Phenotype | Issue 27 | Trinity Term 2017

PHENOTYPE www.phenotype.org.uk

Issue 27 | Trinity Term 2017

THE BODY’S BOUNCERS

The immune system in focus

Iron regulation A weapon against pathogens Page 10

Fancy winning a book?

Enter Phenotype’s crossword competition Back cover

Diabetes

What is the beta-cell’s sweet secret?

Page 18

Careers Alternatives to academia

Page 36

Want to see your image here next issue?

OXFORD Enter Phenotype’s research image UNIVERSITY competition BIOCHEMICAL Page 42

SOCIETY

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Issue 27 | Trinity 2017

EDITORIAL Welcome to the 27th edition of Phenotype! This term we are taking a look at the immune system; how it works, why it is important, and what happens when things go wrong. If you are new to this field, you can check out a comprehensive introduction to the immune system and the history of immunology in Anne Wolfes’ infographic on page 6.

Immune systems defend a host organism from intruders, known as pathogens.The human immune system has evolved over millions of years into a highly complex system, involving the participation of many different cell types in order to carry out its function.

However, even the most simple, single-celled organisms possess mechanisms to protect themselves from environmental pathogens.

In this edition we look at the important cell types and processes that are involved in an immune response. On page 8, Julian Buchrieser tells us about how our understanding of macrophage development is evolving, and on page 24 Janina Nahler explains how new discoveries regarding T cell plasticity are shedding light on how the immune system adapts to different threats. Digging deeper into the biochemical functioning of immune cells, Professor Hal Drakesmith provides insight into the importance of iron in immunity on page 10, and on page 14 Pradeep Kumar highlights the importance of CRAC channels and calcium signalling in immune cell transcriptional response.

Diseases of the immune system come in many shapes and forms. On page 13 Olivia Murray explains the effects of leukaemia on the immune system, and on page 16 I discuss the biology behind coeliac disease and the ongoing efforts that are being made to develop treatments.

On page 21, Emma Thomas delves into the world of plant immunity, and takes a look at the different tactics that are being employed to engineer pathogen-resistant crop strains that will provide greater food security. Also on the theme of food production, Luiz Guidi takes a look at how big data and urban agriculture may be used to combat the growing worldwide demand for food.

In addition to our research articles, do check out our Regular articles and the Science & Society section, where you can find highlights from the recent OUBS careers day on page 36.

Additionally, don’t forget to enter our competitions! Check out the winner of our Snapshot Image competition on page 42 and enter this term’s competition for the chance to get your image on the next cover, as well as to win a £50 voucher from Oxford University Press. On the back cover you will find our crossword, which includes some immune system themed answers; enter this for the chance to win one of the books reviewed on pages 40-41.

Finally, it is with a heavy heart that I tell you that this issue will be my last as editor-in-chief, as I will soon be moving on from the University. I have thoroughly enjoyed this role, and will leave you in the capable hands of Stephanie Kapsetaki.

I hope that you enjoy reading this term’s edition as much as I have enjoyed putting it together!

Heather Booth Editor-in-Chief If you are interested in getting involved with Phenotype, please contact Stephanie Kapsetaki at stefania.kapsetaki@new.ox.ac.uk.

OXFORD UNIVERSITY BIOCHEMICAL SOCIETY Phenotype is also available to read online via our website: www.phenotype.org.uk

Issue 27 | Trinity 2017

In foc

us:

CONTENTS

The immu syste ne m

features

regulars

8

3

Editorial

5

Research highlights

6

Infographic: the immune system

38

5’ with... Professor Simon J. Davis

39

Featured seminar: Ivan Ahel

40

Book reviews

42

Snapshot image competition

44

Crossword

10

Mammalian macrophages: origin and development Infectious diseases and immunity: the importance of being ironic

13

A cancer that kills the immune system

14

CRAC channels: bridging intracellular calcium micro-domains

16

Science vs. gluten: emerging therapies for coeliac disease

18

The beta-cell’s sweet secret

science & society

20

Big data and urban agriculture

30

Cortex Club: the birth of a student-run discussion forum

21

Engineering plant immunity

32

24

Helper T cell plasticity: adaptation requirement or therapy hindrance?

Public engagement: why should you get involved?

34

Stand out from the crowd and boost your career

36

Highlights from the OUBS careers day

26

28

Cracking the cis-regulatory code: how close are we?

Blood-brain barrier modelling: an essential challenge

On the cover The immune system in action: an antibody (blue) binds an antigen (orange).

Read more on page 42!

EDITORIAL TEAM EDITOR-IN-CHIEF Heather Booth DPAG CO-EDITOR-INCHIEF Stephanie Kapsetaki Zoology REGULARS EDITORS Vasiliki Economopoulos Oncology Matthew Cooper DPAG William Broad Plant Sciences

FEATURES EDITORS Stephanie Kapsetaki Zoology Michael Song DPAG Inês Barreiros DTC Beatrice Tyrrell Biochemistry SCIENCE & SOCIETY EDITORS Mariangela Panniello DPAG Anne Turberfield Biochemistry

DESIGN & PRODUCTION Oleg Sitsel Biochemistry Katharina Schleicher DPAG SPONSORSHIP Hok Fung Chan NDM CROSSWORD Daniel Scott Pathology alumnus

COPY EDITORS Amy Flaxman Ana Carolina Barros Anne Hedegaard Burcu Anil Kirmizitas Caroline Woffindale Conor Kelly Dharamveer Tatwavedi Elsie Hodimont Fran van Heusden Isaac Wong Ivan Candido Ferreira Jessica Hardy Karyna Mishchanchuk Katharina Schleicher Kevin Ray

Lauren Chessum Lewis Arthurton Luiz Guidi Marcella Brescia Matt Kelly Osman Tack Rebecca Hancock Rosemary Chamberlain Rosemary Wilson Sandra Ionescu Shelley Harris Shi Yu Chan Stuart Keppie Sonia Muliyil Sebastian Vásquez-López Stefania Monterisi

A selection of recent life sciences research from the University of Oxford

RESEARCH HIGHLIGHTS by Matthew Cooper Chang VT et al. (2016) Nat Immunol 17(5):574-582.

Initiation of T cell signaling by CD45 segregation at ‘close contacts’ Binding of foreign antigens to T cell receptors (TCRs) can transduce signals across the T cell plasma membrane; however, the relative contributions of kinases and the tyrosine phosphatase, CD45, to the signal transduction process are unclear. One model, termed the kinetic-segregation model, proposes that in resting conditions, an equilibrium of kinase and CD45 activity restricts TCR phosphorylation; however, antigen binding results in steric exclusion of CD45, shifting the equilibrium in favour of kinases and promoting receptor phosphorylation. Whilst supported by experimental evidence, several aspects of this model need to be verified, including mechanisms by which CD45 and kinases are segregated, and whether this segregation affects receptor triggering, and signal transduction.

Chang et al. investigated CD45 using structural and biophysical methods. Crystal structures of the CD45 extracellular domain (ECD) revealed the domain to be large and rigid, thanks in part to high levels of disulfide bonding.

The large size of the ECD means that all forms of CD45 are likely to be excluded from sites of TCR-ligand engagement when in an upright orientation. Studying CD45 organisation in live T cells with fluorescent anti-CD45 antibody fragments revealed that ligand exposure resulted in the exclusion of CD45 from, and ring-like arrangement of the phosphatase surrounding, points of contacts. Furthermore, resultant TCR signalling was dependent on segregation of CD45 and tyrosine kinase, Lck, at these T cell-ligand close contacts, as large chimeric forms of Lck were excluded from these contact sites and prevented signalling.

In conclusion, the study demonstrates that the CD45 structure promotes its segregation from T cell receptors. It also supports the kineticsegregation model where steric exclusion of CD45 at close contacts disturbs the kinasephosphatase equilibrium and promotes TCR phosphorylation.

The lifelong persistence of human immunodeficiency virus (HIV) in a latent state in long-lived CD4+ T cells is a major obstacle to complete eradication. A recently proposed therapeutic approach involves combining latency-reversing agents (LRAs) with immunomodulators to reactivate and subsequently eliminate HIV-harbouring long-lived T cells. Effective clearance of reactivated cells requires agents sensitive to low levels of antigen. To this end, Yang et al. have developed immune-mobilizing monoclonal T cell receptors against viruses (ImmTAVs). These molecules redirect CD8+ T cells to kill HIV-infected CD4+ T cells.

To ascertain the efficacy of these agents, Yang et al. engineered ImmTAVs specific for the HIV-1 Gag p17 epitope, SL9, with two demonstrating particularly potent affinity. CD4+ T cells were infected with HIV IIIB, and then cultured with CD8+ T cells in the presence or absence of ImmTAVs. Compared to CD8+ cells alone, ImmTAV presence in culture reduced infected CD4+ T cell frequency by up to 85%, indicating a potent antiviral response. The effect was dependent on ImmTAV concentration and CD8+ to CD4+ T cell ratio. Expanding on this result, when resting CD4+ T cells from anti-retroviral therapy-treated HIV-infected patients were reactivated by LRAs in vitro, co-culture with CD8+ T cells in either the presence or absence of ImmTAVs showed that exposure to ImmTAVs leads to complete abrogation of viral recovery in cultures from 4 out of 5 patients.

By comparing multiple cell culture conditions, it was shown that expression of caspase-3 (a component of the apoptosis process) was notably elevated in infected CD4+ cells conjugated to CD8+ cells in the presence of ImmTAVs, as compared to infected CD4+ cells not treated with ImmTAVs and CD8+ cells. By blocking HIV spread using a reverse transcriptase inhibitor, ImmTAV/CD8+ T cell co-treatment was shown to eliminate up to 80% of infected CD4+ cells within 48 hours, further suggesting that the antiviral activity of ImmTAV’s is dependent upon CD8+ T cell-mediated killing of infected cells. The efficiency of elimination correlated with expression levels of HIV-1 Gag.

Le Got tu a s t ne w w @ O res eet pap xP ea yo er he rch ur O ou no ! x t? fo ty rd pe

Using a non-signalling mutant form of CD48 to initiate formation of close contacts, spontaneous CD45-Lck segregation was found to induce strong TCR triggering even in the absence of a ligand. This aligns with previous reports that suggest genetic or pharmacological alteration of Lck and CD45 activity ratios induce ligand-independent T cell signalling. This indicates that the TCR is not strictly dependent on ligand-induced oligomerisation or conformational changes for activation; however, in vivo, ligands may increase the probability of signalling, purely by promoting formation of close contacts that favour Lck interaction.

Yang H et al. (2016) Mol Ther 24(11):1913-1925.

Elimination of latently HIV-infected cells from antiretroviral therapy-suppressed subjects by engineered immune-mobilizing T cell receptors

Overall, the study shows that immunemobilising HIV-specific TCR fusion proteins promote the killing of HIV-infected CD4+ T cells by CD8+ cells in vitro upon reactivation of the latent virus.

Trinity 2017 | PHENOTYPE | 5

Immune cells and proteins are everywhere in the body, but most circulate through the blood & lymph before entering tissue

All immune cells derive from hematopoietic stem cells in the bone marrow

Cytokines are secreted by immune cells as the immune system’s messaging system Antibodies can directly inhibit viral function, or signal to the innate or adaptive system to kill pathogens

Complement proteins attract neutrophils to infected sites, but some can also kill bacteria by puncturing their cell walls

IMMUNE 1st line of defence: Physical barriers Mucous membranes

Microorganisms try to enter and replicate in the body

Stomach acid

Skin Endothelial tissue

2nd line of defence: Innate immunity

Granulocytes:

“eat” mostly bacteria and fungi, form pus, numerous in blood, migrate to infected sites within minutes,

Newborns have a fully functional innate immune system and antibodies from their mother but only start making their own antibodies weeks after birth

eosinophils

neutrophils

basophils

Monocytes reside in the spleen and blood and in the tissue develop into macrophages and dendritic cells Natural killer cells

kill infected cells

Susumu Tonegawa won the Nobel prize in 1987 for deciphering the genetic principles of antibody diversity

16

8

Luc Montagnier & Françoise Barré-Sinoussi won the Nobel prize in 2008 for discovering and isolating the retrovirus HIV

3rd line of defence: Adaptive immunity Adaptive immune cells are “trained” in the bone marrow (B cells) or thymus (T cells):

B cells produce antibodies

# Immune system Nobel prizes = 15% of all Nobel prizes in Physiology or Medicine!

4

de fe n di se al se m l as ec ha e m n ec ism ha s ni s im ms m un ity

0

created by Anne C. Wolfes 6 | Oxford University Biochemical Society

1786, England: Edward Jenner introduces smallpox vaccination

kill fungi and some bacteria, interact with T cells

T cells kill virus-infected cells, attack foreign tissue (transplants)

1949, Australia: Macfarlane 1894, Belgium: Burnet & Frank Jules Bordet Fenner develop discovers self vs. non-self complement and theory antibody activity for fighting 1891, Germany: bacteria 1948, Sweden: Paul Ehrlich proposAstrid Fagraeus 1944, England: finds that plasma es that antibodies Peter Medawar cause immunity B cells produce proposes hypothesis antibodies of allograft rejection

1884, France: Ilya Mechnikov discovers phagocytosis and establishes cellular theory of immunity

1878, France: Louis Pasteur confirms germ theory of disease, develops chicken cholera vaccine

SYSTEM

590,000 papers on PubMed since 1823:

allergy

SPLEEN: filters blood, hosts T and B lymphocytes and monocytes BLOOD: circulates immune cells and proteins

Vaccines contain or mimic parts of disease-causing agents, which the adaptive immune system recognises and uses to build up future immunity

2010 1983, France: Luc Montagnier & Françoise Barré-Sinoussi discover HIV

1958, USA: Henry Kunkel finds first auto-antibody; autoimmune diseases are recognised

27%

autoimmune

immunity

26%

vaccine

Self vs. non-self recognition

LYMPH: circulates mostly immune system components

1968, Australia: Jacques Miller (photo) & Graham Mitchell distinguish between B and T cells, find that they interact

21%

TONSILS & other LYMPH NODES: host T and B lymphocytes

BONE MARROW: origin of all immune cells (from hematopoietic stem cells)

Lymph nodes and the spleen facilitate communication between immune cells

immune system

11%

BRAIN: has its own specialised immune cells, microglia THYROID: centre for T lymphocyte training LIVER: contains phagocytes, produces complement system proteins

15%

All cells in the body have cell-surface proteins that are recognised as ‘self’ (healthy). Microbes, transplants, or other material entering the body (e.g. pollen) are recognised as ‘non-self’ (foreign).

During pregnancy, the body’s immune system is suppressed to avoid the foetus (non-self) being rejected - making pregnant women more susceptible to infections such as colds.

Autoimmune diseases & allergies When the immune system can’t distinguish ‘self’ from ‘non-self’, autoimmune diseases may arise.

Autoimmune diseases can have genetic or environmental causes, and can occur due to dysfunction in any part of the immune system.

Allergies occur when the immune system becomes hypersensitive to non-disease-causing agents, e.g. pollen or certain foods. In extreme cases, this triggers an anaphylactic shock, which can be life-threatening.

2016

2005, Australia: Ian Frazer & Jian Zhou develop human papilloma virus vaccine

1994, USA: Polly Matzinger develops danger hypothesis of immune responsiveness

FDA approves three classes of immune check-point inhibitors to treat different forms of cancer, using the body’s immune system

At least one person suffers from an allergy or intolerance in 1/4 of UK households In 2014, more than 150 million people in Europe suffered from allergies

1 in 100,000 babies born has severe combined immunodeficiency (SCID), having little or no immune system

Trinity 2017 | PHENOTYPE | 7

Mammalian macrophages: origin and development by Julian Buchrieser

M

acrophages are highly plastic, multifunctional immune cells that play an essential role in mammalian tissues. They can broadly be classified into tissue-resident macrophages, which reside in the tissue, and infiltrating macrophages, which are recruited from the blood as monocytes and differentiate into transient effector macrophages in the tissue.

Tissue-resident macrophages play mainly housekeeping and tissue-specific roles, whereas infiltrating macrophages are recruited to play a specific role often linked to inflammation, infection and pathology. Historically, both tissue-resident and infiltrating macrophages were thought to derive from monocytes and differentiate into non-proliferating terminally differentiated macrophages, however recent research has drastically changed this view.

This article focuses on the new concepts of macrophage development.

engulf a solid particle into an intracellular compartment) and are present throughout all tissues, in vertebrates and most invertebrates (1). In addition to their phagocytic function, macrophages also have broader roles in metabolism, development, and tissue homeostasis and repair (2), and can differentiate into various tissuespecific subtypes (Figure 1A). These subtypes perform a wide variety of tissue-specific functions, including neuronal patterning, adipose tissue generation and bone morphogenesis (2).

