcome in Human Gliomas. BioMed Research International, 2015, 1-10. doi:10.1155/2015/413897 14. Tschirdewahn, S., Panic, A., Püllen, L., Harke, N. N., Hadaschik, B., Riesz, P., Horváth, A., Szalontai, J., Nyirády, P., Baba, H. A., Reis, H., & Szarvas, T. (2019). Circulating and tissue IMP3 levels are correlated with poor survival in renal cell carcinoma. International journal of cancer, 145(2), 531–539. https://doi.org/10.1002/ijc.32124 15. Hoffmann, N. E., Sheinin, Y., Lohse, C. M., Parker, A. S., Leibovich, B. C., Jiang, Z., & Kwon, E. D. (2008). External validation of IMP3 expression as an independent prognostic marker for metastatic progression and death for patients with clear cell renal cell carcinoma. Cancer, 112(7), 1471–1479. https://doi.org/10.1002/cncr.23296

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The Role of Narrative-based Advocacy in Advancing the Talanoa on Climate

by Natalie DeVito (VI) “Talanoa is a traditional word used in Fiji and across the Pacific to reflect a process of inclusive, participatory and transparent dialogue.”

Key Question: What factors currently prevent unified action against climate change and its impacts? What is the power of community advocacy based in narrative to surmount humanity’s patchwork of differences and lead us to a response to the threat of climate change?

Climate change is the most pressing existential threat facing humanity in the 21st century. It is often seen as an interminably advancing physical process, but a community’s experience of climate change cannot be encompassed solely by corporeal changes. It is in fact intimately entangled with the belief systems and religions they follow (1), with the gradually accrued cultural contexts and perceptions that surround them (2), and with the resources and privileges available to them, which determine their relative share in the “unequal ‘distribution of pain’” wrought by climate change (2). The first part of this research will examine the disparate patchwork of differences which so fragment the response of our species to climate change. Myths and religious narratives ascribe meaning and morality to climate events, leaving room for individuals to deny or ignore the reality of anthropogenic climate change. Issues of inequity and access ensure a further fragmentation of experience of climate change and undermine a sense of solidarity among different peoples. Social contexts such as history, political messaging, and models of understanding and action shape our perceptions and attitudes, and limit the range of options our communities are likely to pursue. Within this context, scientists, advocates, politicians and religious leaders have all struggled to find a cohesive, universally accessible plan to halt climate change. The second part of this research will propose that advocacy steeped in emotional expression and personal narrative can reframe the above factors which complicate a response to the indisputable threat that climate change poses. It can leverage the far reach of religion to help communities adapt, and raise questions about the allocation and even the definition of resources our communities need to combat climate change. Community climate advocacy can synthesize diverse cultural perspectives and advance concrete action. Community advocacy based in narrative can surmount humanity’s patchwork of differences and create a response to the threat of climate change.

Religions and myths augment the realities of rising sea levels and intense episodic weather with diverse meanings, ranging from moral judgment to proof of our dominion over the earth. For us to understand how these diverse belief systems determine how cultures across the world respond differently to the threat of climate change, we must realize that “the idea of climate exists as much in the human mind and in the matrices of cultural practices” as in the physical world, accord-

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ing to scholars of comparative religion Willis Jenkins, Evan Berry, and Luke Beck Kreider (1).

Storytelling, myth, and religion grew out of our desire for etiological explanations for things we did not understand, and today religious institutions construct a meaning of climate change, no less than they have constructed a meaning of death. Religious agents use the values of a particular religious tradition to determine the relationship between ourselves and our conditions, and therefore the extent of our responsibility for climate change (1). This prismed interpreting of climate change by different religions not only refracts one real issue into a conflict of ideas, it entrenches this conflict in moral prerogative.

By examining the individual religions that have dominated and shaped the histories of different world cultures, we discover deeply fragmented perspectives on climate change. For example, Christianity is “the dominant religion of the North Atlantic societies that developed fossil-fuel industrialism” (1). In light of America’s enormous singlehanded contribution to global CO2 emissions and our long entanglement with Christianity, ascribing moral sense to climate change is threatening. Under a Christian treatment, responsibility for the sin of condemning whole communities, species and ecosystems to extinction will come to rest on our shoulders.

This religious entanglement can help to contextualize the reticence of some to acknowledge and address climate change, and the contention of the issue in the United States overall.

Meanwhile, in Polynesia, where atoll communities like Kiribati are projected to be mostly underwater by the end of the century (3), a reservoir of flood myths shape public perception (4). Myths recorded from Tahiti, Raiatea and the South Island of New Zealand share common elements of punishing gods; virtuous, individualized survivors; and new covenants with the creator. The resulting message that disaster operates according to moral order, and that virtuous behavior can avert such disaster, runs counter to the reality that no one on Kiribati will be left unscathed by a sea level rise of

1.1 to 2 meters by the end of the century. One traditional retelling of the Tahitian flood myth concludes that even after a cataclysmic flood, “[l]ook, Tahiti is still existing, lush and luxuriant (transl)” (4). In the South Island myth, a king’s covenant with the creator god takes the form of a kahukura or rainbow (4). The belief of inevitable renewal of land and life again clashes with scientists’ findings that unless or even if we slash our carbon output immediately, the effects of climate change will be irreversible. This comforting mythic backdrop, however, shapes how Pacific Islanders experience the threat of climate change. A recent survey on Kiribati found that 20% of responders cited faith in God as their reason for being unconcerned about climate change: “the Creator made Kiribati and no one can change that so nothing can drown our islands. It was God’s promise that there won’t be big floods,’’ (5). Religious faith is therefore a potent vessel for avoidant behavior. The belief that God is greater than the real threat of climate change, undermines the urgent need for behavioral change (5).

The I-Kiribati are not the only ones for whom religion is a means of denial. In the United States, some Evangelical Christians undermine the reality of anthropogenic climate change by refuting “the notion that planetary systems are vulnerable to human action” (1).

They believe in God’s gift of a world impervious to the destruction that science proves we have wrought around us. With real consequences, faith and dogma influence whether we accept, ignore, or deny climate change.

Even attempts at interreligious unity on climate have their own flaws. Most major religions agree on a shared stewardship role over the Earth, entrusted to people by God (1).

Representatives of Indigenous belief systems point out, however, that if the wrong message of stewardship is amplified, it can entrench anthropocentric arrogance and the commodification of the Earth (1). A seemingly incontrovertible claim of responsibility is exposed as a disastrous assertion of ownership, which

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legitimizes exploitation. Another common and controversial theme in religious treatments of climate change is the apocalyptic. Biblical literalist Christians believe that the world will end with the apocalypse detailed in the Bible (1). This valence presents Evangelicals with a narrative of preordained cataclysm which severely undermines the perceived self-efficacy of their actions, and even frees them not to act. At the interplay of climate reality and its religious significance is the potential for denialism, anthropocentrism, false hope, and the fragmentation of climate intentions and goals.

Financial barriers to adaptation or relocation, initial effects as disparately felt as they are extreme, and the lack of preparedness of institutions responsible for the vulnerable further fragment humanity’s experience and attitude towards climate solutions. For Native Alaskans struggling to relocate away from eroding coastlines, institutional constraints make the process of relocation prohibitively expensive (6). Kiribati islanders also cite cost as the main factor preventing them from transitioning to more sustainable water resource routines (5). Poverty is therefore a factor in fragmenting our preparation for climate change which spans the global North and South. The best strategies for the preservation of at-risk ways of life, relocation and anticipatory adaptation, are contingent upon wealth, a resource largely withheld from the most at-risk communities.

Experience of the effects of climate change also hinges on the relative privilege of communities. Through the long association between race, wealth, imperialism, and patterns of settlement, indigenous and poor communities have emerged overrepresented in the “10% of the world’s population . . . 10 m or less above current sea level” (6). A sea level rise of upwards of a meter by the end of the century seems unavoidable, and the “land facing inundation is racialized land—land that has been appropriated, settled, cultivated, and distributed through a long history of deeply racialized projects” (7).

Marginalized peoples all over the world have

been effectively condemned to weather the most immediate impacts of climate change on the front lines of sea level rise, while those in wealthy nations like the United States will largely not be affected until long after I-Kiribati and Alaskan Natives have been displaced.

The disproportionate impact of immediate climate change falls on communities whose adaptive capacity and whose role in solutions is lower due to larger inequities. Many I-Kiribati community members recognize the futility of asking Kiribati, where annual CO2 emissions per capita in 2016 were 0.463 metric tons in comparison to the United States’ 15.564 (8), to decrease its own carbon footprint: “What can we do about sea level rise? . . . . Australia and USA don’t even want to sign up to Kyoto. It’s all lip service but no action, this is capitalism” (5). They have minimal influence in world politics, and over the warming temperature and rising sea level that will determine their future.

Rather than advocating doubly hard for I-Kiribati interests, the United States and other big emitters seem to marginalize them all too often.

Meanwhile, Alaska Native communities that once shifted around to follow the bowhead whale and other staple prey, have become settled during the 20th century. Their resulting vulnerability and dependence on government solutions enmires the prospect of relocation, which would once have been simple and natural for them, in bureaucracy and frustration (6). Both the set of resources available to them as they adapt, and their ability to effectively leverage those resources (5), relies on inchoate extensions of support from institutions such as FEMA and the Alaskan and federal government.

Institutions, meanwhile, are stunningly unprepared to act as dynamically and collaboratively as the threat of climate change demands.

Around the world, traditional humanitarian responses to crises are medical relief and refugee aid, along with long term provisions for the return of infrastructure and economic activity to an affected site. Both of these are futile against a chronic geophysical process such as

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climate change; relocation may be the only viable option (6). The current post-disaster legal framework, however, cannot handle that reality. FEMA’s Stafford Act does not cover gradual biophysical properties like erosion and sea level rise, and even in those cases where FEMA attempts to intervene, financial resources are channeled into futile avenues (6).

This absence of necessary bureaucratic structure, “of clear guidelines and criteria . . . [cause] distrust and frustration with state and federal government authorities” (6). As shown, a community’s historical privilege, access to wealth, and place in institutional power structures contribute to determining its individual experience of climate change, and whether it receives the external support necessitated by this historical inequity can negatively impact its experience of solidarity and unity with other cultures and institutions battling climate change.

As well as by religion and inequity, communities’ response to climate change are steeped in the cultural context of recent history, political messaging, communal values and models for understanding and action. How a society responds to natural disasters is not static; it has evolved over centuries of experience and change. For example, Americans’ view of nature as a resistant frontier is a fundamental product of our historical context: our fervent pursuit of manifest destiny and the Wild West (2). Around the world, as for Americans, communal values forged throughout history shape and often limit responses to climate change.

For example, Alaskan Natives struggling to find a site for relocation must consider cultural criteria (6). The I-Kiribati, on the other hand, cite cultural affinity for their homeland in their strong reluctance to leave Kiribati at all: “I don’t want to leave my land and go to a big country” (5). When culture ascribes such value to tradition and permanence, it undermines a community’s ability to adjust to changes in behavior and location, even for their survival.

Political and societal messaging also impact a community’s response to climate change. In

Kiribati, the government has done well to communicate the severe and dramatic impacts of climate change, such as sea level rise. Messaging on the changes in behavior needed at the individual level in order to anticipate these impacts and weather them with resilience, is far less comprehensive. As a result, “existing climate change discourses in Kiribati place responsibility on the government . . . reasserting their paternalistic role” (5). Incomplete political messaging has downplayed the I-Kiribati responsibility to adapt, and left the community inadequately prepared for the threat of climate change.

Shared paradigms for action and understanding are perhaps the most decisive determinant of individual and communal response to climate change. Through climate adaptation specialist Natasha Kuruppu’s detailed study of adaptive behavior in Kiribati, it has become clear that individuals’ “belief in their own abilities to manage water stress play a crucial role in driving intentions to adapt” (5). Belief that one cannot determine one’s own experience leads to fatalism, denialism and avoidant behavior in the face of threat. Increased feelings of self-efficacy, however, promote the intention to adapt and form new behaviors (5). Whether or not one perceives oneself to have a meaningful impact on one’s own experience of the threat of climate change determines how one will act. However, in Kiribati, Kuruppu found that there is limited understanding of what specific behavioral changes can help families prepare for climate change. Under this model of action, without self-efficacy feelings, the potential for adaptive behavior is limited.

