Tuesday, 29 July 2014

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.

Monday, 28 July 2014

Vectors in maths, disease and molecular biology: understanding terms in context.

I was asked by a student to explain the logic behind the structure of Buckminsterfullerene, or BF, the C60 "spherical" form of carbon that was recently the focus of an outreach visit to the Widnes Catalyst museum by Sir Harry Kroto, who like me, is an alumnus of the University of Sheffield, but unlike me is a Nobel Laureate! The plastic model was thrust in front of me by a UTC student and I asked: "what else does it remind you off?" (as I stood in front of a 3D print of Bacteriophage T4). "Oh yes, a phage capsid" came the reply. Followed by, "But why is it so stable?". I immediately thought back to my school days and applied maths. My maths teacher had given me a taste for the use of maths to describe and calculate forces, such as friction etc. I said "vectors: it's all a matter of balancing forces". Of course I was then challenged by the comment: "aren't they the carriers of malaria?" And I thought to myself, vectors as agents for delivering recombinant DNA (even in the form of bacteriophage DNA), but I meant the vectors as defined by mathematicians, physicists and engineers:  "A quantity having direction as well as magnitude, especially as determining the position of one point in space relative to another". The word being originally Latin and related to the verb vehere to convey and here it is being used as a noun to mean a carrier.

So the word vector is context dependent. In Maths, it means a number that carries with it direction, in Molecular Biology, it means a plasmid or bacteriophage DNA molecule that carries foreign DNA and in Parasitology, it means an organism that carries a pathogen, in the case of malaria, the plasmodium parasite is carried by a mosquito vector. I like the fact that the word vector can be such a good match for multiple scientific concepts. It is a versatile word, just as the red case of a Swiss army knife houses multiple functions. Indeed, as we have touched on this in earlier blogs; genes and proteins can be used in different contexts to provide different functions. This also reminds me of the use of the word "parsimonious" (or thrifty) in a seminar I attended some years ago by Professor Richard Perham when he described the sharing of protein subunits between the two multienzyme complexes: Pyruvate and 2-oxoglutarate dehydrogenase; both key enzymes in 9OR NEAR) the Krebs Cycle.  The synthesis of a pool of subunits for sharing, is something that is now known to be common amongst multi-protein complexes in human cells in particular. In language, we often use words in a context dependent manner, the same is true of biological molecules!

Returning to the "Bucky Ball" and the bacteriophage capsid for a moment. As I understand it, the occurrence in Nature of regular arrangements of proteins in bacteriophage and viral capsids matches sound engineering principles, just as the architect Buckminster Fuller employed in order to design his geodesic domes (Top left). The adenovirus capsid shown top right, is an example of how, like in BF, a combination of pentameric and hexameric rings of subunits (proteins instead of carbon atoms) provides a stable shell in which the genome of the virus is accommodated until the virus infects the host cell. The sum of all of the force vectors (imagine you are building the frame in the same way that you would assemble a tent from tubular struts) across all of the pentagons and hexagons will equal zero in a stable structure. Thus mathematical vectors underpin the stability bacteriophage cloning vectors and we have squared the circle.....well not quite! 

Sunday, 27 July 2014

Guest Blog!

John Guest FRS
By now, those of you reading my Blog will appreciate (or at least tolerate) my attempt to play on words. In this case, I am writing about my encounter last week with Emeritus Professor John Guest FRS, former Professor in the Department of Microbiology and later, the Department of Molecular Biology and Biotechnology at the University of Sheffield. John retired from "active service" in the University several years ago (and an emeritus title is conferred, through application to the University, upon those who have brought distinction to their institution and who wish to remain professionally affiliated in some way): he, remains a frequent visitor, attending mainly when visiting academics come to deliver research seminars in Molecular Biology, or indeed in wider areas of Science. John was elected a Fellow of the Royal Society in 1986, for his work on bacterial genetics in relation to fundamental aspects of the regulation of metabolism in E.coli. I may come back to John's pioneering work on the genes and enzymes of the Krebs Cycle at a later date, but today I want to focus on his work with Charles Yanofsky, while John was a post-doctoral fellow at the University of Stanford in California. 

