Molecule of the Month for February

This month we celebrate water; I like this quote from one of Hungary's (and in fact the world's) greatest Biochemists (Albert Szent-Gyorgi): "Life is water dancing to the tune of solids". Superficially, water is one of the simplest of molecules found in all living organisms, yet at the same time one of the most versatile; and a molecule that lies at the heart of Biology. As you can see from the diagram left, water in solution makes full use of its Hydrogen Bonding (1) potential, and this is an important aspect of its role in Biology. But first, where does it come from? We ingest water with our food and we produce water as a bye-product of the oxidation of nutrients. The final step in the catabolism of glucose, after glycolysis, the Krebs Cycle and Oxidative Phosphorylation (which generates energy in the form of ATP) is the reduction of oxygen to water.

"Water, water, everywhere, nor any drop to drink". We believe that life first emerged in the oceans (which are mostly water), which is where we inherited this dependency on water for the Chemistry of Life. The evolution of mechanisms to preserve and capture water in desert regions and to maintain liquid water under the ice, are remarkable adaptations that Sir David Attenborough discusses regularly in his books and documentaries on Life on Earth. I am sure you are well aware of these issues. If not, make sure you watch everything he has made! I particularly like his 10 minute radio podcasts on evolutionary anecdotes (look on the Radio 4 website).

Back to chemistry! Water molecules form a structured network owing to the non-linear arrangement of the OH bonds. This leaves water molecules

polarised and therefore able to form a Hydrogen bonded networks (left). However, these are weak interactions that can exchange: so we say that water molecules form a dynamic network of hydrogen bonded interactions: this is partly responsible for its unusual physical properties. The importance of this network is found in protein interactions with ligands: for example enzyme and substrates or antibodies and antigens. In order to appreciate this we have to consider the phenomenon of Entropy. 

Clausius. Gibbs, Boltzman and Maxwell (as well as others) are amongst the early pioneers of our understanding and formalisation of Entropy and the second law of thermodynamics. Entropy is often described a a measure of the disorder of matter or a system. The Universe tends to disorder (a little like an adolescent's bedroom or my research bench) and this is known to be energetically favourable. So, when a small substrate molecule interacts with and enzyme, it has been shown that this is often entropically favoured. Like a dog shaking off the water when it emerges from a pond, as a molecule binds to a protein, water is dispersed, giving a net, favourable Entropic change and thus promoting the interaction with the substrate.

Water not only provides the aqueous environment that solubilises cellular molecules, it also provides the foil to oily compounds that provides the cell with its plasma membrane. The favourable interactions between hydrophobic molecules and their "rejection" of water molecules, are central to the folding of proteins: where a soluble protein is polar on the surface and oily in the core. However, a cell membrane comprise polar phospholipids, that are oily in the core and polar on the outside and inside surfaces. Think about the proteins that straddle these membranes, they tend to be the inverse of the soluble proteins: they are oily on the outside and polar in the middle! They often provide a polar channel for communication between the inside and outside of cells. We call these channels, they include channels for ions and hormones, and sometimes even water: such as Aquaporin, which is found in the kidney for obvious reasons? This is a taste of the role of water in Biology, I haven't touched on water in a more global context, or indeed as a key molecule sitting in between metal ions in the active sites of enzymes, it is a truly exceptional molecule and one that underpins most aspects of the Life Sciences.

Work Flow Diagrams as an aid to planning

If you set out to bake a loaf of bread, go on a holiday, get ready to run a marathon or revise for an examination, there is always an element of planning and organisation involved. The better prepared you are the more likely the outcome will be under your control. So if you are travelling to the USA for the first time, you will need to organise tickets, a passport, a visa, dollars or some form of credit card, accommodation, onward travel and possibly arrange tickets for the opera etc. If you want to travel to the USA at short notice, then there are a number of consequences. Without a passport, you will be unable to travel at all, unless you organise a 1 day passport, but for normal passport applications, this may take between 2-8 weeks. You will need a visa, but if you have internet access and online credit you can organise this the day before. As you can see, some things require planning and organisation. 

