Author Archives: Paula

Donating hope – a success for parents in danger of transmitting mitochondrial disease

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 by Prof Bob Lightowlers

It was 2pm on a Tuesday afternoon and there I was watching television in my office. An unusual experience (honestly) but it was to be a truly momentous occasion. For the next 90 minutes, members of parliament would be debating whether to sanction the procedure of mitochondrial donation. What on earth does that mean ? Well, mitochondria are essential compartments (or organelles) that provide numerous key functions for the cell. They also have their own genome, called mitochondrial DNA – mtDNA – that contains the information to make up just 13 proteins, all of which are important in their function of producing main energy source of the cell, ATP. This is why mitochondria are often referred to as the cell’s batteries or the powerhouse of the cell. It is important to note that mtDNA is very small when compared to the nuclear DNA component: 16 thousand mitochondrial nucleotides (that is, the “letters” in the genetic code) vs more than 3 billion nucleotides in the nuclear DNA.

OK, but why would you want to donate mitochondria?

Mitochondrial DNA is strictly passed down via our mothers, unlike nuclear DNA which is comes from our father and mother. Almost 2,500 women in the UK have mutations in some or all of their mtDNA that can cause disease. Defects of this mitochondrial genome, can be responsible for a wide spectrum of mainly muscle and neurodegenerative disorders for which there is no treatment. By identifying those women with defective mtDNA, we could potentially prevent transmission of their unhealthy DNA by substituting their mitochondria for organelles from a donor. Simple on paper!

What’s the problem?

There are two major barriers. First, how safe would any technique be for mitochondrial donation ?

The pronuclear transfer method: Embryos are shown with mitochondria carrying normal (green) or mutant (red) mtDNA. As the embryos begin to develop, pronuclei become visible. Pronuclei from the normal donor embryo are removed (blue, top panel ‘enucleation’) and are replaced with the nuclear DNA from the patients (red, karyoplast). The resultant embryo carries nuclear DNA from the patients and mtDNA from the donor (mitochondrial donor zygote).

The pronuclear transfer method: Embryos are shown with mitochondria carrying normal (green) or mutant (red) mtDNA.
As the embryos begin to develop, pronuclei become visible. Pronuclei from the normal donor embryo are removed (blue, top panel ‘enucleation’) and are replaced with the nuclear DNA from the patients (red, karyoplast). The resultant embryo carries nuclear DNA from the patients and mtDNA from the donor (mitochondrial donor zygote).

The technique being championed in Newcastle is that of pronuclear transfer. The idea is to take the nuclear DNA from a fertilised embryo and transfer the DNA to a donor with no nuclear DNA, ie, where the nucleus was removed. The newly made embryo then has nuclear DNA from the mother and father but has only mtDNA from the donor.
As you can tell, there is quite a lot of tricky manipulation here. Further, although the mtDNA that has been replaced carries only a very small number of genes, could these somehow be incompatible with the nuclear DNA?
After many years of very careful analysis, there is no evidence to suggest that this procedure is unsafe. The question of incompatibility would also seem to be highly unlikely. After all, nature has been performing the experiment of mixing and matching nuclear and mtDNA since the evolution of Homo sapiens. The idea that, for example, there would be something wrong with the child of an aboriginal woman and a inuit man due to the substantial differences in their mitochondrial DNA would seem laughable.

In addition, does the replacement of DNA, albeit the complete mitochondrial genome, constitute genetic manipulation? This is a contentious issue and a strict definition of what constitutes genetic manipulation, particularly when concerning mtDNA, is difficult to agree on. It must be remembered that mtDNA is completely separate from nuclear DNA and needs to be considered as such.

A separate issue is that many people are ethically uncomfortable with this process. Can embryo manipulation ever be acceptable? Certainly, many religious people have a deeply felt objection to this.
Even when we accept that these methods could offer such an immeasurable benefit for many couples, it is clear that there were many questions to be tackled before we could consider the prospect of mitochondrial donation. For this reason, experimentation and public consultations had to be initiated.

The Parliamentary Under-Secretary of State for Health, Jane Ellison, when presenting the vote on Tuesday highlighted the measures taken to date to assess the safety and ethical concerns surrounding mitochondrial donation.

