MRC Clinical Sciences Centre - Latest News

Senescence Surveillance: Cancer Immune Response

7 Feb 2012 GMT

Professor Lars Zender of the Helmholtz Centre for Infection Research in Braunschweig, Germany has an interest in liver cell carcinoma – the most common type of malignant cancer worldwide. Together with collaborators at several institutes in Germany and here at the Clinical Sciences Centre, he has shown that cells shocked into a state of suspended animation by signals sent out by their cancerous neighbours are then closely monitored by 'T' helper immune cells [cells that assist in immunological processes]. The suspended animation state called senescence is something that eventually happens to all cells after they have gone through 50 replications, but it can be brought on earlier with a shock from neighbouring cancer cells. Senescence slows down the growth of cancers: “the body prevents senescent cells from further changing and growing into a cancer,” explains Professor Zender. “We could see the immune system launches a strong reaction against these cells…it then removes them from the body after a couple of weeks”.

The researchers identified the surveillance mechanism in mice engineered to have deficient T helper immune cells. Senescent liver cells in these mice grew into a cancer, while the surveillance mechanism in mice with fully-functioning T cells removed the senescent cells before cancer could develop.

Jesus Gil, who heads the Cell Proliferation group at the CSC and was a contributor to the study, said: "This work by the Zender group is an amazing breakthrough that help us understand how the immune system cooperates with senescence for tumour suppression. Currently we are engaged with Lars in finishing additional experiments to find out which molecules senescent cells use to signal not only to the immune system, but also other cells in the lesions and tumour microenvironment to keep them from dividing."

This newly-identified mechanism also indicates that HIV-positive patients are also at greater risk of liver cancer. The researchers followed this up by measuring the number of senescent cells in the livers of hepatitis C patients who were HIV-positive, and compared these with hepatatis C patients who didn’t have HIV. The first group showed an increase in senescent cells, because their bodies’ T cells have impaired function and cannot remove the senescent cells effectively. The second group didn’t, however.

“This clearly shows how important T cells are in the immune system’s monitoring of senescent cells,” commented Zender.

SJ

This work appears in Nature
Kang, T.-W., Yevsa, T., Woller, N., Hoenicke, L., Wuestefeld, T., Dauch, D., Hohmeyer, A., Gereke, M., Rudalska, R., Potapova, A., Iken, M., Vucur, M., Weiss, S., Heikenwalder, M., Khan, S., Gil, J., Bruder, D., Manns, M., Schirmacher, P., Tacke, F., Ott, M., Luedde, T., Longerich, T., Kubicka, S., Zender, L. (2011). Senescence surveillance of pre-malignant hepatocytes limits liver cancer development. Nature advance online publication.

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Microtuning

22 Jan 2012 GMT

Now, a group at the CSC has found evidence that small molecules called microRNAs ‘fine tune’ PRC1 following transcription, tailoring its component subunits to suit the job in hand. “This is the first study that looked at how PRC1 components can be regulated by micro RNAs during ESC [embryonic stem cells] differentiation,” confirms Jesus Gil, the study’s lead author.

PRC1 comprises four protein subunits, with two, three, five and six versions of each subunit in turn, giving 180 variants of PRC1 (2x3x5x6). The complex acts on the promoters of transcription factor (TF) genes. TFs are protein signallers, which switch genes on or off, priming a cell for differentiation.

The CSC team wanted to identify which of the protein subunits of PRC1 interacted most closely with the molecular tags in the transcription factors associated with pluripotency. They found that one subunit – called Cbx7 – was always bound to the pluripotency tags in mouse stem cells, indicating its crucial role in maintaining the state. To test this, the team performed a ‘knock down’ of Cbx7. “We found that the cells spontaneously began to differentiate,” explains Gil, “whereas when we switched on Cbx7 (called ectopic expression) differentiation was inhibited, promoting stem cell self-renewal.”

