Malaria, Cancer, Alzheimer's, Influenza, Measles, Ebola, AIDS, Cystic Fibrosis.....How do we decide what to investigate?

As you start your new year at the UTC, you will be no doubt be aware of the theme that will pervade the first half term: the Global Challenge of Malaria. I am sure that most of you will have a picture in your mind of African villagers dealing with a health crisis in challenging circumstances. I wont think of the prospect of catching malaria while you sleep in your own bed, but you might wonder if you are likely to catch measles from a classmate, or more likely the flu or a cold. We know that certain diseases are restricted to some parts of the world (by restricted I should really say that they are much more prevalent), some are dangerous to infants and some, like Alzheimer's disease are rarely found in adults under the age of 60. It is really important that you develop a perspective on the "significance" of a health problem. This is the theme of this Blog: I want you to be able to decide for yourselves with evidence, which areas of Medicine and Science are important. I also want to alert you to the ways in which new discoveries arise, the impact they might have on the quality of our own and the lives of others across the world. I also want you to understand why sometimes we need to investigate things out of pure curiosity, since it is from this route that many of our current technologies arose in the first place. However, ultimately, research costs money and it is the tax payer that decides how much money we invest in research. And as Benjamin Franklin said: "In this world nothing can be said to be certain, except death and taxes".

The scale of a health problem often dictates the priority given to research and financial investment. As a new University lecturer, a Head of Department will have recruited you partly because you have impressed at interview, but increasingly because you are working on a problem that is seen to be fundable. So what are the priorities of the UK funding agencies? Life Sciences in Universities and Research Institutes in the UK are funded by the Biotechnology and Biological Sciences Research Council (BBSRC), the Medical Research Council (MRC), the Natural and Environmental Research Council (NERC) and the Wellcome Trust. There are differences in their priorities but they all agree that Human and Animal Health are a priority, with new antibiotics and resistance mechanisms, together with ageing research, receiving a great deal of attention. Cancer Research is the primary focus of Cancer Research UK (CRUK), with different Research Centres specializing in the various forms of cancer, eg lung cancer, bowel cancer, pancreatic cancer etc. You will notice that I haven't mentioned malaria or ebola. This is where the challenge of obtaining funding becomes more complex. 

The history of Tropical Disease Research in the UK is an interesting one, and one in which Liverpool has been centre stage. Over 100 years ago, shipping merchants who relied on the health of their crew for their business successes, identified "tropical diseases" as a major threat. In those days Britain's influence was just as global as it is today, but it was in the context of the British Empire, which required a radically new solution for protecting the health of commonwealth citizens, and in particular the various professionals and troops who governed countries that became lucrative sources of produce and raw materials for the increasingly industrialised "West". This led to the foundation of the Liverpool and then London Schools for studying and developing methods for preventing and treating rare, Tropical Diseases. These institutions were also critical in supporting the health of the armed forces during the two major (and many minor) wars of the last century.Today, they play a key role in both educating and providing advice to international organisations such as the World Health Organization as well as controlling the global spread of such diseases, in the context of international travel which is now commonplace in the West. In short the stability of the population of the world as a whole isn't a National issue, it is global.

We mustn't just consider research funding at academic institutions. What are the economic factors behind the growth of the Pharmaceutical sector into one of Britain's largest employers and most successful businesses? (I have touched on this issue elsewhere on these pages, and at my Molecules to Market Blog page). Clearly, just like any other "for-profit" business, a drug company must make sufficient money from the whole process of drug discovery from sales, and this is determined by the cost of development, meeting the regulatory requirements (through clinical trials) and subsequently marketing the drug at a suitable price. Given that the cost of bringing a drug to market can be several billion dollars, the pharmaceutical industry is in many ways a challenging one. It may not surprise you to learn that the focus for new drugs is driven by market size and ability of that population to pay for such high cost new drugs. It is thanks to major interventions by high profile advocates like Bill Gates, that diseases like malaria are now on the agenda of drug companies who would otherwise see malaria treatment as a high risk market.

So research into diseases is driven in part my national and international agenda in different ways and in addition, we rely on the drug companies to find ways of short circuiting the discovery and development phases, in order to keep their costs down. What room is there for serendipity in Life Science research? In respect of diseases, the work on ebola, that I discussed in my previous blog illustrates how a disease caused by a potent virus has begun to unearth new concepts in fundamental molecular biology. The disease affects a very small number of individuals annually, and is a very minor cause of death in comparative terms. It would therefore be unlikely to receive the same kind of funding as cancer research. Nevertheless, I think the work on ebola illustrates that if your ideas are sound and creative, it is still possible to attract the necessary funding, in the right environment, to make important, non-mainstream, fundamental discoveries. It is important for you as our future scientists that you are as well informed as possible, not only about how to do experiments, but why they should be done and to be aware of the forces that drive scientific discovery and remember as Louis Pasteur said: "Chance favours only the prepared mind".

