Research Features - Issue 104

ISSN 2399-1542 ISSUE 104

NIAID: DR ANTHONY FAUCI

CAHRU: CHILD HEALTH

Features 6 PROFESSOR ROBERTResearch WINSTON

Dr Anthony Fauci, the Director and man in charge, spoke to Research Features about NIAID’s recent research successes, outlining where science currently stands on preventing infectious disease onset worldwide.

Dr Jo Inchley, the Assistant Director, discusses CAHRU’s collaboration with WHO, highlighting the partnership’s impact on child and adolescent health research, particularly within the HBSC study.

The leading fertility expert at Genesis Research Trust updates us on the organisation’s current research advances and shares his views on maternal health.

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WELCOME

This Issue

TO ISSUE 104

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urious, committed and conscientious. The researchers we spoke to this issue have diverse backgrounds but they are all joined by their commitment to improving the lives of others.

Whether that is by developing new technologies, exploring the power of nutrition, or developing novel statistical methods, either at the bench or in the business sphere, these researchers are working hard to answer fundamental questions about our health.

Dr Anthony Fauci, Director of NIAID, made time in his busy schedule to discuss his career, outline the recent successes of the agency and look forward to the preparations being made for possible future epidemics.

We were delighted to hear from Dr Jo Inchley, Assistant Director of CAHRU, on the key areas in child and adolescent health. Professor Robert Winston also contributed his views on infant and maternal health as the leading fertility expert at Genesis Research Trust.

The Weatherall Institute of Molecular Medicine was created to help clinicians and scientists work together and share information. Professor Jim Hughes and Steve Taylor, senior researchers at the WIMM, spoke to us about life at the institute and their current project, CSynth.

From molecular biology to adolescent health, via the immune system and the inner ear - this issue has been a journey around the human body.

Join our global readership to find out more about the exciting research happening in health science and the scientists behind the work.

Published by: Research Publishing International Publisher: Simon Jones simon@researchfeatures.com Editorial Director: Emma Feloy emma@researchfeatures.com Editorial Assistant: Patrick Bawn patrick@researchfeatures.com Editorial Assistant: Miranda Airey miranda@researchfeatures.com Junior Editor: Luna Dewey luna@researchfeatures.com Designer: Christine Burrows design@researchfeatures.com Head of Marketing: Alastair Cook audience@researchfeatures.com Project Managers: Annie Venables annie@researchfeatures.com John French John@researchfeatures.com Julian Barrett Julian@researchfeatures.com Kate Rossiter Kate@researchfeatures.com Contributors: Barney Leeke, Alex Davey, Ella Gilbert, Kate Feloy, Petra Kiviniemi, Peter North, Kate Porter

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CONTENTS

34 06

Spotlight on NIAID: Challenging the variable paradigm of infectious diseases

10 14

Unveiling the human immune response

Cystatin 9: the key to effective treatment for bacterial lung disease?

26

18 22

38 42 46

26

50

Tick Tock: a new test for rapid Lyme disease diagnosis Spotlight on CAHRU: Advancing the debate on child and adolescent health

Paving the way: converting scientific research into substance abuse treatment methods

Fixing uterine fibroids with flaxseed

Emerin and the making of muscle Protect and serve: communitydriven superfund science takes aim at asbestos exposure

Spotlight on MRC WIMM: All under one roof – a pioneering vision for research success

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54

34

58

Making connections: statistical methods to investigate healthrisk behaviours in adolescents

Spotlight on Genesis Research Trust: The champion for maternal health

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62 66

Maintaining the equilibrium: inner ear hair cell regeneration Communicating science: Animation

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Solutions set in stone: an innovative, nutrition-based approach to managing kidney stones

Biomedicine: speeding up sickle cell treatment 5

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Thought Leadership

NIAID: Challenging the variable paradigm of infectious diseases The National Institute of Allergy and Infectious Diseases (NIAID), part of the US National Institutes of Health (NIH), has been a key player behind the creation and success of the US President's Emergency Plan for AIDS Relief, the biodefense strategy following 9/11 and the research efforts to find new ways to prevent and treat infectious diseases. Dr Anthony Fauci, the Director and man in charge, spoke to Research Features about NIAID’s recent research successes, outlining where science currently stands on preventing infectious disease onset worldwide.

O

nly a very select number of people in the world have been awarded the Presidential Medal of Freedom. Even fewer have been asked directly by the President himself to set up a government-funded initiative to combat the HIV/AIDS pandemic. And yet, Dr Anthony Fauci, Director of the National Institute of Allergy and Infectious Diseases (NIAID), has received both of these prestigious honours.

Back in 2003, when George W. Bush was President of the United States, he came to Dr Fauci asking him to work with White House staff to set up what came to be known as PEPFAR – the President's Emergency Plan for AIDS Relief. In the thirteen years that have followed, the initiative has been a huge success, resulting in millions of HIV-infected men, women and children being provided access to lifesaving antiretroviral treatment.

In 2008, Dr Fauci was deservedly awarded the Presidential Medal of Freedom for his groundbreaking scientific work at NIAID. NIAID’s focus is not solely on HIV/AIDS though. As the name suggests, their research

is aimed at a whole variety of different allergies and infectious diseases – including the recent Ebola and Zika virus outbreaks.

Dr Fauci, responsible for managing NIAID’s $4.6 billion annual budget, recently spoke to Research Features about his institution’s current areas of clinical study, shedding light on some of the successes and challenges he has encountered during his time as Director.

What does your role involve as Director of the National Institute of Allergy and Infectious Diseases (NIAID) and what kind of responsibilities do you have?

I’m responsible for setting the institute’s scientific agenda, providing scientific and administrative leadership, outlining the strategic plan of where we’re going, and managing our budget. Currently, we have a budget of $4.6 billion dollars per year, most of which we award in grants and contracts.

As Director, it is my overall responsibility to plan and implement the research, both intramurally with our scientists here at the NIH, and at universities, centres, and institutions throughout the country and the world. In fact, about 80% of our budget is directed to these external organisations through the grant process.

In order to develop and implement an appropriate research agenda for a disease like HIV, you have to be global. Our international collaborations are inherent to what we do here www.researchfeatures.com

NIAID is renowned for its collaborative work worldwide. Why are these global collaborations so important for scientific research?

Science overall, and particularly science involving infectious diseases and global health, really transcends any national boundaries. The economy has become global, security has become global and science has always been global. So collaboration is important in order to implement the kind of science that we are responsible for, but it is also necessary to answer the questions that we need to answer.

Let me give you some examples. A major part of our research portfolio is involved with HIV/AIDS which, as you know, is a global problem – 67% of HIV-infected individuals are in Southern Africa and more than 90% are in the developing world outside of the United States. In order to truly develop and implement an appropriate research agenda for a disease like HIV, you have to be global.

Other good examples include malaria, tuberculosis, neglected tropical diseases, and influenza, which are also important parts of our research portfolio. Influenza by its very nature spreads globally, starting in different parts of the world. The last flu epidemic we had, the 2009 H1N1 influenza, started in Mexico and the United States, so I think our international collaborations are inherent to what we do here at NIAID. Do you mainly work in South African countries and areas that are more affected by infectious diseases, or are your collaborations mainly America-focused?

Our research is global because infectious disease is global. Let me give you an example. Several years ago, in collaboration with the Thai government and the United States Department of Defence, we did a vaccine trial for HIV in Thailand. It was only modestly protective with a 31% efficacy, which really is not ready for the primetime.

From this collaboration and the partially successful results of this trial, we have now recently started a follow-on HIV vaccine trial in South Africa called HVTN 702, in the hopes of building upon that earlier modest success in Thailand, to create an even better HIV vaccine. We are conducting the study in South Africa because that is where a safe and effective vaccine to prevent HIV infection is most needed, but certainly an effective vaccine would have worldwide implications for HIV prevention.

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Thought Leadership

We frequently collaborate with the South African government and the Medical Research Council there because of the terrible burden of HIV in certain Southern African countries, particularly South Africa. But we also conduct and fund research in the US and throughout other parts of the world.

Will that HIV vaccine trial be a follow on from the President's Emergency Plan For AIDS Relief (PEPFAR) that you had such a big part of setting up?

Yes, it will be indirectly connected. PEPFAR was not a research endeavour but instead is something that I got involved in developing with President Bush. It is a programme measured in many billions of dollars for the prevention, treatment and care of HIVinfected individuals, and is not predominantly research-focused. I got involved in it after President Bush asked me to go to Africa and put together a programme that would help deal with the ravaging HIV/AIDS pandemic in Africa. It was not really a research issue but more of an implementation and treatment issue to help prevent new HIV infections and provide treatment and care for those individuals infected with HIV. What kind of impact have you seen from that initiative?

It has been extraordinary. PEPFAR ranks among one of the most important things the United States has ever done in global health. It is certainly a major contribution to President George W. Bush’s legacy. What he has done with PEPFAR and what he allowed me to do to put it together, is something that is truly quite transforming. It has literally saved millions of lives.

In terms of modern day research do you think infectious diseases are still as recognised as they should be, especially in terms of funding?

As researchers have developed effective vaccines and therapeutics to guard against infectious diseases, such as polio and smallpox, there has been a misperception in the developed world that infectious diseases were essentially well-controlled and would soon be a thing of the past. That was a failure on the part of the developed nations to look beyond their borders. Even though certain infectious diseases are prevented or very well controlled through vaccines and drugs in many parts of the world, in other places, infectious diseases are a leading cause of death.

In addition to the established infections, such as malaria, tuberculosis, and HIV/AIDS,

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there is always a threat of emerging and re-emerging infectious diseases. We have experienced two major occurrences in just the last three years – Ebola in West Africa in 2014 and now Zika in the Americas.

There is a continual challenge with infectious diseases because of the global nature of our society and the ever-evolving nature of infectious diseases. The idea that infectious diseases are something we no longer have to worry about is a really dangerous misperception.

In terms of NIAID’s goals, is vaccine research the main aspect of your work or do you focus on other areas as well, such as education?

That is a good question. Vaccine research is a key focus of our research portfolio because an effective vaccine can be so transformative in its ability to end or significantly curb disease. However, we do a lot more research on infectious diseases than just vaccines. We conduct research aimed at understanding the epidemiology and pathogenesis of infectious disease-causing organisms, as well as the development of diagnostics, therapeutics, and, as you correctly mentioned, vaccines.

When I first started taking care of HIVinfected patients in 1981, we did not even know what the virus was, and we certainly did not have any treatments for it. Over the past 35 years we have played a major role, in fact the major role, in the research and development of most of the drugs that are now used to save the lives of HIV-infected individuals. The fundamental underlying research that lead to the development of these drugs came largely from NIAID-funded research conducted by scientists at the NIH and all over the world.

In terms of the future, which direction do you see NIAID’s research going in the next ten years or so? Do you think we will be able to find cures for particular infectious diseases?

Oh, I think so. For example, some of the work that we are most proud of went into finding treatments for HIV infection; however, NIAID was also instrumental in the development of curative drugs for Hepatitis C – the leading reason for liver transplantation in the United States and the developed world.

Dr Anthony Fauci was appointed Director of NIAID in 1984. He oversees an extensive research portfolio of basic and applied research to prevent, diagnose, and treat established and emerging infectious diseases such as HIV/AIDS, Ebola and Zika virus.

Credit: NIAID

We hope to get a vaccine to prevent HIV infection, so that we can turn the trajectory of the epidemic. That would be an enormous accomplishment and we are making our way closer to that goal. Whether or not we will be able to find an absolute cure for HIV is still an open question, but we are learning more about the virus all the time.

We also hope to develop a universal influenza vaccine that can be administered maybe once every ten years that can protect against multiple strains of influenza. Not only would this mean that people would not need to get an annual, seasonal flu shot, but the universal vaccine could presumably protect against a new influenza strain to which people had not been previously exposed. Those new influenza strains are the ones that can cause global flu pandemics.

The PEPFAR initiative has been extraordinary. It ranks among one of the most important things the United States has ever done in global health www.researchfeatures.com

Dr Nancy Sullivan of NIH’s National Institute of Allergy and Infectious Diseases discussing Ebola research with President Barack Obama as NIAID Director Dr Anthony Fauci and HHS Secretary Sylvia Burwell look on. Credit: NIH

to understand the latest developments in a given field and how to apply them. As you highlighted in your first question, science really is a global issue.

From a more personal perspective, your research into HIV/AIDS has led to numerous awards over the years, and you are often described as America’s Man on Infectious Diseases. How does it feel to be such an internationally-recognised peer of infectious disease research, and what does winning these awards mean to you?

It is always nice to be recognised for what you have done, but I have to tell you, when there is so much unfinished business and so many challenges, I spend very little time savouring awards. I mean, it is very nice – it is always gratifying to be recognised for your work – but you cannot take your eye off the ball when there are so many different emerging and re-emerging infectious disease challenges that remain. Being recognised is a great thing, but it is not the reason I do it.

As you mentioned, with the Ebola outbreak occurring in 2014 and the Zika epidemic happening just last year, are there concerns that a future epidemic could be just around the corner? Is there a plan in place in case another outbreak occurs next year?

The most important thing that you can predict about an emerging infectious disease is that it will be unpredictable. No one expected that Zika would have such a devastating effect throughout the Americas.

In order to prepare for it, you try to develop vaccines and diagnostic platforms that can be rapidly extrapolated to the next disease that emerges. You also want to have a good surveillance system in place to see if there are emerging threats that go unnoticed. That surveillance aspect is more the responsibility of the US Centers for Disease Control and Prevention than that of the National Institutes of Health (NIH), but we do get involved in developing the diagnostics that support their surveillance plans.

After 9/11, NIAID was involved in the biodefense research following the attacks.

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Could you tell us some more about this?

Our institute led research efforts to find counter-measures against anthrax, amplify the supply of smallpox vaccine and develop antidotes against botulism and other potential biodefense weapons. After 9/11 and the subsequent anthrax attack, the United States government invested about a billion and a half dollars in NIH, asking us to develop counter-measures against potential bioterror threats.

So having that governmental backing must be very important?

Yes – it is absolutely vital to our research.

NIAID conducts and hosts many scientific events, seminars, and lectures. Why are these so important to progressing research?

Science not only involves discovery but also incorporates communication – giving ideas to others and receiving ideas from them. Communicating with scientists through lectures, events and seminars is very important to establish the kind of scientific information exchange necessary

• NIAID is one of the 27 institutes that make up the National Institutes of Health (NIH).

For over 60 years, it has supported basic, clinical and translational research to improve scientific understanding on how to better treat and prevent infectious, immunologic and allergic diseases. Under Dr Anthony Fauci’s directorial leadership, NIAID has improved the health of millions of people worldwide, developing countless new vaccines, therapies and diagnostic tests through research.

Contact NIAID Office of Communications and Government Relations 5601 Fishers Lane, MSC 9806 Bethesda, MD 20892-9806 USA E: ocpostoffice@niaid.nih.gov T: 866-284-4107 W: www.niaid.nih.gov /niaid.nih @NIAIDNews

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Unveiling the human immune response Dr Clemencia Pinilla is a pioneer in the field of combinatorial chemistry, which involves the chemical synthesis of up to millions of compounds in a single process to produce libraries of compounds. For the past two decades Dr Pinilla and her team have been leading the way in developing and utilising positional scanning combinatorial libraries for T-lymphocyte antigen discovery. Her research has had a groundbreaking impact on our understanding of the human immune response in a wide array of diseases including infections, autoimmune disorders and cancer, with far reaching implications for medical progress.

Immunology

A

t the Torrey Pines Institute for Molecular Studies (TPIMS), California, Dr Clemencia Pinilla is an Associate Member leading research that investigates antigens recognised by T lymphocytes (T cells). T cells play crucial roles in both pathological and physiological immune responses. As well as defending against infectious organisms, T cells respond against cancerous cells and are involved in autoimmune responses. Therefore, the identification of the specific antigens that T cells recognise during an immune response is of vital importance for gaining insight into a vast range of human diseases, as well as for the development of effective vaccines.

T cells recognise antigens on antigen presenting cells (APC) by interaction with the T-cell receptor. The antigens are short peptides; the APC processes proteins into fragments to create the peptides, which are then presented on the APC surface. It is these antigens and the associated epitopes (the part of the antigen recognised by the T cell) that Dr Pinilla’s research aims to identify.

DEVELOPING THE TOOLKIT Dr Pinilla’s research in the field of immunology began with her investigating the potential of positional scanning libraries for the study of T-cell specificity and devising the concept for the use of the system for this application.

These compound libraries are comprised of up to trillions of peptides and allow unbiased identification of T-cell antigens that stimulate the T-cell clones analysed. She cites the importance of collaboration to her work and, in particular, the contributions of Drs Roland Martin and Mireia Sospedra from Universitatsspital Zurich.

Following successful early studies demonstrating that the method is highly effective in identifying antigens recognised

by T cells, Dr Pinilla and her team went on to work on further expanding the method’s capacity for antigen discovery by combining the technique with biometrical analysis. This brought together the results obtained using positional scanning libraries to identify T-cell antigens from protein sequence databases.

The techniques have proved to be extremely powerful when combined. Positional scanning-based biometrical analysis can systematically integrate the results of the positional scanning libraries composed of trillions of peptides with protein databases and predict and identify with high accuracy peptides and their corresponding antigens

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MHC-antigen-TCR

T cell

TCR peptide

MHC APC Coreceptors

TCR: • Recombinant proteins • Overlapping peptides • Positional Scanning Libraries • Immunogenic MHC: • Prediction and binding • Elution • Immunogenicity needs to be determined

recognised by disease relevant T cells.

Through this unique and innovative approach, both native and cross-reactive sequences for the T cells can be elucidated, for clones of both known and unknown specificities. This is important because specific T-cell responses for many diseases, ranging from infections to cancer, remain largely unknown due to a lack of identified antigens.

the fact that approaches have relied on preselection of pathogen proteins or peptide antigens, followed by assessment of whether they elicit a positive T cell response. Dr Pinilla and her team have developed a novel “T-cell driven” approach to investigating antigens recognised by T cells, which they have recently refined, focusing on the immune response involved in Chagas diseases.

A NEW ERA FOR VACCINE DISCOVERY Historically, vaccines typically consisted of mixtures of attenuated or inactivated causative agents. In recent decades, molecular techniques have enabled increasingly refined vaccine design by using immunogenic (causing a response from the immune system) protein antigens in recombinant vaccines. These vaccines use the antigens from a protein to stimulate an immune response, instead of the causative agent itself; knowledge of antigen epitopes recognised by the immune system during disease is therefore of critical importance.

The discovery of candidate antigens to inform vaccine development had been limited by

INVESTIGATING IMMUNE RESPONSE TO EXOGENOUS AGENTS Chagas disease results from infection by the protozoan Trypanosoma cruzi and is a major health problem across the globe, resulting in a larger healthcare burden than malaria due to a lack of therapeutic and protective vaccines. However, through Dr Pinilla’s research in collaboration with Dr Karina Gomez at the Institute of Genetic Engineering and Molecular Biology, INGEBI in Buenos Aires, the identification of the immunogenic antigens recognised by the human immune response to infection with the parasite is helping to open doors towards the discovery of effective Chagas vaccines.

Dr Pinilla and her team have developed a novel “T cell driven” approach to investigating antigens recognised by T cells, which they have recently refined 11

Immunology

Multi-well seeding of memory CD4+ and CD8+ T cells

1

Amplified T cell library

Pathogen Specific T cell Libraries

Pathogen stimulation Selection of Pathogen Specific Wells

PBMC

2

PHA + IL2 stimulation Amplified T cell library

Generation of B cell lines for each donor

Prior to her research focus on Chagas disease, Dr Pinilla worked on another virus that is used in the vaccine for smallpox, vaccinia virus (VACV). She identified vaccinia-specific T-cell antigens from immunised humans, contributing to our knowledge of the immune response to immunisation with vaccinia, which could lead to the development of new improved smallpox vaccines. Another of her earlier projects employing positional scanning-based biometrical analysis led to the identification of the previously unknown

How did your background in microbiology lead to you developing combinatorial chemistry techniques for discovering antigen specificity?

In 1986, I was working in Colombia with Dr Manuel E Patarroyo in the identification of antigens recognised by antibodies from patients with malaria and tuberculosis and I had the opportunity to meet Dr Richard A Houghten, at the time an investigator at Scripps Clinic. Dr Houghten had recently developed the simultaneous multiple peptide synthesis method, and he invited me to be a postdoctoral fellow in his laboratory. I joined in 1987 to work on a project that at the time was named 64 million peptides and resulted in our Nature 1991 publication, in which we described the synthesis and use of a hexapeptide synthetic combinatorial library for basic research and drug discovery. In other words, since my early days in science I have been interested in the identification

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Pathogen Specific T cell Clones

Strategies for examining memory T cells from patients with Chagas disease

peptide specificity of CD4+ T cells that occur during Lyme disease. Dr Pinilla has also worked on projects investigating the antigen specificity of T cells involved in the human immune response to cytomegalovirus (CMV), which is responsible for more congenital birth defects than any other virus, and HIV-1.

It is not only infections that can elicit an immune response. Alongside Dr Andrew Fontenot from University of Colorado Anschutz Medical Campus, Pinilla has also worked on a project that led to the first

ever discovery of an antigen involved in metal-induced hypersensitivity. Chronic beryllium disease (CBD) results from a genetic predisposition to the hyper-sensitive reaction on exposure to beryllium metal (Be).