They found that when a cell differentiates, Cbx7 levels go down, but levels of other PRC1 subunits, Cbx2, Cbx4 and Cbx8 increase. Notably, an increase of these subunits was previously reported in studies of PRC1 interaction with transcription factors in cancer cells.

With such a clear role for a decrease in Cbx7 being important for cell differentiation, the researchers wanted to identify the mechanism behind it. They used a functional screen to look at the interplay of miRNAs with Cbx7, and found that two, called miR-125 and miR-181, directly regulate Cbx7 expression, the first time this has been shown.

Gil concludes: “Taken together, our results suggest distinct, context-dependent roles for individual PRC1 subunits and highlight the importance of Cbx7 and its regulatory miRNA network in ESC self-renewal and differentiation.”

SJ

What do PcG enzymes do?

Cells all start out the same – as stem cells – each with the same DNA sequence. Wrapped around histone proteins, the DNA within the nucleus is highly folded. As a cell matures, polycomb group (PcG) enzymes resculpt its genomic architecture. This impacts on cell fate by making some of the cell’s genes more accessible to the protein machinery that transcribes and translates them than others. The more accessible genes have the lead in telling the cell what to become (a heart, brain or liver cell, for instance).

Reference:
O'Loghlen, A, Muñoz-Cabello, AM, Gaspar-Maia, A, Wu, HA, Banito, A, Kunowska, N, Racek, T, Pemberton, HN, Beolchi, P, Lavial, F, Masui, O, Vermeulen, M, Carroll, T, Graumann, J, Heard, E, Dillon, N, Azuara, V, Snijders, AP, Peters, G, Bernstein, E, Gil, J (2012). MicroRNA Regulation of Cbx7 Mediates a Switch of Polycomb Orthologs during ESC Differentiation. Cell Stem Cell 10, 33–46.

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How the Brain Makes Maps

22 Jan 2012 GMT

“The video aspect lends an entirely new dimension to Virtual Lab,” comments Emma on the new experiment. Previous successful pilot experiments allowed students to engage with real research, but the images that made up the experimental results were static.

Dr Spiers' work is all about movement, because his work focuses on navigation. He wants to understand how the brain constructs a mental map of the world from sensory input and memory. The techniques he uses range from brain imaging and neuropsychological testing to virtual reality and eye-tracking. He has studied the brains of London’s cabbies, but in this new experiment he is interested in how rats use visual cues to help navigate towards a goal.

“It’s really important that school students have the chance to work with real research and interact with practising scientists,” says Dr Spiers, “– in helping with this analysis, students provide a fresh pair of eyes and may even notice things I don’t”.

Watch a video interview with Dr Hugo Spiers

The experiment is now live at www.virtuallab.co.uk. The resource is free, so if you know any secondary school students at GCSE or A-Level who are looking to bolster their Educational Record for application for further science study, please encourage them to sign up and take the challenge. PEMG is also interested in hearing from MRC students who want to become Virtual Lab Ambassadors. Email virtual@csc.mrc.ac.uk if you are.

SJ/EN …

Unsticking DNA 'Shoelace Ends'

22 Jan 2012 GMT

Telomeres are like shoelace ends from a structural perspective though mechanistically they’re somewhat less mundane: acting as buffers for the transcriptional enzymes that read their DNA. They actually stop those enzymes falling off too soon, helping to ensure that all the important bits of DNA are read. And this special role in DNA replication may point to their fundamental involvement in such mysteries as ageing; see At a Loose End in the box below.

Luis and his group describe in Nature Cell Biology how Cdc14 [enzyme] helps most telomeres to separate in yeast. Yeast as a model organism tells us a lot about fundamental biological processes in humans. The group previously reported (Nature 2008) that Cdc14 inhibits RNA Pol I [ribosomal RNA-making enzyme]. Its action in this context stops transcription at the ribosome [protein factory] and allows chromosomes to condense so the cell can divide. Their recent findings show Cdc14 also targets RNAP-II [messenger RNA-making enzyme]. This enzyme fabricates messenger RNA strands, which when dispatched to the ribosome tell it what protein to make.

