JUST Volume I Issue I FALL 2016

Volume I Issue I

BadgerLoop: Innovation in Future Transportation

CRISPR-Cas9: An Introduction to Genetic Engineering

Drug Discovery: The Price of Pharmaceuticals

LETTER FROM THE EDITORS Dear Reader,

It is with great honor and excitement that we present the inaugural issue of the Journal of Undergraduate Science and Technology (JUST). This publication was founded to create a platform to showcase the cutting-edge research being conducted by undergraduates at UWMadison. JUST also strives to fulfill the Wisconsin Idea by making this research more accessible to members of the Madison community. UWMadison is a world leader in academic research and with over 10,000 undergraduates involved in research on campus, we believed it was time to create a way for undergraduates to share their work.

We began this journey as two researchers with an idea and have been delighted to see numerous members of the UW-Madison community step up and support the mission of the journal. Many undergraduates, graduate students, and faculty members across multiple disciplines have been instrumental in establishing JUST and producing this publication. Our heartfelt appreciation goes out to each and every person that has offered their time, resources and talent towards making this dream a reality. We are especially indebted to every undergraduate member of the JUST team. From our teams of Editors and Designers, who spent countless hours working on the content you are about to read, to our Marketing and Finance teams, who spread the word and connected us with the campus support we needed, each and every member of JUST has been absolutely essential in helping the journal get off the ground and creating what you see today.

As you read through the pages ahead, we hope that you are as inspired as we are by the ingenuity and determination of the UW-Madison faculty and students who are leading the way in creating technology and exploring solutions to solve some of today’s biggest problems as well as those who have strived to share this information with you in a meaningful way. We believe that UW-Madison is home to some of the greatest researchers in the world. Fueled by the passion of such researchers, we are excited to share with you what extraordinary discoveries have been made at UW-Madison. The publication of this journal marks the first step into an exciting future for JUST and research on campus, and we can’t wait to see where the journey will take us.

Sincerely,

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Stephanie Seymour

Edward Ruiz

Founder, Co-Editor in Chief

Founder, Co-Editor in Chief

TEAM Managing Editors Lucy Kohlenberg Bailey Flanigan Submission Manager Eva Shelton Content Editors Jeremy Mandel Gregory Zilberg Cecilia Moog Meng Lou Kelsey Nemec John Lynch Samuel Tesch Mansu Kim Madelyn Goedland Evan Hernandez Ryan Prestil Luke Valmadrid Tianxiao Han Alex Waldman Joshua Hicks Noah Johnson Claire Holesovsky Science Communication Editors Bailey Spiegelberg Emma Hazel Copy Editor Ruiqi Yan Design Director Alice Walker-Iampani Design Editor Anastasia Savelyeva Webmaster Tobin McGilligan Director of Interactive Media Coda Phillips Marketing Director Shelby Kuenzli

Marketing Staff Member Niki Patel Finances Director Zuf Wang Treasurer Amanda Padlo Peer Reviewers Alyssa Joachim Claire Holesovsky Emma Geiduschek Eric Clapper Evan Hernandez Helene Altmann John Lynch Joshua Hicks Kelsey Nemec Kendra Taylor Madelyn Goedland Mirelle Goetz Noah Johnson Ruiqi Yan Sam Tesch Samantha Sison Scott Odorico Sydney Walker Tianxiao Han Faculty Reviewers John Puccinelli Paul Wilson Grant Petty David Baum Advisory Board Jessica Newman Brianna Marshall Timothy Kamp Dietram Schueffele We would like to sincerely thank the Office of the Dean of L&S, the Associated Students of Madison, the Holtz Center for Science and Technology Studies, and the Johnson Fund for Innovation and Collaboration for financially supporting the production of JUST’s inaugural issue. Thank you!

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LETTER FROM THE EDITORS

2

TEAM

3

SPOTLIGHT FRANCIS HALZEN

6

EDITORIAL BADGERLOOP

8

Emma Hazel, Cecilia Moog

Luke Valmadrid, Meng Lou

10

FEATURES DRUG DISCOVERY

14

INSIGHTS QUANTUM COMPUTING

18

Tianxiao Han, Ryan Prestil

John Lynch

Noah Johnson

REPORTS REVIEW BIOMATERIALS

24

ARTCILE AFROCARPUS

29

ARTICLE CLIMATOLOGY SATELLITE

33

ARTICLE ARABIDOPSIS

39

Joshua Powel

Alex Goke

Jessica Gartzke

Kara Chung, Brendan Drackley, Emily Lynch, Matthew Pereyra

TABLE OF CONTENTS

FEATURES CRISPR CAS9

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Photo by: Cody LaCrosse

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/SPOTLIGHT

INTERVIEW WITH FRANCIS HALZEN Francis Halzen had no intention of staying at the University of WisconsinMadison; a six month visit turned into a 44 year long career as a professor and researcher here, a university Halzen describes as “one of the greatest, if not the greatest, universities in the world to do science.” Originally born in Belgium, Halzen earned his PhD and already had 3 years of work at the Cern Hadron Collider in Geneva, Switzerland behind him when he came to Wisconsin. He has since enjoyed a successful career as a professor of physics, but his greatest achievements are those in the research field of theoretical particle physics, specifically working with neutrinos.

A neutrino is a non-charged particle very similar in size and mass to an electron. Because they are not subject to electromagnetism that comes with having a charge, they can pass through matter. Other than these simple properties, there is not a copious amount known about neutrinos, making them intriguing to scientists and researchers of particle physics. Halzen is a pioneer in the field, having developed a unique and innovative technology to detect these neutrinos.

The system, called the IceCube Neutrino Observatory, is contained within a kilometer cube of ice almost 2 miles deep under south pole. This diagram depicts the massive and intricate nature of the system. This ice is hundreds of thousands of years old and is so completely pure that light can travel in it for over 100 meters, a quality that proved vital in the success of such a system. Within this ice, Halzen and his team placed thousands and thousands of tiny light sensors. When a neutrino is produced, there is a chance, however minute, that it will travel to earth and collide with the nucleus atom. This collision creates a small nuclear reaction, which in turn produces a blue light. This blue light is detected by the light sensors embedded in the ice, and can be used to map the source of the neutrinos. This data is then used to create a sort of ‘map’ of the galaxies; they provide informations from events such as exploding stars or black holes. The IceCube collaboration is the first system of its kind and one of the most important contributions to research on neutrinos ever made. Halzen and his team recently

“Science is still the domain where contributions are made by young people." celebrated 5 years of data collecting with this incredible system after 20 long years of conceptualizing and constructing. He shared with us an anecdote of the first round of data the IceCube system every collected: “We found 28 neutrinos, that was our first time. It’s like a digital picture with 28 points on it. And in fact you first collect this data, we know we have 28 events. And the first one we reconstructed….came 1º from the center of our own galaxy. And so, I went home totally depressed. Because, you know, to build this telescope and what I see is the sun! And so I went home completely depressed. But it turns out, it takes a about day of computing time to analyze these events, and so after a month of having looked at this, we found actually it didn’t really come from the center of our galaxy; they don’t even come from our galaxy at all. They come from outside the galaxy. We have good ideas where they come from, but not definite yet. And so that’s the excitement, we are accumulating data and getting more and more neutrinos,

and eventually we will see what we will see.

There’s no doubt about that.”

Professor Halzen is proof that scientific success happens as you go; as he and his team set out at more than 20 years ago, he had no idea if this idea would work.

He remembers walking up and down the hallway outside his office asking coworkers if they wanted to be a part of this idea he just had, and being met with overwhelming skepticism. However, today the IceCube is the leading data collection system in the world for this sort of information.

Professor Halzen also shared with us wise words of advice for young scientists and researchers everywhere: “Science is still the domain where contributions are made by young people. So my advice is, don’t wait until you’re old. It’s the only job you can do where you have a big advantage being young, because you haven’t been ‘brainwashed’. Once you’ve been brainwashed you think the same way as 10,000 other physicists.”

So when it comes down to it, if you have an idea go out and make it happen; you never know where you might end up.

- EMMA HAZEL AND CECILIA MOOG

IceCube.wisc.edu

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/FEATURE | Engineering

BADGERLOOP

Undergraduate Innovation in the Future of Transportation It’s rare for a student organization to achieve international success. It’s even rarer for such an organization to be primarily composed of undergraduates.

However, on our very own campus at UWMadison, one group has attained that rarer than rare status—the UW BadgerLoop.

Fueled by their passion for innovation, BadgerLoop was founded in response to a challenge, the SpaceX Hyperloop Pod Competition. Based on the concept by inventor, Elon Musk, SpaceX announced “an open competition, geared towards university students and independent engineering teams, to design and build the best Hyperloop pod” (Hyperloop, SpaceX).

Initially introduced in Elon Musk’s paper, Hyperloop Alpha, in 2013, the Hyperloop concept consists of a high-speed pod inside a low-pressure environment that is capable of traveling at speeds up to 760 mph. Representative of future cutting-edge transportation, the Hyperloop theoretically overcomes the limitations found in current conventional travel (e.g. automobiles, trains, planes).

In the summer of 2015, two seniors at UW-Madison, Brett Sjostrom and Tieler Callazo, eventual co-presidents of BadgerLoop, answered a call from the College of Engineering. Since the call, they assembled a founding team of tenacious undergraduates dedicated to tackling Musk’s challenge. By the time SpaceX released the competition guidelines in August, BadgerLoop was officially ready to begin designing their pod. Working endlessly, the team put in hours, then days, then weeks into the creation of their pod. Months passed, and by January, BadgerLoop was ready to showcase

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their design at the SpaceX Hyperloop Pod Competition Design Weekend in Texas. Facing against teams from MIT, Stanford, Delft, among others that were mainly composed of graduate students, BadgerLoop proudly returned to Madison as third place winners, securing them a final spot on SpaceX’s Hyperloop test track this summer in California.

Though it was primarily composed of mechanical engineers, BadgerLoop now has almost 100 members from various fields and 12 faculty advisors. With members from computer engineering, electrical engineering, physics, business and more, BadgerLoop not only broadened their diversity, but also their scope of vison. As the structure of the organization is enhanced, the group is able to dedicate

various factions to multiple areas of pod design and implementation.

On top of its success, BadgerLoop also offers a rich source of hands-on experience for its student members. Many student organizations promise leadership and administrative guidance, however, few can also act as a curricular medium.

BadgerLoop team member, Cole Pierce, admitted, “Honestly, it’s probably one of the best industry-learning experiences. I realized that you’re gonna learn a lot of stuff in classes, but as far as getting your hands dirty...I’ve learned more in the club than everything combined.”

Besides providing an enriched individual experience, BadgerLoop works on highlighting the importance of teamwork, a value that’s grossly underrated in the

Team member, Sidney Smith, presenting at SpaceX compeition.

Photo by Eric Schirtzinger, originally published on News.wisc.edu

View from inside Badgerloop’s Virtual Reality experience. Created by Felix Tsao engineering industry, as stated by Sjostrom himself. This is only strengthened by BadgerLoop’s diversity in background and experience. Although disagreement is inevitable in any true collaboration, Sjostrom emphasizes that “…clashing is what leads to such a beautiful design.” Like a match that can’t produce a flame without friction, creativity can’t be produced without discord.

In addition to embracing diversity and teamwork, BadgerLoop serves as a source of inspiration for the students. Despite the fact that not all members have extensive engineering knowledge, they all have one thing in common—the desire to make a difference. The empowering feeling of accomplishment and their absolute dedication to their passion is what drives the members of BadgerLoop in their mission It is the attitude and mindset members like this, along with its achievements that make BadgerLoop so desirable to

sponsors. The organization boasts an impressive list of backers, including Cirrus Aircraft, UTC Aerospace Systems, and Saietta. Sjostrom noted, “These companies are what make it possible to build this thing [the Hyperloop pod]. They see our passion for this project and our drive for innovation.”

BadgerLoop attributes their success as an organization to its group personality.

Sjostrom insists, “We’re not afraid to be different from everyone else. In fact, we want to be different.” Members of the group may be young, but they carry themselves with a drive and confidence that make BadgerLoop function as a mature and seasoned team. As the members continue perfecting their Hyperloop pod, UWMadison, and the future of transportation, roots for BadgerLoop and its bright minds and even brighter future.

Citations: Hyperloop. (n.d.). Retrieved March 25, 2016, from http://www.spacex. com/hyperloop

Keep yourself updated with the BadgerLoop squad: Facebook: @BadgerloopTeam Twitter: @badger_loop Instagram: @badgerloop/ Support BadgerLoop: Contact, membership, and donation information can be found on https://badgerloop.com.

- LUKE VALMADRID AND MENG LOU

Experience Badgerloop yourself by downloading BadgerloopVR on your smartphone.

Image and app created by Felix Tsao Badgerloop's Pod Design Photo by: BadgerLoop

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/FEATURE | Biotechnology

The sharpest tool in the biotech shed An Introduction to CRISPR -Cas9

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OVERVIEW | In the last three years,

the gene editing tool CRISPR-Cas9 has permeated nearly every crevice of the biological sciences and has revolutionized the way researchers think about the genetic code. However, the history behind CRISPR-Cas9 stretches much longer than three years, and it is proving to be a much more flexible platform than was ever imagined. At the same time, a few ethical controversies have been raised about the use of CRISPR-Cas9 in engineering human life, particularly the germline. As science moves into an era of personalized medicine and new approaches to cellular and gene therapies, CRISPR-Cas9 will undoubtedly solidify its place at the forefront of these exciting technologies. Following is an overview on the development and applications of CRISPR-Cas9 and the main ethical questions currently under discussion.

DEVELOPMENT OF CRISPR-Cas9 |

Since the discovery of the role of DNA in the 1940s, the ability to manipulate the genome in a precise manner has been a dream of many researchers.

However, CRISPR-Cas9 is only the latest breakthrough in a long line of tools designed to achieve this dream. In the 1990s, the first tools were developed to induce a double-stranded break at a specific place in the DNA molecule in living cells. Reengineering Zinc Fingers, a type of protein found in many organisms which is known to bind specific DNA sequences, allowed specific targeting and cutting of a locus. The damaged cell then uses its own innate repair mechanisms to fix the break, leading to one of two distinct results. First, and most often, the cell performs non-homologous end joining (NHEJ) which typically leads to extensive and unpredictable mutations. Because it is so error-prone, NHEJ is ideal for gene knockout research, in which a gene is disrupted in order to assess the resulting effect on the cell or organism. However, it is too inexact for more precise applications.

As an alternative, a strand of “donor DNA” with sequences that match both sides of the cut site can be incorporated.

