iScientist Volume IV by iScientist

About The iScientist

GUIDE TO THE ISCIENTIST The iScientist publishes 5 types of articles:

ISCIENTIST | 2019

About The iScientist

Original Research

Literature Review

History of Science

Articles in the A detailed review A historical format of original of previous perspective of a research research on a topic within science publications in specific topic or technology academic journals

Mathematical Paper

Letter to the iScientist

Studies regarding theoretical considerations or applications of mathematical concepts

Short pieces that discuss scientific research or argue an opinion

Our articles are divided into 15 disciplines:

Ecology

Earth Sciences

Kinesiology

Biophysics

Mathematics

Biochemistry

Chemical Biology

Chemistry

Pedagogy

Neuroscience

Health Sciences

Molecular Biology

Astronomy

Physics

Psychology

DEAR READERS It is with great pleasure that we present to you Volume IV of McMaster University’s undergraduate interdisciplinary science journal, The iScientist. In this volume of The iScientist, our authors explore the use of tannins as pesticides, advancements in breast cancer treatments, and mass determinations of planets, among other exciting and current topics. In spite of their diverse backgrounds, each article serves as a testament of effective science communication and collaboration within different fields. After reading the articles, we hope readers develop an appreciation for the various forms that scientific inquiry can take. Especially given the multifaceted problems faced in today’s society, we need scientists capable of embracing a holistic perspective in order to generate multidisciplinary solutions.

We would like to acknowledge many individuals at McMaster University whose support allows for the continued publication of The iScientist. First, we would like to extend our gratitude to our advisors and editors-in-chief, Andrew Colgoni and Dr. Sarah Symons, for their invaluable advice and consistent motivation. We must also thank all members of our editorial board and periphery board, who worked tirelessly to maintain the journal’s operations. A special thank you to members of our peer review panel. It is their constructive criticism with every submission that helps establish The iScientist’s reputation and high quality of work. We are also grateful to have received funding from the Student Initiative Fund, which allowed us to print copies of our journal for distribution, as well as host workshops. Thank you to McMaster Science Society for their support in promoting The iScientist and its workshops. Finally, we are eternally grateful to the School of Interdisciplinary Science faculty, students, and alumni. The community has been very receptive to our journal and has allowed for our expansion.

Letter From The Editors

This year, we expanded The iScientist beyond its roots in the Integrated Science program by opening positions on our editorial board and peer review panel for all students in the School of Interdisciplinary Science. In partnership with the McMaster Science Society, we also continued hosting science literacy workshops in efforts to advocate for science communication. The first workshop was an introduction to the peer review process, presented by Dr. Sarah Symons. We later hosted a workshop on non-traditional careers in science, led by Dr. Katie Moisse, a science journalist with a neuroscience background. We hope that The iScientist workshops will continue to provide students with the resources and information needed to advance their pursuits in science communication. We look forward to hosting more workshops in the upcoming year.

Ultimately, thank you to our readers for engaging with our content and events. We sincerely hope our journal has sparked a passion for interdisciplinary research. As always, we welcome any suggestions or comments. Without further ado, please enjoy Volume IV of The iScientist. Happy reading!

Sabrina Macklai

Senior Editors of The iScientist, Volume IV

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Tanya Daniel

TABLE OF CONTENTS Improved Surface Gravity and Mass Constraints for Substellar Objects from Spectral Line Profile Measurements at High Resolution in the Near-Infrared JESSICA SPEEDIE, DAVID LAFRENIERE ……………………………………………………………………………………….. 3 Epidemiology of Fear: Investigating Societal Responses to Pathogenic Invasion ALEXI DOAN ……………………………………………………………………………………………………………………………………………. 15 Tannins as a Pesticide: The Impact of Tannin Acid on the Growth Rates of Myzus Persicae and Arapidopsis thaliana

Table of Contents

DANA PRICE, SHAMA PIRBAY, LELIA WEILAND, JULIET ZHU, ELYSIA FULLERTHOMSON ……………………………………………………………………………………………………………………………………………. 26 Diceros bicornis longipes as a Tool to Test Species De-extinction using Somatic Cell Nuclear Transfer EMILY LALONDE, HANNAH MAHONEY ……………………………………………………………………………… 36 Triple Negative Breast Cancer and Synthetic Lethality RACHEL ELLIS AUBREY, MARYANNE OKETCH ………………………………………………………………… 47 Transcytosis and the Neurophysiological Complications Involved in the Delivery of Drugs Across the Blood-Brain Barrier in Alzheimer’s Disease COULTER MONTAGUE, CHEN CHEN …………………………………………………………………………………… 55

Overcoming Resistance to Anti-Breast Cancer Drugs Targeting Kinases MICHAEL CHAN, JOSEPH D’ERCOLE ………………………………………………………………………………….. 68

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Radioresistance: Implications for Astrobiological and Medical Research

JAMES LAI ………………………………………………….……………………………………………………………………………………. 75

ORIGINAL RESEARCH

Improved Surface Gravity and Mass Constraints for Substellar Objects from Spectral Line Profile Measurements at High Resolution in the Near-Infrared JESSICA J. SPEEDIE1, DAVID LAFRENIERE2

1. Integrated Science Program, Class of 2020, McMaster University 2. Institut de Recherche sur les Exoplanètes, Département de Physique, Université de Montréal

SUMMARY

Received: 11/09/2018

Accepted: 02/14/2019

Astronomy

Observed extra-solar systems within the Milky Way are comprised of stars and substellar objects (planets and brown dwarfs). Standard methods for measuring the mass of isolated brown dwarfs and directly-imaged giant planets are indirect, and rely heavily on largely uncalibrated theoretical models. Consequently, current mass estimates for many of these substellar objects are highly uncertain. With the arrival of new high resolution instruments such as SPIRou comes opportunity for new methods and improved constraints. We present an observational method to constrain the mass of substellar objects precisely, and demonstrate its feasibility on simulated SPIRou observations. We use a cross correlation technique to find the average shape of the absorption lines in an object’s spectrum, and determine its surface gravity to high precision through quantitative comparison to reference models. The average line profile width has the properties of being dependent on surface gravity and independent on the choice of reference model. Our results suggest that by using the average line profile, surface gravity can be constrained to better than 5%, and mass can subsequently be estimated to a precision of 10-15%. Performing our method on real high-resolution observations will provide the ultimate test. Published: 11/17/2019

Keywords: brown dwarfs: atmospheres - line profiles - methods: observational - planets and satellites: individual (2MASS 2322-6151)

To know the mass of an object in the sky is to know its nature. Astronomers classify most astronomical bodies into planets, brown dwarfs, or stars depending on how massive they are. Brown dwarfs and planets are not massive enough to sustain nuclear reactions in their cores, and as such are deemed “substellar.” The environment in which a planet, a brown dwarf or a star can be found is of great interest for understanding the

formation and evolution of the diverse celestial population. For example, discovering a sub-stellar object (a giant planet or a brown dwarf) in isolation indicates it may have actually formed how a star does. The mass distribution of the isolated objects is called the initial mass function, and the lower end of the distribution is the focus of much research. On the other hand, the mass distribution of companion objects, those that orbit a star, can answer questions about the formation and evolution of an

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INTRODUCTION

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Astronomy

astronomical system. Mass as a function of separation of host star, known as the population demographics of companion objects, serves as input for theories concerning the interactions between the bodies in a single system over time. Planets may migrate toward or away from their star, gravitationally scatter off one another, and have observable effects on the circumstellar disks from which they formed. Mass is the key piece of information necessary for insight into all of these processes.

However, it is difficult to determine the mass of substellar objects, and many of the estimates currently available for known directly-imaged giant planets and brown dwarfs (both isolated and companion) are uncertain. Mass cannot be measured directly, and is generally estimated based on an object's observed luminosity and age, in comparison to what theoretical evolution and spectral models predict. It is possible, in the case of binary systems, to measure the mass of substellar objects more precisely using dynamics (Bowler et al., 2018; Dupuy et al., 2018; Dupuy & Liu, 2017). However, the method applies more easily to heavier objects and does not serve as a general one. Both evolution and spectral models, on which most mass estimates are based, carry significant uncertainties. The initial conditions for the formation of a brown dwarf or giant planet pertinent for evolution models are unconstrained observationally; the energy transferred to a protoplanet by infalling gas may be fully retained (the “hot-start” scenario) or it may be shocked back into space (the “cold-start” scenario), depending on the physics of accretion (Burrows et al., 1997; Marley et al., 2007; Szulágyi & Mordasini, 2016). The subsequent evolution of an object's observable properties after a “hot-start” is quite different to that after a “cold start”. This introduces large uncertainties when using evolution models to infer properties such as mass for an object of a given age based on how it is observed today. Uncertainties in spectral models, on the other hand, originate from incorrectly modelling the physics in the atmosphere. Whatever the amount of heat trapped in the interior from formation may be, it will escape through the atmosphere in the form of radiation. The term “dex” is often used by astrophysicists as a shorthand for “order of magnitude.” For example, 0.1 dex ≡ 10$.& − 10$.( , where “5” could be any number, and the difference would still be 0.1 dex. *

Creating spectral models of the emergent radiation is a difficult task due to the complex interactions between molecules in the atmosphere, and the unknown formation or spatial distribution of dust grains and clouds (Homeier et al., 2004). The uncertainties in the spectral and evolution models that result for the reasons given above are translated into uncertainties in mass estimates by the use of methods that rely heavily on their accuracy. To give examples of a few giant planets or brown dwarfs with poorly constrained masses, we quote the best currently available mass estimates. ROXs 12 b is 12-20 Jupiter masses (Santamaría-Miranda et al., 2018); 51 Eri b: 2-12 MJup (Rajan et al., 2017); CT Cha B: 14-24 MJup (Wu et al., 2015); 2MASS 0103+1935: 12.82 ± 8.43 MJup (Faherty et al., 2016). Notably, since the dividingline between a planet and a more massive brown dwarf is 13 MJup, it happens that astronomers don’t confidently know what these objects truly are. In this work, we aim to ameliorate this situation, with the help of new technology. We demonstrate an observational method that can be applied generally for constraining the mass of substellar objects more tightly than methods that rely on evolution models, using high resolution nearinfrared spectroscopy. We present our method in the context of the Canada-France-Hawaii Telescope's new high resolution (R=73,500) IR spectrograph: SPIRou. This revolutionary instrument became operational in mid-2018, and it is only with high resolution spectra from instruments like SPIRou that our method is possible. We take the approach of first determining the object's surface gravity, and subsequently using it to find the mass. It is well known that the thermal emission spectrum is sensitive to surface gravity (Hedges & Madhusudhan, 2016; Cruz et al., 2009a). Many previous works have estimated surface gravity using low resolution spectroscopic observations by comparison with models (Schneider et al., 2016; Chilcote et al., 2015; Bonnefoy et al., 2014; Currie et al., 2013; Bonnefoy et al., 2010); however, the resulting accuracy is seldom better than 0.5-1.0 dex.* Our method uses the information afforded by the high resolution of instruments like SPIRou, namely the line profile of thousands of individual molecular lines, to

constrain the surface gravity more precisely. From this, we obtain a correspondingly precise estimate of the mass. In the following section, we describe our observational method, the collection of spectral models we use, and our process for creating simulated SPIRou observations. We then introduce the Average Line Profile, describe how it can be quantified, and demonstrate its two crucial properties. Next, we perform our method on simulated SPIRou observations of a real object, 2M 2322-6151, and present the mass uncertainty obtained. Finally, we discuss implications, limitations and future work.

gravity are known. Expressed as a fraction of the mass measurement, we find

which we obtained from the usual simple variance formula.

METHODS ANALYSIS APPROACH

The strength of the gravitational field on the surface of an astronomical object, g in cm s-2 (often quoted as log10(g)), is stronger for denser objects and weaker for less dense objects. It is defined as:

where G is the gravitational constant, m is the object's mass and r is its radius. An object's luminosity, the amount of energy it emits per unit time, is a basic observable quantity and depends on its surface area and effective temperature:

The uncertainty associated with a given mass measurement, dm, thus depends on the precision with which luminosity, temperature and surface

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where Ď&#x192; is the Stefan-Boltzmann constant. Equating the radii terms in Equations 1 and 2 yields an object's mass as a function of those aforementioned three properties:

Figure 1: The fraction uncertainty in mass as a function of the fraction uncertainty in surface gravity (given by Equation 4) for the case where temperature and luminosity are known to a precision of 3% and 5%, respectively. A low uncertainty in surface gravity yields a nearly equally low uncertainty in mass (with diminishing returns after a point). The one-to-one line (dashed) is shown for reference. Inset: Constraints on temperature and luminosity tighter than the 3% and 5% used to create the track shown would be necessary in order to reach a precision on mass better than 13%. Main point: If the surface gravity of a brown dwarf or giant planet can somehow be determined and well constrained, then the object's mass can in turn be found to a precision that is only slightly worse.

Astronomy

The mass of a brown dwarf (or any self-luminous astronomical object) can be expressed as a function of only three properties: its effective temperature, its luminosity and its surface gravity. There already exist precise methods of determining the effective temperature and luminosity, and it is our hope to provide a robust determination technique for the surface gravity.

We can estimate the potential rewards of our approach by calculating the uncertainty in mass for varying constraints on luminosity, temperature and surface gravity. Figure 1 shows the relative uncertainty in mass as a function of how precisely surface gravity is known, for the hypothetical scenario wherein luminosity and temperature are known to a commonly-achieved precision of 5% and 3%, respectively. A tighter constraint on an object's surface gravity yields a tighter constraint on its mass. If the log(g) of a giant planet with mass M = 10 MJup could somehow be determined to a precision of, for example, 10%, then the uncertainty with which its mass could be known would be ± 16%, or ± 1.6 MJup, a tight constraint compared to many current estimates quoted in the literature.

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Astronomy

SOURCING THERMAL EMISSION SPECTRA

An object's atmospheric conditions determine the features present in its thermal emission spectrum, including the shape of the continuum and the shape of molecular absorption lines. The purpose of atmosphere models is to describe the features produced under given conditions, so that we can understand the observations, and predict what is observable. We consider here two different sets of brown dwarf thermal emission models: the publically available precomputed grid of PHOENIX BTSETTL atmosphere models (Allard et al., 2011) and the unpublished atmosphere models of Tremblin et al. (2015, 2016). We have sourced eight of the former (at two temperatures and four surface gravities) and have access to only four of the latter (at two temperatures and two surface gravities). Table 1 outlines their respective properties. As the premise of this paper is to demonstrate the feasibility of an observational technique, we consider the Tremblin et al. (2015) spectral models to describe the atmosphere of a hypothetical, illustrative target brown dwarf. We employ the PHOENIX BT-SETTL models to be our set of “reference models”, for no reason other than that they are available in a more comprehensive range of temperatures and surface gravities. The biggest difference between the models is the treatment of clouds. PHOENIX BT-SETTL models use a parameter-free cloud model to account for dust formation (following Rossow,

Table 1: Properties of our reference models (denoted with a, Allard et al., 2012) and notional brown dwarf models (denoted with b, Tremblin et al., 2015): local thermodynamic equilibrium (LTE); effective temperature (Teff); surface gravity (log10g); spectral resolution (R); metallicity by log10 number density with respect to solar values ([M/H]). PHOENIX BTSettl a

Tremblin b

Local Thermal Chemical Equilibrium Teff (K) 900, 1000 900, 1000 log10(g) 4.0, 4.5, 5.0, 5.5 4.0, 5.0 R 99,406 338,616 [M/H] 0.0 0.0 Source https://phoenix. Personal ens-lyon.fr/ communication 1978) and incorporate microphysical atmospheric processes such as settling of dust under the influence of gravity. The Tremblin et al. (2015) models are cloud-less. Designed to reproduce reddening in cold T and Y dwarfs, they incorporate vertical mixing, quenching of ammonia and a slightly reduced temperature gradient in the atmosphere.

CREATING SIMULATED SPIRou OBSERVATIONS We create simulated SPIRou observations of model spectra, specifically the Tremblin et al. (2015) illustrative models, by mimicking two key processes: 1. The interaction of the target's light with Earth's atmosphere on its journey to the telescope; 2. The behaviour of the photons within the observing instrument. The Earth’s atmosphere absorbs light coming to us from the object we want to observe. The effect it has on observations is known as telluric absorption, and needs to be corrected for. We simulate the effect of telluric absorption by

multiplying our target model spectrum with the TAPAS model telluric spectrum (Bertaux et al., 2014), compounded by airmass:

where Fint is the target's intrinsic spectrum before passing through Earth's atmosphere, Fobs is what enters the telescope to be observed, T is the telluric spectrum (∈ [0,1]) raised to the power of a, which represents the airmass* at the time and location of observation (taken to be 1, for ideal conditions). We oversample the target model spectrum to the resolution of the TAPAS model (R= λ/dλ~ 960,000, where dλ is one spectral resolution element) before multiplication in order to best approximate the resolution at which telluric absorption occurs in reality (theoretically infinite).

Airmass is a measure of the amount of Earth atmosphere traversed by the incoming light given the object’s elevation in the sky. It is largest on the horizon and at a minimum directly overhead. *

Surface gravity influences the ambient atmosphere conditions and thus, the absorptive behaviour of the molecules. The wide spectral interval of SPIRou (0.94 - 2.35 µm) enables us to view many thousands of spectral absorption lines, which we can then combine to create a single average absorption profile. We create our average line profiles by cross correlating the spectra with an artificially constructed “stick spectrum” - a mask. Figure 2 demonstrates the process, which is described below. 1. We begin with a set of reference model spectra of four different surface gravities. We resample (ie. convolve and interpolate) each spectrum to common wavelength pixels, if necessary, or to those of the observing instrument. 2. Fit an akima spline function to each spectrum over wavelength regions excluding absorption lines to find its “pseudo-continuum” (roughly speaking, the large-scale behaviour of the spectrum). 3. Normalize every spectrum by its respective pseudo-continuum to obtain solely the absorption lines. 4. Define an array of zeroes on the common wavelength pixels (to become the stick spectrum). 5. Identify the local minima in flux of the spectrum in the set of lowest surface gravity (*./0(2)45.6 ) and +,set the value of the stick spectrum at those wavelength pixels to 1 (or any non-zero constant). 6. Reject those sticks where the absorption lines of each spectrum do not demonstrate different behaviour as a function of surface gravity. In our case, we keep the sticks at those wavelength pixels

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Finally, we make efforts to emulate the correction process undertaken for real observations, namely telluric correction. We divide the simulated observation and the associated noise of each flux value by the aforementioned TAPAS model, resampled onto the same wavelength pixels. We discard those regions of the target spectrum of low flux and high telluric absorption where the resulting flux and associated noise blow up (around 1.4 and 1.8 µm), leaving us with fewer spectral lines than are present in the full spectrum.

The high resolution of the observed spectra provided by spectrographs like SPIRou (R = 73,500) gives opportunity for creativity and should be exploited fully. The molecular absorption line width for a slow-rotating (period ~ 12 h; Artigau, 2018) brown dwarf of 1 RJup, roughly 10 km s-1, is well within SPIRou's capability to resolve.

Astronomy

When propagated through a telescope and spectrograph, a target's photons are diffracted, resulting in monochromatic peaks registering on the detector over a range of wavelengths as broader distributions. The smallest wavelength interval separating two lines that SPIRou can resolve as such, known as 1 resolution element, is 4.1 km s-1 and corresponds to 2 pixels on the detector. We mimic this process by convolving the telluricmultiplied target model spectrum to the line spread function (LSF) of SPIRou, for which we use a Gaussian with FWHM equal to one resolution element. We then interpolate the value of the convolved spectrum onto a new vector of wavelength pixels that represents the resolution of the instrument (R=73,500). Photon noise is the main source of uncertainty in the signal (scaling like its square root), though we include dark noise and read-out noise as well.

FINDING THE AVERAGE ABSORPTION LINE PROFILE

where *./0(2)45.6 <*./0(2)45.7 < *./0(2)47.6 < *./0(2)47.7 and +,+,+,+,set the rest to zero. 7. Convolve the stick spectrum with a Gaussian profile of σ=2. 8. Cross correlate each spectrum with the stick spectrum individually, in doppler shift space (ie. as a function of radial velocity), from -50 to +50 km s-1. 9. Normalize the resulting cross correlation functions to be between 0 and 1 (ie. (yi = yi - ymin) / (ymax - ymin)) to obtain their absolute shape.

requirements. However, since we cross correlate each model spectrum to the same stick spectrum, the generated CCFs can be meaningfully compared and we are at liberty to impose any criteria. We compute the cross correlation values at increments in radial velocity of 20 m s-1, which yields an effective resolution of R = c/∆v = 15,000,000. With this method, we can effectively probe the average line profile of a spectrum at a much higher resolution than that with which we actually observe it.

RESULTS: THE AVERAGE LINE PROFILE

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Astronomy

In this section we apply our cross correlation algorithm described in Section 2.4 to the reference (PHOENIX BT-SETTL) and illustrative (Tremblin et al., 2015) model thermal emission spectra, at their original resolution. This is done in order to understand what is possible to achieve in theory, and what the observations can aim for.

QUANTIFYING THE AVERAGE LINE PROFILE

Figure 2: Top: Four of the PHOENIX BT-SETTL reference model spectra (at 1000K) and their respective pseudo-continua (pale colours) over a small portion of the SPIRou wavelength interval. Bottom: The same four model spectra, pseudocontinuum normalized, and the Gaussian sticks yielded by our algorithm in that region (black). Main point: Our algorithm for computing the average line profile for a given set of reference model spectra involves cross correlating an artificially constructed stick spectrum with the spectra's absorption lines. Constructing the stick spectrum is more of an art than a science, and the resulting cross correlation functions are highly sensitive to the chosen

We present the resulting average line profile yielded by our cross correlation algorithm for one of the eight PHOENIX BT-SETTL reference model spectra in Figure 3. In an effort to quantifiably distinguish between the average line profiles of atmospheres at different surface gravities, we tried to fit them with functions whose shape resemble those of spectral absorption lines - namely a Gaussian, Lorentzian and Voigt profile. In addition to their parameters of characteristic width (σ, γ, and both σ and γ, respectively), we employ a horizontal stretch (s) and a vertical translation factor (lift, l). We find that the best fit to the average line profiles is given by the Lorentzian function centered about x=0:

where x is radial velocity in km s-1 and γ is the HWHM of Lor(x, γ), ie. what the half width at half maximum would be without the added parameters of stretch and lift. Therefore, it is necessary to set

& the function to half its maximum value, &9(: ; + = ), and solve for the characteristic width, in km s-1:

occurring on the molecular level in a brown dwarf or giant planet atmosphere, and how they depend on the strength of the gravitational field. Higher surface gravity would lead to increased ambient pressure and temperature, resulting in collisional and thermal broadening of molecular absorption

Table 2: Characteristic widths (given by Equation 7, in km s-1) for the Lorentzian fits to each of the eight reference and four illustrative model spectra. Quoted uncertainties are for x(γ, s) = γ/s, not x(γ, s, l) (= Equation 7), because the former yielded higher uncertainties. log(g)

Table 2 presents these values for each model spectrum shown in Figure 3.

PROPERTIES OF THE AVERAGE LINE PROFILE

This result is indeed entirely contrived by our algorithm (which selects only those absorption lines obeying the marching order with surface gravity); however, consider the physical processes

BT-Settl 4.0 (reference models) 4.5

5.71 ± 5.96 ± 0.02 0.02 6.37 ± 6.67 ± 0.02 0.02 5.0 7.32 ± 7.45 ± 0.03 0.03 5.5 8.43 ± 8.65 ± 0.04 0.05 Tremblin 4.0 5.52 ± 5.87 ± (models) 0.03 0.04 5.0 7.13 ± 7.56 ± 0.02 0.05 lines in the object's emission spectrum. In this sense, step 6 of our algorithm (Section 2.4) is not arbitrary – in fact, therein lies the method's utility and merit. Enough absorption lines in the reference model spectra (roughly 2500) follow the trend such that the resulting average line profiles are clearly differentiated. Thus, the set can be used as a reference basis, against which one can compare the average line profile of an observed object whose surface gravity is unknown. Provided that the average line profiles of both the observed spectrum and model spectra are created using the exact same stick spectrum, the observed object's surface gravity can be determined relative to that of the reference models. Next, we apply our cross correlation algorithm to our second set of thermal emission models, the four Tremblin et al. (2015) spectra. We find that, for a given surface gravity, the average line profiles

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As is apparent numerally in Table 2 and visually in Figure 4, the width of the average line profile is dependent on surface gravity. The trend suggests that atmospheres at lower surface gravities exhibit narrower absorption lines, on average, while the molecular lines of atmospheres at higher surface gravities tend to be broader.

1000 K

Astronomy

Figure 3: An average line profile, given by our cross-correlation method, and its Lorentzian fit given by Equation 6. The fit quality of the other seven BTSETTL reference models is similar. This line profile represents the average shape of, in this case, 2736 individual spectral absorption lines. Main point: A Lorentz profile (upside-down and vertically translated) describes the average line profile well.

900 K

Astronomy ISCIENTIST | 2019

Figure 4: Top: The average line profiles of four PHOENIX BT-SETTL reference model spectra (light colours) and two Tremblin model spectra (dark colours) computed at BT-SETTL resolution (R=99,406). Middle: The Lorentzian fits of the average line profiles of two BT-SETTL reference model spectra (light green, light blue) and two Tremblin model spectra (dark green, dark blue). Bottom: The full flux vs. wavelength spectra (R=1000) of the models in middle panel. Left: 900 K; Right: 1000 K. Main point: Models of the same surface gravity look vastly different on the large-scale (the shape of the continuum, R=1000, bottom panel), but extremely similar on the small-scale (the average line profile, Reff=15,000,000, top and middle panels). of BT-SETTL and Tremblin et al. (2015) agree remarkably well. Figure 4 shows that both the raw

average line profiles and the Lorentzian fits of the Tremblin et al. (2015) models overlap with that of

the reference models for log(g)=4.0 & 5.0 at both 900 & 1000K, despite the fact that their respective spectra model shows very different atmospheres. To emphasize this, we show the PHOENIX BTSETTL and Tremblin et al. (2015) model spectra over the full SPIRou range, resampled to R=1000, on the same axes in the bottom panel of Figure 4.

width of the mean observed line profile, 8.34 ± 0.05 km s-1, agrees with the 8.27 ± 0.02 km s-1 of the log(g)=5.0 reference model. By fitting a degree3 polynomial to the four HWHM-log(g) points of the reference models (see Figure 6) and evaluating it at the HWHM of the mean observed line profile,

These spectra are the same temperature and surface gravity, and yet, due to differences in how they treat important microphysical atmospheric processes affecting the shape of the continuum, they look very different. That their atmospheres are in fact experiencing the same surface gravity is apparent only when one examines the deviations from the continuum – namely the absorption lines.

CASE STUDY: 2MASS 2322-6151

The small spread in the 100 simulated observations indicates that observational noise has an effect smaller than that of an object's surface gravity. Therefore, even in the presence of noise, one can measure log(g) to a better precision than 0.5 dex. As shown in Table 3, the characteristic

we obtain a surface gravity estimate of 5.015 ± 0.02 dex. In cm s-2, this is a relative uncertainty of only 4.6%. Thus, not only can we recover the average line profile from observed noise, but we can compare it to reference models and conclude the correct surface gravity to high precision.