Macrophages are core cells of the innate immune system and one of the first lines of defence against invading pathogens. They are best known for being professional phagocytes (phagocytosis is the capacity of a cell to

In line with their multifunctional nature, the dysregulation of macrophages is involved in many pathological conditions such as atherosclerosis, type 2 diabetes, fibrosis, osteoporosis, obesity and cancer

Figure 1. Schematic representation of tissue-resident macrophage development. (A) Non-exhaustive list of tissue-specific resident macrophages and how they are maintained in the adult mammal. (B) The three hematopoietic waves, their anatomical locations and the macrophage populations they generate. While most results are derived from mouse studies, there is an increasing amount of data from human studies and human embryonic/induced pluripotent stem cell studies suggesting a very similar picture. M: Mouse, H: Human. E: Day post coitum.

8 | Oxford University Biochemical Society

(3). These immune system cells can derive either from circulating blood monocytes, which differentiate into infiltrating macrophages following tissue invasion, or from embryonic precursors derived during the first weeks of embryonic development. Over the last five to six years, there have been many reports showing that the majority of tissue-resident macrophages are developed and maintained in the tissue itself, independent of circulating monocytes (4). The ‘non-monocyte’ origin of most tissue-resident macrophages was a major paradigm shift, as the previously accepted theory was that in the adult organism all tissue-resident macrophages are maintained by a constant supply of blood monocytes, even under homeostatic conditions (5). In line with this discovery, it is now clear that macrophages can proliferate and selfmaintain within the tissues. This raises many questions regarding the possibility of macrophage ‘stem cells’ and the ‘stemness’ of macrophages.

While concepts of macrophage development are constantly evolving, the main currently accepted theory is that the three hematopoietic waves generated during mammalian development contribute differently to the adult tissue-resident macrophage pool (Figure 1B). The three waves of hematopoiesis are classically referred to as the ‘primitive’, ‘transient-definitive’ and ‘definitive’(6).

The first two waves are derived before development of the major adult hematopoietic organs such as the bones marrow, thymus or spleen. These waves have a strong bias towards erythrocytes, platelets and macrophages, with very little lymphopoiesis.

Early erythrocytes play a key role in oxygenation of the growing embryo, platelets in preventing haemorrhage and macrophages in ‘housekeeping’ and developmental functions (angiogenesis, growth factor production, apoptotic cell clearance, etc.). Until recently, the primitive and transient-definitive waves of hematopoiesis were both considered to be critical for the correct development and oxygenation of the developing embryo, but not thereafter as the definitive wave was thought to entirely take over after birth.

This view has now changed (4), as all three waves have been shown to contribute to the adult tissue-resident macrophage pool, with different waves giving rise to distinct populations of adult tissue-resident macrophages (Figure 1B). For simplicity, macrophages arising from the primitive and transient-definitive waves of hematopoiesis will be referred to as ‘embryonic’ and those arising from the definitive wave will be termed ‘adult’.

The macrophages present in different tissues are maintained through different mechanisms. Microglia, the macrophages of the brain, potentially due to their seclusion behind the blood brain barrier, derive almost exclusively from embryonic macrophages. At birth, microglia expand rapidly in the brain by in situ proliferation, after which they are self-maintained throughout life by proliferation in response to injury or neurodegenerative conditions. Langerhans cells, the macrophages of the epidermis, which are derived from embryonic macrophages, are subsequently maintained in adulthood through low-level proliferation, independent of monocytes. After tissue irradiation, Langerhans cells replenish the tissues by proliferation.

Macrophages in the lung, liver and heart all derive from embryonic macrophages but under steady state conditions they are gradually replaced by adult monocyte-derived macrophages with age. Macrophages in the intestine and dermis are rapidly replaced by recruitment of adult monocytes to maintain the macrophage population. Thus, for the gut and dermis, tissue-resident macrophages are quickly replaced by monocyte-derived macrophages (7).

The recent advances in macrophage development, combined with the mounting data showing that tissue-resident and infiltrating macrophages can have a differential impact on pathological conditions such as cancer and infectious diseases, make the study of macrophage origin of critical importance. Understanding how and if their developmental origin impacts their function will greatly help the development of suitable in vitro tissue-resident macrophage models for disease modelling, drug discovery and regenerative medicine.

References 1. Okabe Y & Medzhitov R (2015) Tissue biology perspective on macrophages. Nat Immunol 17(1):9–17.

2. Gordon S & Martinez-Pomares L (2017) Physiological roles of macrophages. Eur J Physiol 469(3):365–374.

3. Murray PJ & Wynn TA (2011) Protective and pathogenic functions of macrophage subsets. Nat Rev Immunol 11(11):723–737.

4. McGrath KE, et al. (2015) Early hematopoiesis and macrophage development. Semin Immunol 27(6):379–387.

5. Gordon S (2007) The macrophage: Past, present and future. Eur J Immunol 37(SUPPL. 1):9–17.

6. Palis J (2016) Hematopoietic stem cell-independent hematopoiesis: emergence of erythroid, megakaryocyte, and myeloid potential in the mammalian embryo. FEBS Lett 590(22):3965–3974.

7. Belhareth R (2015) Macrophage populations and self-renewal: Changing the paradigm. World J Immunol 5(3):131.

Julian Buchrieser is a postdoctoral researcher in William James’ research group at the William Dunn School of Pathology.

Trinity 2017 | PHENOTYPE | 9

Infectious diseases and immunity: the importance of being ironic by Hal Drakesmith

I

ron supply is critical for the virulence of many pathogens, and indeed enormous effort is made by some microbes to scavenge iron from their host. For example, a significant chunk of the genome of plague-causing Yersinia pestis encodes for a variety of mechanisms that specialise in iron piracy (1), and Neisseria and Haemophilus species encode proteins that capture the mammalian iron-binding protein transferrin as a source of iron.

The importance of this interaction for virulence has been sufficient to drive the evolution of both the host and pathogen proteins, in a cat-and-mouse game of iterative evasion and re-adaptation (2).

What does all this have to do with immunology? It turns out that iron is an important element in both innate and adaptive immune responses to infections.

When the innate immune system detects an infection, pro-inflammatory mediators and acute-phase proteins are mobilised. Amongst these proteins are several that regulate iron, for example the iron storing protein ferritin, as well as hemopexin and haptoglobin, which bind haem and haemoglobin respectively. Haem and haemoglobin can be discharged into blood plasma via the cell death and tissue damage that results from infection. Once in the bloodstream, they can cause further damage by generating reactive oxygen species, or be captured by infectious pathogens to fuel their growth. Therefore, hemopexin and haptoglobin provide important protective mechanisms against infection; indeed the upregulation of hemopexin was recently shown to be required for the control of systemic Citrobacter invasion (3).

Our laboratory’s research focuses on another component of the iron regulatory mechanism that turns out to have a crucial role in multiple infections: the iron hormone

Duodenal absorption (~1mg Fe /day)

Plasma: iron‐loaded transferrin

Muscle, brain, etc

Hepcidin acts by blocking the export of iron from cells into the bloodstream. Duodenal enterocytes and some types of macrophages express high levels of ferroportin, the only known iron exporter protein. On enterocytes, ferroportin releases iron obtained from food, and on macrophages, ferroportin releases iron derived from the breakdown of senescent red blood cells. Hepcidin binds ferroportin and causes its degradation, thereby inhibiting the uptake of iron from the diet and recycling of iron from haemoglobin (Figure 1).

Inflammation enhances transcription of hepcidin and suppresses transcription of ferroportin, which together leads to iron export being greatly attenuated (5). This has profound effects on serum iron levels. For example, by monitoring blood samples from individuals in a controlled

HEPCIDIN Iron control hormone + acute phase peptide: Inhibits ferroportin

Iron in diet FERROPORTIN Iron exporter on enterocytes, macrophages

hepcidin (4). As a rough analogy, hepcidin is to iron what insulin is to glucose; that is, increases in serum iron lead to release of hepcidin into the circulation, and the action of hepcidin is to decrease iron levels, returning the system to equilibrium. Hepcidin is also regulated by innate immunity. Indeed, iron appears to be the only nutrient that is controlled by a hormone that responds both to nutrient levels and to infection. Furthermore, hepcidin is evolutionarily related to microbicidal defensins that target bacteria and yeast infections.

Macrophage uptake of senescent RBCs

Iron recycling by macrophages (~25mg Fe /day)

Erythropoeisis in bone marrow

10 | Oxford University Biochemical Society

Red blood cells

Figure 1. Hepcidin and the iron cycle. Iron in food is absorbed through duodenal enterocytes (~1 mg Fe/day) and loaded onto a dedicated iron binding protein, transferrin. Soluble transferrin delivers iron to cells and tissues, with the majority going to the bone marrow where the iron is incorporated into haem during red blood cell synthesis. Aged red blood cells are phagocytosed by macrophages (typically in the splenic red pulp or liver), which then recycle around 25 mg Fe per day back into circulation. The release of iron into serum from enterocytes and macrophages is via the iron exporter protein, ferroportin (dark red circles). Hepcidin (green) is the iron regulatory hormone, synthesized and secreted by the liver, which binds ferroportin and inhibits iron export. Hepcidin levels increase in response to iron, to maintain homeostasis, but also increase in response to inflammation. Hepcidin is both a hormone and an acute phase response peptide.

A

B

C

D

human Typhoid infection, we observed a strong hepcidin increase concomitant with inflammation, and a drop in serum iron to almost undetectable levels (Figure 2) (6).

This ‘hypoferraemia of inflammation’ provides protection against growth of blood-borne pathogens that obtain their iron from serum and could otherwise cause fatal sepsis. In this way, hepcidin, while not directly antimicrobial like its defensin cousins, does possess an innate immune function that is critical to control some pathogens, and as such hepcidin may have efficacy as a treatment for some infections (7).

A particularly important infection in which hepcidin plays a key role is malaria. A mosquito bite transfers a Plasmodium sporozoite into the bloodstream, which rapidly homes to the liver and subsequently settles in a hepatocyte. The sporozoite then rapidly proliferates, generating tens of thousands of daughter merozoites within days. These exit the depleted hepatocyte in a form specialised to invade red blood cells, and the rounds of replication within and destruction of red blood cells cause clinical malaria. The inflammation that accompanies blood-stage malaria increases the production of hepcidin, restricting absorption of dietary iron and locking it into macrophages. As a result, the amount of iron bound to transferrin in the bloodstream decreases. Because this transferrin-bound iron is mainly incorporated into haemoglobin as red blood cells develop, the increase in hepcidin, and decreased iron availability, may contribute to malarial anaemia. However, there is an unexpected second effect of increased hepcidin levels. By locking iron into enterocytes and macrophages, hepcidin decreases the availability of iron to other cells, including hepatocytes.

The rapid proliferation of Plasmodium merozoites within hepatocytes carries with it a strong nutritional requirement, including a need for iron. Accordingly, high hepcidin levels decrease growth of the liver-stage infection. Mice harbouring an ongoing blood-stage

Figure 2. The hypoferraemia of inflammation. (A) Temperature, (B) serum hepcidin concentration, (C) serum iron, and (D) transferrin saturation were measured in individuals who received typhoid diagnosis following oral challenge with Salmonella Typhi. Values are plotted relative to the day of typhoid diagnosis, TD = day 0; since not all individuals were diagnosed on the same day post-challenge, baseline samples from the day of challenge (Chall) are considered together, as are data from the final day 14 visit (Final). Data show that as individuals become inflamed (increased temperature), serum hepcidin levels increase and serum iron and the percentage of occupied iron-binding sites on transferrin (transferrin saturation) decrease (6).

infection, accompanied by high hepcidin, resist a superinfection initiated at the liver-stage; sporozoites infect hepatocytes, but fail to thrive and do not progress into the blood-stage. Therefore, through a hepcidin-mediated iron effect, an ongoing blood stage infection protects its ‘niche’ from the threat of competitors (8). This may have beneficial effects for the host by restricting potentially harmful multiple Plasmodium infections.

There is one final but important twist to the iron/malaria story. Insufficient iron is the world’s most common micronutrient deficiency, and is responsible for about half of all anaemia, which in turn affects a quarter of the world’s population, especially young children and pregnant women (9). For decades, a mainstay of aid programs designed to combat anaemia has been the provision of dietary iron supplements. However studies have shown that iron deficiency is actually protective against malaria and that iron supplements can exacerbate malaria, as well as other infections (9). Resolution of this global health dilemma will be challenging—how can anaemia on such a large scale be alleviated without undesirable effects on infection?

While work on how innate immunity regulates iron to fight infection is ongoing, an unforeseen critical role of iron for adaptive immunity has recently become apparent. Lymphocytes, once activated in lymph nodes, expand remarkably, with T cells able to divide every six to eight hours. A frequently used marker of lymphocyte activation is the transferrin receptor (CD71), which is the means by which cells obtain iron from serum.

The majority of transferrin receptors in the body are expressed in the bone marrow by developing erythroblasts, to allow acquisition of iron for incorporation into haemoglobin—the dominant steady-state use of iron in the body. Surprisingly however, a mutation in

Trinity 2017 | PHENOTYPE | 11

A: Iron replete

Tf Fe

Tf Fe

Tf Fe

Adaptive immune response

B: Dysfunctional transferrin receptor

Tf Fe

Tf Fe

Tf Fe

Compromised adaptive immunity

C: Iron deficiency

Tf

Tf

Tf

?

Figure 3. Iron and adaptive immunity.

(A) After priming, activated T cells upregulate expression of the transferrin receptor CD71 through which they acquire iron from serum, and then proliferate and differentiate to mount an adaptive immune response. (B) A mutation in the transferrin receptor gene that impairs the acquisition of iron causes a defect in proliferative capacity in T cells (and B cells) and leads to severe immunodeficiency. (C) Iron deficiency, which decreases serum iron availability, is extremely common especially in developing world contexts, but its effects on adaptive immunity are not well understood.

Immune response?

transferrin receptor that impairs its internalisation, which compromises cellular iron acquisition, was found to cause severe immunodeficiency and susceptibility to infection in children, rather than anaemia (10). Defects in both T and B cells were observed; both defects could be rescued by supplying iron in a non-transferrin bound form. This finding demonstrates that the demand for iron during an adaptive immune response by a rapidly expanding population of T cells is considerable and vital (Figure 3A,B). Furthermore it suggests that adaptive immune responses may be vulnerable to inefficient iron acquisition in comparison to erythropoiesis (at least at steady-state). This seminal finding raises many questions, for instance, in what way is iron important for lymphocytes? Is iron acquisition linked to the metabolic reprogramming events that occur during T cell differentiation? Are particular lymphocyte subsets more affected than others by defects in iron acquisition?

The high prevalence of iron deficiency, particularly in children in the developing world, is also relevant for this issue. Babies are born with an endowment of iron from their mothers, but the relative lack of iron in breast milk, and the utilisation of iron during growth, means that by six months of age serum iron levels are frequently very low. Especially in developing world scenarios, iron deficiency in mothers, and inflammatory hypoferraemia due to a high infectious burden, may further restrict serum iron levels in infants. Under such circumstances, are adaptive immune responses compromised (Figure 3c)? Importantly, are responses to vaccines impaired?

In summary, iron is a critical nutrient that plays a key role in host-pathogen interactions. It is a resource required by almost all infectious organisms for virulence, Hal Drakesmith is an Associate Professor of Immunology at the MRC Human Immunology Unit,Weatherall Institute of Molecular Medicine.

12 | Oxford University Biochemical Society

and is defended, albeit not entirely successfully, by the host innate immune response. Genetic variants that alter either host or pathogen iron metabolism influence outcome of infection, and manipulating host iron trafficking may have some therapeutic promise.

Understanding the way in which iron is important for adaptive immunity is only just beginning, but is likely to be illuminating.

References 1. Parkhill J, et al. (2001) Genome sequence of Yersinia pestis, the causative agent of plague. Nature 413(6855):523–527.

2. Barber MF & Elde NC (2014) Nutritional immunity. Escape from bacterial iron piracy through rapid evolution of transferrin.

Science 346(6215):1362–1366.

3. Sakamoto K, et al. (2017) IL-22 controls iron-dependent nutritional immunity against systemic bacterial infections. Science Immunology 2(8):eaai8371.

4. Drakesmith H & Prentice AM (2012) Hepcidin and the ironinfection axis. Science 338(6108):768–772.

5. Drakesmith H, Nemeth E, Ganz T. 2015. Ironing out Ferroportin.

Cell Metab 22(5):777–787.

6. Darton TC, et al. (2015) Rapidly Escalating Hepcidin and Associated Serum Iron Starvation Are Features of the Acute Response to Typhoid Infection in Humans. PLoS Negl Trop Dis 9(9):e0004029.

7. Arezes J, et al. (2015) Hepcidin-induced hypoferremia is a critical host defense mechanism against the siderophilic bacterium Vibrio vulnificus. Cell Host Microbe 17(1):47–57.

8. Portugal S, et al. (2011) Host-mediated regulation of superinfection in malaria. Nat Med 17(6):732–737.

9. Pasricha SR, et al. (2013) Control of iron deficiency anemia in low-and middle-income countries. Blood 121(14):2607–2617.

10. Jabara HH, et al. (2016) A missense mutation in TFRC, encoding transferrin receptor 1, causes combined immunodeficiency. Nat Genet 48(1):74–78.