In homology-directed repair (HDR), this donor DNA recombines with the cut DNA strands to be inserted into the genome.

Researchers have used this method both to repair a mutation by providing the correct DNA sequence and to insert an entirely new gene into a specific host locus, such as a fluorescent protein.

Meanwhile, CRISPR-Cas9 was discovered in bacteria as an immune system against invading viral DNA. When a virus inserts itself into a bacterium, the bacterium

"Crystal Structure of Mn-bound S.pyogenes; Cas9" by Nevit Dilmen is licensed under CC BY 3.0 Courtesy of Creative Commons recognizes it as foreign and incorporates a short segment of viral DNA into its own genome. The bacterium then produces RNA transcripts from this segment, which are used by the Cas9 protein to recognize foreign DNA. When the RNA transcript matches invading viral DNA, the Cas9 protein cuts both strands of the DNA, effectively degrading the virus’s DNA while protecting its own host DNA. The “CRISPR” in CRISPR-Cas9 refers to the bacterial immune system as a whole, and Cas9 is the protein itself which cuts the DNA. In early 2013, the CRISPR system was adapted for use in mammalian cells by making an artificial single-guide RNA (sgRNA) to match with the target sequence in the cell’s genome. While Zinc Fingers require a new protein to be made for each individual cut site, the Cas9 protein couples with any custom sgRNA, drastically reducing the time to design and complete a new edit and allowing largescale genetic screens to be performed.

Additionally, CRISPR-Cas9 can be used to edit cells both in a dish (in vitro) and in full organisms such as mice (in vivo).

But CRISPR-Cas9 is not perfect. Because the sgRNA targets 20 base pairs, similar sequences within the genome can result in off-target cuts. In rare cases, these cuts occur within another gene, leading to unintended effects. However, with over one trillion possible combinations of twenty A, C, T, and G bases, these off-target effects are rare, and many computational tools have been developed to screen the genome and provide “scores” for potential sgRNA

targets. Additionally, the effectiveness of CRISPR-Cas9 to access the target site depends on variations in the target sequence itself, as DNA sequences with additional G and C base pairs are more tightly bound than those with additional A and T base pairs, and large segments of DNA are inaccessible due to binding to packaging proteins. A good score for an sgRNA incorporates all of these factors.

An emerging area of research is mutating the Cas9 protein in order to increase its specificity or alter its function. For example, modifying the part of the protein responsible for cutting each strand does not disrupt the ability of Cas9 to bind to the sgRNA or to localize to the target sequence in the DNA. Such a change can stop the Cas9 from cutting one strand, causing a single-stranded “nick” in the DNA which can be exploited for enhanced specificity. Likewise, altering both cutting sites can completely eliminate cutting activity, known as a “nuclease-dead” Cas9, or dCas9. When dCas9 is localized to a gene, it physically impedes innate DNAto-RNA transcription, effectively silencing the gene. This can be an efficient method to understand the function of a gene without permanently altering its sequence.

Transcriptional activators can also be conjugated to a dCas9, which results in gene transcription when using an sgRNA targeting a promoter region for a gene, even one not normally expressed.

Biomedical and Biotechnology Applications |

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Empowered by the efficient gene editing tool of CRISPR-Cas9, biomedical researchers are now able to apply many new techniques that hold great promises in advancing human health. Gene insertion and deletion allows researchers to manipulate the expression of important proteins and metabolites in cells, while controlling gene expression permits researchers to better understand the

copies of normal or ‘healthy’ genes, biomedical researchers have proposed that CRISPR-Cas9 is sufficiently efficacious, accurate, and adaptable to perform true therapeutic genome editing and fix a native locus. Dr. Timothy Kamp, a physicianscientist on campus, emphasizes that CRISPR-Cas9 has the potential to become a powerful therapeutic tool for treating many genetic diseases, such as muscular

"CRISPR-Cas9 has enabled modeling of numerous diseases and processes, including cancer, neurodegeneration, and cardiovascular disease." function of these genes and the complex biology of the cell. Moreover, as a nearly universal gene editing tool, CRISPRCas9 greatly broadens the applicability of common model organisms and enhances the efficiency of engineering new model systems.

Besides applying CRISPR-Cas9 for understanding the fundamental genetics of an organism, biomedical researchers use CRISPR-Cas9 gene editing as a powerful tool for understanding human diseases. By introducing genes known to be implicated in disease into cells in vitro, CRISPR-Cas9 serves as a powerful tool for disease modeling. For example, researchers use CRISPR-Cas9 in conjunction with human pluripotent stem cells to create organoids that express disease-specific genes in a culture dish.

CRISPR-Cas9 has enabled modeling of numerous diseases and processes, including cancer, neurodegeneration, and cardiovascular disease. In addition to being able to observe and to understand the progression of disease through these models, CRISPR-Cas9 is also opening new avenues for the development of novel drugs. Pharmaceutical companies are testing the safety and efficacy of new drug candidates in these disease models, facilitating faster, more cost-effective, and safer drug development.

CRISPR-Cas9 is not only efficient in introducing disease genes into cell models in vitro, but it also holds great promise for the insertion of genes to correct deleterious mutations. The human genome consists of approximately 25,000 known genes, of which more than 3,000 are linked with diseases when mutated; these genes either lack necessary functionality or generate malfunctioning proteins and metabolites with deleterious effects. While conventional gene therapy aims to introduce additional

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dystrophy and cystic fibrosis.

Apart from editing human cells for biomedical applications, the adaptability of CRISPR-Cas9 enables scientists to edit cells of nearly all organisms, ranging from bacteria to simple animals to complex vertebrates. It is thus possible to harness these organisms for their desired properties in nearly any biomedical or biotechnology application. For example, CRISPR-Cas9 has been used in yeast and in plants to engineer biofuel and pharmaceutical production. Industrial companies have

CRISPR-Cas9 gene editing complex from Streptococcus proposed the use of CRISPR-Cas9 in the food industry, with applications ranging from engineering the bacteria that convert milk into yogurt to engineering the dairy cows themselves.

Another revolutionary application of CRISPR-Cas9 is the gene drive. The gene drive increases the probability that an offspring will inherit a particular copy of an allele. Over a few generations, the gene drive system spreads the allele in the natural population. One key application of the gene drive is to eliminate pest-borne diseases such as malaria and dengue fever by targeting the disease vectors, namely

mosquitos. Conventional gene engineering tools such as homologous recombination have very low efficiency in engineering the desired allele in mosquitoes. CRISPR-Cas9 allows feasible gene drive engineering, which has been effective in driving the spread of particular genes in laboratory animals such as fruit flies. Moreover, the gene drive has been proposed to reverse pesticide and herbicide resistance in pests and weeds in the agricultural industry and to reduce invasive species, aiding in the conservation of native and rare species.

Ethical Concerns |

Just like many other new technologies, there are serious concerns about CRISPRCas9 applications. As an efficient tool for editing the genome of almost any organism, the use of CRISPR-Cas9 has been criticized as scientific overreach.

Critics have questioned both the safety of modifying human genomes for medical applications and the safety of genetically modified organisms in agricultural applications.

The overarching ethical question surrounding CRISPR-Cas9 is the extent to which scientists should manipulate nature.

Since CRISPR-Cas9 makes gene editing relatively fast and efficient, thus reducing the technological barriers in creating new transgenic organisms, scientists today have more power to manipulate living organisms than ever before. For example, biologists recently proposed the use of the gene drive to fight the spread of Zika virus, which has been linked to thousands of severe neurological birth defects over the last year. Dr. Kate O’Connor-Giles, a biomedical researcher on campus who studies fruit fly genetics through CRISPRCas9, has indicated that the recent Zika outbreak could be a trigger for scientists to consider using the gene drive to combat mosquito-borne diseases. Dr. O’ConnorGiles suggested that there are more ethical complexities apart from the extent of the manipulation considered, and that the individuals involved in these processes are a crucial component. In her words, “Who should make that decision? Should scientists, bioethicists and citizens in Zikaafflicted areas have more say?” CRISPRCas9 itself does not give answers to these questions. Therefore, the ethical question of manipulating nature while utilizing gene editing and CRISPR-Cas9 is presently being debated.

Human genome editing by CRISPR-Cas9 is associated with additional ethical concerns. Dr. Krishanu Saha, a biomedical engineering researcher on campus, attended the International Summit on Human Gene Editing in December 2015.

According to Dr. Saha, the major concern surrounding CRISPR-Cas9 is the editing of human germline cells and human embryos.

Last year, a group of researchers at Sun Yat-sen University in China reportedly used CRISPR-Cas9 in unviable human embryos to demonstrate its ability to correct genetic diseases. However, the group concluded that there were too many unpredictable and variable effects to use CRISPR-Cas9 in viable embryos at this time. Furthermore, at the Francis Crick Institute in London, a group of researchers in February 2016 managed to obtain approval to edit viable human embryos using CRISPR-Cas9 to investigate early human development, under the stipulation that they may not keep the embryos longer than seven days.

Importantly, neither of these studies have allowed genetically engineered embryos to implant and develop. Doing so would

to create potentially harmful biological agents. However, the majority of the scientific community does not foresee these threats in the short term. The current regulations of gene editing are undergoing intensive revision to address the long term implications of the technology.

Despite such concerns, CRISPR-Cas9 has already enabled incredible scientific advancement and continues to hold great promise, and with continued development of CRISPR-Cas9, national and international regulations will be needed to address ethical concerns and the future of the field.

The scientific community has been actively responding to some of the above concerns.

For example, the National Academy of Sciences and the National Academy of Medicine collaborated on a gene-editing initiative to inform both researchers and the general public about the emerging

"As an effecient tool for editing the genome of almost any organism, the use of CRISPR-Cas9 has been criticized as and decisions surrounding scientific overreach." technology its use. While risks will always exist, raise many additional ethical issues, such as the potential misuse of the technology for eugenics or enhancements, and the inequity of access to the technology for people of different socioeconomic statuses. Therefore, the consensus by the International Summit participants was that gene editing in human embryos and germline cells, while controversial in its own right, should be restricted to research purposes and these embryos should not be allowed to develop beyond early stages.

The MIT Technological Review recently reported that gene editing was added to

managing CRISPR-Cas9 through proper regulations and oversight will minimize these risks while permitting CRISPR-Cas9 to benefit society tremendously. CRISPRCas9 has expedited biotechnological advances within the last few years, enhancing understanding of organismal development and disease progression, and in the next few decades, ideally it will be commonplace to receive truly curative treatments for genetic diseases.

- TIANXIAO HAN AND RYAN PRESTIL

Acknowledgements

We would like to thank Dr. Timothy Kamp, Dr. Kate O’Connor-Giles, and Dr. Krishanu Saha for the knowledgeable conversations during our interviews.

1.

2.

the list of threats posed by “weapons of mass destruction and proliferation” in the Annual Worldwide Threat Assessment Report of the U.S. intelligence community.

The development of CRISPR-Cas9 as an efficient gene editing tool has made gene editing much easier because of the low cost and relative ease of use.

Therefore, this concern arises from the potential for CRISPR-Cas9 to be used

3.

4.

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Cell stem cell. 2013 Dec 5;13(6):659-62.

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/FEATURE | Pharmaceuticals

Discovery: The Price of Pharmaceuticals The industry of modern medicine dreams of once-a-day cure-alls for every ailment imaginable. An orally available, minimally harmful substance that relieves a patient of their ills is the end goal of a lengthy, costly, brutally bureaucratic, scientific endeavor.

Modern pharma has given society hope against infections, HIV, and many cancers. Simultaneously, the modern pharmaceutical industry has come to be one of the most profitable sectors ever. Profits from “wonder drugs” like the Gilead-developed hepatitis C treatment, Harvoni, are projected in the billions of dollars [1]. With the majority of hepatitis C patients being of the Baby Boomer generation [2], this cost is indirectly shifted on to taxpayers through Medicare.

Drug pricing routinely comes under attack by both the public at large and politicians in D.C., and currently, the poster child for high costs is the

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aforementioned Harvoni, which costs an estimated $94,500 for a round of treatment [3]. Gilead defends the pricing by pointing to the clinical data that shows an efficacy rate of 94-97% and some evidence of near 100% cure rate when combined with another anti-viral drug, ribavirin [4]. This,

combined with the avoidance of the older pegylated interferon treatments, with their corresponding need for constant injections and harsh flu-like side effects, is claimed as a justification of the price. While the debate rages on for the foreseeable future, let’s dig into what goes into bringing a drug to market.

The first step in drug development is biological research. How does an illness or disease work against the host? What are the symptoms? What is the biochemical interaction that causes it? In some cases, this may be well-understood and studied; in others, further work needs to be done, and this is not always a process with a clear finish line. One can look to the history of cyclosporine, an immunosuppressant used for decades in organ transplants. Cyclosporine indirectly inhibits a signaling pathway in T cells by binding to one protein—

cyclophilin—which, in turn, inhibits calcineurin. Cyclosporine was approved for use in 1983 after clinical studies headed by Dr. Thomas Starzl at the University of Pittsburgh [5]. The biology was not fully understood until nearly a decade later when a group led by Dr. Stuart Schreiber of Harvard and Dr. Gerald Crabtree of Stanford published results showing cyclosporine and a similar immunosuppressant, FK-506 (now known as Tacrolimus), formed a complex with a calcineurinbased signaling pathway in immune system cells [6]. Once the biology is

Fig 1. Cyclosporine (yellow) bound to Cyclophilin A. understood to a reasonable degree, small-molecule designing can begin.

There are two main approaches with drug design, the combinatorial approach (sometimes called indirect or ligand-based), where large amounts of variations on a core structure based on known binders to the target in question are tested in assays, or structure-based design built around the understanding of the biological target. Requiring specific knowledge and workflows, both approaches have their strengths and weaknesses.

Ligand-based design requires a team to research similar, small molecules that bind to the target. If there are known ligands, it is a matter of combinatorial chemistry to find new leads. Combinatorial chemistry is the systematic search of “chemical space”, i.e. every combination of different functional groups a team can install on the lead structure. Structure-based design requires an intimate knowledge of the target itself. For example, knowing the complete structure and active site of a targeted enzyme would be vital to designing a smaller number

of potentially potent ligands. While structure-based could conceivably lead to less overall chemistry, it requires a large amount of investment upfront in structural biology research rather than in the synthetic chemistry. The benefit of structural biology over synthetic chemistry is mostly cost. If the target is understood well, the only biology to be done is in testing new compounds.

Additionally, structure-based design utilizes biophysical protein-ligand simulations to provide more biological

These rules provide a good basis for estimating how robust a compound may be in vivo. As assay information is gathered, a more complete picture of the optimal ligand is formed, and eventually, a class of promising compounds is amassed.