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Figure 5 shows the resulting average line profiles of the 100 simulated observations and four reference models. Due to telluric absorption, our observations have only enough remaining flux values to make a stick spectrum with 605 lines, and the geometry of the average line profile suffers for it. Nevertheless, we see that the observed profiles overlap with the reference model of the same log(g) as the Tremblin model from which they were created nearly perfectly.

Figure 5: Top: The average line profiles of 100 simulated observations of the Tremblin et al. (2015) model spectrum at log(g)=5.0 and T=1000 K (grey; mean in black) and of the four BT-SETTL reference model spectra (colours). These line profiles represent the average shape of 605 individual absorption lines. Bottom: The Lorentzian fits of the above (observed mean only). Main point: The average line profile method leads us to conclude the correct surface gravity for our simulated SPIRou observations, and it is an effective way to determine surface gravity to high precision.

Astronomy

In this section, we apply our method to 100 individually created simulated SPIRou observations of the Tremblin et al. (2015) model thermal emission spectra. We show the most successful of the four cases (that of log(g)=5.0, T=1000 K), noting that in two cases (log(g)=4.0, T=1000 K and log(g)=5.0, T=900 K), the average line profiles of the 100 simulated observations did not consistently align with that of the correct reference model. We scale our simulated observations to the brightness of the brown dwarf 2MASS 23225299-6151275 (hereafter 2M 23226151), J=13.55±0.06 mag (Cruz et al., 2009b and set the exposure time to 1. The resulting 100 spectra have a mean signal-to-noise per resolution element of ~14. We discard those flux values of the observations and reference models where observed SNR <15 or where the TAPAS telluric absorption is >0.8.

Combining this result of dg/g = 0.046 with the constraints on luminosity and temperature (dL/L = 0.03 and dT/T = 0.03) yields an uncertainty in mass of 13.7% (Equation 4).

DISCUSSION AND LIMITATIONS

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Astronomy

Brown dwarf spectral observations made with low-resolution instruments (such as GPI, R~70) render subsequent analyses somewhat uncertain, because the method of fitting the continuum

Figure 6: Empirical relationship between the characteristic width of the average line profile and the surface gravity of the model from which it is derived, found using the four reference models. The surface gravity of the mean observed line profile (black) is found to be 5.015 ± 0.02 dex. Horizontal error bars in HWHM are smaller than the width of the points. Table 3: Characteristic widths (given by Equation 7, in km s-1) of the mean of 100 simulated SPIRou observations of the Tremblin et al. (2015) model at log(g)=5.0 & T=1000 K, and of the Lorentzian fits to each of the four reference model spectra also at 1000 K (see Figure 5). Uncertainty in the “observed” width is the standard deviation in the characteristic width of 100 fits; uncertainties in the reference model widths were obtained with the simple variance formula. BT-Settl (reference models)

Tremblin (observed)

log(g) HWHM 4.0 6.08 ± 0.02 4.5 6.80 ± 0.02 5.0 8.27 ± 0.02 5.5 10.28 ± 0.05 5.0 8.34 ± 0.05

observation to a library of model spectra could easily result in the wrong conclusion of surface gravity. That method requires the reference models to describe the same atmosphere as the observed object actually has, which is unlikely to be the case. We have shown, however, that using the spectral information present in high resolution observations made with instruments such as SPIRou and comparing average line profiles can yield the correct conclusion of surface gravity, regardless of whether the reference models correctly describe the observed atmosphere. The shape of the average spectral line in the PHOENIX BT-SETTL models, which contain clouds, is the same as that of the cloud-free models of Tremblin et al. (2015). It is remarkable that the shape of the average line would be the same for different atmospheres. Perhaps this hints at which physical process is dominant in the atmosphere. Future work should be concerned with what is perhaps the largest caveat to this work: spectral line broadening due to the object's rotation. A fastrotating brown dwarf's broadened spectral lines could lead it to be mistaken for an object at higher surface gravity. Efforts to decouple broadening from gravity and rotation could include an alteration to the artificial stick spectrum criteria to be sensitive to chemical species that live low in the atmosphere, and not those in the upper layers. Perhaps the mechanism-dependence of line shape could also lend itself to differentiating the effects; we have found a Lorentzian-shaped profile for non-rotating objects, whereas rotation-broadened profiles are described as a sine curve with the edges clipped due to limb-darkening. If the object’s rotation speed can be constrained by some other means, then the reference model spectra can be correctly rotationally broadened and, thereon, our procedure could be applied as described. The impact of this “procedural detour” on the precision attainable would need to be studied.

More work is needed to investigate the reasons why two of the four sets of 100 simulated observations did not yield average line profiles that agreed with reference models. The procedure should also be performed with a larger variety of atmosphere models, such as the PHOENIX COND and DUSTY, to confirm the modelindependence of the average line profile. Ultimately, using our method on real highresolution spectral observations, such as those SPIRou is starting to provide, will be the definitive test for efficacy.

CONCLUSIONS

ACKNOWLEDGEMENTS The authors thank both anonymous reviewers for thoughtful comments which greatly improved the manuscript. JJS acknowledges funding from iREx's Trottier Excellence Grant. We thank F. Allard et al. for making their model spectra publically available, J. Bertaux et al. for publication of their atmospheric transmission spectrum, and P. Tremblin et al. for access to their model spectra, without which this research would not be possible.

SOFTWARE SciPy, (Jones et al. 2001); NumPy, (van der Walt et al. 2011); pandas, (Mckinney2010); Jupyter, (Kluyveretal.2016); astropy (Collaboration et al. 2013); Matplotlib (Hunter 2007).

Astronomy

The average shape of spectral absorption lines in thermal emission spectra of brown dwarf atmospheres at different surface gravities were found to have similar shapes and be described by the same function. The characteristic width of the function, by way of our selection criteria for the artificial stick spectrum used in our cross correlation, depends on surface gravity in a simple monotonous fashion. By describing the trend with

some empirical relation, we can determine the surface gravity of an object whose spectrum we observe. This work indicates that it should be possible to constrain surface gravity to better than ~5% dex using the average line profile and that this could yield mass measurements with 10-15% precision.

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ORIGINAL RESEARCH

Epidemiology of Fear: Investigating Societal Responses to Pathogenic Invasion ALEXI DOAN1

1. Integrated Science Program, Class of 2019, McMaster University

SUMMARY

Received: 11/03/2018

Accepted: 03/12/2019

Psychology

Human history is punctuated by numerous deadly epidemics. The social environment associated with epidemics often elicits secondary ‘epidemics’ of fear, stigmatization, and moralization. If not properly addressed, these secondary epidemics have the potential to threaten public order. Hence, it is vital to develop policy specifically designed to anticipate and quell human responses to epidemics. In order to predict how societies will respond to future epidemics, it is necessary to analyze historical records of disease contained within media discourse. As such, a total of 216 examples of media discourse pertaining to three major historical epidemics—the Second Cholera Epidemic (1831-1833), HIV/AIDS (1981-1983), and the West African Ebola Outbreak (WAEO) (2014-2015)—were collected in the form of newspaper articles and twitter ‘tweets’. The type of language used was then categorized by valence. It was noted that media discourse pertaining to the HIV/AIDS pandemic was frequently inflammatory towards marginalized groups and was more associated with negative valence than news discourse for the other two case studies. There was also a stark disparity between valence levels for social media and news discourse; social media discourse was more often associated with negative valence than all other news discourse. In fact, the language used on social media was highly inclined towards sensationalism. In all, fatal disease has—and always will—be part of human society, and it is vital that past responses inform future endeavors to develop effective response policies. Published: 11/17/2019

Keywords: disease, epidemic, psychology, ebola, HIV/AIDS, cholera

INTRODUCTION

smaller psycho-social sub-epidemics: fear/ suspicion, stigmatization, and moralization/ explanation (Strong, 1990). Individually, each subepidemic can be conceptualized as a distinct phenomenon which influences all aspects of public life during an epidemic. Epidemic psychology is as much of a social phenomenon as it is biological; just as diseases spread from person to person, so do their associated social and psychological consequences. Most societies are slow to acknowledge the presence of an epidemic, and do so only when consequences become explicitly visible (Rosenberg, 1989). Before epidemics are publicly

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Humans and disease are inherently intertwined; there has never been one human society that has not experienced disease. When a disease is first introduced into a population, it elicits unique social responses—particularly if it is novel or especially fatal. Ever since the HIV/AIDS pandemic of the 1980’s and 1990’s, there has been increased interest in studying public reactions to epidemics. From this interest, the field of epidemic psychology has been established. Epidemic psychology attempts to understand the initial public reactions to fatal epidemics (Strong, 1990). Generally, initial public reactions manifest as three

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Psychology

recognized, there is typically a period of social tension building where people come to realize the serious nature of the disease they are faced with (Rosenberg, 1989). Individuals then tend to act in ways which reaffirm their culture’s pre-existing social values and norms (Strong, 1990; Rosenberg, 1989). In essence, this exposes the innate institutional values of a society, which are used to justify the sub-epidemic of stigmatization (Strong, 1990; Rosenberg, 1989). Coupled with the propensity of the general public to be irrational and uncooperative in emergency situations, this poses a potential threat to public order (Glass and Schoch-Spana, 2002; Strong, 1990). Although the threat to public order remains theoretical, it is a consideration which must be taken into consideration when delineating reactive and proactive health policies to combat epidemics (Glass and Schoch-Spana, 2002).

Once an epidemic is acknowledged, the initial public response is typically widespread and drastic—eliciting collective panic and, extremely, fostering a ‘culture of fear’ (Rosenberg, 1989). Each of the three sub-epidemics of fear/suspicion, stigmatization, and moralization/explanation then become apparent and evoke unique public responses. The psychosocial epidemic of fear, for example, frequently overshadows the reality of the epidemic itself. Individuals tend to be excessively cautious, out of the fear that they will become infected, even if their apprehension is baseless and illogical (Murray, 1991). Simultaneously, the epidemic of stigmatization isolates both victims and perceived ‘carriers’ of the disease (Strong, 1990). These individuals are then often the subjects of aggression, segregation, and persecution regardless of whether they actually carry the disease (Strong, 1990). The combined effects of the three psychosocial sub-epidemics creates the ideal conditions to facilitate a ‘perfect storm’ of public unrest. The goals of the present study are twofold. Firstly, the study attempts to analyze how the initial public response to notable epidemics has changed over the past 200 years. Secondly, this study will also identify how advancements in media, particularly the dawn of the 24-hour news cycle and the epoch of social media, influence initial responses to notable epidemics. To analyze this, three case studies of notable historical epidemics were chosen—the Second Cholera Pandemic (SCP), the HIV/AIDS pandemic, and the West African Ebola Outbreak (WAEO). Those specific case studies were chosen because they represent some

of the most notable outbreaks of disease in recent history. Further, each case study also represents a different point in the evolution of public media. Media pertaining to the SCP, for example, is exclusively newspaper-based, as other forms of public communication had not yet been invented. Conversely, the HIV/AIDS pandemic coincided with the introduction of the 24-hour television news cycle and the WAEO occurred during the age of widespread social media usage, which both drastically influenced the way information was disseminated in the media. To accurately identify initial public responses to historical epidemics, a sampling of media discourse was collected for analysis. Scrutinizing media discourse is a common methodology for research of similar caliber, due largely to media’s inherent relationship with public opinion (Lupton, 2008; Alvarez Amorós and José Antonìo Álvarez, 1992). Media discourse and public opinion are cyclically related—media discourse educates public opinion, and public opinion educates media discourse (Gamson and Modigliani, 1989). As such, media discourse functions as one of the most reliable indicators of public opinion at various points in history (Gamson and Modigliani, 1989). Further, news discourse can be conceptualized as a carefully constructed version of reality ‘packaged’ in accordance with governing institutions as well as public opinion (Wall, 1997). Media collected in this study can then function as the basis for which both public opinion as well as institutional values can be surmised. Rather than analyze news articles in their entirety, the present study only analyzed headlines. Headlines function as an interpretive framework for which an event is conveyed to the public (Fang, 2001). This framework is both consistent with the body text that follows as well as indicative of the social and political predispositions of the publication it appears in (Fang, 2001). Hence, a thematic understanding of news articles can be garnered thorough headline analysis without the need to fully analyze the news article itself. Further, headlines are also comparable in length to Twitter ‘tweets’ which, up until September of 2017, were restricted to 140 characters. As such, adopting a headline analysis allows for both news discourse and Twitter tweets to be considered on an equal level. To analyze how initial public response to epidemics has changed over time, the current research gathered a corpus of 150 news articles and 66 tweets. Headlines from news articles and

text from tweets were compiled and analyzed according to the methodology proposed by Kozareva and colleagues that investigates word usage frequency of condensed news headlines (Kozareva, Navarro, Vázquez and Montoyo, 2007). Condensed headlines were both inserted into online ‘word cloud’ generators as well as randomly presented to supplementary data analyzers to determine valence levels. It was predicted that discourse pertaining to the HIV/AIDS pandemic would have the highest frequency of negative valence ratings and frequently mention marginalized groups, such as gay men and Haitian immigrants, due to the particularly polarizing nature of the disease when it was first introduced. Further, under the assumption that news media headlines were designed to be apathetic, it was also predicted that news discourse would more frequently be rated as neutral valence than other examples of media discourse.

To analyze whether primary public responses to epidemics have changed throughout history, a

In all, 150 articles of news discourse and 66 tweets were collected for analysis. News discourse was gathered from online databases—particularly the British Newspaper Archive and ProQuest News & Newspapers. Discourse representing the SCP originated from a wide variety of different newspapers such as The Morning Post, The London Evening Standard, and The Globe.

Conversely, news discourse collected for the HIV/AIDS pandemic and the WAEO originated from either the New York Times, Wall Street

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Figure 1: Word cloud results from: (A) 50 headlines from the Second Cholera Pandemic (1831-1833), (B) 50 headlines from the HIV/AIDS pandemic (1981-1983), (C) 50 headlines from the West African Ebola Outbreak (2014-2015), and (D) 66 tweets from the West African Ebola Outbreak (2014-2015).

Psychology

METHODS

sampling of historical media discourse was collected. All historical discourse pertained to one of three case studies. The case studies chosen— the SCP (1831-1833), the HIV/AIDS pandemic (1981-1983), and the WAEO (2014-2015)—were each assigned subjective time ranges. The time ranges associated with each case study were representative of when the disease was first introduced into Western societies, namely the United States of America and the United Kingdom, to best encapsulate initial public responses.

Journal, or Washington Post. In addition, tweets pertaining to the WAEO were collected using Twitter’s advanced search. Only tweets which met four specific criteria were included in this analysis: tweets had to be written in English, be comprehensible, mention Ebola in the body text or hashtag, and not originate or link to an existing news article. Further, only media discourse published within the delineated time periods were compiled.

emotion. These four separate categories were graphed separately as per case study (Figure 2), as well as per type of discourse (Figure 3).

RESULTS WORD CLOUD ANALYSIS Frequency of word usage starkly differed across case studies (Table 1). Specifically, the word “fear” was popular in both HIV/AIDS as well as WAEO headlines. Overall, “fear” was the third and second most frequently mentioned word for all headlines concerning the HIV/AIDS pandemic and the WAEO respectively. Notwithstanding, the frequency that “fear” was mentioned was numerically low; “fear” was only mentioned a total of 9 times in HIV/AIDS headlines, and 6 times in WAEO headlines.

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Psychology

After collection, headlines and tweets were prepared for analysis. Each headline and tweet was condensed to eliminate superfluous words and punctuation, and minor grammatical and spelling adjustments were made for consistency. Headlines associated with each case study were then inserted separately into an online word cloud generator to produce three word clouds (Figure 1). In addition, one supplementary word cloud was constructed using condensed tweets as well (Figure 1). The top five most frequently mentioned words, for all three case studies and tweets, were also Table 1: Top five most frequently recorded separately (Table 1).

To identify the valence associated with different samples of media, seven individuals were recruited as supplementary data analyzers. Respondents were presented with 40 randomly selected headlines—ten from each of the three case studies (30 in total) as well as ten tweets about the WAEO—and were asked to classify the type of emotion they felt the random headlines conveyed. Individuals indicated their emotional response towards specific headlines as: joy, happiness, humor, surprise, apathy, sarcasm, anger, fear, or sadness. Importantly, respondents were not made aware of the source of the headline they were presented with—they were only informed of what disease the headlines pertained to. Respondents were also unaware of which emotions were associated with each category of valence at the time of data analysis. After responses were collected, they were compiled into one table (Table 2). Emotions were then broadly classified into four categories of valence: positive (joy, happiness, and humor), neutral (surprise, apathy, sarcasm), negative (anger, fear, or sadness), and no

mentioned words of each case study and type of discourse. DISEASE Second Cholera Pandemic

HIV/AIDS Pandemic

West African Ebola Outbreak (News)

West African Ebola Outbreak (Twitter)

WORD

FREQUENCY

Cholera

England

Progress

Precautions

Morbus

AIDS

Homosexual

Fear

Disease

New

Ebola

Fear

West

Nurse

Africa

Ebola

Dead

Virus

Zombie

Africa

Headlines pertaining to the HIV/AIDS pandemic frequently proclaimed the novel nature of the disease—a sentiment which was not shared among

the other case studies. Collectively, the word “new” was the fifth most frequently mentioned word among all 50 HIV/AIDS headlines. However, similarly to the word “fear”, the numerical frequency itself was quite low—only 7 headlines specifically mentioned the word “new”. Headlines pertaining to the SCP were noticeably more conservative than other case studies. Specifically, words such as “fear”, “death”, or “new”, which were common among other case studies, were seldom mentioned in SCP headlines. Rather, word choice remained relatively constrained; most news articles had headlines such as “The Cholera” or “Cholera Morbus”.

Psychology

Figure 3: Valence levels associated with emotions indicated by respondents for: (A) all news discourse collectively (n=21) and (B) social media discourse (Twitter ‘tweets’) pertaining to the West African Ebola Outbreak (2014-2015) (n=7).

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Figure 2: Valence levels associated with 10 randomly selected news headlines pertaining to: (A) the Second Cholera Pandemic (1831-1833) (n=7), (B) the HIV/AIDS Pandemic (1981-1983) (n=7), and (C) the West African Ebola Outbreak (2014-2015) (n=7).

Table 2: All ratings of emotional value collected, along with the associated valence levels of each emotion (n=7). POSITIVE VALENCE

NEUTRAL VALENCE

Psychology ISCIENTIST | 2019

NO EMOTION

Joy

Happiness

Humor

Surprise

Apathy

Sarcasm

Anger

Fear

Sadness

No Emotion

CHOLERA

HIV/AIDS

EBOLA (NEWS)

EBOLA (TWEETS)

Further, the word “precautions” was both popular as well as unique to headlines pertaining to the SCP. On a similar note, however, the popularity of the word “precautions” is numerically small; only 2 out of the 50 news articles collected mentioned it in their headlines.

NEGATIVE VALENCE

There was a noticeable difference across the two different types of WAEO discourse—tweets and news. For example, the word “dead” was the second most frequently mentioned word among all curated tweets and was mentioned a total of 15 separate times. News headlines for the WAEO were less inclined to mention “dead”, and it was not at all present within the top five most frequently used words. Further, “zombie” was frequently mentioned in tweets about the WAEO—it was the fourth most frequently mentioned word, stated 10 separate times. Understandably, “zombie” was not present whatsoever in any news headlines pertaining to the WAEO. The one similarity in word usage between tweets and news headlines for the WAEO was in the use of the word “Africa”. In both news headlines and tweets, it was the fifth most frequently used word, appearing 4 and 8 times respectively.

VALENCE RATINGS Valence ratings were subject to stark fluctuation across case studies (Figure 2). The SCP, for example, had the highest instances of headlines rated as conveying ‘no emotion’ (38% of total responses). Valence ratings of “no emotion” for headlines concerning the HIV/AIDS pandemic (24%) as well as the WAEO (12%) were much less frequent. Conversely, headlines pertaining to the

WAEO had starkly more instances of positive valence ratings (14% of total responses) when compared to the HIV/AIDS pandemic (2%) and the SCP (3%) whose ratings of positive valence were approximately similar. The frequency of neutral valence ratings remained relatively similar across all case studies, albeit HIV/AIDS news discourse entertained a slightly higher frequency of neutral valence ratings (37%) than SCP or WAEO discourse (31% and 30%, respectively). The most prevalent difference in valence levels across case studies arose when negative valence levels were considered. By far, the WAEO had the highest proportion of negative valence ratings (44% of total ratings assigned). The HIV/AIDS pandemic had the next highest ratings of negative valence (37%) followed by the SCP (28%). When both news and social media discourse pertaining to the WAEO were compared, there were several clear differences in valence frequencies. The most notable differences in valence arose when negative and neutral valence ratings were considered. Headlines from WAEO news discourse were more frequently rated as representing negative valence (44% of total headlines) than tweets about the WAEO (34% negative valence ratings). Further, ratings of neutral valence were more frequently granted to tweets (39%) than news discourse (30%). The frequencies of positive and ‘no emotion’ ratings remained generally static irrespective of whether the sample originated from tweets or news headlines. Both types of discourse were described as representative of no emotion 12% of the time, whereas ratings of positive valence were assigned at approximately the same frequency for news discourse (14%) and tweets (15%).

Collectively, there were noticeable variations in valence between the two different types of discourse that were analyzed (Figure 3). News discourse headlines, irrespective of time period, tended to be 10% more frequently rated as conveying ‘no emotion’ than WAEO tweets. Further, frequencies of positive valence ratings were more apparent in tweets (15%) than all collected news discourse (7%). Rating of neutral valence, although slightly different across discourse methods, remained relatively similar (33% for news and 39% for tweets). Similarly, the frequency of negative valence ratings was relatively similar between tweets (34%) and news discourse (38%).

DISCUSSION

One of the most notable results was the sheer frequency that the word “zombie” was mentioned in Twitter tweets (Table 1). At the time of the tweets, online news outlets were promoting stories claiming deceased Ebola victims were reanimating and terrorizing local villagers (Doloquee, 2014). Although the story was first publicized by the Liberian newspaper The New Dawn and

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DISCOURSE WORD CHOICE

Misinformation in the media, for which the fraudulent Ebola zombie fiasco is a prime example of, exerts a very large influence over perceptions of information validity. Overall, two-thirds of all adults in America used social media as a primary source of news in 2017—an increase of 5% since 2016, and compliant with a decade-long trend (Shearer and Gottfried, 2017). Hence, the presence of “Ebola zombies” and other instances of fallacious news reporting possess an everincreasing influence over the collective public consciousness. When making judgments of truth, individuals rely on two separate measures— recollection and familiarity—which supplement any previous information about the topic that they may possess (Begg, Anas and Farinacci, 1992). Familiarity increases automatically when individuals are exposed to repeated statements, ultimately resulting in the illusory truth effect (Hasher, Goldstein and Toppino, 1977). According to the illusory truth effect, individuals are more likely to believe statements which they have heard before, over other novel statements (Begg, Anas and Farinacci, 1992; Hasher, Goldstein and Toppino, 1977). Even when individuals are aware that repetition does not imply truthfulness, the illusory truth effect remains present (Bacon, 1979). Hence, when individuals stumble across several tweets affirming the presence of an “Ebola zombie apocalypse” they become increasingly familiar with that same notion regardless of how outrageous the claim may seem. Further, recollection also influences judgments of information validity. Individuals are more likely to

Psychology

Media discourse is often the most popular source of information used by the public when educating themselves on pertinent societal issues. The media itself is subject to constant evolution, which greatly influences the means by which it is consumed by the masses. The evolution of media may also affect the way in which individuals conceptualize different global events—namely epidemics. In accordance with epidemic psychology, the initial public response to epidemics is often associated with secondary psychosocial epidemics of fear/suspicion, stigmatization, and moralization/explanation. Changes in public media, coupled with alterations in the ways that individuals initially conceptualize different epidemics, could potentially exacerbate the severity of the secondary psychosocial epidemics. In accordance, this study attempted to analyze historical representations of three notable epidemics—the SCP, HIV/AIDS, and the WAEO—through performing a headline analysis. Both word usage and overall emotional valence were found to vary across both the three case studies of disease as well as the different types of media analyzed (newspaper vs twitter).

subsequently shared by the obscure online news outlet Big American News, it quickly gained traction in more well-known news outlets such as The Mirror and Newsweek in the following days (Dovey, 2014; Bond, 2014). The article shared by Big American News featured a picture from the 2013 science fiction film “World War Z”, as well as a Good Morning America news segment which featured footage of an Ebola victim that was mistakenly believed to be dead (Goodman, 2014). While the initial story was quite obviously a hoax, it went viral on both Twitter and Facebook and remained a topic of discussion for several days after its initial posting. Although it was obvious some twitter users were aware of the fraudulent nature of the ‘Ebola zombies” story when tweeting, others appeared to express genuine fears and some even linked to the original posting from Big American News. It was quite clear that the fallacious nature of the ‘Ebola zombie apocalypse’ was lost on some Twitter users.

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Psychology

rate novel information as true if it corroborates other previously known information (Begg, Anas and Farinacci, 1992). Although the first claims of an “Ebola zombie apocalypse” originated from Big American News—an outlet neither respectable nor well-known—its subsequent promotion in Newsweek and other more eminent news outlets helped to validate the claim. In accordance, despite the obvious implausibility of an Ebola zombie apocalypse, some individuals genuinely believed the claim to be truthful due to the familiarity and repetition fostered by the viral nature of the story. Word choice in news headlines, especially those pertaining to the HIV/AIDS pandemic and WAEO, tended to resemble each other— particularly through their frequent use of the word “fear”—which alludes to similarities in the way that both diseases were initially framed in the media. All news headlines generally adhere to one of four reference frames when reporting an event. Some reflect a conflict frame, and focus on a disagreement between two clashing groups (Pan and Kosicki, 1993). Similarly, other headlines may adopt a crisis frame, and subtly single out a particular group as the party responsible for the situation at hand (Pan and Kosicki, 1993). Others may adopt more of a human interest perspective to emphasize sentimental aspects of the story, while some headlines may neglect the human experience altogether and focus purely on the economic repercussions related to a particular event (Pan and Kosicki, 1993). Headlines pertaining to the HIV/AIDS pandemic and the WAEO, which strongly featured the word “fear”, adhered mostly to the human-interest perspective. Through their liberal use of the word “fear”, headlines emphasized the emotional nature of the disease and the repercussions it brought to specific groups of people. Further, given the fact that that headlines are often the only information individuals may see, they are often used to educate public opinion concerning a particular subject (Smith, 1999). These perspectives help to subtly influence the reader’s own opinions about the subject at hand, and even shape the way they respond to the disease in the future (Stewart, 2005). Through assuming a human-interest perspective, and liberally using the word “fear”, headlines for the HIV/AIDS pandemic and WAEO catalyzed individuals to initially conceptualize the disease similarly—as a formidable entity worthy of fear. Headlines from both the HIV/AIDS pandemic as well as the WAEO tended to isolate groups of

people perceived to be of highest risk of contracting the diseases. As such, HIV/AIDS headlines specifically adhered to a crisis reference frame. The word “homosexual” was the second most commonly mentioned word across all HIV/AIDS headlines, and “nurse” and “Africa” were tied as the fourth most frequently mentioned word among WAEO headlines (Table 1). Those individuals, along with others who were not as frequently mentioned in headlines, were initially believed to be of highest risk of contracting HIV or Ebola. Broadly, this represents the repercussions of the epidemic of stigmatization and the marginalization of already denounced groups (Strong, 1990). Although the isolation of vulnerable groups is a well-studied theme among HIV/AIDS discourse (Lupton, 2008; St. Lawrence et al., 1990; Devine, Plant and Harrison, 1999), stigmatization during the WAEO is a topic of notably less research (Davtyan, Brown and Folayan, 2014). Theoretically, both diseases presented to Western society in similar manners— both were life threatening, without recognized cures, and had initially unidentified modes of transmission. Although sources of stigma differed, similarities lie in the fact that the disease was predominately attributed to groups whom society had already marginalized. The early years of the HIV/AIDS pandemic saw the disease being attributed to gay men and Haitian-American immigrants, while the WAEO was initially accredited to poverty-stricken Africans and African-American immigrants (Davtyan, Brown and Folayan, 2014). Due to the stark parallels between initial HIV and WAEO discourse, it can be inferred that the initial presentation of novel disease has remained relatively similar. Even in modern societies, marginalized group are still blamed, at least initially, as the cause for novel outbreaks of disease.

