PCR Spring 2018 Issue

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PINGRY COMMUNITY RESEARCH JOURNAL

SPRING 2018

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TABLE OF CONTENTS RESEARCHER ARTICLES

REPORTER ARTICLES

3D MOTION TRACKING MICROSCOPE Akash Kumar

TESLAS IN SPACE AND MAN ON MARS Noah Bergam

CREATING POINT MUTAIONS IN BRCA2 Andrew Brosie

COULD YOUR NEXT GADGED BE INSIDE OF YOU? Aneesh Karuppur

INVESTIGATING THE elF4E-4EBP1 SIGNALING PATHWAY AND ITS EFFECTS ON THERAPY RESISTANCE Benjamin Zhou, Parth Patel ,Jessica Li, Shail Avasthi, Morgan D’Ausilio, Phillip Geter

HOW NICOTINE AFFECTS TEENS’ BRAINS Ryan Geller

DESIGNING AND ESTABLISHING A WAKSMAN STUDENT SCHOLARS PROGRAM RESEARCH CLUB Joshua Metzger, Ethan Blum INVESTIGATING DJ-1 MUTATIONS IN PARKINSON’S DISEASE Natalie Lucciola, Jack Smith

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Editor-in-Chief: Allie Riddell, Class of 2019

THE FIGHT AGAINST “SUPERBUGS” AND HOW PHARMACEUTICAL COMPANIES ARE WORKING TO CREATE A FULLY-EFFECTIVE VACCINE Hannah Gruber THE DIFFICULT SEARCH FOR TRUTH IN THE 21ST CENTURY Jacob Gruber, Mr. Coe THE FLU VIRUS Ameera Ebrahim BIASED BOTS Jessica Yatvitskiy

Head Copy Editor: Felicia Ho, Class of 2019

Copy Editors: Darlene Fung, Class of 2019 Brian Li, Class of 2020 Art Editor: Isabelle Sheyfer, Class of 2020

Head Layout Editor: Praesana Danner, Class of 2019 Layout Editors: Leo Zhu, Class of 2019 Noah Bergam, Class of 2021 Aneesh Karuppur, Class of 2021 Faculty Advisor: Mr. David Maxwell 2

PINGRY COMMUNITY RESEARCH JOURNAL Spring 2018 Edition

WINTER 2017

RESEARCHER ARTICLES

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3D Motion-Tracking Microscope

by Akash Kumar, Sean Wang, Aditya Gollapudi, Lily Cao, Ethan Chung, Will Zhang, Azadeh Samadani

has the ability to scan the environment and move toward a more favorable light condition for photosynthetic growth (Bennett). The finely tuned phototactic nature of the algae derives from its rhodopsin based eyespot which allows it to detect and move towards light sources and optimize light absorption (Bennett).

While many previous studies have focused on qualitative aspects of the phototactic response (Polin), our goal was to provide a more quantitative description of Chlamydomonas’s phototactic and swimming behavior.

Abstract A microscope is a tool used to magnify objects and make them visible to the human eye. This magnification is accomplished with a series of lenses, curved pieces of glass that bend the light rays passing through them. For our project, a traditional 2D microscope would not fit our needs as we sought to measure the swimming patterns of Chlamydomonas reinhardtii, a single-celled algae about 10 microns in diameter that swims in helical pattern using two flagella. A 2D microscope can track the movement of the C. reinhardtii back and forth, but cannot sense how the organism moves up and down (the third dimension). The goal of our project was to build a microscope that can measure the third dimension and track the full extent of the Chlamydomonas’ swimming patterns.

In order to gain information on the third dimension, we employed diffraction, which is the way light rays bend when hitting an object. We examined the diffraction “rings” created by the impact of the light on Chlamydomonas. The rings changed size as the organism moves up and down and we used that movement to map the size of the ring to the third dimension. This allowed us to extrapolate a 3D trajectory and successfully track the swimming movement of the organism.

Introduction Chlamydomonas reinhardtii, unicellular, bi-ciliated algae approximately 10 microns in diameter, 4

Figure 1: Diagram of a standard microscope

Figure 2: Our Microscope

The diffraction pattern resulting from a uniformlyilluminated circular aperture has a bright region in the center, known as the Airy disk, which together with a series of concentric bright rings surrounding it is called the Airy pattern (Polin).

WINTER 2017 the z dimension.

Figure 3: Airy pattern of a uniformly illuminated Chlamydomonas cell

Materials & Methods Parts of the microscope and their functions: Light Source: Provides the light for the microsope Collector Lens (2): Condenses the light so it is more coherent Condenser and Field Diaphragms: Creates more uniform intensity and narrows the scope Objective: 40x magnifying objective Camera: Captures the image for the computer Methods Cell Growth

Stationary cells were grown on solid TAP media, composed of TAP Salts (NH4Cl, MgSO4 (NH4Cl, MgSO4, 7H2O, CaCl2, and 2H2O), a phosphate solution (K2HPO4, KH2PO4, and H2O), Hutner’s Trace Elements (contains various compounds), acetic acid, water, Tris, and agar.

Cells were spread across the media and grown in an incubator that ran on a continuous 18/6 hour sun/light schedule. Stationary cells were then transferred to tubes filled with liquid TAP media (does not contain agar) on a shaker that ran continuously. The shaking motion helped to develop the cells’ flagella and enabled them to move.

Radial Averaging

Location of the centroid will give the (x,y) coordinates of the cell and the size of the most outer visible ring. This will give the position of the cell in

Figure 4: Radial Averaging Example

Image Analysis

Thresholding

In order to reduce background noise, we used multiple thresholding methods that separated low and high intensity pixels. One method employed Otsu’s method, a thresholding algorithm that minimizes the intraclass variance of the black and white pixels, making the image pixels look more uniform (MathWorks). In addition, we also used a global threshold value, which involved using the same thresholding value across the entire image.

Lastly, we used an adaptive threshold method, the opposite of a global threshold constant, which set multiple thresholding constants over several clusters of surrounding pixels.

Centroid Finding

In order to locate the cells, we needed to find both the center of the Airy disks and the radius of the first ring. While we used radial intensity averaging to get a final answer, we approximated it first in order to isolate the cell. We first used the darker pixels to find the boundaries of all shapes in the pictures. Then we compared the perimeters and areas to find the circular ones and find the 5

PCR centroid by doing a center of mass calculation. We approximated the radius using the perimeter.

Analysis Protocol (see Figure 5)

Since the original images obtained were quite blurry and unfocused, we switched to using MATLAB to isolate the cells’ diffraction patterns, the light waves reflected off of the cells.

(A) We only look at blue light.

(B) A cell’s centroid is selected via the approximation above and a new image based around the center is created.

(C) The background ‘noise’ of the cropped image is found by calculating the average pixel color value for every pixel with a large resolution.

(D) The background noise is subtracted from the cropped image to create a image with only data and no noise.

(E) The seemingly all black image is a result of subtracting the background noise, leaving behind an image with very low pixel intensity values, we scale the brightness up to create (F) An image on which we can do radial averaging in order to obtain the intensity plots.

Figure 5: Image Analysis Process

Intensity Plot

In order to calculate the average intensity per radial ring, we used a radial average function, given the center of the cell. The result was a graph with the y-axis representing the average intensity of a pixel R units away from the center. The x-axis represented R. 6

Figure 6: Intensity Plots

Discussion We have gathered significant data to create preliminary images and plots with single-cell tracking. We hope to apply this to group dynamics and experiment with different wavelengths of light to get a better understanding of cell movement.

We used our analysis of the Airy disk to find preliminary 3D trajectories for the movement of the chlamydomona. We found the x and y positions from the centroid of the ring and extrapolated the z coordinate from the size of the outermost visible ring, determined from the graphs on the left. The z coordinate is not yet in scale with the x and y coordinates as we have not yet created a calibration pattern. We had some trouble this year with this aspect due to camera issues and this will be one of our first items to resolve next year. We hope to start experimenting with different colors of light soon and see the impact on the phototaxis of the organism.

WINTER 2017 References 1. Bennett, Rachel R., and Ramin Golestanian. “A Steering Mechanism for Phototaxis in Chlamydomonas.” Journal of The Royal Society Interface. The Royal Society, 06 Mar. 2015. Web. 28 Apr. 2017.

2. Nave, C. R. (2012). Circular Aperture Diffraction. Retrieved March 28, 2017, from http://hyperphysics.phy-astr.gsu.edu/hbase/phyopt/cirapp2.html 3. Polin, Marco, Idan Tuval, Knut Drescher, J. P. Gollub, and Raymond E. Goldstein.

“Chlamydomonas Swims with Two.” Science. American Association for the Advancement of Science, 24 July 2009. Web. 28 Apr. 2017.

4. “Otsu’s Method.” MathWorks. N.p., n.d. Web. 28 Apr. 2017.

Acknowledgments The Pingry School Colleen Kirkhart Morgan D’Ausilio David Maxwell

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Creating Point Mutations in BRCA2

by Andrew Brosie

Figure 1: Sequencing of BRCA2 S492A show the presence of a T→ G mutations at nucleotide position 1473 bp. The mutation was confirmed in BRCA2 Mutated DNA 2-6. BRCA2 Mutated DNA 1 showed sequencing of the wild-type BRCA2 gene.