Gaining FDA approval is timeconsuming and costly for a company and requires pharmacological and toxicological data; therefore, animal testing is commonly undertaken to measure the safety of promising

"Virtual screening methods are cheap and easy thanks to advances in highthroughput and high-performance drug leads. Usually, large dogs are computing." information to the medicinal chemistry team. Using these in silico, virtual screening methods are cheap and easy thanks to advances in high-throughput and high-performance computing [7]. Unfortunately, this also requires substantial knowledge of the target’s structure. If the structure is known from previous research, excellent! If not, it may take a talented team of structural biologists valuable time to elucidate.

Once the assays have found “hits” against the target, the compound is optimized by the team. Many factors influence an optimum compound from lipophilicity to the pKa of the compound. As oral administration is ideal, having an air-stable compound that can survive the acidic environment of the stomach and be absorbed into the body, reaching the target with few issues, while remaining active and having high bioavailability is key.

The oft-cited “Rule of 5” from longtime Pfizer chemist Charles Lipinski [8] gives rules of thumb that help determine the “drug-likeness” of a compound and the likelihood of it being orally active. The four rules are: 1. No more than 5 hydrogen bond donors.

2. No more than 10 hydrogen bond acceptors.

3. A molecular mass less than 500 g/ mol.

4.

And a logP not greater than 5.

employed and pharmacokinetic data is gathered along with tests for possible toxicities. Liver or neurotoxicities are especially worrisome as they can scuttle a drug’s hope for phase I trials. If results still seem promising, an Investigational New Drug (IND) application will be filed with the FDA. This document includes all of the clinical data gathered up until that point through animal testing as well as the planned clinical trial protocols and manufacturing information.

This last category can be especially troublesome as a large amount of the possible drug is needed. Scaling up from milligrams to kilograms is a daunting proposition as the FDA is looking for proof of compound purity while a company is trying to balance that with an efficient process. If a compound has solubility issues in vivo, different delivery matrixes must be tried in order to optimize bioavailability [9].

If the IND application is approved, the lead compound is submitted for clinical trials. The three phases are distinct and focus on specific goals: Phase I involves testing for safety issues within a small group (usually under 100 healthy volunteers). More pharmacokinetic data is gathered and possible dosing regimens are tested and verified. 35.5% of compounds that enter this phase do not move on [10].

Phase II brings in a few hundred

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patients with the disease in question and compares them to a control in a double-blind study. Normally, this control is either a placebo or the currently approved standard treatment.

Efficacy and safety are evaluated through activity and possible side effects. Dosing regimens are optimized further in preparation for both Phase

on average, this is a monumental undertaking for both the company to gather and the FDA to analyze all of the data. The FDA will look to answer some fundamental questions from the NDA such as how safe is the drug? Is its efficacy high enough to justify any risks associated with treatment? Does the proposed label reflect the data

"Biologists, toxicologists, chemists, doctors, and other healthcare professionals work in tandem for years to prove the efficacy and safety of the compounds that keep society healthy."

III and final approval. Phase II has the highest attrition rate with 67.6% of compounds failing to advance [10].

Phase III is the most expansive in terms of patient numbers, and therefore, most expensive, phase.

Substantial amounts of data are collected on each patient at hundreds of testing sites across the country. With such a large number of participants, Phase III is often used as a stress test for the manufacturing process devised by the company as progress from this point forward will require a full-scale industrial production. The statistics lean slightly in the compounds favor with 60.1% of compounds succeeding [10].

There are several ways that this process can be fast-tracked by the FDA for breakthrough therapies. The fast track has been utilized in the past for HIV treatments and more recently for Alzheimer’s treatments.

Compounds that receive this designation are subject to increased scrutiny in addition to more lenient regulations, such as a rolling review of the application rather than reviewing the entire application post-Phase III trials [11].

If, and only if, the compound passes all three phases, a New Drug Application (NDA) can be filed. This application involves every piece of data collected over the development cycle. As drug development takes twelve years

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gathered through the clinical trials? Is anything conspicuously missing? As this is a matter of public health and safety, the documentation on how to construct an NDA is extraordinarily thorough and comprises at least 14 separate sections, depending on if the compound in question is novel or a previously approved formulation seeking a new designation. If the FDA is satisfied with the NDA, the compound is approved. At this point in development, there are few hurdles left, so the approval rate jumps to 83.2% [10]. With an approved compound in hand, a company must move on to selling its latest product.

An army of sales representatives is trained, insurance companies are negotiated with, and presentations are made to doctors across the country informing them of why they should prescribe it over any other available treatments.

Taking in this development cycle, the $2.6 billion or so estimate from Tufts Center for the Study of Drug Development [12] does not seem as far-fetched as it originally did. A small army of biologists, toxicologists, chemists, doctors, and other healthcare professionals work in tandem for years to prove the efficacy and safety of the compounds that keep society healthy. The FDA realizes the enormous undertaking it asks of researchers and provides market exclusivity on top of patent protection of new compounds in order to ensure

profitability. A new chemical (NCE) is standardly awarded five years of exclusivity. For a previously approved compound now seeking a new label for treating a new disease, three additional years is given. A third type of exclusivity, the orphan drug (ODE), is both one of the more controversial designations and most sought after as it gives seven years of exclusivity [13] [14]. Orphan drugs are drugs for diseases with small patient populations such as rare genetic disorders. These years of exclusivity are to reward companies for their hard-work and effort in bringing a compound to market in profits. Calls for more price regulations are regular but, for better or worse, the system in place is built around the idea of consumer safety first and foremost rather than price regulation.

The FDA does indeed focus on trying to bridge gaps in treatments as evidenced by the Orphan Drug Act of 1983 that created the ODE designation above. The intent of the act was to incentivize research in areas that would not normally be profitable due to factors such as low patient numbers. Clinical trials are subsidized and tax credits may be awarded along with modifications to the normal approval process [14].

For example, a possible drug for a

disease that affects less than 200,000 people is given leniency in Phase III due to a limited patient population.

This same act also highlights some of the ways companies follow the letter of the law rather than the spirit. Enter the Unapproved Drug Initiative (UDI) of 2006. The UDI was conceived as a way to encourage testing of older compounds for efficacy and safety, thereby filling in gaps for treatment

options and helping to remove unsafe products from the shelves [15]. Coupling the Orphan Drug Act with the UDI, URL Pharmaceuticals sought exclusivity on colchicine for treatment of gout. Colchicine was used as far back as ancient Greece but had never been through the FDA approval process. Instead, URL received exclusivity to colchicine for three years based on a review of previously collected data and some preliminary pharmacokinetic data they themselves collected. Utilizing the Orphan Drug Act, URL was able to acquire seven years of exclusivity on top of the gout exclusivity when colchicine was approved to treat familial Mediterranean fever. Consequently, the price for colchicine increased from $.09 a pill to $4.85 [16]. Many readers will recognize this as a similar tactic to the Turing Pharmaceutical incident last year involving a toxoplasmosis treatment. Many would lump Gilead’s behavior with Harvoni’s price in with Turing and URL, but that would be insulting to the contingent of researchers who worked on the science behind the scenes.

While abuse of the regulatory system does occur, it stands to reason that high drug prices are a consequence of the rigorous studies they are subjected to. While Gilead may be lambasted for the sticker price on Harvoni, pulling back the curtain to see the inner workings of development shines light on their rationale.

When it takes twelve years and $2.6 billion dollars on average to bring a compound to market, some reward is needed, but how much reward is the question. Opponents lambast high prices as naked profiteering off of human suffering while a company like Gilead sees the price as recouping their investment, considering that only 19% of all programs make it through to approval [17]. The research and development costs alone for a pharmaceutical company like Pfizer, Merck, or Abbvie grow seemingly insurmountably; real drug research costs real money, and unfortunately, someone has to foot the bill.

-JOHN LYNCH

•

• •

• •

1.

2.

3.

4.

5.

6.

7.

8.

Further Readings Werth B. The billion-dollar molecule: one company's quest for the perfect drug. New York: Simon & Schuster; 1994.

Werth B. The antidote: inside the world of new pharma. New York, NY: Simon & Schuster; 2014.

Kerns EH, Di L, Kerns EH. Drug-like Properties: Concepts, Structure Design and Methods.

Academic Press; 2008.

Thomas JR. Pharmaceutical Patent Law. 3rd ed. Arlington, VA: Bloomberg BNA; 2015.

Stevens E. Medicinal chemistry: the modern drug discovery process. Upper Saddle River, NJ: Pearson; 2013.

9.

10.

11.

12.

13.

References

Gilead Sales Soar on Hepatitis Drugs. WSJ. Dow Jones & Company; 2015. http://www.wsj.com/articles/ gilead-sales-soar-on-hepatitisdrugs-1438114949 Hepatitis C: Why Baby Boomers Should Get Tested. HEPATITIS Why Baby Boomers Should Get C Tested.

Centers for Disease Control and Prevention; 2015. http://www.cdc. gov/knowmorehepatitis/media/pdfs/ factsheet-boomers.pdf Ledipasvir-Sofosbuvir (Harvoni).

Hepatitis C Online. International Antiviral Society-USA; http://www. hepatitisc.uw.edu/page/treatment/ drugs/ledipasvir-sofosbuvir U.S. Food and Drug Administration.

FDA approves first combination pill to treat hepatitis C. U.S. Food and Drug Administration; 2014. http:// www.fda.gov/newsevents/newsroom/ pressannouncements/ucm418365.htm Starzl TE, Klintmalm GBG, Porter KA, Iwatsuki S, Schröter GPJ. Liver Transplantation with Use of Cyclosporin a and Prednisone. N Engl J Med. 1981;305: 266–269.

Schreiber SL, Albers MW, Brown EJ. The cell cycle, signal transduction, and immunophilin-ligand complexes. Acc Chem Res. 1993;26: 412–420.

Shoichet BK. Virtual screening of chemical libraries. Nature. 2004;432: 862–865.

Lipinski CA, Lombardo F, Dominy BW, Feeney PJ. Experimental and computational approaches to estimate solubility and permeability in drug discovery and development settings.

Advanced Drug Delivery Reviews.

14.

15.

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1997;23: 3–25.

Kerns EH, Di L. Drug-like properties: concepts, structure design and methods: from ADME to toxicity optimization. Amsterdam: Academic Press; 2008.

Hay M, Thomas DW, Craighead JL, Economides C, Rosenthal J. Clinical development success rates for investigational drugs. Nat Biotechnol.

2014;32: 40–51.

U.S. Food and Drug Administration.

Fast Track, Breakthrough Therapy, Accelerated Approval, Priority Review.

2014. http://www.fda.gov/forpatients/ approvals/fast/default.htm Dimasi JA, Grabowski HG, Hansen RW. Innovation in the pharmaceutical industry: New estimates of R&D costs.

Journal of Health Economics. 2016;47: 20–33 Eisenberg RS. Patents, Product Exclusivity, and Information Dissemination: How Law Directs Biopharmaceutical Research and Development. Fordham Law Review.

2002;72: 477–491.

Patents and Exclusivity. U.S. Food and Drug Administration; 2015. http://www.fda.gov/downloads/ drugs/developmentapprovalprocess/ smallbusinessassistance/ucm447307. pdf U.S. Food and Drug Administration.

Unapproved Prescription Drugs: Drugs Marketed in the United States That Do Not Have Required FDA Approval.

U.S. Food and Drug Administration; 2015. http://www.fda.gov/drugs/ cecomplianceregulatoryinformation/ enforcementactivitiesbyfda/ cementactionsonunapproveddrugs/ default.htm Kesselheim AS, Solomon DH. Incentives for Drug Development — The Curious Case of Colchicine. New England Journal of Medicine N Engl J Med. 2010;362: 2045–2047.

Scannell JW, Bosley J. When Quality Beats Quantity: Decision Theory, Drug Discovery, and the Reproducibility Crisis. PLoS ONE. 2016;11.

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/INSIGHTS | Quantum Computing

THE COMPUTER THAT KILLED SCHRÖDINGER’S CAT We have reached a point in the technological development of computers where the logic gates and wires, which make up the circuits that perform all of the operations that run computers, can be etched onto integrated circuit silicon chips of less than a micron across (0.001 millimeters). Following Moore's Law, an observation (not a physical law) by Intel cofounder, Gordon Moore, which estimates that the number of transistors on an integrated circuit chip, an approximate measure of microprocessor complexity, doubles roughly every 18 months, it is inherent that in the near future these same logic gates will soon be on the scale of single atoms. As a result, we will need quantum mechanics to describe these new “quantum computers”. The basis of quantum information is qubits which are fundamentally different than bits, the basic units of classical computational information, due to the property of quantum superposition. While bits can only be prepared in one of two physical states (ex. 0 or 1), qubits can exist in a superposition of both states. This means qubits can exist with certain probabilities of being in each individual state, 0 and 1, at the same time, because of this principle of quantum superposition n qubits can store 2n values at once compared to n values in n bits.

Figure 1. Bloch Sphere, a geometric representation of quantum superposition. Each qubit state is a point on the surface of the sphere.

Courtesy of Creative Commons

Due to superposition and other properties of quantum mechanics, we can run quantum algorithms at a much higher efficiency than their classical analogs; although, as of now this is only true for some algorithms. One example is Peter Shor's famous paper (1) proving that a quantum algorithm could find prime factors of an integer in polynomial time compared with much slower sub-exponential time for a classical computer Another enhanced quantum algorithm was depicted in a recent paper concerning the reduced steps with which a quantum algorithm could solve a system of linear equations compared to a classical algorithm (2). Many popular encryption schemes depend on the extremely long time for computers to factor huge prime numbers and as a result, Shor’s algorithm has tremendous effects on security. Beyond practical applications, quantum computers also give an insight into the realm of testing the theory of quantum measurement and the possibility of simulating quantum systems which classical computers fail to do. We have reached a monumental point in scientific knowledge where we can apply one of our newest discoveries in physics, quantum mechanics, to one of our newest scientific fields, computer science, to develop a computer which could have unbelievable possibilities for solving groundbreaking problems or, possibly, on the other hand, show a discrete barrier to our knowledge.

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However, at this point in time we have a much greater understanding of the theory of quantum computation than the experimental understanding required for building a large scale quantum computer. This discrepancy is a result of the difficulty of extracting data and performing operations on single qubits in addition to the amount of possible error in quantum information due to deconstructive interaction of qubits with the environment. This phenomenon, known as quantum decoherence, prevents a quantum system from interfering with itself and, as a result, destroys the superposition of a quantum system and causes a loss of information. The challenge of building an instrument capable of quantum computer operations depends on roughly five requirements: 1) welldefined two level quantum states, or qubits, 2) reliable preparation of these bits in different states, 3) low decoherence, 4) accurate quantum gate operations, and 5) strong ability to make measurements of stored information (3).

