Fall 2014: Volume 7, Issue 1

The Amherst

ELEMENT Volume 7, Issue 1

Fall 2014

Letter From The Editor Thank you for picking up a copy of the Fall 2014 edition of The Amherst Element! Thank you to all the writers and editors who contributed this semester.

This semester, we have an article on the importance of “blue skies” science research (Blue-Skies Science: The Importance of Fundamental Research by Carolina Carriazo ‘18), the controversy over Pluto’s status as a planet (Pluto’s Status Questioned Once Again by Alifayaz Abdulzahir ‘17), a look at the science behind various popular “challenges,” including the ALS Ice Bucket challenge (The Science Pertaining to #IceBucket and Other Challenges by Xiao Xiao ‘16), a new understanding of cell growth and differentiation (Pulling the Strings of Your Cells

by Nouraiz Falik ‘18), new ways to manipulate DNA (Cut-and-Paste DNA: The Story of CRISPR by Nathan Yao ‘17), RNA sequencing (RNA Sequencing Analysis of Neurons by Kevin Mei ‘16), two articles on different aspects of the STAT5 signaling pathway in immune function (A Not-So Evil Mini-Me by Thomas Savage ’15 and Constitutively Active STAT5 Protein To Enhance T-Cell Proliferation by Minjee Kim ‘17), and a look at jet lag and its applications (Unlikely Match: Melatonin and Jet Lag by Ashley Montgomery ‘16). Kevin Mei ’16 has provided cover photos from research he did on neurons over the summer.

We hope you enjoy reading this edition!

Thomas Savage

News-In-Brief Thomas Savage ‘15 Advances in Image Recognition Software Scientists at Google and Stanford have independently designed artificial intelligence software that mimics humans in describing the content of pictures. The Google software applies two “neural” methods. The first, image recognition software, learns how to identify entire scenes, and the second, natural language processing, then writes a caption of the image. These neural networks, inspired by how our brains work, are able to recognize patterns and similarities that even their human guides may not be able to see. The scientists trained their programs on a small number of annotated pictures and the programs were then able to accurately caption a subsequent photo, writing “A group of young men playing Frisbee.” More growth in the field could help describe the world to the blind and may help catalogue the millions of photos and videos online.

The First Comet Landing We think of comets as dangerous, scary, fast objects that streak past the earth, sometimes colliding with the earth. Scientists from the European Space Agency have managed to land a space craft on the surface of a speeding comet. The comet, known as Comet 67P/Churyumov-Gerasimenko and 2.5-miles wide, was moving faster than 40,000 miles an hour. This mission is the first to rendezvous with and orbit a comet, and now, is also the first to deliver a probe to a comet’s surface. In addition to the proof of principle that humans can reach comets, scientists hope to use data from the probe to explore if the earth’s oceans are made of melted comets, among other things.

About the Cover Photos

These are dissociated cell cultures of primary human cortical neurons. For the front cover, in blue is a stain against the protein TUJ1 which should reveal the entire cytoskeleton. It’s a pan-neuronal protein so we should be able to visualize all neuron types. In green and red are stains for CTIP2 and TBR1, which are transcription factors and why we see localization to the nucleus. They label cortical cell layer V and VI neurons, respectively. For the back cover, in red is a strain for Nestin, which also labels the cytoskeleton and only appears in neural progenitor cells. What we see in red then are cells that can become glial cells or any of the cortical neuron types.

Unlike in the front cover, where we’re only looking at neurons, we can see some great shapes for neural progenitor cells, like the one in the middle of the page. CTIP2 is again in green, while yellow is a stain for SATB2, also a transcription factor, that labels cortical layers II, III, and IV neurons.

Kevin Mei

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Table of Contents

The Amherst Element Staff Editor-in-Chief Thomas Savage

Advisor to the Editor Kevin Mei

Associate Copy Editors Alifayaz Abdulzahir Carolina Carriazo Nouraiz Falik Minjee Kim Kevin Mei Ashley Montgomery

Layout Carolina Carriazo Get Involved!

Send questions, comments, letters, or submissions to theAmherstElement@ gmail.com.

Call For Writers!

The Amherst Element is looking for more writers and contributors!

We’re always looking for diversity! We welcome writers from all backgrounds from computer science and math to the humanities.

Please feel free to submit articles regardless of your background!

If thesis students would like to be write about their research or are willing to be interviewed, please contact us!

Cover Features

1 Pan-Neuronal Stain

Kevin Mei ‘16 24 Neural Progenitor Stain

Kevin Mei ‘16

Features

2 News-in-Brief

Thomas Savage ‘15

Letters

4 Blue-Skies Science: The Importance of Fundamental

Research

Carolina Carriazo ‘18 6 Cut-and-Paste DNA: The Story of CRISPR

Nathan Yao ‘17 9 Constitutively Active STAT5 Protein to Enhance T-Cell

Proliferation

Minjee Kim ‘17 12 Pulling the Strings of Your Cells Nouraiz Falik ‘18 14 Pluto’s Status Questioned Once Again

Alifayaz Abdulzahir ‘17 16 RNA Sequencing Analysis of Neurons

Kevin Mei ‘16 18 The Science Pertaining to #IceBucket and Other Challenges

Xiao Xiao ‘16 20 A Not-So-Evil Mini-Me

Thomas Savage ‘15 22 Unlikely Match: Melatonin and Jet Lag Ashley “Monty” Montgomery ‘16

The opinions and ideas expressed in The Element are those of the individual writers and do not necessarily reflect the views of The Element or Amherst College. The editorials are a product of the opinions of the current editor-in-chief of The Element. The Element does not discriminate on the basis of gender, race, ethnicity, sexual orientation, scientific background, or age.

Research findings published in The Element are not intended for wide distribution or for the reader’s profit. As a member of the Amherst community, please use the information and data presented in The Element judiciously.

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Blue-Skies Science: The Importance of Fundamental Research Carolina Carriazo ‘18

Figure 2: The Tyndall effect as seen in opalescent glass.

Figure 1: The ‘pyramids of solid fire’, as Tyndall may have seen them.

Early on the morning of August 16th, 1871, an Englishman named John Tyndall looked quietly out on the Alps from the top of a mountain. Dawn came – and with it, all the majesty of the region’s natural beauty. He wrote of his regular expeditions, and described the mountains on this morning as “pyramids of solid fire, while here and there long stretches of crimson light drawn over the higher snow-fields linked the summits together.”1 Tyndall’s name lives on today not because he was merely a romantic, but because he was a physicist. He discovered what we now call the Tyndall effect –a cousin of the Raleigh effect– which explains how light scattering by gas molecules gives the sky its characteristic blue color. The Tyndall effect, however, describes the scattering of light by larger, colloidal particles, like dust and other atmospheric impurities.2 These impurities were present in the alpine horizon and produced the glorious sunrise that Tyndall so admired. His singular curiosity for this natural phenomenon led to experimentation that resulted in the eventual discovery of colloidal light scattering.

John Tyndall inspired generations of scientists that followed to pursue curiosity-driven research, or ‘blue-skies’ research, as he did; and the results of scientists pursuing serendipities often prove the most important, as with Alexander Fleming, who grew particularly interested when a rare strain of Penicillium fungus accidentally decimated his laboratory culture. However, as technology progresses at an exponential rate, society has pressured

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and continues to pressure the manufacturers of technology to abandon their ‘irrelevant’ inquiries for the sake of efficient, highly industrialized research. We can see the ramifications of this attitude with a quick glance at the job market: biomedical engineering is expanding at an incredible rate of 27%, while medical science and microbiology lag behind at 13% and 7%.3 4 5 Now science for the sake of understanding teeters on the periphery; science for the sake of technology is a weapon, and an unapologetically narrow one.

Nowhere is this more evident than in the pharmaceutical industry. It is the quintessential example of targeted science – technicians run millions of experiments in succession to create an effective drug for the market and to progress towards a very specific and (admittedly) highly profitable goal. But in the chaos of medical testing, any drug that does not immediately produce a positive result for the project at hand is discarded; for every step forward, thousands of potential discoveries for other applications remain ignored. Johnson & Johnson, America’s largest pharmaceutical company, rakes in $71 billion a year6 in revenue, and industry continues to be a comfortable career goal for scores of the nation’s researchers-in-training.

