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@sugar-complexity
RSC Video Competition Finalists - Please Vote for "Burning the Calories"
Our very own Ellis O'Neill and Mike Rugen from The John Innes Centre are finalists in the RSC video competition http://www.rsc.org/science-activities/human-health/take-1-competition/videos.asp enjoy their video and please do not forget to vote for them!
Ever wondered how much energy is in a biscuit (in this case a Hobnob)? In this video, Mike Rugen and Ellis O'Neill from The John Innes Centre show how much energy is chemically released.
Stop motion animation by Marissa Maciej from The University of Manchester about her current PhD research project, labelling bacteria with azido sugars. Yes it is all hand drawn!
Cell Invaders Game - Play and Vote Now!
Here are the links to our Cell Invaders game:
PC http://sse.royalsociety.org/2013/games/
ipad https://itunes.apple.com/gb/app/cell-invader/id664340722?ls=1&mt=8
Please enjoy and don't forget to VOTE FOR US and send the links to as many contacts, family and friends as you have. We want to win!
Take a look at our official exhibit video!
http://sse.royalsociety.org/2013/exhibits/sweet-complexity/
Due to popular demand. Here's our brilliant animation again explaining all about "The Complex Life if Sugars".
The next video from the JIC "sugar snake"
The Great Seed Give-Away Video and Written Instructions!
Part of our exhibit involves looking at sugar structure in plants, algae and animals under the microscope. In plants, the main sugar structure which keeps the plant upright is called cellulose and this is made out of long chains of glucose molecules. If you visit our stand "The Complex Life of Sugars" and get involved with this experiment then we will give you a random packet of seeds. The seeds will either be wild type OR cellulose deficient mutant arabidopsis seeds. This video tells you how to grow and spot which type of seed you have and also has a nice catchy song to teach you how to say Arabidopsis!!
http://www.youtube.com/watch?v=AqP-IWG2irg&feature=youtu.be
We look forward to seeing you in London 1-7th July!
How to grow Arabidopsis – A beginner’s guide (written instructions)
Prepare a pot by filling it with potting compost and saturating the soil with water, while allowing any excess water to drain away. We use 10cm x 10cm square pots, but pot size is not really important.
You will have a tube with ~ 50 seeds THEY ARE VERY SMALL. You will need to spread them on the surface of the soil. It might be easier to first tip them onto a piece of folded card and then tap the card to get the seeds to roll down the fold onto the soil.
You need to cover the pot to keep the humidity high. We use a propagator lid, but you can use some Clingfilm or tape on a clear plastic bag. Leave them in the light somewhere quite warm, a window sill or even in the garden in summer.
It takes a least a week before the small seedlings become visible. Once you can see the first 2 seed leaves (cotyledons) remove the covering (see top left image).
All you have to do is wait now and water from time to time. The biggest problem is usually over-watering. Wait until the surface of the soil starts to dry before watering them again. This might be a couple of weeks. It is convenient to water the pots “from underneath”, by which we mean put the pot in a tray with no holes or a saucer which should then be filled with around 2cm of water. The pots should absorb most of the water. If there is still water in the tray after two hours tip it out or remove the pot from the tray.
Continue to water as necessary as the surface of the soil dries. This will be more frequent as the plant grows and up to twice a week once the plants are flowering.
The difference between wild type and mutant are obvious after a few weeks the mutant plants are smaller and darker green (see middle left image and middle right image).
Once the plant flowers the affects of the mutation are much more obvious. Mutants are unable to grow upright. They lack cellulose in the woody (secondary) cell wall. Cellulose acts like the steel in reinforced concrete to give the plants their strength (see bottom left image).
What is Arabidopsis?
Arabidopsis, it is probably in your garden, but you will not notice it. It’s a small weed that likes to grow in dry places so you will find it on bare ground or between cracks in the path or pavement. The bottom right image was taken outside the local primary school. Arabidopsis likes dry places and here it has grown between a brick wall and the pavement.
Why use Arabidopsis?
It does most of the things that all plants do, but it is convenient to study because it is small, generates a lot of seeds very quickly and has only a small amount of DNA in every cell (small genome). We can even learn a lot about trees by studying Arabidopsis.
What do mutants tell us?
They help us to understand what different parts of an organism do. In this case the plants makes no cellulose in its woody (secondary) cell wall and the plants cannot stand up straight and are extremely weak. This tells us cellulose is an essential part of the composite material that makes up the plant woody cell wall. It can also help us to find which genes make cellulose and help us to understand how it is made and how to alter/improve cellulose biosynthesis.
What is cellulose?
It is a linear chain of glucose just like starch, but the (b) linkages allow the chains to form a ribbon like structure. Cellulose is made by a very large enzyme complex that makes many (at least 18) glucose ribbons at the same time. These ribbons bond together to form a rigid fibril that forms the basis of all cellulose found in plants and is very strong.