Welcome to the new intake of students and welcome back Y11s and 13s!

This week sees the UTC operating with a "full house" for the first time and I thought I would take this opportunity to welcome you all to the Innovation Blog site, where the activities in the lab and in the wider world of Science are discussed to help you on your journey. My blog allows me to pass on news and thoughts, but it also contains a regular feature: "Molecule of the Month", in which I choose a mixture of topical and fundamental molecules that form an important part of understanding Molecular Biology. You can flick through earlier blogs on the right (RHS) to give you a taste of the kinds of topics discussed. Please pass on the link to friends and family so they too, can see what is going on. I will be posting on the theme for the first half term this week, which centres on the global threat posed by Malaria (the red colour on the globe, top left), and how Science, Medicine, Healthcare and Economics  are being mobilised to address this threat to mankind.

I look forward to your feedback!

Lessons in fundamental Biology from infectious diseases. Ebola part 2

You will no doubt be aware of the developments over the Ebola outbreak in Africa. The arrival of a British nurse back in the UK after contracting the virus, in a high-tech plastic tent (LHS), reinforces the fear associated with such diseases. However, it also serves to highlight the ignorance that pervades about infectious diseases: from simple hygiene, through politics and cultural diversity, to our understanding of the molecular basis of viral and bacterial infections. I want to use this Blog however to draw your attention to the lessons we can learn form such devastating diseases and infectious agents. 

In 1972, Christian B. Anfinsen (RHS) received a share in the Nobel Prize for Chemistry "for his work on ribonuclease, especially concerning the connection between the amino acid sequence and the biologically active conformation". Anfinsen and his colleagues proposed, through a series of elegant experiments, that the information encoded by the primary structure of a polypeptide (i.e. the sequence of amino acids in a protein), determined the three dimensional structure of a given protein. Therefore, if chemical structure determines function, then the amino acid sequence will determine biological function. This is the basis of much of our interpretation of genome data, when we associate the sequence of a gene with its deduced amino acid sequence and then its function. This relationship between primary structure and biological function (or rather conformation) is called the "Thermodynamic hypothesis" or "Anfinsen's dogma" and it has stood the test of time rather well. There are two important caveats: some proteins need a little help along the way to fold into their stable conformation in good time; this is facilitated by a class of proteins called "Chaperones". Secondly, the protein sequence encoded by the gene is not always the same as the sequence of the mature protein, in its functional form. However, I am not going to discuss these two important issues today.

To out the problem a protein faces into some perspective, imagine a polymer chain of 100 amino acids (this logic applies to any polymer) in which there are 99 peptide bonds (linking the amino acids together). Each peptide unit has two bonds around which there is freedom of rotation (I will show you this with a molecular model in the lab): they are called phi and psi (see here for more detail). The probability of the protein "sampling" all of the possible conformations (or as it is referred to: "conformational space"), is so high in terms of possibilities, that it led Cyrus Levinthal in 1969 to suggest that this paradox must imply that protein folding is in some way "guided", since to sample all options would take a time that is longer than that of our Universe. Since E.coli, as I always remind you can complete the process of replication in around 20 minutes, during which time all of its functional proteins will have been expressed, properly assembled and will have completed their function. This poses an interesting question for the biophysicist to explain!

Anfinsen has demonstrated that proteins have a unique conformation that is necessary for function. But what if the protein could adopt two structures with very similar thermodynamic properties? This possibility seems to exist for the prion protein (of mad cow fame). Here, the protein shown right, adopts a stable form which is found in many cells in a normal individual, but which equilibrates to a misfolded (or infectious form) which is also stable. Currently, there is no reason to pin diseases such as scrapie and CJD on anything other than a misfolded protein, which in turn promotes the conversion of the safe prion structure to the infectious form. In short prions defy Anfinsen: there are two acceptable folded states: one is harmless and the other infectious. If we look at the literature in the first half of the 1960s, we also find some landmark work on the ability of proteins to adopt more than one conformation, specifically following the addition of a small molecule. The work of Monod, Wyman and Changeux in France and Koshland Nemethy and Filmer in the US, supported by Max Perutz's elegant structural studies on Haemoglobin at Cambridge, had opened the door for a new way of thinking about proteins and their control (I wont cover post-translational modification here). This work simply shows that a stable conformation can be shifted in the presence of a ligand, but a single (energetically) stable conformation exists in the absence of the ligand (under a give set of conditions). We refer to this as allostery.