A metal-induced hyper-sensitive immune response occurs, causing an influx of CD4+ T cells specific to beryllium (Be) into the lungs. Pinilla’s research found that there is an interplay between antigenic peptides and Be in the generation of the immune response that occurs in these cases.

of the antigens that are recognised by the immune system during infectious disease.

The later development of the decapeptide combinatorial libraries in 1994 and the understanding that T cells’ ‘unique identifier’, the T-cell receptor, recognises a linear peptide led to our efforts to understand how combinatorial libraries could be used for the identification of peptides recognised by T cells. Our first collaborator was Dr Roland Martin at NINDS, NIH. He had generated a number of CD4 clones that became the model systems that we used for the development of the biometrical analysis in collaboration with Drs Richard Simon and Yindong Zhao at NCI, NIH.

the positional scanning libraries and the identified peptides.

What early challenges did you face in applying the positional scanning libraries technique for identifying T-cell antigens?

The early challenges remain the same. They are, firstly, to have a clear rationale and sufficient evidence to be sure that the T cell clones to be studied are relevant to the disease being investigated and secondly, to expand clonal cells in sufficient numbers (at least 30–40 million) for the screening of

What aspects of your research have you found the most exciting in your career?

The collaborations with investigators with different scientific expertise and interest on the elucidation of T-cell receptor specificities are very rewarding and exciting to me. The elucidation of peptides that activate each of the T-cell receptors that we have studied has taught us something new.

Every time that we identified individual peptides that activate a T cell as a result of the testing of mixtures of billions of peptides and the deconvolution of their activities, I am delighted at how well the process works. Finally, the increase in the efficiency of the methodologies of T-cell culture and screening and therefore the knowledge of T-cell specificity has encouraged us to continue our work that will lead to a better understanding of the immune response, the development of diagnostics, the modulation and intervention in autoimmune diseases, and the development of vaccines.

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FURTHERING OUR UNDERSTANDING OF AUTOIMMUNE DISORDERS In addition to this Dr Pinilla has also focused her efforts on several diseases involving an immune response that are not known to result from a specific exogenous agent. Her research has discovered the epitopes recognised by T cells in numerous cancers and she has carried out extensive work unravelling the destructive immune response that occurs during multiple sclerosis (MS).

CD4+ T cells from the cerebrospinal fluid (CSF) of MS patients and used positional scanning-based biometrical analysis to investigate the samples. They found that several of the T cells exhibited high levels of cross reactivity with different peptides, as well as a lack of specificity for variants of APCs associated with T cell interaction. Overall, these findings amounted to a lower degree of specificity than had ever before been identified for these cells, which could account for some of the pathology of MS in patients.

MS is an autoimmune disease and neurological condition that affects the central nervous system (CNS) resulting in damage to the coating surrounding nerve fibres, called myelin. The condition is thought to be due to a CD4+ T-cellmediated autoimmune response, with the disease developing in genetically susceptible individuals in combination with environmental triggers. Relapses often occur following viral infections and it is suspected that viruses play a role in the disease.

Looking to the future, the team’s work developing the positional scanning combinatorial libraries can be used to help create and refine vaccines against multiple diseases. In particular, Pinilla and her team are planning to generate libraries and clones of memory CD4+ T cells from patients infected with Trypanosoma cruzi. These can then be used to identify the antigen specificities using positional scanning libraries, which will further contribute to the design of novel vaccines against Chagas disease. With a health burden greater than that of malaria, a vaccine for this disease could have positive health benefits for millions across the globe.

Dr Pinilla, in collaboration with Drs Roland Martin and Mireia Sospedra, has investigated

How important has team work and collaboration been to your work so far?

Team work has been essential to all our studies; I find that it is when we really try to understand a scientific problem from different points of view that we are able to develop solutions. I have worked closely with the chemists at TPIMS (Drs Richard A Houghten and Marc A Giulianotti) on devising procedures to improve the analysis and reproducibility of the synthesis of the complex mixtures that comprise the positional scanning libraries.

Currently, I work with Dr Radleigh Santos, a mathematician by training, on continuing to improve the biometrical analysis and to develop analysis platforms for the analysis of immunomonitoring results of immunotherapy clinical studies. Also, I very much value all of our collaborators and their interests in T-cell specificity in different diseases. We apply what we have learned from our previous studies and continue to let the screening data and the most unbiased methodologies of deconvolution guide the peptide and antigen identification process.

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Are there any other potential applications of combinatorial chemistry techniques within the field of immunology?

For any molecular interaction where a ligand is needed, TPIMS combinatorial libraries would be useful. If the interest or need is to identify naturally occurring peptides, the L-amino acid peptide positional scanning libraries in combination with the biometrical analysis would be useful tools. We think that studying the specificity of the T cell response to large pathogens using positional scanning libraries is a clear application of TPIMS combinatorial libraries, since other T-cell driven approaches would be more difficult to use. Similarly in cancer, the identification of neo antigens would be another potential application. On the other hand, if a small molecule was desired to block or activate a molecular interaction, TPIMS small molecule libraries in positional scanning format would be the libraries that I would suggest.

Detail RESEARCH OBJECTIVES Dr Pinilla’s pioneering work in the field of positional scanning combinatorial libraries focuses on elucidating the human immune response to pathogens.

The method allows for the discovery of antigens recognised by T cells which can then be used in the development of vaccines. Her work has spanned multiple diseases and disorders and she is now focused on the identification of antigens using a T cell driven approach that could contribute to the development of a vaccine for human Chagas disease.

FUNDING NIH: NIAID COLLABORATORS • Jon R Appel • A ndrew Fontenot • Richard A Houghten • V aleria Judkowski • M arc A Giulianotti • R oland Martin • Radleigh G Santos • M ireia Sospedra BIO Dr Pinilla is an Associate Member at TPIMS. She is recognised as a pioneer in the field of combinatorial chemistry with over 25 years of experience, particularly in the use of positional scanning libraries for the identification of ligands for a wide range of targets. These studies have involved collaborative projects with multiple investigators at TPIMS and other institutions worldwide resulting in more than 130 peer-reviewed publications.

CONTACT Clemencia Pinilla, PhD Associate Member Torrey Pines Institute for Molecular Studies 3550 General Atomics Ct San Diego, CA 92121 E: pinilla@tpims.org T: +1 858 597 3883 W: http://www.tpims.org/our-scientists/ california-team/clemencia-pinilla /clemencia-pinilla-29655a6

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Immunology

Cystatin 9: the key to effective treatment for bacterial lung disease?

Dr Tonyia D Eaves-Pyles’ current research focuses on the role of cysteine proteinase inhibitors called cystatins in the control of immune inflammatory responses in the lungs. These inhibitors may hold the key to limiting the damage done by an overzealous immune response to pathogens in diseases such as pneumonia and influenza.

L

ung infections can be devastating illnesses, and are particularly lethal to those who are already vulnerable, such as children, the elderly and those with already weak immune systems. Pneumonia and lung damage caused by infection with drug-resistant pathogens are also associated with high costs for healthcare services. In the US, pneumonia and influenza are the eighth most significant causes of mortality and in 2005 cost the US economy an estimated $40 billion plus $6 billion in indirect costs.

THE IMMUNE SYSTEM OVERREACTS WHEN REGULATION IS ABSENT The immune system normally deals with pathogens via the process of inflammation, but without regulation the response can be too intense and can cause cell damage. In addition, for some pathogens, inflammation provides the perfect conditions for them to infect their hosts, therefore increasing the level of infection.

Pneumonia and influenza are associated with dysregulated inflammatory responses and the break-down of the pulmonary epithelial cell lining, such as the air sacs (alveoli) that exchange oxygen and CO2 and oxygenate the blood. This causes the membranes to become more permeable and inflamed, and therefore more susceptible to disease. One of the factors that contribute to this overreaction is caused by enzymes called matrix metalloproteinases (MMPs), which break

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down the cellular scaffolding present around cells known as the extracellular matrix (ECM).

CYSTATINS: THE REGULATORS MMPs are enzymes that form part of the endopeptidase subfamily that hydrolyses collagens and elastin. Under normal circumstances, these enzymes control tissue remodelling such as cell propagation and differentiation. MMPs are under tight regulation by cysteine proteinase inhibitors known as cystatins.

Some infections cause an imbalance between MMPs and cystatins resulting in excessive tissue breakdown and immune cell activation at the site of infection. This can lead to unrestrained inflammation resulting in acute lung injury, acute respiratory distress syndrome, and multiple organ failure. Sometimes the inflammation allows pathogens to infect healthy tissue, therefore increasing infection throughout the body.

The restoration of normal cystatin levels, however, has a powerful modulating effect, down-regulating the breakdown of cells and tissues by cysteine proteinases and regulating the immune response so that it does not become over-zealous and trigger excessive inflammation.

CST9 Dr Eaves-Pyles, in conjunction with collaborators Drs Rick B Pyles and Bernard Arulanandam, has been investigating the

immunomodulatory effects of cystatin 9, also known as CST9. They have identified it as an effective tool to control the inflammatory process to ensure that it is strong enough to combat infection but not so rigorous that it disrupts tissues.

The team has shown that CST9 not only modulates host immune responses but can directly affect the viability of deadly human pathogens such as Francisella tularensis, which causes pneumonic tularemia.

Specifically, CST9 disrupts various metabolic

In the US, pneumonia and influenza are the eighth most significant causes of mortality a human context, rather than in rats or mice.

This therefore allows further exploration of the role of CST9 in a more complex environment that is closer to that of the human body.

USING CST9 IN TREATMENT Eaves-Pyles’s work has shown that the pathways that mediate inflammation in the immune system are complex. However, she hopes to develop CST9 into a useful therapeutic intervention that will improve the survival of patients suffering from deadly lung infections and diseases.

The restrained inflammatory response mediated by CST9 may be useful therapeutically because it regulates inflammatory responses through key proteins and pathways in a variety of cells such as macrophages. The identification of these key proteins modulated by CST9 could be harnessed to further temper damaging inflammation and used to prevent death from infection and/or sepsis.

FUTURE APPLICATIONS Other cystatins have been demonstrated to have anti-tumour properties (cystatins C, B and E/M) and protective properties against neurodegenerative diseases such as Alzheimer’s (cystatin C). However, less is currently known about the role of cystatins in combatting bacterial infection. Dr EavesPyles’ work is therefore extremely pertinent. pathways of F. tularensis affecting its ability to thrive and replicate in the lungs resulting in enhanced killing by macrophages (immune cells that engulf and destroy foreign cells).

To do this, they have employed a variety of techniques. They carried out tests in vitro to study the effects of CST9 on macrophages, as well as in vivo using various mouse models of infection. In mice treated with CST9, Dr Eaves-Pyles observed modulation of inflammation, decreased lung damage and a lower bacterial load in vital organs

following a pulmonary F. tularensis infection resulting in improved mouse survival. This suggests that the administration of cystatin 9 can restore crucial immunomodulatory capabilities to the body’s immune responses to fight against invading bacteria.

Further work involves the use of a novel human lung model developed by Dr Joan Nichols, which uses rat lungs removed of all rat cells then re-populated with human pulmonary cells. This method provides unique insight into the interaction of CST9 in

Dr Eaves-Pyles’ studies have shown that cystatin 9 is a strong candidate ideally suited to develop as a promising therapeutic treatment for deadly lung diseases, which have a considerable health impact worldwide. Her results demonstrate the diverse impacts of CST9 on inflammation and bacterial virulence (e.g., multi-drug resistant K. pneumoniae, as well as various other deadly bacterial pathogens) that may prove to be a novel intervention against pulmonary pathogens.

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What are the relative advantages and disadvantages of the methods you used to investigate the immune response?

Advances in technology combined with a better understanding of the innate immune system have made it possible to dissect different parts of the immune responses, their pathways and mechanisms that are mobilised to fight against bacterial infections. We measure cytokine secretion from infected lung cells using a human and mouse inflammatory cytokine array, which can detect up to 43 different chemokines and cytokines. This type of protein multiplex array is highly sensitive, quantitative and offers a rapid, high-throughput system that can identify proteins to pursue studies of cell signalling pathways, cell migration, and cell–cell communication and potential therapeutic targets. For our purposes, the mulit-plex array has been a crucial tool in addressing which chemokines and cytokines are modulated by cystatin 9 during infection. We also use flow cytometry in our in vivo studies to detect and quantify what population of immune cells infiltrates the lungs following cystatin 9 treatment of bacterial pneumonia and helps track the course of residential infection.

The level of immune cell infiltration into the lungs can correlate with inflammatory responses (cytokine array) and tissue damage (lung histology).

against deadly bacterial pathogens allows us to observe individual immune events in a single cell population as well as to elucidate the features of inflammation in multiple cell types that interact and communicate to form responses that are beneficial to the host. It is like pieces of a puzzle; each piece of the puzzle works together to form a complete picture. And although there are limitations and challenges when using experimental in vitro and in vivo models, such as discordance between findings in experimental models and translation to results in humans; these models are crucial to bridge the gap from bench (basic science) to bedside (clinic).

However, even with all the advances in technology, one of the best, most straightforward methods to measure the amount of bacteria load is old-fashioned plating to determine colony forming units.

Bacterial loads and immune responses can be determined at various time points after cystatin treatment and infection by culturing a portion of the organ homogenates on agar plates and analysing the remainder by cytokine array.

The problem is that invading bacterial pathogens can cause an overactive, prolonged inflammatory response that is destructive to tissues and organs. In fact, typically we are in more danger from our responses to the invading bacteria than the bacteria itself. Therefore, I am striving to harness specific features of the immune response that will prevent excessive inflammation that destroys healthy tissue, and will maintain a balanced, active immune response that can effectively eliminate harmful invaders and preserve tissue/organ integrity. This may represent a realistic opportunity to fine-tune immune responses and design new immunotherapeutic interventions that could profoundly impact various disorders and diseases.

Because of technological advances we are able to generate large amounts of raw data, which can be a challenge, raising more questions than it answers. It is the analysis and interpretation of the data that takes time to allow the data to lead to the next logical step(s). The methods that we utilise in our studies to evaluate the immunomodulatory effects of cystatin

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How does the body fine-tune the inflammatory response to get it just right?

Wow, I wish I had the answer to this question!!

This is a simple question but the answer is very complicated and continually evolving. The inflammatory response is a complex system of checks and balances that is a functional network involving numerous components.

Therefore, fine-tuning the inflammatory response involves complex interplay between host cells and the products they produce leading to stimulatory and inhibitory signals that require tight regulation. There are many systemic cell types (neutrophils, monocytes), tissue host factors (Toll-like receptors, resident immune cells) and dietary components involved in balancing inflammatory responses.

Is cystatin 9 the only cysteine proteinase inhibitor that has this effect?

We have discovered that another of the type 2 cystatin superfamily, alone and in combination with cystatin 9, affords unprecedented protection against multi-drug resistant bacterial pneumonia as well as other deadly pulmonary pathogens. Interestingly, the two cystatins differentially modulate various features of the innate immune system and in combination; they work synergistically to improve morbidity and mortality outcomes in an in vivo model of pneumonia.

How easy is it to separate the effects of specific actors in the immune system from one another?

Investigating the immune system in a laboratory setting inevitably presents certain benefits as well as challenges. One model that we employ to investigate specific features of the innate immune response is a respiratory tissue culture model using human lung epithelial and/or immune cells. This model gives us an opportunity to examine the bacterial-induced immune responses and intracellular communication networks of individual cell types in the lungs. We can analyse the signalling pathways and secretions from the same cell type to identify what types of inflammatory cytokines/ factors are produced as well as how much they are producing. The level of bacterialinduced inflammation can be correlated with cell damage by microscopy analysis of cell histology. This will allow us to pinpoint harmful, damaging inflammation then attempt to develop therapeutic approaches to restrain it. However, in the intact biological system, cells do not typically function independently of one another but in a complex multilevel interaction network. In other words, it is a team effort to fight against invading pathogens. As such, the “first responder” cells will secrete inflammatory cytokines that can stimulate and activate surrounding cells and call in circulating immune cells to the lung to rise up and fight.

To mimic this cell–cell communication as it occurs in the immune system network, we utilise a co-culture model where immune cells such as human alveolar macrophages, dendritic cells or neutrophils, are added at physiological levels to respiratory epithelial cells during infection. The co-culture system allows us to better understand the biological

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Immunology

Detail RESEARCH OBJECTIVES Dr Tonyia D Eaves-Pyles’ work focuses on pathogen-induced inflammatory responses that cause damage and/ or death to the host. Her research investigates ways in which unrestrained immune responses can be moderated to successfully fight against deadly pathogens and avoid damage from excessive inflammation.

Immune Responses: The Role of Foe

Overreaction of immune responses Invading bacteria Immune system overreacts to invading bacteria inducing harmful, excessive inflammation Our response to invading bacteria can cause: • Tissue Damage • Multiple Organ Failure • Disease Development

Immune Responses: The Role of Friend

Tempers damaging inflammation Promotes beneficial immune responses Prevents tissue damage

Directly interferes with bacterial virulence and replication

Successful Resolution of Infection function and cross-talk between cells in the event of a bacterial invasion.

The advantage of this ex vivo human lung model system is that it is controlled and reproducible, revolutionising the ability to test new and existing therapeutic approaches to treat lung dysbiosis caused by pathogenic bacteria.

www.researchfeatures.com

What is the next step for your research?

The next step of our research is to translate the protective effects of cystatin 9 against pneumonia from our experimental in vivo model of pneumonia to a human bioengineered lung model (as developed by our collaborator, Dr Joan Nichols) that will allow translation to drug development in patients.

FUNDING NIH: NIAID COLLABORATORS • Dr Rick B Pyles (Professor at University Texas Medical Branch, Galveston, TX) • Dr Bernard Arulanandam (Professor, Assistant Vice President for Research and Director of the South Texas Center for Emerging Infectious Diseases at University of San Antonio, TX) • Dr Joan Nichols (Professor at University Texas Medical Branch, Galveston, TX) BIO Dr Tonyia Eaves-Pyles received her PhD in microbiology from the University of Cincinnati School of Medicine. Currently, she is a tenured Associate Professor in the Department of Microbiology and Immunology at the University of Texas Medical Branch and adjunct faculty at Shriners Hospitals for Children, Galveston, Texas.

CONTACT Dr Tonyia D Eaves-Pyles, PhD Associate Professor The University of Texas Medical Branch (UTMB) MRB, Route 1070 Department of Microbiology & Immunology 301 University Boulevard, Galveston, Texas, 77555-0144 USA E: tdeavesp@utmb.edu T: +1 (409) 772-9429 W: https://microbiology.utmb.edu/ faculty/Eaves-Pyles.asp /eaves-pyles-tonyia-6a000a21

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Technology

Tick Tock: a new test for rapid Lyme disease diagnosis With a wealth of experience in Lyme disease (LD) research, Dr Maria Gomes-Solecki from the University of Tennessee has turned her attention and expertise to LD diagnosis. A new device, developed by Dr GomesSolecki (Immuno Technologies, Inc) in collaboration with Dr Sam Sia (Columbia University), provides rapid diagnosis of LD at the point of care, allowing prompt prognosis decisions and treatment delivery.

L

yme disease (LD) is the most common tick-borne illness in the US and is also highly prevalent throughout Northern Hemisphere temperate regions, including Europe, Mongolia and China. As reported by the Centres for Disease Control and Prevention, the spread of LD is a rapidly growing problem, affecting more people year on year. Cases of the disease are estimated to have doubled in the US over the last decade, and an estimated 300,000 people contract the disease there each year.

Sufferers of LD can experience a wide range of non-specific symptoms that develop gradually as the disease progresses from early to late stage. The disease can affect the skin, joints, heart and nervous system and symptoms often overlap with other illnesses, presenting significant difficulties in diagnosis. If progressed to late stage, LD can result in permanent damage to the musculoskeletal and nervous systems, antibiotic-refractory arthritis and posttreatment Lyme disease syndrome (ongoing fatigue, pain, and joint and muscle aches that persist after treatment is completed).

A HISTORY OF SUCCESS Dr Gomes-Solecki has an impressive track record when it comes to LD research. In a previous project, an oral bait vaccine developed in Dr Gomes-Solecki's laboratory

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was shown to break the cycle of tick–mouse LD transmission. In 2014, Dr GomesSolecki was awarded the prestigious MBQ Innovation Award for this important contribution to LD research. More recently, her continued focus in LD diagnosis led to the development of a rapid point-of-care diagnostic test.

A MATTER OF TIME Lyme disease is caused by Borrelia burgdorferi, a bacterial species transmitted by ticks. Current diagnosis commonly uses a two-tiered serological approach, a series of lab-based tests that measure the B. burgdorferi-specific antibodies in a patient’s blood sample. Typically, the first assay is comprised of a very sensitive ELISA (Enzyme-linked immunosorbent assay) – which, if positive, is followed by Western blot analysis, an assay that is much more specific than ELISA. Although these tests display excellent sensitivity (96–100%) for late stage III Lyme disease, the test sensitivity is significantly reduced in earlier stages of the disease (35–56% for early stage I).

The current approach to testing also has significant limitations.