Abstract The pathway between BRCA2 and cancer is unclear; however, it is known that when BRCA2 is mutated, there a potential for breast cancer to develop.

The goal of this project was to create single mutations to modify phosphorylation of BRCA2 through site-directed mutagenesis. I was able to successfully mutate the BRCA2 gene at the S492A site (Fig. 1,2).

This signifies that we will be able to introduce the mutated gene into a cell and see how the mutation changes the function of the gene.

Introduction Mutations of the BRCA2 gene are commonly found in patients with breast cancer (6). BRCA2’s role in the human body is to 8

repair double stranded DNA breaks through homologous recombination. Unmutated BRCA2 serves as a tumor suppressor, but when a mutation occurs in the gene, breast or ovarian cancer can arise (10).

It is unclear exactly how many BRCA2 mutations in the gene can lead to cancer. Dr. Ryan Jensen is examining how the single, double, and triple phosphomutants of the BRCA2 gene affect DNA damage repair in cancer cells. BRCA2 is regulated by phosphorylation. The sites of phosphorylation in BRCA2 have been identified; however, the importance of many of these sites has yet to be investigated.

Materials and Methods We created two phosphomutants that turn phosphorylation on and off. Each phosphomutant is a

double mutant. The mutations that turn phosphorylation on were created at sites T491A/S492D and S646D/ S648D. The mutations that turn phosphorylation off were created at sites T491A/S492A and S646A/S648A. To make the double mutants, the primers that were previously made by Emily Known for the BRCA2 project last year will be used (9). The QuikChange SiteDirected Mutagenesis Kit was used to create point mutations to change the structure of the protein. After the vector was completed in the thermocycler, the plasmid was treated with Dpn 1 and placed in an incubator for one hour. Dpn 1 is used to degrade the extra parental DNA template that remains in the PCR tube following the thermocycler. After the Dpn 1 was used, only the double mutated plasmids remained.

WINTER 2017 Following site-directed mutagenesis, the mutated plasmid DNA was transformed using the High Efficiency Transformation Protocol with NEB 5-alpha Competent cells. Once the transformation was complete, the cells were plated and cultures grown. Following site-directed mutagenesis, the mutated plasmid DNA was transformed into bacteria. The plasmid was isolated and purified.

After purification, the plasmid was sent out for sequencing at Genewiz LLC for the presence of the S492A mutation.

Results and Discussion: The results of the sequencing shows that the S492A mutation of BRCA2 was successful in five of the six plasmids. This specific mutation changes the serine to alanine at amino acid 492. The mutation successfully changed the amino acid in the protein.

This means I was able to change the structure of BRCA2. Further research will be conducted to determine whether the function of the protein changed. By transfecting the BRCA2 S492A into DLD1 cells, we will be able to see if the function of BRCA2 was changed by the mutation. Transfection works similarly to transformation, except it uses eukaryotic cells instead of bacterial cells.

Transfection will determine whether the S492A mutation has changed the function of BRCA2 inside of a cell. It will be of value to see if the mutation changed the function of the protein, if it helped or hurt the function, or if the function of the protein completely changed. The results of transfection will hopefully lead to an answer of what this mutation does to the function of BRCA2.

Figure 2: Chromatogram of the mutation.

Literature Cited: (1) Biolabs, New England. “High Efficiency Transformation Protocol (C2987H/C2987I).” High Efficiency Transformation Protocol (C2987H/C2987I) | NEB. N.p., n.d. Web. 02 Feb. 2017.

(2) Biolabs, New England. “Q5® Site-Directed Mutagenesis Kit Protocol (E0554).” Q5® SiteDirected Mutagenesis Kit Protocol (E0554) | NEB. N.p., n.d. Web. 02 Feb. 2017.

(3) Biolabs, New England. “Quick Protocol for Monarch® Plasmid Miniprep Kit (NEB #T1010).”

Quick Protocol for Monarch® Plasmid Miniprep Kit (NEB #T1010) | NEB. N.p., n.d. Web. 02 Feb. 2017.

(4) Biolabs, New England. “TransPass P Protein: Transfection Protocol.” TransPass P Protein: Transfection Protocol | NEB. N.p., n.d. Web. 02 Feb. 2017.

(5) Biolabs, New England. “Western Blot (NEB #E8033).” Western Blot (NEB #E8033) | NEB. N.p., n.d. Web. 02 Feb. 2017.

(6) Dephoure, Noah et al. “Mapping and Analysis of Phosphorylation Sites: A Quick Guide for Cell Biologists.” Ed. David G. (7) Drubin. Molecular Biology of the Cell 24.5 (2013): 535-542. PMC. Web. 7 Fed. 2017.

(10) Jensen, Ryan B. “BRCA2: One Small Step for DNA Repair, One Giant Protein Purified.” The Yale Journal of Biology and Medicine 86.4 (2013): 479– 489. Print.

(11) Kwon, Emily. “Creating Point Mutations in BRCA2” The Pingry Community Research Journal.

PCR, Volume 4: Spring 2016 (12) Rass, E., Grabarz, A., Bertrand, P., and Lopez, B. S. (2012) Cancer Radiother 16, 1-10 Acknowledgements: Morgan D’Ausilio Ph.D. The Pingry School David Maxwell Ryan Jensen Ph.D. Alexandra Logerfo

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Investigating the eIF4E-4EBP1 Signaling Pathway and Its Effects on Therapy Resistance

by Benjamin Zhou1, Parth Patel1, Jessica Li1, Shail Avasthi1, Morgan D’Ausilio1, Phillip Geter2

Eukaryotic translation initiation factor 4E (eIF4E) is an essential component of the cellular machinery facilitating mRNA translation into proteins. eIF4E recruits ribosomes to the capstructure of mRNA and is the rate-limiting component of the eukaryotic translation apparatus. eIF4E activity is regulated by its counterpart, 4E binding protein 1 (4E-BP1), which in turn is regulated through phosphorylation by mammalian target of rapamycin complex 1 (mTORC1). The deregulation of this pathway is heavily seen in therapy-resistant, estrogen receptor positive (ER+) breast cancer strains which comprise approximately 23.3% of breast cancer cases. In this paper, we investigate the process of 4EBP1 phosphorylation. We plan to introduce valine mutations at three separate phosphorylation sites (Threonine-70, Serine-83, 10

Serine-112) on 4E-BP1. We will then run a protein pulldown assay to analyze the effects on eIF4E-4EBP1 binding affinity. We hope to find heightened binding affinity between eIF4E and 4E-BP1 when negating potential phosphorylation sites.

Figure 1: Visual representation of estrogen receptor function and tamoxifen’s competitive inhibition of pathway

Introduction In 2016, approximately 231,840 new cases of breast cancer were diagnosed in the United States(4). Around 70% of breast cancers are

WINTER 2017 estrogen receptor positive (ER+), meaning that the cancerous cells express estrogen receptors on their surfaces. The binding of estrogen to these receptors triggers a downstream protein cascade that promotes cellular growth and proliferation.

Common hormonal therapies often introduce medications that inhibit estrogen from binding to estrogen receptors through competitive inhibition (Figure 1). One such medication is tamoxifen, which reduces the risk of contralateral breast cancer by 47% over a five year treatment. However, 33% of patients develop resistance to tamoxifen during the five-year treatment(2).

Figure 2: Simplified elF4E-4EBP1-mTORC1 pathway. elF4E is inhibited by 4E-BP1 which is inhibited by phosphorylation via mTORC1.

Previous studies have implicated overactive or deregulated eukaryotic translation initiation factor 4E (eIF4E) in the development of tamoxifen resistance(5). eIF4E functions as a key cog in the initiation of mRNA translation into protein. It binds to the mRNA cap structure and recruits ribosomal machinery to start the process of translation(3). eIF4E activity is regulated by 4E Binding Protein 1 (4E-BP1), which binds to eIF4E and thus prevents its function. Similarly, 4E-BP1 is also regulated by another protein, mammalian target of rapamycin complex 1 (mTORC1(1). mTORC1 phosphorylates 4E-BP1 and changes its structure, thereby impeding its function in regulating eIF4E (Figure 2).

Phosphorylation of 4E-BP1 by mTORC1 occurs at several amino acid sites on 4E-BP1.

Because our lab does not have the equipment necessary to cultivate a sufficient amount of human

cells necessary for protein expression, we must introduce our genes into bacterial expression vectors. Prior to this year, we had successfully isolated eIF4E and 4E-BP1 DNA genes from their mammalian expression vectors. Our work this year concerned restriction digesting pTXB1, a bacterial expression vector, and subsequent ligation with our genes of interest.

Materials and Methods: eIF4E and 4E-BP1 genes were received from the Sackler Institute at NYU in mammalian expression vectors. Plasmids were transformed into NEB 5-α Escherichia coli cells for replication. Five mL of LB+Amp with transformed cells were inoculated and incubated at 37°C on a shaker at 225 rpm for 16 hours. Next, we mini prepped using a Qiagen Miniprep Kit. We stored the mini prepped plasmids at -80 oC. PCR was performed on our samples using a standard 50 μL procedure. Tubes were then transferred to a thermocycler and followed the following protocol: Initial denaturation at 98°C at 30 seconds 30 cycles of 98°C for 10 seconds, 62°C for 20 seconds, and 72°C for 25 seconds.