The Wisconsin Institute for Quantum Information, a coalition of research groups within the physics department here at the University of Wisconsin-Madison, is performing research at the forefront of quantum computing. The coalition consists of three different experimental efforts working on the physical realization of quantum information processors along with a myriad of theoretical physics professors who work together with these three groups. The three branches include the McDermott group, which focuses on implementing superconducting circuits as qubits, the Eriksson group, which focuses on using semiconductors to create qubits in the form of Silicon quantum dots, and the Saffman and Walker groups, which both work on multiple approaches towards using trapped neutral atoms as qubits.

Professor Robert McDermott performed research in quantum computing as a post doctorate researcher under John Martinis at the University of California at

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Santa Barbara and has continued within the field here at Madison.

The McDermott group focuses their research on implementing qubits with superconducting circuits, which are macroscopic circuits that display quantum mechanical behavior, usually displayed only in atoms. In a paper he wrote, while working as a post doctorate researcher under Dr. Martinis McDermott, Robert McDermott helped to open up the possibility for full characterization of quantum gates made up of multiple qubits by making measurements on coupled superconducting qubits, a step necessary for implenenting quantum algorithms (4).

Professor Mark Eriksson, who works closely with theorist Susan Coppersmith, leads a group that focuses on implementing silicon quantum dots, which are small semiconductor devises that are constructed by confining electrons tightly in silicon, as qubits. In 2014, Eriksson and a group of other graduate students and PhD. physicists, most of whom work here at Madison, published a paper in Nature where they demonstrated quantum operations of double quantum-dot qubits with a speed of 100 picoseconds, corresponding to over an order of magnitude faster than any other double-dot qubit previously controlled (5).

While the other two groups are working on solid state realizations of qubits, Saffman and Walker are looking into the possibility of using trapped neutral atoms as qubits. After all, quantum mechanics was developed because of the need for new science to explain atoms and their structure! And remarkably, Saffman and his group just recently demonstrated the largest number of individually controllable qubits, in neutral trapped atoms of Cesium, for which quantum logic gate operations have been characterized on (6).

The professors spearheading this research effort in the Wisconsin Institute for Quantum Information has helped to solidify the university as one of the top programs focused on

quantum information devices research in the world. Their research, along with the efforts of other universities and companies across the world, are helping to bring us closer to the physical realization of a quantum computer one atom at a time.

-- NOAH JOHNSON References: 1.

2.

3.

4.

5.

6.

Shor P. Polynomial-Time Algorithms for Prime Factorization and Discrete Logarithms on a Quantum Computer.

SIAM J Comput. 1997 October; 26 (5): 1484-1509.

Harrow AW, Hassidim A, Lloyd S. Quantum Algorithm for Linear Systems of Equations. Phys. Rev. Lett. 2009 October 9;103 (15): 150502.

Loss D, DiVincenzo DP. Quantum Computation with Quantum Dots.

Phys. Rev. A 1998 January; 57 (1):120-6 McDermott R, Simmonds RW, Steffen M, Cooper KB, Cicak K, Osborn KD, Oh S, Pappas DP, Martinis JM. Simultaneous State Measurement of Coupled Josephson Phase Qubits.

Science. 2005 February 25: 307;12991302 Kim D, Shi Z, Simmons CB, Ward DR, Prance JR, Seng Koh T, King Gamble J, Savage DE, Lagally MG, Friesen M, Coppersmith SN, Eriksson MA. Quantum Control and Process Tomography of a Semiconductor Quantum Dot Hybrid Qubit. Nature.

2014 July 3: 511;70-4 Xia T, Lichtman M, Maller K, Carr AW, Piotrowicz MJ, Isenhower L, Saffman M. Randomized Benchmarking of Single-Qubit Gates in a 2D Array of Neutral-Atom Qubits. Phys. Rev. Lett.

2015 March 13;114:100503

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REVIEW | THE CLINICAL FUTURE OF POLYHYDROXYALKANOATES

24

REPORTS

Recently, researchers have been developing novel medical applications such as dissolvable sutures made up of a unique type of polymer called Polyhydroxyalkanoates (PHAs). PHAs hold promise to advancing exciting polymer material technologies in the fields of tissue engineering, drug discovery, and drug synthesis.

REPORT | Molecular clock dating of São Tomé and Príncipe floral endemics:a case study in Afrocarpus By using modern biotechnologies, scientists are discovering new information about various plants and trees dispersed throughout the world. A specific tree native to the island nation of São Tomé in the Gulf of Guinea, Afrocarpus manii, has been found to have undergone a long-distance trek from continental Africa to its current location.

Such findings may offer key solutions toward the challenge of improving conservation efforts in the wake of climate change and local environmental disruptions that threaten biodiversity.

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REPORT | Comparison of AIRS Satellite, ERA Model and Radiosonde Convective Available - Potential Energy Data in the Southern Great Plains Region

33

Severe weather is extremely destructive to our civilization and can commonly lead to human fatalities. Therefore, forecasting severe weather events is an important safety endeavor, and research on forecasting methods must take high priority. One way to predict severe weather is to calculate the Convective Available Potential Energy (CAPE), which is an indicator of the atmospheric instability. New satellite technology allows us to obtain a greater quantity of near real-time data on our atmosphere and thus could provide faster forecasting. Understanding errors in the calculation of CAPE from this new data and how to fix those errors will greatly benefit severe weather forecasting.

39

REPORT | CHRONIC NACL EXPOSURE INDUCED SIGNIFICANT IONIC STRESS TOLERANCE IN ARABIDOPSIS THALIANA AS INDICATED BY INCREASED GROWTH FOLLOWING AN ACUTE IONIC STRESS The growing world population requires increases in agricultural food and fuel production. As a non-biological, or abiotic stress factor, high soil salinity promotes microtubule depolymerization, therefore threatening plant cellulose synthesis. Cellulose production is necessary for plant growth, development, and overall biomass. Stress tolerance induced in Arabidopsis thaliana through chronic salinity exposure suggests that agricultural crop losses can be lessened with plant adaptation to abiotic stressors.

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THE CLINICAL FUTURE OF POLYHYDROXYALKANOATES BY| JOSHUA POWELL

ABSTRACT Polyhydroxyalkanoates (PHAs) have long been known to scientists, but due to high costs and low market demand, they have not yet been successfully commercialized. PHAs are a group of biodegradable polymer produced by bacterial cells in the same way humans produce fat. Recently, they have been suggested as being appropriate for a number of medical applications, such as sutures and tissue engineering due to their high biocompatibility with the human body. Along with sutures and tissue grafts, PHAs have been suggested as a vehicle for controlled release drug delivery as the different types of PHA mean that the properties and degradation rates can be manipulated to suit a particular purpose. While little research has been done into the field of drug synthesis with respect to PHAs, it is suggested that drug synthesis using PHAs be explored since the optical purity of PHAs would allow for a more effective drug. Some PHAs also have therapeutic properties and could be used as a drug to treat a range of mental illnesses.

REPORTS

INTRODUCTION | The emerging field of polyhydroxyalkanoates (PHAs) has a vast range of potential applications. While today’s environmentally conscious world is continually searching for environmentally friendly alternatives to petrochemicals for applications such as packaging, office supplies, and household uses (1), novel applications that can be tailored to PHAs are often be overlooked. In the past decade, PHAs have been proposed as a highly useful tool for the medical field due to their biocompatibility and biodegradability. While some of these clinical uses are still in our future, companies in the United States and Japan have already begun pilot plant studies and are undertaking research into PHA based tissue engineering and implants (1, 2). There are a wide range of PHA monomers, such as 3-hydroxybutyrate (3HB) and 3-hydroxyvalerate (3HV) (3). These monomers can also be bonded to one another, to create copolymers such as poly(3hydroxybutyrate-co-3-hydroxyvalerate) (PHBV) (3) [Figure 1].

FIG. 1 | 3HB and 3HV Monomers and PHBV Unit

PHA technology is commonly cited as a future for medical applications such as dissolvable sutures and skin grafts (4).

PHAs also have potential to act as a carrier for drugs to help target certain parts of the body or to delay the release of a drug into the body. Finally, while the technology may be several years away, due to certain unique properties of PHA formation, PHAs could be used as an alternative, more

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efficient, drug synthesis mechanism. Due to the unique properties of PHA polymers, such as their biocompatibility, optical purity, and rate of biodegradation, PHA technology has the potential to surpass existing technologies in the future and become a benchmark in the fields of bioengineering and medicine.

SUTURES AND TISSUE ENGINEERING | One of the most commonly proposed clinical applications for PHAs is use as sutures or skin grafts (4). PHAs are able to biodegrade in a very wide range of conditions, including in seawater and the human body (2). Because of this property, PHAs have been proposed as an effective dissolvable suture.

According to Bhubalan, Lee (5), intramuscular sutures made from P(3HB) and P(3HB-co-15 mol% 3HV) were found to be of comparable strength to traditional silk and catgut sutures. The PHA sutures were of sufficient strength across the full healing time of a muscular facial wound in rats. No significant adverse effects were observed in the study (5).

As sutures are required to be moderately elastic, P(3HBco-3HV) would be the better polymer to use from these two since PHBV copolymers have a greater elongation to break than PHB homopolymers (6). The more expensive polyhydroxyhexanoate (PHHx) would also be suitable for suture applications as it also has a high elasticity (5).

Due to the way they biodegrade in the body, PHAs have also been suggested as scaffolds for skin grafts and tissue engineering (1). Biocompatibility is a complex concept, as the original materials, degradation rates, and degraded materials must all be considered. Kok and Hasirci (3) reported that PHB and PHV, the two most common types of PHA, are highly biocompatible, with very few adverse effects being exhibited when implanted in rabbit tissue. Other tests using rabbits and sheep have shown that P(4HB) and the less common P(3HB-co-3HHx) and P(3HO-co-3HHx) copolymers are also suitable as tissue scaffolds, especially in the circulatory system (5). In 1995, a Swedish team successfully used P(3HB) as a tissue scaffold in human patients following cardiovascular surgery (7). The team found that the P(3HB) patches lowered the rate of postoperative adhesion and that the patch was successfully biodegrading in the body after a

THE CLINICAL FUTURE OF POLYHYDROXYALKANOATES anaerobic environments (5).

One of the biggest opportunities in the field of PHA drug delivery is delayed, or controlled release, drugs. PHA degradation in the body is slower than the degradation of more commonly used drug delivery matrices, such as poly(lactide-co-glycolide), and can be altered based on the molecular weight of the polymer (3). One of the biggest problems with anticancer drugs is their propensity to kill tumor tissue at the expense of contaminating the entire body.

Vauthier, Fattal (12) proposed one method to combat this problem, which is the use of nanoparticles. However, PHAs may be another alternative. Due to their slow degradation rate, PHA encapsulated drugs would not wreak havoc on the body on their way to the targeted tumor tissue. Work is being done to develop highly specific cancer targeting drugs (13), so PHA encapsulated drugs could travel through the body towards the tumor without degrading and contaminating the body.

Another opportunity in PHA drug delivery is controlled release systems to achieve a certain drug concentration in the body over a period of time (5).This would be highly beneficial to Type I diabetics, as it would allow a single injection every day, rather than regular injections after sugar consumption. Previous studies have shown that release rates can be controlled by drug concentration and PHA molecular weight (5). This would allow for tailoring of drug release rates depending on the patient’s individual needs.

Some of this technology is already being developed and patented. Using a combination of P(3HB) and polylactic acid (PLA), another biodegradable polymer, a controlled release drug capsule containing celiprolol, a blood pressure regulator.

It was proven that altering the ratios of celiprolol to PLA to P(3HB) could alter the release rate of the drug into the body (14). In 2005, Metabolic Inc. also successfully applied for a patent in this field. Researchers had found that incorporating 2- hydroxyacids, which are smaller than PHA monomers, could speed up degradation by making the polymer chain more susceptible to enzymatic attack (15).

A NOVEL IDEA: DRUG SYNTHESIS | A particularly unique property exhibited by PHAs is their optical purity. Several studies have observed that only the R enantiomer of a PHA will form (16). While it is not considered an important property for many present uses, this property can open the door to a brand new clinical application for PHAs: drug synthesis.

One of the biggest problems for the pharmaceutics industry is the presence of different enantiomers, or optical isomers. While different enantiomers have identical physical and thermal properties, they can be identified by their rotation in polarized light. Different enantiomers also react differently with other chiral molecules, such as those found in our bodies (17). This concept was the cause of the thalidomide tragedy during the late 1950s and early 1960s. One enantiomer of

REPORTS

period of 6-24 months (7).

PHA production is currently more expensive than the production of the conventional petrochemical plastics that are currently being used. However, recent advances in lowering the costs of common PHAs (8) have turned this application into a very feasible one. For clinical applications, cost is often less important than the performance of the material, making it an ideal stepping stone for PHA production to develop as an industry.

Purity of the polymer is a barrier in clinical applications in general. To date, it is very difficult to purify PHAs enough to gain regulatory approval for use in most clinical applications (3, 9). Unfortunately, there is, as of yet, no official standard for PHA purity set by medical regulatory bodies, so researchers do not have a target purity to aim for.

Some studies involving clinical trials of tissue grafts have been approved, but others have been rejected on the basis of purity (5). There are a number of ways to purify PHA polymers, each of which achieves a varying level of purity.

Each method also has other practical considerations such as cost, yield, and industrial scale up prospects, which have been summarized by Koller, Niebelshutz & Braunegg. Based on their findings, alkaline digestion appears to be the most promising and commercially viable method to purify PHAs to a medical grade. This method gives a high purity, while keeping costs down and remaining viable for industrial scale-ups (10). It is clear that high purity and potential for industrial scale-ups rarely coincide, so it will be necessary for the appropriate regulatory boards to set a standard for purity before a purification method is decided upon. Unfortunately, the alkaline digestion method does not work for all bacterial strains (10), so this cannot be performed on cheaper, mixed culture production methods.

DRUG DELIVERY | Some work has also been done in the field of PHAs as a drug delivery method (1). PHAs, like most biodegradable polymers, are broken down via a mechanism known as chain cleavage. This is where a long polymeric chain is broken down into smaller chains, and then eventually into individual monomers. In the case of PHAs, chain cleavage is cause by microorganisms secreting depolymerase, a chemical which is naturally produced in bacteria to break down PHAs for consumption during times of famine. There are two main types of degradation patterns for biodegradable polymers in general, bulk erosion and surface erosion (11).