Luis explains: “We thought Cdc14 might promote telomere separation by interfering with RNAP-II at sites right next to the telomeres. When first found, Cdc14 was as part of a trio of proteins in a protein complex called RENT, which inhibits RNAP-II transcription of non-coding DNA between ribosomal genes. The two other subunits of the complex were shown to individually regulate RNAP-II activity, but no-one knew if Cdc14 did. We were able to determine that Cdc14 dephosphorylates two specific serine amino acids in the heptapeptide repeat of the C-terminal region in the main subunit of RNAP-II. So we know Cdc14 regulates RNAP-II.”

At a Loose End
Every time DNA replicates, less and less of the telomere is copied, because its function is to act as a buffer. This slow but relentless degradation continues until the telomere is too short to allow complete DNA replication. Steady telomere shortening is inextricably linked to ageing and to senescence, the cellular barrier to tumour development; greatly shortened telomeres are often observed in cancerous cells. The fact that telomeres are there on the ends of chromosomes helps to distinguish them from breaks in DNA that form during normal cellular processes; marking those ends as belonging to their separate chromosomes and preventing them being joined together by overzealous repair enzymes.

So the group wondered if this activity might extend to telomeres. Using yeast mutants for Cdc14, they confirmed their theory, although it only acts on telomeres that contain sub-telomeric Y’ elements, accounting for two-thirds of yeast telomeres. Physically, Cdc14 mutants couldn’t separate their telomeres, although the team had to find out why.

“So we used a drug to inhibit RNAP-II,” explains Luis, “and this resulted in greater separation of telomeres in the mutants. This evidence strongly suggests that Cdc14 inhibits RNAP-II transcription. We think this allows the loading of factors that change chromosome shape during mitosis, allowing them to separate.” This is the first time that Cdc14 – a protein phosphatase and transcription inhibitor – has been shown to play a role in telomere separation.

Cdc14 is differentially expressed in human cancer cells. In fact the group has already confirmed that human version Cdc14 also inhibits RNAP-II acting through the same molecular switches as in yeast. This means the finding is of medical significance.

SJ

This work was published in Nature Cell Biology:
Clemente-Blanco A, Sen N, Mayan-Santos M, Sacristán MP, Graham B, Jarmuz A, Giess A, Webb E, Game L, Eick D, Bueno A, Merkenschlager M, Aragón L. (2011) Cdc14 phosphatase promotes segregation of telomeres through repression of RNA polymerase II transcription. Nat Cell Biol, in press. doi: 10.1038/ncb2365.

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White Matter Matters

22 Jan 2012 GMT

12 January 2009

Conventional MRI has so far failed to detect structural abnormalities that can fully account for the impairment seen in these cases. However, there have been indications that structural deficits in brain white matter, suggested by diffuse signals from the tissue in preterm individuals, are linked to neurological impairment.

Serena Counsell of the CSC's Imaging Physics and Engineering Group led research published in Brain, which demonstrated a link between the directional dependence of water diffusing in brain white matter and neurodevelopmental impairment in two-year old children born prematurely. The technique used, called diffusion tensor imaging, measures Brownian motion of water in tissue: Water moves more freely along white matter fibres than across them. Previous work had shown that the degree of directional dependence of water diffusing in white matter, termed the ‘FA’ (fractional anisotropy) value, increases during adolescence and can be correlated to increased working memory and reading age. FA values are indicators of the tissue microstructure of brain white matter, being dependent on fibre coherence, axonal density, cell membrane and myelination.

Counsell and coworkers have shown that FA values in two-year olds, all born preterm (median birth at 28 weeks), correlate to their performance the Griffiths mental development scale, which measures a number of factors including cognitive performance and hand-eye coordination. Lower FA values were associated with a lower performance score.