When I heard the news about Ebola, I looked up the work of Erica Ollman Saphire, a name that is both unusual, and memorable! One of my old friends from my early days at Sheffield, now at the Scripps Institute in California had published jointly with Erica. I realised that the protein VP40 (called a viral matrix protein) also defied Anfinsen! Using X-ray crystallography, Saphire's group showed recently in a lovely paper in Cell, that VP40 is capable of forming different stable conformations for the infection and replication stages of its life cycle. Put simply, it is able to economise on function by making use of the same primary structure. Similarities jump out in respect of prion diseases, but also begin to pull the rug away from long in the tooth Biochemists like me! Of course, when you read the work, it becomes clear a priori, that conformational equilibria needn't always be to the left or right, but that the sustainable evolution of protein function will make such phenomena the exception rather than the rule.

I recall over a year ago Dr Robert Harrison at the Liverpool School of Tropical Medicine telling me how a relatively small number of proteins in snake venom could immobilise an adult in minutes through haematological or neurological mechanisms. I thought this was pretty amazing. But Ebola virus has only 7 proteins with which to disable a human being who will typically express around 20 000 genes in a complex, interrelated and highly regulated manner! By drawing on this knowledge, perhaps we can turn these potential killers into drugs for the elimination of tumours? In the case of ZMapp (shown as a molecular model on the RHS), the antiserum that was supplied in advance of human trials, was produced, somewhat ironically from transgenic tobacco leaves. It is a cocktail of three humanized monoclonal antibodies raised against key viral components. At the moment the details are sensitive, but the fundamental work of Saphire and colleagues elsewhere, as well as the development of plants for the expression of therapeutic antibodies shows how powerful Science can be in facing a health crisis. In my view this is a shining example of how fundamental research on challenging areas of Biology can be justified and must be supported by countries in the developing world. Since we are as a community of Scientists struggling to identify new therapeutic strategies for infectious diseases, work on pathogens should be prioritized, since they have Darwin on their side and can teach us new tricks that could in turn be our salvation!

From Malaria, AIDS and Ebola, may Good Science deliver us

The title of this blog refers to the line in the Beggar's Litany from around 1600, which I am fond of quoting to my many Yorkshire friends and colleagues:

"From Hull, Hell and Halifax, may the Good Lord deliver me"

The guillotine was in use regularly in Halifax over 400 years ago: not just in Republican France! In fact it was used by the monarchy (well the Royal Mint) to stamp out counterfeiting. When I have time I will tell you about Sir Isaac Newton's other job for the Royal Mint! Getting back to diseases....You will all be aware of the human tragedy playing out on the West coast of Africa, where over a thousand people, young and old have died following infection by the Ebola virus. The outbreak is all too familiar to some of us who watched as the AIDS/HIV story played out in the 1980s

And of course until very recently, Malaria held the inauspicious position as the world's biggest killer. The World Health Organisation (WHO) have only recently determined that Malaria has been overtaken by by cardiovascular disease (that's a topic for later!). Nevertheless, despite the many advances in modern medicine the human race remains highly vulnerable to parasitic diseases and viral infections. I constantly keep thinking that Achilles at least had only two heels that removed his invincibility in battle!

Students returning and newcomers to the UTC will be "welcomed" by an ambitious thematic programme that incorporates experimental classes and enrichment in the Innovation Labs, aimed at providing you with the opportunity to build your scientific skills both technically and intellectually around some of the most challenging Health related problems facing the world today. Before I give you a taster of what is coming, let me say a few words about Ebola virus since it is in the news at the moment.

The picture on the left is one that the BBC are using a lot at the moment. It is a very high magnification image of ebola virus, which has been coloured artificially to enhance our understanding of its properties. Compared with the bacteriophage particles that we have come to know and love (humour me!), the shape is odd, it is more like an elongated bacillus, or rod-shaped bacterium, it is about 10% of the length of many mammalian cells, so it is pretty long. (the AIDS virus (below right, the green spiky sphere ) is quite different, almost spherical with a typical radius of 100nm, which is 10 times shorter than the length of ebola. A typical cell is around 10 000nm. Both viruses have an RNA genome, which means they have to trick the host cell into converting the genome into DNA first before they can begin to replicate, assemble and produce more infectious viral particles. 

The ebola virus has only 7 genes, making it a very efficient beast! The AIDS virus has a few more. The closest relative of ebola is called Marburg virus, which is equally lethal. Why then can these viruses cause so much of a problem. There are two main reasons, one is Biological, but the second reason is just as important, if not more critical and that is Cultural practices. The virus attaches to cells found in the blood stream, fusing with the membrane and then executing a prolific programme of gene expression and replication, leading to an explosive infection that gives rise to a massive bleeding episode, from which recovery is challenging. 

