Trinity Student Scientific Review | Vol. I

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Trinity Student Scientific Review Vol. I

TSSR

Trinity Student Scientific ReviewVolume I

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Trinity Student Scientific Review

TSSR

Volume I

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Published by the Trinity Student Scientific Reviewc/o Faculty of Engineering, Mathematics and Science,

Trinity College, Dublin 2Republic of Ireland.

All rights reserved.Copyright © contributors to the

Trinity Student Scientific Review 2015

All views expressed herein are those of the authors and do not necessarily reflect the views of the editors or sponsors.

This journal claims no special rights or privileges.

Printed by Brunswick Press Ltd.

Cover by David CorishPhotography by Gearóid Gibbs and Benedict Shegog.

The TSSR logo was created by Ger Walsh at Magnet Design in 2014.

The Trinity Student Scientific Review is also available online athttp://trinityssr.com/

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The Trinity Student Scientific Review is kindly sponsored by the

Dr Patrick Prendergast, Provost of Trinity College Dublin

The Faculty of Engineering, Mathematics and ScienceTrinity College Dublin

TSSR


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General Managers: Johnny Deane Georgia O’Sullivan

Biology Editor: Dan McDermott

Chemistry Editor: Elaine Kelly

Physics Editor: Dylan Scully

Production Manager: David Corish

Launch Manager: Luke McGuinness

THE TRINITY STUDENT SCIENCTIFIC REVIEW COMMITTEE 2015

Back Row (L to R): Dylan Scully, Dan McDermott, Luke McGuinness, David CorishFront Row (L to R): Johnny Deane, Georgia O’Sullivan, Elaine Kelly

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Prize-Winning Essays of The Trinity Student Scientific Review 2015

Best Overall Essay:A Quantam Model of Olfactory Reception

Oskar Ronan

Best Biology Essay:A Fourth Dimension in the Bacterial Defence

Against Bacteriophage InfectionsMatthew Dorman

Best Chemistry Essay:Targeting ligands and Nanovehicles:

A Novel Approach to Lung Cancer TherapyShelley Stafford

Best Physics Essay:Graphene-Sulfur Composites: A Novel Method in

Developing Advanced Lithium-Sulfur BatteriesAndrew Selkirk

Best Freshman Essay:The Uses and Limitations of Absorption Spectroscopy

in the Development of Non Invasive Blood Glucose Monitoring Systems

Kate Reidy

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Endorsements

“As Dean of the Faculty of Engineering, Mathematics and Science, it is my pleasure to support the Trinity Student Scientific Review (TSSR), a challenging and innovative initiative by our Science Undergraduate Students. Trinity College is committed to the enhancement of the learning experience of each of its students and TSSR provides a platform in the Faculty for our students to develop the critical academic and literary skills of modern day scientists. TSSR showcases undergraduate academic talent and encourages the recognition of this talent among peers and staff.

This review journal demonstrates the ability of the Editorial Board to collaborate effectively with both academics and industry to reach a successful conclusion and gain invaluable insights into scientific publishing and editing. The production of TSSR demonstrates a combination of enthusiasm, inspiration and organisation that Trinity College actively encourages among its student body.

Science progresses through publication, and TSSR has the potential to stimulate students to pursue further research that ignites their curiosity.”

Prof. Clive WilliamsDean of the Faculty of Engineering, Mathematics and Science

Trinity College Dublin

“I would like to strongly endorse this concept of an undergraduate produced student review journal. It is great that undergraduate students get the opportunity to write scientifically in a serious way at an early stage in their career. It can only help them progress academically and this early exercise of technical writing will be of immense benefit for the rest of their careers.

We should remember that a professional scientist should not only be technically competent and be widely knowledgeable about their chosen subject, but should also aim to be a professional writer. First class written communication is essential for success in getting Grant funding and getting papers published and, more importantly, cited. Writing does not come easy to many. Practice makes it easier.So in short, TSSR is a great idea which has arrived on the scene at an

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Endorsements

appropriate time. The editorial Committee are to be commended for bringing this all together”

Professor Mike LyonsAssociate Professor, Physical Chemistry and SFI Principal

Investigator in the School of Chemistry, Trinity College Dublin

“The TSSR is an exciting initiative run by a talented bunch of enthusiastic third-year science undergraduates for the benefits of their fellow students. It provides participants with the invaluable opportunity to experience the peer review process. Students who are successful in getting their original work published at this stage in their career will be at a distinct advantage when it comes to applying for future PhD positions, which is a process that is becoming more and more competitive.”

Dr Rachel McLoughlinAssistant Professor in Immunology

Trinity Biomedical Sciences Institute

“I’m delighted to be involved with the Trinity Scientific Review. The publication is a great opportunity for students to hone their writing and share their ideas with the world. It’s also a brilliant way for students on the editorial board to gain organisational skills that will be invaluable throughout their future careers.”

Dr Natalie CooperAssistant Professor

Macroecology and Macroevolution Research GroupTrinity College Dublin

“This is a great idea, highlighting exciting developments in science from the students’ point of view”

Professor John McGilp Professor of Surface and Interface Optics



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“I am very happy to support the Trinity Scientific Review. The more we can communicate the science we do the better - for our colleagues, students and the broader community. I look forward to reading the interesting reviews that will appear.”

Professor Luke O’NeillChair of Biochemistry


“The Trinity Student Scientific Review is a great idea for helping students to research and write up topics they are interested in. In this way they can experience the pleasure of sharing with others their enthusiasm for science. For over 40 years I’ve shared with Trinity students my own enthusiasm for physics, and encourage all students to give this initiative their support. I wish the Review and all those involved every success”

Dr. Eric FinchFellow Emeritus and Senior lecturer in Physics


The Trinity Student Scientific Review was kindly supported and endorsed by the Science Gallery, Trinity College Dublin.

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Modules (1 year full time)

• Basic Immunology

• Immunological Techniques

• Communicating Science & Critical Analysis

• Immunogenetics

• Microbe Detection and Evasion

• Clinical Immunology

• Parasite Immunology

• Tumour Immunology

• Global Infectious disease

• Immunotherapeutics & Product Development

• 12 week Immunology Research Project

Application Website

www.tcd.ie/apply/mscimmunology

Contact

Director: Cliona O’FarrellyCo-ordinator: Nigel StevensonEmail: [email protected]: http://www.tcd.ie/Biochemistry

M.Sc. in Immunology School of Biochemistry & Immunology, TCD Commences September

Admission

This M.Sc. is designed for graduates aiming to pursue careers in academic research, medicine or the pharmaceutical industry, for which a thorough grounding in immunology, immune-mediated pathogenic mechanisms and immunotherapy is required. Applicants are required to hold at least an Upper Second Class Honours degree (2.1) in Medicine, Veterinary Science, Dentistry, Molecular Biology, Genetics, Immunology, Biochemistry or any Biological Science.

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TSSR

Trinity Student Scientific Review

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PreludeWelcome MessageJohnny Deane & Georgia O’Sullivan, General Managers

BiologyLetter from the EditorDan McDermott

Best Biology Essay:A Fourth Dimension in the Bacterial Defence Against Bacteriophage InfectionsMatthew Dorman, Junior Sophister

Lysine Methylation of the p53 Tumour Suppresor: Analogies with Histone BiologyHayley Porter, Junior Sophister

DNA VaccinesJeremy Dover, Junior Sophister

Thromboxan A2 Receptor (TPRα): A Potential Human Drug TargetJudy Conmey, Senior Sophister

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Contents

Man’s best friend? Human-Canid Conflict in the 21st centuryHolly English, Senior Sophister

Immunoglobin Binding Proteins of Staphylococcus AureusKelly Murray, Junior Sophister

Natural Killer Cells - More than Meets the EyeKaren Slattery, Junior Sophister

First Line Treatment of Chronic Lymphocytic Leukaemia and the Impact of Genetic Abberations in ChemotherapyTristan Kwan, Junior Sophister

ChemistryLetter from the EditorElaine Kelly

Best Chemistry Essay:Targeting ligands and Nanovehicles: A Novel Approach to Lung Cancer TherapyShelley Stafford, Junior Sophister

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Best Freshman Essay:The Uses and Limitations of Absorption Spectroscopy in the Development of Non Invasive Blood Glucose Monitoring SystemsKate Reidy, Senior Freshman

Production of Hydrogen and its Role in the Transition to a Clean Energy EconomyDavid Madden, Senior Freshman

Minor DNA Groove Binding Agent OptimisationRory Scanlon, Senior Freshman

Developments in Metal Organic Frameworks for Utilisation in Catalysis Joseph Eiffe, Junior Sophister

Physics

Letter from the EditorDylan Scully

Best Overall Essay:A Quantam Model of Olfactory ReceptionOskar Ronan, Junior Sophister

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Contents

Best Physics Essay:Graphene-Sulfur Composites: A Novel Method in Developing Advanced Lithium-Sulfur BatteriesAndrew Selkirk, Junior Sophister

Enceladus: Plume Composition and Interactions in the Saturnian System Jessica Erkel, Junior Sophister

Metamaterials - Future of CloakingKatarzyna Siewierska, Junior Sophister

Implications of a 125 GeV Higgs Boson for the Minimal and Next-To Minimal Supersymmetric Standard ModelBrían Ó Fearraigh, Junior Sophister

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WelcomeOn behalf of the Editorial Board and the Committee, we would like to welcome you to the first ever edition of the Trinity Student Scientific Review.

It has been a pleasure working with our dedicated team of students and academics over the past few months in producing this publication, and we are so pleased to be able to showcase such a high standard of academic talent from our fellow students. This standard meant our editorial teams had their work cut out for them when it came to choosing the best articles, and the following pages are a testament to the quality and talent of Trinity students.

Our aim for the TSSR was to provide Trinity science undergraduates with an opportunity to bridge the gap between their degree and the research-driven world of science by engaging in an area of particular interest to them. In writing this it is important to acknowledge the Student Economic Review here in the University as a source of inspiration for the TSSR. Professor John O’Hagan and his team of students from the Department of Economics succeed in producing a wonderful journal every year, providing their students with the opportunity to be published as undergrads. The SER has become part of the fabric of academic and student life in the department, which is something we hope for the TSSR to achieve.

The establishment of the TSSR this year would have been impossible without the overwhelming encouragement and enthusiasm of the academics we initially approached, and we are very grateful to Dr Andrew Jackson of the Department of Zoology, Professor Werner Blau of the Department of Physics, and Dr Mike Southern of the School of Chemistry. Without them, we would not have accomplished half of what we did. The Trinity Student Scientific Review will grow and develop with them as continuing academic advisors. To the postgraduate students and academics who made the editorial process run smoothly, efficiently and fairly, we are grateful for your time and your invaluable contributions.

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None of what we have achieved this year would have been possible without the generous sponsorship we received from the University itself. We would like to sincerely thank Dr Patrick Prendergast, Provost of Trinity College Dublin, for recognising the potential of the TSSR and ensuring we went to print this year. To Professor Clive Williams, Dean of the Faculty of Engineering, Maths and Science, we are extremely grateful for your immediate and full support from the very start. To the wonderful staff in the Faculty office, we are indebted to you and your patience when we were running up and down the stairs to knock on your door.

If we were to list everyone who has gone out of their way to offer invaluable advice these past few months we would have a publication twice the size it is now, so to keep this as short and as sweet as possible know that we are indebted to every single one of you.

Our fellow founding members of this publication deserve our biggest thank yous; Dan McDermott, Dylan Scully, Elaine Kelly, Luke McGuinness and David Corish, you made our jobs easier by being so good at yours. It was a pleasure, a privilege and a lot of fun working with you.

We are looking forward to seeing the TSSR grow to become a firm fixture in the undergraduate academic calendar. We hope that the work we have done these past few months has laid the foundations for the Trinity Student Scientific Review to go on and become a platform for Trinity science students to explore their own research interests, and we know these exceptional students that come through Trinity College Dublin will ensure the success of the TSSR for years to come.

Johnny Deane & Georgia O’SullivanGeneral Managers Trinity Student Scientific Review 2015

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Editorial

Letter from the EditorThe study of biology for me has always been about the unfathomable complexity of life, from the intricate inner workings of a cell to the majesty of the plants and animals that roam our planet. Biological sciences at Trinity College Dublin came from humble beginnings in 1851 as a moderatorship in experimental science and has expanded to encompass the range of disciplines it does today, and the following articles from my fellow students in the biology section are reflective of the broad scope of study available to us now. Biology disciplines progress together hand in hand towards a better understanding of life, whether it be advances in biomedical research, a greater knowledge of conservation ecology and how to protect the natural world around us, or uncovering more secrets of the genetic code.

The response to the establishment of the TSSR from students of biological disciplines here in the University was overwhelmingly positive. We received articles from all undergraduate years, and almost all courses. It has been a privilege for me as Biology Editor to work on the inaugural year of the Trinity Student Scientific Review. From the academics, PhD candidates, students and the team, working with you all has been one of the most enjoyable experiences of my time here. I could not have completed my work without your help so I just have to say thank you to everyone for that.

I would also like to thank and congratulate every student who took the time to research and prepare a submission for publication. The huge response from students of biology is reflective of the sort of attitude and academic drive our University fosters. I will finish by offering my congratulations to all our successful writers, and I hope that you enjoy reading the following articles as much as I did.

Dan McDermottBiology Editor Trinity Student Scientific Review

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Best Biology Essay

A Fourth Dimension in the Bacterial Defence Against Bacteriophage Infections –

Transcriptional Regulation of Horizontally-Acquired Genes

MicrobiologyMatthew Dorman

Junior Sophister

The systems used by bacteria to resist infection by bacteriophages are diverse and well-studied. Three broad categories of destructive anti-phage strategies have been characterised; restriction-modification, CRISPR/Cas-mediated immunity, and abortive infection. Each system is used by bacteria to destroy phage genomes upon their injection into the cell, depriving a phage of the opportunity to undergo lytic replication, which would eventually kill its bacterial host. However, each of these systems is time-sensitive, and if an infected bacterium does not defend itself quickly, phage genes will be transcribed, and the cell will begin to manufacture phage particles. Time is therefore an important commodity to infected bacteria. Since the transcription of phage genes is essential for a phage to hijack bacterial systems and to replicate, repressing the transcription of phage genes may “buy” bacteria time for their other anti-phage systems to act. Phages have co-opted and hijacked bacterial transcriptional regulation, suggesting that reciprocal phage/host co-evolution is occurring at the level of transcriptional control, and that the silencing of bacteriophage genes by bacterial repressors has contributed to the process of bacterial speciation. Therefore, the temporal control of phage gene transcription can be considered to be as important as restriction-modification and CRISPR/Cas in the

bacterial defence against phage attack.

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IntroductionApproximately 1020 bacteria are believed to exist on Earth, in almost every conceivable environment, and are believed to be part of one of the most successful and prolific evolutionary taxa (Whitman et al. 1998). These prokaryotes are preyed upon by viruses, termed bacteriophages, or “phages”. Phages are obligate intracellular parasites, like all viruses, and are typically plentiful in any environment in which bacteria exist. In the oceans, for instance, phages outnumber bacteria 10:1 (Bergh et al. 1989, Angly et al. 2006). Phages infect bacteria by injecting their chromosomes into the cell. If a phage is “lytic”, upon injection, genes encoded by these chromosomes are transcribed, translated, and eventually produce the components necessary to manufacture new virus particles. New phages enter the environment by lysing their host cell, killing the bacterium (Ackermann & DuBow 1987). A second life cycle, the lysogenic cycle, exists for “temperate” viruses such as bacteriophage lambda - these phages can infect a cell but may refrain from entering the lytic cycle until they receive suitable inductive stimuli such as exposure to DNA-damaging ultraviolet light or mitomycin C (Ackermann & DuBow 1987, Weinbauer & Suttle 1999).

Fierce competition exists between phage and bacterial populations. Phages must infect bacteria to replicate. However, if all of the individuals in a bacterial population are infected and subsequently lyse, the remaining phage particles have no host to infect and can no longer propagate. A similar scenario results if a bacterial population evolves total resistance to an infectious phage. Here, the virus loses all potential hosts, and cannot survive. Phages must therefore defend against bacterial anti-phage systems, and bacteria must reciprocally mutate these systems to “stay ahead” of phage survival strategies. This model led to the suggestion that bacteria and phage co-evolve according to the Red Queen Hypothesis (Stern & Sorek 2011), in which phages and bacteria constantly mutate and evolve in order to ensure their respective survival (Van Valen 1973).

Three main systems have been identified by which bacteria defend against attack by phages (Labrie et al. 2010, Samson et al. 2013, Westra et al. 2014). A summary of each system is


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provided in Table 1. Two of these systems, restriction-modification (R-M) and CRISPR/Cas-mediated immunity, defend individual cells against bacteriophage predators. The third strategy, abortive infection (Abi), refers to a form of bacterial suicide in response to infection by a phage. However, Abi does not allow single bacterial cells to resist a phage infection; rather, it causes an infected cell to lyse, denying the phage access to replicative machinery (Labrie et al. 2010). This has been characterised as an altruistic action by infected cells, designed to ensure the survival of the bacterial population as a whole by depleting the environment of viable infectious phages (Blower et al. 2012).

Table 1: Summary of commonly-characterised bacterial anti-phage strategies. An overview of some characteristics of three commonly-cited bacterial defences against phage infection. Examples of Type II CRISPR/Cas and R-M systems are described. REase: restriction endonuclease. MTase: methyltransferase. crRNA: CRISPR-RNA. Cas: CRISPR-associated gene protein product. TA: Toxin-antitoxin. (Data derived in part from Labrie et al. 2010, Blower et al. 2012, Westra et al. 2012, Samson et al. 2013, Samson & Moineau 2013, Westra et al. 2014).

Restriction-modification (R-M)

CRISPR/Cas-mediated immunity

Abortive infection (Abi)

Defends: Single bacterium.

Single bacterium. Bacterial population (altruistic).

Recognition basis:

DNA epigenetics and sequence.

DNA or RNA sequence.

Varies.

Molecules involved:

REase, MTase, may require specificity subunit (Type I R-M only).

Mature crRNA, Cas endonu-clease, crR-NA-maturation proteins.

Varies. May involve TA system.

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Phage escape strategies:

Modified phage DNA epigenetics or sequences.

Phage-encoded CRISPR-silenc-ing transcrip-tion factor, phage-encoded anti-CRISPR locus.

Various. Phage-encoded antitoxins (for TA system).

Example system:

HindII / MTase II (Hae-mophilus influenzae).

Type II Cas9 (Streptococcus thermophilus).

ToxIN (Pecto-bacterium atrosepti-cum).

Example applica-tions in molecular biology:

Restriction enzymes; DNA methylases; restriction mapping of genomes.

Gene disruption by site-spe-cific editing; gene silencing; transcriptional activation.

Engineering of phage-re-sistant bacteria in fermenta-tion cul-tures.

Many excellent reviews and descriptions of these anti-phage systems exist. However, a consideration of current opinions in this field suggests that a fourth system, the control of phage gene expression, may not be considered to contribute to bacterial anti-phage defence to the same extent as R-M or CRISPR/Cas immunity. This review argues that the temporal control of phage gene transcription can be considered an important fourth strategy used by bacteria to resist phage infections.

Restriction-modification and CRISPR/Cas - two bacterial anti-phage survival strategiesOne way by which a bacterium can defend itself against infection by phages is to use endonucleases to destroy phage chromosomes before they can be transcribed. However, these enzymes must be capable of discriminating between “foreign” phage genomes and bacterial DNA, lest the cell’s chromosome or plasmids be damaged accidentally. Consequently, bacteria have evolved at least two


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systems by which they can determine the provenance of a molecule of DNA. One of these, R-M, discriminates between “self” and “non-self” DNA on the basis of sequence and epigenetic modifications (Figure 1). Endonucleases exist in bacteria which recognise specific DNA sequences, but will only cleave DNA if the target has a specific pattern of modification, usually involving methylation (Landy et al. 1974, Old et al. 1975, Roszczyk & Goodgal 1975). Host DNA sequences are specifically modified by S-adenosyl methionine-dependent enzymes. In theory, phage-derived DNA will have a different methylation pattern to that of host DNA, causing it to be recognised as non-self, and to be digested by an endonuclease. These endonucleases and methylases are often referred to as R-M pairs, and it is from these pairs that Type II restriction endonucleases, now commonly used in research laboratories for purposes such as restriction digests and gene cloning, are routinely isolated (Smith & Welcox 1970, Roy & Smith 1973a, Roy & Smith 1973b, Old et al. 1975, Roszczyk & Goodgal 1975, Loenen et al. 2013)

Figure 1: Simplified overview of the activities of Type II REase-mediated and Type II CRISPR/Cas-mediated restriction of phage DNA.

Type II REase and Type II Cas9-dependent CRISPR systems are depicted recognising their respective targets on a phage chromosome. REases recognise specific sequences in DNA, and determine if the sequence is foreign on the basis of the epigenetic markers harboured by the foreign sequence such as the methylation pattern of the foreign DNA molecule (Labrie et al. 2010). In this example, EcoRI’s recognition sequence (capitalised) is methylated,

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inhibiting EcoRI-digestion of this nucleic acid. In contrast, CRISPR loci on the bacterial chromosome are transcribed into mRNA, or pre-crRNA, which is then processed by the Cas9 endonuclease into short crRNA sequences. crRNA complexes with Cas9 direct the endonuclease to homologous regions of foreign DNA molecules, the RNA-DNA hybridisation will bring Cas9 into close proximity with the DNA causing cleavage of the phage genome (Barrangou & Marraffini 2014).

Recent studies have revealed that bacteria can also use RNA guidance to specifically target endonucleases to foreign genome sequences. Just as R-M systems rely on a methylase and an endonuclease, so too do these sequence-specific systems rely on two components; regions of clustered, regularly interspersed short palindromic repeats on the bacterial chromosome (CRISPR loci), and CRISPR-associated, or cas genes. Our current model of how these so-called CRISPR/Cas systems defend against phage attack is as follows: CRISPR loci comprise short regions of sequences derived from phages, plasmids, or other horizontally-acquired nucleic acids (Bolotin et al. 2005, Mojica et al. 2005, Pourcel et al. 2005). These loci are transcribed into a long mRNA, which is then cleaved into shorter CRISPR-RNA (crRNA) molecules by cas-encoded proteins, which associate with endonucleases encoded by cas genes (Sontheimer 2010, Westra et al. 2012, Marraffini &, Samson et al. 2013, Gasiunas et al. 2014). crRNA molecules are homologous to the sequence of foreign DNA from which they were derived, and hybridise with that sequence if it is present in the cell. This brings the crRNA-associated Cas endonuclease into close proximity with the foreign DNA, causing it to be degraded (Figure 1). However, the true power of CRISPR/Cas immunity lies in its ability to be expanded dynamically as a form of bacterial adaptive immunity. This involves the integration of a protospacer, a DNA sequence derived from foreign nucleic acids flanked by protospacer-adjacent motifs, or PAMs, into the ends of CRISPR loci. Once a protospacer is integrated, the bacterium is able to produce crRNAs homologous to the protospacer sequence, targeting Cas nucleases to that sequence and defending the cell, and all of its daughter cells, against re-infection by that DNA molecule (Barrangou et al. 2007, Barrangou & Marraffini 2014).


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The Silencing of Horizontally-Acquired Genes Defends Bacteria Against Phage AttackR-M and CRISPR/Cas are well-defined bacterial anti-phage strategies, but they are time-sensitive. In order for a phage to successfully propagate within a bacterium, it must first have its genes translated by the cell’s own molecular machinery. Successful defence against phage infection therefore requires that phage genomes be degraded before they are transcribed. Inhibiting phage gene transcription presents an opportunity to prevent a phage from taking over the bacterium – silencing phage genes will prevent the unwanted synthesis of phage proteins, until such time as R-M or CRISPR/Cas nucleases can degrade the phage genome.

One system which bacteria use to delay the expression of phage genes involves the histone-like nucleoid-structuring protein (H-NS). This protein is a global regulator of bacterial gene expression in Gram-negative bacteria (Dorman 2004, Grainger et al. 2006, Lucchini et al. 2006, Navarre et al. 2006). H-NS forms multimers, which bind to AT-rich regions of DNA and spatially loop DNA, similar to those formed by the lac operon repressor, LacI (Lewis et al. 1996, Shin et al. 2005). These structures deny RNA polymerase access to promoters, preventing transcription; hence, H-NS is referred to as a transcriptional repressor (Schroder & Wagner 2002). The affinity of H-NS for DNA of specific base composition is significant. Since DNA which is imported horizontally into bacteria is often of a different AT content to the host’s chromosome, it is conceivable that host and foreign DNA could be discriminated on the basis of nucleotide composition.

It has been observed that DNA which has a high AT content relative to the infected bacterium’s own chromosome experiences selective gene silencing by H-NS (Lucchini et al. 2006). This observation suggests that bacteria have evolved mechanisms to determine the provenance of DNA on the basis of its nucleotide composition. This has significant implications for the evolution of bacterial species. For instance, Salmonella enterica serovar Typhimurium is believed to be a descendant of Escherichia coli. In addition to a genome which is similar to that of E. coli, S. Typhimurium possesses at least five “pathogenicity islands”, regions of DNA of

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a higher AT composition to the rest of the cell chromosome, and which are specific targets for repression by H-NS (Ochman et al. 2000, Oshima et al. 2006, Navarre et al. 2006). It is hypothesised that some of these islands were acquired by Salmonella from the virulence plasmid in Shigella flexneri, another closely-related bacterial species, through a horizontal gene transfer event which may have involved transduction via a phage (Groisman & Ochman 1997, Ochman et al. 2000). The specific silencing of horizontally-acquired DNA is believed to have accelerated the evolution of Salmonella by silencing laterally-acquired genes, the uncontrolled expression of which may negatively affect cell fitness. Strains of H-NS-deficient Salmonella suffer from decreased fitness relative to wild-type Salmonella, due in part to the uncontrolled expression of pathogenicity genes. These mutants increase their relative fitness back to wild-type levels by acquiring loss-of function mutations in their pathogenicity island genes, or by mutating an H-NS paralogue, StpA, to restore H-NS-like repressive function to the cell (Ali et al. 2014). This suggests that uncontrolled expression of genes known to be phage-derived negatively affects Salmonella fitness, and in order for an H-NS-deficient cell to regain wild-type fitness, it must halt transcription of laterally acquired genes, either by silencing the transcription of these genes (via StpA) or by disrupting the sequences of these genes. This implies that transcriptional silencing can be applied to bacteriophage genomes upon their injection into the cell, to prevent the synthesis of phage proteins which may negatively impact on cell fitness (Skennerton et al. 2011).

Phage gene silencing by H-NS is complicated by the fact that CRISPR loci, which must be transcribed in order for the CRISPR/Cas immunity system to operate, are produced from laterally-acquired DNA sequences and are therefore AT-rich. Consequently, transcription of CRISPR loci is inhibited by H-NS, a hypothesis which is supported by the observation that an hns E. coli mutant overexpresses CRISPR/Cas system components (Pougach et al. 2010, Westra et al. 2010, Yosef et al. 2011). In addition, the LeuO anti-repressor, known to alleviate the effect of H-NS repression (Stoebel et al. 2008), has been shown to induce CRISPR/Cas synthesis (Westra et al. 2010). It can be concluded that phage genomes, as well as CRISPR/Cas systems, are under tight, dynamic, and complex


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transcriptional control.

Strategies Employed by Phage to Defeat Bacterial Transcriptional RegulationAlthough phage genes and CRISPR/Cas systems are under careful transcriptional regulation, a number of strategies have been described by which phages can co-opt these control systems, to such an extent that H-NS silencing of phage genes has been described as a bacterial “Achilles’ Heel” (Skennerton et al. 2011). For instance, bacteriophages have been described which contain hns-like genes in their genomes (Doyle et al. 2007, Skennerton et al. 2011). These phages therefore encode proteins which are likely to silence the transcription of their genes. Although this may seem unhelpful to the phage, these proteins also act to silence transcription of AT-rich CRISPR loci and cas genes on the bacterial chromosome, rendering CRISPR/Cas useless for the resistance of a phage infection (Skennerton et al. 2011). This suggests that phages have evolved mechanisms to evade anti-phage systems by simply repressing their transcription.

Other recent studies further increase the complexity of phage/host transcriptional regulation. For example, bacteriophage T7 encodes the 5.5 protein, which has been demonstrated to bind to the H-NS oligomerisation domain. 5.5 protein binding inhibits the formation of H-NS multimers and reduces the effect of H-NS-mediated gene silencing (H-NS repression is dependent upon oligomerisation), increasing the probability that T7 genes will be transcribed in a host cell (Ali et al. 2011). Similarly, T4 bacteriophage possesses the Arn protein which is believed to sequester H-NS from the bacterial cytosol. One Arn domain mimics the 3-D structure and the chemistry of AT-rich H-NS nucleic acid binding sites, and causes H-NS to specifically bind to Arn. Once sequestered by Arn, H-NS has no repressive activity, and this depletion of active H-NS means that T4 genes will not experience H-NS-mediated repression (Ho et al. 2014). The effects of selected phage anti-H-NS-silencing strategies are summarised in Figure 2. Plainly, phage and bacterium have evolved to interact with one another at the level of transcription, although the downstream effects of these interactions on global

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transcription in bacteria have not yet been studied.

Figure 2: Summary of three potential transcriptional interactions that may take place between phage and bacterium. Phage DNA is injected into the bacterial cytosol through the cell membrane. A: Transcription from phage promoters is repressed by H-NS (Lucchini et al. 2006), allowing the bacterium time to destroy the invading genome using REases and crRNA-guided Cas endonucleases. B: The phage genome may contain a gene encoding a protein which is biochemically and physically analogous to DNA regions to which H-NS can bind. If this protein is synthesised, phage-encoded proteins will sequester H-NS, reducing the repressive effect on the transcription of all other phage genes (Ho et al. 2014). C: The phage genome may also encode an H-NS orthologue, which is capable of binding to bacterial promoters associated with bacterial CRISPR loci. Upon binding, this pseudo-H-NS molecule represses the transcription of CRISPR loci, inhibiting the ability of the bacterium to mount a CRISPR/Cas-mediated defence against the phage genome (Ali et al. 2011).

ConclusionsBacteria possess several systems to resist a phage infection. R-M and CRISPR/Cas immunity allow cells to identify a foreign nucleic acid on the basis of its sequence and its epigenetic modifications. However, time is a dimension of significance in considering these resistances. Given sufficient time and opportunity, it is plausible


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to expect that a single infected cell could defend itself against a phage using these endonuclease-based strategies. Upon injection of a phage genome into a bacterium, the cell must have time for its R-M or guided Cas endonucleases to reach the phage molecule, recognise it as non-self, and to destroy it, all before phage genes can be transcribed by bacteria RNA polymerase. Since the beginning of phage gene transcription will eventually cause bacterial lysis, it is logical that delaying the transcription of phage genes using a repressor such as H-NS would increase the time that the cell has to resist the phage infection. Since H-NS is constitutively expressed in the cell (Kröger et al. 2013), its inherent ability to delay phage gene transcription may allow the cell time to upregulate transcription of CRISPR/Cas or REase genes in response to an infection.

Phage/host co-evolution is known to have been driven by interactions through R-M and CRISPR/Cas immunities (Westra et al. 2012, Samson & Moineau 2013, Westra et al. 2014), but there is evidence that co-evolution has also occurred at the level of transcriptional control. Phages appear to have co-opted bacterial transcriptional repressors to prevent the transcription of CRISPR loci into RNA. Phages are also capable of sequestering bacterial repressors to ensure the transcription of their own genes, which may have a secondary effect on the bacterial transcriptome. Evidently, bacteria need time to resist a phage infection, and transcriptional repression of phage genes may facilitate this. It is therefore in the interest of phages to inhibit the activity of bacterial repressors, ensuring the successful and rapid synthesis of phage-encoded proteins. Hence, the role played by transcriptional regulation in maximising the time available to bacteria to destroy phage DNA should be considered a bacterial strategy for resisting phage attack in the same context as R-M, Abi, and CRISPR/Cas.

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AcknowledgementsThe author wishes to thank Dr.’s K. M. Devine, N. Ní Bhriain, S. C. Dillon, and C. J. Dorman for helpful discussions, and acknowledges recent personal research support from the Society for General Microbiology, the Society of Biology, and the Wellcome Trust.

Components of this manuscript have been adapted from an essay “Bacterial Protection Mechanisms against Phage Attack: An Evolutionary Perspective” for course GE3M31, submitted in January 2015.

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Lysine Methylation of the p53 Tumour Suppressor:

Analogies with Histone Biology Genetics

Hayley PorterJunior Sophister

p53 is an essential mediator of cellular responses to genotoxic stresses. As aberrant p53 activity is highly linked to cancer incidence and oncogenic activity, understanding the regulation of p53 is crucial to our ability to understand and fight many different cancers. Post-translational modifications of p53 mediate its ability to respond to the various genotoxic stresses experienced by cells. These modifications have been found to be strongly related to the post-translational modifications of histone proteins. This review will discuss identified similarities between lysine methylation events on both proteins and how the extent of these similarities can be used to expand our current knowledge surrounding the regulation of p53 activity. Furthermore, formation of a ‘p53 code’, analogous to the established ‘histone code’, will be discussed, in which, the versatile nature of p53 modifications, including methylation, has altered consideration of post-translational modifications from ‘on/off switch’ mechanisms, to highly interactive, fine-tuning mechanisms. The limitations of experimental techniques are important issues to be taken into consideration when evaluating past discoveries and when anticipating future perspectives within the field. Many in vitro systems are subject to aberrant conditions, resulting in abnormal activity of p53. Despite organismal differences, in vivo verification in mouse models or physiologically relevant cell culture environments represents a key method of minimising these shortcomings. Some key p53 modifications still require physiologically relevant in vivo verification, such

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as, characterisation of the interaction between p53K382me1 and the L3MBTL1 effector protein.

IntroductionThe p53 tumour-suppressor protein acts to protect cells from malignant transformation following exposure to various stresses. This is achieved via p53-dependent induction of either cell cycle arrest or apoptosis. Differential induction of cell cycle arrest genes, such as p21/CDKN1A, or pro-apoptotic genes¸ such as Bax, Puma, and Noxa, mediate this decision and result in the distinct cellular outcomes of the specific response pathway (el-Deiry et al. 1993, Oda et al. 2000, Vousden & Lu 2002). The precise mechanism by which damaged cells choose between these distinct responses, following varied malignancy-inducing stresses, is not yet fully understood, but is generally thought to be mainly influenced by post-translational modifications of p53 (Gu & Roeder 1997). Post-translational modifications of histone proteins are well documented, with each type of modification having been observed to affect chromatin via one of two distinct outcomes. Acetylation¬ and phosphorylation of histone proteins have been recognised as modifiers of the charge density of histone tails, ultimately resulting in physical alteration of chromatin’s open/closed state. Contrastingly, methylation, neddylation, sumoylation, and ubiquitination do not physically affect the open/closed state of chromatin, but instead act to modulate interaction with ‘reader’/histone-binding proteins, thus controlling the effects of these cellular factors. For example, methylation of lysine 9 on histone H3 (H3K9) has been shown to create a binding site for heterochromatin protein 1 (HP1) family members, allowing these effector proteins to act on chromatin, changing it into its heterochromatin state (Strahl & Allis 2000, Lachner et al. 2001). These ideas underlie the ‘Histone code’ hypothesis, proposed by Strahl and Allis (2000), which brings together the interplay of modifications, combining direct chromatin alterations and modulation of protein-protein interaction to form a powerful mechanism of regulating chromatin structure and hence, transcriptional activity (Jenuwein & Allis 2001). By analogy with the ‘histone code’, a ‘p53 code’/‘protein code’ has been proposed to explain the functional consequence of


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p53 modifications (Toledo & Wahl 2006, Scoumanne & Chen 2008, Sims & Reinberg 2008). This review will explore current knowledge surrounding post-translational modification of p53 by lysine (K) methylation, through the idea of a ‘p53 code’ of modifications, which regulate the distinct cellular pathways induced by p53 in response to varied forms of stress. Parallels between the ‘histone code’ and ‘p53 code’ will be investigated in consideration of potential p53 methylation events, alongside consideration of the effectiveness of current experimental techniques.

Lysine Methylation of p53: How Histone Biology Carved the Way Similar to the methylation sites of histone H3 and histone H4, p53 has an unstructured terminal tail that contains a number of lysine residues capable of accepting methyl groups. Lysine methylation of histones modulates the surface architecture of the protein, promoting or inhibiting modular binding by effector proteins (West & Gozani 2011). For example, monometylation of histone H4 lysine 20 is seen frequently in condensed chromatin, as this modification allows interaction of the histone with L3MBTL1, a chromatin compaction factor (Kalakonda et al. 2008).

Major progress in the field of p53 lysine methylation was made following the discovery of Set7/Set9 methyltransferase (Set7/9) activity towards p53 (Chuikov et al. 2004). Methyltranferases are examples of so called ‘writer’ proteins, which modulate the surface architecture of their substrates via addition of methyl groups. Specifically targeted lysine residues can enter a narrow channel in the core of the Set7/9 protein, where addition of a methyl group is then catalysed (Wilson et al. 2002, Kwon et al. 2003). Set7/9 was first identified as a methyltransferase with specificity for lysine 4 of histone H3 (Wang et al. 2001, Nishioka et al. 2002a). Subsequently, lysine residue 372 (K372) of p53 was identified as a Set7/9 target. Comparison with the established Set7/Set9-H3 complex led to the identification of a putative Set7/Set9-p53 complex, with structural and binding similarities helping to verify the p53 methylation event. This methylation event was in fact found to be required for

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p53 stabilisation, and although the mechanism of this methylation-induced stabilisation was not established, comparison with mechanisms identified in histone biology allowed hypothesis of yet-unidentified factors binding to the methylated p53 and interfering with Mdm2-dependent ubiquitination (Chuikov et al. 2004).

This hypothesis was supported by the findings of Ivanov et al. (2007) in which stabilization of p53 and promotion of its transcription factor activity was seen to occur after methylation by Set7/9, with no effect on p53’s DNA-binding activity observable. To test if this methylation event induced stabilisation via direct elimination of subsequent ubiquitination (a modification known to label p53 for degradation), wild-type and K372R (lysine-to-arginine) mutant ectopic p53 were tested for relative efficiencies of in vivo ubiquitination. However, similar ubiquitination levels were observed, nullifying this hypothesis. Comparison with histone biology revealed an alternate, indirect mechanism for interference with ubiquitination. Similar to methylation of K4 residues increasing K9 and K14 acetylation in histone H3, it was demonstrated that lysine methylation of p53 is imperative for downstream acetylation at proximal C-terminal lysine residues (Zegerman et al. 2002, Ivanov et al., 2007). While use of small hairpin RNA (shRNA) to silence Set7/9 expression was found to have no effect on histone H3 methylation, methylation of p53K372 was diminished, along with DNA damage-induced acetylation of p53. Thus, methylation of p53K372 stimulates acetylation, providing a mechanism for the obstruction of ubiquitination. This methylation-acetylation interplay not only stabilises and activates p53 by blocking its ubiquitination, it also promotes acetylation of histone H4 at, at least, the promoter of the canonical p53 target gene, p21. Jointly, these events up-regulate p21, ultimately leading to cell cycle arrest.

Complexity of Methylation-mediated Regulation Suggests a ‘p53 Code’Aside from this complex interplay with other modifications, Set7/9-mediated methylation of p53K372 has also been shown to


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be an activating event, due to its inhibitory role in the prevention of Smyd2-mediated methylation of lysine 370, a monomethylation event with a repressive effect on p53 activity (Huang et al., 2006). This web of interacting modifications is suggestive of a ‘p53 code’, as no one event is acting as an independent on/off switch for p53 activity.