The Parliamentary Under-Secretary of State for Health, Jane Ellison, when presenting the vote on Tuesday highlighted the measures taken to date to assess the safety and ethical concerns surrounding mitochondrial donation.

To cut a very long story short, both have been carried out for many years, including supportive public consultations and independent review by the Human Fertilisation and Embryology Authority (HFEA) reporting that the procedure was not dangerous, as we’ve covered before (here and here). Following these procedures, Professors Turnbull, Herbert and numerous members of the Wellcome Trust Centre for Mitochondrial Research were instrumental in persuading the government to finally hold a debate in the House of Commons.

The vote scheduled for Tuesday afternoon would decide whether the HFEA would have the right to offer a licence to perform mitochondrial donation, a first for the UK and the world. The week leading up to the debate was a white-knuckle ride. Letters of support were published in leading newspapers from Nobel Laureates and other eminent scientists. Just when it appeared that the tide was turning, the Church of England announced it could not support mitochondrial donation. This was a great disappointment and rather a shock, as they had been involved throughout the lengthy consultation processes and had not indicated their level of concern.

Now a back-benh MP, former Minister for Science, David Willets, made a clear case in support of mitochondrial donation.

Now a back-bench MP, former Minister for Science, David Willetts, made a clear case in support of mitochondrial donation. You can read the whole debate here.

Back to me watching the television. At 3:45pm, the members had cast their votes and the count was in – 382 for the motion, 128 against! This is a fantastic result. It gives couples who may be at risk of having a baby with mitochondrial disease the chance to choose whether they want to try mitochondrial donation, just like couples have been able to choose in vitro fertilisation since the 1970’s.

Obviously, this result has gathered lots of media interest and even I was rolled out to perform a couple of interviews.

There is still more to be done, however. Further important research to support the safety of the procedure is currently in review but it must be recognised that every clinical procedure carries a risk. What this vote does is to empower the HFEA to licence this procedure in the UK, but this is still an important barrier and many issues are still to be addressed. And it also needs to be discussed by the House of Lords, of course.

Regardless, today’s vote was a wonderful day for anyone who has been touched by mitochondrial disease in any form. Twenty years ago, Doug Turnbull and I used to discuss this idea. He and his colleagues have done a remarkable job to make this pipedream a reality.

ECRs at ICaMB – Green transcription: how studying Cyanobacteria could change the world

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In the latest of our series on the Early Career Researchers of ICaMB we asked Dr Yulia Yuzenkova to tell us about her research and the route that took her from a PhD in Moscow to being awarded a Royal Society University Research Fellowship in Newcastle.

Yulia
Dr Yulia Yuzenkova

My early training was in Moscow, firstly as an undergraduate at Moscow State University and then for a PhD from the Russian Academy of Sciences. However, during my PhD I moved to the USA to study at the Waksman Institute (Rutgers University) with Prof Konstantin Severinov, where I was also a postdoc. My work in the laboratory was dedicated to the molecular mechanisms of inhibition of bacterial transcription by antibacterial peptides and small proteins from viruses. Transcription is the first step and critical regulatory checkpoint of gene expression. In all living organisms transcription is performed by multi-subunit RNA polymerases (RNAP). The central role of transcription in cellular metabolism and the presence of domains specific for bacteria make RNAP an obvious target for antibiotics. Yet, for decades, only one inhibitor of RNAP, rifampicin, has been used in the clinic to treat tuberculosis, while only recently, in 2011, lipiarmycin (fidaxomicin) was approved to eradicate Clostridium difficile. Two is a very small number, but this indicates that there is a good probability of finding new drugs that also work by targetting RNAP. Apart from their clinical significance, antibiotics and inhibitors of transcription in general have been proven to be very efficient molecular tools (see “RNAP details” below for more info).

