Research Features - Issue 102

MIB: BEAUTIFUL BIOTECHNOLOGY

ANTHONY NOLAN: STEM CELLS

PROFESSOR ROBERT WINSTON

Professor Nigel Scrutton, the director at the helm of Manchester Institute of Biotechnolgy, shares the ground-breaking research carried out during their first ten years and the future strategic direction for their work.

Professor Alejandro Madrigal speaks about his twenty years as Scientific Director of the Anthony Nolan Research Institute and the direction he sees stem cell research going in the future.

Genesis Research Trust’s leading fertility expert, Professor Robert Winston, discusses the history and heritage ofResearch his institution, Features 3 before highlighting the current challenges facing maternal health.

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WELCOME

This Issue

TO ISSUE 102

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his issue is packed full of articles bringing the work of inspiring researchers to your attention. The world of Health Science is diverse and fascinating and the research in this issue reflects that.

We have heard from Professor Robert Winston at Genesis Research Trust and Professor Neena Modi, President of the Royal College of Paediatrics and Child Health, both leaders in the world of Child Health. We also have features on four scientists whose work is helping to ensure that our knowledge of this area continues to expand.

Elsewhere in the magazine, our view of Health Science remains as broad as ever, with articles featuring the work of researchers in Women’s Health, Novel Drug Development, Molecular Biology, Epidemiology and Neurology. Dr Nigel Scrutton, director of the Manchester Institute of Biotechnology, speaks to us about his institute’s ground-breaking research and their plans for the future, while Professor Alejandro Madrigal shares his insights as scientific director of Anthony Nolan, the world’s first bone marrow and stem cell donor register.

I am also delighted to introduce Women’s Worldwide Web (W4) as our chosen partner charity – their valuable work supports girls and women everywhere to access the opportunities presented by Information and Communication Technologies.

It was a pleasure to create this issue and I am sure you’ll enjoy reading it. So get stuck in and join our worldwide readership (including people in research stations in Antarctica!) – with many in the science community unsure what Trump’s success will mean for the sector, now may be a good time to remind ourselves of the valuable work that researchers in North America and beyond undertake every day.

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 Junior Editor: Chris Barrell editorial@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: John French John@researchfeatures.com Julian Barrett Julian@researchfeatures.com Kate Rossiter Kate@researchfeatures.com Contributors: Ella Gilbert, Simon Heptinstall, Petra Kiviniemi, Barney Leeke, Peter North, Kate Porter /researchfeatures /ResearchFeature researchfeatures

Copyright © 2016 by RPI Ltd

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All rights reserved. No part of this publication may be reproduced, distributed, or transmitted in any form or by any means, including photocopying, recording, or other electronic or mechanical methods, without the prior written permission of the publisher.

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What if ?

W4.ORG CROWDFUNDING FOR GIRLS’ & WOMEN’S EMPOWERMENT

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WomensWorldWideWeb

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CONTENTS

06 06

Spotlight on Genesis Research Trust: The challenges of maternal health

10 16

Three ways to reduce pain in early-life medical procedures

Shining a light on the hidden viral infection that causes birth defects

20 24 30

Cutting the cord: babies benefit from a delay

Understanding the mechanisms of language comprehension

30 38 42

Gene research sheds new light on ALS

Spotlight on Anthony Nolan: The novel techniques of stem cell research

46 50 54

Universal donor cells – a revolution against rejection

New windows into cellular kinase function

58 62

O so simple... How blood group O fights off malaria Spotlight on MIB: Manchester: The home of beautiful biotechnology

66 70

Family matters: drinking patterns in Inuit mothers and adolescents

Women sleep on Venus; men sleep on Mars

Therapeutic cocktails – the drink that protects against liver disease

Spotlight on RCPCH: Pioneering global collaboration in children's health research

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Don’t get overexcited! Restoring inhibition in neurological disorders

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Spotlight

Genesis Research Trust

The challenges of maternal health

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Genesis Research Trust’s leading fertility expert, Professor Robert Winston, recently met with us at Research Features to discuss the history and heritage of his institution, before highlighting the current challenges facing maternal health – in more ways than one.

rofessor Robert Winston is a well-known figure in the world of science communication, having featured in numerous science documentaries and TV shows over the years. These have included his groundbreaking documentary Child of our Time, which looked at how child growth is affected by the interaction between genes and the environment, and his multiple BAFTA award-winning series The Human Body.

Away from the camera though, Professor Winston is the leading fertility expert at Genesis Research Trust – a charitable research institution within Imperial College London. This institution funds the largest collection of UK-based scientists and clinicians dedicated to researching the causes and cures for reproductive conditions that affect women, their families and their babies.

Professor Winston recently sat down with us at Research Features to discuss the institution’s background and heritage in more detail, before highlighting the importance of its research in the modernage.

Hello Robert! Could you tell us some more about Genesis Research Trust and, in particular, the research it has been involved in over the years?

The Genesis Research Trust has really evolved from what used to be called The Institute of Obstetrics and Gynaecology

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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.

One of the earliest workers here that I can recall was Ian Donald – the man who invented ultrasound. This provided one of the cheapest ways of looking at tissues, which was truly a massive breakthrough and one of the firsts that this place was responsible for.

Another person who immediately comes to mind is Erica Wachtel who was a refugee from Nazi Austria and was a key person in getting the cervical cancer smear test running. These two remarkable inventions have shaped our history and heritage here.

Antenatal care has also been very much developed here by McClure Brown. I suppose more recently though we’ve been interested in miscarriage, stillbirth, ovarian cancer and we’ve also made breakthroughs in uterine cancer. Many of the improvements

in in vitro fertilisation (IVF) were also made on this site at Hammersmith Hospital, which is now a part of Imperial College.

For Genesis Research Trust especially, we’re really interested in trying to promote women in science. One of the big issues for us is to make sure that women get a fair deal and if you look around our department, you’ll probably see that there are more women than men working here.

What do you think the challenges are that women might face if they try to pursue a career in science and how do you at Genesis Research Trust protect against this?

I think the challenges for women scientists are invariably when to consider having a family. In my view, doctors are very ready to pontificate about how we should try to get pregnant earlier or how we should manage our career, but to my mind that doesn’t make sense.

We established many, many years ago a ‘women for women’ programme where women raised money to help female scientists have a family and continue working as scientists concurrently – by offering them

Genesis Research Trust has 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 www.researchfeatures.com

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Spotlight

the opportunity for part-time or flexi-time working hours.

The Genesis Research Trust is interested in the social side of what we do so, at the moment, we run the women’s health unit at Queen Charlotte’s Hospital and the research that goes on there. We also have a collection of PhD students who I’d say are the backbone of British science – generally if you go to any lab in Britain, the best research is being done by the doctoral students. We fund all of our doctoral student’s research materials and we’re rather proud of that – we think that’s one of the reasons why this is such an important place.

We look for young people who have energy, work well in a team and are capable of doing really innovative things. We try not to be too disciplinary as well, as we like to let them have freedom when researching. That’s part of the ethos here, and a very large number of our doctoral students are also women.

How important is collaboration with other scientific researchers to you at Genesis Research Trust?

All science groups, if they’re any good, collaborate around the world. Over the years we have collaborated with Harvard, Caltech, University of California, Cambridge University and many other world-class institutions. There was a time in Britain when this place was responsible for more than half the professors in our field in the United Kingdom. Certainly in my own lab, I used to have a map of all the countries in the world which I would cover with hundreds of pins to represent the nationalities of the people in the lab. There was one time when we could even count 65 different languages being spoken in the lab.

I think the influence of institutions such as ours is not fully understood and that’s one of the reasons xenophobia about immigration is such a negative thing. Places like the Genesis Research Trust understand that we have a responsibility to make sure we are training people from outside the UK and we’ve always done that very well here – people come here to do their own research and then go back to their own country to develop it more.

A lot of our research is also taken up in the poorest countries in the world. We have projects that have influenced perinatal care in every African nation and, in fact, most developing nations. For instance,

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prematurity is a huge problem in Africa and the greatest cause of maternal death is preeclampsia. By working with communities in the poorest parts of the world, we can help improve the health for both women and their babies. There is a general recognition for how important international collaboration is, not just with scientists, but with communities who have the needs we would like to support.

Do you think Brexit will significantly impact the research activities of Genesis Research Trust?

It’s difficult to say but it’s quite likely that it might do. When I last looked at the people working in this laboratory, we had people from the United States, Finland, Russia,

There is a general recognition for how important international collaboration is, not just with scientists, but with communities who have the needs we would like to support

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Holland, Belgium, Israel, Africa and quite a few people from Asia. Of course, those people will not be directly affected by Brexit itself, but what they will be affected by is the hardening of attitudes to immigration and allowing people into the country. I think Brexit was the result of a wrongful reaction to immigration, and what we haven’t done is really explain why those people who come into labs like these are actually a huge benefit to the UK. I don’t know how it will affect us at the Genesis Research Trust in particular, but I think generally all research groups are concerned – there is a worry that we won’t be able to attract the high quality teams we currently have.

That is a real shame. It sounds like Genesis Research Trust has a fantastic code of ethics in place though – you promote equality and a collaboration between scientists from different backgrounds and nationalities which is fantastic to hear.

Going back to the research side of things, and with the recent commercialisation of IVF treatment, do you think there is now more than ever a need for clear, accurate and unbiased information for women who are having problems conceiving?

Well one of the things we have at Genesis Research Trust is the ‘Ask Robert’ section of the Genesis Research Trust website where people can contact me directly with a question, anonymously, and we try to give them unbiased information.

We’re not prepared to recommend a clinic to them, and we won’t recommend a particular doctor, because we don’t think that would be anything other than biased, but we try to give information. I think we should generally be doing more of that within a whole range of women’s health issues, but it is definitely needed within fertility.

Trust runs. Could you tell us some more about these?

We started doing challenges to raise money for this building, which was built and opened by the Prime Minister in 2002. The first big challenge we did was a ride from Jordan through Israel into Egypt, which was a massive test at the height of summer. That challenge was for women only, as we wanted to promote women doing something for their own gender that would help women scientists researching women’s health. That then developed into what was called the women-for-women challenge, which we have now done a number of over the years. We went from Gallipoli to Izmir in Turkey one year, we went right along the Nile for two years running, we’ve been to Cuba, we’ve been to Rajasthan, we’ve been to China many times, we’ve been to Cambodia, we’ve been to Vietnam, and we’ve been to other parts of the world as well, including the Baltic and Peking. Our next big rides are to Burma and an amazing trip to Madagascar which we’ve already got close to 100 people signed up for.

These challenges are deliberately arduous – we don’t make it easy – but women of all shapes and sizes do them and they have been very successful. The trouble really is that when we started this we were one of the first charities doing this kind of thing.

Now of course there is a massive market for them, but we still think our charity is rather special because our cause is such a good one – reproductive medicine generally only receives 0.2% of charitable giving. It’s easy to think of a neighbour or a relative who has watched a baby die of a genetic disease that could have been treatable in a unit like this – it’s important to remember that when you’re pedalling up a hill in unbearable humidity.

How successful has that service been?

We get lots of letters of deep gratitude and people who think that it’s wonderful we do it. It’s very time consuming though, and it can be very expensive, because each answer takes 30 – 40 minutes to really make sure that the information is accurate. While we don’t ask for a donation on the site, we hope that more people will donate for this work as it really benefits the charity.

Like you say, these challenges are for a fantastic cause but it seems like not many people have heard about them. Are Genesis Research Trust hoping to get more people involved in the future?

We haven’t advertised enough – that’s been an issue – so I think we could do much more about promulgating what we do. We’ve used a few of the television programmes I go on, like This Morning and so on, and Fern Britton has been a wonderful patron. We’ve also had Charlotte Holmes, a former Miss England, who has been one of our patrons too, but we haven’t done enough of that sort of outreach.

We at Research Features love the fundraising challenges Genesis Research

We can also probably do more challenges inside the UK. Fern Britton has twice done a

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challenge from the very tip of Scotland right down to Land’s End over two weeks. It can be quite a big time commitment though so we’re looking for slightly shorter rides to get people interested and hopefully generate more custom. But you’re right – not enough people have heard about the work we’re doing.

What do you think taking part in these challenges has taught you?

I’ve been on most of the challenges and I think it’s given me a massive respect for the women who are prepared to take part. Very often you start off with a group of people who are strangers to each other, but by the end of the ride, five or six days later, they are almost a different group – they are in each other’s arms, they’re crying, they’ve made permanent friendships. There’s an extraordinary camaraderie about that and I find that very uplifting.

• Genesis Research Trust are always grateful for donations of any amount to help continue their vital work. If you would like to donate to the charity generally, or contribute specifically to a supporter’s fundraising target – or even would like to take part on one of their future cycle rides or treks – please visit their website at www.genesisresearchtrust.com.

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

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Child Health

Three ways to reduce pain in early-life medical procedures Dr Denise Harrison is a nursing professor who, over the past 15 years, has published many studies about pain relief for infants undergoing painful procedures in their first few months of life. Her research aims to narrow the gap between research evidence and practice. This includes encouraging the implementation of three simple, costeffective and well-studied strategies to reduce pain in babies during needles and other procedures.

Your research into the prevention and management of pain and stress in healthy and sick newborn babies and young children has clear benefits for parents worldwide. What drives you to do this important work and educate others?

As a neonatal intensive care nurse and a midwife with over 25 years of experience, I have seen, helped with and done thousands of painful but necessary procedures on sick and healthy babies and young children. We used to be taught that young babies did not feel pain, or that they did not remember pain, yet it was obvious that this was not the truth, and that babies, no matter how preterm and sick, suffered immensely during painful procedures.

I was driven to research how to improve pain treatment for sick babies after helping a physician place a drip in a very sick baby boy with chronic lung disease due to his prematurity. The little guy became so distressed and after a few weak cries, he turned blue, then grey, and we needed to give him extra oxygen and help him breathe, then still had to put the drip in. It was at that point I became very sad for this little baby, who I can still picture clearly, and angry that we were doing such painful procedures with no pain relief. So I set out to discover how to reduce pain during these painful needles in sick babies.

What are the risks of poor pain management during infant procedures?

More and more, researchers are discovering that poorly-treated pain during the many painful procedures sick babies need as part of their care, is harmful to the growing brain. The scenario described above tells of the short-term effects of harm, with the baby being unable to catch his breath and then needing us to resuscitate him. We now know that the number of painful procedures is the strongest predictor of poor brain development – which means

higher risk of poor learning, higher risk of poor physical strength and coordination, and higher risk of behavioural problems (Ranger & Grunau, 2014). If we consistently use effective ways to reduce pain, during all necessary needles and other painful procedures, we may be able to reduce the risk of these poor outcomes.

You describe breastfeeding and skinto-skin care as some of the best ways to reduce pain. Why are these simple measures so frequently ignored by doctors, nurses and midwives who care for newborn babies and young children?

The good news is that, if feasible and possible, supporting mothers to breastfeed, or supporting mothers, fathers or their family or friends to hold premature babies skin-to-skin, during painful procedures, is so simple to do and so effective. It not only helps the babies, it is a very powerful way for parents to help and support their role as protectors of their babies. If this cannot occur, very small amounts, just a drop, of sugar water given to babies to suck, is also profoundly calming for small sick babies and babies up to a year old.

Why are these simple measures not being consistently used? There may be many answers to this question. Again, the good news is these treatments are being used more often than they were. The World Health Organization has now publicly recommended that babies breastfeed during early childhood vaccination, which is a huge step in reducing vaccination needle pain. There is more and more awareness of the poor outcomes of very sick babies, and ways we can give far more gentle care, reduce the number of painful procedures and support parents in their role in caring for their sick babies.

However, we certainly know that many babies, both sick and healthy, still have painful procedures with no pain treatment.

In some cases, this is due to unfounded

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Child Health

myths about using breastfeeding or skin-toskin care, including that the baby will choke (this has not been reported in any trials), the baby will associate the mother with painful procedures (this makes no sense, as a baby will breastfeed about 8 times/day, every day, and during only a couple of these feeds will a procedure be performed), or that mothers do not want to be there for their baby’s needles. When mothers are asked this question, they tell the opposite, that they do want to do everything they can to help their babies. Other reasons may be due to lack of knowledge of the effectiveness of breastfeeding, skin-to-skin or sucrose for painful procedures, or workload issues. For example, standard practice in a unit may be that babies are taken away from mothers for blood work, or that parents are asked to leave the room when procedures are done.

Could you explain the importance of collaboration in disseminating the evidence behind your work and suggest what more international organisations could do to improve painful procedures for infants?

Although there are 100s of published scientific trials and large systematic reviews (the most recent Cochrane systematic review of sucrose for analgesia in newborn babies is 360 pages long), and extensive clinical practice guidelines, these documents are not easy to read, especially for parents of babies. We need to show health care providers and parents how they can help, and clearly show both groups the profound calming benefits to the babies of using breastfeeding, skin-to-skin or sucrose during painful needles. Widely disseminating clear visual messages in the form of ‘usable evidence’ to health care providers and parents around the world will help spread the messages. Providing videos which are free, easily accessible, and in many languages helps to ensure the evidence is able to be shared and used internationally.

What could international organisations do to improve pain treatment?

Some organisations are already working on this. ChildKind International focuses on reducing pain and suffering in organisations that care for children. The Pain in Child Health (PICH) consortium has supported training of a large community of health care researchers aimed at improving knowledge about pain and reducing pain in children around the world. The Council of International Neonatal Nurses, with their

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aim of unifying neonatal nurses globally, shares the BSweet2Babies video series from their site for worldwide dissemination to nurses caring for newborns. We can, and need to, do more, but most knowledge translation begins at our own doorstep: working with our local units, hospitals and health care centres to use such simple strategies consistently is key to changing practices; ensuring that routine practices, such as newborn screening, are performed when mothers are able to breastfeed, or someone is able to hold the babies skin-toskin; ensuring that non-urgent blood work is done when parents are able to be present, rather than at 4am in the morning – a very difficult time for parents to be with their baby in a NICU, yet this is routine practice in many organisations; truly partnering with parents in our pain care and facilitating their presence and their support; working with our organisations to ensure easy availability of sweet solutions – sucrose or glucose.

These are small steps yet they are essential for babies who do not have a voice or a say in the matter.

In addition to the guidelines outlined

in this article, do you have a key message for parents who must put their babies and young children through painful procedures?

My key message would be: ‘ask your nurse or doctor, about ways you can help your baby and young child during painful procedures’. Request to breastfeed, based on the recommendations of the World Health Organization. If you are not breastfeeding, request to hold your little baby skin-to-skin, or your toddler upright and front to front. If your baby is in the NICU, and able to be held, request that non-urgent bloods are done only when you, or your partner / family member / friend is there and able to hold your baby skin-to-skin. You are the ones who will comfort your little toddler when they stumble and graze their knees, when they fall off their bikes and need stitches and whenever they have a ‘hurtie’ or an ‘owie’. Start now, when your baby is just born. You can partner with us – your doctors, nurses, midwives and lab techs – to help us all with your baby’s care during painful procedures.

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Social media [is] an effective way of communicating and disseminating information about neonatal pain reduction strategies

Breastfeeding is a very effective way to keep an infant calm during painful procedures such as a heel lance or venepuncture

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any newborn infants must undergo painful procedures such as a heel lance or venepuncture for blood extraction, or immunisations. Premature or sick infants need more invasive and painful procedures, because they are more likely to need screening and testing to help them recover and become healthy children. After working for many years in neonatal and paediatric intensive care units Dr Denise Harrison began her research career with the question, ‘how can we reduce pain during painful procedures in sick babies?’

Pain early on in life can have a negative effect on cognitive development and have long-term adverse effects on brain development that means infants have altered responses to pain. For instance, painful immunisations and injections early in life can cause a fear of needles, with negative consequences later on.

REDUCING PAIN IS SIMPLE Harrison and her team highlight three simple, cost-effective ways to reduce pain in infants. Firstly, breastfeeding before and during painful procedures can minimise distress to babies. Secondly, skin-toskin care, also called ‘kangaroo care’, is

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recommended for babies who are not able to breastfeed. This involves the baby being held against the skin of a caregiver before and during painful procedures. Finally, the baby can be given small amounts of a sweet-tasting solution, either sucrose or glucose, before and during a procedure.

All three methods are easily administered and can be applied in diverse settings, such as neonatal intensive care units, healthy newborn baby units and immunisation clinics worldwide.