From a cultural perspective, the virus is spread through contaminated food: fruit bats are ebola resistant and therefore they spread the virus through fruit and droppings. Equally, infected animals eaten as "bush meat" are also sources of infection. Finally, families in the major hot spots in Africa have a culture in which sick members of the family are cared for at home, increasing the risk of spread. Poor education and even worse poor adherence to what we would consider normal hygiene routines, is probably at the heart of the spread of infection. The combination of the speed of pathology and the habits of the victims makes ebola, like AIDS before it a major challenge. You may be aware of the difficulty in persuading families to sleep under insecticide treated bed-nets, which would dramatically improve the prevention of malaria! So you must consider these diseases from both the Science and Social aspects.You must decide whether you think it is only appropriate to develop drugs and vaccines that are cost effective, typically for use in the West. In contrast, these potent diseases are prevalent in the poorest parts of the world, consequently resources are generally mobilised after a crisis! Compare that with the recent reaction to swine flu in the UK. If you are interested take a look at web sites like the Bill and Melinda Gates Foundation for more information.

The programme that awaits you at the UTC in September will give you an introduction to the Biology of parasites and infectious diseases, the socio-economic aspects and the opportunity to develop your laboratory skills by exploring model systems first hand. It is through these approaches that we have begun to understand the disease mechanisms, the interplay of their genomes and, importantly, to set some of you on the road to discovering a new generation of treatments for these virulent diseases. I am looking forward to helping you on this journey soon!

The UTC Skills Passport: Capturing Lab Skills

During the first year of the UTC, having welcomed a cohort of new students between the ages of 14 and 16, I wanted to establish first hand just what they were capable of in a laboratory. After all, I had left school over 30 years earlier and my lab class experience, like that of most University academics, has been in delivering undergraduate practicals to new first years and second years in Biochemistry and Molecular Biology for over 20 years (on and off). The Innovation Labs at the UTC are as good as it gets  (and generally better) in comparison with those UK University labs I have experienced, and they meet Industry standards at a level just below GMP (as you can see and from the UTC web site). The rationale behind the UTC movement in general, but in particular at Liverpool, includes the establishment of a programme of lab work that prepares students for the world of Science. The National Curriculum provides students with a knowledge base, whether it is in Maths, Physics, Chemistry or Biology, that is then examined first by GCSE and then A levels. This is clearly an important part of their journey into the world of Universities and employment, but the Liverpool Life Sciences UTC has a wider remit. We are here to produce the Scientists of the future and this, in my view, requires the provision of a set of experiences that are not commonly provided by conventional schools. Moreover, as the current debate over the inclusion of practical science in the National curriculum rages, we are very fortunate at the UTC, to be in the vanguard of those educational institutions where laboratory classes are integrated into our timetable and are additional to the Curriculum classes.

Having organised the equipment and lab space, I wanted to see how the students and staff would respond to an open ended lab challenge: validating the Beer Lambert Law. After all, relationship between the colour of a liquid (or some other measurable property such as fluorescence or radioactivity) and the molar concentration of the chromaphore form the basis of all diagnostic tests. From my own perspective, the class experience would allow me to see how the students responded to the challenge. To give you more of an idea of the lab, there are around 100 students (either 14 year olds, Y10s, or 16 year olds, Y12s). They are allocated a bench between 3/4 and they have access to the necessary reagents (in this case a copper sulphate solution or a microbiological dye such as crystal violet at a known concentration). The students are asked to use pipettmen (colloquially we call them Gilsons (old habits!, but they are actually Finnpipettes from Thermo Fisher), tips ad any appropriate vessels they might need from the lab, to validate the relationship using a spectrophotometer. They are given no detailed instructions, but are asked to devise an experiment (essentially a standard curve) based on the relationship, which is on a screen in front of the class. They are given half a day to carry out the task and teachers and two of my PhD students were on hand to advise.

Success!

The outcome of this was extremely positive. The students naturally "bounced off the walls" a little at first, but a group momentum soon took over, and by the end of the day, all students had performed the task (with a range of levels of achievement, as expected). I wont go into details, but I was able to satisfy myself that both Y10 and Y12 students could work in small groups, they could plan an experiment and they could make measurements in an appropriate manner without being overwhelmed by the challenge. Importantly, their first exposure to physical chemistry equations of this kind and plotting data appeared not to be a major barrier (which I had thought would be the case). With this experience in hand, I decided that I could be more ambitious  (especially at the Y12 level) in my planning and classes could be more challenging than I had originally anticipated. 