Recently there has been an effort to beat back targeted science and allow blue-skies research to thrive once again. In Britain, the Medical Research Council (MRC), in partnership with several of the nation’s leading universities, is embarking on a project called The Francis Crick Institute. Due to open in 2015, the Institute will be a biomedical research center with the added priority of “discovery

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Figure 3: A plant in a Johnson & Johnson pharmaceutical laboratory. without boundaries”; it will allow its scientists to independently probe the questions underlying human biology while still satisfying the public demand for pharmaceutical progress. Researchers from 1200 different disciplines will come together to solve fundamental issues surrounding embryology, immunology, neurobiology, etc., but all within the context of studying human diseases, so that while they make important advances in practical medicine, they can also pursue related, but more theoretical, tangents.7 Many consider the already-established Large Hadron Collider (LHC) in Switzerland to be a massive blue-skies undertaking based on the interests of the physicists stationed there. The experimental discovery of the Higgs-Boson and the confirmation of the Standard Model of particle physics in recent years has proved the LHC to be an invaluable asset to the field.8 The total budget of the LHC is 7.5 billion euros, or about 9.5 billion dollars – and yet that’s only a fraction of the U.S. drug industry’s annual spending in the early 2000s on research and development, which at its most conservative estimate is 20 billion dollars (not adjusted for inflation).9 10 In our modern, fast-paced world, it appears at first glance that the old tradition of fundamental research in science for the sake of science – rather than science as the means to an end of an immediate application – is long forgotten. Many argue that the fine-tuned system of today’s research simply cannot allow for the indulgence of these pursuits, and that they belong in the past, in a time before vigorous industrialization and specialization. But if the course of history can show us anything, it is that successes rarely come from original, deliberate aims. Newton’s famed speculation on an apple falling from a tree led to his discovery of gravity; William Henry Perkin’s failed synthesis of quinine at 18 led to the creation of Victorian England’s most beloved color, mauve; and in 1945, Percy Spencer’s chance run-in with a microwaveemitting radar set that melted the chocolate bar in his pocket led to a revolution in the American kitchen. The importance of these ‘accidental tangents’ remains an open controversy – but without the liberty of free, curiosity-driven pursuit, we may just overlook the one discovery that would change our world forever.

REFERENCES 1. Tyndall, J. (1897). IX. The Weisshorn. In Hours of Exercise in the Alps. New York: D. Appleton and.

2. Douma, M., curator. (2008). Blue and Red. In Cause of Color.

Retrieved October 6, 2014, from http://www.webexhibits.org/ causesofcolor/14B.html.

3. Bureau of Labor Statistics, U.S. Department of Labor, Occupational Outlook Handbook, 2014-15 Edition, Biomedical Engineers. Retrieved October 6, 2014, from http://www.bls.gov/ ooh/architecture-and-engineering/biomedical-engineers.htm .

4. Bureau of Labor Statistics, U.S. Department of Labor, Occupational Outlook Handbook, 2014-15 Edition, Medical Scientists.

Retrieved October 6, 2014, from http://www.bls.gov/ooh/lifephysical-and-social-science/medical-scientists.htm .

5. Bureau of Labor Statistics, U.S. Department of Labor, Occupational Outlook Handbook, 2014-15 Edition, Microbiologists.

Retrieved October 6, 2014, from http://www.bls.gov/ooh/lifephysical-and-social-science/microbiologists.htm .

6. Johnson & Johnson Reports 2013 Fourth-Quarter and FullYear Results. (2014, January 21). Retrieved October 6, 2014, from http://files.shareholder.com/downloads/ JNJ/3332014217x0x719867/185e8986-d100-445a-88a16fd4691a8786/JNJ_News_2014_1_21_Earnings.pdf .

7. Pursue discoveries without boundaries | The Francis Crick Institute. (n.d.). Retrieved October 6, 2014, from http://www. crick.ac.uk/strategy/pursue-discoveries-without-boundaries/ .

8. Higgs boson: Scientists 99.999% sure ‘God Particle’ has been found. (2012, July 4). Retrieved October 6, 2014, from http:// www.telegraph.co.uk/science/large-hadron-collider/9374758/ Higgs-boson-scientists-99.999-sure-God-Particle-has-been-found. html .

9. CERN Ask an Expert Service. (2007). Retrieved October 22, 2014, from http://askanexpert.web.cern.ch/AskAnExpert/en/ Accelerators/LHCgeneral-en.html#3 Figure 1: http://farm5.staticflickr.com/4147/5071415339_ bd5e09604f_b.jpg Figure 2: https://farm1.staticflickr.com/41/112909824_ d4c86088e2_o.jpg Figure 3: http://www.industry.siemens.com/topics/global/ en/magazines/process-news/pharmaceutical/johnson-early planning/PublishingImages/Johnson_pharma_site_458x272.jpg

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Cut-and-Paste DNA: The Story of CRISPR Nathan Yao ‘17

Imagine a world where you could manipulate genes –really any sequence of DNA – at will: DNA selectively inserted or cut out and, if desired, replaced altogether. In many cases, all you would really need are some donor DNA and some proteins (and maybe a short sequence of RNA, depending on the system being used).

Of course, developing and utilizing such proteins, identifying a good target site for insertion, and achieving full manipulation efficiency throughout a multicellular organism all present their own difficulties; nonetheless, all this is possible. These mechanisms are called Genome Editing via Engineered Nucleases (GEEN), and they have tremendous potential in both basic and clinical research; in the two decades that these methods have been around, there has been steady improvement.

For the most part, these GEEN systems require a nuclease, or a protein that breaks down bonds in strands of DNA, and a donor strand of DNA. Engineered nucleases introduce donor sequences into a target DNA strand by initiating a break in both strands of target DNA, triggering cellular DNA repair mechanisms to fix the break by including the donor DNA. The differences between GEEN systems, however, have been in the relative flexibility of each system in binding to targets, in efficiency of cutting DNA, as well as other factors such as in binding and cutting at sites other than the target (toxic off-target effects).

In the 1990s, the first GEEN method emerged: DNA targeting via hybrid meganucleases. These meganucleases naturally occur in microbial species, and contain long recognition sites that allow these proteins to target very specific DNA sequences prior to cutting.

There is one major downside, however: it is extremely difficult, if not impossible, for a hybrid meganuclease to target an arbitrary sequence. While attempting to manipulate genes, researchers will inevitably encounter random sequences in organisms being studied; however, meganucleases are currently not easily modified to target a new sequence. While some research is being done to allow for more customization of these meganucleases (which have a relatively low toxicity in cells and lower potential for off-target effects) the general inflexibility of this method has led to the rise of other GEEN methods.

By the late 1990s, scientists began examining Zinc-Finger Nucleases (ZFNs) for GEEN applications, and by the late 2000s Transcription Activator-Like Effector Nucleases (TALENs) were being considered as well. Structurally, these two systems are quite similar: they rely on fused proteins consisting of a cleavage domain (generally the restriction enzyme FokI) for cutting DNA and a modular DNA binding domain that can be configured to match a particular target sequence. With these constructs, the custom

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protein can bind to the target DNA sequence through the binding domain and cut DNA with the cleavage domain. For ZFNs, zincfinger protein domains each bind to three or four base pairs in target sequence; for TALENs, the binding domain consists of modularly assembled TAL Effector proteins (TALEs) from Xanthomonas bacteria, with each TALE binding to one base pair.

Target sequences are generally at least 18 base pairs or so, to ensure specificity in a large genome; thus, between four to six zinc-fingers might go into a well-constructed ZFN, and around 18 TALEs (the length of the target sequence) might go into a well-constructed TALEN. In late 2011, a paper1 by Jinek et al. initiated the latest generation of GEEN with the development of CRISPR/Cas9.

This system uses a short sequence of RNA (which researchers have termed “short guided” RNA, or sgRNA) to base-pair with target DNA and guide the Cas9 protein to cut DNA adjacent to the site where sgRNA paired with the target DNA. Incidentally, Cas9 protein comes from Streptococcus pyogenes (the same bacteria responsible for strep throat). CRISPR/Cas9 has had great popularity in the few years since its inception, as it requires a single universal protein, Cas9, that is readily available commercially, and sgRNA, which, with the appropriate sequence, can be easily synthesized with RNA polymerase. Other GEEN methods have required the synthesis of custom proteins, which can be both labor- and time-intensive; the relatively facile construction of CRISPR/Cas9 alongside the high efficiency in multiple studies across different organisms has sparked excitement in the scientific community.

This past summer, I conducted a Summer Undergraduate Research Fellowship (SURF) project at the California Institute of Technology on CRISPR/Cas9 in Strongylocentrotus purpuratus, also known as the purple sea urchin. To illustrate how popular this method was just at Caltech, to my knowledge at least seven other labs there have been conducting research using CRISPR/ Cas9, with subjects ranging from the nematode C. elegans to human muscle cells. The lab I worked in, headed by Dr. Eric H. Davidson, focuses on Gene Regulatory Networks (GRNs) in the purple sea urchin. At its core, GRNs involve the regulation of the expression of some target gene(s) by some regulator gene(s).