BREASTFEEDING Although on its own breast milk in small quantities is not a very effective analgesic, the entire process of breastfeeding during procedures is highly valuable. The benefits are multifactorial: it combines maternal holding, sucking, a slightly sweet taste and endorphins. More than anything, it is comforting to babies because it is associated with maternal care. This provides effective pain reduction while a nurse takes blood samples or delivers immunisations. If a mother and baby are able to breastfeed, this method costs nothing because it requires no additional resources. It is suitable for non-urgent procedures and can be used in almost any context.

SKIN-TO-SKIN CARE If breastfeeding during painful procedures is not possible, research shows that skin-to-skin care (SSC) also reduces babies’ pain.

Holding newborns to a parent or caregiver’s chest, on their skin, helps to keep the baby relaxed and calm during blood tests or injections. This is especially useful for premature babies, and babies who cannot breastfeed, and means that fathers or other carers can also provide this care. SSC is most effective if started around 15 minutes before a procedure. Nurses, doctors and lab techs can help parents do SSC during painful procedures by doing non-urgent blood tests when parents are able to be present.

SWEET SOLUTIONS Sweet-tasting solutions are the most commonly studied pain treatment in babies. Very small amounts (just a few drops) of sucrose or glucose, reduce pain during procedures for newborns and babies up to one year of age. The pain reducing effects are due to distraction, because of the sweet taste, and more importantly, an orally mediated sweet-taste-induced opioid response. For best effects, small amounts of sucrose or glucose are given to babies on their tongue, with or without a soother, just before and then during painful procedures.

The concentration needs to be sufficiently sweet, therefore at least 20% glucose or sucrose is needed.

MEASURING PAIN Due to extensive research by nurses, doctors, psychologists and scientists over the last 20 years, Harrison’s team have good ways to measure pain. For babies, they use the Premature Infant Pain ProfileRevised (PIPP-R) and for older babies and young children, they have the FLACC index

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Child Health

(Face, Legs, Activity, Cry, Consolability).

The most commonly used indicator in pain measurement is facial expressions of pain. It is clear that for studies measuring pain in babies during procedures, babies given no treatment have higher pain scores compared to babies breastfed, held with SSC or given sucrose or glucose.

BARRIERS TO IMPLEMENTATION Harrison emphasises the importance of working at translating knowledge into action, and tries to identify facilitators and barriers to the implementation of pain reduction strategies. It seems counterintuitive that these methods are not widely used, because they are easily administered and incur few financial or time costs. In one study, where they examined YouTube videos of infant immunisations, Harrison’s team concluded that there were very few cases where any pain relief was used, with consequent impacts on the babies’ discernible level of distress.

In a later study exploring the barriers to the implementation of pain reduction strategies, Harrison’s team found that the main reasons that measures were not taken were related to the knowledge and education of healthcare practitioners, and practical factors related to the medical procedure and organisational structure.

Further constraints, such as the attitudes of healthcare practitioners and the preference to complete procedures without parents present were also identified.

OVERCOMING BARRIERS Understanding the reasons why pain reduction strategies are not widely adopted in neonatal contexts is the

first step to breaking down the barriers to their implementation. After many years of research, educating healthcare practitioners (nurses, doctors, lab techs) and encouraging adoption of pain management strategies in clinical settings, there has been little actual uptake. This led Harrison to try a different approach. She now also partners with parents of babies, and targets the communication of her findings at parents, advising how to assist during procedures, and how to advocate on behalf of their babies.

As part of her campaign ‘Be Sweet to Babies’, Harrison and her team have produced Youtube videos to promote pain management strategies to parents, and to demonstrate how to deliver them.

She sees social media as an effective way of communicating and disseminating information about neonatal pain reduction strategies. They have also produced simple posters outlining the three techniques that can be used, which aim to encourage parents and healthcare practitioners to adopt them. These methods of communication can also be extended to other settings, such as the developing world, or clinics with poor resources or training.

Harrison also identifies the need for a coordinated and uniform approach to reducing neonatal pain, involving participation from healthcare professionals, parents, frontline carers and the leaders of organisations and professional associations.

Clear guidelines for clinicians on pain management techniques are necessary, but advice for parents is also needed. The evidence is clear that parents can help reduce babies’ pain with simple strategies – Harrison wants to help healthcare professionals work together with parents to put this evidence into practice.

Detail RESEARCH OBJECTIVES Dr Harrison’s program of research, called ‘Be Sweet to Babies’, focuses on pain management in neonates, infants and children. Her studies include efficacy, effectiveness, safety and utilisation of sucrose in diverse neonatal, infant and child populations; systematic reviews of sucrose for pain management; pain prevalence in diverse clinical settings; pain management in NICUs and community settings during immunisation; knowledge translation; and ethics of conducting clinical trials in vulnerable patient populations.

COLLABORATORS • Sandy Dunn from BORN • Baby Friendly Initiative • Bonnie Stevens at SickKids and University of Toronto • Paula Forgeron and The Pain Hub at University of Toronto • And all parents of babies and young children BIO Dr Denise Harrison is the Chair in Nursing Care of Children, Youth and their Families at the Children’s Hospital of Eastern Ontario (CHEO) and CHEO Research Institute and an Associate Professor at the University of Ottawa.

Her research program “BSweet2Babies” focuses on improving pain management for sick and healthy babies and young children. Her research includes using innovative ways to move pain treatment knowledge into action.

CONTACT Faculty of Health Sciences 538-540 KE, Ottawa, ON, K1N 6N5 Canada E: Denise.Harrison@uOttawa.ca T: +1 613 562-5800 W: www.cheori.org @dharrisonCHEO BeSweetToBabies To see Denise’s work for yourself check out her videos on YouTube

Shining a light on the hidden viral infection that causes birth defects Researchers at the University of California are paving the way in discovering potential targets to help us fight the devastating impacts of infection with the birth defect virus, human cytomegalovirus (CMV).

Dr Lenore Pereira is leading the current research into this common, yet relatively unfamiliar, virus.

Child Health

What inspired you to focus your research on human cytomegalovirus (CMV)?

When I began studying CMV, little was known about the protein composition of virions, the breadth of target cells for human infection and the mechanisms of infection and transmission during pregnancy leading to birth defects in the foetus.

What have been the biggest challenges you have faced conducting this research?

The extreme species specificity of human CMV requires that only human cells be used for infection, making it difficult to model congenital disease except for experiments using cells from the human placenta and explants of the earlygestation, developing placenta.

Why do you think there is so little awareness of CMV in comparison to other viruses?

The public is most aware of viruses featured in newspapers, i.e., epidemics (Ebola and Zika viruses), annual seasonal outbreaks (influenza virus), local outbreaks that occur in unvaccinated children (measles,

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mumps), and sexually transmitted viruses and those spread by drug use (human immunodeficiency and hepatitis C viruses).

In contrast, CMV causes a relatively benign infection, without a rash and with a usually mild fever. Nor does infection cause complications in healthy children and adults. Hopefully, this lack of awareness will soon change. The internet has created a new venue for families whose children were born with congenital CMV to communicate with one another at the national and international level to raise awareness about testing newborns and alerting pregnant women about birth defects from first-time congenital infection.

What will be the next steps for the development of therapeutics based on your findings?

Our work, and that of other groups, has focused on understanding the role of maternal immunity that can reduce infection in the developing human placenta, especially in the first trimester when the foetus is most severely affected.

We found that potent neutralising antibodies, to specific CMV proteins in the virion envelope, effectively block infection

hy is there still no treatment or vaccine for congenital CMV infection? At the University of California in San Francisco, Dr Lenore Pereira leads a research team that endeavours to understand how a common virus can cause intrauterine infection in some women that spreads to the developing placenta and foetus and causes congenital birth defects worldwide.

is essential for foetal development. It has been known for over 50 years that CMV infection during pregnancy can result in damage to the foetus. However, there are still no treatments available for infection during gestation. Currently, there is also no vaccine to prevent infection, and although several possible candidates have now been identified, they are still under investigation or in early clinical trials for organ transplant recipients, where CMV infection can involve severe complications.

Human cytomegalovirus (CMV), a member of the Herpesviridae family, is highly specific and infects only humans. In pregnancy, it specifically infects placental cells and impairs their differentiation that

THE MAJOR VIRAL CAUSE OF BIRTH DEFECTS AND HEARING LOSS Between 0.2% and 3.0% of all newborns in the US are infected with CMV and 5 to 10% of those may have poor clinical outcomes

of cytotrophoblasts (target cells in the placenta). Hopefully, in the near future, novel vaccines and antiviral products based on monoclonal-antibodies to these proteins can be developed to prevent or reduce CMV infection in early pregnancy.

How do you see your research progressing in future?

In recent studies of placentas from mothers who deliver congenitally infected babies with intrauterine growth restriction, we found persistent CMV infection in epithelial cells of the amniotic sac (foetal membranes). We also discovered persistent infection in membranes of babies born with symptomatic and asymptomatic infection.

We plan to dissect the molecular processes that lead to persistent infection in these special cells and identify the route of virus spread in late gestation. This could be different from early gestation and explain why infection in the third trimester is usually benign. In addition, we recently reported that the Zika virus infects the human placenta and proposed two routes of virus transmission from mother to baby.

We hope to decipher the mechanisms that target Zika infection to specific cell types.

or hearing loss. A child is born affected by congenital CMV every hour, outnumbering disabilities caused by other conditions routinely screened for in the US. Birth defects that result from CMV infection also exceed those caused by other more widely known conditions, such as Down’s syndrome and foetal alcohol syndrome. Despite the severity of congenital disease and the cost of resultant healthcare estimated to be nearing 2 billion dollars in the US, CMV has been neglected with regards to medical research. This can be partly attributed to the complexity of the viral infection and the high degree of variation in clinical outcomes. The molecular and cellular basis for its impacts and the role of the placenta have until recently been poorly understood.

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Dr Pereira has been at the forefront of work to understand the biology of infection during pregnancy and her ground-breaking research in the human placenta has led to several promising avenues for the development of novel therapeutics and vaccines.

The most severe outcomes of infection occur when a woman contracts the virus for the first time during early pregnancy. This is known as primary maternal infection and poses the highest risk of birth defects for the foetus.

These are unpredictable and range from severe motor and neurological disabilities such as microcephaly and cerebral palsy, to levels of vision impairment and hearing loss. Out of primary maternal infections, the majority of cases resulting in disability occur when a mother is infected during her first trimester of gestation. However, infection can occur at any stage of pregnancy and even late infections can sometimes cause birth defects. Foetal transmission rate from primary maternal infection in the first trimester is 30–40% and a quarter of those infected will suffer from permanent birth defects. Some infants also die soon after birth or their life expectancy is greatly reduced due to complications of congenital disease.

INVESTIGATING THE INFECTION Dr Pereira and her team’s investigation has been focused on the effects of the virus on placental development and function, as this is the major route of transmission to the foetus. They also suspected that placental infection and subsequent injury could be a cause of intrauterine growth restriction (IUGR). Dr Pereira’s team analysed biopsy samples for pathology, patterns of viral proteins in infected cells, and the presence of viral DNA. They found not only evidence that CMV infection affects development and impairs the function of the placenta, but also that placental infection in isolation, without foetal transmission, can also result in negative outcomes. These had previously only been associated with transmitted infection with symptomatic outcome and it was thought that a maternal infection not passed onto the foetus did not have substantive clinical manifestations. However, the pathologies found interfere with placental function during gestation and disrupt the transport of substances between the foetal and maternal blood, therefore restricting growth of the infant in the womb.

To investigate this further, the researchers studied the virus both in vitro, in cell cultures, and ex vivo, in placental tissues. Due to

Figure 1 - Changes in the amniotic epithelium

Figure 2A - Chorionic villus explant from a placenta at 8 weeks gestational age infected ex vivo with a pathogenic clinical CMV strain.

Figure 2B Cytotrophoblasts (red) in villus explant expressing a CMV protein in the nuclei of infected cells (green).

The virus is a major cause of permanent disabilities in children with a child born affected by congenital CMV every hour

Detail RESEARCH OBJECTIVES Dr Pereira’s current research goal is to gain a deeper understanding of the biology of human CMV infection and its mechanisms of pathogenesis of the placenta. Sha also recently began to study Zika virus infection in the human placenta in collaboration with Dr Eva Harris at University of California, Berkeley.

Dr Pereira (left) and her research team

the species specificity of CMV and the relative uniqueness of the development and architecture of the human placenta, studying viral infection in vivo is difficult. Therefore, Dr Pereira and her team devised a novel model for studying the human placenta. Xenografts (a surgical graft of tissue from one species to an unlike species) of placental tissue were implanted into severely immunocompromised mice as a model system. The cells differentiate, remodel blood vessels in the mouse kidney, and facilitate development of new lymphatic vessels like those found in the uterine wall in pregnancy. Using this model system, they were able to show that factors released from infected placental tissue can impair functions in the model system.

Placental structures through which the exchange of substances takes place between maternal circulation and foetal blood vessels are known as chorionic villi. Progenitor cells in the chorionic membrane differentiate into fully mature trophoblasts in chorionic villi.

The researchers found that the virus halts the development of these cells at one of the earliest stages. This impairs placental development, resulting in pathology and a failure to compensate for functional deficiencies they had observed in placentas with severe congenital infection.

In addition to these findings, their research has also led them to another clinically important discovery. They found that adding antibodies to CMV proteins to infected cells suppressed viral replication and rescued the cells’ capacity to differentiate into healthy placental cells. Therefore, treatment with

antibodies could enable intervention to reduce the risk of the virus being transmitted to the foetus and help facilitate continued development for damage the placenta already sustained.

NEW HOPE IN FIGHTING CMV Excitingly, the team’s most recent project has unveiled another promising line of enquiry for developing interventions to fight against congenital CMV infection. Their recent study has shown that the virus suppresses the production of antiviral proteins made by host cells and activates pathways that increase the production of proteins that block selfdestruction of infected cells in the amniotic sac allowing the virus to continue with a persistent infection. Targeting interventions to the signalling pathways that contribute to CMV persistence, and enhancing those with the capacity to suppress it, could also lead to novel treatments.

As a result of the studies on infection of the human placenta led by Dr Pereira, researchers are now equipped with a deeper understanding of how CMV can lead to IUGR and several promising strategies to improve the outcome of cases of primary congenital infection. Further work into developing antibody-based therapeutics based on their findings could yield interventions to reduce the number of babies affected by severe congenital disease. Finally, by tailoring the design of new vaccines to protect the placenta from infection, the future looks brighter for pregnant women and the health of their babies.

FUNDING Primarily the National Institutes of Health, in particular, National Institute of Allergy and Infectious Diseases, Eye Institute, Heart Lung and Blood Institute, Institute of Dental Research, Institute of Child Health and Development, as well as the National Science Foundation, March of Dimes Birth Defects Foundation, Thrasher Research Institute, Fight for Sight, University of California San Francisco, Academic Senate and various corporate research sponsors COLLABORATORS • T akako Tabata, Matthew Petitt & June Fang-Hoover from University of California San Francisco (Human CMV and Zika virus) • E va Harris, Daniela Michlmayr & Henry Puerta-Guardo from University of California Berkeley (Zika virus) BIO Dr Lenore Pereira graduated with a BS in Biology from Marquette University, MS in Microbiology from the University of Illinois and Dr rer. Nat./PhD in Microbiology from the University of Frankfurt am Main, West Germany. In 1985, she joined the faculty at the University of California San Francisco as Associate Professor in the School of Dentistry where she is currently Full Professor in the Department of Cell and Tissue Biology.

CONTACT UCSF School of Dentistry Department of Cell and Tissue Biology Medical Sciences Building S667/S675 513 Parnassus Ave, San Francisco CA 94143, USA E: Lenore.Pereira@ucsf.edu T: +1 415 476-8248 W: http://profiles.ucsf.edu/lenore.pereira Lenore Pereira

Cutting the cord: babies benefit from a delay Research by Dr Judith Mercer of the University of Rhode Island shows that babies who remained connected to their umbilical cord for longer had healthier blood days and months later.

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Why is it common practice to cut the umbilical cord so swiftly?

The main reason is for expediency. Also, people think that the neonatologist can 'fix' whatever is wrong with a newborn.

Many studies are showing that these infants would do better if they received their cord blood before breathing and cord clamping.

Should an expectant mother be worried about the timetable of her birth?

She should take steps to ensure that her infant will receive delayed cord clamping or cord milking at birth. The baby’s father can help to confirm that a delay happens at the birth.

Is a delayed clamping always better?

Yes. This is basic blood volume that

the infant was using for respiration in the uterus. S/he needs it after birth for independent respiration. Also, it is filled with many stem cells that have been shown to help with healing the newborn and may be extremely important over one’s lifetime.

Can a mother request a delay?

Yes, she can and she should.

Are there still risks associated with a delay?

No. We have three meta-analyses involving delayed cord clamping with term infants, preterm infants and both with cord milking.

A meta-analysis combines data from many studies. None of the meta-analyses, or any of the individual studies show harm to the infants.

Many studies are showing that these infants would do better if they received their cord blood before breathing and cord clamping

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Normal western practice has been to cut the baby’s cord within a minute of birth. In the US, it tends to be quicker, unless the baby is premature. In the UK, the current advice is to cut it after a minute.

EFFECTS OF A FIVE MINUTE DELAY WITH INFANTS PLACED ON THE MATERNAL ABDOMEN Mercer and her team compared a random sample of babies that had an immediate cord clamping operation (ICC, within 20 seconds) with babies that continued to receive oxygenrich blood via an intact umbilical cord. In these cases, the cord was not severed until five minutes after the birth, allowing ongoing placental transfusion of vital red blood cells.

If the provider felt they could not wait, they were instructed to milk the cord.

Striking new evidence suggests that a five-minute delay in cutting the umbilical cord could be very beneficial to the health of newborn babies. The recent study by Dr Judith Mercer, from the University of Rhode Island in the US, could have major implications for the way we deliver babies in the future.

RESULTS TWO DAYS LATER Her researchers monitored the effects of the two different strategies. Even 48 hours later, the babies that experienced delayed cord clamping (DCC) had significantly higher blood levels of the protein haemoglobin.

Haemoglobin contains iron, a critical element for early development. A higher haemoglobin

hen a baby is born, how quickly should the midwife cut the umbilical cord? For decades, scientists and health care professionals have had very different views on exactly when this procedure should happen.

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level is usually a proxy for more blood volume.

During birth, babies’ lungs must change in function from an organ that produces fluid to one that allows gas exchange. To facilitate this, the proportion of blood flowing to the lungs increases from 8–10% to around 45%.

ICC reduces the amount of blood available to the newborn, meaning that the baby’s body draws blood from other reservoirs, potentially at the cost of other organs, which may consequently underperform.

One of the reasons that some of those who currently practise ICC do so is to minimise the risk of severe jaundice in the baby (80% of all newborns experience some jaundice).

This is caused by excess bilirubin in the blood. Bilirubin is naturally produced by the body as it breaks down old red blood cells and is protective at normal newborn levels. However, excessively high levels can be extremely harmful, and may even lead to brain damage. Some doctors had been worried about other possible harmful effects on the newborn’s blood but Mercer’s experiments (and others) found no evidence that DCC increased the risk of conditions such as polycythaemia, hyperviscosity, or transient tachypnea, all of which, like jaundice, are supposedly avoided by implementing ICC. Mercer’s study found no increase in bilirubin levels in two-day old babies who had undergone DCC, suggesting that DCC does not increase the risk of hyperbilirubinaemia (excessively high levels of bilirubin).

FOUR MONTH RESULTS At four months, infant ferritin levels were measured. Ferritin is the main iron-storage protein in the body so testing ferritin is a good indication of iron levels. They also underwent MRI scanning using a novel myelin-imaging technique that can quantify the amount of myelin (wrapping of nerve cells) in the brains of newborns. Mercer’s team was looking for any association between higher ferritin levels and myelin content. All MRI scans were done in the evening during non-sedated sleep.

Infants in the DCC group had higher ferritin levels and a positive relationship between ferritin and myelin content in areas that are important for sensory processing and motor function. Babies who had had DCC had more myelin in these same areas than the infants with ICC. This suggests that delayed cord clamping may have continuing benefits

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Delayed cord clamping may have continuing benefits for the early development of white matter for the early development of white matter (brain tissue important for learning and brain function).

PLANS FOR ONE AND TWO YEARS Mercer plans to assess the study children at one and two years of age with repeated MRI scans and development testing. All children will have reached one year of age by November 2016. That data should be ready for publication in the spring of 2017. The

same follow-up is planned for two years of age as well.