During my second PostDoc in the lab of Prof Nikolay Zenkin in Newcastle, I have focused on the mechanisms controlling the fidelity of transcription. The copying of genetic information by RNAP is far from being absolutely precise, and RNAP whimsically dislikes reading some DNA sequences, resulting in ‘pauses’. RNAP is able to correct its own mistakes using a proofreading mechanism and the help of small proteins called transcript cleavage factors (explained in detail below). It seems to be important to have at least one cleavage factors; otherwise, the RNAP molecules stop, resulting in “traffic jams” as trailing molecules keep moving and bump into it.

Batch culturing of cyanobacteria

Batch culturing of cyanobacteria

It was therefore a big surprise for me to learn that one large group of bacteria, cyanobacteria (details below), do not encode anything even remotely resembling cleavage factors. I started to look for an explanation for this extraordinary fact. Are there any factors that might compensate for the absence of cleavage factors? Is cyanobacterial RNAP so accurate and at the same time processive that it does not need them? In searching for the answer, I realised that almost nothing is known about the molecular details of transcription in cyanobacteria, and so I decided to apply for a Royal Society University Research Fellowship to try to answer these questions.

Microscopy image of membrane-stained cell of Synechocystis sp 6803

Microscopy image of membrane-stained cell
of Synechocystis sp 6803

In performing preliminary experiments for my application, I became fascinated with cyanobacteria, as they are truly amazing organisms to work with. They are the only prokaryotes that exhibit the classic circadian clock, and are the bacteria with the most complex intracellular organisation. Moreover, they are one of the most ecologically important groups on Earth. They live everywhere where sunlight is available and produce 30% of atmospheric oxygen; some can even convert inert atmospheric nitrogen into a digestible form.

 

The 2D projection of the membrane structure of cyanobacterium reminds me of a labyrinth

The 2D projection of the membrane structure of cyanobacterium reminds me of a labyrinth

With my Royal Society University Research Fellowship I am planning to investigate the molecular details of the transcription machinery and will look for novel transcription factors that regulate this process. I am also going to test the metal requirements of cyanobacterial transcription, because metal composition of cyanobacterial cells is very different from other bacteria to suit the need of photosynthesis. Another fascinating question is how fast in the cyanobacterial cell, which is tightly packed with photosynthetic organelles, can molecules find their way through the membrane labyrinth.

 

 

The nitty-gritty science:

RNAP in detail:

Inhibitors of RNAP as molecular tools for understanding its functions. A wide range of targets of known inhibitors is mapped on the structure of bacterial RNAP. We contributed to understanding the modes of action of inhibitors marked in bold.

Inhibitors of RNAP as molecular tools for understanding its functions. A wide range of targets of known inhibitors is mapped on the structure of bacterial RNAP. We contributed to understanding the modes of action of inhibitors marked in bold.

Studying the antibiotics and inhibitors modes of action have helped us and other groups to discover previously unknown functions and structural domains of RNA polymerase. For example, work on rifampicin shed light on geometry of the RNA exit path, long before the crystal structure of RNAP was solved. Moreover, streptolydigin led to the discovery of the novel catalytic domain, while microcinJ25 confirmed the proposed entry channel for substrates and tagetitoxin provided insight into the mechanisms of RNAP translocation along the template.

Newly synthesised RNA participates in the proofreading  in a ribozyme-like manner. This method of proofreading, probably a remnant from the distant past, is extremely slow. To accelerate proofreading (and to escape from pauses), all 3 domains of life encode non-homologous, but very similarly folded, small proteins called transcript cleavage factors. In E.coli, GreA is an example of a protein that fulfils this role. Some bacteria have several homologs of GreA (in E.coli there are at least 6). It seems to be important to have at least one, because if these cleavage factors are depleted in the cell, the RNAP molecules on the actively transcribed genes stop, obstructing transcription, but also the chromosomal replication machinery moving along the same DNA.

Cyanobacteria:

Cyanobacteria have been hailed as future photobioreactors.  Indeed, when supplied with little more than tap water and light, engineered cyanobacteria can produce all sorts of compounds from sunscreen to biofuels. Cyanobacteria can also be used for environmental applications such as greenhouse gas fixing and cleaning water from industrial pollutants. These initiatives, however, are compromised by slow growth of cyanobacteria and limited knowledge of their basic biology. By putting more effort into research, the potential abilities of cyanobacteria can eventually be harnessed on the industrial level. With this we could make a giant leap towards a future “greener” economy. With a little bit of imagination, it is not hard to envisage cyanobacteria helping humanity to colonise new worlds, and to permit them to inhabit the first lunar and martial greenhouses in not so distant future.