Tests are unable to differentiate between early- and late-stage LD and therefore cannot be used to predict prognosis.

Furthermore, the current tests are lab-

Flow Direction Flow Direction Technology Inlet

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(A) The Claros1 Analyzer (OPKO) is a benchtop device that can be used to power fluid flow, control temperature setpoints and detect signals from the microfluidic cassette. (B) Automated delivery of reagent sequence. Sera/plasma samples as well as all washes, gold-labelled secondary antibodies and silver amplification reagents are loaded in PE tubing and delivered automatically in sequence, passing over the five detection zones of the microfluidic channel. (C) BSA is used as a blocking agent for an internal negative reference, various recombinant and synthetic proteins serve as the Lyme antigens for capturing anti-Lyme antibodies and anti-goat antibody for capturing gold-labelled secondary antibodies as an internal positive reference.

dependent, resulting in significant delays between obtaining a blood sample and receiving results. Given the risks associated with later stages of LD, rapid diagnosis and treatment is vital to limit serious injury and optimise treatment outcomes.

Dr Gomes-Solecki has recently developed in collaboration with Dr Sam Sia (Columbia University) a pioneering new device that uses ultrasensitive microfluidics technology to diagnose LD. Called the mChip-Ld, this device facilitates rapid diagnosis at the point of care, eliminating the need for laboratorydependent tests.

THE FUTURE OF LYME DISEASE DIAGNOSIS The mChip-Ld device consists of two main components: a microfluidic plastic cassette which is the base for all the biochemical steps and a light-detection device. Bound to the surface of the cassette are five Lyme antigens, substances that induce the production of an immune response in the body. When a patient blood sample is added, any Lyme-specific antibodies present in the sample will bind to the respective antigen on the cassette. Preloaded washes, gold-labelled secondary antibodies and silver amplification reagents are then delivered. The binding of these antibodies and reagents affects silver ion production on the surface of the cassette.

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Silver reagents

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Lyme Ag#1

Lyme Ag#2

Lyme Ag#3

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Flow of goldlabelled goat anti-human lgG/igM Ab Flow of silver reagents Negative reference

results? Signal to cutoff plot for one sample panel?

The mChip-Ld facilitates rapid diagnosis at the point of care. It helps the doctor make a decision regarding best course of treatment in the clinic www.researchfeatures.com

Detail The number of cases of Lyme disease in the US has rapidly increased over the last decade. What are the main factors contributing to this rise?

In the past, there was about 10x the amount of under-reporting of the disease by doctors who patients. Recently, awareness of the disease has become much greater. In addition to growing awareness of LD, there has also been recent expansions of tick habitats into new areas.

Why is it difficult to diagnose LD in the very early stages of the disease?

Most diagnostic assays for LD detect the presence of the bacteria indirectly by assessing immunoglobulins made by the immune cells in the blood. It usually takes 7–21 days for the body to produce enough immunoglobulins (G and M) for detection.

Therefore, if the patient presents to the doctor the same day as he/she was bitten by a tick, there is a high likelihood that the patient’s serological assay will be negative.

The detection component consists of an LED paired with a photodiode for each antigen. Light emitted from the LEDs passes through the cassette and is detected by the photodiodes. As it is transmitted through the cassette, light is absorbed by silver ions present on the cassette. Thus, greater silver production is inversely proportional to light detected by the photodiodes. Quantification of the light sensed by the photodiodes allows simultaneous detection of multiple Lyme-specific antibodies, and subsequent diagnosis for LD. Importantly, to carry out the analysis, the cassette is inserted into a benchtop analyser (OPKO Diagnostics LLC). This analyser controls fluid flow, temperature and signal detection. In addition, the delivery of samples, washes, antibodies and reagents is programmed to occur automatically and sequentially, allowing the mChip-Ld to be used by non-specialist practitioners.

LONG-TERM BENEFITS In a series of tests, the mChip-Ld device was found to have comparable performance to traditional diagnostic methods. In addition, it

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At what stages of disease progression can the mChip-Ld device detect LD? The mChip-Ld device can detect early disseminated (convalescent) and late Lyme disease.

The mChip-Ld device can identify the presence of multiple different antibodies. What can these results tell us about LD in a patient?

Knowing which antigens induced a positive immune response can give indications of the stage of the disease at that time. OspC, for example, is a good indicator for early Lyme Disease. Other proteins can also detect early Lyme as well as late Lyme.

What are the next steps to getting the mChip-Ld device used regularly in clinical practice?

The assay needs to go through optimisation (early product development) and FDA approval.

also provides a broad spectrum of associated benefits. Primarily, the mChip-Ld is portable and facilitates point-of-care diagnosis in 10-15 minutes. A rapid diagnosis allows for prompt treatment and a subsequent reduction in the risk of developing serious and chronic illnesses associated with late stage LD. Furthermore, the separation of antigens and their corresponding detection zones allows for future differentiation between early and late stage LD and informs antigen-based diagnostic decisions. Lastly, at about $1.50 the cassettes are low-cost and the automated nature of the device means it can be used by medical practitioners with minimal training.

RESEARCH OBJECTIVES Dr Gomes-Solecki has a long track record in research and development of vaccines and diagnostic assays for Lyme disease.

FUNDING NIH: NIAID and a Saving Lives at Birth transition grant (United States Agency for International Development; Gates Foundation; Government of Norway; Grand Challenges Canada; and the World Bank) COLLABORATORS Sam Sia, PhD, Columbia University BIO Originally from Portugal, Maria earned her DVM from the University of Lisbon in 1992. After completing her Fellowship at the National Institute of Technology in Lisbon, she moved to New York, where she completed her postdoctorate work at Stony Brook University.

CONTACT Maria Gomes-Solecki, DVM Associate Professor Department of Microbiology, Immunology and Biochemistry The University of Tennessee Health Science Center 858 Madison Ave, suite 301A Memphis, TN 38163 E: mgomesso@uthsc.edu W: www.immunotechnologies.com /maria-gomes-solecki-0224091 http://oportuguesliberal.blogspot. com/2014/02/from-lab-bench-to-storenear-you.htm

The ability to rapidly diagnose Lyme disease, at the point of care, will have extended benefits. While the wider research community strives to reduce LD transmission, a rapid diagnosis, together with rapid treatment, is key to ensuring the best possible outcomes for the many people who contract LD each year. Dr Gomes-Solecki, in collaboration with Dr Sia, has developed a device that is all about saving time, but the benefits to LD patients will reach far into the future.

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Thought Leadership

CAHRU: Advancing the debate on child and adolescent health The Child and Adolescent Health Research Unit (CAHRU) in the School of Medicine at the University of St Andrews has grown a reputation for pioneering research into improving child and adolescent health. This has not gone unnoticed and, in 2013, the World Health Organization designated the School of Medicine as their international collaborating centre for child and adolescent health policy. We spoke to Dr Jo Inchley, who told us how important this partnership has been for advancing child and adolescent health research.

S

ince being founded at the turn of the millennium, CAHRU has quickly established itself as one of the leading, global institutions within child and adolescent health research.

Their work focuses on promoting health among children and adolescents at local, national and international levels, by monitoring the behaviour, health and wellbeing of children and young people.

Fast-forwarding 13 years, WHO recognised CAHRU’s pioneering work, and designated them as their collaborating centre within this field of research back in 2013.

An important element of this partnership is CAHRU's role as the International Coordinating Centre of the Health Behaviour in School-aged Children (HBSC) World Health Organization Collaborative Cross-national Study which incorporates 45 countries across both Europe and North America.

Research Features recently sat down with Dr Jo Inchley, the Assistant Director of CAHRU, to discuss this collaboration further, highlighting the partnership’s impact on child and adolescent health research, particularly within the HBSC study.

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What does your role involve as Assistant Director of the Child and Adolescent Health Research Unit (CAHRU) at the University of St Andrews?

My role is quite diverse. Within CAHRU, I am involved in developing new research ideas, managing existing projects, as well as the strategic direction and longterm sustainability of the Centre. Within the wider School of Medicine, I sit on the Research Ethics Committee, contribute to teaching and supervise undergraduate and postgraduate students.

I am also International Coordinator for the Health Behaviour of School-aged Children (HBSC) Study, a large cross-national study of adolescent health and wellbeing, conducted in collaboration with WHO. I have overall responsibility for the scientific leadership of the study and coordination for our network activities.

Could you tell us some more about CAHRU’s background and the kind of research that is done there?

CAHRU was established in 2000 by the current Director, Professor Candace Currie, as a specialist research centre to undertake research into the health of young people from early childhood to late adolescence. The Unit moved to the School of Medicine at the University of St Andrews in 2011 and currently comprises 12 staff in academic, research, specialist and research administration roles. CAHRU hosts the HBSC International Coordinating Centre which provides scientific leadership and support to the HBSC international network of over 350 adolescent health experts from 45 countries in Europe and North America. This includes experts from a wide range of disciplines such as clinical medicine, epidemiology, biology, paediatrics, pedagogy, psychology, public health, public policy, and sociology. The

Through both the HBSC International Coordinating Centre and WHO Collaborating Centre, CAHRU has played a leading role in raising awareness of child and adolescent health within Scotland and internationally www.researchfeatures.com

study therefore enables cross-fertilisation of a range of perspectives that has resulted in an innovative scientific framework which captures the contextual environment in which young people live thus allowing us to gain an insight into determinants and possible mediators and moderators of young people's health.

Our research aims to improve understanding of child and adolescent health in Scotland and globally, through collecting primary data on health, health behaviours and their social determinants, monitoring trends, designing and evaluating effective interventions, and using the evidence gathered to influence policy and practice.

We study young people’s health in the context of: biological and other developmental processes associated with puberty; social inequalities stemming from family, school, neighbourhood, gender, age, and social-economic conditions; local and national services and policies; school as a setting for health promotion; and international comparisons.

In 2013 the World Health Organization designated the University of St Andrews School of Medicine as its Collaborating Centre for International Child and Adolescent Health Policy (WHO CC). Could you tell us some more about this? How did this collaboration first come about?

The designation resulted from a successful collective effort within the population and behavioural sciences group in the School of Medicine to bring international distinction to the School and the University. Professor Candace Currie and Aixa Aleman-Diaz, from our team in CAHRU, led this effort to further our relationship with the WHO European Regional Office in Copenhagen, with a unique focus on child and adolescent health policy. This prestigious appointment endorses the international aspect of our research and evidence-based policy work.

The WHO CC’s workplan brings together three independent research teams within the School under a common vision to improve child and adolescent health worldwide. This vision is achieved through several strands of work related to social determinants of health and prevention of health inequalities, reduction of youth violence, and prevention of risk behaviours, such as drug use. The WHO CC has been a leading partner in increasing HBSC’s policy footprint, and aims to increase the School’s impact and influence in the international

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Thought Leadership

arena, especially through building links with leading policy-making bodies. Its focus is to enhance the School’s knowledge transfer efforts, specifically to advance the debate on adolescent health.

Which areas of child and adolescent health research are CAHRU currently working on?

We have just undertaken a major review of the HBSC international protocol in preparation for the next survey round in 2017/18 and are finalising topics for inclusion in the Scottish survey. We try to hold in balance the need to remain current in terms of addressing new and evolving priorities in adolescent health whilst retaining important trend data over the last 25+ years.

We are working with WHO on a new report on Childhood Obesity, highlighting international trends in inequalities in obesity, eating behaviours, physical activity and sedentary behaviour.

The report will be published as part of the WHO Health Policy for Children and Adolescents (HEPCA) series and will be used to make the case for action on obesity among European member states. We are also collaborating with UNICEF on the latest in their series of Report Cards which focus on the wellbeing of children in industrialised countries. This builds on successful previous collaborations which used HBSC data to highlight inequalities in children’s health and wellbeing.

Our work also includes evaluations of interventions. For example, CAHRU staff are currently collaborating on a large-scale longitudinal study assessing the impact of measures in The Tobacco and Primary Medical Services (Scotland) Act 2010 on young people’s exposure to tobacco advertising, their attitudes towards smoking and ultimately their smoking behaviour.

CAHRU is leading one of four research strands involving an annual survey of secondary school children in selected communities within Scotland.

From a more personal perspective, what are your main research interests within child and adolescent health?

Broadly speaking, I am interested in social determinants of health, health inequalities and international comparisons in young people’s health. Much of my work has focused on physical activity and sedentary behaviour, nutrition, school health and health promotion. Currently, I am very interested in

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Dr Jo Inchley presenting her findings at the HBSC Lisbon 2015 Autumn meeting

mental health which is emerging as one of the most pressing public health concerns for adolescents today. Recent HBSC findings, for example, have shown marked increases in mental health issues particularly among older adolescent girls, which may be linked to issues such as academic pressure and social media use. Understanding the reasons for these trends is essential to informing effective prevention strategies. I am also working with international colleagues on some relatively new areas within HBSC such as spiritual health, disability and chronic conditions, stress, and sleep.

furthering our understanding of the social determinants of health and health inequalities, and promoting evidenceinformed policy and practice. In Scotland, we work closely with national funders and partners such as NHS Health Scotland to ensure that our research is used to inform policy development. Examples include the Scottish Physical Activity Strategy ‘Let’s Make Scotland More Active’ (2003), the Schools (Health Promotion and Nutrition) (Scotland) Act 2007, and the Scottish Government’s Pregnancy and Parenthood in Young People Strategy 2016-2026.

What impact do you think CAHRU has had on child and adolescent health research since it was first established in 2000?

Through both the HBSC International Coordinating Centre and WHO Collaborating Centre, CAHRU has played a leading role in raising awareness of child and adolescent health within Scotland and internationally,

Internationally, HBSC provides a unique dataset on adolescent health and findings from the study are widely used by national governments and international agencies such as WHO, OECD and UNICEF. In collaboration with WHO, CAHRU initiated a series of WHO/ HBSC Forums to maximise the use of HBSC data across Europe, to promote discussion

While there have been some recent positive trends in adolescent health, such as reductions in drinking and smoking, key challenges remain.

These include non-communicable diseases, obesity, sedentary lifestyles and mental health www.researchfeatures.com

infections and teenage pregnancy and other health indicators worsen such as eating habits, physical activity and stress.

CAHRU has a strong emphasis on translation in all our research. Since our inception in 2000, CAHRU has invested in dissemination and knowledge-exchange activities designed to reach and engage policy makers, programme developers, schools and young people, as well as academic audiences. Good research alone is not a lever of change until it becomes an integral part of the policy discourse; in St Andrews we are aiming to make sure our research affects the way we as a society think, talk and invest in the health of our young people.

among international partners and to facilitate the translation of research findings into effective policy-making and practice.

Most recently, HBSC data were presented at the WHO European Ministerial Conference on the life-course approach in the context of Health 2020, held in Belarus in 2015. HBSC data also informed the development of Investing in Children: the European Child and Adolescent Health Strategy 2015-2020 and are being used to monitor implementation of the strategy across WHO European member states.

Why is advancing the debate on child and adolescent health so important and how does CAHRU go about doing this?

Adolescence is a critical transitional period including biological changes associated with puberty and the need to negotiate key developmental tasks, such as increasing independence and normative experimentation. Adolescence is also the time in which many behavioural patterns are established which help to determine not only their current health status but also future health outcomes.

Although it is generally considered to be a healthy stage of life, several important social or public health problems start or peak during the adolescent years, such as substance use, mental ill health, sexually transmitted

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Do you think research on child and adolescent health receives as much funding and attention as it should?

Recent international publications have highlighted the importance of the “second decade” as a critical stage of life course, including UNICEF’s State of the World’s Children report in 2011 and their report card on adolescents in 2012, the Lancet series on adolescent health in 2012 and the WHO Health of the World’s Adolescents Report in 2014. Globally, we’ve seen huge progress in the early years, such as a reduction in the under-5 mortality rate, but this has led to a shift in mortality and disease burden from childhood to adolescence.

Adolescents now make up more than a quarter of the world’s population but the health of adolescents has improved less than that of younger children over the past 50 years. As a result there has been a call for greater investment in the adolescent years to protect public health investment in early child development and help rectify problems arising during the first decade.

Many of the research teams involved in HBSC are facing funding difficulties due to lack of available research funding at national level.

Investment in child and adolescent health requires investment in research to provide reliable data to underpin evidence-based policy making.

as reductions in drinking and smoking, key challenges remain. These include noncommunicable diseases, obesity, sedentary lifestyles, and mental health.

There is also a need for better understanding of the impact – both positive and negative - of digital technologies, and social media in particular, on young people’s health and wellbeing. Tackling health inequalities must remain a top priority. Targeted interventions are needed to reduce the social gradient in health and improve opportunities and outcomes for the most disadvantaged groups of young people. This can only be achieved through intersectoral action addressing the social, economic and environmental determinants of health.

Global initiatives such as the new Lancet Commission on Adolescent Health can help ensure that young people’s voices are heard and effective action is taken to address their needs.

I hope that young people themselves will also have an increasing role to play in shaping policies and programmes which affect their lives.

• If you would like to keep up to date with the latest developments from the HBSC study, please visit the HBSC website at www. hbsc.org. Further information about the WHO Collaborating Centre is available at www.whoccstandrews.org.

Contact Child and Adolescent Health Research Unit, University of St Andrews Medical and Biological Sciences Building North Haugh, St Andrews Fife KY16 9TF E: cahru@st-andrews.ac.uk T: +44 (0)1334 461732 W: http://www.cahru.org/

How do you see the landscape of child and adolescent health changing over the next ten years?

Improvements in child health are likely to continue, and it is important that these gains are not lost during the adolescent years. While there have been some recent positive trends in adolescent health, such

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Paving the way: converting scientific research into substance abuse treatment methods With over 27 years of combined experience, Dr Jaime Mulligan and Dr Ashli Sheidow from Training Support System (a division of Sheidow Consulting, Inc.) have dedicated their recent research to ensuring scientifically proven methods are translated into clinical practice. Their new web-based training and support system is paving the way for improved treatment outcomes for substanceabusing adolescents.

Adolescent Health

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espite significant recent advancements in drug abuse treatment methods, adolescent substance abuse remains a serious problem. The 2011 National Survey on Drug Use and Health found 7% of American youths between the ages of 12 and 17 met criteria for substance (illicit drug or alcohol) dependence or abuse.

A DIFFICULT PATH AHEAD Although the issue of substance abuse is prevalent across multiple age groups, it presents specific risks and challenges in adolescents. Substance use in early teenage years is associated with higher levels of substance abuse and dependence later in life, often with deleterious effects on educational, social, physical, employment and mental health outcomes. Together, Mulligan and Sheidow have developed a new webbased system that is breaking new ground in counsellor training. The system provides training on a proven treatment method and delivers long-term support for counsellors to ensure that adolescents with substance abuse disorders receive effective treatment.

BEST FOOT FORWARD Evidence-Based Practice (EBP) is a relatively new approach that has rapidly been gaining popularity and traction in the clinical world, and particularly in substance use treatment.

EBP is widely defined as ‘the integration of the best available research with clinical expertise in the context of patient characteristics, culture and preferences’ (Levant, 2005). In practice, an EBP approach uses treatment methods that have been found to have positive effects on patients’ progress in randomised clinical trials. As awareness of EBP spreads, these research-supported methods are being demanded not only by funding bodies and federal agencies but also by those individuals seeking treatment. EBPs are also gaining popularity from counsellors themselves, prompting research into how to ensure these treatments are delivered and sustained for optimum patient outcomes.

One such treatment method is Contingency Management for Youth Addiction (CM-YA)

which combines behavioural and cognitive behavioural principles into a family treatment to target drug use. CM-YA is based on the principle that drug use is an operant behaviour and can therefore can be controlled by the individual with proper supports in place. Practically speaking, the principle of CM is that positive achievements such as negative urine tests or achievement of treatment goals are reinforced with immediate rewards such as vouchers or small cash prizes.

CM has been found to have highly successful results in many studies, including those focused on youth addictions. For instance, Azrin et al (1994) used a randomised trial to compare CM-YA to supportive counselling, and found that youths receiving CM-YA were eight times more likely to abstain from drug use. Furthermore, CM-YA has been found to be safe to implement, produce positive outcomes across a range of addictions and work effectively alongside other available treatments (Carroll, 2004). In addition, community-based outpatient programmes currently provide the majority of substance use treatment. Due to its combination of behavioural therapy, cognitive behavioural therapy, and caregiver involvement, a CM-YA treatment model is particularly applicable to patients in this setting. This encouraging base of evidence has prompted Mulligan and Sheidow to focus on CM-YA treatment for their recent studies.

A GAP IN THE ROAD Despite the wealth of evidence supporting EBPs, and specifically CM-YA, it is clear that there are critical barriers between attaining knowledge and translating this knowledge into actual treatment practices. Although most practitioners support the use of EBPs, very few substance-use patients actually receive any evidence-based care. This gap between science and service can, in part, be attributed to the past stigmatisation of substance abuse issues, resulting in the development of treatments originating outside of mainstream healthcare. However, there are a range of additional factors that are inhibiting the uptake of EBPs in the modern medical world. Systemic barriers, such as cost and a lack of resources to learn a new

Contingency Management (CM) has been found to have highly successful results in many studies, including those focusing on youth addictions 27

Contingency Management for youth addictions (CM-YA) is based on the principle of immediately rewarding positive progress. Can you expand on how this is delivered practically in the context of substance use treatment?