As well as establishing that monomethylation of K370 (K370me1) represses p53 function, this collaboration also revealed that dimethylation of K370 (K370me2) activates p53 function (Huang et al., 2007). Although the distinct methyltransferase responsible for this event was not identified by their work, discovery of the first incidence of demethylation of a non-histone protein was shown in this study, with demonstration that the histone lysine-specific demethylase LSD1 demethylates p53 at K370. While in vitro study showed demethylation of K370me1 and K370me2, LSD1 was shown to have a strong preference for demethylation of K370me2 in vivo. According to knowledge of the consequences of different lysine methylation states in histones, effector proteins of the K370 methylation states were investigated. Using a GST protein domain microarray, the tandem Tudor domains of the p53 co-activator p53-binding protein 1 (53BP1) were found to bind preferentially to K370me2 (compared with K370me0, K370me1 or K370me3). Strong evidence of LSD1-mediated modification of p53K370me2, and not p53K370me1, resulting in regulation of p53 activity via obstruction of p53-53BP1 interaction, was supplied via direct assay experiments, adding to both the known complexity of methylation-mediated regulation of p53 and to the idea of post-translational modifications acting as a fine-tuner for p53 activity (Huang et al., 2007).

Lysine methylation of p53K382 & the Drawbacks of Current Experimental TechniquesSET8/PR-Set7 (Set8) is a histone methyltransferase that has been established to monomethylate histone H4 tails at K20 (Fang et al. 2002, Nishioka et al. 2002b). SET8-mediated regulation of p21 and PUMA, in response to DNA damage, has been determined to be due to direct activity as a p53 methyltransferase, and not due to

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H4K20 methylation effects. Lysine residue K382 was identified as SET8’s target methylation site, and confirmed in vitro via ablation of SET8-mediated methylation, solely upon substitution of K382, and in vivo via decreases in p53K382me1 after SET8 knockdown by RNA interference (RNAi). p53K382me1 levels decrease following DNA damage, corresponding with functional characterization as a mark that contributes to the inhibition of p53-mediated transcriptional activation (Shi et al. 2007). MBT domain-containing proteins have been demonstrated to show preferential binding for monomethylysines on histone proteins (Kim et al. 2006). As lysine methylation alters the surface architecture of p53, and thus should achieve this regulatory activity via influence on interaction with a distinct protein binding partner, interaction with a Malignant Brain Tumor (MBT) domain-containing protein, was hypothesised to explain the molecular mechanism by which p53K382me1 acts to repress p53 (Shi et al. 2007).

L3MBTL1 is an established member of the polycomb group proteins. As a transcriptional repressor it promotes compaction of chromatin, enhancing inaccessibility of areas enriched for mono- and dimethylated histone marks (Kalakonda et al. 2008). Sequence similarity between the L3MBTL1-target H4K20 and p53K382 led to subsequent identification of L3MBTL1 as this MBT-containing protein. Addition of a single methyl moiety at K382 promotes p53-L3MBTL1 interaction, resulting in inhibition of p53 transcriptional activation by subsequent stabilization of L3MBTL1 occupancy at the p21 (and PUMA) promoter. This phenomenon was shown in the absence of DNA damage, to ensure no effects from alternate mechanisms of p21 regulation were recorded. Upon depletion of L3MBTL1 by RNAi, up-regulation of p21 transcript levels was found in p53-containing cells, but not in the absence of p53. Thus, p53 target gene repression was suggested to be achieved due to p53-mediated stabilisation of L3MBTL1 at chromatin locations of these genes. Accordingly, upon DNA damage, reduction in p53K382me1 levels abolishes the p53-L3MBTL1 interaction, resulting in disassociation of L3MBTL1 from the p53-high response targets, allowing immediate activation by the adjacent, hitherto quiescent p53 (West et al. 2010).


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In contrast to L3MBTL1 Flag-immunoprecipitates (IPs), it was noted that the P53-L3MBTL1 interaction was not affected by DNA damage in whole cell extracts prepared from 293T cells. 293T cells contain the simian virus 40 (SV40) larger T antigen, which stabilises and inactivates p53, resulting in high levels of endogenous p53 (Fu et al. 2010). It is unclear whether p53 levels are unaffected by DNA damage in these cells due to this altered p53 activity or whether unknown interactants in the whole cell extract dampen this DNA damage response. To avoid such issues, p53 activity should, ideally, be investigated in cell lines in which p53 activity is not already altered. Furthermore, the decrease in occupancy of L3MBTL1 at the p21 promoter is ~15-20 lower than the increase in p53 occupancy. The reason for this discrepancy is not discussed and presents an area of further study, in which determination of the mechanism behind this amplification of p53 occupancy may shed further light on the p53-L3MBTL1 relationship.

Contrary to the functional characterisation of SET8 as a negative regulator of p53 activity, SET8-dependent p53-K382me1 binding on the promoter of GADD45 was found to be up-regulated in response to DNA damage. GADD45’s role in DNA repair thus indicates that methylation of K382 may play a more complex role than a simple on/off switch. Further studies are needed to explore its proposed role as a mechanism for predisposing p53 to mild cellular stresses, allowing a shift in p53-mediated transcription towards DNA repair without initiation of apoptosis (Shi et al. 2007).

Demethylation of p53: A Knowledge VoidApart from LSD1, no additional p53 demethylases have been conclusively defined. Reduction of p53K372me1 levels have been recorded during p53 activation, suggesting this mark is subject to demethylation (Huang & Berger 2008). While the histone lysine demethylase JMJD3/KdM6b has been suggested to be responsible for this demethylation event, direct evidence of p53 demethylation by JMJD3 was not shown in this study (Ene et al. 2012). Furthermore, Williams et al. (2014) were unable to show demethylation of p53 peptides by JMJD3 in vitro, suggesting JMJD3 does not act as a p53

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demethylase, but may instead act on p53 responsive elements, via interaction with p53. Alternatively, demethylation of currently unidentified p53 methylation sites by JMJD3 cannot be ruled out by either of these studies. As shown by Cao et al. (2013), new methods of in vivo large-scale surveys may yet identify many more lysine methylation sites in p53, making the likelihood of an unknown JMJD3 demethylation site in p53 much more probable.

JMJD2c and PLU-1, like JMJD3, are members of the JmjC domain-containing protein family (He et al. 2012). Both proteins have been characterised as histone demethylase, with JMJD2c responsible for H3K9me3/me2 demethylation, and PLU-1 responsible for demethylation of H3K4me (Cloos et al. 2006, Yamane et al. 2007). Changes in the expression of JMJD2c and PLU-1 have both been found to have no substantial effect on p53 levels, indicating that these demethylases are not likely to demethylate p53, even through currently unidentified methylation sites (Yamane et al. 2007, Ishimura et al. 2009). Therefore, expansion of current knowledge surrounding p53 demethylation must look beyond these histone demethylases to untested demethylases or to completely novel regulators of p53.

ConclusionsIt is clear that post-translational modifications regulate p53 and histones via the same set of underlying principles. Just as methylation regulates the activity of histone proteins via alteration of their ability to interact with a range of effector proteins, so too does methylation regulate the activity of p53 via modulation of its capacity to associate with a range of interaction partners. These modification processes are also highly interconnected, lysine methylation ‘writer’ proteins, including Set7/Set9, Set8, and LSD1 show functionally overlapping activity, as do the ‘reader’/effector proteins L3MBTL1 and 53BP1. Thus, we can see that apparent parallels allow elucidation of the function of unannotated modifications in p53, by drawing comparison to partner histone modifications. While this technique may not yet have resulted in successful characterisation of additional p53 demethylase proteins, examination of untested histone demethylases presents an area where future investigation


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is likely to yield some significant results. It is also important to note that the discovery of novel p53 methylases or demethylases could uncovered unidentified histone-targeting proteins.

The limitations of experimental techniques can beget inconsistent or inconclusive, results as evident in the characterisation of p53-L3MBTL1 interaction. Moreover, investigation of p53 in vitro can lead to incorrect characterisation of modifications or their interactants. Transfection stress, aberrant ratios of p53 and its regulators or targets, and forcing of nonphsiologic interaction can result in the deviant phenotypes from which these characterisations arise. These phenomenon can occur due to overexpression of one or more proteins within the system, due to the selective pressure of cell culture environments, or simply from the use of cancer cell lines, such as the 293T cell line (Toledo & Wahl 2006, Love & Grossman 2012). Validation of L3MBTL1’s association with p53, and of any putative modification, by investigation of activity in in vivo mouse models or physiologically relevant cell culture environments, must become an essential component of experimental procedure in order to best circumvent these shortcomings. As assay development and mouse models become more sophisticated, including the development of human p53 knock-in ‘HUPKI’ mouse models, this requirement will become less of a challenge, as even physiological distinctions between humans and mice can be overcome (Luo et al. 2001).

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AcknowledgementsThis work was adapted from an essay “Post-translational modifications of the p53 tumour suppressor” for course GE3M31.

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DNA VaccinesMolecular Medicine

Jeremy DoverJunior Sophister

In order to stay ahead of ever changing pathogens, vaccinology must forever be moving forward. Eliciting potent, targeted, safe immune responses against pathogens is a challenge that can be addressed in many ways. DNA vaccines have been touted as the next step forward, the “third generation” of vaccines. However challenges of poor immunogenicity still need to be addressed.

Several methods of doing this are reviewed here.

IntroductionOne of the major hopes for future vaccine development is the prog-ress of DNA vaccines, touted the “third generation” of vaccine tech-nology. DNA vaccination involves the introduction of genes encod-ing the vaccine antigen, to a host, whose cells then expresses the antigen, inciting an immune response. The concept was first shown in 1992, when Tang et al. (1992) injected the genes coding for a pro-tein into mice and elicited the same immunological response as in-jecting the pure protein. There are many advantages to this form of vaccination over current methods: pDNA (plasmid DNA) is cheap, easy to manufacture and work with. Unlike conventional vaccines, they do not require refrigerated transport and storage, a huge bo-nus for the distribution of vaccines to certain regions, such as rural African areas. This is due to DNA being a relatively stable biomol-ecule, compared to other molecules used in vaccines such as vari-ous proteins (McMichael et al. 1983). Another attractive feature is its safety. As they only encode certain antigens, there is no danger of the vaccine reverting to its viral form, as can happen in attenuated vaccines. Attenuated vaccines can also be harmful to immunocom-promised recipients (Jean-Philippe et al. 2007), and some pathogens

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are considered too risky to deliver even if attenuated, DNA vaccines can safely deliver immunity to a broader range of pathogens. DNA vaccines can be constructed chemically, instead of from potentially dangerous virulent sources, making safety another plus for DNA vaccination.

Perhaps the biggest advantage of DNA vaccines is their ability to generate a cellular as well as humoral immune response, spanning all areas of adaptive immunity (antibodies, helper T cells, cytolytic T-lymphocytes (CTLs)) through the activation of both MHC class-I and class-II molecules. A vaccine such as this, which can generate CTLs, is very desirable as highly conserved epitopes across various strains tend to be internal or functional proteins, making access difficult for antibodies. Generating a CTL immune response against these epitopes is important in delivering heterologous immunity, as the antigens that can be reached by antibodies, are likely to mutate causing pathogen resistance to the vaccine. CTLs can reach the highly conserved epitopes, resulting in a broader range of immunity. It has been shown that CTLs can provide immunity against viral challenges, even if pre-existing antibodies to the specific strain of virus do not exist (McMichael et al. 1983). This CTL response cannot be stimulated by normal vaccines, as upon injection of the exogenous protein or inactive vaccine, the particles are guided down the endolysosomal pathway, activating major histocompatibility complex II (MHC II) on antigen presenting cells (APCs), which in turn stimulate CD4+ (helper T) cells. These produce cytokines and trigger B cells to produce antibodies. In order to produce a substantial CTL response, intracellular production of the foreign antigens needs to take place (such as during viral infection of a cell). These antigens are processed by proteasomes and picked up by nascent MHC class I molecules. The complex is then transported through the Golgi apparatus onto the cell surface (Fig. 1). Immature CD8+ (CTLs) bind and are activated, begin dividing, and start destroying cells presenting the antigen. The ability to deliver a gene encoding antigens straight into cells, which will then be processed through the endogenous pathway described above, is the main motivation behind the development of vector based vaccines. In particular the ability the generate CTLs is particularly exciting in the area of therapeutic cancer application, “breaking” the bodies


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tolerance towards the cancer cells. The area of cancer vaccines, in particular DC vaccines will be explored later in this article.

Initial trials demonstrating the safety and immunogenicity of DNA vaccines, lead to great excitement at the potential for the technol-ogy. Early fears that the DNA vector could elicit an autoimmune response (Klinman et al. 2000) proved to be unfounded, and DNA vaccines are now being developed to treat autoimmune conditions. There are currently four DNA vaccine products licensed for use in animals (Liu 2011), however the majority of clinical trials in humans to date, have resulted in lower than anticipated immune respons-es. The mechanism by which immune responses are generated from DNA vaccines proved more complex than initially thought. The next section will deal with methods used to enhance immune response, to a clinically relevant level.

Figure 1: Plasmid DNA entry via viral vector/electroporation. Encoded antigens are then expressed and presented for immune response from T+B cells (Liu 2011).

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Methods of Enhancing Immune ResponseVarious methods of enhancing the, until now, disappointing im-munogenicity of vector vaccines, have been researched. The use of adjuvants, both native and foreign, the method of administration, the construction of the DNA vector with various promoters and enhancers, different vector types (viral, bacteria etc.) have all been shown to be effective.

Cytokine AdjuvantsThe method that seems to be showing the most promise is the addi-tion of genes encoding adjuvants to the vaccine. Adjuvants are com-pounds that potentiate/modulate the immune response of a vaccine towards an antigen in a desired direction. Molecules such as cyto-kines, growth factors, TLR ligands and enzymes have all proved to be effective adjuvants. Cytokines will be dealt with in detail here as their clinical use as adjuvants has been well established. Purified recombinant cytokines are very expensive and there are dangers as-sociated with their use in vaccines, so Raz et al. (1993) developed a new approach, injecting cytokine encoding genes straight into the muscle, resulting in in vivo production of cytokines. This effect was shown to last several weeks, showing that administration of vectors encoding cytokines can regulate humoral and cellular responses. Cytokines also have disadvantages as adjuvants, they have a rela-tively short half-life in the body, and one type of cytokine can mod-erate the activities of another (Trinchieri 2003), leading to an unpre-dictable immune response. Despite this, the immunodulatory effects of cytokines as a way of amplifying DNA vaccine effects have been well established. When the cytokine, IL-2, was administered as an adjuvant alongside a DNA vaccine against HIV-1, the HIV-1-spe-cific delayed-type hypersensitivity response and CTL activity were significantly amplified (Xin et al. 1998). Further studies found that the effect of the cytokine IL-2 as an adjuvant was improved by the creation of an IL-2/immunoglobulin fusion protein, due to the in-creased half-life of the new protein. This protein augmented cellu-lar immune responses in HIV-1 (Barouch et al. 1998). These results have been carried forward to human trails with promising results.


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There are two main subtypes of cytokines, type 1 which enhance the proliferation, stimulation and/or function of T cells (INFγ, TNFα, etc.), and type 2 which favour the enhancement antibody responses through the recruitment of APCs, increasing their ability to prime adaptive immune response. The timing of the administration of the plasmid cytokine in relation to the DNA vector, as well as dosage, an individual’s genome and combination of adjuvants used, all have to be considered.

Delivery MethodsThe traditional method of administering vaccines (intramuscular injection) may not be the most effective form of delivery for DNA vaccines. The bulk of the vectors injected do not actually succeed in transfecting any cells (Dupuis et al. 2000). The majority of those that do, end up transfecting skeletal muscles cells (monocytes), instead of the desired professional APCs. Monocytes are at best are only partially effective at presenting antigens, and do not have any MHC molecules while under physiological conditions (Wiendl et al. 2005). This makes them unable to directly activate CTLs. Instead these cells produce the antigen encoded by the vector, and then by the process of cross presenting, the antigen is transferred to an APC, and the appropriate MHC class-I CTL response elicited. Needle type, size and speed of injection all have effects on optimisation of vaccine ef-fects (Bins et al. 2013). The high speed implantation of vaccine load-ed polymer films, to carry pDNA and bio degradable polycations into the immune cell rich epidermis has been shown to induce up to 140 times more gene expression than a straight forward intradermal DNA injection, in primate skin. This “multilayer tattoo” type of nee-dle injection is used widely in animal studies but not yet in a human clinical context (DeMuth et al. 2013).

The biolistic particle delivery system, or “gene gun” meth-od, originally designed for plant transformation procedures, has been shown to be by far the most efficient way of delivering DNA vaccines (Fynan et al. 1993). It uses compressed helium to accelerate heavy metal particles coated with pDNA directly into cells (direct transfection), as opposed to the extracellular space, where a normal

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injection will deliver its load. The heavy metal tends to be gold, as it is stable and its high molecular weight lends itself to the task. As with the similar “multilayer tattoo” method, the delivery is directed to skin, where many APCs are present, through particle-mediated epidermal delivery (PMED). This direct delivery system means that far less pDNA needs to be used (1-3μg in mice, has to be scaled up depending on organism) compared to a saline injection (2-20μg) (Feltquate et al. 1997). The potency of the technology was also demonstrated when a gene gun delivered DNA vaccine, induced antibody production in 12/16 subjects that had failed to produce a response to a licensed conventional attenuated vaccine (HBsAg vac-cine) (Rottinghaus et al. 2003). However the procedure needs to be performed several times in different places to overcome the weak immunogenicity of pDNA, and also to counteract the small amount of pDNA that can be delivered per bead. Instability of the DNA on the beads themselves is also a hindrance to the development of this technology.

Perhaps the single strongest method of increasing immuno-genicity of DNA vaccines is the Electroporation (EP) method. One of the reasons for the poor immunogenicity of DNA vaccines in cells is the lack of uptake into cells of the foreign DNA molecules. Transfection of DNA into cells has long been aided through the use of EP, where an electrical field induces a voltage across the mem-brane, opening temporary pores, allowing pDNA (or any desired macromolecule) to pass through inside. The cell reseals slowly (sec-onds-minutes) after the destabilisation. It is still debated whether the DNA passes through by electrophoretic facilitation or passive diffusion. This EP method is used both in the lab and also on the macro scale, where it is used alongside the administration of the pig gene therapy product described in a previous section. Indeed this method has shown to increase antigen delivery by up to 1000 fold compared to delivery of naked DNA alone (Sardesai & Weiner 2011). EP induces a large Th1 response, with an immune response more than 10 times that of other pDNA methods. The use of EP is an exciting area of investigation, and can be used in conjunction with other forms of transport to incite an even larger immune response. Currently there has been at least one phase 1 clinical trial using the EP method (Heller & Heller 2010).


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Vectors

Artificially constructed plasmids are used as vectors for the purpos-es of DNA vaccination. A vector is a DNA molecule that can be used to transport foreign DNA into cells. The very makeup of the vectors used in a DNA vaccine can be altered to elicit better immunogenicity. A good vector design is important in maximising gene expression. Various factors need to be considered when developing a vector for use as a DNA vaccine, and gene capacity, safety, simplicity to work with and expression of heterologous protein all need to be taken into account. The consideration of the immune response to the vector itself was, and still is, not fully understood. Using DNA encoding for HIV-1 envelope proteins (gp120 and gp41) as model antigens, various vector manipulations and their effects have been studied. Viral gene transcription and translation in host mammalian cells can be hindered by biased codon usage (the favouring of one particular codon across the genome over others that code for the same amino acid) (Wang et al. 2006)2006 . Codon optimisation, from wild type codons to codons with a higher frequency of mammalian tRNA, has been carried out successfully on gp120 encoding sequences. In vi-tro expression of the HIV envelope protein was greatly increased, in vivo, it resulted in increased CTL reactivity and antibody titres (André et al. 1998)1998. Thus optimising codon usage on synthetic vectors represents a workable way to increase efficiency of expres-sion in DNA vaccines.

The significance of Toll-like receptor (TLR) driven adaptive immune response has resulted in TLR ligands emerging as attractive adjuvants to DNA vaccines, expressed through the vaccine vector. TLRs recognise broadly persevered molecules across pathogens, known as pathogen-associated molecular patterns (PAMPs). TLRs are expressed on various APCs, with 13 different subtypes having been discovered so far. Activation of TLRs leads to proinflammato-ry immune responses. Of specific interest in relation to DNA vac-cines is TLR9, the only TLR to recognise DNA. TLR9 is activated by unmethylated CpG (cytosine –phosphate- guanine) motifs. This is used to alert the immune system to the presence of viral or bacterial infection, both of which have DNA with high levels of unmethylat-ed CpG, in contrast to low levels present in mammalian genomes.

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Oligonucleotides containing CpG motifs have shown to induce sig-nificant B-cell immune response (Krieg et al. 1995)1995. Cytokine secretion is also enhanced. Generally DNA plasmids are produced on bacterial vectors, and hence have large amounts of these CpG motifs. It would thus be naïve to think that the plasmid backbone is immunologically inert and has no effect on the immunogenicity of the vaccine. The activation of the TLR leads to triggering both innate and adaptive immune response. Initial phase 1 and 2 trials, of the ef-fects/effectiveness of oligonucleotides containing CpG motifs as vac-cine adjuvants in humans have been promising. Subjects given the motif compound showed higher IgG (class of antibody) levels then subjects who had been given just the vaccine and saline (used as a control). The test subjects given higher doses of the motifs also tend-ed to have higher CTL levels compared to control groups (Cooper et al. 2004), only relatively mild side effects were observed during the study. This demonstrates the potential for exploiting these CpG motifs, as anti-tumour agents (through the stimulation of CTLs) and as standard vaccine adjuvants.

Another Toll-like receptor, TLR5, has also been the subject of research for enhancing DNA vaccine immunogenicity. TLR5s are unique as they are the only TLR with a protein as their antagonist. They are stimulated by flagellin, the primary protein building block of bacterial (both gram positive and negative) flagella (Hayashi et al. 2001). Stimulation results in maturation of DCs and production of cytokines. DNA vaccines encoding for this protein on the vec-tor have been shown to function well as adjuvants (Applequist et al. 2005)2005. A transmembrane bound version of Salmonella flagel-lin, FLiC was used in this study. The in vivo expression of FLiC by mammalian cells activated monocytes, inducing local inflammation. Significant antibody and cellular responses were also noted.

Prime Boost ImmunisationAn interesting, if slightly confusing observation is the increased po-tency of DNA vaccines when used as part of a mixed modality or prime-boost immunization. A mixed modality vaccine is where two different types of vaccine are given sequentially, the priming shot,


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such as a DNA vaccine, followed up with a boost shot of a different vaccine modality such as a viral vector or protein boost. This results in greater immunogenicity compared to one type being given on its own, regardless of the dosage (Liu 2011).

Interestingly, the sequence of the shots has a large effect on the resulting potency of the treatment. J Schneider et al. (1998) showed this, while at the same time bringing about the very concept of heter-ologous prime-boosting , when they found that injecting mice with a plasmid DNA followed by a boost consisting of a recombinant viral vector vaccine (encoding the same antigen), resulted in very high levels of CD8+ T cell response. When they reversed the order of ad-ministration, this effect was not observed. The initial prime-boost sequence generated immune response greater than either of the vac-cines given alone, or in reverse order, leading to better protection to malarial challenge (Schneider et al. 1998). This was exciting news in the search for greater DNA vaccine immunogenicity even if the exact immunological reason for the observation remains unknown. There has been a few explanations proposed, with the true reason proba-bly being a combination of these and other, as of yet undiscovered, clues. The use of DNA vaccine as the prime, as it only presents the antigen of interest, may focus the immune response, and eliminate vector specific immunity, which would reduce immune response (de Mare et al. 2008). Another possibility is that, while DNA vaccines are excellent at eliciting cellular immune responses, other pre-exist-ing technologies have had better results in relation to humoral im-munity, producing larger antibody responses. This has been seen in numerous studies, using a recombinant protein to greatly increase antibody immune response (Otten et al. 2005). Bacterial/viral vectors may also be used as boosts, they may be advantageous to use due to the innate and adaptive immune responses of the organism to the vectors (Liu 2010). There have been numerous trials of this prime boost technology, the largest being a 16,000 human phase 2 trial for a HIV vaccines in Thailand, with results showing “modest benefit” with vaccine efficacy (reduction in incidents of disease among vacci-nated subjects) around 31.2%, but show exciting possibilities for the future (Rerks-Ngarm et al. 2009). A follow up study a few years later showed that the vaccination did not affect the clinical course of AIDs once the subject was infected (Rerks-Ngarm et al. 2013). Another tri-

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al, a prime-boost vaccine for malaria, carried out on rhesus monkeys showed that while total dosage of the DNA prime was unimportant, the amount of DNA injections and interval between prime and boost were critical in optimising vaccine efficacy. Intervals ranged from 7-21 weeks (Weiss et al. 2007). The most important thing to take away from this study is the finding that shorter intervals between boost and prime lead to less protection, something that must be applied to a human clinical context.

ConclusionsThe use of prime boost technology is probably the most likely way for DNA vaccines to initially make their way into mainstream phar-maceutical use in humans, given that trails have already been car-ried out on thousands of humanoid subjects. Given the efficacy the prime boost system lends to DNA vaccines, along with the fact that they are being used in conjunction with established, licensed, prov-en vaccines, this approach seems the most likely way forward. How-ever none of these methods should be considered in isolation. The benefits of DNA vaccines are too much to ignore, the possibility of a cancer vaccine, not to mention possible treatments of diabetes and allergies (Isakovic et al. 2014, Tasyurek et al. 2014), the ability to elicit meaningful CTL response and the lack of transport issues are just some of them. Combinations of vector design, delivery methods, use of suitable adjuvants along with the use of prime boosts, are the best bet for delivering maximum DNA vaccine immunogenicity in the future.


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AcknowledgementsAdapted from mini review “Vaccines of the Future” – for BI3020

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Thromboxan A2 Receptor (tprα): A Potential Human Drug Target

BiochemistryJudy ConmeySenior Sophister

The human thromboxane A2 receptor (TPRα), a member of the superfamily of G protein-coupled receptors, is involved in platelet activation and vasoconstriction in response to binding of thromboxane A2 (TXA2). However, over-activation of TPRα can lead to the development of thrombotic cardiovascular events such as heart attacks and strokes. Currently the prevention of secondary cardiovascular events is carried out by the administration of aspirin. The TPRα is an important target for drug development due to its role in platelet aggregation and vasoconstriction. This review will focus the potential of TPRα

as a human drug target.

IntroductionThromboxane A2 is a prostanoid that activates vasoconstriction and platelet aggregation through interaction with the thromboxane A2 receptor (TPRα). The official name of this receptor is the thromboxane A2 receptor (Magrane M. and the UniProt consortium 2011), but it is also known as the thromboxane/prostaglandin H2 (PGH2) receptor or the prostanoid TP receptor (Coleman et al. 1994, Ting et al. 2012). For the purpose of this review, thromboxane A2 and the thromboxane A2 receptor will be referred to as TXA2 and TPRα, respectively.

TPRα is a G-protein coupled receptor (GPCR) expressed in human blood platelets and plays a vital role in platelet activation through coupling to the G-protein Gq (among others) (Ting et al. 2012). Although it binds both TXA2 and PGH2, the ligand with greater affinity for this receptor is TXA2 (Devillier & Bessard 1997).

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Activation of TPRα by TXA2 triggers cellular signalling responses which result in platelet adhesion, vesicle trafficking, and changes in the cytoskeleton (Ting et al. 2012), all essential for haemostasis. However, if TPRα is over-activated serious thrombotic cardiovascular diseases may develop such as acute myocardial infarction, hypertension and unstable angina (Halushka & Halushka 2002).

Aspirin is currently used to inhibit platelet TXA2 synthesis as a means of treating a variety of thrombotic cardiovascular diseases, such as acute myocardial infarction, unstable angina and secondary prevention of myocardial infarction (Morinelli & Halushka 1991, Mousa 1999). Aspirin inhibits cyclooxygenases (COXs), key enzymes involved in the biosynthesis of TXA2 (Fig. 1) (Mousa 1999).

However, as mentioned, TXA2 is not the only endogenous ligand for TPRα. Therefore aspirin inhibits just one of the many pathways that can result in platelet aggregation (Mousa 1999). Aspirin, despite its success in the treatment of secondary acute myocardial infarction, has efficacy issues. There is a proportion of the population who may experience “aspirin resistance”, with explanations for this resistance still inconclusive (Halushka & Halushka 2002, Gasparyan et al. 2008). Furthermore, low doses of aspirin carry a risk of increased upper gastrointestinal side effects, such as bleeding and ulcers, which could become life-threatening (Cryer & Feldman 1999). Despite Bousser et al. (2011, p. 2013) concluding that aspirin is still “the gold standard anti-platelet drug for secondary stroke prevention in view of its efficacy, tolerance, and cost”, it is responsible for a high rate of people being hospitalised due to a bad reaction to the drug (Pirmohamed et al. 2004).

Thus, the development of a new anti-platelet drug is necessary for the treatment of numerous diseases where platelet activity is promoted. The three-dimensional structure of TPRα at atomic resolution would aid the design of effective anti-platelet agents by providing a molecular insight into how the receptor functions.

This review aims analyse what is known about TPRα in current literature and highlight some considerations when targeting TPRα.


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Figure 1: The human TXA2 biosynthetic pathway. The chronological steps of thromboxane A2 (TXA2) synthesis and the stages where drugs act, affecting TXA2 function, are shown. TXA2 is synthesised from arachidonic acid (AA) which is released from the membrane by phospholipase A2 (PLA2). Cyclooxygenases (COX-1 and COX-2) convert AA into important biological mediators known as prostanoids, the first being prostaglandin H2 (PGH2), a precursor of thromboxane A2 (TXA2). PGH2 is converted to TXA2, 12-L-hydrooxy-5,8,10-heptadecatrienoic acid (HHT) and malondialdehyde (MDA) by thromboxane synthase (TXS). Thromboxane B2 (TXB2) is a product of TXA2 hydrolysis that is biologically inactive and removed from the body. (Nakahata 2008)

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1. Functions of Thromboxane A2 and Thromboxane A2 Receptor 1.1. Biosynthesis of TXA2

TXA2 is an unstable product of the cycloxygenase (COX) mediated metabolism of arachidonic acid (AA), catalysed by phospholipase A2 (PLA2). COXs synthesise the endoperoxide prostaglandin, prostaglandin H2 (PGH2), from AA which is in turn converted to TXA2 by thromboxane synthase (TXS). TXA2 has a short half-life of about 30 seconds (Fig. 1) before being converted into the inactive TXB2 (Hamberg et al. 1975). Thus, TXA2 acts on neighbouring cells in close proximity to the cell where it is produced in an autocrine or paracrine manner (Nakahata 2008).

1.2. TXA2 and the TPRα Signalling Pathway

Binding of a TXA2 to TPRα elicits several molecular responses which, overall, result in the elevation of intracellular Ca2+ levels which is the key to platelet activation (Fig. 2) (Brass et al. 1997).

Several G-protein isotypes are stimulated by TPRα upon ligand binding. In 2000, Perry V. Halushka outlined that there are at least nine G proteins which couple to TPRα; Gαq, Gαi2, Gαs, Gα11, Gα12, Gα13, Gα15, Gα16 and Gh. The Gq isotype is of interest as TPRα-mediated platelet aggregation primarily associated with Gq (Offermanns et al. 1994). The Gq family activates Phospholipase C beta (PLC-β) which cleavesthe phospholipid phosphatidylinositol 4,5-bisphosphate (PIP2) to form second messengers inositol 1,4,5-triphosphate (IP3) and diacylglycerol (DAG) (Ting et al. 2012). IP3 releases intracellular calcium stores and DAG activates protein kinase C (Fig. 3) (Berridge & Irvine 1984, Kikkawa et al. 1986). Overall, these responses lead to platelet activation (Huang et al. 2004).

http://en.wikipedia.org/wiki/Phospholipid

http://en.wikipedia.org/wiki/Phosphatidylinositol_4,5-bisphosphate



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Figure 2: G protein coupling of thromboxane A2 receptor and signal transduction When ligand bound, TPRα can couple to several different G proteins. When TPRα is bound to Gα/11/h, phospholipase C-β (PLC-β) is activated, which converts phosphatidylinositol 4,5-bisphosphate (PIP2) into 2nd messengers inositol 1,4,5-trisphosphate (IP3) and diacylglycerol (DAG). IP3 releases intracellular calcium stores and DAG activates protein kinase C (Berridge & Irvine 1984, Kikkawa et al. 1986). (adapted from Nakahata 2008).



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Figure 3: TXA2 and TPRα intracellular signalling. Gq protein activates phospholipase C, which hydrolyses phosphatidylinositol 4,5-bisphosphate (PIP2) to produce inositol 1,4,5-triphosphate (IP3) and diacylglycerol (DAG). IP3 elevates the cytosolic free Ca2+ concentration by inducing vesicular release of Ca2+ into cytosol. DAG activates protein kinase C and activates phospholipase A2 stimulation of phospholipase A2 is associated with the activation of glycoprotein IIb/IIIa complex, leading fibrinogen cross-linkage by binding to glycoprotein IIb/IIIa receptors present on each platelet (Jeng et al. 1998). Activation of PLA2 also amplifies the signal transduction message by the generation of more TXA2 from arachidonic acid by cyclooxygenase (COX). Aspirin inhibits COXs, down-regulating TXA2 and thus platelet aggregations and clot formation. (Shiau et al. 2012)

1.3. Physiological/Pathophysiological role of TXA2 and TXA2 Receptors in cardiovascular diseases

Platelet activation is a significant physiological response when TXA2 binds TPRα. Platelet activation leads to alterations in platelet shape, aggregation and secretion. This in turn stimulates thrombus formation (Ally & Horrobin 1980, Dorn & DeJesus 1991, Spurney et al. 1991, Katugampola & Davenport 2001). TXA2-induced platelet aggregation and vasoconstriction can lead to acute myocardial


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infarction (Martin 1984, Hiraku et al. 1986, Dorn et al. 1990, Katugampola & Davenport 2001). These disease states are currently treated by TXA2 synthesis inhibitors but not TPRα antagonists. TPRα antagonists could in theory be an effective means of treatment, but to date development of this class of drugs has been unsuccessful. TXA2 is a ligand well-known to exert its actions through TPRα, but it is not the only endogenous ligand to do so. There is current evidence that suggests isoprostane 8-iso-prostaglandin F2α (8-iso PGF2α) is another TPRα stimulant (Montuschi et al. 2004). Isoprostanes are peroxidative products of AAs, and are more stable when compared to TXA2 (Montuschi et al. 2004). Therefore, TXA2 may not by the primary ligand responsible for the cardiovascular diseases associated with TPRα-mediated signalling.

2. Physiological/Pathophysiological Effects of Targeting TPRα-Mediated Signalling 2.1. Marketed Drugs which Target TPRα-mediated Signalling

An effective antagonist targeting TPRα-mediated signalling has not been synthesised to date due, in part, to the fact not enough is known about what happens structurally when TPRα becomes activated. If the crystal structure was determined then it is possible that an effective drug could be synthesised based on the structure of the receptor. As the antagonist would be specific for the TPRα, it should not, in theory, have any serious side effects. To further qualify it as effective and successful in clinical trials, the proposed TPRα drug should be suitable for oral or intravenous administration, like that of aspirin. Also, the length of time of which the drug acts in the body should be adequate to therapeutically treat the disease state (Mousa 1999) i.e. it must be stable and not breakdown into a potentially dangerous by-product in-vivo.

The class of drugs which target TXA2 production itself have been very successful to date (aspirin and dazoxiban). Aspirin and non-steroidal anti-inflammatory drugs (NSAIDS) are cycloxygenase inhibitors which inhibit TXA2 at the early stages of biosynthesis.

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These inhibitors stop TXA2 production by blocking arachidonic acid conversion to PGH2 (Fig. 1). There are three cycloxygenase (COX) isoenzymes identified; COX-1, COX-2 and COX-3. Platelets express COX-1 which can be inhibited non-selectively (i.e. by COX inhibitors which target both COX-1 and COX-2). Aspirin inhibits COX-1 by irreversibly acetylating a serine residue in the catalytic channel (Roth et al. 1977). At higher concentrations, COX-2 may also be inhibited by aspirin (Cipollone et al. 1997). This action leads to long-term down-regulation of TXA2 synthesis in platelets as they don’t possess a nucleus and cannot make their own COX-1.

Low dose aspirin remains the best standard of secondary prevention after myocardial infarction or stroke and more than 80% of such patients take aspirin regularly (Campbell et al. 2007). Despite the success of this class of drugs, none of the current anti-platelet agents available to the public meet all of the requirements outlined, as their lack of selectivity result in undesirable side-effects (Mousa 1999).

A negative side-effect of the non-selectivity of aspirin is that it may also inhibit PGI2 synthesis, along with several other prostaglandins (Fig. 4) (Coccheri 2010). PGI2 is a lipid essential for the maintenance of the endothelium and its inhibition can lead to deleterious side-effects such as disruption of the gastrointestinal lining. The ability of aspirin to inhibit PGI2 production is believed to be relevant to explaining why some people are sensitive to aspirin while others are resistant (Pusch et al. 2008, Coccheri 2010). It is for these reasons that research efforts have shifted to focus on other targets in the signalling pathway.


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Figure 4: The cyclooxygenase (COX) and prostanoid biosynthetic and signalling pathways. Arachidonic acid (AA) is released by phospholipase A2 (PLA2) and is acted on by COX enzymes and synthase enzymes (prostaglandin synthases and thromboxane synthase). These enzymes synthesise their respective prostaglandins and they move out of the cell through prostaglandin transporter (PGT). When aspirin blocks COX-1, it invariably inhibits the all of these signalling cascades. (adapted from Ruan et al. 2011)

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2.2. Thromboxane Synthase (TXS) Inhibitors

Thromboxane synthase (TXS) inhibitors were developed to block conversion of PGH2 to TXA2. These drugs proved successful as they reduced TXA2 synthesis in platelets and improved TXA2-related pathophysiological conditions (Dogne et al. 2005). Many drugs of this class were designed but unfortunately failed at clinical trials due to the accumulation of PGH2, which increases platelet activity by acting as an agonist for TPRα (Ting et al. 2012). If a drug was produced which targeted the downstream receptor as opposed to the TXA2 synthesis itself, it would reduce the effects of TXA2 but PGI2 production would be unaffected.

The added advantage of direct antagonism of TPRα over TXA2 synthesis inhibition is that such drugs would block other ligands which have agonistic effects on the receptor, such as endoperoxides and isoprostanes (Gresele et al. 1987, Fiddler & Lumley 1990). There is evidence to show that TPRα mediates signalling through multiple effectors, including phospholipase C (PLC), some small guanosine triphosphate hydrolases (GTPases) and adenylyl cyclase (AC) (Ting et al. 2012). Furthermore, TPRα can recognise endogenous ligands other than eicosanoid thromboxane A2 (TXA2), such as the endoperoxide PGG2 isoprostanes (Khasawneh et al. 2008). It will be important to considering the implications of the downstream pathways of these ligands when antagonising/blocking receptor.

Unfortunately, little progress has been made with successfully targeting these receptors through development of drugs due to problems with efficacy and toxicity. Most antagonists synthesised have not passed phase I/II clinical trials (Ting et al. 2012). In 2011, Bousser et al. studied terutroban. They interpreted no safety or efficacy advantages with terutroban versus aspirin. However, this study is inconclusive as it was prematurely stopped by the Data Monitoring Committee as it didn’t meet the predefined criteria for non-inferiority (Bousser et al. 2011). Non-inferiority trials are intended to show that the effect of a new treatment is not worse than that of an active control, aspirin in this case, by more than a specified margin (Snapinn 2000). Therefore, research in developing drugs to target TPRα would benefit from the high resolution structure


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determination.

3. Structural Features of TPRα 3.1. G-Protein Coupled Receptors

TPRα is one of five prostanoid receptor classifications recognised (Coleman et al. 1994). TPRα is a member of a large superfamily of transmembrane proteins, GPCRs, that are implicit in various cellular signals (Moreira 2014).