The incidence of preterm birth in developed countries is increasing while mortality rates are also falling. It is hoped that early assessment using FA as an indicator may assist in prognosis and that FA can be used as a biomarker for therapeutic studies.

Stefan Janusz

This work was published in Brain

Regions mapped by DTI (green) and General Quotient versus FA value

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Ready for Action

22 Jan 2012 GMT

The study focused on the urokinase-type plasminogen activator (uPA) in human HepG2 cells. Here it fits the 'standard model' of gene activation: sitting silently on the nuclear territory of chromosome 10 until treatment with an inducing compound switches on its expression. When activated, the majority of uPA genes relocate outside of their chromosome territory.

The observation that gene movement outside chromosome territories is associated with expression has led some researchers to propose that genes which do not move will remain silent, because they have less access to RNA polymerase factories. However in this study, Carmelo Ferrai and Sheila Xie show that the genes are already interacting with transcription factories, even before activation and while still located inside the chromosome territories.

The team led by Dr. Ana Pombo in collaboration with Dr. Massimo Crippa investigated whether relocation of the uPA gene during activation was accompanied by changes in chromatin structure that might facilitate transcription. Surprisingly, they found that even before the gene was chemically induced, the regulatory regions of uPA were already open: histone modifications associated with gene activity were present and the regions were bound by the RNA polymerase transcription complex.

Two critical phosphorylation targets control RNA polymerase II activity during transcription: serine-2 and serine-5. When only serine-5 is phosphorylated, the polymerase is poised – inactive but ready. When serine-2 and serine-5 are both phosphorylated, the gene is expressed. Before induction, most of the uPA genes within the chromosome 10 territory were only phosphorylated on serine-5, and therefore characterised as poised. Once chemically activated, the gene bound the fully active polymerase and moved out of its territory. Surprisingly, the interior of the territory was also accessible to the double phosphorylated polymerase, in contrast with current models that assume that the transcriptional machinery has limited access to the interior of chromosome territories.

The authors also found that after activation, the frequency with which uPA alleles are transcribed was the same whether the gene was inside or outside of the territory, again in contrast to the standard model. However, before activation genes inside the territory were largely silent – those few on the outside were transcribed at a rate equal to that of the induced gene. This study provides novel insights into the mechanisms of repression of poised genes prior to activation.

This research appeared in PLoS Biology

Ferrai, C., Xie, S. Q., Luraghi, P., Munari, D., Ramirez, F., Branco, M. R., Pombo, A., Crippa, M. P. (2010). Poised Transcription Factories Prime Silent uPA Gene Prior to Activation. PLoS Biol. 8(1): e1000270

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Jesus Gil Comments on Senescence Finding

22 Jan 2012 GMT

Scientists at the Mayo Clinic in the USA have announced results apparently showing that killing senescent cells [cells that have stopped replicating having done so more than the 'natural limit' of about 50 times] can slow the signs of ageing. From Gallagher's report:

Scientists at the Mayo Clinic, in the US, devised a way to kill all senescent cells in genetically engineered mice.

The animals would age far more quickly than normal, and when they were given a drug, the senescent cells would die.

The researchers looked at three symptoms of old age: formation of cataracts in the eye; the wasting away of muscle tissue; and the loss of fat deposits under the skin, which keep it smooth.

Researchers said the onset of these symptoms was "dramatically delayed" when the animals were treated with the drug.
...
The study raises the tantalising prospect of slowing the signs of ageing in humans. However, senescent cells cannot be just flushed out of human beings.
...
Dr Jesus Gil, from the Medical Research Council's clinical science centre, said the findings needed to be "taken with a bit of caution. It is a preliminary study".

However, he said it was a fascinating study which "suggests if you get rid of senescent cells you can improve phenotypes [physical traits] associated with ageing and improve quality of life in aged humans".