VALENCE RATINGS Valence levels for media discourse pertaining specifically to the WAEO differed across the two types of discourse—news and social media— present within this analysis. Specifically, ratings of negative valence were more common among news discourse than tweets, and ratings of neutral valence were more common among tweets than news discourse. Although many tweets were blatantly sarcastic, there were just as many which could have been interpreted as both sarcastic and non-sarcastic, which introduced a contextual ambiguity. This ambiguity in contextual

This research, through analyzing headline word usage patterns and valence ratings, provides

STUDY LIMITATIONS Regardless of the reliability of media as an indicator of public opinions, there is no way to conclusively identify public opinion at different points in history. Hence, there could be minor inconsistencies between how some diseases were conceptualized by the public and the ways in which they were presented in the media. Although this limitation is negated in the WAEO case study, since public tweets from personal accounts were analyzed, it remains present for both the SCP and the HIV/AIDS pandemic. Specifically, the nondescriptiveness of SCP headlines presents the largest obstacle to identifying public opinion. Public opinion regarding the SCP likely differed from the relatively constrained headlines—whose emotional value was virtually nonexistent. To negate this limitation, future research could include samples of personal discourse, such as journals and letters, to garner individual thoughts regarding the SCP and obtain a more accurate representation of public opinion. However, in the scope of the current media discourse analysis, this limitation was unavoidable.

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There was distinct cross-case study variation in valence determinations. Namely, the SCP was more frequently rated as conveying no emotion than the HIV/AIDS pandemic and the WAEO. This was likely due to the non-descriptive nature of SCP headlines with respect to HIV/AIDS and WAEO headlines. Generally, headlines at the time of the SCP were relatively undescriptive—often detailing the news-worthy situation in only 5 words or less (Cohen and Vandello, 1998). As newspapers evolved, so did their associated headlines; headlines became gradually more sensationalistic, meaningful, and moralizing the closer to the mid-19th century they were published (Cohen and Vandello, 1998). As such, headlines at the time of the SCP were not as indicative of the interpretational framework unique to its associated newspaper—this quality arose when headlines increased in descriptiveness (Fang, 2001; Cohen and Vandello, 1998). As such, the concise nature of headlines at the time restricted the ability of news outlets to convey emotional meaning through headlines.

preliminary insight into the complex interplay between disease and the mass media. By understanding how past diseases have been framed by the media, it provides a unique opportunity to inform future disease responses. Given that media is the foremost means by which the general public informs themselves about outbreaks of disease, it is imperative that it does so realistically, without sensationalizing (Glass and Schoch-Spana, 2002). The current research used case studies of some of the most notable outbreaks of disease in modern human history and identified that contemporary media tends to follow a specific interpretational framework when initially presenting outbreaks of disease. Specifically, this framework adheres to the innate principles of epidemic psychology—namely the sub-epidemics of stigmatization and fear—which has theoretical repercussions on public order (Strong, 1990). By knowing how the public is presented with diseaserelated information, governmental institutions can surmise how the public will conceptualize—and later react to—disease. Anticipating how individuals react to disease is invaluable towards forming effective disease-response policies whose explicit goal is to preemptively quell the theoretical panic and fear characteristic of epidemic psychology.

Psychology

perception could have been due to the difference in literary construction between news headlines and tweets. Twitter carries its own unique ‘language’—specific abbreviations, references, and literary connotations—conveyed in tweets that is not present in other, more traditional forms of discourse (Rajadesingan, Zafarani and Liu, 2015). As such, the language of Twitter is subject to constant fluctuation in accordance with shortlived pop culture trends (Rajadesingan, Zafarani and Liu, 2015). Due to the changing nature of twitter ‘language’ conventions, individuals could be led to erroneously perceive sarcasm, even when it is absent from the tweet itself (Rajadesingan, Zafarani and Liu, 2015). Further, sarcasm can also be conceptualized as a linguistic phenomenon as well as a literary phenomenon. In order to perceive sarcasm, individuals rely on tonal and contextual cues, which are absent from tweets (Kunneman, Liebrecht, van Mulken and van den Bosch, 2015). In the absence of such cues, judgments of sarcasm exclusively fall to constructional cues which, due to the fluid language of Twitter, can be vague and uninformative for many (Kunneman et al., 2015; Rajadesingan, Zafarani and Liu, 2015). Individuals were more apt to judge tweets as sarcastic, due to the unique ‘language’ of Twitter being unhelpful towards sarcasm detection, which fostered a perceptual ambiguity.

Psychology ISCIENTIST | 2019

There were also several other limitations innate to the methodology of the current study. One of the most apparent limitations of this research concerns the limited scope of discourse collection. Due to both time and resource constraints, the current study limited discourse collection to 150 news articles and 66 tweets. Future research should consider amassing a larger corpus of discourse to uncover trends that were not apparent in the current study’s limited analysis. Secondly, at the time of data analysis, all supplementary data analyzers had received, or were in the process of receiving, a post-secondary education. Previous research has identified education as a factor that greatly influences news consumption. Specifically, higher education is associated with increased voluntary news exposure, differential news source engagement, and a better ability to discern factual information from invalid information (Ksiazek, Malthouse and Webster, 2010; Baum, 2003; Allcott and Gentzkow, 2017). Accordingly, level of education could also influence headline valence ratings— which future research should consider analyzing. Lastly, news articles possess a wealth of subliminal meanings in a multitude of different areas which were neglected in the current study—namely the body text, picture selection, and story placement respective to the overall newspaper (Lupton, 2008). Hence, future research into initial public responses to epidemics should undertake a more comprehensive analysis of news discourse.

CONCLUSION Given the inevitable presence of disease in society, anticipating how humans will react to epidemics is a topic of great importance. This analysis has attempted to understand how past responses to notable case studies of disease have changed across both time as well as type of media. As such, a total of 66 tweets and 150 examples of news discourse were collected from online databases, whose headlines were analyzed with respect to word choice and valence. The analysis yielded instances of the illusory truth effect, stigmatization of already marginalized groups, and timedependent changes in valence level across both media sources as well as case studies. Comprehensively, this research identifies preliminary trends indicative of how mass media initially frames deadly disease—which can be used by governmental institutions to anticipate and quell the mass public fear that often accompanies disease. Future research should consider broadening the scope of analysis to include both more instances of discourse as well as a more comprehensive analysis of all aspects of news discourse to offer further insight.

ACKNOWLEDGMENTS The author would like to thank Dr. Chad Harvey for both his guidance in planning this research and his valuable supervision throughout the research period.

REFERENCES Allcott, H. and Gentzkow, M., 2017. Social media and fake news in the 2016 election. Journal of Economic Perspectives, [e-journal] 31(2), pp.211–236. 10.1257/jep.31.2.211. Alvarez Amorós, J.A. and José Antonìo Álvarez, 1992. Language in the news: Discourse and ideology in the press. Atlantis, [e-journal] 14, pp.285– 292. https://doi.org/10.1017/S0008413100020132. Bacon, F.T., 1979. Credibility of repeated statements: Memory for trivia. Journal of Experimental Psychology: Human Learning & Memory, [e-journal] 5(3), pp.241–252. http://dx.doi.org/10.1037/0278-7393.5.3.241. Baum, M.A., 2003. Soft news and political knowledge: Evidence of absence or absence of evidence? Political Communication, [e-journal] 20(2), pp.173–190. 10.1080/10584600390211181. Begg, I.M., Anas, A. and Farinacci, S., 1992. Dissociation of processes in belief: Source recollection, statement familiarity, and the illusion of truth. Journal of Experimental Psychology: General, [e-journal] 121(4), pp.446–458. http://dx.doi.org/10.1037/0096-3445.121.4.446. Bond, A., 2014. Ebola victims in African village ‘rise from the dead’ causing panic and fear among locals. Mirror UK. [online] 25 Sep. Available at: <https://www.mirror.co.uk/news/world-news/ebolavictims-african-village-rise-4320414>. Cohen, D. and Vandello, J., 1998. Meanings of Violence. The Journal of Legal Studies, [e-journal] 27(S2), pp.567–584. 10.1086/468035.

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Lupton, D., 2008. Archetypes of infection: People with HIV/AIDS in the Australian press in the mid 1990s. Sociology of Health & Illness, [e-journal] 21(1), pp.37–53. https://doi.org/10.1111/1467-9566.t01-1-00141.

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Glass, T.A. and Schoch-Spana, M., 2002. Bioterrorism and the people: How to vaccinate a city against panic. Clinical Infectious Diseases, [e-journal] 34(2), pp.217–223. https://doi.org/10.1086/338711. Goodman, A., 2014. Africa Confirms 3rd Ebola Victim Rises From the Dead, Releases Picture of First “Ebola Zombie” Captured. Big American News. [online] 30 Sep. Available at: <http://bigamericannews.com/2014/09/30/africaconfirms-3rd-ebola-victim-rises-from-the-dead-releasespicture-of-first-ebola-zombie-captured/>. Hasher, L., Goldstein, D. and Toppino, T., 1977. Frequency and the conference of referential validity. Journal of Verbal Learning and Verbal Behavior, [e-journal] 16(1), pp.107–112. https://doi.org/10.1016/S0022-5371(77)80012-1. Kozareva, Z., Navarro, B., Vázquez, S. and Montoyo, A., 2007. UA-ZBSA: A headline emotion classification through web information. In: Proceedings of the 4th International Workshop on Semantic Evaluations. [online] Prague: Association for Computational Linguistics, pp.334–337. Available at: <https://dl.acm.org/citation.cfm?id=1621546> [Accessed 23 Mar. 2018]. Ksiazek, T.B., Malthouse, E.C. and Webster, J.G., 2010. News-seekers and avoiders: Exploring patterns of total news consumption across media and the relationship to civic participation. Journal of Broadcasting & Electronic Media, [ejournal] 54(4), pp.551–568. https://doi.org/10.1080/08838151.2010.519808. Kunneman, F., Liebrecht, C., van Mulken, M. and van den Bosch, A., 2015. Signaling sarcasm: From hyperbole to hashtag. Information Processing & Management, [e-journal] 51(4), pp.500–509. https://doi.org/10.1016/j.ipm.2014.07.006.

Pan, Z. and Kosicki, G., 1993. Framing analysis: An approach to news discourse. Political Communication, [ejournal] 10(1), pp.55–75. 10.1080/10584609.1993.9962963. Rajadesingan, A., Zafarani, R. and Liu, H., 2015. Sarcasm detection on Twitter. In: Proceedings of the Eighth ACM International Conference on Web Search and Data Mining WSDM ’15. New York, New York, USA: ACM Press, pp.97–106. 10.1109/ICEMIS.2017.8272990. Rosenberg, C.E., 1989. What is an epidemic? AIDS in historical perspective. Daedalus, [e-journal] 118(2), pp.1–17. https://doi.org/10.1017/CBO9780511666865.014. Shearer, E. and Gottfried, J., 2017. News Use Across Social Media Platforms 2017. [online] Washington. Available at: <http://www.journalism.org/2017/09/07/news-useacross-social-media-platforms-2017/> [Accessed 26 Mar. 2018]. Smith, E.J., 1999. Leadlines may be better than traditional headlines. Newspaper Research Journal, [e-journal] 20(1), pp.55–65. https://doi.org/10.1177/073953299902000105. Stewart, C.O., 2005. A rhetorical approach to news discourse: Media representations of a controversial study on ‘Reparative Therapy’. Western Journal of Communication, [e-journal] 69(2), pp.147–166. https://doi.org/10.1080/10570310500076858. Strong, P., 1990. Epidemic psychology: A model. Sociology of Health and Illness, [e-journal] 12(3), pp.249–259. https://doi.org/10.1111/1467-9566.ep11347150. Wall, M.A., 1997. The Rwanda crisis: An analysis of news magazine coverage. International Communication Gazette, [e-journal] 59(2), pp.121–134. https://doi.org/10.1177/0016549297059002003.

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Davtyan, M., Brown, B. and Folayan, M.O., 2014. Addressing Ebola-related stigma: Lessons learned from HIV/AIDS. Global Health Action, [e-journal] 7(1), p.26058. 10.3402/gha.v7.26058.

Dovey, D., 2014. Video: Ebola Victim Thought Dead Comes Back to Life Minutes Before Cremation. Newsweek. [online] 3 Oct. Available at: <http://www.newsweek.com/ebolazombie-comes-back-life-minues-cremation-275149>.

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ORIGINAL RESEARCH

Tannins as a Pesticide: The Impact of Tannic Acid on the Growth Rates of Myzus persicae and Arabidopsis thaliana DANA PRICE1, SHAMA PIRBAY1, LELIA WEILAND1, JULIET ZHU1, ELYSIA FULLER-THOMSON1 1. Integrated Science Program, Class of 2021, McMaster University

Ecology

SUMMARY We completed a study on the effectiveness of natural pesticides in lieu of artificial pesticides with the purpose of finding a more environmentally friendly alternative to pesticides. We investigated the interactions between the plant, Arabidopsis thaliana, and the herbivore, Myzus persicae, as well as the effect of tannic acid on these interactions. Our goal was to answer whether a dose of tannic acid would positively or negatively affect both the plant and the animal. This paper aims to determine if there are alternatives to pesticides which may be safer for the environment. We determined that, if the pesticide generated a positive or neutral effect on the plant and a negative effect on the herbivore, it could be considered an effective insecticide. After completion and analysis, it was determined that tannic acid is not an effective insecticide for our given study system at 0%, 1% and 3% concentrations. It had no effect on aphid population growth, and at a 3% concentration in the presence of aphids, a negative effect on plant growth, which presents itself as a possible future herbicide. Received: 11/08/2018

Accepted: 03/12/2019

Published: 11/17/2019

Keywords: pesticide, insecticide, tannic acid, Arabidopsis thaliana, Myzus persicae, plant defence

ISCIENTIST | 2019

INTRODUCTION

Pesticides are chemical compounds that humans have used as a convenient method to kill undesired plants, as a herbicide; or insects, as an insecticide (Aktar, Sengupta, and Chowdhury, 2009). Over two million tonnes of pesticides are used annually worldwide in the 3.342 trillion dollar agricultural industry, the majority of which are used to target insects (Worldbank, 2017). This subcategory of pesticides is called insecticides and indirectly cause an increase in the yield and quality of crops (Aktar, Sengupta, and Chowdhury, 2009). Despite these short-term benefits, the long-term effects of these artificial chemical pesticides are greatly concerning. For instance, many pesticides contain toxins that are dangerous to both humans and organisms

beyond their intended target species (Nicolopoulou-Stamati, et al., 2016; Aktar, Sengupta, and Chowdhury, 2009). Insecticides can also damage many non-target organisms in the environment (Ansari, Moraiet, and Ahmad, 2014). For example, insecticides can mix with main water systems through surface runoff, leading to contamination of aquatic ecosystems and drinking water (Mahmood et al., 2015; Aktar, Sengupta, and Chowdhury, 2009). This can negatively impact both aquatic organisms and humans. The chemicals in pesticides can also leach into soil and negatively affect many beneficial microorganisms that contribute to soil fertility, such as nitrogen-fixing bacteria. Furthermore, volatiles from the large-scale spraying of pesticides

can also lead to air contamination (Aktar, Sengupta, and Chowdhury, 2009). There is also evidence that the wide use of insecticides has led to insecticide resistance in many insects (Ansari, Moraiet, and Ahmad, 2014). Another alarming fact is that pesticides can have an impact on humans, negatively affecting health immediately. In fact, around 26 million people suffer from pesticide poisoning annually (Ansari, Moraiet, and Ahmad, 2014). Many workers in insecticide manufacturing report experiencing symptoms such as nausea, vomiting, and fatigue regularly (Mahmood et al., 2015). With long-term or frequent exposure, the chemical compounds can act as endocrine disruptors, leading to immune system suppression, reproductive abnormalities, and cancer (Mahmood et al., 2015; NicolopoulouStamati et al., 2014). As of 2013, Canada is still using more synthetic pesticides than naturallyderived ones (Figure 1) (Government of Canada, 2013). For this reason, it is crucial to find alternative pesticides for commercial use in Canada that are safer for the environment and human health.

extract tannic acids from plants may be great (Karchesy, Kelsey, and González-Hernández 2018). They are commonly found in several different types of plant life, including Castanea sativa (sweet chestnut), Quercus infectoria (Aleppo oak), and Rhus spp. (sumach) along with several other organisms (European Safety Authority, 2014). The tannins can be extracted easily using solvents such as 80% acetone or an alcohol ether mixture (Karamać et al., 2007; Gino, 1951). There are several studies that demonstrate the benefits of using tannic acid. For instance, humans are already accustomed to metabolizing tannin-rich foods, so it will have a minimal impact on human health, especially at low concentrations (Chung et al., 1998). There is also evidence that tannins are a more environmentally friendly alternative to bactericides when treating leather (Zengin et al., 2014), and it has already been proposed to be an alternative fungicide (Forrer et al., 2014).

Tannic acid is already used as a fungicide, inhibiting bacterial growth (Forrer et al., 2014). Therefore, we set out to investigate tannic acid’s potential as an insecticide against herbivores, such as the green peach aphid (Myzus persicae), a commonly found pest. While tannic acid has been shown to reduce fitness in grasshoppers, little has been done to demonstrate its effect on aphids, and

ISCIENTIST | 2019

We propose the use of tannic acid (C76H52O46) as a naturally derived alternative to insecticides. It has the potential to be a good alternative to artificial insecticides since tannic acid is already naturally produced by many woody plants therefore not requiring chemical manufacturing. However, one must also consider that the cost and effort to

Ecology

Tannins are naturally produced by plants as a defence mechanism against plant-feeding insects (War et al., 2012). The chemical defends against insects through a variety of mechanisms. Firstly, tannins oxidize within the guts of insects due to its highly acidic climate, then bind to various essential amino acids, such as important digestive enzymes, within the insect’s gut (Barbehenn & Constabel, 2011). Tannins can cause significant nutrient loss by binding to many lipids and carbohydrates that insects ingest, leading to decreased digestibility of these molecules (Barbehenn and Constabel, 2011). In addition, there is evidence that tannins inhibit insect development by causing the formation of midgut lesions (War et al., 2012), likely due to the semiquinone and quinone free Figure 1: A diagram comparing the percentage of radicals released when tannins chemical and natural pesticide usage in Canada oxidize (Barbehenn and Constabel, from 2007 to 2013 (Government of Canada, 2013). 2011). Furthermore, tannins also act as feeding deterrents to insects due to their bitter taste (War et al., 2012). Tannic Acid

Ecology

especially how it jointly interacts with both plant and pest. Unlike many other pesticides, it does not present a considerable risk to human health, such as having no mutagenicity in vivo, or carcinogenic sources (Onodera et al., 1994). Due to its natural presence in the environment, there is not a major concern for it to be harmful for the environment (European Safety Authority, 2014). The purpose of our study was to investigate the relationship between a pesticide (tannic acid) and the health of both an animal (Myzus persicae) and the plant (Arabidopsis thaliana). We hope to answer the question: what is the effect of different concentrations of tannic acid on M. persicae population growth rate and A. thaliana plant growth rate? Our hypothesis is that, by treating A. thaliana with varying tannic acid concentrations, the overall health of the plant will not be affected. As well, the tannic acid will affect M. persicae negatively, decreasing population growth rate over time. Conversely, our null hypothesis is that neither the growth rates of A. thaliana nor M. persicae will be affected by tannic acid concentrations. This experiment was conducted to investigate whether tannic acid could act as a natural and environmentally friendly pesticide. More specifically, as tannic acid is used by some plants as a defence mechanism, we wanted to demonstrate whether applying this compound to the surface of A. thaliana could aid in its defence against M. persicae.

MATERIALS & METHODS

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STUDY SPECIES: MYZUS PERSICAE

This section aims to provide background information on the species M. persicae, commonly referred to as the green peach aphid. It is a model organism for research regarding plant and animal interactions. M. persicae are known to feed on over 40 different families of plants, including A. thaliana (CAB International, 2018). Illumina sequencing technology has been used to compare the genomic differences between adult and nymph M. persicae. The life stages of M. persicae vary in cuticle formation, detoxification, hormone production,

and metabolism, which has resulted in 2244 genes being expressed differently between the two stages (Ji et al., 2016). This can affect the aphid’s feeding and migratory patterns on the plant. M. Persicae has several different life stages; egg, nymph, and adult. The adult stage can be divided into two subgroups: viviparous and alate. The viviparous are unwinged and able to give birth while alate is the winged morph. A study by Srinivasan and Brisson discusses phenotypic plasticity, the process in which aphid females send signals that can affect the development of their offspring (2011). The life cycle of M. persicae must be taken into consideration during this study, as different stages of life may affect plant growth differently. The youngest life stage is nymphs, which are green or green-yellow in colour. They can grow into viviparous adults that are capable of asexual reproduction (Capinera, 2001). This is an effective model system for studying the effects of environmental cues (e.g. tannic acid concentrations) on animal growth patterns and phenotypic plasticity (Srinivasan and Brisson, 2011). We obtained our aphid population from other A. thaliana plants hosting aphids in a laboratory at McMaster University, under the direction of Dr. Chad Harvey.

STUDY SPECIES: ARABIDOPSIS THALIANA The plant species chosen for this study is A. thaliana, commonly used in a variety of biological experiments. It is an ideal plant for our purposes, as there is extensive background already known about the plant’s genome and structure (Van Norman and Benfey, 2010). The plant’s size ranged from 1.1 cm to 5.7 cm tall, allowing for many plants to be stored in limited space. Furthermore, the life stages are easy to distinguish through observation. Our study examines plants in rosette, bolting, and flowering stages. Also, the life cycle of A. thaliana is short, at approximately six weeks, allowing for easy study of how various factors affect plant growth over time (Koornneef and Meinke, 2010). This species is very straightforward to maintain and can be found in many locations across North America, Europe, Asia, and Africa (Clarke, 1993).

INTERACTIONS BETWEEN A. THALIANA AND M. PERSICAE

TANNIC ACID TREATMENT All plants used for the experiment were separated by applied insecticide amount: 0%, 1%, and 3% w/w tannic acid, whereby each group had equal number of aphid and no aphid treatments. These treatment concentrations were chosen to investigate the lowest concentration of tannic acid which would influence our study species. Following methods similar to Bien (2016), three solutions were dissolved using pure, powdered tannic acid in water. They were then administered to the plant by spraying each plant three times from a spray bottle approximately 5 cm away. This treatment was administered only once, at the beginning of the experiment.

Thirty-six A. thaliana plants were used in this experiment. To minimize potential block effects that could be caused by the separation of the plants into three covered trays, we randomized the placement of the different treatments. Individual trays received two plants of each treatment type: each with different acid concentrations with either

This may have contributed to observer bias; however, it is believed that since our observations were numerical in nature, this would not have affected our data to a great extent. The 1% tannic acid was made through serial dilutions of the 3% stock solution. We varied the concentration to both analyze the effects of different concentrations as well as to potentially propose an optimal concentration for the most effectiveness.

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EXPERIMENTAL DESIGN

Figure 2: There are six treatment groups within our experiment. The blue labels represent the control treatment (i.e. no tannic acid), the green labels indicate a 1% tannic acid solution was applied, and a red label indicated that a 3% solution of tannic acid was applied. The yellow labels represent the aphid treatment. These six treatment groups were placed in three different trays in a randomly arranged mixing of the categories seen above.

Ecology

In the case of A. thaliana and M. persicae, the herbivore requires the plant as a habitat and source of nutrition in the context of our study. The plant, however, does not require the herbivore and is constantly trying to ensure that the herbivore does not kill it, as herbivores regularly do to plant species. This is consumption, therefore, since the plant does not require or benefit from the presence of the herbivore. In fact, the plant is consistently trying to eliminate the aphids from its environment through its own defenses. A. thaliana has mechanical defenses, specifically trichomes, to protect itself from herbivores (Bruner, 2009). The trichomes are modified epidermal cells, specially designed to eliminate herbivores. This is evident when we observe where M. persicae resides on the plant and see that it abides mostly under the leaves and on the stem where trichomes are rare. We are also aware that if plants are invaded by herbivores, their jasmonic acid pathway will be suppressed by the active salicylic acid pathway (Capinera, 2001). This would decrease the defense against bacterial pathogens while increasing defense against herbivores.

no aphids or three aphids initially. A random number generator (Accessed through website www.random.org) was used to decide in which position each plant would be placed within the tray. Each possible position for the plant was assigned a number. Then, through the number generator, we randomly designated a position for each plant. Then, cardboard dividers were placed to separate the plants from one another, spanning from the bottom of the tray to around plant-height to avoid blockage of sunlight. This experiment design method was selected to minimize any potential block effects and ensured each treatment would have the same probability of being affected by confounding factors such as proximity to light source, proximity to plants with aphids, and potential temperature differences. The plants were labelled based on the aphid treatments (yellow tape to indicate presence of aphids or orange tape to indicate absence of aphids) and the tannic acid concentrations (blue tape to indicate 0%, green tape to indicate 1%, and red tape to indicate 3% tannic acid) (Figure 2).

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Ecology

After the plants had been inoculated with three aphids on each plant with orange tape, they were placed in similar lighting conditions: indoors, beside natural light, and covered in a transparent lid to prevent cross-contamination of aphids. Unfortunately, cross-contamination could not be prevented within trays. Over the course of 12 days (September 20th, 2018 to October 2nd, 2018), maximum plant height, maximum plant diameter, and aphid count, along with qualitative observations of leaf discolouration and bolting on each plant were measured and recorded only 9 of those days due to limited access to the laboratory room. More specifically, observations were taken on the day they were inoculated (day 0), then day 1, 4, 5, 6, 7, 8, 11, and 12. After 7 days of the experiment, each plant was watered with 6 mL of water directly into the soil so as to not interfere with the tannic acid treatment on the stem and leaves.

Aphid population growth rate was determined by calculating the slope of the aphid count over time for each individual plant. This was necessary to analyze due to the uneven time separation of each aphid count measure. This method of calculating aphid population growth rate can account for that potential confounding factor and minimize measurement imprecision that could occur day to day. These imprecisions were caused by different observers and the possibility for counting error. Plant growth rate was determined first by calculating plant conical volume, using both plant height and diameter in methods similar to those proposed by Pontailler et al. (1997) (Box 1). Both plant height and plant diameter were determined to be crucial to consider because there were some healthy plants that did not bolt but experienced width growth, and there were also healthy plants that bolted and did not grow in diameter. A slope was then determined from plant conical volume change over time for each individual plant.