Studying the genes and enzymes required to synthesise the amino acid Tryptophan in E.coli, may seem a little esoteric, but this field has given us many applications in Biotechnology, and arguably the work that John published during his time in Charles Yanofsky's laboratories ranks amongst the most significant in the field of Molecular Biology. The key paper can be found at this link, where the full text is free to download and read! You wont find an extensive page on wikipedia about this work, but I believe the experiments published between 1964 and 1967 are fundamental to our understanding of the nexus of nucleotide chemistry and protein chemistry in the evolution of life. It is also important to mention the Nobel Prize winning work discussed by Rich Roberts in his guest blog, that led Roberts and Sharp to independently demonstrate that there is an interruption in this colinear relationship between nucleic acid coding information and the primary structure (amino acid sequence) in higher organisms. These interruptions give rise to exons (coding sequence) and intervening sequences (introns). In fact only 10% of our own genome appears to code for proteins or RNA molecules.

Back to some chemistry and the fact that in the 1960s it was immensely challenging to obtain the sequence of a protein: no mass spectrometry in those days: in fact John and his colleagues would have to rely on a semi manual approaches that were developed by pioneering protein chemsits including Stein and Moore in the USA and Fred Sanger in the UK, which involved complex analysis of proteolytic fragments (see RHS). Another important piece of work carried out in the laboratory of Vernon Ingram using this powerful technique of protein fingerprinting was to  establish the molecular basis of sickle cell anaemia in haemoglobin. Worse still it would be another 10 years at least before DNA sequencing would become possible (thanks in part to Fred Sanger again!), so the team had to use recombination frequencies in order to match the gene and the protein sequences. All in all, this was an experimental and intellectual tour de force and it came along as the genetic code was cracked and a few years before Masayasu Nomura reconstituted the bacterial ribosome: the site of protein synthesis. The complete set of papers published by John in while he worked in the Yonofsky lab can be found here. I recommend those of you interested in the intellectual development of modern molecular biology take a look.

John kindly emailed me an example of his original peptide fingerprinting data from his time in Yanofsky's lab. It is shown below.

I dont think you need to be an expert in protein chemistry to see that this experiment reveals a significant difference in the peptide patterns (purple spots) between the wild type protein (left) compared with the mutant (right). This is a lovely example of how a carefully planned and executed experiment can be so revealing. John has commented on the Blog below.

The story doesn't end here though. Placing this work in context is important. Previous landmark experiments had demonstrated the relationship between genes and proteins: the classic work of Beadle and Tatum on Neurospora had laid the main foundations. Crick, Brenner, Khorana (a PhD graduate of the University of Liverpool), Ochoa, Nirenberg and collaborators (see Wiki site) were all making significant progress on the determination of the Genetic Code: the relationship between the nucleotide sequences in mRNA (and the role of tRNA) in defining the sequence of amino acids in a protein chain. By the end of the 1960s, a robust model existed for the bacterial processes of replication, transcription and translation, the mechanisms underlying the central dogma were well established: DNA makes RNA makes protein, and John was a major contributor. By the end of the 1970s, there were discrepancies emerging in respect of higher organisms: Roberts and Sharp had discovered the phenomenon of splicing, in which the message (mRNA) is first processed into a mature form before the process of translation can begin. Moreover, another 10 years later, it was observed (particularly in ancient bacteria) that protein sequences are sometimes spliced! It had been discovered that some proteins lose their ends before they become fully functional, but the discovery of a mechanism in which internal sequences of protein are chopped out and the chain re-joined, was unexpected. In fact many of the DNA Polymerases used in PCR are processed in this way. The excised segments are called inteins (and the protein equivalent of exons are known as exteins). We also now know that some sequences of DNA can encode more than one protein, achieved by starting translation in a different "reading frame" or register. In order to confound us further some organisms (such as protozoa) use a normal stop codon to encode the aromatic amino acid Tryptophan (bringing us full circle to the Trp operon work in Yanofsky's lab!). 


Despite these anomalies, we still observe the main findings that John made in Charles Yanofsky's lab hold true for the vast majority of gene:protein relationships, with the caveat that splicing is generally required in higher organisms. When you carry out a BLAST search to compare your sequences, it wouldn't work if we didn't have a robust understanding of the Genetic Code and the co-linearity of the gene and protein. I hope you agree with me that the experiments of the pioneering molecular biologists after the Watson and Crick discovery represent some of the most impressive in all of Science.