Currently, the identification of all the genes within these GRNs often involves manipulation of genes in an embryo. Assume there is a system with a suspected regulator (A) and a suspected target gene that it regulates (B); after altering the functionality or expression of A, the expression of B is measured to confirm that A indeed regulated B. Current non-GEEN methods, however,

Letters

Figure 1: CRISPR/Cas9 digestion of three different genomic sequences as well as controls. With “1” being the gel lane adjacent to the leftmost gel ladder, the lanes are: 1 and 2: Pre- and post-Cas9 digestion of target 1 (FoxA) with mutations induced 3 and 4: Pre- and post-Cas9 digestion of target 2 (Hox11/13b, first target sequence) with mutations induced 5 and 6: Pre- and post-Cas9 digestion of target 3 (Hox11/13b, second target sequence), with mutations induced 7 and 8: Pre- and post-Cas9 digestion of control for target 1 (FoxA, without mutations induced) 9 and 10: Pre- and post-Cas9 digestion of control for targets 2 and 3 (FoxA, without mutations induced) For reference, a gel shows the size of fragments of DNA by its vertical position as compared to a gel ladder; in this particular gel, the ladder is in the leftmost and rightmost lanes, with about 14+ distinct bands) as a reference. From bottom to top, respectively, the bands stand for the following sizes (in base pairs, bp): 100, 200, 300, 400, 500, 600, 700, 800, 900, 1000 (or 1.0 kilobases, kb), 1200 (or 1.2 kb), 1500 (1.5 kb), 2000 (2.0 kb); the others, relatively indistinguishable in this gel, are at 3, 4, 5, 6, 8, and 10 kb. have limitations due to off-target effects, and they can be generally cumbersome and difficult to work with; furthermore, current nonGEEN methods are more limited in the range of genes that it can examine compared to GEEN methods.

Through GEEN methods, and especially the promising CRISPR/Cas9 method, GRNs could be constructed by altering the functionality of suspected regulators on a large scale. I attempted to induce Non-Homologous End-Joining (NHEJ) in embryos, where, in contrast with the cellular repair mechanism described earlier, no external donor DNA is included; in the absence of external DNA, cellular repair mechanisms often can insert, invert, or delete nucleotides near the site of the DNA break, leading to mutations. The purpose of this project was to determine whether or not CRISPR/Cas9 could work in sea urchin embryos to cut DNA at a given site and to introduce mutations at those sites.

My co-mentor and I expected, however, that CRISPR/Cas9 is not yet 100 percent efficient, meaning that some copies of the target DNA would remain uncut and thus not mutated (wild-type).

After doing PCR to amplify both mutated and wild-type strands and allow for base-pairing between mutated and wild-type strands in the process, mismatch of base pairs at the site of mutation could be detected and cleaved by a special endonuclease, T7e1.

By gel electrophoresis, the presence of two smaller cut strands of DNA (in addition to the larger intact strand) would confirm that cleavage by T7e1 had occurred, suggesting that mismatch and thus

NHEJ by CRISPR/Cas9 had occurred.

The gel in Fig. 1 contains both successful and unsuccessful T7e1 assays: for example, in lanes 1 and 2, a before and after comparison of T7e1 on target 1 is shown for the single band at around 1.2 kb. In lane 1, the band has not been digested by T7e1.

In lane 2, the band has been digested by T7e1 into two bands at approximately 750 and 550 bp. The fact that target 1 could be digested into two bands by T7e1 shows that mutation had occurred in this sample and that CRISPR/Cas9 was effective. An example of unsuccessful T7e1 digestion is shown by gel lanes 7 and 8, also representing before and after: while there is some smearing in the “after” lane as a result of some nonspecific cleavage in T7e1, the absence of distinct bands in both lanes (apart from the original at 1.2 kb) shows that no mutations have been induced. These two lanes were controls that were not treated with CRISPR/Cas9, so this was to be expected; this showed conclusively that the CRISPR/Cas9 treatment was responsible for inducing mutations, which resulted in T7e1 digestion.

Figure 2 is a gel picture of PCR-based genotyping to confirm the presence of mutations in samples. This particular method relies on the mutations that can result from the DNA break, such as random insertions, deletions, or inversions the site of the DNA break. PCR primers are chosen within this region, and where the primer can bind successfully (due to an absence of mutations), DNA is amplified. Where PCR primers cannot bind successfully, The Amherst Element, Vol 7, Issue 1. Fall 2014

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Figure 2: PCR-based genotyping of target 1 (FoxA) across ~96 bacterial colony samples. Yellow circles denote where a band would be expected but does not appear due to successful introduction of mutations in the sample. no amplification of DNA occurs, and nothing appears at that position in the gel. As can be seen above, there are very few yellow circles relative to the whole array of bacteria tested, meaning that there was very low efficiency (9%) observed in this bacterial assay; taken as an analogue for efficiency across cells in sea urchin embryos, there would be around a 9% efficiency expected for inducing mutations in sea urchins via CRISPR/Cas9.

Applying this method of CRISPR/Cas9 to research in GRN construction for sea urchins is still in its early stages; without a doubt, a key issue to address will be overall efficiency.

Other literature suggests that this is quite feasible, with CRISPR efficiencies above 50% observed in other model organisms. Such work would certainly supplement basic, theoretical research; meanwhile, progress in other organisms, including various human cell lines, suggest that vast medical applications in gene therapy are forthcoming. In fact, preliminary trials suggest that CRISPR/ Cas9 may be able to respond to diseases such as sickle-cell anemia, HIV, and cystic fibrosis; it may also shed light on conditions such as autism and schizophrenia.2 There is still much work to be done in the scientific community to improve the CRISPR system as a whole; nevertheless, the sheer potential it has to offer is readily apparent, and its researchers seem understandably optimistic.

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References 1. Jinek et al. “A Programmable Dual-RNA–Guided DNA Endonuclease in Adaptive Bacterial Immunity.” Science. August 2012.

2. Young, Susan. “Genome Surgery.” MIT Technology Review. February 2014. http://www.technologyreview.com/ review/524451/genome-surgery/

Letters

Constitutively Active STAT5 Protein to Enhance T-Cell Proliferation Minjee Kim ‘17

Cancer immunotherapy is one of the cancer therapies that receive the most attention from the media largely due to its potential from previous clinical trials on leukemia and lymphomas.

T cells are one of the key players in our immune system, and they have the ability to recognize specific antigens (toxins or foreign substances) through surface proteins called receptors (proteins expressed on these immune cells’ surfaces). Cancer cells often overexpress certain proteins that are normally present in cells. By engineering artificial T cell receptors, scientists are able to instruct the patient’s immune system to attack specific antigens or proteins overexpressed on cancer cells’ surfaces.

Cancer immunotherapies with chimeric antigen receptors (CARs), or engineered T cell receptors, have been tested in several clinical trials. In one of these studies, several of the patients, who had B-Cell Acute Lymphoblastic Leukemia, had engineered T cells effectively attack cancer cells (Terakura et al). In B-Cell Acute Lymphoblastic Leukemia, cancer starts from the B cell, a type of lymphocyte in the bone marrow, and spreads quickly to the rest of the body. In the study, the patients’ T cells were extracted from their blood, and their T cells were modified to express the CARs.

Figure 2 (above). In presence of IL-2, phosphorylated STAT5 protein shows up in all samples. In absence of IL-2, only the CA-STAT5 sample shows phosphorylated STAT5. To make sure that my protein loading level was equal, I also stained for ß-actin, a protein that is present in all T cells.

Figure 1 (above): figure showing the STAT5 molecule; when phosphorylated, it promotes transcription of growth-related genes. Adopted from Waldmann (2006).

While these engineered T cells have a promising future for cancer treatment, there are several flaws in the adoptive T cell therapy, one of them being the short life cycle of the T Cells once they are re-injected into the patient. The engineered T cells need cytokines, or non-antibody proteins secreted by cells upon antigen recognition, to survive. One example of such a cytokine is interleukin-2 (IL-2). The levels of IL-2 needed for the engineered T cells to survive, however, are toxic to patients because IL-2 not only binds to T cells but to many other molecules, and overexpression of these other molecules is toxic (Boyman et al). To relieve this problem, scientists have identified various other proteins, such as growth factors and transcription activators that enhance T cell growth and proliferation. These proteins could be transduced into the T cells, in conjunction with the engineered receptors, so that the cancer-specific T cells survive and show high proliferation and growth without the associated toxicity of IL-2. This past summer, The Amherst Element, Vol 7, Issue 1. Fall 2014

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Figure 3: Growth Curves and Viability Curves of T cells. The T cells extracted from healthy donors were cultured for 12 days prior to the removal of IL-2. These cultured cells were split into two plates, and in one of the plates IL-2 was removed. The T cells were counted at three additional time plots using the hemocytometer: 14 days, 17 days, and 19 days. The control Mock group did not have any virus transduced, and – and + indicate whether IL-2 was added or not.

I worked with one of these transcription activators to enhance T cell growth: the STAT5 molecule.

The Signal Transducer and Activator of Transcription 5 (STAT5) is one of the proteins that have been shown to stimulate T cell growth (Hand et al). In normal T cells, IL-2 binding triggers tyrosine phosphorylation of STAT5. This activated phosphorylated-STAT5 protein turns on the transcription of T cell growth-related genes. By using point mutations, or by mutating a specific sequence of the STAT5 gene, scientists have engineered the normal STAT5 molecule to a constitutively-active (CA) STAT5 molecule; this CA-STAT5 protein, when inserted into T cells, has shown the ability to stimulate cell proliferation to a greater extent than normal (wild type) STAT5 (Ariyoshi et al).