You can read James Gallagher's report here:

http://www.bbc.co.uk/news/health-15552964

You can also read more CSC stories on senescence here:

Cholesterol and Cellular Retirement

Senescence Barrier to Stem Cells

Behind the Barrier

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Professor Anne Soutar Retires from Research

22 Jan 2012 GMT

To mark the occasion, colleagues at the MRC Clinical Sciences Centre organized a seminar and reception in her honour on Monday 31st October. The programme featured talks from past scientific collaborators. Professor Geoff Gibbons presented a history of cholesterol, and Professor Steve Humphries discussed the development of our understanding of familial hypercholesterolaemia (FH), while Dr Mafalda Bourbon, an ex-PhD student, described setting up genetic diagnosis of FH in Portugal. Anne finished off with a whistlestop tour of some highlights of her research career exploring the ins and outs of the low-density lipoprotein (LDL)-receptor.

When Anne arrived at the MRC Lipid Metabolism unit in the 1970s, it had been postulated – largely through the pioneering work of American scientist, Ancel Keys – that replacing saturated by unsaturated fat in the diet would reduce blood cholesterol and the incidence of coronary heart disease. What was soon to be discovered, later resulting in a Nobel Prize for Joseph Goldstein and Michael Brown, was that individuals suffering from FH (one in 500 people) had defects in the biological machinery that regulates blood cholesterol.

Family studies revealed that individuals homozygous for the defect suffer severe FH, which while rare (one in a million), results in death by the age of 30. The heterozygous condition is much more common (one in 500 people) though less severe, resulting in early onset of coronary heart disease. Goldstein and Brown linked the high blood cholesterol in these patients to faults in the LDL receptor protein. They identified the function of LDL receptors in the uptake of cholesterol – in the form of low-density lipoprotein (LDL) – from the blood.

Low-density lipoprotein (LDL) is one of the five major groups of lipoproteins that transports cholesterol in the blood. High levels of LDL can cause health problems and cardiovascular disease, so it's often referred to as 'bad cholesterol'. Levels of LDL in the bloodstream become elevated if LDL-receptor molecules are in short supply. The receptors, located on cell surfaces, bind LDL and effectively remove it from the blood. In the absence of sufficient receptors, plaques can form in the walls of arteries, eventually leading to heart attack or stroke.

In patients with severe FH, functional LDL-receptors are partially or completely absent. Anne’s research programme involved biological investigations of the disorder by collecting blood samples and cells from families affected by FH. She recounted early attempts to clone the LDL receptor and contrasted the available techniques in the 70s for cloning and sequencing with the extremely powerful methods in use today. She and her team identified a mutation in a homozygous FH patient (Soutar et al 1989), which was later discovered to be prevalent throughout his large extended Pakistani family (Soutar et al 1991), as well as being a recurrent mutation in the UK (Bourbon et al, 1999). Genetic diagnosis of FH, in which her group identified more than 100 different mutations in patients attending the Hammersmith Lipid Clinic (Tosi et al 2007), was to some extent a by-product of her basic research. This focussed on the underlying mechanisms that regulate LDL receptor function and the identification and characterisation of other novel genetic defects that cause FH by affecting the LDL receptor pathway (Eden et al 2002, Herbert et al 2010, Sun et al 2011).

Today almost a thousand such mutations have been identified in the LDL receptor gene of FH patients, which helps to explain why this condition is so common. Understanding the biology of cholesterol and the regulation of its metabolism not only offers new treatments possibilities for FH sufferers, but is also of relevance to the general population in regards to the influence of diet on health. While cholesterol is essential to 'fluidize' cell membranes, uptake of excess dietary cholesterol discourages cells to make their own, which impacts on LDL receptor availability and the level of 'bad cholesterol' in the blood.

BM …

Bumpology: Perinatal Imaging

22 Jan 2012 GMT

The article highlights some of the important science behind the Perinatal Imaging group's use of MRI, especially the advantages of MRI over the more conventional ultrasound in illuminating detailed foetal physiology and neurology during the later stages of pregnancy.