The GPCR superfamily is comprised of over 800 members, making it one of the largest protein families in the human genome. Not surprisingly it is associated with numerous human diseases and 30% of marketed drugs target GPCRs (Fredriksson et al. 2003, Hopkins & Groom 2002). Phylogenetic analysis has identified five main families that classify human GPCRs; rhodopsin-like (672 members), secretin (15 members), glutamate (22 members), adhesion (33 members) and frizzled-taste 2 (36 members) (Moreira 2014). Of these, TPRα belongs to the rhodopsin-like, also known as sub-family A.

The topology of sub-family A GPCRs comprises seven transmembrane α helices (TM1-7) in addition to an extracellular N-terminus and an intracellular C-terminus (Moreira 2014). These helices are connected by three extracellular (ECL) and three intracellular (ICL) loops.

3.2. Coupling of TPRα to G proteins

GRCR signalling involves the coupling of the activated receptor to a heterotrimeric GTP-binding protein (G-protein) (Jastrzebska et al. 2010). In the case of TPRα, binding occurs to the G-protein Gq among others. TPRα can only be associated with one G-protein at a time and receptor recycling regulates the specificity of the G-protein (Ting et al. 2012). The Gq protein is divided into two functional units, the guanine nucleotide-binding Gα subunit and the Gβγ dimer (Fig. 5). When activated, the functional units dissociate and these in turn regulate a large number of downstream effects through association with enzymes and effector proteins (Hamm 1998). As

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these receptors are so influential with regards to signal transduction in the cell in which they are expressed, it is important to structurally characterise them. However, only recently was the first GPCR/G-protein complex structure characterised (Rasmussen et al. 2011). This was a significant breakthrough as this was the first high-resolution view of transmembrane signalling by a GPCR in an activated state (R*) and in complex with its endogenous G-protein. The active state of a GPCR can be defined as that conformation that couples to and stabilises a nucleotide-free G protein (Rasmussen et al. 2011). Having a picture of the transmembrane protein in an active conformation provides crucial information of how ligand binding affects structure and thus enables design of better agonists/antagonists of the target protein.

Therefore, it would be beneficial to solve the structure not only of TPRα, but also that of TPRα-Gq complex. If the TPRα-Gq complex structure was determined then one would have a picture of the TPRα in R* state and it might be possible to study how TPRα responds to ligand binding and how it this changes the conformation of the receptor by comparison of the co-complex structure to that of the receptor on its own.

Figure 5: Structural representation of Heterotrimeric Gq-protein (PDB ID code 3AH8). Generated using Pymol. Gαi/qβγ heterotrimer orientated with respect to the plasma membrane. Gαi/q consists of the GTPase and the helical domains connected by two linker


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regions, Linker 1 and Switch I (Linker 2) (Nishimura et al. 2010).

ConclusionsTPRα is not a “potential” drug target but it is in fact already a current human drug target for treatment of multiple thrombotic disorders. There has been a great deal of interest in regulating this receptor’s activity as an alternative to circumvent the side-effects observed with aspirin (Halushka & Halushka 2002). Aspirin’s side-effects are observed due to its lack of selectivity as it antagonizes both COX-1 and -2. Aspirin inhibits PGI2 production, which is essential for maintenance of the endothelium which explains why patients who regularly take aspirin develop disruption of the gastrointestinal lining e.g. stomach ulcers. The next target in the pathway is thromboxane synthase (TXS), but inhibitors of this enzyme have been unsuccessful in clinical trials due to accumulation of PGH2 which increases platelet activity (Ting et al. 2012). In theory, direct inhibition of downstream receptor itself, TPRα, presents many advantages as a drug target over the blocking of its upstream regulators. The advantage of an antagonist specific for TPRα is that all ligands which up-regulate the receptor would be blocked, not just TXA2. Furthermore, PGI2 production would be unaffected, thus limiting the possibility of side-affects, and PGH2 accumulation would not be an issue. Unfortunately, the vast majority of compounds targeting TPRα failed to pass clinical trials or toxicity screenings as mentioned previously (Ting et al. 2012). The determination of the high resolution structure of TPRα may be a deciding factor to its success as a drug target as it will aid in rational, structure based drug design. While none of the antagonists developed for targeting TPRα to date have made it into clinical use, it may still prove a lucrative investment both intellectually and financially.

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AcknowledgementsThis work was adapted from a mini-review “Thromboxane A2 Receptor (TPRα): A Potential Human Drug Target” for course BI3015: LITERATURE SKILLS.

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Man’s Best Friend? Human-Canid Conflict in the

21st CenturyZoology

Holly EnglishSenior Sophister

Despite our close relationship with the domestic dog (Canis fa-miliaris), humans have frequently been in conflict with other members of the canid family. Due to the adaptable nature of the canids, they have been capable of living in close proximity to us in a human dominated landscapes for centuries. This prox-imity has contributed to our complicated relationship with this family. Though many canid species are no longer vilified and persecuted to the extent they were in the past, public perception of these animals remains an important factor to consider in their conservation. The predation of domesticated animals, from cows to chickens, is a significant source of much of the negative sen-timent directed at canids, and continues to be a source of con-flict. Educating the public about these species in order to dispel myths, and setting up and maintaining systems in which people are compensated for costs associated with living in proximity to wild canids, such as reimbursement for livestock losses, are inte-gral to the successful co-existence of humans and canids. Here I look at the African Wild Dog (Lycaon pictus) and the Grey Wolf (Canis lupus) as case studies on how public perceptions of wild canids have changed in recent years, and what further change is required to safeguard these species for centuries to come.

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IntroductionThe domestic dog (Canis familiaris) is often referred to as man’s best friend. This species was the first to be domesticated and so we have shared a close relationship with these canines for over 15,000 years (Hughes & Macdonald 2013). Dogs are highly valued in many cul-tures as pets but have also gone on to perform a number of import-ant roles in human communities. Dogs aid our police forces in the detection of illegal substances, search for missing persons and lead the blind. Recent research has even pointed towards dogs fulfilling a role in medicine, using their highly effective sense of smell to detect cancer in patients; a study by Cornu et al. (2011) found that dogs can be trained to detect prostate cancer with a significant success rate by smelling urine samples. But despite our general appreciation of the domestic dog, we do not necessarily extend this same fondness to its wild relatives.

Members of the Canidae family (order Carnivora) are ad-pated for the cursorial pursuit of prey, typically in relatively open environments. To achieve this, they are equipped with lithe bodies and long limbs with digitigrade, four-toed feet (Sillero-Zubiri 2014). Most of the smaller canid species are opportunistic omnivores, while the larger group-living species tend to be more strictly carnivorous. Due to the inherent versatility of the canids, many thrive in human landscapes such as farms and cities where they may prey upon live-stock and game species (Sillero-Zubiri 2014).

Where there are large carnivores and humans sharing a space, conflict typically follows. The conservation needs of large carnivores frequently clash with human interests, making the conservation of members of the order Carnivora particularly challenging. Though in recent years, public perceptions towards canids have been improv-ing. Historically, the majority of people felt negatively about these animals. This negativity often stemmed from an ingrained fear of the larger canid species (Sillero-Zubiri et al. 2004) Here I will review two of these larger species as case studies.


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Case Study in the Developing World:Lycaon pictus – The African Wild Dog

Three million years of divergence make the African Wild Dog (Lyc-aon pictus) a distinct member of the canid group, and one belonging to a monotypic genus (Creel & Creel 2002). Wild dogs have a pro-pensity to avoid areas heavily used by lions (Panthera leo), which often causes them to move outside of protected areas and come into conflict with humans (Becker et al. 2013).

Wild dogs were actively persecuted throughout much of the 20th century (Creel & Creel 2002). In Zimbabwe for example, a minimum of 3,404 wild dogs were shot between 1956 and 1975, for the purpose of “vermin control”. The species had been declared vermin at the start of the 20th century as farming and ranching ex-panded across Africa and rewards were offered for each individual destroyed (Childes 1988). Wild dogs were intensely disliked for a multitude of reasons. The main reason for the scorn directed at this species was its method of killing its prey. As wild dogs are small rel-ative to the size of many of their prey species, they kill their target by disembowelment (Creel & Creel 2002). It was also widely believed that wild dogs kill more prey than they require and “terrorise” their prey needlessly (Woodroffe & Ginsberg 1999).

As a result of this intense persecution throughout much of the 20th century, African Wild Dogs have been eradicated from 25 of the 39 in which countries they were originally found (Woodrof-fe et al. 2005). By the 1980s, this active persecution of African Wild Dogs was slowing as a greater number of studies were conducted on their behaviour and ecology. The species also received legal protec-tion in the six countries where its populations were highest, namely; Botswana, Kenya, South Africa, Tanzania, Zambia and Zimbabwe (Creel & Creel 2002).

For years African Wild Dogs lived in the shadows of Afri-ca’s better known carnivores, but recently the fate of wild dogs has looked more certain as the species has become the focus of a number of studies and conservation projects scattered across the continent (Creel & Creel 2002). Wild dogs range widely, meaning that even

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packs living in protected areas come into contact with humans at re-serve borders. Over half of wild dogs found dead in protected areas have died due to human activity; namely shooting, snaring, poison-ing and traffic accidents, with domestic dogs also contributing to the mortality rate as disease vectors (Woodroffe et al. 1997). Many of these deaths are not the result of direct persecution, as African Wild Dogs are frequently caught in snares set to catch other species. Snare survivors occurred in 67% of wild dog packs studied in a population Luangwa Valley, Zambia (Becker et al. 2013), showing just how fre-quent snaring incidents are. Whether these snaring incidents, as well as other anthropogenic causes of wild dog mortality, are intentional or accidental, human activity remains a significant threat to this spe-cies.

In South Africa, there is only one remaining viable popula-tion of African Wild Dogs, which exists in Kruger National Park. Current conservation efforts in South Africa are therefore focused on maintaining a metapopulation of wild dogs across a series of iso-lated reserves. However, Lindsey et al. (2005) suggest that there is potential to conserve wild dogs in situ on ranchland. Cattle ranching has now been replaced by game ranching across much of southern Africa. There are approximately 4,000 game ranches in South Afri-ca alone (Hearne & Mckenzie 2000). Wild dogs have been seen to recolonise parts of their former range in game-ranching areas, but the long-term survival of these packs depends on the willingness of people to tolerate their presence. Lindsey et al. (2005) investigated the attitudes of South African ranchers to African Wild Dogs. Their results indicated that wild dogs are the least popular large carnivore among ranchers, but younger ranchers were more positive than old-er ranchers, suggesting that tolerance to this species may increase. Attitudes of ranchers who already had wild dogs on their land were more positive than those without, so increased exposure to the spe-cies may also improve the public perception of the African Wild Dog, as the negative stereotypes surrounding them may be more perceived than real.

Wildlife tourism is now a significant source of income for many people across the African Wild Dog’s range. Often left in the shadows of other large carnivores in the past, wild dogs may now be


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considered one of the most economically and ecologically important large mammal species in many areas (Becker et al. 2013). Although significant progress has been made, more than half of the mortality recorded among adult African Wild Dogs is still directly caused by human activity (Sillero-Zubiri et al. 2004).

Case Study in the Developed World:Canis lupus – The Grey Wolf

There are few if any animals so enshrined in myth and fairytale as the Grey Wolf (Canis lupus). Although some stories portray wolves in a positive light (such as the founders of Rome Romulus and Remus, being raised by a wolf), for the most part wolves have been demon-ised in folklore for centuries (Sillero-Zubiri 2014).

Perhaps linked to this, few large carnivores have been so per-secuted as the wolf, despite its status as the direct ancestor of our close companion, Canis familiaris. Originally, the Grey Wolf was the most widely distributed mammal in the world (Sillero-Zubiri et al. 2004) but now it has been extirpated from much of Western Europe, the United States of America and Mexico. The species’ original range has been reduced by approximately one third due to human perse-cution and habitat fragmentation (Hunter & Barrett 2011). However, while conflict between humans and wolves remains topical, recent years have seen enhanced legal protection and reintroduction pro-grammes for wolves, as well as natural recolonisation (Ripple et al. 2014).

In the developed world there have been changes to livestock husbandry practices in areas where large carnivore populations were greatly reduced or eradicated all together in the past. Livestock are no longer guarded by people or domestic dogs across much of Eu-rope and North America, making them easy targets for recolonising carnivores (Thirgood et al. 2005). A study conducted in Slovakia by Rigg et al. (2011) found that the use of livestock-guarding dogs with sheep flocks lead to significantly decreased predation from wolves.

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Wolves are currently found in 28 European countries with an estimated total number of over 12,000 individuals (Chapron et al. 2014). The species is thriving in human-dominated landscapes and is largely found outside of protected areas, with most populations having been either increasing or stable in recent years (Chapron et al. 2014). Chapron et al. (2014) suggest that there is reason for “cautious optimism” as wolves and other large European carnivores thrive in areas densely populated by humans, thus “representing an often underappreciated conservation success story.” Europe’s wolf pop-ulation is over twice that found in the lower 48 states of the U.S.A. despite being little over half the size and over twice as densely pop-ulated (Chapron et al. 2014).

In recent years, the role of the wolf as a top predator in its ecosystem has been more widely appreciated. Wolves have a pro-found influence on the biological communities they inhabit through predation, interspecific competition and triggering trophic cascades (Treves & Karanth 2003). Studies carried out in Yellowstone Na-tional Park, U.S.A., have looked at the trophic cascade involving wolves, elk (Cervus canadensis) and trembling aspen (Populus tremu-loides); these studies have shown that the movement patterns of elk are shaped by wolf distribution (Fortin et al. 2005). Elk do not avoid travelling through high-wolf-use areas, but they do adjust their behaviour as a response to the predation risk. As a result of this, some areas are browsed more heavily than others (Fortin et al. 2005). Wolves can also impact mesocarnivores such as the Red Fox (Vulpes vulpes), and therefore structure ecosystems along multiple food-web pathways (Ripple et al. 2014).

The Co-Existence ModelThe co-existence model is the alternative to the separation model, which has often been regarded more highly in the past. The sepa-ration model argues that large predators can only thrive in protect-ed areas or wilderness, away from human activity. However, this may well be a result of previous policies which aimed to exterminate these species (Chapron et al. 2014). Linnell et al. (2001) found that wolves do not require vast wilderness areas to thrive. They can per-


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sist in heavily modified habitats in close proximity to humans, even when both their prey and the wolves themselves are being harvest-ed, provided this harvest is well regulated to ensure sustainability. The recolonisation of wolves across much of Europe and the return of wild dogs to parts of their former range in ranchland areas indi-cate that humans and large canids can co-exist if appropriate man-agement strategies are in place.

One such management strategy is the provision of financial compensation to those affected negatively by carnivore presence, and this can also have important benefits in terms of reducing pov-erty in developing countries (Dickman et al. 2011). Carnivore popu-lations can generate considerable revenue. However, many existing revenue streams in developing countries are diverted externally in-stead of going to the local community (Dickman et al. 2011). This poor cost-benefit ratio leads to the extirpation of large carnivores from human-dominated land (Dickman et al. 2011). This issue needs to be addressed and improved as a matter of urgency, as large areas of the remaining ranges of threatened carnivores is on human-dom-inated land.

ConclusionsThe conservation of canids can be a contentious topic due to inher-ent human biases. The issues discussed here are widely applicable to large carnivores in general but wild canids in particular seem to engender negative attitudes (Lindsey et al. 2005). Education of local communities living with canids is of crucial importance but conser-vationists must be willing to be educated themselves in turn. People who suffer costs due to canid species tend to oppose conservation agendas. The critical challenge facing conservationists is to develop workable measures to reconcile human activity with the needs of canids, particularly when the species in question is threatened (Sil-lero-Zubiri et al. 2007).

Large carnivores pose one of the most pressing current con-servation issues due to the striking declines in their geographic rang-es and population sizes, and also because of their roles as umbrella

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and flagship species for wider biodiversity (Dickman et al. 2011). Conservation in the 21st century should not focus solely on the pres-ervation of vast wilderness areas. Though reserves remain hugely important, the successful conservation of canids should be fostered as co-existence between man and the family of his supposed “best friend”.

Recent decades have seen great changes in the way we per-ceive wild canids. Gradually, fear of the “big bad wolf” appears to be transitioning into respect for keystone species. Whatever the current status of wild canids, the future is sure to pose new issues as the human population continues to grow and expand. Whether the implications of this on canids are direct or indirect, education about these wide ranging carnivores is critical. Like humans, canid species require a lot of space. They have demonstrated that they can live with us, despite the monumental changes we make to the land around us. Perhaps the 21st century will be the time we show that we can live with them too.


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Immunoglobulin-binding Proteins of Staphylococcus Aureus

MicrobiologyKelly MurrayJunior Sophister

Antibiotic resistance is developing faster than the rate of discovery of effective new treatments against Staphylococcus aureus (S. aureus) infections, meaning research into the virulence factors of S. aureus is more important than ever. The four Immunoglobulin-binding proteins of S. aureus play a crucial role in virulence, as well as enabling the pathogen to evade the host immune defences. Infection with S. aureus does not result in lasting immunological memory, due to the B-cell superantigenic activity of immunoglobulin-binding protein A. By understanding the roles these immunoglobulin-binding proteins play in infection, we can devise potential treatments

and preventative strategies against them.

IntroductionStaphylococcus aureus is a Gram-positive pathogen that causes recurrent skin and soft tissue infections (SSTI) and more serious infections such as pneumonia, sepsis, endocarditis or bacteremia (Blot et al. 1998). 20-30% of the human population are carriers of S. aureus on their skin and nasal cavity, however it only poses a threat when it enters the body, as it is an invasive pathogen. Due to the overuse of antibiotics, S. aureus strains have become resistant to most treatments, and are known as methicillin-resistant S. aureus (MRSA). MRSA has become a major public health problem, as even with treatment the survival rates are poor. To devise new treatments and preventative strategies, it is therefore crucial to understand how S. aureus evades the host immune defences and to determine the

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virulence factors involved. The immunoglobulin-binding proteins of S. aureus are Protein A, Staphylococcal binder of immunoglobulin and Superantigen-like binding proteins 7 and 10. All of these proteins play a crucial role in virulence in S. aureus infection (Yamamoto et al. 2013, Zecconi & Scali 2013, Foster et al. 2014). Protein A in particular, is crucial in pneumonia and sepsis. Here we review how these four immunoglobulin-binding proteins evade the complement system. Even though a recently discovered antibiotic has no known resistant mutants, it is not the end of the era of antibiotic resistance (Ling et al. 2015).

Immunoglobulin-binding Proteins

Protein AProtein A (SpA) is a surface protein of S. aureus with two functionally distinct halves, the c-terminal domain and the N-terminal domain. The C-terminal domain anchors SpA to the surface of the cell wall via LPXTG motif (Fig1). The N-terminal contains five protein binding domains, each consisting of three helices: E, D, A, B and C (Schneewind et al. 1992). Recently, the crystal structures of these five binding domains have been established (Deis et al. 2014). These crystal structures confirmed that a single domain can bind multiple partners at the same time, such as Fab and Fc. (Graille et al. 2000). SpA is involved in both adhesion to host cells and evading innate immune responses relating to immunoglobulins and opsonisation. SpA is a major microbial surface recognizing adhesive matrix molecule (MSCRAMM). Experiments with spa-deletion mutants, show decreased adhesion which corroborates this (Hartleib et al. 2000, Foster et al. 2014). SpA also acts as a B-cell superantigen as it can interact with the Fab region of VH3 type B cell receptors on IgM. This causes the proliferation of B-cells by super clonal expansion and apoptosis of activated B-cells (Goodyear & Silverman 2003, Palmqvist et al. 2005). The destruction of B-cells prevents the host from developing immunological memory.


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SbiStaphylococcal binder of immunoglobulin (Sbi) is both a secreted and cell bound protein that binds to IgG, like SpA (Smith et al. 2011). Sbi is composed of four globular domains (D1, D2, D3, and D4). Domains 1 and 2 are the immunoglobulin binding domains (IgBD), similar to SpA (Fig.1). Experiments have shown that the IgBD of SpA and Sbi share a similar amino acid sequence (Atkins et al. 2008). While Sbi is cell envelope associated, IgBD 1 and 2 are on the cell surface, which allows them to bind the Fc regions of IgG, like SpA. When Sbi is in secreted form, domains 3 and 4 have been found to interact with the antigen recognition of B-cells. The pathogen remains undetected and as a result, opsonins are not produced and the immune response isn’t triggered (Toapanta & Ross 2006, Smith et al. 2012). Unlike SpA, Sbi doesn’t have a C-terminal LPXTG sorting signal. This is important, as it shows that Sbi is attached to the cell envelope differently than the LPXTG-anchored SpA. Sbi has been found to bind to lipoteichoic acids (LPA) in the cell envelope and that some Sbi is secreted across the membrane (Smith et al. 2012).

Figure 1: Schematic diagrams of Sbi and SpA. Domains 1 and 2 of Sbi bind IgG, while domains 3 and 4 are involved in interfering with the C3 convertase of activation pathways. Sbi contains a variable region Y involved in membrane binding while SpA contains an LPXTG, a wall anchoring motif and transmembrane domain M. The Wr and Xc regions are proline rich. Domains 1 and 2 share structural homology with regions E, D, A, B and C of SpA. Adapted from Smith et al. (Smith et al. 2011)

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Superantigen-like ProteinSuperantigen-like proteins (SSL), formally known as staphylococcal exotoxin-like proteins, are family of exotoxins. SSL proteins have no superantigenic activity, even though they are structurally similar to enterotoxins, which do show superantigenic activity. SSL7 and SSL10 are the only SSL proteins that bind to immunoglobulins, therefore these will be the focus of our discussion. SSL7 binds to IgA while SSL10 binds to human IgG and this may be beneficial in preventing FcR-mediated leukocyte activation (Langley et al. 2005). SSL10 only binds to primate IgG, specifically to the Fc part through its N-terminal oligonucleotide binding fold domain (Patel et al. 2010). When bound to IgG, SSL10 has been found to act as an anticoagulant, however, more research is needed to establish if this plays a role in S. aureus virulence (Itoh et al. 2013). SSL10 also binds to phosphatidylserine (PS) to the surface of apoptotic cells, and may interfere with the clearance of these cells (Itoh et al. 2012). Additionally, both SSL7 and SSL10 appear to act as virulence factors in S. aureus infection by interfering with the classical pathway in the complement system.

Immunoglobulin-binding Proteins in Evasion of ComplementThe complement system is comprised of a family of proteins and proteolytic fragments. Complement proteins bind to the surface of the pathogen to identify it as a target for opsonisation by macrophages. The activation of complement can occur through three different pathways; classical, alternative and lectin pathways (Fig. 2). All of the pathways activate C3 by cleaving it into C3a and C3b. In the Classical and lectin pathways, C4bC2a also called C3 convertase, cleaves C3 whereas C3bBb is the C3 convertase in the Alternative pathway.

The Classical pathway is initiated by the binding of antibodies to the antigens displayed on the surface of the pathogen, and a complement complex (C1S,C1q,C1r) attaches on to the Fc region of immunoglobulins. SSL10 competes with C1q to bind to IgG and in


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doing so can inhibit activation of the complement pathway (Itoh et al. 2010, Patel et al. 2010).

SpA aids the pathogen in evading the host immune defences by binding the Fc receptors on Immunoglobulin G (IgG).This acts as a decoy by binding the complement binding portion of the IgG the wrong way round and coating it with antibodies, so it can’t trigger the complement system. This prevents the S. aureus being killed by opsonophagocytosis. SpA binding to IgG is essential to avoid the host immune defences as mouse models with a SpA deficient strain of S. aureus suffered less severe arthritis and septic death. This indicated that SpA is a virulence factor (Palmqvist et al. 2002). SpA can also bind to von Willebrand factor (vWF) which is a large glycoprotein, that when bound to SpA is involved in the adhesion to platelets. This is very important in S. aureus caused endocarditis, as it allows for the adherence of the pathogen to the host platelets (Hartleib et al. 2000)

Sbi is involved in disrupting the alternative pathway by causing the sequestration of C3. The function of C3b is to attach to the surface of the pathogen through thioester bonds, which causes them to be phagocytosed. The crystallized structure of Sbi has shown that it binds to C3 via domain 3 and 4 which prevents it binding to its target, thus preventing the initiation of phagocytosis (Atkins et al., 2008). Sbi works in tandem with extracellular fibrinogen-binding protein (Efb) to sequester C3 with plasminogen. Alternatively, the C3b protein can from a complex with C4b2a to form the C3/C5 convertase, causing the formation of the membrane attack complex (MAC). The MAC causes a pore to form on the pathogens surface, resulting in cell lysis. Sbi also prevents the formation of the MAC by binding to it, preventing it reaching its target surface and form a complex with C4b2a (Laursen et al. 2010).

SSL7 binds both IgA and complement C5. The C5 convertase cleaves C5 to give C5a and C5b. Both of these molecules are a threat to the survival of S. aureus as C5a stimulates a proinflammatory response and attracts leukocytes, while C5b is responsible for the formation of the MAC. The crystal structure of SLL7 interacting with IgA, showed that the C-terminal β–domain binds to C5 and that the OB domain interacts with IgA (Laursen et al. 2010). Although IgA

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bound SSL has shown enhanced inhibitory activity in complement systems in mice, it is not essential that they are bound to each other (Lorenz et al. 2013)2013. SSL 7 binding to C5, has been shown to prevent the cleavage of C5 by the C5 convertase, which inhibits both the classical and alternative pathway.

Figure 2: Complement cascade pathways; A is the Classical pathway which is initiated by the binding of antibodies to antigens present on the surface, Ig then binds to this complex. Subsequent arrows represent cleavage events by which the next complement protein becomes activated in the cascade. B is the Lectin pathway, which is initiated by mannose binding. It follows the same mechanism of action as the Classical pathway once C3 is cleaved. C is the alternative pathway which isn’t discussed, but it provides an amplification loop binding C3b to the bacteria surface which increases the production of more C3b until the pathogen is covered with them, which attracts macrophages to destroy them. This diagram was taken from ’’Immune evasions by Staphylococci’’ (Foster 2005)


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SpA induced signalling pathways in pneumonia and sepsisProtein A is a virulence factor in both S. aureus induced pneumonia and sepsis (Palmqvist et al. 2002, Bubeck Wardenburg et al. 2007, Kim et al. 2012). SpA is multifunctional as it can aid S. aureus in evading the complement system and establish infection, but it can also regulate inflammation. SpA is thought to act as a virulence factor by interacting with signalling pathways, such as Tumour necrosis factor receptor 1 (TNFR1) and epidermal growth factor receptor (EGFR) (Gomez et al. 2004). SpA can use its IgG binding domains to bind with TNFR1, which activates the expression of chemoattractant cytokines, such as interleukin-8 (IL-8).This interaction also initiates the production of CXCL10 in airway epithelial cells, and attracts type 1 interferon (IFN) (Ahn et al. 2014). This results in inflammation and tissue damage to the lungs.

Figure 3: The induction and repression of inflammation. A, binding of SpA to the TNFR1 receptor results in the production of IL-8 through a

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signalling cascade, which attracts neutrophils, resulting in inflammation. B, TNF-α is neutralized upon TNFR1 binding as the TNFR1 receptors on the cell surface have been cleaved off by SpA induced ADAM17 and EGFR resulting in reduced inflammation. Adapted from Gomez et al. (Gomez et al. 2004).

TNFR1 signalling can also be activated when TNF-α binds to it. TNF-α is a pro-inflammatory signalling cytokine that binds to its receptor (TNFR1) present on the surface of cells, which triggers the production IL-8, recruits neutrophils, and results in inflammation (Gomez et al. 2004, Gomez et al. 2007). TNFR1 can be positively or negatively regulated. Negative regulation of TNFR1, in relation to pneumonia and sepsis, is carried out by an enzyme called ADAM17 (also known as TNF-α converting enzyme, TACE). ADAM17 cleaves the TNFR1 off the surface of cells so TNF-α has less receptors to bind to, which dampens the inflammation response (Giai et al. 2013). EGFR is triggered by SpA, and activates ADAM17 to cleave off the TNFR1 on the surface of macrophages (Fig. 3). This suggests that SpA can promote inflammatory events as well as inhibit them, depending on which receptor it binds to. In pulmonary infections such as pneumonia, SpA normally binds to TNFR1 which signals for the production of IL-8, causing a proinflammatory event in the lungs (Giai et al. 2013). Early action of TNF-α is very limited in the lung as airway epithelial cells don’t produce significant levels of it so SpA binds to TNFR1. However, in other S. aureus infections, such as sepsis, there are plenty of immune cells that can generate TNF-α. A novel mechanism has been suggested to account for this, and suggests that soluble TNFR1 may neutralize circulating TNF-α, by causing the early shedding of TNFR1, which reduces inflammation. This has been shown using SpA deficient mutants, and tnfr1-/- in mice (Gomez et al. 2004).

Preventative Therapies and Treatments Successful vaccination is dependent on the induction of immune memory. SpA has been found to prevent the production of specific antibodies to other staphylococcal antigens (Pauli et al. 2014), so for vaccination to be successful , SpA must first be supressed. However,


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experiments using SpAKKAA mutant, which lacks the Fab and Fc binding regions, elicited antibody production against SpA in mouse models (Kim et al. 2010).This mutant also resulted in partial immune protection from the wild-type SpA. However, it is important to note that past clinical vaccine trials have all failed and that they all used a single antigen target, be it SpA or any of the immunoglobulin-binding proteins. It is highly likely that a successful vaccine strategy would incorporate multiple antigens as different types of S. aureus caused infections contain different virulence factors that aid its evasion of the host immune system (Bagnoli et al. 2012). Experiments in mice have already shown that vaccines with a combination of protein targets, provide more protection. Four surface proteins; IsdB, IsdA, Sdr+ and SdrE provided better protection to mice than any single component vaccine (Stranger-Jones et al. 2006, Kim et al. 2012, Rauch et al. 2014).

Recent experiments using phage therapy in mouse models found promising results for the use of phage therapy as a treatment to septicaemia, and potentially against MRSA pneumonia. Mice infected with lung derived septicaemia and then treated with S. aureus phage S13, showed lower levels of the pro-inflammatory cytokines, IL-6 and TNF-α which implies reduced inflammation and disease severity (Takemura-Uchiyama et al. 2014).

Treatment of S. aureus infections with antibiotics is becoming increasing difficult, due to the rapid rate of strains developing resistance. However the recent discovery of a new antibiotic that so far has no detectable resistant strains, is showing hope as a treatment to resistant S. aureus strains. In MRSA caused septicaemia mouse models, with a 90% mortality rate, all mice treated with teixobactin survived (Ling et al. 2015). Further experiments also showed that teixobactin is nontoxic to human cells, which indicates it may be a successful therapeutic agent in humans against S. aureus infection. Patients suffering from MRSA infections are currently treated with vancomycin, but it has poor bactericidal activity, which often leads to patient death (Kollef 2007). Teixobactin has excellent bactericidal properties as it is thought to interfere with the formation of Teichoic acid, which is responsible for anchoring autolysins. Teixobactin binds to lipid II which is the precursor for peptidoglycan formation,

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and lipid III which is the precursor for cell wall teichoic acids. This mode of action results in excellent bactericidal activity against S. aureus, as it frees the autolysins which then digest the cell wall and cause bacterial cell lysis (Ling et al. 2015). Teixobactin is only effective against gram-positive bacteria, so it may also be used against Clostridium difficile and Bacillus anthracis.

ConclusionsIn this review I have described the major strategies in which S. aureus uses its immunoglobulin-binding proteins to evade the host immune system, particularly the complement system. SpA is the most studied, and also appears to carry out multiple functions involved in immune evasion, such as inflammation, prevention of opsonisation and platelet adhesion. However, there is little known about the more recently discovered immunoglobulin-binding proteins SSL10 and SSL7. Further research is needed to determine their roles in immune evasion and to confirm that they are involved in the prevention of leukocyte activation.

Although teixobactin has no resistant strains yet, it is thought that some may evolve over time. It took more than 30 years for vancomycin resistant strains to appear so resistant strains of teixobactin may eventually emerge (Ling et al. 2015). This recent antibiotic discovery suggests that there may still be more compounds with potential therapeutic affects against S. aureus, waiting to be discovered in nature. The recent finding that phage therapy is successful in mouse models, needs to be confirmed by other studies, but it is promising. It is accepted that a possible S. aureus vaccine would need to incorporate multiple antigens in a single vaccine to target a specific S. aureus induced infection. This indicates that a universal vaccine against S. aureus will not be possible until we fully understand the different virulence factors and their mechanisms of action in each S. aureus infection.


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AcknowledgementsThis work was adapted from an essay “Immunoglobulin-binding proteins of Staphylococcus aureus “for course MI3MO3.

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Natural Killer Cells:More than Meets the Eye

Molecular MedicineKaren SlatteryJunior Sophister

Natural killer cells are cytotoxic cells critical to the innate immune response. Their key roles are in detection and killing of virally infected and transformed cells. Since their discovery in the 1970s, they have been shown to have many functions involving a variety of biological processes in the body, other than the killing of their traditional target cells. This includes the regulation of angiogenesis in the reproductive tract during pregnancy and bone remodeling during osteoclastogenesis. More recently, experiments in mice have shown the presence of memory NK cells, an unprecedented discovery that has raised many questions concerning the idea of immunological memory and the innate immune response. Accumulating evidence also supports the existence of organ-specific functions in subsets of NK cells. A key example of this phenomenon can be seen in the liver, where hepatic NK cells have distinct phenotypic and functional characteristics in comparison to their counterparts in the blood. As a result, hepatic NK cells play a significant role in the regulation of liver diseases such as liver fibrosis. As the importance of NK cells grows, so does their potential as a therapeutic target. NK cell-based immunotherapy is becoming increasingly significant and can be applied to a huge number of infections and diseases, particularly cancer. This review will discuss the non-traditional functions of NK cells and how they

can be regulated clinically.

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Introduction to NK CellsNatural Killer (NK) cells have classically been described as innate lymphoid cells that function in the protection against virally infected and transformed cells. The innate immune response is the body’s first line of defense and provides a non-specific, short-lived response to a pathogen (Tosi 2005). NK cells respond rapidly to their targets by killing them via the induction of apoptosis or cell lysis, and the production of appropriate pro- or anti-inflammatory cytokines such as IFN- γ and TGF- β. Cytokines are small signaling proteins that are secreted to regulate many biological processes including the immune response (Dinarello 2007). In addition to their role in the innate immune response, NK cells have also been shown to function in a range of diverse processes in the body, including those as complex as angiogenesis and immunological memory (Moffett-King 2002).

NK cell recognition in humans is through the CD56 marker, a glycoprotein routinely involved in cell-to-cell adhesion. It defines two subsets of NK cells based on its relative surface expression; CD56bright and CD56dim (Poli et al. 2009). In the peripheral blood (PB), CD56dim NK cells constitute 90% of all NK cells and are characterized by their high cytotoxicity. The remaining 10% are CD56bright NK cells which are characterized by their cytokine production, such as tumor necrosis factor- α (TNF- α) production (Cooper et al. 2001).

The function of NK cells is primarily in the protection against viral infection and cancer. A wide variety of mechanisms are used by NK cells to kill their targets, including cytolytic granule release of toxins such as perforin and granzymes, antibody-dependent cell-mediated cytotoxicty (ADCC) and expression of cytotoxic molecules such as TNF-related apoptosis-inducing ligand (TRAIL). TRAIL is released by NK cells and binds to specific death receptors on target cells, triggering a caspase-8 dependent signaling pathway that results in programmed cell death (Zamai et al. 1998).

NK cells also use cytokines to affect the functions of other cells and shape the adaptive immune response. IFN-γ is one of the key cytokines produced by NK cells and has many immunomodulatory,


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anti-viral and anti-tumor properties (Schroder et al. 2004). TNF-α has pro-inflammatory effects with functions including the regulation of the inflammatory response via activation of transcription factors, control of cell proliferation and differentiation and activation of apoptotic caspase cascades (Krueger et al. 2011). TGF-β and IL-10 are examples of anti-inflammatory cytokines produced by NK cells (Yu et al. 2006, Mehrotra et al. 1998).

The importance of these functions results in tight regulation of NK cells, which is mediated primarily through the balance of activating and inhibitory receptors. The main groups are the killer immunoglobulin-like receptors (KIR) and the NKG2 receptors. Both are a mixture of activating and inhibitory receptors and interact with a variety of immunoregulatory ligands such as the major histocompatibility complex (MHC) class 1 molecules, HLAs (human leukocyte antigens), and stress induced ligands such as MHC class I polypeptide-related sequence A and B (MICA and MICB) and UL16-bringing proteins (Huntington 2014).

Although the traditional view of NK cells is that they are innate lymphoid cells that protect against cancer and viral infections, research from the past 30 years has revealed that there is a lot more to NK cells than meets the eye. This review will focus on the important functions of NK cells that are often overlooked, with particular emphasis on recent developments and the potential for clinical application of these features.

NK cells and PregnancyUterine NK cells (uNK cells) are critical to angiogenesis of the maternal spiral arteries during pregnancy. Angiogenesis involves the assembly and disassembly of the endothelial lining of the blood vessels. Extravillous trophoblast cells (EVT) grow out from the placenta and penetrate the decidualised uterus (Rossant & Cross 2001). They ensure that there is sufficient blood flow to the intervillous space by infiltrating deep into the uterine wall, encircling the walls of the maternal spiral arteries and destroying them if an increased blood supply is required by the fetus (Pijnenborg 1990). It

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is essential that the EVT cells infiltrate correctly to avoid any of the common pregnancy disorders that can arise such as pre-eclampsia or miscarriage.

EVT cells express and secrete soluble HLAs, the ligands of the regulatory KIRs expressed by NK cells (Pijnenborg 1990). Along with the fact that uNK cells are always abundant among EVT cells, this suggests that uNK cells may be involved in controlling the extent of placental invasion by EVT cells. Hiby et al. (2010) showed that the presence of maternal genes encoding activating KIRs in combination with HLA-C alleles of the fetus correlated with successful pregnancy, with reduced risk of preeclampsia and recurrent miscarriage. It is believed that typically, upon ligation of the KIRs with the soluble HLA molecules secreted by EVTs, uNK cells are stimulated to secrete various pro-angiogenic cytokines (Moffett-King 2002). These include vascular endothelial growth factor C (VEGFC), which stimulates angiogenesis by binding to the VEGFR2 receptor on the vascular endothelial cell. VEGR2 is a transmembrane tyrosine kinase receptor which initiates a signal cascade involving many secondary messengers such as PIP2 and DAG (Holmes et al. 2007). Placental growth factor (PIGF) binds to the VEGFR1 receptor and functions via a signal cascade similar to that of VEGFC (Luttun & Carmeliet 2002). Angiopoietin 2 (ANG2), another vascular growth factor produced by uNK cells, is an agonist for ANG1 and promotes endothelial cell migration & proliferation in the presence of VEGF (Lim et al. 2004). Rajagopalan & Long 2012a) reported that binding of trophoblast-derived HLA-G with its uNK cell receptor induced a senescence-associated secretory phenotype that lead to the sustained secretion of pro-angiogenic factors by uNK cells. More recently, Kieckbusch et al. (2014) showed that MHC-dependent inhibition of uNK cells impedes fetal growth and decidual vascular remodeling in mice.

Overall, this suggests that uNK cells are involved in the regulation of angiogenesis of the maternal vasculature by detecting signaling ligands released from EVT cells, and responding via the production of various cytokines involved in angiogenesis regulation. The regulation of angiogenesis during pregnancy is vital in order to achieve appropriate blood flow to the growing fetus. This involvement provides interesting insights into the immunology


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of reproduction and also supports the idea of NK cells as multi-functional effectors.