I reaffirmed the efficacy of CA-STAT5 protein by comparing it to the wild type (WT) STAT5 protein. The wild type STAT5 protein was needed as a control, so that we know that it is specifically CA-STAT5 that helps T cell proliferation and viability.

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In the Ben Towne Center for Childhood Cancer Research in Seattle, the lab already had a CA-STAT5 gene cloned. I cloned the WT-STAT5 gene into a DNA backbone so that WT-STAT5 could be put into a virus. Then, I put the CA- and WT-STAT5 genes into two different viruses. After isolating T cells from healthy donors’ T cells, I transduced the T cells and analyzed the phosphorylated STAT5 protein levels with and without IL-2, and tracked the growth and viability of the T cells. My control was cells without any virus transduced (Mock).

To look for phosphorylated-STAT5 levels without IL-2, I cultured the T cells without IL-2 for 3 days. After 3 days, I lysed the cells and extracted proteins from the T cells for western blot analysis. In a western blot analysis, the extracted proteins are run on a gel and are separated by their size. These proteins are then stained with antibodies that bind specifically to the proteins. I stained for phosphorylated STAT5, to see whether T cells had any activated STAT5 proteins without IL-2.

Letters Additionally, I tracked the cell growth and viability by counting them with a hemocytometer. The cells were grown with and without IL-2 for seven days. T cells with transduced CASTAT5 have the greatest growth and the highest viability, both in the presence and absence of IL-2.

My work did not show whether CA-STAT5 enhanced the engineered T cells’ elimination of cancer cells. However, it demonstrated that CA-STAT5 is a possible protein for our engineered T cell proliferation experiments, as T cells proliferate faster and are more viable when transduced with CA-STAT5. Our research institute will continue to explore the efficacy of CASTAT5 in our engineered T cells by making a virus containing both CA-STAT5 and our engineered receptor. We’ll track the viability and growth of each of these cell lines, as well as test their efficacy in killing tumor cells. This would allow T cell specific immune responses against cancer cells without the need for toxic levels of other proteins, enhancing cancer immunotherapy without toxic side effects.

References Ariyoshi et al. Constitutive Activation of STAT5 by a Point Mutation in the SH2 Domain. Journal of Biological Chemistry.

(275: 24407-24413) http://www.jbc.org/content/275/32/24407. long Boyman et al. Insight into Mechanism of IL-2-Induced Toxicity Provides Rationale for Improved Treatment Strategy using IL-2/ mAb Complexes. Journal of Immunology 182(38.8) http:// www.jimmunol.org/cgi/content/meeting_abstract/182/1_ MeetingAbstracts/38.8 Hand et al. Differential effects of STAT5 and PI3K/AKT signaling on effector and memory CD8 T-cell survival.

PNAS 107(38) http://www.ncbi.nlm.nih.gov/pmc/articles/ PMC2944719/ Terakura et al. Generation of CD19-chimeric antigen receptor modified CD8+ T cells derived from virus-specific central memory T cells. Blood 119(1) http://www.bloodjournal.org/ content/119/1/72.long?sso-checked=true Waldmann et al. The biology of interleukin-2 and interleukin-15: implications for cancer therapy and vaccine design. Nature Reviews Immunology 6(595-601) http://www.nature.com/nri/ journal/v6/n8/full/nri1901.html

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Pulling The Strings of Your Cells Nouraiz Falik ‘18

Throughout our years of high school and college biology courses, we’ve always been taught that genes are the key to everything. We’ve heard genetics being associated with phrases like “code of life,” and there is truth in such statements. Genes instruct cells on how to make proteins, which perform tasks for the cell, including determining what kind of cell it becomes. However, researchers working under Steffano Piccolo at the University of Padua in Italy have found that there may be ways for cells to differentiate, independent of proteins.1 The discovery of this new mechanism of cell differentiation has new implications for cancer and stem cell research.

Piccolo writes in his article that scientists working in the 1970s were the first to note a correlation between physical forces – namely, pulling and compression - and the subsequent biological response during cellular reproduction. The cells that were grown on a larger substrate (a fancy name for surface or surroundings) were able to stretch and flatten out. On the other hand, the cells that were grown on a smaller area rounded up and either started differentiating or dying; crucially, they did not divide. Unfortunately, the scientists were not able to provide an explanation for their findings. This is where Piccolo’s team steps in. His team discovered that two proteins, YAP (Yes-associated protein) and TAZ (transcriptional coactivator with PDZ binding motifs)2, are responsible for turning a physical force into a biological response. Piccolo’s research

team’s hypothesis was that YAP and TAZ are always present in the cytoplasm (the jellylike part of the cell that contains organelles, nutrients, and other assorted chemicals) and when a force, such as a stretching force, is exerted, YAP and TAZ end up closer to the nucleus because the inner framework of the cell (the cytoskeleton) had to rearrange itself. This proximity allows the YAP and TAZ to enter the nucleus and bind to certain sections of DNA, resulting in the transcription of genes that create cell growth proteins, which round the cell and even it out. If the cell is unaffected by any stretching or pulling, YAP and TAZ are degraded in the cytoplasm.

You may read this and ask, “Well, is that all YAP and TAZ are useful for? To keep my cells round?” The answer is no. First and foremost, we lose cells every day. The reason your skin is not starting to peel and your organs are not dying is because of YAP and TAZ, which ensure that new cells are made to take the spot of old ones. YAP and TAZ are also vital when you get injured.

The cells near the wound sense that they have more wiggle room so they stretch out leading to the process described above.

In this case, YAP and TAZ trigger production of proteins that promote healing through cell reproduction. For example, Duojia Pan at Johns Hopkins University showed that YAP and TAZ are responsible for regeneration of intestinal lining in mice with colitis, which is inflammation of the lining of the colon.3 There is also a drawback that was observed: the concentrations of YAP and TAZ

Figure 1: YAP and TAZ in two different circumstances. On the top is a diagram of what happens to them when the cell is not stretched. The bottom displays what happens when the cells stretch: YAP and TAZ enter the nucleus and the cells proliferate.

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Letters in the cells have to be just right. Too much of these proteins could cause uncontrolled cell growth, aka cancer, and too little will have minimal effect, meaning your cut will not heal.

Scientists are currently working with mesenchymal stem cells, a special type of cell found in adult organs that are responsible for healing injuries. They have discovered that both the substrate and the levels of YAP and TAZ contribute to determining what kind of cell the stem cell becomes. Adam Engler and Dennis Discher at the University of Pennsylvania discovered that mesenchymal stem cells differentiate into different cell types based on surrounding rigidities.4 For example, a stem cell in a substrate that mimics the stiffness of brain tissue becomes a nerve cell. A member of Piccolo’s team, Sirio Dupont found differing rigidities also change the levels of YAP and TAZ in the stem cell, and both of these factors in unison change the cell.

This work is especially important because of the medical applications of stem cells. Stem cells could be used to repair damaged tissues as well as damaged organs. For example, stem cells could be used to replace weakened muscle in patients suffering from muscular dystrophy. Currently, scientists are trying to work on creating organs outside the human body using equipment that mimics the conditions and pressure of the body. The cells can thus develop as they would in a person, and hopefully the organ will come out functional. This sort of work is going to require much trial and error because the conditions have to be exactly right.

Scientists are also working on using YAP and TAZ to create large populations of stem cells.

As mentioned before, YAP and TAZ are not your best friends if they show up with more than their plus ones. Too much YAP and TAZ leads to tumor development, and thus, cancer. The work on YAP and TAZ by Piccolo and others supported the notion that cancer is a product of disturbed genes and the microenvironment. There are genes that lead to an increased chance of getting cancer, like BRCA which is related to breast cancer, but one does not necessarily have to have these genes to get cancer.

Once the “microscopic architecture” is disturbed, one is more susceptible to cancer. Thus, scientists are working on using YAP and TAZ inhibitors, which would prevent certain types of cells from growing uncontrollably. These drugs may inhibit normal cell proliferation as well, however, which would prevent normal wound healing and the like. Finding some way to remedy this problem may help to put a dent in cancer’s spread.

References 1. Piccolo, Stefano (October 2014). Twists of Fate. Scientific American, Vol. 311, No. 4; pages 74-81 2. Badouel, C., Garg, A., McNeill, H. (December 2009) Herding Hippos: regulating growth in flies and man. Retrieved from http://www.sciencedirect.com/science/article/pii/ S0955067409001835 3. Retrieved from http://humangenetics.jhmi.edu/index.php/ faculty/duojia-pan.html 4. Discher, D.E., Engler, A.J., Sen, S., Sweeney, L. Matrix Elasticity Directs Stem Cell Lineage Specification. http://www. cell.com/abstract/S0092-8674(06)00961-5 Figure 1: http://physrev.physiology.org/content/94/4/1287. figures-only

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Letters

Pluto’s Status Questioned Once Again Alifayaz Abdulzahir ‘17

Pluto was first discovered in 1930 by Clyde W. Tombaugh at the Lowell observatory in Flagstaff, Arizona. Scientists had predicted that the solar system might have a ninth planet, which they referred to at the time as Planet X, because something massive was affecting Neptune’s orbit. Tombaugh discovered this ‘Planet X’ after a yearlong observation of two photographs which were taken two weeks apart. He noticed that an object appeared to jump when comparing one photo the other. They called this ‘Planet X’ Pluto, the name of the Roman god of death. It was a name suggested by an eleven year old school girl in Oxford, England.