Dr Yulia Yuzenkova’s ICaMB website: http://www.ncl.ac.uk/camb/staff/profile/yulia.yuzenkova

Royal Society University Research Fellowship: https://royalsociety.org/grants/schemes/university-research/

Antisense Science: A Science Blog by Students

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Blogs are now a widespread science communication tool, with many researchers taking to the blogosphere to discuss the latest scientific discoveries, explain the basic concepts in their research field to a wide audience or just talk about science and scientists. Our 3rd year bioscience students have done just that, and this week we have a guest post prepared by them.

 

by Antisense Science

Antisense Science is a science blog founded by a group of 3rd year bio-scientists from Newcastle University. As a team, we recognise that science is not as accessible to the general public as it should be.  We therefore make it our primary aim to translate complex scientific principles and research articles that interest us personally, into topical, thought provoking blogs accessible to everyone.

Our project is small but our ambition is big! Since our founding in October 2013 we have published 58 articles covering psychoactive baths salts, human evolution and the neurobiology of love, to name a few, and with a growing following (we’ve had over 6000 hits since inception) we were thrilled to be given the opportunity to guest post on ICaMBlog. As Newcastle University students, our interest in research was stirred by the professors at Newcastle University, including those who founded this blog. With planned guest posts focusing on research by Prof Rick Lewis among others, maybe YOU will feature on Antisense Science in the near future! We foresee (we hope!) that our blog can form a bridge between researchers here at Newcastle and the student body, raising awareness of what is actually being discovered right under our noses. By forming mutually beneficial collaborations, we hope to diversify and grow our following and expose our current readers to a continual stream of stimulating articles which never fail to pique the interest of the curious.

Meet the Antisense Science team

Meet the Antisense Science team

Currently, we have a total of 7 writers, all of whom enjoy sharing the intrigue of the latest developments in science, from biochemistry to microbiology (and even physics) and we have no plans of stopping. Although many of us will be moving on from our BScs to ever greater things, Antisense Science will remain and we are even in the process of recruiting further up and coming bio-scientists as writers (keep your eyes peeled for blogs from first year students Bethany Lumborg and Lucy Gee, as well as our fellow third year Emily Lawson and a multitude of guest posters from across the student body). The future looks bright and we’re very glad with the progress we have made thus far!

For an example of what we do, here is an article on depression written by our very own Joe Sheppard. We hope you enjoy it!  If you ever want to be involved in any of our projects feel free to drop us a message.  We also have Facebook (www.facebook.com/antisensescience) and Twitter (@AntisenseSci) so there are plenty of ways to keep in the loop.

 

The Confounding Contradictions of Depression

If you currently have or have had depression then you may already be able to tell your MAOIs from your SSRIs, but if you haven’t then what you read here might actually help. Knowledge is power and I believe learning a little something about depression could contribute a bit of control to an otherwise daunting and often underestimated medical condition.

Depression is perhaps the ultimate “common complex disorder”. Unlike pathogens or mutations that affect physical body tissue, depression is a condition that alters the very consciousness and emotional state of an individual making it a truly unique affliction. Throughout the course of our lives 1 in 5 of us will experience depression or anxiety of some kind, yet the majority of people conceive depression  simply as a disease of “sadness” when the truth is much more complex. “Anhedonia“, an inability to feel joy in anything and “congruent memory bias”, the inability to remember or altered recall of specific memories, are extremely common cognitive behaviours in depression that we often inflict upon ourselves on a day to day basis.

It may surprise you to know that modern science can say with little certainty what neuro-physiological changes initiate depression, and linking those that we do think are involved to the broad psychiatric manifestations seen in cases of depression is even harder as human experience and consciousness is beyond the understanding of molecular neuroscience (and by extension, definitely me). But from the murky depths of our own minds patterns do emerge, and as such there are a few good theories out there.