PHAs only exhibit surface erosion (5), which is where the polymer degrades from the outside. This erosion pattern has been suggested as the better pattern for use in controlled or targeted drug delivery devices. The rate of surface erosion is dependent on surface area and thickness of the polymer (11), which allows manipulation of release times to be fairly simple. PHAs degrade in aerobic environments to produce carbon dioxide and water, with the addition of methane in

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the drug was highly effective in curing morning sickness, while the other enantiomer was later found to cause severe birth defects. Following the withdrawal of thalidomide from the market, tougher legislation was introduced to prevent another similar situation from happening in the future (18).

Currently, extensive tests are done to isolate enantiomers and test them individually to ensure there are no adverse effects from the inactive enantiomer (17).

In addition to their optical purity, the type of PHA formed is dependent on the feeding substrate that is provided and the strain of bacteria (19), which results in over 150 different

FIG. 2 | Synthesis pathway of PHB from glucose.

PHAs having been observed and even more having been postulated (2). Sudesh & Doi further explain that bacteria from the genus Pseudomonads are particularly versatile and can produce a wide range of branched, aromatic, and even halogenated PHAs containing 6-14 carbon atoms (2005).

While 6-14 carbon atoms do not seem to be large numbers, several common drugs, most notably the painkiller and muscle relaxant, ibuprofen (Figure 2), contain a number of carbon atoms within this range. Significantly, the R enantiomer of ibuprofen is the active enantiomer, hence it may be possible to synthesize an optically pure form of ibuprofen as a PHA from bacteria (Figure 3).While producing ibuprofen in this way may be expensive, it may appeal to pharmaceutical companies due to its lesser waste and the simplicity of the process. The more commonly used Boot’s synthesis is a process which requires several steps (20)

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FIG. 3 | Ibuprofen Molecule with R-Configuration Highlighted

(Figure 4). The multi-step process can drive production costs up, as numerous reaction vessels may be required.

The Boot’s synthesis also produces a number of waste products, which, while largely harmless, are a concern for production companies, as a disposal procedure must be considered. Furthermore, an optically pure drug would mean that half of the active ingredient is necessary for an effective drug, hence capsules or tablets could be smaller. When developing new drugs, pharmaceutical companies can also save time and money in research and regulatory approval as only one enantiomer will need to be tested and approved.

It has also been observed that some moderately common PHAs, such as P(3HB-co-4HB) and P(4HB), have some useful therapeutic properties. 4HB units (Figure 5) are known to be an anesthetic that can affect the brain quickly without disturbing other systems in the body (5). Less common is the knowledge that 4HB units have been observed to be effective at treating psychological disorders, such as schizophrenia, narcolepsy, and drug and alcohol withdrawal.

These polymers are commonly found in mammalian brain cells (2), though little is understood about the mechanism by which 4HB units can resolve or prevent such psychological disorders. It is recommended that this interaction be studied more in-depth to further explore the possibilities for using 4HB units as treatment for these disorders.

CONCLUSIONS AND RECOMMENDATIONS | PHAs are a hugely versatile material that has enormous potential for use in the medical market. While purification is currently a problem, dissolvable sutures and tissue graft scaffolds have the potential to surpass existing technologies, such as silk or catgut, because of their exceptional biocompatibility. Some clinical trials have already been undertaken, which have shown that P(3HB) patches are superior to traditional technologies, but it is recommended that a standard be set for medical grade PHA so researchers can better develop the technology.

Using PHA as capsules or microcapsules for controlled or delayed release drug treatments is also being explored.

PHA capsules or microcapsules can protect the body from harmful side effects of drugs such as chemotherapy drugs by delaying the release of the drug. They can also help

THE CLINICAL FUTURE OF POLYHYDROXYALKANOATES

FIG. 5 | 4HB Monomer

PHAs can allow pharmaceutical companies to save huge amounts of money and time, as they won’t have to isolate and test two different enantiomers when developing new drugs. It can also save money producing existing drugs as less waste will be produced and less active ingredients will need to be synthesized. Some therapeutic effects have also been observed in 4HB units, found naturally in mammalian brains, such as the ability to prevent psychological disorders such as schizophrenia and drug and alcohol withdrawal.

Little is known about the mechanism by which 4HB units affect the brain’s chemistry, so it is recommended that further research be conducted to understand what the units do and how they can be used commercially to treat mental illness.

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FIG. 4 | Boots Synthesis of Ibuprofen showing waste products

maintain constant levels of a drug in the body, such as insulin for Type I diabetics. While little has been seriously considered in the area of drug synthesis, PHAs can provide a novel method for producing drugs. The optical purity of

REFERENCES | 1. Shen L, Haufe J, Patel MK. Product overview and market projection of emerging bio-based plastics.

Heidelberglaan: Utrecht University, 2009 June.

2. Sudesh K, Doi Y. Polyhydroxyalkanoates. In: Bastioli C, editor. Handbook of Biodegradable Polymers. GB: Smithers Rapra; 2005. p. 219-56.

3. Kok F, Hasirci V. Polyhydroxybutyrate and Its Copolymers: Applications in the Medical Field. In: Yaszemski MJ, Trantolo DJ, Lewandrowski K-U, Hasirci V, Altobelli DE, Wise DL, editors. Tissue Engineering and Novel Delivery Systems. New York: Marcel Dekker Inc.; 2004. p. 543-62.

4. Thorstenson JB, Narasimhan B. Combinatorial Methods for the High-Throughput Characterization and Screening of Biodegradable Polymers. In: Mallapragada S, Narasimhan B, editors. Handbook of Biodegradable Polymeric Materials and Their Applications. 2.

Stevenson Ranch, CA: American Scientific Publishers; 2006. p. 1-11.

5. Bhubalan K, Lee W-H, Sudesh K. Polyhydroxyalkanoate.

In: Domb AJ, Kumar N, Ezra A, editors. Biodegradable Polymers in Clinical Use and Clinical Development.

Hoboken: John Wiley & Sons; 2011. p. 247-315.

6. Arcos Hernandez MV, Laycock B, Donose BC, Pratt S, Halley PJ, Al-Luaibi S, et al. Physicochemical and mechanical properties of mixed culture polyhydroxyalkanoate (PHBV). European Polymer Journal. 2013; 49:904-13.

7. Duvernoy O, Malm T, Ramström J, Bowald S. A

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Biodegradable Patch used as a Pericardial Substitute after Cardiac Surgery: 6- and 24-Month Evaluation with CT. The Thoracic and cardiovascular surgeon. 1995; 43(5):271-4.

Laycock B, Pratt S, Halley P, Werker A, Lant P, editors.

Biodegradable polymers from pulp and paper wastewater streams. Appita Annual Conference; 2013.

Laycock B. Personal Communication. In: Powell J, editor. St Lucia; 2014.

Koller M, Niebelshutz H, Braunegg G. Strategies for recovery and purification of poly[(R)3-hydroxyalkanoates] (PHA) biopolyesters from surrounding biomass. Engineering in Life Sciences.

2013; 13(6):549-62.

Jain JP, Ayen WY, Domb AJ, Kumar N. Biodegradable Polymers in Drug Delivery. In: Domb AJ, Kumar N, Ezra A, editors. Biodegradable Polymers in Clinical Use and Clinical Development. Hoboken: John Wiley & Sons; 2011. p. 3-58.

Vauthier C, Fattal E, Labarre D. From Polymer Chemistry and Physicochemistry to Nanoparticulate Drug Carrier Design and Applications. In: Yaszemski MJ, Trantolo DJ, Lewandrowski K-U, Hasirci V, Altobelli DE, Wise DL, editors. Tissue Engineering and Novel Delivery Systems.

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Frazer I. Professor Ian Frazer. Bulmer M, editor. St Lucia; 2014 October 17.

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Boca Raton, Fla; Cambridge: CRC Press; 2005. p. 32-56.

Tessier.Synthesis of Ibuprofen. Akron: University of Akron; 2010.

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MOLECULAR CLOCK DATING OF SÃO TOMÉ AND PRÍNCIPE FLORAL ENDEMICS:A CASE STUDY IN AFROCARPUS BY | ALEX GOKE

ABSTRACT The island nation of São Tomé and Príncipe in the Gulf of Guinea hosts an incredible diversity of floral species relative to its size and distance from larger land masses. The occurrence of Afrocarpus mannii (Hook.f.) C.N.Page on São Tomé, in particular, illustrates a classic example of long distance dispersal from the continental distribution of the rest of the African genus, providing a unique opportunity to study diversification patterns in a disjunct Afromontane population. Phylogenetic analysis of Afrocarpus is carried out using numerous publicly available genomic sequences. Molecular clock dating indicates a time of divergence for A. mannii at approximately 3.5 MYA. The findings presented here suggest a pre-Pleistocene speciation event discordant with historical assumptions. Further study will likely reveal similar diversification patterns for other island endemics. These data support the need for conservation action to protect a rare and informative species in one of the most biologically diverse regions of the planet. Afrocarpus mannii at approximately 3.5 MYA (1.2996-8.6183 95% HPD) well after the volcanic formation of São Tomé and Principe. Lack of extant Afrocarpus on nearby mainland habitat implies an extreme longdistance dispersal event towards establishment on the island. The findings presented here indicate a previously undocumented and extremely rare dispersal event to one of the most biologically diverse, yet often overlooked, regions of the planet.

This information will assist in evaluating the status of atrisk species and distributions in the region and highlight the importance of understanding diversification histories as they relate to conservation.

METHODS AND MATERIALS | ------HH---VISUALIZING DISTRIBUTION OF AFROCARPUS ---Occurrence records for Afrocarpus were obtained on 2 March Occurrence records for Afrocarpus were obtained on 2 March 2015 from the Global Biodiversity Information Forum [7]. One hundred and ninety-one georeferenced occurrences were available, representing all species except A. gaussenii. Distributions were visualized within the DIVAGIS platform and then generalized in Photoshop CC v. 2014.

Distribution of A. gaussenii was inferred from the literature.

PHYSICAL AND BIOLOGICAL DESCRIPTION OF SÃO TOMÉ AND PRÍNCIPE | A literature review was conducted for information relevant to the biological and geological history of the island nation of São Tomé and Príncipe, as well as the larger Gulf of Guinea region. Specifically, conclusions about the geologic origins of the island, its spatial relations to mainland Africa through time, the modern biotic structure of the island, and factors influencing biodiversity, including regional aeolian and fluvial pollen circulation, were researched. Principal findings from the review were incorporated in this study in order to contextualize phylogenetic results.

PHYLOGENETIC ANALYSIS | ----Partial sequences of the maturase K (matK) gene, 5.8S ribosomal RNA gene, 26S ribosomal RNA gene, and ribulose1,5-bisphosphate carboxylase/oxygenase large subunit (rbcL) gene were obtained for the family Podocarpaceae, and NEEDLY and phytochrome P (PHYP) gene partial sequences were obtained for the order Pinales [8-11]. Sequences were selected for reliability and ease of acquisition. GenBank accession numbers for all taxa evaluated can be found in

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INTRODUCTION | Afrocarpus (Buchanan-Hamilton & N.E.Gray) C.N.Page is a poorly-described genus of two to six tree species of the family Podocarpaceae (Pinales) [1, 2]. Assumed to be of Pleistocene origin, members of the genus are characteristic of the distinctive Afromontane floristic region which includes over 4000 species, nearly three-quarters of which are endemic. [3, 4]. Afrocarpus is restricted to the eastern and southern high-elevation forests of the African continent with the exception of A. mannii (Hook.f.) C.N.Page and A. gaussenii Woltz C.N.Page, which are found endemic to the island of São Tomé off the coast of Equatorial Guinea and eastern Madagascar, respectively [3].

A. mannii occurs only on the upper slopes (1,300m+) of mountainous Pico de São Tomé, confined to a mere 25km2 area of cloud forest where it faces eradication through deforestation and stochastic events. These threats have earned the population a “Vulnerable” designation on the International Union for Conservation of Nature Red List of Threatened Species [5]. It is the only gymnosperm representative on the island [6].

The continental members of the genus, including A. dawei (Stapf) C.N.Page, A. falcatus, A. gracilior (Pilg.) C.N.Page, and A. usambarensis (Pilg.) C.N. Page, maintain stable populations throughout the scattered Afromontane regions stretching from South Africa to Ethiopia [5].

With this study, I seek to describe the timing of diversification in Afrocarpus that led to the eventual establishment of an endemic A. mannii population over 250km from continental Africa and 2,400km from the rest of the genus. Molecular clock dating will grant insight into the diversification history of Afrocarpus, and not only will it aid in explaining contemporary spatial patterns of diversity in the genus, but of São Tomé and Príncipe as a whole.

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Appendix I [12, 13].

Genetic sets containing multiple data for any one taxon were reduced in an arbitrary fashion as to contain only one sequence per species. Sequences were then aligned via MAFFT v. 7.017 using an auto alignment algorithm and 200PAM/k=2 scoring matrix with a 1.53 gap open penalty and a 0.123 offset value within Geneious v. 8.1.4 [14-16]. Aligned sequences were then concatenated in Geneious and delivered to the CIPRES Science Gateway v. 3.3 on 28 April 2015 for phylogenetic tree inference using RAxML-HPX Blackbox algorithm v.

8.1.11 and run for two hours with default input parameters [17, 18]. The output was reduced so that 162 taxa (including select Podocarpaceae, Araucariaceae, and Cupressaceae as outgroup) were retained for analysis from 305 to reduce processing time and to focus results (Appendix I).

FIG. 1 | Contemporary distribution of Afrocarpus across continental and oceanic Africa.

Evolutionary rate of the best maximum likelihood/rapid bootstrapping result tree was assessed with reference to estimated divergences of Microcachrys tetragona (x=23.5 MYA, s=1), Nageia (x=47 MYA, s=4), and Cupressaceae (x=245 MYA, s=15) [19-21]. Parameters for analysis were set in BEAUTi v. 1.7.5 then delivered to CIPRES for Bayesian analysis using BEAST v. 1.8.0. Ten million generations were evaluated and recorded every 1,000 generations under GTR, Yule speciation, nucleotide substitution models, and a log-normal relaxed clock algorithm [22-24]. The posterior distribution was extracted and summarized using TreeAnnotator v. 1.8.2 and then visualized with FigTree v.

1.7.4.