Wednesday, 23 July 2014

"It was the best of times, it was the worst of times, it was the age of wisdom, it was the age of foolishness..."

The opening lines of "A Tale of Two Cities" by Charles Dickens, seem to capture my thoughts as we enter the last week of the first year since the Liverpool Life Sciences UTC first opened. Not only are the opening lines apt, but this year has seen me negotiate between the two cities of Liverpool and Sheffield: as I have tried to combine my new role at the UTC, with my role as an academic at the University of Sheffield. I'll cover both the successes (the best of times?) and also the challenges that lie ahead, since next year will probably be more important than the year behind us. It is of course understandable that for our students, their parents, and those who will employ or educate them further, examination results will be the major focus, but I shall concentrate on the activities in the Innovation Labs, which have been my primary concern.

September saw the doors open, shortly after the paint had dried and about 20% of the equipment had been set up in the labs. The sun was shining and the sense of anticipation at the UTC was stronger even than it would be at the end of the month, when freshers would arrive at Sheffield. Why? A complete team of new teaching staff, new support staff and no time for a dry run! I decided to play safe and combine some elementary Bioinformatics with the basic principles of visible light spectroscopy. Easy enough? Well, actually, yes. Of course there were some blank expressions, anxious looks, nerves etc., but the students were OK, that was just me and the teachers! By the end of the month I was feeling confident that the Y10s and Y12s would be capable of considerably more than I imagined. So I decided to get more ambitious. I was also very conscious that the planning of classes had to be almost spontaneous in order that I could react to students on a weekly basis. However, this made it somewhat challenging for the teachers, but thanks to their patience (and I like to think a level of enjoyment!) we managed to muddle through, while the students grew in confidence on a daily basis.


By October, I was using my regular walk from Lime Street station to Greenland Street, to plan the day's activities, with further reminders of the Dickens theme in view (I don't think we get to know Uriah's father's name, but he had endowed within Uriah the positive (in his eyes) value of being a yes man, and this was of course something I wanted to make sure didn't happen with UTC students). I decided I could easily challenge the students a little more than I had originally intended and importantly, that rather than use the tried an tested model of structured class practicals, I would begin to empower the students and teach them the value of failure. The lab classes were now combining skills such as  spectroscopy, microbiology, chromatography, electrophoresis and Bioinformatics, with planning, teamwork and the need to organise and label samples. All of which prepared the Y10s for proteomics of milk and the Y12s for a Biotechnology project with the aim of purifying recombinant Green Fluorescent Protein. Before I leave October, I have to say that emotions ran high (in me) when the entire lab broke into a round of applause when the Nobel Prize for Physiology or Medicine was announced over the lab screen via a live stream from Stockholm!


The dark nights were upon us, it was now November, but I was brightened by the prospect of delivering a tube of Green Fluorescent Protein. GFP has not only proved to be a major help in elucidating a range of Biological phenomena from the early stages of cell division, to the protein mediated cues in animal development, but has also brought high visual impact to Molecular Life Sciences, in a way that I decided could capture the imagination of the students in Y12. In fact the project that was associated with our partners Eden Biodesign incorporated an understanding of the mathematical basis of cell growth, the principles of gene repression and induction, the fundamentals of affinity chromatography and the power of spectroscopy. By the end of term, we were well on the way to the "Green" Prize.

Through November and December, the students were alerted to the requirement to present their results in front of staff and representatives from our partner industries. Eden Biodesign kindly sent a a team of senior research staff to judge our Y12 presentations, while staff judged the Y10 presentations. In the latter case, we had developed the use of supermarket milk as a vehicle for exploring the analytical methods used to analyse the proteins present in milk (see left). We also investigated one of the earliest forms of Biotechnology: cheese production. When I mentioned "...the worst of times", the quality of the cheese produced may be the best candidate in this respect. It looked like a reasonable French soft cheese, but I am afraid that's were the similarity ended. Nevertheless, the students managed to pool together their results and background reading and I was incredibly proud of their presentations. Leaving me with Great Expectations for 2014! The presentations from Y12s to Eden, filled me and the teachers with enormous pride. This was the first real sign that we had shifted the aspirations and capabilities of students of this age into one that made the panel raise a few eyebrows in pleasant surprise. I must also say a special thanks to Geoff Dickinson in the Central Teaching Labs at the University of Liverpool for his unwavering help in providing last minute equipment to bail me out of what could have been a few embarrassing moments in the lab!