What can we use cellulose for?
It already forms most to the material that we use to make paper, cotton fibres are nearly pure cellulose, it is one the most abundant components in all our vegetables. It could be used to make biofuels if we were able to easily break down plants cell walls into individual sugars.
What make cellulose hard to breakdown?
Plants cell walls are the first line of defense. They are designed to be indigestible and so not to be broken down by invading microbes. The nature of the cellulose microfibril also makes it hard to breakdown. Enzymes first have to strip individual cellulose chains from the microfibril surface before it can be broken down into to individual sugars.
Why worry about cellulose?
There is a lot of it! It makes up around 50% of most plant material. It is probably our most abundant biopolymer and the yield per unit area of land is huge and would be a huge source of renewable chemical and energy if we could use it efficiently.
Oobleck Video
Take a look at Mike Rugen and Ellis O'Neill from The John Innes Centre performing the oobleck experiment! http://www.youtube.com/watch?v=sXucOdwr_HM
Here's the production line in action!
Production Line!
The Complex Life of Sugars were very busy yesterday aliquoting 100,000 seeds into 3,000 micro tubes as give-aways for our exhibit. There will be Arabidopsis wild type and cellulose deficient mutants. Our next blog piece will tell you how to grow and spot which type you have! Thanks to everyone for your help, only 3,000 labels to fix to the envelopes now!
Screaming Jellybaby Video
Dr Mike Rugen and Dr Ellis O'Neill at The John Innes Centre have been hard at work creating a series of exciting sugar related chemistry videos. This first one is now available to watch here http://www.youtube.com/watch?v=z5rgPOnBqFA Enjoy!
The Complex Life of Sugars Video
Find out more about The Complex Life of Sugars with this excellent video http://www.youtube.com/watch?v=DHCpbEZ3kEg&feature=youtu.be
How Cell Surface Sugars Can Help Treat Disease and Injury
Heparan sulphate is a complex long chain sugar (a polysaccharide) strategically positioned at the cell surface (Pic 1 – top left). Heparan sulphate is critical for regulation of the growth and differentiation of stem cells by protein growth factors. It binds selectively to proteins such as growth factors and enzymes and controls their activities via a code of specific structures (Pic 2 – top right). It often acts as a “molecular matchmaker” that influences the assembly of multiple protein complexes. These mechanisms of action are central to a wide range of normal biological processes, and are relevant to many diseases and medical conditions. Recent studies have provided new insights into this area of biology, and have real potential for translation into novel treatments for diseases including neurodegeneration, cancer, inflammation, infections, and also for tissue repair. Chemical methods for synthesis of these complex sugars have recently been developed, including compounds found to be potent inhibitors of an enzyme central to causing Alzheimers disease (AD)1. These compounds could be developed to treat the underlying cause of AD for the first time. In spinal cord injury, such compounds have the potential to reduce scarring reactions which prevent nerve regrowth (Pic 3 – middle left), and improve the efficacy of cell transplantation therapies designed to aid nerve repair2. Mechanisms for natural repair of muscle injury depend on stem cells present in muscle tissue (Pic 4), and proper activation of these cells depends on heparan sulphate3. This has the exciting potential for development of drug-based therapies to enhance muscle repair, and for treatment of muscular dystrophies.
Pic 1 – top left - Mouse embryonic stem cells stained for cell surface heparan sulphate (green), with additional staining for a marker of stem cells, (red) and nuclei (blue).
Pic 2: top right - Heparan sulphate – a long chain sugar with specific protein binding sites that controls protein functions and regulates biological processes relevant to many disease processes.
Pic 3: middle left - Schwann cells, a supporting nerve cell type, can be transplanted at sites of spinal cord injury to enhance repair, but this can cause scarring reactions which prevent nerve regrowth. However, as this image shows, Schwann cells (green) and astrocytes (red), which normally form a distinct boundary between each other in the scarring response, can be induced to mingle freely after treatment with a low sulphated modified heparin. Such compounds have exciting potential for enhancing repair of spinal cord injuries.
Pic 4: middle right - Muscle stem cells are tiny cells normally resident in a dormant state attached to muscle fibres. In response to injury, these stem cells become activated, divide and then repair damaged muscle fibres. In this fluorescent picture the red staining identifies Syndecan-4, a protein with heparin sulphate side chains,expressed on the surface of muscle stem cells (indicated here by an arrow). The two muscle stem cells arrowed are the result of a recent cell division and are activated (as shown by green staining indicating high levels of phospho-tyrosine (note: the sum of red and green appears orange in fluorescence microscopy).