Individuals’ responses to climate change are limited not only by their existing models of action, but by models of understanding. Among those surveyed, Kuruppu found that “a majority (75%) had no idea of the causal drivers behind changes to local climatic patterns” (5).

Many I-Kiribati seemed to need to see it to believe it: “They said in the paper that global warming is caused by a blanket covering the atmosphere but because I can’t see the blan-

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ket, it makes it hard for me to fully understand and believe in global warming” (5). In fact, most I-Kiribati respondents did not even identify “anthropogenic climate change” as a factor in changing climate patterns, instead blaming factors that were local and easier to conceptualize, such as their neighbors’ farming practices. A majority were aware of climate change and its consequences, but understanding of its causes and possible solutions, and its impact on water resources, was weak (5). As shown, inadequate models of knowing and understanding can leave a community feeling uncertain and limit its ability to respond to climate change with informed and confident action.

Social and cultural factors such as religion, inequity, and cultural context add a dizzying dimension to the search for a cogent response to climate change. Advocacy based in emotion, expression and personal narrative can engage that same social and cultural complexity to probe for a solution. Historian Uwe Lübken argues that the accelerating effects of climate change “demand a unique mode of historical understanding appropriate to their dramatic pace” (1). Similarly, the growing multitude of subjective factors which makes agreeing and acting in unity on climate so difficult, requires a unique cultural and personal solution to unify them.

Our understanding and response to natural disasters already hinges on subjective emotion and social behavior: “The social dimension of natural disasters is evident . . . in the waves of solidarity and readiness to help that regularly accompany them” (1). Just as solidarity and support are our natural response to natural disasters, an outpouring of expression and narrative will be our way to overcome conflict due to beliefs, resources and contexts, and advance the discussion on climate. We can “integrate ecological integrity and societal well-being”, reaching people and reaching a consensus while aiming to protect the climate from further change (6).

The art of storytelling and the potency of personal narrative can reconcile communities to rapid change and reach those unsure how to

proceed. In Kiribati, women are shrugging off the paradigm of ‘extreme victimization by climate change’, choosing instead to “use their experiences and their voices to tell stories with the aim of creating change on social and political levels” (9). When women in Kiribati employ storytelling as a tool for advocacy, they affirm the truth of the narrative paradigm; every human uses narratives to connect with others and comprehend the world. By communicating its reality in a universally accessible medium, storytelling can share the moral impetus and urgency of climate change with others. The reciprocal exchange of stories is often key. One female I-Kiribati advocate recounts how “instead of telling them, this is climate change, we went out there and said: ‘Did you notice any changes … in your island?’” (9). By sharing narratives and accepting personal stories in return, these women cultivate connections of reciprocity and trust, and transmit knowledge and comfort to those unsure how to proceed.

Another female advocate shares, “I tell personal stories, and those are powerful stories,’ . . . . I don’t like telling the story that Kiribati is hopeless . . . . I also want to tell people that we can do something” (9). To these advocates, storytelling, especially when based in emotion and experience, can transmit hopeful versions of the future, leaving listeners with the sense of self-efficacy which has been discussed as essential for individual behavioral change, especially when cultural paradigms for action fall short.

For the Iñupiat of Alaska, storytelling is of value to whole communities as well. Since worsening erosion mandated the relocation of Point Hope Iñupiat to “New Town”, a settlement two miles from their ancestral coastal village, storytelling has been “a way of maintaining a connection to a disappearing place” (10). In a culture for whom sense of home and place are vital to life, the ability to maintain an identity based no longer in the physical place but in the continually renewing stories of it, is self-preservation.

Storytelling is a critical form of cultural adaptation in the face of existential change, a way for the Iñupiat to turn their surroundings “into

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humanized and inhabited places” (10). Thus, with narrative, they can establish a hardy sense of place in an unfamiliar home. Familiar stories of “the drowning home unified the people into one place . . . . the symbolic relationship between the villagers and the environment remains close to the heart of the telling” although the environment itself has changed (10). In Pacific Island communities, advocates are using “the story of our journey to . . . shape a narrative that paints us not as victims of the climate crisis but as the leaders, the healers . . . we are” (11).

Storytelling can communicate truth and forge trust, project a positive future, shape physical places and even redefine the roles of the marginalized in the fight against climate change.

In advocacy, communities use narrative to react to and typify the urgency and uncertainty of the climate change realities they face. Climate change-related sea level rise threatens indigenous cultural identities by erasing whole landscapes. With that destruction, “what’s going on is [the] destruction of the oldest continuously known people and place. You’re looking at them right now” (10). In this urgent context, when every incremental rise in sea level submerges not only artifacts and areas but the tangible proof of a people’s history, the critical role of storytelling is to declare a community’s continued existence. For Pacific Islanders, this narrative power can manifest itself in a simple statement: “We are not drowning” (11).

Through narrative, they respond to the threat of oblivion with a firm refusal to fade away.

As well as cultures’ fight for survival, storytelling reveals unprecedented changes playing out as communities respond to the encroaching threat of climate change. Stories told since the Point Hope Iñupiat’s relocation to New Town highlight the evolving relationship between themselves and their spiritual surroundings.

Burdened by extreme existential anxiety, the generation that made the move to New Town told stories of restless spirits. As the Iñupiat adjust to New Town and mourn cultural losses, the evolution of their stories mirrors the uncertainty of their environment: “as the Arctic environ-

ment becomes more unpredictable, so do the distribution and behavior of its spirit beings” (10).

Today, the evolution of the Iñupiat people’s adaptive storytelling has entered a new stage. As revealed in emerging folk stories, Alaskan Native communities are, for the first time, “making conscious and unconscious alliances with spirit beings in order to remain rooted in their land” (10). This is a new and necessary bond, absent from previous tales. A spiritual context once regarded as mischievous and alien, perhaps unfriendly, has become a reservoir of comfort and familiarity to help Alaskan Native communities contend with the external stress of climate change. Able to adapt and provide new comfort in the face of extreme change, storytelling maintains a “viable place of cultural survival” for the Iñupiat and others when climate change threatens not only the physical places they know but the cultural traditions better adapted to those vanishing environments (10). Storytelling allows communities to respond to urgency with determination, and adapt dynamically to change.

Storytelling and other narrative modes of advocacy are notable for the way they use emotion and empathy to advance action. In Kiribati, advocates believe that emotions, chiefly communicated through folk songs, can mitigate feelings of helplessness and induce change (12). I-Kiribati songs are emotionally evocative, frequently pairing emotional appeals with calls to action.

On a related note, “the land, in the cultural logic of the I-Kiribati, is . . . an indissoluble, ideal unity” with its people, and thus the vernacular terms for “land” and “people” are the same (12).

Love of both land and people are key to the narrative advocacy of the I-Kiribati. These links between emotions and actions, between people and their homeland, are evident in the way advocates have repurposed the song “Koburake!”.

Its lyrics describe a frigate bird searching for its drowned home, an extended metaphor for the uncertain future of the I-Kiribati. The song, as advocates employ it, first elicits empathy for the I-Kiribati people, then presses the world to translate that emotional concern into

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meaningful action by doing all it can to protect their homeland. A common refrain found in I-Kiribati narratives around climate change is nanoanga, a word which means “giving heart/ feelings/thoughts”, or empathy (12). For them, empathy is foundational to action. I-Kiribati advocates display emotional vulnerability in music and narrative, then “call on other nations to show empathy too” by taking action (12). The title of the song, koburake!, means “rise up!”, a phrase which doubles as an encouragement for the I-Kiribati and a mandate to the rest of the world. For Kiribati and its people must be “lifted up” in part by others. They ask for empathy and action from the world-- and external audiences do feel compassion and motivation to act in response to songs like “Koburake!”.

I-Kiribati performers touring the United States in 2011 reported a “reaction in audiences . . . . “the audience really feel the song” . . . . even moved to tears.” What the I-Kiribati conclude from experiences like these is that external audiences do, in fact, develop empathy and will come to the aid of the land of Kiribati” (12).

Lastly, advocacy based on the emotions and narratives of a community is anti-hegemonic.

It demands that work towards a climate solution be driven equally by all stakeholders in the future of our climate. In Kiribati, external and internal discourses on climate change have mounted up, each canvassing projected consequences. The Kiribati Adaptation Program (KAP), launched in 2003, aims to encourage people to adapt accordingly (12). The most basic discourse, however, remains this: rising sea level is dramatic and inevitable. The I-Kiribati perceive a “hegemonic status, demanding consensus” between the KAP idea of cautious adaptation, and the change climate change will wreak (12). Ultimately, many feel powerless. In Kiribati culture, messages of the future and of coming danger are perceived to have unique power, and the I-Kiribati feel their power being stripped by the discourses inundating them (12). These competing discourses of adaptation and destruction, both, to the I-Kiribati, pervaded with power, are “generally denying

agency to local actors. Accordingly, they combine their critique [of external discourses]. . . with a plea for more attention to be paid to what the people of the Pacific region actually think, say and do” (12). Their advocacy is an anti-hegemonic response not only to the threat of climate change but to the idea of external powers determining the I-Kiribati future.

Ironically, musical composition was once a carefully guarded knowledge on Kiribati, reserved for expert, ritual composers who were endowed with the power to project the future through their music (12). Now folk songs, the opposite of guarded institutional power-knowledge, are being recast as commentary on climate change (12). “Koburake!” has paradoxically been endowed with the future-projecting significance once reserved for elite compositions.

Thus, folk music, the very medium of I-Kiribati advocacy, combats hegemony and champions inclusion. Through musical expression of this sort, the I-Kiribati can “engage with globally circulating discourses of climate change and sea level rise” on their own terms (12). They both express acceptance of scientific projections of existential change, and affirm a determination to act against hegemonic knowledge structures. Community narrative and advocacy such as that of the I-Kiribati demands and allows for the involvement of all voices.

Community advocacy imbued with emotion and personal experience reframes religion as a tool for communities to leverage as they adapt, not as an obstacle to unity and change. In frontline communities, evidence such as the role of adaptive storytelling in Alaska and the link between land and people in Kiribati demonstrates that behaviors exhibited by resilient communities in the face of climate change are rooted in spiritual, if not religious, connections to place.

In the above cases, spiritual value placed on a homeland necessitates the adaptive storytelling or aggressive advocacy which have served to keep both places “alive” (in vibrant and dynamic stories if not physically, in the case of the Point Hope Iñupiat). Community-level adaptive

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behaviors such as advocacy and storytelling are therefore both linked to and fed by spiritual prerogative. Formal statements of such spiritual prerogative ask for behavioral change on a global scale instead, but also carry the narrative and emotional weight that makes community advocacy so potent. Pope Francis demands “an inward change, within the human spirit . . . to hear “both the cry of the earth and the cry of the poor” (1). If spiritual prerogatives both formal and informal are after all linked with narrative and emotion, those elements can strengthen religion into a tool to aid communities in adapting.

Indigenous faith-based responses to climate change are another instance where religion does not serve to fragment, but rather strengthens a response to climate change in which elements of narrative and emotion are present.

Because colonial and imperial systems are the embedded causes of climate change and also of indigenous suffering, “indigenous climate statements tend to show less interest in presenting themselves in alliance with statements of other religious bodies” (1). For indigenous climate advocates, who find themselves and their communities uniquely imperiled by changes such as rising sea level, the question of religious responsibility for climate change is integrated with that of their political and cultural survival (1). So they argue not always for unity but for fairness, for indebtedness and amends.

These arguments are emotionally driven, influenced by the personal and spiritual narratives of indigenous people, and uniquely potent.