With the opportunity to reflect on the first term over Christmas and of course enjoy the festivities, it wasn't long before Christmas was behind us, and my main undergraduate teaching at Sheffield too. The New Year provided an opportunity to introduce the Y12 students to experimental logic of drug discovery and the Y10 students to meal worms and practical genomics. This involved the introduction of microbial plate based and broth assays for the detection of bacterial growth inhibition. 


Recalling the discovery of penicillin, we set out in Y10 to investigate the effects of bleach and detergents compared with some common antibiotics. This provided a vehicle for sneaking in the use of log graph paper plotting exponential relationships, which in turn fired my enthusiasm for making maths a focus of lab classes wherever possible. Around this time I also started using Twitter to try and engage a wider audience in the innovation lab activities. Possibly my most unlikely idea (a result of the sight of ten pomegranates in a box at Tesco) to teach the metrics of viral infection using fruit and veg, proved fun and took us into unexpected avenues of Greek maths and science. A bit of a eureka moment in teaching methods for me! I think I have developed a reputation for science on the cheap and I am sure the students will fondly recall the first tub of 99p dried mealworms and the 6 pack of Poweraid: my best source of a negatively charged brilliant blue dye in chromatography classes!

February saw the students dealing with extraction methods from plants and fruit through to meal worm larvae, either dried or fresh. These experiments took us into unknown territory for the first time, with students being expected to decide and evaluate the methodology that they would employ to obtain the most active extracts. In the case of the plants, we were looking at the use of different solvents and cell breakage methods, with activity against E.coli plate growth as our assay. In the case of the mealworm, we utilised a range of techniques and managed to settle on the use of a glass-teflon homogenizer (kindly donated by the University of Sheffield teaching labs) whilst keeping the larval extracts ice cold. This generated protein extracts and both DNA and RNA, which will form the basis of our meal worm "omics" project. Dr. Moore and I thought we would try our luck with the Royal Society in view of the students' success and bid for research funding. The grant was submitted and a decision would follow in April....


 In March we hosted as usual, a number of visitors  and amongst them we were fortunate to host  Professor Phil Ingham FRS, from Singapore who  chatted to students and staff. Phil, kindly provided  our first guest Blog (which you can find earlier in  the series). I was absolutely delighted by the  success of the Y12 group in taking on a challenge  from Dr. Doug Cossar at Croda. Lyndsay and I had  been impressed by the warm welcome at Croda, and in particular the suggestion for a project in synthetic biology, one of the most exciting, emerging research areas in Biotechnology. Doug asked if we could obtain a set of genes and their proteins from Blue Green algae, that related to the synthesis of tri-peptides. Recalling a colleague worked on Synechocystis and having my lap top on hand to carry out a quick BLAST search, I decided we should give it a go. What you can see top left is the result of the first UTC, mass PCR experiment. Importantly on Synechocystis genomic DNA isolated by a method developed by the class. We finally completed the amplification of the genes and several Y13 students will move onto the next stage as part of a small project team and produce recombinant proteins in order to establish whether we can synthesise tri-peptides in this way.


April saw the arrival of Easter and exams! This enabled us to a little time out fro lab classes to expand our interest in the use of 3D printing in the labs. George had given us a tantalising insight into the power of 3DP, but it was only when we began to design and print lab equipment, that we realised the significance of this new technology. The Greenland Biodesign team warmed to the opportunity of incorporating 3DP into the lab, and soon we were able to print our own lab equipment which now ranges from column chromatography racks, through to a fully operational, battery operated microcentrifuge: all for a fraction of the price! George is keen to establish the labs as part of the Fab (for fabrication) Lab network and, with the award of a number of further printers in the new academic year, I can see us becoming much more dependent on this technology in the future. Another important development on the Blog site was the contribution from Dr. Antal Kiss from the Hungarian Academy of Sciences; exposing students to the wider community of scientists and promoting the UTC as part of a new venture in Science, which s of course a major part of the UTC's mission. Recognising our place in the wider Scientific and Educational communities is something that will continue over the coming years. (All of the earlier Blog which contain more detail can be accessed on the RHS) 



 By May, the examinations were in the air for the  Y12s, but the most exciting news was the award  from the Royal Society of our first grant application!  The mealworm, or Tenebrio molitor, to give it its  full scientific name, was now our very own model  organism and we completed a set of ambitious  experiments aimed at developing a proteomics   approach to understanding a range of Biological phenomena using contemporary methods. As this was going on, discussions were afoot with the University of Liverpool Genome Centre to begin to sequence the mealworm genome (we are currently carrying out preliminary DNA extractions for this and hope to get started in the new academic year). 