Last week, we decided to repeat the bio-prospecting experiment using your plant extracts, since the top agar plates were, in many cases, less than perfect, which made it very difficult to interpret your results. So, by repeating the experiment, hopefully this week we shall see an improved outcome. It was while we were starting the day in the lab, (the time I ask you all to discuss the day's lab work with your colleagues and come up with a plan), that I realised a Work Flow Diagram (WFD) might be the way to ensure you organise your experiment and in addition, provide you with a framework for noting down any observations as you go through the day's protocols. I was surprised to find that you all rushed into the experiment without any serious planning: this is a recipe for disaster!

I went through the general principles with a number of groups and realised that a Work Flow Diagram should form the first activity in the morning. As you know by now, I don't run the innovation labs like a structured class practical, in which all reagents and solutions are provided, rather you have to plan your experiment and obtain the materials. To help you with this we shall make use of the WFD in every lab session and it must be approved before you get started.

What should a good WFD look like? The diagram should include a brief title, the overall aim of the experiment(s), the sequence of steps which should include any key pre-incubations (e.g. pre-heating agar plates, thawing frozen samples, preparing fresh reagents, ensuring you have tubes ready for aliquoting samples etc.) You might construct a Table to help you organise the composition of multi-component assay mixtures (where appropriate), and you might work out the dilutions of samples in order to ensure the data that emerge cover the appropriate numerical range. 

I am keen for you to develop a style of your own, but as you will appreciate, a typical WFD will have some common features and some specific features that relate to the experiment and the materials. There should also be space on the WFD for noting observations, which may include suggestions for improvements, or steps that can be eliminated etc. It should be a live document! I will be asking you to produce a WFD in all classes, so start thinking about the way you will approach this. We will then try and migrate the best ideas to an iPAD format, in collaboration with the Studio.

The importance of experimental failure

It is only natural for us to want to succeed in our endeavors, but sometimes failure is more valuable. As Sir Winston Churchill once commented: "Success consists of going from failure to failure without loss of enthusiasm". Replace "Success" with "Science" and you have your future as a Scientist in a nutshell! I am sure that Andy Murray would agree that this philosophy applies just as much to sport, or indeed any creative activity. Failure is simply an outcome that is unexpected and often unwanted. But it may give you an insight into your lack of appreciation of the complete set of variables associated with your experiment.

1         2         3        m

When we set out last week to investigate the composition of the mealworm larvae, the outcome of the manual grinding and extraction was a little disappointing. I expected that grinding would release a significant amount of protein, and indeed the first centrifugation looked very promising. After centrifugation, the Falcon tubes had 4 layers (from the bottom up): exoskeleton (brown), disrupted cells (pale grey), around 30ml yellow supernatant and finally a fatty layer floating on the supernatant. Typically, a viscous yellow supernatant would be protein rich. However, looking at the samples run on SDS PAGE (right) suggests that the protein concentration is low, with one high molecular weight protein predominating (see lanes 1 and 2). Those of you who took spectral readings noted an absorbance maximum around 450nm. So whey was the "proteome" so simple and the total protein concentration so low? 

Some of you attempted to fractionate the sample using a Q Sepharose column. A brown colouration at the top of the column at low ionic strength (PBS buffer) was displaced by 1M NaCl. I thought this again looked promising, but SDS PAGE revealed a protein concentration too low to easily detect by Coomassie Blue staining. So what do you do under these circumstances?. Well this apparent failure, led us to see if we could concentrate the protein using ammonium sulphate and acid precipitation. As you can see left. this did do the trick (to some extent, and ideally, we should have dialysed the samples before loading, to neutralise the pH and remove the salt), and indeed confirmed a lower than expected protein concentration in the extract the supernatant, as before. We still see a major band at high molecular weight and little in the body of the gel.