Box 1: The equation for plant conical volume is used to combine the variables of plant diameter and plant diameter (Pontailler et al., 1997).

STATISTICAL METHODS USING R Before statistically modelling the effect of tannic acid on plant growth rate and aphid population growth rate, another variable that must be considered is the potential interacting effect of plant growth rate and aphid population growth rate. This was investigated using a linear regression between aphid population growth rate and plant growth rate of all the treatments combined. A two-way analysis of variance was used to determine the possibility of tannic acid treatment levels and aphid treatment levels interacting to influence plant growth rate. Next, a Tukey HSD post-hoc test was performed to isolate the differences of means and investigate significance. Next, the relationship between aphid population growth rate and tannic acid treatment levels was investigated using analysis of variance solely in the plant group which was inoculated with aphids. The effectiveness of the tannic acid treatment as a pesticide can be determined by analyzing the difference between the means of all three levels of acid treatment on the aphid population growth rate. A post-hoc test was not performed since there was no significant correlation.

RESULTS OBSERVATIONS Over the course of 11 days, some visual changes were observed in the plant and aphid population growth. Over time, 27 of the 36 plants developed yellow or shriveled leaves. Aphids were often found on the stem of the bolted plants or under the leaves for plants in both the bolted and rosette stages (i.e. where there was little or no tannic acid treatment if applicable). The greatest density of aphids was often found at the base of the stems. The aphids found were most often nymphs; however, several viviparous adults and several alates were found as well. As a result, these winged aphids would have been able to migrate and colonize other plants in the vicinity, including plants that originally had no aphids (Figure 3). This was observed when plants which initially were not inoculated with aphids were found to have aphids by the end of the experiment.

Many of the plants exposed to the tannic acid treatment, both at 1% and 3% concentrations, had dark brown spots visible on the leaves. These were only visible on the leaves which were already present at the time of treatment (i.e. none of the new leaves were affected). These brown spots were visible after one day of treatment on several plants; however, they were more apparent later in the trials. Furthermore, the plants with the 0% tannic acid treatment did not have these dark brown spots for the duration of the experiment. Almost all of the plants with the 3% concentration treatment had brown spots on their leaves.

PLANT GROWTH

(M=0.86 cm3/day, SE=0.43) levels of tannic acid on plant growth (p>0.1). There was a significant difference between 3% and 0% for the aphid treatment (p<0.05). The 0% tannic acid aphid treatment (M=2.92 cm3/day, SE=0.96) had a mean plant growth rate that was 4.38 cm3/day higher than the 3% tannic acid aphid treatment (M=-1.46 cm3/day, SE=1.15). Furthermore, the negative mean growth rate shows that, on average, the plant volume decreased over time under the presence of tannic acid. However, there were no significant results between 0% and 1% (M=0.72, SE=0.65) for the aphid treatment (p=0.2438275) as well as 1% and 3% for the aphid treatment (p=0.4287740) (Figure 5).

APHID POPULATION GROWTH The growth rate of the aphid population was not found to be affected by the growth rate of the plant, so aphid population growth and its connection with tannic acid was examined independently of plant growth solely in the group

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Plant growth rates were not significantly related to aphid population growth rate within the study (R2=0.01363, F(1,16)=1.235, p>0.1). This was investigated solely on the aphid treatment level because even though the non-aphid treatment had some aphids due to contamination, there were too few to appropriately consider this relationship. This allows us to investigate plant growth along with aphid treatment levels independently of aphid population growth rate (Figure 4).

Plant growth was significantly affected by the interacting effect of tannic acid and the aphid treatment (F(2,30)=5.794, p=0.00745). In the nonaphid treatment, there were no significant differences between the 0% (M=0.61 cm3/day, SE=.32), 1% (M=2.37 cm3/day, SE=0.64) and 3%

Ecology

Figure 3: This Arabidopsis thaliana plant was treated with 3% tannic acid solution and populated by aphids. It has visible damage such as dark brown spots, yellowing, and loss of structure to the leaves that was common to plants of this treatment group.

Figure 4: Within the aphid treatment and all three acid concentrations, plant growth rate and aphid population growth rate were plotted together and found to not have a significant correlation. Tannic acid concentration is categorized in percentage by weight (0%, 1%, and 3%).

Mean Plant Growth Rate (cm cubed per day) which was inoculated with aphids. Aphid population growth rate was not significantly related to tannic acid concentration at any concentration (F(2,15)=0.2227, p=0.8029) between any of the concentration levels: 0% (M=0.61, SE=0.32), 1% (M=2.37, SE=0.64), and 3% (M=0.86, SE=0.43) (Figure 6).

Mean Aphid Growth Rate (cm cubed per day)

Ecology

Figure 5: The effect of tannic acid concentration on mean plant growth rate. There is a significant difference of means between 0% and 3% for the aphid treatment. The mean plant growth rate of 3% was 4.38 cm3/day less than the 0% concentration. A negative growth rate is seen solely for the 3% tannic acid within the aphid treatment. * denotes significance of p <0.05.

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CONFOUNDING FACTORS

Several unforeseen elements of our experiment may have affected our results. Moss was present on the soil of some of the samples. Alates were able to migrate between plants, and consequently, likely colonized other plants, as most of the plants that were not inoculated with aphids had aphids at the end of the experiment, and therefore our nonaphid treatment was determined to have been contaminated.

DISCUSSION As indicated by the results of our experiment, tannic acid is not a suitable pesticide within our study system, though many plants also use tannic acid as a chemical defense (Clausen et al., 1992).

Figure 6: There is no effect of tannic acid on mean aphid population growth rate. All the aphid population growth rates are positive, demonstrating that aphids increase in population over time. Tannins are naturally a difficult chemical to digest for herbivores, decreasing the nutritional value of the plant for the herbivore. This is one of the main reasons we wanted to test tannins on the plant. Tannins negatively affected the plant invesitaged in our study, even though they naturally occur in many other plants and are used to harm herbivorous predators. The aphids in our study were not negatively affected by tannins, contrary to

what was expected. Perhaps they were able to overcome this issue because commercialized tannins have been known to have different dietary effects on herbivores (Hagerman et al., 1992). In fact, due to insecticide overuse, some species of insects have evolved gut adaptations, such as extreme pH tolerance and preventing tannins from interacting with digestion (Martin, Rockholm, and Martin, 1985). However, it must be noted that tannins are a digestibility reducer and have not been found to affect sucking insects, such as aphids, in the same way as they affect insects which chew on plants (Stienezen, M., et al., 1996). Finally, topical application such as in our experiment interacts differently with the aphids as would typical ingesting of tannins that come from within the plant itself.

FUTURE STUDIES

Since the tannic acid was only effective as a herbicide when in the presence of aphids at higher populations, it would be of interest to test stronger concentrations of the acid without aphids. This may provide a simpler solution to a herbicide with

From our study, we have been able to conclude that tannic acid is not an effective insecticide for the plant A. thaliana with respect to the herbivore M. persicae. Although the acid did appear to be effective under the condition that it was at a high concentration (3% in this study) and inhabited with pests, it still results in the conclusion that the tannic acid negatively affected the plant growth. This is not ideal for an insecticide as the plant would die while the pest would thrive. It may be possible that in another study system, this pesticide would prove effective. For example, the acacia plant utilizes tannins as a natural form of defense against herbivores (Elgailani and Ishak, 2014). It may be hypothesized that if tannins were to be sprayed onto an acacia plant, the plant may have an increased defense against herbivores. If this were to occur, the tannins would be considered an effective natural insecticide. This would encourage further studies into tannins and other study systems. These results, however, should not discourage further research for other natural pesticides, even those which are used in plant defenses. One could look into another plant defense mechanism which would enhance the plantâ&#x20AC;&#x2122;s defense against aphids and not harm the individual plant. Studies could also be designed to test if higher concentrations of tannic acid could damage the plant enough to kill it without the presence of aphids. We have determined that tannic acid could be a potential effective herbicide instead of insecticide at the tested concentrations. This could lead to new ideas in the field of invasive species management, as it is naturally produced and not harmful to plant or human health. For example, if we aimed to be rid of an invasive species, we could send a herbivore (such as M. persicae) onto the plant and then spray the plant with tannic acid. From our results, the tannic acid must be in the presence of aphids to damage the plant enough that it dies. These conclusions allow room to create new studies into the efficacy of tannic acid or if there is another natural compound which would more effectively impede plant growth rate.

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One of the main confounding factors in this study was contamination of aphids on the no aphid control group. In future studies, it would be beneficial to create an environment where the individual A. thaliana samples are completely isolated to better analyze the independent effects of aphids and tannic acid concentration on plant growth rate. This experiment also did not account for the effects of tannic acid wearing off over time. The tannic acid spray was not reapplied as it might have affected the aphid populations on the plants. It would be interesting, instead, to find a mechanism to maintain a constant level of tannic acid present on the plant without disturbing the aphids.

CONCLUSIONS

Ecology

Tannic acid did not increase plant growth but instead damaged the plants when coupled with an aphid attack. The aphids induced a stressful environment in which the plant is taxed for resources. This occurs when a tannic acid concentration of at least 3% is also added to the plant; consequently, it cannot handle the extra stress and its growth rate substantially decreases. These results support the Plant Stress Hypothesis, whereby stresses, such as insect herbivory, act as a resource sink so that the plant is not able to properly protect itself from the tannic acid and crop damage ensues (Joern and Mole, 2005).

the intent of eliminating unwanted plants, such as invaders, or even weeds in a garden. The resulting herbicide would be a natural one, with fewer negative effects on the environment and humans than an artificial pesticide (Okuda, 2005).

Based on our finding that 3% tannic acid damages plants and decreases plant growth rate when in the presence of aphids, we suggest that tannic acid has the potential to be used as a herbicide instead of an insecticide. If a population of plants is infested by aphids, applying tannic acid to the leaves would decrease the plant growth rate if the desired solution to the infestation is to kill the plants.

ACKNOWLEDGMENTS

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Ecology

We would like to acknowledge Dr. Chad Harvey for his teachings about plant-animal interactions as

well as his guidance in the experiment, Dr. George Dragomir for his clarification of growth rates, and Noah Houpt for his helpful advice and support throughout the process.

Author Contributions All authors contributed to design of study, research, statistical analysis, methods, materials, and manuscript writing.

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Ecology

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through: Springer <https://www.springer.com/gp/book/97803064425 20> [Accessed 02 October 2018].

LITERATURE REVIEW

Diceros bicornis longipes as a Tool to Test Species De-extinction using Somatic Cell Nuclear Transfer EMILY LALONDE1, HANNAH MAHONEY1

1. Integrated Science Program, Class of 2020, McMaster University

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Molecular Biology

SUMMARY

The need for a solution to species extinction has become more crucial following the official extinction of the West African black rhinoceros in 2011 and the more recent extinction of the North African white rhinoceros on March 18th, 2018. This literature review explores the de-extinction of the West African Black rhinoceros through somatic cell nuclear transfer, commonly known as cloning. This idea was developed by analyzing the literature related to the several methods of de-extinction: back breeding, somatic cell nuclear transfer, and genetic engineering. Furthermore, the role of the West African Black rhinoceros within its ecosystem before and after extinction, as well as its genetic variation as a subspecies of the black rhinoceros, is discussed. Common concerns pertaining to deextinction and cloning are also examined in order to justify potential future initiatives. Received: 11/09/2018

Accepted: 03/12/2019

Published: 11/17/2019

Keywords: West African black rhinoceros, de-extinction, genetic cloning, somatic cell nuclear transfer, genetic engineering, conservation, invasive species, ethics

INTRODUCTION As of March 2014, the International Union for Conservation of Nature (IUCN) had assessed 71,576 terrestrial and fresh-water species; of this, they classified 860 as extinct or extinct in the wild, and 21,286 were deemed threatened with 4,286 described as critically threatened. These extinctions cannot simply be explained based on past extinctions in Earthâ&#x20AC;&#x2122;s history: humans must be negatively affecting a large portion of species. This theory is further supported by comparing pre-human extinction rates established by the IUCN of 0.1 extinctions per million species years (E/MSY) to present extinction rates of approximately 100 E/MSY (Pimm et al., 2014). Based on these findings, the prevention and solution to extinction seem to be of the utmost importance. A recent strategy is the concept of deextinction which involves resurrecting extinct species using back-breeding, somatic cell nuclear transfer (SCNT), and genetic engineering. These techniques utilize current technologies and

resources to represent a new field of scientific research. While de-extinction events have occurred and will be discussed in more depth throughout the review, no organism born from a de-extinction event has lived for more than a few days. This is expected to change, as the field of genetics is one of the fastest advancing scientific fields in the world (Eisenstein, 2015). The question then arises of how using de-extinction as a means of conservation and study will impact the environment of the cloned organisms; moreover, if there will be an impact on extant animals. This review aims to use the literature surrounding the West African Black rhinoceros (WABR), a subspecies of the African black rhinoceros, as a qualitative tool to help hypothesize the effects of de-extinction, as the subspecies was declared extinct in 2011 (Moodley, 2017). By summarizing how the loss and reintroduction of the subspecies may impact its environment and the discussion around species de-extinction, the WABR is an excellent example that could be used to elucidate the de-extinction of other species that have been

extinct for similar periods of time and eventually ones that have been absent for longer.

METHODS

Search terms were used consistently when searching all resources to ensure relevant articles were found. For example, in order to find scientific articles pertaining to SCNT, the search terms included: SCNT, cloning for de-extinction, and somatic cell nuclear transfer extinct species. All searches conducted involved using more than two search terms to ensure that a wide range of articles on the subject was included.

BACK-BREEDING The concept of back-breeding as a method of deextinction was made popular by the Heck brothers. Between the 1920s and the 1930s, the brothers attempted to recreate the auroch (Bos primigenius), a breed of large wild cattle that was domesticated approximately 2,000 years ago and became extinct over 400 years ago (Stockstad, 2015). The brothers attempted to resurrect the auroch by cross-breeding different types of cattle and selecting the desired traits for their offspring. The result of such experiments found the Heck cattle to not be very morphologically similar to the auroch (van Vuure, 2002). Despite this, the experiments introduced the concept of selective breeding to recover distinct ancestral phenotypes within a population. While the new species may be similar to the extinct species phenotypically, the exact gene pool remains extinct with the original species (Price, 2006). Still, back-breeding provides an interesting means of filling previously empty ecological niches and allows for the new species and the ecosystem to re-evolve naturally (van Vuure, 2002).

SOMATIC CELL NUCLEAR TRANSFER Unlike back-breeding, somatic cell nuclear transfer (SCNT) aims to create a replica that possess identical nuclear DNA to its donor. Cloning by SCNT (Figure 1) has been successfully accomplished in 23 mammalian species as of February 2018 (Lui et al., 2018). The first mammalian example of SCNT was Dolly the Sheep in 1996. Sir Ian Wilmut of the Roslin

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It was important to ensure that only relevant articles were used to create this literature review. Therefore, articles chosen had to abide by a list of inclusion criteria; however, the criteria varied between topics. For example, the age of the articles pertaining to species de-extinction did not matter as this section is meant to include all relevant instances of species de-extinction. Whereas, articles pertaining to the population decline and extinction of the WABR were published prior to 2007, as the species was declared extinct on March 18th, 2018. Moreover, information relating to rhinoceros had to be constrained to articles based on work in Africa, and more specifically West Africa. However, all articles had to be peer-reviewed and directly related to the topic.

There are currently three techniques that are used and explored as solutions to species extinction. These techniques include back breeding, somatic cell nuclear transfer (SCNT), and genetic engineering. Their specific methodologies and applications are discussed in the following subsections.

Molecular Biology

Articles used in this systemic review were found using online journal databases including, but not limited to, Web of Science. The Web of Science database was used as the primary database as it includes articles from a variety of disciplines. This is essential for this literature review because a range of different topics are covered. For example, scientific topics such as the methodologies of deextinction (e.g. back-breeding, SCNT, and genetic engineering) as well as ecological concepts surrounding the WABR and its reintroduction are discussed. However, subjects that are more related to social sciences are also reviewed. The topics discussed in Ethics and other Considerations, which delve into the ethics and other considerations of species de-extinction, fall more within this category.

TECHNOLOGIES OF DEEXTINCTION

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Molecular Biology

Institute accomplished this by using a mammary gland of a six-year-old female sheep in her third trimester of pregnancy. These experiments are significant because the results proved that a mammalian adult cell can be completely reprogrammed (Wilmut, 1997). Dolly the sheep was the product of a multistep process involving three female sheep. One provided the genetic material that was inserted into an unfertilized and enucleated oocyte provided by a different female. After undergoing fertilization, the oocyte became an embryo and was implanted into a third female surrogate (Campbell, 1996). Since the birth of Dolly the sheep, technical improvements and increased knowledge of cellular reprogramming have enhanced the efficiency of SCNT. Despite this, the overall ability of SCNT to produce a live offspring is only on the order of 1-3% (Booth et al., 2001).

Figure 1: A nucleus from an adult somatic cell is injected into an enucleated oocyte (egg cell with the nucleus removed). Together they are reprogrammed. During reprogramming, the adult somatic cell becomes an undifferentiated stem cell which upon fertilization develops in the same manner as an embryo (Schorschski, 2007). In terms of de-extinction, SCNT is more attractive than back-breeding because it holds the potential to truly resurrect an extinct species (on the nuclear level) rather than just produce a new species that possess similar traits. However, obtaining intact living cells is not simple with an extinct species as DNA within tissues begin to decay, damage, and fragment shortly after death (Lindahl, 1993).

GENETIC ENGINEERING Genetic engineering utilizes advances in the fields of ancient DNA extraction and genome editing to combat extinction. These advances create living cells that possess extinct genes, which then can be used for SCNT (Yu & Bradley, 2001). Genetic engineering provides a means of de-extinction for animals that have been extinct for a long time, unlike the WABR. For this reason, it will not be discussed in great detail in this review.

THE WEST AFRICAN BLACK RHINOCEROS The West African Black rhinoceros (Diceros bicornis) (WABR) is a subspecies of the African Black Rhinoceros species. They are distinguishable from the white rhinoceros by their hooked upper lip, which allows them to browse and feed on leaves from bushes and trees. Like all rhinos, the WABR is a megaherbivore and plays an important role in the health of its native environment (Cromsigt et al., 2014). Norman Owen-Smith, a highly regarded ecologist whose work pertaining to rhinoceroses will be referred to throughout this review, defined megaherbivores as plant-feeding animals that typically attain an adult body mass in excess of 1,000kg (Owen-Smith, 1987). With both male and female WABRs easily reaching weights up to 1,300kg, the species fits well into this definition. The important role of megaherbivores can be summarized by the Keystone Herbivore Hypothesis, which states that large animals are necessary ecosystem engineers, opening up densely forested regions, and thus food resources, for smaller ungulates (Mills et al., 1993). The term â&#x20AC;&#x2DC;keystone speciesâ&#x20AC;&#x2122; is poorly defined and broadly applied with possible applications to organisms on all trophic levels, but it is generally used to describe any species other organisms largely depend upon. The removal of such species results in drastic changes to the environment. The extinction of a keystone species like the WABR results in a trophic cascade, thus affecting the entire ecosystem. As a keystone herbivore, rhinos maintain the diverse grasslands in which other, smaller organisms depend upon (Cromsigt et al., 2014). Their diet aids in the reduction of woody

plants and bushes, which allows grasses to grow in their place (Kotze and Zacharias, 1993). This benefits other animals such as small mammals and ungulates who depend on grasses for food and shelter, thereby increasing the biodiversity of the area. In 1987, Norman Owen-Smith conducted a study which used extant megaherbivores (rhinos, elephants, giraffes) to extrapolate information on the importance of Pleistocene 39 Megaherbivores. The study suggested the removal of megaherbivores would be a mechanism for the loss of approximately half the mammalian genera within a shared ecosystem (Owen-Smith, 1987).

RHINO SUBSPECIES AND GENETIC VARIATION

By 1988, the population of the WABR was in the hundreds (Kiffner, 2017). While habitat loss and trophy hunting were still partly responsible for this decrease in population, they could not account for the speed in which it occurred. Poaching was the main reason for this unprecedented loss in the WABR population and it continues to be one of the greatest threats to megaherbivores and other animals globally (Tilman et al., 2017). A 2006 field study conducted by Isabelle and Jean-Francois Lagrot attempted to assess the population of what was believed to be the last existing WABR population in Cameroon (Lagrot et al., 2007). Over 46 field patrols were organized in the area situated roughly between Faro National Park on the western border and Bouba Ndjida National Park on the eastern border of the country, totaling over 2,500 km of patrol effort. Using historical data, information from a network of villagers, and cooperation with trophy-hunting guides, the fieldwork illustrated that no reliable sign of rhinoceros presence was found to attest to the survival of the WABR. The study concluded the extinction of the WABR five years before it was officially declared extinct by the IUCN.

WEST AFRICAN BLACK RHINOCEROS POST EXTINCTION

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In a study by M.K Swart and J. Ferguson in 2002, genetic relationships between the subspecies East (D.b. michaeli), West (D.b. longipipes), South-Centre (D.b. minor), and South-West (D.b. bicornis) of African Black rhinoceroses were determined (Moodley, 2017). F-Statistics in the study found significant difference between the populations, with no evidence of interbreeding, although the WABR (D.b. longipes) was determined to have the lowest genetic diversity of all subspecies tested. After years of population crashes due to poaching (refer to Somatic Cell Nuclear Transfer), the dangerously low genetic diversity was to be expected (Wilson and Peter, 1988). The WABR was also found to be the most genetically distinct of the black rhino subspecies (Harley et al., 2005; Moodley, 2017).

Historically, the territory of the WABR extended through the savannah zones of Central-West Africa, an area which includes Tanzania, Zambia, Zimbabwe, and Mozambique (Moodley, 2017) (Figure 2). For much of the 1900â&#x20AC;&#x2122;s, the WABR had the highest population of all the subspecies with approximately 850,000 individuals (Cohn, 1988). During this time, the WABR population began to decline due to the high volume of trophy and big game hunting, but the populations began to rise again after conservative efforts were implemented in the 1930â&#x20AC;&#x2122;s (Kiffner, 2017). However, as the population increased conservation, actions were no longer strictly enforced, and the population once again declined due to poaching (Kiffner, 2017). Between 1970 and 1992, the WABR experienced a population decline of 96%. It was during this period that the rates of poaching started to increase more rapidly (Emslie, 2013).

Molecular Biology

In 1992, Norman Owen-Smith published Megaherbivores: The Influence of Very Large Body Size on Ecology which identified seven subspecies of black rhinoceros (Owen-Smith, 1987). While this is technically true, the intraspecific variation of black rhinos has been discussed by various authors and still remains unsettled. The most accepted scheme consists of seven subspecies but published work by Kes Hillman-Smith and Collin Grooves in 1994 argued the existence of an eighth subspecies due to geographical range overlap and the existence of small isolated populations (Hillman-Smith, 1994). Due to the extinction of three subspecies, D.b. longipes, D.b. brucii, and D.b. bicronis, and critical endangerment of D.b. chobiensis (possibly one surviving individual), confirmation of an eighth subspecies has been near impossible. Even with advances in genetic testing, only four subspecies have been tested for genetic differences and variations (Moodley, 2017).

POPULATION DECLINE AND EXTINCTION OF THE WEST AFRICAN BLACK RHINOCEROS

The removal of megaherbivores from an ecosystem is hypothesized to result in the loss of

Molecular Biology ISCIENTIST | 2019

Figure 2: Historic range of four black rhino subspecies as determined for the study by Yoshan Moodley and his team. The historic habitat of the WABR (D.b.longipes) is shown in pink (Moodley et al., 2017). approximately half of the mammalian genera sharing the ecosystem (Owen-Smith, 1987). While the WABR was important for reducing the population of woody plants in their habitats, and thus increasing the food availability to other smaller herbivores, they shared their habitat with other megaherbivores that played similar roles. The territory of savannah elephants overlaps in some areas with the historical territory of the WABR (Pimm et al., 2014). Savannah elephants are also known for pushing down woody trees to obtain fruits and leaves and to open space to reach wild grasses. In that sense, the loss of the WABR simply left an open niche for another megaherbivore to fill (Pimm et al., 2014). How fast the niche space is filled depends entirely on the

competition between organisms for that position in the ecosystem, and whether another organism can fill it (Flannery, 2015). While the elephant and rhinoceros have been known to occupy similar niches, many elephant species are also at risk of extinction by poaching, and the niche left the WABR has yet to be entirely filled (Flannery, 2015). Although its absence has minimally affected smaller herbivores, the WABR played an integral part of an ecological relationship with large predators and scavengers (Ripple et al., 2015). Megaherbivores not only facilitate the hunting success of large predators by opening up densely forested regions and making smaller prey more

vulnerable, their carcasses also yield more nutrients to a wider suite of scavengers than those of smaller species. Large carnivores tend to consume less of large carcasses, thereby leaving more for other species (Ripple et al., 2015). Megaherbivores are not immune to predation especially since their young are often in the preferred size range of predators. Overall, the WABR influenced the predator-prey dynamics either as an individual or by facilitating the hunting of other mammals. The loss of the WABR depleted the prey availability of large predators such as lions sharing the habitat (Ripple et al., 2015). Overhunting of large herbivores in West Africa has reduced the prey availability, which, at least in part, has caused regional lion populations to become critically endangered (Ripple et al., 2015).

To successfully clone a WABR through SCNT, an available resource of genetic material and a sufficient surrogate mother and oocyte donor need to exist. Preferably, genetic material would come from sampling a WABR when the creature was still alive, and the material properly stored to avoid degradation (Harley et al., 2005). Fortunately, measures have been taken to preserve the genetic material taken from a live WABR; cell cultures collected from ear clippings of the rhino are stored in nitrogen gas at the University of Cape Town (Harley et al., 2005). While this genetic source likely offers the best quality DNA, other sources may also be utilized, such as stored WABR horns.

Using cell cultures from ear clippings is the most feasible method of resurrecting the WABR because they possess DNA. Another approach would be to perform SCNT using the keratin within the horns of the WABR. Extracting DNA from keratinous materials requires the use of buffers (DTT, proteinase K, and detergent) and then the purification using methods involving organic materials or silica-column (Campos & Gilbert, 2012). This sort of technique would apply to prehistorically extinct species, such as the woolly mammoth.

INVASION

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Apart from genetic material, SCNT requires a surrogate mother. An adequate surrogate mother for this process depends on the genetic relationship between the two species; the species of the mother and the species of the clone. Having a genetically similar species as a surrogate mother and/or oocyte donor is stated as ideal across literature defining SCNT (Folch et al., 2009). For extant species, this means surrogate mothers are from the same species, usually chosen from a pool of healthy female individuals. When attempting to accomplish de-extinction, there is no perfect surrogate mother. In the case of the bucardo the surrogate mother was a domesticated goat, a distant relative of the bucardo. Differences in uterine environments, prenatal development, and

For the WABR, the ideal surrogate mother would be of the black rhino subspecies, whichever was the most closely genetically related. As of today, the WABR has been genetically compared to three other subspecies and determined to be the most genetically distinct subspecies (Moodley, 2017). This means there is no strong genetic relationship between the WABR and the East, South-West, and South-Centre African Black rhinoceroses. Even though it may be difficult to determine the subspecies of black rhino which is the closest relative to the WABR, using another subspecies would still be the best option as an oocyte donor and surrogate mother. The conservation status of the black rhinoceros species as a whole is considered critically endangered, except the South-Western subspecies (Diceros bicornis occidentalis) which is considered vulnerable (Grooves and Grubb, 2011). It would be redundant to use an individual from a critically endangered subspecies as a surrogate mother for a WABR de-extinction event because the process would take away valuable time in which the mother could produce offspring of her own subspecies. Based on the lack of known genetic relationships linking the black rhinoceros subspecies, it is reasonable to suggest a surrogate mother and oocyte donor be taken from the South-Western subspecies, as they are not as severely endangered.