RESULTS |

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VISUALIZING DISTRIBUTION OF AFROCARPUS | Figure 1 depicts the continental and oceanic distribution of Afrocarpus. As shown, A. mannii exists solely on the main island of São Tomé and Príncipe, and approximately 2,400km from the rest of the genus. A. dawei persists in the highlands of East-Central Africa, circumnavigating Lake Victoria through Uganda, Tanzania, and Kenya. A. falcatus is located mainly in South Africa, though extends somewhat into Southern Mozambique with a disjunct path through central Tanzania. A. gaussenii, as previously stated, is isolated to the montane eastern regions of Madagascar. A. gracilior maintains a similar distribution pattern to A. dawei, though extends further north into Ethiopia. Finally, A. usambarensis inhabits the gap between recorded A. dawei, A. falcatus, and A. gracilior populations through northern Tanzania.

Afrocarpus has not been recorded in West Africa [3].

PHYSICAL AND BIOLOGICAL DESCRIPTION OF SÃO TOMÉ AND PRÍNCIPE |São Tomé is the larger of the two oceanic islands of São Tomé and Príncipe. It rises ca. 5,000m from the ocean floor in the Gulf of Guinea, providing 964km2 (with Príncipe) of habitable surface. The island is one of four that constitutes the oceanic sector of the Cameroon Volcanic Line (CVL), which extends from the southern island of Annobón to the plateaus of Eastern Nigeria/Northern Cameroon [25].

The exact timing of formation of the CVL oceanic islands is a contentious issue. Competing theories place formation from between 66 to 80 MYA and 124 to 140 MYA [26]. Discussion of these debates is beyond the scope of this study, and therefore all theories seemed rational to consider in analysis.

While the origins of the volcanic chain are still being debated, the fact that São Tomé is of volcanic origin is authoritative, as the island remains volcanically active. Argon isochrome sampling indicates lava flow as recently as 0.03 MYA [27].

São Tomé has never made contact with mainland Africa or other oceanic CVL land-masses. The relative distance between masses and depth of the ocean that surrounds them (ca. 1,800m) prevented land bridges from forming during historical glaciation events [28].

São Tomé has a constituent flora of 602 species, of which 96, including an entire genus, are endemic [29]. Other estimates have placed rates of endemism as high as 20% [30].

Montane elements are particularly prone to endemism [31].

Between the CVL oceanic islands, only 16 species are shared, indicating each island received its biota independently from each other [28]. The recognized uniqueness of the biota has earned the island World Wildlife Foundation “Center of Plant Diversity” designation, though only 35% of the 750km2 forest is protected to some degree and continues to be encroached upon by expanding plantation activity [31, 32].

Regional dispersal events to the island are rare. Movement of pollen into the Gulf from extant podocarps in the western

MOLECULAR CLOCK DATING OF SÃO TOMÉ AND PRÍNCIPE FLORAL ENDEMICS:A CASE STUDY IN AFROCARPUS forests of Cameroon represents less than 1% of grain deposition. On a larger scale, long-distance dispersal to the Gulf of Guinea of eastern and southern African species, such as continental Afrocarpus, has only been observed through Asteraceae Tubuliflorae and Combretaceae-Melastomataceae pollen grains, which represent minor contributions to the marine sediment pollen bank of the Gulf [33, 34]. Afrocarpus as a whole is considered to be severely dispersal-limited [3].

Together, these factors make recent or continued dispersal events of Afrocarpus highly unlikely, particularly in the face of prevailing south-westerly monsoon winds [35].

before the beginning of the Pleistocene. Other members of the genus appear more recently from 2.8 to 0.7 MYA. These data suggest repetitive subjection of a non-diversifying A. manni lineage to habitat fragmentation and continental aridification throughout the Quaternary, while other members underwent further diversification as assumed through the Pleistocene.

This would seem to indicate pre-Pleistocene dispersal to São Tomé where A. mannii continues to persist, supporting in part the standing hypothesis of an “old” Afrocarpus distribution [3].

FIG. 3 | Divergence in higher Podocarpoids (red bars indicate 95% HPD interval at ancestral nodes, values indicate mean age).

PHYLOGENETIC ANALYSIS | ----Bayesian analysis generated trees that substantially conform to those presented by larger studies [e.g. 8-11, 36, 37]. The best phylogenetic estimate is shown in Figure 2. Molecular clock analyses of Afrocarpus yielded divergence times of ancestral node to A. mannii at 3.5 million years ago (MYA) with a 95% highest posterior density interval of 1.3 to 8.6 MYA (Fig. 3).

Complete divergence data are listed in Appendix I. DISCUSSION | São Tomé, Príncipe, and other Gulf of Guinea island masses are largely agreed to be of volcanic origin, and with no land bridges forming between either island and continental Africa, their constituent flora are entirely the result of long distance dispersal or human-mediated establishment. Time of divergence for A. mannii is established at 3.49 MYA, well

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FIG. 2 | Divergence in interior Pinales (red bars indicate 95% HDP interval at ancestral nodes, estimated ages available in Appendix I).

The resolution of these data are limited by the number and confidence of fossil reference taxa and completeness of molecular sequences. Further analysis with additional sequence and reference data would likely corroborate these findings and strengthen our understanding of this vastly understudied genus. Additionally, diversification patterns in Afrocarpus, being the only gymnosperm representative on the island, may not be congruent with those of other regional flora as suggested, though a few studies in to angiosperm groups have shown similar results [e.g. 38].

Knowledge of the biogeographical history of São Tomé and Príncipe endemic flora, particularly that of Afrocarpus mannii, has far-reaching implications in the contemporary understanding of patterns in Afromontane diversification and distribution resulting in regions of endemic diversity .

Specifically, the findings here invoke theory of Afromontane pre-Pleistocene paleobiotic movement and diversification events and confirm the merit of phylogenetics in reaching such conclusions. Additionally, awareness of the severity of endemism in the region, coupled with the rarity of longdistance dispersal events, such as the one introducing Afrocarpus to the island, can inform and direct conservation efforts, knowing that rescue effects will not support the local population in the face of land use change, exploitation activities, and climate change effects that threaten the biodiversity of the island. Further study of the flora is needed to complement the findings provided here in order to support the immediacy of action needed to conserve one of the most

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MOLECULAR CLOCK DATING OF SÃO TOMÉ AND PRÍNCIPE FLORAL ENDEMICS:A CASE STUDY IN AFROCARPUS unique and diverse biological hotspots in the world.

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COMPARISON OF AIRS SATELLITE, ERA MODEL AND RADIOSONDE CONVECTIVE AVAILABLE - POTENTIAL ENERGY DATA IN THE SOUTHERN GREAT PLAINS REGION BY | JESSICA GARTZKE ABSTRACT High priority must be given to research for Earth remote sensing applications, especially relating to the forecasting of severe weather, due to its destructive consequences. Convective Available Potential Energy (CAPE) is routinely used to characterize convection as having moderate or severe potential. Relating a climatology of CAPE to near real time observations from meteorological sensors on new weather satellites is a valuable tool in assessing the risk of severe weather. Satellite data products from AQUA AIRS were used to compute a ten year climatology for the Southern Great Plains region. CAPE was computed from vertical profiles of pressure, temperature, and dew point temperature from high vertical resolution AIRS soundings (101 levels) using the SHARPpy algorithm used by the National Weather Service Storm Prediction Center (NWP).

It was found that numerical estimates of CAPE are sensitive to the vertical smoothing of the temperature and moisture profile. In addition, error in the surface parcel dew point estimate degrades the accuracy of CAPE, but can be corrected when the satellite estimated surface dew point is limited to agree with the radiosonde measurement to within 1 °C. Further improvements in estimating CAPE will allow us to take full advantage of this satellite data for near-real time forecasting.

FIG. 1 | NASA Aqua MODIS imagery May 31, 2013 at 18:30 UTC (1:30 pm local time)

FIG. 2 | CAPE computed using SHARPpy from model ECMWF ERA-Interim at 18 UTC (top) and from NASA AIRS L2 v6 18:30 UTC (bottom). Circle symbol marks Nonnan, Oklahoma near El Reno. Star symbol marks the location of the DOE ARM SGP site.

We then look at the ability of satellite observations to characterize the CAPE probability distribution function at the 1:30 am/pm overpass times as a function of distance from the SGP site.

The ability to measure vertical profiles of water vapor from space at times when ground-based upper air soundings are not available can fill an important need in short-range weather prediction. New satellite observations allow for the retrieval of water vapor measurements with higher vertical resolution than was previously available. In order to demonstrate the advantages of these new data opportunities, it’s important to look at a practical application. Supercell thunderstorm events, like the El Reno, Oklahoma tornado on May 31, 2013, are examples of just how dangerous and unpredictable tornados can be. Fig. 2 demonstrates the severe convection from this disastrous storm. The El Reno case study

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INTRODUCTION | A common goal of the severe storm science community is to obtain accurate information in a timely manner regarding atmospheric stability. This information can be used to communicate predictions of severe weather events. To better inform how to make these predictions, a ten year record of upper air sounding profiles from the Department of Energy Atmospheric Radiation Measurement (ARM) Southern Great Plains (SGP) site was used to create a climatology of Convective Available Potential Energy (CAPE) [11]. CAPE is a measurement of the amount of energy a parcel of air contains, which is effectively the buoyancy of that parcel. CAPE is commonly used by meteorologists as an indicator of severe weather. The seasonal variation in CAPE and dew point is shown in Fig. 1.

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will be used to illustrate the potential value of estimating CAPE values from satellite soundings.Fig. 3 compares the regional CAPE for the El Reno case study between European Centre for Medium-Range Weather Forecasts (ECMWF), ERA-Interim model fields, and NASA Atmospheric Infrared Sounder (AIRS) L2 v6 satellite observations.

tornado showing the close proximity to the radiosonde launch site. Fig. 4 provides an example comparison of the vertical soundings of temperature and dew point temperature at the DOE ARM SGP site (just north of El Reno) at about noon on that day. Note that the radiosonde profile has much higher vertical resolution than either the NWP model or the satellite retrieved profile from AIRS. National Weather Service (NWS) forecasters are currently using Geostationary Operational Environmental Satellite (GOES) sounder products for a range of applications, for which they are obtaining positive results. These products include estimates of total precipitable water vapor (TPW) and atmospheric stability indices, such as convective available potential energy (CAPE) and lifted index (LI).

Infrared observations from geostationary orbit capture the diurnal cycle of surface skin temperature with data collected over the continental United States every hour.

These geostationary data profiles can contribute to weather warnings [10] . However, the limited number of infrared spectral channels fundamentally limits the vertical resolution of the existing GOES sounder thermodynamic products, and utilizing other sensors could fill this gap. Unlike the current GOES sounder, new high spectral resolution infrared sensors on polar orbiting weather satellites (POES) can sense the atmospheric boundary layer at specific times of day (about 10:30 am/pm and 1:30 am/pm). Forecasters could make use of CAPE estimates from operational satellite sounders, such as CrIS and IASI, on JPSS and METOP platforms during the most unstable daytime period considering there are no operational ground level observations during this time.

FIG. 3| Temperature and dewpoint temperature vertical profiles from ARM Vaisala RS92 Radiosonde (red), ECMWF ERA-Interim (black) and NASA AIRS L2 Version 6 (blue) at the DOE ARM SGP site on 31 May 2013 at about 18:00 UTC (noon).

The ECMWF reanalysis is a European climate model that assimilates observations to give a numerical description of the atmosphere. While AIRS and ERA data are very similar to each other, there are notable differences in the precise location of the most extreme CAPE values with the satellite observation of peak CAPE slightly west of the model analysis. Note that El Reno is just west of Norman, Oklahoma and is where the supercell formed that produced the El Reno

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FIG. 4| Seasonal variation in dewpoint (top) and surface CAPE (bottom) at the ARM SGP site.

For example, the Atmospheric Infrared Sounder (AIRS) has been used to provide quantitative information about the

COMPARISON OF AIRS SATELLITE, ERA MODEL AND RADIOSONDE CONVECTIVE AVAILABLE - POTENTIAL ENERGY DATA IN THE SOUTHERN GREAT PLAINS REGION lower atmosphere [4]. Software to process AIRS data in near real-time has been included in the IMAPP direct broadcast software package [1] [13]. Near real-time data assimilation of polar orbiting advanced sounder products into rapidly updated NWP models have the potential to provide a positive impact for future warnings on forecasts [10]. The objective of this paper is to make a quantitative assessment of CAPE derived from high spectral resolution infrared sounders.

Satellite overpasses of the Department of Energy (DOE) Atmospheric Radiation Measurement (ARM) program Southern Great Plains (SGP) site provide the validation dataset needed to make this assessment [8]. This analysis makes use of a combination of research radiosondes launched from the DOE ARM SGP site, ECMWF model reanalysis fields, and coincident NASA AIRS satellite observations.

A characterization is made of the temporal, spatial, and measurement accuracy of CAPE derived from hyperspectral infrared weather satellites. An analysis of CAPE sensitivity to these errors will help outline whether or not utilizing near real-time hyperspectral satellite soundings of temperature and water vapor from direct broadcasting instruments is a plausible path for accurate severe weather forecasting.

where g is the acceleration due to gravity, LFC is the level of free convection, EL is the equilibrium level, Tparcel is the temperature of the parcel, and Tenv is the environmental temperature.

The forecasted parcel is a parcel estimate at the expected time of convection. This paper utilizes the surface parcel method for calculating CAPE. In particular, this study uses the SHARPpy software routines described in Halbert [7].

SHARPpy is a python software library that can be used by the research community and is derived from the SHARP software used operationally at the Storm Prediction Center [7].

RESULTS | An analysis was performed to understand the ARM radiosonde, ERA, and AIRS CAPE sensitivity to spatial, temporal, vertical, and measurement error.

SPATIAL SAMPLING ERROR | For the satellite product, a spatial sampling error can exist when the AIRS profile coincident with the SGP site location is invalid (e.g. overcast and the closest valid profile is some distance away). To quantify this issue an analysis was created for CAPE values within a radius of the ARM SGP site. The spatial sampling error for the ERA interim can be neglected because the model grid is continuous over the domain of interest [7]; however, the ERA was analyzed in the same circular region for consistency with the AIRS analysis. There is no significant spatial sampling error in the AIRS product due to invalid retrievals when using the quality control criteria given in Table 1 ( Supplementary figures).