M      1       2       3

I was bothered that the larvae contained a major protein and little else and I also wondered why the supernatant was so viscous. The lipid was largely removed, but I wondered about nucleic acids: DNA and RNA. So Michael ran an agarose gel and stained for nucleic acid with ethidium bromide. The first impression is that there is high molecular weight DNA (top of the gel) and a smear of RNA (which at this stage I expect might be rRNA, but this needs further work). Since our starting material was freeze dried,  the high molecular weight of the DNA is a bonus (it is indicative of minimal degradation), but the RNA looks to be degraded. The salt in the samples is probably the reason for the "half smile" of the fuzzy RNA bands. Lanes 1, 2 and 3 contain different amounts of sample, nonetheless, it is clear that the supernatant contains significant amounts of nucleic acids as well as protein. This now presents us with an opportunity to use the mealworms for a wide range of experiments in proteomics and genomics.

We have systematically begun to investigate the potential of the mealworm, Tenebrio molitor as our model organism. I believe the results are very encouraging, but I will now look at similar experiments in Drosophila and mosquito for comparisons and methods. The most important aspect of these experiments, is the approach to research in which an experiment is carried out, the results evaluated and experience and knowledge brought to bear on the outcome. The follow up experiments we carried out begin to bring insight into the investigation, but the following questions remain. Why is the proteome so limited? Do we have levels of degradation as a result of using lyophilised material? What is the origin of the brown colouration? The spectroscopy measurements may give us a clue. These are the questions we shall investigate in the coming weeks. 

The message from this is that we failed to obtain high yields of a diversity of proteins from the larvae, but it may be that our methodology is inefficient or our source material is degraded (or prone to degradation through the day). It is also possible that the protein content of the larvae is limited and the high molecular weight band is a storage protein with some developmental role. The unexpected outcome is that the larvae are a good source of genomic DNA, which opens the door to Molecular Biology based studies. This is an example of serendipity, which I shall discuss in due course.

What's in a genome?

I was discussing the difficulty in using digital sequence data to distinguish one mammal from another in the lab classes last week. The power of the BLAST algorithm is that you can look at individual genes and see how similar or different they are across wide ranges of sequenced genomes. In fact we often use BLASTP (where P stands for protein), in order to compensate for codon usage differences, rather than BLASTN (where N stands for nucleotide, or nucleic acid). If we BLAST any histone sequences, we immediately confirm the close similarities between eukaryotes, or we say that these proteins are highly conserved in Nature. We can also see patterns of conservation distributed evenly throughout a sequence in some cases; examples of almost identity followed by a region of almost a total mismatch are also found in some searches. These patterns of conservation and divergence provide clues to gene and protein function and the shared functions of genes and proteins that form a key part of the story of Evolution.

However, if Darwin had only used Bioinformatics, rather than observation of living and preserved organisms, maybe it would have taken longer to deduce his theory of Natural Selection in order to propose an explanation for the Origin of the Species? And maybe George Orwell had a good point when he wrote in Animal Farm: "The creatures outside looked from pig to man, and from man to pig, and from pig to man again; but already it was impossible to say which was which"

It is the combination of experimental observation and in silico analysis that brings us the greatest insights into the relationships between living organisms. The similarities and differences observed in a BLAST search, should encourage us to think of the significance in Organismal, physiological, behavioural and molecular terms. After all as Scientists, trying make sense of the complexity of Life and the Universe, we need all the help we can get!

This brings me to Synthetic Biology. If we are to harness Biology in a systematic way, we must use the rational methods of Synthetic Biology (where we combine Engineering standards and protocols with recombinant DNA methodology) in order to test ideas and hypotheses not only to create new opportunities for improvements to to our Health, our economy and to improve the quality of our lives in general, but also to expose our ignorance of Biology, so that we can establish the truth in Nature.

Monkey Business: what does it all mean?