Although religious communities may be in conflict over the moral sense of climate change, the most successful religious climate advocates are those who use personal narrative and emotion in their attempts to integrate religious morality with climate change. Confessional thought and literature aims to make climate change matter within the morality of a particular religious community. For Jews, it may be a matter of pursuing justice, aiding the vulnerable, and repaying a debt incurred to earth (1). Muslims may see anthropogenic climate change as disrespectful

to Allah’s revelation of creation, and work to remedy the insult (1). Those Evangelicals who accept that anthropogenic climate change is possible, may see it as God’s ruthless judgment of our extractive society, and what we deserve for believing in a fantasy of “secular salvation” through mastery of nature (1). What these approaches share is an attempt to present responsibility for climate change in a way that can be easily incorporated into religious individuals’ personal narratives. Constructive thought and literature, which attempts to make secular moral sense of climate change by drawing from religious elements, but need not be limited to believers, is similarly effective because it presents moral ideas that appeal to and are incorporated into individuals’ personal narratives.

Again, it is an interplay of religious reasoning and personal narrative which can advance climate action in individuals and communities.

Returning to the religious theme of stewardship discussed earlier, we can examine spiritual traditions where personal narratives reinforce the stewardship role. In these traditions, the idea of stewardship is far more nuanced. The indigenous sense of stewardship and generational responsibility for the planet is not the same as that of Judeo-Christians, who passively received the stewardship role from God. Instead, indigenous communities perceive themselves as in relationships of reciprocity and interdependence with their natural environment.

Furthermore, they reaffirm this commitment through personal narratives, empirical stories of reciprocal and respectful interactions with the world around them. Therefore, personal narratives, not arbitrary prerogatives from God, are the basis of humanity’s role as careful and respectful steward of the earth. The crude idea of God-endowed stewardship which threatens to “reinscribe colonialist ideologies into global environmental politics, . . . objectify, commodify, and put a monetary value on the sacred” lacks the narrative component which supports indigenous traditions of stewardship (1).

Personal narrative can even direct a less fatal-

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istic interpretation of the theme of the apocalyptic in climate change. Inasmuch as climate change is an apocalypse for the anthropocene era, individuals can choose to craft a story of virtue and choice. They can construe the climate change apocalypse not as the inevitable end of time, rather as a time of revelation, decisive action and responsibility, when “God begins the final overthrow of extractive empires, perhaps by drawing all Earth into violent judgment on a corrupt civilization”, and real change away from exploitative capitalism is possible (1).

The cultural experience of climate change presents a significant enough threat to humanity as to be almost religious in nature. The destructive reality of anthropogenic ecological destruction “seems to represent at once the apotheosis of humanity and its eclipse” (1). That we can wreak such major change both advances us almost to the status of gods, and exposes our sinful and destructive nature. To choose to ascribe one of these two possible meanings to climate change, then, is a form of adaptive storytelling to oneself. “To establish the moral value of Earth’s living systems, then, it is … doing a kind of religious work” but also a work inseparable from subjective emotion and personal narrative (1).

Ultimately, religion has fragmented humanity’s experience of climate change and introduced a complicating moral dimension. At its best, however, when religious thought on climate change engages with personal narratives and subjective emotions, it can be a valuable tool.

Narrative advocacy has the potential to combat inequity by reallocating and redefining resources which determine communities’ response to climate change. In coastal Alaska, where neither the Alaskan nor federal government have institutions in place to allocate funds for Native villages to relocate, communities have turned to creative means to raise funds (6). In one village, “efforts to raise additional relocation funds from a climate-change lawsuit against oil, coal, and gas companies” ultimately proved unsuccessful (6), but the attempt is an example of narrative advocacy, which is interested in

emotional issues of justice and fairness, identifying an opportunity for resource advancement Community advocacy around the world urges us to view cultural assets we may not have considered before as meaningful resources in the fight against climate change. For example, indigenous weather prediction strategies, which rely largely on holistic observation, are proving to be more useful as rapid climate change makes years of carefully recorded climate data obsolete in predicting future patterns. While these and other such “diverse knowledge systems of the third world are claimed as heritage that belongs to all humanity, the knowledge about how to apply this diversity is often exclusive to the domain of the people who have developed it” (13). This knowledge, often portrayed as backwards and superstitious, is a resource. Once realizing this, we must “integrate the collective wisdom” of humanity, both formal and informal, in the fight against climate change (13).

The specific sphere of influence of women in least developed nations, while evidence of inequity in and of itself, also proves to be a resource.

In Kiribati, women hold critical knowledge of daily water routines, and how to integrate adaptive behaviors into these routines to carefully steward water resources. As well as their knowledge, though, women’s membership in cohesive community networks such as church and shared chores gives them a means of sharing such critical information (9). Women’s knowledge, and access into community networks, is another resource in the fight against climate change.

Lastly, community advocacy is the kind of advocacy which is strengthened, not stymied, by diverse cultural perspectives. It asks for an inclusive and participatory approach to climate work, with “a shift in paradigm from “top down” strategy to a “bottom up” one in order to value the ideas of those who often go unheard (13). This approach leaves room for different models of action and understanding.

The Alaskan planning group most successful in relocating a Native Alaskan village so far is

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“unique in Alaska in its multidisciplinary and multijurisdictional structure . . . . all voluntarily collaborate to facilitate Newtok’s relocation” (6).

Interaction between different disciplines and models of thought, then, is a strength of such groups. Under the systems for which this sort of climate advocacy calls, diverse perspectives can exist in tandem, thrive on shared knowledge, and adjust for each other’s weaknesses. In Kiribati, one community advocate shares, “we are advocating because we feel there is a need for the international community to hear our voices in Kiribati … our work in Kiribati is really on adaptation, our work internationally is on advocacy for mitigation” (9). Formed out of the emotions, goals and narratives of individuals, such advocacy is uniquely attuned to perceive subtle differences in the way audiences will respond to its work. This type of advocacy can dynamically lace together the different goals necessitated by the different perspectives existing across its areas of work, such as promotion of adaptation strategies internally, and demand for reduced emissions globally, into one comprehensive plan for action and advocacy. Where cultural preparation and context falls short of advancing action, community advocacy savvy enough of these contexts can move the needle.

The human patchwork of myths and religions, differing access to resources, and cultural contexts seems at first to prevent a unified response to climate change. Community advocacy and emotional personal narratives can transmit knowledge and sense of place, typify uncertainty and reveal change, reach others through emotion, and counter hegemonic structures of knowledge and power. We can also begin to address our fragmented perceptions and attitudes toward climate change by searching for a solution in the intersection of religion, inequity, and cultural contexts with the power of narrative and community advocacy.

To humanity, climate change presents “‘a question of ultimacy,’ in the sense that it is a site of conflicting interpretations about what it means to be human” (1). Above, multiple pos-

sible meanings for humanity and our role in anthropogenic climate change have been proposed. There are indigenous narratives, supported by storytelling and contemporary indigenous advocacy, of an interdependence and indebtedness to the earth gone awry. This framing waits only for our commitment to alter the course and carefully steward the earth once again. Unfortunately, there are also the Evangelical interpretations of climate change as an impossibility in the perfect and unchanging world gifted to us by God, or as a pre-ordained apocalypse. There are I-Kiribati villagers who trust that God will protect their island from the coming flood, and others who struggle to believe that their planet is being smothered by a blanket of gas which they can neither touch nor see. There are Americans, whose obsession with the frontier and the subjugation of nature frames anthropogenic climate change as just another barrier broken, another test of the limit of human power over the natural world. For them, how far is too far? What evidence of their irreversible, willful destruction, if any, will be enough to advance meaningful policy or behavioral change? Whichever of these interpretations emerges dominant, it will shape our policy and action during the critical window we have to attempt to mitigate climate change. It is crucial that we understand the different factors at play in fracturing and especially unifying humanity’s response to anthropogenic climate change, if we hope to find that unity, first in consensus on some small shared goal, and eventually, in a way forward that unites humanity against climate change.

Appendix A

Matagi Mālohi

By Fenton Lutunatabua You are matagi mālohi. Strong winds. A symbol of our movement blurring identities, validating purpose and strengthening stewardship over this vanua we are called to protect.

You are stained bark cloth for skin, saltwater chants dancing with the moon and reimagined dreams pacing with the tides.

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You are matagi mālohi. Strong winds from sacred places and revered spaces.

A spiral kaleidoscope of broken coral and memory called to collect, curate and reconcile.

You are matagi mālohi. Strong winds rising up and villaging children.

Brown bodies moulding brown minds. Moving between healer and warrior, you are future ancestors carving visions of liberation we can’t even imagine.

You are matagi mālohi. Strong winds seeking frontline truths in this transcendent talanoa of knowledges.

Masculine culture, feminine consciousness, woven together, lose’d together- wai….donu….

You are matagi mālohi. Strong winds from the four pillars of our fale.

The same ancestors that are calling you to belief will also call you to unbelief, so your faith can take a new form. So you can return to the source and be reminded of the commonality of our plurality.

You are matagi mālohi. Strong winds listening, nourishing, transforming.

We are stewards of gifts from our old people.

Noqu wasawasa, era sa vura, era sa vura, era sa vura (my ocean, they have emerged) Appendix B https://www.youtube.com/watch?v=xOcMLWVNIms Works Cited 1. Jenkins, Willis, et al. “Religion and Climate Change.”

Annual Review of Environment and Resources, vol. 43, 2018, pp. 85-108. Annual Reviews, https://doi.org/10.1146/annurev-environ-102017-025855. Accessed 26 July 2021.

2. Lübken, Uwe, and Christof Mauch. “Natural Disasters in Transatlantic Perspective: River Floods in German and U.S.

History.” Bulletin of the German Historical Institute, no.

35, fall 2004, pp. 99-111, www.ghi-dc.org/fileadmin/publications/Bulletin/bu35.pdf. Accessed 26 July 2021.

3. Ray, Caleb. “Rejecting Reality: Kiribati’s Shifting Climate Change Policies.”

4. Bucková, Martina. “Deluge in Polynesian Mythology.”

Asian and African Studies, vol. 13, no. 2, 2004, pp. 191-97, www.sav.sk/journals/uploads/040214187_Buckova.pdf. Accessed 26 July 2021.

5. Kuruppu, Natasha, and Diana Liverman. “Mental Preparation for Climate Adaptation: The Role of Cognition and Culture in Enhancing Adaptive Capacity of Water Management in Kiribati.” Global Environmental Change, https:// doi.org/10.1.1.457.8121. Accessed 26 July 2021.

6. Bronen, Robin, and F. Stuart Chapin. “Adaptive Governance and Institutional Strategies for Climate-induced Community Relocations in Alaska.” Proceedings of the National Academy of Sciences of the United States of America, vol. 110, no. 23, June 2013, pp. 9320-25, https://doi. org/10.1073/pnas.1210508110. Accessed 26 July 2021.

7. Hardy, R. Dean, et al. “Racial Coastal Formation: The Environmental Injustice of Colorblind Adaptation Planning for Sea-level Rise.” Geoforum, vol. 87, Dec. 2017, pp. 62-72. ScienceDirect, https://doi.org/10.1016/j.geoforum.2017.10.005.

Accessed 27 July 2021.

8. European Commission. Joint Research Centre., et al.

Fossil CO2 and GHG Emissions of All World Countries.

Luxembourg, European Union, 2017. Core, https://doi. org/10.2760/709792. Accessed 27 July 2021.

9. Haughton, Pippa. Women’s Climate Change Advocacy in Kiribati: Vulnerability, Agency and Storytelling. 2020.

Malmö U, MA thesis. Malmö University Electronic Publishing, Malmö University, ls00012.mah.se/handle/2043/32621.

Accessed 26 July 2021.

10. Sakakibara, Chie. “’Our Home Is Drowning’: Iñupiat Storytelling and Climate Change in Point Hope, Alaska.”

Geographical Review, vol. 98, no. 4, Oct. 2008, pp. 456-75.

JSTOR, www.jstor.org/stable/40377348. Accessed 26 July 2021.

11. Woodward, Forest, et al. “Matagi Mālohi : Strong Winds.”

Facebook, 20 Sept. 2020, www.facebook.com/350Pacific/videos/matagi-m%C4%81lohi-strong-winds/621328961898500/.