Molecular Biology

POTENTIAL DE-EXTINCTION OF THE WEST AFRICAN BLACK RHINOCEROS

imperfect genetic material made successful pregnancies rare, with only two of 57 trials resulting in a live birth (Folch et al., 2009). Although the reborn bucardo went extinct again within hours, the birth suggests that streamlining techniques could make success rates significantly higher.

Invasion ecology interfaces with a variety of distinct sub-disciplines including ecology

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Molecular Biology

economics, environmental law, and epidemiology (Lockwood et al., 2013). This review has chosen to focus only on the ecological impacts pertaining to the biological invasion of non-native species. While all sub-disciplines of invasion ecology are important, this review aims to simplify the definition of invasion and use this to determine whether the de-extinction of an organism should be considered an invasion event.

Creating a general yet precise definition of invasive species has been a topic of discussion and controversy amongst conservationists and largely depends upon the viewpoint of the observer (Moutou & Pastoret, 2010). For the purpose of this review, the definition of invasiveness will follow the stage-based approach, a common methodology used to elucidate the parameters surrounding invasion of a species (Arim et al., 2006). Specifically, this review will define an invasive species as any species introduced to a non-native environment in which it is able to overcome environmental resistance and proliferate to a point in which it reduces the fitness of native species. As well, this review will define a non-native environment as an environment that may have once been a native location to a species, but post-extinction, the environment has changed significantly in the sense the species is no longer required to play an ecological role. Environmental resistance, as mentioned by Charles S. Elton in his work The Ecology of Invasion by Animals and Plants, is the number of factors within an environment that restrict the biotic potential of an organism to proliferate (Elton, 2000). One of the main tenets of invasion biology is that most species introduced to a new environment do not survive. This is largely due to environmental resistance. Many species are highly adapted to the specific environment from which they evolved, and introduction to a new area in which they have not specialized or adapted to means they are unlikely to become an invader (Elton, 2000). If an organism is brought back by a de-extinction process, the organism must be able to survive, whether as an invader or not, in the environment in which it is placed. For an organism that went extinct recently like the WABR, the organism should have no unforeseen ill effects after being reintroduced to their native habitat. The habitat has likely not changed in a way that would make it difficult to reintroduce. Like the reintroduction of wolves into Yellowstone National Park the reintroduction of a keystone species like the

WABR will likely result in an increase to the health and biodiversity of the ecosystem. But what about a case in which a long-extinct species is reintroduced into an environment that has drastically changed since their extinction? As will be discussed later, there has been a growing interest in the cloning of a woolly mammoth since the discovery of well-preserved specimen in Siberia (Palkopoulo, 2015). The woolly mammoth has been extinct for 10,000 thousand years and would be reintroduced into a completely different ecosystem compared to the one it left (NoguĂŠsBravo, 2008). Some extinct species may not have been invasive during their lifetime; however, they may severely disturb the dynamics of the present day. Whether a de-extinction event is considered invasive remains unclear, as there has been limited research in this area. In the case of the WABR, not enough time has passed for its native ecosystem to change in a way that makes it ecologically redundant. Such as in the case of the Yellowstone wolves which will be discussed, the environment has not adapted to the absence of the WABR and reintroduction would likely increase the ecological health of the environment (refer to Genetic Engineering). Species redundancy, assuming natural extinctions, depends on the role a species played in an ecosystem (Gitay, 1996). If a species was one of many that played similar ecological roles, its extinction may not have a large impact on the surrounding environment, but since many modern extinctions involving species like the WABR do not occur naturally, it is reasonable to assume all recently extinct species are not ecologically redundant, and that reintroduction would not be invasive. Recently extinct, as defined by the IUCN, refers to any species that has gone extinct since 1500 CE but for the purpose of this review recently extinct will refer to any species that has gone extinct since 1760 (Feinstein, 1998). It was this time that the Industrial Revolution began, and human impact on animal ecosystems increased exponentially (Feinstein, 1998).

ETHICS AND OTHER CONSIDERATIONS With any emerging innovation in science, there must be consideration for the potential ethical, ecological, societal, and economical issues that might arise from such a project. Within this literature review, both cloning and de-extinction can be subject to a lot of scrutiny for a multitude

of reasons based on their fairly recent emergence. The following section aims to explore the considerations that must be made prior to implementing a reintroduction or de-extinction project. As well, common fears and concerns surrounding cloning and de-extinction will be discussed.

CONSIDERATIONS REGARDING SPECIES REINTRODUCTION AND DE-EXTINCTION

In terms of the reintroduction or de-extinction of the WABR, these examples provide insight into some of the considerations that must be investigated prior to any project of this sort. The research that was conducted before, during, and

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As a result of the loss of the wolves, Yellowstone National Park witnessed an overall decline in its biodiversity. Due to the loss of the apex predator within this ecosystem, the local elk (Cervus elaphus) population proliferated, causing a trophic cascade and reducing vegetation and thus habitats and food resources for smaller organisms (Beschta & Ripple, 2010). After the reintroduction of the gray wolf, elk populations decreased again, allowing new plant growth. Changes such as increased

The reintroduction of the gray wolf into Yellowstone National Park was a 65-year process that required a lot of deliberation. On a similar topic, a roundtable session in 2004 addressed some of the requirements for reintroduction of species that have gone locally extinct. Specifically, it considered the reintroduction of noble crayfish (Astacus astacus), back to their native habitat (Taugbøl, 2004). Noble crayfish were native to the Glomma and Halden waterways in Norway. In 1987 and 1989 the waterways were struck by crayfish plague (Aphanomyces astaci) and the populations underwent complete fatality. (Taugbøl, 2004). During the roundtable session, several scenarios were provided for the appropriate reintroduction of crayfish: (1) to restore a population that was recently lost, (2) to help return a native species to its historic range, and (3) to preserve genetic diversity of that species by creating new or confined populations. After considering these guidelines for reintroduction, it was also imperative to assess whether the reason for local extinction remains present- in this case crayfish plague. Reintroduction would not be justified nor successful if the threat still existed (Taugbøl, 2004). Reintroduction projects began in Norway in 1989 and 1995, and in 2001 the waterways were surveyed to track the progression of the project. The researchers found that density of the crayfish populations were much lower than densities observed pre-plague. However, they related this slow population growth with similar observations from other crayfish being introduced into a new environment in Norway (Taugbøl, 2004).

Molecular Biology

Historically, the motivation for species reintroduction has been to restore a species in an ecosystem and not to restore the ecosystem itself. However, reestablishing a specific species in a region where it had gone locally extinct will affect the dynamics of an ecosystem, especially if said species is a keystone species as in the case of the gray wolf (Canis lupus) to Yellowstone National Park. The eradication of the gray wolf began during the colonization of North America by Europeans because they were considered to be a danger to livestock (Ripple et al., 2013). By 1930, populations were completely eliminated from Montana, Idaho, and Wyoming (Bangs & Fritts, 1996). A recovery pact was set in 1974, in hopes of eventually removing the gray wolf from the Endangered Species Act in the Northern Rocky Mountains of the United States. However, wolf reintroduction was viewed as controversial because it was thought it would most likely significantly affect the human environment. Specifically, it was theorized to greatly disrupt agricultural and local practices. Therefore, a revision to the recovery pact was made in 1987 (finalised in 1994) entitled the Environmental Impact Statement 1987 (Bangs & Fritts, 1996). Throughout the winters of 1995 and 1996, wolves from British Columbia, Canada were introduced into the three states. Of particular interest are the wolves that were introduced into Yellowstone National Park and their impact on the environment.

height growth in willow, aspen, cottonwood, and other types of vegetation were observed (Beschta & Ripple, 2007). The recent rise in riparian vegetation helped to decrease the erosion of river channels, thus changing the flow of rivers and replenishing sources of wood, leaves, dissolved organic carbon, and nutrients that are required by aquatic ecosystems (Beschta & Ripple, 2010). Beavers, also previously locally extinct in the park, returned, and this encouraged the return of otters, muskrats, and reptiles (Beschta & Ripple, 2010). The return of the wolves to Yellowstone National Park is considered one of the most successful acts of conservative ecology in the 20th century (Beschta & Ripple, 2010).

Molecular Biology

after the reintroduction of the gray wolf into Yellowstone National Park is a true success story for initiatives of this nature. It acts as an additional motivator for the reintroduction of the WABR, and as the WABR is also a keystone species, its successful reintroduction -like the wolves of Yellowstone- could benefit its ecosystem in a similar manner (refer to Genetic Engineering). Moreover, the roundtable session provides parameters that could be applied in determining whether the reintroduction of the WABR to its natural habitat is justified. In this case of deextinction, it is important to assess whether the reason they no longer persist in their past environment remains a factor, just as the roundtable session considered the presence of crayfish plague in the rivers prior to reintroduction. If the illegal and legal practices of rhinoceros hunting persist, then the risk of history repeating itself would be inevitable. Additionally, it is important that the de-extinction of the WABR is not for exploitative purposes and in the best interest of the species and its ecosystem. This concept will be discussed further below. Despite current laws and restrictions regarding illegal rhinoceros hunting, poaching remains at a level of crisis in many countries. In 2017, the South African government reported that 1,028 rhinoceroses were illegally killed. While this figure is less than 2016 levels it is still very high compared to levels recorded in 2007 (Environmental Affairs, 2018). These statistics imply that history would in fact repeat itself, thus some solution would need to be reached before such a de-extinction project. It is important to note that these examples suggest that species reintroduction always benefits the ecosystem; however, this is not always the case.

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ETHICS OF CLONING AND DEEXTINCTION

The concept of cloning and de-extinction is a highly debated topic. Scientists wish to advance the field of cloning extinct animals for the sake of their research. Whereas, bioethicists, policy makers, and the media, strongly protest the idea (Fiester, 2005). Arguments against de-extinction are concerned with animal welfare, human health, environmental (refer to Genetic Engineering), political, and moral issues (Sherkow, 2013). With such concerns, the question arises as to whether opposition is based off of the fear of the unknown. The following subsection aims to provide enough context to form an opinion on the matter.

A major concern with de-extinction is animal welfare. The animals that are used as surrogates in SCNT may suffer due to complications during their pregnancies as well as during surgeries to implant embryos and to remove failed attempts (Carter, 2002). The offspring may die as high levels of genetic abnormalities and chronic diseases are common. Due to the risks of SCNT, The Animal Welfare Act (enacted in 1966) restricts such treatment (Animal Welfare Act, 1966). Moreover, it is imperative that there is a distinct reason for animal de-extinction and that it is not to serve as a public attraction, as is the case of zoos and other institutions that exploit animals (Sherkow, 2013). The cloning of animals can also cause problems that hold the potential to eventually affect humans more directly. Of the utmost concern is the prospective cloning of humans or commercial cloning that may arise upon perfection of cloning techniques (Fiester, 2005). This idea sparks even more controversy than the concept of deextinction. Furthermore, if cloning becomes common practice in the food industry, the livestock may be unsafe to consume due to unexpected results from genetic modification such as increase in allergens (Fiester, 2005). These issues do not particularly relate to the topics discussed in this paper, however, they help to provide context to the overall fears that surround cloning. The moral issues regarding cloning and deextinction are the most relevant to the project proposed in this paper. However, these concerns are the most ambiguous. They demand an answer that is on the same order of the divine power. It is somewhat inconceivable to imagine reversing the â&#x20AC;&#x2DC;naturalâ&#x20AC;&#x2122; course of world. Some questions include whether things happen for an ultimate reason, or what determines if such a cause is almighty enough. In terms of assessing the moral validity of such an initiative, there is no singular answer that encompasses every instance of cloning or deextinction. It depends on the situation and a costbenefit analysis should be conducted for each project (Fiester, 2005). With regards to animal and human welfare in the context of the cloning and de-extinction of the WABR, the benefits seem to outweigh the costs. This initiative would not support the exploitation of animals, their population and the entire ecosystem holds the potential of prospering, and humans are not directly affected.

CONCLUSION & FUTURE DIRECTIONS Understanding the current literature pertaining to de-extinction is pivotal to making informed decisions on what will likely be a part of future conservation ecology and bioethics. While still an emerging and controversial topic, the use of gene technology in species conservation and de-extinction is likely to be an unavoidable result of advancements in the field of genetics and conservation ecology. While no longer sciencefiction, the de-extinction of animals may take years to perfect, but many groups are working together to make de-extinction a reality for a variety of species.

As the discussion on de-extinction turns away from ‘if’ and towards ‘when’, the argument of which organisms to bring back becomes more prevalent. While bringing back extinct mammals

ACKNOWLEDGMENTS The authors would like to thank Dr. Chad Harvey for both his guidance in planning this research and his valuable supervision throughout the research period.

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Reviving the woolly mammoth has also been a future target in the field of biotechnology and genetics. The goal has spanned multiple research teams, including a team from McMaster University. In 2015, a team of international researchers at McMaster sequenced the genome of two Siberian woolly mammoths (Palkopoulo et al., 2015). With multiple sources of woolly mammoth DNA available from a number of well preserved, frozen specimens found in glaciers and Arctic permafrost, researchers from Harvard suggest that a viable mammoth clone could be produced in as soon as two years (Shapiro & Seddon, 2016).

It is also suggested that more tangible and experimental research be conducted pertaining to the re-introduction of previously extinct species. Researchers would do well to accumulate data pertaining to the impacts and reintroduction of recently extinct organisms before the reintroduction of long extinct organisms such as the woolly mammoth or other species. There is limited experimental data that can be used to quantify the impacts of de-extinction on ecology and justify it at the same time. This is why the WABR would likely be an excellent choice as a deextinction candidate. As a recently extinct mammal that would not be considered invasive after reintroduction, the WABR would help elucidate existing questions and concerns about de-extinction.

Molecular Biology

A group of Australian Scientists from the University of New South Wales in Sydney, Australia, have been focused on completing a successful de-extinction event since 2013 (Stone, 2013). The researchers have been attempting to revive the Australian gastric brooding frog (Rheobatrachus silus) using donor eggs from a distant relative and DNA from a specimen preserved since 1970. Using SCNT, the researchers have been successful in reviving the genome of the extinct frog by allowing the cells from SCNT to divide and form embryos. Although the embryos of the frog did not survive for more than a few days, they were all confirmed to contain the genetic material from the sample specimen (Stone, 2013). The team suggest fixing technical and methodological issues would make success more likely.

such as the woolly mammoth would be a huge advancement, future aspirations should not forget about recently extinct organisms and preserving the genetic diversity of a species not only as live individuals, but also as cultured genetic samples. The future of de-extinction has a multitude of implications for conservation and protection of critically endangered species and could be used to avoid cases 44 such as those of the North African White rhinoceros (Ceratotherium simum cottoni). Considered endangered since 1909, the North African White rhino subspecies was declared functionally extinct as of March 19th, 2018 after the death of the last male specimen (Gross 2018). Although two female North African White rhinos still exist, they were genetically related to the male and any further breeding attempts would result in inbred offspring. A genetic library of the species DNA could have aided in the production of genetically viable offspring, but with less than 500 individuals existing in 1975, the species had already bottlenecked to a point of genetic decline (Gross, 2018). While de-extinction should not be treated as a priority over conserving extant species and keeping them from becoming extinct, it will offer the possibility of introducing genetically healthy populations of once lost species back into the environment.

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LITERATURE REVIEW

Triple Negative Breast Cancer and Synthetic Lethality RACHEL ELLIS AUBREY1, MARYANNE OKETCH1

1. Integrated Science Program, Class of 2020, McMaster University

SUMMARY

Received: 09/11/2018

Accepted: 03/20/2019

Published: 11/17/2019

Keywords: synthetic lethality, triple negative breast cancer, PARP inhibitors, MYC pathway, RNA interference, CRISPR-Cas9, chemical screens

INTRODUCTION In 2017, there were an estimated 206,200 new cases of cancer in Canada, and this number continues to rise (Canadian Cancer Society, 2017). With the increase in individuals requiring cancer treatment, the development of more effective and lower risk methods is of great necessity. Currently, cancer treatments impact not only the rapidly dividing cells within the body, but are also proven to be toxic to normal cells, and are therefore characterized as having a low therapeutic index (Kaelin, 2005). Thus, research into novel cancer therapies is of great importance.

BACKGROUND TRIPLE NEGATIVE BREAST CANCER Breast cancer is the most common type of cancer in women. Current treatment methods are usually radiation therapy and chemotherapy, however new treatments are beginning to surface (Li, Uribe and Daling, 2005). Breast cancer is characterized by different combinations of the expression or underexpression of estrogen receptors (ER), progesterone receptors (PR) and human epidermal growth factor receptors 2 (HER2) (Li, Uribe and Daling, 2005). One important type of breast cancer is triple negative breast cancer (TNBC) and its nomenclature is due to the lack of expression of ER, PR and HER2 within the tumors (Chavez, Garimella and Lipkowitz, 2010). TNBC is a

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In general, the development of carcinogenic tumors is attributed to biological characteristics, referred to as the six hallmarks of cancer. More specifically, this refers to the cellâ&#x20AC;&#x2122;s resistance to death, constant proliferation, ability to induce angiogenesis, capacity to metastasize to other locations within the body, the evasion of growth suppressors, and the presence of replicative immortality (Hanahan and Weinberg, 2011). Each of these six hallmarks are initiated by mutations in genomic DNA and allow for the

characterization and classification of carcinogenic tumor cells.

Molecular & Cellular Biology

Triple negative breast cancer (TNBC) is characterized by the lack of expression of estrogen receptors (ER), progesterone receptors (PR), and the human epidermal growth factor receptor 2 (HER2). Despite the recent and extensive search for new therapeutics, few have been proven successful. This review article investigates synthetically lethal interactions within TNBC involving poly(ADP-ribose) polymerase (PARP) inhibitors and the overexpression of the MYC pathway. It also explores the screening for synthetic lethality by utilizing RNA interference, Cas9 libraries, and chemical screens. Limitations of synthetic lethality to treat cancer was also considered. By performing a holistic review of the current known synthetic lethal interactions, conclusions can be made about the implications of this research and the potential for future use in the treatment of cancer patients.

Molecular & Cellular Biology ISCIENTIST | 2019

heterogeneous disease and is known for its distinct metastatic patterns, and lack of targeted therapies (Aysola et al., 2013). Approximately 170,000 of the one million annual worldwide diagnoses of breast cancer are TNBC (Wahba and El-Hadaad, 2015). Using cDNA expression analysis, the majority of TNBC cell lines have been classified as basal or luminal based, with approximately 75% being basal-like (Chavez, Garimella and Lipkowitz, 2010; Wahba and El-Hadaad, 2015). Both Basal and luminal are two of the five main subtypes of TNBC, including luminal A, luminal B, HER2/Neu overexpressing, basal-like (BL1,BL2) and normal-like, each being differentiated by their relative level of expression of hormone receptor genes (Aysola et al., 2013, Xu, Chen and Olopade, 2010). Each subtype has different characteristics and molecular pathways, which is reflected in the manner in which they are treated. Luminal and basal are often classified as being the more aggressive subtypes, making treatment methods more challenging to formulate. Since patients with TNBC do not contain the target receptors ER, PGR, or HER2, the method of hormone or trastuzumab based therapy is not beneficial (Wahba and El-Hadaad, 2015). This leads to surgery and/or chemotherapy as the only available methods of treatment against the cancerous tumors. Due to the aggressive nature of TNBC, rapid resistance development, and low therapeutic index of chemotherapy, both surgery and chemotherapy are seen to be ineffective treatment methods. Thus, the development of a more effective treatment for TNBC is essential (Chavez, Garimella and Lipkowitz, 2010). Angiogenesis inhibitors, epidermal growth factor receptor targets, Src kinase, and mTOR inhibitors have all been unable to provide feasible data (Nowsheen et al., 2012). However, the concept of synthetic lethality involving PARP inhibitors or the overexpression of the MYC pathway has been proven to be promising methods of therapy with less adverse side effects (Aysola et al., 2013).

OVERVIEW OF SYNTHETIC LETHALITY Synthetic lethality is when the co-occurrence of two genetic interactions results in cellular or organismal death (Nijman, 2011). In terms of cancer, an interaction is considered synthetically lethal when a mutation occurs within a cancer specific protein and the tumor cell requires the activity of the synthetic lethal partner for gene

viability (O’Neil, Bailey and Hieter, 2017). Within the field of synthetic lethality, there are three main types that can create a feasible interaction. The types include a double mutation resulting in loss of cell viability, the mutation of one gene coupled with the induced inhibition of another, and finally synthetic dosage lethality, where the overexpression of a gene and induced inhibition of another causes cell death (Figure 1) (O’Neil, Bailey and Hieter, 2017).

Figure 1: A simplified diagram showing various scenarios of synthetic lethal interactions. Mutation of either gene A (blue) or B (orange) is viable, or the overexpression of both (Case A). In comparison, a mutation in (Case B), the inhibition (Case C) or the inhibition and overexpression (Case D) of the genes causes synthetic lethality (O’Neil, Bailey and Hieter, 2017). Continual activation of the downstream mutant can result in drugs of the target being rendered ineffective. Similarly, if the mutant is upstream of the target, resistance to drugs can be attained by a mutant bypassing a protein in the signalling pathway, such as ERBB2 activating AKT3 rather than PI3K (Miller, Goulet and Johnson, 2016).

METHODS Preliminary information was obtained by searching Google Scholar, and peer reviewed papers were used to provide the information given in this paper. While searching for articles, the terms used varied depending on the section. Often both the terms “synthetic lethality” and “triple negative breast cancer” were used throughout in order to direct the research to the specific method of treatment and breast cancer. Otherwise the terms: “PARP inhibitors”, “MYC pathway”, “RNA interference”, “CRISPR-Cas9”, and “chemical screens” were used for the various sections. The pathways that are discussed in the paper were chosen based on the amount of research being conducted on the particular pathway. A pathway was considered as a lead if the pathway was used by cancerous cells, and if the pathway selected had a role in the nucleic function of a cell.

In terms of TNBC, two known synthetically lethal interactions include PARP inhibitors and the MYC pathway (Lord and Ashworth, 2017; Yang et al., 2010). PARP inhibitors are an example of the mutation of a gene coupled with the inhibition of another, while the MYC pathway is an example of synthetic dosage lethality.

Of great interest to the field of synthetic lethality is the poly(ADP-ribose) polymerase (PARP) inhibitors, as they were the first clinically approved drug type to exploit the use of synthetic lethality (de Lartigue, 2013, Lord and Ashworth, 2017). The DNA damage response (DDR) is the process with which cells defend themselves against DNA damage (Lord and Ashworth, 2017). PARP is responsible for the detection of the DDR, and more specifically, the associated single-strand DNA breaks (Lord and Ashworth, 2017). Single stranded breaks (SSBs) cause conformational changes to the damaged DNA and create a binding site for PARP (Lord and Ashworth, 2017). Once bound, a conformational change occurs, causing the activation of PARP’s catalytic function (Lord and Ashworth, 2017). This catalytic activity causes the PARylation of PARP proteins, initiating DNA repair by attracting DNA repair effectors and altering the chromatin structure (Lord and Ashworth, 2017). After the repair is complete, autoPARPylation occurs, and PARP is released (Lord and Ashworth, 2017). Within cancerous tumors, PARP inhibitors (PARPi) stimulate nicotinamide adenine dinucleotide (NAD), the enzyme cofactor, and inhibit PARylation (de Lartigue, 2013). When inhibition occurs, PARP is no longer able to be released through autoPARylation, causing the buildup of PARP in a process known as PARP trapping (de Lartigue, 2013). This prevents DNA replication initiation, and homologous recombination repair (HRR) is required to restore regular nucleic function to the affected cell (Figure 2) (Lord and Ashworth, 2017)Within the PARP pathway, the synthetic lethal interaction is a result of mutations within the BRCA1 and BRCA2 genes (Lord and Ashworth, 2017). Mutations in BRCA1/2 result in an increased chance for the development of breast and ovarian cancer (Mehrgou and Akouchekian, 2016). These genes are required for HRR and without the presence of HRR, alternative methods of DNA repair thrive, although these methods can be the cause of numerous DNA mutations (Lord and Ashworth, 2017). In combination with the mutations in either BRCA1 or BRCA2 and induced PARP inhibition, the synthetically interaction can be used to prevent the repair of DNA breaks and induce tumour cell death (Lord and Ashworth, 2017). Thus, PARP

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PATHWAYS ILLUSTRATING SYNTHETIC LETHALITY POTENTIAL

PARP INHIBITORS

Molecular & Cellular Biology

The field of synthetic lethality has been proven to have potential as it allows for the targeting of nondruggable cancer mutations after the identification of the second mutation site or inhibitor synthetic lethal target. Currently the concept of synthetic lethality has been proven to be very effective within budding and fission yeast, engineered cells, and transgenic mouse models (Kaelin Jr, 2005; Ooi et al., 2006; Yang et al., 2010). Since a vast amount of information is known about these organisms and the manner with which a synthetically lethal interaction can be screened for, synthetic lethal interactions have been easy to identify (O’Neil, Bailey and Hieter, 2017). In terms of human cell cultures, however recent advances are being made in the field of RNAi and CRISPR technology to increase the ease of finding synthetic lethal interactions.

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Molecular & Cellular Biology

inhibition is an example of a mutation (BRCA1/2) and inhibition, causing synthetic lethality.

Figure 2: The PARP cycle. PARPi disrupts the cycle in its fourth stage, causing the repair process to halt (Lord and Ashworth, 2017).

(Horiuchi et al., 2012). MAX can either bind to an activator such as Myc, or a repressor such as MXD1-4. MXD1-4 competes with myc for MAX binding. Within TNBC, myc is overexpressed and the regulation of myc expressions is altered (Fallah et al., 2017, Horiuchi et al., 2012). More specifically, the Myc activators are up-regulated while the MXD1-4 is significantly down-regulated. This reduces the competition, leading to the overexpression of the myc pathway (Horiuchi et al., 2012). In most cancers, myc expression is deregulated, although in breast cancer it is overexpressed in 30-50% of high-grade tumors (Fallah et al., 2017). In order to treat cancer progression, inhibition of this pathway at a specific target is a potentially viable therapeutic route. Within TNBC, a direct target of Myc remains unknown. Currently, research is being conducted on potential targets that can be paired with the overexpression of MYC to produce a synthetically lethal interaction (Fallah et al., 2017).