TEMPORAL SAMPLING ERROR | Temporal sampling error can be an important error in CAPE estimation given the rapid boundary layer changes due to surface heating during the day and cooling after sunset. For this reason, operational radiosondes launched at 0 and 12 UTC (6 am and 6 pm) are not ideal for of assessment of CAPE during mid-day in the SGP region. The ARM SGP site was chosen for this study of AIRS CAPE because research grade radiosondes are launched at 6 and 18 UTC (about midnight and noon local time). The afternoon satellite overpass is at about 1:30 pm (19:30 UTC) with some variation from day to day. The radiosonde launch time and Aqua satellite overpass time difference is typically less than 2 hours. To further minimize this relatively small temporal sampling error, the

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METHODS | To obtain a statistically useful range of CAPE values in the U.S. Southern Great Plains, vertical profiles of pressure, temperature, and water vapor were obtained for the time period January 1, 2005 to December 31, 2014 for a region centered on the ARM SGP central facility. The ARM site was chosen for this study because routine radiosonde launches at 6 Coordinated Universal Time (UTC) and 18 UTC are within 1.5 hours of the nominal satellite overpass times of the AQUA satellite. Values of CAPE were computed for each vertical profile using software consistent with that of the NWS Storm Prediction Center (SPC) [7]. Only cases with radiosonde profiles having CAPE greater than 50 J/kg were included in the analysis. This threshold was used to eliminate the large number of zero (or small) CAPE values that are not relevant for severe weather. To investigate spatial sampling issues, CAPE values from AIRS and ERA were selected within a radius of 50, 150, and 250 km of the ARM SGP central facility. The selected CAPE values for a time and space region are used to create histograms using a uniform bin size of 50 J/kg. Normalizing by the sum of the histogram creates a probability distribution function (PDF).

PDF at the 25th, 50 th, 67 th, 75 th, 95 th and 99th percentiles were tabulated to quantify the characteristics of each CAPE distribution shown in Fig. 5.

To analyze the dependence of CAPE on the vertical resolution of the temperature and water vapor profiles, the radiosonde profiles were smoothed with a vertical boxcar

function at width values (1 3 5 7 9 15 21 27 35 41 47 53) of 75 meters per layer. The CAPE computed from the smoothed profile was differenced from the original radiosonde CAPE for each profile. The following equation defines CAPE [2] [9],

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COMPARISON OF AIRS SATELLITE, ERA MODEL AND RADIOSONDE CONVECTIVE AVAILABLE - POTENTIAL ENERGY DATA IN THE SOUTHERN GREAT PLAINS REGION radiosonde data was interpolated to each Aqua satellite overpass times. The ERA Interim analyses are available only at 0, 6, 12, and 18 UTC. For this study, the 6 and 18 UTC ERA analysis fields are used without time interpolation and thus represent the atmospheric state about 1 to 2 hours prior to the satellite overpass, but they are time coincident with the ARM SGP radiosonde launches.

VERTICAL RESOLUTION ERROR | The ARM radiosondes were used to investigate the dependence of CAPE on vertical resolution of the temperature and moisture sounding. In the ARM product file used for this study, the radiosonde data has been interpolated to 200 height bins with a spacing of 75 meters and two additional bins at 2 m and 30 m. A boxcar smoother was applied to each vertical profile for a range of boxcar full widths, and CAPE was recomputed for each smoothed profile. Results are summarized in Fig. 5. There is an 11% reduction in CAPE for a full-width vertical smoothing of 1,000 meters.

A vertical smoothing of 2,000 m causes a reduction in CAPE values of about 18%. AIRS has a reduction of 17% at the 50th percentile, which is roughly consistent with the expected vertical resolution of about 2 km for retrieved water vapor profiles inherent to the hyperspectral infrared [2]. The ERA model 50th percentile is biased by –23% relative to radiosonde profiles, which may be due to a vertical smoothing inherent in the NWP model, particularly with respect to the vertical layering of water vapor. This is apparent in the dew point profiles of the case study examples shown in Fig. 4.

Vertical smoothing of the radiosonde profiles can occasionally lead to a temporary increase in CAPE values as indicated by the positive outliers. Fig. 7 illustrates the effect of smoothing a profile containing a nocturnal temperature inversion. The surface parcel temperature increases with smoothing in this case, which leads to an increase in the CAPE value computed from the smoothed profile. Investigation of these anomalous cases of increasing CAPE shows they are all profiles at night containing a temperature inversion.

MEASUREMENT ERROR | The case study analysis revealed a sensitivity of CAPE to surface parcel temperature and dew point temperature (i.e. an error in the surface parcel estimate could cause errors in the computed CAPE). In order to quantify this error, the ARM radiosonde surface temperature and dew point temperature were used as references to compute the error in the surface parcel estimates from the closest AIRS retrieved profiles and ERA reanalysis profiles to the ARM SGP launch site.

The mean differences are less than 1 °C in each comparison

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FIG. 6| Vertical smoothing error by smoothing width.

FIG. 7| An example of a smoothed soundings and an original radiosonde sounding on August 27, 2005 at the ARM SGP site containing a nocturnal temperature inversion.

with a standard deviation of about 2 °C; however, this good, statistical agreement in the mean disguises an error when CAPE is non-zero. Table 2 (Supplementary figures) shows the result of analyzing the 10-year matchup dataset for the subset of cases with CAPE greater than a minimum cutoff.

The most notable feature in Table 2 is how the error in surface dew point changes from near zero for all CAPE values to –2 °C for the subset with CAPE greater than a minimum value of 50 J/kg. This is error is the same for both AIRS and ERA. As the CAPE minimum threshold increases, the error grows

COMPARISON OF AIRS SATELLITE, ERA MODEL AND RADIOSONDE CONVECTIVE AVAILABLE - POTENTIAL ENERGY DATA IN THE SOUTHERN GREAT PLAINS REGION

DISCUSSION | In previous works, several authors validated AIRS retrieved temperature and moisture vertical profiles [5] [6] [3] [12]. Only a limited study has been published previously on the accuracy of CAPE derived from AIRS profiles relative to radiosondes [13]. That study commented on the lack of correlation of CAPE derived from AIRS to the CAPE derived from the small number of radiosonde profiles considered, but no explanation was provided for the result. For the current study, a long time series of AIRS and radiosonde matchups was created to characterize the systematic biases and random characteristics of the hyperspectral infrared satellite retrievals estimations of CAPE. As shown in Fig. 5, the AIRS and ERA CAPE distributions share similar characteristics, including a smaller median value relative to ARM radiosondes. This under-estimate is consistent with the lower vertical resolution of the satellite and NWP products.

The relatively poor correlation of AIRS and ERA CAPE, with matched ARM SGP radiosondes (0.35 and 0.5, respectively), is explained by an error in estimation of the surface parcel dew point temperature.

The systematic bias found in the AIRS derived CAPE and the ERA-Interim derived CAPE is consistent with the known reduced vertical resolution of NWP and satellite retrievals compared to radiosondes. However, the scatter in the AIRS and ERA-Interim CAPE values relative to radiosondes was shown to be primarily due to error in the estimate of the surface parcel dew point temperature. To account for this error and develop a correction method, a comparison between ASOS automated surface observations at Ponca City, OK (near the ARM SGP site) and AIRS retrieved surface temperature and dew point was conducted. A time series plot was created to see the seasonal variation in the two sets of data as shown in Fig. 9.

FIG. 8| AIRS vs. ARM radiosonde all data with AIRS cloud fraction less than 0.8 (upper left) and a subset with surface dew point within one degree (upper right), all data with AIRS cloud fraction less than 0.1(lower left) and a subset with surface dew point within one degree (lower right).

When comparing surface temperatures, AIRS is seeing higher temperatures in the winter than ASOS. In summer, when CAPE is high, there is a fair amount of scatter but no bias for AIRS 2 meter temperature as seen in Fig. 10. In contrast, the AIRS estimated dew point is drier than the ASOS estimated dew point by several degrees in the summer. This is consistent with what we found at the ARM site. The next step is to use the ASOS surface temp and dew point in updating the satellite CAPE calculation. Future work includes the use of ASOS surface temperature and dew point observations coincident with AIRS soundings to improve CAPE estimates

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for both ERA and AIRS with ERA exceeding –5 °C and AIRS exceeding –7 ° C for the CAPE values greater than 2500 J/ kg. Surface air temperature error also grows with increasing CAPE, but the error is less than half as large as the dew point temperature error. Table 2 shows very similar behavior between ERA and AIRS for CAPE up to 1500 J/Kg. For higher CAPE values, the AIRS bias error exceeds that of ERA although both have equally large standard deviations.

To characterize the extent to which errors in the surface parcel estimates lead to error in the derived CAPE estimates, a correlation coefficient was computed between AIRS and ARM radiosonde for nonzero CAPE values with a range of quality control criteria. Decreasing the AIRS cloud fraction cutoff increases the correlation with ARM radiosondes from 0.35 to 0.5, however the highest correlation (>0.8) is achieved only when the surface dew point of AIRS is within 1 C of the ARM site radiosonde. This is illustrated in Fig. 8. The left hand panels show the variation with cloud fraction, from < 0.8 to < 0.1, while the right hand panels show the additional effect of restricting the subset to dew point temperature with agreement better than 1 C. This demonstrates that the scatter in the matchup between AIRS and ARM SGP radiosonde CAPE values is primarily driven by an error in the estimation of the surface parcel dew point temperature. The comparison of the right column of Fig. 8 shows that a high correlation (>0.84) can be obtained even for AIRS cloud fractions up to 0.8 as long as the surface dew point estimate is within 1 degree of the truth. Fig. 8 also shows the comparison of AIRS and ERA with a similar restriction on cloud fraction and surface dew point error. The correlation coefficient between AIRS and ERA increases from 0.37 to 0.88 when the surface dew point temperatures agree to within 1 ° C independent of AIRS cloud fraction.

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CONCLUSIONS | A comparison of CAPE was made for the U.S. Southern Great Plains region using a combination of DOE ARM radiosondes, ERA model reanalysis fields, and AIRS satellite observations. CAPE estimates were evaluated for spatial, temporal, vertical resolution, and measurement errors. Numerical estimates of CAPE are sensitive to the vertical smoothing of the temperature and moisture profile.

A vertical smoothing of 1-2 km leads to a reduction in the 50th percentile of CAPE by 10-20 percent. In addition, error in the surface parcel dew point estimate is found to degrade the accuracy of CAPE. For CAPE values greater than 50 J/kg, both AIRS and ERA-Interim surface dew point temperatures are dry by 2 degrees compared to the surface radiosonde observations. This error increases to more than 5 °C for CAPE exceeding 2500 J/kg. Improvements of surface parcel dew point temperature can be expected to improve the CAPE estimates derived from both hyperspectral infrared satellite observations and NWP forecasts. This suggests that merging surface station meteorological data and available boundary layer profiling with satellite profiles could greatly improve the utility of the hyperspectral satellite sounding products and the NWP model fields. Timely and useful information on the evolution of the vertical structure of the atmosphere is available from the satellite overpasses at 10:30am (EUMETSAT/METOP) and at 1:30pm (NASA Aqua and NOAA/JPSS) and should be exploited for NWS forecasting applications.

See Supplementary Figures online at justjournal.org to view Table 1 and Table 2 of this manuscript.

ACKNOWLEDGEMENTS | The author wishes to thank Genevieve Burgess for assisting in the acquisition of AIRS products and discussions with Greg Blumberg on the use of the SHARPpy software. We acknowledge the support of NOAA grant NA10NES4400013. In addition, acknowledgment is made for the contribution of Dave Turner for the ARM data obtained from the DOE archive at http://www.archive.arm. gov.

REFERENCES | 1. Al SET. Dual-Regression Retrieval Algorithm for RealTime Processing of Satellite Ultraspectral Radiances. J Appl Meteor Climatol. 2012; 1455–1476. doi:10.1175/ JAMC-D-11-0173.1 2. Bedka, S., Knuteson, R., Revercomb, H., Tobin, D., & Turner D. An assessment of the absolute accuracy of the Atmospheric Infrared Sounder v5 precipitable water vapor product at tropical, midlatitude, and arctic ground. J Geophys Res Atmos. 2010; 181–202. doi:10.1175/2009JAMC2286.1

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3. Botes D, Mecikalski JR, Jedlovec GJ. Atmospheric Infrared Sounder ( AIRS ) sounding evaluation and analysis of the pre-convective environment. J Geophys Res Atmos Geophys Res,. 2012;117: 1–22. doi:10.1029/2011JD016996 4. Chahine MT. Improving Weather Forecasting and Providing. Bull Amer Meteor Soc. 2006; 911–927. doi:10.1175/BAMS-87-7-911 5. Divakarla MG, Barnet CD, Goldberg MD, McMillin LM, Maddy E, Wolf W, et al. Validation of Atmospheric Infrared Sounder temperature and water vapor retrievals with matched radiosonde measurements and forecasts. J Geophys Res Atmos. 2006;111: 1–20. doi:10.1029/2005JD006116 6. Fetzer EJ, Lambrigtsen BH, Eldering A, Aumann HH, Chahine MT. Biases in total precipitable water vapor climatologies from Atmospheric Infrared Sounder and Advanced Microwave Scanning Radiometer. J Geophys Res Atmos. 2006;111: 1–14. doi:10.1029/2005JD006598 7. Halbert KT, Blumberg WG, Marsh PT. SHARPpy: Fueling the Python Cult. Prepr 5th Symp Adv Model Anal Using Python. 2015; 1–3.

8. Hart, J. A., J. Whistler, R. Lindsay and MK. NSHARP, version 3.10. Storm Predict Center, Natl Centers Environ Predict. 1999; 9. Rasmussen EN, Blanchard DO. A Baseline Climatology of Sounding-Derived Supercell andTornado Forecast Parameters. Weather Forecast. 1998;13: 1148–1164. doi:10.1175/1520-0434(1998)013<1148:ABCOSD>2.0.

CO;2 10. Stensrud DJ. Convective-scale warn-on-forecast system. Bull Amer Meteor Soc. 2009; 1487–1499. doi:10.1175/2009BAMS2795.1 11. Stokes GM, Schwartz SE. The Atmospheric Radiation Measurement (ARM) Program: Programmatic Background and Design of the Cloud and Radiation Test Bed. Bulletin of the American Meteorological Society. 1994. pp. 1201–1221. doi:10.1175/15200477(1994)075<1201:TARMPP>2.0.CO;2 12. Tobin DC, Revercomb HE, Knuteson RO, Lesht BM, Strow LL, Hannon SE, et al. Atmospheric Radiation Measurement site atmospheric state best estimates for Atmospheric Infrared Sounder temperature and water vapor retrieval validation. J Geophys Res Atmos.