A cancer that kills the immune system L

by Olivia Murray

eukaemia is a group of cancers that affect the blood cells; it starts in the bone marrow and results in a high number of abnormal white blood cells (WBCs), known as blast cells. There are many different types of leukaemia, including those that affect the myeloid or lymphoid lineages which can develop quickly (acute) or progress slowly (chronic) (2). Despite their high concentration, the leukaemic WBCs are not able to fight infection, and impair the ability of the bone marrow to produce red blood cells (RBCs) and platelets, leading to the blood being overloaded with non-functional WBCs.

Neutrophil

Natural killer cell

Figure 1. The maturation paths of the myeloid and lymphoid lineages. Figure by Oleg Sitsel.

Basophil

Plasma cell

Eosinophil

B lymphocyte

Monocyte/ macrophage

Myeloblast

Blood stem cell

Lymphoblast

Platelets T lymphocyte Red blood cells

The role of white blood cells in the immune system A population of stem cells that gives rise to blood cells are made in the bone marrow; they constantly divide and multiply. Some new cells remain as stem cells and others develop into mature blood cells before being released into the bloodstream. The parent stem cell is called a pluripotent stem cell, which means it has the ability to develop into either a lymphoid or myeloid stem cell and give rise to the different lineages (Figure 1) (1).All of the WBCs in our immune system are crucial for our survival.

Normal working granulocytes have prominent cytoplasmic granules containing reactive substances that kill microorganisms (1). The most abundant of the granulocytes is the neutrophil, which is specialized in the capture, engulfment and killing of microorganisms (known as phagocytosis).

Macrophages also phagocytose, and eosinophils defend against parasites. Within the lymphoid lineage, T cells can be either cytotoxic T cells that kill infected cells, or helper T cells that secrete cytokines which help other cells of the immune system, like antibody producing B cells, to become activated.

What happens when things go wrong in one of these cells?

Well, to start with, things are constantly going wrong in many of our cells. Mutations often occur during DNA replication, but luckily the vast majority of these mutations either have very little, if any effect on the cell’s functionality.

Many mutations are detected by DNA repair enzymes and either the DNA is repaired, or the abnormal cell is destroyed. So why can we develop cancer when we have such good mechanisms in place to protect us from it? Well, sometimes these mutations aren’t detected and repaired, and sometimes the mutations affect the DNA repair enzymes directly. Although our bodies are very efficient at detecting abnormal cells, often cancer cells will express

and display the same oligopeptides on their surface as normal healthy cells, resulting in our immune system being unable to distinguish the abnormal cells from our healthy cells, and so they evade destruction. When a cell accumulates multiple mutations in genes concerned with the control of cell division and survival, it can cause it to replicate uncontrollably, ultimately becoming cancerous.

The link between leukaemia and the immune system As WBCs make up the basis of our immune system and protect us against infection, it is no surprise that when these cells cease to function properly, it has a major impact on our health and immune defences. Leukaemia cells are immature and aren’t able to develop into normal functioning blood cells. They cannot fight infections and disease efficiently which means any infection contracted is likely to last longer and be more severe. Due to the lack of platelets and RBCs, patients also feel tired from anaemia, have shortness of breath, bruise easily, and struggle to stop any bleeding that occurs (2). In patients who cannot be cured of leukaemia, their death is not directly caused by the presence of leukaemia cells, but is due to the establishment of infections that their body cannot then fight off - a product of immune system failure.

References 1. Parham P (2009) The Immune System 3(8-18):489–494.

Okabe Y & Medzhitov R (2015) Tissue biology perspective on macrophages. Nat Immunol 17(1):9–17.

2. American Society of Hematology: Leukemia. http://www. hematology.org/Patients/Cancers/Leukemia.aspx [Accessed 16th February 2017]

Olivia Murray is a Part II student in Professor Chris Schofield’s research group at the Department of Organic Chemistry.

Trinity 2017 | PHENOTYPE | 13

CRAC channels: bridging intracellular calcium micro-domains during regulated gene expression by Pradeep Kumar

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alcium signalling is one of the most intensely researched areas in the biological sciences because of the involvement of calcium in a wide variety of cellular functions. These range from cell contraction, migration, growth, and differentiation, to cell death and many more. The main backbone of calcium signalling is the strength and pattern of the calcium signal generated in the cytoplasm after a stimulus reaches a cell. This signal can be an electric pulse in a neuron or a chemical messenger reaching a muscle, eventually culminating in neurotransmitter release or muscle contraction respectively.

The increase in calcium concentration is attributed to the opening of many types of calcium channel present on different cellular membranes. The most important are those present on plasma membranes, which lead to calcium flow from the extracellular fluid into the cytoplasm. In excitable cells such as neurons and muscle cells, multiple voltagedependent and ligand-dependent calcium channels have been characterised. In non-excitable cells, one of the most important channels is the CRAC (calcium releaseactivated calcium) channel, which will be the focus of this article.

“Early studies highlighted CRAC channels as an integral part of the immune response.”

The idea of store-operated calcium channels, now known as CRAC channels, was first proposed by James W. Putney in 1986 in a review to explain the phenomenon of receptormediated calcium signalling (1). This involves activation of calcium channels on the plasma membrane upon release of endoplasmic reticulum (ER) calcium stores. These channels essentially consist of two parts: a sensor to detect ER calcium and a pore-forming domain in the plasma membrane. It was only in 2006 that siRNA screens in Drosophila and HeLa cells led to the identification of proteins constituting CRAC channels (2). ORAI1 was shown to be a plasma membrane pore-forming unit and STIM1 an ER calcium sensor. Calcium was known to be essential in immune system function as gene linkage analysis in a severe combined immunodeficiency disease (SCID) patient revealed a mutation in the ORAI1 protein. CRAC channel-forming ORAI proteins have three paralogs in humans (ORAI1-3) and STIM has two: STIM1 and STIM2 (2). These early studies highlighted CRAC channels as an integral part of the immune response but the molecular mechanisms involving CRAC channels were still unknown. Studies from Anant Parekh’s group at the University of Oxford showed that calcium signals local to these calcium channels were necessary and sufficient to induce the activation of transcription factors, such as NFAT and c-FOS, in order to mount a proper immune response (3). Lack of external calcium and inhibition of CRAC channel function leads to exhaustion 14 | Oxford University Biochemical Society

and reduced responsiveness of immune cells. Sustained or unnecessary activation of CRAC channels can lead to allergies. More recently, the Parekh group and others have shown that these channels work in conjunction with active mitochondria to carry out these functions.

It is now clear that the functioning of CRAC channels requires interaction of at least three organelles: ER, mitochondria and plasma membrane.

How local and global increases in calcium signals activate transcription factors is an interesting problem which is fundamental to the normal function of cells.

Binding of an extracellular signalling molecule to its membrane receptor produces the calcium mobility second messenger inositol 3-phosphate (IP3), which releases calcium from the ER. In turn this activates CRAC channels (3). Calcium entry through CRAC channels leads to activation of enzymes which constantly produces IP3, allowing a calcium signal to be sustained for tens of minutes. Highly localised calcium microdomains also form around active CRAC channels, activating transcription factors. Calcium entry through CRAC channels activates the calcium-dependent phosphatase calcineurin. Calcineurin dephosphorylates its target transcription factor NFAT to expose the nuclear localisation signal, otherwise hidden under phosphate groups. This leads to nuclear translocation of NFAT (Figure 1). Recent work from the Parekh group has revealed new mechanisms whereby calcium can selectively recruit different NFAT isoforms.

In mast cells, activation of leukotriene receptor type 1 using leukotriene C4 (LTC4) evokes physiological calcium signalling. This signalling pathway is part of the inflammatory response associated with allergies and asthma. LTC4 signalling recruits STIM1 and ORAI1 which are necessary for downstream gene activation. It was demonstrated through carefully designed experiments that ORAI1 is ‘special’ in terms of recruiting signalling molecules and creating a signalling platform involving calmodulin, calcineurin and the scaffolding protein AKAP within a few nanometers of the channel pore, providing an efficient and specific response to the local calcium (4). Another CRAC channel protein, ORAI3, has a weaker ability to assemble signalling molecules and therefore does not activate the same downstream pathway. Activation of LTC4 signalling causes nuclear

Figure 1. Activation of receptors generates IP3 molecules and initiates calcium signalling from multiple domains inside a cell. CRAC channels primarily allow calcium entry, which produces local calcium micro-domains around the plasma membrane (depicted through dotted lines). IP3 receptors on ER (not shown) provide calcium pool from ER and IP3R type1 receptor in the inner nuclear membrane provide nuclear calcium pool.

Concerted activity of these multiple calcium micro-domains regulate activation of NFAT isoform for regulated gene expression (6).

translocation of different isoforms of NFAT, NFAT1 and NFAT4. Further work in mast cells using fluorescently tagged NFAT isoforms was performed to understand the kinetics of their translocation into and out of the nucleus. NFAT1 and NFAT4 migrate to the nucleus with the same time constant but their exit rates, once the agonist is removed or CRAC channels are inhibited, are very different (5). NFAT4 exits with a time constant which is ten times faster than NFAT1. Considering that both of these transcription factors respond to the local calcium concentration, it was surprising to observe such a divergence in nuclear exit rates.

It was later shown that the local calcium inside the nucleus is necessary for slowing NFAT4 exit rates. These two transcription factors respond to the same calcium signal for activation, but only NFAT4 requires another source of calcium (nuclear calcium) for improved residence time in the nucleus. What is the source of the nuclear calcium rise? Electron microscopy, immunofluorescence and nuclear fraction blotting showed the presence of type1 IP3 receptor in the inner nuclear membrane (6).

Buffering nuclear IP3 by expressing nuclear specific IP3 binding protein prevented NFAT4 migration while NFAT1 remained unaffected (6). These observations provide a rationale for the response of different genes even when the stimulus is the same.

The primary function of CRAC channels is to facilitate calcium entry into cells. Calcium signalling can occur in different locations inside the cell at the same time and this helps to achieve a highly cell specific function. A cell, albeit very small, is like a big city and things that happen in localised pockets can have huge effects on the function of the entire cell. Unravelling these localised pockets and their interactions is one of the biggest challenges in the calcium field and beyond.

References 1. Putney JW (1986) A model for receptor-regulated calcium entry. Cell Calcium 7(1):1–12.

2. Hogan Patrick G, et al. (2010) Molecular basis of calcium signaling in lymphocytes: STIM and ORAI. Annu Rev Immunol 28:491–533.

3. Di Capite J & Parekh AB (2009) CRAC channels and Ca2+ signaling in mast cells. Immunol Rev 231(1):45–58.

4. Kar P, et al. (2011) Selective activation of the transcription factor NFAT1 by calcium microdomains near Ca2+ release-activated Ca2+ (CRAC) channels. J Biol Chem 286(17):14795–14803.

5. Kar P & Parekh AB (2015) Distinct spatial Ca2+ signatures selectively activate different NFAT transcription factor isoforms.

Mol Cell 58(2):232–243.

6. Kar P, et al. (2016) Control of NFAT isoform activation and NFAT-dependent gene expression through two coincident and spatially segregated intracellular Ca2+ signals. Mol Cell 64(4):746– 759.

Pradeep Kumar is an EMBO long-term postdoctoral fellow in Professor Anant Parekh’s research group at the Department of Physiology, Anatomy and Genetics.

Trinity 2017 | PHENOTYPE | 15

Science vs. gluten: emerging therapies for coeliac disease by Heather Booth

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oeliac disease is an autoimmune disorder that occurs in response to ingestion of gluten, a class of proteins found in wheat, barley, and rye, and it is thought to affect around 1% of the world’s population (1). Currently, the only available treatment for coeliac disease is a lifelong, gluten-free diet, which can often be difficult to manage.

Researchers are working on developing new interventions that they hope will improve the quality of life of millions of people with coeliac disease around the world.

Symptoms Coeliac disease has historically been characterised by gastrointestinal symptoms and weight-loss, due to intestinal damage that is inflicted by the immune system in response to gluten. However, it has more recently been recognised that most patients with coeliac disease do not experience these issues. Many present with other symptoms including tiredness, headaches, anaemia, osteoporosis and other related autoimmune conditions (1). There is evidence to suggest that patients who do not adhere to a gluten-free diet have an increased risk of developing certain types of cancer such as lymphoma, and a decreased life expectancy (1).

corresponds to the level of contamination that may occur when gluten-free food is served with utensils that a glutencontaining food has touched. In many areas of the world, gluten-free food is not necessarily readily available, and is often expensive.

Disease mechanism A component of gluten, called gliadin, is considered the main culprit in the initiation of an autoimmune response in people with coeliac disease (Figure 1).

Gliadin is particularly resistant to enzymatic digestion.

In healthy people, when gliadin peptides reach the small intestine, they are bound by IgA secreted by cells in the intestinal wall. This complex then binds to transferrin receptors on enterocytes in the intestinal wall and is taken up into the cells, where it is degraded. In coeliac disease, this degradation is incomplete and intact gliadin peptides are transported across these cells into a region of the intestinal wall known as the lamina propria (2).

Once gliadin peptides reach the lamina propria, tissue transglutaminase (tTG) removes amide groups from the gliadin peptides. This deamidated gliadin is then picked up by dendritic cells, which display the deamidated gliadin molecules at their cell surface, on MHC molecules (2). Coeliac disease is more common in people who have HLA-DQ2 and HLA-DQ8 variants of MHC molecules (1). These variants have been shown to have a high affinity for binding deamidated gliadin peptides, which results in increased levels of antigen presentation to the immune system (2). T cells then recognise the gliadin molecules and initiate an inflammatory cascade, which can result in injury to the cells in the intestinal wall, which in turn promotes atrophy of the intestinal villi, resulting in impaired nutrient absorption.

One approach uses a polymer (BL-7010) that binds to gliadin in the digestive system, inhibiting its digestion and therefore the formation of immunogenic peptides. This polymer has been shown to prevent intestinal damage in gluten-sensitised mice that were fed with gluten (4), and was demonstrated to be safe in a phase I/II clinical trial that ended in 2014. It is currently undergoing further development (5).

The gluten-free diet Treatment of coeliac disease with a lifelong gluten-free diet can be difficult to manage as consumption of food containing more than 20 ppm of gluten can trigger an immune response (3). To put this in perspective, 20 ppm 16 | Oxford University Biochemical Society

Ongoing clinical trials Researchers are currently working to develop various new treatments to complement the gluten-free diet, to make it more manageable by preventing an immune response to the low levels of gluten that are ingested when food is crosscontaminated. A number of new therapies are undergoing clinical trials to establish their effectiveness in patients.

A second approach is the use of enzymes that have been identified in microbial species that can digest gliadin in the acidic environment of the stomach. A combination of two proprietary enzymes (ALV003) has completed phase I and II clinical trials in patients with coeliac disease, where it was shown to prevent intestinal damage in response to ingestion of two grams of gluten per day for six weeks (6). It is expected that ALV003, under the new name of IMGX-003, will enter phase III clinical trials in the near future.

Another option that is being explored is the possibility of training the immune system to tolerate gluten. A vaccine (NexVax2), consisting of three highly immunogenic gliadin peptides that contain HLA-DQ2 epitopes, has been developed and shown to be safe in a phase Ib clinical trial (7). As this vaccine is specific for those who carry the HLA-DQ2 variant, it is only appropriate for around 80-90% of patients. Nevertheless, NexVax2 is the only treatment currently in clinical development that may negate the need for dietary management of the disease.

Promising preclinical strategies The immune cascade that occurs once gliadin has reached the lamina propria is also a potential target for new therapies.

Figure 1. Coeliac disease pathogenesis. Gliadin peptides form a complex with secretory IgA and the transferrin receptor on the surface of enterocytes. This complex is taken up by the cell and gliadin peptides are released into the lamina propria.

The peptides are then deamidated by tissue transglutaminase (tTG) and taken up by dendritic cells.

These cells present the deamidated gliadin peptides to T cells via MHC molecules HLA-DQ2 or HLA-DQ8.

This results in pro-inflammatory cytokine release, and the production of autoantibodies by B cells, both of which cause damage to the intestinal wall.

Ex vivo experiments using small intestinal biopsies from patients with coeliac disease have demonstrated an enhanced T cell response to de-amidated gliadin peptides compared to biopsies from healthy controls (3).

Researchers have shown that inhibition of tTG results in a reduction of this response, indicating a possible new avenue for therapeutic intervention.

Another potential therapeutic target in coeliac disease is the peptide-binding site of HLA-DQ2/8. Antagonistic peptides that bind to HLA-DQ2, but do not elicit an IFN-γ stimulatory effect, have been developed (7).

Furthermore, certain classes of dimeric and cyclic gliadin peptides have been shown to antagonise HLA-DQ2 without inducing T cell proliferation in vitro.

The pocket gluten detector These promising treatments for coeliac disease are all at the preclinical stage or in clinical trials, so for now a gluten-free diet is still the only available treatment. In the meantime, one recent development aiming to help patients is the Nima sensor. This device allows people to test a pea-sized portion of their food to determine whether it is gluten-free. The technology is antigenbased and utilises an antibody against gluten to provide an optical readout within three minutes. The sensor is designed to detect gluten at levels greater or equal to 20 ppm, the level above which a reaction may be triggered in people with coeliac disease.