Final extension of 72°C for 2 minutes Hold indefinitely at 4°C. PCR products were then transferred to 4°C refrigerator. The products were then purified using a standard QiaQuick PCR Purification kit. For a 60 μL reaction, two μl of each of our two restriction enzymes, six μL of CutSmart 10X NEB Buffer, 47 μL of PCR purification product were added and then filled to 60 μl with water. The restriction digest mixture was incubated at 37°C in a water bath for 2 hours. The restriction digest product was then transferred to the freezer. These steps were then repeated with pTXB1 in preparation for ligation.

Both eIF4E and 4E-BP1 were ligated into our pTXB1 expression vector using the standard NEB Quick Ligase Protocol. When ready, our ligation product was immediately transformed into NEB 5-α highefficiency cells. A test restriction digest was then run on our ligated plasmids to verify the results of our ligation. At numerous points in our procedures, a standard 1% 50 ml agarose gel with a SYBR Safe dye was utilized.

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Results and Discussion: A gel of our test restriction digest reveals that one of our products, our fourth 4E-BP1 ligation sample, yielded the desired results (Figure 3). The lanes between the first two ladders are eIF4E+pTXB1 samples. None show cut DNA fragments, indicating that the colonies picked did not have properly ligated plasmids. The lanes between the second two ladders are 4EBP1+pTXB1 samples. Lane 4 shows a cut fragment, with one band at approximately 2400 base pairs and another at approximately 400 base pairs, which is presumably 4E-BP1. This result confirms that our ligation of 4E-BP1 into pTXB1 succeeded.

To check our results, a PCR on the restriction

using site-directed mutagenesis, run a pulldown binding assay, and possibly introduce proteins into human breast cancer cells to analyze the effects of dephosphorylation in vivo.

In the immediate future, we plan to transform our 4E-BP1 ligation sample into NEB 5-Îą high-efficiency cells for amplification and later sequencing. We are also in the process of finding a new expression vector for eIF4E, as it must express a protein tag distinct from the intein tag expressed 4EBP1+pTXB1, a necessary prerequisite for later protein binding assays.

In our long term planning, we hope to be able to express 4E-BP1 and introduce mutations using site-directed mutagenesis. We would also like to express eIF4E and run a pulldown binding assay.

Finally, we are also looking into the possibility of introducing our proteins into human breast cancer cells to analyze the effects of the mutations in vivo.

Literature Cited: 1 Gingras, Anne-Claude, et al.

Figure 3: Test restriction digest of ligation products

digested sample was run that showed bands with mini prepped 4E-BP1 as a control. Both express bands at approximately 450 base pairs (Figure 4).The agreement of results confirms that our ligation of 4E-BP1 into pTXB1 succeeded.

We plan to introduce mutations at phosphorylation sites at Threonine-70, Serine-83, and Serine-112.

By mutating these amino acid residues into valines, we can simulate dephosphorylation in order to ascertain the effects of phosphorylation of 4E-BP1 at different sites. We plan to sequence our genes in plasmids, express our protein, introduce mutations 12

Figure 4: PCR of test restriction digest 4E-BP1 sample and miniprep sample

“Regulation of 4E-BP1 phosphorylation: a novel

WINTER 2017 two-step mechanism.” Genes & Development, PDF ed., vol. 13, 20 Apr. 1999, pp. 1422-37.

2 Jiang, Quan, Shilong Zheng, and Guangdi Wang. “Development of New Estrogen Receptor-Targeting Therapeutic Agents for Tamoxifen-Resistant Breast Cancer.” Future medicinal chemistry 5.9 (2013): 10.4155/ fmc.13.63. PMC. Web. 27 Jan. 2017.

3 Mamane, Yaël, et al. “eIF4E- from translation to transformation.” Oncogene, vol. 23, 2004, doi:10.1038/ sj.onc.1207549.

4 “US Breast Cancer Statistics.” Breastcancer.org, 2017, www.breastcancer.org/symptoms/understand_bc/ statistics.

5 Youtani, Takeshi, et al. “Regulation of Human eIF4E by 4E-BP1: Binding Analysis Using Surface Plasmon Resonance.” Life, PDF ed., vol. 49, 2000, pp. 27-31.

Acknowledgements: Thank you to Philip Geter, PhD candidate at NYU, for mentoring this team. Thank you to Andrew Verdesca and Katie Coyne for their past leadership on this project. Thank you to Morgan D’Ausilio for supervising and guiding the project. Thank you to the Science Department at Pingry for their ongoing support.

The Pingry School, Basking Ridge, NJ, United States; 2Sackler Institute of Graduate Biomedical Sciences, NYU School of Medicine, New York, NY, United States 1

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Designing and Establishing a Waksman Student Scholars Program Research Club

by Joshua Metzger and Ethan Blum The mantra of the Waksman Student Scholars Program, “learning science by doing science,” guides the course of the research project.

This program was divided into two portions: a bioinformatics step and a lab portion. In the lab component of the club, we were able to achieve our goal of properly sequencing duckweed clone’s cDNA by growing the clones in a bacterial culture, performing polymerase chain reactions (PCR), running a gel electrophoresis on the fragment to determine its size, performing a plasmid DNA miniprep, and running a restriction digest on the plasmid DNA to corroborate the fragment size determined in the PCR step. During the bioinformatics component, students used the DNA Sequence Analysis Program (DSAP) to find species within the National Center for Biotechnology Information (NCBI) database that have similar DNA or amino acid sequences. Students that were able to find a new and unique DNA sequence of duckweed and completed all necessary steps within the program protocol had their clone published on the NCBI website as official authors. We were able to develop key leadership and organizational skills mentoring younger students in their developing science research lives.

The Waksman Students Scholars Program (WSSP) investigates the chemical and biological properties of Landoltia punctata, a small, freshwater plant that grows in slow-moving fresh water. As a potential biofuel source, one study obtained yields of 24.06 g/L ethanol and 680.36 mg/L of isopentanol from duckweed using acid hydrolysate. This is 15 times higher than what could be obtained through fermentation by yeast (8). In the end, the acid hydrolysate can be converted to sugar for fermentation. Duckweed can serve as a viable energy source while not posing any ecological or environmental liability, as it does not compete with 14

crop production (3).

L. punctata can also be used for bioremediation of contaminated soil and groundwater (4). Duckweed traps contaminated materials such as lead or arsenate in their roots and fronds, and extracts nitrogen and phosphate from the water (4). All of these advantageous aspects of duckweed make it a very beneficial species to research and looking for novel proteins within its genome could help us cultivate these qualities (10).

This project has a dual purpose. We will also be mentoring sophomores and juniors that voluntarily enroll themselves into The Pingry School’s WSSP club. While we gain experience in supervising and management, these aspiring researchers will gain the fundamental benchwork that will serve as critical tools in their future science lives (1). This project will enhance students’ understanding of genomics, bioinformatics, protein modeling, and microbiology. Because Waksman is based on the understanding that students “learn science by doing science,” we expect to see their experience in the lab work and bioinformatics steadily become more proficient (5). A longer-term goal will be structured on the ability to set up future members for at least the next two years and have two individuals succeed me as president of the club.

Materials and Methods The Pingry chapter of the program was reinstat] ed by us under the supervision of Mr. David Maxwell as part of our AP Biology Project. After the 2014-2015 school year, the club lacked the necessary leadership and supervision in order to function in the following school year. Mr. David Maxwell, wishing to revive the club, proffered the opportunity to have students lead the club as part of their project. As we completed the program our sophomore year, we were more than eager to take on the opportunity and pass down our knowledge and experience to younger students.

Before working on their own clone, students must complete practice clones PC1.16, PC2.16, and

WINTER 2017

Figure 2: Membership of Waksman Student Scholars Program throughout the 2016-1017 school year.

PC3.16 using the DNA Sequence Analysis Program (DSAP). Each PC requires the student to edit a DNA waveform, perform a search of the National Center for Biotechnology Information (NCBI) database using the Basic Local Alignment Sequence Tool (BLAST), and answer analysis questions. We analyzed practice clone sequences, in which we identified the start and end of the nucleotide sequence. After editing the sequence for missing or additional base pairs, ambiguous peaks in the waveform, and miscalled bases, we initiated the BLAST stages.

We searched for matches for these sequences within the nucleotide collection and protein databases. In comparing the matches, we recorded the accession number, definition, organism, query start, query end, and E value of three different organisms.

After defining the open reading frame (ORF), we completed the analysis stages, in which we determined the name and function of the protein,

found if a similar protein exists in Homo sapiens, searched the PDB database to determine if there is a homologous protein in which the structure has been determined, performed additional BLAST searches, and concluded what experiments could be done next to further research the protein.

Results

The wet lab lab portion of the Waksman Student Scholars Program has been delayed until the fall of 2017. In the meantime, students will analyze and evaluate the clones that have already been sequenced by the staff at the Rutgers Institute of Microbiology and given to our club at Pingry. In the beginning of the year, our club consisted of 14 members, 12 of which continued through the end of the year, a retention rate of 85.6%. These 12 members started off the year working on the practice clones, which would help them build up 15

PCR some of the skills mentioned and prepare them for the work that they would be doing on their individual clones. 6 students submitted PC1.16, 6 students submitted PC2.16, and 3 students submitted PC3.16. In terms of completion, ultimately 3 clones have been marked as “reviewed and correct” by three different students.