2006;111: 1–18. doi:10.1029/2005JD006103 13. Weisz E, Sr WLS, Smith N. Advances in simultaneous atmospheric profile and cloud parameter regression based retrieval from high-spectral resolution radiance measurements. J Geophys Res -Atmospheres. 2013;118: 6433–6443. doi:10.1002/jgrd.50521

CHRONIC NACL EXPOSURE INDUCED SIGNIFICANT IONIC STRESS TOLERANCE IN ARABIDOPSIS THALIANA AS INDICATED BY INCREASED GROWTH FOLLOWING AN ACUTE IONIC STRESS BY | KARA CHUNG , BRENDAN DRACKLEY, EMILY LYNCH, MATTHEW PEREYRA ABSTRACT To accommodate increasing agricultural demands associated with increasing population, efforts are being made to uncover and improve native mechanisms plants use to cope with abiotic stresses. One type of abiotic stress, high soil salinity, has been shown to rapidly depolymerize microtubules. The cellulose synthase complex, which is responsible for production of cellulose in plants, relies on microtubules for functionality. The complex includes a recently discovered group of companion proteins that accelerate microtubule repolymerization following an ionic stress-induced degradation. Given their compensatory nature, we would expect that an upregulation of companion proteins would increase a plant's tolerance to ionic stress. If regulated by ionic stress, we would also expect to observe an increase in companion protein expression while exposed to high saline conditions. Therefore, we hypothesized that chronic exposure to ionic stress will lead to increased tolerance for subsequent ionic stress. This study showed that prior exposure was sufficient to improve tolerance as shown by increased growth and microtubule repolymerization after an acute ionic stress, relative to plants without prior chronic ionic stress. Although not conclusively shown, this study supports upregulation of companion protein transcription in response to ionic stress. demonstrating that the enzymatic activity of CesA is guided by microtubules (Paredez et al. 2006). Depolymerization of microtubules has been shown to lead to the internalization of the CSC and inhibit cellulose production (Zhong et al. 2002).

In a 2015 study by Endler et al., two proteins in the cellulose synthase complex (CSC) of Arabidopsis thaliana were identified. Subsequently, these proteins, CC1 and CC2, have been termed companions of cellulose synthase proteins (CC).

These CC’s exhibit numerous characteristics that warrant further investigation. Activity of both CC’s were shown to be induced primarily under saline conditions (Endler et al.

2015). Furthermore, CC’s were found to be unnecessary for plant survival under non-saline conditions, but increase salt tolerance and survival under saline conditions (Endler et al.

2015). Redundancies were found amongst both companion proteins such that only one is required for adequate tolerance to acute NaCl stress (Endler et al. 2015). In case studies on cc1cc2, double mutants exposed to acute NaCl stress, adult plants contained significantly lower levels of cellulose, and, thus, a decreased rate of growth as compared to controls (Endler et al. 2015). Further research analyzed the mechanism by which companion proteins confer NaCl stress tolerance.

Exposure of plant cells to a NaCl solution induces both ionic and osmotic cellular stresses (Endler et al. 2015).

However, it has been found that microtubules disassemble rapidly only after exposure to ionic stress (Komis et al. 2014).

As noted previously, depolymerization of microtubules results in internalized CSC and ceases production of cellulose. As shown in Fig. 1, the companion proteins act to tether cellulose synthase to microtubule subunits. Upon depolymerization, CC’s promote microtubule reassembly.

Enhanced microtubule reassembly enables faster recovery of cellulose synthesis during times of ionic stress.

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INTRODUCTION | It is not without justification to say that agriculture makes up a substantial component of the world’s economy (Holcomb et al. 2014). The biomass of plants provides the world with many essential products that are used daily. Plant biomass primarily consists of plant cell walls, which provide the major sustainable resource for many human products including feed, food, and fuel (Somerville et al. 2010). The primary mass contribution of plant cell walls is cellulose, which is also the most abundant biopolymer on Earth (Endler et al. 2015). Consisting of microfibrils formed by linked glucan chains and stabilized by intra- and intermolecular hydrogen bonds, cellulose is essential for plant development, directed cell growth, and total plant structure, as the main source of cell wall tensile strength (Heredia et al.

1995; McFarlane et al. 2014). ---Environmental conditions play a large role in determining numerous characteristics of plants, such as rate of development, size, and overall structure. Abiotic environmental stresses, such as salinity, drought, and cold, can result in severe yield losses for all major plant crop species (Endler et al. 2015). In fact, it is estimated that the yield loss due to abiotic stresses may be as large as 50% for various crop species (Boyer 1982). Products that reduce or eliminate such yield losses make up a large industry in itself, with US farmers spending over $29 billion on fertilizers and protectants annually (Holcomb et al. 2014).

Given that cellulose is such an integral component of the cell wall and plant biomass, an understanding of the cellular machinery that produces it is essential for improving tolerance to abiotic stresses. Cellulose is synthesized by a microtubule-guided cellulose synthase complex (CSC) embedded in the plasma membrane (Endler et al. 2015). The keystone of the CSC is the cellulose synthase (CesA) enzyme itself. Using Arabidopsis thaliana as a model, fluorescently labeled CesA proteins have been observed as motile foci,

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FIG. 1 | Application of NaCl rapidly depolymerizes microtubules (Endler et al. 2015). The cellulose synthase complex (synthase (CesA) and companion proteins (CC)), requires microtubule organization for mobility and enzymatic activity (Heredia et al. 1995). CC’s help repolymerize microtubules following depolymerization due to ionic stress (Agrios 2005).

While the role of companion proteins have been investigated in cases of acute NaCl stress, little is known about how long term exposure to NaCl stress affects their production and the total plant adaptation to ionic stress.

Given sustained exposure to a stressor, organisms can adapt by upregulating production of proteins involved with tolerating the stressor, allowing it to survive exposure (Boyer 1982). Furthermore, in the event that adaptive tolerances are upregulated, the organism could withstand subsequent exposures at a higher level than was previously tolerable (Boyer 1982). This study investigated such a mechanism with regards to CC1 and CC2 in Arabidopsis thaliana.

As shown in Fig. 2, we hypothesized that chronic conditioning of Arabidopsis thaliana to 25mM NaCl would increase the rate of microtubule repolymerization and leaf area growth following a 4 hr 200mM NaCl acute stress test, as compared to those without chronic conditioning.

FIG. 2| Chronic NaCl exposure suspected to induce upregulation of CC which will increase tolerance to ionic stress. Tolerance of conditioned plants were assessed by observing growth after exposure to an acute ionic stress. CC2 protein image generated used Phyre2 web portal for protein modeling (Schindelin, J. et al. 2012).

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METHODS | We grew Arabidopsis thaliana plants in a peat/vermiculite mix supplemented with Jack’s nutrient solution (~MiracleGro®).

The plants were split into three conditioning groups: 25mM NaCl, 50mM sorbitol, and no conditioning (control). The NaCl group was grown with a modified version of Jack’s containing 25mM NaCl (concentration taken from Choi et al. 2013) for 14 days, which simulated a low level of ionic stress. The sorbitol group contained Jack’s with the addition of 50mM sorbitol. Since NaCl also induces osmotic stress, and we were only interested in how the plants adapt to long term ionic stress, the sorbitol was used to induce the equivalent of 25mM NaCl osmotic stress. This allowed us to attribute differences in growth or microtubule repolymerization in part to the CC’s involved in ionic stress rather than a physiological change associated with osmotic stress, like vascular adaptations. In order to simulate an acute ionic stress, we exposed all 3 groups (control/NaCl/sorbitol) to 200mM NaCl for 4 hours after the 2 week conditioning period (concentration and duration taken from Endler et al.

2015). GROWTH ASSAY | Before and after the acute NaCl stress, we measured the average leaf area of the Arabidopsis plants.

We imaged and measured leaf area in FIJI (Schindelin et al.

2012) as an indication of size and therefore growth, as shown

by Weraduwage et al. 2015. Tukey-Kramer t-tests were used to compare mean leaf area by treatment, before and after acute stress test.

ROOT INTEGRITY ASSAY | The roots were stained with calcofluor-white (CW) fluorescent dye, as described by Galbraith 1981. CW binds specifically to cellulose and not glucose or any of the other sugars comprising the cell wall(s) making it an ideal stain for measuring cellulose. Calcofluor excitation occurs at 360 nm, while emission can be observed from 430-550 nm, which allowed us to separate its wavelength from the GFP-TUA6 on the microtubules. Cellulose was visualized with Zeiss Axiozoom, and average RFU/pixel measured in FIJI image analysis software (Schindelin et al.

2012). Independent t-tests were used to compare treatment groups before and after acute stress.

MICROTUBULE ASSAY | We grew Arabidopsis thaliana with GFP-TUA6 marked microtubules (Gilroy lab, University of Wisconsin-Madison) in order to visualize the de- and re-polymerization of the microtubules after an acute NaCl stress. We used the Zeiss LSM510 confocal microscopy at the Newcomb Imaging Center, Department of Botany at the University of Wisconsin-Madison to observe the GFP labeled microtubules. GFP-TUA6 excitation occurs at 488 nm while emission can be observed from 285-365 nm. We observed the microtubules immediately before treatment and then every

CHRONIC NACL EXPOSURE INDUCED SIGNIFICANT IONIC STRESS TOLERANCE IN ARABIDOPSIS THALIANA AS INDICATED BY INCREASED GROWTH FOLLOWING AN ACUTE IONIC STRESS hour for 4 hours after the acute stress in order to visualize and compare the breakdown and repair of the microtubules between conditioning treatments.

RESULTS | Average leaf areas between all conditioning treatments before stress were not significant (p= 0.87, 0.92, 0.99, respectively, Tukey-Kramer HSD). NaCl conditioned plants showed higher average leaf area following the acute stress test as compared to the control, however no significant difference was observed between NaCl-sorbitol or sorbitol-control (p=0.05, 0.12, 0.92, respectively, Tukey-Kramer HSD).

Results are depicted in Fig. 4.

As depicted in Fig. 3, GFP-labeled microtubule images in the chronically conditioned plants showed faster microtubule reorganization and repolymerization upon exposure to acute NaCl stress than the control. Due to low sample size of images and inability to quantify repolymerization, statistical analysis was not performed. All results represent the average of the overall observed characteristics for each treatment group at a given time.

As shown in Fig. 5, the root integrity assay exhibited no statistical difference in the cellulose or structural composition of the roots before or after acute NaCl stress (t(20)= 2.03, p=0.25; t(20)= 2.03, p=0.87, two-tailed, respectively).

DISCUSSION | --GROWTH ASSAY | A significant increase in leaf area growth, after an acute NaCl stress, was observed in plants exposed to chronic NaCl stress as compared to plants not exposed to chronic NaCl stress. This implies that prior conditioning with NaCl leads to increased ionic stress tolerance, which could be related to an increase in the expression of factors

FIG. 4 | Microtubule repolymerization in A. thaliana over time after acute 200mM NaCl stress test in salt and control conditioning groups. Scale bar represents 20Âľm. Confocal imaging was performed using the Zeiss LSM510 at the Newcomb Imaging Center, Department of Botany, UW-Madison.

include the companion proteins described in Endler et al.

2015. However, analysis of transcription levels following ionic stress would need to be performed in order to directly support the regulatory mechanism outlined in Fig. 2.

MICROTUBULE REPOLYMERIZATION ASSAY| Due to the highly complex nature of microtubules, quantification of the results obtained from the microtubule repolymerization assay was not feasible. However, the images obtained qualitatively suggest that the group chronically conditioned with NaCl exhibited increased microtubule repolymerization and stabilization as compared to the control. This qualitative assessment is demonstrated by the greater density of microtubule organization seen in the time course images of the NaCl conditioned group.

ROOT INTEGRITY ASSAY | Any damage to the roots of a plant would hinder its ability to absorb micronutrients and water, ultimately, affecting the overall growth of the plant.

In order to control for any difference in growth due to root damage by chronic or acute NaCl exposure, we examined the health of the roots by microscopy using the cellulose stain

REPORTS

FIG. 3 | Microtubule repolymerization in A. thaliana over time after acute 200mM NaCl stress test in salt and control conditioning groups. Scale bar represents 20Âľm. Confocal imaging was performed using the Zeiss LSM510 at the Newcomb Imaging Center, Department of Botany, UW-Madison.

involved in recovering cellulose synthesis. Potential factors aiding in recovery, that could be upregulated by ionic stress,

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CHRONIC NACL EXPOSURE INDUCED SIGNIFICANT IONIC STRESS TOLERANCE IN ARABIDOPSIS THALIANA AS INDICATED BY INCREASED GROWTH FOLLOWING AN ACUTE IONIC STRESS

Fig 5. a) Root integrity in A. thaliana assessed before and after acute NaCl stress by measuring calcofluor fluorescence with Zeiss Axiozoom. Box plots represent average fluorescence per pixel. Conditioned plant roots did not differ significantly from control before acute NaCl stress (t(20)= 2.02619, p=0.2504, two-tailed) and after (t(20)= 2.03452 p=0.8740, two-tailed). b) Sample exa1nination ofroot health by calcofluor stain. Average fluorescence measured 1nun from root tip using FIJI image analysis software.

REPORTS

calcofluor white in the root integrity assay. No significant difference in root health between treatment groups was observed, suggesting that the observed difference in leaf growth was not due to root damage.

CONCLUSIONS | These results allow us to support our hypothesis, suggesting that prior chronic NaCl stress does induce an adaptation to deal with acute ionic stress, as measured by leaf area and microtubule repolymerization.

Due to complexities inherent in the repolymerization of microtubules, analysis is currently restricted to qualitative observations. In order to more accurately quantify a change in the rate of repolymerization, a more thorough understanding of microtubule formation and degradation is needed. The ability to apply statistical analysis would allow for greater support of the ionic stress recovery mechanism. Direct quantification of CC protein production and accumulation would also be needed for elucidation of the recovery pathway.

The ability for plants to upregulate companion protein production represents an evolutionary advantage to environments in which fluctuations in salt stress are a possibility (Boyer et al., 1982; Wang et al., 2007). Companion proteins confer adaptive resistance to salt stresses and other ionic stresses, and would have likely evolved a mechanism for regulation in response to a sustained stress exposure.

Studying the underlying mechanisms of salt stress adaptation could be useful for agricultural purposes. Overall, this study offers new insights into the regulatory properties of companion proteins of the CSC, and enables further research

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into manipulation of these regulatory properties.

ACKNOWLEDGMENTS | We would like to thank Simon Gilroy and Sarah Newcomb of the Gilroy Laboratory, University of Wisconsin-Madison and Michelle Harris, Seth McGee, and Josh Pultorak from the Biology Core Curriculum Program, University of Wisconsin - Madison for their contributions in executing this research study.

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