I am sure many of you read or heard about the new technological breakthrough that enables scientists to create apes with specific genetic modifications. As the Guardian put it:

         Genetically modified monkeys created with cut-and-paste DNA

So what is "cut and paste DNA" and how does this differ from the experiments to clone Dolly the sheep? The phenomenon that has been harnessed for modifying monkeys is called CRISPR which stands for Clustered Regularly Interspaced Short Palindromic Repeats. So what does this mean? It is a genetically programmed system found in most bacteria that allows specific DNA sequences (genes) to be inserted at a specific location in a chromosome, this means that a gene for a particular disease can be engineered into an organism that is very similar to humans. This allows us to test drugs in a better controlled setting. There will be those who consider this a dangerous step and those who see it as a major breakthrough. We looked at the history of model organisms in an earlier blog, so how does this development compare? What are your own views?

The problem with the use of model organisms or cultured human cells in the evaluation of new drugs lies in the differences between species and not the similarities. While there may be some drugs that target functions that are common to mice and men, there are some diseases that are organism specific. This is particularly true for pathologies associated with the nervous system and in particular the brain. Clearly, any unnecessary use of animals is to be avoided, but if we are to rely on data from animal experiments, we must be sure that we do not repeat the mistakes of the past: recall that thalidomide was successfully tested on rats and rabbits and given the all-clear. Any advances in developing a deeper and robust understanding of human pharmacology is to be welcomed.

How did Dolly the sheep get cloned. In Dolly's case, the nucleus of a set of breast cells grown in culture was carefully removed and the egg cell, reprogrammed with nuclear DNA. Shortly after, it was clear that this approach worked, but did epigenetics cause the cells to lose their DNA: Maybe we may never know? Dolly grew past pregnancy, but ended up with major chromosomal defects, caused in part by a loss of epigenetic signals and abnormal activities of the chromosome maintenance enzyme: telomerase, which prematurely aged Dolly. 

Molecule of the month January 2013

This month's molecule isn't in fact a single molecule, but rather a class of molecules referred to as both antibodies and Immunoglobulins, or Ig for short. In fact Ig molecules come in several categories (or classes): IgG, IgA, IgM, IgE and IgD. However, they share some common structural and functional features. The classic structure of an antibody molecule (IgG) is shown on the left and is often considered a Y shape. The top arms of the Y provide two surfaces for interaction with the antigen, or invading material which can be a cell, a virus, a foreign object, as long as it can be "recognised" by the so called Fab region (which stands for Fragment antigen binding). The Fc region (of Fragment crystalline) is part protein and part carbohydrate (or Glycan) and is the part of the molecule (or moiety) that interacts with blood cells called B cells or B lymphocytes. The B refers to the receptor proteins on the cell surface that bind to the Fc region of an antibody when the antibody is engaged with an antigen.


The immune response is triggered when an antibody recognises an invading antigen. The two arms embrace the antigen and the IgG (typically) then has an increased affinity for the B cell surface receptor (right). A series of reactions ensue and the antigen is eliminated. (This will be the topic of a later Blog). The antibody molecule is an excellent example of structure related to function. The arms recognise the antigen and the Fc region recognises the cells that eliminate the antigen: antibody complex.

But, I hear you ask, don't we need an antibody for every invading antigen? Re we born with a set of antibodies that protect us from influenza,E.coli, asbestos fibres, cholera etc? Well, we are born with a "library" or antibodies, but only a few molecules of each and we acquire antibodies from our mother's milk. But we also have a machinery that allows us to generate more antibodies than we originally inherited. This is a complex area of Biology and I will only give you a taster. First of all, when an antibody binds to an antigen and then to a B cell. the B cell (which is an antibody factory) produces lots of that specific antibody (we call this clonal expansion). This is one important part of vaccination (more in the future!). We are also able to mix and match segments of the genes that encode the antigen recognition component of an antibody and add it to the common Fc region. So like the chap on the left, we have one head and body (Fc) and a whole set of different hats (each one having a different appearance and recognising different antigens.


What about IgM, Ig E and IgA. These are more specialised antibodies and they look a little different. IgE looks like IgG, but is responsible for your allergic response, recognising pollen grains and triggering histamine release from Mast Cells. Ig A is two copies of an IgG (a dimer) that is found in mucous, saliva, tears and milk. Finally, IgM is a monster (see right) it is a pentamer of Igs, which forms rosettes of antigens. The fundamental unit is a little like IgG, but it assembles into this five membered ring. It is thought to be responsible for the early stages in the immune response, arriving on the scene before IgG.