The counsellor works with the youth and their family to develop a list of rewards that motivate the youth. These can be monetary rewards or non-monetary such as privileges, time with family or friends, favourite activities, or getting out of a chore. Once the list of rewards is finalised they are tied to points. At each session, points are earned for a negative drug screen or lost for a positive drug screen.

Once the youth has enough points, they can select a reward off the menu when they have a negative drug screen.

The treatment is divided into 3 levels. As the youth achieves consecutive weeks of abstinence (tested by drug screens), they move up the levels – they earn more points and rewards become bigger. This level system helps maintain motivation in treatment. In addition, the youth and family identify a Most Valued Privilege. The youth earns this MVP with every negative drug screen. The MVP ensures they always earn a large reward with each negative drug screen, in addition to the points that they can cash in for rewards. The MVP ensures that the youth has a clear, meaningful reward on their mind when they are faced with peer pressure or triggers to use. They can make a decision in the moment – if they want to use, they lose the MVP; if they resist peer pressure and manage triggers, they earn the MVP – and they earn points on top of getting the MVP! Is CM applicable to all drug and alcohol problems? Do different drugs require different treatment approaches?

CM has been found to be successful with all drug and alcohol problems. The only difference in treatment would be the frequency of drug testing. Drugs that exit the system quickly will require more frequency and random drug tests.

Why is the CM family-therapy approach particularly effective for adolescents?

It includes family in every stage of treatment by teaching them how to recognise signs of drug use, effectively supervise the youth, reward them for making good decisions, and help identify triggers so that they can prompt the youth to use their skills to manage the triggers without resorting to drug use. In addition, the interventions target the unique developmental needs of adolescents.

Typically family involvement in treatment is rare. The TSS trains counsellors in the importance of family involvement, the processes behind family treatment and how to engage parents in the treatment. The ABC assessment process also demonstrates to the youth the clear link between their triggers, their drug use, and the negative outcomes in their life in a very concrete and meaningful way. The point-and-level system provides a concrete way for achieving success and monitoring progress, which is important for adolescents still learning how to link their decisions to their behaviours.

Treatment is often provided in a community-based outpatient setting.

Can you explain what this is and the implications for practitioner training?

Community-based outpatient settings are typically the first, and lowest-cost, treatment option provided to a teen. With this option the youth and family come to the office to meet with the counsellor once or twice a week for about one hour each visit. For the counsellor, the focus becomes

The Training Support System (TSS) for CM-YA is an entirely unique, web-based training and support platform that offers a cost-effective and accessible alternative to conventional training methods 28

maximising the number of sessions each week to ensure revenue, which leaves little time for training and development.

Typically, counsellors will do the minimum training required to maintain their licence.

Our TSS addresses this by making training convenient and meaningful and by providing the research demonstrating that when you use an EBP, and you implement it with fidelity, your patients are more likely to show up for their session and will stay in treatment longer. In addition, insurance companies will often pay a counsellor more money per hour if they can prove they are implementing an EBP. This reimbursement model is often called “Pay for Performance” and it is increasingly common that insurance will either not pay, or pay you less money per hour, if you do not implement an EBP. How can CM-YA ensure long-term effects even when the rewards have ceased? a) Treatment focuses on the concrete skill deficits and environmental factors that are triggering the drug use. The youth earns rewards for negative drug screens, but most treatment is updating the ABC assessments (functional analyses) so that we have a current, working understanding of the individual teen’s drug use patterns.

This allows us to focus intensively on their specific needs. This skill development drives long-term abstinence. b) Family involvement is critical. The family learns how to support their teen and monitor them once treatment is over. c) As treatment progresses, the rewards in the point-and-level system are replaced entirely by natural rewards and activities which the family provides. Teens struggling with substance use often have legal problems, have educational issues, or are not permitted to have a job or participate in sports or activities. In addition, much of their time is spent getting money for drugs, obtaining the drugs, using the drugs, and hiding their use. As the teen decreases drug use, they have more time to engage in activities they once enjoyed and they can access privileges that were once forbidden because of the drug use. This return to “normal life” is highly motivating and sustainable.

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Adolescent Health

Detail RESEARCH OBJECTIVES Mulligan and Sheidow focus their research efforts on how best to train practitioners in adolescent substance abuse to ensure that best practice research findings make their way into real-world treatment.

FUNDING NIH: National Institute on Drug Abuse COLLABORATORS Aspects of the TSS for CM-YA grew from Dr Sheidow’s work with Scott Henggeler, PhD, and others.

EBP create practical obstacles in EBP uptake.

Furthermore, personal barriers such as an unwillingness to accept that long-practised methods may not be best and resistance to change also play an important part. Alongside these factors, one of the key points to come out of numerous studies is that treatment models provide vastly improved clinical outcomes if they are delivered with fidelity (remain true to the model) and competency.

This has served to shift the spotlight onto how best to train and monitor practitioners, alongside monitoring patient progress.

BRIDGING THE GAP To overcome the barriers mentioned above, and thus deliver better patient treatment, Mulligan and Sheidow have developed the Training Support System (TSS) for CM-YA. This is an entirely unique, web-based training and support platform that offers a cost-effective and accessible alternative to conventional training methods. Training and support are delivered in a three-tier design as below: 1) Basic initial training in CM-YA is provided through the Contingency Management Computer Assisted Training (CM-CAT) that counsellors can complete at their own pace.

This basic initial training includes a training companion toolkit and connects counsellors via the web with a Lead Trainer who monitors progress and provides 1:1 support, practice opportunities, and resources. Importantly, this web-based approach overcomes the financial burden faced by many communitybased agencies and prevents scheduling and travel barriers. Additionally, it has proven to be popular with users: in a recent trial, 97% of people who completed the CM-CAT described it as “extremely helpful� and an increase in CM knowledge was observed following the training.

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2) The second stage is the provision of ongoing feedback, Lead Trainer availability, and professional networking on both an individual and agency-wide level. Feedback relating to the understanding and use of CM-YA is provided through knowledge tests and coded session tapes. In addition, confidential monthly surveys from caregivers provide counsellor-specific and agency-wide feedback on adherence levels. These Key Performance Indicators are summarised quarterly in an implementation report provided to the counsellor and agency with recommendations for improving outcomes and adherence to the treatment model.

3) Training is sustained within the TSS with the availability of ongoing support and mentoring as well as access to up-to date literature and tools. A CM-YA expert Lead Trainer is assigned to each agency and, through this trainer, counsellors have access to resources, feedback and training that is tailored to their individual needs.

This coaching can take multiple forms but may consist of elements such as web-based booster training sessions or role-play.

Thus, the TSS provides a training method that attends to all the possible barriers of dissemination, as well as providing comprehensive and ongoing support that continues well after the initial training is delivered. This will have vast positive implications for substance-abusing adolescents receiving treatment from practitioners trained through the TSS and sets a new level for how training methods can ensure both competency and fidelity.

BIO Jaime Mulligan, PsyD (formerly Jaime Houston) has over 14 years of experience implementing evidence-based programmes in the community. Dr Mulligan is currently the Vice President and Clinical Director for Training Support System and the Principal Investigator for current NIDA-funded studies evaluating ways to effectively train professionals to use evidence-based treatments.

Ashli J Sheidow, PhD, is a Senior Research Scientist at the Oregon Social Learning Center (OSLC) and President of Training Support System. She is NIDAand NIMH-funded to research treatments for mental health and substance abuse problems in adolescents and emerging adults, particularly those who have co-occurring problems. Her research also focuses on effective dissemination of evidence-based practices, particularly training practices for community-based counsellors.

CONTACT Jaime L Mulligan, Vice President Training Support System (a Division of Sheidow Consulting, Inc.) 3003 West 11th Ave, #168 Eugene, OR 97402, USA E: jmulligan@tssarena.com T: +1 717 253 4556 W: www.trainingsupportsystem.com MORE INFO: http://trainingsupportsystem.com/meetour-team-2

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Making connections: statistical methods to investigate health-risk behaviours in adolescents

Adolescent Health

Clinical psychology research is increasingly looking to use information from multiple sources to understand the processes behind known relationships. Dr Ginger Lockhart, Assistant Professor of Quantitative Psychology at Utah State University is developing innovative statistical methods to investigate the mechanisms by which adolescents develop health-risk behaviours.

to choose their friendship groups based on common alcohol intake. However, this study also found that adolescents do not clearly assimilate their alcohol intake to that of their friends. This study implies that prevention programmes targeting alcohol use among young people would benefit from a focus on preventing the formation of negative friendships.

n the world of clinical research, there is currently a great interest in establishing not only relationships between variables, but also how and why one variable may affect another. A good understanding of such causal processes allows effective prevention methods to be identified and can enhance our understanding of how treatment methods operate. Statistical mediation analysis investigates how an independent variable (X) influences a mediator (M), which in turn affects a dependent outcome variable (Y). This method explores the underlying mechanism behind a known relationship and is employed when there appears to be no definite connection between the independent and dependent variables.

DISORDERED EATING In order to examine the relationships between weight stigmatisation and disordered eating, Dr Lockhart tested the presence of two distinct mediated pathways: stress as a mediator between weight stigmatisation and emotional eating; and social withdrawal as a mediator between weight stigmatisation and dietary restraint.

The hypothesised pathways were tested with a path analysis followed by a random sampling method, known as bias-corrected bootstrap mediation analysis.

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In clinical psychology research, the use of multiple informant (MI) data is considered best practice to assess variables X, M, or Y. An informant can be anyone who reports on a case, for example a patient (self-report), parent, or healthcare worker. Thus, MI data describes the combination of reports from multiple sources. This approach is widely used and deemed best methodological practice as it allows for the collection of different perspectives and the determination of convergent validity (the level of agreement between reporters).

In her research, Dr Lockhart aims to develop innovative statistical techniques that can be used to address complex problems in prevention and intervention science.

Her current work combines MI data with statistical mediation analysis to discover the mechanisms through which adolescents develop health-risk behaviours. Results from Dr Lockhart’s research have important implications for informing prevention programme strategies.

UNCOVERING THE LINKS Dr Lockhart’s research on statistical methods

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has a wide range of applications. However, the primary focus is how these methods can be used to understand how young people develop health-related habits. In a number of previous studies, Dr Lockhart has applied varying statistical approaches to identify the processes by which adolescents develop a range of health-risk behaviours.

TV AND UNHEALTHY SNACKS In one such study, Dr Lockhart used a single, multilevel multivariate path model to explore connections between adolescents’ personal characteristics, technology use and food cravings. The study found that ethnicity was a significant factor, with TV exposure in non-Hispanic youths associated with cravings for sweet snacks, sweetened drinks and salty snacks more so than for Hispanic youths. Hispanic youths showed stronger associations between phone messaging and sweet snack, sweetened drink and salty snack cravings. Gender was also found to be a significant mediating factor, with stronger associations between video games and salty snack cravings shown for males.

ALCOHOL INTAKE A separate study investigated how adolescent alcohol use is influenced by the dynamics of social networks. In this study, Dr Lockhart used a network dynamic model (longitudinal stochastic actor-based model) to differentiate between peer selection and social assimilation. Adolescents were found

Results showed that both stress and social withdrawal partially mediated the pathways between weight stigmatisation and emotional eating and dietary restraint, respectively. As weight stigmatisation is a widespread social issue, targeting these person-level mediating factors is a practical way of addressing disordered eating in the study population.

RISK BEHAVIOURS A further study published by Dr Lockhart earlier this year examined how self-worth mediates between adolescent attachment security and three distinct risk behaviours (physical fights, weapon carrying and substance abuse). A longitudinal study was employed to examine how these variables interact over time in a study group of adolescent impoverished African Americans.

Results were obtained using a method of

Dr Lockhart aims to develop innovative statistical techniques that can be used to address complex problems in prevention and intervention science 31

Adolescent Health

investigation (ordinal logistic path analysis) which simultaneously specified the three mediated pathways linking attachment security to the three risk behaviours. The results confirmed the hypothesis: greater attachment security is associated with future higher self-worth and subsequent lower substance use and weapons carrying later in adolescence, although the findings for violence were inconclusive. The confirmation of these mediated pathways suggests that adult mentorship is an important component for effective prevention programmes.

COMBINING METHODS Whilst efforts have previously been made to combine MI data with mediation analysis, this task presents many methodological and practical challenges and previously developed approaches have significant limitations. One commonly used approach involves taking the average of scores from multiple informants and using this average to create a composite score. The mediation analysis is then performed on this new composite score. Although this approach is simple, it does not allow for the determination of convergent validity and it assumes all informant scores should be equally weighted. An alternative approach reports separate mediation analyses for each informant type, providing interesting informant-level details but failing to provide a single comprehensive statistical model.

A third commonly used approach takes the observed scores from informants and uses them as indicators of a latent (unobserved) variable. The modelling of mediating effects is carried out on the latent variables. This has the advantage of allowing different informant reports to be given different factor loadings. However, these models are susceptible to overestimations of random error variance and underestimation of the reliability of observed variables, resulting in bias in the model.

In order to address these limitations, Dr Lockhart has developed an innovative new approach for combining MI into statistical mediation analysis. The new approach is based on a CT-C(M-1) model. Within a CT-C(M-1) approach, each informant type reports on each trait (e.g., X, Y and M for mediation studies). Multiple observed variables are used as latent variable indicators for each trait and each informant type. When MI data is combined with this approach, one informant serves as the reference informant. Non-reference reports are contrasted against reference reports,

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enabling researchers to examine how well informants’ reports converge to the ‘gold standard’ reference report.

Dr Lockhart’s new approach expands on the CT-C(M-1) model by combining it with mediation models that are already commonly used in path analysis. This latent variable approach allows for the correction of random error. Furthermore, it allows trait effects, informant effects and measurement error to be separated, facilitating the quantification of convergent validity, informant specificity and informant reliability. Each informant type and trait has multiple indicators allowing the study of whether discrepancies between informants are shared across different informants or generalised for different traits. Another advantage of this method is that differences

in wording or content that result in method effects can be properly accounted for due to the use of variable-specific trait factors. In addition, the model provides the flexibility to be used in situations where either all or only some constructs (X, Y and M) are assessed with MI. STEPS TO PREVENTION Dr Lockhart’s new approach provides a methodology that is appropriate to the analysis of mediating effects in the context of a MI study and will have significant impacts for adolescent health-risk prevention programmes. The successful dissemination of these results will provide crucial knowledge for those working in the field of prevention when designing effective interventions and informing prevention programme strategies.

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Detail Your research focuses on adolescents, what particularly interests you about this study group?

This developmental stage fascinates me because of the rapid brain and social changes that are taking place. These changes create a complex web of both strengths and vulnerabilities that challenge us to optimise positive health outcomes as adolescents transition into early adulthood and beyond.

When assessing how a given variable may affect a certain behaviour, why is it important to consider reports from multiple informants?

With few exceptions, considering only selfreport of a measure such as depression introduces bias, because people (particularly kids), tend to do a rather poor job of reporting on their own psychological and behavioural functioning. Moreover, it gives us the opportunity to discover the extent of overlap vs non-overlap between reporters and across contexts, which can be interesting in and of itself. For example, some kids may show more outwardly obvious signs of depression, and they may therefore show strong agreement in their depression scores as provided by their teachers and parents. Other kids may behave differently at school vs home and show disparate scores between reporters.

These differences in reporters’ overlap are clinically meaningful because they may uncover the extent to which psychological problems are consistent across contexts or specific to a situation (e.g., a child shows depressive symptoms at school but not at home).

You have shown that, for many healthrisk behaviours, the cause and the outcome are linked by a mediating variable: M. When conducting a study, how do you choose M?

The primary reason to perform a mediation analysis in the context of prevention science is to identify variables in a causal sequence that can potentially be acted upon to effect change in an outcome. Mediators must therefore be malleable, meaning that they have the potential to change if targeted in an intervention, and they must be intermediate in that causal sequence, such that changes in a predictor happen first, which then cause changes in the mediator, which in turn cause changes in a health-risk behaviour.

Why is it effective to focus on the mediating variable in prevention programmes?

This theory-driven approach to preventing health problems is effective because it is typically not possible to directly change a health behaviour; practitioners are not in a position to physically keep kids from smoking or using drugs when they are living their daily lives. But we can identify malleable variables, that, if changed in a healthy way, can therefore exert changes in their behaviour. For example, we have found that working on kids’ self-worth reduces the likelihood of substance use over time.

How do you ensure the successful dissemination of your research findings so that they can be employed practically in prevention programmes?

We publish our findings in applied journals with a wide reach covering both researchers and practitioners and present our work at clinical conferences.

Beginning this year, we will begin producing animated video shorts to disseminate through social media to communicate our findings to an even wider audience.

Dr Lockhart’s new approach is set to have a significant impact on adolescent health-risk prevention programmes www.researchfeatures.com

RESEARCH OBJECTIVES Dr Ginger Lockhart has been developing innovative statistical methods to investigate the ways in which adolescents develop health-risk behaviours.

FUNDING National Institute of Drug Abuse and United States Department of Justice COLLABORATORS Lab Associates: • Tyson Barrett • Amanda Hagman, MS • Morgan Kawamura • Emily Long, MEd • Melissa Simone, MS Collaborators: • John Bolland, PhD • Laurie Chassin, PhD • Rick Cruz, PhD • Christian Geiser, PhD • Nicholas Ialongo, PhD • Michael Levin, PhD • David MacKinnon, PhD • Greg Madden, PhD • Kim Reynolds, PhD • Michael Twohig, PhD BIO Ginger Lockhart is an Assistant Professor of Quantitative Psychology at Utah State University. Her research is driven by two primary goals: firstly, to discover the processes through which young people develop health-related habits and secondly, to develop innovative statistical techniques to answer complex questions in prevention science.

CONTACT Ginger Lockhart, PhD Assistant Professor Department of Psychology Program in Quantitative Psychology Utah State University E: ginger.lockhart@usu.edu W: lockhartlab.org https://www.researchgate.net/profile/ Ginger_Lockhart

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Thought Leadership

The champion for maternal health Genesis Research Trust’s leading fertility expert, Professor Robert Winston, discussed maternal health and the organisation’s current research advances with Research Features.

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ollowing numerous TV appearances and countless years of medical research, Professor Robert Winston has become the friendly face of science in the modern era.

However, away from the limelight, he is not only a massive advocate for charity-based scientific research at Genesis Research Trust, but he is also a huge ambassador for women and infants’ health.

He sat down with us at Research Features to discuss how women’s health has changed throughout his time as a researcher, while highlighting any areas where he feels further work is required.

Hello Robert. Thank you ever so much for agreeing to talk with us today. Could you tell us a bit about the work you are currently doing at the Genesis Research Trust related to maternal and infant health?

The most dangerous journey we all take is the four inches down the birth canal, as that is actually where most people get damaged or die. That damage can have a very longterm effect on future health even when you’re 60 or 70 years old, so the research we do at Genesis Research Trust is vital to understanding how we can prevent this.

The Genesis Research Trust has really evolved from what used to be called The Institute of Obstetrics and Gynaecology which was founded after the Second World War. It’s always been a major hub for research and is probably the leader across Europe for the work it does in all aspects of women’s health. This involves pregnancy, fertility, delivery, early growth of babies and cancers which only affect women. My work at the moment is mainly involved with fertility and I am currently looking into embryology and at how sperm develop.

Are there any other areas of medical research that are beneficial to your current research?

Definitely. Embryology has more complex and advancing aspects to it that are not just involved with fertility but involve the whole of health and medicine. Recently, I’ve been very interested in using gene modification techniques within animals, such as mice and pigs, to create organs that can be used for human transplantation without the risk of rejection. Although the pigs would be completely normal and totally healthy, these techniques could allow us to find new hearts, new livers, new lungs and new kidneys to use safely within humans.

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Thought Leadership

That would be incredible! It is amazing how far and fast science is progressing – which areas of maternal and infant health do you think have changed the most in your time as a researcher?

When I first started working here back in 1970, the test for pregnancy was to put a pregnant woman’s urine in contact with a toad to see whether it ovulated. Nowadays, however, we can actually test the specific hormone in minute quantities – even in only a single droplet of urine. It’s remarkable.

I think the early detection of cancer has been one of the greatest advances in medicine.

Great strides have been made in the understanding of pre-cancer in the cervix, in conditions such as cervical intraepithelial neoplasia, and this has been due to the cancer smear test – one of the best pieces of early cancer detection in women’s health.

These tests have allowed us to find that cervical cancer can be caused by viruses, and brought on by particular irritants.

Since they were first introduced, smear tests have become more and more sophisticated, so we can now look in much more detail at individual cells to treat specific areas of the cervix in a way that prevents the disease from developing. Ultrasound has also been a major improvement in women’s healthcare because you can look at the ovaries, the uterus and the baby, but also screen for tumours as well. It’s advances such as these which have changed people’s lives.

Are there any areas of women’s health you feel need further research or development?

It’s difficult to say really but at the moment there is a lot of interest in stillbirth.

One of the great tragedies is to go right through pregnancy and then have a dead baby at the end for no apparent reason.

Maternal death is now very rare, but there are still too many babies dying early.

The most common cause for this is premature labour – if a baby is born before they’re ready at 40 weeks of pregnancy, this could not only increase the likelihood of early baby death, but could also lead to the baby suffering from a severe disability or potentially even brain death.

