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.
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!
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.
What do guinea pigs, Saccharomyces cerevisiae, zebra fish and Escherischia coli have in common? They are all used experimentally to investigate Biological phenomena. We refer to them collectively as model organisms. So why Drosophila melanogaster and not Musca domestica? It usually comes down to practical reasons. Drosphila, or fruit flies have been used for scientific discovery for over 100 years. They are fast breeders, they eat cheap food and they are easy to store and maintain. From a genetics point of view, this fits the bill for mutational studies. Hence we have a rich history in using the fruit fly to explore the relationship between genotype and phenotype in general, but in particular phenomena such as eye colour and more recently development. In general, Biologists use the simplest organism they can, until a more complex one is required to investigate a phenomenon.
Horses for courses I. In my opinion, some of the greatest experiments in Molecular Biology have been carried out on bacteriophage and E.coli. The great ideas of Salvador Luria and Max Delbruck on evolution were a result of a statistical analysis of the frequency of mutations that led to bacteriophage resistance. So what does all that mean! Darwin's ideas suggested that mutations arise spontaneously in populations and it is only when those mutations give rise to some benefit to the organism, that they become fixed in a population, eventually out-competing the wild type organisms. In order to confirm this hypothesis, Luria and Delbruck turned to bacteria and bacteriophage (these are viruses that attack bacteria). Because the numbers of bacteria in a culture are in the millions, and the phage (as they are called) are even greater in number; and, doubling times are 20 minutes, it became possible to carry out a statistically controlled analysis. This demonstrated experimentally that Darwin's ideas were correct. This was only possible using this model organism.
Horses for courses II. Bacteria can provide us with insight into many molecular processes, but unlike human cells, bacteria have no nucleus (hence the name prokaryote). However, there
are many aspects of human biology that would not be appropriate to try and explain from a study of bacteria alone. So, for example, if we want to understand the way in which humans develop, we turn to fruit flies, zebra fish and mice. The genes that are responsible for laying down the body plan for a fruit fly are essentially the same as those that dictate the positioning of our limbs and the polarity of our body (head and toes). These genes (including hox genes) are often called master regulators, since they coordinate sets of other genes and in particular their spatio-temporal patterns of expression (or simply when and where they are expressed).
I have chosen to focus on the simple beetle Tenebrio molitor (or the mealworm), since it is easily obtained as a living larva, it is sold cheaply as dried food (for birds and humans), which simplifies the biochemistry; and it can be readily matured in the lab, producing a black beetle. Work in a number of genome labs have begun to explore the response of T. molitor to bacterial infection, and related genomes are available: the mealworm genome will be completed soon and we can already use its transcriptome to access information about its genetic makeup. Lets see if we can find out something new in our very own post-genomic study.
My school Chemistry text book told me that atoms are like the solar system: in which electrons orbited the central nucleus just like the planets in our own solar system orbit the sun. The truth is much more complex, and the recent announcement that the Higgs Boson (a particle that confers mass on other subatomic particles) has been "seen" using a high energy particle accelerator doesn't really fit with the simple model that I used to get me through my chemistry exams. But that's OK because it was an early stage in my journey to making discoveries of my own (or more accurately with colleagues) and I developed a level of understanding that enabled (or empowered) me to question established facts and concepts and to make up my own mind. This can only be achieved by looking at the "evidence" that has been used to justify a particular conclusion. So what is evidence and how do we evaluate it?
The Oxford English Dictionary defines "evidence" as follows:
"The available body of facts or information indicating
whether a belief or proposition is true or valid"
There is a problem with this definition, since a fact can be challenged by an experiment, which often happens as new technology emerges. So, until about 1985, we believed that all enzymes were proteins. Tom Cech and Sidney Altman, independently demonstrated that RNA, previously thought to be an information molecule, could also catalyse certain biological reactions. Not only has this subsequently been confirmed by many labs, but it has opened the way for a new way of thinking about genes and gene therapy.
In this case the facts changed and therefore new evidence was used to displace the protein-centric idea. Facts in Science only remain set in stone after they are subjected to rigorous investigation. The double helix of DNA, now an iconic symbol, was thought to comprise 3 strands, until Watson and Crick (not forgetting of course Rosalind Franklin and Maurice Wilkins) demonstrated unequivocally, that it was a double helix...until of course we discovered Z DNA and quadruplexes at the tips of our chromosomes. But don't panic, double helical DNA is here to stay, it is just that when solid evidence is presented that challenges the universality of this structure it often explains things that were a little awkward to accommodate, in this case recombination and chromosome crossing over.
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| Geoff Schatz |
So how do we deal with the "moving target" or "shifting sand" that is the scientific evidence base? My first suggestion is to embrace it: always challenge a concept or fact by asking yourself this question. What is the definitive (or most compelling) experimental result that led to the formulation of a hypothesis? When Geoff Schatz was head of Biochemistry in the Biozentrum at Basel (Switzerland) he transplanted short segments of amino acids that he suggested were responsible for targeting proteins to the mitochondria (the organelle that generates cellular energy) onto cytoplasmic proteins. The result was that these proteins were now targeted to the mitochondria. This is referred to by Molecular Biologists as "an elegant" experiment: it cut to the heart of the problem and yet is very simple in concept. Of course Schatz was drawing on on years of experience, great insight, formidable technical skills and of course he was a bit of a genius! This concept remains robust, but there are exceptions: the important thing is that Schatz's (and others similar scientists) provided us with a model that allowed us to move forward in the lab in a logical and constructive manner. If the Schatz rules need to be overturned or modified by new and perhaps more compelling evidence, then so be it. This is how we progress in Science. Next, think about who judges the quality and significance of the evidence?