It wasn’t until the discovery of its moon, Charon, in 1978 that scientists were able to accurately measure Pluto’s mass. And by knowing its mass, scientists predicted the diameter of Pluto to be approximately 2,400 km (1,500 miles). This is quite a small diameter considering it’s a planet. It’s even smaller than Russia’s diameter, which is approximately 4,135 km (2,567 miles). However, it was still thought to be the largest object beyond Neptune’s orbit.

As technology advanced, new and more powerful telescopes were invented, completely changing our previous understanding of the solar system. Larger and larger objects were discovered in the Kuiper Belt, which is a disc shaped region beyond the orbit of

Neptune that contains many icy bodies including Pluto. Finally, in 2005, an object larger than Pluto was discovered in the Kuiper Belt. It was officially named 2003 UB313, later called Eris, and is approximately 2,600 km (1,600 miles) in diameter. This makes Eris about 25% more massive than Pluto. This brought about lots of controversy and raised many questions about the definition of a planet such as whether Eris is another planet or just another object in the Kuiper Belt? And if it not a planet, then why is Pluto, which is smaller than Eris, considered a planet?

This was a question for the XXVIth (26th) General Assembly of the International Astronomical Union to answer, which met from August 14 to August 25, 2006 in Prague, Czech Republic.

Leading astronomers and scientists from around the world had the chance to vote on a new definition of a planet. One proposed definition would have increased the number of planets in our solar system from 9 to 12, another would have reduced the number of planets from 9 to 8, and a third position would have kept the number of planets at 9 without any scientific rationale. In the end, the astronomers from the assembly chose the second definition and reduced the number of planets to 8.

The International Astronomical Union (IAU) came up with

Figure 1: An artist’s depiction of the Kuiper Belt and Pluto’s orbit in relation to it.

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Letters three criteria that an object needed to fulfill in order to be considered a planet: 1. It needs to be in orbit around the Sun. 2. It needs to have enough gravity to pull itself into a spherical shape.

3. It needs to have “cleared the neighborhood” of its orbit.

Pluto was demoted from planet status because it didn’t meet the third criterion. But what exactly does it mean to have “cleared the neighborhood”?

According to the IAU, it means that a planet needs to be significantly larger than the objects in its vicinity, and since Pluto has several similar sized bodies around it, it has technically not “cleared the neighborhood.”

Any object that meets the first two but not the third criterion is considered a dwarf planet. Therefore, Eris Pluto, and several other similar sized bodies in the Kuiper Belt have been labeled as dwarf planets.

According to the Director of the Planetary Science Figure 2: Relative sizes of a number of dwarf planets in the Kuiper Belt.

Division at NASA, there are over 1,000 objects in the References Kuiper Belt that are considered dwarf planets.

However, a debate was held between Harvard science historian Cain, Frasier. (January 5, 2012). Why Pluto is No Longer a Planet.

Own Gingerich, a chair of the IAU planet definition committee, The Universe Today. Retrieved from http://www.universetoday.

Gareth Williams, an associate director of the IAU’s Minor Planet com/13573/why-pluto-is-no-longer-a-planet/ Center, and Dimitar Sasselov, director of the Harvard Origins of Life Initiative, on September 18, 2013. This debate sparked Overbye, Dennis. (August 24, 2006). Pluto Is Demoted to ‘Dwarf the discussion over Pluto’s planetary status once again. It was Planet’. The New York Times. Retrieved from http://www. held in an auditorium-like setting with each expert providing a nytimes.com/2006/08/24/science/space/25pluto.html PowerPoint of his or her argument. The audience consisted of scientists, teachers, and civilians. Gingerich and Sasselov argued Axelson, Ben. (October 3, 2014). Is Pluto a Planet Again? that “a planet is a culturally defined word that changes over time,” Harvard Reignited Debate. Retrieved from http://www. and so, Pluto should be a planet. Williams, on the other hand, syracuse.com/news/index.ssf/2014/10/pluto_planet_debate_ argued that Pluto does not meet the official definition of a planet harvard.html and therefore, cannot be a planet. It was a heated debate and they even asked the audience to vote on Pluto’s planetary status. The Figure 1: http://scienceblogs.com/startswithabang/ audience responded with a resounding yes, giving Pluto a chance files/2013/10/Kuiperbelt-1.jpg Figure 2: http://d1jqu7g1y74ds1.cloudfront.net/wp-content/ to become a planet once again.

Nevertheless, Pluto will remain a dwarf planet and until the uploads/2008/04/800px-eighttnos.png IUA decides to change its definition of a planet once again. The next IAU General Assembly meeting will take place in Honolulu, Hawaii in August, 2015.

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Letters

RNA Sequencing Analysis of Neurons Kevin Mei ‘16

Immediate Early Genes Neurons meet at synapses, where signaling occurs; the pre-synaptic neuron releases signaling molecules called neurotransmitters that will bind to receptors on the postsynaptic cell. Normally, the cell membrane of neurons is held in a polarized state, with negative charge inside and positive charge outside. The binding of neurotransmitters depolarizes the postsynaptic cell and allows cation entry. When neurons stimulate each other, signaling pathways, mediated by calcium, trigger gene transcription.

Immediate-early genes are those that are first transcribed, before late-response genes. They usually code for transcription factors that will then go on to activate the late-response genes, whose downstream effects will work at the synapse. As an example, calcium-dependent signaling pathways activate or deactivate transcription factors, by phosphorylation or dephosphorylation of those proteins, and initate transcription of neuronal pas domain 4 gene (NPAS4), an immediate-early gene1 (Figure 1). NPAS4 itself is a transcription factor and will initiate transcription of brain derived neurotrophic factor gene (BDNF), a late-response gene.

BNDF protein then goes back to the synapse where it modulates the activities of receptors.

This past summer, I worked in Michael E. Greenberg’s lab (via the HST-BIG program). Michael Greenberg is well-known for discovering immeadiate early genes. Previously, it hadn’t been known that there was this fast and transient initial wave of immediate-early gene transcription... genes being quickly turned on to perform a function before being quickly turned off. To study these genes in the brain, my mentors had taken human embryonic cortical neurons and exposed them to potassium chloride (KCl) treatment. In KCl solution, positively charged potassium ions (K+) will flow into neurons, depolarizing them and activating any immediate- early genes that would’ve been activated by normal physiological stimulation. Neurons 0 hours, 1 hour, and 6 hours after KCl treatment were then sent for RNA sequencing. By 1 hour after KCl treatment, we expected that RNA sequencing would capture the immediate early genes that had been transcribed and by 6 hours after KCl treatment, we expected that RNA sequencing would capture the late response genes transcribed.

RNA Sequencing and Analysis RNA in the cells is extracted, then fragmented into pieces of equal length and converted into its complementary DNA (cDNA) by reverse transcriptase. This collection of cDNA fragments, called a library, represents the total RNA from a cell population.

Ribosomal RNA and transfer RNA (tRNA) together make up

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Figure 1 In this case, glutamate, an excitatory neurotransmitter, released from the presynaptic neuron binds to receptors (NMDAR, AMPAR, mGluR) on the postsynaptic neuron and causes depolarization. This depolarization allows calcium to enter the cell. Calcium-dependent signalling to the nucleus activates an initial round of immediate early gene transcription, whose gene products then activates a second round of late response gene transcription. The products of these genes will have effects back at the synapse. (Ebert and Greenberg 2013). around 90% of the total RNA in a cell, but messenger RNA (mRNA), the RNA that represents the majority of the genes in a cell, is what we’re interested in. After the RNA extraction from the

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Figure 2 Expression bar plot for the NPAS4 gene. Because NPAS4 is an immediate-early gene, we see an upregulation in expression by 1 hour, but down-regulation by 6 hours after KCl treatment. Notice the incredibly high error bars. cells, rRNA is usually depleted from the sample. The cDNA library is then modified and sent for sequencing.

Out of RNA sequencing, we get many, many short sequences, termed “reads.” Analysis takes several steps: quality control, alignment, and differential expression. In quality control, we preprocess the raw sequencing data, which includes running statistics to measure variables such as the GC content. Sequencing is not always accurate; what the sequencer says is an A may actually be a G, so a confidence score is associated with every nucleotide.

Sequences with poor confidence scores are usually thrown out.

The reads are then aligned to a reference genome. This itself is an algorithmic problem because in the mRNA, introns have been spliced out, so one read may span several exons, but these exons are separated in the genome. When reads have been aligned as best as they can, the analysis program has to decide which are transcripts (the mRNA transcribed from one gene) and how many transcripts of a gene are in the sample. A single gene can have many splice variants and when a read is mapped to an exon, the analysis program has to guess which splice variant transcript the read comes from.