Figure 1: The role of NK cells in vascular remodeling during pregnancy. Fetal trophoblast cells secrete soluble HLA-G (sG) during pregnancy that is endocytosed by KIR2DL4 receptor. Endosomal signaling results in a sustained proangiogenic secretory response resulting in vascular remodeling and increased blood flow to the fetus (Rajagopalan & Long 2012b).

NK Cells and Bone RemodelingIn addition to their role in vascular remodeling during pregnancy, NK cells are believed to play an important role in bone remodeling during osteoclastogenesis, which is associated with rheumatoid arthritis (RA) (Söderström et al. 2010). Osteoclasts are monocyte-derived cells which break down bone in the presence of the receptor activator of NF-kappaB ligand (RANKL) and macrophage colony-stimulating factor (M-CSF) (Nakagawa et al. 1998). Söderström et al. (2010) showed that NK cells and monocytes are frequently found in excess at the site of the inflamed joint and that these NK cells express RANKL and M-CSF. Coculturing of NK cells extracted from the synovial fluid of mice with monocytes resulted in differentiation of the monocytes into osteoclasts, and depletion of NK cells resulted in a decrease in the severity of arthritis and prevention of bone destruction (Söderström et al. 2010). Overall, this suggests that

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NK cells are capable of initiating bone remodeling during RA by triggering the development of bone destroying osteoclasts and activating them via production of RANKL and M-CSF.

Anti-TNF drugs are often used in treatment of RA. For example, infliximab is a monoclonal IgG antibody that binds to TNF-α, one of the key pro-inflammatory cytokines produced by NK cells. Administration of infliximab during randomised, double blinded clincial trials produced significant improvements in all measures of rheumatic disease activity when compared with the placebo resulting in its FDA approval in 2000 (Elliott et al. 1994). Perhaps inhibition of NK cells at the site of inflammation would decrease production of these receptor activators and pro-inflammatory cytokines, reducing the severity of the disease and the need for costly drugs such as infliximab. Many experiments have shown that alteration of NK cell activity is a feasible therapeutic strategy. For example, Binyamin et al. (2008) reported that antibody-mediated KIR blockade significantly increased NK cell ADCC in human cell lines, while Romagné et al. (2009) augmented NK cell killing of tumor cells using the novel monoclonal antibody 1-7F9, which also binds specific KIRs ( Binyamin et al. 2008, Romagne et al. 2009). By adjusting these novel antibodies to block NK cell activating receptors, inappropriate NK cell activity could be stopped and autoimmune disorders such as RA could be treated effectively.

Memory NK CellsAlthough it was originally believed that immunological memory was a feature unique to the adaptive immune response, recent evidence suggests that certain subsets of innate immune cells may also have memory. Immunological memory refers to the ability of the immune system to respond more effectively and rapidly to a pathogen upon secondary exposure, due to prior development of antigen specific lymphocytes. O’Leary et al. (2006) studied the immune response of T and B cell-deficient mice exposed to haptens. Haptens are small molecules that become immunogenic when bound to a carrier molecule (Lemus & Karol 2008). They found that adoptive transfer of hepatic NK cells from a sensitized donor resulted in a hypersensitive


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response upon exposure to specific antigens at least four weeks later (O’Leary et al. 2006). Subsequent work found that hepatic NK cells, but not splenic or naïve NK cells, developed memory to vaccines containing antigens from influenza, vesicular stomatitis virus (VSV) and HIV-1, and that it was dependent on the chemokine receptor CXCR6 (Paust et al. 2010a). The exact role of CXCR6 in NK cells has not yet been investigated, however it appears to regulate the effector functions and survival of hepatic memory NK cells. Interestingly, NK cells do not express recombination-activating gene (RAG) proteins which are required by T and B cells for their ability to produce the huge numbers of antigen-specific receptors they do (Paust et al. 2010b).

This revelation raises many questions concerning the traditional view of NK cells and the innate immune response. The most important mysteries lie behind the molecular mechanisms NK cells use to mediate this antigen-specific response independent of RAG proteins, yet a similar phenomenon has been reported in sea lampreys and hagfish, where recombinatorial assembly of gene segments is used to produce variable lymphocyte receptors (Cooper & Alder 2006). As immunological memory is the primary aim of any successful vaccine, memory NK cells may be used as a novel approach in future vaccine developments, particularly in patients suffering from T or B cell deficiencies, such as in HIV. This comes at a time where vaccine development is one of the top priorities in biomedical research, with increasing antimicrobial resistance and decreasing antibiotic reliability (Davies & Davies 2010).

Tissue-Specific Functions of NK CellsThe distribution of certain subsets of NK cells varies dramatically throughout the body (Carrega & Ferlazzo 2012). One of the key examples of this phenomenon is in the liver. The liver represents a unique immunological environment and has a cellular repertoire enriched with lymphoid cells such as NK cells, NKT cells and γδ T cells (Crispe 2009). Hepatic NK cells are located in the hepatic sinusoids, often adhering to the endothelial cells where they are under constant exposure to gut-derived antigens. While NK cells

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normally constitute 5-15% of the total number of lymphocytes in the PB, they can reach up to 25-40% of the total number of lymphocytes in the non-diseased liver (Tian et al. 2013).

Hepatic NK cells contain a unique composition of markers on their surface. In contrast to NK cells in the PB where 90% are CD56dim NK cells, CD56dim NK cells & CD56bright NK cells are expressed in equal amounts in the liver (Krueger et al. 2011). While the PB CD56bright NK cells are characterized by potent cytokine production, the hepatic CD56bright NK cells have been shown to display increased cytotoxicity & degranulation in response to target cells. This was shown in functional studies carried out by Moroso et al. (2010) on NK cell subsets during human liver transplantation. They also showed increased expression of the toxic molecules perforin & granzyme in CD56bright cells via intracellular staining. This, along with upregulated TRAIL expression, results in hepatic NK cells having increased cytotoxicity towards target cells in comparison to those in the PB.

The importance of this increased cytotoxicity can be seen in the role of hepatic NK cell in liver fibrosis. Liver fibrosis is an innate tissue repairing mechanism that involves the formation of excess fibrous connective tissue. When a tissue is damaged, the immune system reacts by producing a variety of immune cells, such as macrophages, that secrete pro-inflammatory and pro-fibrogenic cytokines, leading to the activation and proliferation of hepatic stellate cells (HSCs) (Pellicoro et al. 2014). These are induced to transdifferentiate into myofibroblast cells, which are characterized by secretion of extracellular matrix (ECM) proteins such as collagens and glycoproteins. Upon persistent tissue damage, such as in HCV infection or alcohol abuse, fibrosis can progress to a pathological state involving scar formation and impaired organ function. Hepatic NK cells protect against fibrosis by killing early-activated HSCs via mechanisms including cytolytic granule mediated apoptosis and release of TRAIL. They also produce the anti-fibrotic cytokine IFN- γ, which works by antagonizing the effects of the pro-fibrotic cytokine TGF- β (Weng et al. 2007, Dooley & ten Dijke 2012). It does this by inducing the expression of inhibitory proteins, known as inhibitory Smads, involved in the TGF- β signaling pathway, and


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also by the inhibition of TGF- β dependent collagen expression through competition with the transcriptional coactivators p300 and CBP (Ghosh et al. 2001). IFN- γ also induces HSC cell cycle arrest and amplifies NK cell activity via the upregulation of the activating NKG2D receptor and TRAIL expression.

This feature of NK cells can be seen throughout the body, in organs such as the lungs, the spleen and the kidneys, and suggests that the microenvironment of NK cells has a strong influence in the role they play in the immune response. Little is known about the exact mechanism behind this, yet it is likely that there are a multitude of factors involved. For example, macrophages have been shown to influence the phenotypes and functions of lung and splenic NK cells, while gut NK cells may be regulated by signals from dendritic cells, epithelial cells and the luminal contents (Reynders et al. 2011, Michel et al. 2012). Further research is required to clarify the origin and actions of these tissue-specific NK cells.

NK Cells and ImmunotherapyAs the mechanisms that control NK cell cytotoxcity towards infection and disease have been revealed, a wide range of NK-cell based therapies have become possible (Farag et al. 2003). Initial trials attempted to use in vitro generated NK cells and in vivo cytokine therapy, such as IL-2 therapy, to treat cancer, yet the results were not substantial and so researchers are now looking in different directions.

Monoclonal antibodies that bind to cell surface antigens on tumor cells have been developed which aim to attract NK cells to the site of tumorigenesis and induce ADCC. For example, FDA approved rituximab and ofatumumab binds to CD20 on tumor cells, while trastuzumab binds to Her2 (Adams & Weiner 2005). Another potential approach is to block the interaction between NK cell inhibitory receptors and their ligands. Koh et al. (2001) reported that blocking of Ly49C and I inhibitory receptors in mice using monoclonal antibodies increased NK cell cytotoxicity towards tumors and decreased tumor growth, suggesting that NK cell

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receptor blockade is feasible and would be effective in the treatment of cancer.

Finally, the ability to modulate the balance of activating and inhibitory NK cell receptors and ligands would be a powerful tool in the treatment of disease. Santourlidis et al. (2002) showed that KIR expression is controlled epigenetically via DNA methylation. They then used the gene-demethylating agent decitabine to induce the rapid and stable transcription of all KIR receptors in NK cell lines, NK cell clones and freshly isolated NK cells. These advances come at an important time, as the development of resistant tumors and increased relapse has diminished the effectiveness of common cancer treatments such as chemotherapy and ionizing radiation. Each of these approaches also has the potential to be adapted to treat numerous pathological diseases, including fibrosis and viral infections.

ConclusionsIts is clear that defining NK cells simply as innate lymphoid cells may not be an accurate description of these multifunctional cells. The diverse roles of NK cells range from the killing of tumor and virally infected cells, to producing long lasting antigen specific receptors independent of RAG proteins, and even stretch as far as regulating tissue remodeling and repair, as seen in angiogenesis, osteoclastogenesis and fibrosis. The more we discover about the role of NK cells, the less clear the line between the innate and adaptive immune response becomes.

With this increase in the functions of NK cells comes an increase in their potential as a therapeutic target. The possibilities of NK cell based therapies are growing and are relevant to a huge number of infections and diseases, including the one with the most sought after treatment, cancer. Future research must concentrate on determining the underlying mechanisms used by NK cells to carry out these functions, so that diseases can be diagnosed effectively, understood at a molecular basis, and treated using novel immunotherapies.


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Acknowledgments This work was adapted from an essay “The Role of Hepatic Natural Killer Cells in Local Tissue Remodeling and Repair” for course BI3020.

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First-Line Treatment of Chronic Lymphocytic Leukaemia and the

Impact of Genetic Aberrations in Chemotherapy

Molecular MedicineTristan KwanJunior Sophister

B cell chronic lymphocytic leukaemia has been historically thought of as a benign disease with no effect on the patients’ livelihood. The exact cause of the disease is currently unknown as the molecular basis of the disease continues to be unveiled. The diseases occurs in people over 50 years. Interestingly, the incidence of the disease is mostly only found in Western countries. Research in the past decade has shown the variable clinical course of the disease with the identification of several important prognostic factors. Many of these factors include genetic aberrations observed and are used in determining which treatment strategy should be implemented. Of these, genetic aberrations involving the Ataxia Telangiectasia Mutated and p53 genes are most important in predicting the behaviour of chemotherapeutic agents used in treatment. Also, IGHV gene mutations are useful in identifying poor-prognosis patients. Chemotherapeutic agents currently under study are fludarabine-based, taken as a cocktail with cyclophosphamide (FC), or with cyclophosphamide and rituximab (FCR) which can be administered orally or intravenously. This review will discuss the behaviour of such chemotherapeutic agents and how specific genetic abnormalities affect the efficacy of such agents.

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IntroductionB cell chronic lymphocytic leukaemia (CLL) is characterised by the monoclonal expansion of CD19+, CD5+, and CD23+ B cells which do not produce effective antibodies (Chiorazzi et al. 2005). The rate of incidence is around 4.2/100000/year increasing to >30/100000/year at an age of >80 years, ranking this type of leukaemia as the most common in the Western world (Chiorazzi et al. 2005, Eichhorst et al. 2011). The median age at diagnosis is 72 years with only 10% of CLL patients reported to be younger than 55 years (Eichhorst et al. 2011). Discovery of the disease is usually through routine health check-ups by the elderly.

CLL is known to be a clinically heterogeneous disease that originates from B cells that may differ in activation, maturation state or cellular subgroup (Chiorazzi et al. 2005). Some patients with CLL survive for many years without the need for treatment and eventually succumb to an unrelated disease while others have a fatal disease despite undergoing treatment (Chiorazzi et al. 2005). The age of patients diagnosed with CLL is deemed to be an insignificant prognostic factor in determining the clinical course of the disease (Rai et al. 1975, Binet et al. 1977). Because of this, well-defined parameters for clinical staging are required.

There are two staging systems used in clinical settings today. Rai et al. (1975) proposed a staging system which is now commonly used in the United States. The Rai system was based on the fact that CLL is a disease of progressive accumulation of incompetent B lymphocytes: stage 0, peripheral and bone marrow lymphocytosis (>15,000/mm3); stage I, lymphocytosis with enlarged lymph nodes; stage II, lymphocytosis with enlarged spleen (splenomegaly) or liver (hepatomegaly) or both (hepatosplenomegaly); stage III, lymphocytosis with anaemia (haemoglobin (Hb) <11 g/dL); and stage IV, lymphocytosis with thrombocytopenia (platelet count <100,000/mm3) (Rai et al. 1975). From this, Binet et al. (1977, 1981) aimed to simplify the Rai system, describing the system to have too many defined stages. The Binet system is now widely used in Europe and was proposed after isolating important prognostic factors at diagnosis and defining three prognostic groups: group A (Binet A), no anaemia, no thrombocytopenia, less than three


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involved areas (Rai stage 0, I and II); group B (Binet B), no anaemia, no thrombocytopenia, three or more involved areas; group C (Binet C), anaemia (Hb < 10 g) and/or thrombocytopenia (platelet count < 100,000/mm3, Rai stage III and IV) (Binet et al. 1977, Binet et al. 1981).

At present, important cellular markers that are useful in predicting the aggressiveness of the disease have been identified. Most of these markers are genetic aberrations found in patients with the disease and predicts which treatment strategies serves to be most beneficial (Cheson 2011). It is important to realise that treatments readily available do not serve to cure the disease. Rather, they aim only to increase the period of remission for patients (Chiorazzi et al. 2005).

First-line TreatmentTreatment of CLL poses a challenge due to its heterogeneous clinical progression. Prognostic subgroups in CLL is defined by chromosomal abnormalities and this becomes a factor in choosing the effective treatment (Juliusson et al. 1990, Zenz et al. 2011).

The discovery of chlorambucil in the 1950s as the first-generation of alkylating agents remained the standard treatment for chronic lymphocytic leukaemia as well as other haematological malignancies, including chronic myeloid leukaemia and myeloma (Catovsky et al. 2011). However, the drug was replaced by fludarabine (a purine analogue) after several phase III trials found that the rate of complete remission (CR), the rate of response, the duration of the response and the progression-free survival (PFS) was significantly better among patients treated with fludarabine than with chlorambucil (Rai et al. 2000). Fludarabine showed better results to chlorambucil with regards to overall response (OR) rate in early stages Binet A and B but not in advanced Binet C. As such, fludarabine-based combination treatments have become the new standard in patients (Eichhorst et al. 2009).

Cyclophosphamide is a drug that is commonly used in combination with fludarabine (FC) in the treatment of CLL. This combination of drugs yielded improved CR, OR and PFS in phase

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III trials in patients with previously untreated CLL compared with those treated with fludarabine alone (Flinn et al. 2007). Similar results were observed by Eichhorst et al. in younger patients with combination therapy resulting in significantly higher CR and OR rate than treatment with fludarabine alone (Eichhorst et al. 2006).

Rituximab, a chimeric, monoclonal antibody, that targets the CD20 cell surface antigen, is further agent used in combination with fludarabine and cyclophosphamide. The agent binds CD20 on B cells, triggering natural killer (NK)-cell mediated antibody-dependent cellular cytotoxicity (ADCC). Laser scanning confocal microscopy shows that rituximab causes CD20 to cap at the B cell surface. Proteins, including intercellular adhesion molecule 1, and moesin are then recruited to the cap leading to the microtubule organising centre (MTOC) becoming polarised towards the cap. This polarisation increases NK cells’ ADCC activity on B cells by 60% compared to unpolarised B cells (Rudnicka et al. 2013). In an international, multicentre, randomised trial of 552 patients with Binet stage A (1%), B (59%), or C (31%) disease, patients were randomly assigned to receive rituximab with FC (FCR) or FC alone. The FCR treatment significantly improved PFS in patients with previously treated CLL (median, 30.6 months for FCR versus 20.6 months for FC). The event-free survival, response rate, CR rate, duration of response, and time to new treatment or death were also improved. (Robak et al. 2010). Further tests were completed on relapsed CLL patients i.e. patients that achieve CR or partial remission but, after a period of 6 months or more, demonstrate the presence of disease progression and refractory CLL patients which include patients that had treatment failure (eg. nonreponse, stable disease or progressive disease) (Hallek et al. 2008a). The addition of rituximab to FC in this control group led to a observed response rate overall of 74% with 30% CR, indicating the addition improved the quality of response (Badoux et al. 2011).

Genetic Aberrations in Patients with CLLThe identification of specific gene aberrations and mutations in patients with CLL is crucial for distinguishing biological and clinical


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subgroups of patients. Such aberrations act as prognostic markers that allow further understanding of the disease and ensure better management of patients with regards to treatment. Conventional cytogenetic analysis is only able to detect chromosomal aberrations in 40 – 50% of cases; due to the low mitotic activity of cells in vitro (Dohner et al. 2000). The most common aberrations found with this type of analysis are trisomy 12 and chromosome band 13q14 abnormalities (Juliusson et al. 1990). Fluorescence in situ hybridization (FISH) can detect these genomic aberrations in about 80% of CLL patients, not only in dividing cells, but also in interphase nuclei (interphase cytogenetics) (Dohner et al. 2000). The most frequent chromosomal aberrations in CLL include a deletion of 13q (55%), a deletion in 11q (18%), trisomy 12q (16%), a deletion in 17p (7%) and a deletion in 6q (6%) (Dohner et al. 2000). Defining subgroups of patients that show each genetic abnormality is important in predicting the disease progression as well as the effect of the treatment chosen. Of these chromosomal aberrations, 11q23 deletion, 17p13 deletion and p53 mutation have significant clinical consequences with regards to treatment (Table 1).

11q23 Deletion and MutationDeletions of the long arm of chromosome 11 (11q) are one of the most frequent chromosomal aberrations in various types of lymphoproliferative disorders. However in CLL, 11q deletions accounts for about 18% of patients chromosomal change, making it the second most genomic aberration present in CLL (Dohner et al. 2000). 11q deletions were seen to have a negative prognostic effect primarily in younger patients (Dohner et al. 2000).

Interphase cytogenetic study have identified a new clinical subset of CLL that is defined by the deletion of a genomic region in chromosome bands 11q22.3-q23.1 (Dohner et al. 1997). This chromosomal region includes the Ataxia Telangiectasia Mutated (ATM) gene in almost all cases. ATM is a serine/threonine protein kinase involved in DNA repair, recombination and the regulation of the cell cycle (Negrini et al. 2010). It is also the principal activator of the P53 protein (Austen et al. 2005). Based on the status of the

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residual ATM allele, 11q deletion in CLL patients can be divided into two subgroups: those with wild-type residual ATM allele and those who have acquired mutated residual ATM allele in the progression of the disease. FC treatment in vitro leads to the phosphorylation of downstream ATM targets only when the wild-type ATM gene is retained. Therefore, complete loss of the ATM gene has defective responses to chemotherapy which in turn leads to a poorer clinical outcome. This poor outcome has also been associated with the mutated genes defective phosphorylation of downstream ATM targets (Austen et al. 2005, Austen et al. 2007).

17p Deletion and Mutation of p5317p deletion occurs in as much as 7% of the cases of CLL, frequently found in patients with a more advanced disease (Dohner et al. 2000, Oscier et al. 2002). Patients in the groups with 17p as well as 11q deletions exhibit rapid disease progression with a median time from diagnosis to first treatment of 9 and 13 months, respectively (Dohner et al. 2000). The deletion almost always includes the P53 gene. P53 is a tumour suppressor protein involved in regulating cell growth and death and, as such, has been known to be the ‘guardian of the genome’ for its role in preventing the replication of altered DNA, which could eventually lead to tumourigenesis (Sionov & Haupt 1999). Around 80-90% of patients with 17p deletions that have P53 mutations were identified in older patients (Zenz et al. 2010). Mutations involving P53 is a characteristic present in almost all types of cancer. The residues most frequently mutated are at positions 175, 179, 248 and 273 of the amino acid sequence (Zenz et al. 2010). Rare tumour-derived mutations at residue 175, such as 175Ser, 175Leu and 175Pro, have shown loss of the protein’s apoptotic function (Rowan et al. 1996).

Response to chemotherapy was significantly worse in patients with P53 mutations or 17p deletion. Those with P53 mutation showed a median PFS of 23.3 months compared to those without P53 mutation where PFS median is 62.2 months (Zenz et al. 2010). Assessment of potential treatment effect was achieved by entering patients into a randomized CLL4 trial to compare F versus


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FC as first-line treatment. The study suggested FC as the potential treatment for patients without P53 mutations with CR found in 22.1% of patients compared to 5.6% CR rate in the F arm (Zenz et al. 2010). However, there was 0% CR in each treatment arm for patients with P53 mutation and further research has yet to be conducted in this area (Zenz et al. 2010).

IGHV MutationsSome CLL cells have somatically mutated immunoglobulin heavy chain V-III region VH26 (IGHV) genes, indicating that the cell of origin has passed through the germinal centre (Hamblin et al. 1999). ATM mutations have been shown to be associated with unmutated IGVH genes. However, the prognostic effect of an ATM mutation is independent of IGVH mutation status suggesting that mutations in the ATM gene alone lead to reduced overall and treatment-free survival periods (Austen et al. 2005). P53 mutations were also shown to be associated with unmutated IGHV region genes (Zenz et al. 2010).

Patients with unmutated IGHV genes exhibit a more malignant disease and have much shorter survival than those with mutations, which is consistent with the fact that those with ATM and P53 mutations also show a more malignant disease (Hamblin et al. 1999, Austen et al. 2005, Zenz et al. 2010). On the other hand, the mutated gene seems to be more favourable. In a multivariate analysis of prognostic factors in CLL, patients with mutated IGHV genes showed to have superior survival compared to those with unmutated IGHV genes (median survival of 310 and 119 months, respectively) (Oscier et al. 2002).

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Table 1. Summary of genetic prognostic factors and respective potential treatment approach in patients with CLL and 1st line treatment indication. (Zenz et al. 2011).

ConclusionsClinical treatment of CLL is a challenge due to the many genetic aberrations found in patients (Zenz et al. 2011). As previously mentioned, combination treatment with fludarabine, cyclophosphamide, and rituximab behaves differently from patient to patient depending on factors which include such genetic abnormalities but also, whether patients have stable, relapsed or refractory type of the disease (Austen et al. 2007, Zenz et al. 2010, Badoux et al. 2011).

Toxicity and other side-effects are major problems in treatment strategies. No excessive toxicity was found when FC combination treatment was implemented (Flinn et al. 2007). However, more thrombocytopenia and leukopenia was caused by FC treatment in younger patients (Eichhorst et al. 2006). FCR treatment was also well-tolerated with no significant detrimental effects found on quality of life in previously treated CLL patients (Robak et al. 2010). However,


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in a study of the effects of FCR in previously untreated patients with advanced CLL, early death was observed due to toxicity and secondary malignancies caused by the treatment. Increased cases of neutropenia and leukopenia were also found. (Hallek et al. 2008b).

Learning from all of this, a pharmacogenomic approach to treatment would be more beneficial than going with the more generalised approach. Moreover, better correlation between chemotherapeutic and chemoimmunotherapeutic agents with the clinical history of patients and which agents would suit best for a given clinical history would prevent toxicity and genetic abnormality issues from arising in the care of CLL patients.

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AcknowledgementsThis work was adapted from a mini-review “The role of T cells in chronic lymphocytic leukaemia” for course BI3020

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HALLEK, M., CHESON, B. D., CATOVSKY, D., CALIGARIS-CAPPIO, F., DIGHIERO, G., DÖHNER, H., HILLMEN, P., KEATING, M. J., MONTSERRAT, E. & RAI, K. R. (2008a) Guidelines for the diagnosis and treatment of chronic lymphocytic leukemia: a report from the International Workshop on Chronic Lymphocytic Leukemia updating the National Cancer Institute–Working Group 1996 guidelines. Blood, 111, 5446-5456.

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Editorial

Letter from the EditorChemistry has always been a broad and multidisciplinary area of science, encompassing biochemistry, environmental science, materials science, and drug synthesis, to name just a few of its facets. For me, it’s the variation in content and applications which makes chemistry so exciting. It’s hard to think of a technological or medical advancement that doesn’t have its foundation in chemistry. It’s wonderful to see this diversity reflected in this year’s TSSR chemistry submissions. The authors themselves come from varying backgrounds- nanoscience, medicinal chemistry and chemistry – and the submissions, ranging from hydrogen production to glucose monitoring, reflect the multidisciplinary nature of this field.

I would like to thank the staff in the chemistry department who have contributed to the review process of these articles and for their general advice regarding the TSSR. Without the help of Dr. Mike Southern, Prof. Sylvia Draper, Prof. Mike Lyons, Prof. Isabela Rozas and Dr. Wolfgang Schmitt the chemistry section of the TSSR would not have come to fruition.

I also found it interesting to note that various TCD chemistry lecturers co-authored several of the papers referenced by the TSSR chemistry authors in their reviews. This is a great display of students taking interest in their lecturers’ research work, and is also an indication that Trinity staff are at the forefront of research in their field.

Perhaps the most important people to thank are the TSSR authors themselves. Whether successful in their submission or not, they are to be commended for using their free time to research and write on topics interesting to them, and for producing high quality and engaging review articles. I am very appreciative of the time and effort put into each submission and I believe that this volume is an excellent representation of the incredible talent and drive within students in Trinity College.

Elaine Kelly Chemistry Editor Trinity Student Scientific Review 2015

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Best Chemistry Essay

Targeting Ligands and Nanovehicles:

A Novel Approach to Lung Cancer Therapy

Chemistry Shelley Stafford

Junior Sophister

Huge advances made in the field of nanotechnology over the past twenty years have opened a wide range of possibilities for innovative new cancer treatments. Ligand-decorated drug delivery nanovehicles are a novel treatment that can offer both therapeutic and diagnostic effects simultaneously. Not only this, but they also bypass the healthy cells due to the active targeting caused by the ligands, and so one does not experience the negative side effects associated with traditional chemotherapy. In this review, a variety of these novel drug carrier systems are examined and compared, as well as another possible diagnostic use of ligands in lung cancer – as a liquid biopsy to quantify circulating tumour cells. This review summarises the effect of the targeting ligand, the synthesis of the nanovehicles, liquid biopsies and the dual purpose of inorganic cores in these nanoparticles, illustrating their potential as viable cancer treatment options in

the near future.

IntroductionLung cancer is the most common cause of cancer death in both Ireland (O’Regan 2014) and the UK (Cancer Research UK 2014). There are two main types of lung cancer, small cell lung cancer (SCLC) and non-small cell lung cancer (NSCLC) – the second of which is

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more common (87%). The high mortality rate is primarily due to the difficulty in effective treatment methods currently available. This review explores an innovative form of lung cancer treatment, which involves the inhalation of drug-loaded nanoparticles directly into the lungs. In conventional cancer treatment, when the drug is administered it can attack both cancer and healthy cells without specificity, resulting in the side effects one might ordinarily associate with chemotherapy, such as hair loss, nausea and fatigue. However, the drugs encapsulated in nanovehicles will still be internalised by both cancer and healthy cells. It was in the late 1890’s that Paul Ehrlich (Nobel Prize for Physiology 1908) proposed the idea of ‘magic bullets’ – drugs that have a special affinity for the disease cells they are designed to treat. Combining Ehrlich’s idea with the recent advancements in the field of nanoscience, it is now possible to accessorise drug loaded nanovehicles with ligands that will bind preferentially to the cancer cells. This process is termed ‘active-targeting’. It is the means by which these ligand decorated drug loaded nanoparticles accumulate and bind to tumour cells only, thus preventing the unwanted side effects observed in traditional chemotherapy (Ehrlich 2014).

This review examines the use of the epidermal growth factor receptor ligand as an active targeting moiety on the surface of the nanovehicles, the nanoparticles chosen as drug delivery systems and the use of the folate receptor ligands as a liquid biopsy in the diagnosis of NSCLC. Lastly, the new and innovative manipulation of nanovehicles as a theranostic device will be reviewed. In this case, theranostics refer to the means by which a nanovehicle can act as both a drug delivery system, and a biological imaging agent.

The use of active targeting are two vital components – the first is the attachment of an active targeting ligand, to the surface of the nanoparticles, such that it will bind selectively to receptors present on cancer cells. The second step of this treatment involves the internalisation of the nanoparticles into the cancer cell, whereupon it releases the drug it has been carrying in order to kill the cell itself.

The ligand most commonly used in the design of nanoparticles for the treatment of lung cancer are EGFR (Epidermal Growth Factor Receptor) ligands, as it is the EGFR that is expressed at significantly


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higher levels in NSCLC cells compared to healthy lung cells, according to Fujino et al. (1996) and Rusch et al. (1996). The EGFR ligands are covalently bound to the outer surface of the nanoparticle drug vehicle so that the drug carriers will only interact with tumour cells. The use of other ligands, such as the Folate Receptor (FA) and its use in capturing circulating tumour cells (CTC) in the early stages of NSCLC will also be examined. Similar to EGFR, the folate receptor is produced at high levels on lung cancer cells, and at much lower levels on healthy cells (Thomas et al. 2013).

Nanoparticles can be used as drug delivery systems (DDS) in the treatment of lung cancer, and can be administered intravenously or directly into the lungs by inhalation. The use of inhalation as a drug delivery method is suited especially to lung cancer, as the drugs are being directly applied to the area they are required to treat. However, the toxicity of the nanoparticles themselves presents an issue. Silica, a material used in a number of the nanovehicles discussed in this review, is toxic at both high dosages and high concentrations (Lin et al. 2006). Problems may arise with residual nanoparticulate material, in which silica nanoparticles remain in the system and prove toxic to healthy cells. Long term effects of the nanoparticles in the body have not yet been addressed, and long running clinical trials are needed in this area.

A variety of these nanovehicles are described in this review, each with similar biochemistry, showing the versatility in this method of treatment. An interesting subset of these nanoparticles that feature inorganic cores which have imaging capabilities will also be looked at. These special nanoparticles can be used in a new branch of medicine – theranostics – in which a drug may be administered to treat a disease, and simultaneously track the progression and effectiveness of the drug.

The Use of EGFR as an Active-Targeting Moiety Epidermal Growth Factor Receptor is overexpressed on the surface of NSCLC cells, and only in small quantities on healthy cells, (Fujino et al. 1996). In designing ligands that will bind selectively to EGFR

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and attaching them to nanovehicles containing an anticancer drug, such as paclitaxel or doxorubicin, cancer cells alone can be targeted. The use of the targeting ligands prevents drug expulsion occurring at any other site in the lungs or body – the result of a random drug release could be that the cancer cells will develop resistance to the anti-cancer agent, through a mechanism known as multi-drug resistance (Persidis 1999). There are a number of variations of the ways in which EGFR can appear as an active targeting ligand on the surface of nanoparticles.

In a study by Tseng et al. (2007), biotinylated EGF is used on a gelatin nanoparticle to target A549 adenocarcinoma cells in mice. The GP-Av-bEGF drug was nebulised to generate aerosol particles which were then inhaled. The mice were then sacrificed at different time points and their lungs examined using Xenogen IVIS. Tseng et al. (2009) conducted a further study in which they attached the same EGF ligands to gelatin nanoparticles; however, in this case they investigated the capabilities of the nanovehicle to deliver cisplatin (CDDP) via aerosol to the lungs of nude mice. Both experiments drew the same conclusion – the cancer cells alone were targeted and the nanoparticle was internalised into the cell, which was easily observable using bioimaging techniques (Fig. 1).

In another study conducted by Sundaraaj et al. (2014), a variation of the EGFR receptor ligands were bound to silica nanorattles. The ligands in this case were generated by using anti-EGFR in EDC (1-Ethyl-3-(3-dimethylaminopropyl)carbodiimide) to activate the carboxylic acid groups in the epidermal growth factor receptor antibody (EGFRAb). These were consequently bound to the porous gelatin surface of the nanorattles. In addition to these ligands, pyrrolidine-2 was loaded into the pores of the nanoparticles, which acts as a cPLA2α inhibitor (cPLA2α is an enzyme secreted primarily by cancer cells (Sundaraaj et al. 2014).) with the intention of more accurately targeting cancer cells. The effect of these ligands was observed, with the H460 lung cancer cells internalising significantly more than the healthy L-132 lung cells. This was determined using confocal microscopy (Fig. 2). As is evident from the results in each of these studies, and similar experiments, the use of targeting ligands, such as EGFR, greatly enhances the specificity of cells attacked.


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Figure 1: The effect of the targetiang EGF ligand on the lungs of healthy (N) SCID mice, and mice inoculated with A549 tumour cells (T). a and b correspond to different time points (0.5hr and 24hr incubation respectively). The uptake of the drug by A549 cells is clearly illustrated by the spread of the fluorescence throughout the lung. As is seen in the healthy cells, the nanoparticles stay localised in the central cavity of the lung and do not get internalised. (Tseng et al. 2009)

Figure 2: The internalisation of EGFRb/cPLA2α decorated nanovehicles into healthy L-132 cells vs H460 cells. The nanovehicles were labelled with FTIC, RTIC and observed using confocal microscopy. (Sundarraj et al. 2014)

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Folate-Receptor Ligands as Bio-Markers for CTC in the Early Stages of Lung CancerIn the early stages of any cancer, some tumour cells will be shed from the primary tumour and make their way into the bloodstream – these are known as circulating tumour cells (CTCs). Although these CTCs are relatively rare, they can be used as a ‘liquid biopsy’ – a means of diagnosing cancer non-invasively, resulting in less trauma for the patient (Alix-Panabie & Pantel 2013). This diagnosis can be achieved through the use of folate receptor ligands. Folate receptors are produced at high levels on NSCLC CTCs and at negligible levels on healthy cells (Parker et al. 2005, Thomas et al. 2013).

It was proposed that these FR-positive cells could be biomarkers in NSCLC (Yu et al. 2013), based on a polymerase chain reaction (PCR) that arises from targeting the ligand. It was found that this method was far more effective in diagnosing CTCs than the five more commonly used bio-markers, CEA, NSE, cyfra-21-1, CA125 and SCC Ag, and was second to only cyfra-21-1 in the detection of CTCs in the early stages of NSCLC. This illustrates that it is a satisfactory and viable biomarker. Similar results were found by Lou et al. (2013), although they also compared the results obtained with EpCam enrichment method of quantifying CTCs and found that the FR ligands had a significantly higher sensitivity. However, they also noted that Cyfra-21-1 was still the most sensitive detector of CTCs, although it is still not sensitive enough to quantify CTCs in the early stages of NSCLC. Discrepancies between different studies also came to light – while the study by Lou et al.(2013) found that folate was produced in similar levels across both squamous cells and adenocarcinoma cells. It was found in significantly higher levels on adenocarcinoma cells by O’Shannessy et al. (2012) and also by Nunez et al. (2012). This is indicative that greater research and broader based clinical trials are necessary in this novel method of diagnosis. However, these results illustrate the possibility of using and developing folate receptor ligands as biomarkers in NSCLC.


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Ligand Decorated Nanovehicles as Drug Delivery Systems Many of the side effects associated with chemotherapy are associated with the fact that it is administered freely (i.e. intravenously and not encapsulated) , and so has the capacity to attack and kill any fast growing cell it comes into contact with. One way to achieve the desired specificity and thus by-pass the unwanted side effects is to encapsulate the drug in ligand-decorated nanovehicles. The storage of the drugs within a nanoparticle is what enables the attachment of the ligands, and thus the active targeting. A number of possible nanovehicles have been investigated, including gelatin nanoparticles, silica nanorattles and gold nanoparticles. Gelatin is often chosen as a component of the nanoparticle due to its biocompatibility (Vandervoort & Ludwig 2004). Furthermore, treatment of the gelatin surface with Avidin enables the bonding of a number of biotinylated ligands to the surface of the particle (Coester et al. 2000).

This was the premise of a study conducted by Tseng et al. (2007), who bound biotinylated EGFR ligands to the surface of a gelatin nanoparticle through the use of NeutrAvidin (Fig. 3). The internalisation of these nanoparticles into adenocarcinoma cells and healthy cells was then compared – it was found that the adenocarcinoma cells that over-expressed EGF (92%) had a much higher uptake rate than healthy lung fibroblast cells (2.2%), and that the internalisation occurred due to a receptor- mediated endocytosis pathway. However, this huge uptake of the nanovehicles by the adenocarcinoma cells may also be partially attributed to EPR (enhanced permeability and retention – a common occurrence in cancer cells). An issue with these nanoparticles is their relatively large size – 220 nm (ideally a nanoparticle must be 100nm or less in order to circulate longer and avoid being destroyed by macrophages), however, this problem can be overcome by delivering the drug via inhalation. The use of inhalation as a drug delivery method is suited specifically to lung cancer, as the drugs are being administered directly to their site of action. This was tested, and found to be as satisfactory as the internalisation achieved by administering the drug intravenously.

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Gelatin nanoparticles were also used in a similar study conducted by Tseng et al. (2009), except in this study the nanoparticles were infused with CDDP (cisplatin). FTIR spectroscopy was used to examine the chemical composition of the encapsulated drug, and to verify it had not undergone polymorphism. These results indicate that although there is some bonding interaction between the cisplatin and the silica shell, the bond is easily reversible and does not affect the anti-cancer effect of the drug. It was ultimately found that this method of drug delivery using cisplatin was superior to the administration of the free drug (Fig. 1), illustrated by the IC50 (the concentration of the drug required to inhibit half of the cancer cell activity) being 2.1 times lower than that of the free drug. It is clearly evident that the encapsulation of anti-cancer agents has a variety of benefits, and, as such, deserves further investigation and clinical trials.

Figure 3: An overview of the synthesis of EGF-decorated silica nanoparticles. (Tseng et al. 2007).


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Figure 4: The superior anti-tumour effect of cisplatin encapsulated in an EGF decorated silica nanovehicle. (Tseng et al. 2009)

The Use of Nanoparticles as Imaging and Drug Delivery AgentsThe use of an iron oxide core in the ligand decorated nanovehicles opens the door to a new realm of clever medicine – theranostics. Super paramagnetic iron oxide nanoparticles are an excellent MRI contrast agent, particularly in the diagnosis of solid tumours, due to their low cytotoxicity, high magnetic signal strength, low sensitivity to surrounding water molecules and long lasting contrast abilities (Rosen et al. 2012).

The idea to incorporate iron oxide into ligand decorated nanovehicles was first tested by Chen et al. (2010). The use of iron oxide as a rattle-type centre in mesoporous silica nanoparticles enables the simultaneous delivery of drugs, and imaging of the tumour masses. The functional nanocore also introduces the possibility of magnetically targeting the tumour, further focusing the drug delivery on the cancer cells only. These nanoparticles exhibit a high loading capacity of about 20%, and, in this experiment, the mesopourous

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nanoparticles were loaded with doxorubicin hydrochloride (DOX). The enhanced loading effect is due to electrostatic interaction between the silica and the DOX molecules, as well as the large pore size and large surface area. In the same way as many other of the nanoparticles observed in this manner of treatment, it is suspected that the NPs enters the cell via endocytosis, resulting in a higher accumulation of DOX within the cell than one would find from the administration of free DOX. The iron oxide NPs were found to be incredibly biocompatible.