"With ultrasound you have a relatively limited view, and beyond 32 weeks you can only really see parts of the body moving," says Mary Rutherford in the article. In comparison, MRI can produce high resolution images, elucidating fine structures while the baby continues her 'work-out' in the womb.

One interesting finding highlighted in the article is that the difference between the foetal acrobatics of a 20 week-old and a 40 week-old are minimal, suggesting that these 'kung-fu' moves are actually the result of "fairly primitive brain processes".

Mary Rutherford added: "People had thought that as gestation developed, babies' movements would become more complex as a sign of higher brain activity, but that doesn't seem to be the case."

Linda Geddes' article appears on the NewScientist website here

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Reprogramming & Chromatin

22 Jan 2012 GMT

Group Head Petra Hajkova joins us having previously worked under Azim Surani at the Gurdon Institute in Cambridge, a group whose research interests centre on genetic and epigenetic regulators of the germ line and pluripotency.

Petra's interest in epigenetics began before she was an undergraduate at Charles University, Prague, where she later received her MA for work on transcriptional silencing of integrated retroviruses. Her research in the field was then furthered at the Max Planck Institute in Berlin, where she was awarded her PhD. She then joined Surani's group at the Gurdon Institute, which specialises in cancer research and developmental biology.

Reprogramming and Chromatin group page …

Behind the Barrier

22 Jan 2012 GMT

Senescence is a barrier to cancer.
Graphic from Narita, M & Lowe, S (2005). "Senescence Comes of Age", Nature Medicine, Vol. 11, No. 9, pp. 920–922. Used with the Author's permission.

Senescence was first observed over 40 years ago as a mechanism that stops cells from dividing more than a finite number of times – usually about 50. It marks the onset of cellular degradation and is often seen as a cellular parallel to ageing in organisms.

In addition to this in-built replicative redundancy, cells can also be shocked into senescence prematurely by stresses, such as an oncogenic ‘insult’ sent out by a cancerous cell. This at least temporarily slows the multiplication of cancerous cells, so the study of senescence could potentially have far-reaching implications for the treatment of cancer.

Workers in the CSC’s Cell Proliferation Group, headed by Jesús Gil, have previously identified a gene called CXCR2 which mediates senescence. By inhibiting the gene, they have shown that cells can be made to live for longer. Now, they have identified the mechanism by which a locus called INK4a/Arf is derepressed by oncogenic signals to push the cells towards a senescent state.

Previous studies by other workers has shown that when the function of INK4a – normally silenced by Polycomb Group (PcG) proteins – is induced, the cells enter a senescent state prematurely.

In order to elucidate the mechanism by which this occurs, the researchers developed an ‘inducible’ system that allowed them to introduce oncogenic signals to human and animal cell cultures in vitro. Cells receiving an ‘insult’ developed characteristics of senescence, followed by a reduction in the methylation levels of a particular amino acid – ‘lysine 27’ on histone H3 – which must be methylated for the PcG proteins to bind and repress INK4a. Demethlyation of the amino acid ultimately leads to derepression of INK4a in human breast fibroblast cells, eventually leading to cellular arrest and the transmission of the ‘feedback’ senescence signals to stop growth. A slightly different pathway in mouse embryonic cells, where both INk4a and Arf are activated by the insult, echoed the result in humans, but showed that there are important differences between human ARF and its counterpart in mice.

The team reasoned that, besides nucleosome exchange, the reduction in methylation levels of the Ink4a/Arf locus could result either from a reduction in the levels of the methyltransferase enzyme EZH2 – which adds methyl groups to lysine 27 and normally facilitates PcG binding – or by an increase in either UTX or JMJD3 demethylase, which remove methyl groups and is important for cell differentiation. They followed the levels of these proteins by immunofluorescence, and determined that levels of JMJD3 increased over a period of days, whereas EZH2 increased. UTX level did not change, ruling out the involvement of the protein in the senescence pathway.