Biological Variability and Other Problems My main problem this summer was biological variability in the data. We had five different cortical samples (from different individuals) as our biological replicates to make sure that the results were consistent across samples. If we saw that one gene seems to be more highly expressed at 6 hours than at 1 hour, we want to be sure that this is so for all five replicates. However, the compositions of the five cortical samples differed from each other in their proportions of neural progenitor cells, endothelial cells, and glial cells. The concept of biological replicates is that they should all be similar to each other and in this case, it wasn’t so, so the program I used (Tuxedo suite2) couldn’t tell what the true expression level of a gene was. Figure 2 shows incredibly

high error bars for the expression level of NPAS4 because the expression level varied across replicates.

That, combined with my greenness in computer science and statistics, was discouraging. I had only taken Intro Comp Sci I and Intermediate Statistics here at Amherst but wanted to “get my feet wet” with “dry lab.” I didn’t know to run statistics on the sequencing data or how. Despite the rRNA depletion after RNA extration, there are still variable levels of rRNA in the samples sent for sequencing. I made the error initially of not controlling for rRNA in the data and had to find the sequences of every known rRNA and mask them from my analysis. Differences in how sequencing facilities handle samples also skewed analysis.

Moreover, there’s simply an abundance of analysis software because they aren’t standardized or consolidated. I mentioned that I used the Tuxedo suite, but on Wikipedia alone3, you can find a long list of programs with overlapping functions but perhaps using different algorithms. Which do you use? Why? How? The biostatistician in the Greenberg lab wrote many of his own analysis software code and the same is true in many labs.

RNA sequencing is incredibly powerful. DNA sequencing, say, of the genome, has expanded our compendium of known genes, but unlike RNA sequencing, doesn’t tell you which genes are being expressed or the level of their expression. Moreover, RNA-seq allows us to identify new genes and new isoforms or splice variants and compare gene expression profiles across conditions. In my summer program alone, my peers worked on finding ways to keeps track of data sets as they’re being manipulated, which actually is a very serious problem. File names can be changed and what you do with data is not always recorded. Another person worked on finding ways of distinguishing out which cell type data may have come from (a glial cell or a neural progenitor cell or so on), which would’ve solved my biological variability problem. Rather than being discouraged, I’ve been inspired to take more computer science. Computational methods are an incredibly important tool in solving biological problems.

References 1. Ebert, D. H., & Greenberg, M. E. (2013). Activity-dependent neuronal signalling and autism spectrum disorder. Nature, 493(7432), 327–37. doi:10.1038/nature11860 2. Trapnell, C., Roberts, A., Goff, L., Pertea, G., Kim, D., Kelley, D. R., … Pachter, L. (2012). Differential gene and transcript expression analysis of RNA-seq experiments with TopHat and Cufflinks.

Nature Protocols, 7(3), 562–78. doi:10.1038/nprot.2012.016 3. “List of RNA-Seq Bioinformatics Tools.” Wikipedia. Wikimedia Foundation, 31 Oct. 2014. Web. 25 Nov. 2014.

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Letters

The science pertaining to #IceBucket and other challenges Xiao Xiao ‘16 Introduction Fads and trends come and go, spreading like a virus, then disappearing like the fall breeze in the Pioneer Valley. With social media, fads both spread faster and reach more people. In the case of Internet and social media platforms, the speed of the spread of a phenomenon is akin to that of a virus. Despite the widereaching capabilities of trends like Cronut and Doge, it is rare to find something that carries a weight. Then enters the Amyotrophic Lateral Sclerosis (ALS) Ice Bucket challenge, an icy wild fire that burned for a long time and had a great deal of impact.

The Ice Bucket Challenge The challenge has two different options: The participant either 1) dumps a huge bucket of icy water on top of his or her head and donates $10 to the American ALS foundation or 2) donates $100 without the splashing of water. Helpers record the challenge on video, and at the end of the splashing the participant nominates three others to continue by social media tagging. Those tagged repeat the challenge within 24 hours and further nominate other people.

The ALS foundation hopes to raise awareness of the ALS disease, a neurodegenerative disorder that causes muscle spasticity.

Afflicted individuals experience progressive weakness that leads to difficulty in speaking, swallowing, and breathing. The pouring of icy water mimics the loss of muscular control. The extreme and sudden change in the surface temperature of the skin induces a temporary local paralysis, which is especially noticeable in the exposed neck region when the participants bend their heads forward due to the momentum and weight of the falling water.

By participating in the Ice Bucket Challenge, participants and ALS patients establish a common understanding of the difficulty faced without muscular control.

The challenge caught the Internet community by storm with participants ranging from Bill Gates to Homer Simpson. It also created spinoffs like the rice bucket challenge in India and Bangladesh and the toilet water challenge, courtesy of celebrity Matt Damon, to raise awareness for other social issues such as food shortage and waste disposal system mismanagement, respectively.

As of August 2014, the ALS foundation has received more than $100 million in additional donations compared to the same period (June-August) last year.

The Salt and Ice Challenge Closely related but far more light-hearted is the salt and ice challenge. The participant places cubes of ice on his or her bare skin and sprinkles salt in the nearby region. When that is done, he or she simply endures for as long as possible. A typical length

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falls within a minute or two, but enduring up to five minutes is not unheard of. Similar to the ice bucket challenge, a helper will film the process and upload that to video hosting sites such as YouTube or DailyMotion. Unlike the ALS challenge though, the motivation for this endeavor is purely trivial. Participants hope to outlast one another just for bragging rights and Internet fame.

However, the danger behind this challenge is real. Salt is soluble in water and the initial body heat transforms part of the ice into water. As salt dissolves, it lowers the freezing point of the water. This, in turn, causes more ice to melt yet the temperature of that ice-water-salt mixture dips below zero Celsius. As the skin temperature lowers, the participants will feel numb as the nerve loses its ability to sense. The sub-zero temperature also leads to frostbite and burns that participants often ignore due to the loss of sensory ability that region. Since the emergence of this challenge and the reports of multiple incidents of serious injuries, various doctors and hospitals have stood up to denounce this challenge.

The Flame Tummy Challenge In a similar but opposite spin, there is the fire challenge, or the fire stomach challenge as the stomach is the most commonly used part of the body. Again, this challenge exists without any association towards serious social issues, like the salt and ice challenge. Participants douse the chosen part of their bodies, usually the stomach, with flammable liquid like rubbing alcohol.

Then they start the fire and allow it to spread before putting it out a split second later. There is, of course, the requisite filming and uploading.

The danger is apparent and immediate. First- and seconddegree burns are just a couple of the many ways to cause irreversible harm. The uncontrolled spread of the fire could lead to panic and the participants could easily pass on the fire to other flammable material in the vicinity. Also, since the source of the fire is so close, participants risk inhaling super-heated air that might injure their respiratory system. Again, physicians spoke out collectively against the challenge, this time joined by firefighters and police.

The Cinnamon Challenge It is customary to include this challenge for any article of this topic simply because of its sheer history and popularity.

Predating the three challenges mentioned before, the cinnamon challenge first occurred as early as 2001. The simplicity and ease of preparation, at that time, meant that anybody who had access to a grocery store could have done it. Participants simply try to consume a tablespoon of dry cinnamon powder. Adding to the appeal is the aftermath reaction, as participants cannot endure the burning sensation and bellow out waves of beige plumes.

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Figure 1: The ill-advised Flame Tummy Challenge.

Due to its chemical composition, cinnamon powder is caustic.

That means that once it dissolves in water, the mixture will be alkaline. When the challenge is attempted, the mucus membrane and saliva in the throat provide plenty of water. This causes the chemical environment in the throat to turn basic. In extreme cases, the corrosive nature of the mixture will scar the throat or induce pneumonia. Mechanistically, a sudden influx of dry powder not only presents a choking hazard but also siphons out moisture from the esophagus via osmosis. This leaves a parched feeling in the participant that can lead to gagging, vomiting, and coughing.

The Saltine Cracker Challenge Far less dangerous but equally difficult is the saltine cracker challenge. Despite how harmless a single piece of saltine cracker looks, completing this challenge requires some serious work. To begin, participants just have to consume six saltine crackers within a minute, but it has to be done without any liquid, be it water or other beverages. The difficulty lies in the dryness of the crackers and their ability, similar to the cinnamon powder, to siphon out moisture from your throat.

Fitting six crackers within the mouth is seemingly easy but the mass of crumbs quickly exhausts the saliva available. Even though 60 seconds is more than enough to chew the crackers to break them down, the challenge lies after. Physiologically, saliva acts as a lubricant to bring food deeper down the digestive system. A lump of semi-dry cracker will not pass through the throat without either more liquid to moisten the lump or tremendous will power.

It is no wonder that in North Dakota, the annual cracker eating competition record champion is somebody with an extra salivary gland.