CHAMPIONING DELAYED CORD CLAMPING Mercer has been an advocate of DCC for much of her career. 'I adopted the practice of delaying cord clamping to ensure a more gentle birth and have used it for more than 30 years,' she has previously told the Science and Sensibility website. Mercer was awarded the

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LACK OF IRON Mercer’s research is supported by other recent studies across the world that hint at DCC’s far-reaching benefits. It has been suggested that higher iron content in early life is connected to better coordination and social skills in later life and that developmental issues like ADHD are linked to less brain myelin.

Iron is crucial to early brain development, particularly in areas responsible for sensory processing and motor function. It is essential for the formation of oligodendrocytes in the brain. Oligodendrocytes are cells that are key for myelin formation, the development of the fatty sheath around brain cells that is essential to their function.

While questions about the timing of cord clamping linger, in premature deliveries DCC has been shown to lower the risk of bleeding in the brain as well as the need for transfusions.

Preterm infants are very susceptible to the harmful effects of low blood pressure in the first 24 hours of life. DCC has been shown to raise blood pressure in these tiny babies during that critical period.

highest honour of the American College of Nurse-Midwives, the Hattie Hemschemeyer Award, in 2014.

She recounts an early clinical experience with a baby boy born with his umbilical cord wrapped tightly around his neck. In this instance, the baby was born pale and limp. In a hospital, the tight cord would have been cut immediately and the baby put on a warmer or resuscitaire to be resuscitated. Instead, Mercer simply unwrapped the cord and allowed the highly oxygenated blood to flow naturally.

'In about one and a half minutes, he flexed his extremities, opened his eyes and took a gentle breath. He nursed very well and was a normal child at one year of age when I last saw him,' she said.

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Although the long-term effects of ICC on the infant were not studied until recently, Mercer showed an advantage to preterm infants at 18 months of age in an earlier study and Dr Ola Andersson from Sweden found advantages for children at 4 years of age who had DCC as babies. Many experienced midwives had been advocating DCC in order to promote a more relaxed, natural transition from the pre-natal world. In the past this attitude has been frowned upon in some hospital settings, says Mercer.

The importance of the timing of cord clamping is shown by the physiological figures: delaying by just three minutes allows an extra 100 ml of blood to travel along the cord to the baby. Relatively speaking, this is equivalent to just under 2 litres of blood for an adult; the effect of leaving the cord intact at such a vital time could be very significant.

Detail RESEARCH OBJECTIVES Dr Mercer’s experience as a midwife has informed her research into cord clamping. Her work shows that a 5-minute delay in cord clamping can have long-lasting benefits to the term infant.

FUNDING National Institutes of Health (NIH) COLLABORATORS • R esearch partner: Dr Debra EricksonOwens • D r Sean Deoni • D r James Padbury • D r William Oh • D r Betty Vohr • R ichard Tucker, BA BIO Judith Mercer was educated at University of Maryland, Columbia University, and Catholic University of America.

Her first career as a midwife (30 years) led to her second career as a funded researcher studying the effects of delaying cord clamping on the newborn. She is a research scientist at Women & Infants Hospital in Providence, Rhode Island and Professor Emerita at the University of Rhode Island.

CONTACT Judith Mercer, PhD, CNM, FACNM Women & Infants Hospital Pediatric Research 101 Dudley St Providence, Rhode Island, RI 02905 E: jsmercer@uri.edu T: +1 401 480 1542 W: http://online.uri.edu/professorprofile_judith-mercer.aspx http://cordclamping.info/

'Birth is perhaps the most dramatic physiologic event any human will experience,' says Mercer. 'How it is conducted may have effects that will last a lifetime. Evidence is building that the current practice of immediate cord clamping is creating harm.'

More information: http://cordclamping.info/

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Understanding the mechanisms of language comprehension Dr Julie A Van Dyke, a senior research scientist in the field of psycholinguistics at Haskins Laboratories, Connecticut, USA, is helping to unravel the mechanisms underlying language comprehension, including the processes that lead to poor understanding when reading or listening.

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L

anguage comprehension is one of the most automatic tasks that humans perform. Yet it is also one of the most complex, requiring the simultaneous integration of many different types of information, such as knowledge about letters and their sounds, spelling, grammar, word meanings, and general world knowledge. In addition, general cognitive abilities such as attention monitoring, inferencing, and memory retrieval are used in order to organise this information into a single meaningful representation.

For the most part, we take the ability to accomplish this task for granted. However, for those with language-based disabilities – including developmental disabilities (such as dyslexia, specific comprehension impairment, or speech deficits) and acquired disabilities (such as language impairment after brain injury) – assembling all this information accurately is a major challenge.

While clinicians and educators are on the frontlines in helping individuals to overcome these challenges, scientists in the field of psycholinguistics are conducting the basic research that investigates: how the brain processes spoken and written language; what brain functions go awry in the case of language disability; and how to most effectively remediate deficits when they occur.

Dr Julie A Van Dyke and her team at Haskins Laboratories are working to develop a computationally precise model, referred to as a cognitive architecture, of the mechanisms that support language comprehension. This work requires methods that reveal information both about the real-time processes associated with comprehension, and about how the products of comprehension are later accessed and retrieved for subsequent use. This is necessary to address questions such as: Are all words, or word meanings, equally likely

to be retrieved? Which type of information (phonological, grammatical, semantic) is used first, or has more influence over how a person understands a text? What processes do people use to correct themselves if they realise that they’ve misunderstood a text?

What if they don’t realise when this happens?

For the most part, readers and listeners are completely unaware of how their brain is managing these tasks, yet the answers to questions like these provide crucial clues about the nature of the cognitive architecture for language processing.

A major part of language comprehension is integrating new information with what is already known. Thus, Dr Van Dyke’s work focuses especially on memory processes.

Her research is driven by two hypotheses: the first concerns the potential contribution of poor memory retrieval to reading disability, through failure to integrate the right information at the right time during comprehension; the second concerns the role that interference from similar representations of words (or phrases or clauses) plays in inefficient memory retrieval.

For example, in collaboration with Dr Brian McElree (New York University, USA), she showed that when a reader’s attention is directed towards a group of ‘fixable things’ (e.g. table, sink, truck), he or she has a more difficult time processing the verb ‘fixed’ in a sentence like, ‘It was the boat that the guy who lived by the sea fixed in two days’.

This is because readers have a harder time focusing on fixing ‘the boat’ (the correct interpretation) because they become distracted by the other ‘fixable’ things that are also prominent in their memory.

This type of interference occurs when retrieval cues become associated with other similar items in memory. In this example, association was along semantic lines (‘tables’, ‘sinks’, ‘trucks’, and ‘boats’ are all ‘fixable’).

However, interference can also arise along phonological lines (for example, words that rhyme: ‘hat’ > ‘mat’, ‘sat’) or lexical lines

HASKINS LABORATORIES Haskins Laboratories is a private, not-for-profit research institute founded in 1935, with a scientific mission to investigate the biological basis of speech, language and reading, and their related disabilities. Together with long-standing collaborators from University of Connecticut, Yale University, and over 40 international partners, Haskins has pioneered the scientific theories that guide current clinical and educational remediations for speech and reading disabilities. The overarching mission of the Laboratories is to leverage cutting-edge science to enable those with language impairments to participate more fully in society.

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How close is your research to facilitating the development of remediation strategies that can help poor readers improve their comprehension skills?

While the scientific basis in support of word reading remediations is extensive, research on effective methods for teaching comprehension is weak and inconsistent. This is partly due to the complexity of the comprehension task. A much greater hindrance, however, is the lack of detailed process models of how comprehension takes place. For example, one of the most influential models of reading comprehension – known as ‘The Simple View of Reading’ (Gough & Tunmer, 1986) – suggests that once a student learns to decode words, then reading comprehension follows automatically from their oral language comprehension ability. Yet it is exceedingly common for a child with perfectly normal oral language abilities to exhibit difficulty comprehending written material. In order to provide effective remediations for such children, it is crucial to have a clear understanding of how the processes of comprehending written texts differ from comprehending oral language. This is one of the primary goals of my research. Methods that track comprehension processes as they unfold in real time, together with targeted experimental manipulations enable us to identify which types of linguistic knowledge and/or general cognitive abilities (such as retrieving information from memory) matter most when reading specific types of texts (i.e., narratives, expositions, discourses, etc.). Data of this sort can be directly translated into classroom practices that can improve how children are taught to read.

How similar are the reading disabilities that you focus on to the more wellknown reading disabilities such as dyslexia?

Dyslexia is a disability at the word reading level, and is present in roughly 10% of the general population. Obviously, dyslexia can produce poor comprehension; when individual words cannot be efficiently

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recognised, comprehension will be poor.

The remediation in this case should be at the word level, focused on improving decoding ability. In contrast, since our focus is specifically on comprehension processes, we are more interested in those who have normal word decoding ability but still struggle to comprehend. This is an extremely common occurrence, and for this reason much of our research does not target individuals with diagnosed disabilities. However, at the extreme end of this spectrum are those who have perfectly normal decoding abilities but who have reading comprehension abilities that are at least two standard deviations below the level expected for their age.

These individuals have been referred to as ‘Specifically Poor Comprehenders’ (SPC) and they have a prevalence similar to that of dyslexia (roughly 10% of the general population). When our goal is to focus on a specific clinical population, this is our group of interest. Recent work with Dr Nicole Landi (University of Connecticut) suggests that SPC do not notice when they retrieve incorrect information, and hence cannot correct themselves. In contrast, skilled comprehenders realise when they’ve experienced retrieval interference and do correct themselves.

What are the main implications of adopting a retrieval–interference approach in the study of comprehension difficulties?

Our research represents a major paradigm shift in the field of reading and language comprehension. Traditionally, the primary explanation for poor comprehension was low working memory capacity, with an associated assumption that a person’s working memory capacity was an innate and immutable trait. From this perspective, remediations are only weakly motivated, since the core-deficit is viewed as part of an individual’s genetic makeup. The breakthrough associated with the cue-based retrieval approach is the understanding that the mechanisms of language processing (including reading comprehension) require only the most minimal memory capacity.

This means that a person’s innate memory ability need not constrain their language (or reading) comprehension. According to our theory, comprehension ability is determined by whether information can be retrieved efficiently when it is needed. To achieve this, two major components must be in place: individuals must have high quality linguistic representations and they must have the ability to resist interference from distracting information. These two components lead directly to specific interventions that can more efficiently target the causes of comprehension difficulty.

Is there conclusive evidence for interference in encoding and, if so, how does it affect language comprehension?

Language is apprehended phonologically – even written text is transformed into ‘silent speech’ as we read. This means that phonological features play a fundamental role during encoding: if an individual cannot perceive important phonological distinctions, those distinctions will not be part of the individual’s memory representation.

For comprehension, however, a word’s phonological form serves mainly as a ‘handle’ that allows access to its meaning.

This meaning, together with the word’s grammatical context, is what matters most in comprehension. We examined this idea in a study led by Dave Kush (Norwegian University of Science and Technology), in which we asked whether interference from phonologically similar items held in memory (i.e., coat, wrote, note) would affect how participants read ‘It was the boat that the guy who drank coffee sailed in two days’.

We found definite effects of interference from the distracting words at the point of reading ‘boat’. However, these differences had no bearing on how well participants comprehended the sentence. What seems to matter more is whether readers can perceive distinctions along semantic or syntactic dimensions, and this is entirely dependent on the quality of their semantic and syntactic encodings. Interestingly, data from a recent fMRI study show a qualitatively different pattern of brain activation for encoding

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Figure 1 Poor readers (panels C, D) display different eyemovement patterns to good readers (Panels A, B), particularly in sentences with increased syntactic processing demands (panels B, D).

interference (Panel A in Figure 3) versus retrieval interference (Panel B in Figure 3).

In your research you refer to the idea of a ‘cognitive architecture’. Could you explain this term?

The use of the term ‘architecture’ is a very deliberate choice. Physical architectures create human thoroughfares by specifying how structures are supported and defining how spaces are separated. Similarly, a cognitive architecture specifies a thoroughfare for information. Just as a wall will separate people, with doorways to control how they interact, so too will a cognitive architecture separate types of information, and include constraints on how and when information interacts. Thus, when we speak of a cognitive architecture we are referring to a particular processing model that incorporates hypotheses about the mechanisms that support the structure (e.g., memory storage and retrieval, attentional control), what types of information enters into the structure (e.g., lexical, grammatical, semantic, pragmatic) and when, and how these components work together, both in normal and impaired processing. For example, in our model a crucial architectural question is about how different types of retrieval cues are weighted during retrieval. If a distractor matches the retrieval cues on their semantic dimension, but not on their syntactic dimension, will it be retrieved? Or more narrowly, which of several different syntactic cues matters more in a given context? These questions are akin to asking whether the door between active memory and passive memory is like a sliding door, through which anything could pass, or whether it is more like a revolving door, which imposes a particular order on the entities that pass through it. Highly articulated models of this sort play a crucial role in language research, and especially in disability research, because they capture the underlying regularities in how the brain manages information, and reveal ‘pressure points’ in the system where breakdowns may occur.

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Figure 3 Both encoding and retrieval interference invoked increased activation in dorsal-lateral pre-frontal cortex, which has been associated with conflict control. However, retrieval interference was uniquely associated with posterior activations, which have been related to the integration of syntactic structures.

Figure 2 Event-related brain activity in good and poor readers while reading short passages. Good readers are better at realising when additional information must be retrieved (increased P300), and at integrating that information into a coherent meaning representation (increased P600).

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(words that have an associated meaning: ‘hat’ > ‘hair’, ‘wig’). Dr Van Dyke has suggested that retrieval interference such as this is pervasive in language processing, and is one of the main factors affecting comprehension ability.

GATHERING THE DATA Dr Van Dyke and her team employ a range of experimental methods in their research.

The real-time behavioural measurement techniques they use include: eye-tracking technology to study people’s eye movements while they are reading (see Figure 1); speed–accuracy tradeoff (SAT), which tracks changes in response accuracy with the accumulation of information over time; and electroencephalography (EEG), which records the electrical activity of the brain as it processes information (see Figure 2). These techniques allow the language processing architecture of the brain to be characterised with precision, and are complemented by magnetic resonance imaging (MRI), which allows the team to characterise the neurobiological foundations of skilled and disabled reading.

Dr Van Dyke’s research is also rare in its effort to create an individualised skill profile for all study participants. To accomplish this, her team administers a broad range of cognitive assessments which provides information on language-related abilities such as phonological processing, word decoding, knowledge of syntax, vocabulary, and reading experience, as well as on more general cognitive capacities such as IQ, processing speed, and visual memory.

A particular emphasis in her project is to include data from individuals who are not high-school graduates and who do not (and may not plan to) pursue higher education.

This group has been largely overlooked in previous language research, but is essential for providing a representative sample of reading ability in the general population.

Because the scale of such datasets is large – they are more comprehensive than is typical in psycholinguistic research – Dr Van Dyke and her colleagues have pioneered the use of innovative analysis techniques. With Dr Kazunaga Matsuki and Dr Victor Kuperman (McMaster University, Canada), Dr Van Dyke has demonstrated that a machine-learning technique known as Random Forests can determine which assessments are most important for explaining variation in her experimental measures. The outcome of these studies can provide critical guidance about which instructional techniques are

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Dr Van Dyke and colleagues have shown that both poor and skilled readers alike employ efficient, direct-access retrieval during listening comprehension best matched to particular subgroups of readers.

MANAGING INFORMATION A functional cognitive architecture supporting language comprehension must accommodate the fact that linguistic input – words, phrases, sentences, texts – contains many different kinds of information, but can only be processed serially (e.g., in a word-by-word fashion). This is the essential challenge for the language comprehension system: to reconcile the hard constraint of the serial experience of linguistic information, with the simultaneous processing and comprehension of its multi-dimensional features. In some cases, this is not so challenging. For example, understanding a phrase like, ‘the dog waited by the door’ is straightforward, because each word can be easily integrated with what came before it. However, in many cases, words that must be understood together are separated from each other,

as here: ‘the dog, which had barked at the cat in the neighbour’s yard all morning, waited by the door’. Understanding this sentence puts more emphasis on a person’s ability to organise information in memory and to make appropriate connections: in this case, to realise that it is the dog that waited, and not someone or something else that can ‘wait’, such as the cat. To do this, a comprehender must accurately encode the grammatical relations in the sentence, and use these grammatical cues to select the appropriate animate noun for the verb ‘waited’.

Figuring out how these processes occur is important because the human ability to manage and process several separate pieces of information concurrently is limited: some studies suggest we can only hold between one and four items of information in memory at a time. Therefore, a complex sentence cannot be understood in its entirety as one block piece of

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Detail RESEARCH OBJECTIVES Dr Van Dyke explores the cognitive architecture that supports online reading and language comprehension processes, with a special focus on memory retrieval as a key component of skilled and disabled language use.

FUNDING National Institute of Child Health and Human Development COLLABORATORS • M organ L Bontrager (Haskins) • A ndrew A Jahn (Haskins) • C linton L Johns (Haskins) • Victor Kuperman (McMaster) • D ave Kush (Norwegian University of Science and Technology) • K azunaga Matsuki (McMaster) • A shley G Lewis (Haskins) • N icole Landi (University of Connecticut and Haskins)

information. Rather, there is a memory mechanism that encodes and stores partially processed information, which is later retrieved and patched together into a (hopefully) coherent understanding.

With her colleagues, Dr Van Dyke has demonstrated that a direct-access information retrieval mechanism that operates based on retrieval cues is the key to skilled adult reading. Such a mechanism can compensate for limited memory capacity, explain individual variation in language comprehension, and account for specific difficulty encountered in understanding certain complex grammatical constructions. In principle, retrieval cues can be virtually anything, such as a smell, a sound, a colour or a place, that acts as a guide to what a person is supposed to remember. In the case of written or spoken language comprehension, it is a person’s encoding of the linguistic dimensions (e.g., phonology, grammar, meaning) that matters most.

People who have comprehension difficulties tend to have low-quality word representations stored in memory. These poor representations can lead to confusions among similarly encoded items, and poor comprehenders can have difficulty retrieving the correct representations when they need them.

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FINDING EFFECTIVE REMEDIATIONS It may seem logical to assume that poor readers do not possess the cue-based retrieval system used by skilled readers, perhaps instead relying on inefficient, exhaustive searches of the contents of memory.

However, with Dr Clinton Johns (Haskins Laboratories), Dr Van Dyke has shown that both poor and skilled readers alike employ efficient, direct-access retrieval during listening comprehension. This means that poor readers possess the same basic language-processing architecture as skilled readers. Potential remediations should therefore focus on reducing poor readers’ sensitivity to interference from distracting information. Dr Van Dyke’s work points to the importance of using instructional techniques that encourage learners to draw distinctions between word meanings and grammatical functions, as well as direct their attention to appropriate linguistic cues when processing a sentence.

Beyond helping children and adults learn to read, this research may also provide a new perspective for understanding acquired language deficits (e.g., due to stroke or brain injury), including potentially identifying networks of brain regions associated with managing different types of interference (see Figure 3).

BIO Dr Julie A Van Dyke is a Senior Research Scientist at Haskins Laboratories, and holds adjunct Professorships at McMaster University and the City University of New York Graduate Center. She earned her PhD in Cognitive Psychology from the University of Pittsburgh, a MSc in Computational Linguistics from Carnegie Mellon University, and BA in Computer Science from the University of Delaware. When not in the lab, Julie enjoys spending time with her husband and four daughters. Her research is inspired by mothering a child with spoken language impairment and dyslexia.

CONTACT Dr Julie A Van Dyke Haskins Laboratories 300 George Street, Suite 900 New Haven, CT 06511 E: HaskinsCompLab@gmail.com T: +1 203-865-6163 x 214 W: http://www.haskinslabs.org/research/ research-areas/haskins-complab /drjvandyke /HaskinsCompLab @HaskinsCompLab

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Pioneering global collaboration in children's health research Professor Neena Modi is the President of the Royal College of Paediatrics and Child Health – a charity that, as one of its main aims, champions advocacy on behalf of children worldwide to ensure their fair representation in medical research. She recently spoke to Research Features about this aspect of the Royal College’s current and future strategy, to highlight the importance of global research collaboration.

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he Royal College of Paediatrics and Child Health (RCPCH) have been at the forefront of promoting children’s health research since their inception 20 years ago. They work tirelessly to improve outcomes for children in scientific research, ensuring clinical paediatricians are trained effectively to deliver care to those who need it most.

Today in 2016, collaboration is the word of the day and, as such, RCPCH have made it their mission to ensure that children’s health

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charities work together wherever they are in the world. Professor Neena Modi, the President of RCPCH, recently spoke to Research Features about the institution’s latest campaigns, seeking to guarantee a fair representation for children in research, while also ensuring effective training to paediatricians both in the UK and worldwide.