Discussions with University academics, employers and parents as well as the students, of course have led to one very simple conclusion: the skills we teach in the Innovation labs must prepare students for life. For example, Life Scientists in the Pharmaceutical Industry are just as likely to need to prepare samples for mass spectrometry or X-ray crystallography as they are for enzymatic analysis or an ELISA assay. For this they will use Gilsons, microcentrifuge tubes (Eppendorfs), bench top centrifuges and sterile/filtered and high quality reagents. And they may often have to assemble reactions in a total volume of 10 microlitres (for the uninitiated, this is about the volume of a pin head). It is not unusual for new Molecular Biologists to question whether there is anything in the tube (myself included years ago!). For all of these manipulations, the operator must take great care, must learn how to calibrate their instruments and must understand how to obtain accurate results in a reproducible manner. 

There are some significant differences in the approach employed in the Innovation Lab programme, which I have given the acronym REAL: for Research Enhanced, Active Learning (the addition of Active was kindly suggested by my good friend Dr. Rob Rule). In short, students learn by planning and conducting every experiment as a research project in which they are responsible for all stages of the work. I have provided students with a sample planning template (the work flow diagram Template in the sidebar) and a series of skills sessions in which they are introduced to methods, standard and advanced items of equipment, safety aspects, sample storage advice, Bioinformatics and literature/Internet searching and quantitative data analysis methods. The skills training is embedded in the lab sessions (although a period of induction for new students forms part of the start of each academic year), but it also forms the core of our "Skills Passport", which provides the student with a portfolio of skills that s/he can take away from the UTC upon "Graduation". The Skills Passport is the culmination of the experiences in the first year at the UTC and follows consultation with a range of Academic and Commercial Scientists and Science managers. I shall explain it in a little more detail below.

The use of micro-methods is at the heart of contemporary laboratory science, but it is not the only difference between school science and that practised in University research labs and Industry (which I will refer to as real world science for simplicity). Contemporary separation sciences that have (mostly) replaced crystallization and filtration including liquid chromatography (simple and high performance), electrophoresis on thin gels and the use of recombinant DNA technology to clone genes and obtain customised fermentation products for research or for Biotechnological and Biomedical applications. Then there are some more generic skills: the use of ICT, the ability to communicate through presentations and reports and a range of extraction procedures, microscopic methods, many of which have become standard practice in the real world, but for many reasons are not taught or demonstrated in schools. 

In consultation with senior experimental and theoretical scientists around the world, including a number of Nobel Laureates, Fellows of the Royal Society, Senior Team Leaders in Industry and authors of high profile Science Texts, I have distilled down the key skills that UTC students will take with them, following a programme of assessments over 2-4 years. These are summarised in a document that also gives a summary of some of the concerns expressed by employers and academics, it is in the sidebar under Skills Passport Background. I would welcome any feedback. But I would like to thank the following for their (often lengthy and always constructive suggestions, ideas and comments. Without their input the Liverpool Life-sciences UTC Skills Passport would have been much less comprehensive. In addition to this independent group, I am also grateful for all of the suggestions from our sponsors and partners, who provide an invaluable and constant stream of suggestions and advice.

The details of our REAL programme and project plans for the coming year will feature in two subsequent Blogs

The core skills are summarised (and taken from the linked document) below:

Foundation Skills

• Keeping a legible lab notebook
• Planning experimental work
• Constructing and testing a hypothesis
• Understanding precision, accuracy and the need to repeat experiments
• Behaving appropriately, observing health and safety rules and regulations
• Understanding the key concepts in chemistry: gravimetrics, volumetrics, molarity and pH
• Computer literacy
• Oral, visual and written communication

Core Lab Skills 

• Using and maintaining a "Gilson" (or equivalent)
• Safe use of a centrifuge
• Sterile technique: microbes and cultured cells
• Understanding the storage requirements for experimental samples
• Visible and UV spectroscopy Using and calibrating a pH meter
• Using and respecting the cleanliness of a balance (top pan and fine balances)

Advanced Laboratory skills

• Preparation of nucleic acids (genomic DNA, plasmids and RNA)
• Microbial cell disruption for protein preparation
• Nucleic acid and protein gel electrophoresis
• Chromatography (ion exchange, affinity, gel filtration and HPLC
• Basic plasmid transformation and mini-prep methodology
• Designing PCR primers and carrying out PCR
• Knowledge of a method of molecular cloning and restriction analysis
• Use of a light microscope and fluorescence microscopy

Professional Awareness

• Appropriate and traceable storage of reagents and materials
• Standard COSSH regulations
• General Laboratory safety
• Safe use and disposal of hazardous materials including radioisotopes
• General laboratory management and design

Thanks to those who are not affiliated to our partners, they are (in no particular order) and apologies for any omissions, there were  some inevitable compromises!