Rather confusingly the best fitting theory of depression is actually based on the drugs WE ALREADY USE to treat it, not a common theme in medicine, I might add. “The monoamine theory of depression” states that depressed brains have reduced signalling between neurons via a group of specific chemical neurotransmitters called monoamines. Two in particular called 5-hydroxytryptophan (hereafter referred to as serotonin) and dopamine are released into a synapse to induce electrical signals between neurons in the midbrain. These two neurotransmitters and the resulting electrical signals are most notably perceived as feelings of joy, euphoria, reward and attention. And there is some evidence to back this up: depletion of tryptophan, an amino acid essential for serotonin synthesis in the brain, caused mood congruent memory bias, and altered reward-related behaviours. Biochemical evidence exists too, abnormalities of the protein that binds serotonin in the brain called the 1A receptor have been noted in multiple brain areas of major depressive disorder (MDD) patients. Why does serotonin decline in patients with depression? Well, search my pockets, you will find no answers.

Rather reassuringly this hasn’t stopped treatment of depression at all, and several drugs for which the theory is named are all targeted at increasing serotonin, and so good feelings, within the brain. The so named selective serotonin re-uptake inhibitors (those “SSRIs” I snuck in earlier, such as fluoxetine and sertraline) are the most prescribed group of antidepressants and work in a way best aided pictographically:

Neuron

Schematic representation of neuron activity

Serotonin is synthesised in the presynaptic neuron from tryptophan, from here it is packaged into vesicles and upon nerve impulse firing (see previous article “ shedding light on neural networks”) is released in the synaptic cleft (the space between neurons).  Serotonin then binds to receptors on the postsynaptic neuron, stimulating a similar nerve impulse. However, serotonin is also able to control its own release: By binding to the 1A receptor on the presynaptic neuron it prevents continued release of serotonin, allowing the proposed channel protein SCL6A4 to re-absorb serotonin in the presynaptic neuron to be destroyed.

This is where SSRIs come in. Believed to bind to SCL6A4 and prevent the re-absorption of serotonin, it allows serotonin to remain in the synaptic cleft for longer and therefore stimulate nerve impulses in the post synaptic neuron for longer, and so increase the degree of monoamine signalling.

Let’s not forget our friend dopamine:

Dopamine too is a monoamine consistently found reduced in the blood of patients with depression, as a result of decreased synthesis and degradation in the brains of these patients. Neurons that signal using dopamine (as opposed to serotonin) are found in a region of the brain called the substantia nigra that degenerates during Parkinson’s disease. Interestingly the motor impairment (shaking) in this disease is often preceeded by major depressive disorder in 50% of Parkinson’s cases!

This brings me quite nicely to my final point. Since so little is known about the basis of psychiatric disorders their treatment has been almost purely symptomatic for the last 60 years. Thomas Insel, director of the US National Institute for Mental Health was quoted in 2013 as saying “In the rest of medicine, this would be equivalent to creating diagnostic systems based on the nature of chest pain, or the quality of fever.” This was following the release of the 5th edition of the diagnostic and statistical manual of mental disorders (DSM-5), that diagnoses psychiatric conditions based on common symptoms presented by each condition. Despite the strong agreement with Insel by many psychiatrists that this method is outdated, the shift to a more objective and molecular diagnostic is years away given the outstanding complexity of these diseases. But keep the hope, recent booms in neuroscience research are a sure step towards a less archaic means of treating depression and other mental disorders.

A new approach spearheaded by the same Thomas Insel called “Research Domain Criteria” or RDoC is already doing just that by utilizing genomic sequencing technology, fMRI imaging techniques and cognitive science to develop an entirely new platform for diagnosis based on new data being attained all the time, rather than the laboured DSM-5 classification. While still in its infancy this new approach aims to start looking at broader groups for diagnosis rather than classification of different disorders by the symptoms they present. For instance, by looking at a group of patients with different disorders but all experiencing anhedonia will not only allow a greater insight into a unifying cause for these symptoms, but also speed up treatments in the future.

Of course, there are always two sides to each argument and depression does in some sense stand alone from other psychiatric disorders such as schizophrenia in that it imparts a greater emotional influence on the sufferer – if “precision medicine” were able to prescribe a pill for the treatment of emotional conditions, would you want it to? Of course this is a quandary we won’t face for some time, but worth a thought.