Probably the most significant outcome of the month was my quest for the key skills (sorry but the artful dodger's craft was the best Dickens reference to skills training I could think of!) needed by an experimental Life Scientist. It is easy to assume that after over 35 years as a trainee Biochemist, I would know the answer to this, but it is important to remember that scientists depend on collaboration and communication; so I decided to ask as many people as I could, ranging from our partners to research scientists from all over the UK, the USA, Europe and indeed the East Asia. What do they think are the skills that a contemporary Life Scientist should possess? The answers came in two waves: immediate responses, Sir Rich Roberts being an early responder as always! Followed by a steady stream of response (still coming in!). This information, combined with a detailed survey of our regional partners, forms the basis of our Skills Passport. More than a piece of paper, the aim of this initiative is to empower UTC students by preparing them in a robust manner for experimental Science in the 21st century. However, this does not mean that we only focus on the methods of the day, we include in our initiative key transferable skills in Science (and indeed in Life in general), such as project planning, sample tracking, analysis of data, computational skills, numeracy, literacy all of which are essential not only for understanding Science, but also in communicating it.


With June came the sun, the Minster for Business  and Skills and the return of the students to the  Innovation Labs. It is also examination season for  me at University and this includes my job  examining final year undergraduates at the  Universities of Lancaster and Cambridge. So Miss  Hudson kindly stepped into the breech for the Y10  students and delivered a phenomenal class on the  anatomy, physiology and genomics of the locust. The locust has been used widely in Biological education, owing to its rapid breeding, its size (making it easy to observe and to dissect) and its reproductive organs contain easy-access and easy to see chromosomes. More recently the locust genome has been published and the opportunity to combine classical biology with contemporary bioinformatics represents a superb example of how Life Science has developed over the last 10 years and this lab class illustrates the power of the Liverpool Life Sciences UTC in being able to deliver such a confident experimental programme through our teachers. 

The Y12s, unfortunately had to contend with me and the dreaded exercise of "primer design". My old PhD supervisor (actually he looks younger and is certainly still more active than me!) wrote in his undergraduate text on Enzyme Kinetics, that Enzyme Kinetics (the study of the reaction rates and intermediates in enzyme catalysis) is a little like "Latin and cold showers". Whilst the curriculum has changed somewhat since he and I attended school, it is true to say that "primer design" has been the cause of many tears in the lab! The logic is simple: DNA is made of a sequence of 4 bases (GATC), knowing the sequence of one strand allows you to predict that of its "complement". So an A on one strand will have a T on the other, a G pairs with a C and so forth. The direction of the strands is polarised in the opposite direction, and this is where the confusion comes (hence the Twist top left!), since the enzyme we use to incorporate the synthetic primer into DNA for the "forensic" technique of PCR (the Polymerase Chain Reaction is a means of amplifying minute quantities of DNA for analysis in all areas of Science) can only run in one direction. I am pleased to say that everyone (in the end!) cracked the primer design problem. We then used this predictive logic to design primers to amplify genes from a Blue Green algae to round off our Synthetic Biology project. Presentations will follow after summer, since we have many students away on placements.

Student ideas are incredible

July marked the time for students to consolidate their skills passport and to design their own Y11 and Y13 projects. Once again, more surprises were in store for me! My year in the Innovation Lab has taught me many things about Science, but I think the most important lesson I have learnt is never (ever) underestimate the ability of young people. Science projects, in which individuals or small teams of students design plan and execute a piece of research based on some observations of others (or sometimes their own observation) is the cornerstone of Science: it is the way that we "stand on the shoulders of giants". However, at University, students usually carry out their first research projects in the lab after their second year of study. I decided (as you can read in an earlier Blog) to ask students to design their own projects (something that used to be the way PhDs were managed) and the ideas that came back were exceptional. Of course the use of gamma rays to explore fungal responses to DNA damage may not be easy to accommodate in the Innovation Labs and we may not be switching on the first mass spectrometer designed and built in a school, but we will work within our limitations to accommodate these ideas. I have most of the project ideas in hand and over the summer I shall be obtaining resources finding research mentors from Industry and Academia and I am confident that by this time next year I will not only be writing up research papers from class experiments, but from research groups within the UTC. This represents a major shift in Science education in schools and will be a defining moment for the Liverpool Life Sciences UTC