Discussion The club can improve in several key areas. At the beginning of the year the students were very excited to start working on the clones, but they lost this enthusiasm as the year progressed. We would recommend addressing this by having the lab portion of the program occur first to keep the students motivated to officially publish their clones.

In this way, we can boost morale and increase our retention rate.

What we learned as Waksman leaders throughout this whole process is how to manage a class and teach valuable research skills. Throughout the year we have been able to build these skills by helping our underclassmen on anything from their practice clones to the lab portion itself. Not only is the Waksman Student Scholars Program supposed to lead to the sequencing of duckweed, more importantly it supposed to give the students a strong foundation and the basic skills they need to be able to conduct research of their own in the future.

Works Cited 1. Barab, Sasha A., and Kenneth E. Hay. “Doing Science at the Elbows of Experts: Issues Related to the Science Apprenticeship Camp.” Barab - 2001 - Journal of Research in Science Teaching - Wiley Online Library. JRST, 9 Jan. 2001. Web. 27 Jan. 2017.

2. Carr, Edward A., Jeff Charney, WIlliam Sofer, and Andrew K. Vershon. “An Integrated Molecular Biology Research Project for High School Students.”

BioOne. National Association of Biology Teachers, Oct. 2006. Web. 27 Jan. 2017.

3. Chen, Qian, Yanling Jin, Guohua Zhang, Yang Fang, Yao Xiao, and Hai Zhao. “Improving Production of Bioethanol from Duckweed (Landoltia Punctata)

by Pectinase Pretreatment.” MDPI. Energies, 10 16

Aug. 2012. Web. 27 Jan. 2017.

4. Fang, Yun Ying, Zed Rengel, Olga Babourina, Xiao E. Yang, and Pei Min Pu. “Ammonium and Nitrate Uptake by the Floating Plant Landoltia Punctata.”

Oxford Academic. Annals of Botany, 4 Jan. 2007.

Web. 27 Jan. 2017.

5. Heppner, Frank. “Learning Science by Doing Science | The American Biology Teacher.”Learning Science by Doing Science | The American Biology Teacher. The American Biology Teacher, 6 Sept.

1996. Web. 27 Jan. 2017.

6. Lemke, J. L. “Missing Context in Science Education.” Missing Context in Science Education.

AERA, Apr. 1992. Web. 27 Jan. 2017.

7. Moreland, John L., Apostol Gramada, Oleksandr V. Buzko, Qing Zhang, and Philip E. Bourne.

“The Molecular Biology Toolkit (MBT): A Modular Platform for Developing Molecular Visualization Applications.” BMC Bioinformatics. BMC Bioinformatics, 6 Feb. 2005. Web. 27 Jan. 2017.

8. Su, Haifeng, Yun Zhao, Juan Jiang, Qiuli Lu, Qing Li, Yao Luo, Hai Zhao, and Maolin Wang. “Use of Duckweed (Landoltia Punctata) as a Fermentation Substrate for the Production of Higher Alcohols as Biofuels.” Energy & Fuels 28.5 (2014): 3206-216.

Web. 27 Jan. 2017.

9. Vershon, Andrew. “NEWSLETTER.” RCSB PDB Newsletter. RCSB PDB, 2012. Web. 27 Jan. 2017.

10. WSSP Project Overview. Prod. Andrew Vershon.

N.p., 2012. Web. 7 Feb. 2017. <https://wssp.rutgers. edu/videos>.

Acknowledgments Thank you to the following people for their support, guidance, and assistance throughout our research project: The Pingry School David Maxwell Waksman Institute of Microbiology Andrew Vershon, Ph.D Sofia Briones Brian C. Wahler Anthony Weinkopff

WINTER 2017

Investigating DJ-1 Mutations in Early-Onset

by Natalie Lucciola and Jack Smith Abstract The cause of Parkinson’s Disease is unknown.

However, some studies suggest mutations in the DJ-1 gene may play a role Parkinson’s Disease. In order to recreate the mutated gene seen in these patients, we performed Site-Directed Mutagenesis on the E18 amino acid in DJ-1. With future research, we hope to express proteins of the mutated gene in E.coli to see if our mutation was successful.

Introduction More than 10 million people are living with Parkinson’s Disease worldwide, and 60,000 new cases are diagnosed in America each year (8). Men are one and a half times more likely to develop the disease (8). Parkinson’s Disease appears to manifest from a variety of different genetic mutations and protein alterations (4).

DJ-1, also known as PARK7, is a gene that is often mutated in cases of early-onset Parkinson’s Disease (3). DJ-1 plays an important role in regulating oxidative stress and cell death. In the body, it acts as an oxidative stress sensor, redoxsensitive chaperone, and protease (9). Although a few mutations have been shown to alter the functionality of the DJ-1 protein, the effects of most mutations are unclear. However, we do know that when DJ-1 is mutated, it can result in a decrease in enzymatic activity and a lack of regulation of cell death (6). We conducted our research in order to study how a mutation of the E18 amino acid residue in the DJ-1 gene might decrease the enzymatic activity of DJ-1.

Materials and Methods Our first step in this experiment was to clone the DJ-1 into a vector capable of expressing and

allowing for purification of the protein. We removed DJ-1 from the original vector pET3a, which was not usable for future protein isolation, by digesting the plasmid with BamHI and NdeI. We then ligated the gene into pET28a plasmid, which allows for His-Tag purification once the protein is expressed. The next step was performing a transformation of the ligated plasmid into DH5α E. coli cells using selective media (LB agar) and sequencing tools like GeneWiz. Our gene has been successfully cloned into the pET28a expression vector.

Results and Discussion DJ-1 was successfully cloned into the pET28a vector.

We designed the primers below to allow us to perform site-directed mutagenesis on our plasmid. We then sequenced the PCR site-directed mutagenesis plasmids. Once we confirm the mutation of our cloned DJ-1 plasmids, we will be able to express the mutated protein in BL21 cells and isolate the proteins using His-Tag purification.

Further research will examine oxidative stress of the mutated DJ-1 gene, as well as study the mutated DJ-1 gene with a treatment, 17

PCR Pyrroloquinoline quinone (PQQ) (7). This drug would counteract the effects of the mutation, lowering the oxidative stress. In order to see the effectiveness of the treatment, we will study the proteins expressed and analyze them to see whether we were successful in restoring the function.

Literature Cited 1. Abdallah, Jad, and Renee Kern. “CLONING, EXPRESSION AND PURIFICATION OF THE GENERAL STRESS PROTEIN YhbO FROM ESCHERICHIA COLI.” Semanticscholar.org. N.p., n.d. Web. 06 Jan. 2017.

2. Biolabs, New England. “Site Directed Mutagenesis.” Site Directed Mutagenesis | NEB. N.p., n.d. Web. 06 Jan. 2017.

3. Huang, Pei, Xiao-Dong Yang, Sheng-Di Chen, and Qin Xiao. “The Association between Parkinson’s Disease and Melanoma: A Systematic Review and Meta-analysis.” Translational Neurodegeneration. BioMed Central, 03 Nov. 2015. Web. 17 Jan. 2017.

4. Klein, Christine, and Ana Westenberger. “Genetics of Parkinson’s Disease.” Cold Spring Harbor Perspectives in Medicine. Cold Spring Harbor Laboratory Press, Jan. 2012. Web. 17 Jan. 2017.

5. Maita, Chinatsu, Hiroshi Maita, and Sanae Iguchi-Ariga. “Monomer DJ-1 and Its N-Terminal Sequence Are Necessary Mitochondrial Localization of DJ-1 Mutants.” Plos One. Plos One, Jan. 2013. Web. 27 Jan. 2017.

6. Nunome, K., S. Miyazaki, and S. Aguchi-Ariga. “Pyrroloquinoline Quinone Prevents Oxidative Stress- induced Neuronal Death Probably through Changes in Oxidative Status of DJ-1.” Biological & Pharmaceutical Bulletin. U.S. National Library of Medicine, n.d. Web. 27 Jan. 2017.

7. Nunome, Kana, and Shin Miyazaki. “Pyrroloquinoline Quinone Prevents Oxidative Stress- Induced Neuronal Death Probably through Changes in Oxidative Status of DJ-1.” Jstage.jst.go. N.p., n.d. Web. 04 Jan. 2017.

8. “Parkinson’s Disease Foundation (PDF).” Statistics on Parkinson’s | Parkinson’s Disease Foundation (PDF). N.p., n.d. Web. 17 Jan. 2017.

9. Saito, Yoshiro. “Oxidized DJ-1 as a Possible Biomarker of Parkinson’s Disease.” Jstage.jst.go. N.p., n.d.

Web. 08 Dec. 2016.

Acknowledgments We would like to thank Alexandra Logerfo and David Maxwell for all of their help and knowledge to get us to accomplish what we have this year on our project.

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REPORTER ARTICLES

WINTER 2017

19

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Teslas in Space and Man on Mars

by Noah Bergam

SpaceX

was founded 15 years ago by entrepreneur Elon Musk with the intent to lower rocket costs and, in time, lead the human race to colonize Mars.