The Milk Gallon Challenge Too much liquid is also undesirable. For the milk gallon challenge, participants have to down a gallon of milk (3.78 litres for the metric user) within the time span of an hour. Milk is the most preferred liquid of choice, though use of other drinks such as water or fruit punch is also possible. Aesthetically, the cream color of milk appears the most dramatic when participants begin to vomit, regardless of the participant’s tolerance level of lactose.

Figure 2: In the midst of the milk gallon challenge.

The challenging aspect of this is due to the physical limitation of our digestive system. The stomach capacity of an average adult is a mere half a gallon. The stomach lining has receptors to detect if the content is approaching that limit. If it is, then the receptors trigger a vomit reflex. Milk also contains considerable fat and protein. Hardwired by evolution, humans cannot pass the stomach content downstream without absorbing some of that protein and fat first. Thus, a continuous inflow and a stopped outflow create a dilemma that ultimately leads to vomiting.

Conclusion No matter how fun some of those challenges sound, especially in a party setting, don’t try them without seriously considering the consequences. You have been warned. There is a reason why doctors band together against the some of them. In between laughter-filled periods of fun with friends, please take a moment to ponder the emergence of social media in publicity and outreach. Appreciate the power and ingenuity (or in some cases the lack thereof) of the masses. (PS donate to the ALS society.)

References

1. Steel, Emily (August 17, 2014). “’Ice Bucket Challenge’ Has Raised Millions for ALS Association”. The New York Times.

2. Rao, Mallika (August 26, 2014). “’Rice Bucket Challenge’ Reminds World How Scarce Clean Water Is In India”. Huffington Post.

3. Ewing, Samara. “Cinnamon Challenge Game Has Serious Health Consequences”.

WUSA9.

4. “What The Hell Is A ‘Fire Challenge,’ And How Could It Not Go Horribly Wrong?”. Huffingtonpost.com.

5. Walsh, Mary (2006-01). “Mission Impossible: A Gallon of Milk in an Hour.”

POiNTS iN CASE. The Fine Print of College Life.

6. Shipman, Dustin (2008-04-29). “‘Dr. Food Science’ mixes bananas and Sprite, conducts other questionable food experiments”. The Joplin Globe.

7. Bechtel, Mark (2004-01-19). “Broom At The Top ; Baby, it’s cold outside, so North Dakotans like to curl up with a good CURLING tournament”. Sports Illustrated.

8. Kwak, Janet. “Ice-and-Salt Challenge Fires Up Health Officials | NBC Southern California”. Nbclosangeles.com.

9. SHESINGSALONG. Chugging Milk. http://shesingsalong.com/2013/04/22/ chugging-milk.

10.http://www.sodahead.com/fun/does-saltice-challenge-hurt/ question-3264585/

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Letters

A not-so evil Mini-Me Thomas Savage ‘15

In the Austin Powers trilogy, Dr. Evil creates Mini Me, a miniature version of himself who copies everything Dr. Evil does. Mini Me is bald and wears the same drab, grey jacket as Dr. Evil. He is a slightly diminutive clone of Dr. Evil, but nonetheless we get the sense that he might grow up to be just as evil as Dr. Evil himself. Scientists have created Mini-Me immune systems: miniature versions of human immune systems in mice. The mice possess the full variety of human immune cells and those human cells are functional.1 They are capable of mounting immune responses against foreign tissue, such as transplanted skin.

By transplanting human hematopoietic stem cells (precursors to blood cells) along with thymus tissue (crucial for T cell development), researchers reconstitute immuno-compromised mice with an immune system identical to that of the donor.1 Although the model– termed the Personalized Immune model, or PI model –may have its limitations, (these are mice, after all) the advantages are numerous: researchers can study the immune system’s development through experimentation, which is ethically impractical with humans.

A group of researchers at the Columbia University Medical

Center uses the PI model to study the immune system of people with type 1 diabetes (T1D): they compare the development of the T1D immune system to that of healthy controls. T1D is an autoimmune disease characterized by cell-mediated destruction of the pancreatic beta cells. These cells produce insulin, which controls blood glucose levels, and without them, patients with T1D must rely on insulin injections, which has complications such as kidney failure, heart disease, and blindness. One possible solution to this problem is to transplant a new pancreas for T1D patients, similar to the way kidney transplants are performed to treat dysfunctional kidneys. (Many groups of scientists are currently investigating this.) This method does not investigate the causes behind T1D, i.e. the process by which the immune system becomes dysfunctional.

What is known about T1D is that T cells and B cells are responsible for the immune response that kills the pancreatic beta cells. Generally speaking, T cells and B cells recognize very specific proteins, called “antigens,” as foreign and try to destroy anything with that antigen that they encounter. Following recognition of antigen, B cells and most T cells proliferate, producing an immunologic response that eliminates the antigen entirely. One group of T cells, however, stifles the immune response to their antigen. These cells, called regulatory T cells (Tregs), play a crucial role in the maintenance of self-tolerance, preventing the immune system from attacking its host; with dysfunctional Tregs, mice develop autoimmune diseases.2 When an immune response is initiated, immune cells produce interleukin-2 (IL-2), which causes conventional T cells to proliferate. One way Tregs regulate the immune response is by binding IL-2 with a higher affinity than conventional T cells so that less IL-2 is available. Tregs, it turns out, also need IL-2 to maintain their regulatory function: IL-2 activates the STAT5 signaling pathway in Tregs that results in transcription of a protein called Foxp3, which is responsible for maintaining the regulatory function of Tregs.

Figure 1 (Left): The IL-2 signaling pathway in Tregs. IL-2 activates the STAT5 pathway (in green), which eventually results in expression of Foxp3 (in box outlined in green).

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Letters Figure 2: This is a representative stain displaying how I identified Tregs, and then among those Tregs, levels of pSTAT5 when stimulated with IL-2 and when not stimulated.

Tregs express the high-affinity IL-2 receptor, CD25, and the immunoregulatory protein, Foxp3, whereas conventional T cells do not. I identified Tregs by high expression of CD25 and Foxp3, and then I found high levels of pSTAT5 following IL-2 stimulation but not when unstimulated.

A group in Seattle has found that, following IL-2 stimulation in Tregs of T1D patients, there is defective activation of the STAT5 pathway and decreased levels of Foxp3.3 Thus, it is possible that T1D results from defective Tregs that are not suppressing autoimmune responses (T cells and B cells that recognize their host as foreign). The Seattle group identified this defect in circulating blood from humans, but could not determine whether the defect was caused by, or resulted from, the disease.

I worked at Columbia University Medical Center this past summer studying IL-2 signaling in T1D Tregs. I used a flow cytometry-based assay that looked for activation of the STAT5 pathway following IL-2 stimulation. Flow cytometry is a lab technique that looks at large numbers of cells and identifies subpopulations and their characteristics.

We were looking for defective activation of the STAT5 pathway in T1D Tregs, as this would indicate a defect in regulatory immune cells, a possible explanation of T1D. Activation of the STAT5 pathway results in phosphorylated STAT5 (pSTAT5), which can be identified by flow cytometry. Therefore, to identify defective activation of the STAT5 pathway, we would hope to see low levels of pSTAT5 following IL-2 stimulation.

I used flow cytometry to identify Tregs and then looked at levels of pSTAT5 in those Tregs. This would give us insight into the ability of IL-2 to activate the STAT5 pathway by comparing levels of pSTAT5 with and without IL-2 stimulation. Without any stimulation, there should be low levels of pSTAT5, as the STAT5 pathway should not be activated. Using the flow cytometry assay, I was able to identify Tregs and find activation of the STAT5 pathway following IL-2 stimulation. (See figure 2.)

We hope to study the IL-2 signaling pathway in Tregs as they develop in the PI mice. If the Tregs from T1D patients display defective activation of the STAT5 pathway, there is likely an intrinsic defect in T1D patient blood cells. The PI mice do not develop T1D, thus it is impossible for the disease to cause anything, including the defect in Tregs. Therefore, if the Tregs from T1D develop this signaling defect, the defect must occur prior to the development of T1D. (The disease would occur after the defect already exists.) This provides a possible explanation for the pathogenesis of the disease, as the Tregs fail to suppress the selfreactive T cells, and a possible therapeutic target.

Therefore, as much as we might love to experiment on Dr.

Evil, he is a living, breathing human; unfortunately we cannot do anything to him. We can study characteristics of him, but not what caused those characteristics. We can create our miniature versions of him: the PI mice which, while not as cute as Mini Me, are analogous in function. We can perform experiments on the MiniMe version of the immune system, but not the Dr. Evil version.

Miniature versions of humans can provide more than just comedic relief from evil. They can provide us with a great understanding of a disease that affects a wide variety of people.

In the future, researchers using this model can study autoimmune diseases other than Type 1 Diabetes, such as rheumatoid arthritis, and diseases of immune dysfunction, such as cancer. The model would give a deeper understanding of the disorders, as well as perhaps the best therapeutic option.