Concluding remarks The future for treatment of coeliac disease is looking bright. Multiple potential therapies, each targeting different components of the disease mechanism, are under development. The majority will still require adherence to a gluten-free diet; however, the extra safeguards that they will provide against crosscontamination will be very welcome in the gluten-free community.

References 1. Rostom A, et al. (2006) American Gastroenterological Association (AGA) Institute technical review on the diagnosis and management of celiac disease. Gastroenterology 131(6):1981–2002.

2. Escudero-Hernandez C, et al. (2016) Immunogenetic pathogenesis of celiac disease and non-celiac gluten sensitivity.

Curr Gastroenterol Rep 18(7):36.

3. Makharia GK (2014) Current and emerging therapy for celiac disease. Front Med (Laussane) 1:6.

4. McCarville JL, et al. (2014) BL-7010 demonstrates specific binding to gliadin and reduces gluten-associated pathology in a chronic mouse model of gliadin sensitivity. PLoS One 9(11):e109972.

5. BiolineRX (2015) Drugs in development: BL-7010. Available at http://www.biolinerx.com/default.asp?pageid=13&itemid=34 [Accessed 9th March 2017].

6. Lahdeaho ML, et al. (2014) Glutenase ALV003 attenuates gluten-induced mucosal injury in patients with celiac disease.

Gastroenterology 146(7):1649–1658.

7. Rossi M (2015) Vaccination and other antigen-specific immunomodulatory strategies in celiac disease. Dig Dis 33(2):282– 289.

Heather Booth is a DPhil student in Professor Richard WadeMartins’ research group at the Department of Physiology, Anatomy and Genetics, and is editor-in-chief of Phenotype.

Trinity 2017 | PHENOTYPE | 17

The beta-cell’s sweet secret by Maria Rohm

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iabetes now affects over 500 million people worldwide, and numbers are still on the rise. There are three different types of diabetes: type 1, type 2 and neonatal. The most common form is type 2 diabetes, which often correlates with obesity and lifestyle, and develops later in life. However, with a growing number of overweight children, the disease now also increasingly affects younger patients. Type 1 diabetes occurs earlier in life and is independent of obesity. Neonatal diabetes is defined by its occurrence in the first six months of life and is usually caused by genetic defects.

Common to all forms is a dysfunction in the secretion capacity of the insulin-producing beta-cells, located in the pancreatic islets. Since insulin controls cellular glucose uptake and usage, it represents a critical factor for maintaining normal metabolism. Lack of insulin results in hyperglycaemia, a dramatic rise in blood glucose levels.

This has severe long-term effects such as an increased risk for heart disease, stroke, kidney damage, eye damage and even cancer. A recent study has now revealed a novel pathway by which hyperglycaemia may damage beta-cells, thereby worsening the secretory defect and, ultimately, diabetes.

Beta-cells secrete insulin upon stimulation by glucose.

The present study by Brereton et al. (1) uses a mouse model of neonatal diabetes, in which glucose stimulation fails to induce insulin secretion, in order to study how beta-cells react to high ambient glucose levels. In this model, diabetes can be activated in adult mice by inducing the expression of a gene that is commonly mutated in neonatal diabetes, which switches off insulin secretion and results in hyperglycaemia within 24 hours. Analysing beta-cell metabolism, the authors of the study found that the diabetic beta-cells, in addition to no longer secreting insulin, also no longer respond to glucose stimulation by increasing their metabolic rate. Under normal conditions,

glucose stimulates the production of the cellular energy currency, adenosine triphosphate (ATP). This reaction was completely abolished in the diabetic beta-cells. This finding led to the assumption that incoming glucose could no longer be used efficiently. This was confirmed by high-throughput gene expression screening using microarrays, which demonstrated a decrease in the expression of genes involved in ATP production.

If glucose cannot be metabolised efficiently in diabetic beta-cells, despite a large amount entering the cell, where does it go? In an earlier study, the authors had described a large amount of unidentified material in diabetic beta-cells (2). Now, they could demonstrate that this previously unknown substance was in fact glycogen.

This was a surprising finding, since glycogen was not previously believed to play a role or even be produced in beta-cells. Glycogen is the major glucose storage form in the liver and muscle. It is built up during times of sufficient energy supply, i.e. feeding, and rapidly used during times of energy need, such as during fasting or exercise. Patients with defects in glycogen storage develop severe muscle weakness and cramps, liver disease and growth retardation. In these specialised tissues, glycogen represents an inert form of sugar storage that protects cells from the detrimental effects of free glucose, while Figure 1. Glycogen staining of pancreatic tissue. (A) PAS staining of islets of (left to right) control, 1 day diabetic, 2 weeks diabetic, and 4 weeks diabetic mice. (B) Electron microscopy image using special lead-citrate fixation to preserve glycogen, 4 weeks diabetic mice; compared to conventional fixation methods (right). N nucleus, G glycogen, U unstructured area. (C) PAS staining of Ins1 beta-cell line cultured at low (left) or high (right) glucose levels. (D) PAS staining and electron microscopy of islets of a type 2 diabetic organ donor. Arrows and G indicate glycogen. The images in this figure were originally published in Nature Communications (1).

18 | Oxford University Biochemical Society

Figure 2. Proposed mechanism of glycogen-associated cell death in beta-cells at high glucose concentrations. (A) Glucose metabolism and insulin secretion in a normal beta-cell. (B) Hyperglycaemia-induced metabolic dysfunction and glycogen accumulation, leading to impaired insulin secretion and cell death by apoptosis.This figure was originally published in Nature Communications (1).

still serving as a rapid fuel source. In the past 30 years, its role in the beta-cell has largely been overlooked, although early reports have described its appearance in diabetic rodents (3). However, numerous studies have previously reported empty or unstructured cells. The reason for this is surprising: glycogen is lost during most modern tissue preparation techniques and cannot be detected unless samples are specifically examined for it. Brereton et al. used different tissue staining techniques to show the presence of glycogen: special fixation and electron microscopy, and periodic acid-Schiff (PAS) staining, which stains complex carbohydrates. . They showed that not only was glycogen present in the beta-cells of their diabetic mouse model, but also in a rat beta-cell line cultured in high glucose conditions (Figure 1), and in isolated islets cultured in high glucose conditions. Interestingly, when using PAS staining, larger empty areas in the cells appeared where glycogen should be detected, which is again owed to the difficulties in preserving glycogen during staining.

In addition to the rodent model, the authors also found glycogen accumulation in beta-cells of patients with type 2 diabetes and badly controlled blood glucose levels (Figure 1).

Despite this recent study, it is still unclear why or how the beta-cell stores glycogen. The authors showed that the accumulation was reversible when blood glucose levels were reduced to normal, for example by sulphonylurea (a drug used to treat neonatal diabetes) or insulin treatment. This may be an additional reason why glycogen accumulation has not recently been found in patients, since modern medication controls blood glucose levels much more reliably than in the past.

What are the consequences of increased glycogen accumulation in a tissue that is not normally meant to store glycogen? Initial insights came from studies in the brain, where increased glycogen accumulation in neurons has been shown to cause cell death (4). Additionally, kidneys of diabetic rats have been shown to accumulate glycogen, which is again linked to cell death (5). Brereton et al. demonstrated that this idea also holds true for betacells. Increased glycogen accumulation in islets of diabetic

mice was associated with increased caspase 3 levels, a marker of cell death by apoptosis. In addition, beta-cell numbers were dramatically reduced after only 2 weeks of diabetes. Inhibiting glycogen accumulation without altering glucose levels, for example by metformin treatment, inhibited caspase 3 expression and prevented cell death. Therefore, the presence of glycogen accumulation in islets under hyperglycaemic conditions may contribute to beta-cell failure in diabetes (Figure 2).

The study described here has established a novel pathway by which hyperglycaemia alters beta-cell function and survival, and reveals the beta-cell’s formerly overlooked sweet, but deadly secret. The authors show that glycogen accumulation is common in various forms of diabetes across species, and that it is linked with cell death, further aggravating the disease. Preventing beta-cell glycogen storage may thus represent a novel target for future treatment options.

References 1. Brereton MF, et al. (2016) Hyperglycaemia induces metabolic dysfunction and glycogen accumulation in pancreatic β-cells. Nat Commun. 26(7):13496.

2. Brereton MF, et al. (2014) Reversible changes in pancreatic islet structure and function produced by elevated blood glucose. Nat Commun. 22(5):4639.

3. Graf R & Klessen C (1981) Glycogen in pancreatic islets of steroid diabetic rats. Carbohydrate histochemical detection and localization using an immunocytochemical technique.

Histochemistry 73(2):225–232.

4. Duran J, et al. (2014) Deleterious effects of neuronal accumulation of glycogen in flies and mice. EMBO Mol. Med. 4(8):719–729.

5. Bamri-Ezzine S, et al. (2003) Apoptosis of tubular epithelial cells in glycogen nephrosis during diabetes. Lab. Invest. 83(7):1069– 1080.

Maria Rohm is a postdoctoral researcher working in Professor Dame Frances Ashcroft’s research group at the Department of Physiology, Anatomy and Genetics.

Trinity 2017 | PHENOTYPE | 19

Big data and urban agriculture by Luiz Guidi

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he Food and Agriculture Organisation of the United Nations estimates that the world’s food production will need to increase by 70% before 2050 if we are to feed the entire population as it reaches around 9.5 billion and diets become more ‘Westernised’. Many of the efforts to close this ‘food gap’ focus on increasing the plant yield by using genetic modification, but climate change is likely to limit their success. With urbanisation growing, and arable land disappearing from over-cultivation, new agriculture start-ups combining ‘big data’ and the ‘internet of things’ have been sprouting to make farming ‘smarter’ and more sustainable by bringing them into cities.

Figure 1. Lettuce heads grown indoors within cities can reach restaurant tables within hours of being harvested.

Urban agriculture is nothing new. The Aztecs, for example, had floating food gardens around their lakes. However, technology now allows crops to be grown anywhere, from shipping containers, to supermarket rooftops or green skyscrapers. In these ‘vertical farms’, plants are stacked in layers using hydroponics, a technique used to grow plants without soil that uses nutrient-rich water tray solutions and artificial light. In a globalised economy, growing food within cities means a drastic cut in the environmental and economic cost of food transport (1). Instead of travelling long distances across countries, food can be grown ‘hyperlocally’, virtually at the point of sale. For example, Growing Underground, a London-based start-up, has converted old air-raid shelters built during World War II into farms, and now delivers fresh herbs to restaurants within four hours of harvesting. Because they are vertical, these farms also maximise the use of ‘land’ as some can produce as much as 100 times more per area than ‘horizontal’ farms. The promise of urban farms has also caught the attention of Elon Musk’s brother Kimbal, who has initiated an incubator program in Brooklyn called Square Roots, where 10 aspiring entrepreneurs are developing their vertical farms inside shipping containers. The key to urban farms is growing food indoors in highly controlled environments. Using a plethora of sensors embedded in chambers and trays, huge masses of data can be collected for almost every plant. For example, plant scientists from AeroFarms, the biggest of its kind in New Jersey, can monitor growing conditions with their ‘smart farming’ system using a phone app which tracks 24/7 around 30,000 data points in a single harvest. This data-rich approach is combined with manipulation of the plant’s local Luiz Guidi is a DPhil student in Zoltan Molnar’s research group at the Department of Physiology, Anatomy and Genetics.

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conditions with extreme precision to optimise growth and quality by controlling standard variables such as temperature, humidity, lighting and airflow, but also more sophisticated ones, such as dissolved oxygen levels and electrical conductivity (2). Manipulating each element to minute detail affects the expression of genes in plant cells and, ultimately, determines the crop’s texture, colour, size, shape and taste – to produce mouth-wateringly crunchy lettuces for example. This ‘precision agriculture’ eliminates the impact of unpredictable or extreme weather and also reduces the need for chemicals, so the resulting product is ‘post-organic’. Caleb Harper, a principal investigator at MIT, wants to use these innovations in agricultural data and technology to drive the ‘Fourth Agricultural Revolution’ with his Open Agriculture Initiative and create the farmer of the future with Food Computers, an opensource software/hardware platform scalable from office table top to warehouses for new commercial ventures (3).

This system uploads all data on the local environment and plant quality to create a database of ‘climate recipes’.

Caleb’s team is playing with growing conditions and plant stress in search of the tastiest recipe by measuring how different conditions affect the molecules present in plants, using basil as a model organism. Despite the buzz, the numbers do not yet add up, as the operational and environmental costs of these indoor farms outweigh the benefits, primarily due to artificial lighting. The range of crops available is also very limited and restricted mainly to herbs and salads. However, with technology advancing at an exponential rate, in a sort of ‘vertical’ way, these indoor farms may stack up.

A more in-depth look at this topic was originally published on the Science Innovation Union website (http://scienceunion.org/).

References 1. Santo R, et al. (2016) Vacant lots to vibrant plots: a review of the benefits and limitations of urban agriculture. Available at http:// www.jhsph.edu/research/centers-and-institutes/johns-hopkinscenter-for-a-livable-future/research/clf_publications/pub_rep_ desc/vacant-lots-to-vibrant-plots-a-review-of-the-benefits-andlimitations-of-urban-agriculture.html [Accessed 31st March 2017].

2. MIT Sloan School of Management (2016) Front row at a vertical farm. Available at: http://mitsloan.mit.edu/newsroom/articles/frontrow-at-a-vertical-farm-aerofarms/ [Accessed 31st March 2017].

3. MIT Media Labs (2016) Open Agriculture Initiative. Available at: https://www.media.mit.edu/groups/open-agriculture-openag/ [Accessed 31st March 2017].

Engineering plant immunity by Emma Thomas

A

alt Dijkhuizen once said “We cannot feed tomorrow’s world with yesterday’s technology”.

Plant diseases are a huge challenge to global food security. Disease and pests account for a substantial 35% of preharvest crop losses. Today, almost two billion people in the world do not get sufficient nutrients, and this issue will only worsen with the world’s population predicted to double in the next 50 years (1). Food production will need to at least double to keep pace with this growth.

Reducing crop losses to disease is an obvious target to boost food production per land area. Agriculture grows large monocultures of crops that are a desert of genetic diversity. This means crops are more susceptible to new emerging strains of pathogens. To get an idea of the scale of the problem, around 100% of wheat in the world is under threat from rust diseases, with 80% of bananas threated by Panama disease and 20% of citrus crops by citrus greening (2).

Engineering plant immunity could not only save the bread and bananas on your table, it could also make them cheaper. Disease control is a huge cost for food production and a substantial challenge for smaller holdings famers.

In 2014 alone, $14 billion was spent just on chemical control of crop disease (costs from Steve Byrne, Merrill Lynch 2015 Global agriculture inputs primer), and this was not even an effective means of eradication.

A number of strategies have arisen to engineer plant immunity, including engineering genetically modified (GM) crops and transient methods of RNA application.

These strategies offer promising opportunities for future treatments.

Transfer of key genes in immunity A pathogen’s weapons in infection are genes known as effectors, that subvert defences of the host, enabling the pathogen to evade detection and promote disease.

These genes are highly specific to the host-pathogen system and are able to deviously manipulate molecular processes in the plant. However, the plant is not helpless to the pathogen manipulation. In every cell of the plant, resistance (R) proteins of the plant can recognise specific effectors, producing a powerful immune responses against the pathogen (effector-triggered immunity, ETI).

“Disease control is a huge cost for food production.”

Transferring R genes that recognise effectors from resistant wild species to susceptible crops has been a prevalent strategy to engineer immunity. However, this has not proved successful in the long term. Strong selective pressures drive a molecular arms-race between

plants and pathogens. As a result, effectors are rapidly evolving genes which can accumulate mutations, or even be jettisoned from the genome, to subvert recognition over short timescales. Therefore, careless usage of this strategy can be a recipe for disaster. A project in Bangladesh to introduce the GM late blight resistant GM potato harbouring only one additional R gene (RB) is a prime example (3). Whilst in the short term this should help control the devastating losses of potato crop to Phytophthora infestans, within a few years resistantbreaking strains will most likely emerge. Resistance breaking is well documented in both agriculture and

“Often resistance emerges in a matter of a few years in the field.” field trials. Often resistance emerges in a matter of a few years in the field. For example, in one study transgenic potato varieties (Atlantic and Bintje) containing a single additional R gene were already immediately susceptible to some P. infestans strains (4). Bad publicity from the ill-considered use of GM crops, such as Bangladesh RB potato, harms the widespread adoption of GM crops that would benefit farmers.

One strategy to overcome this issue is by using the technique of R gene ‘stacking’ (4), where multiple R genes are transferred together to target a suite of effectors. If the pathogen evolves to evade the initial R gene mediated immunity, more must be overcome before resistance breaks down.

Increasing resistance durability In the plant, the first layer of defence is the ability to detect invaders. Some plant species detect highly conserved small molecules, known as pathogen associated molecular patterns (PAMPs) and triggers an immune response, similar to ETI. PAMPs, such as fig22 peptide from bacterial flagella or the oligosaccharide chitin component of fungal cell walls, are critical for the organism’s survival.