Recently, the company launched the Falcon Heavy into space. Holding up to 141,000 lbs, it is a greater capacity than any rocket ever developed since the Apollo missions’ Saturn V. The rocket sent Elon Musk’s Tesla Roadster inside, and the car is now cruising past Mars on an unexpectedly long path, playing David Bowie’s Space Odyssey on its stereo.

This launch begs the question: how do we launch rockets into space? How do we control a rockets’ orbit?

Why haven’t we launched people to the Moon or Mars?

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WINTER 2017

Rockets propel themselves by burning fuel and oxygen in their rear, sending high pressure gas outwards and in turn causing an equal and opposite reaction that pushes the rocket forward, as per Newton’s Third Law. The force with which rocket pushes forward is known as thrust.

Usually, the thrust created by the rocket propellants is just not enough to get the rocket as far as, say, the International Space Station. So, the rocket is split into different fuel compartments. When those compartments run out, they simply fall off the rocket, making it lighter and therefore letting it go faster when the next compartment starts running.

Speed is key for entering the earth’s orbit. See, gravity is always pulling things towards earth’s surface, so in order to enter the realm of satellites without falling back down to Earth, a rocket must have a sideways thrust equivalent to the force of gravity. Thus, as it falls down, it moves “out of the way” of the curved planet surface such that it never actually changes its distance to earth.

Would that mean the rocket has to keep running its fuel to keep up the speed? Thankfully not -- since there are very few particles in space to cause friction (unlike on earth, with all our fancy air) the rocket simply would need to reach orbit speed, and it would stay at that speed. It is a perfect example of Newton’s First Law -- an object in motion will stay in motion unless acted upon by an outside force.

T o reach further planetary objects like the moon, you would need to have a thrust sufficient enough to break out of earth’s gravity and enter the moon’s.

Getting to Mars requires even more speed and efficiency. Getting to Mars with people requires even more. SpaceX, however, has boldly set its goals to bring humanity to Mars.

Make no mistake, SpaceX has made history before the launch of the Falcon Heavy.

In December 2015, the company’s Falcon 9 rocket became the first to run a successful landing, and on April 8th, 2016, another Falcon 9 became the first to make a successful sea landing. The rocket landed by running against its orbit trajectory in order to fall towards the surface, and carefully angling itself to slowly make contact with a landing pad. The significance of this landing is twofold.

Firstly, landing rockets enables space companies to reuse materials and save tremendous amounts of money and work. Secondly, Falcon 9 was developed quickly and for relatively cheap by SpaceX alone, as opposed to most other rockets developed by governments.

In short, it was just one small step to land and one big step towards efficiency in the future.

Now more current news -- SpaceX’s Falcon Heavy rocket, as stated earlier, is not only the first rocket to send a car into space, but also the most powerful rocket in the world currently, developed at an absurdly low price.

The rocket definitely undercuts its governmentdeveloped counterparts, like NASA’s Space Launch System (SLS). To put it in perspective, NASA has spent $7.8 billion in tax money for three years developing the SLS, an amount of money that equates to launching 86 Falcon Heavy Rockets into space.

It is but another sign that the future of space travel will be dominated by commercial developers as opposed to governments. And who knows? With this efficiency, perhaps SpaceX will be the first to put humans on Mars.

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Could your next gadget be inside of you?

by Aneesh Karuppur

In our day and age, we find it pretty impressive when a new gadget comes out boasting of amazing features.

“Lightest, thinnest, smallest, fastest, and best” are all words that companies use to describe their latest products. However, is there a limit to how small or thin an electronic device can be? Researchers at Harvard University’s Wyss Institute for Biologically Inspired Engineering do not see a limit in sight, and their latest paper documents how they created a complex biological computer using the 22

ribonucleic acid (RNA) of an E. coli bacterium.

The Wyss Institute is all about bioengineering projects like this, such as molecular robotics and nanoarchitectures for air purification. So, it makes sense that Alexander A. Green, Jongmin Kim, Duo Ma, Pamela A. Silver, James J. Collins & Peng Yin developed this idea there. The paper, “Complex cellular logic computation using ribocomputing devices,” was recently published in Nature. It explains how to construct a series of logic gates (the foundation of computer logic) using RNA. RNA is typically used to carry out instructions based on deoxyribonucleic acid (DNA).

The foundation of the ribocomputer is using RNA as inputs and proteins as the output. There are three basic logic gates: AND, OR, and NOT. The ribocomputer mimics the function of a two input AND gate, where the result is only activated if both inputs have an “on” status.

When two RNA strands are fed

into the ribocomputer’s AND gate, the modular sensor inside is only activated when both of the RNA can base-pair together.

Then, the result can be translated by a ribosome into the desired output protein. For a two-input OR gate, where the result is activated if one or more of the inputs is “on”, another sensor module allows two RNAs to interact with themselves through base-pairing, and a ribosome once again translates the result into a protein. The RNA inputs can individually trigger protein synthesis, which makes them perfectly suitable for the one-ormore “on” inputs needed for an OR gate. The NOT gates, which output the opposite of the oneinput, work a little differently in the ribocomputer. Two, instead of the usual one, input RNAs are required: one to negate the first (once again, through basepairing). The ribosome then translates the result into an output protein.

WINTER 2017

This figure demonstrates the various parts of the three logic gates and how they use RNA to trigger protein synthesis.

The sensing module in the ribocomputer is a new invention that is called a toehold switch. These special switches only translate an output gene when a trigger RNA is expressed in the cell (in this case, in the E. coli bacterium). The trigger RNA then interacts with another RNA to begin expression of the gene.

This figure demonstrates the various parts of the three logic gates and how they use RNA to trigger protein synthesis.

In this figure taken from the original paper, we can see exactly how the researchers created their logic gates. In order to quantify their results in a visible way, they set the output protein to be Green Fluorescent Protein (GFP). GFP is usually used to signal that a certain gene has been expressed by making the place in which the GFP is expressed glow fluorescent green.

So what does this all mean for us? Although a computer inside of an E.coli seems fairly

limited in practicality, the model and strategy of implementation can be applied to other organisms.

For example, this could allow for instantaneous information processing of our bodily functions. We could receive real time feedback about the way that our bodies are managing things like diseases and different climates. It could also help us predict possible future conditions based on the way our body is functioning. Additionally, ribocomputers implanted in the brain could interpret neural signals of those who are unable to speak or move, allowing them to communicate with everyone.

While the iPhone may not go out of vogue any time soon, donâ&#x20AC;&#x2122;t be surprised if a gadget you buy a few years down the road will be inside of you.

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How Nicotine Affects Teens Brains

by Ryan Geller

Nicotine is a naturally occurring compound found in tobacco plants. It is a toxic plant alkaloid that can be

absorbed into the bloodstream through smoking, vaping, chewing, or snorting tobacco products. It can even be absorbed through contact with the skin. Similar to caffeine, it is a stimulant; however, it is also a relaxant. Stimulants cause increases in alertness as well as concentration, whereas relaxants cause relaxation and sleepiness. Its affects make it almost as addictive as heroin and cocaine. Nicotineis one of six-hundred ingredients present in cigarettes, but it is not the only one responsible for addiction. However, nicotine has an extremely harmful effect on the brain.

The brain is the primary target of nicotine. The brain is made up of millions of nerves which send messages through neurotransmitters, which bind to nerve receptors. The nerve receptors form the nervous system, which controls all the muscles in the body. Nicotine is absorbed into the bloodstream and reach the brain almost ten seconds after its inhaled. It stays in the bloodstream for approximately four hours. Inside the brain, nicotine behaves similarly to neurotransmitters and binds with receptors. Specifically, nicotine acts like an acetylcholine. Acetylcholines are released by nerves to send signals. Nicotine will attach to acetylcholines and release hormones throughout the body which control mood, memory, and 24

muscles. The quick release of hormones causes an immediate relaxation followed by prolonged pleasure. Similar to marijuana, nicotine targets dopamine. Dopamine is a major cause of addiction in smokers. In addition to dopamine, nicotine releases glutamine which controls one’s memory.

The release of glutamine by nicotine implants the past experience of nicotine and causes individuals to crave more, which resorts in cigarette addiction.

Today, almost 85% of smokers have admitted that cigarette addiction started during adolescence, an important time in an individual’s life, when a teenager’s brain and body are maturing. As a matter of fact, an individual’s brain doesn’t fully mature until their mid-twenties. Teenagers are flooded with extreme emotions and teens are more susceptible to anxiety, depression, and addiction.

Therefore the introduction of nicotine during teenage years is extremely dangerous as it starts a cycle of dependence. Once an individual is addicted, his life will never be the same. Furthermore, teenagers are more susceptible to the effects of nicotine. It does not take as many cigarettes to become dependent on nicotine to feel happy.

Interestingly, teens worry less about risks and consequences, as they are more susceptible to peer pressure. Adolescent brains have a higher chance of developing more nicotine receptors than adults.

Ultimately, nicotine changes the functions of the brain.