In the same study, spherical gold nanoparticles replaced the ellipsoidal iron oxide nanoparticles in mesoporous silica structures, which were again loaded with DOX. These nanoparticles were also grafted with GD-SI-DTPA, and combining the optical properties of gold nanoparticles (surface plasma resonance enhanced light scattering) with those of GD-Si-DTPA (common MRI contrast agent), dark field light scattering cell imaging can be utilised, as well as MRI (Fig. 5). This was the first in a promising new world of lung cancer treatments.

Figure 5: Dark field optical microscope images of HeLa cells before (a) and after incubation with Gd-Si-DTPA grafted gold nanoparticles (b). The optical scattering induced by the presence of the gold centred in the nanocapsules can be clearly seen in in second image, showing its possible use in theranostics. (Rosen et al. 2012)

ConclusionsLigand decorated nanoparticle drug delivery systems looks to be the future of lung cancer treatment. With the massive advancements made in the field of nanotechnology over the


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past few years, drug vehicles such as these are becoming easier and easier to synthesise, and will one day hopefully lead to the effective treatment of lung cancer, and a sharp decline in mortality rates. The use of targeting moieties on the surface of the nanovehicle means that healthy cells are highly unlikely to be incorporate them, as they will bind to receptors present primarily on cancer cells. A number of these possible drug delivery systems also had the potential to be nebulised and administered via inhalation, thus minimising discomfort to the patient and targeting the lung directly. This method showed incredibly satisfactory results. A number of these drug delivery systems were rattle-type silica nanoparticles with a functionalised core, and this opened the door to a range of imaging possibilities. The use of iron oxide in these drug delivery systems means that the therapeutic effect of the drug could be measured as the drug was administered. Furthermore, there also exists a possibility that the drug could be further guided to the tumour masses using magnets. The light scattering properties of a functional gold nanocore were also examined, and demonstrated to be of great use. However, there are a number of issues associated with this form of treatment. There have been very few long term toxicity studies conducted with these DDSs, and there is a need for more clinical trials which test the action of the anti-cancer drugs after their encapsulation into the nanovehicle. A number of specifically acting drugs, such as Iressa and Erlotnib, which act as EGFR inhibitors, are known and widely used in the maintenance therapy of advanced lung cancer. However, resistance to these drugs is an issue (Pao et al. 2005). The problem of drug resistance is by-passed through the encapsulation of the drugs, and the decoration of the nanovehicles with targeting ligands ensures the drug will only be released to cancer cells, thus minimising the possibility of drug resistance taking place. The majority of recent research in this area has been conducted using nude mice or in vitro cell cultures, with very few clinical trials taking place. However, the future of ligand decorated nanovehicles for use as drug delivery systems looks promising, and will hopefully result in more efficient treatment and longer life-spans for lung cancer patients.

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CHEN., Y, CHEN, H., ZENG, D., TIAN, Y., CHEN, F., FENG, J., SHI, J., (2010) Core/Shell Structured Hollow Mesoporous Nanocapsules: A Potential Platform for Simultaneous Cell Imaging and Anticancer Drug Delivery. ACS Nano, 4(10), 6001-6013.

COESTER, C., KREUTER, J., VON BRIESEN, H., LANGER, K., (2000) Preparation of avidin-labelled gelatin nanoparticles as carriers for biotinylated peptide nucleic acid (PNA). Int. J.Pharm, 196(2), 147-149.

FUJINO, S., ENOKIBORI, T., TEZUKA, N., ASADA, Y., INOUE, S., KATO, H., MORI, A. (1996) A comparison of epidermal growth factor receptor levels and other prognostic parameters in non-small cell lung cancer. European Journal of Cancer, 32(12), 2070-2074.its ligands as therapeutic targets in human tumors. Cytokine & Growth Factor Reviews, 7(2), 133-141.

LIN, W., HUANG, Y., ZHOU, X., MA, Y. (2006) In vitro toxicity of silica nanoparticles in human lung cancer cells. Toxicology and Applied Pharmacology, 217(3), 252-259.

LOU, J., BEN, S., YANG, G., LIANG, X., WANG, X., NI, S., HAN, B., (2013) Quantification of Rare Circulating Tumor Cells in Non-Small Cell Lung Cancer by Ligand-Targeted PCR. PLOS ONE, 8(12), Available at: http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0080458

NUNEZ, M.I., BEHRENS, C., WOODS, D.M., LIN, H., SURAOKAR, M., KADARA, H., HOFSTETTER, W., KALHOR, N., LEE, J.J., FRANKLIN, W., STEWART, D.J., WISTUBA, I.I. (2012) High Expression of Folate Receptor Alpha in Lung Cancer Correlates with Adenocarcinoma Histology and EGFR Mutation. Journal of Thoracic Oncology, 7(5), 833-840.

O’REGAN, E. (2014) Lung cancer deaths rise in women as smoking takes its toll. Irish Independent, 18 December 2014. Available at: http://www.independent.ie/irish-news/health/lung-cancer-deaths-rise-in-women-as-smoking-takes-its-toll-30846278.html . [18 January 2015]

O’SHANNESSY, D.J., YU, G., SMALE, R., FU, Y., SINGHAL, S., THIEL, R.P., SOMERS, E.B., VACHANI, A.(2012) Folate Receptor Alpha: A New Tool in the Diagnosis and Treatment of Lung Cancer. Oncotarget, 3(4), 414-425.

PAO, W., MILLER, V.A., POLITI, K.A., RIELY, G.J., SOMWAR, R., ZAKOWSKI, M.F., KRIS, M.G., VARMUS, H.(2005). Acquired Resistance of Lung Adenocarcinomas to Gefitinib or Erlotinib Is Associated with a Second Mutation in the EGFR Kinase Domain. PLOS Medicine, 2(3), 225-235.


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PARKER, N., TURK, M.J., WESTRICK, E., LEWIS, J.D., LOW, P.S., LEAMON, C.P. (2005) Folate receptor expression in carcinomas and normal tissues determined by a quantitative radioligand binding assay. Analytical Biochemistry, 338(2), 284-293.

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Best Freshman Essay

The Uses and Limitations of Absorption Spectroscopy in the

Development of Non Invasive Blood Glucose Monitoring

SystemsNanoscience, Physics and Chemistry

of Advanced MaterialsKate Reidy

Senior Freshman

Type I and II diabetes rates and their associated mortality rates are increasing day by day. Careful, constant glucose monitoring is paramount in treating the disease and the development of an accurate, easily implementable and non-invasive technique is currently in the research spotlight. Absorptive spectroscopic techniques are at the forefront of research into viable solutions to this problem. For diabetes sufferers’ reliability, simplicity and ease of use are of critical importance. NIR spectroscopy is currently seen as the most promising technique however its limitations include extreme sensitivity to outside and physical factors and lack of a suitably accurate data calibration method. MIR spectroscopy has often been discarded due to its low penetration power and subsequent inability to obtain measurements through the skin, however, with new advances in attenuated total reflection (ATR) technology this method could definitely be reconsidered as a viable future method of detection. MIR spectroscopy shows both less sensitivity to outside factors and less need for calibration than NIR methods. There are advances to be made before absorptive spectroscopic techniques can be implemented on an industrial scale as feasible and accurate alternatives to the fingerpick method. This paper

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presents a review of the principles of these and their limitations, focusing on current and future research directions in order to combat these limitations and create a viable, non-invasive glucose monitoring technique which would be viable for future

use.

IntroductionDiabetes mellitus is a metabolic disorder and one of the most prevalent diseases in the world today. About 4-5% of the world’s Caucasian population suffer from it and it is estimated that about 40% of all blood tests are related to it (Steiner et al. 2011). In Type 1 or ‘early onset’ diabetes the pancreas does not produce any insulin. In Type 2 diabetes either it under-produces insulin or the cells in the body do not react to insulin. In both cases the sufferer develops abnormally high blood glucose levels (hyperglycemia) which can result in many devastating complications such as cardiovascular disease (Morrish et al.. 2001), neuropathy, blindness (WHO Geneva 2012), kidney failure or amputations (Roglic et al. 2005). Frequent and accurate blood glucose monitoring is paramount in the treatment of diabetes in order to delay and sometimes even prevent these complications.

Currently, the most popular and effective blood glucose monitoring technique is an invasive ‘fingerprick’ technique which is an electrochemical enzyme based method of detection. Devices usually consist of two carbon-based electrodes, a thin dry reagent layer and enzymes such as glucose oxidase or glucose dehydrogenase. The blood is placed in a capillary volume strip and mixed with the dry reagent in an enzymatic reaction to form a chemical product which is proportional to the blood glucose level. This capillary strip is inserted into the meter and the working electrode is polarised which generates an oxidation process that is converted to an electric signal by an electrochemical transducer to be read and outputted by the meter. This method is effective and accurate however the pain and risk of infection caused by frequent self-testing, along with possible nerve damage, discomfort and callousing from long term monitoring (Pickup et al. 2005) often deters patients from monitoring


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glucose levels as often as recommended and also causes problems monitoring very young patients, elderly or less dexterous patients (Turner et al. 2005).

With 347 million people worldwide suffering from diabetes (Danaei et al. 2013) and glucose biosensors comprising over 85% of the estimated $5 billion market for biosensors worldwide (Newman et al. 2004) it is therefore not surprising that many resources are being put into the development non-invasive blood glucose detection methods to improve the lives of people suffering with the disease by providing an easy, painless and non-invasive method of glucose monitoring.

FigURE 1: An overview of the available glucose monitoring techniques, from invasive, to minimally invasive (interstitial fluid) to non invasive techniques. (Adapted from diagrams and information included from So et al. (2012), Poddar et al. (2006) and Amaral & Wolf (2008).) The shaded subsections are the spectroscopic methods detailed in this review.

This review will focus specifically on absorptive spectroscopic techniques as an alternative to invasive techniques in measuring in vivo glucose levels and will provide a chemical background to these techniques and a complete critical analysis of their limitations. Optical analysis is currently the most promising and researched technique of non-invasive in vivo glucose analysis, with over 100 small companies and universities working on monitoring devices

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using these methods (Coté, 2001). Within the range of optical analysis techniques, near infrared (NIR) spectroscopy has been the primary area of interest and focus (McNichols & Coté, 1998, Gabriely et al. 1999), with mid infrared (MIR) spectroscopy being researched in particular cases (Heise et al. 1989, Yu et al. 2014) as a very promising glucose monitoring technique.

Spectroscopic Techniques

Near Infrared Spectroscopy

PrincipleNIR involves the focusing of a beam of light onto the sample which penetrates to a depth of 1 - 100 mm (Vashist, 2012). Light of wavelengths of 1050 - 2450 nm are used for most studies (Malin et al. 1999) although NIR wavelengths are defined as the set of wavelengths between 800 -2500 nm (Sileoni et al. 2013). NIR measures the absorption of light caused by the rotation or vibration (Teixeira et al. 2009) of excited aliphatic, aromatic or alkene carbon-hydrogen bonds, amine nitrogen-hydrogen bonds, and oxygen-hydrogen bonds(Reich, 2005). Glucose (C6H12O6) scatters and absorbs the incident light resulting in characteristic interactions and absorption spectrums determined by the transmitted light (Stuart 2004, Poddar et al. 2006)NIR uses the overtones and vibrational modes of a compound to obtain physical and chemical information of the sample and shows the characteristic peak of glucose.

Most apparatus consists of a deuterium-halogen light source (Fernández-Novales et al. 2008), NIR spectrometer and bundles of fiber optic cables which are arranged systematically around the site to be tested (Amaral & Wolf, 2007). The equation used to determine the attenuation of light is I = Ioe-u*d , in accordance with the light transport theory where Io represents the incident light intensity, I the reflected light intensity, d the path length of the beam in the tissue and u* representing the effective extinction coefficient which can be calculated as a function of both the absorption and scattering coefficients (Tura et al. 2006)


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Although the overtones and vibrational mode spectra often overlap, making it harder to distinguish glucose peaks, the NIR range is still extremely suitable for the evaluation of compounds in human tissues due to the high concentration of water molecules. At these shorter infrared wavelengths water absorption is weak (Amaral & Wolf, 2007) allowing the spectrum of glucose to be distinguished. If longer wavelengths are chosen, the absorption of water would be the most prevalent signal and the spectral bands of glucose would overlap and be hidden by the high water absorption band. Conversely, if the wavelengths were shorter, the spectrum would transverse into the visible spectrum in which visible light would be absorbed by the pigments in skin. (Tura et al. 2006)

The apparatus must then be calibrated using results from both diabetic and healthy patients with varying BG levels. There is no general consensus for a universal calibration method, which is a drawback to the NIR technique. Multivariate data analysis techniques such as principal component analysis (PCA) and partial least squares fit (PLS) are used to mathematically separate the desired spectra from the matrix (Oliviera & Neves et al. 2012). This is a huge advantage of NIR treatment over UV or visible light spectroscopy which mostly require a manual separation of the sample from the matrix, mainly in the form of dissolution (Cozzolino et al. 2006). PCA eliminates variables by analysing the data in terms of its most prominent components instead of the normal x-y axis, whereas PLS eliminates variations by transforming the data into a linear model based on only a small number of orthogonal variables. These techniques begin to break down when there are a large number of variables present in the sample as it can no longer be considered a linear model.

Limitations and AdaptationsPossibly the most challenging aspect of the development of NIR spectroscopy is determining the site at which to measure glucose levels. Due to the fact that NIR measures the vibrational modes and overtones of compounds instead of the fundamental absorption spectra found in MIRS it is very susceptible to changes in skin

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structural properties, for example: dryness, depth, fat content and environmental factors.

It was shown by Sibbald et al (1996) and Monnier et al (1999) that diabetic patients can develop a characteristic ‘thick skin’. The incongruity between the structural properties of skin in diabetics and healthy patients could in turn lead to incorrect calibration, difficulty in analysis due to thickening of skin and a long term instability of the monitoring process. Hyperglycemia also affects the thermal properties of skin (Yeh et al. 2003) which greatly distorts the spectral measurements. A breakthrough occurred with the work of Burmeister and Arnold (1999) who conducted a study of six measurement sites: cheek, lower lip, upper lip, nasal septum, tongue and finger webbing. This study found that the tongue was the most feasible site for NIR measurements, demonstrating the highest signal to noise ratio. This is due to the fact that the tongue is a purely muscular structure, eliminating deviations due to the thermal and structural properties of skin (Burmeister & Arnold 1999). The tongue also exhibits low levels of fatty tissue and a very high blood supply which allows accurate glucose analysis without the need for deeply penetrating wavelengths. Their work, however, did not address the dependence on environmental factors.

Many environmental and physical factors can affect the results of calibrated NIR spectroscopic data, including background conditions - temperature, humidity, atmospheric pressure - and interference variables - varying CO2 concentration, fats, proteins and hemoglobin levels present in human tissue. (Zhang et al. 2013). Multivariate calibration and data analysis is therefore necessary to avoid unreliable data sets when using the apparatus in a variety of conditions that do not correspond to those of original calibration. Methods have been examined to correct background variation: multiplicative scatter correction (MSC), polynomial fitting (Komsta 2011) and weighted least squares fit (Zhang 2009). In addition, alternative methods such as uninformative variable elimination (UVE) (Cai 2008) and randomisation tests (Xu 2009) have been developed to deal with the effect of interference variables. There are, however, no methods which successfully accomplish both of these tasks simultaneously. Researchers Li et al. (2014) succeeded in


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devising a hybrid algorithm (a combination of UVE and least squares fit approaches) for multivariate calibration that could combine these two factors. They applied it successfully in a glucose level analysis on 35 diabetic and 11 healthy subjects. The human tongue was used as a sample test site and it was found that the correlation coefficient of prediction increased 31.21 % and root mean square error of prediction decreased 34.56 % from the originally calibrated methods (Li et al. 2014). This illustrates the possibility of hybrid analysis techniques to create feasible NIR spectroscopic devices which overcome variations in background conditions and are able to precisely determine glucose levels without being influenced by other compounds present in the tissue. Unfortunately the major assumption of these algorithms is that the data can be fitted to some sort of linear model, which proves inaccurate and untrue in most cases (Li et al. 2014).

Mid Infrared Spectroscopy

PrincipleThe principle and methodology of MIR spectroscopy is very similar in nature to that of NIRS, but the MIR spectrum is defined as wavelengths in the 2500 - 10,000 nm range (Khalil et al. 2004), although many studies are carried out in the 8382 - 9708 nm range (Martin et al. 2002, Malchoff et al. 2002). Much less research has been carried out on this method due to its limited light penetration capabilities of only a few micrometers (Brancaleon et al. 2001) which greatly hinders the ability to use transmitted light data. As a result, only scattered light can be considered. However MIRS results in more distinct glucose peaks than NIRS. This is due to the higher absorption coefficients (Landgrebe et al. 2010) and the fact that MIRS ranges are able to induce an excitation and fundamental vibration spectrum whereas NIRS spectra consist of the overtones of a fundamental vibration, resulting in more distinct, less overlapping spectra (William & Norris 1987, Rhiel et al. 2002). The diffusion of light in the MIR range is also much less than in the NIR range, which creates less noise in the signal, making MIR data much less skewed by factors which affect the diffusion of light, for example porosity or

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hydration of the tissue (Bellon-Maurel & McBratney 2011). The need for developing accurate multivariate data analysis is therefore much less. Some treatment of the data is still required, however it does not constitute as much of a limitation as in the NIR range.

Limitations and AdaptationsMany researchers have dismissed MIRS as a viable spectroscopic technique for the measurement of blood glucose levels due to its poor penetration qualities (Tura et al. 2006) however Sandor et al (2013) in a comparative study of non-invasive monitoring using both NIRS and MIRS methods argues that MIRS is the most accurate technique in monitoring a single analyte as it had a root mean square error (RMSE) of 0.16 g/L where as NIRS had a RMSE of 0.36 g/L. However, during their experimentation the MIR probe had to be purged with nitrogen gas at 20 mL/min to prevent unwanted absorption bands of CO2 and water vapour, which greatly limits the adaptability of this technique. (Sandor et al. 2013).

Harvey & McNeil (2006) found that the problem of limited penetration could be circumvented by the use of attenuated total reflection (ATR). This method uses total internal reflection of a light beam through a crystal which is placed on the skin. The reflected light creates an electromagnetic field which can then reach the dermis (location of most of the skin glucose) (Thennadil 2001). The beam absorption will change, depending on the levels of blood glucose. However this requires more expensive and fragile fibre optic MIR probes (Roychoudhury et al. 2006). Saiko et al (2014) successfully developed a MIR attenuated total reflection glucose monitoring system using a hollow optical fibre probe. Owing to the results of Burmeister and Arnold (1999) mentioned earlier the tests were carried out on oral mucosa where the capillaries are found very close to the surface, allowing for precise glucose measurement. Their results indicated that the accuracy and reproducibility of the results were high enough to deem this a viable blood glucose monitoring instrument (Saiko et al. 2014).


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Conclusions The principles, limitations and adaptations of absorptive spectroscopic techniques have been discussed in this review. It highlighted NIR as the most promising technique for feasible implementation in terms of efficiency and cost of the sensing probe. However a more complete and accurate method of data calibration and analysis is necessary before this method can be implemented. This limitation was partially solved by Li et al. (2014) in creating a hybrid data analysis model although an even more highly sophisticated technique is required if these devices are to be implemented as an accurate alternative to invasive methods. MIR has the advantage of having much more distinguishable peaks and therefore requires less final data calibration, however, the sensing techniques are not as advanced as it has been discarded as an area of research by many companies and research groups. This is mainly due to the poor penetration of the MIR spectrum. Recent ATR advances (Saiko et al. 2014) have greatly increased the penetration power and combining this with the work of Burmeister and Arnold (1999) for determining the most effective monitoring site, may generate a successful MIR glucose monitoring device, making MIR a candidate to be strongly reconsidered in the future to provide a more effective solution to NIR.

In the area of blood glucose detection, the reliability and accuracy of results are of paramount importance because inaccuracy or misdiagnosis of blood glucose levels can have extreme ramifications for patients. It is challenging to create a method which meets all bio-compatibility specifications while maintaining high stability, robustness and accuracy which is not influenced by outside factors. Absorptive spectroscopic techniques could become a viable future solution with a few advancements and adaptations. The race for the next generation of commercially available, user friendly and painless methods of glucose detection has begun. Absorptive spectroscopic techniques are not currently feasible for industrial implementation due to the limitations discussed here but are definitely forefront candidates for a possible solution in the not too distant future.

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AcknowledgementsThis work was adapted from an essay “The Role of Nanosensors in Glucose Detection” for the Broad Curriculum Project in course CH2202.

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Production of Hydrogen and its Role in the Transition to a

Clean Energy EconomyNatural Sciences David MaddenSenior Freshman

This review examines how hydrogen, an energy vessel, may be produced for the transition towards a renewable energy economy. Incorporation of hydrogen into coal-energy extraction by means of IGCC is assessed. IGCC technology is compared environmentally and economically with the current coal extraction method - PC plants. Challenges for industrial adoption of this technology is discussed. This details a means to clean hot syngas in the process of electricity generation. The role of hydrogen as an energy store for fluctuating renewable energy is evaluated. Photobiological production of hydrogen from Chlamydomonas reinhardtii offers a means to harness solar energy in an effective, environmentally friendly manner. The metabolic process is briefly outlined, focusing on the role of hydrogenases in the production of hydrogen, and how oxygen damages these enzymes. Methods to prevent this damage and hence increase hydrogen yield are discussed, namely sulfur deprivation, copper addition and genetic engineering. The production of hydrogen from electrolysis is assessed, focusing on methods to minimise the use of expensive precious metals. These include adoption of nickel or iron based catalysts. The use of Nafion polymer or carbon nanotubes as a support for anodic catalysts and their effect on decreasing the anodic overpotential for the evolution of oxygen, the most costly aspect of electrolysis,

is assessed.

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IntroductionMankind is faced with a difficult challenge: identification and extraction of a sustainable energy capable of meeting global demands. Presently, fossil fuels are the dominant source of energy (BP 2014). However these are finite and produce harmful by-products that exacerbate global warming (O’Neill & Oppenheimer 2002; Thomas et al. 2004; IPCC 2014). Hydrogen fuel has been identified as a possible alternative (Dandajeh 2014). When hydrogen is burned with oxygen it releases energy, and water being the only by-product. Despite hydrogen being an abundant element, it’s not found in its molecular state, H2, on earth, but in compounds such as water or methane. Energy must be supplied to extract hydrogen. Hydrogen isn’t a form of fuel but an energy carrier, comparable to electricity. Efficiencies in production and storage are essential for the adoption of a hydrogen economy (Kim et al. 2014). A swift change over to a hydrogen economy would be unattainable. Challenges facing this include devising efficient production methods, constructing a hydrogen infrastructure including transportation, storage and safe handling of hydrogen (Simbeck 2003). This review focuses on hydrogen production, transitioning from fossil fuels to sustainable energy. Integrated gasification combined cycle (IGCC) incorporates hydrogen into energy extraction. While this relies on coal, it’s cleaner than the current method of energy extraction; pulverised coal (PC) fired boiler plants. PC involves combusting powdered-coal in the presence of air. Unlike steam reforming, the main industrial method of producing hydrogen, IGCC technology uses coal, the most globally abundant fossil fuel (IEA 2014). This method isn’t sustainable, but acts as a progression towards a clean energy economy. Photobiological production of hydrogen offers the opportunity of a renewable, clean energy carrier. Chlamydomonas reinhardtii, a type of green algae, integrates hydrogen production into its metabolic processes. However the wild species produces low yields (Kosourov et al. 2002). Sulfur deprivation, copper enrichment and genetic engineering has allowed for greater yields, however this technology currently fails to compete economically with fossil fuels. With further advancement it may provide a method for effectively harnessing solar energy in the future (NREL 2007). The splitting of water (electrolysis) offers a means to produce hydrogen.


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Precious metal catalysts are currently used, however these metals are expensive. Nickel or iron based catalysts offer an alternative to precious metals. Innovations in the oxygen evolution reaction (OER) for electrolysis are important, as this is the most expensive step of the reaction (Marinia et al. 2012). Use of Nafion, a polymer with unique thermal/mechanical stability, and single walled carbon nanotubes (SWNCTs) as a basis for hydrous iron oxide catalysts results in lower overpotentials for the reaction, making the process more efficient (Doyle & Lyons 2014).

IGCC TechnologyIGCC is a process that combines gasification of coal, resulting in syngas (a mixture of hydrogen, carbon monoxide and carbon dioxide) that is then purified and combusted, powering gas and steam turbines. This occurs at 1500-1600, and at pressures of 2.5-4.5 MPa (De Graaf 2008). Currently steam reforming, using natural gas, is the primary method for producing hydrogen, however IGCC uses the most abundant fossil fuel and is the only comparable technology that reaches the environmental standards of a natural gas-based reforming plant (Collodi et al. 2009).

IGCC is environmentally cleaner than current PC plants, which generate 50% of the USA electric supply (Zhai et al. 2011). EPA (2005) have conducted an assessment of IGCC, and concluded that this technology offers greater thermal efficiency, requires less water and generates less waste than PC plants, for the majority of fuel and system configurations. Further environmental targets may be met through carbon sequestration. This involves the capture and storage of carbon dioxide. In the case of IGCC plants, a pre-combustion process may capture CO2. This involves a water-gas shift reaction of the syngas with steam, converting carbon monoxide to carbon dioxide and hydrogen. This requires energy, so the IGCC plant must increase its fuel consumption per kWh by 14-25%. To capture CO2 generated by current PC plants by post-combustion capture, a 25-50% increase in kWh is required, depending on the specific type of plant. The cost of implementing the carbon capture process is significantly less for IGCC plants, as is its maintenance

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relative to PC plants (IPCC 2005).

IGCC results in hot syngas at temperatures up to 1600 (De Graaf 2008). Before it enters gas and steam turbines, contaminants must be removed. These include; sulfur oxides (SOx) and nitrogen oxides (NOx) (Rand & Dell 2008). Currently the syngas must be cooled below 100 before processing by ‘candle’ filters. This cooling and subsequent reheating to power turbines is environmentally unfriendly. There is general consensus that the development of rigorous hot-gas cleaning systems would result in greater adoption of IGCC technology (Cargill et al. 2001; Simbeck 2002; Woolcock & Brown 2013). The major contaminants of syngas are sulfur oxides (Rand & Dell 2008). Progress has been made on hot-gas desulfurization technologies, by using copper based sorbents, at temperatures up to 600 (Slimane & Abbasian 2000). A sorbent gathers molecules of another substance together. The contaminants may be isolated as by-products. When the challenge of hot gas cleaning is met or stringent CO2 emission taxes become widespread, IGCC technology may become an economically viable option for delivering cleaner energy from fossil fuel (Kim 2008).

Photobiological Production of HydrogenSolar energy to produce biofuels is the one of the most environmentally friendly technology still at infancy in research (Dismukes et al. 2008). Chlamydomonas reinhardtii, a type of algae, has been shown to produce hydrogen gas under specific conditions (Hemschemeier et al. 2008). These photosynthetic microorganisms split water into hydrogen ions, H+, and electrons. Through metabolic processes involving hydrogenase enzymes, hydrogen gas is produced. Low hydrogen yields makes this method unfeasible for industry; ‘Economic H2 production requires high H2 production efficiencies at low capital and operating costs’ (Rupprecht et al. 2006). A number of techniques have been proposed to remedy this issue.

Oxygen is toxic to hydrogenases (Stripp et al. 2009). Researchers have identified novel ways to remove oxygen from the green algae by sulfur deprivation. Anaerobic green algae deprived


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of sulfur results in reversible inhibition of oxygenic photosynthesis. The electrons that feed this pathway are directed towards the Fe-hydrogenase pathway. The Fe-hydrogenase complex receives electrons from reduced ferredoxin and efficiently donates them to protons, resulting in hydrogen evolution (Greenbaum 1988; Peters et al. 1998). Zhang et al. (2002) identified that maximum hydrogen evolution was reached at the 4/5 day mark after initial sulfur deprivation. From here the Fe-hydrogenase pathway becomes completely inhibited. The algae must return to oxygenic photosynthesis in order to recuperate expended metabolites. This cycle may be repeated (Ghirardi et al. 2000) however, initial sulfur deprivation results in the normal ellipsoid-shaped cells becoming spherical and enlarged. Extended deprivation results in mass algae reduction (Zhang et al. 2002). This harms the viability of this method for industrial hydrogen production. The key to hydrogen production in C. reinhardtii is blocking oxygen production; while sulfur deprivation accomplishes this, copper addition has a key advantage. Surzycki et al. (2007) found that copper addition turns off Cyc6 promoter, causing the Nac2 gene to be supressed. This inhibits photosystem II (PSII), a protein complex that is integral in oxygenic photosynthesis. While this method yields slightly less hydrogen than sulfur deprivation, the algae cells remain healthy. However, the cyc6 promoter is not turned off permanently, due to anaerobiosis, and so the algae returns to oxygen production. Like sulfur deprivation, this process can be repeated in cycles. These approaches, sulfur deprivation and copper addition, circumvent oxygen production as it is lethal for hydrogenases, and in turn hydrogen production. Development of oxygen-tolerant hydrogenases is considered essential for industrial hydrogen producing technologies to compete with current fuels (Friedrich et al. 2011).

A strain of C. reinhardtii, STm6, has been genetically engineered and tested against the wild form of green algae. This new mutant form resulted in a four-fold increase in hydrogen evolution during sulfur deprivation (Volgusheva et al. 2013). This is attained by the absence of the MOC1 gene, which is involved in the assembly of the mitochondrial respiratory chain (Schönfeld et al. 2004). Melis and Mitra (2010) identified suppression of the Tla1 gene resulted in greater efficiency in hydrogen production. Large light harvesting

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antennae allow capture of light in murky conditions. However in industrially processes this negatively impacts hydrogen production, as excess light is used and there is no need for such large antennae. Suppression of the Tla1 gene results in antennae reduction.

Water ElectrolysisRenewable energy sources are variable by nature and require a storage mechanism to accommodate daily and seasonal changes. This energy must be used immediately or stored (Ter-Gazarian 2011). Hydrogen acting as an energy vessel can accommodate these variances. Renewable energies generate electricity that may be used to split water into hydrogen and oxygen. When this energy is needed, hydrogen is incorporated into a fuel cell. Current methods for generating hydrogen from renewable resources use PEMEs (Proton exchange membrane electrolysers). These require precious metal catalysts and continuous high densities of current, which is difficult to achieve with fluctuating renewable resources. ‘The replacement of platinum with inexpensive materials is critical to the large-scale utilization of hydrogen as a clean energy vector’ (Artero & Fontecave 2005).

Hoffert et al. (2013) has identified a nickel based catalyst, [Ni(P(Ph)2NC6H4OH2)2CH3CN)](BF4)2 which dissociates to [Ni(P(Ph)2NC6H4OH2)2]2+, that has a high turnover of hydrogen at low overpotentials. Overpotential indicates the extra voltage required at the electrode to initiate an electrode reaction. The p-hydroxyphenyl groups of the complex are hydrophilic and allow the catalyst to dissolve in solutions of up to 75% water and 35% acetonitrile, CN3CN. Acetonitrile acts as a polar aprotic solvent. The amine pendants (the nitrogen components) have been shown to act as the proton donor, H+, whereas the nickel centre acts as the hydride donor, H-, in the formation of hydrogen gas (O’Hagan et al. 2012). This process has remarkable hydrogen turnover, due to the efficiency in proton transfer from the aqueous environment to the endo sites. The aqueous environment, in this case water-acetonitrile, has been identified as integral in the rate of catalysis (Stewart et al. 2013). Use of sterically smaller acids after addition of water allows


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for greater access to the endo sites (Shaw et al. 2013).

Figure 1: A graphical representation of the reduced form of the catalyst complex, [Ni P(Ph)2NC6H4OH2)2]2+

Source: Drawn using ChemDraw.

The most energy intensive step of electrolysis is the generation of oxygen at the anode (Marinia et al. 2012). The large overpotential of the oxygen evolution reaction (OER) at the anode site during electrolysis hinders hydrogen production. Current anode materials, RuO2 and IrO2, express low overpotentials at operational current densities (Michas et al. 1992). They are expensive and obstruct adoption of this technology (Gao et al. 2014). Doyle and Lyons (2014) investigated hydrous iron oxide composite films, using Nafion and single-walled carbon nanotubes (SWCNT) films, and identified the importance of Nafion and carbon nanotubes (CNT) for increased OER efficiency. The incorporation of these composite materials results in a dispersed jelly-like structure, increasing the surface area. The inner surface of the oxide layer is anhydrous and compact, whereas the outer layer is hydrated and microdispersed, forming extending rope-like structures (Burke & O’Sullivan 1981). The jelly-like structure of the catalyst acts as a combination of a solid and liquid state. Due to this unique nature, the reaction may be described as a hybrid of homogeneous and heterogeneous catalysis as the reactant, water, is liquid. The catalyst and the reactant are in the same state.

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ConclusionsIGCC will play an integral role in the progression to a clean energy economy. It must be seen as a step, not a conclusion, of meeting energy demands. This technology is not far from large-scale industrial capabilities and will serve as a means to adopt a potential hydrogen infrastructure. The value of IGCC is that it may produce electricity directly from syngas or undergo purification to isolate hydrogen, which may be used in other industrial processes, acting as a byproduct of IGCC. Hot gas cleaning methods warrant further investigation, as the development of rigorous and inexpensive processes will lead to greater efficiency, thus making IGCC an economically viable alternative to the current PC plants.

The metabolic production of hydrogen from Chlamydomonas reinhardtii serves as a reminder of hydrogen’s versatility - capable of being produced chemically and biologically. Identification of the metabolic pathways of these algae has been essential for the understanding of how hydrogen is produced. This discovery has allowed the identification of the hydrogenase family as central for the production of hydrogen. From this, methods to protect the oxygen sensitive enzymes has been devised, namely sulfur deprivation and copper addition. While the former results in greater hydrogen production, its damaging effects on the algae during long term use makes this method impractical for industrial purposes, unless a strain of algae is developed that may endure sulfur deprivation. Copper enrichment offers a solution, ensuring the algae remains healthy throughout cycles of oxygenic and anaerobic photosynthesis, however it too poses issues, specifically the resulting hydrogen yield is small. Development of new strains by genetic engineering will be crucial if this technology is to become a feasible option. Targeting of the MOC1 gene specifically hinders development of mitochondrial respiratory chain features, while the suppression of the Tla1 gene results in reduced antennae size, diminishing the metabolic energy input required for their maintenance. These genetic engineering techniques target the metabolic processes of hydrogen production. Ultimately, the development of oxygen tolerant hydrogenases that can thrive in bright environments will be essential if this technology is to offer a viable means of hydrogen production. Further research


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specifically identifying, or genetically engineering, oxygen tolerant hydrogenases is recommended.

As an energy store, hydrogen provides a means to deal with fluctuations in renewable energy. The use of nickel-based catalysts in the electrolysis of hydrogen is a progression from the current, yet economically unfeasible, precious metal catalysts. The detailed analysis of, [Ni(P(Ph)2NC6H4OH2)2]2+ a nickel based catalyst, has resulted in greater efficiencies, including the use of sterically smaller acids, more capable of donating protons to the endo sites. Novel solutions have been devised to tackle the OER at the anode, the most energetically costly aspect of electrolysis. By the incorporation of hydrous iron oxide composite films embedded in Nafion or SWNCTs, the surface area of the electrode is increased, resulting in an increase in potential reaction sites. This reduces the overpotential of the reaction, minimising the cost of the most expensive aspect of electrolysis. The value of this technology is that it results in very pure hydrogen, which may be directly incorporated into a fuel cell. While these methods collectively increased hydrogen production while diminishing the energetic and financial input, electrolysis faces a number of challenges. Further research on increasing efficiency, particularly cost effective electrocatalysts for OER is recommended.

Hydrogen will perform an important role in the transition to a clean energy economy. Its versatility in production, from fossil fuel derivation, photobiological synthesis and electrolysis allows the benefit of a steady energy vessel while energy sources remain dynamic. When the highlighted challenges are met, hydrogen will aid the transition from the ‘hydrocarbon economy’ to the ‘renewable energy economy’.

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Minor DNA Groove Binding Agent Optimisation

Medicinal Chemistry Rory ScanlonSenior Freshman

A current mode of action for antitumor drugs is the non-covalent binding of drugs to the minor groove of DNA in order to deregulate the replication of the cell. The principal compounds that are used for this pathway are Distamycin and Netropsin. The traits of these structures which are thought to be useful are the amount of hydrogen bond donors and acceptors present as well as their positions. The main limitation of these compounds is their lack of anti-tumour activity. This has resulted in the development of many hybrid derivatives to optimise binding and specificity as well as to introduce anti-tumour activity. Drugs that bind to the minor groove often target cancer and have been proposed to be useful in disrupting the DNA of Protozoal diseases like Human African trypanosomiasis (HAT) and plasmodium diseases. The compounds that are useful for this pathway, as well as possibly cancer chemotherapy, are guanidine 2-aminoimidazoline diaromatic derivatives, which are related in structure to furamidine. This review focuses on some of the general compounds whose mode of action is to bind to the minor groove of DNA in a non-covalent manner and attempts to discuss optimum design by looking at the interactions of these ligands with the aim of increasing activity against protozoic

diseases and cancer.

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IntroductionDNA is often the target of drugs trying to interfere with basic cellular mechanisms, such as cell division, and for years DNA has been the target for chemotherapeutic action. (Henderson, D. et al. 1995). Often diseases such as cancer or HAT interfere with cellular processes at the DNA level and therefore treatment of such diseases employs the use of minor groove binding drugs. There are three main modes of action for drugs to bind to DNA - electrostatic interactions, van der Waal forces and hydrogen bonding. They mainly target the minor groove of DNA to cause cytotoxic effects (Khan G.S. et al. 2012). Small crescent shaped molecules are often the basic framework that is used in the design of these drugs (Khan, G.S. et al. 2012). This is because the concave shape of the ligand fits into the convex shape of the groove (Neidle, S. 2001). Hunt, R. et al. (2011) showed that linear ligands also bind in the minor groove, using analogues of DB921.

N

N HN

N

NH2 O

O

MeN

N

Schematic view of hydrogen bond donors and acceptors in the A:T base pair, adapted from Neidle S. (2001)

The minor groove is regarded as being devoid of many molecules compared to the major groove and is therefore a great target for these drugs (Khan G.S. et al. 2012). Drugs which target the minor groove bond favourably in this position because of an entropic increase due to the displacement of water bound in the minor groove. (Neidle, S 2001). Distamycin and Netropsin are often the primary compounds used and they have been shown to preferentially bond to the adenine and thymine base pairs of DNA (Wartell, R.M. et al. 1974). Their mode of action is to bind to the minor groove temporarily and alter the structure of DNA, thus changing


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its function. This has an advantage over covalent bonding as the binding is temporary and minimizes risk of damage to the DNA (Khan, G.S. et al. 2012).

Main BodyWhen a compound binds to the minor groove it displaces the spine of hydration in the groove. This is driven by hydrophobic forces and results in an increase in entropy. In response to this DNA counter-ions are released to maintain electric neutrality and the compound is incorporated into the minor groove. The counter-ion release manifests as a large entropic increase that favours binding. Once this occurs van der Waal forces and hydrogen bonding are exhibited between the compound and A-T sequences. Netropsin is unable to bind to G-C pairs because of the amino group at position two on guanine and binds exclusively to double stranded DNA. This demonstrates the specificity and shape optimisation of such compounds. (Wartell, R.M. et al. 1974).

N

O

NH

HN

N

O

H2N

NH2

N

HN

O

NH

H2N

adapted from Khan et al (2012) 0

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Netropsin is a molecule which consists of two pyrrolecarbamoyl units with an amidino group at each end of the molecule (Khan, G.S. et al. 2012).