THE MYC PATHWAY In the pathogenesis of numerous human cancers, the myc proto-oncogene is important within multiple signal transduction pathways (Dang, 2012). Myc is a regulatory gene coding for transcription factors required for many cellular processes, such as ribosome production, metabolism, and the cell cycle - all of which govern the growth rate of the cell (Hsieh and Dang, 2016). Alterations to the regulation of myc can cause the pathways to bypass normal control checkpoint mechanisms (apoptosis and senescence), ultimately contributing to the uncontrollable cancerous cell growth commonly observed in tumors (Gabay Li and Felsher, 2014). Within cancerous cells, the myc pathway becomes a means for controlling the induction of stemness and blocking cellular senescence and differentiation (Gabay Li and Felsher, 2014). It is also responsible for controlling the microenvironment of tumors, the activation of angiogenesis, and suppression of the host immune response (Gabay Li and Felsher, 2014). On a molecular level, the myc transcription factor contains a basic helix-loop-helix zipper (bHLHZ) motif and is regulated by the binding of another bHLHZ protein, termed MAX (Figure 3)

Figure 3: The MYC proto-oncogene is found within many receptor signal transduction pathways, a few of which are depicted in the figure. The MYC gene codes for the transcription factor Myc, and after binding with MAX and the target DNA sequences regulation of transcriptional genes important to cell growth and proliferation occurs (Dang, 2012). In the exploration of synthetic lethality interaction, a few possible targets have been identified through the use of varied screens. Synthetic lethality between the overexpression of the MYC pathway and the inhibition of cyclin-dependent kinases (CDK) has been illustrated in engineered cells and mouse models (Goga et al., 2007). CDKs are

In cells overexpressing Myc as a result of the inhibition of Cdk1 using synthetic lethality, an increase in a proapoptotic protein, Bim of the Bcl2 family, has been observed (Bcl-2 interacting mediator of cell death) (Horiuchi et al., 2012). BCL-2 family members control an initial step within apoptosis, the permeability of the outer mitochondrial membrane (Shamas-Din et al., 2013). Horiuchi et al. (2012) observed that Bim was upregulated after MYC overexpressing cells were treated with purvalanol A. Thus, with an upregulation of Bim in cancerous cells, an increase in apoptosis occurs (Horiuchi et al., 2012).

SYNTHETIC LETHAL INTERACTIONS-SCREENS

RNA INTERFERENCE (SIRNA) RNA interference (RNAi) is a biological response to double-stranded RNA (dsRNA) that allows a cell to silence a gene by inhibiting RNA translation. This is done by using short interfering double stranded RNA (siRNA) (National Center for Biotechnology Information, 2017). One strand

To screen for synthetic interactions between genes, the precursor of siRNA - specifically pooled shRNA libraries - are typically used (Diehl, Tedesco and Chenchik, 2014). This allows multiple shRNAs to be tested at the same time (Kampmann et al., 2015). To perform a dropout viability shRNA screen, a library of shRNA is introduced to a population of live cells by a single large-scale transduction, and the shRNA is integrated into the genomic DNA of these cells (Diehl, Tedesco and Chenchik, 2014). The transduced cells are then left to incubate for at least six doublings, which allows the shRNA to be expressed inside the cells, and thereby cause the cells to display the associated phenotype of the gene in question in the presence of potentially protein-inhibiting shRNA (Kampmann et al., 2015). After the growth period is complete, highthroughput sequencing is used to determine the relative shRNA levels in the cell population (Diehl, Tedesco and Chenchik, 2014). If there are shRNAs that are underrepresented in the cell population, it is because those shRNAs killed or inhibited the growth of the cells. These shRNAs can then be considered hits for potentially viable drugs, and their mechanism of action can be elucidated.

CRISPR-CAS9 Clustered regularly interspaced short palindromic repeats - CRISPR associated protein 9 (CRISPRCas9) is a genome-editing system that is used to introduce DNA double-strand breaks (DSBs) at a genomic area of interest (Sรกnchez-Rivera and Jacks, 2015). The CRISPR-Cas9 system is a targetspecific single-guide RNA (sgRNA) used in conjunction with the Cas9 endonuclease (Dhanjal, Radhakrishnan and Sundar, 2017). The sgRNA, bound to the Cas9 enzyme, binds to the specific sequence complementary to the sgRNA in the host genome (Redman et al., 2016). After the specific sequence is found, Cas9 cleaves the DNA at that location, introducing a DSB (Sรกnchez-

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Once a pathway that cancerous cells are dependent on is targeted, the next step in treatment development is to discover a mode to disrupt its function through synthetic lethality, while leaving healthy cells relatively unharmed. This can be done by performing screens for interactions that cause the cancerous cells to die. This process is known as screening (Barbour and Xiao, 2006). For TNBC, there are three main types of screens that are of interest for human cells: RNA interference, CRISPR-Cas9, and chemical (Nijman, 2011).

of siRNA then binds to a protein-coding mRNA complex that has a complementary nucleotide sequence to the siRNA. This complex binds to mRNA, which blocks translation and thus silences the gene (Carthew and Sontheimer, 2009; National Center for Biotechnology Information, 2017). RNAi has been used to induce synthetic lethality in multiple cancers (Dai, Fang and Roth, 2009). More specifically, it has been used to target EGFR in lung cancer, and HER2 in breast cancer.

Molecular & Cellular Biology

essential in the progression to the next stage of each step in the cell cycle; numerous varieties exist for the different stages of the cycle. For instance, Cdk1 is a subfamily responsible for centrosome maturation and separation, chromosome condensation, and entry into the mitotic phase after the breakdown of the nuclear envelope (Malumbres, 2014). Extending this idea to TNBC, it has since been observed that MYC overexpression and the inhibition of CDK successfully halts the growth of TNBC within xenografts (cells from patient tumour transported into immunodeficient mice). More specifically, through the use of a purvalanol A and dinaciclib CDK inhibitors a synthetic lethal interaction has been found in xenografts (Horiuchi et al., 2012).

Molecular & Cellular Biology ISCIENTIST | 2019

Rivera and Jacks, 2015). This DSB is usually repaired by non-homologous end joining (NHEJ) (Sánchez-Rivera and Jacks, 2015). NHEJ repairs the DSB without using template DNA as a reference for the correct genome sequence (Sánchez-Rivera and Jacks, 2015). This causes the repaired DNA to have a higher chance of random insertions and deletions – called indels (Ma et al., 2017). These cause a frameshift that disrupts gene function (Dhanjal, Radhakrishnan and Sundar, 2017). If this gene was integral to the cell’s survival, the cell will die (Sánchez-Rivera and Jacks, 2015). This is how a lethal interaction is induced with Cas9. When the interaction is found, the gene can then be used as a target for developing inhibitory drugs to recreate the interactions.

CHEMICAL SCREENS Another way to test for synthetic lethality is to utilize chemical screens. In this method, the goal is to find chemical leads that can be further developed into anticancer drugs that induce a synthetically lethal interaction (Dahlin and Walters, 2014). Chemical screens are the preferred screening method by some researchers since they can lead to the development of a drug faster than other screens, since a high amount of chemicals can be screened in a short amount of time compared to other types of screens (Barbour and Xiao, 2006). There are two methods that can be used for chemical screens: high throughput screens (HTS) and fragment-based screens (Dahlin and Walters, 2014). In HTS, the goal is to screen as many molecules as possible in an attempt to identify active compounds that have potential therapeutic activity (Bressan, 2014). HTS are executed in an assay format. Depending on the setup, there are many detection methods to validate the efficacy of the molecules (Wildey et al., 2017). After a screen, there can be multiple molecules identified that may interact with a desirable target (Bleicher et al., 2003). These are narrowed down to a smaller number of molecules that will continue in the drug discovery process (Gupta et al., 2009). The selected molecules are optimized for qualities that function best and a select few will then go on to clinical trials for further drug development (Bleicher et al., 2003). The utilization of fragment-based screens has increased in popularity since its conception in

1996 (Rees et al., 2004). Fragments are defined as organic molecules which have a low molecular weight, are moderately lipophilic, and are highly soluble (Kumar, Voet and Zhang, 2012). The concept of fragment-based screening is simple – small fragments are put through a screen, and molecules that interact with the target are noted (Kumar, Voet and Zhang, 2012). The small molecules are then combined to create larger ones that can greatly inhibit their target (Rees et al., 2004). The size restriction of the fragments decreases the number of molecules that are screened compared to HTS, with a maximum of a few thousand molecules in one screen (Rees et al., 2004). The interactions are then quantified and validated using nuclear magnetic resonance (NMR) spectroscopy, since NMR chemical shifts are sensitive to ligand binding, and compound interference can be solved with spectral editing to isolate only the spectrum corresponding to the ligand binding (Hajduk and Greer, 2007; Wang et al., 2003). Another advantage of fragment-based screens is that a high proportion of the atoms in the fragment directly interact with the target allowing each fragment to efficiently bind to it (Hajduk and Greer, 2007; Rees et al., 2004).

DRAWBACKS OF DISCUESSED TECHNOLOGIES As illustrated above both the PARP and MYC pathway have known successful synthetically lethal interactions, thus the number of molecules that need to be screened for potential synthetically lethal interactions decreases. Although ideal for cancer treatment due to selective cell death towards tumor cells, a limitation of synthetic lethality is that it is dependant on a singular pathway. In the case that the cancer being treated develops a mutation allowing the bypass of the exploited pathway, then the drug can become ineffective (Dai, Fang and Roth, 2009). Not only can the cancer cells develop an additional mutation to resist the drug but also they can potentially develop an alternative pathway by amplifying the targeted gene (Pao et al., 2005; Gorre, 2001). Upon further investigation of this synthetic lethality these limitations would have to be further explored in order to determine the potential risks involved in using this method in the treatment of TNBC.

LIMITATIONS

ACKNOWLEDGEMENTS Thank you to Dr. John Hassell for supervising and advising our research and paper. Thank you to Dr. Sarah Symons for reviewing the paper in its early stages and ensuring that we were on track stylistically.

AUTHOR CONTRIBUTIONS CONCLUSION If a synthetic screen is found to be viable, the molecule that is found can then be further developed into a drug to help combat TNBC. When analyzing all aspects of synthetic lethality and the current knowledge of successful interactions, it can be assumed that there are more lethal interactions that have yet to be discovered.

R.A. wrote about TNBC, the MYC pathway in TNBC, RNA interference and contributed to the conclusion of the paper. M.O. wrote about synthetic lethality, the PARP pathway, CRISPRCas9 and chemical screens. Both Rachel and Maryanne conducted preliminary research on triple negative breast cancer and synthetic lethality, as well as combined the research from the various sections to write the future directions section.

Molecular & Cellular Biology

One limitation in the methodology was the lack of anticancer drugs that utilize synthetic lethality as their mode of mechanism. While researching, there was only one FDA approved drug that used synthetic lethality. Due to the substantial amount of research on drugs that are still in the developmental stages, most of the data that may have been beneficial for our research, was unavailable to access. For future steps, a metaanalysis can be conducted on the data available to directly compare the current potential drugs showing synthetically lethal effects. Comparisons could be made on their adverse side effects and overall effectiveness for treatment of TNBC in attempt to propose the most beneficial drug.

This progressing shift in traditional cancer treatment raises the chance of survival for many cancer patients. Treatments based on findings of synthetic lethality may not be widespread currently, but it opens a door for new, more specific, cancer treatments.

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Molecular & Cellular Biology

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LITERATURE REVIEW

Transcytosis and the Neurophysiological Complications Involved in the Delivery of Drugs Across the Blood-Brain Barrier in Alzheimer’s Disease COULTER MONTAGUE1, CHEN CHEN1

1. Integrated Science Program, Class of 2020, McMaster University

SUMMARY

Received: 10/06/2017

Accepted: 04/04/2019

Published: 11/17/2019

Alzheimer's disease (AD) is a neurodegenerative disorder that currently affects more than 40 million people worldwide, and this number is expected to increase exponentially in the coming decades (Esquerda-Canals et al., 2017). AD is characterized primarily by cognitive impairment

and neurodegeneration - the result of synaptic damage and subsequent neuronal loss. At a molecular level, this results from the formation of amyloid-β-containing plaques and neurofibrillary tangles composed of hyperphosphorylated tau protein (Crews and Masliah). These features then manifest themselves on a spectrum from early

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Keywords: Alzheimer’s disease, blood-brain barrier, drug delivery, nanotechnology, transcytosis, antibodies, absorptive-mediated transcytosis, receptor-mediated transcytosis

INTRODUCTION

Neuroscience

The medical field relies heavily on the collection and development of knowledge required to design novel drugs, as well as formulate innovative and effective methods of drug delivery, particularly with reference to neurophysiological diseases. Alzheimer’s is one of many chronic neurodegenerative diseases and impacts more than 40 million people worldwide. Diseases such as Alzheimer’s can be devastating for a patient and those caring for them, and when coupled with other ailments common to aging populations they can significantly decrease quality of life. While some treatments currently exist for Alzheimer’s disease, they serve only as short-term symptom relief by causing an increase of acetylcholine in the brain. Extensive research is currently underway to identify and design new drugs capable of sustaining long-term slowing of, or arrest Alzheimer’s disease progression. One of the main challenges to successfully delivering such a drug, however, is traversing the blood-brain barrier. Therefore, the aim of this literature review is to examine two methodologies of drug delivery through the blood-brain barrier currently undergoing development: adsorptive- and receptor-mediated transcytosis. These methods focus on the interactions of the molecular carrier and the blood brain barrier and its chemical and physiological characteristics to assist in drug delivery. This review also investigates the stage of development that each of these delivery techniques are currently in, assesses the potential for the delivery method to be used in the active treatment of neurological diseases, and evaluates the benefits and disadvantages of each method.

memory changes to functional dependence, and eventually death (Neugroschl and Wang, 2011). Currently, AD is treated symptomatically, however such treatments exhibit limited success since they can only counter surface-level neurotransmitter imbalances (Yiannapoulou and Papageorgiou, 2013). There have recently been significant strides made in the research of disease-modifying drugs which attempt to treat AD at its source. Unfortunately, these new drugs must overcome one main obstacle - traversing or circumventing the blood-brain barrier (BBB) to effectively reach the target areas of the brain.

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Neuroscience

BLOOD-BRAIN BARRIER

The brain is a highly important organ that, despite its dense vasculature, is extremely sensitive to circulating compounds (Herve, Ghinea, and Scherrmann, 2008). Thus, there needs to be constant and strict control over which substances are allowed to come into direct contact with the brain. The BBB is known to perform this function.

The BBB is defined by most researchers as a nonfenestrated microvascular endothelium with incredibly narrow tight junctions, few alternate transport pathways, and elevated levels of degrading enzymes (Herve, Ghinea, and Scherrmann, 2008; Rocha, 2013). This barrier serves to maintain homeostatic stability in the environment created by the brain parenchyma through the control of blood vessels and selective transport systems (Betsholtz, 2014). Almost all water-soluble compounds are prevented from entering the brain via the usual paracellular pathway - through aqueous channels - and this, unfortunately, includes peptide drugs among many others. Hence, the BBB is one of the main obstacles for the successful delivery of drugs to the central nervous system (CNS), which is essential for the treatment of neurodegenerative diseases such as AD (Banks, 2012). Not all the mechanisms that facilitate the function of the BBB are fully understood, and therefore it is a greater challenge for researchers to circumvent the mechanisms and processes which stop most drugs from passing through it (Betsholtz, 2014). As such, this literature review focuses on existing and better

Table 1: A list of search terms used to search for literature used in this review, categorized by section in review. General Background

Absorptive-mediated Transcytosis

Receptor-mediated Transcytosis

Blood-brain barrier

Negative blood-brain barrier

Receptor-mediated transcytosis

Blood brain barrier

Absorptive mechanisms

Receptor ligand systems

Alzheimerâ&#x20AC;&#x2122;s disease

Absorptive-mediated transcytosis

Transferrin Receptor Antibody

Alzheimer

Adsorptive-mediated transcytosis

Transferrin receptor

Structure

Putrescine cationization

Targeting ReceptorMediated Transport

Function

Cationized albumin

Transferrin-mediated transcytosis

Acetylcholinesterase inhibitor

Cell-penetrating peptide

Delivery system Alzheimerâ&#x20AC;&#x2122;s disease Drug delivery CNS

understood transport systems capable of being harnessed as novel and effective drug delivery methods to treat neurological diseases such as AD, namely methods of transcytosis.

TRANSCYTOSIS

ABSORPTIVE-MEDIATED TRANSCYTOSIS

Another transport mechanism of interest is receptor-mediated transcytosis (RMT) (Preusch, 2007). RMT is the receptor-mediated uptake of a ligand on one side of the cell, vesicular transport across the cell, and exocytosis of vesicle contents on the opposite side, much like AMT. A consequence of RMTs molecular process and function of acting as a key transport system at the BBB is the availability of this system for ‘hijacking’ to facilitate cerebral biologic delivery (Rip et al., 2010). RMT makes use of so-called ‘trafficking machinery’ on and within BMECs to deliver a range of proteins including transferrin (Tf), insulin, leptin, and lipoproteins to the brain (Dehouck et al., 1997; Descamps et al. 1996; Duffy and Pardridge, 1987; Golden, Maccagnan and Pardridge, 1997; as cited in Lajoie and Shusta, 2015). The RMT process involves four steps (Figure 1). Initially, a circulating ligand binds to a cognate transmembrane receptor expressed on the luminal (blood side) plasma membrane. Endocytosis then occurs through membrane invagination and the formation of an intracellular vesicle that contains the receptor-ligand complex (Parkar et al., 2009, as cited in Lajoie and Shusta, 2015). The newly formed intracellular vesicle can be directed to various destinations across the endothelial cytoplasm through the use of the cell’s vesicular and endolysosomal trafficking machinery (Brooks, 2009; Rodriguez-Boulan, Kreitzer and Müsch, 2005, as cited in Lajoie and Shusta, 2015; Sharma et al. 2016). In the case of transcytosis, the vesicle is shuttled to the basolateral (brain-side) membrane where exocytosis occurs, releasing the vesicular contents into the parenchyma (Strazielle

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There are multiple forms of transcytosis that occur across the BBB to transport certain blood plasma constituents to the parenchyma. One of the most fundamental types is absorptive-mediated transcytosis (AMT), which have been shown to have the potential to facilitate successful biologic delivery (Herve, Ghinea and Scherrmann, 2008). AMT involves the endocytosis of a cationic molecule at the luminal surface of the BBB through interactions with anionic particles on the plasma membrane, followed by exocytosis from the abluminal surface (Herve, Ghinea and Scherrmann, 2008). Moieties expressed on the luminal surface of BMECs are suited for interactions with ligands, that are ideally polycationic, which leads to binding, membrane invagination, and their eventual uptake (Herve,

RECEPTOR-MEDIATED TRANSCYTOSIS

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The BBB maintains cerebral homeostasis by admitting or preventing particular substances in the bloodstream from entering the brain (Weiss et al., 2009). The transport of nutrients, such as amino acids, glucose, or other small molecules through brain microvascular endothelial cells (BMECs) is done through the process of transcytosis - the vesicular transport of macromolecules from one side of a cell to the other (Lajoie and Shusta, 2015; Tuma and Hubbard, 2003). This is done in order to enable and ensure proper neuronal and supporting cell function and growth. The BBB’s selectivity is based on the existence of specific surface receptors that are expressed on BMECs in order to bind and signal the endocytosis of ligands carrying desired molecules or minerals (Yu, Li, Tao and Wang, 2015). It is evident based on recent literature that the advancement in our understanding of endogenous transport at the BBB will support and cultivate the development of successful procedures and nanoparticle-based strategies for transporting and delivering biologics capable of treating neurological diseases to the brain. These particles may be between 1 and 100 nanometres in diameter.

Ghinea, and Scherrmann, 2008). The BBB is already suited for AMT due to the many membrane surface regions of the luminal face of BMECs that are negatively charged at physiological pH (7.4) (Herve, Ghinea, and Scherrmann, 2008). Negative charges stem from the sialo-glycoconjugates and heparan sulfate proteoglycans which make up the glycocalyx of the BBB’s luminal surface (Herve, Ghinea, and Scherrmann, 2008; cited as Vorbrodt, 1989). In addition, since this is an active transport process which requires metabolic energy in the form of adenosine triphosphate, the BBB is an ideal location, having five times more mitochondria than any other peripheral endothelium (Herve, Ghinea, and Scherrmann, 2008).

and Ghersi-Egea, 2013, as cited in Lajoie and Shusta, 2015).

ABSORPTIVE MEDIATED TRANSCYTOSIS VIA CATIONIZATION OF PEPTIDE DRUGS The initiation of AMT from cationic particles binding to anionic particles in the plasma membrane can be harnessed by drug delivery systems. There are several possible ways to cationize a peptide drug to enhance theraputic delivery, a number of which involve carbodiimidemediated amidation of carboxylic acid groups to directly deliver the peptide without a cationic import carrier (Herve, Ghinea, and Scherrmann, 2008).

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CATIONIZATION OF AMYLOIDBETA ANTIBODY

Cationic molecules have displayed improved uptake to the brain in several experiments using

mice. For example, cationized bovine serum albumin-PEG-PLA demonstrated a significant increase in transport to the brain, compared to non-cationized albumin-PEG-PLA (Lu et al., 2005). In a study related to AD, Syvänen, Edén, and Sehlin (2017) investigated the effect of cationization of an antibody fragment with the polyamine putrescine as a means to increase the surface charge of the antibody and potentially improve its interaction with the luminal face of endothelial cells to penetrate the BBB. The researchers used an amyloid-beta protofibril selective antibody h158, which was cleaved enzymatically to a F(ab’)2 fragment then cationized by cross-linking it to the polyamine putrescine using 1-ethyl-3-(3dimethylaminopropyl) carbodiimide. The percentage of fragments that reached the brain was determined using radiolabeling with iodine-125. After comparing the unmodified antibody fragment to the cationized fragment in the brains of the mice, it was demonstrated that cationization led to lower concentrations of the antibody remaining in the blood, whereas the concentrations in the brain were at the very least similar, and at times increased (Syvänen, Edén, and

Figure 1: An illustration of RMT in a BMEC. (i) Initially, a ligand binds to a cognate transmembrane receptor on the luminal plasma membrane. (ii) Endocytosis occurs through membrane invagination, which generates receptor-ligand containing intracellular vesicles. (iii) The intracellular vesicle can be directed to various destinations across the endothelial cytoplasm via the cell’s vesicular and endolysosomal trafficking machinery. (iv) During transcytosis, the vesicle is shuttled to the basolateral membrane where exocytosis occurs. (v) Vesicles can also be sent to a lysosome to be degraded (Lajoie and Shusta, 2015).

ABSORPTIVE-MEDIATED TRANSCYTOSIS WITH CELLPENETRATING PEPTIDES If the drug itself is not directly cationized postdevelopment, researchers may find cellpenetrating-peptides (CPPs) as appropriate choices to aid transport into the brain by AMT. CPPs are short peptides, less than thirty amino acids long, with a net positive charge and amphipathic characteristics that are capable of entering living cells without inducing cytolysis (Chen and Liu, 2012). These peptides successfully interact with lipid membranes by adopting a certain secondary structure upon contact. In fact, CPPs increase doxorubicin transport into rat brains thirty-fold (Chen and Liu, 2012). In an in vivo experiment with a murine brain microvascular endothelial cell model, CPPmodified liposomes (Figure 3) significantly increased the transport ratio of rivastigmine solution to the CNS (Yang et al, 2013). Rivastigmine is an FDA- and Health Canadaapproved acetylcholinesterase inhibitor drug currently used to treat Alzheimer’s disease. The study also demonstrated no significant difference between parameters such as nasal mucosa morphology and cilia movement in rivastigmine CPP-liposome formulations and normal saline, indicating that the drug system did not cause notable toxicity (Yang et al, 2013). The most promising type of CPP in this study was found to be L-penetratin, though there are other popular peptides such as TAT and the Syn-B vector as well. Similar to cationization, several studies cite the success of CPPs to enhance the systemic delivery of other particles through the blood-brain barrier, namely polymeric nanoparticles (Patel et al., 2012).

TARGETING BIOLOGICS AT THE BRAIN VIA RECEPTOR-MEDIATED TRANSCYTOSIS The method of manipulating receptor-mediated pathways to allow for the delivery of biologics to the brain involves the conjugation of a receptortargeting signal molecule with a therapeutic of interest (Broadwell et al., 1996; Friden et al., 1991; Pardridge, Buciak, and Friden, 1996, as cited in Lajoie and Shusta, 2015). The use of RMT has

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Figure 2: The coronal view of SPECT images obtained of the third ventricle in the brain of 17–18 month old tg-ArcSwe mice two hours after injection with either (A) [125I]pF(ab’)2-h158 or (B) [125I]F(ab’)2-h158. (C) Coronal, sagittal and transverse images obtained from the frontal cortical area of the mouse injected with [125I]pF(ab’)2-h158. The blue crosshair indicates the same position in all three images. A legend assists in qualifying the level of radioactivity represented by the image, proportional to accumulation of antibodies, which demonstrate increased uptake of the antibody fragment when cationized (Syvänen, Edén, and Sehlin, 2017).

antibodies were able to be transported successfully to the brain.

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Sehlin, 2017). Moreover, the cationized fragments were ostensibly present in the third ventricle and front cortical region of mice brains using single photon emission computed tomography imaging (SPECT) (Figure 2C), which were known to contain large amounts of amyloid-beta plaques (Syvänen, Edén, and Sehlin, 2017). The cationized fragments did not visualize beta-amyloid plaques (Figure 2B and 2C), but were hypothesized to represent the soluble amyloid-beta species around them. In addition, enzyme-linked immunosorbent assays allowed researchers to determine that cationization of the F(ab’)2 antibody fragment did not alter its binding affinity to amyloid-beta protofibrils (Syvänen, Edén, and Sehlin, 2017). Therefore, cationized antibody fragments, and perhaps other cationized peptide drugs, present a viable option for improved therapeutic uptake. Another experiment conducted in by Agyare et al. (2008) supported the claim that putrescinecationization can transport several types of particles through the BBB, as chitosan nanoparticles coated with putrescine modified

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Figure 3: A Schematic diagram of (A) liposomes and (B) CPP-modified rivastigmine liposomes (CPP-Lp) carrying a rivastigmine displaying membrane bilayer and other attached constituents (Yang et al., 2013).

been developed to allow for the delivery of many different biologics, including monoclonal antibodies (mAbs), recombinant proteins, RNA, DNA, and nanomedicines (Lajoie and Shusta, 2015). There are generally two methods of coupling the biologic to the RMT-targeting moiety, the first of which involves a direct chemical linkage or molecular fusion, and the second the generation and use of loaded nanoparticles such as liposomes (Jones and Shusta, 2007, as cited in Lajoie and Shusta, 2015).

TRANSFERRIN One of the first RMT system proteins to be studied and exploited for neurological drug delivery was the transferrin receptor (TfR). Binding experiments have shown that the TfR, a glycoprotein that is approximately 80-kDa in length, is highly expressed on BMECs of both rats and humans, and that the transport of iron to the brain in both species involves the RMT of Tf (Cabezón et al, 2015; Descamps et al. 1996; Fishman et al., 1987; Lajoie and Shusta, 2015; Zuchero et al., 2016). In iron transport, after the binding of diferric Tf to the TfR, the receptorligand complex is endocytosed into the cell where a lysosome fuses with and causes dissociation of the iron, which can subsequently be transferred elsewhere in the cell for use or storage (Descamps et al. 1996). There still exists a dispute over the level of Tf recycling by the BMECs and the rate at which Tf is transcytosed (Clark and Davis, 2015; Descamps et al. 1996; Wiley et al., 2013; Yu et al., 2011). Recent studies have explored new variations in the manipulation of this trafficking

machinery, which have encouraged new and ongoing investigations involving the RMT pathway.