Professor Sir Richard Roberts FRS and Nobel Laureate (USA)
Professor Steve Yeaman (Newcastle)
Dr. Clive Price (Lancaster)
Professor Emeritus Nick Price (Glasgow)
Professor Phil Ingham (Singapore)
Professor David Coates (Dundee)
Dr. Nick Brewer (Dundee)
Professor Andy Sharrocks (Manchester)
Dr. Paul Shore (Manchester)
Dr. Iain Mattaj (EMBL)
Dr. Doug Gjerde (Phynexus, USA)
Professor Chris Smith (Cambridge)
Professor Kathryn Lilley (Cambridge)
Professor Tony Wilkinson (York)
Dr. Mark Dickman (Sheffield)
Dr. Mark Paine (Liverpool, LSTM)
Professor Neil Hunter FRS (Sheffield)
Professor Jeff Green (Sheffield)
Professor Emeritus John Bryant (Exeter)
Professor Malcolm Press (Birmingham)
Professor Peter Nixon (London, Imperial College)
Professor Simon Oldfield (Leicester, DMU)
Dr. Gareth Lycett (Liverpool, LSTM)
Dr. Antal Kiss (Hungary)
Professor Chris Barratt (Dundee)
Professor Emeritus David Lloyd (Cardiff)
Professor Alister Craig (Liverpool LSTM)
Professor Richard Pleass (Liverpool LSTM)
Dr. Paul Andrews (Stem Cell Solutions, Dundee)
Dr. Simon Baker (Bioline/Meridian)
Dr. Mark Powell (MP Scientific)
Dr. David Dryden (Edinburgh)

Personalized medicines, allergy and a renaissance for thalidomide?

This week's announcement that funding will be made available to sequence 100,000 cancer genomes, comes after the announcement two years earlier that 100,000 individuals would have their genomes mapped in Saudi Arabia. The logic is that an knowledge of the complete genome sequences of large cohorts of both healthy and sick individuals will significantly improve clinical decision making in respect of the prescribing of drugs. This approach has become known as personalized medicine. Currently the treatments available for serious illnesses are administered on an empirical basis, using patient assessment procedures that are often viewed as inadequate, with close monitoring of side effects essential in order to evaluate the efficacy of the medication. You will be familiar with the "allergy" that around 10% of the population exhibit towards penicillin: this is one example of how differences between the "genomes" of individuals can impact on the mode of action of a drug. The solution is in this case to prescribe an alternative class of antibiotic.


The most effective antibiotics and antiviral drugs (I wont deal with vaccines here) are targeted at the invading organism or virus or a distinctive feature of their molecular pathology. Thus penicillin interferes with essential steps in bacterial cell wall metabolism, ciprofloxacin targets the terminal stages of genome replication in bacteria and others act to specifically block bacterial protein synthesis. Importantly, these drugs have no effects on the related processes in us. Perhaps the best known antiviral drug is aciclovir (often marketed as Zovirax), used to treat cold sores and chickenpox. This compound acts by inhibiting the replication of the viral genome (targeting the DNA polymerase). However, because antibiotics and antivirals target the infectious agent and not our own physiological processes, they are somewhat special and although the issue of resistance to antibiotics is topical here, I will not discuss these drugs further in this Blog.

Drugs that we take to relieve pain (e.g. aspirin and paracetamol) are considered safe enough to be purchased over the counter (although some individuals can suffer adverse reactions, and paracetamol must be taken at doses that accommodate the rate of its metabolism in the liver). Many of these types of drug are derived from flora that have been found over many years to bring relief to a wide range of illnesses and allergies. It was around 60 years ago that drug discovery became a more "serious" commercial venture and many of you will have heard of the drug Thalidomide. This was a molecule that was launched onto the market for the alleviation of the discomfort (often extreme) associated with the early stages of pregnancy. This kind of medication is referred to as an anti-emetic (i.e. it stops you vomiting). It seems timely to remind you of the Thalidomide story, since an exquisite set of molecular structures have recently been published in the journal Nature, which shed considerable light on the mechanism of action of this somewhat "notorious" drug.