To synthesise new compounds which were better in their affinity for minor groove binding, the number of pyrrole units were increased up to six frames. This provided larger specificity for longer chains which are high in amounts of A-T sequences and thus increased a drug’s potency (D’Allessio, R. et al. 1994, Bailly, C. et al. 1998, Baraldi, P.G et al. 2007). An increase by six units appears to be the limit as in an experiment by Bailly, C. et al. (1998) the twisting of the DNA helix didn’t allow a seventh unit to be attached to the compound in the hopes of increasing binding. To overcome the amino group on guanine - which acts as a hydrogen bond donor (HBD) - a solution was to incorporate a hydrogen bond acceptor (HBA) in place of one of the pyrrole units. The most effective functional group was an imidazole where the nitrogen that was not bound to the methyl unit would function as the HBA (Lown, J.W. 1988).

Distamycin is distinguished by the presence of only one cationic guanidinium group in its structure - which has at least one more pyrrolecarbamoyl unit - and by having a longer binding site in DNA (Neidle, S. 2001). In Distamycin A, the compound is characterized by three oligopeptidic pyrrolecarbamoyl units ending with an amidino group. It has a lack of antitumour activity but specifically binds to AATT segments.

N

HN

O

NH2

HNH

O

NHHCl

n

(Distamycin A), n=3 1n=4n=5

Adapted from Baraldi P.G. et al (2007)


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Baraldi, P.G et al. (2007) used Distamycin A and a homologue with four pyrrole units as a vector. They attached various alkylating agents to improve its chemotherapeutic action. These include a pyrrolo[2,1-c][1,4] benzodiazepines (PBD) group attached at the formyl end of a variable pyrrole chain unit of Distamycin (Baraldi, P.G et al. 1999).

O

CH3O N

N

O

HH3COOC

2

pyrrolo[2,1-c][1,4] benzodiazepines (PBD) group. Adapted from Baraldi P.G. et al 1999

NNHHN

NH2

O

HN

O

O

CH3O N

N

O

H

n

n=1, 3an=2, 3bn=3, 3cn=4, 3d

(PBD) group attached at the formyl end of a variable pyrrole chain unit of Distamycin. Adapted from Baraldi P.G. at al (1999)

HCl

These compounds were shown to have a much lower IC50 (µM) in vitro against T-lymphoblastoid Jurkat cells and human chronic myeloid leukaemia K562 cells than Distamycin A itself. The substituent chains showed a lower IC50 when they were longer. This meant that there were extra possibilities for hydrogen bond donation (Baraldi, P.G et al. 1999).

This minor groove binding property of compounds has been considered not only for drugs looking to treat cancer but also for those aiming to treat parasitic diseases such as HAT. Little investment has been made into new clinical drugs that combat HAT and other tropical diseases. In 2003, less than 1% of all new chemical substances distributed treated tropical parasitic diseases,

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with some minor exceptions (Fairlamb, A.H 2003). HAT is thought to be one of the most neglected diseases in terms of distribution, according to the WHO. Plasmodium derived species, which cause diseases such as malaria, are one of the exceptions to the distribution problem but there has been development of resistant strains of these diseases. In both of these cases new drugs need to be developed to respond to the problem. Pentamidine has historically been used to treat HAT but has been seen to have levels of toxicity that proved it largely unsatisfactory for treatment (Fairlamb, A.H 2003). Even the arsenic based drug melanosporol, which treats late stage HAT when it breaches the CNS, is toxic (Tabernero, L. et al. 1996). 2,5-Bis(4-guanylphenyl)furans were seen to have antiprotozoal activity against the acute form of HAT, Trypanosoma brucei rhodesiense (T.b.rhodesiense), much like Pentamidine but with higher levels of activity (Das, B.P et al. 1977) and also lower levels of toxicity (Neidle, S. 2001).

ONH

NH2

HN

H2N

Furamidine

OO

NH

NH2

NH

H2N

Pentamidine

Adapted from Rozas et al 2013

4

5

The 2,5-Bis(4-guanylphenyl)furans’ main mode of action is to bind to A-T rich sequences, which causes an inhibition of key DNA-dependant enzymes. The principle enzyme to be inhibited is topoisomerase which replicates the kDNA within the Protozoan


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mitochondria. The specific mode of inhibition is reported to be at the first stage of topoisomerase action - site recognition - and it has been hypothesised by Dykstra, C.C et al. (1994) and Shapiro, T.A. et al. (1990) that the drugs that cause this inhibition linearize the DNA.

Bis guanidine and bis 2-aminoimidazolinediphenyl compounds have been shown to be analogous to 2,5-Bis(4-guanylphenyl)furans structurally and, as a result, are similar to Distamycin and Netropsin in the function of groove binding (Rodríguez, F. et al. 2008). Both the guanidine and 2-aminoimidazoline groups function as cations. The ligands tested in a study by Rodríguez, F. et al. exhibited activity against T.b.rhodesiense as well as Plasmodium Falciparum (P.Falciparum). The pathway taken was through the P2 transporter into the protozoan, a new vector, which has previously been resistant to most of this class of compounds, and minimizes the development of cross resistance. The importance of some binding interactions of parts of the ligands tested were demonstrated by testing the activity of different scaffolds which differed by the presence of single parts (Rodríguez, F. et al. 2008).

X

X

X

Y

Scaffold

A

B

C

Cation =NH

NH3

H2NHN

NH

HN

or

Adapted from (Rodríguez, F. et al 2008)guanidine (GUA) 2-aminoimidazoline (IMI)

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Table 1Com-pound

Scaf-fold

Cation (R)

X Y IC50(µM)T.b.rh-ode-siense

P.Fal-ci-parum

Cyto-toxici-ty L6-cells

6a A GUA NH - 0.022 0.018 0.656b B GUA NH - 4.8 1.6 49.56c C GUA NH2 H 311.2 >26 131.87a A GUA CH2 - 0.161 0.032 2.87b B GUA CH2 - 15.9 3.8 43.97c C GUA CH3 H 163.7 >27 >4848a A IMI CH2 - 0.897 0.0157 63.68b B IMI CH2 - 4.9 1.7 738c C IMI CH3 H 262.4 >23 >425

Adapted from (Rodríguez, F. et al. 2008)

Molecules with scaffold A had IC50 values that were nanomolar in range but the other scaffolds (B and C) had values that were only micromolar, even when the same atom was used for (X), which indicated reduced activity. The bridging groups (X) that produced the lower IC50 values were amine, amide and ethane bridges, which were all similar in IC50 value. This was postulated to be due to their ability to function as similar HBDs, although the ethane bridging group is afforded more rotation and therefore has less ability as a HBD (Rodríguez, F. et al. 2008). Compounds with the 2-aminoimidazolinium cations showed higher selectivity based on the activity against T.b.rhodesiense than the corresponding guanidinium ones by having higher selective indexes for DNA consisting of A-T oligonucleotides. The 2-aminoimidazolinium derivatives had higher DNA denaturation temperature (∆Tm) values and a relationship was elucidated between this and their DNA affinity (Rodríguez, F. et al. 2008). Many sources agree that higher ∆Tm values are characteristic of a molecule showing an increased affinity for binding to DNA. This is characteristic of stronger attractive forces (Dardonville, C. et al. 2006, Rodríguez, F. et al. 2008, Nagle, P.S et al. 2009, Nagle, P.S et


170

al. 2010)

Along this line of research new groups were later tested for their DNA binding affinity. These groups included a series of asymmetric diaromatic guanidinium/2-aminoimidazolinium dications. They all follow the basic shape of furamidine with synthetic changes made to the cationic arms of the molecule and bridging atom (X) (Nagle, P.S et al. 2009).

X

NH

NH2

NH

NH

HN

H2N

Adapted from (Nagle, P.S et al. 2009)

Again, the highest changes recorded for ∆Tm for diaromatic guanidinium/2-aminoimidazolinium dications were ones that had nitrogen atoms as the bridging group (X), along with a carbonyl group. The specificity was tested in a UV titration with separate carbonyl and nitrogen bridges. With a mixed amount of sequences both samples had UV shifts as the DNA was added, indicating the disappearance of the free substance. Addition of A-T oligonucleotides produced a stronger shift with the nitrogen bridging group representing the complex, implying a stronger binding (Nagle, P.S et al. 2010).

Nitrogen is capable of forming strong hydrogen bonds by acting as a HBD. Oxygen and sulphur atoms function as HBAs. Therefore addition of more hydrogen bond donor groups could have helped increase affinity of these molecules (Nagle, P.S et al. 2009). SPR (Surface Plasmon Resonance) of these compounds with a methylene and nitrogen link confirmed the nitrogen atom as having a stronger binding constant for AATT oligonucleotides, which is what showed the best agreement with changes in denaturation temperature. The carbonyl linker also showed a lower binding constant for AATT but a higher one for CG sequences (Nagle, P.S et al. 2010).

A comparative experiment was performed on Netropsin which showed a two-fold increase in affinity compared to the tested compounds. The large number of donor groups as well as acceptors

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is thought to be responsible for Netropsin’s performance. Repeated experiments in comparative conditions with increased amounts of A-T composition showed even bigger increases in temperature, revealing the increased affinity for these nucleotides (Nagle, P.S et al. 2009). In the case of methylene and ethylene, ethylene showed greater affinity from a higher temperature increase. The rotation afforded by the extra CH2 could have been a contributing factor in increasing affinity (Nagle, P.S et al. 2009).

Based off this work of asymmetric diaromatic guanidinium/2-aminoimidazolinium derivatives, aminoalkyl derivatives of diaromatic guanidines were measured for their strength as DNA binders (McKeever, C. et al. 2013). The idea was to introduce an aliphatic chain on one of the aromatic groups which would help binding by displacing water molecules present in the minor groove. These hydrophobic interactions improve binding which could increase the antiprotozoal activity of the compounds (McKeever, C. et al. 2013).

X

NH

O

NH

NH2

NH

n

H2N

HCl HCl

n=1 X=CH2, CH2CH2, O, CO, NH 9a-en=2 X=CH2, CH2CH2, O, CO, NH 10a-en=5 X=CH2, CH2CH2, O, CO, NH 11a-en=8 X=CH2, CH2CH2, O, CO, NH 12a-en=9 X=CH2, CH2CH2, O, CO, NH 13a-e

Adapted from McKeever, C. et al 2013

X

NH

NH

NH2

NH HClNH

H2N

HCl

X=CH2, CH2CH2, O, CO, NH 14a-e

X

H2N NH

NH2

NH HCl

X=CH2, CH2CH2, O, CO, NH 15a-e


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Data presented by Nagle, P.S et al. (2009) agree that while most compounds had an increase in the ∆Tm when tested in a random assortment of nucleotides, the figures reported for these derivatives were generally lower than those reported for the asymmetric diaromatic guanidinium/2-aminoimidazolinium derivatives. (McKeever, C. et al. 2013). I would suggest the aminoalkyl chain is not a suitable replacement for the aminoimidazole group if the objective is to bind to DNA. McKeever, C. et al. (2013) tested compounds without the amino alkyl side chain instead using guanidine group (14a-e). Some of this class reported the highest levels of binding in all the tested compounds. Compounds 15a-e, monoguanidinium-like derivatives in which an amino group replaces the aliphatic side chain showed very poor affinity toward DNA, probably due to their monocationic nature. This could imply the chain as the causative agent of this increased DNA binding in some cases. McKeever, C. et al. (2013) used wild type salmon sperm DNA so the specificity of these compounds for certain nucleotides wasn’t tested. The highest value (∆Tm=6) had oxygen and NH as their bridging groups and a chain of four methylene units (McKeever, C. et al. 2013). The optimum length of chain used is thought to be ones of four methylene units with n=2. These reported the best results for chained compounds and adding more units appeared to interfere with binding (McKeever, C. et al. 2013).

ConclusionsConsidering the relationship between structural differences and DNA binding, one can see that the parts of the ligand that attach to DNA are important in determining the binding strength of the ligand and its therapeutic action. It is still a matter of discussion as to what interactions are the most important between electrostatic interactions, hydrogen bonding and van der Waal forces. Looking at the difference in IC50 based on the scaffolds, it would imply that the absence of a cation is more important than an aromatic ring capable of van der Waal interactions. 6b reported 218 times less activity than 6a whereas 6c was only 64 times less active (Rodríguez, F. et al. 2008). Looking at figures provided by McKeever, C. et al. (2013) and comparing the strength of binding between hydrogen bond

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donor rich ligands (14a-e) and ones that have more van der Waal opportunities instead (9-13, a-e), it appears that maximising the number of hydrogen bonds present in a ligand is more important than the van der Waal forces supplied by an aliphatic chain (Tabernero, L. et al. 1996). While Rodríguez, F. et al. (2008) see ligand cationic groups as an important factor for binding, Tabernero, L. et al. (1996) say that the ligand cationic groups did not show a preference for hydrogen bonding. In this case the electrostatic interactions in the groove could be a compromise between both observations. In this review I mainly focused on compounds that bind specifically to A-T nucleotides. However they are not the only type that should be investigated. As discussed above, Lown, J.W. (1988) developed a range of compounds, termed lexitropsins, which allow binding to G-C pairs. Knowledge of how to optimise the right type of interaction is going to be important to elucidate ligands that may one day function as antitrypanosomal and antitumor drugs.


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References BAILLY, C., & CHAIRES, J. B. (1998). Sequence-specific DNA minor

groove binders. Design and synthesis of netropsin and distamycin analogues.Bioconjugate chemistry, 9(5), 513-538.

BAILLY, C., & CHAIRES, J. B. (1998). Sequence-specific DNA minor groove binders. Design and synthesis of netropsin and distamycin analogues.Bioconjugate chemistry, 9(5), 513-538.

BARALDI, P. G., BALBONI, G., Cacciari, B., Guiotto, A., Manfredini, S., Romagnoli, R., G. Spalluto G., Howard P.H., Thurston D.E., Bianchi N., Rutigliano C., Mischiati C. & Gambari, R. (1999). Synthesis, in vitro antiproliferative activity, and DNA-binding properties of hybrid molecules containing pyrrolo [2, 1-c] [1, 4] benzodiazepine and minor-groove-binding oligopyrrole carriers. Journal of medicinal chemistry, 42(25), 5131-5141.

BARALDI, P. G., PRETI, D., FRUTTAROLO, F., TABRIZI, M. A., & ROMAGNOLI, R. (2007). Hybrid molecules between distamycin A and active moieties of antitumor agents. Bioorganic & medicinal chemistry, 15(1), 17-35.

D’ALLESSIO, R., GERONI, C., BIASOLI, G., PESENTI, E., GRANDI, M., & MONGELLI, N. (1994). Structure-activity relationship of novel distamycin A derivatives: synthesis and antitumor activity. Bioorganic & Medicinal Chemistry Letters, 4(12), 1467-1472.

DARDONVILLE, C., BARRETT, M. P., BRUN, R., KAISER, M., TANIOUS, F., & WILSON, W. D. (2006). DNA binding affinity of bisguanidine and bis (2-aminoimidazoline) derivatives with in vivo antitrypanosomal activity. Journal of medicinal chemistry, 49(12), 3748-3752.

DAS, B. P., & BOYKIN, D. W. (1977). Synthesis and antiprotozoal activity of 2, 5-bis (4-guanylphenyl) furans. Journal of medicinal chemistry, 20(4), 531-536.

DYKSTRA, C. C., MCCLERNON, D. R., ELWELL, L. P., & TIDWELL, R. R. (1994). Selective inhibition of topoisomerases from Pneumocystis carinii compared with that of topoisomerases from mammalian cells. Antimicrobial agents and chemotherapy, 38(9), 1890-1898.

FAIRLAMB, A. H. (2003). Chemotherapy of human African trypanosomiasis: current and future prospects. Trends in parasitology, 19(11), 488-494.

HENDERSON, D., & HURLEY, L. H. (1995). Molecular struggle for transcriptional control. Nature Medicine, 1(6), 525-527.

HUNT, R. A., MUNDE, M., KUMAR, A., ISMAIL, M. A., FARAHAT, A. A., ARAFA, R. K., SAY, M., BATISTA-PARRA, A., TEVIS, D., BOYKIN, D. W. & WILSON, W. D. (2011). Induced topological changes in DNA complexes: influence of DNA sequences and small molecule structures. Nucleic acids research, 39(10), 4265-4274

KHAN, G. S., SHAH, A., & BARKER, D. (2012). Chemistry of DNA minor groove binding agents. Journal of Photochemistry and Photobiology B: Biology, 115,

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105-118.LOWN, J.W (1988). Lexitropsins: rational design of DNA sequence reading agents

as novel anti-cancer agents and potential cellular probes. Anti-Cancer Drug Des. 3, 25

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NAGLE, P. S., QUINN, S. J., KELLY, J. M., O’DONOVAN, D. H., KHAN, A. R., RODRIGUEZ, F., BINH, N. W., WILSON D., & ROZAS, I. (2010). Understanding the DNA binding of novel non-symmetrical guanidinium/2-aminoimidazolinium derivatives. Organic & biomolecular chemistry, 8(24), 5558-5567.

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RODRÍGUEZ, F., ROZAS, I., KAISER, M., BRUN, R., NGUYEN, B., WILSON, W. D., GARCÍA, R.N., & DARDONVILLE, C. (2008). New bis (2-aminoimidazoline) and bisguanidine DNA minor groove binders with potent in vivo antitrypanosomal and antiplasmodial activity. Journal of medicinal chemistry, 51(4), 909-923.

SHAPIRO, T. A., & ENGLUND, P. T. (1990). Selective cleavage of kinetoplast DNA minicircles promoted by antitrypanosomal drugs. Proceedings of the National Academy of Sciences, 87(3), 950-954. Linearize kDNA with pentamidine.

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Developments in Metal Organic Frameworks for Utilisation in

Catalysis

ChemistryJoseph EiffeJunior Sophister

Metal organic frameworks (MOFs) are crystalline materials comprised of metal centres co-ordinately connected to each other by polydentate ligands. With the recent increase in research into MOFs the interesting properties of these materials become apparent. Most notable of these properties are the very high specific surface area and very high porosity. These two properties make MOFs interesting materials for catalysis. Developments on a number of methods have been made in making MOFs catalytically active. These have ranged from the use of catalytically active metal nanoparticles located in the pores of the material to the use of MOFs for the heterogenation of homogenous catalysts. It has been found however that the MOFs are sensitive to the conditions that they are place it (i.e. pH, temperature). This need for mild environments makes MOFs industrially non-comparable to robust nature of the common place zeolite catalyst. Therefore MOFs, for the moment at least, are constrained to small scale research in academic research laboratories until new MOFs can be developed that are resistant

to these extreme conditions.

IntroductionMetal organic frameworks are crystalline compounds that are comprised of both organic molecules and inorganic atoms or clusters. The organic molecules are comprised of polydentate ligands that bridge between two or more metallic nodes. The metallic nodes are

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comprised of single transition metal atoms or clusters. Due to the large selection of the metallic nodes and polydentate ligands the properties of the MOFs can be easily adapted to suit the required properties. By interchanging the ligands and metal nodes the structure and properties of the MOF change. These changes can be drastic from just small changes to the molecular makeup of the MOF.

Some of the most notable features of MOFs are the extremely high porosity and high specific surface area of the molecular structures. The pores in the MOF are large in size and can be connected together to form tubular networks in the material. The pores can range in size from a few angstroms to several nanometres (Kuppler et al. 2009). This allows for very high selectivity of the size of molecules that can penetrate into the material and the ability to trap molecules within the structure upon formation of the MOF. The high specific surface area of the MOFs arises due to this highly porous network. This high specific surface area allows for large quantities of gases to bind to the surface of the material in a process known as physisorption as well as allowing access to a large number of catalytic sites within the crystalline structure. These properties make MOF ideal structures for materials used in catalysts, gas storage, drug delivery and molecular/ionic sieves, among many other useful applications (Kitagawa et al. 2004, Kuppler et al. 2009)


178

Figure 1: MOF-5, a zinc – 1,4’-benzoic acid MOF. (a)Stereogram of unit cell (b) Pore size represented by spheres.

The structure of the MOF can be described as an infinite metal organic crystal (Hotskins et al. 1989) Depending on the structure of the organic struts used in the MOF (i.e. bond angles, denticity, length of ligand) and the metal nodes (coordination number, oxidation state) three different topologies can be made. These are one dimensional rods, two dimensional sheets and these are often stacked to create a three dimensional structure similar to graphite and 3D crystals. In catalysis, and therefore most pertinent to this review, the most commonly used structure is 3D crystals.

Initial research of MOFs was found to be slow due to poor methods of creating the structures. However recent developments have now made it possible to create the MOF with relative ease. The most commonly employed method today is the solvothermal process. This involves the addition of the ligands and metal slats to a boiling polar solvent. These reactions occur at relatively low temperatures (>300oC ). Other recently developed methods include the use immiscible solvents and microwave solvothermal syntheses. Microwave solvothermal syntheses is an exciting new methods as

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it allows for control over a number of features, such as temperature and rate of crystallisation, during the reaction. Control over these features helps determine the particle size and macro-structure of the crystals and therefore the type and number of catalytic sites within the crystalline material (Kuppler et al. 2009).

Mechanisms for Catalytic Activity:MOFs can act as catalysts through a number of methods. These range from the unique binding nature between the supporting ligands and the metallic nodes to the heterogenation of homo catalysts. In this section the different methods will be discussed and as will their potential areas of application:

1. Metal Nodes as Catalytic Centres:As the name suggest in these types of catalysts the metal node itself acts as a catalyst in the reaction. There are two main methods in which the metal node can act as a catalyst.

Unsaturated metal node:The structure of this kind of MOF is based on the three-unit building approach, the units being the inorganic node, the structural bridging ligands and labile ligands. During the activation step of the catalytic process these labile ligands detach from the metal centre leaving open a vacant coordination site and this causes a change in the number of electrons on the metal centre. (Chiu et al. 1999). This allows for the metal node to be catalytically active in oxidation addition/reductive elimination catalytic cycles and the unfilled orbitals on the metal can act as strong Lewis acids and accept an electron pair. This method of activating the metallic node by the removal of labile ligands is similar to that of transition metal homogenous catalysts.

These vacant coordination sites, and therefore unfilled orbitals, have a strong affinity for electrons and hence behave as strong Lewis acids. This allows the vacant coordination sites to catalyse reactions that can be catalysed by Lewis acids. Henschel et al. (2008) demonstrated the presence of these active sites in the MOF


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as during their study of MIL-101 the metal node was observed to be the active site in the reaction of cyanosilylation. This was attributed to the presence of Cr(III) sites in the MOF. By comparing two MOFs - one that contained Cr(II) and another that contained Cr(III) - it was found that the MOF that contained Cr(III) had higher activity in the catalysis of the reaction. This can be contributed to the fact that Cr(III) is much stronger Lewis acid than that of Cu(II). This is due to the higher oxidation state of Cr(III) than Cr(II). With less electrons and more positive charge Cr(III) will more readily accept electrons than Cr(II) and hence has a higher reactivity. This increase in reactivity by the stronger Lewis acid indicates that it is the metal centre that is acting as the catalytic site in the MOF.

Horike et al. (2008) demonstrated that the majority of the catalytic activity by the MOF occurs in the tubular channels of MOFs. This allows selective catalysis to be exploited. On their study of a Mn3[(Mn4Cl)3(BTT)8(CH3OH)10] MOF it was found the MOF to could catalyse the cyanosilylation of aromatic aldehydes. The structure of the MOF was found to contain pores of 10Å. Upon using a variety of sizes for the substrates it was discovered that the highest conversion rates occurred with the smaller sized substrates. This preference for the smaller size substrates indicates that the majority of the catalytic activity occurs within the channels. This “sieve effect” can be used to selectively catalyse specific molecules in a solution.

Saturated metal node:At these metal centres all coordination sites are occupied. In order for these metal nodes to act as a catalyst there must be a change in the coordination number around the metal centre. This change in coordination causes the formation of vacant sites. In this case however, the ligand that disassociates from the metal is one of the structural ligands. Due to the limited movement of the structural ligand, it remains close to the metal node.

One example of this type of catalysis is done by Llabres & Xamena et al. (2007). They created a palladium based MOF which was found to have similar catalytic properties to that of other palladium homogenous catalysts. The MOF was found to be an effective catalyst for Suzuki cross-coupling reactions and aerobic

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oxidation reactions. For the Suzuki cross-coupling reaction it was found that 85% conversion was achieved with a ~99% enantiomeric excess.

It was discovered that this catalytic activity is due to the change in oxidation state of palladium from Pd(II)-Pd(0). This change in oxidation state causes a change in the molecular geometry around the palladium metal centre. This change in geometry causes the crystal structure around the metal centre to break down with the dissociation of a structural ligand. The structural ligand has limited movement as it is still bound to other parts of the framework and so it is highly favourable for the structural ligand to re-bond to the metal node upon the metal node returning to its original structure. Some imperfections may occur upon returning to the original coordinated structure - structural ligands may not bind correctly, reactants may not disassociate and free the coordination sites. These potential defects will lead to the breakdown of the crystalline structure. Without this crystalline structure and the re-formation of the catalytically active metal node the catalyst will eventually fail. If the breakdown of the structure is total the catalyst may dissolve into solution.

2. Ligand Struts as Catalysts:The polydentate bridging ligands within the MOF -also known as ligand struts - may also play a role in the catalytic activity of the MOF. The first method of ligand struts having a contribution to catalytic activity was to employ the use of homogenous catalysts as the ligand struts. The homogenous catalysts were modified to be able to act as the supports in a MOF.

Research done by Cho et al. (2006) was done on a manganese MOF. The MOF was designed to have similar catalytic activity to the catalyst used in the Katsuki–Jacobsen epoxidation reaction. The nature of the Katsuki–Jacobsen raection catalyst made it straightforward to convert the catalyst to a viable ligand strut. This was done by using two different ligand struts in the MOF, 4,4’-bipehnyldicarboxilate and salen. One notable difference between the Kastuki-Jacobsen catalyst and the catalytic ligand strut is the use of pyridine instead


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of 4-tetrabutyl-benzene in the complex. This allowed the catalyst to act as a structural ligand within the MOF (Kastuki 1995, Palucki et al. 1998). It was found that the reaction rate for the MOF catalyst was only slightly slower than that of the original Katsuki-Jacobsen catalyst variation of the catalyst. By comparing the two catalysts it was shown that incorporating catalytic activity into the ligand strut is a viable method for MOF in use as catalysts, the benefits of which will be discussed later.

Figure 2: (a) Mn containing ligand. (b) Katsuki-Jacobsen catalyst.

A secondary metal containing organic ligand that has seen a great deal of research is the metaloporphyrin ligands. The porphyrins can be easily converted into ligand struts within a MOF. The key area in catalysis for metaloporphyrins - a free porphyrin with a metal centre, is in the oxidation of alkenes to epoxides. The metaloporphyrins are used as homogenous catalysts, however this method however may cause a number of problems. The catalysts

(b)(a)

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may react intermolecularly which would deactivate the catalyst, or the porphyrins may not be soluble in the solvent. These along with the similar problems associated with homogenous catalysts make a heterogeneous variant more favourable (Nakagaki et al. 2013). These problems were found to be mitigated by placing the metaloporphyrin in a MOF. By placing the metaloporphyrins within a MOF the metaloporphyrins are fixed in position with regular spacing. This eliminates the intermolecular interaction between the metaloporphyrins that cause the formation of the deactivated catalyst as well as allowing the metaloporphyrin to be fully solvent accessible.

Work was done by Brown et al. (2015) to place manganese porphyrins within a zirconium MOF. It was found that there was minimal deactivation of the catalyst while the activity of the catalyst remained high. The reactions were performed on small alkenes as this would allow the molecules to fit within the pores of the material and therefore have full access to the activation site of the manganese porphyrin.

Figure 3: Epoxation of Phenylethene using MOF-525-Mn and molecular oxygen (Brown et al. 2015)


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Non-Metal containing Ligands:These ligands may have functional groups that do not bind to the metal nodes. Similar to the MOFs mentioned before organic variants of these catalysts also benefit from the large number of catalytic sites within the material. While research has been done in this area, the methods of which the ligands may act as catalysts are limited when compared other methods. This is due to the requirements for a molecule to act as a ligand strut. Organic catalysts tend to be bulky in nature and will therefore occupy a large fraction of the internal volume that would be otherwise occupied the reactants and solvent. These limitations reduce the number of viable active sites that may be used in MOF by reducing the accessibility to the catalytic site. There is, however, documented work done by organic catalysts in MOF. The most notable work is done by Kitagawa et al. with the development with ligands that contain free amide groups. These act as stable bases for the Knoevengal condensation reaction.

The amide groups of 1,3,5-benzenetricarboxylic acid tris-N-(4-pyridyl)amide (4-BTPDA), when part of a MOF with cadmium, catalysed the reaction between benzaldehyde and malonodinitrile. It is interesting to note that in this case the homogenous version of the catalyst did not catalyse the reaction. This was due to hydrogen bonding that occurs intermolecularly between the 4-BTPDA molecules. This was one of the first instances where a molecule was unreactive in its free form but once contained with a MOF acted as an efficient catalyst (Kumar et al. 2004)

3. MOFs as Supports for Catalysts.MOFs, with their crystalline nature and high surface area, make excellent materials as supports for a wide range of secondary catalyst. This method of placing a catalyst in or onto the surface of a MOF is known as a “host-guest” network. When a homogenous catalyst is placed in a MOF it can described as the heterogenation of a homogenous catalyst. It is important to note however that this is different than the aforementioned use of a metal containing ligand as a structural component of the MOF. In this case the catalyst is located in the pores and tubular channels of the MOF.

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Similar to the work done by Brown et al. on the use of metaloporphyrins in MOFs Alkordi et al. (2008) worked on the use of encapsulated metaloporphyrins as catalysts. The metaloporphyrins were encapsulated in the pores of a MOF of the formula [In48(HImDC)96]. The pores in this MOF were large enough to house both the metal porphyrin and the reacting compound while the gaps within the surrounding structure that encapsulates the pores, were small enough for the metaloporphyrins to remain within the pores. Through the use of charged porphyrins and electrostatic techniques during the syntheses of the MOF it was possible to increase the porphyrin content of the MOF beyond 60%. By using a post-synthetic modification technique of adding the metal ion to the porphyrin after the formation of the MOF, it was possible to create a large variety of metal porphyrins from the same MOF. These metal porphyrins were found to be effective catalysts for the oxidation of alkanes, similar to that of the use as porphyrins as the ligand struts.

Figure 4: (a) Crystal structure of rho-ZMOF (left) and schematic presentation of [H2TMPyP]4+ porphyrin ring enclosed in rho-ZMOF R-cage (right, drawn to scale). (b) Cyclohexane catalytic oxidation using Mn-RTMPyP as a catalyst at 65 ◦C (Alkordi et al. 2008)

A similar method was used for the heterogenation of a cationic homogenous catalysts. Developed by Genna et al. (2013) the technique involves the cationic exchange of guest molecules in the framework of the MOF. The MOF was comprised of an anionic framework with stabilizing cations located in pores in the MOF. Upon addition of the cationic catalyst to a solution containing the host MOF a partial cationic exchange of the cationic catalysts and the indigenous cations occurred. It was found that up to 34% mol exchange occurred in the MOF (Genna et al. 2013)


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Comparing the MOF encapsulating catalysts, ZJU-28-[Rh(dppe)(COD)]BF4, to the free homogenous catalysts it was found that the two catalysts had similar reactivity and turnover rates. It was found however, that the heterogeneous variant of the catalysed could be isolated and recycled four times before there was minimal loss in reactivity. The homogenous variant could only be used one additional time, this was was with only a further 200 addition turnovers by the catalyst. This was then followed by a total breakdown of the catalytic material (Genna et al. 2013).

Figure 5: (a) cationic exchange of transition metal complex with endogenous guest cation. Hydrogenation of 1-octene to n-octane in acetone catalysed by ZJU-28- [Rh(dppe)(COD)]BF4 (circles) and homogeneous 1d (squares). Conditions: 0.5 M 1-octene in acetone, 0.0013 mmol [Rh] (Genna et al. 2013)

Methods for the deposition of a heterogeneous catalyst on to the surface of the MOF have also been developed. It was found that palladium could be deposited onto the surface of a zinc MOF via a metal-organic vapour deposition technique. Upon deposition of the palladium metal it was found that the surface are of the MOF drastically reduced from 2885m2/g to 985m2/g. Upon analysis of the catalytic activity for the catalyst, for the hydrogenation reaction, it was found to comparable to that of the industry standard of Pd/C catalyst. It is noted however, that this MOF system was unstable at room temperature (Sabo et al. 2007).

By using deposition techniques nanoclusters of pure metals can be impregnated into the pores of the MOF. This technique involves the absorption of vaporous organometallic compounds into the pores of the MOF. Once inside the pores of the MOF the precursor

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compounds breakdown to create pure metallic nanoparticles. By using this technique a number of different nanoparticles were formed within the MOF. A broad range of reactions have been attempted by using various nanoparticles, each of which have been found to useful for catalysing various reactions e.g. Ru-nanoparticles for the oxidation of benzyl alcohol to benzaldehyde (Hermes et al. 2005)

ConclusionDevelopments in the use of MOFs for catalysis are still in their infancy with the majority of papers in this field having only been published in the past five years. The main area of research is for these materials to perform efficiently under more stringent conditions such as high temperature, acidic or basic conditions or under a range of pressures. Currently under such conditions the MOF breaks down due to a weak metal node to ligand strut bond, compared to that of a covalent bond. Improvements in these aspects will then allow MOF to be used in industrial applications. With all the advances in the past few years MOF still have yet to be able to compete with zeolite equivalent catalysts. It appears the key area of catalysis where MOF will play their role is in catalysis under mild conditions.

With the wide range of possible mechanisms for catalysis by MOFs, or MOF derivatives, it appears that MOF will be able to act as viable catalysts for a whole host of reactions. The main advantage of MOFs is their activity and mechanism being finely controllable by careful selection of metal nodes and ligand supports. The number of possible configurations of ligands and metal nodes makes the number of possible MOFs appears endless. The versatility of MOFs and their capacity to act as catalysts is has been an area of academic research for the past few years but due to the limitations described above MOFs are currently only feasible for small scale industrial application under mild conditions.


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References

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AFSHARI M., S. H. CHO, HUPP J. T., GADZIKAW T., MA B., NGUEN S. T., (2007) [Bis(catechol)salen]Mn III coordination polymers as support-free hetero-geneous asymmetric catalysts for epoxidation. Eur. J. Inorg. Chem 31, 4863-4867

ALKORDI M. H., EDDAOUDI M.,EUBANK J. F., LARSEN R. W., LIU Y., (2008) Zeolite-like Metal−Organic Frameworks as Platforms for Applications: On Metalloporphyrin-Based Catalysts, J. Am. Chem. Soc. 130, 12639–12641

BROWN J. W., BOUCHARD L-S., GLÖGGLER S., JARENWATTANANON N. N., NGUYEN Q. T., OTTO T., (2015) Epoxidation of alkenes with molecular oxygen catalyzed by a manganese porphyrin-based metal–organic frame-work, Catalysis Commun., 59, 50-54.

DAS A., DASTIDAR P., KUMAR D. K., JOSE D. A., (2004) Nonpolymeric Hydro-gelators Derived from Trimesic Amides, Chem. Mater. 16, 2332.

DINCA M., HORIKE S., LONG J. R., TAMAKI K., (2008) Size-Selective Lewis-Ac-id Catalysis in a Microporous Metal-Organic Framework with Exposed Mn2+ Coordination Sites, J. Am. Chem. Soc. 130, 5854-5855

FANG Q-R., KUPPLER R. J., LI J-R., MAKAL T. A., TIMMONS D.J., YOUNG M.D., YUAN D., ZHAO D., ZHOU H-C., ZHUANG W., (2009) Potential appli-cations of metal-organic frameworks Coord. Chem. Reviews, 253,3042-3066

FINNEY N. S., GULER ML., ISHIDA T., JACOBSEN EN., PALUCKI M., POSPISIL P. J., (1998) The Mechanistic Basis For Electronic Effects On Enantioselec-tivity In the (Salen)Mn(Iii)-Catalysed Epoxidation Reaction J. Am. Chem. Soc. 120, 948-954

FRODE H., KASKEL S., HENSCHEL A., KLEMMB E., SABO M., (2007) Solution in-filtration of palladium into MOF-5: synthesis, physisorption and catalytic properties, J. Mater. Chem. 17, 3827- 3832.

GENNA D. T., MATZGER A. J., SANFORD M. S., WONG-FOY A. G., (2013) Heter-ogenization of Homogeneous Catalysts in Metal−Organic Frameworks via Cation Exchange, J. Am. Chem. Soc., 135,10586-10589

GEIDRICH K., HENSCHEL A., KASKEL S., KRAENHERT R., Catalytic properties of MIL-101, (2008) Chem. Commun.,35, 4192-4194.

HERMES S., SCHROTER MK., SCHMID R., KHODEIR L., MUHLER M., TISSLER

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A., FISCHER RW., FISCHER RA., (2005) Metal@MOF: Loading of highly porous coordination polymers host lattices by metal organic chemical va-por deposition, Angew. Chem., Int. Ed. Engl. 44, 6237 -6241

HOSKINS B.F., ROBSON R., (1989) Infinite polymeric frameworks consisting of three dimensionally linked rod-like segments, J. Am. Chem. Soc., 111 (1989) 5962-5964

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Letter from the EditorPhysics, as a discipline, attempts to answer the most fundamental questions we have about the nature of the world we live in. It stems from the profound and innate desire to pursue knowledge, order, and reason that has always been an intrinsic part of what it means to be human. Curiosity is what drives us as scientists. The Physics papers presented here are varied in theme, content and scope. Some are practical, putting forwards plausible solutions to very real problems we face today and reflecting the research interests of Trinity students. Some are more mathematically based, attempting to write the laws that govern the universe on a piece of paper. Others are more imaginative, with the authors presenting new and fresh ideas and dreaming of what might be possible in the future. It is clear however, that what each piece has in common is tangible evidence for the underlying curiosity, which fuels our quest for reason.

Nature possesses beauty on every scale – be it the stunning complexity of our universe, the nuances and subtleties of the atom, or the elegant symmetries of pure mathematics. All the authors presented here have attempted to provide us with a glimpse of this beauty on some level. The standard of submissions was exceptionally high this year and I’d like to take this opportunity to congratulate the successful candidates and thank everyone else who submitted, as well as all the academics involved in the review process. In particular, I’d like to thank Prof. Werner Blau for his advice, guidance and constant willingness to help.

People often question the relevance of basic research, and doubt the need to pump money into research facilities such as CERN’s Large Hadron Collider or NASA’s space exploration missions. There is no immediate or obvious benefit to the economy. While it’s true that conducting primary research may lead to many beneficial spin off inventions (the lightbulb, x-ray machine, and the computer to name but a few), this is never our motivation. We do not ask questions about our universe with a specific result in mind. We ask questions simply because we are curious. We ask questions simply because we want to understand more about the world around us, and that in my

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opinion, is the most noble of causes. In light of this, I cannot overstate how proud I am to be involved in a publication such as the Trinity Student Scientific Review, which provides student scientists with a platform to engage with the research-driven world of academia, possibly for the first time. I relished every second of working with my fellow committee members and academics.

I invite you now to read the successful Physics submissions. I’m sure you’ll find them enriching, intriguing and thought provoking.