Immunity is therefore not easily overcome by the loss, or modification, of PAMPs. Transferring PAMP receptors from wild species to crops is a promising method to engineer durable immunity to multiple pathogens in one shot.

Trinity 2017 | PHENOTYPE | 21

Indeed, work on the PAMP receptor Elongation Factor Tu (EF-Tu) Receptor (EFR) from Brassicaceae (cabbage family) has yielded promising results. EFTu is essential for mRNA translation elongation in all bacteria, and therefore recognition confers resistance to a broad spectrum of phytopathogens. EFR has been successfully transferred from the model species Arabidopsis thaliana to solanaceous tomato (Solanum lycopersicum), increasing its resistance to a range of bacteria from different genera (5).

This strategy has already been replicated in crops, generating transgenic wheat with enhanced resistance to the bacterial pathogen Pseudomonas syringae pv. oryzae (6). The relative ease of transferring a single gene between distantly related species makes this strategy highly attractive. Currently, apple, cassava, citrus, kiwi, lettuce, corn, potato, rice, and strawberry transgenic crops are also being tested.

Engineering new host recognition proteins However, the strategy of receptor transfer is limited by the existing natural diversity of receptors. In a recent paper, the Innes Lab tackled this by engineering new specificity of the immune response (7). The group focused on R gene rps5 and reprogrammed the perception system to respond to a virus instead of bacteria. Rps5 monitors the state of another plant protein, PBS1, which is cleaved by a bacterial protease effector, AvrPhpB. By changing the amino acid sequence recognised by the protease, PBS1 was no longer cleaved by the bacterial protease but was recognised by a viral tobacco etch virus protease. RPS5 still monitors the cleavage state of PBS1, yet this is now only cleaved by the virus. In this way, only one component of immune response has been altered to engineer resistance to a completely unrelated pathogen.

RNA interference to silence pathogen genes Other innovative ways of engineering plant immunity are also being tested. Instead of producing GM plants, new strategies take advantage of RNA interference.

RNA interference is a gene silencing mechanism that relies on the pathogens’ endogenous system of controlling RNA transcript levels. The double stranded RNA (dsRNA) structure is recognised, cleaved, and used to target complementary RNA transcripts for degradation. Remarkably, application of dsRNA to crops has proved effective at controlling the fungal

Emma Thomas is a DPhil student in Professor Renier van der Hoorn’s research group at the Department of Plant Sciences.

22 | Oxford University Biochemical Society

pathogen Fusarium graminearum (8), a key threat to barley. dsRNA specific to crucial fungal growth genes, for example biosynthetic enzymes for ergosterol, were sprayed onto leaves and showed efficient reduction of fungus at both local and distal sites. As GM plants are, frustratingly, still up against a wall of public opposition, alternative tools are desirable for many countries with strict anti-GM laws.

There is no silver bullet for the engineering of plant immunity. For each crop and pathogen, different strategies will be more effective. Engineering durable resistance is still a significant challenge that will need us to continue developing even more innovative methods to tackle.

References 1. Suweis S, et al. (2015) Resilience and reactivity of global food security. P Natl Acad Sci USA 112(22):6902–6907.

2. 2Blades Foundation: 2Blades Foundation The Unmet Need.

Available at http://2blades.org/the-unmet-need/ [Accessed 28th January 2017].

3. Ahmad R (2017) The Daily Star: Second GM crop ready for release. Available at http://www.thedailystar.net/frontpage/ blight-resistant-potato-save-tk-100cr-year-1341061 [Accessed 27th January 2017].

4. Jo K-R, et al. (2014) Development of late blight resistant potatoes by cisgene stacking. BMC Biotechnol 14(1):50.

5. Lacombe S, et al. (2010) Interfamily transfer of a plant pattern-recognition receptor confers broad-spectrum bacterial resistance. Nat Biotechnol 28(4):365–369.

6. Schoonbeek H, et al. (2015) Arabidopsis EF-Tu receptor enhances bacterial disease resistance in transgenic wheat. New Phytol 206(2):606–613.

7. Kim SH, et al. (2016) Using decoys to expand the recognition specificity of a plant disease resistance protein. Science 351(6274):684–687.

8. Koch A, et al. (2016) An RNAi-Based Control of Fusarium graminearum Infections Through Spraying of Long dsRNAs Involves a Plant Passage and Is Controlled by the Fungal Silencing Machinery. PLOS Pathog 12(10):e1005901.

9. Dodds PN & Rathjen JP (2010) Plant immunity: towards an integrated view of plant–pathogen interactions. Nat Rev Genet 11(8)539–548.

10. Lotz B (2015) Resistance genes from wild relatives of crops offer opportunities for more sustainable agriculture worldwide.

Available at http://www.wur.nl/en/newsarticle/Resistancegenes-from-wild-relatives-of-crops-offer-opportunities-formore-sustainable-agriculture-worldwide-.htm [Accessed 10th April 2017].

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Helper T cell plasticity: adaptation requirement or immunotherapy hindrance? by A Janina Nahler

central question concerning immunologists is how the immune system manages to raise appropriate immune responses to the highly diverse repertoire of pathogens encountered during a lifetime. Tailoring responses to the particular type of challenge lies at the heart of a protective and efficient immune system.

In simple terms, following the detection of invading organisms, innate immune cells, such as neutrophils, dendritic cells and macrophages, act as initial defenders by secreting a variety of destructive antimicrobial molecules, and by directly engulfing and ingesting invaders. Antigenpresenting cells process the pathogen into antigens that they subsequently present to antigen-inexperienced naive CD8+ and CD4+ T cells, thereby initiating a highly specific and tailored adaptive immune response.

Following the identification of two separate CD4+ helper T cell clones in 1986 (1), the Th1-Th2 paradigm was proposed. It states that naive CD4+ T cells can differentiate into these two distinct effector lineages, characterised by the expression of master transcription factors and signature cytokines. This fate decision is believed to be primarily determined by polarising signalling molecules called cytokines. Since then, the helper T cell paradigm has been extended, integrating additional CD4+ T cell lineages including Th17 and regulatory T cells (Tregs).

Helper T cell lineages are known to exhibit distinct functions (Figure 1A): Th1 cells fight bacterial and viral infections, Th2 cells expand in response to helminths that are commonly known as parasitic worms, Th17 cells

protect us from fungal infections and Tregs are essential for immunological self-tolerance and homeostasis (2).

The concept of terminally differentiated CD4+ T cell effector lineages has provided a very useful framework for studying and understanding the induction and regulation of specialised immune responses. However, emerging evidence of helper T cell plasticity and transdifferentiation is now forcing immunologists to think outside the box (Figure 1B).

CD4+ T cell plasticity is here referred to as the ability of individual helper T cells to transition between phenotypes characteristic of specific subsets.

Reprogramming between distinct CD4+ T cell subsets was first observed in in vitro polarisation assays.

Conversion of Th1 cells into Th2-like cells, Tregs taking on both Th1 and Th17 characteristics, and Th17 cells repolarising into Th1-like and Treg-like phenotypes have brought our understanding of lineage stability into question (3). Do stable CD4+ T cell subsets actually exist or do they simply represent distinct activation states? Reports of lineage instability were initially met with doubt and scepticism due to the artificial nature of

Figure 1. Models of helper T cell differentiation. (A) Traditionally, it has been believed that stably committed helper T cell lineages are characterised by the expression of master transcription factors and signature cytokines. (B) It is becoming apparent that helper T cells display a degree of functional and phenotypic plasticity regarding their expression of both cytokines and master transcription factors. For simplicity, other helper T cells including follicular helper T cells, Th9, Tr1 and Th22 cells are not included here. Figure adapted from (2).

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in vitro polarisation experiments. However, a growing number of in vivo studies are now supporting this idea. New technologies, such as fate mapping studies, are allowing us to trace the expression of molecules of interest in specific cell types. By tracing the expression of IL-17, it was found that Th17 cells acquire alternative fates in vivo depending on their microenvironment. In the context of chronic inflammation, Th17 cells switched off their IL-17A production and instead produced the type 1 cytokine IFN-g, acquiring an ‘ex-Th17’ phenotype.

In contrast, they retained their original Th17 phenotype during an acute inflammatory response (4).

In addition to cytokine composition in the local microenvironment, mechanisms underlying phenotypic plasticity have been suggested to involve epigenetic modifications and small non-coding RNAs. Bivalent chromatin modifications exhibiting both transcriptionally activating and repressing marks might mechanistically explain the occurrence of a ’Th1+2’ phenotype characterised by simultaneous production of both IL-4 and IFN-g (5).

How could this plasticity be physiologically relevant?

Plasticity might be considered a double-edged sword: while it allows adaptation in the face of changing environmental circumstances, it is detrimental if dysregulated. Intuitively, it seems advantageous for our immune system to retain a degree of phenotypic flexibility in order to appropriately react to the wide variety of pathogens and insults encountered, and to concurrently minimise the extent of collateral damage inflicted.

As Darwin said, “It is not the strongest of the species that survives, nor the most intelligent that survives. It is the one that is most adaptable to change.” As an example, let’s consider immune responses in the context of helminth infection. Helminths represent a particular challenge to the immune system as they are too large to be engulfed by phagocytes and inflict extensive tissue damage while migrating through the body. In the fight against them, anti-inflammatory type 2 immune responses are believed to be the most effective, as they promote the expulsion of intestinal worms and are associated with the induction of rapid tissue repair mechanisms (6).

In this scenario, Th1 and Th17 cells transdifferentiating into a Th2-like phenotype might not only contribute to host protection but also prevent the establishment of pro-inflammatory responses associated with potentially deleterious injury to the host. In contrast, inappropriate helper T cell reprogramming has been associated with a range of immune-mediated diseases. In particular, Treg instability has been identified as a contributing factor to

autoimmune diseases, allergies and chronic inflammatory disorders.

In recent years, focus has been placed on designing ways to therapeutically harness the immune system for the treatment of cancer and immune-mediated diseases.

One of the main therapeutic avenues explored for the restoration of self-tolerance in autoimmune diseases is the isolation, ex vivo expansion and subsequent reinfusion of Tregs (3, 7). Does helper T cell plasticity threaten the success of cell-based immunotherapy?

The risk of transferred Tregs gaining inflammatory properties is highly concerning (7). Meanwhile, the potential of manipulating pro-inflammatory helper T cells such as Th17 and Th1 cells to convert to a regulatory phenotype opens up new opportunities for the development of novel therapeutic interventions (3).

It seems that it might be time to review our understanding of helper T cell subsets. It has become apparent that CD4+ T cells exhibit a substantial degree of phenotypic flexibility and might not represent stably committed lineages. Though the initial discovery was alarming, as it put the success of cell-based immunotherapies at risk, immunologists are now starting to realise that T cell phenotypic transitions could be important for the adaptability of our immune system (8) and might aid in the treatment of immune-mediated diseases (3).

References 1. Mosmann TR, et al. (1986) Two types of murine helper T cell clone. I. Definition according to profiles of lymphokine activities and secreted proteins. J Immunol 136(7):2348–2357.

2. O’Shea JJ & Paul WE (2010) Mechanisms underlying lineage commitment and plasticity of helper CD4+ T cells. Science 327(5969):1098–1102.

3. DuPage M & Bluestone JA (2016) Harnessing the plasticity of CD4+ T cells to treat immune-mediated disease. Nat Rev Immunol 16(3):149–163.

4. Hirota K, et al. (2011) Fate mapping of IL-17-producing T cells in inflammatory responses. Nat Immunol 12(3):255–263.

5. Hegazy AN, et al. (2010) Interferons direct Th2 cell reprogramming to generate a stable GATA-3+ T-bet+ cell subset with combined Th2 and Th1 cell functions. Immunity 32(1):116–128.

6. Allen JE & Wynn TA (2011) Evolution of Th2 immunity: a rapid repair response to tissue destructive pathogens. PLoS Pathog 7(5):e1002003.

7. d’Hennezel E, & Piccirillo CA (2012). Functional plasticity in human FOXP3+ regulatory T cells: implications for cell-based immunotherapy. Hum Vaccin Immunother 8(7):1001–1005.

8. Hirahara K, et al. (2013) Mechanisms underlying helper T-cell plasticity: implications for immune-mediated disease. J Allergy Clin Immunol 131(5):1276–1287.

Janina Nahler is a DPhil student in Professor Graham Ogg’s research group at the Weatherall Institute of Molecular Medicine.

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Cracking the cis-regulatory code: how close are we? by I Ivan L. Candido-Ferreira

n multicellular organisms, transcriptional regulation controls precise patterns of gene expression. Recent studies suggest the existence of a combinatorial code encrypted in the regulatory genome, with similar properties to the genetic code. This article summarises the recent progress in understanding how multicellular eukaryotes regulate their transcription, focusing on Drosophila as a model.

Multicellular organisms contain large genomes that encode exact information for when and where genes should be switched on and off. Such information is encrypted in cisregulatory sequences known as transcriptional enhancers.

These are key determinants of embryonic development as they integrate multiple inputs from programming modules, which control the precise and complex spatio-temporal patterns of gene expression (1). Thus, enhancers really function as microprocessors, computing the regulatory logic of development in multicellular organisms.

To address this gap in understanding, one model is particularly attractive given its conceptual similarity to one of the most famous endeavours of biochemistry, the deciphering of the genetic code. Because of the combinatorial nature of the TF inputs that enhancers integrate, it has been proposed that the eukaryotic genome encodes a cis-regulatory code (2) similar in many ways to the genetic code.

In the genetic code, different combinations of nucleotide triplets encode information for amino acid translation.

Whereas in the cis-regulatory code model, nonoverlapping combinations of TFs binding to enhancers encodes information for spatio-temporal patterns of transcription (3). Therefore the underlying regulatory code can be understood from studying TF binding site motifs found in endogenous enhancers. By extension, some key properties of the genetic code thus apply to the regulatory code, such as combinatorics, redundancy, modularity, grammar, and, to some extent, universality.

In contrast to the cis-regulatory sequences of unicellular organisms, which are located proximal of their target genes such as the lac operon, transcriptional enhancers of multicellular organisms can be located virtually anywhere in the genome. This feature turns their identification into a real challenge. Additionally, eukaryotic DNA is highly condensed as chromatin, so an efficient molecular system is necessary to make enhancers accessible to transcription factors (TFs). However, a complete understanding of how transcriptional precision is achieved during embryonic development is still elusive.

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Figure 1. A fully synthetic enhancer recapitulates the stripe 2 pattern of gene expression, indicating key architectural properties of transcriptional regulation in multicellular eukaryotes. (A) Top: The complex pattern of even-skipped (eve) expression in early Drosophila embryonic development, which plays a primary role in establishing segmentation. Seven stripes are produced by different enhancers, each integrating different combinatorial codes of TFs. Bottom: the eve locus displaying all seven stripe-specific enhancers. (B) The endogenous eve stripe 2 modular enhancer and (C) the synthetic stripe 2 enhancer. Transcriptional repressors are depicted as circles; activators as squares, and the pioneer factor Zelda as stars.

Note the overlapping binding sites for transcriptional activators and repressors.

(D) Gradients of transcriptional activators and repressors. Zelda is ubiquitously expressed. A, synthetic activator; BC, Bicoid; Gt, Giant; Hb, Huntchback; Kr, Kruppel; R, synthetic repressor; Z, Zelda.

An example of such regulatory patterns can be observed by the even-skipped (eve) stripes in Drosophila development , which are comprised of seven stripes, each of them being regulated by a stripe-specific enhancer that integrates different regulatory logics (Figure 1A). eve establishes early segmentation within the anteriorposterior axis, thus being important for patterning the body plans in Drosophila embryos (1, 2, 4).

A major leap forward in cracking the genetic code was achieved by the Nirenberg and Matthaei experiment in 1961, resulting in the discovery of the first codon. They used synthetic poly-U RNA in a cell-free extract, which produced a protein entirely composed of phenylalanine, thus revealing the first codon as UUU. As the eve stripes in Drosophila embryos have remained as a paradigm of transcriptional control in multicellular organisms (1, 2), demonstrating a way to synthetically produce these stripes would similarly be the first step towards deciphering the regulatory code.

Recently, a major breakthrough has been made by the Stern lab at the Janelia Research Campus, Howard Hughes Medical Institute. David Stern and colleagues envisioned a fully synthetic transcriptional toolkit (4, 5) composed of few modules useful for probing transcription properties in Drosophila (Figure 1BD), which allowed them to create a synthetic pattern with seven stripes (4). The key modules are stretches of transcriptionally inactive DNA and reprogrammable synthetic TFs, known as Transcription activator-like effectors (TALEs) that were designed and fused either to a transcriptional activator (VP64) or a repressor (Hairy).

These synthetic activator (TALEA) and repressor (TALER) TFs can be engineered to bind virtually any sequence, and thus they function as reprogrammable TFs in a sequence-independent manner (4, 5).

This elegant work can be best exemplified by their efforts to recreate the stripe 2 (5). Gradients of these synthetic TFs were created, mirroring the endogenous gradients of stripe 2 activator TF inputs (Figure 1C, E). Whereas combinatorial binding of transcriptional activators was sufficient to recreate a synthetic stripe 2 pattern, precise patterns of gene expression were only achieved when activator and repressor binding sites were overlapping.