What does your role involve as President of the Royal College of Paediatrics and Child Health (RCPCH)?

The presidency of the RCPCH is an elected post which involves all of the charity’s 17,000

members. My responsibility is to lead the development of RCPCH’s strategy and really be the outward face and spokesperson for them, overseeing the streams of work and advocacy that the royal college is involved in. It’s an extremely interesting role and it’s a great honour to be in it.

Could you tell us some more about RCPCH’s background and the kind of work into children’s health that is done there?

The RCPCH is one of a small number of royal colleges. We are unique because we are not solely a royal college of paediatricians, but we are a Royal College of Paediatrics and Child’s Health. Inherent in our title is that our primary responsibility is supporting improvements in children’s health. We are a membership organisation predominantly comprised of paediatricians, but we also have affiliate memberships for non-paediatricians as well. One fifth of our 17,000 members also come from abroad so we are an international organisation.

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Spotlight

We were actually first established in 1928 but at that time we existed as the British Paediatrics Association (BPA), which was a part of the Royal College of Physicians (RCP).

We separated from the RCP 68 years later in 1996 when we received our royal charter – so we have quite a long history.

There are a number of things we are extremely proud of. The formation of the BPA was really a recognition that children’s health required its own specialty and expertise in demonstrating that children were not just small adults. That was a vital evolution of the way in which we care for children and is really the first and foremost thing we are proud of here.

As the years have gone on, and particularly when we became a royal college, we took over examinations for paediatricians – so that has been another major achievement. The main examination we run is the Membership of the Royal College of Paediatrics and Child’s Health which trainees complete once they have committed to becoming a paediatrician. It isn’t an exam they do when they finish their training but instead they do it at the point on which they commit to being a paediatrician. It’s an exam that shows they have fulfilled the knowledge-based requirements for becoming a paediatrician.

We are here to advocate and improve children’s health – and we do that in a number of different ways.

First of all, we contribute to the education and training of paediatricians because it is them, of course, who are largely responsible for delivering child health. The second thing we do is promote science and research that benefits children, and the third thing we do is improve the children’s health service by ensuring that the taught paediatricians deliver high quality healthcare. We have also established growing global footprints from the number of programmes we have overseas. Inherent in everything though, is our primary objective: to advocate on behalf of children.

What impact do you think RCPCH has had on children’s health since it first received its Royal Charter in 1996? Are there any accomplishments you are particularly proud of?

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We are also proud to continually produce large numbers of training materials to support paediatricians in the work they do for children’s health. Overall though, we are hugely proud that we have advocated very, very hard for children’s science research.

And how about on a more personal level?

I’m only 18 months into my role as President so it may be a bit premature to be saying this, but one of the things I am really pleased about is having established something called the Children’s Research Fund. This is particularly aimed at supporting the professors of paediatrics of the future – the people who are really going to push forward paediatric science in the future.

During 2012, RCPCH released a report called Turning the Tide, on which you were one of the lead authors. What was this report about?

This was a report we released because we are very keen to ensure that children benefit from science and clinical research. What we wanted to do in the first instance was to define the extent to which children were represented in ongoing medical research, by determining really hard objective facts and figures. What this report showed was that children were under-represented in science and research.

One of the headline figures we found was that, on average, each UK adult had £50 a year spent on affiliated research, compared to children who had only £10 spent on them.

We also established lots of other facts and figures which proved that children were under-represented in scientific research, so having that document meant we had a basis on which to advocate for improvements.

What kind of influence has the report had on children’s health since it was released?

A number of things have happened since that report which I am also very pleased about. First of all, we established something called a Children’s Research Collaboration, which was a forum that brought together a large number of children’s research funders – foundations who are very hard working but have a relatively small annual turnover. Most clinical trials these days cost upwards of £1.5 million, and the training and academia required for a paediatrician costs an estimated £1 million – so substantial sums of money are required to support clinical research. We established the Children’s Health Research Collaboration to bring together these smaller charities so that they could collaborate with each other. This involves not only the bigger charities like the MRC and the NIHR, but also lots of the smaller charities such as Action Medical Research and Sparks. So we can now provide a forum and mechanism for these charities to work together.

The presidency of the RCPCH is an elected post which involves all of the charity’s 17,000 members. My responsibility is to oversee the streams of work and advocacy that the royal college is involved in 31

Spotlight

The next thing I’m really proud about is the development of a children’s research charter.

This was developed in April this year and really enables children and young people to speak in their own voices about how they want to be involved in research. And then the third thing that I’m really proud of is the creation of the Children’s Research Fund, as I mentioned earlier.

So these are three really big things that have emerged subsequent to the Turning the Tide report. We have actually just received some very preliminary data from the National Institute of Health Research as well showing that the overwhelming majority of acute trusts, around 90% of them, are now recruiting children into clinical studies – so there has been a very positive effect since the report was released.

The RCPCH have produced many campaigns over the years on key issues within children’s health. Can you give some examples of your current campaigns?

Well the major one that’s involved us over the past few months has been children’s obesity, which is a huge issue. We’ve been campaigning quite heavily on that, as indeed have a lot of other children’s organisations.

The main issue with this has been the disappointment with the UK government’s children’s obesity strategy. This was originally supposed to be a 50-page document, but was reduced to just 10-pages when it was released, and many of the things that we had hoped to see have not transpired, apart from the sugar tax, which we welcome.

The other big issue for us at the moment is the treatment of child refugees. This is a very acute and ongoing issue, and we are concerned about the fate of the children who are waiting to enter the UK. We feel that the UK should be more welcoming of child refugees and we really want to see them having access to the full range of support they need when they do get into the country. Our recent campaigns have been about, firstly, enabling them to get into the country, and then, secondly, the care that they receive when they get here.

What impact have these campaigns had?

Many of our campaigns have a positive effect – the children's obesity story is by no means over yet because we really do need to turn around the epidemic. We were very strongly engaged in all sorts of collaborative

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advocacy work with other organisations to combat this issue and we are a member of the children’s obesity alliance. We’ve got a lot of collaborative campaigning that is very much still ongoing.

Aside from being President of the RCPCH, you are also the Professor of Neonatal Medicine at Imperial College London. Not only that, but you run clinical duties as an Honorary Consultant in Neonatal Medicine at the Chelsea and Westminster NHS Foundation Trust. How does it feel to be such a recognised peer of children’s health and how do you find the time to balance each of these positions?

As I say, it’s a great privilege and a great honour to be in this position. How do I find the time? Well I guess I find the time!

It’s a lot of work but nobody comes into this kind of job without recognising that. I don’t get much spare time but that is by no means a complaint, because it’s all extremely enjoyable work

I have three main roles: first of all, I have a clinical role because I’m a clinician. Secondly, I have an academic role and I lead a big research group of about 22 people in a huge

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RCPCH is renowned for its collaborative work worldwide, with programmes in Africa, Asia and the Middle East. Why are these global collaborations and programmes so important to you?

These programmes are largely what our international members request of us. We run examinations overseas, where countries approach us to deliver the RCPCH membership examination for paediatric trainees. We also support the training of child health staff in different countries so, for example, we provide training in the emergency treatment of children and newborn babies to paediatric staff overseas.

Not only that, but we also run all other sorts of training programmes. It is mainly education-based training but we also ask to support paediatricians who wish to establish their own specialty organisations, so we are moving into the development of research programmes abroad.

Skilled multi-disciplinary child health workforce

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Children’s health is often an area of ethical dispute within science. How does the RCPCH work within these ethical boundaries during its research?

Ethical conduct in research is absolutely paramount – we want children to benefit from research but we want research to be conducted to the highest possible standards. RCPCH has been at the forefront of really developing a lot of the ethical guidance available, and we have produced a lot of publications for children’s researchers and ethics committees who are considering children’s health research.

This has allowed us to really make sure that the voices of children and young people are heard. We have also done this via our children’s research charter.

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It is a lot of work but nobody comes into this kind of job without recognising that.

Both my trust (Chelsea and Westminster NHS Foundation Trust) and my university (Imperial College London) have been extremely supportive. I also have an extremely supportive husband and my kids are both grown up so my time is my own.

I don’t get much spare time – there is no denying that there’s a lot to do – but that is by no means a complaint, because it’s all extremely enjoyable work.

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research programme at Imperial College London. And my third role accounts for all of my wider professional activities as the RCPCH president.

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Figure 1: RCPCH's priorities wheel highlights their 2015-2018 three-year strategy

in relation to the UK child health workforce, as there are a substantial number of doctors and nurses in the NHS who are non-UK based. We are really worried about some of the language that is being used about UK doctors and we’re concerned that the UK won’t be seen as welcoming to doctors who have trained overseas. One in ten doctors are EU nationals and 5% of the total NHS England workforce are from the EU, so the NHS depends on doctors from overseas.

How do you see the landscape of children’s health changing over the next ten years?

That’s a great question – thank you for asking me that. What I hope to see is a much greater recognition that if we don’t get child health right, then we’re not going to get adult health right. If we want people to live long and healthy lives, and to be healthy in old age, then we really have to start thinking about this from the moment of conception.

We know that so many of the long-term problems – the problems that start to afflict us in old age – actually have their origins in uterine life. So, for example, a mother who smokes, or a baby who is exposed in utero to atmospheric pollutants (air pollution), will be at a much greater risk of developing respiratory disease in adult life.

• If you have a particular interest in child health and would like more information on how to become a member of the RCPCH or about any of the campaigns Professor Modi mentioned, please visit their website at www.rcpch.ac.uk.

Contact Royal College of Paediatrics and Child Health 5-11 Theobalds Road London WC1X8SH

T: 020 7092 6000 E: enquiries@rcpch.ac.uk W: www.rcpch.ac.uk

@rcpchtweets

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Don’t get overexcited!

Restoring inhibition in neurological disorders Dr Davies and Professor Moss from Tufts University have collaborated to successfully uncover a new way of treating one of the underlying causes of neurological disorders such as epilepsy and Fragile X Syndrome. Restoring the lost inhibitory action of GABAA receptors, their work combats the overexcitation of neurones associated with these conditions.

Novel Drug Development

What first drew you to the investigation of neuronal disorders such as Fragile X Syndrome?

I have had a long standing interest in how neurosteroids modulate GABA ARs, especially extrasynaptic GABA ARs. When I started my collaboration with Steve, he was already examining how neurosteroids change the phosphorylated state of extrasynaptic GABA ARs and we saw a rapid change in the surface expression of functional channels leading to a rise in tonic inhibition. At the same time, there were reports on a reduced tonic inhibition in neuronal developmental disorders such as Fragile X and we thought that boosting the expression of GABA ARs would be a novel therapeutic approach. Traditionally, the aim would be to allosterically enhance the channels that are already there in the membrane but we are seeking to increase the number of receptors back to normal levels.

How has collaboration helped in moving the research forward?

We have distinct and overlapping skills: Steve is known for his pioneering work on GABA AR phosphorylation and trafficking; I have experience on recording the currents that flow through GABA AR ion channels and examining subunit-dependent changes in function and pharmacology. Together, we have the experience to examine the broader picture of how changes to protein phosphorylation affect neuronal excitability and how brain circuits are altered.

Why has it taken so long from the discovery of GABA to uncover these aspects of its regulation?

As with most experimental breakthroughs, it has taken so long because it is experimentally challenging to isolate and examine the individual steps involved. For our work we needed to identify the amino acid residues that are phosphorylated and then generate specific antibodies to identify changes

in phosphorylation. Then, designing the experiments and experimental conditions to properly measure changes in receptormediated currents and neuronal excitability can take time to achieve through trial and error, luck, and insight.

Because of the complex signalling mechanisms that regulate GABA AR trafficking and function there is still much that is unknown.

How did SAGE Therapeutics become involved in the work?

SAGE had an interest in how their neuro-active steroids were modulating synaptic and extrasynaptic GABA ARs. When we identified a novel mechanism of neurosteroids changing the phosphorylated state of extrasynaptic GABA ARs to increase the trafficking of the receptor, we, together with SAGE, became interested in knowing whether synthetic neuro-active steroids could also work through this mechanism or if it was just naturally occurring neurosteroids that could phosphorylate GABA ARs. Discovering that certain synthetic compounds can enhance tonic inhibition through a trafficking mechanism differentiates these SAGE compounds from the typical allosteric modulator often used clinically to control excitation.

What is the next step in realising NAS as a therapeutic agent?

Firstly, we are awaiting results from clinical trials of some neuroactive steroids that are currently underway. However, we still do not understand the mechanistic pathway of how neuroactive steroids alter the phosphorylated state of extrasynaptic GABA ARs. Once we know more about the pathway then we could identify more selective compounds. In order for that to happen we are examining different pathways and different interacting proteins. Once the pathway(s) have been identified we could generate novel animal models which will demonstrate the specificity of the compounds generated before going forward into clinical trials.

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Novel Drug Development

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he human brain is a complex organ and scientists have spent long careers attempting to elucidate the mechanisms underlying its activity. Central to this is the regulation of synaptic activity. A synapse is the point of communication between brain cells, called neurones, as well as their connection to other tissues such as muscle. Each of these synapses is bristling with receptors, proteins inserted into the cell membrane to sense the extracellular signalling molecules which are the messengers of the body. Gammaaminobutyric acid (understandably shortened to GABA) was known of as long ago as the 1950s as one of these powerful neuronal transmitters, but its mechanism of action, and particularly the role of the receptors it stimulates, is still a matter of intense research.

GABA is almost exclusive to the brain, with only trace amounts found in other tissues.

Couple this with the fact that almost all neurones are sensitive to it and it is clear that its function is vital to normal brain functioning. It has been estimated that about 30% of all the synapses in the central nervous system (CNS, consisting of the brain and spinal cord) utilise GABA as a transmitter; its role is to inhibit the effect of other transmitters which are exciting the neurones, providing an important regulatory mechanism

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The GABAA receptor is a target for therapeutic drugs due to its ability to inhibit excitatory signals from other cells in neuronal activity. Each transmitter has its own receptor – in the case of GABA there are two, designated GABA A and GABAB. The first inhibits the reception of signals from other cells (post-synaptic), the second inhibits the release of signalling molecules to other cells (pre-synaptic). The GABA A receptor is a target for therapeutic drugs due to this ability to inhibit excitatory signals from other cells, the uncontrolled activity of which is found in disorders such as epilepsy. Anaesthetics and barbiturates have long been known to affect the intrinsic activity of the receptor, but less is known about how their physical presence at the synapse is regulated.

THE BENEFITS OF COLLABORATION Professor Moss and Dr Davies have been collaborating since 2008, using their electrophysiological and molecular biochemistry skills to explore the role of GABA A receptors in neuronal inhibition.

A key focus of their research has been the movement of these receptors within the cell, known as trafficking. Receptors are proteins which are created from the template held in the cell’s DNA. They are then often modified by cellular machinery before being moved into position on the cell membrane. Receptor populations are then managed through a process of continual recycling as new receptors are created and others degraded.

Prof Moss and Dr Davies are currently examining how errors in this trafficking are implicated in disorders such as anxiety, neurodevelopment disorders, and epilepsy.

The researchers have recently discovered that a form of protein modification, known as phosphorylation, is critical for the correct localisation of the GABA A receptor at the synapse. In phosphorylation, one or more phosphate groups are added to the protein to achieve its final conformation (shape) and

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the potential to increase activity of the enzyme responsible for phosphorylation and are associated with improved membrane insertion of alpha-4 and beta-3 sub-units. Unfortunately, endogenous NAS, those naturally occurring in the brain, are relatively low. Administering additional supplies has been limited by problems around bioavailability (the proportion of the administered substance which enters circulation and is able to exert its effects) but Prof Moss and Dr Davies have been greatly assisted by the development of a novel NAS by SAGE Therapeutics. SAGE is a biopharmaceutical company which specifically develops novel therapeutics for CNS disorders. Their compound, SGE-516, has the equivalent efficacy as endogenous NAS but with radically improved bioavailability from oral administration. This opens up the possibility of new treatments for a multitude of neurological disorders, including epilepsy and anxiety.

activity. Their experiments have elucidated the precise location of phosphate groups on the receptor, made up of multiple smaller proteins called sub-units. They believe that the phosphorylation of the alpha-4 and beta-3 sub-units is particularly critical, as it increases both receptor insertion into the membrane and its inhibitory activity once inserted.

KNOWLEDGE IS POWER Taking this knowledge forward, the team identified neuro-active steroids (NAS) as potential therapeutics for the mis-trafficking of GABA A receptors within neurones. NAS have

Fragile site

Normal X chromosome

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Fragile X chromosome

THE FRAGILITY OF LIFE Of particular interest to Prof Moss and Dr Davies’ team is the role of mis-trafficking of the GABA A receptor in Fragile X Syndrome (FXS). FXS is an autism spectrum disorder, and the most common form of inherited intellectual disability. Deficits in neuronal inhibition by GABA A receptors are frequently implicated in FXS, along with a reduction in expression of a protein associated with the disorder, known as fragile X mental retardation protein (FMR1). A reduction in neuronal inhibition results in complex intellectual and behavioural issues in those with the genetic condition. An increased incidence of epilepsy is also a factor affecting individuals with the condition, and thought to be directly related to the deficits in neuronal inhibition.

Using a mouse model of Fragile X in which the FMR1 protein has been ‘knocked out’ through genetic modification, the researchers have shown that it is indeed the mis-trafficking of GABA A receptors which is responsible for the reduction in inhibitory activity at these neurones. It is the discovery of this novel mechanism by the team which has allowed for the investigation of high bioavailability NAS, with the aim of reversing the problem and restoring correct regulation of excitatory signals in the CNS. This is a breakthrough in the treatment of FXS, but the identification and successful modulation of this therapeutic target has created the possibility of addressing a much wider range of neurodevelopmental disorders.

Detail RESEARCH OBJECTIVES Dr Davies and Professor Moss focus their work on the action of GABA A receptors in the brain. Their current research investigates how neuroactive steroids interact with the receptors and their potential use as a therapeutic in conditions where neuronal inhibition is deficient.

COLLABORATORS •P rofessor Stephen Moss, Tufts University School of Medicine •D rs Mike Ackley and James Doherty of Sage Therapeutics, Inc. http://www.sagerx.com/index.php BIO Dr Davies and Prof Moss have been collaborating since 2008. They have combined their electrophysiological and molecular biochemistry skills to explore how trafficking and pharmacological modulation of GABA A receptors impacts neuronal inhibition. They are currently examining how GABA A receptors are mistrafficked in disorders such as anxiety, neurodevelopment disorders, and epilepsy.

CONTACT Dr Paul Davies Tufts University School of Medicine Department of Neuroscience 136 Harrison Avenue Boston, MA 02111 USA E: Paul.Davies@tufts.edu T: +1 617 636 3866 W: http://sackler.tufts.edu/Academics/ Research-Faculty-and-Staff/Paul-Davies @neurorunner Paul Davies

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Gene research sheds new light on ALS Despite short repetitive DNA sequences having been linked to numerous neurodegenerative diseases, our understanding of the mechanisms that underlie their pathology has remained in its infancy. Dr Haeusler’s research investigating the first sequence repeat to be linked to ALS-linked diseases has uncovered the core mechanism through which the mutation results in cellular damage.

Neurology

What led you to focus your research on the C9orf72 repeating sequence in particular?

My PhD work focused on the dynamics of nucleic acid protein complexes in the context of genetic regulation. I wanted to extend this work into disease models, and I was drawn to nucleotide repeat expansion diseases and ALS, which converged with the discovery of the C9orf72 mutation in ALS and FTD. Why do you think research looking at repetitive DNA sequences has been so limited, despite there being so many of these regions in the human genome?

Unfortunately, it can be technically difficult to sequence through highly-repetitive DNA regions. Furthermore, most sequencing technological approaches rely on aligning relatively short DNA reads that may not span the entire repeat region. Therefore, if the repeat region was long enough, we could be unable to read through and properly assemble the entire region due to the redundancy of the sequence and the inability to stitch the short reads of DNA sequence back together correctly.

How far away do you think we are from having effective therapies available for ALS and other neurodegenerative diseases that can attenuate the pathology of the C9orf72 NRE? It only takes one great scientific breakthrough to find a cure. However, it is difficult to put a time on when this will occur. I believe the ALS field has made substantial progress in understanding

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lthough the Human Genome Project deciphered the human DNA sequence about fifteen years ago, our understanding of the function of all that is encoded in the sequence remains far from complete. Dr Aaron Haeusler, an Assistant Professor in the new Jefferson Weinberg

disease-linked mechanisms recently, and the discovery of the C9orf72 gene has been truly exciting in an already invigorated field of research.