One of the lesser known works of Charles Dickens is the "Battle of Life", published as a metaphor (which reminds me of the discussion with Mr. Harries about the origin of electro-phor-esis!) for the struggles of domestic life and life and death on the battlefield. Life Science is all about our struggle to understand where we come from, how we "tick", why we sometimes get sick and how we can manage to maintain a good quality of life as we get older. The UTC aims to provide its students with an unprecedented opportunity to be a key player in this journey and I feel delighted that with our first steps, the next year will bring even more challenges, but I am looking forward to sharing them with you and the new teachers and students!

Have a great summer, thanks to all the students, teachers, support staff and partners for helping me over the last year, but most of all Mr. Ward for giving me the opportunity. It has really been the Best of Times!

Monday, 14 July 2014

Following your nose can be educational! The value of serendipity?

During the preliminary investigation of our proteomic "hits" from the fractionation of the soluble proteins isolated from mid phase Tenebrio larvae (ie 1-2 weeks old), we discovered a number of proteins whose primary structures (inferred amino acid sequences) suggested a possible role in melanization. This provided a nice opportunity to demonstrate the power and the limitations of proteomics and bioinformatics, and as always, I was delighted by the students' collective findings. I provided them with the Mass Spec data files in which the most significant matches to the analysed peptides were top of the list. They were asked to consider the validity of the computer prompted similarities and to "follow their noses" by searching the Internet for information relating to the biological/biochemical properties of the matches. 


For those of you without a background in this area it is simply a pattern matching exercise. For example, you might be looking at a pair of eyes which provide a strong indication of the "owner". The picture top left of the Mona Lisa's eyes are a real giveaway. Or are they? It is sometimes more appropriate to consider the data as a set of jigsaw pieces: instead of a complete section of sequence data, there are fragments that are matched against global databases of proteins in order to identify the best match. The image on the right is probably the most common scenario: fragments give us clues to the identity of the whole face in this case. It must be stressed that these matches with, in this case the Mona Lisa, are only tentative at this stage, something that is often forgotten! In other words other reference points are needed to establish the true context of the information and therefore interpret it correctly. 

The data that one recovers from a mass spectrometry experiment are shown below to illustrate what I mean. The red letters are amino acids detected and the black letters corresponds to the gaps in the jigsaw, filled in by the computer program.