One of the most extraordinary advancements in reproductive medicine has been the discovery of fetal-programming.

We’ve known for a long time that a mother who smokes, drinks excessively or takes

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When women miscarry nobody really talks to them or considers how serious it is on their mental health recreational drugs during pregnancy can damage the foetus – however, we now realise that other factors such as extreme stress or poor nutrition during those nine months can also affect the unborn baby’s life-chances.

Intervention at the very early stages of human development could save a generation of people from diseases affecting not only their mental health but also their physical health.

And how about with issues surrounding miscarriage?

Miscarriage is really important because it’s

extremely common. One in four pregnancies are lost nowadays which, to the woman losing her baby, is a massive catastrophe.

This can lead to mental health issues such as anxiety which could be missed by clinicians because of how common miscarriages have become. When women miscarry they just go to hospital, have their womb scraped out, and nobody really talks to them about it or considers how serious it is on their mental health, because everyone’s busy doing supposedly more important medical research. Very often, miscarriage gets completely neglected – people don’t look for the cause of it and don’t have any clear ideas about how different causes can be treated.

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That sounds encouraging. In terms of healthy baby deliveries though, do you think the emotional ill-health of pregnant women is sufficiently recognised by practitioners? Or should there be more to raise awareness around this?

I think the biggest problem is in depression after delivery and that’s now a very common issue. There are a large number of women who are really quite self-sufficiently unhappy after they deliver – they can’t breastfeed and they’ll have an increased response to anxiety around them. We are now beginning to understand the hormonal basis to that – but that is only one part of the story.

Do you feel more research needs to be done then, focusing on how to prevent miscarriages?

Well, my early work was actually largely involved with miscarriages. What we were doing 25 years ago was really the first treatment for people who were miscarrying repeatedly – this allowed us to screen the embryo for the right genes and for the right chromosomes, which tied with my other research looking at the detection of genetic disease in embryos. More recently though, we have made great strides towards understanding the cause of miscarriages and we now know that certain bacteria are vital for changing how and when women go into labour. This, as I’ve already mentioned, can be important in preventing premature birth and the potential disability and mortality problems that come with that.

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With all of this emerging research, how important is it to consider the ethics behind the potential treatments, particularly in areas of women’s health related to fertility issues like IVF? We need to be very much aware of the ethics of all these treatments, and not just in IVF – I would say we’re probably too aware of the ethics of IVF in the wrong sort of way.

We understand very well the need to be highly cautious when making sure people don’t get the wrong sperm or eggs, and to make sure people aren’t treated as if they’re ridiculously old or too young. I think a much bigger responsibility is in the commercial process, but that we can’t control.

the life support machine off or do you hope it might get better, even though you can’t predict what’s going to happen? These are the problems our clinicians are faced with every single day and they are very difficult to handle.

If you would like to see the full video interview with Robert Winston, please visit http://researchfeatures.com/

Contact Genesis Research Trust, Hammersmith Hospital, Du Cane Road, London, W12 0NN T: +44 (0)20 7594 2192 E: genesisevents@imperial.ac.uk W: www.genesisresearchtrust.com/ /GenesisResearchTrust /Genesis Research Trust @ProfRWinston @GenesisTrustUK

And what about the ethical issues from a clinician’s perspective when dealing with miscarriages or stillbirth?

There are a number of huge ethical issues a clinician has to consider, especially when caring for a new-born child that may not survive and, even if it does survive, could live its life with brain damage. Do you switch

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Women's Health

Fixing uterine fibroids with flaxseed

Dr Romana Nowak and her team, from the University of Illinois, focus their research on determining the physiological causes of uterine leiomyoma formation and investigating potential treatments for this disease.

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terine leiomyomas (fibroids) can be detected in an extraordinary number of women: projected numbers range from 20–80% being affected by the age of 50. Uterine leiomyomas are thought to arise as a response to local inflammation in the myometrial tissue, which is the muscle layer of the uterus. Ovarian hormones, such as oestrogen, influence uterine fibroid formation by stimulating smooth muscle cell proliferation and collagen deposition.

Fibroid dimensions vary considerably, from microscopic to grapefruit-sized and many women may be asymptomatic and unaware that they have this disease. This can be dangerous as uterine leiomyomas can ultimately cause extensive uterine bleeding, anaemia and, most devastatingly, infertility and miscarriages.

Hormonal therapy, surgical procedures (such as myomectomy or hysterectomy) and arterial embolism are the main therapeutic options currently used to treat uterine fibroids. In the USA alone, approximately 200,000 hysterectomies and 30,000 myomectomies are performed annually. However, these treatments can have severe side-effects. Hormonal therapy can cause menopausal-like symptoms including osteoporosis and hot flushes, and hysterectomies render patients infertile. Furthermore, these treatments are expensive – they cost the USA 5.9–9.34

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billion dollars annually. Clearly, there is a compelling case to develop cost-effective and efficient alternative treatments.

As one of the most common gynaecological problems, it is shocking that we know so little about the specific physiological pathways regulating the development of these tumours. Dr Nowak's research focuses on improving our understanding and investigating preventative therapeutic options to inhibit fibroid formation, reducing the risk of lasting damage and the potential need for surgical intervention.

RESEARCHING PATHOLOGIES USING ANIMAL MODELS Animal models are common research tools used to further our knowledge of certain human pathologies – and uterine fibroids are no exception. Dr Nowak and her team have confirmed that the domestic laying hen is the most appropriate animal model used to study uterine fibroid formation because hens suffer from oviductal leiomyomas (the oviduct is a long tube where the egg becomes fully formed) that are histologically and physiologically very similar to uterine fibroids found in women.

WHAT DOES THE 'HEN MODEL' SHOW? Dr Nowak and her team performed a histological analysis on 263 sexually mature hens and found that over the four year reproductive lifespan of hens, the incidence

of oviductal fibroids increased dramatically from 2.5% in year one to 77.3% in year four. Furthermore, the team confirmed that oviductal leiomyomas increased in frequency and size in the older population.

Immunohistochemical analysis indicated that, like uterine fibroids, hen oviductal leiomyomas are comprised of smooth muscle cells and collagen. Gene expression analyses supported this finding – the tumour cells showed an increased expression of oestrogen/progesterone receptors and fibrosis genes, such as collagen type 1.

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Overall, these results support Dr Nowak's hypothesis that uterine fibroid formation results from inflammation or damage caused by menstruation or pathogen exposure (uterine fibroid incidence in pathogen-free hens was 10% – significantly lower compared to pathogen-exposed hens). Resultant inflammation within the uterine myometrial tissue reduces blood supply, causing oxidative stress and hypoxia, contributing to fibroid formation.

TREATMENT USING FLAXSEED Using this hypothesis, Dr Nowak's research

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Dr Nowak's research has shown that year-three laying hens given a diet of 10% flaxseed exhibited uterine fibroids that were 40% smaller on average compared to those found in control hens 39

Women's Health

Dietary Intervention with Flaxseed

team are now focused on identifying effective treatments to decrease the inflammatory response and hence reduce the number and severity of uterine fibroids.

In fact, initial research suggests that dietary intervention could offer a revolutionary new type of treatment.

Dr Nowak's research has shown that year-three laying hens given a diet of 10% flaxseed exhibited uterine fibroids that were 40% smaller compared to those found in control hens. This may be because flaxseed is rich in bioactive components such as omega-3 fatty acids which have significant anti-inflammatory activity.

REDUCTION IN INFLAMMATION This exciting finding was supported by genetic studies which analysed the mRNA expression levels of cyclooxygenase-2 (Cox-2) encoded by the prostaglandin synthetase 2 gene. Cox-2 is an enzyme involved in prostaglandin production which triggers inflammation. It was found that hens fed on flaxseed had significantly reduced expression levels of Cox-2, compared to the control hens, because the omega-3 fatty acids inhibit the Cox-2 enzyme, thus suppressing prostaglandin production and reducing inflammation.

Dr Nowak has also shown that curcumin – the active component of turmeric – is also anti-inflammatory and, when fed to hens, similarly reduces Cox-2 expression.

LOWERED OESTROGEN ACTIVITY Flaxseed also contains a phytoestrogen called lignin that has been shown to reduce oestrogen activity by inhibiting the aromatase enzyme (which converts androgens to oestrogen) and blocking oestrogen receptors located on the surface of fibroid smooth muscle cells. Oestrogen inhibition may reduce the proliferation of the smooth muscle cells and collagen deposition further limiting fibroid formation and growth.

Dr Nowak has also shown that treating human fibroid smooth muscle cells in culture with mushroom polysaccharides reduces smooth muscle cell proliferation, and inhibits collagen type 1 and aromatase gene expression.

OTHER THERAPEUTIC BENEFITS OF FLAXSEED Interestingly, flaxseed could also be an effective therapeutic option for diseases other than uterine fibroids. Currently, Dr

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Flaxseed

toestrogen lignan Phy Omega-3 fatty acids

Prostaglandins Oxidative stress Inflammation Angiogenesis Anti-apoptosis Fibrosis

Oestrogens Receptor-mediated proliferation Collagen production

Hypothesis: Lignan and omega-3 work synergistically to combat fibroids

Nowak and her team are investigating the effects of flaxseed on chronic gutassociated diseases such as Crohn’s colitis and inflammatory bowel disease (IBD).

Microbiota activity is extremely important for gut health and Dr Nowak is looking to investigate the effects of flaxseed on the microbiome composition. This will be achieved by feeding hens a diet of 10% flaxseed and comparing their gut microflora against control-diet-fed birds. The team hypothesises that the anti-inflammatory properties of flaxseed may also reduce the severity of these gut-associated diseases by altering the gut microbiome.

FURTHER RESEARCH Finally, Dr Nowak and her team are investigating other dietary factors that

could be used as potential treatments, comparing their efficiency against flaxseed.

For example, they are currently looking at vitamin D (known to have potent antiinflammatory properties) and halofuginone (an anti-fibrotic drug). Initial research indicates that both of these may inhibit cell proliferation and collagen deposition.

Overall, preliminary results arising from Dr Nowak's various studies indicate that flaxseed could offer a promising alternative to existing therapies which may have unpleasant side effects or involve invasive surgery. Although further research is needed to validate these results, Dr Nowak's work could lead to a radical change in how we treat inflammatory disease, including uterine leiomyomas, through dietary intervention.

Dr Nowak and her team have confirmed that the domestic laying hen is the most appropriate animal model used to study uterine fibroid formation www.researchfeatures.com

Detail Why could dietary intervention be a preferred route for treating uterine leiomyomas?

There are currently very limited options for nonsurgical treatment of leiomyomas.

While arterial embolisation is successful for a certain proportion of women with leiomyomas, this nonsurgical treatment cannot be used for everyone. Current drug therapies primarily target ovarian steroid hormone action and often have significant side effects. The potential advantage of dietary intervention is that this would be a more natural method involving the use of dietary components normally present in specific foods that would most likely have minimal side effects and would not be an invasive treatment.

How promising is flaxseed as a preventative treatment option for uterine leiomyomas?

Flaxseed and other dietary components that are known to have anti-inflammatory effects are being investigated by several different research groups as potential treatments for uterine leiomyomas.

However, studies carried out to date have been performed in different animal models or with human cells cultured in vitro. The results have been very promising but it is important that well controlled clinical studies be carried out in human patients before definite conclusions or recommendations can be made. Such studies will probably be undertaken in the next 1–3 years in research hospital settings.

In terms of physiological mechanisms underlying uterine fibroid formation, what areas require additional research?

The mechanisms underlying the pathogenesis of uterine fibroids are still not well understood. Some of the most important areas that still need to be investigated include: the identification of novel genes that may cause fibroids; the role of genetic vs lifestyle factors leading to the greatly increased incidence of symptomatic fibroids in African-American/ black women; the cellular mechanisms linking inflammation to abnormal fibrosis in leiomyoma smooth muscle cells; and

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the potential role of endocrine disruptors and environmental toxicants on incidence of uterine leiomyomas.

What further tests/trials need to be conducted before flaxseed is approved for clinical use?

There is a need for well controlled clinical trials with human patients to confirm both efficacy and safety of flaxseed as a potential dietary intervention for uterine leiomyomas. This is true for any potential dietary intervention strategy. Such clinical trials require participation of large numbers of patients and would involve multiple sites. Different levels of flaxseed would need to be tested with varying lengths of treatment time. Such trials would also involve testing the effects of combined treatment with more than one dietary component such as, for example, flaxseed and vitamin D. However, flaxseed is currently a readily available food supplement utilised by many as an excellent source of anti-oxidants and a means to improve digestive and overall health. Flaxseed is not at present a recommended treatment for uterine fibroids, but it would not require the same level of regulatory oversight as a new drug.

Where do you see your research focus in 5 years’ time?

Our research group will continue to investigate the potential benefits of dietary components as treatment for uterine leiomyomas and we anticipate collaborating with our physician colleagues in clinical trials over the next few years. We are also now beginning to investigate the role of environmental endocrine-disrupting chemicals in the pathogenesis of both uterine leiomyomas and endometriosis. In particular, we are interested in understanding how exposure to such chemicals may promote inflammation, alter the gut microbiome, and contribute to the development of chronic pain. We will be using domestic animal models, epidemiologic data, and in vitro model systems to study the mechanisms involved.

RESEARCH OBJECTIVES Dr Nowak’s work uses the domestic hen model to study the beneficial effects of a flaxseed-based dietary intervention on uterine fibroids. Her research could point to an alternative non-invasive and lowcost treatment for a condition that can cause bleeding, infertility and miscarriage.

FUNDING NIH COLLABORATORS • Dr Buck Hales, Southern Illinois University • Dr Janice Bahr, University of Illinois BIO Dr Romana Nowak is Professor of Animal Sciences and Member of the Institute for Genomic Biology at the University of Illinois. Her research focuses on the study of women’s reproductive diseases using human and domestic animal models and has garnered over $7 million in NIH funding over the last 10 years.

She is very actively involved in mentoring students through several NIH-funded training grants including a Summer Translational Biomedical Research programme for veterinary students; a Reproductive Toxicology Training programme for graduate students; and a Summer Undergraduate Research programme for underrepresented minority students.

CONTACT Dr Romana A Nowak, PhD Professor of Animal Sciences Member of Institute for Genomic Biology University of Illinois 1207 W. Gregory Drive Room 310 ASL Urbana, IL 61801 USA E: ranowak@illinois.edu T: +1 217 244 3902 W: http://ansci.illinois.edu/labs/repro-toxlab /nowak-romana-3930a346

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Emerin and the making of muscle ONM

rin Eme INM

1 AN M

in er Em

2 1/ ir n sp Ne

3/ 4

Emerin

Emerin HDAC3

Btf

Repr e LAD ssed S

Chromatin

Nucleus

Lamins

The role of cellular machinery in the regulation of gene expression is currently a field of intensive study. Dr James Holaska of Philadelphia’s University of the Sciences is leading the way in uncovering the role of emerin in the development and maintenance of skeletal muscle and pathology of muscle disease. This work has the potential to revolutionise the understanding and treatment of muscle diseases, including various types of muscular dystrophy and cardiomyopathy.

merin is a relatively small protein made up of just 254 amino acids. Despite its small size, it is fundamental to the correct formation of muscle tissues and their maintenance and repair. Rich in the amino acid serine, responsible for the catalytic function of many enzymes, it is clearly designed to interact with other molecules. Couple this to

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LAP2 Ă&#x; HDA C3

Repressed

7 LMO

BAF

E

Sun 1/2

Nuclear Pore Complex

Actin

le bu u t cro Mi

Sun 1/2

Ne sp rin

Cytoplasm

the fact that it is highly conserved (the identical protein exists in many other species) and it has all the hallmarks of a vital element of living organisms. Knowing it is important is not enough, however, and Dr Holaska and his team are determined to get to the bottom of its molecular activity and identify the mechanisms by which it exerts its influence on gene expression and molecular pathways.

IT’S ALL IN THE GENES Every cell of an organism contains the entire genetic code of that organism, stored in the form of the now well-known deoxyribose nucleic acid (DNA). However, not every cell needs all of it. Skin cells need to know how to make the proteins necessary for skin, nerve cells those needed to transmit signals, and muscle cells those needed to make muscles move. For this reason, in any one cell, a vast proportion of the genome is silenced or repressed and will never be translated into proteins. How this is achieved is a major question for modern science, particularly considering recent work with stem cells (undifferentiated cells with the potential to become any cell type).

Much of the regulation is thought to be achieved by structural differences in the

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Molecular Biology

ER

Figure 1 – The Nuclear Envelope Emerin, Lap2β, and MAN1 are integral inner nuclear membrane (INM) proteins of the nuclear envelope where they perform diverse functions within the nucleus. INM proteins bind to lamins to form a proteinaceous network called the nuclear lamina that is required for nuclear structure and function.

Emerin regulates gene expression by regulating chromatin architecture, by binding to and regulating the activity of a number of transcription factors, and interacting with components of multiple signalling pathways. ONM, outer nuclear membrane; INM, inner nuclear membrane; ER, endoplasmic reticulum.

Em erin

Em erin ß-Catenin

TCF -LEF

GCL E2F -DP 3

arrangement of DNA in the nucleus (the compartment of the cell where DNA is stored and managed). The entire DNA of a human cell, if stretched out, would be about two metres long. For this reason, it is instead tightly wound around support molecules called histones. This histone/DNA complex is called chromatin and is further packaged into units called nucleosomes. DNA cannot be transcribed for translation into proteins whilst it is stored in this way, so which sections are in storage and which unwound is an important regulatory mechanism in gene transcription.

ENTER EMERIN There are many diverse proteins associated with the regulation of gene expression in the nucleus, some key members of which are identifiable by the disorders caused when they are not correctly expressed. Dr Holaska’s

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Mutations in the genes which code for emerin have been linked to the development of Emery-Dreifuss muscular dystrophy research team have found that emerin is key to the expression of a number of genes important in the maintenance of muscle tissue; mutations in the genes which code for emerin have been linked to the development of Emery-Dreifuss muscular dystrophy (EDMD) as well as certain cardiomyopathies (diseases of the heart muscles). How this modest protein exerts these significant effects is the subject of intense research by the Holaska laboratory.

EMERIN AS THE ARCHITECT Dr Holaska and his colleagues have identified a number of ways in which emerin regulates the architecture of both the nucleus and the nucleosomes which hold the DNA within it. Perhaps the most important of these is emerin’s regulation of histone deacetylase 3 (HDAC3), one of a group of enzymes which alter chromatin structure and regulate transcription factor access to DNA. The team showed that emerin activates HDAC3 and localises it to the wall of the nucleus, where it is able to repress genes by restricting access to the DNA. The researchers have successfully shown that these two molecules work together to localise and activate or repress specific sections of the genome during muscle formation and development. In investigating the structure of emerin itself, Dr Holaska and his colleagues identified the regions of the protein required for specific purposes. These enable the protein to be transported into the nucleus, and for it to then interact with both the nuclear membrane and the underlying matrix of structural proteins called lamins, which supports it. This structural function has been evidenced by showing nuclear membrane dysfunction and increased cell death in emerin-deficient cells.

EMERIN AS THE FACILITATOR The researchers have repeatedly shown that emerin does not work alone, rather it recruits and regulates a variety of other molecules. The most significant findings in terms of EDMD and related myopathies is emerin’s relationship with a specific transcription factor in muscle cell differentiation called Lmo7. The process of muscle cell differentiation is complex and requires particular genes to be ‘switched’ on and off in a time-critical manner. Dr Holaska

and others have shown that emerin is able to regulate the activity of Lmo7 through competitive binding, preventing the factor from initiating expression of genes in a way that is vital for correct differentiation of the cell.

Having identified Lmo7 as a target of emerin’s activity, the team then demonstrated that a mouse model which lacked the gene to make Lmo7 exhibited similar symptoms to those of EDMD. The mice exhibited growth retardation, decreased muscle fibre size and impaired skeletal and cardiac muscle function.

The team were therefore able to confirm that it is this interaction between emerin and Lmo7 which is important for regulating the gene transcription and signalling pathways which are the underlying mechanism of EDMD. In cultures of myogenic progenitor cells (undifferentiated cells which will evolve into skeletal muscle), Dr Holaska and his colleagues have shown that the removal of emerin results in significant perturbation of four critical signalling pathways for cell differentiation.

They propose this is caused by emerin interacting with other molecules involved in the regulation of these pathways such as betacatenin (a dual function protein that regulates gene expression and cell adhesion), and microRNA (short sections of transcribed DNA which interfere with other RNA to prevent translation into proteins). The mechanisms are complex, however, and the team recognise that more work is needed to fully elucidate the pathways involved.

DRAWING OUT THE MERITS OF EMERIN Dr Holaska’s group continues to use the latest molecular techniques to probe the mechanisms underlying nuclear envelope architecture and the role of the proteins that interact with it. To date their work has made a significant impact on the understanding of emerin’s function in both gene expression and the architecture of the nucleus which supports it. By observing normal function and comparing it to that in disease states, they have contributed much to the understanding of EDMD in particular and other myopathies generally. Their research so far shows that there is much more still left to discover in this complex field.

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What led you to begin investigating Emery-Dreifuss muscular dystrophy?