Dylan ScullyPhysics EditorTrinity Student Scientific Review 2015

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Best Overall Essay

A Quantum Model of Olfactory Reception

Physics Oskar RonanJunior Sophister

The exact mechanism by which we recognise different scent molecules is one of the great mysteries of our physiology. While a complete explanation of our sense of smell may fall under the purview of biology, the fundamental physical phenomena that we are in fact detecting might be only explainable by quantum mechanics. This theory, that olfactory receptors respond not to the shape of a molecule (as is the case in the majority of biological reactions), but its vibrations, was first put forward in 1938 by Malcolm Dyson. However, a plausible mechanism for detection of these vibrations by olfactory receptors wasn’t fully developed until 1996 by Luca Turin. He postulated that the receptors in the nose acted as spectroscopes, detecting quantised atomic vibrations (phonons) by means of inelastic electron tunnelling. This vibrational theory reconciled many of the paradoxes of the traditionally accepted “lock-and-key” theory of odorant recognition, such as how different shaped molecules can smell the same, and similarly shaped molecules can have radically different scents. This has been a highly controversial theory in the olfaction community, due to the fact that many experiments designed to test it cannot conclusively prove whether or not it is correct. We would need to know the detailed atomic-scale structure of the olfactory receptor in order to confirm or dismiss

the competing theories.

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IntroductionThe exact process by which odorant receptors detect individual molecules remains to this day, unclear. Structure based theories, which argue that recognition of molecules is determined by the shape of the molecules alone (informally referred to as “lock-and-key” models) are insufficient to explain odorant detection, due to a multitude of exceptions that cannot be reconciled with the theory. Molecules of radically different structures have been shown to exhibit remarkably similar odours, and minor structural differences can change the scent of compounds completely. The fact that the bitter almond scent is shared by benzaldehyde, hydrogen cyanide and up to 70 other chemicals, and that structurally similar chemicals like different metallocenes smell completely different cannot be explained by such a theory. They also fail to account for the fact that specific functional groups within molecules can give a characteristic odour, regardless of the structure of the rest of the molecule. The best known example of this would be the unique ‘sulphurous’ scent shared by any odorants containing a thiol (SH) group. The other problem with structure-based theories is that most of them suggest that each receptor would respond to a specific molecule shape. The fact that olfactory receptors can respond to chemicals that they have never encountered before suggests that there is no way they could have evolved to identify specific molecules (Brookes et al. 2012). They are far more versatile, and can detect new chemicals that they have never been exposed to before.

The quantum mechanical based theory (Turin 1996) suggests that olfactory organs can somehow detect molecular vibrations. Turin’s theory of detecting molecular vibrations explains many of the anomalies of structure-based theories of scent.

The principle behind how these molecules are detected could be explained by inelastic electron tunnelling spectroscopy (IETS). IETS works by exploiting the quantised set of vibrational energy levels within a molecule, separated by an arbitrary energy equal to E=ħω0, (ω0 = resonant frequency of a particular vibrational mode) (Jacklevic et al. 1966, Adkins et al. 1985). In laboratory IET spectroscopes, two metal contacts are positioned, with a narrow insulating gap between them. A bias voltage is applied between


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the contacts, and as a result, the energy levels on either side of the gap are different. When the gap is empty, electrons are capable of tunnelling across the gap at a constant energy.

When a molecule (simplified in the diagram as a simple harmonic oscillator) is present in the gap, it can absorb the energy of the incident tunnelling electrons, and excite a phonon in the molecule. The electrons then tunnel to the second metal with less energy than the incident electron. IET spectroscopes are capable of scanning a range of bias voltages, and thus, can build up a complete vibrational spectrum of the molecule present in the gap (Figure 1). If the second derivative of the current measured across the junction is plotted against the bias voltage, clearly defined peaks appear as Dirac delta distributions, and show the energies of the vibrational transitions of the molecule under inspection. (Adkins et al. 1985). This can be thought of as analogous to an ohmic junction, where the resistance value changes for each transitional state.

Figure 1: (Fu et al., 2010) Schematic of basic principal of inelastic electron tunnelling spectroscopy, showing excitation of a phonon within the molecule, a V-I curve, and the resulting Dirac delta distribution pinpointing the energy of the transition.

The quantum model for olfaction posits that a form of biological IETS is responsible for the sense of smell. The theory put forward by Turin describes such a theoretical bio-spectroscope. A source and removal mechanism for electrons at well-defined energies would need to be present in the receptor; electron transfer within biology is universally accepted, acting by a series of oxidation and reduction reactions across biological molecules (Rawson et al. 2002), so this is a reasonable assumption to make in an organism. One side of the gap could have an electron donor (such as NAD(P)H), and the other, an acceptor. For the type of tunnelling described above

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to occur, an odorant with a vibrational energy matching the energy difference between the energy levels of the donor and acceptor would need to be present in the gap. Electrons could then tunnel across the gap, and reduce a part of the G-protein coupled receptor known to be part of the structure of olfactory receptors (Axel et al. 1991), in a form of biological signal transduction that is well understood (Rawson et al. 2002) (Figure 2). The receptor protein could then act as a spectrometer designed to detect a specific quantised vibration, related to this energy difference.

Figure 2: (Turin 1996) Turin’s proposed schematic for a biological IET spectroscope for olfactory reception, showing the G-protein coupled receptor, and inelastic electron tunnelling.

Is Such A Quantum Model Viable?The question is, are Turin’s ideas viable from a physics standpoint? There is evidence that correlates with Turin’s assertions, and that this could indeed be the process by which odorants are detected, but definitive evidence for the theory has yet to be established.

Obviously, metallic junctions and a range of bias voltages are absent in biology, so a biological analogue to this arrangement is


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argued. The theory simply constructs a hypothetical arrangement of a biological IETS, constructed from molecular components known to be present elsewhere in other biological systems (Rawson et al. 2002). Performing IETS with proteins would require a number of prerequisites.

A biological IETS could not scan over a range of frequencies, but would instead build up a spectrum piecewise (Turin 1996), using receptors tuned to a range of different frequencies. The scanning range is limited only by the bias voltage. As the vibrational energies of most molecular bonds found in odorants range from 0 to 0.5eV (0- 4000cm-1), typical biological voltages of 0.5V could easily sample the necessary range. A number of types of receptor would be required, each one tuned to a different range of the vibrational spectrum. This is analogous to the fact that the sense of vision uses a number of types of receptor to cover segments of the complete spectrum.

So, if a biological IET spectroscope is the method by which scent molecules are detected in organisms, the vibrational modes of the molecules, along with their partial charges will affect the perceived scent of the chemical. (Partial charges would be involved in electron scattering within the molecule, and thus alter the energies of the electrons as they tunnel across the gap.) (Turin 1996)

Recent computational work by Brookes et al. (2006) has tested the physical viability of Turin’s model. They estimated the timescales involved for IETS at that scale, to see if it could indeed occur in the same time frame that olfaction occurs. Olfaction generally occurs in the course of a few milliseconds, so the estimate breaks down olfaction into its constituent steps:

1. Time taken for electron carrier to diffuse through the cytoplasm to the donor site

2. Time taken for electron to pass to donor site

3. Time taken for electron to elastically or inelastically tunnel across the gap

4. Time taken for electron to pass from acceptor site

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The assumption was made that the electron had to diffuse through an aqueous medium, and that the odorant is already present in the receptor site. Using a standard method for computing diffusion of material through a solution and the Stokes-Einstein relation for the diffusion coefficient, diffusion time was estimated to be:

where η is the viscosity of the fluid, nX is the concentration of X, T is the temperature (Kelvin), & kB is the Boltzmann constant. Substituting values typical of biological systems into this equation yielded a value for diffusion time to be in the range from 0.01-1ms.The values for electron transport were assumed to be typical values of electron transport rates in other biological systems, ranging from 0.001-1ms for each one.

For approximating the rate at which electrons tunnel across the gap, Fermi’s Golden Rule was used:

where Ψi is the eigenfunction of the initial eigenstate, Ψf is the eigenfunction of the final eigenstate, & P is the density of final states. Equations for the complete system, described by the Hamiltonian Ĥ based on a quantum oscillator, were derived by Brooks et al. (2006)

Again, values typical of biological systems were chosen for the parameters of their derived equation, and the results gave values that indicate elastic tunnelling times (τT0)≈100ns, and inelastic tunnelling times (τT1)≈0.1ns. The fact that τT1<< τT0 indicates that inelastic electron tunnelling could indeed be a viable mechanism for the detection of odour molecules. This validation of the physics behind the theory only proves that it is plausible, as the total time for all of these processes to occur could theoretically be within the order of milliseconds required (Brookes et al. 2006).

If Turin’s theory is correct, then certain predictions can be made based on the model. Firstly, it is possible to alter the vibrational modes of molecules without altering the chemistry by


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altering the mass using stable isotopes of the constituent atoms in the molecules. Theoretically, the physics behind the model can be explored directly through this method. Also, we should be able to use predicted vibrational spectra of molecules to predict their smell. If these criteria cannot be observed experimentally, Turin’s model cannot be the case.

Experiments were conducted in 2004 to determine the effect of isotopes in the scent of a molecule (Keller et al. 2004). They conducted a double-blind smell test, comparing acetophenone, and its deuterated counterpart acetophenone-d8. The vibrational spectra of acetophenone and acetophenone-d8 are noticeably different, and vibrational olfaction theory predicts that they should smell very different. They concluded from the experiment that the theory had no basis in scientific fact, as none of the test subjects were able to distinguish between the original molecule and the deuterated molecule.

However, the experiment was repeated in 2013 to confirm the results (Gane et al. 2013). Turin was involved in the conduction of this experiment, and they also found that subjects could not discriminate between acetophenone and acetophenone-d8. However, they conducted a second set of experiments using deuterated versions of much larger molecules, noteably musks such as cyclopentadecanone. These molecules contained up to three times as many hydrogen atoms to deuterate, and subjects were able to detect a noticeable difference between the deuterated and undeuterated musks. Other experiments on human subjects have been carried out to examine if there is a noticeable smell difference between different isotopes of benzaldehyde (Fortin et al. 2001), which there were. Clearly there is a discrepancy in the theory if humans only perceive this change in vibrational energies in certain cases.

Further experimental evidence that isotopes can be detected by scent alone has been carried out using common fruit flies (Drosophila melanogaster) as the subjects (Franco et al. 2011). The drosophila were trained to have an aversion to either acetophenone, or acetophenone-d8, and were placed in a t-shaped maze with a sample of one of the molecules at each end. The drosophila were able to discriminate between acetophenone and acetophenone-d8,

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where humans could not. Furthermore, the drosophila trained in this experiment to avoid the deuterated compound were able to detect deuterium in other, structurally unrelated molecules. This provides strong evidence for the vibrational theory of olfaction.

The theory also postulates that the smell of chemicals can be predicted from their vibrational spectra, despite their structural characteristics. An example of this is the distinct ambergris note shared by the following collection of molecules:

Figure 3: A selection of molecules of different structural form, yet all share the same “ambergris note” in their smell.

When the spectral data were examined by Turin’s CHYPRE algorithm (Turin 1996), their vibrational spectra agree with the similarity of their smell (Figure 4).

Figure 4: Convolved vibrational spectra of the molecules shown in Figure 3. The similarity of the spectra may be the explanation as to why they all smell of ambergris. (Turin, 1996)


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Further evidence supporting the theory comes from work done by Flexitral Inc. (of which Turin was CTO.)(Turin 2004). In an attempt to find a molecule that had the same scent as coumarin (1-benzopyran-2-one), a new molecule with an identical scent was discovered. Using the vibrational spectrum of coumarin, a molecule with an almost identical spectrum was theorised, and subsequently produced. This was benzo[4,5]thieno[3,2-b]pyran-2-one, or ‘Tonkene’®; this molecule smells identical to coumarin (Figure 5). While accurately determining a molecule’s scent from its theorised vibrational spectra is not conclusive evidence for the theory, it strongly suggests that vibrational energies are involved in some way in how smell is detected.

Figure 5: Coumarin, a sweet-smelling, warm, tobacco-like odorant; Tonkene®, a molecule theorised by its vibrational spectrum to have the same scent as Coumarin. Despite structural differences, both molecules smell identical.

Many antagonists of the vibrational theory state that it cannot reconcile the fact that many enantiomers smell different. The classic example of the carvone molecule is often debated. S-carvone smells of caraway, while R-carvone is the flavourant in spearmint confectionary (Figure 6). Both molecules obviously have identical vibrational spectra, but smell nothing like each other. A simple model of vibrational olfaction cannot explain this (Dyson 1938).

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Figure 6: The two enantiomers of carvone (2-methyl-5-(1-methylethenyl)-2-cyclohexenone), smell nothing like each other, despite having obviously identical vibrational spectra.

Turin has attempted to explain this experimentally, by stating that due to the different geometry of the enantiomers, some of the vibrational modes are not being detected by the olfactory receptors. In the specific example of carvone, he has postulated that it is the carbonyl stretch (C=O stretch) that is going unnoticed by the receptor, due to the molecule sitting in the wrong orientation. To prove his theory, he suggested that if one were to “add” an additional carbonyl stretch frequency (at ħω0=0.223eV) to R-carvone by smelling another molecule with the C=O stretch frequency simultaneously, one could recover the caraway scent of S-carvone. In fact, a mixture of R-carvone and simple ketones (like pentanone) is indistinguishable from S-carvone (Turin 1996).

ConclusionsTurin’s model of a quantum mechanical based method for odour recognition is interesting, and is gaining more traction in the scientific community as experimental evidence mounts in support of it. Combining this theory with our understanding of biological signal transduction from nasal receptors could lead to a far more complete understanding of human olfaction than has ever been compiled previously. The exact interaction of the odorants with the


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receptors is yet to be fully explored by biologists, and more study is still needed in the fields of physiology and neurobiology to solve this problem; however the underlying physics of the detection method seems to be sound (Brookes et al. 2012).

There is also a noticeable flaw in the model, which is also present in structure-based theories of olfactory recognition; neither theory can fully explain why the scent of some molecules are concentration dependant. This is a problem well known to perfumers, and yet unexplained (Gross-Isseroff et al. 1988).

Though still not fully proven, biological mechanisms based on quantum mechanics are gaining more and more attention, and could encourage further research into the trans-discipline field of quantum biology. (Al-Khalili & McFadden 2014).

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ReferencesADKINS, C.J.; PHILLIPS, W.A. “Inelastic Electron Tunnelling Spectroscopy” (1985) J.

Phys. C: Solid State Phys., 18 pp1313-1346.

AL-KHALILI, J. & MCFADDEN, J. (2014) “Life on the Edge: The Coming of Age of Quantum Biology” Bantam Press. Ealing.

AXEL, R; BUCK, L. (1991) “A NOVEL MULTIGENE FAMILY MAY ENCODE ODORANT RECEPTORS: A MOLECULAR BASIS FOR odor recognition” Cell. 65. pp175-187

BROOKES, J.C.; HARTOUTSIOU, F.; HORSFIELD, A.P.; STONEHAM, A.M. (2006) “Could Humans Recognise Odor by Phonon-assisted Tunnelling?” Phys. Rev. Lett. 98, pp038101-

BROOKES, J.C.; HORSFIELD, A.P.; STONEHAM, A.M. (2012) “The Swipe Card Model of Odorant Recognition” Sensors., 12 pp15709-15749

DYSON, M.G. (1938) “The Scientific Basis of Odour” Chem. Ind. 57 pp647-651

FORTIN, J.; HAFFENDEN, L.J.W.; YAYLAYAN, V.A. (2001)“Investigation of vibrational theory of olfaction with variously labelled benzaldehydes” Food Chemistry. 73 pp67-72

FRANCO, M.I.; MERSHIN, A.; SKOULAKIS, E.M.C.; TURIN, L. (2011) “Molecular vibration-sensing component in Drosophila melanogaster olfaction” Proc. Nat. Acad. Sci. 108 pp3797-3802

FU, Q.; LUO, Y.; YANG, J.; HOU, J. (2010)“Understanding the concept of randomness in inelastic electron tunnelling excitations” [image] Phys. Chem. Chem. Phys. 12 pp12014

GANE, S.; GEORGANAKIS, D.; MANIATI, K.; RAGOUSSIS, N.; SKOULAKIS, E.M.C.; TURIN, L.; VAMVAKIAS, M. (2013) “Molecular Vibration-Sensing Component in Human Olfaction” PLOS ONE. 8 e55780

GROSS-ISSEROFF, R.; LANCET, D. (1988) “Concentration-dependent changes of perceived odour quality” Chem. Senses. 13 pp191-204.

JAKLEVIC, R.C.; LAMBE, J. (1966)“Molecular vibration spectra by electron tunnelling” Phys. Rev. Lett. 17, pp1139-1140

KELLER, A.; VOSSHALL, L.B. (2004) “A Psychophysical Test of the Vibration Theory of Olfaction” Nature Neuroscience. 7 pp337-338

RAWSON, N.E.; GOMEZ, M.G. (2002)“Cell and Molecular biology of human olfaction” Microsc. Res. Tech. 58, pp142-151.

TURIN, L. (1996) “A Spectroscopic Mechanism for Primary Olfactory Reception.” Chem. Sens. 21. pp773-791.

Turin, L. “Fragrance compositions comprising benzo[4,5]thieno[3,2-b]pyran-2-one” U.S. Patent 10/552,459, April 8, 2004

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Best Physics Essay

Graphene-Sulfur Composites:A Novel Method in Developing

Advanced Lithium-Sulfur Batteries

Nanoscience, Physics and Chemistry of Advanced Materials

Andrew SelkirkJunior Sophister

Graphene-based sulfur composites are currently the subject of a great deal of scientific study, due to their ability to significantly improve the performance and efficiency of Lithium-Sulfur battery systems. This article reviews the recent advances made in the field, with some discussion on the future and practicality of the development of these new composites. There are many potential applications to these composite battery systems, including more efficient large-scale energy storage, longer-lasting rechargeable electronic devices, and the improved proposition for electrical vehicles as a popular mode of transport. It is thought that many of the issues that Li-S battery systems currently have, including the high resistivity of sulfur and the loss of polysulfides into the electrolyte, can be improved upon or solved using nanomaterials such as graphene. Many different methods have been explored in an attempt to integrate graphene into the sulfur cathode. These include the creation of sulfur-graphene nanosheets; the development of a functionalized graphene-sheet sulfur (FGSS) nanocomposites, the use of graphene oxide, and the integration

of graphene into MOF derived nanoporous carbon.

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IntroductionIn recent times, scientific interest has grown considerably in the area of energy storage, in particular large-scale rechargeable storage systems that can be utilized over an extended period of time. These systems, if developed sufficiently, will enable major advances in areas such as renewable energy distribution, and practical electric vehicular transport (Bruce et al. 2008). One such rechargeable system that has garnered recent attention is the lithium-sulfur (Li-S) battery. This is due to the fact that elemental sulfur has the highest theoretical energy capacity (the amount of electrical charge it can deliver at a specific voltage) (1672 mA h g-1), and power density (2600 W h kg-1) of all discovered cathodes (Wang et al. 2011). In addition to this, sulfur is naturally abundant and would therefore be a highly cost-effective battery material, in addition to it being non-toxic and environmentally friendly.

Despite these major advantages, however, there are a number of challenges in Li-S batteries that must be overcome before any functional, long-term prototype can be developed. These include the high resistivity of elemental sulfur (which hinders ion transport between the electrodes and reduces the battery capacity), and the high solubility of polysulfide ions that are discharged into the electrolyte as the cathode is cycled (charged/discharged) (Cao et al. 2011). This loss of sulfur ultimately leads to a gradual decrease in the battery capacity, as less sulfur atoms are available for the electrochemical reaction. The tendency for the sulfur component to change volume as it is converted to polysulfides is also a major issue, as this leads to the gradual damage of the cathode from repeated expansion and contraction (Ji et al. 2011). These problems lead to a decrease in initial battery capacity and efficiency, as cycling progresses.

Much research over the past few decades has been conducted in an attempt to address these problems, in particular through the integration of sulfur into chemical structures known as composites. These are essentially materials that are comprised of two or more constituent materials that when combined, yield a novel material with different chemical and physical properties to its components. Such research includes the investigation of conductive polymers in


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improving the conductivity of sulfur composites (Liang et al. 2009, Ji et al. 2009, Lai et al. 2009). As well as this, significant progress has been made recently in the use of sulfur-carbon composites as the cathode in Li-S batteries, in an attempt to reduce the dissolution of polysulfides (Liang et al. 2009).

Previously, research into other types of electrochemical storage systems, including lithium-ion batteries (Yoo et al. 2008, Wang et al. 2009, Wang et al. 2010) has shown that graphene has the correct chemical properties to improve the lifetime and quality of certain battery systems. This is due to its high conductivity, chemical resilience, large surface area (approx. 2600 m2 g-1) and wide-ranging electrochemical window (Choucair et al. 2009).

In the case of lithium-sulfur batteries, a number of novel methods have been approached in recent years in an attempt to integrate graphene into the structure of the sulfur cathode, in the form of a composite. Current research and study into this area is of huge interest to many areas of the scientific community, due to the potential applications of such systems. The question should therefore be raised as to how successful these methods have been at overcoming some of the fundamental issues of Li-S batteries. A number of these recent methods will now be analyzed in detail.

The use of Sulfur-Graphene Nanosheets (S-GNS)One particular strategy at integrating the useful properties of both graphene and sulfur is the creation of what are known as Sulfur-Graphene Nanosheets (S-GNS). These consist of elemental sulfur that has been physically mixed into graphene nanosheets (GNS), and heated to allow the infiltration of sulfur into the graphene layers. Wang et al. (2010) successfully synthesized a S-GNS composite, using a graphene-sulfur weight ratio of 1:1.5. Using various methods of spectroscopic analysis, such as Raman spectrum (fig. 1), it was found that the sulfur particles coated the graphene in a uniform fashion, which is ideal for ensuring that the sulfur has as much exposure to the graphene interface as possible, and maximum electrical conductivity is achieved throughout the cathode. This

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was confirmed by SEM images (fig. 2(c)), which show that the sulfur particles had melted, and had evenly distributed throughout the GNS.

During this experiment, a number of performance tests were conducted on the resulting S-GNS cathode, in comparison to a conventional cathode of pure sulfur. One of these tests involved the measurement of the cycling stability of the cathode. This is defined as a measure of the ability of a battery to retain a fraction of its initial capacity over a number of charge-discharge cycles. When the measurements were conducted (using a lithium anode, with a constant current density of 50 mA g -1), it was found that the S-GNS nanocomposite retained a higher level of capacity as the cycles progressed (see fig. 3) (Wang et al. 2010). It was also noted that the initial capacity of the S-GNS electrode was significantly higher than

Figure 2: SEM imagery of pure sulfur (a), the GNS (b), and the S-GNS composite (c). It can be observed that the solid structure of pure sulfur has entirely disappeared in the S-GNS composite, indicating excellent dispersion. (Wang, J. Z. et al 2010).

Figure 1: Raman Spectrum of S-GNS composite. No characteristic peak for sulfur was observed (normally found at 259, 412 cm-1 (White, S. N., 2008)), indicating that the sulfur was in a highly dispersed state throughout the composite (Wang, J. Z. et al 2010).


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that for pure sulfur (1611 mA h g-1 for S-GNS in comparison to 1100 mA h g-1 for pure sulfur), indicating greatly improved conductivity. It can therefore be concluded from these results that the S-GNS nanocomposite leads to a significant performance and lifetime improvement, when compared to a bare sulfur electrode.

Improvement of GNS capacity through development of Functionalized Graphene Sheet-Sulfur (FGSS) nanocompositeThe experimental results obtained using a S-GNS nanocomposite are clearly a major improvement on the conventional Li-S battery type. However, another experimental report (Cao et al. 2011) notes that the sulfur loading (i.e. the amount of sulfur that is successfully absorbed into the composite) is extremely low, at approximately 17-22 wt% (Wang et al. 2010). It also notes that there is a large decrease in the capacity of the S-GNS battery as the charge/discharge cycles progress (approx. 1100 to 600 mA h g-1 in 40 cycles, see fig. 3), which is partially due to the dissolution of polysulfides into the electrolyte. It is therefore clear that a more efficient method to integrate sulfur into the composite should be explored, and to better protect the polysulfides from dissolving into the electrolyte.

One particular type of composite that has shown improvement in these areas is a functionalized graphene sheets-sulfur nanocomposite (FGSS) (Cao et al. 2011). This consists of sheets and stacks of functionalized graphene (graphene with various oxide

Figure 3: Capacity of pure sulfur, S-GNS battery systems vs. cycle number. It can be observed that the S-GNS composite had a higher initial capacity than the pure sulphur, and maintained better cycling stability. (Wang, J. Z. et al 2010)

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groups attached) (Schniepp et al. 2006, Wang et al. 2011), with interlocking layers of sulfur atoms. This is commonly referred to as a sandwich-type layout (see fig. 4). The major advantage of this arrangement is that the sulfur loading is much higher, and the distribution of sulfur nanoparticles is more uniform. An experiment by Cao et al. (2011) analyzes the effectiveness of this arrangement, and finds that although the performance of the battery is improved, there is still significant migration of polysulfides into the electrolyte.

Interestingly, in order to solve this issue of polysulfide loss the FGSS composite was also coated with Nafion®, a highly conductive polymer which is intended to act as a protective film to hinder polysulfide dissolution. This is shown to have a significant effect on the capacity and cycling stability of the battery system (see fig. 5). It was found that the uncoated FGSS electrode had 52% of its initial capacity remaining after 50 cycles. On the other hand, the Nafion coated FGSS electrode retained 79.4% of its original capacity after 50 cycles, and approx. 74.3% after 100 cycles (Cao et al. 2011) (at constant rate of 0.1 C, see fig. 5). In addition to the Nafion acting as a protective barrier against polysulfide dissolution between

Figure 5: Graph of cycle performance of pure FGSS, and with Nafion coating. Current kept at 0.1 C. The improvement capacity and cycling stability when the Nafion coating is added can be observed (Cao, y. et al, 2011).

Figure 4: TEM images of Nafion-coated FGSS nanocomposite, at scales of 100nm (c) and 50nm (d). The sandwich structure of interlocking dark (sulphur) and bright (graphene) lines can clearly be seen (Cao, y. et al 2011).


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the graphene sheets, this is also due to its ability to act as a cation exchange material. This arises due to the polytetraflouroethylene component of the polymer, which allows the Li+ cations to enter the cathode, while simultaneously preventing the polysulfides from leaving due to static repulsion. This particular use of a graphene-based composite, in combination with the protective polymer coating, yields significant improvement in battery performance and capacity. It should be noted that when compared to the previous method (Section 2), the FGSS design is a far better candidate for integration into a practical battery system. The multilayered structure is far more efficient at sulfur loading, and performs significantly better at protecting the dissolution of polysulfides.

Use of Oxides of Graphene as Polysulfide TrapsAs mentioned previously, finding a method to hinder the escape of polysulfides from the cathode is of extreme importance in the attempt to develop an efficient Li-S battery system, with minimal capacity fading. One such method that has been attempted makes use of graphene oxide (GO) as a sulfur immobilizer (Ji et al. 2011).

The basic idea behind this concept is that the hydroxyl groups (and various other oxygen-based functional groups that are present on the graphene oxide) positively contribute to the chemical bond between the carbon and sulfur atoms, which significantly hinders any polysulfide discharge from the cathode into the electrolyte. It has been observed previously that the GO composite also has the capability to withstand the volume changes that occur as sulfur is converted into the polysulfides, and as it reverses back to elemental sulfur upon recharging of the battery (Lai et al. 2009, Chen et al. 2011). The expansion and contraction of the sulfur can lead to the disintegration of the composite as cycling progresses. The larger surface area of the graphene oxide (due to the functional groups) also provides a well-established electron transfer network between the ions, facilitating better conductivity. In an experiment conducted by Ji et al. (2011), a graphene oxide-sulfur (GO-S) nanocomposite is synthesized, through the

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use of chemical deposition to deposit sulfur atoms onto layers of 2D graphene oxide. The resulting composite is then heat treated to remove any excess sulfur atoms that were not attached to the composite. A notable advantage in using this method of synthesis is that it is cheap and environmentally friendly, which enables a higher suitability for mass production.

The cycling efficiency of the composite was experimentally tested as a cathode of an electrode system, with a lithium anode. It was found that after 50 cycles (at a constant rate of 0.1 C), the capacity of the system remained constant at approximately 954 mA h g-1. This consistency indicates that there is very little dissolution of polysulfides occurring in the system, and even surpasses the levels of polysulfide protection achieved in method 3. The Coulumbic efficiency (the efficiency by which charges are transferred during an electrochemical reaction) was measured to be around 96.7% (Ji et al. 2011). This indicates that the cathode is highly stable as the reversible electrochemical reactions progress with each cycle (see fig. 6) These results show that using functional groups as immobilizers in the GO-S composite is an effective way of confining sulfur to the conducting matrix, and hindering any dissolution into the electrolyte.

Figure 6: Graph depicting the cycle performance at constant rate of 0.1 C. Can be seen that the capacity remains near initial levels, even during the final cycles (Ji, L. et al 2011).


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Hybridization of Graphene and Microporous Carbon-based Metal Organic Frameworks (MOFs)As of recently, another promising method that has been developed for the containment of polysulfides is a process that involves the use of Metal Organic Frameworks (MOFs). These porous, crystalline structures are composed of coordination bonds between metal ions, and multidentate organic ligands (Nature publishing group, 2014). Under the correct conditions, they are able act as a template in the formation of nanoporous carbon, which supports sulfur diffusion throughout its structure (Xi et al. 2013).

This nanoporous carbon structure, derived from a MOF, is highly suited for containing polysulfides. This is due to the small pore sizes of the carbon matrix that trap the sulfur atoms by adsorption onto the surface. This allows the cycling stability of the system to be maintained at a high level for an extended period of time. Previous scientific study has also found that variation of the pore size of carbon-based MOFs affects their electrochemical suitability as a cathode in a Li-S battery. Smaller pore sizes (<2 nm) are found to improve the cycle stability of the cathode, while larger pore sizes (2-50 nm) cause an increase in initial discharge capacity (Xi et al. 2013). Carbon-based MOFs also have a large surface area, which greatly encourages efficient ion transfer during the electrochemical reaction.

It has been found, however, that the capacity of the MOF derived microporous carbon is quite low. This is due to the fact that there are many grain boundaries and interfaces throughout the composite (Chen et al. 2014). These lead to poor electrical conductivity, as there are many possible electron scattering centers that impede the efficient transition of ions throughout the cathode.

One particular study has taken a novel approach to addressing this problem (Chen et al. 2014). Due to the low resistivity of graphene, it can be used as a structural intermediate to increase the electrical conductivity between each component of the composite. In this study, a composite known as graphene sheet-sulfur/zeolithic imidazolate framework-8 derived carbon (GS-S/

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CZIF8-D) was synthesized. This consists of a carbon host that is derived from a MOF known as Zeolitic Imidazole Framework-8 (ZIF-8). The carbon host is then infused with sulfur, to produce a composite. The resulting composite is then wrapped with graphene sheets (see figure 8 for illustrated production process). Due to the high flexibility of graphene, it easily wraps around the S/CZIF8-D. This ensures that the improved conductive effects are well applied throughout the composite (see fig 9 for SEM image) (Chen et al. 2014).

Figure 8: SEM image of graphene-wrapped MOF derived microporous carbon framework. Due to the flexibility of graphene, it can easily wrap around the GS-S/CZIF8-D nanocomposite. (Chen, R. et al, 2014)

Figure 7: Synthesis process for GS-S/CZIF8-D nanocomposite (Chen, R. et al, 2014)


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The resulting composite was performance tested as a cathode in a Li-S cell, in comparison to the same composite without the graphene wrapping. It was found that the initial discharge capacity of GS-S/CZIF8-D was significantly higher than that of the S/CZIF8-D composite (1171 mA h g-1 vs. 982 mA h g-1 respectively). This initial discharge value is also significantly higher than any of the previous methods (2-4). The S/CZIF8-D composite was also found to have both a lower capacity and cycling stability than that of the graphene wrapped composite over 120 cycles (see figure 10) (Chen et al. 2014). This improvement is due to the increase in charge-transfer speed, due to the highly conductive graphene sheet network. It should also be noted from these results that when compared to the previous methods, this is the most efficient composite system. The improvements made in this experiment, through the use of graphene as a conductive intermediate, clearly indicate that such a structure is a very promising design for a rechargeable Li-S electrode system.

Figure Cycling performance of GS- S/CZIF8-D composite compared to the S/CZIF8-D composite. Can be observed that the capacity retention is significantly improved for the GS- S/CZIF8-D composite compared to the S/CZIF8-D. (Chen, R. et al 2014).

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ConclusionsIt can be seen from the experimental results above that the integration of graphene-based composites into Li-S batteries leads to a significant improvement in performance and efficiency, when compared to conventional Li-S designs. The varieties of novel approaches that have been developed, which incorporate graphene into their composite structure, indicates how important the nanomaterial has become in the development of composite battery systems. It should also be noted, however, that the sheer variety of experimental methods highlights how the integration of 2D nanomaterials such as graphene into these systems is still highly experimental. Therefore, assuming current research continues at a similar pace, even more efficient composite systems will be produced in the very near future, which will quickly surpass the achievements of these recent developments.

In terms of the production of practical applications using these composite batteries, these are still a number of years away. This is due to the rapidly changing and purely experimental nature of current developments, both in graphene synthesis and Li-S composite battery design. One issue that will likely arise is how these methods will be industrialized for mass production, in particular the current issue of the large-scale manufacturing of advanced nanomaterials (Kim et al. 2009). However, as noted previously the fast pace of current research in the area indicates that it may not be long before Li-S batteries become an attractive alternative to conventional battery systems already in use, and could one day revolutionize many important energy systems of the modern world.


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Enceladus: Plume Composition and Interactions in the

Saturnian System

Physics and AstrophysicsJessica ErkalJunior Sophister

Enceladus is Saturn’s sixth largest moon and orbits at a distance of approximately 4 Saturn radii. The recently discovered plume of the South Polar Region ejects vast amounts of material into the E ring and its own torus which have been shown to have a significant effect on the entire Saturnian system. The general composition, structure and other features of the plume is discussed in this review. Also, Enceladus’ interactions with Saturn’s magnetosphere and the effect on E ring will be

discussed.

Introduction“The discovery of a tenuous atmosphere and a plume of water, carbon dioxide, methane, and possibly nitrogen erupting from warm and tectonically and volcanically active regions near Enceladus’ south polar terrain confirms that Enceladus ranks alongside Io (and possibly Triton) as one of the few currently geodynamically active satellites in the solar system.” (Barr & McKinnon 2007).

A small, icy satellite of Saturn, known as Enceladus, orbits at a distance of 3.95 RSat (where RSat is one Saturn radius = 60,268km). Enceladus orbits Saturn at a speed of 12.6 km/s and is accompanied by a plasma torus corotating with Saturn at a speed of 39 km/s. This positioning allows the corotating plasma flow to remain unaffected by solar wind. (Kivelson 2006, Jia et al. 2010). The Cassini spacecraft

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made three close flybys to Enceladus in 2005 and an additional four in 2008 to observe the Saturnian system (Khurana et al. 2007, Jia et al. 2010). Observations from Cassini (CAPS, CDA, CIRS, INMS and UVIS instruments are used) suggested that Enceladus was geologically active (Barr & McKinnon 2007). Enceladus is accompanied by a plasma torus which is mostly composed of water group ions (Jia et al. 2010).

Figure 1: Schematic diagram of Saturn’s magnetosphere with Enceladus and other moons shown. The plasma torus and E ring are also shown (Sittler et al. 1983).

The outermost E ring of Saturn is dominated by water vapour and ice particles. It is suspected that the tiny moon, of radius 250 km, is the source of these particles and is considered to be a major replenishing source of the E ring (Postberg et al. 2007). In 2005, large water vapour plumes were discovered in Enceladus’ south polar region (Matson et al. 2006, Hansen et al. 2011). The Cassini flybys confirmed the existence of such plumes and also observed that such a plume is expelling approximately 1028 gas particles per second into the inner Saturnian system. These particles become ionised and join the plasma disk of Saturn’s magnetosphere (Jia et al. 2010).

The Saturnian system is complex and intriguing. There is a vast range of interactions between Saturn’s magnetosphere and its moons: “from the addition of significant quantities of gas, dust and plasma causing significant consequences for the dynamics and energetics of the entire Saturnian magnetosphere, to the simple


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absorption of plasma and energetic particles by the icy moons” (Jia et al. 2010). Also, it has long been suspected that the E ring is replenished by the material ejected from Enceladus’ plume. Evidence for this comes from the higher particle density that has been observed at and near Enceladus than in any other region of the E ring (Postberg et al. 2007).

The aim of this review is to establish and discuss the relationship between Enceladus’ plume and the Saturnian system. This will be done via two parts: one explaining the general structure, composition and other features of the plume, the other discussing the interactions between Enceladus and Saturn.

Composition and Structure of Enceladus’ Plume and Some Implications for Enceladus’ Internal StructureVarious models and simulations have been used to investigate the composition and structure of the plume at Enceladus’ south pole. It is believed that this plume is centred at Enceladus’ south pole with a latitudinal distribution of 10o ~ 15o (Jia et al. 2010). From Cassini’s Infrared Spectrometer (CIRS), observations of the ‘tiger stripe’ fissures of Enceladus’ south pole led to surface temperature estimates of 170 K with gas thermal velocities greater than 450 m/s (Hansen et al. 2011). However other sources (Cassini CIRS instruments) have shown different observed temperatures, as shown below in Figure 2.

Figure 2: Schematic of observed temperatures on Enceladus with a view of the south polar region. As mentioned above, some sources estimate a higher temperature than here. (Enceladus Reveals Geological Activity, 2006).

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It has been observed that during Enceladus’ slightly eccentric orbit of 1.37 Earth days, gravitational variations, due to Saturn’s gravitational pull, can affect the amount of material ejected into the Saturnian system (Hedman et al. 2013). This supports the idea that Saturn’s tidal and gravitational forces affect the activity at Enceladus’ south pole, even if the exact mechanism is still unclear (Barr & McKinnon 2007). There has been much debate about the source of heat for this region of the icy moon, and also about the origin of the plume and how this affects the interior structure and composition of Enceladus (Halevy & Stewart 2008).

Tidal dissipation at Enceladus should be distributed as a function of longitude and latitude. It is well known that the plume is centred near the south pole of Enceladus. This may be due to a large density perturbation in the ice shell or rocky interior that may have caused the moon to ‘roll over’ so this area settled onto the rotation axis. It is possible that tidal dissipation is a cause of density perturbations, or that some thermal, compositional or mechanical anomaly is present in Enceladus’ ice shell to localize the heating to the south pole. The source of heat for the plume and volcanism of Enceladus is believed to be tidal dissipation which is maintained by the 2:1 mean motion resonance with Dione (Barr & McKinnon 2007). This means that for every one orbit of Dione, Enceladus orbits Saturn twice.

Burger et al. (2007) discuss the shape of the Enceladus plume and further evidence for the southern polar region as the plume’s location. The Cassini INMS and UVIS instruments made observations of the water vapour of the plume, finding that the source is nonuniform. A nonuniform source would be expected to be asymmetric which is what is observed for the Enceladus water vapour plume. However, the peak is symmetric about the south pole, displaying a maximum density here indicating the location of the source.


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Figure 3: (a) shows the shape of Enceladus’ plume and contour lines showing the radius of particles found at each line. (b) shows the percentage of salt-rich ice as a function of logarithmic altitude above the south pole. (Postberg et. al. 2011).

The inner magnetosphere (near the region of the E ring) contains a neutral cloud of H2O, OH and O and H molecules which are believe to originate from Enceladus’ water vapour plume (Khurana et al. 2007). Data recorded in 2005 by the Cassini INMS instruments revealed that the plume is composed of H2O (91 ± 3%), N2 or CO (4 ± 1%), CO2 (3.2 ± 0.6%), CH4 (1.6 ± 0.4%) with possible trace amounts of ammonia, acetylene, hydrogen cyanide and propane (Matson et al. 2006). The vast quantities of H2O being ejected by the plume, and as found throughout Saturn’s E ring, lead to debate about the origin of this water. As one may suspect, it is difficult to erupt liquid water onto (or past) the surface of an icy satellite, due to the fact that liquid water has a higher density than solid ice. However, the thickening of the ice shell may increase the pressure in a subsurface ocean. This excess pressure may not be sufficient enough to allow the eruption of liquid water on larger satellites, such as Europa, but on a small moon such as Enceladus, ocean pressure can increase enough to cause an eruption of liquid water. These volume and pressure changes may be caused by coupled orbital and thermal evolution of the moon’s interior or secular cooling (Manga & Wang 2007). Evidence for a subsurface ocean at Enceladus comes from Cassini’s CDA observations of grains in the plume containing sodium salts.