The most well known long term consequence of nicotine use is addiction, a major problem around the world. Nicotine addiction is especially difficult to stop. As mentioned earlier, it is as hard to quit as heroin and cocaine. Furthermore, the brain is deeply affected. Adolescent brains have smaller prefrontal cortex which contributes to less concentration and a weaker memory. Through

WINTER 2017 smoking, nicotine impairs concentration, memory, and impulse control. The prefrontal cortex is extremely altered by adolescent nicotine exposure, and the brain fails to mature properly. Additionally, the brain will not function properly as an individual ages. It could contribute to loss of memory, hearing, nervous system breakdown, and other problems in the long-run. Similar to coffee, it also stunts your growth. In conclusion, adolescent brains cannot withstand the effects of nicotine, no matter its form.

Today, vaping is increasing in teen popularity throughout the world. Vapes are more popular in middle school and high school than cigarettes.

Vaping and e-cigarettes are more popular now than ever before. Thus, the number of nicotine addicts has risen since 2011. Companies advertise vaping and e-cigs as an alternative to smoking and tell consumers that it is a less harmful alternative. Vapor is considered to be harmless by teens. Researchers have not done enough experimental trials to conclude how dangerous vaping is for individuals. However, it contains a higher level of nicotine without the added tobacco. Higher levels of nicotine have worse effects on adolescent brains. In 2015, it was reported that one in four high schools students have vaped while one in fourteen middle schoolers have also vaped. Nicotine use in cigarettes, e-cigarettes, and vapes are an epidemic among teenagers in the United States.

Adolescent smoking needs to be addressed. Nicotine has many long-lasting effects on the brain and body. While there has not been enough research about vaping to understand the extent of the damage it may have to your health, the high levels of nicotine are detrimental to the brain and lead to decreased cognitive skills.. Vaping is not safe no matter how you look at it. Although vaping is not a carcinogen, it is the same as cigarettes. Nicotine is one of the most dangerous chemicals in cigarettes or vapes.

Sources: “Nicotine.” Psychology Today, Sussex Publishers, 17 Apr. 2017, www.psychologytoday.com/conditions/ nicotine.

“How Does Nicotine Act in the Brain?” NIDA for Teens, NIDA, 14 Feb. 2018, teens.drugabuse.gov/teachers/ mind-over-matter/nicotine/how-does-nicotine-act-brain.

Alban, Deane. “Nicotine: An Unlikely Brain Enhancing Drug.” Be Brain Fit, 18 Feb. 2018, bebrainfit.com/ nicotine-brain-enhancing-drug/.

Goriounova, Natalia A., and Huibert D. Mansvelder. “Short- and Long-Term Consequences of Nicotine Exposure during Adolescence for Prefrontal Cortex Neuronal Network Function.” Cold Spring Harbor perspectives in medicine 2.12 (2012): 10.1101/cshperspect.a012120 a012120. PMC. Web. 12 Mar. 2018.

Blakeslee, Sandra. “NICOTINE: HARDER TO KICK...THAN HEROIN.” The New York Times, The New York Times, 29 Mar. 1987, www.nytimes.com/1987/03/29/magazine/nicotine-harder-to-kickthan-heroin. html?pagewanted.

Trimarchi, Maria, and Ann Meeker-O’Connell. “How Nicotine Works.” HowStuffWorks Science, HowStuffWorks, 2 Jan. 2001, science.howstuffworks.com/nicotine2.htm.

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The Fight Against “Superbugs” and How Pharmaceutical Companies are Working to Create a Fully-effective Vaccine

by Hannah Gruber

“Superbugs” have killed around 23,000 people in America over the past year, and doctors have little ability to prevent or treat the diseases. These

mysterious “superbug” disease have been cited to the New York Times as “flesh-eating bugs”, and can be contracted from hospitals or nursing homes. This threat has not diminished, and now pharmaceutical companies need to find a solution quickly.

Johnson and Johnson is now researching if one of their current medications for tuberculosis, Bedaquiline, is an effective vaccine for the new superbugs. Currently, it has proven to be effective, but with the wide range of superbugs, it is impossible to create a single vaccine that could cure each one at once. However, their innovative idea of using antibodies that most people should have already is revolutionary. Since most people already have the tuberculosis vaccine in their body, a new vaccine that builds off that specific antibody can be more effective for treating superbugs. In addition to Johnson and Johnson, there are currently 28 new antibodies that are in the process of becoming FDA approved from pharmaceutical companies across 26

the world.

A different approach has been identified by Dr. Timothy Lu from the Massachusetts Institute of Technology. Dr. Lu indicates that a way to stop the bacteria from infecting the entire body is to treat it at a molecular level and turn away from antibiotics.

And the way to do this is by Crispr-Cas9, a biological tool that can cut away specific sequences of DNA, or genes, and kill the bug at the same time. In addition to Crispr-Cas9, bacteriophages are necessary to carry the Crispr-Cas9 to the specific site of the bug.

This system has its advantages because the DNA in a bug is vital to its survival, and eliminating specific genes eliminates the bug as a whole. However, it has its disadvantages as well, because the process to use Crispr-Cas9 is nowhere near its final stages.

Additionally, as superbugs evolve, there is a chance that this method could be deemed useless. Thus, scientists from around the country are working to find solutions, but the complexity of their ideas mean that a solution may not be found for many years.

Many of the superbugs that are currently in circulation around the world are resistant to antibiotics. For this reason, the people who have contracted these diseases are dying at extremely high rates. Many factors can contribute to a microbe being antibiotic resistant. A person could be taking an antibiotic at the same time they contract the superbug and not have the available antibodies to combat the bug. Also, it is not clear to researchers what type of microbe is being affected by the superbug because the medical history of the patient is needed to understand why their body is not responding to the bug in the first place. A theory that has been introduced is that when a person has taken a specific antibody many times, the microbe in their body, necessary to combat the superbug, as become resistant.

To prevent the deadly superbugs from entering you or your family, the Center for Disease Control and Prevention (CDC) has released methods for protection. The first, and arguably the most important, is to be sure to wash your hands. Most of the superbugs are transmitted through touch or breath, so you could potentially be breathing and touching the same things that a person with a superbug is. Also, it is important to have the necessary vaccines and to keep up-to-date with

WINTER 2017 them. The CDC also makes it clear that if you are already sick, make sure you know exactly what your antibiotic is doing to your body and how it would be affected by the introduction of a new vaccine. There may be case in which some illnesses do not need to be treated by antibiotics, so it is imperative to rule out all other options before turning to antibiotics.

Links: https://www.nytimes.com/2018/01/23/health/antibiotic-resistence-glaxo-johnson. https://fairfarmsnow.org/wp-content/uploads/2017/01/Hero2_1200x650.jpg

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The Difficult Search for Truth in the 21st Century

by Jacob Gruber and Mr. Coe

The truth is often elusive when analyzing any given claim about the nature of the physical universe.

Searching for the truth of various claims isn’t as difficult as before, but finding the truth is harder than ever. The Internet provides a vast realm for claims, discoveries, and theories on the sciences, but how does one determine what is true and what is false? This idea of what should be accepted as true dominates much of the discussion in modern society and is a challenge worth researching in and of itself.

In order to discover the truth of a claim, one must be able to distinguish pseudoscience, myth, and various other forms of “fake-science” from genuine science when exploring resources found on the web and elsewhere. It’s important to note that the process of weeding out the “non-science” can be expedited in a myriad of ways. Finding truth is the essence of science, and we should seek the truth to preserve the integrity and authenticity of science, which has so much impact on our daily lives.

Searching out the truth is more difficult than ever due to the immensity of the Internet and the lack of editorial control of its contents. The copious amount of claims and beliefs passing as “truth” or “science” can be investigated through various manners. Finding sound evidence may be the most important factor in determining the validity of a claim. In practice, the evidence would ideally be backed up by research, data, images, and many other forms of convincing detail. In 28

the Stanford Encyclopedia of Philosophy, an article entitled Scientific Objectivity describes the importance of evidence in science. It states, “no matter how desirable, it is clear that our ability to use scientific claims to represent all [facts] and only facts about the world depends on whether these claims can unambiguously be established on the basis of evidence.” It is noteworthy that the word “unambiguously,” is used to show how evidence in supporting a claim is paramount. The article also stated that, “we test scientific claims by means of their implications, and it is an elementary principle of logic that claims whose implications are true need not themselves be true. It is the job of the scientific method to make sure that observations, measurements, experiments, tests—pieces of the scientific evidence—speak in favor of the scientific claim at hand.” By giving “implication and evidence” priority over claim itself, one can discover the truth more easily. For example, if how a belief could be true doesn’t make sense, we should be skeptical of that belief—we may be looking at anecdotal, isolated examples or simply at outliers. This involves the concept of causation and method: for something to be generally true there must be a plausible explanation for how it occurs.

The search for underground water (and for other objects, often metal) by holding a wooden pointer and watching how it moves is called dowsing. As bizarre as this process sounds, 38% of Americans believe in its authenticity (according to a Harris Poll). The support for a scientifically valid method by which dowsing would work doesn’t make sense; it’s not like a metal detector at an airport. That is the first sign of the likely baselessness of dowsing. The rod that a dowser uses could be wood, nylon, or a selection of suitable materials. As far as we know today, water and wood, as well as water and nylon, share no scientific linkage. Electrical metal detectors use scientifically established magnetic properties of alloys to find hidden objects made of metal. The assumption that a wood rod could find water is equivalent to saying a plastic cup could detect underground insects—there is just no credible linkage between them.