This configuration is similar to the native eve stripe 2 enhancer, (Figure 1B, C) thus suggesting that binding competition is essential not for switching transcription on and off but to regulate the sharp boundaries of gene expression (5).

However, the synthetic enhancer sequence was inactive in the endogenous chromatin context, as DNAse hypersensitivity I assays suggested that chromatin was inaccessible to the synthetic TFs. Ectopic expression of the stripe 2 pattern could however be restored when flanking binding sites for the generic pioneer factor Zelda were included in the synthetic enhancer sequence. As the name suggests, pioneer TFs are capable of being the initial TFs that bind to nucleosomal DNA and recruit chromatin remodelers, making the DNA accessible to other TFs. This suggests that binding of pioneer TFs are key for stage-specific activation of enhancers.

Therefore, precise patterns of gene expression such as that illustrated by the endogenous stripe 2 enhancer seem to be dictated primarily by the combinatorial binding of a certain number of activators. Overlapping binding sites for repressors seem to be necessary for tissue-specificity, and binding sites for pioneer factors are essential for making the chromatin accessible to TFs. Taken together, these results indicate that the aforementioned elements are the minimal modules that need to be identified in order to crack the regulatory code.

In summary, these elegant experiments reveal underlying principles of the functional architecture of enhancers and illustrate a pipeline of how the cis-regulatory code may be deciphered in the near future. Although transcriptional research is still in its early stages, it has just entered a new era resembling one of the most exciting periods of modern biochemistry. The emerging systems understanding of transcription, brilliantly illustrated by the work of David Stern and colleagues, will likely impact fields as diverse as developmental and synthetic biology.

References 1. Levine M (2010) Transcriptional enhancers in animal development and evolution. Current Biology 20(17):R754–R763.

2. Yanes-Cuna JO, et al. (2013) Deciphering the transcriptional cis- regulatory code. Trends Genet. 29(1):11–22.

3. Brown CD, et al. (2007). Functional architecture and evolution of transcriptional elements that drive gene coexpression. Science 317(5844):1557–60.

4. Crocker J, et al. (2016) Quantitatively predictable control of Drosophila transcriptional enhancers in vivo with engineered transcription factors. Nat Genetics 48(3):292–8.

5. Crocker J, et al. (2017) A Fully Synthetic Transcriptional Platform for a Multicellular Eukaryote. Cell Reports 18(1):287–296.

Ivan L. Candido-Ferreira is a DPhil student in Professor Tatjana Sauka-Spengler’s research group at the Weatherall Institute of Molecular Medicine.

Trinity 2017 | PHENOTYPE | 27

Blood-brain barrier modelling: an essential challenge for neuroscience to overcome by Isobel Parkes

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he blood-brain barrier (BBB) is primarily composed of microvascular endothelial cells. Brain microvascular endothelial cells differ from peripheral endothelial cells in a number of ways including that they are mechanically coupled by focal adhesions, known as tight junctions. Tight junctions prevent leakage of molecules into the space between the cells, the brain interstitium. This defines the endothelium as the principle barrier for the transport of molecules across the BBB.

Brain microvascular endothelial cells are functionally and anatomically coupled with pericytes, astrocytes and neurons to form the neurovascular unit (NVU) (Figure 1). As arterioles penetrate deeper into the brain, the endothelium basement membrane comes into direct contact with astrocytic end-feet, and these intracerebral arterioles and capillaries are encapsulated by pericytes.The coordination of the above cells in response to injury, and the concept of neurovascular coupling—the relationship between neural activity and cerebral blood flow—led to the notion that these cells comprise an integrated unit.

This unit serves as the main interface between the blood and brain tissues, providing the first line of defence against the detrimental effects of neurotoxic molecules in the systemic circulation.

“Cells discern and respond to the dimensionality, topology and rigidity of their environment, and these qualities are not modelled by the two dimensions of a flat dish.”

Neuroscience needs an anatomically accurate and reproducible in vitro model of the BBB for two principal reasons. Firstly, the disruption of the BBB plays an important role in cellular damage in many neurological diseases, including vascular dementia and multiple sclerosis. The molecular mechanisms underlying BBB disruption remain largely elusive, and the lack of a robust in vitro model is a major hindrance to understanding the cellular biology responsible for how the BBB behaves in diseased states. Secondly, an in vitro model of the BBB that accurately replicates the organisation of cell populations and the restrictive nature of the barrier would revolutionarise drug discovery by allowing economical, noninvasive and reproducible screening of novel drug candidates. Drug screening is complicated, time-consuming, and requires significant investment, with clinical trials often failing late in the process. In the last five years, the number of drugs developed by large pharmaceutical companies for nervous system disorders decreased by 50% (1). This reduction is partly due to 28 | Oxford University Biochemical Society

the lack of understanding of BBB neurobiology, which limits the development of new drug molecules that can effectively permeate the BBB and achieve the correct therapeutic concentrations in the brain (2).

Traditionally, in vitro models of the BBB have been established from animals, such as mouse, rat, pig, and bovine models. Primary cell lines derived from these animals are still the most prevalent used to construct BBB models. However, there are inherent differences between the BBBs of different species, such as differential expression of enzymes, transporters and tight junction proteins. Since in vitro models are vital for translation to humans, cells derived from humans would be a better model than animal-derived cell lines.

In the past two decades, many human in vitro models of the BBB have been developed. These models tend to employ immortalised or primary cells. Primary cell lines are considered superior, as immortalised cell lines, which have acquired mutations that ensure unlimited life-span, tend not to recapitulate normal physiology. One of the most conventional and best-characterised cell types used are human umbilical vein endothelial cells (HUVEC), due to their ready availability and simplicity to culture.

Nevertheless, the use of human-derived primary cell lines is still not ideal for BBB modelling as human material is often not suitable due to ethical reasons, limitations in obtaining the tissue and the finite proliferative lifespan of primary human cells in culture before permanent growth arrest. Moreover, results reported from models established from primary cell lines suggest that they do not form adequately restrictive tight junctions and thus fail to accurately model brain microvascular endothelial cells (3).

Despite such shortcomings of in vitro models of the BBB, significant progress has been made in the utilisation of induced pluripotent stem cells (iPSCs), which can be differentiated into any cell type of interest. Human iPSCs are derived from skin fibroblasts and reprogrammed to iPSCs using the Sendai virus to introduce Yamanaka factors. iPSCs are increasingly relevant in vitro models for analysing BBB dysfunction in disease because of their ability to retain the genome of the person from whom they are derived. This enables the study of disease phenotypes in cells derived from patients. Moreover, iPSCs have a highly proliferative nature, which makes

closely modelling the in vivo anatomical organisation of the NVU. However, to date, in vitro models fail to explicitly reproduce the unique characteristics of brain microvascular endothelial cells, most importantly the restrictive behaviour of the BBB due to poor integration of the endothelial cells and surrounding cells making up the NVU.

Figure 1. Schematic of the human neurovascular unit, composed of neurons, astrocytes, brain microvascular endothelial cells, pericytes and extracellular matrix components.

Figure by Oleg Sitsel.

iPSC-derived models suitable for high-throughput drug screening.

The most basic in vitro iPSC-derived BBB models are 2D monolayers, which have significant limitations.

These models exhibit low trans-endothelial electrical resistance (TEER) values and high permeability to often-impermeable marker molecules, alongside low expression of key membrane transport proteins such as the P-glycoprotein efflux pump. Similarly, the short-term viability of cells cultured in static models substantially reduces their application as a drug screening tool.

Standard 2D in vitro models also poorly replicate in vivo tissues because of their static nature. Cells discern and respond to the dimensionality, topology and rigidity of their environment, and these qualities are not modelled by the two dimensions of a flat dish. Therefore, 3D in vitro models are becoming more prevalent since the cellular biology and behaviour is more comparable to that found in vivo. Most contemporary 3D models involve either co-culture or tri-culture of multiple cell types. The most common approach uses transwell inserts to mimic the BBB (4), which allows the culturing of different cell types on either side of a microporous membrane. This approach enables the coupling of the NVU to be modelled through differentiation of iPSC-derived endothelial cells, pericytes, neurons and astrocytes within the in vitro model (5), thereby more

The current state-of-the-art and future of in vitro modelling of the BBB lies in the utilisation of iPSCs in the nascent field of microfluidic devices. The design of microfluidic models ranges from a single-cell model to the intricacy of the multicellular ‘organ-on-a-chip’ format. In normal physiological conditions, shear stress is generated by the flow of blood through blood vessels, and significantly affects tight junction and transporter expression, including the function of the endothelial barrier. Microfluidic devices allow the model to be dynamic by incorporating both shear stress and other cell types by culturing endothelial cells and astrocytes on the inside and outside of hollow tubules, thereby mimicking the complex cellular microenvironment of the NVU in a simple device. Contemporary ‘organ-on-a-chip’ models have been successfully engineered with cells derived from human embryonic stem cells and therefore, by extrapolation, the potential for using iPSCs is clear (6).

There is still some way to go in constructing the ideal in vitro model of the BBB, but significant progress has been made, and iPSC technology has provided new invigoration into building an anatomically accurate model of the BBB. References 1. Herper H (2015) The coming boom in brain medicines. Available at http:// www.forbes.com/sites/matthewherper/2015/02/11/brainboom-the-drug-companies-bringing-neuroscience-back-from-thebrink/#3e0b344152a2 [Accessed 22nd February 2017].

2. Wang JD, et al. (2016) Organization of endothelial cells, pericytes, and astrocytes into a 3D microfluidic in vitro model of the bloodbrain barrier. Mol Pharmaceutics 13(3):895–906.

3 Wekslet BB, et al. (2005) Blood-brain barrier-specific properties of a human adult brain endothelial cell line. FASEB J 19(13):1872– 1874.

4. Banerjee J, et al. (2016) In vitro blood–brain barrier models for drug research: state-of-the-art and new perspectives on reconstituting these models on artificial basement membrane platforms. Drug Discovery Today 21(9):1367–1386.

5. Yamamizu K, et al. (2017) In vitro modelling of blood-brain barrier with human iPSC-derived endothelial cells, pericytes, neurons, and astrocytes via Notch signaling. Stem Cell Reports 8(3):1–14.

6. Van der Meer, et al. (2013) Three-dimensional co-cultures of human endothelial cells and embryonic stem cell-derived pericytes inside a microfluidic device. Lab Chip 13(18):3562–3568.

Isobel Parkes is a Neuroscience undergraduate in Professor Zameel Cader’s research group at the Weatherall Institute of Molecular Medicine.

Trinity 2017 | PHENOTYPE | 29

Cortex Club: the birth of a studentrun discussion forum by Samuel Picard

T

he Oxford University Cortex Club is a unique forum for discussion and debate, dealing with significant, challenging contemporary issues in neuroscience. Entirely run by a different group of students each year, it now organizes more than twenty events per year, ranging from small intense debates with local neuroscientists to large seminars featuring world-leading researchers—always followed by further discussions over a drink in the pub.

The Cortex Club was established only eight years ago— since then, it has grown into one of Oxford’s leading neuroscience discussion forums. As this year’s copresident, I feel very fortunate that I can build on all the hard work that previous committees have put into establishing the Cortex Club’s reputation. Indeed, over the last year, it’s been a real thrill to see some incredibly influential scientists accept our invitations.

How can a simple but ambitious idea, started by a small group of curious DPhil students, grow into something so established and well attended in just a few years’ time?

To answer this question, I thought it would be good to get some historical perspective. I therefore decided to have a chat with our co-founder and first president, Abhishek Banerjee, who is now a Marie Curie fellow at the University of Zürich.

“The time was just right for us to go beyond the classical seminar format.”

Could you tell us about where the idea for the Cortex Club came from?

We started in the spring of 2009. Oxford Neuroscience, the platform that is now coordinating neuroscience research across the university, had not yet taken the shape that it has today. As a result, all neuroscience-related research groups were more scattered, and people would often stick to their own departmental seminars. Some of us, however, would enjoy hanging out after seminars. In doing so, Dennis Kätzel and I—both DPhil students in neighbouring labs—felt that there was an empty space to be filled. Indeed, together with recordings and imaging, many scientists were starting to manipulate circuits using novel methods like optogenetics, and cross-disciplinary collaboration was becoming more and more essential.

Now looking back, I think the time was just right for us to go beyond the classical seminar format, and to create a novel platform for discussion and debate.

What did your first events look like?

The first Cortex Club session ever was held in May 2009.

Daffodils were up about 10 cm and the snowdrops were 30 | Oxford University Biochemical Society

in bloom. Jesper Sjöström came over from UCL—he had carried out important work on synaptic coincidence detection. The discussion was moderated by Ole Paulsen, now Chair of Physiology at Cambridge, who was my DPhil supervisor at the time. The audience was relatively small, but nevertheless it was a fantastic session. It felt like the perfect balance between a roundtable discussion and a fully-fledged talk. We decided to continue with this idea and see where it would take us.

“Because the issue was so fundamental to neuroscience, the discussion quickly grew into a very passionate debate” What is the best memory you have of that first year?

We had several very interesting sessions, but I have particularly fond memories of a discussion panel, which perfectly captured the spirit of collaboration between Cortex Club members. It was a debate on rate coding versus temporal coding, with students and post-docs from Andy King’s and Andrew Parker’s labs leading the discussion. Because the issue was so fundamental to neuroscience, the discussion quickly grew into a very

passionate debate, involving everyone who was there.

Looking back, it’s not surprising that many of the DPhilstudents who were in the audience at the time ended up taking part in future Cortex Club committees.

Did this transition from a discussion group, led by you and Dennis, to a fully-fledged university society happen naturally?

It definitely required some executive decisions! At the end of the first year, we decided to introduce a voting system to be able to elect a new committee each year.

Although it was hard to hand over my ‘baby’ to someone else, I felt it was important to allow the Club to develop under another person’s leadership. Therefore, I organised a hand-over to a fantastic new president, Blake Richards.

Looking back, I think this was one of the key events in the short history of the Cortex Club. Before the new committee took the lead, Dennis and I wrote the constitution, Zoltan Molnar became our Senior Member, and we enrolled the club with the university. Although Dennis and I were probably the driving forces behind the Cortex Club, the participation of several people around us was already very important at that stage.

In the beginning, you weren’t able to take advantage of the reputation that Cortex Club has now. How did you manage to successfully invite renowned neuroscientists to come and discuss their work?

To be honest with you, at the time it was important to us to invite those people whose work connected with the questions we were exposed to all the time in Oxford. We quickly understood the following: if you are seriously interested in a person’s work, and you have read what they do, they will often be convinced just from the way you interact with them. Just imagine how many invitations they get, you have to be genuinely enthusiastic to make any impact at all. Nowadays, it is probably much easier for Cortex Club; everyone likes to speak here!

That is true, but a positive reply to an invitation is usually not enough—you need to be able to fund a speaker’s visit too!

I agree with you, but keep in mind that at the beginning we were very poor! We would try to offer some wine to the speakers and the audience after each talk, but the best bottles often came from Zoltan Molnar himself.

Also, local speakers did not always need accommodation or travel refunds, and plenty of our guests were kindly hosted by enthusiastic PIs in their colleges. I remember how ecstatic we were when Edith Sim told us that we were going to get a few hundred pounds for our activities! Last but not least, several key figures within Oxford Neuroscience, including Gero Miesenböck, Peter Somogyi, Colin Akerman, Colin Blakemore, and above all Zoltan Molnar, were extremely supportive in our early days.

“The most important thing is to find controversial, provocative topics that many people can relate to.”

Luckily, we are now generously funded by several departments and have recently made a sponsorship deal with a wonderful nanomedicine company, Precision NanoSystems. This has allowed us to have more frequent events and to invite more international speakers. Do you think this is a good thing?

Although it’s great that there is such a breadth of activities now, in my opinion it’s important to make sure that there isn’t too much happening either. Dennis and I made a conscious decision to have events happening quite sparsely, so people would be eagerly awaiting each event. The most important thing is to find controversial, provocative topics that many people can relate to. It’s great that Cortex Club has grown so much, but it is important to keep the spirit of small-scale, student-led debate, which is what makes the Club unique!

Thanks so much for sharing your thoughts, and for all your hard work in founding the Cortex Club!

I am extremely happy and proud that all of you, through your hard work, kept it alive and made it into a great success. Good luck with everything!

For more information about the Cortex Club, visit http://www.cortexclub.com/

Samuel Picard is a DPhil student in Professor Andrew King’s research group at the Department of Physiology, Anatomy and Genetics.

Trinity 2017 | PHENOTYPE | 31

Public engagement: why should you get involved? by Amy Flaxman

I

f you are passionate about science, it’s very rewarding to share your knowledge with others and for them to ask questions, and learn from you. I hope to convince you, through my experience as a STEM Ambassador, that any scientist can and should try their hand at public engagement.

Firstly, what exactly is public engagement? “Public engagement describes the myriad of ways in which the activity and benefits of higher education and research can be shared with the public. Engagement is by definition a twoway process, involving interaction and listening, with the goal of generating mutual benefit.” (1) And why should we bother with it? “Research Councils UK (RCUK) believes that engaging the public with research helps empower people, broadens attitudes and ensures that the work of universities and research institutes is relevant to society.”