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This is consistent with an ocean at Enceladus being in contact with its rocky core (Hansen et al. 2011).

The most abundant species observed at or near Enceladus and the E ring is H2O with smaller amounts of other species mentioned above. Cassini’s observations allow for an estimate of velocities of these particles to be calculated. With Cassini at a distance of 175km from the surface of Enceladus and water detected at least 1000km from the surface, an estimated velocity of 225 m/s is found. This is slightly less than the escape velocity of 250 m/s for a body the size of Enceladus. It then seems obvious how a large portion of the ejected water particles can escape fully from Enceladus and end up in Saturn’s E ring (Matson et al. 2006, Burger et al. 2007).

Matson et al. (2006) conclude that the composition of Enceladus’ plume indicates the production of N2 below the icy surface as a derivative of ammonia at a temperature above the melting point of water. This indicates that Enceladus’ interior is composed of a “rich, warm, aqueous, organic ‘soup’” which may imply that the environment is favourable for the production of other organic components such as methanol or amino acids, this however is beyond the scope of this review.

Interactions in the Saturnian SystemIt is the effect on the magnetic field which indicates the presence of the plume (Kivelson 2006). Cassini observations indicate that the plasma torus emanating from Enceladus spreads to fill the Saturnian magnetosphere. The magnetosphere of Saturn is strongly dominated by neutral water group atoms, with a magnetic field strength of 330 nT at Enceladus’ orbit (Bagenal 2007, Jia et al. 2010). Saturn’s magnetodisk is unique and is between that of Earth and Jupiter. The magnetodisk is bowl-shaped and bent upward from the equator toward ecliptic north and seasonally tilts toward the Sun (Gombosi & Ingersoll 2010).

Jia et al. (2010) applied a 3D magnetohydrodynamics (MHD) model to examine the flow around a body that is simpler than Enceladus and then by adding processes to occur simultaneously,


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a relatively accurate model of Enceladus is simulated. It was found that the plume expels approximately 1028 gas particles per second into the Saturnian system. Enceladus was found to be the main mass-loading source in the Saturnian magnetosphere and the main source for E ring replenishment.

Kivelson (2006) finds that the ejected particles affect local properties of Saturn’s inner magnetosphere. As gas particles are ejected from Enceladus’ plume, they become ionized and are added to the plasma disk of Saturn’s magnetosphere. The particles may undergo one of three types of ionization: photoionization, electron impact ionization or ion-neutral charge exchange. The combination of these processes changes to mass and momentum of the plasma flow and so the effect is considered as mass loading to the Saturnian plasma (Khurana et al. 2007, Jia et al. 2010).

Jia et al. (2010) use a ‘self-consistent’ MHD model to examine the steady state interaction between the torus flow, Enceladus and its plume with the aim of creating a 3D plasma environment of Enceladus to compare with magnetometer data. It was found that nearer to the surface of Enceladus, mass-loading is less efficient. This effect is seen between the surface at the south pole and a radius of 2.8 Enceladus radii. There are a number or possibilities which could cause this: where neutral density is high inside the neutral jets, hot electrons may be depleted and so less electron impact ionization occurs; the ejecta may not be fully vaporized or; the model used here cannot fully predict the density concentration in thin jets closer than 2.8 Enceladus radii. It was found that approximately half of the loss of momentum in the plasma can be contributed to the charge exchange between torus ions and neutrals while this exchange between the neutrals and newly added ions account for the rest of the momentum loss. It was concluded in the paper by Jia et al. (2010) that the ionization processes result in a 50% increase of ion density in the mass-loading region of the plasma torus and also that the charge exchange effect contributes to 90% of magnetic field perturbations in the Saturnian system. It is also found that a global circulation of plasma is set up around Enceladus’ orbit as follows: incoming flow with a radial force balance is slowed after and interaction with neutrals from the plume. After this interaction there is a decrease

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in centrifugal force and a mostly unaltered inward magnetic stress which leads to the plasma ‘falling’ inward toward Saturn.

Khurana et al. (2007) suggest that the newly formed ions are added to the magnetospheric plasma flow and extract momentum from the flow. This slowing of the plasma creates thermal and pressure gradients and curvature in the magnetic field which can change the direction of the flow to be diverted around the mass-loading region. By using magnetic field data from the Cassini flybys, it was found that the total mass picked up by the plasma within 5 Enceladus radii is <3 kg/s, with additional pick up most likely occurring in the neutral torus outward of the orbital location of Enceladus.

ConclusionsWhile the paper by Manga & Wang (2007) assumes a subsurface ocean exists at Enceladus, more recent findings and observations now give conclusive evidence for a subsurface ocean. With this in mind, their assumption that there is an ocean beneath the icy shell of Enceladus is valid and allows us to make definite conclusions about the mechanisms for eruptions of liquid water at Enceladus.

All papers discussed above show strong evidence for a plume at Enceladus which ejects primarily H2O into the E ring of Saturn. Various models, such as Monte Carlo and magnetohydronamics models, are used to investigate the effect the plume has on the Saturnian system.

It can be concluded that the particles ejected from the plume are the main source for replenishment of the E ring of Saturn. Evidence for this comes from the observation of water vapour and H2O particles throughout the E ring, but also in higher densities nearest to Enceladus. This water vapour plume has produced a unique environment for these interactions which involve various states of matter (solid ice, liquid water, gaseous material etc.). This system is coupled with the Saturnian magnetosphere by bending the magnetic field, causing magnetic perturbations.


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From Cassini’s observations and the results of various simulations it is concluded that Enceladus is a unique and intriguing satellite in the solar system with a huge effect on the Saturnian system.

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JIA, Y.-D., C. T. RUSSELL, K. K. KHURANA, G. TOTH, J. S. LEISNER, AND T. I. GOMBOSI (2010a), Interaction of Saturn’s magnetosphere and its moons: 1. Interaction between corotating plasma and standard obstacles, J. Geophys. Res., 115, A04214, doi:10.1029/2009JA014630.

JIA, Y.-D., RUSSELL, C. T. KHURANA, K. K.MA, Y. J.NAJIB, D.GOMBOSI, T. I., (2010) Interaction of Saturn’s magnetosphere and its moons: 2. Shape of the Enceladus plume, Journal of Geophysical Research: Space Physics, J. Geophys. Res., 115, A4, 2156-2202, http://dx.doi.org/10.1029/2009JA014873, 10.1029/2009JA014873, A04215

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MetamaterialsFuture of Cloaking

Theoretical Physics Katarzyna Siewierska

Junior Sophister

Metamaterials are one of the most exciting research areas in physics. The current knowledge and technology allows us to design, simulate and con-struct artificial materials that exhibit properties not found in any natural material. In the field of metamaterials, the freedom of design can allow sci-entists to construct materials that only existed in science fiction books. The work of the two pioneers, Victor Veselago and John Pendry, laid down the theoretical framework for the construction of the first negative refractive index materials. Such materials can manipulate electromagnetic radiation in new ways, giving rise to invisibility cloaks. Soon scientists went further and began pondering the possibility of designing metamaterials to cloak objects from thermal, acoustic, seismic and even matter waves. As a result, many interesting designs and properties have been discovered, although not all have yet been tested experimentally. The future of metamaterials is a fascinating one. Their applications will revolutionise our world. Scientists

are constantly exploring new designs and applications.

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Introduction

Metamaterials are the frontier of nanotechnology. These artificial ma-terials can be specially designed to have many exotic properties. Neg-ative refractive index metamaterials (NIMs) are just one class of ma-terials in the general family of metamaterials. These are impossible tofind in nature and have the potential to be used to fabricate invisibil-ity cloaks. This review paper will focus mainly on the development,applications and future of NIMs. The two pioneers that ignited thecuriosity driven research into metamaterials, NIMs in particular, areViktor Veselago and Sir John Pendry. In 1967 Veselago published apaper in Russian entitled ”The Electrodynamics of Substances withSimultaneously Negative Values of ε and µ”. It was translated intoEnglish in 1968. He hypothesised that a material can possess a nega-tive index of refraction if two parameters, electric permittivity (ε) andmagnetic permeability (µ) are both negative. These two parametersare determined by the electrical and magnetic properties of substances.Figure 1 gives a visual summary of the ranges of values of ε and µ as-sociated with different naturally occurring materials.

Figure 1: Summary of the ranges of values of ε and µ associated with differentnaturally occurring materials.

3Veselago investigated the properties of materials with ε < 0 and µ < 0in great detail. He provided the theoretical basis and the physicalrealisation of these materials.

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The theory begins with an equation for the refractive index n

n2 = εµ (1)

From equation 1, it may seem that the simultaneous change of sign ofboth ε and µ has no effect, but Veselago goes on further to show thatsetting ε < 0 and µ < 0 will make the material behave in ways onlydescribed in Science Fiction. To do this Veselago turns to equationswhere ε and µ appear independently. Now consider two of the famousMaxwell equations in matter, with no free charges and currents

∇× E = −1

c

∂B

∂t(2)

∇×H =1

c

∂D

∂t(3)

whereB = µH (4)

D = εE (5)

Now apply these to the simplest case of a monochromatic plane wave,propagating through an isotropic, homogeneous medium, with electricand magnetic components given by

E(ω,k) = E0e(ik·r−iωt) (6)

H(ω,k) = H0e(ik·r−iωt) (7)

where ω is the angular frequency and k is the wave vector. The aboveequations yield

k× E =ω

cµH (8)

k×H = −ω

cεE (9)

From equations 8 and 9, it is clear that for ε > 0 and µ > 0 the threevector quantities E, H and k form a right handed set, where for ε < 0

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and µ < 0 the vectors form a left handed set.Now consider the Poynting vector S defined as

S =c

4πE×H (10)

It follows from equation 10 that in right-handed materials, S and k areparallel and for left-handed materials S and k are antiparallel. Since kpoints in the direction of the phase velocity vp of the wave, it followsthat in left-handed materials the direction of vp is opposite to thedirection of S, the energy flux.

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Figure 2: a)Illustration of negative refraction at the interface of positive and nega-tive refractive index materials. Used from (Skullsinthestars,2009).b)Diagram demonstrating refraction in left- and right-handed materials.Used from (Harshpaul 2011).

Figure 2(a) illustrates how waves behave at the interface of right-handed and left-handed media. Veselago goes on to derive the form ofSnell’s law for two media with opposite handedness, using the diagramshown in Figure 2(b)

sinφ

sinψ=

n2

n1=

p2p1

√ε1µ1

ε2µ2(11)

For a right handed medium, p = 1 and for a left handed mediump = −1, hence in the case where medium 1 is right handed and medium2 is left handed, it follows that p2

p1= −1 and that medium 2, which is

left handed with ε < 0 and µ < 0, has a negative index of refraction.(Veselago 1968)

Veselago predicted that NIMs will have many interesting propertiessuch as reversed Doppler effect. From then on the challenge was todesign the dream material. The first recipes were proposed by JohnPendry (Pendry et al. 1996, Pendry et al. 1999). These were found tohave many new properties, including negative refractive index (Shelby2001). The applications of NIMs include perfect lenses (Pendry 2000)and invisibility cloaking (Pendry et al. 2006). The concepts of cloakingin electromagnetism can be applied to different areas. These materi-als can have many real-world applications that could transform ourlives.(Kadic et al. 2013)

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Figure 2: a)Illustration of negative refraction at the interface of positive and nega-tive refractive index materials. Used from (Skullsinthestars,2009).b)Diagram demonstrating refraction in left- and right-handed materials.Used from (Harshpaul 2011).

Figure 2(a) illustrates how waves behave at the interface of right-handed and left-handed media. Veselago goes on to derive the form ofSnell’s law for two media with opposite handedness, using the diagramshown in Figure 2(b)

sinφ

sinψ=

n2

n1=

p2p1

√ε1µ1

ε2µ2(11)

For a right handed medium, p = 1 and for a left handed mediump = −1, hence in the case where medium 1 is right handed and medium2 is left handed, it follows that p2

p1= −1 and that medium 2, which is

left handed with ε < 0 and µ < 0, has a negative index of refraction.(Veselago 1968)

Veselago predicted that NIMs will have many interesting propertiessuch as reversed Doppler effect. From then on the challenge was todesign the dream material. The first recipes were proposed by JohnPendry (Pendry et al. 1996, Pendry et al. 1999). These were found tohave many new properties, including negative refractive index (Shelby2001). The applications of NIMs include perfect lenses (Pendry 2000)and invisibility cloaking (Pendry et al. 2006). The concepts of cloakingin electromagnetism can be applied to different areas. These materi-als can have many real-world applications that could transform ourlives.(Kadic et al. 2013)

7Engineering materials with ε < 0 and µ < 0

The flexibility of design in metamaterials allows for the constructionof components, with a size smaller than the wavelength of radiation.Collectively, these produce artificial responses which results in a mate-rial with new properties. NIMs are constructed such that the artificialelectric and magnetic responses yield negative permittivity and per-meability. Pendry et al. (1996) described a structure that consistsof infinitely long and very thin wires arranged in a simple cubic 3Dlattice structure. In this structure the average electron density is re-duced and hence the effective electron mass is greater, because of theself-inductance of the structure. The plasma frequency is depressedinto the far infrared, which has been confirmed by simulations. Thecalculations show that the system can be described using the Drude-Lorentz model (Jackson 1999), hence the effective relative permittivityis given by

εr,eff(ω) = 1−ω2p,eff

ω(ω + iγeff)(12)

with the effective plasma frequency and effective damping factor givenby

ω2p,eff =

2πc2

a2 ln(ar )(13)

γeff ≈ 0.1ωp,eff (14)

where a is the lattice constant and r is the wire radius. From equation12, it follows that ω < ω2

p,eff implies εr,eff < 0.

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Pendry et al. (1999) proposed structures with effective permeabilitythat can be tuned to values not found in natural materials. One suchstructure is a split ring resonator (SRR), composed of two concen-tric split metallic rings,e.g. copper, with their openings positioned asshown in Figure 3.

Figure 3: The split ring resonator structure that defines c,d and r. Used from(wikipedia, 2012).

The SRR is equivalent to an LC circuit. The magnetic flux passingthrough the SRR induces a strong current that generates a magneticmoment, in accordance with Faraday’s law. If this magnetic response issufficiently strong, it is possible to achieve a negative effective relativepermeability µr,eff . The expression for µr,eff is given by

µr,eff(ω) = 1− Fω2

ω2 − ω20 + iΓω

(15)

where F is the filling ratio of the SRR

F =πr2

a2(16)

ω0 is the resonant frequency

ω0 =

√1

LC=

√3dc20π2r3

(17)

and Γ is the damping term

Γ =2

rσµ0(18)

9where r, a and d are defined by Figure 3, c0 is the speed of light invacuum, σ is electrical conductivity of the metal and µ0 permeabilityof free space (Pendry et al. 1999).

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When the two kinds of meta-structures, one with εr,eff < 0 and theother with µr,eff < 0 in the same frequency range, are combined, it ispossible to construct a material with neff < 0. Smith and colleaguesconstructed the first metamaterial composed of a periodic arrangementof long wires and SRRs as described above. This material exhibitednegative ε and µ simultaneously for a narrow frequency range (Smithet al. 2000). Shelby, Smith and Schultz (2001) also constructed similarstructures that exhibited a negative refractive index from 10.2 to 10.8GHz. The image of their metamaterial structure and results are shownin Figure 4. Smith et al. (2002) reported on another material composedof SRRs and wires that exhibited a negative refractive index in thefrequency region from 8.5 to 9.0 GHz.

Figure 4: Components of a negative refractive index metamaterial. These consist ofcopper SRRs and copper wire strips an a fibre glass circuit board materialassembled in an interlocking lattice. Each unit cell has dimension of5mm. Used from (Johnson 2008).

After the achievements in the field of metamaterials and their possibleapplications, interest in this field grew rapidly. Now the goal was tomanufacture materials that can couple to light at microwave and op-tical frequencies. Isotropic NIMs that can manipulate light at higherfrequencies, with a broadband response and low energy losses requiredmuch smaller components and different material designs. Such mate-rials could have many interesting applications such as perfect lensing,invisibility cloaking and many more. In this review paper, the focus is

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on cloaking using negative refractive index materials. ( Pendry 2000,Pendry 2004, Pendry et al. 2006, Liu 2010).

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Cloaking with Electromagnetic NegativeRefractive Index Materials

Figure 5: a) Two dimensional cross section of the cloaking system showing the di-rection of rays inside and outside the metamaterial. Rays are divertedaway from the cloaked circular area and emerge undisturbed from theiroriginal course, just as if it propagated through free space. Used from(Dodson 2013).b) A three dimensional view of the process described in a).Used from (Sambles).

Pendry et al. (2006) proposed an approach for designing electromag-netic cloaks, based on transformation optics. Figure 5 shows how aspherical shell constructed from a negative refractive index metamate-rial can alter the electromagnetic fields. The light rays come in and aredeflected smoothly around the object and emerge in the same directionjust as it would if it passed through empty space. No radiation entersor leaves the cloaked volume. Hence any object placed into the vol-ume in the middle will appear invisible to the outside observer. Thiscloak does not depend on the shape or size of the object, as long as itfits inside the cloaked volume. The limitation in this case is that thiscloaking can only be fully effective for one frequency.

This approach was then used by Schurig et al. (2006), who provided thefirst experimental proof of cloaking. The cloak was constructed frommetamaterials that operated most effectively at 8.5 GHz. The materialconsists of copper SRRs in rectangular unit cells. These SRRs were

13

specially designed to exhibit negative ε and µ. The metamaterial’soverall structure is shown in Figure 6. The object being cloaked was acopper cylinder. The results of the simulations and measurements arepresented in Figure 7.

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Figure 6: The structure of the two dimensional cylindrical cloaking structure. Theshape of SRRs varies gradually in the structure at every cylinder. Usedfrom (Gbur 2011).

Figure 7: Diagrams of steady-state electric field pattern with stream lines(a - c) indicating the direction of the Poynting vector. The cloak lies inthe region between the two concentric circles. Inside the smaller circle liesa copper cylinder. The right-hand scale shows the instantaneous intensity.a)Simulation of the cloak with exact material properties. b)Simulation ofthe cloak with reduced material properties. c)Experimental measurementof the copper cylinder. d)Experimental measurement of the cloaked cylin-der.Used from (Schurig 2006).

While this design gives excellent results, it is only valid for a very nar-row frequency band. Also, this design is not practical for engineeringof optical metamaterials.

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Valentine et al.(2009) reported on the first demonstration of opticalcloaking using a carpet cloak. This material consists of isotropic di-electric materials, which allows it to operate for a broad frequency bandand produce low-loss invisibility for a wavelength range of 1400-1800nm. The carpet cloak is formed by etching holes in a silicon slab usinga focused ion beam (FIB). The carpet cloak was used to transform amirror with a bump, into a virtually flat mirror. Any object placed be-hind the bump will be invisible because the reflected beam will appearto have bounced off a flat mirror surface. The structure of the carpetcloak is show in Figure 8. The results show that a beam reflected off aperfectly flat surface has a gaussian intensity profile. The light hittingthe mirror with a bump scatters and the profile has three lobes. Thelight reflected off the cloaked bumped surface has a profile very similarto the gaussian profile of a perfect mirror.

Figure 8: a) Diagram of the structure of the carpet cloak. The triangular region(C2) has a uniform hole pattern and serves as a background mediumwith constant effective refractive index. The rectangular region C1 has avariable index profile. b) SEM image of the structure of the carpet cloak.Used from (Yaris 2009).

The work on creating better metamaterials and cloaks, operating ateven higher optical frequencies is on-going. The results so far havebeen very promising. In the future we can expect the making of bulkmetamaterials on much larger scales. We will not only have invisibil-ity cloaks, but also perfect lenses, perfectly absorbing materials, etc.These materials and many more will transform the world.

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Beyond Electromagnetic Cloaking

The concepts of cloaking in electromagnetism can be applied to otherareas of physics to give rise to thermodynamic, acoustic, elastody-namic and, in principle, matter-wave cloaking metamaterials. Thesematerials could have scientific and real world applications and henceit is an important topic of research. Principles analogous to those oftransformation optics can be used to produce cloaking devices that willmanipulate waves in matter, e.g. thermal, acoustic, seismic, etc. Sincematter itself is a wave, theory suggests that in principle, matter cloak-ing is possible. This last section will briefly outline the work done onthe theory and design of such metamaterial cloaks and their possibleapplications in that future.(Kadic 2013)

Figure 9: Principle of cloaking. a)A simple square grid. b)After applying an ap-propriate coordinate transformation, a distorted square grid with a holein the middle is obtained. Used from (Leonhardt 2011).

Thermal, acoustic and seismic cloaking are based around a similarprinciple. In thermal cloaking, the grids in Figures 9 (a) and 9 (b)are composed of thermal and electrical resistors. Conductivity locallyvaries radially and azimuthally, however a measurement of heat fluxtaken by an observer outside of the region marked with the blue cir-cle would show that the two constructions indistinguishable. Hencean object placed inside the region marked with the red circle can becloaked. Using this principle Schittny et al. (2013) constructed andcharacterised the first thermal cloak. Certainly such cloaks could have

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many applications in technology and industry. In acoustic cloaking,the grids in Figures 9 (a) and 9 (b) are composed of springs. Theidea is then exactly the same as with the thermal cloaks, just nowthe springs represent the bulk modulus. Once again the bulk modulusvaries radially and azimuthally, however outside the blue circle line thetwo constructions are indistinguishable because phase velocity of thesound wave measured outside the blue circle will be the same for bothconstructions. The design for a 2D acoustic cloak was first proposed byCummer & Schurig (2007), but 3D model was also proposed (Chen &Chan 2007). Acoustic metamaterials could be used to create a seismicshadow zone. In this scheme, the seismic waves could be converted tosound and heat (Kim & Das 2013). A seismic cloak constructed fromisotropic heterogeneous thin plates, that could deflect seismic wavesaround buildings has also been investigated(Farhat et al. 2009). Theobvious application of seismic cloaks is to shield buildings from earth-quakes, which would save peoples’ lives and protect buildings fromdamage. Another possible application could be to deflect shock wavesin car accidents, which could also save people from serious damage anddeath. One type of cloaking that has not yet made a definite transitionfrom science fiction into science fact is the quantum-mechanical matterwave cloaking. If an object is cloaked by, say an optical cloak, one wayof finding it would be to throw an object in its direction and watchhow it scatters. In principle, once could cloak themselves from the pro-jectile by generating a special potential-energy distribution. Howeversuch a cloak could only work if the projectile had a particular incidenceenergy(Kadic et al. 2013). However Greenleaf et al.(2008) proposed away of approximate quantum cloaking using a potential. If a potentialis surrounded by an approximate cloak, it would be unaltered and un-detectable to particles. At some energies, matter waves could becometrapped in the cloak and this could have applications in beam switchesand magnetically tunable ion traps.

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Conclusion

Since Veselago gave his mathematical description of negative refractiveindex, the scientific world had to wait almost thirty years for Pendryand others to come along and turn metamaterials into the exciting areaof research it is today. At the beginning of the new millennium, thetheoretical work of Veselago and Pendry was verified experimentallyby Smith and others. Since then many more metamaterials with exoticproperties were designed and some were constructed. Invisibility cloaksmade the transition from science fiction to science fact. Possible ap-plications in the real world will transform our society and technology.Electromagnetic cloaks can smoothly guide radiation around objects.Acoustic and seismic cloaks could protect houses from the damages ofthe powerful shock waves produced by earthquakes. Thermal cloakscould shield objects from heat. The metamaterial world is just waitingto be further explored.

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Implications of a 125 GeV Higgs Boson for the Minimal and Next-

To Minimal Supersymmetric Standard Model

Physics and Astrophysics Brían Ó Fearraigh

Junior Sophister

In March 2013, CERN tentatively announced the discovery of a mass ≈ 125 GeV/c2 Higgs boson. While the discovery of the Higgs boson is a great triumph for the Standard Model of particle physics, no supersymmetric particles were found, and the mass of 125 GeV has left many physicists at a crossroads as to whether or not Supersymmetry is a valid extension of the Standard Model. The implications of this discovered mass for the Minimal and Next-To-Minimal Supersymmetric Standard Model are investigated. It was found that the MSSM was heavily constrained, although can still effectively describe the 125 GeV mass, whereas the NMSSM is much less restricted and appears to be the most viable candidate as an extension to the Standard

Model.

IntroductionThe Standard Model (SM) of particle physics is a theory which classifies all currently known subatomic particles and attempts to combine the electromagnetic, weak and strong nuclear forces. It has gained continuous credence due to the discoveries of the top quarks, tau neutrino and the most recent Higgs boson. The Higgs boson of the SM is an excitation of the Higgs field, a fundamental field explaining why some elementary particles have mass; in particular why photons and gluons are massless and why the W and Z bosons (elementary particles mediating the weak nuclear force) are heavy. One problem

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arising from this proposal is known as the hierarchy problem, the discrepancy between the weak and gravitational force; namely why the weak force is 32 orders-of-magnitude larger than gravity and why the Higgs boson is much, much lighter than the Planck mass. As of yet, the best-motivated solution to this problem is Supersymmetry (SUSY), an extension to the SM which proposes a superpartner for every elementary particle - that is, for every fermion there is a superpartner boson with the same mass and internal quantum numbers excluding spin, which differs by a half-integer; and similarly for bosons (Nilles 1984). Note the superpartner of a particle is named by placing the prefix ‘s’ before the particle name. SUSY results in mass cancellations from both fermionic and bosonic particles in the calculation of quantum corrections to the Higgs mass squared. Thus far, the variation of SUSY which has appeared to be most promising is the Minimal Supersymmetric Standard Model (MSSM), wherein two Higgs doublet fields (the minimum number needed) are required to break electroweak symmetry and for radiative corrections. However, with the announcement of a mass 125.36 ± 0.37 (stat) ± 0.18 (syst) GeV (/c2) Higgs boson (CMS Collaboration 2012, ATLAS Collaboration 2014) found at CERN (via the ATLAS and CMS detectors) through the use of the Large Hadron Collider (LHC), the MSSM has been under heavy scrutiny as of late, and as shall be discussed, this has resulted in severe constraining of the model. The Higgs mass discovery has strongly hinted at new dynamics beyond the MSSM, especially the Next-to-Minimal Supersymmetrical Model (NMSSM) which will be shown to have less constraint placed upon it. Both models will be compared and contrasted throughout the course of this review.

Implications for the MSSMMuch work has gone into re-defining the MSSM due to the 125 GeV mass of the Higgs boson discovered. Note that all papers cited have utilised programmes such as Fermilab, FeynHiggs, etc to re-create the MSSM under the constraint of this mass (which can all be easily found online are included in the relevant cited papers). All equation


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manipulation is carried out through these various programmes; however it is useful to define the formula for the one-loop Higgs mass: one-loop Higgs mass:

m2h = m2

Zc22β +

3m4t

4π2v2

(log

(M2

S

m2t

)+

X2t

M2S

(1− X2

t

12M2S

))

where

1. mh is the Higgs boson mass.

2. c22β = cos2(2β), where tan(β) is the ratio of the vacuum expectationvalues (the average value of an operator in a vacuum) of the Higgsdoublet .

3. mt is the top quark mass.

4. Xt is the stop mixing parameter, equal to At − µcot(β), A is the tri-linear coupling and µ is the SUSY mass parameter which gives thesupersymmetric fermionic partner of the Higgs boson its mass.

5. MS = (mt1mt2)1/2 where mt is the top squark mass.

(please see Draper et al. (2012) for the equation above).

There is much general agreement that, due to the 125 GeV of Higgs bosonmass, many of the parameters in this equation of the MSSM are severelyconstrained, notably tan(β), A and the mass of the stop quark (Draper et al.2012 ; Heinemeyer, Stal & Weiglein 2012 ; Ellwanger 2012 ). As argued byDraper et al. (2012), a lower bound is placed on the value of tan(β) of 3.5,while a mild lower bound is placed on MS when the ratio Xt/MS is takeninto account. See Figure 1 for an example of the parameter constraints inaccordance with the Higgs mass (Draper et al. 2012). The lowest value theA-term and MS can take is the modulus of Xt = ≥ 1000 GeV and MS ≥ 500GeV. Light-scalar restrictions limits the A-term to at least 1 TeV. In orderfor SUSY to hold, large A-terms and heavy scalars are necessary to complywith the 125 Higgs mass. Also, (quark) mixing was allowed for, and as agreedby Hall & Pinner (2012) and Kadastik et al. (2012), maximal-stop mixingwas shown to be necessary. This avoids the less-probable multi- TeV stops(Hall & Pinner 2012). Overall, the MSSM is shown to be heavily constrained.Draper et al. (2012) also notes that were some assumptions relaxed, such asallowing for the NMSSM, the results ”could change”.

3

There is much general agreement that, due to the 125 GeV of Higgs boson mass, many of the parameters in this equation of the MSSM are severely constrained, notably tan(β), A and the mass of the stop quark (Draper et al. 2012, Ellwanger 2012, Heinemeyer, Stal & Weiglein 2012). As argued by Draper et al. (2012), a lower bound is placed on the value of tan(β) of 3.5, while a mild lower bound is placed on MS when the ratio Xt/MS is taken into account. See Figure 1 for an example of the parameter constraints in accordance with the Higgs mass (Draper et al. 2012). The lowest value the A-term and MS can take is the modulus of Xt = ≥ 1000 GeV and MS ≥ 500 GeV. Light-scalar restrictions limits the A-term to at least 1 TeV. In order for SUSY to hold, large A-terms and heavy scalars are necessary to comply with the 125 Higgs mass. Also, (quark) mixing was allowed for, and as agreed by Hall & Pinner (2012) and Kadastik et al. (2012), maximal-stop mixing was shown to be necessary. This avoids the less-probable multi-TeV stops (Hall, Pinner & Ruderman 2012). Overall, the MSSM is shown to be heavily constrained. Draper et

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al. (2012) also notes that were some assumptions relaxed, such as allowing for the NMSSM, the results could change.

The constrained parameters for the MSSM are also investigated by Heinemeyer, Stal & Weiglein (2012), using the FeynHiggs and HiggsBounds code. In this case the lower limit of tan(β) was found to be 3.2, lower than the 3.5 value calculated by Draper et al. (2012). A high-sensitivity to tan(β) was found, resulting in a narrowly allowed region for this parameter under the new Higgs mass. The type of Higgs boson discovered at CERN is seemingly that of the SM, a pure scalar of spin 0, with no ’exotic’ properties of a SUSY-like Higgs boson being found (CMS Collaboration 2013 ). The type of Higgs boson discovered, and the likelihood of it being of a certain type, is proposed by Heinemeyer, Stal & Weiglein (2012). Interpreting the discovery as a CP-odd Higgs (CP - charge parity) boson is highly disfavoured. A significant parameter space of the MSSM is found to be compatible with a light CP-even Higgs boson, albeit a heavier CP-even boson could exist allowing for low MA (pseudoscalar Higgs boson mass) and moderate values of tan(β). Although admitting constraints, Heinemeyer, Stal & Weiglein (2012) encourage the MSSM. Carina et al. (2012) agree that some MSSM parameters are severely constrained, and that the results obtained at the LHC concerning photon decay rates corresponds to CP-even Higgs mixing.

Certain constrained MSSM theories are found to be disfavoured by Arbey et al. (2012), such as the minimal anomaly and gauge-mediated SUSY-breaking models (two variations of the MSSM which require specific stop energies, and which predict too-light a Higgs boson mass). Also ruled out in the paper is the non-mixing scenario, predicting a mass of only 123 GeV. Typical mixing scenarios only allow very high tan(β) and MS parameters. In general, many types of the MSSM are taken to be disfavourable, yet gravity- mediated constrained MSSM is still viable with large A and heavy scalar top quarks. This is in contrast to the seemingly much-available parameter space for a 125 GeV Higgs described by Heinemeyer, Stal & Weiglein (2012).


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With regards to neutrino-oscillations, an area unincorporated into the SM alone but which SUSY does attempt to explain, Fukuyama & Okada (2002) studied (solar) neutrino oscillation data and attempted to explain it using a subsection of MSSM, the SO(10) model. A more general case of CP-violation phases was examined, unlike the case where CP is not violated which does not agree with observations. It was found that a heavily constrained parameter region of the MSSM cannot be excluded from agreement with experimental data in predicting the neutrino mass matrix, however all parameters except one were fixed and tan(β) was set to 45, a very high value. Again this tan(β) was shown to be very large in order for the MSSM to fit with current knowledge, as mentioned by previous sources.

The same SO(10) model is investigated by Gogoladze & Shafi (2012), who conclude that perfect t − b − τ Yukawa unification is possible for a Higgs boson of mass 122 - 124 GeV with an uncertainty of ± 3 GeV. However, tan(β) is - as usual - constrained to a high value of 45 - 47.

Conversely, the BABAR Collaboration (2012) produce findings for which the MSSM does not seem to hold. In considering the decay ratios of authors show that the expected results cannot be explained by the presence of a type II charged Higgs doublet, for which the MSSM is a specific case. Thus far, the mass result of the Higgs boson has severely limited the MSSM as a complete extension to the SM.

Implications for the NMSSMThe Next-to-Minimal SM has an extended (scale-invarient) superpotential compared to the MSSM, with a generalised μ-term (Maniatis 2010). Investigations into the NMSSM indicate it suffers far less parameter-constraint than its counterpart. Maniatis (2010) has concluded that the Higgs boson mass of 125 GeV complies with the NMSSM model without much limitation, resulting in less fine-tuning than the MSSM at low values of tan(β). See Figure 2 for the visualisation of the larger parameter-space available in the NMSSM compared to the MSSM. Figure taken from (Bastero-Gil et al. 2000,

phases was examined, unlike the case where CP is not violated which does notagree with observations. It was found that a heavily constrained parameterregion of the MSSM cannot be excluded from agreement with experimentaldata in predicting the neutrino mass matrix, however all parameters exceptone were fixed and tan(β) was set to 45, a very high value. Again thistan(β) was shown to be very large in order for the MSSM to fit with currentknowledge, as mentioned by previous sources.

The same SO(10) model is investigated by Gogoladze & Shafi (2012),who conclude that perfect t− b− τ Yukawa unification is possible for a Higgsboson of mass 122 - 124 GeV with an uncertainty of ± 3 GeV. However,tan(β) is - as usual - constrained to a high value of 45 - 47.

Conversely, Lees J.P. et al. (2012) produce findings for which the MSSMdoes not seem to hold. In considering the decay ratios of B → D∗τ vτ , theauthors show that the expected results cannot be explained by the presenceof a type II charged Higgs doublet, for which the MSSM is a specific case.Thus far, the mass result of the Higgs boson has severely limited the MSSMas a complete extension to the SM.

Implications for the NMSSM

The Next-to-Minimal SM has an extended (scale-invarient) superpotentialcompared to the MSSM, with a generalised µ-term (Maniatis 2010). Inves-tigations into the NMSSM indicate it suffers far less parameter-constraintthan its counterpart. Maniatis (2010) has concluded that the Higgs bosonmass of 125 GeV complies with the NMSSM model without much limitation,resulting in less fine-tuning than the MSSM at low values of tan(β). SeeFigure 2 for the visualisation of the larger parameter-space available in theNMSSM compared to the MSSM (Bastero-Gil et al. 2000 ; Maniatis 2010).Bastero-Gil et al. (2000) also agree that the NMSSM is less restricted thanthe MSSM.

Ellwanger (2012) outlines the main differences of this model in comparisonto Minimal SUSY, with NMSSM giving rise to 3-neutral CP-even Higgs par-ticles. It is concluded that the best way to validate this model is to retrievedata from the LHC which can be explained by NMSSM rather than MSSM.

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Maniatis 2010). Bastero-Gil et al. (2000) also agree that the NMSSM is less restricted than the MSSM.

Ellwanger (2012) outlines the main differences of this model in comparison to Minimal SUSY, with NMSSM giving rise to 3-neutral CP-even Higgs particles. It is concluded that the best way to validate this model is to retrieve data from the LHC which can be explained by NMSSM rather than MSSM. Such findings would include enhanced signal rates in the γγ channels, and the discovery of sparticles of mass which “turn out to be incompatible with the necessarily large radiative corrections to the Higgs mass in the MSSM” and the discovery of the lighter CP-even Higgs boson (Ellwanger 2012, p. 7). The fine-tuning necessary for the NMSSM in order to fit with the 125 GeV mass of the Higgs boson was found the be less straining than the MSSM, with 5-10% fine-tuning needed if the mediation scale is low and stop mixing non- maximal (Hall, Pinner & Ruderman 2012). The lack of need for maximal-stop mixing is in stark contrast to all data gathered from simulations of the Minimal SUSY model.

It is noted by Cao et al. (2012, p.1) that the large At and top squark mass values of the MSSM are “much ameliorated” in the NMSSM. This is in much agreement with King, Mühlleitner, & Nevzorov (2012, p. 28), who investigate the parameters of the model and find that a 125 GeV Higgs is allowed for all mixing below 1 TeV and all stop quark masses, and conclude that “the NMSSM appears to be the best-compromise between naturalness and minimality that

Figure 1: Contour plot of mh in tan(β) vs. Xt/MS plane. The stops were set at mQ = mU = 2 TeV, and the result is only weakly dependent on the stop mass up to ~ 5 TeV. The solid curve is mh = 125 GeV with mt = 173.2 GeV. The band around the curve corresponds to mh = 123-127 GeV. Finally the dashed lines correspond to varying mt from 172-174. The absent dashed contour at left does not exist (light tops and Xi < 0 cannot accommodate a 125 GeV Higgs with 2 TeV stop masses).


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Figure 2: Scatter plot of fine-tuning in (5.35) as a function of the lightest physical Higgs-boson for tan(β) = 3, left, and tan(β) = 5, right. In this scan there are fixed values of mO = 100 GeV, At(ΛG) = 0, M1(ΛG) = M2(ΛG) = 500 GeV, µ = λvS < 0 and varied parameters Mỹ(ΛG), mHd(ΛG) and mHu(ΛG). The dark shaded regions show the fine-tuning values corresponding to the NMSSM and the brighter shaded regions corresponding to the MSSM. The LEP limit on the SM Higgs-boson with mSM <108 mass is also shaded. Figure take from [189].H

can account for a 125 GeV SM-like Higgs boson”. Finally, Gunion & Jiang (2012) also state that a Higgs mass 126 GeV is easily obtained for the parameter space available.

ConclusionsAfter extensive analysis of the topic, it has been shown that the NMSSM is a less-restricted model describing the finding of a Higgs mass of 125 GeV. While the MSSM has been shown to accurately describe the results, albeit it severely constrained, the NMSSM is far less-limited in terms of available parameter space. For example, tan(β) need not be so high in order to incorporate a SM-like Higgs boson. The data indicates towards new dynamics beyond the MSSM, and at the moment the NMSSM appears to be the best candidate as an extension to the Standard Model.

Before the announcement of the Higgs discovery, the MSSM was considered the best model to explain SUSY. More research and computational effort should be directed towards the NMSSM as of now, and physicists and those with a scientific interest alike wait for March 2015 for the second running of the LHC, for which collisions of up to 13 TeV are planned. Hopefully new results will be obtained which will make clear which version of SUSY is correct, if any, because we are currently at a crossroads between the concept of SUSY and a multiverse in our explanation of the physical world.

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Standard Model reviewed’, Int.J.Mod.Phys., A25, pp. 3505 - 3602

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