When I first started my post-doctoral fellowship fifteen years ago, it was our initial interest in the function of proteins within the nuclear envelope. The nuclear envelope was then considered by many to be merely a ‘bag’ that held the genome or a barrier that physically separated the genome from the cytoplasmic machinery. However, the presence of integral membrane proteins on the nuclear side of the nuclear envelope suggested these proteins had functions analogous to proteins on the inner side of the plasma membrane. Evidence over the last 20 years showed that the nuclear envelope is important for regulating many important functions, including cell signalling, mechanotransduction, gene expression, chromatin architecture and cell differentiation.

Mutations in one of these integral membrane proteins caused Emery-Dreifuss muscular dystrophy, demonstrating the importance of emerin in cellular function.

We have been studying emerin and its role in normal cellular function and in EmeryDreifuss Muscular Dystrophy ever since.

What is the role of emerin in this disease and cellular mechanisms generally?

Emerin plays important roles in maintaining nuclear architecture and regulating cell signalling and gene expression. Emerin is thought to be important in regulating the transduction of both chemical and mechanical signals from the plasma membrane to the nucleus. Emerin’s role in gene expression regulation is multi-fold, since emerin is important for regulating the coordinated re-organisation of the genome during stem cell differentiation, emerin binds to and regulates the activity of a handful of transcription factors and emerin regulates the nuclear entry and exit of transcription factors. Thus the exact mechanism(s) underlying how emerin regulates gene expression remain illdefined.

How does the architecture of the nucleus regulate gene expression?

There are two ways to think about nuclear architecture. The first concerns the

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structure of the nuclear envelope and the biophysical behaviour of the organelle as it relates to its elasticity, rigidity, ability to resist forces when stressed and the geometrical configuration of the proteins and lipids that generate these biophysical properties. The second concerns genomic or chromatin architecture, whereby repressed genes localise to specific regions within the nucleus, including the nuclear lamina at the nuclear envelope, to induce, maintain or propagate repressed chromatin. Loss of emerin causes the nuclei to become more easily deformed and alters the biophysical properties of the nucleus demonstrating its importance in nuclear structure. Emerin also binds to proteins of the ‘linker of nucleoskeleton and cytoskeleton (LINC)’ complexes, which also play vital roles in nuclear structure and mechanotransduction. Emerin was also found to be required for the temporal localisation of repressed genomic loci to the nuclear lamina when they are turned off during differentiation. Further, the interaction between emerin and HDAC3 was important for positioning repressed genomic loci to the nuclear lamina to inhibit the expression of genes located here. Because nuclear architecture can affect genomic architecture it will be important to determine the mechanisms for how loss of emerin causes altered nuclear architecture and genomic architecture (i.e., the chicken or the egg?).

The mechanisms of gene expression are very complex; how can you be confident that your interpretation is correct?

We can’t really be sure our interpretations are 100% correct, but we are confident we are on the right path. We are confident that emerin regulation of genomic organisation during differentiation via its regulation of HDAC3 is important for the impaired differentiation in emerin-null myogenic progenitors. First, disruption of the coordinated expression of differentiation genes inhibits myogenic differentiation; this is true for most developmental programmes, whereby transcriptional programmes are tightly regulated and coordinated. Genomic regions containing the differentiation genes Pax3, Pax7, MyoD or Myf5 are all mispositioned during differentiation resulting in their expression at inappropriate times during differentiation of emerin-null cells to impair

Figure 2 – Skeletal Muscle Progenitor Cell Differentiation into Mature Multinucleated Myotubes Skeletal muscle progenitors harvested from mice were cultured in a dish and induced to differentiate into long, multinucleated cells that resemble the skeletal muscle fibers in our body. Myosin heavy chain, a component of mature muscle, is shown in red. The nuclei are stained blue. .

myogenic differentiation. Approximately 35% of the genome associates with the nuclear lamina and we think emerin plays a key role in this localisation by interacting with and activating the activity of HDAC3 and other chromatin-repressive enzymes. Based on our results, we further predict emerin may preferentially interact with more dynamic genomic regions containing developmental genes that are activated or repressed in a temporally coordinated fashion. We plan on testing these predictions in the near future.

However, we do not think this is the sole function of emerin in terms of gene expression regulation, given the many studies showing emerin functions directly in transcriptional regulation. First, evidence suggests emerin regulates nuclear accumulation of transcription factors in response to cell signalling, including β-catenin and MKL1, to regulate their activity. It is unclear whether emerin directly regulates nuclear import, nuclear export, or interactions of transcription factors with its binding proteins or DNA targets in the nucleus; these are active research areas in many labs around the world.

Second, we and others showed emerin binding to transcription factors inhibited their ability to bind directly to their promoter or enhancer elements on DNA to inhibit expression of their target genes. Thus the direct inhibition of transcription factor activity

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Molecular Biology

Detail RESEARCH OBJECTIVES Dr Holaska’s work focuses on emerin and its role within the cell. His work has multiple applications, including a better understanding of Emery-Dreifuss muscular dystrophy.

FUNDING National Institutes of Health, Ellison Medical Foundation, American Heart Association COLLABORATORS • E lizabeth McNally, Northwestern University Feinberg School of Medicine • Karen Reddy, Johns Hopkins University School of Medicine • Tatiana Cohen, Center for Genetic Muscle Disorders, the Kennedy Krieger Institute • T erry Partridge, Children’s National Medical Center, Washington, DC

by emerin binding is another mechanism for emerin regulation of gene expression.

How this functions in differentiating muscle cells or in other cell types in the body remains to be seen. Lastly, emerin can be post-translationally modified on more than 15 different residues. Post-translational modifications are well established in molecular biology to regulate the binding of proteins to their partners, to regulate the activity of proteins and to alter the structure of the protein. Thus, how these many posttranslational modifications, individually and combinatorially, regulate emerin’s functions in nuclear structure, chromatin architecture, gene expression and myogenic differentiation will be important areas of future research in our field. For example, is there a posttranslational modification in emerin that blocks HDAC3 binding to emerin to regulate the association of genomic regions with the nuclear lamina to repress their expression? If so, then the dynamic addition or removal of this modification may occur during myogenic differentiation to regulate the association of MyoD genomic loci during myogenic differentiation to regulate its coordinated expression.

How might your work lead to treatments for skeletal- and cardio- myopathies?

Muscle regeneration is impaired in EDMD. By studying the mechanisms by which

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emerin regulates skeletal muscle stem cell differentiation it is predicted therapeutic targets for treatment will be identified.

Recently, we identified a number of molecular pathways misregulated during differentiation of emerinnull progenitors, for which a few inhibitors exist. We are currently creating myogenic progenitors expressing selected EDMD-causing emerin mutants to confirm these findings. We also plan on using unbiased approaches to identify molecular pathways disrupted in emerin-null progenitors and all EDMD-causing emerin mutant progenitors.

Using this approach we predict we will identify pathways implicated in EDMD. Validation of pathway involvement in the disease mechanism will be followed by small-molecule screening for inhibitors or activators of these pathways to rescue the impaired differentiation seen in the EDMD mutants. Other laboratories, notably Howard Worman’s lab at Columbia University, showed that similar molecular pathways may be involved in the skeletal muscle phenotype and cardiomyopathy.

Thus we predict that molecular pathways identified by studying myogenic differentiation will also be important for cardiomyopathy pathogenesis and any inhibitors identified in our studies will be tested for treating cardiomyopathy.

BIO Dr Holaska majored in biology at St. Joseph’s University and graduated in 1995. After working for a year at a molecular biology startup company, he joined the graduate program in Microbiology at the University of Virginia. Working under Dr Bryce Paschal he received his PhD in 2001. He then started a post-doctoral fellowship in the laboratory of Dr Katherine Wilson, where he began studying the structure and function of the nuclear lamina, which is still the focus of his research today.

CONTACT Dr James (Jim) Holaska, PhD Associate Professor Department of Pharmaceutical Sciences University of the Sciences 600 South 43rd Street Philadelphia, PA 19104-4495 USA E: J.holaska@usciences.edu T: +1 215 596 7153 W: https://holaskalab.com/ https://w.taskstream.com/ts/holaska/ HolaskaLab /jim-holaska-940b7b12

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Protect and serve: community-driven superfund science takes aim at asbestos exposure

Cancer

As a direct response to local community concerns, Dr Ian A Blair and colleagues at the Penn SRP Center are using federal funds to study asbestos exposure pathways that lead to asbestos-related diseases. Dr Blair hopes to identify biomarkers of asbestos exposure with the aim of assessing an individual’s risks of developing asbestos-related diseases.

T

he Center of Excellence in Environmental Toxicology (CEET), part of the Perelman School of Medicine at the University of Pennsylvania, used a $10m grant from the National Institute for Environmental Health Sciences (NIEHS) to set up the Penn Superfund Research and Training Program (SRP) Center: a collaborative reactor for scientists working on superfund research. It brings together all the elements necessary to support world-leading science and has six specific project areas under investigation.

Each of the projects addresses a specific community concern.

made up of microscopic fibrils, easily shed as the material is worked or degraded and readily inhaled deep into the lungs. Its resistant properties then become a major problem as the body is unable to reject the material. This causes inflammation at the site and can ultimately lead to the development of a cancer known as mesothelioma. Those who worked with the material are most at risk, but family members and communities near mines and industrial processing centres are also affected. Its persistence in the environment and potential for disturbance through human activity are also major factors in its toxic legacy.

Superfund sites exist across the United States (US), where federal funds are committed to aid the clean-up of sites contaminated by hazardous materials or pollutants. One such site, the BoRit Asbestos site in Ambler, Pennsylvania, is a cause of concern to local communities who are worried about asbestos contamination from lingering industrial waste. A town built almost entirely on its asbestos mining industry, Ambler suffers the legacy of this once promising mineral which became the pariah of twentieth-century industry.

PAINSTAKING DETECTIVE WORK In communities where exposure levels are difficult to assess with any accuracy, a method of calculating individual risk is a vital tool to direct clinical monitoring and followup studies. Dr Blair is investigating how approaches used to identify biomarkers in other disease states, such as his pioneering work on Friedreich’s ataxia, can be turned to asbestos exposure. Oxidative stress (the response of cells to increased levels of reactive oxygen species) is a prime target and thought to be associated with asbestos exposure. This can be effectively measured in blood samples by analysing metabolites in the serum (the fraction of blood left

A FELONIOUS FIBRE Asbestos is a silicate mineral known for its naturally fibrous nature. It was mined extensively in the early twentieth century and used in building materials, as well as many other applications, because of its desirable mechanical properties. Light, strong and resistant to heat, it was often mixed with concrete and other binders to create panels or fittings with these attributes. Each visible fibre, however, is

A town built almost entirely on its asbestos mining industry, Ambler suffers the legacy of this once promising mineral which became the pariah of twentieth-century industry 47

Cancer

Figure 1

Penn SRP Center projects addressing the six major concerns of the Ambler Community Phytoremediation: Project 1 Environmental Science: Project 2 Epidemiology: Project 3 Drs Blair and Emmett with community leaders (Diane Morgan, Bob Adams and Sharon Vargas) at the BoRit Asbestos Superfund Site in Ambler, PA

after it has clotted and the cells have been removed).

Using advanced chromatography techniques and mass spectrometry to separate out the constituents of the samples, the researchers are able to create a series of chromatograms from healthy serum and compare it to that of the disease state. This technique has provided a list of biomarkers which show lack of normal regulation in asbestos-exposed individuals, potentially providing a template against which unconfirmed cases can be assessed.

CALL FOR BACKUP This is just one of the projects at the SRP which houses six distinct research teams working on different aspects of asbestos toxicity: • Project 1, led by Dr Brenda Casper, researching how plants and fungi can be utilised in the remediation of asbestos contamination in the environment.

• Project 2, led by Professors Douglas Jerolmack and Reto Gieré, investigating the mobility and fate of asbestos in the environment to prevent offsite migration.

• Project 3, led by Associate Professors Frances Barg and Douglas Wiebe, studying epidemiological and ethnographic data on the incidence of asbestos-related diseases in the community.

• Project 4, led by Dr Joseph Testa and Prof Rebecca Simmons, investigating animal models of mesothelioma to understand the genetics of mesothelioma and explore possible remediation strategies.

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Cancer Genetics: Project 4 Chemoprevention: Project 5 Diagnostics: Project 6

Can asbestos be remediated in situ?

Is asbestos transported through soil and water?

Why is there a cluster of mesothelioma among women in Ambler?

Is there a genetic predisposition to asbestos-induced mesothelioma?

Can asbestos-induced mesothelioma be prevented?

Is there a blood test for asbestos exposure and mesothelioma?

The major benefit of the SRP Center is that researchers working on separate aspects of asbestos remediation, toxicity, and exposure can collaborate effectively • P roject 5, led by Associate Professor Melpo Christofidou-Solomidou and Professor Steven Albelda, looking into therapeutic agents for the prevention of mesothelioma in asbestos-exposed individuals.

• P roject 6, led by Professor Ian Blair and Assistant Professor Anil Vachani, studying biomarkers of asbestos exposure.

This means that Dr Blair’s team are not operating alone; the major benefit of the SRP Center is that researchers working on separate aspects of asbestos toxicity can collaborate effectively and share their findings. For example, Project 6 is providing data for Project 4 to use in assessing remediation strategies through animal models of asbestos exposure, while Project 5

uses the same oxidative stress markers identified by the team to investigate the efficacy of flaxseed lignans as potential chemotherapeutic agents.

The Center also brings together datacrunching expertise in the form of the Biostatistics Research Support Core (led by Dr Wei-Ting Hwang), provides administrative functions to free up researchers' time and, perhaps the key component, provides a mechanism for the findings to quickly benefit the local community through the Research Translation Core (headed up by Mr Richard Pepino) and Community Engagement Core (led by Drs Edward Emmett and Fran Barg).

There is also a vital training element to the centre (led by Drs Trevor M Penning and

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Detail Why was the Penn SRP Center formed?

The National Institute of Environmental Health Science funded the Center of Excellence in Environmental Toxicology (CEET), directed by Dr Trevor Penning, in 2008. The Community Engagement and Outreach Director of the CEET, Dr Edward Emmett, then began participating in the community advisory group in Ambler, PA where there is a Superfund Asbestos site known as the BoRit site. The concerns of the Amber community were too extensive for the CEET to deal with and so Dr Penning invited Dr Blair to form the Penn SRP Center in order to address these concerns. The six projects in the Center came directly from questions posed by the community (see Figure 1).

What are the advantages to this model of research establishment?

Research involves bi-directional knowledge exchange in order that the Penn SRP Center can address the Ambler community concerns. This differs from the traditional model in which researchers simply assume that the community will benefit from their research. Community engagement connects the Penn SRP Center’s science with issues that are locally relevant and complements the research strengths and problem-solving goals of the Center.

How will your research help identify atrisk individuals?

Biomarker studies conducted in Project 6 of the Center will identify serum biomarkers of response to asbestos exposure as well

Reto Gieré), which gives physicians valuable insight into the causes and treatment of asbestos-related diseases.

SERVING THE WIDER COMMUNITY This model has shown how research centres, engaging with local communities, can facilitate interdisciplinary collaboration to build creative solutions to local problems with global significance. The problem of asbestos-related diseases, while particularly close to the hearts of the communities around the BoRit site, has international relevance. Due to its ubiquity in twentiethcentury construction, populations of

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as biomarkers for the early detection of mesothelioma. The Amber community is participating in these biomarker studies.

Why are these biomarkers present in asbestos exposure?

Asbestos is a fiber and so exposure cannot be analysed directly in the systemic circulation like a regular chemical. Instead, serum biomarkers of response to asbestos exposure have to be discovered. Asbestos is known to cause oxidative stress and the challenge is to discover biomarkers that differentiate asbestos-induced oxidative stress from other causes of oxidative stress such as cigarette smoking.

Asbestos exposure is the major cause of mesothelioma and so biomarkers for the early detection of mesothelioma will also be useful in identifying a prior exposure to asbestos.

What do you hope the centre will achieve for the local community?

The Penn SRP Center provides an independent body for the community members to express concerns that they feel have not been addressed satisfactorily by government agencies. It also provides a resource for medical intervention if a particular community member is concerned about their risk for mesothelioma. Once a serum biomarker of response to asbestos exposure has been validated, it will provide a reliable way for concerned individuals to find out whether or not they been exposed to asbestos.

previously exposed individuals are common around the world, and in some areas, asbestos extraction and use continues despite the known hazards.

The Penn SRP Center shows how it is possible to produce a truly collaborative research and teaching environment, making it possible to combine effective bi-directional community engagement with innovative scientific research. This in turn produces translational science that can rapidly benefit local communities as well as other populations at risk for exposure and other scientists working in the field.

RESEARCH OBJECTIVES Dr Blair’s research at the Penn SRP Center aims to identify biomarkers of asbestos exposure. Knowing which biomarkers signal previous asbestos exposure will help practitioners establish whether an individual is at risk of developing asbestos-related diseases.

FUNDING National Institute for Environmental Health Sciences (NIEHS) P42ES023720 COLLABORATORS From Penn SRP Center: Brenda Casper, PhD; Reto Gieré, PhD; Douglas Jerolmack, PhD; Fran Barg, PhD; Edward Emmett, PhD; Joseph Testa, PhD; Rebecca A Simmons, MD; Melpo ChristofidouSolomidou, PhD; Steven M Albelda, MD; Clementina Mesaros, PhD; Richard Pepino; Wei-Ting Hwang; Trevor M Penning, PhD. Other key collaborators: David R Lynch, MD, PhD; Garret A FitzGerald, MD, FRS; Kathryn E Wellen; Nathaniel W Snyder, PhD. BIO Dr Blair received his PhD in Organic Chemistry in 1971 from Imperial College of Science and Technology, London, under the mentorship of Nobel Laureate Sir Derek H R Barton.

He was appointed as the A.N. Richards Professor of Systems Pharmacology and Translational Therapeutics at the University of Pennsylvania in 1997 and Vice-Chair of the Department in 2002. He became Director of the Penn SRP Center in 2014.

Dr Blair has published over 370 refereed manuscripts and he has an h-index of 63.

CONTACT Ian A Blair, PhD Systems Pharmacology & Translational Therapeutics University of Pennsylvania 853 BRB II/III 421 Curie Boulevard Philadelphia, PA 19104-6160 E: ianblair@exchange.upenn.edu T: +1 215 573 9880 W: http://www.med.upenn.edu/blairlab/ Penn SRP Center: http://www.med.upenn. edu/asbestos/

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Thought Leadership

All under one roof – a pioneering vision for research success Togetherness is a beautiful thing – Professor Jim Hughes and Steve Taylor, two senior biomedical researchers from the MRC Weatherall Institute of Molecular Medicine at Oxford, discuss the joy of collaboration. In their latest shared project, the pair are developing software that will enable scientists to visualise the three-dimensional structure of a DNA molecule inside a cell.

S

ir David Weatherall founded Oxford University’s Institute of Molecular Medicine in 1989 with what was then a pioneering vision of getting scientists and clinicians to work together, all under one roof. At the time, the two specialists worked separately – scientists in the lab, doctors in the clinic. Housing them in a joint location allowed the relationship between the two fields to be fully appreciated and provided new opportunities for the crossfertilisation of ideas. Twenty-eight years on and the renamed MRC Weatherall Institute of Molecular Medicine (MRC WIMM) is now recognised the world over for its innovative biomedical research. With a mixture of scientists and clinicians working side by side, the institute continues to reap the rewards of a collaborative approach to tackling some of the most important questions in medical research. And Sir Weatherall’s pioneering vision has become a winning formula adopted by research centres across the globe.

MRC WIMM researchers Professor Jim Hughes (Group Leader of the Genome Biology Group) and Steve Taylor (Head of the Computational Biology Research Group) met up with Research Features to discuss life at the institute and to share news on where their own investigative partnership is taking them.

Hello Jim and Steve, welcome to Research Features! Could you tell us briefly about the origins of the MRC WIMM. Jim: The MRC WIMM was set up in 1989 by Sir David Weatherall. David had a clinical background but he also understood the power of molecular biology and genetics.

He thought that clinicians should be tapping

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into the kind of scientific developments that were going on, and they needed a way to be trained in proper scientific methods. He also thought that basic biologists should be using the kind of knowledge that could be derived from human disease. He had the idea to house them in one place where they could work coherently together, located beside a hospital, to keep clear clinical links. It was very novel at the time and I think it’s proven to be a very successful model. Many institutes around the world now work in the same way.

What makes us unique, I think, is that we were the first and we’ve developed it a long way. It can take a lot to get clinicians and scientists to work together effectively and I think we’ve got a very good model for doing that.

Could you tell me about your roles at the institute? Let’s start with Steve.

Steve: I head up the Computational Biology Research Group, which is a core bioinformatics group. I’ve been here for about 12 years now, in Oxford, and before that I was in industry. I’ve always been involved, in some shape or form, in setting up bioinformatics infrastructure and advising people how to do the best bioinformatics analysis.

So what does the Computational Biology Research Group do?

Steve: My team supports scientists, primarily at the Weatherall Institute, but others too.