TRANSFERRIN VECTORS Many variations of nanoparticles, as well as therapeutic drug molecules, can be conjugated to either Tf proteins or TfR-targeted monoclonal antibodies. The TfR antibody (anti-TfR) has been shown to bind to a different site compared to a Tf protein, and as such unlikely to interfere with the endogenous Tf circulating in the blood (Sharma et al. 2016). Therefore, several recent studies have presented, and in many cases developed, improvements to the application of monoclonal antibodies and nanoparticles for targeting the RMT of Tf (Bien-Ly et al., 2014; Clark and Davis, 2015; Wiley et al., 2013; Yu et al. 2011). Although antibodies and antibody-conjugated vectors have been shown to enter, and sometimes fully transcytose across BMECs, there have long been reported caveats to the methodology of RMT manipulation and the use of Tf (Gosk et al. 2004; Lajoie and Shusta, 2015). The primary issue that has perpetuated within the study and manipulation of Tf-RMT, with respect to the delivery of nanoparticles and biologics across the BBB, has been the transfection levels of the Tf-conjugated delivery vectors (Gosk et al. 2004; Lajoie and Shusta, 2015). The high concentration of Tf protein in circulation competes with the transferrin on the nanoparticle system (Girão da Cruz, Simões and Pedroso de Lima, 2004, as cited in Sharma et al. 2016). Fortunately, there have been two different approaches to rectify this issue.

There are other factors to be considered when evaluating the appropriateness of utilizing RMT as a delivery strategy, and these will be discussed later in this review.

MODIFYING MONOCLONAL ANTIBODY AFFINITY

Figure 4: A Model representing the inverse relationship between an antibody’s Affinity for TfR and its RMT to the brain. When a trace dose is administered, higher-affinity antibodies (A) will bind more readily to receptors expressed on the luminal side of BMECs compared to the trace dose of lower-affinity antibodies (C), meaning more high-affinity antibodies are available to enter the brain parenchyma. When a therapeutic dose is administered, the saturation of BMECs will result in antibody binding to receptors on the luminal side of the BBB epithelium regardless of antibody affinity. The dissociation of lower-affinity antibodies (D), however, will be more likely, and result in the higher accumulation of antibodies in the brain parenchyma compared to the higheraffinity antibodies (B) (Yu et al., 2011).

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Traditionally, monoclonal antibody affinities for the Tf receptor were designed to be extremely high in experiments with Tf-RMT and antiTfR antibodies. The use of highaffinity anti-TfR antibodies ensures specific binding to the TfR, which leads to uptake of the antibodies to BMECs even at low blood concentrations. Their high affinity for TfRs, however, has been found to likely reduce the probability of the antibody being released from the cerebral vasculature or TfRcarrying-endosome, and thus potentially prevents accumulation of antibodies in the parenchyma (Figure 4). A study by Yu et. al (2011) was conducted to determine the implications of lowering antiTfR antibodies’ affinity on antibody EC uptake, transcytosis, and potential for these antibodies to function as a successful therapeutic mechanism. Trials involved the

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Numerous studies have been conducted to examine the potential of anti-TfR antibodies to target the TfR and have presented findings revealing the potential for their endocytosis into BMECs. However, whether or not this leads to antibody release and therapeutic accumulation in brain parenchyma has remained controversial. Following this, data has shown high levels of anti-TfR degradation by lysosomes, leading to the break down and expulsion of the antibody, and thus any attached biologic (Lajoie and Shusta, 2015). In order to act as an effective targeting system, an anti-TfR antibody must be able to deliver attached therapeutics to the brain parenchyma at doses high enough to cause a measurable therapeutic effect.

injection of either a trace or therapeutic dose into mice intravenously, and subsequent brain uptake was measured after 1 and 24 hours. It was found initially that while high-affinity anti-TfR antibodies could accumulate in the brain at both trace and therapeutic levels, they remained predominantly in the cerebral vasculature and did not enter the parenchyma (Figure 4A and B). These observations indicate that only minimal transcytosis occurred. Researchers then explored whether lowering the antibody’s affinity for TfRs would allow for greater accumulation in the parenchyma, which was done by introducing

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alanine mutations into the complementaritydetermining regions of anti-TfR antibodies. This generated antibody variants with differing affinities for the TfR, and these variants were then evaluated for uptake after trace and therapeutic intravenous dose administrations. It was found that when delivered in trace doses, anti-TfR antibodies demonstrated a direct correlation between affinity and brain uptake, with loweraffinity antibodies showing the lowest uptake (Figure 4C). Conversely, when therapeutic doses were administered, there was an inverse correlation between affinity and brain uptake (Figure 4D). This represents a higher level of successful transcytosis of lower-affinity antibodies. The antibody with the lowest affinity in the experiment was even found to have a more than a fivefold increase in brain antibody concentration compared to control Immunoglobulin G (IgG) 24 hours after injection.

When comparing the high- and low-affinity antibodies at low blood concentrations, Yu et al. (2011) found that the high TfR affinity antibodies bound tightly to brain endothelial receptors, while low-affinity antibodies were less likely to bind and remain bound to the luminal side of the cerebral vasculature. This led to the conclusion that when BMECs are not saturated with low-affinity antibodies, their RMT to the brain is reduced. At higher doses, however, the luminal TfR would be saturated regardless of antibody affinity, and so there would be similar endothelial uptake for both types of antibodies. In this case, low-affinity antibodies would demonstrate a higher level of brain accumulation through an increased dissociation from TfR and release into the brain. Additionally, it was found that having a lower affinity antibody decreases the probability of receptor-mediated efflux out of the parenchyma, since concentrations in the brain are likely to no longer be saturating. In order to further address the parenchymal distribution of antibodies at therapeutic doses, Yu et. al (2011) compared antibody distribution in the brain for each affinity variant. The results of brain section visualizations from stained fluorescent anti-human secondary IgG were that the high-affinity anti-TfR antibody localized predominantly in the vasculature, but in contrast, lower-affinity variants localized with the neuronal marker neuronal nuclei, suggesting their broad distribution in the brain parenchyma surrounding neurons. This supplemented the observation that the low-affinity antibodies possessed a greater propensity to be transcytosed and distributed through the brain than high-

affinity antibodies. These findings have ultimately demonstrated how the manipulation of antibody affinity can be used to increase their RMT and parenchyma delivery, which may present a partial solution to the issue of delivering therapeutics in such a manner.

MODIFYING NANOPARTICLE AVIDITY The branch of RMT study that analyses the potential therapeutic use of antibodies and antibody-bound biologics is one of many. To the same extent, the investigation into the influence of antibody affinity on transcytosis is just a single focus of research and development with the goal of adapting RMT as a successful therapeutic method for treating neurological diseases. An alternative approach taken to develop a successful method of delivering therapeutic agents across the BBB was examined by Wiley et al. (2013), and Clark and Davis (2015). These studies examined the avidity, or overall stability between antibodies and antigens, of Tf-containing nanoparticles. Avidity is governed by the intrinsic affinity of the antibody for the epitope - the valency of the antibody and antigen - and the geometric arrangement of the interacting components. One of the main goals of research into Tf-RMT is to find or design a nanoparticle with the ability to cross the BBB after a therapeutic dose and deposit a biologic at a concentration high enough to be capable of treating neurological diseases (Yu et al., 2011). Wiley et al. (2013) examined the capability of Tf-containing gold nanoparticles (AuNPs) to cross the BBB. This study found that Tfcontaining 80-nm gold AuNPs with near-neutral zeta potentials were capable of entering the parenchyma after transcytosis when their avidity to TfRs was low, whereas high-avidity AuNPs remained strongly associated with the BMECs of the BBB. Building on the findings from Wiley et al. (2013), Clark and Davis (2015) sought to further develop the avidity-tuned AuNP design and increase the ability of such Tf-containing nanoparticles to reach the parenchyma. To do this, they generated chemical linkages between the nanoparticle cores and Tf that were capable of cleaving at moderately acidic pH levels (Figure 5). The design of these nanoparticles provided them with high-avidity interactions with TfRs on the luminal face of the BBB, as well as the ability to undergo cleavage of the linkage during transcytosis (Clark and Davis,

2015; Mellman, Fuchs and Helenius, 1986, as cited in Clark and Davis, 2015; Sade et al., 2014). This inducible cleavage meant that the nanoparticles could become unbound during the process of vesicle endocytosis into, and transport through BMECs, and would subsequently be readily available for release to the parenchyma. After testing using an in vitro model, the targeted acidcleavable AuNPs demonstrated an increased ability to cross the BBB, and in vivo they were able to enter the parenchyma of mice in far greater amounts after systemic administration than similar high-avidity nanoparticles containing noncleavable Tf.

To further improve their application, Yu et al. (2011) went on to design bispecific antibodies capable of binding with low affinity to TfR, and with high affinity to the enzyme β-secretase (BACE1). BACE1 converts amyloid precursor protein into amyloid-β peptides, including those associated with Alzheimer’s disease (Roberds, 2001; Vassar, 1999 as cited in Yu et al., 2011).

To explore the cellular basis of the improvements made by Yu et al. (2011) previously, Bien-Ly et al. (2014) explored whether TfR antibody affinity alters TfR intracellular trafficking after receptorligand endocytosis. When researchers compared high- and low-affinity TfR bispecific antibodies, it was found that high-affinity binding to TfRs caused a dose dependent RMT and reduction of brain TfR levels. Using live imaging and colocalization experiments in vitro, researchers determined that high-affinity TfR bispecific antibodies facilitated the trafficking of TfRs to

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DEGRADATION

Monospecific antibodies to BACE1 (anti-BACE1) were also tested in this investigation as a means to quantify changes in binding and RMT of the bispecific anti-TfR/BACE1. At trace doses, there was higher RMT and brain accumulation observed with the anti-TfR/BACE1 bispecific antibody compared to the anti-BACE1 antibody. These findings accurately fit models presented earlier in the study regarding antibody affinity, concentration, and RMT. At therapeutic doses, however, the anti-TfR/BACE1 bispecific antibody demonstrated significantly higher RMT compared to anti-BACE1 or anti-TfR alone.

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Figure 5: (A) The proposed mechanism of transcytosis for Tf-containing nanoparticles with acid-cleavable linkages. After endocytosis, the acidification of the endosome causes the separation of Tf ligand from the nanoparticle core, which allows the movement of the nanoparticle to the parenchyma to complete transcytosis. (B) The preparation of acid-cleavable DSS-DAK-PEG-OPSS and the addition to a targeting ligand (the anti-TfR antibody) to create the cleavable conjugate. (C) The addition of the anti-TfR antibody-DAK-PEG-OPSS ligand followed by excess mPEG-SH to prepare targeted gold nanoparticles (Clark and Davis, 2015).

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lysosomes and thus induced the degradation of TfR, an observation which was confirmed in vivo. This ultimately led to the research team determining that high-affinity anti-TfRs alter the process of TfR trafficking and dramatically impacts the ability of TfRs to facilitate BBB transcytosis.

Similar findings regarding the influence of affinity on TfR trafficking were also found by Sade et al. (2015). In this study’s in vitro model of the human BBB using human cerebral microvascular endothelial cells (hCMEC/D3), it was found that low-affinity anti-TfRs were able to transcytose across the hCMEC/D3 cells, whereas high-affinity antibodies were directed to lysosomes. This suggests vesicle trafficking may be affected by the targeting ligand used for RMT. Based on these findings, Clark and Davis (2015) examined whether Tf and anti-TfR antibodies behaved differently when used as targeting agents for nanoparticles with the addition of an acidcleavable linkage - diamino ketal (DAK). The results of this examination indicated that highavidity nanoparticles showed a nearly threefold increase in their ability to reach the parenchyma in vivo after the incorporation of the DAK linker. It was also observed that there existed a direct relationship between surface Tf-DAK content on the nanoparticles and their brain penetration.

DISCUSSION OF USING TRANSCYTOSIS FOR BIOLOGIC DELIVERY ACROSS THE BLOODBRAIN BARRIER Several considerations should be made when examining the potential success of delivering a drug through transcytosis, the first of which is the drug’s ideal environment. For example, the extent of cationization increases as the pH of the protein’s environment decreases (Herve, Ghinea, and Scherrmann, 2008). Therefore, if the peptide drug is only stable in relatively basic conditions, AMT may not be effective at physiological pH. Transcytosis may also alter the function of the drug molecule. Linking cationic groups to a peptide may change its activity at the drug target meaning AMT may not be possible with all drugs (Goulatis and Shusta, 2017). There is no evidence insofar as to suggest this occurs with RMT as well, although the current developmental stage of RMT-technology may not allow comprehensive investigation of this phenomenon.

One of the most important issues with the application of AMT is that the exact mechanisms whereby directly cationized molecules, CPPs, and many receptor-bound ligands are transcytosed to the CNS remain mostly elusive (Betsholtz, 2014; Herve, Ghinea, and Scherrmann, 2008; Mäger et al., 2017). Some studies only claim that endocytosis is always involved (Yang et al., 2013), while almost all are unclear about what occurs after absorptive endocytosis is triggered (Gabathuler, 2010; Chen and Liu, 2012; Kamalinia et al, 2015). Most schematic diagrams illustrate a direct path for therapeutics molecules being internalized into the endothelial cell to exocytosing at the abluminal surface. In reality, however, vesicles are still likely to progress to a lysosomal path in AMT (Banks, 2012). Not elucidating why a vesicle may be directed towards transcytosis versus degradation may ultimately contribute to the reduction of the success of drug delivery techniques. As mentioned earlier, the transfection levels of the Tf-conjugated delivery vectors in RMT is perhaps the most difficult obstacle to overcome in the progression and development of RMT as a method of therapeutic delivery to the brain (Gosk et al. 2004; Lajoie and Shusta, 2015). Of course, the issue that arises while attempting to deliver therapeutics via RMT is the propensity for endosomes to be directed for lysosomal degradation. While research is being conducted to determine how to counter this challenge, it is still a significant limit on RMT’s efficacy as a delivery pathway for biologics. Another consideration with the use of RMT is that, depending on the method of dose administration, there are varying levels of biodistribution within the subject. This level of distribution may vary based on the species and even the individual administered the biologic, as well as based on the carrier or loaded-nanoparticle used. In several studies, antibiotic tagged nanoparticles accumulate in areas of the body other than the brain, such as the spleen, liver, kidneys, lungs, and heart of mice, and in almost all tissue of primates (Friden et al., 1996; Johnsen et al., 2017). Such a distribution of antibodies and nanoparticles is likely due to the variable level of expression of the TfRs throughout the body, since it is not exclusive to BMECs. The use of the TfRs as a means of RMT holds an innate flaw regardless of treatment since iron is required by all tissues in the body in order to collect and endocytosis diferric iron floating in the bloodstream. Similarly, due to its reliance on electrostatic interactions, many researchers believe AMT is not specific

It should be noted that there can be many advantages of selecting AMT as the transport pathway for a drug over RMT. First, the capacity of AMT is significantly greater than RMT. If the volume of a certain therapeutic molecule required needs to be dramatically increased, AMT is an ideal transport process, since it is not limited by the number of receptor proteins expressed on the endothelial surface (Upadhyay, 2014). The anionic and cationic properties of the molecules and membranes in question will also remain relatively consistent, ensuring that AMT is a safe choice for therapeutic delivery.

CONCLUSION While the exploration of AMT and RMT as potential therapeutic drug delivery mechanisms continues to advance, there is still a lack of basic conceptual understanding surrounding these processes that limits their potential for clinical success. There does appear to be progress in terms developing new agents for the treatment of neurological diseases, most pointedly the Genentech and Roche partnership and development of a stage II/III drug to treat AD, though this does not compensate for the lack of suitable current understanding and use of transcytosis for therapeutic delivery to the brain (Boettner, 2014). Continued study and development of technologies and methodologies that make use of transcytosis for brain biologic delivery must persist. AMT is an asset for increasing the therapeutic effect of drugs by reducing their toxicity in peripheral organs, as well as allowing them to successfully transcytose through the BBB. On the other hand, RMT may be able to minimize the influence that a mode of drug delivery has on the efficacy of a drug while also allowing for enhanced transcytosis of therapeutics to the brain. An additional avenue to explore is the combination of several transport pathways to create a more integrated and cohesive method of biologic delivery allowing for greater brain uptake and accumulation. Ultimately, our current understanding of the fundamental properties and mechanisms that govern AD and the BBB is insufficient for troubleshooting many drug delivery technologies that are undergoing development.

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Many of the considerations for RMT discussed thus far are specific to the use of Tf and the TfRs. While RMT is an important and extensively researched method for neurological drug delivery, there are few instances in research and experimentation that demonstrate sufficient RMT to treat a neurological disease. This is perhaps largely due to the lack of data that exists for receptors that are selective and abundant on BMECs, and a focus on only a select few RMT receptors, such as the Tf and insulin receptors, their antibodies, and nanoparticles, such as liposomes (MĂ¤ger et al., 2017). This particular issue in research and literature can be resolved through the search for and study of other receptors, targeting methods, and potentially drug carrying ligands. Such research is being conducted

by MĂ¤ger et al. (2017) who, with the Collaboration on the Optimization of Macromolecular Pharmaceutical Access to Cellular Targets (COMPACT) project, have set up a proteomicsand transcriptomics-based workflow to identify brain microvascular cell surface specific receptors with the aim of using them for biologic delivery across BMECs. Other receptors have the potential to become extremely specific, yet effective means through which therapeutic molecules can be delivered, with much more control and less side effects than AMT. Ultimately, the future clinical application of both AMT and RMT for the delivery of therapeutic agents to the brain must be built on a more solid foundation of fundamental knowledge and understanding.

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enough for therapeutic deliver to the brain and can lead to toxic effects on other surrounding organs (Rocha, 2013; Goulatis and Shusta, 2017). However, the mechanism whereby one specific type of molecule crosses the blood-brain barrier by AMT - diamine or polyamine-modified peptides has not yet been ascertained. Therefore, it is likely that their transport requires not just electrostatic interactions, but carrier-mediated transport as well, which may prove to be more specific (Rocha, 2013). As well, many cationized molecules were observed to have decreased half-lives, which would decrease their bioavailability in the blood. Along with no reports of cationized peptides or CPPs disrupting the BBB, delivering therapeutics through AMT appear much less traumatic and toxic for patients with neurodegenerative diseases. On the other hand, a decreased half-life may also lower the therapeutic window for a drug to a level that renders it ineffective (Yi et al., 2014). Therefore, additional studies should be conducted for each potential drug to evaluate the effect of the local environment, the linkage or transformation process itself, non-specificity, and decreased halflife on its function and activity.

As new drugs are discovered, further investigation will also be required for methods of delivering these drugs to their intended targets as well, though AMT and RMT are both promising endogenous transport pathways that may be harnessed for this purpose in the future.

ACKNOWLEDGMENTS

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We would like to thank Russ Ellis for his supervision, guidance, support, and feedback. We would also like to thank the Integrated Science program for providing us with the resources and opportunity to conduct this review. We would like to acknowledge Dr. Margaret Fahnestock for her insight into current knowledge and research in the

field of study related to molecular neurobiology, and the treatment of neurological diseases.

AUTHORSHIP AND CONTRIBUTIONS Coulter Montague was responsible for conducting a review of the literature and reporting on findings related to receptor-mediated transcytosis. Chen Chen was responsible for conducting a review of the literature and reporting on findings related to absorptive-mediated transcytosis. Both authors were responsible for examining and reporting on literature related to the blood-brain-barrier, and partnered to complete the introduction, discussion, and conclusion sections for this paper.

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LITERATURE REVIEW

Overcoming Resistance to AntiBreast Cancer Drugs Targeting Kinases MICHAEL CHAN1, JOSEPH Dâ&#x20AC;&#x2122;ERCOLE1

1. Integrated Science Program, Class of 2020, McMaster University

Biochemistry

SUMMARY Although there are no definitive cures, breast cancer may be treated through administration of drugs targeting specific protein kinases. Protein kinases are prominent factors in breast cancer, as they are essential for tumor cell proliferation and are highly traceable targets. These kinases are often targeted because of their ubiquity and capacity to become oncogenic. Patients can develop resistance to kinase inhibitors, increasing the risk of recurrence. As kinase inhibitors continue to be developed and used in clinical settings, there is an increased need to overcome resistance. The acquired resistance to kinases responsible for the proliferation of breast cancer cells has rendered many therapeutic strategies obsolete. A further understanding of the mechanisms allowing tumor cells to bypass targeted kinase inhibition can lead to improved treatments and the development of more effective drugs. This literature review provides a summary of the current state of research into therapeutics targeting protein kinases associated with breast cancer. Current protein kinase inhibitors for breast cancer will be discussed, as well as resistance mechanisms associated with these inhibitors. Mechanisms of resistance can be classified into five categories: 1) gene mutations, 2) alternative signalling and transcriptional changes, 3) alterations in transmembrane transportation, 4) off-target drug binding, and 5) cellular transitions. This review aims to summarize the current research in protein kinase inhibitor resistance mechanisms and development of novel therapeutics. Received: 02/13/2019

Accepted: 04/08/2019

Published: 11/17/2019

Keywords: breast cancer, protein kinases, overcoming drug resistance, mutations, alternative signalling, transcriptional adaptation, cell transitions, transmembrane transportation

INTRODUCTION ISCIENTIST | 2019

EPIDEMIOLOGY

Breast cancer is the second leading cause of cancer death in women and presents a major public health concern, with a lifetime risk of developing this disease being approximately 11% for women. This cancer type is heterogeneous in pathology and current statistics suggest that worldwide incidence of breast cancer is on the rise (Tao, et al., 2015). Developing breast cancer is more likely in an older population, as a majority of the cases occur in women aged 50-69 (Statistics Canada, 2015). It is

projected that by 2050, over 3 million new cases of breast cancer will be diagnosed annually (Tao, et al., 2015). These statistics exhibit the magnitude of breast cancer incidence on global health and the increasingly pertinent need for treatment measures. While therapies have been successful in reversing the progression of breast cancer, many questions remain unanswered with regards to the underlying mechanisms that enable tumor cells to develop resistance to current therapeutics.

HALLMARKS OF CANCER Cancer cells function differently compared to normal cells and the “hallmarks of cancer”, developed by Hanahan and Weinberg in 2011, highlight the unique qualities associated with tumors. The hallmarks include uncontrollable cell proliferation, evading growth suppressors, replicative immortality, evading cell death, tissue invasion and metastasis, as well as inducing angiogenesis (Hanahan and Weinberg, 2011).

Kinases within the human body are essential in cell signalling as they play an integral role in regulating cell growth, survival, and apoptosis (Angus, Zawistowski and Johnson, 2017). Given their importance in cellular functions, the deregulation

MECHANISMS OF KINASE INHIBITOR RESISTANCE MUTATIONS OF TARGET PROTEINS If a protein contains a point mutation within its coding region, the mutation will alter the protein’s structure and modify its affinity for certain drugs and ligands. Since mutations can affect the binding of both the drug and ligand, either the drug’s affinity must decrease or the affinity of the ligand must increase for resistance to arise. Mutations have the potential to confer resistance to a given drug. With respect to tyrosine kinases, relevant mutations can be classified according to their location within the protein: 1) gatekeeper mutations, 2) g-loop mutations, 3) a-loop mutations, and 4) αC helix mutations (BarouchBentov and Sauer, 2011).

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PROTEIN KINASES

Acquiring resistance to anti-cancer drugs prior to the administration of therapies is common in cancer cells, though the resistant species may not be dominant within the tumor. Treatments preferentially select for resistant cells as nonresistant species are susceptible to therapies. The mechanisms of tumor resistance to anti-cancer drugs can be classified into five categories: 1) mutations of the target protein, 2) signalling and transcriptional adaptation, 3) alterations in transmembrane transportation, 4) off-target drug binding, and 5) cellular transitions. Though many of these mechanisms may be applied to numerous types of cancers, these have all been documented within breast cancer and some mechanisms, such as cellular transitions, are more specific to breast cancer. This literature review provides an in-depth analysis of these five mechanisms of resistance and how kinases continue to be the driving force behind the progression of breast cancer through resistance to therapeutics. In analyzing the mechanisms of resistance, a further understanding of its relationship to the kinome can provide new strategies and novel pathways for kinase inhibitor therapy.

Biochemistry

Cancer cells escape normal growth control by deregulating the checkpoint signals needed for division. Uncontrollable proliferation is promoted by the overexpression or hyperactivity of growth factor receptors and mutations (Sledge Jr and Miller, 2003). To acquire insensitivity to antigrowth signals, the negative feedback systems that regulate cell growth and proliferation fail to function properly. This indicates alterations in the expression of cyclins, cyclin-dependent kinases, and their inhibitors in order to evade growth suppressors (Hanahan and Weinberg, 2011). The shortening of telomeres is associated with senescence and promotes cell death, however, cancer cells bypass this process by expressing telomerase. Telomerase prevents the progressive shortening of telomeres and allows for the limitless replicative potential of cancer cells (Hanahan and Weinberg, 2011). Moreover, cancer cells with the capability of migrating from their origin to surrounding body tissues are considered to be malignant tumors. Tumors may secrete proteases that degrade surrounding tissues and downregulate the expression of proteins responsible for cell-cell adhesion (Sledge Jr and Miller, 2003; Miller, Goulet and Johnson, 2016). These processes allow for the invasion of cancer into other tissues within the human body, known as metastasis. The metastatic process is dependent on angiogenesis, which provides additional nourishment and aids in the spread of cancer (Hanahan and Weinberg, 2011). These six biological capabilities acquired by human tumors demonstrate the complexity and genetic diversity of cancer, slowing the advancement of breast cancer treatments.

of kinases and uncontrolled kinase activity leads to the pathogenesis of various cancers. The abnormal activity of deregulated kinases drives tumor cell progression and gives them the critical features that make cancer such a difficult disease to cure (Angus, Zawistowski and Johnson, 2017).

Gatekeeper mutations involve the substitution of the amino acid which participates in the entry of

Biochemistry ISCIENTIST | 2019

the drug into the active site of the protein. As such, mutations of the gatekeeper residue can readily lead to resistance, as seen in the proteins SCFR, EGFR, and ERBB2 (Barouch-Bentov and Sauer, 2011). An example of a gatekeeper mutation is that of the T790M mutation within EGFR (Yun, et al., 2008). In approximately 50% of cases in which resistance to inhibitors of EGFR is observed, the T790M mutation is present (Chen and Fu, 2011). The T790M mutation involves the substitution of a threonine, a polar amino acid, with methionine, a larger and nonpolar amino acid. This mutation has been found to result in an increased affinity for the ligand, adenosine triphosphate (ATP) (Yun, et al., 2008). Despite resistance introduced via the T790M mutation, it has been overcome through drugs that irreversibly bind to the C773 residue and drugs binding to other regions (Kwak, et al., 2005; Kobayashi, et al., 2005; Jia, et al., 2016). Similar to gatekeeper mutations, g-loop mutations may reduce drug efficacy against the target protein as the g-loop is involved in ATP binding (Barouch-Bentov, et al., 2009). Drugs targeting the g-loop often competitively inhibit ATP binding, indicating that alterations within the g-loop can impact drug effectiveness. Many mutations involving the g-loop either destabilize the inactive

state or stabilize the active state, leading to hyperactivity (Cowan-Jacob, et al., 2007). Within the protein kinase BCR-ABL, the mutations G255K and G255V both lead to destabilization of the inactive state when bound to an inhibitor. Mutations G250E and Y253H also impact drug resistance, however, whether these stabilize the active state or destabilize the inactive state is more ambiguous. The a-loop and ÎąC helix regions are involved in the transition between the active and inactive conformations and mutations within these regions may lead to changes in kinase activity (Liu and Gray, 2006; Ferguson, 2009; Fabbro, CowanJacob, and Moebitz, 2015). Mutations within these regions tend to confer resistance to drugs by destabilizing the inactive conformation, increasing kinase activity (Barouch-Bentov and Sauer, 2011). A common mutation within the a-loop is L858R, which represents approximately 41% of oncogenic mutations in EGFR and significantly increases EGFR activity (Johnson, 2009). If a mutation causes the activity of a protein to increase, it is likely that the required dosage will increase. Mutations may also result in a protein that preferentially binds to its natural ligands rather than an inhibitory drug, leading to greater activity.