Pic 5: bottom - Muscle stem cells can be isolated from muscle tissue and grown in a petri dish. In this picture individual muscle stem cells are identified by the blue staining in the nucleus; they have proliferated and express on their surface Syndecan-4(stained in green), a protein with heparan sulphate side chains. Cultured muscle stem cells can also differentiate in a petri dish and fuse one to another to form large cells called myotubes, which eventually become mature muscle fibres. The nuclei in myotubes appear only blue, since they do not express Pax7 (stained in red, so that these nuclei become purple). Pax7 is only expressed by the non-differentiated progenitor cells, but still express Syndecan-4, which is now localized to special adhesion structures on the surface of myotube membranes called focal adhesions. In non-differentiated muscle stem cells Syndecan-4 controls and modulates growth factor signals through its heparin sulphate sugar chains, as well as mediating cell adhesion to the extracellular matrix.
Authors: Professor Jerry Turnbull and Dr Dada Pisconti, Liverpool University
References:
1. Schwörer, R; et al., Synthesis of a Targeted Library of Heparan Sulfate Saccharides as Inhibitors of b-Secretase: Potential Therapeutics for Alzheimer’s disease. Chemistry European Journal. 2013, DOI: 10.1002/chem.201204519
2. Higginson, J R; et al., Differential Sulfation Remodelling of Heparan Sulfate by Extracellular 6-O-sulfatases Regulates Fibroblast Growth Factor-induced Boundary Formation By Glial Cells: Implications for Glial Cell Transplantation, Journal of Neuroscience, 2012, 32, 15902-15912. DOI:10.1523/JNEUROSCI.6340-11.2012
3. Pisconti A., et al., Syndecan-3 and Notch cooperate in regulating adult myogenesis. Journal of Cell Biology, 2010, 190(3): 427-41. DOI:10.1083/jcb.201003081
Glycotherapeutics - How Sugars Can Help Us
Sugars are central to biology, controlling many vital processes relevant to health and a broad range of diseases. This provides many targets for therapeutic interventions. Existing sugar-based therapeutics – “glycotherapeutics” - cover a wide range of applications. On cell surfaces, they are attachment points for many disease pathogens, which has led to the development of drugs like Relenza (a neuraminidase enzyme inhibitor to treat influenza1) and anti-bacterial vaccines such as Haemophilus influenzae type B (Hib vaccine). Sugars interact with and control many proteins such as enzymes: the understanding of these mechanisms has resulted in the development of effective drugs such as heparin and low molecular weight heparins (for reducing blood clotting after surgery and for deep vein thrombosis - DVT); acarbose (for diabetes); and hyaluranon polysaccharide (for eye surgery, arthritis and wound healing). Sugars linked to the surface of proteins are also critical for their activity and stability, and this is a vital aspect for quality control of proteins made synthetically by recombinant technologies; these include therapeutic antibodies for cancer and autoimmune diseases, and protein drugs such as erythropoietin (EPO) for anaemia (see picture of EPO showing sites of sugar attachment on the surface). New insights into the biology of sugars are constantly providing new targets, so there is huge potential for new sugar-based drugs for diverse diseases. For example, advanced synthetic chemistry can now produce specific heparin sugars with potential to treat Alzheimers disease2, spinal cord injury3 and cancer, and might be used in the future to enhance muscle repair4, and control stem cells for tissue repair.
Author: Professor Jerry Turnbull, University of Liverpool
References:
1 von Itzstein M; et al. Rational design of potent sialidase-based inhibitors of influenza virus replication. Nature, 1993, 363 (6428): 418–23.
2 Schwörer, Ralf; et al., Synthesis of a Targeted Library of Heparan Sulfate Saccharides as Inhibitors of b-Secretase: Potential Therapeutics for Alzheimer’s Disease. Chemistry European Journal, 2013,DOI: 10.1002/chem.201204519
3 Higginson, Jennifer R; et al., Differential Sulfation Remodelling of Heparan Sulfate by Extracellular 6-O-sulfatases Regulates Fibroblast Growth Factor-induced Boundary Formation By Glial Cells: Implications for Glial Cell Transplantation. Journal of Neuroscience,2012,32, 15902-15912. DOI:10.1523/JNEUROSCI.6340-11.2012
4 Pisconti Addolorata; et al., Syndecan-3 and Notch cooperate in regulating adult myogenesis. J Cell Biol, 2010, 190(3): 427-41. DOI:10.1083/jcb.201003081
Tools for studying cell surface sugars
Cells are coated with a sugar-rich layer called the glycocalyx. Understanding its interaction with other molecules will provide a wide range of opportunities for the development of new foods, medicines and healthcare treatments. As the glycocalyx is very dynamic and changes between individuals or during some diseases, it provides a method for producing advanced diagnostic tools for personalised medicines.