There’s a lot of small research groups who don’t have bioinformatics support and almost every biological experiment these days will require some informatics input. We work in lots of different fields, such as developing databases and custom software. We also deal with proteomics, microarrays and

next-generation sequencing. Some people imagine that as a core bioinformatics group we are in a kind of service role, browbeaten by the scientists, but actually it’s a very collaborative relationship. We work with people to find a solution, and then we get co-authorship on the end result. We also get to collaborate and make interesting tools that we publish in our own right, such as Zegami.

In that example, I started work on image analysis for a project at the MRC WIMM, and we needed tools to help us manage large amounts of images. The Zegami software began as a collaboration with Roger Noble, a computer scientist in Australia, and we published and then ended up setting up a business around it. So essentially the MRC WIMM is a space for coming up with new ideas and getting involved in projects.

www.researchfeatures.com

CSynth allows molecules to be visualised in three dimensions.

It can take a lot to get clinicians and scientists to work together effectively and I think we’ve got a very good model for doing that – Professor Jim Hughes

www.researchfeatures.com

Because we’re the front line of bioinformatics, we tend to get thrown the new, interesting projects. Often we’ll get data that people don’t really know what to do with, because we’ve got a whole range of expertise here, especially in the new MRC WIMM Centre for Computational Biology funded by the Medical Research Council (MRC).

You mentioned Zegami, a spin-out company from your research. I’m interested in the relationship between the research that you’re doing and how that can be applied and turned into a business model. Could you tell me more about that?

Steve: One of the things I’m keen on is transparency in data analysis and providing tools for people to enable that. Zegami allows users to display tens of thousands of

images, documents, movies, 3D objects or dashboards in a single field of view that they can search, sort, filter or group in real time. It’s a very broad project. It’s not just applicable in biology, it’s applicable to any area where you have lots of images and metadata and hence is now being used in areas from plant phenomics to human resource management.

It’s very difficult to get funding in certain areas, especially if you want to develop a new user interface, because you need to see the software in action to understand its value.

A while ago, I was giving a talk about an early version of Zegami and someone from Oxford University Innovation saw me doing a demo and asked me if I was interested in doing a spin-out business based on it. It’s something I hadn’t considered before, but it allowed us to get funding to develop the idea further. Ultimately, we got full funding for the company (http://zegami.com) and there’s a lot of advantages in that. Often in informatics, in the academic field, you will begin developing a software tool, initially backed by funding, which then runs out so you can’t support it even though lots of people may be using it.

But in industry, you’re generating revenue and so the situation can be a lot more stable. Oxford University Innovation have organised it so that some of the money from our development projects flows back to the institute, so that should help fund future projects as well.

So developing your research into a business model can actually provide support for your project and help fund future research?

Steve: I think we’re going to see a lot more of this way of working. I also think that we will have to be more creative about how we get funding in a post-Brexit UK, as I imagine it will become a lot more restricted.

Jim, could you tell me a bit more about your role and what you’re currently working on?

Jim: My role in the MRC WIMM is more of a traditional research group leader. By traditional, I mean in the way that the group is funded, but it’s also pretty non-traditional

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Thought Leadership

in that it’s a fusion. There’s a large part of me that’s computational, but I actually have an equal presence at the bench as well. I was a bench scientist for 20 years but then, out of frustration, I learnt to code to become a bioinformatician.

I continued working at a bench, but I also had a desk in what was the original Computational Biology Research Group. That was how I met Steve. It turned out to be a fantastic collaboration which still exists now because we co-manage this facility with the head of another computational group. MRC WIMM’s crossover of skills allowed us to translate data into code very quickly and I think that’s where our big advantage has been. Outside of this kind of setting, it has been very hard for traditional groups to get their bench data analysed effectively. Often they would have to go and find somebody, try to convince them to analyse their data, they’d analyse it in a way that was not totally correct and then they’d go back; they’d have long conversations to try and literally understand what each other was talking about.

Because I bridged the two fields, my group didn’t have those complications. We developed work at the bench inspired by our computational projects, and then we developed computational projects inspired by the fact that we understood exactly how the assays worked.

So you’re able to bridge both of those areas that have historically been quite difficult?

Jim: Yes. A lot of my work has been about communication. I have sat in a room with biologists and computational people and I literally translate both ways. Barriers have broken down a little further now, but that’s what my original role was in bringing the computational and bench sides together. I was successful in setting up a group in the MRC WIMM itself, a specialist genomics group, interested in how genes are regulated.

That’s a very basic scientific question.

However, because we’re in the MRC WIMM, we’re very, very conscious of the clinical side of this. I’m funded to try and work out how gene regulation intersects with disease propensity. Essentially, we’re using our technology to try and understand the mechanisms behind anaemia, diabetes, and autoimmune diseases. I’m very conscious of how my technologies and abilities intersect with the clinical community, particularly in trying to diagnose mutations which are very inaccessible at the moment. So it’s a mixture of basic science, but informed to help and

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intersect with clinical science which is what the MRC WIMM’s remit is.

You’re both involved in the CSynth (http:// www.csynth.org) project. Developed in collaboration with Goldsmiths University in London, CSynth is a new interactive software which allows users to visualise the threedimensional structure of a DNA molecule inside a cell and will integrate genomics data, super resolution microscopic images and polymer modelling. Could you tell me more about that?

Jim: We were trying to understand how long-range gene interactions happen and so had developed the Capture-C technology.

Using Capture-C allows researchers to take hundreds of high-resolution pictures of the interactions within a given region of DNA, so a much sharper overall picture of the interaction landscape can be built up. Once we’d got that far, it became very obvious that to try and get any traction on the questions we had, we’d have to try and understand the threedimensional structure of the nucleus. It’s very hard to understand any biological question in 2D; we’re essentially 3D animals. For example, it would be very hard to understand how your heart worked in a two-dimensional way. Because Steve and I work very closely together, he was aware of what I was doing on this project.

Steve: I was keen to get involved because this project focuses on visualisation, which I’m really interested in. I’m a microbiologist originally so I’m very interested in biological properties, but I’m also really interested in providing tools that make the science clearer and more obvious.

I attended a visualisation conference a couple of years ago, where Frederic Fol Leymarie was talking about a tool called FoldSynth, which is protein folding software developed by the team at Goldsmiths University. The interesting thing about it was the way the protein was shown as folding dynamically and also the way you could actually see how the 2D interactions related to the 3D space. I got talking to Frederic and that led onto the CSynth collaboration with Goldsmiths. Their group

is involved in computer games programming and I’ve always thought computer games is a brilliant subject for informatics because the interfaces are often very well thought out and making useable tools for complicated data is key. We had a prototype viewer mocked up using FoldSynth and we loaded some data into it to see what would happen.

Jim: Essentially, if you can model the 3D contacts within a protein, there’s no reason you can’t model any other 3D contacts.

The principles are the same. The aim of our experiment was to see what uses it could be put to. We didn’t have any great expectations about how it would work, but the result has been transformational. One problem we had to overcome was that the data was represented statically and of course the system itself is not static; it’s highly dynamic and so we needed some way to try and bring dynamics into it. FoldSynth already did that, so we took the cue from there. That allowed us to try and visualise dynamics in a way that the human mind could interact with. We could turn it around and watch it change. After we started up the collaboration, the first thing they did was try to think of ways to visualise these data in a coherent way, and I think that was really successful, although we’ve got a long way to go in both data generation and code development.

Steve: Yes, integrating all the data together is going to be really instrumental in

This is the sort of research we’re really interested in – trying to push those boundaries to enable scientists to work together – Steve Taylor www.researchfeatures.com

Left: The entrance to the Weatherall Institute.

Right: Professor Jim Hughes (left) and Steve Taylor (right) with some of the equipment from the CSynth project.

had diseases themselves, trying to understand where the research is going and how it will impact on them. The interest we got was eyeopening.

Jim: I have to agree. I felt quite touched a few times. As Steve said, people would come up and talk to us about certain diseases they had. It always throws you slightly, but if you give your knowledge freely and try to explain what the context is, I think it helps people understand why their life is the way it is. To try and get that kind of information from webpages or books is very hard, whereas if you’re just standing there as a human explaining what genetic mutation is, why that would give them a disease, why it may or may not pass on to their children, all those things, just talking about it in a very human way, I think they found that very valuable. It also affected us and clearly brought home to us the human impact of our work.

understanding new things. I’m quite excited that we’re going to be using virtual reality, creating a new way of interacting with data that we haven’t really done before. You’ll literally be able to walk round the molecule and look at it from different angles. You’ll also potentially be able to collaborate remotely on the same 3D model. You’ll be able to walk round something that looks physically present in the room and say, “What do you think of this structure? Do you think this interaction is correct?” I think that could be fantastically powerful. This is the sort of research we’re really interested in – trying to push those boundaries to enable scientists to work together.

How do you see it evolving over the next few years?

Jim: I think we’re probably one of the first to try and do this. There are a few other instances, but instead of being very competitive, we’ve been very collaborative.

It needs to be solved, but it’s not going to be straightforward or easy and CSynth will help clarify the needs and the goals and promote interest.

The Weatherall Institute as a whole really focuses on collaboration. How significant has collaboration been to your work?

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Jim: I’ve been doing this science lark for quite a long time. In the early days, you could spend most of your time in your lab, but I think that’s changed. There are no little islands anymore, it really comes down to collaboration. To get the job done, you need different skillsets and I think any group that’s trying to do something new will realise that, or they simply won’t be able to do it competitively.

I first became aware of CSynth at the New Scientist Live Exhibition. You said it was a very hectic and quite exhausting four days, but how important do you think that kind of outreach work with the public is?

Steve: I think it’s really important. Gone are the days where you’ve got people in white coats just beavering away, working on a research paper that’s going to be published in some academic journal. I think we’ve got to get out there and show the public what their money is being spent on. When we explained CSynth to the visitors at the New Scientist Live Exhibition, in London, we got some fantastic comments back from people from all different walks of society. That really makes you appreciate what sort of impact you’re having. We had people asking about how they could get into the fields of biology and mathematics, and then at the other end of the spectrum we had people come up to us who

Steve: I think it was an inspiring event for the next generation of people interested in the sciences. I’ve been doing computing for nearly 40 years now and when I started I would never have dreamt that I could merge computers and biology. A lot of people were really interested in hearing about that, with regards to their own career paths. I remember it was my nirvana when I found that you can actually do those two things at once.

Contact The MRC Weatherall Institute of Molecular Medicine, University of Oxford, John Radcliffe Hospital, Headley Way, Oxford, OX3 9DS T: 01865 222443 W: www.imm.ox.ac.uk

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Solutions set in stone: an innovative, nutrition-based approach to managing kidney stones The biotechnology company Captozyme is spearheading an innovative approach to nutrition to prevent the formation of painful kidney stones and other complications arising from the absorption of calcium oxalate from food. The company’s research efforts are led by Dr Qing-Shan Li, co-founder and CSO of the company.

Novel Compound Development

O

Image of kidney stones by Kempf EK via Wikimedia Commons https://creativecommons.org/licenses/by-sa/3.0/

xalate, a plant-based dietary substance with no known positive effects on the human body, is responsible for several debilitating health problems. These include the painful condition known as kidney stones. Dr QingShan Li and associates at the biotechnology company Captozyme (www.captozyme. com) are dedicated to the prevention and treatment of these problems.

OXALATE: THE ANTI-HERO Oxalate can be present in high concentrations in many plant foods, including rhubarb, beets, okra, spinach, sweet potatoes, nuts, beans, tea, chocolate, and soy products. It is, therefore, a common component of the average Western diet.

Up to approximately 25 percent of the oxalate consumed in the diet is absorbed into the human bloodstream through the intestines. The unabsorbed oxalate can chelate (bind to) the dietary nutrients magnesium, iron and calcium, both in food and in the gastrointestinal tract, forming salts with these elements and limiting their bioavailability in the body. Thus, oxalate can, effectively, be described as an antinutrient: a substance that works against the nutritional value of food by rendering certain nutrients unattainable.

THE FORMATION OF CALCIUM OXALATE KIDNEY STONES Dietary oxalate can become actively harmful when too much of it is absorbed; oxalate is a component of the most common type of kidney stone. As an illustration, approximately 75% of all kidney stones in patients in the United States consist of calcium oxalate. It is estimated that 1 in 11 adults in industrialised countries will develop a kidney stone, and preventative measures employed to date are of limited efficacy.

Kidney stones arise from an excessive urinary excretion of oxalate, or hyperoxaluria. Hyperoxaluria has two sub-types, primary hyperoxaluria and secondary hyperoxaluria, which differ in their pathogenesis, severity of clinical presentation, and treatment plan. However, both disorders can cause recurrent kidney stones, nephrocalcinosis, and end-stage renal disease, and for primary hyperoxaluria there is no existing treatment. Oxalate can also cause complications in patients who have inflammatory bowel disease (IBD) and short-bowel syndrome.

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Rhubarb

Potatoes & Yams

Swiss Chard

Beets

Spinach

Soy Products & Tofu

Raspberries

High Oxalate Foods

Nuts & Seeds

Chocolate

Black Tea

Beans

Buckwheat Wheat Bran

People consuming typical Western diets tend to have a serum oxalate concentration that is much lower than the saturation concentration (the point at which it will start to solidify out of the solution).

However, water re-absorption in the kidney concentrates oxalate in urine by more than 100 times, with the result that urinary calcium oxalate is often supersaturated.

Calcium oxalate supersaturation is thought to be the primary cause of the formation of calcium oxalate stones, but the process is complex, not well understood, and may involve a number of critical factors.

Reducing the ingestion of oxalate can keep the concentration in urine below the point at which it starts to solidify and therefore prevent the formation of an abnormal level of calcium oxalate precipitate.

EXISTING TREATMENTS AND THERAPIES Humans do not naturally produce enzymes to break down oxalate and so a combination of lifestyle changes and medication is the usual approach to preventing kidney stones. Doctors may recommend that patients drink water throughout the day to produce more urine and flush calcium oxalate out of their systems. Reducing both salt intake and consumption of animal protein, promoting oxalate excretion through increased calcium intake, increasing urine alkalinity, and avoiding dietary oxalate ingestion are also typical methods for reducing the chances of developing calcium oxalate kidney stones and other oxalate-related conditions.

Captozyme has taken the innovative approach of reducing the oxalate concentration in food through adding an oxalate-degrading enzyme 55

Novel Compound Development

A graphical rendering of one type of oxalatedegrading enzyme; oxalate decarboxylase or OxDC. Captozyme specialises in oxalatedegrading enzymes but their research programmes also include other enzymes for a different set of sensitivities and ailments.

However, as oxalate is contained in many different foods, often at different levels in a single food, avoiding oxalate consumption while maintaining a balanced diet is difficult.

Moreover, available therapies do not address the formation of calcium oxalate within the body. Captozyme has addressed these problems, taking the innovative approach of using oxalate-degrading enzymes to remove oxalate in the digestive tract and to reduce the oxalate concentration in food. Their approach tackles oxalate absorption at the first possible moment.

LIMITING THE FLOW Captozyme has developed enzyme products that degrade oxalate, thereby promoting its removal from both the diet and the body.

These enzymes can act throughout the gastro-intestinal tract, from the stomach to the large intestine. Cz2294, is an oxalatedegrading enzyme that is highly stable in acid environments and can remove oxalate in the stomach. In contrast, the enzyme Cz1408 removes oxalate throughout the intestine. This combination means that oxalate is removed throughout the digestive system, thereby limiting the amount of

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Oxalate can be described as an anti-nutrient: a substance that works against the nutritional value of food oxalate that is absorbed in the intestines and reducing the risk of kidney stones without compromising nutritional requirements.

STRAIGHT TO THE SOURCE In addition, Captozyme’s enzymes can be used in a range of food products to remove oxalate from food and beverages before packaging. This processing would degrade the oxalate present in the food, unlocking the potential of the iron, calcium, and magnesium within the regular diet and avoiding the need for dietary restrictions.

Captozyme is also poised to introduce a supplement to address oxalate sensitivity and is developing its proprietary enzymes as stand-alone therapeutics for the treatment of more severe diseases caused by oxalate.

These enzymes are the culmination of almost a decade’s worth of funded research, testing and refinement.

PHILOSOPHY FOR SUCCESS In addition to developing its enzymatic oxalate-degrading products, Captozyme seeks to build on growing positive consumer behaviour towards food and nutrition by providing education about anti-nutrients and simple personal nutrition solutions.

Unlike those requiring a gluten-free or lactose-free diet, Captozyme regards the oxalate-sensitive population as having been ‘completely underrepresented’ by specialty foods and is determined to address this.

The success of Captozyme’s approach in applying research to commercial opportunities exemplifies how the support currently given to small businesses leads to innovative solutions and commercial success.

www.researchfeatures.com

Detail Given that oxalate has always been a naturally-occurring component in our diets, are there specific lifestyle factors that predispose modern-day humans to hyperoxaluria? Or have people always been at the same level of risk?

The level of risk is increasing despite oxalate content in food items staying at the same level. This increase in risk is due in large part to a change in diet and a change in the environment: the majority of people now eat less calcium per day than the recommended daily intake and they do not hydrate well using the best fluid, water. The environment has also changed with warmer temperatures making hydration even more important. Poor hydration and an imbalance in calcium oxalate intake will cause our urine to become saturated in calcium and oxalate, which causes precipitation of calcium oxalate crystals.

Increased presence of crystals in urine, known as crystalluria, has been linked to kidney stones.

How did you become interested in oxalate-degrading enzymes?

I have worked with enzymes and proteins throughout my career ranging from the engineering of enzymes for specific properties to expression of enzymes in E. coli, yeast and mammalian cells.

Enzymes are fascinating for their capacity to catalyse reactions and the vast applications that are possible. Oxalatedegrading enzymes became interesting to me as I was working with an oxalatedegrading organism. This organism, Oxalobacter, has oxalate-degrading enzymes functioning within the cell and I became interested in exploring the characteristics of stand-alone enzymes

with the same capacity of degrading oxalate but outside of a microbe.

What kind of foods can we expect to find in the forthcoming range?

Any food that can be processed to remove oxalate. In the near future, these are beverages such as vegetable juices, teas and beer. Oxalate can also be removed from canned goods and soup. More foods are being evaluated for removal of oxalate but it will require some more development.

Do you foresee your enzyme-based approach transferring into the prevention and treatment of other diet-related disorders?

This approach of using enzymes to allow management of diets and promotion of health is one that I definitely foresee as a promising tool in diet-related disorders.

Awareness around nutrition and how it links to disease is increasing and the scientific community is also learning more about how the gut and diet intake affect our wellbeing. Our diet is a big part of who we are and how we feel and if an enzyme can help us remove anti-nutrients from our diet then that is a good thing that should be shared with all of those who need it.

Where are the enzymes that you use found in nature and are they something that we have lost from our diets over time?

The enzymes we use can be found in nature but in low quantities. In certain diets, they are ingested as part of fungi or fermented foods but this is not generally a common dietary intake.

Our diet is a big part of who we are and how we feel – if an enzyme can help us remove anti-nutrients from our diet then that is a good thing www.researchfeatures.com

RESEARCH OBJECTIVES As Chief Scientific Officer at Captozyme, Dr Qing-Shan Li focuses his research on developing products to help remove oxalate either from the digestive system or from the diet.

FUNDING Captozyme’s research and development efforts have been aided by the company’s participation in the US National Institutes of Health’s CAP programme (Commercialisation Acceleration Programme), specifically by a grant from the National Center for Complementary & Alternative Medicine (for further details see: http://grantome.com/grant/NIH/R44AT006065-03).

COLLABORATORS Victoria Bird, MD at University of Florida University of Illinois at Chicago University of Wisconsin BASi, Mt. Vernon, IN Product Safety Labs, Dayton NJ BIO Dr Qing-Shan Li has over 20 years of experience in protein engineering, expression and fermentation and his work has resulted in 33 papers published in peer-reviewed journals and several patent applications. Dr Li has held research positions at Kyoto University, Stuttgart University, and at the University of Kansas under the direction of Professors Robert Hanzlick and Robert Borchardt. In 2009 he co-founded Captozyme Inc., where he currently acts as Chief Scientific Officer.

CONTACT Helena Cowley, MS, MBA, COO Captozyme Inc 5745 SW 75th Street #298 Gainesville, FL 32608, USA E: helena.cowley@captozyme.com T: 352 672 5320 W: www.captozyme.com

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Technology

Biomedicine: Speeding up sickle cell treatment

An innovative, quantitative test for the mutant blood proteins that cause sickle cell disease, developed by Drs Jason Kim and Xiaoxi Yang of BioMedomics, Inc., could make therapy much more successful.

E

ach year, 300,000 people worldwide are born with sickle cell disease (SCD), a painful and debilitating genetic disorder of red blood cells. SCD is potentially fatal, responsible for 9% of all infant mortality in low-resource regions, but early diagnosis and prompt treatment can save lives. Until now, however, both diagnosis and therapy have relied upon expensive blood tests which can only be carried out by specialised staff in large, centralised laboratories.

THE SCOURGE OF SICKLE CELL Red blood cells are the body’s delivery trucks, transporting oxygen to each and every cell of the body. The key component of red blood cells is haemoglobin, a chemical compound that combines with oxygen in the lungs and releases it again in all tissues of the body. In SCD, sufferers inherit two mutated copies of the haemoglobin gene, causing them to produce haemoglobin S instead of the normal haemoglobin A, and rendering their red blood cells sickle-shaped instead of the usual doughnut-shaped version. Carriers of SCD have one mutated and one normal

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copy of the haemoglobin gene, and do not show symptoms of the disease, but can pass it on to their children. SCD is most common in those of African, Mediterranean, South and Central American, Middle Eastern, Indian and Caribbean descent.