The legacy of thalidomide is somewhat controversial and has recently been

linked to the tragedy of the Nazi inflicted holocaust. There is no doubt that the German company Chemie Grunenthal filed the original patent, but the controversy arises around the similarities between a family of compounds that formed part of the forced human drug trials at a number of Nazi concentration camps. These compounds, it has been suggested, included thalidomide and and the absence of the original "trial" data leave an unpleasant "smoking gun" in the archives. These issues were raised two years ago when the company unveiled a memorial to the victims of their drug. Getting back to the Science, but more specifically the chemistry; this is where the legacy of thalidomide provide key lessons for any future drug development programme. The initial topic was a consideration of the value of personalized medicine. One of the aims of this new approach to therapy is to alleviate (if not eradicate) unpleasant, and the occasional life-threatening side effects of drugs.  Following the dramatic events during the early 1960s, when thalidomide (or in the UK it was marketed by the drug company Distillers as distaval) was linked to birth deformities, investigations began into the cause of these defects. It should not be forgotten that drug companies in Europe, including the UK made a lot of money from sales of thalidomide. However, it was, in large measure the result of a tenacious scientist Frances Oldham Kelsey working for the early stage organisation of drug regulators in the USA, that we now know as the FDA (or Food and Drug Administration), that thalidomide distribution was halted.

Ubiquitin

The compound taken by pregnant women in the early days was a racemic mixture (the word racemus means a bunch of grapes in Latin, so I can only assume it was applied to chemicals whose optical properties were neutral, since they contained an equal amount of the right and left enantiomers, like a your left and right hands, mirror images or opposites) of the two chiral forms of the drug. You can read more on by linking to the nicely concise and well written description of the chiral centre in thalidomide by Brent Iverson at the University of Texas here. The target (i.e. the protein molecule (in this case) that captures the drug) is a protein called cereblon, which is encoded by the gene CRBN in humans. This protein forms part of a larger protein complex which catalyses the addition of a very small protein called ubiquitin (top LH image). As its name suggests, it is present in all cells in eukaryotes and, when attached to a protein in the appropriate way by the enzyme complex ubuquitin ligase (ligation is the process of tying two things together: a ligature in medicine). Cereblon is one component of the ubiquitin ligase called (sorry about this!) cullin-4-containing E3 ubiquitin ligase complex CUL4-RBX1-DDB1, or CRL4 for short! In short, the addition of ubiquitin can target that protein for degradation, relocation or some other changes in function. 


The work from Nicolas Thoma and colleagues published in the prestigious journal Nature combines X-ray crystallography and a range of other Molecular and Cell Biological experiments. They show not only the site of interaction of the drug thalidomide (and two variants), but they also propose a mechanism for its action. The schematic diagram on the left shows thalidomide occupying the CRBN (cereblon) "active" site. The diagram below it, reveals that this is the site into which the protein MEIS2 binds. The presence of thalidomide blocks the MEIS2 interaction (remember from my blog on dissociation constants that most molecules "exchange" from their binding site, so in this case there will be some MEIS2 bound, but depending on the overall dissociation constant, there will be less MEIS2 bound than in a cell without thalidomide. This type of "inhibition" or blocking is called competitive inhibition: the two molecules compete for the same binding site. Recall that ubiquitin ligation can lead to protein degradation. So the inability of MEIS2 to become ubiquitinylated means it hangs around in the cell too long. This protein is a transcription factor and it is therefore firing genes off inappropriately. Therefore the consequence of administration of thalidomide is a wider de-regulation of gene regulation. The authors go on further to show how other regulators are misfiring in the presence of thalidomide and similar drugs. In summary, thalidomide interferes with a master control system in the cell. This system plays important roles on several occasions in both infant and adult life stages. Unfortunately, by administering thalidomide to pregnant mothers, the drug interferes with a a key set of events that are required to programme the genes that determine the normal development of limbs, hence the disastrous consequences. But there is an up side to thalidomide.

Thalidomide is one of those compounds that crops up now and again that has potent effects on Biological systems and it is proving valuable in the treatment of diseases in later life. It has been used in the treatment of leprosy and in certain myeloma cancers. You can hopefully see how a drug can appear effective if the trials are not conducted correctly. In the 1950s and '60s we had only a rudimentary understanding of the relationship between genes and development. As we embark on the sequencing of 100 000 genomes, let us not forget that it isn't the sequence of the genes alone that is important, but it is our understanding of their encoded function. Moreover, it is vital that we continue to investigate the interactions of the encoded products (proteins and RNA) and the relationship of gene function in different tissues and at different stages in physiological development. These challenges are much more daunting than the sequencing experiments (not they aren't in themselves a considerable challenge!) and require experimental creativity combined with Bioinformatics at levels that transcend today's methodologies. This is what makes the education of young scientists so important.