Importantly, the advancements we have made in understanding different mechanisms of ALS, have also revealed that there may not be a single cure for all of ALS, but rather akin to the cancer field, specialised treatment regimens to combat specific patient symptoms within the observed disease spectrum or stage of ALS. What has been the most exciting finding you have been part of during your career in research so far?

I was really glad to be a part of the great work coming out of exploring nucleocytoplasmic transport defects.

I think with this discovery we are on the cusp of greater discoveries and new potential therapeutic strategies.

However, I think the most exciting findings are still to come.

Are there any other avenues you plan to pursue with your research in the following years in relation to NREs? I am fascinated by NRE expansions in disease, and we have learned a lot about the C9orf72 NRE paradigm through the excellent mechanistic work that has accumulated over the years by studying other NRE-linked diseases such as Fragile X, Huntington’s, Myotonic dystrophies, and many of the spinal cerebellar ataxias.

Proceeding forward I will look at these prototypical NREs to understand how the C9orf72 NRE compares.

ALS Center in the Vickie and Jack Farber Institute for Neuroscience at Thomas Jefferson University in Philadelphia, is investigating a specific region of the human genetic sequence. This region varies greatly in size between individuals and has been linked to numerous neurodegenerative diseases. His work is beginning to unravel

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Neurology

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the mechanisms by which the expansion of short repetitive DNA sequences can result in disease.

URGENT NEED TO DISCOVER ALS CURE Haeusler’s research thus far has focused primarily on investigating the association between these sequences and amyotrophic lateral sclerosis (ALS). The symptoms of ALS, also known as Lou Gehrig’s disease and motor neurone disease, result from the death of neurones responsible for controlling voluntary muscle movement. The condition is degenerative with progressive motor neurone loss, which manifests as stiff and twitching muscles with increasing weakness and atrophy, leading to the eventual loss of control of all voluntary movement. There is currently no known cure for ALS and any applicable therapies only extend life by a few months, with assisted ventilation providing some further therapeutic value.

The progression of the disease, which usually begins between the age of 50 to 60, is rapid with an average survival from onset of only three to four years.

There is currently no known cure for ALS and any applicable therapies only extend life by a few months as a nucleotide repeat expansion (NRE).

When less than 30 repeats are present this does not usually result in any problems.

However, the NRE in the gene can contain hundreds to thousands of repeats in individuals. This type of high-number repeat has been identified as the most common genetic abnormality in patients with ALS and frontotemporal dementia (FTD). Similarly, other neurological and neuromuscular disorders have also been linked to the expansion of short repetitive DNA sequences. These include NRE-linked Fragile X, Huntington’s, Alzheimer’s disease and many ataxias.

Dr Haeusler has been investigating a region of DNA that encodes a gene within chromosome 9, open reading frame 72, known as C9orf72. When mutations occur in this portion of DNA code, it can result in varying numbers of repeats of a part of the sequence. This gene mutation was the first of this type to be linked to neurological disease and since being described, a number of clinically distinct disorders have been associated with this genetic defect.

Although the short repeating sequence has been linked to many diseases, the primary mechanism by which this type of mutation results in those conditions has remained unclear. This has hindered the development of therapeutic interventions that could prevent disease progression. However, while working in Dr Jiou Wang’s laboratory as a postdoctoral fellow, Dr Haeusler, in collaboration with other Johns Hopkins ALS laboratories, recently identified mechanisms that may account for the neurotoxicity seen in ALS patients due to the NRE of C9orf72.

UNRAVELLING THE ROLE OF REPEATING DNA SEQUENCES This repeating region of sequence is known

CELLULAR DISRUPTION ON SEVERAL LEVELS Collaborations between laboratories

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at Johns Hopkins, involving Drs Jeffrey Rothstein, Tom Lloyd, and Jiou Wang, studied drosophila, a type of fruit fly commonly used as a model organism in genetic research. They also employed C9orf72 ALS patient brain tissue and neurones derived from stem cells and brain tissue of C9orf72 ALS patients. A focused genetic screen for proteins associated with suppression of neurodegeneration identified a drosophila homologue of a human gene and protein it encodes, known as RanGAP1. This protein is a key regulator of transport between the cell nucleus and the cytoplasm. Their study showed that when C9orf72 DNA is transcribed into RNA, the RNA structures that are formed are also expanded, which collapse in on themselves forming abnormal threedimensional structures. The C9orf72 RNA that results from the NRE mutation interacts with RanGAP1 and transport between the nucleus and cytoplasm is perturbed. It is possible that direct interactions between nuclear transport complexes and the RNA cause this disruption in C9orf72 expressing flies, C9-ALS patient-derived neuronal cells, and brain tissue.

In addition to this, they found that the resulting RNA structures may also interfere with the activity of other components of the nuclear pore complex. These structures

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Detail Cytoplasm

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RNA retention

Nucleocytoplasmic trafficking defects

C9orf72 repeat RNA RBP sequestration

Dipeptide repeats (DPRs) Nucleolar Stress

RNA foci

Altered RNA splicing/ editing Altered autophagy ER stress Increased stress granules

RESEARCH OBJECTIVES Dr Haeusler’s research focuses on neurodegeneration linked to repeating sections of DNA. His work is directly relevant to the pathogenesis of Amyotrophic Lateral Sclerosis (ALS) and related ageing-dependent chronic neurological disorders.

FUNDING National Institutes of Health (NIH) COLLABORATORS • Christopher Donnelly, PhD, University of Pittsburgh • J iou Wang, PhD, Johns Hopkins University • Jeffrey Rothstein, MD, PhD, Johns Hopkins University •T om Lloyd, MD, PhD, Johns Hopkins University • Davide Trotti, PhD, Thomas Jefferson University • Piera Pasinelli, PhD, Thomas Jefferson University BIO

are the channels through which transport of molecules between the nucleus and cell cytoplasm occurs. Therefore, disruption of their activity leads to dysfunctional transport. This collaborative research effort involving Dr Haeusler concluded that it is this disruption of transport at nuclear pore complexes that is a fundamental mechanism for induction of cellular injury in not only ALS but also FTD in C9orf72 repeat-expansion patients. Dr Haeusler hypothesises that this may also be one of the primary causes of the cellular defects associated with other neurodegenerative diseases. He also speculates that similar nuclear transport dysfunction may be responsible for agerelated neurodegeneration in healthy individuals, since it has come to light that many nuclear pore complex components are very long lived and the integrity of these complexes may be compromised as ageing progresses.

RESCUING NEURONAL CELLS DESTINED FOR DESTRUCTION Importantly, they also demonstrated that the deficits in nuclear transport resulting from the C9 mutation could be rescued in both the fly model and cultured human

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neurones. This was demonstrated by using molecules targeted at the abnormal RNA produced from the mutated DNA sequence. Therefore, molecules such as these show great potential in attenuating the defects in nuclear trafficking that occurs in the cells of patients with this mutation.

Dr Haeusler postulates that, as the current human genome is filled with repeat DNA sequences with a high degree of variation between individuals, NREs similar to those of C9orf72 may underlie many other diseases. Identifying these regions and understanding the mechanisms by which they result in disease is therefore essential for the future development of treatments. Dr Haeusler envisions that we are ’at the tip of the iceberg in understanding how repetitive DNA sequences function in normal biology and contribute to human disease’. In the Jefferson Weinberg ALS Center at Thomas Jefferson University, he aims to further expand on our knowledge of the mechanisms by which NREs such as those in C9orf72 result in human disease and to identify therapeutic targets for intervention.

Aaron Haeusler obtained a BS in Biochemistry from Northern Michigan University before attending graduate school under the guidance of his PhD advisor, Jason D Kahn, PhD at the University of Maryland, College Park. A postdoctoral fellow with his mentor, Jiou Wang, PhD followed at Johns Hopkins University, Bloomberg School of Public Health in Baltimore. His work here brought him to his current home in the new Jefferson Weinberg ALS Center at Thomas Jefferson University.

CONTACT Dr Aaron Haeusler, PhD Assistant Professor Jefferson Weinberg ALS Center Vickie & Jack Farber Institute for Neuroscience Department of Neuroscience Thomas Jefferson University 900 Walnut Street, JHN - Suite 410 Philadelphia, PA 19107 E: aaron.haeusler@jefferson.edu T: +1 215 955-8630 W: https://www.linkedin.com/in/aaronhaeusler-57b1aa34

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Spotlight

The novel techniques of stem cell research Professor Alejandro Madrigal has been an integral component of the Anthony Nolan Research Institute’s success throughout his twenty years as Scientific Director. He spoke with Research Features about his time in the role and the direction he sees stem cell research going in the future.

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A

nthony Nolan really do put the stem in stem cell research.

Since becoming established as the Anthony Nolan Register back in 1974, the charity has pioneered vital research into stem cells in the hope of curing leukaemia and developing haematopoietic (blood-derived) stem cell transplantation techniques.

Not only that, but Anthony Nolan have played a massive role in getting stem cell disorders recognised and gathering public support for their UK stem cell register. As of today, they currently have over 620,000 stem cell donors on their UK register – but there is always a need for more.

With this in mind, Professor J Alejandro Madrigal – Scientific Director of the Anthony Nolan Research Institute – recently sat

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down with Research Features to discuss the charity’s newly-released research strategy, while highlighting the areas of stem cell research in need of further consideration.

Hello Alejandro! How would you describe your role as Scientific Director of Anthony Nolan?

I’ve been the Scientific Director of Anthony Nolan since 1996, and my main mission has been to lead research to improve donor provision and outcomes of haematopoietic stem cell transplantation through innovative research.

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Spotlight

Anthony Nolan was born in 1971 with Wiskott-Aldritch syndrome. Thanks to his legacy, the number of registered stem cell donors has grown to 28 million people around the world Could you tell us who Anthony Nolan was and why the foundation was set up in his name?

Anthony Nolan was a young boy born in 1971 with Wiskott-Aldritch syndrome – a rare blood disorder. He needed a bone marrow transplant but at that time there was no register of unrelated donors. His mother Shirley set up the world's first register to match people in need of a transplant with people willing to donate their bone marrow.

Sadly, no donor was found in time to save Anthony, but thanks to his legacy the Anthony Nolan register has grown to over 620,000 people in the UK, with 28 million people on registers around the world.

What impact has Anthony Nolan had on stem cell research since it was first established? What accomplishments are you particularly proud of?

We’ve seen dramatic improvements in donor provision, HLA matching and immune cell therapy. We, at the Anthony Nolan Research Institute, are extremely proud to have been very involved with each of these areas and to have contributed by performing research and collaborating with world leaders in the field.

Back in the early 2000s, Anthony Nolan pioneered new techniques for analysing compatibility genes (HLA genes). Could you tell us more about this and the effect this has had on stem cell research?

HLA typing has been one of the most challenging aspects in donor provision, since these genes are extremely variable (polymorphic) and have thousands of variations on the same gene. Since they were discovered, we’ve been hitting a moving target and have had to invent several techniques. Around the year 2000, we invented an innovative technique, published in Nature Genetics, called RSCA; it allowed for high resolution matching for HLA types to identify more accurate matches than conventional methods.

This year we became the first register in the world to introduce Third Generation

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Sequencing, which we believe is the optimum and most efficient approach to typing all these genes, with no uncertainties. By doing this we can provide patients with the best possible donor.

Stem cells are often an area of ethical dispute. How does Anthony Nolan recognise this in its research? And how have the ethics surrounding stem cells changed since the institution was first founded?

We work with blood stem cells from living adults or with umbilical cord blood. We do not conduct research into embryonic stem cells. Anthony Nolan places the welfare of donors and patients at the heart of our work. All of our research is ethically approved and Anthony Nolan abides by the Human Tissue Act 2004, the Human Tissue Regulations 2007 and associated Codes of Practice. We do not use animals in experiments at the Anthony Nolan Research Institute and, in terms of our umbilical cord blood programme, we comply with all the requirements of the Human Tissue Authority. A major feature of the Human Tissue Act is that it makes consent the fundamental principle for the lawful removal, storage and use of human tissue.

As medical science progresses, new ethical questions will arise and we will approach these in the same way – by complying with best practice and putting the wellbeing of donors and patients first.

Anthony Nolan has recently released its research strategy and aims for future work.

Are there any areas of stem cell research that you are particularly focused on studying?

At the Anthony Nolan Research Institute, we perform translational research. We are focusing on developing innovative techniques for donor provision but we also have a strong component on immune cell therapy. This aims to select specific immune cells that can target tumour cells or modulate immune responses to decrease complications, such as graft versus host disease or infections.

Why is researching into what happens following a stem cell transplant so important?

We need to understand how immune systems and engraftments recover posttransplant. Unfortunately, approximately 50% of transplant recipients die due to relapse or because of post-transplant complications. Studying the reasons behind this will help us to prevent them and improve the survival rates and quality of life for many patients.

How do you see the landscape of stem cell research changing over the next ten years?

Genomics and immune cell therapy are very promising tools that are already changing the way we treat patients.

Pharmacogenomics and personalised medicine will no doubt help us extend

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the benefit of haematopoietic stem cell transplants to many other conditions, such as autoimmunity.

Anthony Nolan has seen a massive rise in the number of stem cell donors over recent years. Is there still an urgency for people to donate their stem cells and, if so, how should they go about it?

There is always a need for people to register as donors as we are only able to find the best match for 60% of patients. However, rather than just adding more people, we are focusing on finding the right people – and that means diversifying the register.

a one in five chance of finding the best donor match. We are also urging more young men to join the register; they are the preferred donors, yet make up just 15% of the register. Joining the register is very easy – if you’re aged 16–30 and in good health, simply visit our website and request a free spit kit. We’ll then send you a kit in the post to collect a saliva sample.

We’ll add you to the register and notify you if you are ever a match for someone in need of a transplant. Ninety per cent of donations take place via peripheral blood stem cell collection, which is similar to platelet donation.

There is a shortage of black, Asian and ethnic minority donors, which means it is a struggle to find a match for patients from these backgrounds. They have just

• If you would like to find out any more information about stem cell research, or how to become a registered stem cell donor, visit www.anthonynolan.org.

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Contact Anthony Nolan 2 Heathgate Place, 75-87 Agincourt Road, London NW3 2NU T: 0303 303 0303 E: press@anthonynolan.org W: www.anthonynolan.org /AnthonyNolan @AnthonyNolan /AnthonyNolan

45

Universal donor cells – a revolution against rejection Dr Claudia Mitchell (CEO of Universal Cells) and her team are focused on the commercialisation of an innovative gene-editing technology to develop universal donor stem cells that are not subject to immune system rejection.

Molecular Biology

P

luripotent stem cells offer huge therapeutic potential. These undifferentiated cells can give rise to almost any specialised cell type, acting as a natural repair system for damaged tissues or as novel therapeutics that can be further engineered to treat diseases.

achieve this goal. Founded by Dr Claudia Mitchell and Dr David Russell in 2013, the company focuses on the use of a pioneering gene-editing technology to develop Universal Donor Cells (UDCs) – modified pluripotent stem cells that are refractory to rejection by a patients’ immune system.

Scientists have exploited the unique properties of stem cells to treat diseases associated with dying or damaged cells. Currently, in Europe, 26,000 patients per year receive stem cell therapy to treat blood disorders and some cancers, including leukaemia. However, currently human donors are the main source of stem cells, limiting the treatment's success due to rejection by the patient's immune system.

In order to achieve the full therapeutic potential of pluripotent stem cells, the issue of rejection must be solved: Universal Cells Inc. aims to

THE MOLECULAR BASIS OF REJECTION Each individual has a unique set of human leukocyte antigens (HLAs) – cell surface protein markers that signal to the immune system that they are that individual's own cells. In the population, the genes that encode HLA molecules are hypervariable – meaning each individual has a specific barcode of HLA molecules that are custom-fit for their immune system. There are 2 classes of HLA molecules: HLA class I molecules, which are expressed on all cells and HLA

class II molecules, which are expressed on a subset of cells associated with the immune system. Both class I and class II molecules are made up of two parts. Class I molecules are formed when the hypervariable HLA class I protein binds another protein called Beta2-microglobulin (B2M), which is required for the HLA molecule to reach the surface of the cell. HLA class II molecules get to the surface of the cell when two hypervariable HLA class II molecules bind together. Both types of HLA molecules serve the role of presenting pieces of other proteins to the immune system.

HLA molecules are closely monitored by the immune system. Differences in either HLA class I or HLA class II proteins lead to the identification and rapid destruction by cytotoxic T-cells (cells that ‘kill’ other cells).

How are Universal Donor Cells (UDCs) developed using recombinant adenoassociated virus (rAAV) technology?

Universal Cells, Inc. uses its rAAVmediated genome-editing technology to engineer the HLA molecules of pluripotent stem cells and thus create cells that are universally compatible. First, a single-chain, non-polymorphic HLA class I transgene composed of the HLA-E molecule fused to the beta 2-microglobulin HLA class I common subunit is knockedin to the beta 2-microglobulin locus. This transgene acts as a decoy preventing lysis of the modified cells by natural killer cells, but allows for the cells to avoid recognition by the immune system. Then, to eliminate all HLA class II expression, another rAAV vector is used to knock out a transcription factor (RFXANK) that is necessary for all HLA class II expression.

In what ways is rAAV technology an improvement over nuclease-based gene

The immune system is also ready to detect and destroy cells that completely lack HLA class I molecules, using a type of immune cell called a Natural Killer (NK) cell. Thus, simply removing HLA molecules will not suffice to create a Universal Donor Cell.

WHAT PREVENTS UDCS FROM BEING REJECTED? UDCs have been genetically modified so they do not express any hypervariable HLA class I or HLA class II molecules. Cytotoxic T-cells are thus unable to recognise UDCs as 'foreign'. Additionally, to avoid rejection by NK cells, UDCs express a polymorphic HLA transgene (HLA-E) fused to the B2M gene, which acts as a decoy to prevent lysis by NK cells. Unlike the hypervariable HLA molecules, the HLA-E transgene is the same throughout the population. Therefore, the UDCs are recognised as 'self' by the immune system.

HOW ARE UDCS DEVELOPED? Universal Cells edits genes using recombinant adeno-associated virus (rAAV)-mediated gene editing, which was first developed at University of Washington by Dr Russell. rAAV is a single-stranded DNA virus, that edits genes

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editing tools?

Recombinant AAV-mediated gene editing is a pioneering approach with lower efficiency / higher fidelity characteristics that greatly minimise the genotoxic risks associated with the use of nucleases. Unlike nucleasebased approaches, rAAV-mediated gene editing does not induce off-target cutting of the DNA, nor does it promote on-target mistakes.

What barriers must be overcome before UDCs can be commercialised?

As for any new therapeutic product, its safety and efficacy need to be proven in clinical trials before this technology can be commercialised. Universal Cells, Inc. expects to have the first in-human clinical trials using UDCs starting within the next 3 years.

How can rAAV be used as a gene therapy to treat genetic diseases?

Besides using rAAV to create UDCs, this technology can also be used to directly

by homologous recombination – a process whereby two similar (homologous) DNA molecules exchange genetic material without introducing any cuts or damage to the DNA itself. The rAAV can be designed to insert any genetic material to a very precise location in the genome.

During UDC manufacturing, the HLA-E transgene described above is encoded within the rAAV and is knocked-in at the B2M gene via homologous recombination. Additionally, in order to knock-out HLA class II expression,

Parent Cells

repair disease-causing mutations in a patient’s own cells. The technology is highly flexible, and can generate any desired genetic modification, including deletions, insertions, and point mutations, and therefore could be used to inactivate genes, knock-in genes at specific loci, or correct mutations anywhere in the genome. Universal Cells, Inc. is currently developing an ex vivo gene-editing therapy based on rAAV to correct mutations that cause immunodeficiencies.

Where do you see Universal Cells in 5 years’ time?

Within the next 5 years, Universal Cells, Inc. expects to have driven our genetherapy product for immunodeficiencies towards the clinic, obtained first in-human proof of concept of UDCs through our strategic partnerships, and have advanced our innovative cellular therapies through rigorous preclinical research and into clinical trials.

another rAAV vector is used to eliminate the expression of a transcription factor that is necessary for the expression of all HLA class II molecules.