Accessed 26 July 2021.

12. Hermann, Elfriede, and Wolfgang Kempf. “2 ‘Prophecy from the Past’: Climate Change Discourse, Song Culture and Emotions in Kiribati.” Pacific Climate Cultures: Living Climate Change in Oceania, by Tony Crook and Peter Rudiak-Gould, Warsaw, De Gruyter, 2018, pp. 21-33.

De Gruyter, https://doi.org/10.2478/9783110591415-003. Accessed 26 July 2021.

13. Pareek, Aparna, and P. C. Trivedi. “Cultural Values and Indigenous Knowledge of Climate Change and Disaster Prediction in Rajasthan, India.” Indian Journal of Traditional Knowledge, vol. 10, no. 1, Jan. 2011, pp. 183-89. Niscair Online Periodicals Repository, nopr.niscair.res.in/handle/123456789/11079. Accessed 26 July 2021.

The University of Texas at Austin, 31 Dec. 2019, sites.utexas.edu/climatesecurity/2019/12/31/kiribati-policy-shift/. Accessed 27 July 2021.

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Understanding Protein Interactions with BACE1

by Sriya Tallapragada (IV), Dr. Marc Tambini1 Rutgers Brain Health Institute, Piscataway, NJ

1

Abstract Alzheimer’s disease is the most common neurodegenerative disease in the world, affecting more than five million Americans and amounting to $148 billion in healthcare expenses in the US alone.

Mounting research has shown that amyloid plaques composed of β-amyloid peptides are a factor in Alzheimer’s.The enzyme BACE1 is an initiating enzyme in β-amyloid peptide formation, making it a prime target for Alzheimer’s treatments. BACE1 works by cutting the peptide bonds of the Amyloid Precursor Protein (APP), the longer protein from which β-amyloid is made, at a specific cleavage site. β-Amyloid peptide formation requires two proteolytic cleavages (the hydrolysis of two peptide bonds in a protein): the first is through β-secretase in APP, and the other is by β-secretase within the hydrophobic lipid bilayer. BACE1 is the principle β-secretase responsible for the first proteolytic cleavage. Since BACE1 is important in additional bodily functions, it is challenging for scientists to find an inhibitor that does not inadvertently interrupt other essential processes.

BACE1 is usually found in the Endoplasmic Reticulum (ER) or Golgi Apparatus, which trims proteins involved in neuronal functions. Structurally, the enzyme has two portions: the catalytic domain (at the start of the protein, its N-terminus) that cleaves proteins, and the portion on the end of the protein embedded in the membrane (the C-terminus), which controls direction and regulates enzymatic activity (N-terminus refers to the amine end of the amino acid while C-terminus refers to the carboxyl end). BACE1’s long tail holds the enzyme on a membranous surface, so it does not float freely throughout the cell. A GPI anchor, a posttranslational lipid attachment, targets the enzyme and holds it to lipid raft domains. It also has a deep active site cleft to hold onto proteins and a pair of aspartate amino acids that cut certain protein chains, which are vital to typical cell function. Neuregulin– a protein that helps control the formation of myelin sheaths around nerve axons, and voltage-gated sodium channels, which are necessary for transmitting nerve signals– is cut by the GPI anchor.

While BACE1 is essential to protein maturation, it is involved in disease processes. BACE1 cuts APPn, breaking the protein chain and releasing a small amount of amyloid peptide. While normal amounts of peptides are crucial for normal function, if BACE1 is hyperactive, this peptide can tangle into amyloid fibers and potentially impact nerve transmission, leading to Alzheimer’s disease.

Results Figure 1 shows the names of the compound bonded with the BACE1, the amino acid sequence of the compound, the number of polar contacts, and the distance between polar contacts. Analyzing the interactions between these compounds and BACE1 interactions helps to understand Alzheimer’s disease pathology and potential drug targets by learning how certain inhibitors binding to BACE1 can affect the way it cuts the amyloid fibers.

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Figure 1: Images of compounds and active site of BACE1. Compounds are in purple, residues within five angstroms of the compound are in green, and water molecules are in red

Figure 2 shows BACE1 protein-protein interactions to better understand its basic biology and contribution to Alzheimer’s Disease pathology.

The figure focused on examining these interactions in terms of hydrogen bonds formed and measured the number of polar contacts formed and the distance between them. The compounds 3, 8b, 7, and 6b have a high number of polar bonds formed. Compounds 3, 4-piperidine, and 6b have a short distance between polar contact, meaning the bond formed would be stronger. Understanding the strength of hydrogen bonds can show how well the compounds work in attaching to BACE1.

PDB number 2OHN seems to have the strongest bond formed in relation to the hydrogen bonds of all analyzed PDB.

Conclusion Based on what could be found from the results, PDB number 2OHN forms a stronger hydrogen bond than the other examples. The strength of the inhibitor affects the way BACE1 is able to release certain amounts of amyloid fibers; in the case of PDB 2OHN, the leaving portion of BACE1 will not be able to cut APPn at all, and will not be able to release amyloid fiber. Thus, while studying these interactions between BACE1 and other inhibitors is helpful in learning more about Alzeihmer’s Disease pathology, relying on the inhibitors is not a viable solution in stopping the amyloid fibers being tangled up. After all, a healthy amount of peptide being released from BACE1 cuts is vital for some particularly important neural function.

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Works Cited 1. Hemming ML, Elias JE, Gygi SP, Selkoe DJ (2009) Identification of β-Secretase (BACE1) Substrates Using Quantitative Proteomics. PLOS ONE 4(12): e8477.

2. Cole, S L, and R Vassar. “The Basic Biology of BACE1: A

Key Therapeutic Target for Alzheimer’s Disease.” Current genomics vol. 8,8 (2007): 509-30.doi:10.2174/138920207783769512 3. “Picking through the Rubble, Field Tries to Salvage BACE Inhibitors.” ALZFORUM, 20 Dec. 2019, https:// www.alzforum.org/news/conference-coverage/pickingthrough-rubble-field-tries-salvage-bace-inhibitors.

Figure 2: Chart with information about bonds formed with each compound. In this project, the x-ray crystal structure of BACE complexed with different compounds is modeled.

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Solar system by Kelly Cao (IV) Astronaut by Ava Khan (III)

Reporter Articles.

23.46 Cents:

Exploring the Development of 12- TET Through History and Understanding the Cosmic Flaw at the Center of all Tuning Systems

by Ryan Arrazcaeta (VI) Most people are familiar with the basics of music theory from years of immersion in popular culture at the very least, if not from formal education in it. The solfege method is taught in grade school, and most know the basics of major and minor scales. These are often taken as objective fact: the notes are organized this way because that is simply the way things are. Very rarely is the mathematical foundation behind tonality ever explained. The truth is, despite music as an art being a subjective experience, its mechanics are based in the fundamental truths of physics.

The math informing music has been studied extensively by many cultures across the world and across all of human history. Thus, by exploring some of the mathematics and how they influenced the way music was made and organized over time, one can arrive at a better understanding of this concept of music as a whole.

Whenever an object vibrates, it creates changes in air pressure, which are interpreted by the ear as sound. The frequency at which the pressure changes determines the pitch, which is the lowness or highness of the sound. In the case of a vibrating string, its lowest frequency can be modeled with the equation f=12LTµ, where L is length, T is tension, and µ is linear density (i.e. mass per unit length). This lowest or fundamen-

tal frequency (often referred to simply as the fundamental) is what the ear perceives as the pitch of the note. However, due to the different modes of a string’s vibration occurring at once, other higher frequencies – known as overtones– occur at integer multiples of the fundamental.

These follow a simple harmonic series, meaning that a given fundamental of frequency n has overtones at 2n, 3n, 4n, and so on (Figure 1).

This multiplicative structure for overtones re-

Figure 1: The above diagram shows the vibrations of several common overtones. These different modes of vibration occur during the superimposition of multiple waves, which causes parts to either cancel (nodes) or reinforce (antinodes). This follows Fourier’s theorem stating that any waveform can be synthesized by combining sine and cosine functions of appropriate amplitude and frequency.

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Figure 3: List of the most commonly used modes in western music.

Figure 2: List of frequency ratios for justly tuned intervals. These ratios are derived from the harmonic series of overtones.

veals the nature of frequency versus pitch: the interval between two pitches, such as a perfect fifth, is not the difference between the frequencies, rather it is their ratio. In the case of a fifth, it’s ratio is 3:2 (Figure 2). In a musical context, imagine an A4, which has a frequency of 440 Hz. The frequency of an A5 (an octave above A4) is 880 Hz, which yields a ratio of 2:1 between an A4 and A5: the frequency ratio of a perfect octave.

One of the earliest instrumentations of this math was by the Pythagoreans, the cult following of Pythagoras of Samos, who believed that frequency ratios based on smaller integers (octaves, perfect fifths, perfect fourths) sounded the most consonant or “in-tune.” Greek music at the time used 7 tones, which were often strung onto a lyre (which had fixed strings).

These were set at the intervals calculated by the Pythagoreans to be the most consonant with each other. The intervals closely resembled a modern major scale in what would now be referred to as a single “key”. While, at first, it seems difficult to grasp that one “key” created diverse enough sound to compose an entire musical tradition, the Greeks had a clever way of filling in the emotional range: modes.

By changing the tonic center of a given piece without retuning the instrument, Greek music could have seven distinct emotional “feels”, rather than only one. This concept of modality remains in western music to this day. The most familiar example of modality is the idea

that a key is Major (Ionian mode) or Minor(Aeolian mode) (Figure 3), although Pythagorean modality was distinct from its modern counterpart and has unfortunately been lost to time.

Looking at the math, an observant reader might begin to notice a glaring issue with the Pythagorean tuning system, and potentially with math as a whole. Mathematicians and musicians alike have grappled with this problem for the past 3 millennia. This is the “cosmic flaw” of the harmonic series and it throws a massive wrench in the plans of anyone hoping to devise a musical system.

In order to fully understand the problem, let’s conduct a thought experiment: imagine a grand piano. It has 88 keys spanning from A0 to C8.

It is known that multiplying a frequency by 32 will yield a pitch a perfect fifth above its fundamental (because a perfect fifth has frequency ratio of 32:1), so by starting on a C1 and multiplying by 32, one should end on a G1. By repeating this process 11 more times, one eventually reaches the C8. A musician would recognize this series as simply going around the cycle of fifths. This is just a geometric progression with a frequency of C1 (33 Hz) as the principal value. Plugging these values into the equation fC8=33*(3/2)12yields a product of 4281.63 Hz for a C8. One can check this work by using octaves instead of fifths. As it takes 7 octaves to get from C1 to C8, and knowing that an octave is a ratio of 2:1, the equation fC8=33*27 should also yield 4281.629 Hz, but the math states otherwise. The second equation yields a slightly flatter 4224.00 Hz. To the untrained ear, this difference of

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23.46 cents, not even a quarter of a semitone, is barely perceptible. But the deviation has immense implications on all aspects of music theory. This gap, known as the Pythagorean comma, means that when tuning an instrument with fixed strings, such as a harp or piano, it is impossible for all frequencies to be in tune with each other because their frequencies do not physically add up correctly (Figure 4). Furthermore, it is only possible to tune an instrument “correctly” for a single key, and playing out of that key will be incredibly dissonant.

tuning was a set of tuning systems that tried to rectify the Pythagorean comma by narrowing the ratio of a fifth. For example, the ¼ comma meantone system tempered fifths out of tune by about ¼th of the Pythagorean comma. This improved major and minor thirds, as well as octaves, but the system still had the same major flaw of Pythagorean tuning: modulation.

While modulation was not quite as impossible as in the Pythagorean system, it was still rarely viable. All hope was not lost, however, as the next century would see an innovation in music the world had never seen before.

The year is 1722. The pirate Bartholomew Roberts has been killed in battle, Benjamin Franklin has just written the Silence Dogood letters, and Johan Sebastian Bach has published the Well-Tempered Clavier, a collection of pieces for solo keyboard written in each of the 24 major and minor keys. Perhaps the most important legacy of the WTC was in the tuning instructions.