ἀνάφάσις: it's all Greek to me? Molecule of the month: the anaphase promoting complex

Paul Andrews/University of Dundee

The remarkable picture on the left was taken by a good friend of mine, who, not long after his PhD in my lab at Sheffield became the microscopy imaging maestro at the University of Dundee, one of the UK's leading Life Science Departments. Paul Andrews now heads up his own Stem Cell consulting company, Stem Cell Solutions. His beautiful image shows migration of sister chromatids, which have just split from their parent chromosomes, to the opposite "poles" of the cell prior to completion of either mitosis or meiosis. You can read about anaphase at the wiki site and the whole sequence of events during the cell cycle here. The pioneering work of scientists that include the 2001 Nobel Prize winning trio of Sir Paul Nurse, Tim Hunt and Lee Hartwell, opened the door to our understanding of a molecular process that hold one of the major the keys to understanding and ultimately managing cancer. The controlled division of cells is at the heart of this disease and considerable efforts have been, and continue to be made, in elucidating the core mechanism as well as the network of associated molecular events in cell division. 

The cell cycle (RHS) is described in more detail at the above links, but briefly comprises 4 major stages. G1 (or the first Gap phase): this is a period of preparation for S phase, in which synthesis of DNA occurs. This is followed by the second Gap phase, which ensures the genome is ready for Mitosis, the phase that leads to cell division. During these phases, there are mechanisms of surveillance or quality control that make sure everything is present and correct for cell division. These processes are called checkpoints and are a major focus of cell cycle research. You can read more here at Boundless. It is the process of Mitosis that we are concerned with today (I will reserve meiosis until another time). Mitosis itself comprises Prophase, Metaphase, Anaphase and Telophase which result in Cytokinesis. Another set of intimidating terms, I'm afraid: Pro comes from the Greek, before (think pro-logue, pro-active). Meta in this context means after, or the following phase (metamororphosis, following a change in shape: you will have heard me use this word in the context of the life cycle of the mealworm, or I can thoroughly recommend the short story of the same name by Franz Kafka). Ana comes from the Greek for up, specifying in this case the polar movement of the chromatids, and finally Telo relates to completion or the end of a process (again from the Greek, remember telomere, the end or tip of a chromosome). All of these words are prefixes that allow us to "organise" the steps of Mitosis, which is itself derived from the Greek for a threading (Mito) process (sis) and for completeness Meiosis means a reducing process since the chromosome number is halved. Finally, Cytokinesis is the result of all of this classical activity and this is derived from Cyto (meaning a container) and kinesis which means movement (think of kinetics from your Physics and Chemistry).

This month, following years of effort and a number of intermediate publications the group of Professor David Barford at the Institute of Cancer Research in London (and now at the Laboratory of Molecular Biology in Cambridge) have determined a molecular structure for the Anaphase Promoting Complex, or APC. The significance of these results have been nicely summarised by Ian Foe and David Toczyski in a Nature News and Views article, and their schematic diagram for this complex is shown left. The protein complex comprises 13 different polypeptide chains and represents not only a major advance in our understanding of this giant enzyme, but also advances our technology in the analysis of large multiprotein complexes in general. The team used insect cells in culture combined with the multibac cloning system that allows the complete set of genes (or more accurately the open reading frames) to be expressed simultaneously and purified for analysis. 


The determination of the structural details of a complex of this size requires the use of many techniques, but one of the most powerful methods is called cryo-electron microscopy, in which single particles are analysed at low temperatures and their resultant contours mapped against data from other X-ray studies and the structures of similar proteins (inferred from sequence and functional similarities). The importance of mass spectrometry, which is becoming a key method in projects of this kind, should also be mentioned in dispatches. The structure on the RHS is too small for you to observe the detail, but if you focus on the representation of structural elements (the coloured cylinders are alpha helices) they are mapped onto an envelope of electron density that is derived from electron microscopy. This not only defines the "molecular envelope", but begins to give us insight into the internal structure of APC. This image is taken from the Nature paper recently published by the Barford lab.

Cancer cell division, Paul Andrews
(University of Dundee)

How does this structural work help? I have always been impressed by the beauty of structural biology and the structure of APC is undoubtedly a biochemical tour de force. Similar landmarks in recent years have included the ribosome and the RNA Polymerase (PolII) machinery from yeast. However, there must be a value in these structures beyond scientific aesthetics. This is now the challenge that must be addressed. The understanding of the sequence of events and the specific role played by each of the APC polypeptides in anaphase will undoubtedly follow. We will then be faced with understanding the catalytic events, which are much more of a challenge, and then we will try and establish whether we can interfere with this process in a predictable manner. Ideally we would like to be able to modulate the cell cycle in a controlled and tissue specific way, in order to inhibit unregulated cell division in cancer (as shown in another or Paul Andrews' lovely images, top left). So, the determination of the structure is indeed a momentous moment, but many would say that this is now where the work begins. As students of molecular biology, we must assimilate and understand these elegant experimental structures, but we must design experiments to test their significance: we must pick up the baton now and run with it. I look forward to watching the impact of this work over the coming years and hope it stimulates an interest in the cell cycle.