The suggestion that JMJD3 may be an important tumour suppressor seems to be backed up by clinical evidence: Elevated levels of the protein are often observed in benign and premalignant lesions, whereas aggressive tumours show a decrease in JMJD3, characteristic of a cancer in which the safeguarding measures of senescence have been overcome by the ravages of cancer growth.

Stefan Janusz

This work was published in Genes and Development.

Barradas, M., Anderton, E., Acosta, J.C., Li, S., Banito, A., Rodriguez-Niedenfuhr, M., Maertens, G., Banck, M., Zhou, M.-M., Walsh, M. J., Peters, G., and Gil, J. (2009). Histone demethylase JMJD3 contributes to epigenetic control of INK4a/ARF by oncogenic RAS.Genes Dev. 23 1177-1182.

Also see: Agger, K., Cloos, P. A., Rudkjær, L., Williams, K., Andersen, G., Christensen, J., Helin, K., May 2009. The h3k27me3 demethylase jmjd3 contributes to the activation of the ink4a–arf locus in response to oncogene- and stress-induced senescence. Genes & Development 23 (10), 1171–1176. (Same issue as above).

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Cohesin Forms Chromosome Loops

22 Jan 2012 GMT

Following DNA replication, sister chromatids are held together by a protein called cohesin, a ‘chromosome glue’ that also facilitates DNA repair.

In recent years, an additional role for cohesin as a regulator of gene expression has emerged from studies in model organisms and from human genetics. Slight disruption of cohesin binding has been implicated in disorders such as Cornelia de Lange Syndrome, which is characterised by potentially severe developmental anomalies. Cohesin therefore seems to be crucial for normal development, but the exact nature of its role, and the mechanism of its action, were unknown.

Last year, the CSC’s Lymphocyte Development Group, headed by Matthias Merkenschlager and Amanda Fisher, were one of four research groups to publish work showing that cohesin is recruited to specific sites by a DNA binding protein called CTCF. CTCF acts at chromatin structure boundaries and ‘insulator’ regions in the genome. The papers showed that cohesin was involved in CTCF’s ability to block interactions between sequences that control gene expression, such as promoters and enhancers. These findings provided a first explanation for cohesin's involvement in gene regulation but raised new questions: firstly, what is the molecular mechanism of cohesin action on gene expression? – and secondly, can cohesin binding also promote gene expression?

To address these questions, a new study by Sue Hadjur and colleagues of the Lymphocyte Development Group focused on IFNG, a gene that contains a cohesin binding site and is situated between two additional cohesin binding sites. IFNG encodes a cytokine central to T helper cell differentiation – one of the best examples of genetically identical cells acquiring distinct expression programmes and biological roles. The team looked at CTCF and cohesin binding around IFNG during T helper cell differentiation and found increased CTCF and cohesin binding in TH1 cells, where IFNG is inducibly expressed. Next, they used restriction enzymes to systematically chop up the chromosome and then measured the ability of the fragments to re-ligate. The further the fragment was from IFNG, the less it was linked to IFNG. The CTCF/cohesin sites, however, showed a marked increase in linking.

The obvious explanation was the 3D organization of the cohesin binding sites: sequence elements that are far apart in the DNA sequence can be in close proximity in three dimensions because of chromosome looping. Interestingly, looping was cell type-specific and occurred preferentially in TH1 cells. But this raised another question: was the interaction due to the looping of a single chromosome, or was the interaction occurring between chromosomes?

Measurements made using 3D-FISH, a technique that reveals the distance between gene loci with fluorescent probes, suggested that the homologous chromosomes were too far apart to interact. TH1 and TH2 cells were also shown to have nuclei of similar size, ruling out the possibility that restricted space might force interactions in one cell type but not the other. Finally, the 3C interactions at IFNG were shown to occur before DNA replication, ruling out the possibility that the interactions occur between sister chromatids. The team concluded that the interactions most likely are between binding sites on the same chromosome, termed ‘in cis’. This is a novel function of cohesin, which was previously only known to hold sister chromatids together 'in trans'. The team speculate that cohesin's ability to impose a cell type-specific 3D configuration of gene loci may be a mechanism by which cohesin influences gene expression.