References 1. Kalscheuer H, et al. A model for personalized in vivo analysis of human immune responsiveness. Sci Transl Med. Mar 14, 2012; 4(125): 125ra30.

2. Kim, J. Cutting Edge: Depletion of Foxp3+ Cells Leads to Induction of Autoimmunity by Specific Ablation of Regulatory T Cells in Genetically Targeted Mice. J Immunol. 2009 Dec 15;183(12):7631-4.

3. Long SA, et al. Defects in IL-2R Signaling Contribute to Diminished maintenance of FOXP3 Expression in CD4+CD25+ Regulatory T-Cells of Type 1 Diabetic Subjects. Diabetes. Feb 2010; 59(2): 407–415.

Figure 1: http://diabetes.diabetesjournals.org/content/61/1/14/ F1.expansion.html

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Letters

Unlikely Match: Melatonin and Jet Lag Ashley “Monty” Montgomery ‘16

Traveling is a huge deal for many of us at Amherst. Many of us are not from this small town and have either traveled to the College or have spent time exploring nearby areas. Some of us tend to go pretty far, spanning across different regions of the United States, the continent, and the world. Despite increasing fare prices, many of us still rely on airplanes to get us where we need to go. Ignoring any impending jet lag, we endure long, cramped plane rides anyway. The good news is that there may be something to help alleviate some of the struggle of these trips in terms of jet lag.

The cure to a difficult trip actually lies in how you sleep.

What Is Jet Lag and Why Do We Get It? So what exactly is jet lag? In the simplest terms, jet lag is a disruption in one’s natural circadian rhythm. The National Institute of General Medical Sciences (NIGMS) defines circadian rhythms as, “…physical, mental, and behavioral changes that follow a roughly 24-hour cycle, responding primarily to light and darkness in an organism’s environment.”1 It may be helpful to think about circadian rhythms as your body’s way of reacting to light and darkness in terms of sleep.

The environment affects your sleep cycle. If a light is shone on you while you are asleep, you will probably wake up. Some studies state that temperature is contributing factor to the regulation of circadian rhythm during sleep time. A study observed the average body temperatures of seventy sailors while they slept, which we can see in figure 1. The researchers found that the sailors were more likely to fall asleep with a lower body temperature than a higher one.2 Therefore, they hypothesized that fatigue reaches its peak late at night when the temperature is at its lowest and the environment is completely dark.

Consequently, it makes sense that people who have stayed up all night, such as students cramming for an exam or writing a paper, feel a “second wind” in the morning. A person may feel as if they have more energy and their body has not been affected by sleep deprivation, when in fact, their circadian rhythm has simply been kicked on but will not function at full power. The temperature is warmer, the sun has risen, increasing the amount of light present, and their circadian rhythms, which remain steady respond to these changes.

Traveling across time zones disrupts this rhythm. When daily routine is thrown off by repeated lack of sleep or traveling, the body’s internal clock that responds to light and dark is thrown off as well. Traveling east perpetuates sleeping trouble because nighttime comes sooner than one is accustomed to. According to John Palmer, author of Living Clock: The Orchestrator of Biological Rhythms,

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“the problem here is obvious, one’s clock is set to, and by, the day/night conditions of your home time zone. When you move rapidly to a new zone, the clock must be reset, and the main resetting stimulus is, of course, the new ambient light/ Figure 12: The average daily body temperadark cycle as ture curve of 70 English seamen who took recorded by their temperatures every hour during the your eyes.”3 day, and at 2-hour intervals at night Conquering jet lag becomes the task of adjusting to the sleep pattern of your new location as soon as possible.

How (and Where) Exactly Can Melatonin Help?

This is where melatonin comes in. Melatonin, chemically known as N-acetyl-5-methoxytryptamine, is a hormone (See Figure 2).4 A pea-sized organ called the pineal gland secretes melatonin into the bloodstream from its location behind the third ventricle in the brain.

The secretion of melatonin into the bloodstream is rhythmic like our circadian rhythms, reaching peak secretion at night because light inhibits the pineal gland from releasing melatonin. According to one study, “The normal level of melatonin circulating in our blood at the peak of rhythm is about 100 picograms per milliliter of blood (a picogram is one trillionth of a gram).”5 These biological effects are a result of melatonin binding in the brain (activating Melatonin 1 (MT1) and Melatonin 2 (MT2) receptors), which regulates our circadian rhythm, through monitoring our reactions in cycles of light and darkness.

In jet-lag reduction experiments, scientists typically give participants 0.3 milligrams to 5 milligrams of melatonin, which reaches its peak concentration in the bloodstream 30 to 60 minutes after ingestion. Most over-the-counter melatonin treatments for

Letters

Figure 2 (left)4: The chemical structure of melatonin.

Figure 3 (right): Underwater Worm Circadian Rhythm (Deep, Dark Secrets of Melatonin in Animal Evolution, Sept 2014)

jet lag are extracted from the “pineal glands of beef cattle or synthesized chemically,” and consist of a sleeping pill. Though many people take melatonin as commercial treatment and it is not known to cause any undesirable side effects, in general, the few studies that have been completed show it has not been consistent in effectively treating jet lag.6 Because of this, alternatives for jet lag reduction include modifications to your schedule before and during travel. Palmer suggests, “Logically then, when we fly rapidly to a new time zone it is imperative that we immediately expose ourselves to as much daylight there as possible during the first few days.”7 We could also attempt to change our sleep patterns (different bedtimes, etc.) a week or so prior to their arrival at that destination.

Melatonin As More Than A Jet Lag Cure The Food and Drug Administration (FDA) has not approved melatonin as a sleep aid but has approved it for other uses. According to a 2005 study, melatonin acts as a “powerful antioxidant” in the brain, protecting the nuclear and mitochondrial DNA.8 Scientists formed their initial hypotheses on the use of melatonin for jet lag reduction from their studies on other animals: “Beginning in the late 1970s it was found that the daily administration (always at the same hour) of melatonin would synchronize the circadian rhythms of birds, lizards, rats, and hamsters to a strict 24-hour period.”9 Today, scientists using the same tactics have found that the key to unlocking more information about the brain and sleep could be determined by studying the role of melatonin in underwater worms.

A study in Cell entitled “Deep, Dark Secrets of Melatonin in Animal Evolution” conducted in September 2014 found numerous chemical melatonin similarities between sea-worms and humans that suggest that our current sleep cycles may be derived from tiny, ocean ancestors from 700 million years ago.10 Like humans, sea worms do not produce melatonin all the time, but only at night

(See Figure 3). The worms also get jet lag. Scientists hope to use the sea-worms to gain a deeper understanding of the human brain.

The scientists who conducted the study hope to learn more about how sleep cuts us off from the world. This might give better therapeutic options for dealing with jet-lag; rather than having to adjust our sleeping patterns a week in advance, we might just have to take the right pill. Perhaps, with even more knowledge about how our brains work, we will all be able to sleep better at night— even if we are jet-lagged.

References

1. Circadian Rhythms Fact Sheet. (2014, August 8). Retrieved October 7, 2014, from http://www.nigms.nih.gov/Education/Pages/Factsheet_CircadianRhythms. aspx 2. Palmer, J. (2002). Human Rhythms. In The Living Clock: The Orchestrator of Biological Rhythms (p. 22). Oxford: Oxford University Press.

3. Palmer, J. (2002). Jet Lag Can Be A Drag. In The Living Clock: The Orchestrator of Biological Rhythms (p. 52). Oxford: Oxford University Press.

4. Melatonin. (2014, August 10). Retrieved October 7, 2014, from http:// en.wikipedia.org/wiki/Melatonin 5. Palmer, J. (2002). Jet Lag Can Be A Drag. In The Living Clock: The Orchestrator of Biological Rhythms (p. 64). Oxford: Oxford University Press.

6. N., B., B., V., R., P., N., H., L., T., L., H., ... T., K. (2004, November 1).

Melatonin for Treatment of Sleep Disorders. Retrieved October 7, 2014, from http://archive.ahrq.gov/clinic/epcsums/melatsum.pdf 7. Palmer, J. (2002). Jet Lag Can Be A Drag. In The Living Clock: The Orchestrator of Biological Rhythms (p. 52). Oxford: Oxford University Press.

8. Boutin, J., Audinot, V., Ferry, G., & Delagrange, P. (2005). Molecular tools to study melatonin pathways and actions. Trends in Pharmacological Sciences, 26(8), 412-9. Retrieved October 7, 2014, from http://www.sciencedirect.com/science/ article/pii/S0165614705001525 9. Palmer, J. (2002). Jet Lag Can Be A Drag. In The Living Clock: The Orchestrator of Biological Rhythms (p. 65). Oxford: Oxford University Press.

10. Tosches, M., Bucher, D., Vopalensky, P., & Arendt, D. (2014). Melatonin Signaling Controls Circadian Swimming Behavior in Marine Zooplankton. Cell, 159(1), 46-57. Retrieved October 7, 2014, from http://www.sciencedirect.com/ science/article/pii/S0092867414009921

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