J.

Some interesting but by no means comprehensive reviews on depression, its far too huge for 3 articles!

Hasler G (2010). Pathophysiology of depression: do we have any solid evidence of interest to clinicians? World psychiatry : official journal of the World Psychiatric Association (WPA), 9 (3), 155-61 PMID: 20975857

Martinowich K, Manji H, & Lu B (2007). New insights into BDNF function in depression and anxiety. Nature neuroscience, 10 (9), 1089-93 PMID: 17726474

Frances, A. (2013) One manual shouldn’t dictate US mental health research
(Accessed: 07/01/14).

Students take the stand: 2nd Year Post-Graduate Research Talks Day

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by Dr Tim Cheek

The annual ICaMB 2nd year PGR Talks Day was held at the Research Beehive in November 2013

Every year, we give our 2nd year Phd students to present their research in a relaxed, informal setting to their peer group and to the incoming 1st year students. The purpose of the event is two-fold:

– provide 2nd year students with a new presentation skill of giving a research talk to an informed but non-expert audience

– give 1st year students  an illustration of the diversity of the science that is done in ICaMB and an idea of where their own research should be in one year’s time when it will be their turn to speak.

This year’s talks on eukaryotic biology ranged from whole organism mammalian physiology (“The effect of neuromodulatory medication on the oral mucosa”, Mustafa Al-Musawi, Jakubovics lab) to single cell yeast genetics (“The role of the CST complex in telomere integrity, Kate Clark, Lydall lab)].

Jack Stevenson presents his work on copper metabolism in S. aureus

A key aim of basic bioscience research carried out in ICaMB is that it underpins our understanding of the molecular basis of disease and disorder and informs future translational biomedical approaches. This aim was strongly evident with talks on mitochondrial translational defects in encephalomyopathy (Maria Wesolowska, Lightowlers lab), the molecular basis of disease caused by non-invasive enteric pathogens (Oli Amin, Kenny lab), the role of the oxidative stress response in mediating fungal infections (Melanie Ikeh, Quinn lab), the molecular basis of “ribosomopathies”- human disorders of ribosome dysfunction (Loren Macdonald, Watkins lab), the effects of Resveratrol (Suzanne Escome, Ford lab) and DNA methylation (Joy Hardyman, Ford lab) on age-related genes, and on the role of membrane transport proteins in renal stone disease (Sarah Rice, Thwaites lab). The theme was extended with talks on how dysfunction in metal ion homeostasis in cells plays a key role in disease and disorder, for example calcium signals in neuroblastoma (Claire Whitworth, Cheek lab) and copper in Alzheimer’s disease (Eliona Tsefou, Dennison lab). Copper metabolism in lower eukaryotes was discussed in a talk on copper trafficking in yeast (Kerrie Brusby, Dennison lab) and in prokaryotes by talks on copper binding sites in Bacillus subtilis (Gianpiero Landolfi, Dennison lab) and Staphylococcus aureus (Jack Stevenson, Waldron lab).

The evolution of eukaryotic cells, their genomes and organelles, is also of great interest to ICaMB researchers who collaborate with, among others, the Natural History Museum in London. One talk described the development of better methods, based upon likelihood and Bayesian approaches, for phylogenetic analysis of molecular data (Svetlana Cherlin, Embley lab). The aim is to improve the reconstruction of phylogenetic trees relevant to understanding the early evolution of eukaryotes and the origins of eukaryotic genes.

Another illustration of the diversity of ICaMB research is that laboratories are also engaged in the development of new technological strategies for biomedical research. This was demonstrated by talks on the development of a primary tissue culture model system that can be used to identify the renal toxicity of new therapeutic drugs (Sarah Billington, Colin Brown lab), a simplified detection process for aggregation in the manufacture of biotherapeutics (Alysia Davies, Lakey lab), and on the use of natural antisense transcripts that may lead to improved gene silencing strategies in clinical applications (Monica Piatek, Werner lab).