The second sign that should promote skepticism with respect to dowsing is the lack of rational evidence and other forms of scientific

WINTER 2017 support to buttress the claim. When scientifically tested under controlled conditions, dowsers have repeatedly failed to find water. According to Robert Carroll, writing in “The Skeptic’s Dictionary”: In 1949, an experiment was conducted in Maine by the American Society for Psychical Research.

Twenty-seven dowsers “failed completely to estimate either the depth or the amount of water to be found in a field free of surface clues to water, whereas a geologist and an engineer successfully predicted the depth at which water would be found in 16 sites in the same field....”. There have been a few other controlled tests of dowsing and all produced only chance results.

I n Amber Kanuckel’s article, Dowsing: Is It Fact or Fake?, she states, “Skeptics would argue that the reason for this is simple: No matter where you drill, as long as you’re willing to drill deep enough, you’re bound to find water eventually” (Kanuckel). Essentially, if a dowser dowses for long enough, he will find water. Confirmation bias would tend to convince dowsers and observers that dowsing works, and produce testimonial evidence from both groups, but finding something that is invariably present doesn’t qualify as evidence a scientist would accept.

For a claim to qualify as science, many boxes must be checked. As stated above, significant evidence must be available to bolster the claim. Experimentation, when applicable, is also a vital component of providing the evidence to demonstrate whether a theory is true or false.

The result of an initial experiment could just be an anomaly, but those who choose not to test their theory again will never find out. If a dowser was to find water on his first try, he should try again, and investigate under controlled conditions to be sure the discovery is not just good luck. The dowser must not assume that the discovery was a result of successful dowsing until he is able to repeat the experiment many times with similar results.

The problem of coming to a conclusion too quickly is common in superstition and pseudoscience. If someone were to perform more experiments to justify a claim, thus taking more time and care, the claim would be scientifically demonstrated to be ostensibly true or false. Qualification for a claim to be scientific is achieved through meticulous experimentation and/or the accumulation of

compelling evidence. Sherrilyn Roush, the author of Tracking Truth: Knowledge, Evidence, and Science, writes that “philosophers of science implicitly assume that we would know what scientific knowledge was, or near enough, if only we knew what makes one statement evidence for another: a true belief is scientific knowledge, roughly, when one has good evidence for it.” While scientists might disagree with Roush about the premise of how philosophers of science qualify a claim as true, she makes an interesting point. When she says, “if only we knew what makes one statement evidence for another,” the reader can consider various beliefs, with their respective evidence, according to her statement. If dowsing rods and the material that dowsers search for share a common bond that allows the one to react to the presence of the other, then dowsing could be true, but that’s only a small piece of evidence supporting a possible mechanism for dowsing. In this hypothetical, we are able to begin to justify dowsing though evidence, thus making “one statement evidence for another.”

Qualifying claims as scientific can only be done through a painstaking search for evidence, often involving experimentation.

Myths in the world of science are common, but one of the oldest and more widely known is the legend of The Lost City of Atlantis, a “utopian” and “highly advanced” society. Atlantis was described to be home to “moral and spiritual” people. Plato, the famous Greek philosopher, was the person responsible for initiating the worldwide attention given to Atlantis. Plato wrote two pieces on Atlantis which are the first historical documented references to the city. The works are “Timaeus” and “Critias.”

Plato spoke of Atlantis in one his works, saying “... your country shone forth, in the excellence of her virtue and strength, among all mankind” (Timaeus 2). He also said that Atlantis “was pre-eminent in courage and military skill” (Timaeus 2). He wrote that the people of Atlantis, however, turned into materialistic and trivial people who lacked morals.

Their gods “became angry because the people had lost their way and turned to immoral pursuits,” writes Charles Orser, a curator of history at New York State Museum. Plato also said, “afterwards there occurred violent earthquakes and floods; and in a single day and night of misfortune all your warlike men in a body sank into the earth, and the 29

PCR island of Atlantis in like manner disappeared in the depths of the sea” (Timaeus 2). Any hunt for Atlantis begins by reflecting upon the very words of Plato. A number of people believe that Plato might have just contrived Atlantis, despite Plato saying that he learned about the city during his travels.

James Romm, a professor at Bard College, was convinced of the fact that Plato invented Atlantis.

He believes he may have done this to display his own philosophical ideas. Romm claims, “He was dealing with a number of issues, themes that run throughout his work...his ideas about divine versus human nature, ideal societies, the gradual corruption of human society—these ideas are all found in many of his works. Atlantis was a different vehicle to get at some of his favorite themes.” If Romm is correct, then the search for Atlantis is a search for nothing.

Claims for the existence of Atlantis are widely available, but they are not necessarily as reliable as they are pervasive. Renowned film director James Cameron directed a documentary on Atlantis that was released in January of 2017.

The documentary first looks at Plato’s works for hints about locating the lost city. Scientists cited by the movie did in fact find ancient anchors where the Pillars of Hercules once stood. What makes this interesting is that the location matches where Plato indicated Atlantis might have stood. Plato stated, that Atlantis had “subjected the parts of Libya within the columns of Heracles” (Timaeus 2). Richard Freund, a professor at the University of Hartford, explains, “‘Plato is writing in a very specific time period, so when he says Atlantis was located at the Strait of Gibraltar—he called them the Pillars of Hercules at the time—every single mariner, every single Greek reader, knew exactly where he was talking about.’” The anchors discovered by the scientists are believed to be 3,000 to 4,000 years old, an age consistent with the possible disappearance of Atlantis. Another compelling fact is that Plato said that there was a port by the Pillars of Hercules, which could easily explain the anchors. While this may seem to some like definitive proof of Atlantis’ existence, the evidence actually lacks substance.

Finding anchors in the ocean is a fairly common occurrence, and isn’t an immediate indication that Atlantis existed where they were found. What is missing from the justification of the existence of 30

Atlantis is an actual record. Plato had no known evidence supporting his story of Atlantis, making its existence even less likely. Evidence (such as the anchor story above) exists to weakly support a claim for the existence of Atlantis, but there is also evidence stacked against the belief. Many scholars who have searched sources contemporaneous with Plato have found no mention of the city, which is unlikely given Plato’s claim that it fought a war with Sparta.

Another more scientific example of how evidence supports the search for truth (even in the face of impassioned opposition) is the discovery of the bacterium that causes peptic ulcer disease, Helicobacter pylori. Barry J. Marshall and Robin Warren discovered that Helicobacter pylori populates the stomach and causes a multitude of problems: chronic superficial gastritis, chronic active gastritis, peptic ulcer disease, and gastric adenocarcinoma. Before their pioneering work, doctors believed that the cause of peptic ulcer disease was stress, diet and lifestyle, and therefore Marshall and Warren ran into a well known problem in science--the concept that scientific communities (and all knowledge-based communities) resist ideas that challenge established beliefs, an attitude the philosopher Thomas Kuhn called an “established knowledge paradigm.” According to Niyaz Ahmed, the writer of 23 Years of the Discovery of Helicobacter Pylori: Is The Debate Over?, “Warren and Marshall rebutted that dogma [that stress, diet, and lifestyle caused and cured ulcers], and it was soon clear that H. pylori, causes more than 90% of duodenal ulcers and up to 80% of gastric ulcers.” However, Warren and Marshall had trouble gaining the support for their claim because it was dramatically inconsistent with the widely accepted scientific paradigm at the time.

They felt they needed to use exceptionally abnormal methods in order to generate sufficient evidence to support their claim. “In 1985, for example, Marshall underwent gastric biopsy to produce evidence that he didn’t carry the bacterium, then deliberately infected himself to show that it in fact caused acute gastric illness” (Ahmed). He then cured himself of the ulcers using an antibacterial agent. Warren and Marshall were initially ridiculed by the medical community, but their convincing demonstration of their claims via Marshall’s unusual measures

WINTER 2017 became widely known as possibly the most extreme example of producing indisputable evidence to support a scientific claim ever. In this case, the uncommon measures were justified, as Marshall and Warren received the Nobel Prize for Medicine in 2005, and countless sufferers of peptic ulcer disease were cured.

When detecting truth, one must utilize an inductive process. Experimentation and the gathering of evidence is crucial, as it allows us to distinguish between between truth and fiction while pursuing information. Questionable beliefs such as dowsing, which nearly all scientists consider pseudoscientific, and stories such as Platoâ&#x20AC;&#x2122;s description of Lost Continent of Atlantis can be explored through a systematic approach involving the search for evidence and controlled experimentation. This exploration is remarkably similar to the methods used by Marshall and Warren in their discovery of H. pylori. With the vast, unverified information on the Internet, truth is harder to detect. However, one can be much more confident of success using the right tools, one of which is the scientific method of gathering and validating evidence.