Bach intended his pieces to be played in equal temperament, a tuning system that claimed to solve the Pythagorean comma dilemma. Although it had early advocates, such as Andreas Werckmeister in the late 1680s, at that point very few people seriously considered such a system.

Figure 4: A visual representation of the Pythagorean comma.

In 1636, a French mathematician named Marin Mersenne published his seminal work Harmonie Universelle in Paris, the largest documentation of music theory at the time, that held hundreds of pages of information on stylistic practices, acoustics, as well as mathematics.

The equation for the fundamental frequency of a vibrating string discussed in the introduction to this report, f=(1/2L)*√(T/µ), was first recorded by Mersenne, who also proposed a potential solution to the Pythagorean comma: meantone temperament. In contrast to the Pythagorean tuning method which emphasized tuning based on the perfect fifth, meantone

But how exactly would the proposed system work? In order for equal temperament to work, it would need 12 equally spaced tones within an octave. Since frequencies are multiplicative, proponents of equal temperament used 2f=f*x12, where f was any given fundamental and x was the common ratio, to find the number to base frequency ratios. Evidently, solving the equation made it clear that using 122 as the basis for equally tempered ratios would work (Figure 5). While equal temperament “fixed” tuning, it presented its own unique set of challenges. As theorized by the Greeks, the psychoacoustic phenomenon of consonance occurs when the overtones of two notes line up. By adjusting the values of intervals ever so slightly, equal temperament made all of the intervals that much less consonant. Equal temperament did not get

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rid of the Pythagorean comma, rather it spread the Pythagorean comma across the octave.

Today, 12 tone equal temperament (12-TET) is the most widely used and taught musical system on the planet. Its presence has influenced centuries of musical composition. Ultimately, the choice of a musical system comes down to an artist’s personal creative vision. With their knowledge of temperaments and musical theory, concert pianists will often request special tunings for their pianos before a performance, as tuning for specific keys will often result in a better sound. For better or for worse, 12-TET’s elegant solution to one of nature’s cruelest jokes stands to represent humanity’s ingenuity and intelligence even in the face of cosmic odds.

Works Cited 1. 1/4-comma meantone / quarter-comma meantone. (n.d.).

Tonalsoft. http://www.tonalsoft.com/enc/number/ 1-4cmt.aspx 2. Beat frequency formula. (n.d.). Toppr. https://www.toppr. com/guides/physics-formulas/ beat-frequency-formula/ 3. Bradley, C. A. (n.d.). Polyphony in medieval paris: The art of composing with plainchant [Google Books].

4. Chapter 4: How scales and intervals REALLY work. (n.d.).

How Music Really Works. https://howmusicreallyworks.com/Pages_Chapter_4/4_2. html 5. Duffin, R. W. (2008). How equal temperament ruined harmony: (and why you should care). W.W. Norton.

6. Equation for sound created from a string. (n.d.). Ron Kurtus’ School for Champions. https://www.school-for-champions.com/science/sound_ from_string_equation.htm#.YGMoFK9KiMp 7. Marin mersenne. (n.d.). Encyclopaedia Britannica Online. https://www.britannica.com/biography/ Marin-Mersenne 8. Russell, D. A., Ph.D. (n.d.). The vibration of a fixed-fixed string. Acoustics and Vibration Animations. https://www.acs.psu.edu/drussell/Demos/string/Fixed.html#:~:text=When%20a%20fixed%2Dfixed%20string,of%20the%20initial%20string%20displacement.

9. Vibrations of a string. (n.d.). The Fact Factor. https://thefactfactor.com/facts/pure_science/ physics/vibrations-of-string-harmonics-overtones/8410/#:~:text=Concept%20of%20Overtones%3A,multiples%20of%20the%20fundamental%20frequency.

10. Winternitz, Emanuel. The Musical Quarterly, vol. 44, no.

4, 1958, pp. 534–540. JSTOR, www.jstor.org/stable/740715. Accessed 30 Mar. 2021.

Figure 5: Table showing differences in frequency ratio between equal-tempered and just-tuned intervals.

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Crime in the Era of COVID-19

by Parth Patel (III) At the onset of the COVID-19 pandemic, concerns about a spike in urban crime rate began to circulate. Potentially, the fear of illness could slow down urban crime or the redirection of the police’s time to enforcing quarantines and curfews could unleash chaos.

In June of 2021, Following the initial surge of Covid-19 cases worldwide, Manuel P. Eisner of The University of Cambridge conducted a global analysis of the effect of COVID-19 stayat-home restrictions on urban crime rates.

Eisner analyzed police records in 27 cities– including, San Francisco, Lima, and London– focusing on the trends for 6 types of crime.

Eisner saw a thirty-seven percent reduction of reported cases of assault, theft, burglary, robbery, vehicle theft, and homicide in the cities.

Conversly, COVID’s impact on homicide and intentional first-degree murder fell by only fourteen percent, which was the smallest decrease out of the types of crime observed. A substantial number of domestic homicides is thought to account for the discrepancy in the crime rates.

Accounts of burglary (entering a building with the intent to steal) declined by twenty-eight percent; however, it is worth noting that some of the cities in the sample do not distinguish between commercial and residential burglary.

When studying the data from the cities that have differentiated the two subsets of burglary, they found that because of the increased presence in homes, residential burglary rates went down significantly. On the contrary, due to the lack of supervision in commercial buildings, there was a smaller decrease in cases of commercial burglary. Reported cases of theft also fell, as they saw a monumental decline of 47% compared to pre-pandemic levels, which is

most likely attributed to less social interaction, therefore less opportunity to steal. As a whole, urban crime rates decreased across all six categories, in spite of less police supervision.

Eisner’s study suggests that preliminary fears of a state of global anarchy due to the Pandmeic are not necessarily warranted. People panicked and worried that if the police fell ill, a reign of lawlessness would increase crime rates. Others believed that fear of falling ill would dissuade large amounts of criminal activity, citing lower crime rates in the winter. With Eisner’s data on decreased crime rates, the question becomes: Why were so many people wrong about this outcome? Why did people expect the worst? Just how can our newfound understanding of crime impact the field of criminal psychology or even the fields beyond that?

Works Cited 1. Nivette, A.E., Zahnow, R., Aguilar, R. et al. A global analysis of the impact of COVID-19 stay-at-home restrictions on crime. Nat Hum Behav 5, 868–877 (2021). https://doi.org/10.1038/s41562-021-01139-z 2. Abrams, David S. “Crime Rates Dropped in 2020—Just as They Did in 1918.” Wired, 14 Jan. 2021, www.wired.com/story/crime-ratesdropped-in-2020-just-as-they-did-in-1918/.

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Gavel by Connor Chen (VI)

Maggot Debridement Therapy

by Sarina Lalin (IV) While most people shudder at the thought of writhing fly larvae, the act of intentionally putting maggots into the human body has led to notable medical advances since some of the world’s earliest civilizations. In maggot debridement therapy (MDT), disinfected maggots are introduced into an open wound with the purpose of eliminating necrotic tissue. Once the maggots are placed onto the wound, they release an enzyme in their excretion that liquefies dead tissue. The maggots then feed on this, leaving behind only healthy, living tissue. As antimicrobial resistance becomes increasingly prevalent, maggot debridement therapy proves to be a promising solution for those suffering from non-healing wounds and diabetic ulcers.

The use of maggots as wound treatment can be traced back centuries to the Aboriginals of Australia and the Mayan tribes in Central America, who regularly used larvae to clean wounds. In 1557, French surgeon Ambroise Paré found that his patients with maggot-infested wounds tended to heal faster and better than those who lacked the larvae. This observation was made several times in the future by surgeons like Baron Dominique-Jean Larrey, who went one step further, discovering that maggots removed dead tissue by feeding on it.

During the American Civil War, John Forney Zacharias purposely exposed maggots to festering wounds after noticing that soldiers with unkempt and maggot-born wounds were more likely to survive. Over the next few decades, interest in maggots and their ability to treat wounds and ulcers heightened, reaching its peak in the 1930s after a study on maggots’ role in removing dead tissue was released by Johns Hopkins University.

This, as scientists call, “golden age of maggots” quickly subsided due to the commercialization of penicillin. Towards the end of the 1980s, maggot therapy gained popularity once again after numer-

Maggots by Allen Wu (VI) ous studies revealed that maggots were a cost-effective alternative to traditional debridement methods. Robert Sherman’s studies, for example, showed that dead tissue was completely removed from 80% of maggot-treated wounds, compared to 48% of surgically debrided wounds. By 2002, maggot debridement therapy was being used by over 2,000 medical facilities, and in 2004, maggots were approved as an official medical device.

From antiquity to the present day, maggots have proven to be a safe and effective method for debriding ostensibly non-healing wounds. Although the stigma surrounding maggots prevents the treatment from reaching its full potential, maggot debridement therapy is an accessible and affordable technology for chronic wounds.

Works Cited 1. Whitaker IS, Twine C, Whitaker MJ, Welck M, Brown CS, Shandall A. (2007, June) “Larval therapy from antiquity to the present day: mechanisms of action, clinical applications and future potential” Postgraduate Medical Journal. https://www.ncbi.nlm.nih.gov/pmc/articles/PMC2600045/ 2. Chan D. (2015, January 17).”Maggot debridement therapy in chronic wound care” Hong Kong Medical Journal. Retrieved from https://www. hkmj.org/abstracts/v13n5/382.htm 3. Gaydos J. (2016, March 30). “History of wound care: Maggots: An extraordinary natural phenomenon” Today’s Wound Clinic. https:// www.todayswoundclinic.com/articles/history-wound-care-maggots-extraordinary-natural-phenomenon 4. Mole B. (2012, December 10). “Maggot Medicine” The

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Scientist Magazine. https://www.the-scientist.com/the-nutshell/maggot-medicine-40066 5. Ngan V. (2005) “Maggot Debridement Therapy” DermNet NZ. https://dermnetnz.org/topics/maggot-debridement-therapy/

6. Medical Maggots™ (maggot therapy, maggot debridement therapy, MDT, biotherapy, biosurgery, biodebridement, larval therapy). (n.d.). Monarch Labs.http://www. monarchlabs.com/mdt

The Incredible Mozart Effect

by Krish Patel (III) In 1993, Frances Rauscher started playing music composed by Mozart to help patients with epilepsy. Epilepsy is a neurological disorder that causes abnormal brain activity that leads to seizures. It impacts one percent of the general population and yet a cure remains elusive. Prior studies have demonstrated that about one-third of epilectic patients experience intracranial interictal epileptiform discharges (IEDs), brief neural discharges that increase the frequency of seizures in people with refractory epilepsy.

The theory presented in the article touches upon the critical emotional response that is able to decrease the IED rate. One truly shocking revelation was that duration of the auditory stimulus actually would determine the reduction of the IEDs. Some directions this study can go in are ponder questions like “Are Mozart K448 and Mozart K545 really the only two songs that elicit the emotional response that combats the IEDs?”, or “Would a playlist of songs that utilize the sixteenth notes be an effective treatment of the future, and would the variEleven researchers from Geisel School of ety of songs have an impact on the IED rate?”.

Medicine at Dartmouth University have shown that Mozart K448 can decrease the frequen- In summation, the experiment demonstrates cy of seizures in refractory eplieptic patients. how Mozart K448 can reduce IED rates in refrac84% of people with refractory epilepsy have tory epilectic patients which decreases the frea significant reduction of IED upon listening quency of seizures. Mozart K448 has been helpto the sonata. In the study, the subjects were ing patients with refractory epilepsy since 1993, split into two groups (Group 15, and Group 90) but its service to these patients can only increase and participated in two different treatments as we learn more about epilepsy and the Mozart that each lasted twenty-five minutes. Using Effect. And maybe, this could be the foundaelectrodes, the scientists tracked the IED dis- tion of a long awaited cure for the one percent. charges. Group 15 listened to the song for fifteen seconds before taking a fifteen second Works Cited break. Group 90 listened to the song for two 1. Quon, R.J., Casey, M.A., Camp, E.J. et al. Musical compominutes, and answered true or false questions nents important for the Mozart K448 effect in epilepsy. Sci for the last 30 seconds, before taking a one Rep 11, 16490 (2021). minute break. The subjects were then played a sound and were asked if it was the same sound as earlier. The researchers found that during the control breaks, there was no significant fluctuation in IEDs. They also found that the IED reduction was dependent on the duration of the auditory stimulus, as the GEE models used by the researchers detailed how Group 90s IED rate decreased more than Group 15s.