Sarah Shapiro discussing her work on gut microbiota

The Centre for Bacterial Cell Biology in ICaMB is at the forefront of research into fundamental aspects of the cell biology and biochemistry of bacteria. Research provides scientific insights crucial for the discovery and development of new antibiotics, as well as providing solutions to a huge range of industrial and environmental problems through the emerging discipline of synthetic biology. This focus was reflected by talks on bacterial cell wall peptidoglycans (Adam Lodge, Vollmer lab) and D-alanine (Karzan Sidiq, Daniel lab), on system noise in the transcription of negatively regulated bacterial genes (Thomas Ewen, Hamoen lab), on the bacteriocin colicin (Daria Stroukova, Lakey lab), on cell wall deficient bacteria (L-forms; James Brown, Errington lab) and on the role of dental plaque bacteria in biofilm formation (Jill Robinson, Jakubovics lab). The theme was advanced with two talks on complex glycan recognition, acquisition and degradation by human gut bacteria (Sarah Shapiro, Bolam lab; Max Temple, Gilbert lab). Results of this research have applications in a number of areas including the development of biofuels derived from plant cell wall material and in personalised nutrition approaches to optimise microbiota function for the benefit of human health.

Students chatting about the talks and their research

This opportunity to talk about science and to engage with peers in a semi-formal environment, with no academic staff, is invariably voted by 1st and 2nd year students as their most favoured ICaMB event of the year. This year was no different. With 26 speakers and around 50 attendees in each of the 4 sessions, ICaMB students are clearly voting with their feet!

 

 

Dr Tim Cheek is the ICaMB Postgraduate Tutor.

If you are interested in applying for a PhD studentship in ICaMB more details can be found at http://www.ncl.ac.uk/camb/study/postgraduate/index.htm

Links

PANIC: the postgraduate network of ICaMB http://www.societies.ncl.ac.uk/panicicamb/index.html

Postgraduate Opportunities at ICaMB http://www.ncl.ac.uk/camb/study/postgraduate/index.htm

 

ECRs at ICaMB: Solving 3D puzzles

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by Dr Paula Salgado

After nearly one year editing the ICaMBlog, the time has come for me to tell you about my science and work since I joined ICaMB almost 18 months ago.

The fact that it has been 18 months since I moved up North to establish my own research group seems to have snuck up on me… Don’t get me wrong, so much has happened that, if anything, it’s surprising it all took place in 1 and a half years. At the same time, the feeling of a new adventure is still there.

Science is a constant adventure to seek new knowledge, to understand new mechanism, to see new things. In my case, to see into the very core of life’s machines: proteins. I use X-ray protein crystallography to probe the structure of proteins. It’s a bit like solving a puzzle: fitting the pieces of information together until we have a 3D view of the protein.

It is actually fitting that my blog post is the first ICaMB publishes in 2014 as this is the International Year of Crystallography. I could write a lot about it, but for now, I’ll leave you with an amazing video made by the Royal Institution that explains it all – in cartoons! If you want to know more about Crystallography, the Ri has a great collection of videos there, including Prof Stephen Curry’s Friday Evening Discourse, which I strongly recommend.

Freezing protein crystals for data collection at Diamond Light Source. H&S warning: liquid nitrogen is a hazard and we do handle it safely. At this point, I was just dipping the crystals into a small volume, all other procedures handling larger volumes involve wearing appropriate protection.

As a protein crystallographer, I’ve always been interested in proteins that have a relevance to human disease and used this technique to understand their structure and function. In the last few years, I’ve worked on proteins from human pathogens associated with hospital acquired infections, particularly Clostridium difficile and Candida albicans. However, protein structures don’t necessarily give us all the answers and they must be complemented with biochemical studies, as well as in vivo experiments. So my long term goal has become to establish a Structural Microbiology group, where we focus on structure determination of key proteins and complexes involved in pathogenicity as well as on their functional in vivo characterisation.