References: Drye, W. (n.d.). Atlantis. Retrieved from National Geographic website: http://www.nationalgeographic.com/ archaeology-and-history/archaeology/atlantis/ Griffiths, S. (n.d.). Are we closer to finding Atlantis? Retrieved from Dailymail website: http://www.dailymail. co.uk/sciencetech/article-4172388/Has-Lost-City-Atlantis-last.html Kanuckel, A. (n.d.). Dowsing: Is it fact or fake? Retrieved from Farmers Almanac website: http:// farmersalmanac.com/weather/2015/07/27/dowsing-fact-fake/ Roush, S. (n.d.). Tracking truth: Knowledge, evidence, and science [e-book]. Retrieved from https://books. google.com/ https://img.gazeta.ru/files3/617/6339617/luxfon-pic4_zoom-1000x1000-69119.jpg

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Biased Bots

by Jessica Yatvitskiy

Did you ever think that it was possible for inanimate objects to be biased?

Recently, scientists have been training machine learning programs to complete tasks such as predicting income, areas of future drug crime, and potential of future recidivism by criminals, and even judging promotion-worthiness! The decisions made by these algorithms have major effects on human lives, so when bias skews the results, we have a problem. Unfortunately, bias in AI is very common, and scientists often do not even realize that their machines’ results are distorted.

So far, many of the machines’ predictions have been mostly accurate, but the biases they have gained from being fed data over the last previous years often causes them to make incorrect, or narrow, predictions that discriminate against certain races, sexes, incomes, etc.

“This is a very common issue with machine learning,” says computer scientist Moritz Hardt of the University of California, Berkeley.

“Even if a programmer designs an algorithm without prejudicial intent, you’re very likely to end up in a situation that will have fairness issues.

This is more the default than the exception.”

mainly reported perpetrators from Non-Caucasian, unprosperous neighborhoods, while public health data of 2011 suggests that crime was indeed much more widespread.

Similarly, the Correctional Offender Management Profiling for Alternative Sanctions program, or COMPAS, made incorrect predictions about white and African American defendants.

Among the defendants who didn’t commit further

crimes in the future, the algorithm had far more often wrongly marked the black defendants as high-risk for future criminal activity than the white defendants. On the other hand, among those who did commit future crimes, whites were much more frequently incorrectly predicted to be low-risk for future crime.

Effects of Bias One example of “skewed results” caused by AI bias is a program that was fed drug crime data from Oakland, California in order to predict areas of future drug crimes. In 2010, this program 32

Dealing with Bias To address the bias in the drug crime predicting machines’ algorithms, scientists suggested

WINTER 2017 changing the factors that the machines use to predict whether an area was mostly non-white or unwealthy. For example, if the programs used zip codes to determine what the prominent race and income status of certain neighborhoods were, the scientists could assign fake numbers to a very large amount of people’s zip codes so that these zip codes would no longer be effective in predicting race.

While the proposed changes have not yet been implemented, the researchers are planning to begin the revised approach soon.

While being optimistic about the results, some, researchers are concerned that accuracy may slightly decrease as a result of this change.

After all, even though the researchers do not want the machines to focus solely on non-Caucasian, poor neighborhoods, these neighborhoods really do encounter drug crime more often than others.

As for the COMPAS algorithm, the researchers suggested switching several of the program’s past decisions until each race is incorrectly marked at the same rate. In addition, they would supplement code to their machine’s programs to ensure that the algorithms dole out errors to those of different backgrounds at equal rates and to make their program account for past mistakes. This approach would ensure that the program will be far more “even-handed” in the future.

When scientists tested their revised algorithm on a dataset containing criminal profiles and recidivism rates (whether or not the criminal reoffended), they found that their approach had been successful in reducing the contrast in incorrect classification rates between the races.

While the “old” program results mislabeled fortyfive percent of blacks and twenty-three percent of whites as future criminals, a revised approach indicated a significant drop to twenty-six percent of blacks and twenty three percent of whites.

Once again, researchers hypothesized that while this approach may be effective in preventing bias, it may also lead to less accuracy in the program’s future decisions; it is a fact that, presently, African Americans are arrested for crimes at higher rates than Caucasians. In addition, it would take a long time to collect enough real outcomes to contrast with the program’s predictions.

A Broader Look at Decreasing Bias Anu Tewary, chief data officer for Mint at Intuit, proposed one way to decrease gender bias in AI algorithms: encouraging more females to become involved with computer science. “To inspire more women to get involved in tech and therefore reduce gender bias in tech products, Tewary started a program called the Technovation challenge in 2009, meant to empower females to see themselves as creators of technology. To date, the global program has been completed by 10,000 girls.”

Perhaps, inspiring more African Americans to join the computer science field will help to reduce racial bias in many algorithms, including a facial recognition program that falsely marks African Americans as gorillas.

Article Sources: Maria Temming, “Fair-Minded Machines”, ScienceNews Magazine, 16 Sept. 2017 https://www.techrepublic.com/article/bias-inmachine-learning-and-how-to-stop-it/ Hope Reese, “Bias in Machine Learning and How to Stop It”, Tech Republic, 18 Nov. 2016 https://www. sciencenews.org/sites/default/files/2017/08/main/ articles/091617_learning-machines_bargraph-730. png Image Sources: https://www.sciencenews.org/sites/default/ files/2017/09/091617_learning-machine_maps_ REV_f6.jpg https://i2-prod.mirror.co.uk/incoming/ article9114083.ece/ALTERNATES/s615b/RobotGavel.jpg

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Outbreak of the Flu Virus

by Ameera Ebrahim

This year’s flu virus was one of the most deadly we have seen thus far in the United States.

From October 1st to January 20th, the peak of flu season, 11,965 laboratory confirmed hospitalizations due to the flu were reported. This year alone there could have been 12,00056,000 flu related deaths. In North Carolina, 140 reported flu-related deaths were reported as of February 9th. However, it is not just elderly people who are dying; there are healthy ten year olds, and even a twenty-one year old bodybuilder who died due to the flu. So what made this years influenza virus so lethal?

The first reason the flu was so intense this year was because of the type of flu virus that was present. The most dominant virus was classified as the H3N2 strain. This strain is typically the harshest and most difficult to treat. In years were the H3N2 strain were most prevalent 34

there aware more reported hospitalizations, deaths, and illnesses due to complications of the flu. People who are hit the hardest by this virus are often the very young and very elderly.

As top CDC flu expert Daniel Jernigan said, “of the viruses we hate, we hate H3N2 more than the other ones.”

Not only is the dominant strain the most deadly, but there were other factors that are attributed to this year’s flu mortality rate. The difference this year compared to past years was the timing of when the virus struck. This was a huge factor in the widespread nature of this year’s flu. In years past there wasn’t much flu activity until January, but this year there were symptoms reported as early as November. The flu came about just as people were gathering for the holidays, making the virus prone to spread faster and to more people. Kristen Nordland, press officer for the Centers of Disease Control and Prevention, explained that “in previous seasons it tends to happen in different parts of the United States at different times. This is one of the first years we’ve really seen that widespread activity is everywhere. Flu is really across the board in every state at the same time.” According to the

CDC, over the past fifteen flu seasons, this season is the only time that all fifty states reported widespread flu activity in the same week.

According to National Geographic, this year’s flu season was not only particularly bad in the USA, but also in other countries such as the UK and Australia. Along with the other issues as described earlier, National Geographic claims the vaccination was not as effective as in years past. They believe that “influenza vaccines are typically grown in chicken eggs and when this year’s vaccine was being incubated, the virus mutated while it was growing and therefore became less effective.” Researchers believe that only thirty percent of the vaccines were effective against H3N2. In Australia, the vaccine was only 10 percent effective.

This meant that compared to previous years, those who chose to get vaccinated were not as well protected. Therefore, people were more susceptible to catching the flu and the lethal symptoms that came along with it.

Unfortunately this year, we just got unlucky. Not only did we get the strain of influenza that is associated with the most deaths and illnesses, but it hit the entire country all at once, lasted for three more weeks, and our vaccination didn’t work as well as in previous years. In order to prevent a repeat of this year’s flu season in the future, it is important to stay home from gatherings when you are sick and, of course, wash your hands.

It is also important to realize

WINTER 2017 that after you get sick once in a season, that doesn’t mean you can’t get sick again. During the flu season we should all be cautious and take all the necessary preventative measures so as to avoid this year’s particularly deadly flu season.

Sources Sun, Lena H. “Here’s What You Should Know about the Flu Season This Year.” The Washington Post, WP Company, 2 Mar. 2018, www.washingtonpost.com/news/to-your-health/wp/2018/01/25/heres-what-youshould-know-about-the-flu-season-this-year/?utm_term=.49b10fa5b735.

Cope, Cassie. “Flu Has Killed 140 People in NC This Season – and Experts Expect It to Get Worse.”

Charlotteobserver, Charlotte Observer, 8 Feb. 2018, www.charlotteobserver.com/news/local/ article199086399.html.

Ryan, Lisa. “Why Is This Flu Season So Deadly?” The Cut, 31 Jan. 2018, www.thecut.com/2018/01/fluseason-2018-deaths-peak.html.

Gibbens, Sarah. “What Makes This Flu Season So Bad.” National Geographic, National Geographic Society, 17 Jan. 2018, news.nationalgeographic.com/2018/01/flu-influenza-h3n2-virus-outbreak-vaccine-spd/. https://cdn10.bostonmagazine.com/wp-content/uploads/2013/10/flu-virus-main.jpg

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