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The Perseverance Rover’s Scientific Instruments

by Evan Xie (IV) On February 18th, 2021, NASA’s Perseverance Rover successfully landed on Mars in the Jezero Crater. Following the Sojourner, Spirit, Opportunity, and Curiosity rovers, NASA’s previous Mars missions, Perseverance is tasked with four key missions: determine if life previously existed on Mars, identify the characteristics of past Martian climates, characterize the geology on Mars, and prepare for future human-led missions. With such difficult and important objectives to complete on a foreign planet, it should come as no surprise that this rover features some of humanity’s most advanced and revolutionary technology. Spending a great amount of time and $2.2 billion designing, prototyping, and building the rover, the scientists at NASA’s Jet Propulsion Lab (JPL) were able to pack an astounding seven scientific instruments, alongside a rock sampling system, a tiny helicopter, and more, within Perseverance’s 2260 pound, 10ft x 9ft x 7ft frame (Figure 1). Each scientific instrument combines different technologies to perform their respective experiments on Mars, which will hopefully broaden our understanding of the Red Planet.

First, Mastcam-Z is Perseverance’s intricate camera system that will capture pictures of the Mar-

Figure 1: Image of the Perseverance Rover from the front

Rocket by Ava Khan (III) tian surface and atmosphere while also helping scientists select rocks to core and study. Mastcam-Z is an upgrade from Curiosity’s camera system in almost every aspect: each individual camera can shoot 20-megapixel colored images, rather than 1-megapixel black and white images, contains powerful zoom capabilities to study smaller objects, and has a wider field of view providing Mastcam-Z with a full 360-degree perspective. With these features, Perseverance can photograph and record different terrain features, rocks, soil, and Martian phenomena (dust storms, cloud motions, etc), providing scientists with invaluable visuals to further their understanding of Mars.

Next, the Mars Environmental Dynamics Analyzer (MEDA) is a group of sensors responsible for measuring different aspects of the Martian environment —namely wind speed, temperature, humidity, dust particle size, and radiation levels. This data will be crucial for future manned missions, as weather predictions will protect astronauts in the dangerous Martian environment and weather. For example, intense dust storms can reduce the amount of sunlight reaching solar panels, decreasing energy production and causing equipment shut-downs, just as the Spirit and Opportunity rovers did. Being warned beforehand would allow astronauts to conserve some

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emits a fluorescent X-ray. This can then be analyzed to determine the object’s elemental composition since each element produces its own unique fluorescent X-ray. With this technology, PIXL will be able to identify 26 different elements, including sodium, sulfur, and iron, allowing it to potentially identify signs of past life.

Figure 2: JPL scientists installing MOXIE (car battery sized) in the rover energy and protect their equipment. Additionally, measurements of radiation levels would allow scientists to better understand signs of past life.

As stronger solar energetic particles (SEPs) and galactic cosmic rays (GCRs), two sources of radiation in space, are capable of penetrating the Martian soil, any signs of life near the Martian surface could have been destroyed by gamma rays. If this is the case, scientists would have to collect samples from deeper in the Martian surface to determine if the planet ever housed living organisms.

The fifth instrument is the Radar Imager for Mars’ Subsurface Experiment (RIMFAX), a ground-penetrating radar (GPR) that can detect and map underground rock layers, water, and ice on Mars. All GPRs work in a similar fashion, sending a series of tiny electrical pulses into the ground and then recording the strength and return time of the reflected signal. After numerous pulses are released in a given area, the collected data can be input into special software that produces depth slices, or horizontal cross-sections, at specific depths underground. In the case of RIMFAX, the emitted electrical pulses vary from 150 MHz to 1.2 GHz, enabling a wide variety of scans; higher-frequency pulses can detect smaller targets but cannot penetrate as deep while the opposite is true for lower frequency pulses. In the end, RIMFAX is capable of scanning up to and potentially even past depths of 10m.

Possibly the most intriguing instrument of the seven, the Mars Oxygen In-Situ Resource Utilization Experiment (MOXIE) is a device theoretically capable of generating oxygen from Mars’s 95% carbon dioxide concentrated atmosphere (Figure 2). Using one-third of the rover’s power, MOXIE will use heat and electricity to split carbon dioxide (CO2) molecules into carbon monoxide (CO) and oxygen (O) molecules. Despite this device’s high energy consumption, it is only capable of producing 6 to 10 grams of oxygen per hour, which is not sufficient to support even a single human. Thus, this technology is currently only in a testing phase and much larger versions of MOXIE will be used for any manned missions.

The Planetary Instrument for X-ray Lithochemistry (PIXL) uses a high-resolution camera and an X-ray fluorescence spectrometer to identify the small-scale composition of Martian materials. When PIXL shines a narrow, focused X-ray beam onto a tiny spot of rock or soil, the object

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Perseverance Rover’s SuperCam by Allen Wu (VI)

The next instrument is quite similar to PIXL, in terms of both technology and purpose. SHERLOC, short for the Scanning Habitable Environments with Raman and Luminescence for Organics and Chemicals device, consists of a Raman and fluorescence spectrometer and a laser. Similar to fluorescence spectroscopy, Raman spectroscopy identifies an object’s chemical structure using a laser. Specifically, the laser light reflecting from that object is analyzed by a spectrometer — only the approximate 0.0000001% of light with different wavelengths to the original laser provides data — which is able to identify different bonds (C-C, C=C, N-O, etc) and groups of bonds (benzene rings, lattice modes, etc). Thus, SHERLOC can determine an object’s elemental composition and chemical structure, and this data can be used to detect minerals, organic materials, and potential biosignatures on Mars.

Lastly, the SuperCam is a collection of instruments capable of identifying rock textures, atomic and molecular compositions, and chemical structures of different rocks and soils. Carrying technology from the Curiosity Rover’s ChemCam, the SuperCam can zap objects up to 20 feet away with a laser and analyze the resulting vapor to determine that object’s elemental composition. Furthermore, it can perform Raman spectroscopy to determine chemical structures, Time-Resolved Fluorescence spectroscopy (TRF) to identify intermolecular interactions and Visible and Infrared reflectance spectroscopy (VISIR) to classify the mineralogy of different soils and regoliths.

Overall, these various instruments that consti-

tute the SuperCam enable the rover to study the molecular structure of different samples as well as search for organic materials, all from afar.

With all the data already sent back by Perseverance, it appears that the rover and its scientific instruments are functioning as planned. Now, it is the rover’s responsibility to explore Mars and complete its tasks as we patiently wait and watch.

Undoubtedly, this mission will be vital for future Mars exploration, from increasing our understanding of the foreign Red Planet to preparing for future manned missions. Hopefully, Perseverance will be a major success that we can look back on in gratitude during future Mars missions.

Works Cited 1. Bell, J., Rodriguez-Manfredi, J. R., Hecht, M., Allwood, A., Hamran, S.-E., Luther Beegle, & Wiens, R. (n.d.). Instruments. NASA. Retrieved March 16, 2021, from https:// mars.nasa.gov/mars2020/spacecraft/instruments/ 2. Gifford, S. E. (2014, February 18). Calculated Risks: How Radiation Rules Manned Mars Exploration. Space.

Retrieved March 16, 2021, from https://www.space. com/24731-mars-radiation-curiosity-rover.html 3. Mars 2020 Mission Contributions to NASA’s Mars Exploration Program Science Goal. (n.d.). NASA. Retrieved March 16, 2021, from https://mars.nasa.gov/mars2020/ mission/science/goals/#:~:text=The%20Perseverance%20 rover%20is%20designed,in%20which%20it%20was%20 formed 4. Redd, N. T., & Wall, M. (2021, March 2). Perseverance rover: NASA’s Mars car to seek signs of ancient life. Space.

Retrieved March 16, 2021, from https://www.space.com/perseverance-rover-mars-2020-mission 5. Wood, C. (2021, March 1). With MOXIE, Perseverance will try to make oxygen on Mars. Popular Science. Retrieved March 16, 2021, from https://www.popsci.com/story/ science/moxie-instrument-on-perseverance-mars/#:~:text=Instead%2C%20MOXIE%20takes%20in%20all

Diagram of the rover’s scientific instruments

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Possible Dinosaur DNA Discovered in Remarkably Preserved Hypacrosaurus stebingeri Cartilage

by Adrian Kurylko (V) A nesting ground filled with disarticulated bones of Hypacrosaurus stebingeri, a herbivorous duck-billed dinosaur, was discovered in Montana during the 1980s. Further studies on several limb and skull elements of the discovery were recently performed, and a piece of calcified cartilage located within a supraoccipital, a region in the back of the skull, differentiated itself from the rest. Fossils typically form when an organism dies and after the fleshy parts of their body decompose; the bones are buried in sediment where they harden into fossils. Molecular changes to bones begin immediately after death, making the bones more thermodynamically stable. Collagen, the main structural protein found in the extracellular matrix, is degraded - fluids can then move through the much more porous bones, causing them to crystallize. However, calcified cartilage contains an extracellular matrix that is much less porous than bone, limiting the entry of water or microbes that would cause biodeterioration. As a result, the cartilage may have been preserved at the molecular level. Ground sections of the supraoccipital presented chondrocytes, or cells associated with bone growth, that consisted of darker spots that resemble a nucleus (Figure 1).The calcified cartilage of the Hypacrosaurus stebingeri was compared with calcified cartilage of a young emu to highlight differences between dinosaur and extant, or living, cartilage.

Figure 1: Calcified cartilage of Hypacrosaurus stebingeri compared with calcified cartilage of a young emu. A) A Hypacrosaurus supraoccipital, or a bone located in the skull. B-D) Ground section of calcified cartilage containing hypertrophic chondrocytes lacunae. In figure 1C, the cells shown by the green arrow appear to be empty while the cells shown by the pink arrow present dark, condensed material consistent with a nucleus. Figure 2D shows material with characteristics consistent with chromosomes in metaphase. E) A young emu skull. F-G) Ground section of emu calcified cartilage exhibiting cells similar to those seen in the Hypacrosaurus cartilage.

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The extracellular matrix of extant cartilage and bone can be differentiated using Alcian Blue, or a stain that reacts heavily with acidic material such as acid mucins and glycosaminoglycans. These molecules are found in cartilage but not in bone. Both dinosaur and emu cartilage were compared to demineralized bone of their respective organisms and results support the notion that there are chemical differences between calcified cartilage and bone (Figure 2).

Figure 2: (A, B, E, F) Unstained bone and cartilage of the Hypacrosaurus and emu. (C, D, G, H) Although shown with less intensity, the Hypacrosaurus cartilage shares similar characteristics with emu cartilage, as the Alcian blue stain highlights the acidic material naturally found within cartilage. Both Hypacrosaurus and emu bone presented a lighter stain.

As previously mentioned, collagen is a protein found in the extracellular matrix of chondrocytes, or cells located in calcified cartilage. Assays using collagen II, or the most abundant protein epitope in extant cartilage, were performed on Hypacrosaurus and emu cartilage in an attempt to generate an immune response (Figure 3). The antiserum containing collagen II consisted of chicken protein in order to enhance immunoreactivity due to the limited phylogenetic distance between dinosaurs and chickens. The results prove that collagen II was preserved within the calcified cartilage, as it is not produced by microbes.

In order to test for DNA, the researchers isolated Hypacrosaurus chondrocytes. When photographed, they appeared to be transparent. Similar results were observed with emu chondrocytes.

The cells were tested with propidium iodide, a stain that inserts itself between every four or five base pairs in DNA and is shown in red under fluorescent light. A limited stain was shown, and in order to confirm these results the cells were also stained with DAPI (which requires at least three successive A-T base pairs as a binding site). This trial produced similar results.