The characteristic sickle-shaped red blood cells of SCD are more rigid than normal blood cells and can block small blood vessels in the body, reducing the blood supply which causes pain and damage to tissues. If this occurs in the brain, it can cause a life-threatening stroke. Other complications of the disease include red blood cell rupture, resulting in chronic inflammation and putting sufferers at risk of infection. Finally, sickle cells are much shorter lived than normal red blood cells – surviving about 16 days rather than 120 – which can cause debilitating anaemia.

The most effective means of therapy for SCD, used in about 90% of cases, is transfusion with normal blood containing haemoglobin A. Repeated transfusions help both to reduce the proportion of haemoglobin S in the body and to prevent stroke, one of the most common

Hemo SCAN-S in use

and damaging complications of the disease. However, transfusion carries risks if too many blood cells are given.

Therefore, monitoring the proportion of the different types of haemoglobin in the bloodstream, before, during and after transfusion, is crucial to successful SCD therapy. Currently, the only way to measure the haemoglobin composition of blood is to send samples away to centralised laboratories, which can take two or three days to deliver results.

Thus, transfusion rates are frequently estimated based on outdated blood test results.

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BIOMEDOMICS STEPS IN An obvious solution to the issues of high cost and long turnaround of blood testing that presently hinder diagnosis and treatment of SCD, is to develop low-cost, portable testing kits that provide immediate results at the patient bedside. This is where BioMedomics, Inc. (http://www. biomedomics.com/) steps in. The company is a world leader in designing unique, rapid tests for blood disorders. Established a decade ago, BioMedomics, Inc., based in North Carolina, combines the expertise of high level researchers in biology, biochemistry and biomedical engineering to

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Monitoring levels of the different haemoglobins in the bloodstream, before, during and after transfusion, is crucial to successful SCD therapy 59

Technology

Above: (from left) Key team members – Taylor Osborne, Dr Xiaoxi Yang, Chris Roberts and Dr Jason Kim Right: Dr Jason Kim and Dr Xiaoxi Yang test Hemo SCAN-S

develop advanced technologies for effective diagnostic testing applicable in both highand low-resource regions.

BioMedomics’ latest innovation, Hemo SCAN-S, has been developed by Principal Investigator Dr Jason Kim and Head of Product Development Dr Xiaoxi Yang. In a remarkable improvement upon existing systems, Hemo SCAN-S delivers accurate measurements of relative haemoglobin levels within fifteen minutes. This breakthrough allows physicians to tailor transfusion therapies precisely to patient needs in real time. Dr Kim hopes that the low cost and portable design of the Hemo SCAN-S system will make it applicable throughout the world, and is working with key US and international partners including Kwaku Ohene-Frempong, MD at the Sickle Cell Foundation of Ghana, Bob Kabugi of the Glorious Church of Kenya and AMPATHKenya, Marilyn Telen, MD at Duke University Medical Center, and Julie Kanter-Washko at the Medical University of South Carolina.

THE HEMO SCAN-S SOLUTION Developed from BioMedomics groundbreaking qualitative test for diagnostic screening of newborn babies, Hemo SCAN-S uses antibodies (highly-sensitive immune-system proteins), which recognise the protein configuration of different types of haemoglobin and can report their relative proportions within as little as fifteen minutes.

For SCD patients subjected to repetitive blood tests during treatment, it is important that testing be as simple and painless as possible; Hemo SCAN-S requires only a tiny drop of blood (five thousandths of a millilitre) which can be obtained by a simple

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In a remarkable improvement upon existing systems, Hemo SCAN-S delivers accurate measurements of relative haemoglobin levels within fifteen minutes finger-prick. The blood is placed onto a cartridge containing special chemicals which break down blood cells, releasing the haemoglobin contained therein. The haemoglobin then flows across a surface impregnated with the antibodies, which capture the different haemoglobin types and report their presence as coloured lines – much like the lines in a pregnancy test.

The truly exciting feature of the Hemo SCAN-S test is that the intensity of the lines is directly proportional to the concentration of the different types of haemoglobin present in the original blood sample. The intensity can be measured by a small, portable, red-light reader which reports results as easily-interpretable percentages.

This enables transfusions to be matched precisely to the patient’s needs at the time of treatment.

ENHANCING QUALITY OF LIFE BioMedomics have carried out extensive testing of the Hemo SCAN-S system and proven it to be highly sensitive to haemoglobin A and haemoglobin S, producing results as accurate and precise as those obtained in the traditional laboratory setting, and resistant to interference from other blood components. Compared to current central lab blood testing, Hemo SCAN-S completely eliminates the need to transport samples away from the patient bedside, thereby drastically reducing patient waiting time and potential over- or undertransfusion. In addition, the technology is simple to use, relatively painless, and of course rapid. For patients with SCD, Hemo SCAN-S – along with other diagnostic tools developed by the BioMedomics team – could revolutionise not just their treatment but their quality of life.

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Detail Why is SCD such a debilitating disease?

In SCD, mutated versions of the haemoglobin gene are inherited from both parents, causing red blood cells to become sickle shaped. These distorted cells die early causing anaemia or block the flow of blood causing severe pain crises. Damage caused by these rigid abnormal cells causes a potentially fatal stroke in a quarter of SCD children and adults by the age of 45. A life-long disease, SCD can negatively affect a person’s growth and harm organs such as spleen, brain, lungs, liver, kidneys, or skin.

How can transfusions help to mitigate the effects of SCD? Blood transfusions limit the complications of SCD and consequently improve the life of an SCD patient. The rate of stroke can be reduced by more than 80% through the implementation of transfusion therapy.

Transfusions can immediately alleviate aneamic conditions and also enhance quality of life for SCD patients with vital organ failure.

Why are current methods of blood testing inadequate?

Current methods used for monitoring transfusion therapy are haemoglobin fractionation in the central laboratory using haemoglobin electrophoresis, high performance liquid chromatography, or isoelectric focusing. These tests require a high equipment cost (>$100,000 USD), a trained technician to operate and maintain the system, and have turnaround times in the range of hours to days.

SCD patients requiring transfusions are guidelined to receive healthy HbA transfusions until their abnormal HbS levels reach <30% of total haemoglobin.

Due to the long turnaround time of a haemoglobin fractionation test, the healthcare provider must estimate the

amount of blood to transfuse using pretransfusion Hb concentration, body weight, and out-dated data from a previous visit. Missing the mark on the right amount of blood can have negative consequences and the burden of increased hospital visits. A patient may get an insufficient amount of healthy blood, and suffer painful, dangerous complications of SCD. Or, a patient may receive too much blood, and instead be faced with iron overdose.

How can a quantitative ‘point of care’ test help?

Our test system aims to produce patient data rapidly – while the clinician is with the patient. Rather than estimating the amount of blood to be applied to the patient using indirect clinical information, the healthcare provider can base decisions directly on the guidelined %HbS. Accurate and current %HbS data would be available before, during, and after the transfusion to monitor the therapy. By applying the optimal amount of healthy blood, SCD patients can limit their disease complications, reduce the burden of hospital visits, and extend and improve their quality of life.

How could Hemo SCAN-S particularly benefit SCD patients in low-resource regions?

The easy-to-use Hemo SCAN-S test system (thousands of dollars) is several orders of magnitude less expensive than a central laboratory system (hundreds of thousands of dollars). The test system has a small desktop footprint and does not require a highly trained technician.

Centres that provide transfusion therapy (US/Europe as well as in developing countries) could increase and improve usage of this life saving therapy through lower cost and ease-of-use.

This breakthrough allows physicians to tailor transfusion therapy precisely to individual patient needs in real-time www.researchfeatures.com

RESEARCH OBJECTIVES BioMedomics, Inc. develops rapid, point-of-care diagnostic tools. Drs Jason Kim and Xiaoxi Yang have focused on creating a replacement test for relative haemoglobin levels to increase the accuracy of essential transfusion treatment for sickle cell disease sufferers.

FUNDING North Carolina Biotechnology Center Small Business Loan Award; NIH Small Business Innovation Research Grants 1R43HL128670-01 and 2R44HL128670-02 COLLABORATORS Marilyn Telen, MD at Duke University Medical Center; Julie Kanter-Washko, MD at the Medical University of South Carolina; Russell Ware, MD at Cincinnati Children’s Hospital; Kwaku OheneFrempong, MD at the Sickle Cell Foundation of Ghana; Bishop and Church Leader Bob Kabugi of the Glorious Church of Kenya; Ampath Kenya BIO Jason S Kim, PhD is the Principal Investigator of both BioMedomics’ NIH NHLBI Small Business Innovation Research awards for point-of-care diagnostic tests for sickle cell disease. Dr Kim is the head of product development at BioMedomics, leading a team of talented engineers and scientists. Dr Kim previously held positions at Nanoentek Korea and the US Naval Research Laboratory in Washington DC. Xiaoxi Yang is a co-PI of the NHLBI grants and director of research at BioMedomics.

She was recruited based on her expertise and pedigree as a rising star in innovative sickle cell diagnostic research. Previously, Dr Yang held positions at the University of Houston and Tulane University.

CONTACT Jason S Kim, PhD BioMedomics, Inc. Six Davis Drive, Research Triangle Park, NC 27709 USA E: jkim@biomedomics.com T: +1 919 558 1210 W: www.biomedomics.com www.sickle-scan.com

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Maintaining the equilibrium: inner ear hair cell regeneration Dr Jennifer Stone, from Virginia Merrill Bloedel Hearing Research Center, of the University of Washington, discusses her latest research which focuses on vestibular hair cell regeneration in rodents.

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Hearing

T

he mammalian inner ear is a labyrinth of incredibly intricate sensory structures. The cochlea, a spiral-shaped cavity, is a vital part of the auditory system and converts vibrations (produced by sound waves) into electrical impulses which communicate to the brain via the auditory nerve.

The vestibular system, also part of the inner ear, is essential in maintaining spatial orientation and balance. It is composed of three semi-circular canals, each orientated in a different plane. Within each canal, fluid moves in response to head motion, triggering tiny vestibular hair cell receptors which send signals to the brain and eyes via the hair cellsâ&#x20AC;&#x2122; innervating neurones. This mechanism gives mammals a sense of balance and coordination. Vestibular hair cells can be further divided into two subcategories: type I and type II. Although knowledge is limited, research suggests that type I hair cells are better suited to detect high-frequency movements than type II hair cells.

VESTIBULAR SYSTEM DEGENERATION The hair cells and innervating neurones associated with the vestibular organ are very vulnerable to degeneration, which increases with age. In fact, approximately 35% of the United States population aged 40 or over are affected by some form of vestibular system disorder and the consequences can be devastating. Patients may experience debilitating bouts of intense vertigo and imbalance. In addition, sufferers often find it difficult to concentrate and physically struggle to perform routine activities, causing great emotional distress.

Current options for treating vestibular disorders are limited and include vestibular rehabilitation therapy, medication and surgery.

Even in a best-case scenario, symptoms are only improved, not cured. The only way in which vestibular function could be fully restored would be via hair cell regeneration and subsequent nerve innervation.

Dr Stone and her team are currently exploring this process in depth and hope to develop more effective therapies to treat vestibular disorders via cellular regeneration.

HAIR CELL REGENERATION Non-mammalian animals respond to vestibular damage by regenerating both type I and type II hair cells. In birds, amphibians, and fish, new hair cells arise via mitosis (cell division) of supporting cells and their subsequent differentiation into hair cells.

Replacement hair cells in non-mammals are also produced via direct transdifferentiation â&#x20AC;&#x201C; a non-mitotic process whereby supporting cells are phenotypically converted into hair cells. In birds, studies have shown that regenerated cells become innervated, restoring function.

However, in a recent study, Dr Stone and colleagues showed that only type II vestibular hair cells can regenerate in mammals. The team destroyed vestibular hair cells in adult mice by inserting the destructive human diphtheria toxin receptor (DTR) gene into the locus for the Pou4f3 gene. Sixty days after treatment, they discovered that hair cell numbers had actually increased significantly, despite little increase in mitotic activity, suggesting that regeneration had occurred via the direct transdifferentiation of supporting cells (rather than mitosis).

Intriguingly, however, there is no evidence to suggest type I hair cells are replaced and Dr Stone and her team are now investigating the properties of type I hair cells to understand whether they can also regenerate.

TYPE II HAIR CELLS' UNIQUE PROPERTIES In order to distinguish some of the differences between the two hair cell types, Dr Stone and her collaborator RĂŠmy Pujol used confocal and transmission electron microscopy (TEM) to study hair cell structure in adult mice.

Unlike type I hair cells, type II hair cells have basolateral processes (processing units) that are in physical contact with each other, forming a delicate network. However, more

Understanding the molecular background underpinning hair cell differentiation and regeneration is vital if we are to develop therapies to treat vestibular disorder www.researchfeatures.com

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Hearing

Inner ear

research is needed to explain this unusual phenomenon â&#x20AC;&#x201C; perhaps the connection is simply for mechanical support, or maybe it enhances communication between hair cells.

A more radical idea is that a direct link between the hair cell population could regulate their homeostasis. Dr Stone, in collaboration with Brandon Cox's lab at Southern Illinois University School of Medicine, has attained evidence that vestibular type II hair cells undergo 'turnover' under normal conditions: individual hair cells are culled from the sensory organs and then replaced via transdifferentiation of supporting cells. In contrast to normal conditions, hair cell destruction causes supporting cells to produce six times the amount of replacement type II hair cells. This plasticity (adaptability) of vestibular organs may help to ensure balance function is retained in adult mammals, perhaps even in humans.

MOLECULAR BASIS OF HAIR CELL REGENERATION Understanding the molecular background underpinning hair cell differentiation and regeneration is vital if we are to develop therapies to treat vestibular disorders.

The 'Notch signalling pathway' is particularly important in the process of hair cell development in embryos. Dr Stone and her team have shown that this pathway inhibits the production of a key basic helix-loop-

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Vestibular organ (utricle, surface view)

Approximately 35% of the United States population aged 40 or over are affected by some form of vestibular system disorder helix transcription factor that activates hair cell differentiation, called atonal homolog 1 (Atoh1).

During embryo development, signalling proteins bind to the 'Notch' receptor, located on undifferentiated cells, activating enzymes which cleave the receptor. Subsequently, the cleaved protein activates genes that encode other proteins that inhibit Atoh1.

To determine whether Atoh1 is reactivated after vestibular hair cell loss, Dr Stone and her team performed a study on adult mice utricles (an organ found in the vestibular system), in which hair cells had been destroyed using neomycin. Interestingly, the team detected Atoh1 expression in the supporting cells 4 days after neomycin treatment. These supporting cells then underwent direct transdifferentiation to form very primitive hair cells.

Additionally, Dr Stone showed Notch pathway inhibition resulted in an increase in Atoh1 levels, and supporting cells progressed to later stages of hair cell differentiation.

Again, however, these new hair cells were not fully functional â&#x20AC;&#x201C; they lacked hair bundle maturation and innervation. Dr Stone is working with a consortium of international scientists, the Hearing Restoration Project, funded by the Hearing Health Foundation, to determine additional signals that regulate hair cell regeneration in adult mice.

FUTURE RESEARCH Dr Stone's research is extremely promising, indicating that mammalian adult hair cells have the potential to regenerate via the phenotypic conversion of supporting cells. However, many questions remain unanswered. For example, what are the molecular processes involved in regulating hair cell maturation? Are there ways in which we can initiate type I hair cell regeneration?

And, what molecular mechanisms underpin vestibular cell diversity?

Overcoming these obstacles will take us one step closer to developing hair cell replacement therapies used to treat vestibular disorders and improve the lives of thousands of sufferers.

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Type I hair cell

Type II hair cell

Detail RESEARCH OBJECTIVES Dr Stone's research focuses on the replacement of vestibular hair cells in mature rodents as a possible means to learn more about cell death in humans and the possibility for regeneration.

FUNDING National Institutes of Health; the Hearing Health Foundation; the Virginia Merrill Bloedel Hearing Research Center

Supporting cell

Vestibular nerve fibers

Vestibular sensory epithelium (utricle, cross-section)

What inspired you to focus your research on inner ear vestibular hair cell regeneration?

Since my graduate training, I have studied auditory and vestibular hair cell regeneration in birds. I wanted to move my lab’s research into a model system that was more like humans, and the best option was mice, since they offer abundant genetic tools. There was already some evidence that replacement of vestibular hair cells might happen in adult guinea pigs, but it was clear it did not occur in the cochlea.

We knew little about vestibular hair cell regeneration in mice at that time. This background, coupled with the knowledge that understanding more about hair cell regeneration in mammals could someday help people with vestibular dysfunction, motivated me to work in this area.

Why do vestibular hair cells degenerate with age?

We don’t know the answer to this. However, hair cells are highly metabolically active all of the time; they never rest! So, they are probably more susceptible to wear and tear than other cell types.

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Why do non-mammalian hair cells have a higher level of plasticity, compared to mammalian?

We wish we knew! Regeneration of multiple tissue types is an enviable property among lower vertebrates, and the predominance of regenerating tissues in a given animal seems to be inversely proportionate to its morphological complexity.

Is there a chance that we could manipulate the molecular pathways that regulate hair cell regeneration for clinical benefit?

We certainly hope so! Research advances in our field each year bring us closer to the identification of molecules that have significant and lasting effects on hair cell replacement in mammals.

Where do you see your research progressing in 5 years’ time?

I hope that we will identify ways to promote replacement of higher numbers and both types of vestibular hair cells in mice, such that balance function is restored in a meaningful and lasting manner. This will be a key step in moving toward a clinical application for our research.

COLLABORATORS • Members of the Hearing Restoration Project, funded by the Hearing Health Foundation • The Virginia Merrill Hearing Research Center, University of Washington • Edwin Rubel, University of Washington • James Phillips, University of Washington • Brandon Cox, Southern Illinois University • Ruth Anne Eatock, Chicago University • Rémy Pujol, University of Montpellier, France BIO Jennifer Stone received her Bachelor of Arts in Biology from Skidmore College and her PhD in Anatomy and Neurobiology from Boston University.

She performed a post-doctoral research fellowship in the Otolaryngology – Head and Neck Surgery Department at the University of Washington, where she is now a Research Professor.

CONTACT Jennifer Susan Stone, PhD Research Professor Dept of Otolaryngology VM Bloedel Hearing Research Center CHDD CD 176 Box 357923 University of Washington Seattle, WA 98195-7923 E: stoner@uw.edu T: +1 206 616 4108 or 206 616 4155 W: http://faculty.washington.edu/stoner/ Stone_Lab/Home.html http://otolaryngology.uw.edu/faculty/ jennifer-stone

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RESEARCH SOCIAL MEDIA NEWS

Communicating Science: Animation Patrick Bawn delves into the science behind using animation to break down complex subject areas and educate the public.

Research suggests that by the time you reach the end of this sentence, your interest in this article will be long gone and you will already be looking at something else.

If you’re still there then good – you’re the exception. I will carry on.

Goldfish Memory In a world where the attention span of a human has decreased to just eight seconds (one second less than a goldfish), it is vital to determine scientific methods that counteract this reduced level of attentiveness, and ensure that our time is put to good effect. This is especially important within education – after all, how can anyone expect to learn anything if they can only hold their attention for eight seconds?

The answer is surprisingly simple, and involves nothing but your eyes and a screen (and an engaged brain ideally).

Numerous studies throughout the years have found that using animated videos as an educational tool is a very effective way of grabbing the viewer’s attention, engaging their brain and educating them about varying topics.

animation. The differences seen were comprehensive, with education via animation proving substantially more effective than the alternative paper format. Not only is animation more interactive and visually appealing, its ability to hold a viewer’s attention and motivate them to learn is what really sets it apart.

So how about animations with a voice over?

Does having a narrative make a difference?

This was particularly investigated by Danton O’Day during a study back in 2007. For this, he looked at the effect narrated animation had on communicating complex biological processes when compared with the same animation, but without a narration on top. Surprisingly, he found that more people could better retain information without a narrative than with one.

So, in other words, having a voice over isn’t as important as the animation itself.

Learning Subconsciously Watching interactive animations is not only great at educating

the viewer, but can be a lot of fun too. The beauty of animated design is the flexibility it can provide – a blank canvas on which creativity can flourish and make education exciting. This level of fun can increase the likelihood of a viewer returning to the animation to watch it again which, in effect, makes the learning aspect a secondary, subconscious process. It’s like when you watch a TV show you’ve never seen before; you don’t deliberately learn each character’s name – you pick them up sub-consciously.

Overall, animation is a fantastic tool for breaking down complex science and explaining it quickly and easily in an engaging, visual format. As research continually makes clear, its function needs to be effectively utilised within the modern day world of teaching and education – especially now our attention span is worse than a goldfish.

Textbook vs. Animation In a study by Soika et al in 2010, they compared the difference between two teaching methods – traditional paper teaching and teaching via computer

The beauty of animated design is the flexibility it can provide – a blank canvas on which creativity can flourish and make education exciting 66

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