It is very difficult to study the sugars of cell surfaces because cells are very small and fragile. We therefore have built new tools that allow us to measure communication events on cell surfaces, and one of these tools are glycoarrays. We use a gold plate because it is conducting and we can connect it up to many analytical instruments used in diagnostics in the clinic. We have developed methods to stick the sugars of the glycocalyx onto these gold surfaces so that is looks like a cell surface and then measure how diverse biomolecules react with these sugars. In the future, this high-throughput technology could be used for the discovery of sugar biomarkers in diseases such as cancer. It will also help us prevent and treat microbial infections, if we know how different microbes such as viruses and bacteria bind to the cell surface sugars. This strategy has recently been shown to be successful in new anti-flu therapies.
Author: Professor Sabine Flitsch, The University of Manchester
References
1. Šardzík, Robert; et al., Chemoenzymatic synthesis of O-mannosylpeptides in solution and on solid phase. J Am Chem Soc, 2012, 134, 4521-4524. DOI:10.1021/ja211861m
2. Noble, Gavin; et al. Accelerated Enzymatic Galactosylation of N-Acetylglucosaminolipids in Lipid Microdomains, J. Am. Chem. Soc., 2012, 134, 31, 13010–13017. DOI:10.1021/ja302506t
3. Rannes, Julie; et al. Glycoprotein labelling using engineered variants of galactose oxidase obtained by directed evolution, J. Am. Chem. Soc., 2011, 133(22), 8436-9. DOI:10.1021/ja2018477
4. Liu Y; et al.,Receptor specificity and cell tropism of D222G hemagglutinin mutants isolated from fatal cases of pandemic A(H1N1) 2009 influenza virus. J Virol. 2010, 84, 12069-12074. DOI:10.1128/JVI.01639-10.
Plant sugars - the next generation of renewable energy
Plant cell walls are important for a whole range of industrially important processes, from food processing to paper production. Recently they have received considerable attention as a potential sustainable source of CO2 neutral biofuels, however, it also possesses particular problems when trying to utilise the constituent components efficiently. Consequently, we are interested both in the fundamental research, but also how we might apply this research to generate an applied output.
Our focus has been on the woody, secondary cell wall since this constitutes the majority of plant Biomass. The secondary cell wall is composed predominantly of three polymers: cellulose, xylan and lignin. While cellulose and xylan may are potential sources of sugars for biofuels production lignin must be removed and utilized separately. While it is possible to identify a number of targets that could improve cell wall quality, such as less cross-linking of carbohydrates to phenolics and greater cellulose content our lack of basic understanding of how cell wall polymers are made, what controls cell wall quantity and composition and what physical properties different polymers confer upon the wall limit our ability to make these beneficial changes.
We identified a series of mutants that were deficient in deposition of cellulose, xylan and lignin. These have allowed us to identify several of the proteins required for cellulose and xylan biosynthesis that had previously not been possible to do using biochemical approaches. One of these xylan mutant appears to greatly increase the rates at which sugars can be released from the cell wall and should enhance our ability to breakdown plant biomass to use for sugar and biofuel production (Brown et al. 2011).
The mutants also allowed us to identify the CESA proteins that are presumed to be the catalytic subunit from the complex that makes cellulose. We are currently combining genetics with biochemistry to understand the composition and organization of the large complex that makes up cellulose with the aim of understanding what regulates the rates of cellulose synthesis and the crystalinity and organization of the cellulose microfibril. While we have used epitope tagging to generate highly pure preparation of oligomers of the catalytic CESA proteins (Atanassov et al. 2009) isolating an intact and functional complex remains one of the most important aims in this field. We are currently exploiting the information gained from epitope tagging to modify our approach and address this question.
Finally, generating large quantities of biofuels or other products from plant cell walls will require huge quantities of biomass. We have been looking at how to increase the number of cells that make these thick woody cell walls and have recently identified a receptor kinase and its ligand that regulate this process. Over expressing these genes gives a huge increase in biomass in the model plant Arabidopsis and we are currently testing how far we can extend these results into more useful plants and particularly trees (Etchells and Turner 2012).
Author: Professor Simon Turner, The University of Manchester
References:
1. Etchells, J.P., et al., Plant Vascular Cell Division Is Maintained by an Interaction between Pxy and Ethylene Signalling. PLoS Genet. 2012, 8, e1002997-e1002997. DOI: 10.1371/journal.pgen.1002997.
2. Brown, D; et al., Arabidopsis Genes Irregular Xylem (Irx15) and Irx15l Encode Duf579-Containing Proteins That Are Essential for Normal Xylan Deposition in the Secondary Cell Wall. Plant J, 2011, 66, 401-413. DOI: 10.1111/j.1365-313X.2011.04501.x.
3. Atanassov, I.I; et al., Elucidating the Mechanisms of Assembly and Subunit Interaction of the Cellulose Synthase Complex of Arabidopsis Secondary Cell Walls. J. Biol. Chem., 2009, 284, 3833-3841. DOI: 10.1074/jbc.M807456200.