The findings of the various experiments performed on the calcified cartilage of the Hypacrosaurus stebingeri prove exquisite preservation at both the histological and molecular levels, with the remnants of chondrocytes and nuclear material consistent with DNA. The discovery of DNA in a fossil suggests that there could be an abundance of the genetic material of a variety of prehistoric animals (such as mammoths or other dinosaurs) throughout the natural world. However, many questions regarding the legitimacy of these findings arise: Could the DNA found belong to bacteria that reside within the bone during fossilization? Are there other molecular aspects of fossilization that account for such a rare discovery? Wouldn’t we have discovered more examples of a phenomenon like this by now? There are still many unknowns, but for now the prospect of obtaining genetic sequences of animals long extinct will become a focus of scientists for years to come.

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Figure 3: (A, B, E, F) The green fluorescence shows antibody-antigen complexes in the extracellular matrix of the calcified cartilage. Immunoreactivity was shown to be more intense in the calcified cartilage of the emu, representing superior functionality. However, antibody activity was decreased after being exposed to collagenase II (C, D, G, H) in both the Hypacrosaurus and emu cartilage, proving that reactivity with collagen II is specific to a protein found in cartilage of both animals. (I, J, K, L) A lab added as a control. Collagen I is not expressed in primary extant cartilage and no binding was observed in either animal.

Figure 4: Hypacrosaurus chondrocytes isolated as individual cells (A), and as cell doublets (B). Both Hypacrosaurus and emu cells displayed positive reactions to PI, a stain that intercalates DNA and highlights it (C,F). DAPI (a different type of stain) produced similar results to PI, with the staining in the emu cells being significantly more intense (D,G).

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Works Cited 1. Keenan, S. (2021). The Timing and Scales of Fossilization.

Fossilization - an overview | ScienceDirect Topics. Retrieved October 8, 2021, from https://www.sciencedirect. com/topics/earth-and-planetary-sciences/fossilization.

2. Bailleul, A., Zheng, W., Horner, J., Hall, B., Holliday, C., & Schweitzer, M. (2020, January 12). Evidence of proteins, chromosomes, and chemical markers of DNA in exceptionally preserved dinosaur cartilage. National Science Review.

Retrieved October 3, 2021, from https://academic.oup.com/ nsr/article/7/4/815/5762999.

3. Black, R. (2020, April 17). Possible dinosaur DNA has been found. Scientific American. Retrieved October 3, 2021, from https://www.scientificamerican.com/article/possibledinosaur-dna-has-been-found/.

Dinosaur by Allen Wu (VI)

Temozolomide in the Treatment of Gliomas

by Mirika Jambudi (V) Accounting for more than 78% of brain cancers (AANS) and almost 15,000 casualties a year (AANS), gliomas are among the most common forms of brain cancer. Characterized as intra-axial tumors (because they interfere with brain tissue), glioma develops in glial cells, including astrocytes, ependymal cells, and oligodendrocytes. Traditionally, these glial cells support and surround nerve cells in the brain (2). The specific glioma cell type involved in the tumor determines the tumor’s genetic characteristics and can help predict various treatments’ efficacy (3). A variety of factors are considered when determining treatment, which may include a combination of chemotherapy, radiation, or surgery. Treatment also depends on the tumor’s grade, ranging from “low grade” or “high grade.” This grade is determined by the tumor’s aggressiveness and growth rate according to a predetermined scale. Developing alternative and targeted therapies has posed a challenge to scientists because glioma tumor cells are protected by the blood-brain barrier

(BBB), which has highly selective permeability.

A typical therapy used for many forms of cancer is alkylating agents. They are used in cancer therapies because they have the ability to prevent a cell from replicating by inflicting damage to the cell’s DNA (3). Alkylating agents are also able to permeate the BBB, making it an optimal choice. Temozolomide is a typical alkylating agent used for treating glioblastoma multiforme, a type of glioma originating in the astrocytes.

Glioblastoma multiforme is the most aggressive form of brain cancer and is characterized by its high invasiveness and mortality rates.

Temozolomide is typically used in conjunction with radiotherapy and then as a monotherapy. It has been a standard chemotherapy drug used to treat gliomas since 2000. Being an alkylating agent, its mechanism of action is its ability to add methyl groups to DNA after conversion into MTIC. Methyl groups are most commonly added on the N-7 or O-6 positions on guanine residues (4). Alkylation of

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Temozolomide chemical composition

the DNA results in damage, which subsequently triggers these malignant cells’ apoptosis (4).

However, some tumor cells can be resistant to Temozolomide’s effect, especially if they contain the MGMT gene (4). MGMT allows the cancerous cells to repair the DNA damage, preventing apoptosis and allowing the damaged cells’ uncontrolled proliferation to continue (4).

Temozolomide is orally administered and absorbed in the small intestine (3). Due to its small size, it can effectively penetrate the blood-brain barrier. As a prodrug, a change in pH triggers Temozolomide to undergo a conformational change via hydrolysis (5).

It turns into the cytotoxic MTIC (3-methyl-triazine-1-y1-imidazole-4-carboxamide), which has a growth inhibitory function (5).

Works Cited 1. American Association of Neurological Surgeons. (2016).

Glioblastoma multiforme. Retrieved March 26, 2021, from https://www.aans.org/en/Patients/Neurosurgical-Conditions-and-Treatments/Glioblastoma-Multiforme#:~:text=Glioblastoma%20multiforme%20(GBM)%20(also,as%20 a%20grade%20IV%20astrocytoma.

2. Johns Hopkins University. (2017). Gliomas. Retrieved March 26, 2021, from https://www.hopkinsmedicine.org/ health/conditions-and-diseases/gliomas 3. Cancer Association. (2018). How chemotherapy drugs work. Retrieved March 26, 2021, from https:// www.cancer.org/treatment/treatments-and-side-effects/ treatment-types/chemotherapy/how-chemotherapy-drugs-work.html#:~:text=Alkylating%20agents%20 keep%20the%20cell,%2C%20multiple%20myeloma%2C%20 and%20sarcoma.

4. Zhang J;Stevens MF;Bradshaw. (2012, January). Temozolomide: Mechanisms of action, repair and resistance.

Retrieved March 26, 2021, from https://pubmed.ncbi.nlm. nih.gov/22122467/ 5. Wesolowski, J., Rajdev, P., Mukherji, S. (2010, September 01). Temozolomide (Temodar). Retrieved March 26, 2021, from http://www.ajnr.org/content/31/8/1383#:~:text=The%20proposed%20mechanism%20of%20action,small%20size%20(194%20Da).

Oncologists and brain cancer researchers have seen that administering the drug with radiation therapy has increased the time before disease progression. A significant drawback of this drug is its very high costs, but this can be offset by the fact that most of Temozolomide’s expenses are covered by governmental health care programs such as Medicaid. Researchers are now looking to investigate where pairing Temozolomide with another pharmacologic agent would increase its cytotoxicity and efficacy. Additionally, after extensive research on creating therapies that can permeate the bloodbrain barrier, alkylating agents have paved the way for new therapy technologies such as nano-particle mediated brain drug delivery (1).

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The blood-brain barrier by Kelly Cao (IV)

Oncolytic Viruses in Cancer Treatment

by Annabelle Shilling (IV) Vaccines have been used to prevent cancer-causing infections such as HPV, but their applications within cancer research extend further than that. While virtually every vaccine in history has been wielded to prevent the acquisition of some infectious disease, oncolytic virotherapy is a novel branch of oncology pertaining to vaccines used not to prevent an infectious disease, but designed to cure a noninfectious one.

An oncolytic virus is one that seeks and destroys exclusively tumor cells. It typically does so by lysing such cells, but this direct cytoreduction is not the only effect of these viruses. A helpful byproduct of the infection is the stimulation of the immune system within the tumor microenvironment. While the viruses go about lysing tumor cells, the tuOncolytic virus particle by Lleyton Lance (VI) mor antigens released induce both an innate and an adaptive immune response (ex. increasing tumor T-Cell counts), the latter of cle arrest after becoming infected with it. With which functions as a lasting immunotherapy. adenoviruses, quiescent cells can be pushed into S phase by E1A proteins after they bind to Oncolytic viruses can be lab-made or naturally retinoblastoma proteins within the cell. These occuring; the former of which ensures the viruses Rb proteins are used to regulate the transition must depend on tumor cells for replication and from G1 to S phase through interactions with its establishment of tumor-specific alterations E2F transcription factors, and the Rb binding for lysis. The Onyx-015 Adenovirus was one of to E1A subsequently causes the release of E2F. the first oncolytic viruses used in clinical trials, E1A, undesirably, causes p14ARF to be released and in spite of its benefit proving slightly unas well, a protein that inhibits Mdm2 (Murine derwhelming, the trials did confirm two things: double minute 2) from degrading p53. The tranoncolytic viruses are safe to be administered scription factor p53 is used to initiate cell cycle to humans and they hold the potential to work arrest or apoptosis as a consequence of signals in conjunction with systemic radiotherapy and revealing damage to DNA—it can also do this chemotherapy as a combined cancer treatment. due to cellular stress. However, if p53 accumulates due to the lack of degradation, the cell cyOnyx-015 is selective, not in the sense that cle arrest or apoptosis that can occur is detriit will not infect healthy cells, but that only mental to the viral life cycle—if the cell dies or healthy cells will undergo apoptosis or cell cyceases growth before the viral cycle is complete,

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it will reduce the amount of viral offspring produced. Adenoviruses contain E1B genes to combat this threat to their reproduction, one of which, known as E1B55K, codes a product that binds to p53 and renders it nonfunctional.

Cancerous cells are those with damaged DNA that continue to replicate and exist. Thus, the lack of functional p53 is universal to almost all known cancers—the damaged DNA and resulting protein does not signal p53 to initiate apoptosis or restrict cellular growth. Onyx-015 was genetically engineered (827 bp deletion and a point mutation--causing an early stop codon-in E1B), preventing the proper expression of E1B55K’s product. Thus, in infected healthy cells, the adenovirus cannot restrict the buildup of p53. The resulting apoptosis or restricted cellular growth interferes with the viral life cycle and thus prevents the infection from spreading among healthy tissues. However, as tumor cells do not possess functional p53, the lack of functional E1B55K does not make a difference in the viral life cycle. The virus can go about replicating itself and ultimately lyse the cell without any risk of apoptosis or restricted cell growth occurring. Thus, Onyx-015 is selective in its ability to replicate in and lyse tumor cells without posing a threat to surrounding, healthy tissues.

As of now, only one therapy using an oncolytic virus has been FDA-approved for cancer treatment: Talimogene laherparepvec, which is used to treat melanoma using a genetically modified herpesvirus. It bolstered the theory that oncolytic viruses can systematically target cancer manifested when tumors not directly injected with the treatment began to shrink.

While none are approved by the FDA, many other viruses are being examined for oncolytic potential–even a genetically modified form of the poliovirus is now in trial for targeting brain tumors. Another treatment in development uses reoviruses capable of crossing the brainblood barrier to target brain tumors as well.

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While these treatments do seem very promising, there are limitations to the capabilities of oncolytic viruses. They are susceptible to neutralization, hypoxic environments, high IFPs (interstitial fluid pressures), being wiped out by the immune system, movement-inhibiting stromal barriers, and transduction errors. That being said, oncolytic viruses remain a possibility for cancer treatment, combined or solitary.

Works Cited 1. Harbour, J. W., & Dean, D. C. (2000). Rb function in cell-cycle regulation and apoptosis. Nature Cell Biology, 2(4), E65-E67. https://doi.org/10.1038/35008695 2. Munhoz, R. R., & Postow, M. A. (2016). Recent advances in understanding antitumor immunity. F1000Research, 5, 2545. https://doi.org/10.12688/f1000research.9356.1 3. Oncolytic virus therapy: Using tumor-targeting viruses to treat cancer. (2018, February 9). National Cancer Institute.