ADVANTAGES OF UDCS AND RAAV TECHNOLOGY There are multiple strategies that can be employed to reduce the rejection of stem cells, including: i) HLA-typed stem cell banks, where hundreds of separate HLA-specific cell lines are produced, ii) allogenic donor stem cells, where a single line is produced and transplanted but

B2M-/E, TK+ HLA Class II +

B2M-/E, TK+ HLA Class II -, Suicide 2

Universal Donor Cell

6 HLA Class I

HLA-E Single Chain

6 HLA Class II

6 HLA Class II

HLA-E Single Chain

HLA-E

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Molecular Biology

Detail RESEARCH OBJECTIVES Universal Cells’ core technology (rAAV-mediated gene editing) allows them to produce customised cells with insertions, deletions or point mutations at any genetic sequence. Using this technology, they are now developing Universal Donor Cells – a line of stem cells that can be used in a therapeutic setting without rejection.

Universal Donor Cells are an exciting product that will enable a new class of therapeutics to treat a variety of life-threatening diseases requires toxic immunosuppressive drugs and iii) autologous induced pluripotent stem cells (iPSCs), where a patient's own cells are isolated and manipulated to behave like a pluripotent stem cell.

there is enormous variability between different preparations of iPSCs, leading to potentially unpredictable cell differentiation potential, manufacturing capacity and ultimately unpredictable clinical outcomes.

A major advantage of UDCs over HLA-typed stem cell banks is that only one cell line is required. This significantly reduces the cost, complexity and time required to undertake clinical trials and eventual commercialisation.

HLA-typed banks also do not solve the problem of HLA class II matching or the presentation of foreign proteins by the matched HLA molecules.

CURRENT AND FUTURE USES OF UDCS UDCs are an exciting product that will enable a new class of therapeutics to treat a variety of life-threatening diseases. For example, Universal Cells, Inc. has recently established partnerships to develop cancer therapies using universal donor T-cells (with Adaptimmune) and universal donor retinal pigment epithelium cells for the treatment of adult macular degeneration (with Healios). Other examples of applications include the development of UDCderived keratinocytes therapies which could be applied to treat skin wounds and burn injuries, or the development of UDC-derived beta pancreatic cells to treat type I diabetes.

Although allogenic donor cells also entail just a single cell line, they require the use of immunosuppressants, which weaken the immune system. This reduces the incidence of rejection but also results in severe side-effects, which complicate the clinical outcome. UDCs, however, do not require immunosuppression, as there are no foreign proteins to trigger an immune response.

Compared to UDCs, autologous iPSCs are very expensive to manufacture. Furthermore,

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COLLABORATORS • A daptimmune, a UK-based company with whom Universal Cells are developing universal donor T-cell therapies for cancer (http://www. adaptimmune.com/) • H ealios KK, a Japanese company with whom Universal Cells are developing universal donor retinal epithelium cells to treat blindness (https://www. healios.co.jp/en/) ABOUT Universal Cells, Inc. is a private Seattle-based biotechnology company developing nuclease-free genomeediting technologies that accurately and efficiently edit any gene, without off-target cutting. Co-founded by Claudia Mitchell, PhD, MBA, and David Russell, MD, PhD, Universal Cells welcomes collaborative opportunities with partners whose expertise and complementary technologies can accelerate the development of stem cell and gene therapies.

CONTACT Claudia Mitchell, PhD, MBA CEO Universal Cells, Inc. 3005 1st Ave Seattle, WA 98121 E: info@universalcells.com T: +1 425 246 5454 W: www.universalcells.com

Overall, rAAV-mediated gene-editing technology and UDCs can have a huge impact in the healthcare industry, overcoming the obstacle of transplant rejection, revolutionising stem cell therapies and changing the way we treat genetic diseases.

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New windows into cellular kinase function Dr Charles J Bieberich is a professor based in the Department of Biological Sciences at the University of Maryland, Baltimore County. There, together with assistant professor Dr Xiang Li, he leads a team of researchers investigating a family of proteins known as kinases and the role they play in cancerous cells.

Molecular Biology

What led you to focus your research on elucidating the substrates of cellular kinases in particular?

Our interest in kinases grew out of our attempts to understand how an important prostate tumour suppressor protein called NKX3.1 is regulated. As a PhD student, Dr Li discovered, using a classical method called an in-gel kinase assay, that protein kinase CK2 phosphorylates NKX3.1. He then came up with the brilliantly simple idea of running that assay backwards' to use it as a kinase substrate profiling tool.

What do you think the biggest challenge to completing a profile of all kinase substrates will now be?

I think the biggest challenge will be to convince others that our approach provides physiologically relevant datasets that can be leveraged. There is a sort of knee-jerk reaction on the part of researchers in the field to say 'Oh, a lot of the substrates identified in RIKAs are probably not real, the kinase would never see this or that protein in a live cell', or 'Kinases are so promiscuous in vitro that they can phosphorylate a lot of substrates that they wouldn't in a physiological context'. While there will no doubt be some false positives, our published data show that for CK2, 97% of the substrates identified in a RIKA respond, in live cells, to a CK2-specific inhibitor. That’s pretty good evidence that most of the substrates are 'real'. In terms of a kinase encountering a substrate in a RIKA that it would never encounter in a cell, we have not been able to think of an experiment to definitively show that two proteins never interact under any physiological circumstance. And we know for a fact that kinases can be mis-localised in diseases, especially cancer. So we’re willing to cast

a wide net, and sort through the potential by-catch, because we firmly believe the 'keepers' are just too important for us to sit at the dock and design the perfect net.

With this knowledge being so crucial for optimal drug development, why has there not been a more concerted effort before now to discover the downstream effects of many kinase inhibitors?

That’s a really good question. Profiling kinase substrates and validating kinase inhibitor biomarkers is hard and expensive basic research. And there are just not a lot of options when it comes to facile enzyme substrate profiling methods.

Will your new technique for measuring inhibitor efficacy in vivo be employed in phase I drug trials?

We are very hopeful that our method of analysing inhibitor efficacy will be incorporated into clinical trials. Right now, many trials employ a single biomarker, usually using an anti-phosphoprotein antibody. We hope to provide a panel of biomarkers, comprised of tens or hundreds of direct kinase substrates.

What plans do you have for the future of your research?

We are already developing the next generation of the RIKA, designing it to work for kinases that are difficult to reactivate by refolding, and for multisubunit enzymes. We are also exploring new approaches for separating and sequestering proteins that will get us away from polyacrylamide gels. But for now, we plan to plough through as much of the kinase 'substratome' as we can, using our current methods.

We hope to provide a panel of biomarkers, comprised of tens or hundreds of direct kinase substrates 51

Molecular Biology

K

inases comprise a large family of enzymes that mediate protein phosphorylation, one of the key mechanisms by which cell functions are regulated.

Disruption of this regulation is associated with hundreds of diseases, including many cancers. It is therefore imperative that disease-related kinases are profiled, in order to inform the development of therapeutics and provide better outcomes for patients.

Despite the great benefits to be gained from understanding how kinases function, little research has been aimed at elucidating their physiological substrates. Professor Bieberich has been working on developing ways of uncovering these details, devising several novel methods to open up the field for research. “sees” further the non-phosphorylated substrate pool

Kinases are a family of proteins that function P as key regulators of cellular function.

The “sees” genes the non-phosphorylated pool P encoding them makesubstrate up one of theP P most functionally diverse and largest gene P P RIKAhuman genome. TheyP are P P P in the P families P enzymes that direct the activity, localisation P P P P P and function of substrate proteins through P P the addition of phosphate groups, a RIKA P P P P process known as phosphorylation. Kinases are essential in many cellular Psignalling P se Prediction ition pathways, which involve a highly coordinated series of reactions to mediate complex functions, such as cell division. Although se over 500 Prediction kinases are known to be encoded ition in the human genome, most remain to be P functionally categorised.

P

P P

P

RIKA

P

FIGHTING CANCER THROUGH KINASE P P INHIBITION Changes in kinase activity are associated P P RIKA with a multitude of devastating diseases, including many cancers. Inhibition P of abnormal kinase activity using small molecule inhibitory drugs has now become the standard mode of therapeutic intervention for numerous cancers, with hundreds of such inhibitors currently in development. These drugs can be very effective as therapeutics, with patient response rates as high as 85%.

Despite their widespread use and potential effectiveness, knowledge of the physiological substrates of the kinases that these drugs target, and the downstream cellular effects of their activity, is limited.

This has resulted in difficulties in accurately measuring the effectiveness of inhibitory drugs due to a lack of biomarkers, contributing to an unacceptably high failure rate in clinical trials. This lack of information

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P

P P

Dephosphorylation

Phosphorylation

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in RIKA

P P

P

Protease digestion

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LC-MS

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P Large scale substrate identification

Does RIKA identify physiological substrates?

Figure 1.

Large-scale identification of kinase substrates using the RIKA. Complex protein extracts are dephosphorylated then resolved on a RIKA gel. After an in-gel kinase reaction, proteins are digested by a protease, and phosphopeptides are analysed by high-resolution mass spectrometry.

P P Does RIKA identify physiological substrates?

P P

CONTROL

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Figure 2.

The RIKA identifies physiological kinase substrates. Only non-phosphorylated substrate molecules become P P labeled in a RIKA (blackened ovals). Kinase inhibition is predicted to increase the non-phosphorylated pool.

This can be demonstrated by treating live cells with a kinase inhibitor, and quantifying the increase in RIKAlabeled peptides by high-resolution mass spectrometry.

also limits the potential to predict possible side effects of a drug and how inhibitor resistance may develop. Furthermore, a better informed approach to kinase inhibitor drug design would greatly decrease time and cost of development.

A NEW ERA FOR KINASE 'SUBSTRATOMICS' Professor Bieberich is leading the way in what he terms kinase 'substratomics', the profiling of kinase substrates. He wants to help uncover what role individual kinases play in cells, which substrates they

phosphorylate and what signalling pathways they are involved in regulating. Despite the wealth of information to be gained from investigating kinase substrates, research efforts to profile them have been limited.

Although the drawing of a complete map of cellular kinases is an enormous task, Bieberich felt it was crucial to start somewhere.

In their quest to uncover kinase substrates, they found there was a need for a novel system for their identification. In 2007, Bieberich, together with Dr Li, developed

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Although over 500 kinases are known to be encoded in the human genome, most remain to be functionally categorised a new approach known as the reverse in-gel kinase assay (RIKA), a powerful tool that has the capacity to rapidly profile kinase substrates. The system works with any kinase that can be incorporated into a polyacrylamide gel and catalytically reactivated after gel electrophoresis (gel electrophoresis is a technique used to resolve a tissue or cell protein extract).

The sample is run through a porous gel matrix with an electric current, which separates components of the sample in the gel according to the molecular weight of each protein. Kinase activity and substrate structure is then restored and an in-gel kinase reaction is carried out. The phosphorylation signal can then be detected to locate potential substrates, which can be sent for mass spectrometric analysis to be identified. In the team’s testing, the RIKA proved to have a much improved signalto-noise ratio when compared to another existing system, as well as having the benefit of being highly accessible due to the simple experimental set-up.

DELVING DEEPER INTO SUBSTRATE PHOSPHORYLATION Since the development of the RIKA, Bieberich and his team have been working on developing a method to enable the precise details of the reactions that occur between kinases and their substrates to be elucidated. There is almost nothing known about the phosphorylation stoichiometry (PS) for kinase substrates in the body; this is the measure of the extent of phosphorylation of a substrate that occurs. Building on the RIKA system by combining it with high resolution mass spectrometry technology, they are devising strategies for broadly measuring the PS of kinase substrates in vivo. The technique utilises mass tagging to differentiate between substrate molecules, allowing for the phosphorylated and total substrate molecules in a sample to be quantified.

The ratio of non-phosphorylated substrate to that phosphorylated by the kinase is then calculated, revealing the substrate’s

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PS. Importantly, the PS can also be established following treatment of the substrate with a therapeutic inhibitor in order to measure drug efficacy.

This new system will provide an unprecedented opportunity for the identification of biomarkers by determining whether or not a defined set of direct kinase substrate responds to an inhibitor in a patient sample and, if so, to what extent. It will allow the development of a comprehensive panel of biomarkers for inhibitor response in cells, enabling accurate measurement of whether an inhibitory drug is effective. The project will also lead to the discovery of new substrate profiles for individual kinases, providing insights into the cellular roles they each play. This will provide the information required to develop combinations of drug therapies that co-target a particular pathway, which would greatly decrease the chances of drug resistance.

REFINING THERAPEUTIC TARGETS In addition to this, many kinases are implicated in a wide range of cellular functions with a multitude of substrates.

Therefore, a complete picture of each kinase’s substrate profile is crucial if we are to develop drugs that will only interfere with a specific cellular mechanism, rather than impairing all of the kinase’s functions.

This would allow for very precise drug targeting with fewer side effects and less toxicity than current drugs that work through global kinase inhibition.

Detail RESEARCH OBJECTIVES Dr Bieberich’s work aims to elucidate the physiological substrates of kinases.

The reverse in-gel kinase assay (RIKA) that his team have developed is a powerful tool that can rapidly profile physiological substrates. This knowledge can be used to inform combination therapies.

FUNDING • Innovative Molecular Analysis Technologies Program • National Cancer Institute • National Institutes of Health COLLABORATORS • Dr Xiang Li, University of Maryland, Baltimore County BIO Dr Bieberich received his PhD from the Johns Hopkins University in 1987 and was a postdoctoral fellow at Yale from 1987–1990. From 1990–1997, he was a scientist at the Holland Laboratory, American Red Cross. Since 1997, Dr Bieberich has been a faculty member in the Department of Biological Sciences, UMBC. CONTACT Charles J Bieberich, PhD Professor of Biological Sciences and The Bearman Foundation Chair in Entrepreneurship UMBC 1000 Hilltop Circle Baltimore, MD 21250 E: bieberic@umbc.edu T: +1 410 455 3125 W: http://biology.umbc.edu/directory/ faculty/bieberich/

With Bieberich and his team’s contributions of novel methods for the discovery and analysis of kinase substrates, the unveiling of full kinase profiles is now more attainable than ever before. With over 500 human kinases to investigate, the concerted efforts of researchers in the field will be essential for us to obtain a complete map of kinase activity in cells and improve on kinasetargeted therapeutics.

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Therapeutic cocktails – the drink that protects against liver disease Dr Sam W French, a distinguished pathologist from UCLA at Harbor-UCLA Medical Center, is investigating how liver disease in alcoholic patients can be prevented by targeting the proteins and genes that play a key role in the causation processes.

Molecular Biology

Why is the liver so difficult to treat?

The liver is difficult to treat because chronic disease is irreversible: the fibrosis that results from chronic liver disease permanently alters the blood supply and replaces the liver cells with scar tissue.

Only a liver transplant using a normal liver restores the normal functions. Also, the liver cells are permanently unable to multiply and regenerate new liver cells with normal function. This is called liver/ cell senescence.

How does cell cycle arrest impact liver regeneration?

Cell cycle arrest permanently prevents liver cell regeneration. The only treatment that will allow liver cell regeneration is injecting granulocytic-colony stimulating factor. This stimulates the bone marrow to generate a supply of liver cell progenitor cells which multiply and regenerate the liver parenchyma and replace the senescent liver cells.

If betaine is added to alcoholic drinks to prevent liver disease from developing, could this encourage alcohol abuse?

No, the alcoholic is addicted and doesn’t modify his/her drinking behaviour because of additives to the alcoholic beverage.

Other than methyl donors such as betaine, do you believe that there are other compounds that could be used to prevent alcoholic liver disease?

Many different treatments for alcoholic liver disease have been tried after the disease is established by chronic alcoholic abuse. So far, no treatment has been found other than by stopping the alcohol abuse. No treatment to prevent alcoholic liver disease has ever been proposed other than abstinence.

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

A clinical trial treating alcoholic liver disease in patients by injecting granulocytic-colony stimulating factor will be my focus. Two studies have already been done in India which changed the death rate due to alcoholic hepatitis from 80% to 20% over a period of six months.

No treatment to prevent alcoholic liver disease has ever been proposed other than abstinence

T

he liver is a remarkable organ.

As the body's natural filter, hepatocytes (liver cells) degrade metabolic waste and detoxify harmful blood compounds, maintaining a homeostatic balance.

Amazingly, the human liver is also capable of regenerating following extensive tissue trauma, even from just 25% of the original healthy tissue.

Despite its resilience, the liver is not invincible and is especially vulnerable to

alcohol abuse. Excessive binge drinking reduces the liver's ability to regenerate, leading to life-threatening diseases such as liver cancer and alcoholic hepatitis, ultimately resulting in cirrhosis (scarring of the liver). In fact, in 2010, 47.9% of all cirrhosis deaths were due to alcohol abuse. Alcoholic liver disease is incredibly difficult to treat – liver transplants often being the only late-stage option. However, a lack of available organs, coupled with a high incidence of immune rejection, limits success. By studying alcoholic hepatitis

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Molecular Biology

liver biopsies, Dr French and his team are elucidating the mechanisms behind alcoholinduced liver damage and evaluating potential preventative therapies.

CAUSES OF ALCOHOL-INDUCED LIVER DISEASE By administering rats with ethanol to mimic the effects of binge drinking, Dr French, in collaboration with a French colleague, has discovered that one of the most significant effects of alcohol abuse on the liver is the inhibition of 26s proteasome. This organelle degrades unwanted or dangerous proteins and has a key regulatory role in many other processes that occur in the liver; its disruption greatly enhances the risk of developing liver disease.

One effect of 26s proteasome inhibition is increased levels of oxidative stress. This is due to an accumulation of unfolded proteins, which are normally degraded by 26s proteasome.

CELL CYCLE ARREST In order to avoid the undesirable replication of oxidised DNA, the cell cycle is arrested, preventing liver regeneration, as no new hepatocytes are being produced. Dr French used RNA sequencing to analyse the abundance of certain proteins in diseased

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Dr French has suggested that in the future betaine methyl donors could potentially be added to alcoholic beverages, preventing the development of alcoholic liver diseases livers and discovered that cell cycle arrest is the result of over-production of five genes: p15, p21, p27, ATM and TGFB. Essentially, the cell cycle is the process whereby a cell divides to produce two genetically identical copies (via mitosis) replacing damaged cells. Prior to mitosis, the cell is in 'interphase' – it is preparing to divide by replicating the DNA. The genes p15 and p21 inhibit the transition to DNA replication, meaning that mitosis cannot occur and the cell cycle is arrested.

By comparing liver biopsies of healthy and diseased livers, Dr French was able to show over-production of p15 and p21.

Interestingly, Dr French and his team have shown that more than 600 genes are over-expressed and 100 genes are underexpressed in alcoholic hepatitis.

MALLORY-DENK BODY (MDB) PRODUCTION Using electron microscopy, Dr French detected an increase in hepatocyte MDBs, due to a lack of 26s proteasome activity. MDBs are aggregations of misfolded, damaged protein. MDBs are particularly associated with alcoholic hepatitis – affecting 70–75% of patients.

The development of MDBs indicates that the main mechanism involved in alcoholic hepatitis is the loss of protein quality control.

ALCOHOL-ASSOCIATED LIVER DISEASE PREVENTION Following research into the molecular basis of alcohol-induced liver disease, Dr French then conducted studies that focused on manipulating these processes, to actually prevent liver disease from occurring. As an example, cell cycle arrest can be overcome

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Detail RESEARCH OBJECTIVES Alcoholic hepatitis has a very poor prognosis and, so far, liver transplant is the only available late-stage treatment. Dr French’s work focuses on understanding the effects of alcohol abuse on the liver and how this can be prevented rather than treated.

FUNDING National Institutes of Health (NIH)

by giving patients a compound to stimulate stem cells (from the bone marrow) to differentiate into new hepatocytes, which leads to liver cell regeneration.

generation. In turn, NAD+ is a molecule that is vital for the functioning of alcohol dehydrogenase, which reduces blood alcohol levels.

BIO Graduating from the University of California Medical School, Dr Sam W French specialised in Pathology. He has worked in the field for over 60 years and is now Distinguished Professor of Pathology at UCLA, California. Dr French sits on multiple editorial boards and has authored or co-authored over 800 publications which have been cited more than 19,400 times. He was awarded a Life Achievement Award by the LA Pathology Society in 2011 and the Gold Headed Cane Award given by the American Society of Investigative Pathology in 2014.

Perhaps, however, the most exciting discovery is that a powerful antioxidant (betaine) can be used to prevent liver damage from alcohol. Dr French has shown that rats administered alcohol containing betaine methyl donors have reduced blood alcohol levels to 100–200mg/% blood alcohol, compared to an anticipated 500mg/% blood alcohol (equivalent of binge drinking). Furthermore, Dr French found that oxidative stress and MDB formation were significantly reduced in ethanol-subjected hepatocytes when treated with betaine in rats and tissue culture of human hepatocytes.