1MGLKKIILVLTALAGLSKAFNTLSPWDKEVIYNWKALVKAGTNLPTSYAS
51HFVLEGKIHVQANSNATHVKITDLSYKLYNGILNHVQEHEAHSVPLHLKE
101LEHVFKIIYDTKGLAIGIVTEAQEKEFSRNIKKAIATIFQMDESVLKQVH
151HHPHSFVIKEHSIYGHFDVDYTIKPHQNEIEVHKLHDMHDPHHLYLNLFS
201NNEAGVCDMGYENPIMHNTKKQYRIVKQDGHHVVKDIHSAGVVFYYPFKG
251QSDHQFIFVNQSMHLTQMVPIQEQFHIQSEHFENHLTYHSFETSQDGSIH
301DVTNGRHVVNYEKLIPNIQHMLSDIHSYLNEDHIHTREPDSKRGQMVNRV
351QRLLQFFSLTKLEELHKKLSGDSLTVFHHILPLVGTKSSTLFIKQLIVNK
401VLSAGVASELLQHFPEHVIEPTVELLAEMEELLHLDESISWNVRKVAVLS
451FASLVQHSHSFVEKTHRNKEFMKNDGVDIHHHETKYSRQQPYEKYVIEYI
501NKLRSSHDYKVQMTYLMGLCNMRLHSIIKHLLPAVYGEWYPDHHFRLLSL
551WAVSTAVVEERDPNTVIETLWPIFTNVDELTEMRTIAFYFIMMTYPNDSR
601LTNMFNYLLMEPNDEVYSFVYTFMHGMMETNEPCHREYAVKVAHLVKLLP
651PRSHGRSFATRHDYKDVNFDFSAGVENYFINGSDSQIYGVVTTSQQFNIY
701HEDRTFFVKIHGVDHHIDPVNMAVEVFQKKIPKITDNKDVHIEVVLLRHG
751HIVNTYFFDQHSLHQLNEVLDFFTKESHSLTQDLLHMSYNEYHRNLIPTD
801LGIPALWEYLMPVVHHNNISVTKETVNKVVHLHVDNRYVNWIHYRHGLSF
851YNPFVDVWQGVNRYHSYDAVWPLHFEITLNTQQQSLQLSWKKDSNPAHNV
901AGFRSHVKQMVFIKDDYNRNILMQSCPQCKDFQIVSHGEKFRNDYLLIQD
951DDLSTGHQWRVGIYDSSQYATNGSFYHQMYLFTQSHSSHSESPLRHFWMS
1001YANWHQYCLLLPEPGSYGLIVRVEPSHQHSVSNIDLSLRLHSDATVDDDY
1051YYAPKGKWSTRMTYSVKNQETSLKTWDVNAILDMNSGHTHNTVKMHVTRI
1101IPGQKDYKVCVDGIKKWTESGVEGHLNVAMSQTPDGKCVKDDTVIDVTMT
1151GQQSHEQEQGHHKYGSCEYVNYYAIAHYHTTQCLVDHTTVRHYVYNVKTT
1201NVPTEFKKVFAHWWDHVKSHYMSYYVFEDEHSDNIADQNLKVDVMYPMQV
1251EEVNLHVTTPQHVYKLTGVPTHYWKWFGVAPDSVEYPEWFQYLHQVGFVE
1301HCTVHHNHYHLNHHDIREAVPNDWTLYVGNAVQNPQKAVYVKQVGDDEIG
1351VKVVDHGHTLEIVPHGDGHQVTIDGQVVPSTSIYTKIGCIVTCLFKILII
1401RLLLIFQHSGIHVEYHHHQAVIVIPKVDQQLHGRCYDHN

I think you will agree the probability of finding all of these red letters in a sequence of the correct mass is a strong indicator of the identity of the protein. If we now look at the proteins that have similar sequence signatures, we see that they are associated with a common "domain" (a term used to define a structural or functional component of a protein), called LPD_N, for Lipoprotein, N-terminal domain. This in turn is associated with proteins that transfer lipid molecules (fats) across a cell membrane: one of the most well studied being the protein called vitellogenin, which you will know since it forms the majority of the protein in egg yolk! The other important piece of information recovered from the computer analysis is the link to a publication in which the authors claim: "Activated phenoloxidase from Tenebrio molitor larvae enhances the synthesis of melanin by using a vitellogenin-like protein in the presence of dopamine". I was intrigued when one student told me that the melanization pathway was a feature of a fungus found on the walls of the Chernobyl reactor. I then found the articles entitled: "Ionizing Radiation Changes the Electronic Properties of Melanin and Enhances the Growth of Melanized Fungi"  and "Ionizing Radiation: how fungi cope, adapt, and exploit with the help of melanin" 

It is vital that the students appreciate how powerful Bioinformatics and the Internet can be, but we also need to keep in mind the role of experimental work in addressing, in this case,  the role of melanization in the meal worm, as we explore the diversity of this Biological phenomenon!  So we have information relating to two types of proteins and one strand of biochemical information: the likelihood that this protein is associated with melanization is reinforced by the students' observations that the protein extracts were pale brown as the larvae were extracted and became darker during the day: a feature common with enzymes of this class. I asked the students to take a look at melanization and its significance in Biology.

A protein closely related
to Vitellogenin (Lipovitellin)
The class are now following the information and preparing their final reports and presentations, while I am wondering where we go next, as the prospect of our genomic sequencing experiment looms. The class are now following the information and preparing their final reports and presentations, while I am wondering where we go next, as the prospect of our genomic sequencing experiment looms. It is vital that the students appreciate how powerful Bioinformatics and the Internet can be, in conjunction with experimental Science. It seems that we are combining the development of scientific and technical skills with curiosity driven research in a way that I never imagined could happen in a school! 

Thursday, 10 July 2014

Pigment of my imagination again!