Retrieved October 7, 2021, from https://www.cancer.gov/ news-events/cancer-currents-blog/2018/oncolytic-viruses-to-treat-cancer Woller, N., Gürlevik, E., Ureche, C.-I., Schumacher, A., & Kühnel, F. (2014). Oncolytic viruses as anticancer vaccines. Frontiers in Oncology, 4. https://doi.org/10.3389/ fonc.2014.00188 4. Zhao, Y., Yu, H., & Hu, W. (2014). The regulation of mdm2 oncogene and its impact on human cancers. Acta Biochimica Et Biophysica Sinica, 46(3), 180-189. https://doi. org/10.1093/abbs/gmt147 5. Lawler SE, Speranza MC, Cho CF, Chiocca EA. Oncolytic Viruses in Cancer Treatment: A Review. JAMA Oncol. 2017 Jun 1;3(6):841-849. doi: 10.1001/jamaoncol.2016.2064. PMID: 27441411.

6. Hemminki, O., dos Santos, J.M. & Hemminki, A. Oncolytic viruses for cancer immunotherapy. J Hematol Oncol 13, 84 (2020). https://doi.org/10.1186/s13045-02000922-1

Transcriptomic Analysis of COVID-19 Patients

by Maya Khan (V) This past summer, I participated in a virtual Transcriptomic Analysis + COVID-19 course run by Milrd, an educational organization in New York City, supported by the Mason Lab at Cornell Medical Center. I was new to transcriptomic analysis, the study of RNA transcripts, but the course gave me a strong foundation and incredible mentors. Through this program, I learned how to analyze a broad collection of data and use specific programs to compare reference genomes. This course would be perfect for anyone interested in computational biology and large-scale sequencing surveillance.

The class focused on a two-step analysis of COVID-19 transcriptomic data. While the genome is a larger collection of all nucleic or mitochondrial DNA, the initial product of genome expression is the collection of mRNA copied from the genes during transcription. The transcriptome measures this complete set of RNA transcripts in the intermediary stage between genes and proteins. For the analysis, cDNA is synthesized from the single-stranded RNA. Using a series of programs to clean and organize the raw sequencing data, we transformed it into an understandable format. Then, using the Integrated Genomics Viewer (IGV), we generated visualizations of the genomic data and identified point mutations.

Over the past decade, more reliable sequencing methods to track and map genes have been

Figure 1: Graphic showing the transcriptome’s variability due to alternative splicing.

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Figure 2: The IGV program was used to compare the cleaned COVID-19 patient sample and the reference genomes.

developed. In response to the COVID-19 pandemic—caused by Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2)—many countries have invested in genome sequencing (a process used in molecular biology to study genomes and the resulting proteins).

However, transcriptomic analysis can further explain the complexity of gene expression.

The nucleotide sequence found in RNA reflects the code in DNA. By analyzing the transcriptome, researchers also investigate the COVID-infected cell gene expression and activity. Because of alternative splicing, each gene may produce more than one type of mRNA. Changes in normal gene activity may signal disease and may help guide vaccine development.

To perform our analysis, we cleaned and organized the data in stages using the Terminal found in most computers. In the first stage, we turned our sets of raw DNA reads into a _strain profile_ of SARS-CoV-2 patient isolate that delineates nucleotide and amino-acid level mutations in the sample. This process had two major steps: quality control and mutation analysis.

During the second stage, we removed human DNA from our samples using a program called Bowtie2 and a human reference genome. Most SARS-CoV-2 patient samples contain human RNA that gets converted to cDNA, but we were not interested in analyzing it. To focus on the important COVID genomic data, we had to get rid of the host cell RNA. We then aligned our data with the indexed SARS-CoV-2 Reference Genome to find mutations. This alignment format gave coordinates indicating where mapped segments had mis-

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matches between the reference sequence nucleotides and our COVID-19 patient sample.

The discovered mismatches between the reference genome and our sample could be due to two factors: (1) a genuine biological mutation in the sample or (2) an error as a result of the sample preparation and sequencing process.

After we had aligned reads in our sample and (hopefully) called a list of high-quality variants, we visualized the results and listed the nucleotide position and associated amino acid mutations. These counted mutations allow genomic epidemiologists to track how a particular virus travels around the globe. We used a genome browser called the Integrated Genomics Viewer (IGV), which is authored and distributed by the Broad Institute of Harvard and MIT. Performing sequencing on viral and host cell genomes is important for many reasons. On average,

Figure 3: Final results, on mutations in the patient sample, displayed by a table.

the SARS-CoV-2 genome accumulates about two changes per month. Sequencing SARS-CoV-2 genomes and identifying subtle shifts help researchers follow how the virus spreads. Most of the mutations don’t affect how the virus functions, but a few may change the virion’s transmissibility or severity. Sequencing is an important tool, especially during this pandemic because it allows lineages and virulent genetic shifts to be tracked around the globe. For example, they have found that the Coronavirus outbreak in New York City stemmed primarily from Europe, while the SARS-CoV-2 genomes sequenced in Washington State indicate a Wuhan origin. Using the data and mutations, scientists can group global outbreaks and build phylogeny “family” trees for viruses. These trees are constantly being updated on GISAID and nextstrain.org.

All in all, this was a great class to take to get an inside look on the tools being used to track and sequence SARS-Cov-2. Taking part in this program is a great way to explore current research being done in computational biology.After applying, I chose from a variety of classes and different sessions.

The experience was virtual, extremely flexible, and allowed me to pursue my interests in science.

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Trisomy Formation Within Cancers

by Annabelle Shilling (IV) Trisomies are the most common cancerous chromosomal abnormalities. As a result, many researchers have attempted to identify how they form. Many consider a dysfunctional spindle assembly checkpoint (SAC) to be the cause of such errors, implying that the improper separation of sister chromatids during mitosis causes the gain of an extra chromosome in some cells; however, researchers studying the childhood kidney cancer nephroblastoma have discovered evidence suggesting otherwise.

Tripolar mitosis is often a distinguishing feature of cancerous tissue. Because bipolar mitosis is the default for many healthy cells, tripolar mitosis, a mitotic division creating three daughter cells instead of two, is a dangerous abnormality (Figure 1). Bipolar mitosis requires two centrosomes to be active, but any quantity greater than two can result in undesired spindle poles. These spindle poles guide the chromosomes to different parts of the cell for replication (anaphase), but when there are three of them active rather than two, the chromosomes are pulled in three different directions. Thus, the cell is preparing to split the chromosomal content into three daughter cells.

Figure 1: Tripolar mitosis

Nephroblastoma models used by researchers at Lund University demonstrated that it is possible for trisomies to form even when the SAC is functional. This formation is possible when triple mitosis begins to occur but fails during cytokinesis, resulting in the production of two cells but with a mismatch in chro-

mosome counts. Multipolar divisions are 16 to 128 times more likely to result in chromosomal aberrations than bipolar segregation errors, and of the 18 initial cells involved in the study that exhibited tripolar mitosis, only two did not fail during cytokinesis. Researchers later found, using a different set of cells, that 80% of tripolar replications fail during cytokinesis, in contrast to a mere 5% of bipolar replications.

These failures in cytokinesis often lead to trisomies in the subsequent binucleated daughter cells. Contrary to popular belief, mutations present in genes involved in the mitotic checkpoint are uncommon in human cancers, thus further debunking the SAC theory. That being said, it is not yet certain how epigenetic components affect this process, so the lack of mutations cannot be taken as complete evidence.

The mechanisms through which cancer is induced are very complex, and there is no standard to be applied to cancer as a whole. Tripolar mitosis has not been proven to cause all trisomies in cancers, and the research discussed above simply discredits the common assumption that the SAC is solely responsible for those trisomies.

Works Cited Gisselsson, D., Jin, Y., Lindgren, D., Persson, J., Gisselsson, L., Hanks, S., Sehic, D., Mengelbier, L. H., Ora, I., Rahman, N., Mertens, F., Mitelman, F., & Mandahl, N. (2010). Generation of trisomies in cancer cells by multipolar mitosis and incomplete cytokinesis. Proceedings of the National Academy of Sciences, 107(47), 20489-20493. https://doi.org/10.1073/pnas.1006829107 Kalatova, B., Jesenska, R., Hlinka, D., & Dudas, M. (2015).

Tripolar mitosis in human cells and embryos: Occurrence, pathophysiology and medical implications. Acta Histochemica, 117(1), 111-125. https://doi.org/10.1016/j. acthis.2014.11.009 Ottolini, C. S., Kitchen, J., Xanthopoulou, L., Gordon, T., Summers, M. C., & Handyside, A. H. (2017). Tripolar mitosis and partitioning of the genome arrests human preimplantation development in vitro. Scientific Reports, 7(1). https://doi.org/10.1038/s41598-017-09693-1

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Treatment of Metastatic Cancer with Statins

by Grace Stowe (IV) In an effort to eradicate cancer, scientists are researching a variety of treatments and possible cures. One such treatment involves a drug previously only used to treat high cholesterol: statin, an inhibitor of HMG-CoA reductase in the mevalonate pathway. When statins inhibit the HMG-CoA reductase, they inhibit the downstream effects of the signaling cascade in the mevalonate pathway: increased membrane integrity, increased protein synthesis, increased cell cycle progression, and increased cell signaling, which all promotes cell proliferation. The inhibition promotes cell apoptosis (programmed cell death), which reduces the risk of metastatic cancer and uncontrolled tumor growth.

While there is no definitive association between statins and the risk of developing cancer, there is promising evidence that statins are efficient at slowing the progression of metastatic prostate cancer. For example, in Coogan’s 2002 study on the association between statin use and incidents of prostate cancer the odds ratio (statistic that quantifies the association between two events, a value greater than one indicated that there is a correlation between the two parameters)of statin users with prostate cancer was 1.2, but in advanced cases, it was 0.9, meaning that there is a correlation between statin use and a decrease in the progression of non-metastatic prostate cancer, suggesting that the usage of statins reduces progression of metastatic prostate cancer. A similar result is seen in Platz’s 2006 report, which demonstrates that as the cancer progressed, the odds ratio decreased.

Platz’s report indicates that the later the stage of prostate cancer, the more effective statins are at decreasing the progression of the disease.

While these promising statistics are seen in the treatment of metastatic prostate cancer, they are not seen in the treatment of breast, colorectal, lung, or reproductive organ cancer, which all had an odds ratio of less than one when tested (no correlation between the risk of cancer and statin usage). As the use of statins for metastatic prostate cancer is new, there is limited data to support the efficacy of this treatment in the long term. In spite of the lack of data for usage over a longer period of time, statins are potentially a promising treatment for metastatic and fatal prostate cancer.

Works Cited 1. Boudreau, D. M., Yu, O., & Johnson, J. (2010, April 9).

Statin Use and Cancer, Risk: A Comprehensive Review.

Retrievedfrom: https://www.ncbi.nlm.nih.gov/pmc/articles/ PMC2910322/#R36 2. Coogan, P. F., Rosenberg, L., Palmer, J. R., Strom, B. L., Zauber, A. G., & Shapiro, S. (2002, May). Statin use and the risk of breast and prostate cancer. Retrieved from: https:// pubmed.ncbi.nlm.nih.gov/11964926/.

3. Kochhar, R., Khurana, V., Bejjanki, H., Caldito, G., & Fort, C. (2005, June 1).

4. Statins to reduce breast cancer risk: A case control study in U.S. female veterans. Journal of Clinical Oncology. Retrieved from: scopubs.org/doi/abs/10.1200/jco.2005.23.16_suppl.514.

5. Platz, E. A., Leitzmann, M. F., Visvanathan, K., Rimm, E. B., Stampfer, M. J., Willett, W. C., & Giovannucci, E. (2006, December 20). Statin drugs and risk of advanced prostate cancer.

6. Retrieved from: https://pubmed.ncbi.nlm.nih. gov/17179483/.

7. Singh, H., Mahmud, S., Turner, D., Xue, L., Demers, A., & Berstein, C. (2009, December). Long-Term Use of Statins and Risk of Colorectal Cancer: A population-Based Study.

Retrieved from: https://insights.ovid.com/pubmed?pmid=19809413.

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