However, once the disease becomes established in humans, SAMe (a methyl donor like betaine) is ineffective. Patients with chronic liver disease were administered 20g of SAMe per day for 6 months and there was no change in their liver histology.

CONTACT Samuel W French, MD Harbor-UCLA Medical Center Department of Pathology 1000 West Carson Street Torrance, California 90509

THE FUTURE OF LIVER DISEASE PREVENTION Dr French has suggested that in the future betaine methyl donors could potentially be added to alcoholic beverages, preventing the development of alcoholic liver diseases. Fortunately, betaine is colourless, odourless, tasteless, soluble, cheap and extremely effective and can reduce high blood alcohol levels by up to 60%. It seems that this is the ideal preventative measure. However, much testing needs to be performed before betaine can be used commercially in alcoholic drinks.

Further research is also needed to further our understanding of how alcohol abuse impacts the liver. Only then can effective preventative measures be developed, saving countless lives.

E: sfrench@labiomed.org T: +1 (310) 222 2643 / 2627 W: http://labiomed.org/samuel-wfrench-md

This is because betaine indirectly increases alcohol dehydrogenase activity – an enzyme that breaks down alcohol. Betaine is a methyl donor and methyl groups are needed for the enzymatic conversion of norepinephrine (noradrenaline) to epinephrine (adrenaline). These hormones are involved in the 'fight or flight' response.

Epinephrine in particular is the 'stress hormone' and is very effective at increasing metabolic rate and increasing NAD+

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Epidemiology

O so simple...

How blood group O fights off malaria Professor Mats Wahlgren’s recent work on the mechanisms and proteins involved in the development of severe malaria has advanced current scientific understanding of the disease and may reveal answers that will allow a vaccine to be developed.

M

alaria is a widely geographically distributed disease – though most common in sub-Saharan Africa – that is estimated by the World Health Organization to affect 200 million people every year, 425,000 fatally. The majority of those who die from malaria are under five years old, making malaria one of the most important diseases to eliminate in order to improve childhood mortality.

The disease is caused by vector-borne parasites in the Plasmodium family, and most cases of fatal or severe malaria are caused

by Plasmodium falciparum. Severe cerebral malaria is characterised by the occlusion of cerebral vessels by tightly packed red blood cells. Blood clots block vessels, causing coma, brain damage and ultimately death.

REVEALING THE MECHANISMS Blood groups are classified according to

the way different antigenic substances are expressed on the surface of red blood cells, the vascular endothelium and serum proteins.

This means that red blood cells of different blood groups react differently to exposure to different proteins because differently shaped complexes are exposed on the cell surfaces.

It was demonstrated in the late eighties and early nineties that those with O type blood are protected from severe or fatal malaria.

However, the exact processes that facilitate this protection had until relatively recently eluded scientists. In research published last year, a team from the Karolinska Institutet in Sweden led by Professor Mats Wahlgren, have pushed current understanding of the underlying mechanisms of malarial pathogenesis one step further.

Some of their work during the early nineties highlighted the importance of ‘rosetting’ as a phenomenon related to malaria. Rosetting is a process whereby uninfected red blood

RIFINs may be prime candidates for targeting in the search for a drug for the treatment of severe malaria www.researchfeatures.com

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Epidemiology

The team from the Karolinska Institutet have pushed current understanding of the underlying mechanisms of malarial pathogenesis one step further

The Mats Wahlgren research group

Rosettes can be composed of more than ten blood cells, and are much larger in infected individuals with blood type A than in those with type O blood.

Rosetting was previously attributed mainly to the P. falciparum erythrocyte membrane protein 1 (PfEMP1), although the recent study suggests that RIFINs are equally, if not more, important in causing blood cells to adhere to each other. This is particularly the case in blood group A: the majority (70%) of RIFINs belong to a subgroup, A-RIFIN, which preferentially cause red blood cells with A antigens to adhere to each other, creating large rosettes. While this mechanism is also seen in O blood group red blood cells, the rosettes generated are much smaller.

cells adhere to red blood cells that have been infected with malarial parasites such as P. falciparum, forming clusters, or rosettes.

Once rosettes are formed, it becomes more difficult for the host’s immune system to destroy the infected cells because they are surrounded by uninfected cells.

In a paper published in The Lancet in 1990, Johan Carlson, Mats Wahlgren and colleagues showed that isolates from children with severe malaria displayed higher rosetting activity compared with isolates from children with milder forms of the disease. Findings such as these emphasised the importance of further research into the mechanisms behind rosetting and the prevalence of anti-rosetting antibodies. Later work revealed that rosetting

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was less pronounced in blood group O than in blood groups B, and particularly A. MAKING PROGRESS Published in Nature Medicine in 2015, Wahlgren’s team’s new research further develops the understanding of the disease. In what Wahlgren describes as a 'conceptually simple' mechanism, they found that polypeptide proteins called RIFINs (repetitive interspersed family of proteins) secreted by parasites move to the surface of infected blood cells, causing them to become sticky and bind together, creating the clots that block blood vessels and cause the symptoms of severe malaria. Interestingly, these proteins bind more strongly to blood cells belonging to blood group A than to those in group O.

The function of RIFINs had not previously been identified, yet they are encoded by 150 rif genes and comprise the largest family of antigenically variable molecules in the P. falciparum parasite that causes severe malaria. The team’s analysis indicates that the incidences of RIFIN and PfEMP1 at the surface of infected red blood cells correlated with one another as well as with rosetting rates, suggesting that both are implicated in the formation of rosettes.

However, they show that A-RIFINs are the major ligand, or binding molecule, of the process in blood group A. This is demonstrated by the continued formation of rosettes in blood group A once PfEMP1 was removed by treatment with the enzyme trypsin or blocked by the addition of PfEMP1specific antibodies. Individuals with blood type O are able to generate antibodies to counteract PfEMP1, which disrupts the rosettes and prevents these individuals developing severe malaria. All of this means,

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Detail How does your research fit into the current drive against malaria?

Forty percent of the world’s population is at risk of malaria, and 214 million acquire the infection annually. An additional 400 million are asymptomatic carriers of malaria parasites. Each year, four million cases of severe malaria occur, of which 10-20% (430,000) succumb to the infection, representing a daily death toll of 1000 to 1500 that is predominantly made up of children. Without treatment, cerebral malaria is invariably fatal.

However, there is a genuine lack of understanding of how severe malaria is brought about, what triggers the pathogenesis of severe malaria?

We therefore both need a detailed knowledge of the pathogenic processes, and adjunct drugs that would decrease the number of deaths and sequel from severe malaria. We are working in these areas and I was one of the founders of Dilaforette Ltd, a company which develops adjunct therapy for severe malaria and sickle-cell disease.

Your project has made some significant steps in explaining how blood group O offers protection against malaria.

Are you proud of your team’s achievement?

We are proud of our work and also happy that a recent study in Nature Medicine also shows that blood group O is selected for in populations where malaria is highly endemic. That strengthens our

simply, that A-RIFINs mediate rosetting in group A, whereas PfEMP1 is mainly implicated in group O rosetting, though rosetting is much more minimal in group O. LOOKING AHEAD The team’s findings fit well in the current body of literature and have been well received. They support the hypothesis that O-type alleles have been evolutionarily selected in areas with high incidences of malarial infection and suggest that RIFINs may contribute to the varying global distribution of ABO blood groups by selecting preferentially for group O.

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data and indeed suggests that both RIFINs (repetitive interspersed family of proteins) and ABO blood groups are at the centre-stage of the pathogenesis of severe malaria.

What were the key challenges that you managed to overcome?

Identifying the RIFINs as the adhesive molecule that binds to the ABO bloodgroup antigens was difficult since there are about 150 different RIFINs and only one of these is relevant, the one expressed. 149 genes are silent.

The malaria parasite can make red blood cells sticky. Your team identified an important mechanism for how this occurs – how might this be applied to other research?

The biochemistry of the molecule is interesting and the role of the molecule in adhesion also argues that other molecules such as the STEVORs are important for the survival of the parasite but also points towards its involvement in the pathogenesis of malaria.

What are the next steps towards applying your work to fight the disease?

It would be of importance to develop a vaccine from the RIFINs that could protect against severe malaria. However, we will only know after further work if that is doable.

As Wahlgren says, 'we can explain the mechanism behind the protection that blood group O provides against severe malaria, which can, in turn, explain why the blood type is so common in the areas where malaria is common. In Nigeria, for instance, more than half of the population belongs to the blood group O, which protects against malaria'.

FUNDING The 2015 study discussed was financed by: • Swedish Foundation for Strategic Research • EU Commission • Swedish Research Council • Torsten and Ragnar Söderberg Foundation • Royal Swedish Academy of Sciences • Karolinska Institutet BIO Mats Wahlgren is a professor of infectious disease control and parasitology at the Karolinska Institutet in Stockholm, where he leads a research group. Their research focuses on the molecular pathogenesis of severe malaria caused

by Plasmodium falciparum, looking particularly at the effect of the disease on the surfaces of infected red blood cells. The team was responsible for linking the rosetting phenomenon with severe malaria, and is currently working with a pharmaceutical company, Dilaforette AB, to develop an antirosetting vaccine. They also collaborate closely with scientists from Makerere University Medical Biotech Labs in Kampala, Uganda, as well as others from numerous other countries.

CONTACT Department of Microbiology, Tumor and Cell Biology (MTC) Nobels väg 16 KI Solna Campus Karolinska Institutet, Box 280, SE-171 77 Stockholm Sweden E: Mats.Wahlgren@ki.se T: +46 8 524 872 77 W: http://ki.se/en/mtc/the-matswahlgren-group @karolinskainst /karolinskainstitutetenglish /mats-wahlgren

Furthermore, the team’s work suggests that RIFINs may be prime candidates for targeting during screening for new drugs and may be the key to unlocking doors in the search for a vaccine for malaria.

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Spotlight

Manchester: the home of beautiful biotechnology The Manchester Institute of Biotechnology was set up in 2006 and quickly became one of the world’s leading institutions within biotechnological research. Professor Nigel Scrutton, the director at the helm, recently spoke to Research Features about the ground-breaking research carried out during their first ten years and the future strategic direction for their work.

B

iotechnology is a field of science which applies the complexity of biological processes to technology for the construction of products.

At the heart of this process, the Manchester Institute of Biotechnology (MIB) have become one of the front-runners of this field, with their work consistently featured in well-known scientific journals like Nature, Cell, Science, Nature Chemistry, Nature Biotechnology, Journal of American Chemical Society – the list goes on.

Their research teams have not only uncovered perfect drug combinations to combat severe diseases, but they have also determined bacterial protein structures that could provide effective drug targets for certain inherited cancers.

First established only ten years ago, MIB has already attracted more than £100 million of investment and has developed a unique collaboration with the young researchers

at the University of Manchester. They first opened their Garside building back in 2006, which quickly became one of the leading features of the Manchester skyline – it really is something to behold.

With its spectacular aesthetic and high-tech interior, it also comprises a huge number of research staff, up to 47 research groups and a large atrium that encourages spontaneous discussion between researchers.

Of those researchers, Professor Nigel Scrutton is the man in charge of it all. From his personal research into enzyme catalysis to his awardwinning dedication to biotechnology, he is responsible for the strategic leadership and operational management of the institute.

He recently sat down with us at Research Features to discuss his role further, while highlighting the current successes and downfalls within the biotechnological world of scientific research.

Challenge-led bioscience requires seamless working across disciplines. It is important to bring under one roof interdisciplinary scientists committed to working in this way to tackle major societal challenges 62

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Spotlight

Hello Nigel! How would you describe your role as Director of MIB? My role is to provide leadership related to the strategic and operational oversight of interdisciplinary research programmes at MIB. This extends from anticipation of, and planning for, future research trajectories to recruitment of research leaders. I'm also responsible for the planning and procurement of major equipment purchases, development of international partnerships, and interactions with all key stakeholders, including funding bodies and industrial partners.

MIB champions team-based approaches to challenge-led bioscience and my role is to create the environment and support for MIB researchers that enables these activities to flourish.

Back when MIB first opened in 2006, it became the first of its kind to represent a university-based, purposebuilt interdisciplinary research institute. Why is having this interdisciplinary approach so important to biotechnology research?

Challenge-led bioscience requires seamless working across disciplines. As such, it was important to bring under one roof interdisciplinary scientists committed to working in this way to create unique teams that tackle major societal challenges. MIB is particularly noted for its capabilities in industrial biotechnology – a University of Manchester research beacon. MIB has assembled teams of chemists, chemical engineers, computational and materials scientists, as well as biologists, to drive major programmes in the industrial biotechnology space, and has also worked with industry to translate basic discovery science to commercial success.

MIB’s Garside building really is something to behold. How important has the design of this building been in the sharing of ideas and research between scientists?

Crucial – the open laboratory environment and breakout spaces enable scientists and lead investigators from different discipline backgrounds to work shoulder-to-shoulder.

This ensures easy exchange of information and ideas at all levels, which is crucial to building successful interdisciplinary teams.

What impact do you think MIB has had on biotechnology research since it was first established and are there any personal achievements you are particularly proud of?

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MIB has had a major impact in its first ten years. It has secured more than £160 million of competitive grant funding, filed numerous patents and established eight spin-out companies, one of which was floated on the London stock exchange (and valued at £33 million). Industrial partners have exploited a number of MIB basic discoveries in several commercial processes. The MIB brand is recognised internationally as a globally leading institute in biotechnology and is often the partner of choice for industry in developing strategic programmes that address industry need.

When MIB first opened it was known as the Manchester Interdisciplinary Centre.

What were the reasons behind changing its name to the Manchester Institute of Biotechnology?

MIB was the first interdisciplinary research institute of its kind at the time of opening and it was right at the time to reflect this in the title of the institute. With time, however, we felt that the name of the institute should communicate more clearly the

strategic direction of the institute’s research programmes. The acronym MIB was instantly recognisable but the words Manchester Interdisciplinary Centre were less so to non-specialists. We felt that the new name was a more effective way of informing nonspecialists about the research direction and purpose of our research programmes.

MIB is unique in having such a strong collaboration with Manchester University.

Why is this relationship so important?

MIB is a flagship institute of the University of Manchester. The institute also draws on the research excellence found elsewhere in the university and many of the institute’s programmes involve specialists located outside the MIB. Also, the MIB community is not fixed, as research hotel investigators from other parts of the university can benefit as institute members from the unique research environment in the institute. Maintaining this flexibility in MIB membership is important as new research challenges emerge, and the right combination of staff can be brought together to form new cooperating research teams.

Welcoming overseas staff to MIB is essential if the institute is to remain a prominent player in the biotechnology space www.researchfeatures.com

MIB is renowned for its international collaborations with researchers. Why is having these worldwide collaborations so significant? Will Brexit have any effect?

Having strong partnerships overseas is crucially important. That way, MIB staff can work with some of the best scientists globally to form unique programmes at the cutting edge. The ability to welcome overseas staff to MIB and for MIB staff to work in the laboratories of overseas partners is essential if the institute is to remain as a prominent player in the biotechnology space.

Rightly, there are major concerns about the potential effects of Brexit on our ability to work effectively with international colleagues.

From a more personal perspective, your work into enzyme catalysis and long-term commitment to MIB has seen you win numerous awards over the years. Does winning these awards make all of your work feel worthwhile or are they just a bonus?

Of course it is nice to see the work of my team recognised through these awards but this is not the motivation. For me, this comes from seeing the science progress from fundamental discovery through to application. Most scientists are driven primarily by their science and the contribution it makes to understanding and knowledge transfer.

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What are MIB’s next steps in terms of future research and business direction?

The burgeoning bio economy is based on our ability to manufacture everyday chemicals, materials and medicines, and to find innovative technologies for energy supply. Synthetic biology is a key enabler in all these areas and will underpin the industrial biotechnology agenda in years to come. We are well positioned in MIB to capitalise on these developments and I look forward to MIB driving this forward with innovative solutions to many of the manufacturing challenges faced by society.

• Professor Nigel Scrutton is the Director of the Manchester Institute of Biotechnology and an awardwinning scientific researcher. After receiving a first class honours degree in Biochemistry at King’s College London, Professor Scrutton went on to gain an ScD and PhD (as a Benefactor’s Scholar) at the University of Cambridge. During his research career, he has published approximately 370 papers and is widely regarded as an expert in the fields of enzyme catalysis, biophysics and biomolecular engineering. He manages a group of around 40 researchers and has amassed numerous awards for his work, including the Biochemical Society Colworth Medal and the RSC Charmian Medal, to name a few.

The MIB brand is recognised internationally as a globally leading institute in biotechnology and is often the partner of choice for industry

Contact Manchester Institute of Biotechnology University of Manchester 3.020 Garside Building, 131 Princess Street, Manchester, M1 7DN T: +44 (0)161 306 8917 E: Nigel.Scrutton@manchester.ac.uk W: www.mib.ac.uk /MIB2006/ @UoMMIB /UoMMIB

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Family matters: drinking patterns in Inuit mothers and adolescents Dr Marilyn Fortin is a postdoctoral fellow at the University of Laval’s School of Psychology, based in Montreal, Canada. Her current research focuses on the psychosocial factors that influence excessive alcohol use in Inuit communities in Northern Quebec, with particular focus on the intergenerational transmission of binge-drinking habits between mothers and adolescents.

Women’s Health

Why did you decide to focus your research on Inuit communities?

After travelling substantially and a number of years studying anthropology, I knew that I wanted to grow in a profession in which I could explore, day after day, the cultural diversity that surrounds us. I see this research project as a natural progression of my own interests in culture and health. To pursue this work, I needed to look beyond my specialisation in sociology and alcohology. I gained new knowledge in psychology, perinatal health, and environmental and population health in order to work cooperatively with a community particularly touched by the harmful effects of alcohol – a community that also calls our vast and beautiful Québec home. In a population with a high rate of binge drinking, it is crucial to identify the social and familial determinants influencing the adoption of this behaviour, especially among women and adolescents. Excessive drinking among women often goes hand in hand with a difficult family situation or the presence of psychosocial risk factors. It is important to raise society’s awareness here.

How did you first become interested in the impact of mothers’ drinking on their children?

Women and children’s health has always been a catalyst for my explorations. During my postgraduate studies, I joined a research group that focuses on the social determinants of health and the effects of context on social inequalities in health, specifically among users of alcohol and psychoactive substances. I quickly saw how my personal interests in health intersected with the needs of these vulnerable populations. And since pregnancy is a happy event, it is all the more exciting to work on an issue associated with one of life’s most extraordinary moments, the birth of a child.

What part of your research findings do you think is the most important for public health?

The goal of this study is to evaluate the contextual familial and psychosocial factors likely to influence excessive alcohol use among Inuit mothers and their children. It will allow for the identification of factors associated with excessive alcohol use among Inuit from Nunavik and will help to formulate recommendations on binge drinking with the hope of reducing its harmful effects on the Inuit population.

By observing whether certain psychosocial and health determinants have an influence on the

mother–child relationship, we will be able to propose prevention strategies and health interventions to help the Inuit community and the most vulnerable. This research uses a multidimensional approach in order to ultimately understand the impact of a child’s family environment on his or her alcohol-related behaviour during adolescence – another extraordinary time of life. Thoroughly understanding the causes and/ or determinants of a mother’s excessive alcohol use could also help to prevent excessive use among adolescents, which is a particularly devastating phenomenon within Inuit populations. All the phases in this study are important because each one is like a piece in a giant puzzle. In the end, we will have a better view of the big picture and will hopefully be able to provide better support to Inuit youth populations.

What impact do you hope your research will have?

We hope to raise awareness among women at risk of excessive alcohol use, as well as those around them, about the harmful effects of drinking during pregnancy and the long-term effects on children’s health. We also hope to demonstrate that explaining the causes of alcohol use is often very complex and that prevention is often an effective avenue for reducing the effects. The expected results are aimed at the operationalisation of interventions targeting excessive alcohol use among Inuit youth in order to limit harmful consequences and to prevent continuation and transmission. Lastly, the study will provide more data about homes in which excessive alcohol use is present in order to identify the direct factors of this behaviour and soften its impact on future generations.

What are the next steps for your research?

Our next step is to continue research on adolescent drinking patterns and to examine how alcohol use among Inuit youth affects the mental health of this population at high risk of psychological distress. Other studies, in collaboration with Inuit communities, are ongoing or will begin in years to come, with the hope of increasing our knowledge about the multiple barriers to achieving an acceptable quality of life in northern Québec and Canada. These studies aim to develop health and social services that are in harmony with Inuit culture and to empower Inuit to manage their own health care programmes.