The results of our Royal Society-sponsored, Y10 mealworm proteomics experiment are now in hand thanks to the help of Dr. Mark Dickman in the CheLsi group at the University of Sheffield (left). Dr. Alison Nwokeoji in Mark's lab, analysed the samples that Michael and I pooled from the extraction and purification of meal worm larvae using ion exchange chromatography. Just to refresh your collective memories, everyone extracted soluble protein, centrifuged to discard the insoluble debris and handed me their supernatants. I pooled them all and passed them through a 10ml Q Sepharose column during the day. I eluted with a series of increasing concentrations of NaCl, culminating in a 2M NaCl elution for the final step. Having collected the fractions, you then used saturated ammonium sulphate to concentrate the proteins, centrifuged and redissolved ready for SDS PAGE analysis. Since the first gels were a little streaky, we dialysed the samples and the students whose samples we chose to send to Sheffield were: Marcus Kennedy and Millie Keegan from the "Crick Bench" .

The objective of the mealworm experiments has been to establish this simple beetle as a school friendly model organism, with which to illustrate contemporary methods of biochemical analysis. For example, investigations into the mechanisms of cancer have moved towards single cell analysis of the proteome and the genome of individual cancer cells.Of course it is not clear at this stage whether this will prove to be the most incisive or fruitful approach to the attack on cancer (or even an understanding of the fundamentals of the disease), but it is the methodology that is current and therefore I feel for this reason alone,  its inclusion in our innovation lab programme is justified. 

In the mealworm proteome project, we set out to develop a simple, reproducible

protocol for the extraction of soluble proteins from the mealworm (larvae at first). The prominent bands isolated from Marcus and Millie's dialysis samples (prepared from the whole class extraction and purification) were analysed by Mass spectrometry in the Sheffield. The whole process represents an example of how proteins are analysed in complex mixtures and simulates contemporary analysis of for example protein biomarkers in blood or other biological fluids.

So, how successful were the students? If we break the workflow down into stages:
1. Mealworm "husbandry" 
2. Protein extraction
3. Protein purification
4. Protein concentration/de-salting
6. Mass spectrometry
7. Data Analysis and interpretation


Then the students collectively score 8/10 (well maybe 7/10 since data interpretation is still in hand). The only step that wasn't completed by a Y10 (just for those who don't know, Y10 students are typically 14-15 years old and have had very little laboratory experience), was the mass spec analysis, since this is a very specialised process. However, the analysis of proteins by mass spectrometry is becoming more common place and is often outsourced by professional laboratories to experts like Dr. Dickman.

What did we discover? The main protein that I want to mention represent the tip
of the iceberg that is the mealworm genome and proteome! The protein identified from the low salt elution fractions of Molecular Weight in excess of 150 000, turns out to be a "melanization" related factor from Tenebrio. It has been seen in related organisms, but how robust is the association of function to this predicted protein sequence? 
It is one thing to say a protein sequence is like another one, whose function is known, but it would be premature to assign function without further research and experiment. Why? Well, assigning a function for a molecule that is identical to a known protein of the same sequence is a good first step in assigning function, but does the protein have the appropriate properties? Well, it did have the characteristics of a phenol oxidase (it appeared to be oxygen sensitive and showed a brown colouration). The molecular weight on the SDS Gel was close to the mass of the best "hit" by MS and previous studies have shown this activity to be present in the larvae.  One pure fraction, remains unedentified until we obtain a complete genome sequence for Tenebrio (and I am working on funding for this), but we will follow up analysis of this by other methods. The other proteins that were identifies are also linked to melanization and so I have an interesting weekend ahead on PubMed! 

These data will form the basis of the first of two publications to emerge from the innovation labs at the UTC. The Y12 work on the non-ribosomal peptide synthetases has led to the amplification of the gene from Synechocystis and we are currently aiming to produce the recombinant protein products in a number of wild type and reconfigured forms for the development of novel specificity enzymes. The summer is the time for working through the data and when we have completed this, I shall let you know the details of our findings. In the meantime, thanks to our collaborators (Mark and Alison), our sponsor, the Royal Society and the enthusiasm of the Y10s. I would also like to single out Jack Webster a member of the Greenland Biodesign team in Y12, who has made all of the mealworm research possible through his dedication to developing and maintaining our UTC colony.