This is a challenge as it means stepping out of my structural biology comfort zone into the world of microbiology and cell biology. Not that I haven’t stepped out of my comfort zone before – if anything, those are areas that featured strongly during my undergraduate training as a Biochemistry student at the University of Porto in Portugal. In those days, choosing to do protein crystallography as my undergraduate project was the big step into the unknown. A trend that continued as a post-doc, when I joined Dr Cota and Prof Mathews group, a Nuclear Magnetic Resonance (NMR) lab at Imperial College, learning a completely different approach to protein structure determination. And just before coming to ICaMB, I worked in Prof Fairweather’s microbiology lab and always tried to learn a bit about the techniques others were using. So the current idea of bringing structural biology and microbiology expertise together in my lab is the natural evolution of these experiences.

C. difficile cells (green rods) lining the microvilli of the human gut. © Wellcome Trust (CC-BY)

Since joining ICaMB, I’ve focused on 2 main projects, both involving proteins from C. difficile. This spore forming strict anaerobe is resistant to most antibiotics and colonises the gut of individuals whose microbiome has been disturbed by these drugs. It is the most prevalent cause of gastrointestinal infections in hospitals and is a major cause of morbidity and mortality in the hospital environment. Despite recent decreases in the number of deaths and infections as hygiene procedures have improved in the UK, over 1600 people died in England and Wales in 2012 due to C. difficile infections (CDI). It also causes a huge burden to health systems, with an estimated €3,000 million per annum costs in the EU.

C. difficile disease symptoms are caused by the toxins it releases in a process that has been extensively studied over the years. However, the mechanisms of colonisation of the gut and spore formation are poorly understood. So we have been focusing on proteins involved in these two mechanisms.

Firstly, I’ve been trying to determine the structure of SlpA, the main protein constituent in C. difficile S-layer. S-layer is a paracrystalline coat that covers the cell and is presumed to act like a defense mechanism, as well as being involved in gut colonisation. This work, initiated a few years ago in Prof Fairweather’s lab is now a joint collaboration between our two labs and Dr Fagan, at Sheffield University.

SlpA crystals viewed under polarised light (protein crystals are birefringent, unlike salt crystals)

 

 

As this protein has tendency to form 2D paracrystalline layers, getting well ordered 3D crystals required for X-ray crystallography has been a challenge, but I have now succeeded in obtaining good crystals. However, other hurdles still need to be overcome to get a structure – but we are getting there!

 

 

Our lab: Adam Crawshaw and Paula Salgado

Last year, Adam Crawshaw joined my group as a BBSRC Doctoral Training Programme (DTP) student and we started a new project, looking at a complex between two membrane proteins that are essential for spore formation. As spores are the infectious agents, revealing the molecular details sporulation is important to understand the pathogenicity and infection cycle of C. difficile.

SpoIIQ and SpoIIIAH localise at the membranes of the forming forespore. Green: Membrane; Red: SNAP-tagged proteins.

When spores are first formed, a small cell (forespore) is engulfed by the larger mother cell, physically isolating it from the environment and nutrients in the medium. So, for the forespore to fully mature, it needs a nurturing channel to the mother cell. The two proteins we are studying  – SpoIIQ from the forespore membrane and SpoIIIAH from the mother cell membrane – create this channel. We have already successfully produced recombinant versions of the proteins and shown their interaction in vitro. In collaboration with Prof Henriques at ITQB, Lisbon, we also established their localisation during C. difficile spore formation. Next: crystals! But we are also investigating potential enzymatic activity both in vitro and in vivo, to bring the structure and biology together.

It has been an exciting year and a half – a steep learning curve with many new tasks, from supervising students to managing a lab and teaching undergraduates and postgraduates. The adventure continues, with new challenges and exciting discoveries ahead.

 

Links

International Year of Crystallography http://www.iycr2014.org/

Royal Institution Crystallography gallery http://richannel.org/celebrating-crystallography

Office for National Statistics (Clostridium difficile data) http://www.ons.gov.uk/ons/rel/subnational-health2/deaths-involving-clostridium-difficile/2012/index.html

European Centre for Disease Intervention and Control on Clostridium difficile  http://www.ecdc.europa.eu/en/healthtopics/healthcare-associated_infections/clostridium_difficile_infection/pages/index.aspx

BBSRC Doctoral Training Programme http://www.ncl.ac.uk/fms/dtp/