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Women’s Health

C

anadian Inuit populations differ from others in numerous ways, including their use of alcohol.

Although Inuit people drink less frequently, they binge drink at a rate three times higher than other Canadians and typically drink well above the threshold defined as binge drinking: five drinks in one session.

Consuming large quantities of alcohol in a short amount of time can have harmful effects on an individual’s health in the short-, mid- and long-term. This is particularly true for Inuit women because the harmful effects of drinking take a greater toll on their daily lives and on the lives of their children.

Indigenous Canadians are particularly at risk of Foetal Alcohol Spectrum Disorder (FASD), caused when a woman drinks during pregnancy, and which is linked to delays in growth and neurodevelopmental issues.

THE FAMILY ENVIRONMENT INFLUENCES ADOLESCENT DRINKING Dr Fortin’s research examines the social context in which drinking occurs. Binge drinking in women who are pregnant or have young children often reveals underlying domestic issues or psychosocial contexts that have triggered excessive alcohol use.

Moreover, family environments where alcohol is consumed excessively are likely to encourage children to binge drink themselves, thus perpetuating alcohol problems.

There is a well-documented link between heavy drinking during adolescence and mental health problems. Dr Fortin aims to determine whether a family context of heavy drinking, and particularly maternal drinking, influences adolescent drinking patterns, and whether this influence varies with the gender of the adolescent.

Adolescent binge drinking is strongly related to increased risk of unintentional injury, attempted suicide, violence, delinquency, and episodes of depression. Binge drinking is also the most common mode of alcohol consumption among adolescents.

Dr Fortin and her colleagues’ main hypothesis is that mothers’ excessive drinking habits are passed on to their children through the family and psychosocial contexts, which influence children’s behaviour. Mothers have a primary role in Inuit households, therefore mother–child transmission of alcohol habits are particularly important to study.

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Adolescent binge drinking is strongly related to increased risk of unintentional injury, attempted suicide, violence, delinquency, and episodes of depression GENDER DIFFERENCES IN DRINKING PATTERNS Dr Fortin has also been examining the effect of the child’s gender on the degree to which they are influenced by their mother’s drinking habits. Gender is a key determinant of identity and behaviour, including drinking habits. Drinking habits differ between men and women because of the distinct social norms, expectations, and rules for people of different genders.

She therefore considers it important to evaluate the effect of gender on alcohol consumption.

STUDYING DRINKING HABITS IN NUNAVIK The study used data from the Nunavik Child Development Study (NCDS) as well

as follow-up interviews with mothers when their children reached 11 years, and with the adolescents themselves when they reached 16. The study allowed the team to examine the connection between excessive adolescent drinking and maternal alcohol use during pregnancy in the Inuit community, as well as the mediating role of certain psychosocial, family and circumstantial factors of drinking on this relationship. Some of the factors identified include age, education, economic poverty, food insecurity, domestic violence, depression, and suicide ideations and attempts.

This is the first trend analysis of longitudinal data tracing the evolution of drinking patterns around pregnancy in

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alcohol despite abstinence at the beginning of the pregnancy. One third of the women sampled reported using marijuana during their pregnancy. Marijuana use was shown to increase the risk of alcohol consumption during pregnancy. This indicates that the concomitant use of substances other than alcohol could be used to identify women who are at higher risk of alcohol misuse during pregnancy.

DEFINING DRINKING PROFILES Estimating alcohol consumption can be tricky. Data can be collected during or after pregnancy, but there are no comparative studies in Inuit populations to suggest which method is better. Additionally, issues can arise with self-reporting because of the stigma associated with alcohol consumption, and therefore social pressures to under-report.

In the next stage of Dr Fortin’s project, the team will use retrospective interviews (11 years on) to define drinking profiles of mothers (abstinent, drinkers who do not drink heavily, and binge drinkers), and analyse personal, familial and environmental factors related to each drinking profile. The analysis of this data will also investigate the effect of paternal drinking on that of the mother and youth. an Inuit community. Dr Fortin’s research investigated alcohol consumption and binge drinking trajectories from the year prior to conception until one year after the birth of the child. This comprehensive picture elucidates the drinking patterns of Inuit mothers around their pregnancy and risk factors of heavy drinking at this time.

DRINKING TRAJECTORIES OF MOTHERS DURING PREGNANCY Fortin’s results demonstrate that alcohol use generally falls during early pregnancy, tending to increase during pregnancy and the following year. Of the 248 women sampled, 18.7% drank throughout the study period, but just 1.6% reported binge drinking. Overall, this shows that women were more likely to abstain from alcohol completely, or to at least stop binge drinking during pregnancy, potentially as a result of public health interventions. Those who were in a couple were more likely to stop drinking, suggesting that family support can be an important determinant of alcohol consumption.

However, some women still consumed

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NEXT STEPS Dr Fortin’s team will soon be ready to evaluate their main research hypothesis regarding the intergenerational transmission of heavy drinking in Inuit families between mothers and adolescents, and assess whether there is a gender dimension to this. They expect to be able to identify the key factors that influence binge drinking, which often co-occurs with difficult family situations or the presence of psychosocial risk factors.

Dr Fortin’s research has been conducted with the collaboration of various indigenous representatives, such as the Kativik Regional Government, President of the Inuit Circumpolar Council (Canada Office) and Executive Board of the Nunavik Regional Board of Health and Social Services, among others. The eventual outcomes will be to reduce the negative health outcomes for Inuit people associated with excessive alcohol use by implementing targeted, evidence-based policies. Hopefully this will enable local government to reduce the impact of alcohol on future generations.

Detail RESEARCH OBJECTIVES Dr Fortin’s research focuses on the relationship between a mother’s drinking habits during and after pregnancy and the subsequent drinking habits of her adolescent offspring.

She is also interested in the effect that gender can have on drinking habits.

FUNDING • N asivvik Centre: http://www.nasivvik.ca • I RSC: http://www.cihr-irsc.gc.ca/e/193.html COLLABORATORS • D r Gina Muckle, Université Laval • D r Richard Bélanger, MD, FRCPC, Université Laval BIO Dr Marilyn Fortin earned her PhD in Sociology of Health from University of Montreal. She received a doctoral research award from the Canadian Institutes of Health Research to work on social determinants of alcohol use among the Canadian population. She is currently a postdoctoral researcher at the School of Psychology, Laval University, and a member of the Population Health and Optimal Health Practices Research Unit of the CHU de Québec and Groupe de Recherche sur l’Inadaptation Psychosociale chez l’enfant (GRIP). She received a postdoctoral research award from the Canadian Institutes of Health Research and from the Nasivvik Centre to pursue her research in the area of social determinants of alcohol use among indigenous populations.

CONTACT Marilyn Fortin, PhD École de psychologie, Université Laval 2875 boulevard Laurier Édifice Delta II Bureau 600 6e étage Québec Québec G1V 2M2 E: marilyn.fortin.2@ulaval.ca T: +1 418-525-4444 ext 46518

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Women’s Health

Women sleep on Venus; men sleep on Mars Dr Ketema Paul is an Associate Professor at UCLA with a background in neurobiology, sleep and circadian rhythms. His current research focuses on sleep disorders and the interactive effects of sex chromosomes on our ability to recover from sleep loss.

Your current research examines the effects of sex chromosomes on our ability to recover from chronic sleep deprivation. What is chronic sleep deprivation and what effects can it have?

Chronic sleep deprivation is when you’re able to sleep on a daily basis but are not getting the sufficient amount of sleep you need. If you sleep substantially less than you need, you build up a sleep debt which can then accumulate. This accumulation negatively affects your brain’s cognitive function – your ability to think properly and remember things.

Can you talk me through the phases of sleep and wake that we normally experience as humans?

First of all, I’d like to dispel the myth of ‘normal’ sleep – there really is no such thing.

Sleep–wake architecture really represents

the different vigilance states you’re in – everyday wake, REM sleep, non-REM sleep – and that is dependent on several things.

Age, location and artificial light can all affect this but my research focuses on the effect gender has on sleep. Not only do men and women sleep differently, but women sleep a lot more dynamically during their lifespan.

How a woman sleeps is dependent on her reproductive cycle, pregnancy, postpartum recovery, and menopause, so sleep-wake architecture depends on these variables.

It can be different for some people – some people need less sleep, some people need more, but that’s for each individual person and their healthcare provider to figure out.

Some people use naps and for some naps are healthy, so it really is about having the self-awareness to recognise the signs of sleep deprivation.

So, gender affects your everyday sleep as well as your response to sleep deprivation?

Gender is a complicated variable – it incorporates psycho-social, cultural, and a lot of different factors. What we wanted to do was isolate the biology of gender, which is sex, to see whether gender differences when sleeping can be traced to biological sex differences. When I was a post-doc we looked at sexual reproductive hormones and found that these had limited effects on what we call sleep homeostasis – the ability to recover from sleep loss. In our rodent models, we found that when you removed the sexual reproductive hormones, there were still sexual differences in the ability to recover from sleep loss. These differences were only subtle but, like I mentioned earlier, sleep debt is cumulative so over time it

Women’s Health

would cause much larger differences to health and well-being.

When we first saw these differences, we began to ask questions and we found that sex chromosomes do in fact affect the ability to recover from sleep loss. Perhaps more importantly though, we also found that during the mid-active phase, the time when we normally get sleepy, mice with a Y chromosome tended to favour sleeping more during this siesta time. That effect came to be increased by sleep deprivation – the less sleep the mice with Y chromosomes had, the more likely they were to sleep during the siesta time.

So, we think that we have found, and we are still working on it right now, a unique sleep regulatory mechanism that promotes sleep loss recovery using a kind of pressure switch that occurs during the mid-afternoon – it’s biological and seems to be related to genes that lie on sex chromosomes.

A large part of your research uses mouse models. How similar are sleep patterns between mice and humans? What are the differences?

Mice sleep differently to humans. Humans have more of a biphasic sleep pattern, where we’re awake throughout the day and we’re asleep at night, whereas mice have a more fragmented sleep pattern. They are still biphasic but they display more wakefulness during the rest phase, which for them is during the day time. However, what we’re really interested in is the genes and the molecules that regulate sleep, and those are pretty well conserved between mice and humans. We know that mice and humans share the same molecular regulatory elements and areas of the brain that regulate sleep so, as researchers, we think that mice are a really effective model to ask these kinds of questions.

One study has shown that major depressive disorders (MDD) affect the homeostatic desire for sleep in a similar way to sleep loss. What relevance do you think your research might have to mental health?

That is what led us to these studies in the first place. I was interested in looking at the impact stress has on sleep as it can dysregulate sleeping patterns and contribute to mood disorders such as post-traumatic stress syndrome. If you could target the sleep though, you might be able to improve outcomes for people with these disorders.

We need to find a way to look at how people

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respond to stress and the effect this has on sleep quality and, in turn, sleep deprivation.

This sleep deprivation is one of the ways that stress acts to dysregulate sleep so we’re trying to figure out how this influences the ability of stress to affect sleep. We think if we can do that then we can better understand how sleep homeostasis works, and perhaps can even use sleep as a target to treat major depressive disorders. We already do that behaviourally, as sleep deprivation is used as a therapeutic treatment for mood disorders, but what we would like to do is make those treatments more effective by understanding how sleep deprivation influences the brain and body.

What are the next steps for your research?

Our ability to understand the dynamics of sleep homeostasis has been sort of limited by the tools we have to measure it, so what we’ve been doing is trying to develop more elaborate tools that measure the ability to recover from sleep loss – like a kind of biomarker. In our mouse models, we’ve been trying to design more effective ways to measure sleep homeostasis so we can map out the dynamics of sleep loss. Over time we know that sleep debt accumulates but what does that look like? Which times of the day is sleep debt likely to be higher or lower, and which times of the day is sleep debt accumulation likely to have a more negative effect on your cognitive abilities? And what are the impacts of sex on those differences?

These are the questions we are asking right now. To answer these, we are actively doing experiments on stress and sleep, particularly looking at social stress in our mouse models.

We’re modelling what we call a ‘social defeat’, caused by a confrontational encounter, to study how sleep deprivation affects resilience and susceptibility to social stress. I think by improving the understanding of the dynamics of sleep loss, we can truly determine whether sleep can be used as a target to build resilience to social stress.

What influence are you hoping your research will have?

This work is very important to women’s health. When I first began the research, it wasn’t because I was interested in sex difference research or women’s health, I was just amazed that there was so much we didn’t know about women’s sleep – women’s sleep is so dynamic and yet we were asking so few questions about it. That really floored me when I was a post doc, so I decided that if I wanted to learn more about sleep, I needed to study it more in females.

I felt that one of the reasons we knew so little about the ability to recover from sleep deprivation was because we included so few females in our studies, so I decided to do the opposite. Women, in several ways, have a biology that is more dynamic and that’s a great thing – it enables the fitness of the human population.

So, music is your other great passion in addition to science. Can you tell me more about the role it has played in your life? How important is creativity to your scientific career?

I think creativity is critical to good science.

The best scientists – the ones who I admire and aspire to be like – come up with the best experiments that answer the most important questions. You need to be really creative to do that. If there’s a really pressing question or need in biomedical science you have to be able to expand your creative outreach to come up with the most effective and conclusive experiments. You then have to communicate those results effectively to reach the largest audience and resonate with as many people as possible. That is what draws me to science, as it provides a creative outlet that really allows me to continue to do creatively the things I do with music. And quite frankly coming up with experiments and completing them, writing them up and communicating them is not much different to producing music.

Creativity is critical to good science.

The best scientists – the ones who I admire and aspire to be like – come up with the experiments that answer the most important questions www.researchfeatures.com

S

leep is essential for human health and the long-term consequences of sleep loss can be different in men and women. Sleep-deprived women have higher rates of affective disorders such as depression.

Moreover, the dynamics of sleep recovery in women are more sensitive to reproductive processes. Dr Paul’s work, in collaboration with colleagues, focuses on picking apart the underlying biological reasons for these gender disparities in order to understand the differences between men and women’s sleep health.

MEN AND WOMEN SLEEP DIFFERENTLY Sleep is controlled by two systems: the circadian system, which regulates the timing of the sleep-wake cycle and consolidates it into phases; and the homeostatic system, which determines the amount and intensity of sleep based on how long the person was previously awake. Reproductive hormones interact with these systems, and therefore may be responsible for some of the differences in sleep between men and women.

During sleep, the body goes through several sleep cycles, each incorporating three separate stages of vigilance – wake, non-REM and REM. REM sleep and the deeper stages of non-REM sleep are very important, as they provide cognitive repair and restoration which, in turn, improves memory and

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This suggests that the sleep homeostat is regulated differently in men and women.

prevents disease. As Dr Paul states: ‘NonREM sleep has several stages. Stage one and two are transitional stages while stages three and four are slow-wave sleep. This slow-wave sleep is restorative and helps you to recover your cognitive ability and restore certain peripheral processes, while also acting to prevent disease and keep you healthy.

REM sleep, on the other hand, is especially important for consolidating memories and maintaining brain fitness.’

In rodent models, as used in Dr Paul’s research, androgens reinforce sleep, whereas oestrogens enhance wakefulness.

Female mice are awake for 1.5 hours more per day, but their sleep patterns are more ‘consolidated’, meaning they are less likely to wake up during sleep, and more likely to stay awake for sustained periods without interruption. Male mice get more non-REM sleep than female mice, as well as more total sleep.

LOSING SLEEP Our response to sleep deprivation is still quite unclear because the underlying mechanisms have not yet been clearly identified. However, men and women respond differently to sleep deprivation, and the differences between the sexes increase as sleep loss increases.

The homeostatic drive to sleep, which increases as the period of wakefulness lengthens, is dissipated during sleep. This pressure, or sleep propensity, is typically measured by looking at slow wave activity (SWA) in the brain during non-REM sleep.

Women seem to have higher basal sleep pressure and slower age-related reduction in SWA. Sleep disorders also exhibit sex differences: for instance, insomnia is more common in women, but obstructive sleep apnoea is more common in men. Disorders like depression can affect the homeostatic sleep drive in a way similar to sleep deprivation (making people with depression want to sleep more often), and affect women and men differently.

SLEEPING IT OFF The reduced ability to recover from sleep loss of women compared to men may explain the dissimilarity in the adverse health effects experienced by men and women.

Dr Paul’s team has shown that reproductive hormones, which differ between males and females, are responsible for some of the sex differences in daily sleep amount.

However, the ability to recover from sleep loss appears to be relatively insensitive to reproductive hormones, therefore Dr Paul and colleagues have hypothesised that sex

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Women’s Health

chromosomes directly affect recovery from sleep deprivation.

In some rodent studies, there were no differences in the male vs. female response to six hours of sleep deprivation, but the difference in SWA between male and female mice in the first two hours of non-REM recovery sleep was heightened, as in humans.

Results also showed no differences in the absolute amount of recovery sleep following deprivation, but that females regained more non-REM sleep than males, while males regained considerably more REM sleep.

Results from fruit fly studies also suggest differences in the recovery from sleep deprivation between sexes.

HORMONAL VS. GENETIC EFFECTS Dr Paul’s team has been working with the four core genotype (FCG) mouse model, which offers some exciting and promising insight. The sex chromosome complement (XX, XY) of mice in this model is independent of their phenotype (male or female), allowing scientists to determine whether differences observed between males and females is genetic, or to do with other sex-specific characteristics such as hormones. The FCG model in mice shows that genetic sex does not determine the difference in baseline sleep propensity. This supports the notion that it is reproductive hormones that affect the differences. However, it also suggests that sex differences in recovery from sleep loss are dependent on something else.

Men and women respond differently to sleep deprivation, and the differences between the sexes increase as sleep loss increases

The effects of hormones can be organisational or activational. Organisational effects are relatively permanent effects on the structure and function of the body, often established during critical moments in development, such as during foetal development or puberty. Activational effects are immediate (and temporary), and depend on the momentary presence or absence of the hormone.

Rodent studies have shown that female gonadal hormones inhibit sleep amount and that these hormones have an organisational effect on sleep architecture. Multiple studies have demonstrated that the female hormone oestrogen reduces the amount of REM sleep or non-REM sleep, or both. Reproductive hormones also influence the ability of circadian rhythms to adapt to light–dark cycles.

OF MICE AND (WO)MEN Models like the FCG mouse model are instrumental in advancing research on sleep loss and recovery and, although they may not be directly comparable, the genes responsible for sleep regulation are ‘well preserved’ between the two species. As Dr Paul explains: ‘We know that mice and humans share the same molecular regulatory elements and areas of the brain that regulate sleep so, as researchers, we think that mice are a really effective model to ask these kinds of questions.’

Dr Paul’s team hopes to use these models, alongside improved techniques to measure SWA in active animals, to increase their understanding of the mechanisms underlying the differences in sleep regulation and recovery between the sexes – but especially in women. Throughout his research, Dr Paul has become an advocate for women’s health and his team now hope to further develop their understanding of how sleep deprivation affects diseases more commonly suffered by women. As he says: ‘I think it’s imperative we understand the things that could increase disease risk, and particularly diseases that are more prevalent in women. I’ve kind of reversed that in my own research since I first started and, because of the work I’ve done, I am now a very large advocate for women’s health.’

Detail RESEARCH OBJECTIVES Dr Paul’s research is helping to determine how sex chromosomes impact on the ability to recover from sleep loss. His work increases our understanding of sex-based differences in sleep deprivation and the mechanisms underlying these differences in sleep control.

FUNDING National Institutes of Health (NIH) COLLABORATORS • Karyn Esser, University of Florida • Gianluca Tosini, Morehouse School of Medicine • Chris Ehlen, Morehouse School of Medicine • Jason DeBruyne, Morehouse School of Medicine BIO

After receiving his bachelor’s degree in biology from Howard University, Dr Paul went on to study neurobiology and circadian biology at Georgia State University in Atlanta, Georgia where he received his doctorate. Dr Paul completed a postdoctoral fellowship at Northwestern University in Evanston, Illinois in 2006 at the Center for Sleep and Circadian Biology, after which he accepted a faculty position at the MSM. Dr Paul is currently an Associate Professor at UCLA. CONTACT Ketema Paul, PhD Associate Professor Department of Integrative Biology and Physiology UCLA Semel Institute for Neuroscience and Human Behavior 740 Westwood Plaza #68-237A Los Angeles, CA 90095 E: ketema.paul@ucla.edu T: +1 (310)794-7755 W: https://www.ibp.ucla.edu/ physcifacultyindiv.php?FacultyKey=11609 LEARN MORE HERE: http://www.ninds.nih.gov/diversity_ programs/success_stories/ketema_paul. htm

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