(2) As staff and students at the University of Oxford, we are often encouraged to participate in Public Engagement events. This is exactly how I first got involved, by helping out at the Oxfordshire Science Festival and Oxford Open Doors in 2015. I enjoyed talking with members of the public about science and was encouraged by the interest they had in our research. I wanted to get more involved in public engagement: in particular, working with young people and becoming a STEM (Science, Technology, Engineering and Maths) ambassador was the ideal platform for me to do so.

STEM ambassador network The STEM network creates “opportunities to inspire young people in STEM” and works with “thousands of schools, colleges and STEM employers, to enable young people of all backgrounds and abilities to meet inspiring role models, understand real world applications of STEM subjects and experience hands-on STEM activities that motivate, inspire and bring learning and career opportunities to life.”

(3) Becoming a STEM ambassador involves registering online, attending a two-hour training session, (often held in Oxford) and obtaining an enhanced Disclosure and Barring Service (DBS) check. The STEM network connects

Figure 1. Introducing myself to Year 6 pupils.

32 | Oxford University Biochemical Society

its ambassadors with local schools via postings on their website and through weekly emails. One week last autumn I came across a request for a STEM ambassador to visit my local primary school to give a talk to Year 6 pupils about evolution. I was quickly in contact with the school’s science co-ordinator and set a date for my talk. Soon enough, I had prepared my presentation and was stood in front of the classroom (Figure 1). This was the first time I had presented to an audience who were seated on the floor! My main concern was pitching my talk to the pupils’ level of scientific knowledge, however this was not really a problem. To keep the students interested I asked them plenty of questions and, to make the session interactive, we drew our own tree of life on the whiteboard. I felt that the students, the teacher and I all benefitted from the talk.

Organising your own activities I enjoyed my talk with Year 6 pupils so much that I wanted to come back to the school for some practical science sessions. Experimental work and discussion about the outcome of our experiments is key in encouraging scientific thinking and enthusiasm in the younger generation. Being a microbiologist, I started with exploring the unseen living world with my students.

Once I had designed the worksheets and poured almost 100 agar plates, I was ready to go! The children worked in pairs to take samples from around the classroom and use their own fingertips to investigate microbial growth. We thought about what differences we expected to see when an antibiotic was present in the agar and after washing hands. A week later I met them again to analyse the results (Figure 2) and determine whether they supported our hypotheses! We found that very few bacterial colonies grew in the presence of antibiotic, as expected (Figure 2B).

However, more colonies grew after washing hands (Figure 2C) than before (Figure 2A). After some troubleshooting, we concluded that after washing their hands, students had to go through several doors to return to their classroom, which may have been a source of additional microbes! We also found varying numbers and types of colonies across different areas of the classroom. Each student counted the number of colonies that grew from the site they had swabbed (Figure 2D). By collating the data from everyone in the class we found that the teacher’s computer keyboard had the most bacterial colonies! I thoroughly enjoyed these microbiology sessions with Year 6 and was pleased to receive very positive feedback from the teachers. In fact, the school has invited me back this summer for a microbiology session with Year 5 children. Although these sessions involve some preparation, it is so encouraging to

see the students enjoy investigating the world around them.

These sorts of experiences will remain with the students and hopefully inspire them to think scientifically and maintain an interest in biology as they progress through their education.

Getting involved As well as the STEM ambassador network, numerous departments in Oxford now have public engagement officers.

You will no doubt receive emails from them about upcoming events. Reply to them—get involved yourself ! It’s great to step away from the lab and interact with an interested audience about your area of expertise, or science in general.

As well as having plenty of fun, I think you will find public engagement a very worthwhile experience, both on a personal and professional level.

Figure 2. Agar plates from one pair of students from my microbiology practical sessions. Students labelled their plates with their initials. Samples were applied to agar plates and incubated overnight at 37°C. (A) Growth from fingertips. (B) Growth from fingertips in presence of antibiotic (Kanamycin added to agar). (C) Growth from fingertips after washing hands. (D) Growth from selected sites around the classroom.

References 1. National Co-ordinating Centre for Public Engagement (2017) What is Public Engagement? Available at: https://www.publicengagement.ac.uk/ explore-it/what-public-engagement [Accessed 27th March 2017].

2. Research Councils UK (2014) RCUK Public Engagement. Available at: http://www.rcuk.ac.uk/pe/ [Accessed 27th March 2017].

3. STEM Learning Ltd (2017) STEM ambassadors. Available at: http:// www.stemnet.org.uk/ambassadors/ [Accessed 27th March 2017].

Special thanks to Victoria Hillier, Science Co-ordinator at Fir Tree Primary School, Wallingford, for facilitating these events.

Amy Flaxman is a DPhil student in Dr. David Wyllie’s research group at the Jenner Institute, NDM.

A CAREER AS A PATENT ATTORNEY . . . . . . a challenging and rewarding option for Oxford Biochemists Training as a Patent Attorney is a career path that will enable you to combine your understanding of biochemistry and related disciplines with legal expertise. You will leave the lab environment yet remain at the cutting edge of science and technology, applying your knowledge and skill in a commercial context. Your role will be to help to protect intellectual property assets and grow businesses.

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Trinity 2017 | PHENOTYPE | 33

Stand out from the crowd and boost your career by Sofia D’Abrantes

D

eveloping as a professional involves more than building your research profile and research skills. As employers are looking for researchers who can ‘add value’ to their organisations, wider professional development is becoming increasingly important. Some of the most soughtafter skills by employers include those in public engagement and impact, with a focus on entrepreneurship and innovation skills. This is precisely why you should attend the novel openaccess bio-entrepreneurship education programme provided by the Science Innovation Union (SIU) and stand out from the crowd.

Our mission The Science Innovation Union is a non-profit organisation led by post-graduate students and young professionals.

Our core focus is to provide a communication, networking and training platform linking academia, industry and society. We aim to inspire innovation and encourage the translation of science into real-world applications.

By connecting scientists and industrial partners across the globe, we are building a global network for the Life Sciences and Healthcare industry.

What we deliver The SIU offers a novel, open-access bio-entrepreneurship education programme aimed directly at the UK’s top scientists and entrepreneurs. Each academic term, we organise a series of high impact evening seminars given by world-renowned industry and academic leaders. During the first academic term of 2016 (Michaelmas Term), we ran our first four sessions of the SIU360 programme, which aimed at helping our participants to develop core skills necessary to succeed in the business world. One of the sessions involved exploring the different areas of risk along the trajectory of a new start up, from gathering funding to choosing the right people for your team.

After the SIU360 programme, we launched our SIUconversations educational programme for the second academic term of 2016 (Hilary Term). We hosted sessions on several topics, from intellectual property to bringing academia and industry together. The talks were delivered by high profile and successful entrepreneurial scientists and business people, including members of the Royal Society and Enterprising Oxford. But the SIU is not just about our education programme: we have also expanded our activities to cover all aspects of translation in science via our networking, consulting and editorial activities. All of our events are free of charge and allow the development of key skills that employers are seeking. We believe our approach can shape the perfect scaffold for establishing a young generation of bio-entrepreneurs that is capable of thinking in terms of both science and business.

Sofia D’Abrantes is an Oxford Interdisciplinary Bioscience DTP DPhil student in Professor Stanley Botchway’s research group at the Central Laser Facility at Harwell, and is also the co-lead for the Education arm of the Oxford SIU division.

34 | Oxford University Biochemical Society

Figure 1. The first SIU360 event of the term—Professor Chas Boutros’s talk about the importance of industryacademia collaborations—at the Saïd Business School in Oxford on October 18th, 2016.

The future As a young organisation, we have big plans for the future! We aim to keep providing our open-access bio-entrepreneurship education programme and train the next generation of scientists and entrepreneurs.

In parallel to our primary central team, we have established regional divisions in Cambridge, London, Frankfurt, Sarasota, Zürich, Berlin and Katowice.

The Science Innovation Union is the first of its kind to interconnect organisations locally in each city, as well as across countries.

We are currently working hard to establish a public engagement arm. Its main aim will be to reach members of the public and discuss the importance of science, as well as the need for translation of science into realworld solutions. We are also planning to organise ‘Impactful’ sessions and debates twice per term, where we will deal with a specific entrepreneurship topic and have a panel discussion with key players form industry, academia, society and government.

At the SIU, we work towards building a world-leading brand and we are inviting you to build it with us. We are rapidly expanding and always looking for the right people to join our teams, so if you want to learn new skills, make new contacts and boost your career, visit http://science-union.org/join-the-team-1/.

For more information about our organisation and about our future events near you, please visit http://science-union.org/thescienceunion/

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RECENT EVENTS Highlights from the OUBS careers day by Burcu Anil Kirmizitas

This year’s OUBS careers day on February 21st brought together professionals from publishing, patent law, science policy, public engagement and biotech. It was inspiring, informative and fun to hear about the journey the speakers—all Oxford alumni—took after getting their degrees in biochemistry.

it’s definitely a plus when it comes to starting salaries.

“A PhD would be hired for an associate editor position” says Katy, “the starting salaries would be around £2530k”. When asked about the work-life balance, Katy told us it’s good since you can work from home for a couple of days a week.

Publishing The first speaker to take to the stage was Katherine Eve, an executive publisher from Elsevier. After finishing her Master’s in Biochemistry, Katy got her first publishing job at Taylor & Francis as a publishing editor, specialising in chemistry. Two years later, she moved to Elsevier to take up a publisher role in geochemistry. In 2013 she assumed her current role, specialising in renewable energy and fuels. She told us that Elsevier is a very transparent company and offers lots of opportunities to its employees to develop themselves professionally.

Public engagement The second speaker was James Brown, an education and public engagement officer at the Biochemical Society.

James has a Master’s in Biochemistry from Oxford and a MEd from Cambridge. After completing his degrees, he worked as a science teacher at secondary schools and started to hold engagement events such as science fairs and competitions. The more he organised these events the more he enjoyed science outreach, so he decided to permanently transition into public engagement. His job is to tell the public about science.

Katy loves her job because she is immersed in science all the time, is always updating herself on the topics she works on, and attends many conferences. She also gave us insight into the world of publishing and the different roles one can take. The industry is vast and versatile so there are always openings. The roles range from editorial, marketing, production, design, sales and distribution, to rights and contracts. According to Katy you don’t need a PhD to get an editorial role in publishing, but

This can be in many different forms, ranging from preparing a movie, holding competitions, and talking to people on the street, to visiting schools and putting together art installations. He told us he was lucky to get the job he has, since there still are not that many permanent jobs of this sort, but that things are changing.

As public engagement is becoming increasingly important, research funding bodies and institutes are increasingly employing public engagement officers.

36 | Oxford University Biochemical Society

For those who want to get involved, James said the Biochemical Society has schemes for undergraduates, and offers membership for £13 a year.

Intellectual property law Next we listened to Daniele Selmi’s talk about his adventures in intellectual property law. Daniele completed his undergraduate Master’s degree in the Department of Biochemistry, graduating in 2005. He then completed his DPhil in physics and undertook a postdoctoral fellowship. Following this, he decided to move into law and obtained his Graduate Diploma in law from Oxford Brookes University. He now works at Three New Square Chambers in London.

He’s never bored intellectually and is involved in many areas of science. It is challenging to quickly come to grips with different types of science when dealing with cases, and he thinks having a DPhil is an advantage for him. He needs to consult experts in the particular field to which a given IP case belongs, so communications skills are important as well. “You can transition into law from any background,” Daniele says “but law is not very international in its nature.” During the Q&A session, we discovered that Daniele was chosen Sportsman of the Year in 2008, when he was a student at Oxford. He highly recommends extracurricular activities for those who want to get into law, as he believes he stood out thanks to his hobbies when applying for his pupillage.

Science policy Giles Robertson, the fourth speaker, told us all about making science policy. Giles received his DPhil in structural biology from Oxford in 2006. Since then he has held different roles as a civil servant. He started off at the Department of Health, working on emergency preparedness, and is now Assistant Director of the Research and Innovation Reform Directorate within the Department for Business, Energy and Industrial Strategy (BEIS). Giles, just like our previous speakers, has to get to grips with different areas of science very quickly. Challenging as it may sound, it is very interesting and fun, Giles stresses.

Over the years he has found himself giving advice on a variety of situations, such as how to proceed with flights when volcano Eyjafjallajökull in Iceland erupted in 2010 and on flood protection measures when the 20132014 winter floods hit the UK. He is now advising in areas including vaccine development, renewable energy, sustainable agriculture and science funding. As with any other job, there are times when he has to put in long hours, and situations he has to adapt to very quickly, but there are many perks to his job too. He loves the variety

of roles he has taken, travel opportunities and the sense of purpose he gets from his job. He had one particularly good piece of advice for future job applicants: “getting hired takes a long time, so don’t give up if you apply and don’t hear back right away, since a reasonable time frame to hear back is actually about 6 months”.

Biotech The last speaker of the day was Nathan Rose from Oxitec. Nathan completed his DPhil in chemistry and worked as a postdoc at the Department of Biochemistry in Oxford until the end of 2015. He is now a group leader at the Abingdon-based company. In the first part of his very interesting talk, Nathan told us about the technology Oxitec developed to combat mosquito-borne diseases such as Zika. Oxitec’s genetically modified male mosquitos mate with wild females, and pass on a gene that prevents their offspring from reaching adulthood.

The company, established in 2002, was acquired by Intrexon in 2015 and now has over 60 employees at their UK site. The mosquitos are bred in a factory in Brazil and released to the wild there.

Nathan is finding his job to be exciting and stimulating and has been learning a lot since he joined the company.

He says that the technical skills he acquired during his time in academia have all been relevant to what he is doing now. He also believes that other skills such as verbal and written communications developed while giving talks and writing papers in academia are extremely important for his job. He found that his collaborations in the past taught him about management, which is a big part of his role as a group leader. The biggest difference between academia and industry, Nathan says, is the pace. In academia people usually work on one or two projects for two to four years but in industry, in his experience, these numbers are seven to nine projects for a few months up to a year. These differences stem from the nature of the goal and project driven work in industry, which means very formal deadlines. Nathan doesn’t work in the lab any more but he is still very much involved in science.

He finds the working hours comparable to the hours he used to put in as a postdoc. Oxitec offers three to twelve month internships to students, and at the moment they are advertising for several senior scientist positions.

At the end of the day, Prof Tony Watts summed up everybody’s thoughts: “Today’s talks have given a fascinating insight into the potential and flexibility that a degree in Biochemistry can offer. For tutors and supervisors like me, these kinds of experiences make our efforts and time all the more worthwhile—I have learnt a tremendous amount”.

Burcu Anil Kirmizitas is a post-doctoral research associate in Professor Neil Brockdorff ’s research group at the Department of Biochemistry.

Trinity 2017 | PHENOTYPE | 37

5’ WITH...

Professor Simon J. Davis by James R. Eaton Professor Simon J Davis is a Senior Research Fellow in immunology at the Weatherall Institute of Molecular Medicine.

He completed his PhD in 1987, working with John Wheldrake at Flinders University in Adelaide. He then came to Oxford as a postdoctoral research fellow at the Dunn School of Pathology where, after seven years, he was awarded a Career Development Award by the Wellcome Trust. Currently, Simon’s work focuses on trying to prove a theory of receptor triggering that he and a colleague, Anton van der Merwe, developed and published back in 1996.

What motivated you to become a scientist?

To be honest, I went through my education and nothing else appealed along the way. John Wheldrake suggested that we undergraduates should all read The Double Helix

by Jim Watson, which showed what fantastic fun making scientific discoveries could be. Later, during my honours year, I found I was quite good at doing experiments, which finally set me on my path. Jim’s book made the very important point that it’s just a question of your brain versus the next person’s. Your history, or where you’re from? Doesn’t matter. A highlight of my career was spending 30 minutes one-on-one with Jim in 2013.

How did you arrive at your research interests?

After reading Jim’s book, I was drawn to structural biology, although I knew nothing about it. As a postdoc I became interested in the structures of proteins at the cell surface, and eventually receptor triggering. The great thing about structural biology, I now realize, is that it puts very helpful constraints on biological reality.

The importance of this became very apparent when Anton and I were thinking about the receptor signalling problem. We realised the old ideas were no good because they just didn’t fit the structure of the receptor we were thinking about (the T-cell receptor). So we knew some new biology would come of it, which is where you want to be.

If you weren’t a scientist, you would be….?

I think I would have loved to be a musician, although I’ve never played an instrument in my life. Or, being an

James R. O. Eaton is a DPhil Student in Dr Akane Kawamura’s and Professor Shoumo Bhattacharya’s research groups at the Departments of Chemistry and Cardiovascular Medicine.

38 | Oxford University Biochemical Society

Aussie, a racing driver if I had any talent for driving. I’m very lucky I found something I was quite good at, and that it carries with it the potential to do something that might last.

What are you most proud of in your career so far?

I would say our theory of signalling for sure. We don’t know if it’s really the answer yet, but 21 years on there don’t seem to be any better ideas out there, and all the tests that Anton and I have done seem to fit it well.