So, what a great set of molecules and of course, at Cambridge and Switzerland, Cesar Milstein and Georges Kohler discovered a way of making specific antibodies in the lab in the early 1980s. These monoclonal antibodies have been harnessed recently to attack cancer cells. They are the biggest selling therapeutic molecules in the world today. Ever since Rodney Porter (at Oxford, left) and Gerry Edelman in New York worked out the building plan of antibodies in the late 1960s, these molecules have gone from interesting Biology, to useful lab reagents to exciting new therapeutic agents. I hope this has whetted your appetite to find out more!

No STEM without roots

The recent visit of Mersey STEM and the teachers' interest group, together with the wide ranging activities aimed at promoting Science, Technology, Engineering and Mathematics in schools, colleges and Universities, made me realise that it is over 10 years since Sir Gareth Roberts published the first National Level Report on STEM. Linking to my discussion on evidence, it made me wonder what, if any, progress has been made since then. In his undergraduate text on Enzyme Kinetics, my PhD supervisor Professor Paul Engel, remarked that undergraduates approached Enzyme Kinetics (quantifying the rates of enzyme catalysis) in the same way that they approached Latin and cold showers. Whilst the comparison is a little dated:  I think it makes the point well and is probably more widely applicable to STEM. 

Over the last 10 years, STEM initiatives to raise awareness and interest in STEM subjects have been funded, implemented and evaluated. So, are more students interested in STEM subjects? Are more students taking STEM examinations? Are more people applying for STEM courses at University? Are employers claiming that there are improvements in numeracy of school-leavers and graduates? Importantly, where do you find the information?

The Government provides a wealth of data on trends in Education, at all levels. So when I looked at the Higher Education Statistics Agency (HESA) data on changes in student courses, there was, for example, between 2010 and 2011, a 2% net increase in students applying for STEM related courses (I include Medicine, Vet Science, Biology, Sciences and Maths and Computing). During 2007-2011 there has been an increase from 1.8 to 1.9m students taking undergraduate courses. So a 2% increase for 2011 translates to around 38 000 new science graduates due next summer. I wondered whether this was a significant impact, given the efforts made at Schools and Universities, to engage students in Science (starting with major activities n 2004, but allowing a period of three years for the impact of these strategies to emerge). So in 2007 there where 557 000 students studying STEM subjects and by 2011 this had risen to 632 000. All good so far, an additional 75 000 Scientists, an increase of around 13%. I then compared this with the total numbers of students registering at Universities in the same period: 1.48m compared with 1.72m, which is an increase of 16%. So my conclusion is that STEM initiatives have failed to change the interests of school students: the proportion of students engaging in Science is not significantly different. 

So what should be done about this? What is most revealing, is the underlying issue: the public are uncomfortable about Science: "In a new HuffPost/YouGov poll, only 36 percent of Americans reported having "a lot" of trust that information they get from scientists is accurate and reliable. Fifty-one percent said they trust that information only a little, and another 6 percent said they don't trust it at all." We believe that this is one of the most compelling arguments for the development of UTCs. Moreover, that as a group of students, interested and enthusiastic about Science, which was expressed so eloquently by Jack and Libby on Thursday, you will be the force that changes this perception. So here are a few suggestions that you should pass on to your friends and family.

Vaccines:                 Polio has been eradicated by Science
Antibiotics:              Infectious diseases are under control
Irrigation/purification:  Imagine life without clean water
Computing:                No Internet or smart phones
Electrons:                No TV

Think of some others yourselves and make it your mission to promote Science as something that is not only vital to our Society, but also both fascinating and fulfilling. Also, whilst antibiotics have been revolutionary, their mis-use is causing problems: so more Scientists are needed and it is our responsibility to communicate these problems clearly with non-scientists using the best evidence.