Figure 1: Known important signalling pathways involving kinases in breast cancer. Mutations in proteins either downstream or upstream of the target can potentially result in resistance to a given drug. If the mutant is downstream of the target protein, as is with PI3K and ERBB2, continual activation of the downstream mutant can result in drugs of the target being rendered ineffective. Similarly, if the mutant is upstream of the target, resistance to drugs can be attained by a mutant bypassing a protein in the signalling pathway, such as ERBB2 activating AKT3 rather than PI3K (Miller, Goulet and Johnson, 2016).

SIGNALLING AND TRANSCRIPTIONAL ADAPTATION Cell signalling is essential for proliferation and growth, though some tumors have evolved alternative methods to maintain communication. Various treatments target cancer by inhibiting signalling pathways. To acquire resistance to treatments, protein kinases may activate pathways downstream of the protein target, resulting in

Many anti-cancer drugs target proteins within the cell rather than surface receptors. For a drug to be effective, it needs to first enter the cell, which may be mediated by transporter proteins. Transporter proteins surrounding the cell membrane selectively choose molecules to pass through the membrane (Bixby and Talpaz, 2009). With respect to drug influx, some drugs may enter the cell with the aid of transporters. The human organic cation transporter (HOCT) is an example of this process, as it allows the import of anti-cancer drugs such as imatinib (Andreev, et al., 2016; Thomas, et al., 2004). It has also been shown that inhibiting HOCT reduces intracellular drug concentrations, implicating a method of resistance. On the contrary, efflux transporters can force unwanted compounds out of the cell and decrease its concentration in the intracellular environment. Efflux pumps reduce the efficacy of a given drug and its cellular bioavailability. This mechanism can be seen in P-glycoprotein efflux pumps (PGP), which excludes drugs from the cell. It has been previously shown that inhibiting PGP restores the functionality of various drugs in otherwise resistant cells (Mahon, et al., 2000; Che, et al., 2002; Kotaki, et al., 2003). Overall, a decrease in the activity of influx pumps or an increase in efflux pump activity result in reduced intracellular drug concentrations and may lower the effectiveness of a drug.

OFF-TARGET DRUG BINDING To avoid the therapeutic effects of anti-cancer drugs in the body, cancer cells can utilize different cellular proteins to interact with drugs. By providing molecules to interact and bind with the drug, cancer cells can effectively reduce drug availability. Certain basic drugs have been observed to interact with proteins, such as α1 acid glycoproteins which are found within blood plasma (Daub, Specht and Ullrich, 2004). When bound to α1 acid glycoproteins, the drug is sequestered and no longer able to work against its target, reducing cellular bioavailability and preventing the intended interaction with the target protein (Smith, et al., 2012). In breast cancer, α1 acid glycoproteins are capable of binding to drugs

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Transcriptional adaptation shares similarities with the concept of signalling adaptation with a distinct mechanism. Adaptation via transcriptional modification involves the activation of signalling pathways and the upregulation of other proteins rather than a mutation in the pathway. This is how the amplification of the hepatocyte growth factor receptor (MET) confers resistance to gefitinib (Engelman and Settleman, 2008). The PI3K/AKT pathway is activated by ERBB2 or by EGFR through the RAS/RAF pathway. MET upregulation presents an alternative pathway by allowing ERBB3 to activate the PI3K/AKT pathway in lieu of ERBB2 and EGFR. Gefitinib, which inhibits EGFR, is no longer effective in this scenario since MET allows for ERBB3 to activate pathways previously activated by EGFR. Therapies would

TRANSMEMBRANE DRUG TRANSPORT

Biochemistry

insensitivity to inhibitors (Angus, Zawistowski and Johnson, 2017). Resistance through downstream activation allows signalling pathways to be activated even in the presence of an inhibitor targeting an upstream protein. For example, the protein phosphoinositide 3-kinase (PI3K) can be upregulated and confers resistance to inhibitors of ERBB2 (D’Amato, et al., 2015). This example may be especially concerning in the case of HER2enriched breast cancer, which is characterized by greater ERBB2 expression (Iqbal and Iqbal, 2014). Continual activation of PI3K removes the need for activation by ERBB2, as PI3K is downstream of ERBB2 (Figure 1). In HER2-enriched breast cancer, ERBB2 is often targeted in therapies, yet PI3K upregulation may render such treatments ineffective. Rather than continual activation of a downstream protein, mutations in upstream proteins may result in resistance. Upstream proteins have the potential to undergo mutations, allowing them to bypass the target protein and altering the signalling pathway to omit the target protein. Bypasses subsequently cause treatments exploiting the target protein to be rendered ineffective and are often documented in HER2 enriched breast cancer (Miller, Goulet and Johnson, 2016).

require inhibition of both EGFR and MET in order to remain effective.

like imatinib in blood plasma, reducing the treatment effects and cellular bioavailability. Also, the concentration of Îą1 acid glycoproteins is greater in patients with breast cancer, amplifying the effect of off-target drug binding (Smith, et al., 2012). This results in a greater dose required for the same effect, increasing the cost of treatment and potentially increasing side effects.

ISCIENTIST | 2019

Biochemistry

CELLULAR TRANSITIONS: EPITHELIAL TO MESENCHYMALLIKE CELLS

A method of gaining resistance to anti-cancer drugs is the transition of a breast cancer cell to a mesenchymal-like cell. By transitioning to a different cell type, the cancer cell acquires a new proteome which can lead to drug resistance, such as those inhibiting the PI3K/AKT pathway (Martz, et al., 2014). Transitional resistance may arise in any molecular subtype of breast cancer, though this tends to be more likely in claudin-low breast cancer due to the similarity of its proteome to that of mesenchymal cells (Miller, Goulet and Johnson, 2016). Claudin-low breast cancer involves expression of AXL and ZEB1, in addition to a lesser expression of e-cadherin and claudin-3. In a cell population which has transitioned to a more mesenchymal-like state, there may be an elevated expression of vimentin and ncadherin. In transitional resistance, overexpression of Notch1, a protein that promotes the transition to mesenchymal cells, has been shown to provide resistance to inhibitors targeting the PI3K/AKT pathway (Fender, et al., 2015; Miller, Goulet and Johnson, 2016). Notch1 also reduces the expression of proteins responsible for intercellular adhesion, enabling cells to migrate through the extracellular matrix. Some mesenchymal transcriptional factors such as Twist, Snail, Slug, and ZEB2, control the expression of AXL and increase the likelihood of metastasis when upregulated. Overexpression of Notch1 has also been observed to cause resistance to EGFR and ERBB2 inhibitors. Due to the resistance conferred to ERBB2 inhibitors, transitional resistance may be particularly harmful in HER2-enriched breast cancer.

POTENTIAL STRATEGIES TO OVERCOME RESISTANCE MUTATIONS OF TARGET PROTEINS In a mutated protein resistant to therapeutics, the protein tends to become hyperactive or exhibit an increased preference for the protein to bind to the natural ligand rather than the drug (Kwak, et al., 2005). Mutations impacting drug binding can be overcome via rational drug design. Based upon the structure of effective anti-cancer drugs against the non-mutated protein, new drugs may be designed. Considering the changes in protein structure that come with mutations, such as the more common T790M mutation of EGFR, this can enable the development of novel drugs. By modifying the structure of a drug and accounting for sterics and functional group interactions, a drug specific to the mutant may be developed, though this would require screening patients for mutants in the event of resistance. Rather than developing new drugs to target the same region of a protein, attacking different portions of a protein may be useful. For example, the T790M mutation of EGFR has been overcome in the past by drugs that irreversibly bind to C773 (Kwak, et al., 2005; Kobayashi, et al., 2005). Alternatively, targeting downstream proteins may minimize the resistance conferred by an upstream mutation. A downstream target would eliminate the need for a drug to be effective against a particular mutant protein.

SIGNALLING ADAPTATION AND TRANSCRIPTIONAL ADAPTATION Both upstream and downstream mutations may be treated through combination therapy. If both ERBB2 and PI3K were mutated to be constitutively active, a combination of drugs that inhibit ERBB2 and PI3K may be an effective treatment. A potential effective treatment could solely target PI3K since it is downstream of ERBB2, provided that ERBB2 does not operate through alternate pathways (Engelman and Settleman, 2008). Given a mutant ERBB2 that bypasses PI3K, therapy involving inhibitors of

both proteins may be necessary. With respect to transcriptional adaptation, combination therapy may also be required. In the case of MET upregulation, inhibitors targeting both MET and EGFR proved to be effective and both were required in treatment, as inhibiting only one allowed for the other to propagate signals (Engelman and Settleman, 2008).

TRANSMEMBRANE DRUG TRANSPORT

OFF-TARGET DRUG BINDING

CONCLUSION The emergence of protein kinase inhibitor resistance causes many drawbacks of breast cancer treatment. It undoubtedly poses challenges for current successful anti-cancer therapies as breast cancer biology remains a diverse field. Protein kinases are common drug targets for cancer treatment as they are important for tumor cell proliferation and survival. However, the six deemed biological traits of cancer and its ability to acquire resistance to kinase inhibitors through protein mutations, signalling and transcriptional adaptation, transmembrane drug transport, offtarget drug binding, and transitional resistance make cancer a challenging disease to cure. Nevertheless, mutations may be overcome thr-ough rational drug design and targeting conserved areas of the target protein. Signalling and transcriptional adaptation could be bypassed by targeting downstream proteins or alternative pathways, as well as combination therapies. Transmembrane drug transportation offers a method to enhance cellular bioavailability and reduce the required therapeutic dose. On the other hand, off-target drug binding increases the therapeutic dose, yet lowering the levels of sequestering proteins can help remediate this. Transitional resistance entails a modified proteome and kinome, providing other pathways that may be exploited in treating breast cancer. Gaining a further understanding allows researchers to continually improve the efficacy of anti-cancer drugs and identify novel target molecules to overcome kinase inhibitor resistance in prospective clinical studies. The overarching goal of this review is to distinguish the key pathways

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Despite the presence of proteins that are able to sequester certain drugs, current treatments can still remain effective. Raising the dosage of a drug known to be sequestered may bypass resistance, though this could be dangerous for the patient receiving treatment. Conversely, the drug could be administered in tandem with a molecule that binds with sequestering proteins, like α1 acid glycoproteins, or those that reduce the concentration of sequestering proteins (Smith, et al., 2012). The anti-cancer drug tamoxifen is known to reduce α1 acid glycoprotein concentrations and may prove useful when given in conjunction with other anticancer drugs (Smith, et al., 2012). By reducing the propensity of α1 acid glycoproteins to drug binding, the sensitivity to the drug may increase and reduce the required dosage, potentially lowering the cost of treatment.

Regardless of the resistance conferred to inhibitors of various signalling pathways, including the PI3K/AKT, EGFR, and HER2 pathways, the transition to mesenchymal-like cells provides opportunities for treatments targeting pathways specific to mesenchymal cells. By transitioning, the cell attains a unique proteome, some of which may be targeted in the treatment of cancer (Martz, et al., 2014). The Slug protein is thought to have antiapoptotic activity (Inukai, et al., 1999). Targeting the Slug protein for inhibition could potentially induce apoptosis, providing a viable treatment for mesenchymal-like cancer cells.

Biochemistry

The presence of efflux and influx pumps provides numerous opportunities for overcoming resistance and reducing the side effects associated with drugs. Influx pumps may have therapeutic value, as activation of these pumps can potentially improve drug efficacy. As inhibition of the HOCT reduces cellular drug uptake, applying an activator of HOCT would increase drug uptake by cells (Thomas, et al., 2004). Similarly, as PGP can reduce intracellular drug concentrations, an inhibitor of PGP would lower net drug efflux and increase overall drug efficacy (Mahon, et al., 2000; Che, et al., 2002; Kotaki, et al., 2003). Though influx and efflux pumps may confer resistance to anti-cancer drugs, this also opens new avenues for treatments to increase cellular drug uptake, which may reduce side-effects. If a specific influx pump is overexpressed within the tumor, a treatment can be made more specific by targeting the influx pump, lowering the therapeutic dosage.

CELLULAR TRANSITIONS: EPITHELIAL TO MESENCHYMALLIKE CELLS

driving resistance for breast cancer cells in order to aid in the design of novel therapies.

ACKNOWLEDGEMENTS The authors would like to acknowledge Dr. John Hassell for his helpful advice on the various topics examined in this review paper.

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Biochemistry

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AUTHOR CONTRIBUTIONS This collaborative piece was divided between M.C. and J.D.. M.C. wrote the introduction, mechanisms of kinase inhibitor resistance and conclusion. J.D. wrote the abstract, mechanisms of kinase inhibitor resistance and potential strategies to overcome resistance.

Kwak, E.L., Sordella, R., Bell, D.W., Godin-Heymann, N., Okimoto, R.A., Brannigan, B.W., Harris, P.L., Driscoll, D.R., Fidias, P., Lynch, T.J., Rabindran, S.K., McGinnis, J.P., Wissner, A., Sharma, S. V, Isselbacher, K.J., Settleman, J. and Haber, D.A., 2005. Irreversible inhibitors of the EGF receptor may circumvent acquired resistance to gefitinib. Proceedings of the National Academy of Sciences of the United States of America, [ejournal] 102 (21), pp.7665–70. 10.1073/pnas.0502860102 Liu, Y. and Gray, N.S., 2006. Rational design of inhibitors that bind to inactive kinase conformations. Nature Chemical Biology, [e-journal] 2 (7), pp.358–364. 10.1038/nchembio799. Martz, C.A., Ottina, K.A., Singleton, K.R., Jasper, J.S., Wardell, S.E., Peraza-Penton, A., Anderson, G.R., Winter, P.S., Wang, T., Alley, H.M., Kwong, L.N., Cooper, Z.A., Tetzlaff, M., Chen, P., Rathmell, J.C., Flaherty, K.T., Wargo, J.A., McDonnell, D.P., Sabatini, D.M. and Wood, K.C., 2014. Systematic identification of signaling pathways with potential to confer anticancer drug resistance. Science signaling, [e-journal] 7 (357), pp.ra121. doi:10.1126/scisignal.aaa1877. Miller, S.M., Goulet, D.R. and Johnson, G.L., 2016. Targeting the Breast Cancer Kinome. Journal of Cellular Physiology, [e-journal] 232 (1), pp.53-60. https://doi.org/10.1002/jcp.25427. Sledge Jr, G.W. and Miller, K.D., 2003. Exploiting the hallmarks of cancer: the future conquest of breast cancer. European Journal of Cancer, [e-journal] 39 (12), pp.1668-1675. https://doi.org/10.1016/S09598049(03)00273-9. Smith, K., Behan, J., Matthews-Smith, G. and M., A., 2012. Alpha-1-Acid Glycoprotein (AGP) as a Potential Biomarker for Breast Cancer. In: Glycosylation. [online] InTech. Available at: <http://www.intechopen.com/books/glycosylation/ alpha-1-acid-glycoprotein-agp-as-a-potentialbiomarker-for-breast-cancer> [Accessed 28 Mar. 2019]. Statistics Canada, 2015. Cancer in Canada: Focus on Lung, Colorectal, Breast, and Prostate. [online] Available at: <https://www.statcan.gc.ca/pub/82-624x/2011001/article/11596-eng.htm> [Accessed 25 March 2018]. Tao, Z., Shi, A., Lu, C., Song, T., Zhang, Z. and Zhao, J., 2015. Breast Cancer: Epidemiology and Etiology. Cell Biochemistry and Biophysics, [e-journal] 72 (2), pp.333338. 10.1007/s12013-014-0459-6. Thomas, J., Wang, L., Clark, R.E. and Pirmohamed, M., 2004. Active transport of imatinib into and out of cells: implications for drug resistance. Blood, 104 (12) pp.3739–3745. https://doi.org/10.1182/blood-200312-4276. Yun, C.-H., Mengwasser, K.E., Toms, A. V., Woo, M.S., Greulich, H., Wong, K.-K., Meyerson, M. and Eck, M.J., 2008. The T790M mutation in EGFR kinase causes drug resistance by increasing the affinity for ATP. Proceedings of the National Academy of Sciences, [ejournal] 105(6), pp.2070–2075. 10.1073/pnas.0709662105.

LETTER TO THE ISCIENTIST

Radioresistance: Implications for Astrobiological and Medical Research JAMES LAI

Michael G. DeGroote School of Medicine, Class of 2019, McMaster University

SUMMARY

Received: 02/24/2019

Accepted: 09/23/2019

Keywords: radioresistance, radiodurans, DNA repair

astrobiology,

INTRODUCTION

Published: 11/17/2019 tardigrades,

Deinococcus

This damage can result in alterations of the nucleobases in the DNA molecule, or singlestrand and double-strand breaks (Figure 1). While many organisms have mechanisms by which they repair damage to DNA (e.g., MacRae, et al. 2015), large amounts of ionizing radiation can cause significant irreparable damage that ultimately leads to mutation or cell death (Sutherland, 2014). While it has generally been thought that high radiation environments are thus extremely dangerous to, and even incompatible with, terrestrial life, there in fact exist some organisms that have been observed to have the capability to survive in high radiation environments. These radioresistant organisms have been the subject of

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Ionizing radiation refers to radiation that contains enough energy to ionize atoms and molecules by raising the energy level of electrons such that they become unbound (Bacal and Romano, 2016). This includes electromagnetic radiation in the high ultraviolet, X-ray, and gamma range, but can also include subatomic particles, such as those that comprise cosmic rays (Nordheim, et al., 2015). In a biological context, ionizing radiation is dangerous to life due to its ability to penetrate living organisms and break chemical bonds in deoxyribonucleic acid (DNA) and other biomolecules (Melott and Thomas, 2011).

medicine,

Health Sciences

Ionizing radiation is commonly thought to be dangerous to, and even incompatible with, terrestrial life, due to its damaging effects on vital biomolecules like DNA. However, there exist organisms such as tardigrades, as well as several bacterial species, such as Deinococcus radiodurans, which have been observed to survive exposure to radiation levels in excess of 5000 Gy. This resistance is accomplished through differing mechanisms that, in general, involve processes to protect or rapidly repair molecules such as DNA that are damaged by high-energy ionizing radiation. In this Letter to The iScientist, the current understanding of the biology of radioresistant organisms is explored through a discussion of tardigrades and D. radiodurans as examples. Subsequently, it is argued that furthering our understanding of these organisms and the mechanisms by which they withstand ionizing radiation have important applications for a variety of fields. These include applications to the astrobiological search for life beyond Earth, as understanding the potential limits of radioresistance may allow for the expansion or constraint of possible environments in which extraterrestrial life may survive, guiding future life detection efforts. At the same time, mechanisms of DNA repair in radioresistant organisms may provide avenues of exploration in the search for interventions that may be applied to addressing DNA damage in humans, a problem associated both with cancer and aging.

Health Sciences

within the phylum, experimentally observed median lethal dosages of radiation survived by one species, Milnesium tardigradum, have been measured to be 5000 Gy of X-rays or 6200 Gy of heavy ions (Horikawa, et al., 2006). In comparison, a 4 Gy dose is considered lethal to a human (Hashimoto and Kunieda, 2017).

Figure 1: When DNA is exposed to ionizing radiation, it may be damaged in several ways, including changes to the nucleobases within the DNA molecule, or breaks in one or both strands of the DNA double helix. study into the mechanisms by which they are able to survive such hostile environments. Examples include the tardigrades, a phylum of animals commonly referred to as “water bears” that are ubiquitous on Earth, as well as several bacterial species such as Deinococcus radiodurans. Here, current knowledge of the radioresistance mechanisms of these organisms is reviewed; subsequently, it is argued that furthering this understanding may prove valuable for a variety of fields, including astrobiological and medical research.

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TARDIGRADES Tardigrades are a phylum of animals comprising various species of “water bears”, microscopic organisms that are ubiquitous on Earth, which have been found in diverse environments ranging from the oceans to alpine habitats (Weronika and Łukasz, 2017). Tardigrades are notable for being resistant to a variety of extreme conditions, having survived desiccation, extreme temperatures, and extremely high radiation experimental environments (Weronika and Łukasz, 2017). While the exact level of radioresistance varies between species

In tardigrades, radioresistance, along with resistance to other extreme conditions, is achieved through cryptobiotic states such as anhydrobiosis, in which the organism’s metabolism rate is significantly decreased and water content is reduced. Tardigrades possess various unique biochemical characteristics that impart increased resistance to these extreme conditions, such as the use of trehalose to stabilize biomolecules in the anhydrobiotic state (Weronika and Łukasz, 2017); these are also of great interest to various scientific disciplines, but fall outside the intended scope of this work’s argument as their relationship to radioresistance are either unclear or minimal. Among the various biochemical adaptations of tardigrades, it has been found that they produce a unique protein, Dsup, which suppresses DNA damage by radiation (Hashimoto, et al., 2016). At this time, however, the exact mechanism by which this protection is conferred is not yet clear. Thus far, it has been demonstrated that Dsupexpressing cells observed immediately following exposure to radiation, before significant repair would have time to occur, suffered less extensive DNA fragmentation, suggesting that Dsup functions to protect DNA from damage rather than being a component of a DNA-repair mechanism. Dsup is known to be a DNA-binding protein, and it has been found that it is necessary for the protein to bind to DNA in order to exert its protective effect (Hashimoto and Kunieda, 2017). Given the many unknowns in this process, research is ongoing to further elucidate the mechanism underlying Dsup-conferred DNA protection, as well as the evolutionary origins of this protein.

BACTERIA While tardigrades are considered extremotolerant in that they possess mechanisms by which they may survive extreme environments for a duration of time, several species of extremophilic bacteria are able to not only tolerate high radiation environments, but are able to thrive and reproduce normally in such situations (Rampelotto, 2013).

One of the most well-studied species of such bacteria is Deinococcus radiodurans. Experimentally, D. radiodurans has been observed to survive a variety of extreme conditions, including acute exposure to 7 kGy of radiation with a lethality rate of only 10% (Slade, et al. 2009); other studies have shown resistance to radiation from 5 to 15 kGy, depending on growth conditions (Minton, 1996). Deinococcus radiodurans possesses a variety of radioresistive mechanisms. First, the bacterium contains four copies of its genome when in the stationary phase, and this can increase further when the bacterium is undergoing active reproduction to eight to ten copies (Makarova, et al., 2001). Furthermore, the bacterium possesses a DNA-repair mechanism that allows it to repair double-stranded breaks, even when they are

numerous, as is the case after exposure to high doses of ionizing radiation. This is facilitated by the protein DdrB, which is hypothesized to function as part of an early stage of repair through 76 a process of single-stranded annealing. It has been shown that DdrB-deficient strains of D. radiodurans do not suffer reduced viability when exposed to lower-doses of radiation, suggesting that the single-stranded annealing process only becomes important at high ionizing radiation levels (De la 76 Tour, et al., 2011). This serves as a unique back-up process to the homologous recombination process facilitated by the protein RecA (Figure 2), which is a common protein to a variety of bacterial species; homologous proteins exist in other domains as well, such as Rad51 in yeast (Rajpurohit, et al., 2016).

Ionizing radiation

DdrB-mediated single-strand annealing

Health Sciences

High dose ionizing radiation

77 RecA-mediated homologous recombination

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Figure 2: A simplified representation of the hypothesized role played by DdrB single-strand annealing in the repair of radiation-induced double-strand breaks in D. radiodurans. The particular pathway mediated by DdrB, and thus unique to D. radiodurans, is highlighted with a dot-dashed line. DdrB begins the repair process in situations in which DNA is fragmented severely by high doses of ionizing radiation, before the products of single-strand annealing enter the RecA mediated pathways.

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Health Sciences

DISCUSSION

Radioresistant organisms and their unique biological processes represent an interesting subject of study for a variety of scientific disciplines. Better characterization of the mechanisms and limits of radioresistance can allow astrobiologists to better understand the range of potential environments in which life may survive on extraterrestrial bodies. While the Earth possesses a protective magnetosphere that shields the surface from ionizing cosmic rays (Chancellor, Scott and Sutton, 2014), and an atmosphere that attenuates much of the high energy ionizing electromagnetic radiation (Thomas, 2009), many other bodies in the Solar System of astrobiological interest do not have these characteristics. Mars, one of the subjects of most intense astrobiological study, no longer possesses a magnetosphere due to the cooling of its core (Lammer, Stumptner and Molina-Cuberos, 2002). As such, its surface is exposed to a high radiation level, estimated to be approximately 0.213 mSv/day, or 77.8mSv/yr (Simonsen and Zeitlin, 2017), compared to the annual dose on Earth of 2.4mSv/yr (Atri and Mellott, 2014). An exact conversion of this value into Gy is difficult to produce because a significant portion of the radiation on Mars is from cosmic radiation, and the Relative Biological Effectiveness (RBE) of cosmic radiation, required to calculate the conversion, is poorly defined. However, the RBE has been previously estimated at around 4 (Aceto, Leith and Baker, 1974), suggesting that considering radiation alone, organisms like tardigrades and D. radiodurans would be capable of withstanding the Martian radiation environment. Examples such as this suggest that a better understanding of mechanisms by which life may resist radiation damage can provide guidance on constraining the environmental parameters in the search for life in the Universe.

Besides aiding the search for life in the Universe, research into radioresistant organisms may produce benefits in medicine as well. Fundamentally, radioresistance is based on protecting DNA from damage, and repairing the damage that does occur when exposed to insults like radiation. DNA damage is also an important component of such processes as malignant transformation in the etiology of cancer, as well as in aging (Hoeijmakers, 2009). As such, a better understanding of the mechanisms by which these extraordinary organisms are able to protect and repair their genetic material may yield benefits in the search for interventions to protect human DNA, and mitigate the development of malignancy and the effects of cellular aging. For example, research on the tardigrade Dsup has shown that human cells transfected to express this protein were much more resistant to radiationinduced damage than wildtype cells (Hashimoto, et al., 2017). Whether such a cell-line is more resistant to DNA damage in general, or only in the case of high radiation exposure, has not yet been investigated, however. Further investigation may reveal whether interventions such as gene therapy involving this pathway or those from other radioresistant organisms like D. radiodurans may prove beneficial to human cells and may thus be of interest to medical research. In conclusion, research into the biochemical processes by which radioresistant organisms like tardigrades and D. radiodurans survive high doses of DNA-damaging ionizing radiation may prove beneficial for a variety fields, including clarifying the range of conditions in which the astrobiological search for life may be conducted, as well as elucidating potential avenues of investigation for medical research on cancer and aging.

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Health Sciences

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