Experimental Science. The journey from a student to becoming a practicing scientist never ends, or at least in my view it shouldn't. One of the things that I find most frustrating is a day spent with students carrying out an experiment while racing against the clock. Under these circumstances there is little chance of acquiring new skills (or even honing old ones) and an appreciation of the over-arching concepts, the significance of each step and ultimately an understanding of the outcome, are all lost in the process. In short the exercise is largely pointless. So, how should you approach your time in the lab? Below are some suggestions.
Listen, make notes, read instructions carefully and discuss your understanding with your group members. I generally find that taking notes during any meeting helps me remember things (and some say it aids in understanding). I recommend that you have a small notebook and pen with you when someone is giving a presentation in the lab or in a seminar (or in any meeting). If you have been given instructions or you are searching for information on the internet, read the instructions carefully. Avoid jumping into an experiment until you have mentally "walked through" all of the steps.
Team-work. As a group you should then make sure you all understand what you have to do. Then allocate tasks and begin to get organised. Gather materials, clarify any uncertainties (after you have attempted to understand) with a demonstrator and write out a schedule for the day: we call these a work flow. This can form the basis of your methodology write up later.
Observation. I cant stress how important it is to keep watching what is happening to your samples, the amount of a solution that you dispense, the colour or appearance of a fraction from a column or a tube in an incubator. Again, make notes of what you observe. Some of these observations may not be important, but treat everything as important as you go through the experiment. It is only later that you can decide which observations should be included in the analysis of your results.
Sample labeling. So far everyone has had an experience of losing their samples from one week to the next. Since we are running lab projects over several weeks, tracking your samples is vital. Think about how you label samples and don't leave it until the last minute. A petri dish labelled on the lid, is an experiment wasted if the lid is accidentally knocked off by you or a colleague in a shared fridge or incubator. Tubes labelled 1,2,3 or A,B,C will become forgotten among the multitude of As and Bs and Cs. If the sample is to be frozen, think about the likelihood of the label being lost. Use the right kind of pen/ink or sticker. Also write down the labels you have used and the location of your sample.
Tidy your bench. Before you leave, allow time to tidy your bench. This is considerate and polite, since someone else will have to do it for you: and that's unfair and unsustainable. Secondly, by tidying, you make sure that your solutions, samples and equipment are kept in the right place and temperature and that the equipment is well maintained. This is not an option.
Writing up your experiments. You have all been given clear guidelines for writing up your experiments. Take your time, think about the significance of what you have done and what you have observed. Experiments do not "work" or "fail", they give you outcomes or results that are influenced by many parameters: the reagent quality, the skill of your manipulations, proper use of instruments from pipettes to spectrophotometers etc. Therefore write up what happened and explain why you think you obtained your particular set of data. As you gain experience, your skills will improve and your ability to obtain robust data will also improve. This is a learning process and it never stops!
Today we have been using a method called BLAST searching. Very simply, we are accessing all of the genes and genomes whose sequences have been deposited in the "Public Domain". As young Life Scientists, this information will feature increasingly in your lives. As future professional scientists you will possibly contribute to our understanding of the significance of this information, in ways that we are unable to appreciate in 2014.
When I first helped sequence the gene encoding the restriction and modification enzyme from the lowly bacteriophage P1 in 1985, I had no idea that 30 years later, Mark Szczelkun at Bristol would use a new single molecule technique with this enzyme, to establish important new rules concerning the trafficking of molecules along the DNA double helix. Obtaining the sequences of genes and genomes was then a time-consuming labour of love. Recently, one of my own PhD students has recently obtained the sequences of 6 bacterial genomes using a University service in a matter of weeks: obtaining the sequence of two genes took 6 of us over one year! We are hoping to find a clue to the origins of a new genetic damage phenomenon which is linked to some cancers: chromothripsis. When we have analysed the data, you will be the first to know!
Returning to genomics and bioinformatics; I explained that the sequences of the genomes of man, mouse and pig were very similar (but each of their brains are quite different!). Interestingly, if Charles Darwin had used BLAST searching, I wonder if he would have developed his unifying theory of evolution. I find it easier to tell a chinchilla from a pig by looking at them, than I would by searching the data base. It is much harder
to spot the difference pair of genes from mouse and pig and man than you might think. It seems that small differences in individual genes combine to determine the appearance, or "phenotype" of the organism (and indeed each cell). In higher organism it is a combination of the genome and our neural networks, that confers our evolutionary advantage.
The freedom to access to this phenomenal resource of biological data is, in my view, one of the greatest achievements of Science in this century. The challenges that lie ahead to interpret these sets of data will form a major part of your lives in Science over the next 50 years. I will leave you by asking you to think about evolution in terms of the complexity of the human genome and the success of the relatively simple genomes that are required to make viruses. Is the virus the most highly evolved? I hope you find time to enjoy searching through the NCBI portal.