What’s very exciting is that the scale of the events we proposed match the new breakthroughs in fluorescence imaging, and we can start trying to see if what we predicted can actually be observed. I’m also very proud to have had a role in the lives of the students I’ve trained, who have all been remarkable people.

What has been the biggest challenge in your career?

Making the leap from post-doc to PI. It’s the same for everyone.

What advice would you give to a young scientist looking to follow in your footsteps?

Read Watson’s book! If you totally get it, and think that making discoveries is the best fun there is, there’s every chance you could be a good researcher. It’s a good test. Another thing is to accept that the key to creative thinking is simply time spent thinking. I find that ideas come seemingly randomly from ‘system I’ of my brain, but only when ‘system II’ is engaged with the problem at hand. I never had a good idea about T cells while I was playing cricket. You need to have read Daniel Kahnemann’s book Thinking Fast and Slow to know what I’m really talking about here. The final thing would be to try to be lucky.

What do you think is going to be the big discovery in immunology in the next five years?

Seeing how far immune checkpoint immunotherapy can be taken.

FEATURED SEMINAR: ADP-ribosylation signalling in regulation of genome stability by Oliver Adams

An OUBS seminar by Ivan Ahel

Ivan Ahel heads the University of Oxford’s DNA Repair Mechanism and Human Disease Group at the Sir William Dunn School of Pathology. His lab combines biochemical, structural and in vivo approaches to investigate the regulatory functions of ADP-ribosylation in maintaining genome stability.

During his Hilary OUBS seminar, Ivan summarised a flurry of interesting ADP-ribosylation functions recently discovered by his lab, from epigenetics to bacterial toxin-antitoxin systems Since its discovery over a half-century ago, ADPribosylation (ADPr) has emerged as a functionally plastic and biomedically relevant post-translational modification (PTM). ADPr modulates target protein activity, subcellular location, and stability through reversible, site-specific conjugation of monomers or polymers of ADP-ribose moieties (the latter termed PARylation).

The majority of chemically reactive amino acids function as ADPr recipients which, combined with variations in PARylation chain structure and ADP-ribose structural complexity, permit ADPr to act in a diverse array of cellular processes. Historically associated with the DNAdamage response, ADPr has become increasingly linked to aspects of chromatin structure, RNA-biology and hostvirus interactions. PARylation is enacted by a network of ‘writer’, ’reader’ and ‘eraser’ proteins. ADP-ribose addition by poly-ADP-ribose-polymerase (PARP) family ‘writer’ enzymes promotes subsequent recruitment of ADPribose binding domain (ARBD)-containing ‘readers’, including macrodomains and PAR-binding zinc fingers.

This process enables host proteins to couple PARylation to different biological phenomena (1). Finally, site-specific ADP-ribose removal is achieved through the cooperative action of multiple ADP-ribose glycohydrolase ‘erasers’.

Ivan’s seminar began with a summary of his group’s research into PARP-1, the prototypical DNA damage-sensing PARP family member. PARP-1 locates and is activated

by DNA strand-breaks. Activation triggers PARP1 auto-PARylation and trans-PARylation of various chromatin proteins (e.g. histones, p53, topoisomerases).

Resultant signalling through ARBD readers prompts DNA repair complex assembly and, in certain cases, apoptosis. Importantly, histone PARylation is thought to aid expansion of compact chromatin, improving access of repair machinery to a DNA lesion. During screening for PARP-1 interacting partners, the Ahel lab identified a promising candidate, C4orf27, that interacts not only with PARP-1 but also with core histones. Further characterisation of C4orf27 revealed that its recruitment to DNA lesions occurred in complex with PARP-1. Absence of C4orf27 sensitised cells to exogenous DNA-damaging agents, reduced histone PARylation, and induced PARP-

1 hyper-automodification. This knockout phenotype led to the renaming of C4orf27 to histone PARylation factor 1 (HPF1). The group’s findings on HPF1 are the first example of a ‘specificity factor’ capable of regulating the ratio of PARP-catalysed cis-to-trans PARylation (2).

Next, Ivan detailed work using mass spectrometry to map the positions of histone residues that were PARylated in response to DNA damage in human osteosarcoma cells. To the group’s surprise, 12 sites were identified, split between the tails of all four nucleosomal histones as well as H1, in which serine was universally the accepting amino acid; this makes H1 the first protein reported to undergo ADPr at serine (3). The Ahel lab was also able to show that HPF1 switches PARP-1 from glutamate to serine auto-modification, in line with its role as a ‘specificity factor’, and that HPF1 is required for in vitro PARP-1/2-mediated serine trans-ADPr (4).

The seminar concluded with discussion of the growing research area of bacterial ADPr, in which Ivan’s group recently published the first example of reversible singlestranded DNA ADPr. Found in bacterial species such as M. tuberculosis, DNA ADP-ribosyl transferase (DarT) catalyses sequence-specific thymidine ADPr, stalling DNA replication and causing bacteriostasis. DarT is sequestered by DNA ADP-ribosyl glycohydrolase (DarG), which is also capable of reversing DarTmediated ADPr. Together, these two enzymes form a toxin-antitoxin system of as-yet-unknown function (5).

Further research into the ADPr interaction network will undoubtedly benefit from the exciting fundamental findings from the Ahel lab.

References 1. Gupte R, et al. (2017) PARPs and ADP-Ribosylation: recent advances linking molecular functions to biological outcomes. Genes Dev 31(2):101–126.

2. Gibbs-Seymour I, et al. (2016) HPF1/C4orf27 Is a PARP-1Interacting Protein that Regulates PARP-1 ADP-Ribosylation Activity. Mol Cell 62(3):432–442.

3. Leidecker O, et al. (2016) Serine is a new target residue for endogenous ADP-Ribosylation on histones. Nat Chem Biol 12(12):998–1000.

4. Bonfiglio J, et al. (2017) Serine ADP-Ribosylation Depends on HPF1. Mol Cell 65(5):1–9.

5. Jankevicius G, et al. (2016) The Toxin-Antitoxin System DarTG Catalyzes Reversible ADP-Ribosylation of DNA. Mol Cell 64(6):1109–1116.

Oliver Adams is an Interdisciplinary Bioscience DTP DPhil student.

Trinity 2017 | PHENOTYPE | 39

BOOK REVIEWS Natural Hazard Uncertainty Assessment: Modeling and Decision Support Karin Riley, Peter Webley, Matthew Thompson (Editors) ISBN: 978-1-119-02786-7 Wiley-Blackwell (2017) 360 pages: Hardback £120 / eBook £108.99 Reviewed by Mantas Krisciunas Common natural disasters have a somewhat strange existence in the popular psyche. On the one hand, their overwhelming potential for devastation makes us fearful and mindful of the fragility of the stable and calm planet we’re accustomed to. However, their impersonal nature and the inability to pin the blame of their destruction to anything but chance leaves us less willing to take on the long-term burdens of making sure their effects are minimised. Nevertheless, wildfires, landslides and floods continually lead to large death tolls, with recovery efforts eating into national budgets already strained by other challenges that are vying for funding.

Key to mitigating the destructive aftermath of natural disasters is our ability to respond to them as fast as possible, using accurate theories about the way the resultant events might unfold. Since computing power has developed, complex models for simulating the evolution of many

Invasion Genetics:The Baker and Stebbins Legacy Spencer C.H. Barrett, Robert I. Colautti, Katrina M. Dlugosch, Loren H. Rieseberg (Editors) ISBN: 978-1-118-92216-3 Wiley-Blackwell (2016) 400 pages: Hardback, £54.95 / eBook, £49.99 Reviewed by Daniel Biggs The original book by Baker and Stebbins, The Genetics of Colonizing Species, initiated the growth of a new field of research. This new field has recently been given the name ‘Invasion genetics’, and can be described as the study of the introduction and spread of non-native species throughout the world, together with their impact on the environment, health and economy. Since the original publication was released, the field has continued to grow and this book recognises the lasting effect of their work.

In this collection of articles, the advances in the field of invasion genetics are traced back to the original work of Baker and Stebbins (1965). The book begins with a brief review of the Baker and Stebbins publication, then moves on to three detailed sections, containing articles written by a number of different authors. The first part of the book concerns itself with evolutionary ecology, and how it can answer some of the questions first posed in 1965. The second section discusses the evolutionary genetics at work 40 | Oxford University Biochemical Society

types of natural disasters have become indispensable for both local and federal authorities.

This volume, edited by Karin Riley, Peter Webley, and Matthew Thompson, tackles the often-neglected topic of uncertainty in judging the effectiveness of the natural disaster models used when informing action. Communicating the size of uncertainty in such complex models is not easy and threatens to undermine the trustworthiness of these models if readers don’t understand its unavoidability. However, the awkward subject of uncertainty cannot be ignored if these models are to be effectively used in fighting the destruction of natural disasters.

Even though the monograph deals primarily with uncertainty in modelling natural disasters, its overall aim is to provide a comprehensive overview of how earth systems science researchers approach uncertainty.

Every chapter highlights specific problems that lead to high uncertainty in the predictive power of the models in question, emphasising the way they could be lessened.

The hope of the editors is that their work will lead to a more widespread and homogenous understanding of uncertainty among scientists dealing with natural disasters.

While not for casual reading, this volume nonetheless provides an approachable way to learn about the realities of natural disaster modelling. In addition, it highlights the importance of uncertainty, an inseparable part of science that is very often ignored.

in the field and investigates invading species as tools for developing genetics models. Finally, the book considers the genomics of colonizing species, something only possible through recent developments in sequencing, functional genomics and advances in bioinformatics.

The book’s format is easy to navigate, with single articles serving as chapters, providing a comfortable route through which one can locate useful references.

The three sections are well defined and cohesive, and contain discussions that bring together the thoughts of the contributing authors on the featured articles.

The book ends with a final chapter shining a light on what the authors are yet to discover and pondering over outstanding questions within the field.

In this modern and continually changing world, it is perhaps more relevant than ever that we should be looking into the ever-changing spectrum of species within the natural world and how they fit together, migrate and establish populations in new areas. Species are constantly vying for territory and resources, and the book helps to shed light on the struggles at a genetic level.

This book serves as a great reference source, with clearly defined articles and an easily navigable layout. It would prove similarly useful for those with interests in either evolution, genetics, or both.

BOOK REVIEWS Cytogenetic Laboratory Management: Chromosomal, FISH and Microarray-Based Best Practices and Procedures Susan Mahler Zneimer ISBN: 978-1-119-06974-4 Wiley-Blackwell (2016) 840 pages: Paperback, £104 / eBook, £93.99 Reviewed by Gabriela Vilema-Enríquez Running a lab requires more than the scientific knowledge acquired in any postgraduate or postdoctoral programme—it requires knowledge of the field of management, in which interpersonal relations, organisation and staff training are essential. Susan Mahler Zneimer, in her book Cytogenetic Laboratory Management: Chromosomal, FISH and Microarray-Based Best Practices and Procedures, guides the reader through several practical concepts on how to develop and implement best practices for laboratory operations. The author has been the director of several cytogenetic laboratories and demonstrates her 25 years of experience by offering a detailed and informative guide for laboratory management.

This interesting book is divided into three sections.

The first section focuses on Good Laboratory Practices and includes guidelines for personnel safety, quality

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management, and preclinical validation studies, which are integral parts of laboratory-developed tests. The second section of the book describes the Best Practices for Staffing and Training. In this section, the author centres the reader’s attention on the necessity of having lab-specific training programmes and comments on the important Six Sigma approach to laboratory improvement. Although accurate results come from good laboratory practices as well as properly trained and competent staff, it is crucial to have Standard Operating Procedures (SOPs) in order to complete tasks or processes safely. This is exactly what the author advises in the last section of the book, which describes SOPs for chromosome analyses, fluorescence in situ hybridization and chromosomal microarray analyses. Zneimer takes into consideration pre-analytic, analytic and postanalytic steps.

From beginning to end, this book provides relevant concepts, procedures and strategies that give the reader a complete overview of good laboratory practices. In addition to providing an excellent guide for setting up a new clinical lab, Zneimer’s book should be considered as a useful guide for any laboratory because it provides a wealth of practical information that can be used on a daily basis. All of the spreadsheets, guides, examples and templates in the book are useful bonus features and represent a valuable legacy of the author’s extensive experience.

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Trinity 2017 | PHENOTYPE | 41

SNAPSHOT

Research Image Competition

This issue’s winner is...

Dr. Peter Canning, Weatherall Institute of Molecular Medicine

This issue’s winner of the SNAPSHOT competition is Dr. Peter Canning!

The winning image is an artistic impression of an antibody (blue) binding an antigen (orange), illustrating a critical component of the immune response in which specifically generated antibodies correctly identify target antigens. The image is based on the crystal structures for Notch1 and immunoglobulin G. The ability of an antibody to recognize and selectively interact with a specific target underpins a large portion of modern biomedical research.

Dr. Canning is a member of Professor Terence Rabbitts’ group in the Weatherall Institute of Molecular Medicine, where the group uses antibodies to develop new therapies, and also to assist in the design of small molecule drugs capable of inhibiting protein-protein interactions within cells.

Win a £50 book voucher kindly provided by Oxford University Press!

SNAPSHOT

Research Image Competition

Do you have an image from, or inspired by your research? Why not enter it in SNAPSHOT? We are now accepting entries for pictures to be featured on the cover of the Michaelmas 2017 issue of Phenotype.

To enter, send images to stefania.kapsetaki@new.ox.ac.uk with a brief description (maximum 100 words). Please get permission from your supervisor before sending any images.

The deadline for the competition is Friday 28th July 2017.

42 | Oxford University Biochemical Society

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Write for Phenotype!

Do you work at the very cutting edge of science? Are you involved in exciting and influential outreach? Are you passionate about communicating your scientific endeavours to others? Then this is the opportunity for you!

• We are looking for contributors of a wide range of stories: research articles, science in society features, career insights, interviews with academics and more!

• The next deadline for article submissions is Friday 28th July 2017.

Work for Phenotype!

We also have a large editorial team, responsible for managing, editing and laying out articles ready for publication - and we are always looking for new members!

Get in touch!

If you’d like to get involved in any aspect of Phenotype, please get

in touch: stefania.kapsetaki@new.ox.ac.uk

Trinity 2017 | PHENOTYPE | 43

crossword

PHENOTYPE Fish challenges you to this latest cryptic crossword! Can you crack it? Answers to last issue’s crossword are given at the bottom of the page. Enter this term’s competition by sending

The winner of the crossword competition will receive their choice of one of the books reviewed in this issue, kindly provided by

your answers to stefania.kapsetaki@new.ox.ac.uk. Entries received before the 28th July 2017 will be entered into a prize draw to win one of the books reviewed in this issue.

ACROSS

DOWN

8. Spokesperson describes part 1. Antibodies recognise Giardia of a trumpet, perhaps? (10) first in mum’s bouillon soup (15) 9. Being impetuous is an irritation 2. A festival of masculine immola(4) tion? (7,3) 10. Disconcerted by having neu- 3. Mix carbon and hydrogen in rons removed (8) container (5) 11. In film, 24’s partner is recast 4. Send telegram to yachts seekas fictional Danish prince (6) ing an electrician’s tool (4,7) 14. The lingo in Kyrgyzstan in- 5,27. Octane combusts in opencludes a pig noise (4) ing scenes of 11, perhaps? (3,3) 15. In a precarious situation, 6. See 23 where one might find an anaesthetised hand or foot? (3,2,1,4) 7. Bleed ill Scot who, confusingly, offers his leukocytes (5,5,5) 18,28. Spooner’s natural spirit has a burden: an accumulation of 12. River running through Pana7 (5,4) ma zone (or maybe Brazil?) (6) 19. Also took off the tail (3)

Answers to the crossword from Issue 26, Hilary 2017: Across: 8.Differentiation 9.Omanis 11.Scenic 13.Testosterone 14.Offhand 17.Dataset 20.Subservience 21.Medial 22.Lie low 26.Bang in the middle Down: 1.Edge 2.If so 3.Teratoma 4.Jedi 5.Diaspora 6.Et cetera 7.Totipotency 10,19.Stem cell 12.Selfrenewal 15.Hustings 16.Nibbling 18.Triploid 19.See 10. 23.Iced 24,25.Wide open 25.See 24.

13. Greek poet’s written about game with no omission: “a chain, 20. Italian underworld figure (one composed of a single subunit, ...” from Monteverdi opera?) sculpt- (11) ed in gold and iron oxide (5) 16. ”. . . used in nice, soft play with 21. EMBO is confused; given new what are combated by 7” (10) pointer to where 7 are produced (4,6) 17. Therefore, my turn to puncture organ where some 7 mature 22. Proton donor will help with (6) absorption of carbon (4) 23,6. Young man to turn in . . . or 24. Pest: one intrudes upon wom- ring for a late-night liaison? (5,4) an (6) 25. ”Loosen up!”: Greek charac26. Signalling molecule produced ter at party (4) following exchange of potassium for sulfur in DNA base (8) 27. See 5

28. See 18 29. During examination, inguinal half-removal of the guts! (10)