The action of cohesin at the IFNG locus

This work was published in Nature

Hadjur, S, Williams, L. M., Ryan, N. K., Cobb, B. S., Sexton, T., Fraser, P., Fisher, A. G., Merkenschlager, M. (2009) Nature, epub 20 May

Background:
Parelho V, Hadjur S, Spivakov M, Leleu M, Sauer S, Gregson HC, Jarmuz A, Canzonetta C, Webster Z, Nesterova T, Cobb BS, Yokomori K, Dillon N, Aragon L, Fisher AG, Merkenschlager M. 2008. Cohesins functionally associate with CTCF on mammalian chromosome arms. Cell 132:422-433.

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White Matter and Schizophrenia

22 Jan 2012 GMT

Now, a paper recently published in Schizophrenia Research by members of the CSC’s PET Methodology and Psychiatric Imaging groups has shown that the relative reduction of grey matter between schizophrenics and healthy control subjects actually diminishes with age, contrary to predictions.

The study by Subrata Bose and co-authors looked at 34 male patients with schizophrenia aged between 27 and 65, and compared the tissue volumes of grey and white matter in their brains with that of 33 healthy male control subjects aged between 18 and 73. Volumes of each tissue type were determined by dividing each scan into an array of voxels (3D pixels) and categorising it as grey matter, white matter or cerebrospinal fluid.

Across the study, it was sound that patients with schizophrenia had overall reduced brain volume (1509 vs 1596 mm³), grey matter ( 755 vs 822 mm³) and white matter volume (525 vs 565 mm³); the latter consists mainly of myelinated axons.

The crucial finding was that total brain matter loss in patients with schizophrenia increasingly comprises white matter as the illness progresses, with 0.67% of white matter lost each year. Contrastingly, the study found that the volume of white matter in the healthy controls actually increased by 0.002% per year; the negligible difference being commensurate with previous studies, which indicate that there is no correlation between age and white matter volume.

The team also found that there was an increase in cerebrospinal fluid in regions adjacent to grey matter loss.

The paper concludes that two pathological effects may be in play: one acting prior to or early on in illness (which reduces grey matter), and another acting as the illness progresses (affecting white matter). This has important implications for understanding the pathophysiology of schizophrenia and related psychological disorders.

Link to the paper in Schizophrenia Research

Ref: Bose, S. K., Mackinnon, T., Mehta, M. A., Turkheimer, F. E., Howes, O. D., Selvaraj, S., Kempton, M. J., Grasby, P. M., May 2009. The effect of ageing on grey and white matter reductions in schizophrenia. Schizophr. Res. (in Press)

Also see: White Matter Matters

This paper has also been written about in:
Medwire
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Integrative Genomics and Medicine

22 Jan 2012 GMT

Enrico's interest in developing approaches to complex systems started when he was an undergraduate at the University of Sassari in Italy, where he studied non-linear interactions during the evolution of complex chemical reactions. His research interests then shifted towards genetically complex systems, studying population and statistical genetics at the University of Pavia where he achieved his Master Degree in Statistical Genetics, as well as multifactorial human diseases working at the University of Sassari and Institute of Population Genetics of C.N.R. in Italy, where he was awarded his PhD in Biochemistry, Biology and Molecular Biotechnologies. He further specialized in integrated linkage analysis and microarray-based expression profiling at the CSC Physiological Genomics and Medicine group.

His unique expertise in statistics, bioinformatics, physics, complex systems and population genetics allows him to pursue multidisciplinary and highly integrated approaches to exploit genomic, phenomic and genetic resources for the study of metabolic and cardiovascular disorders.

Click on image to enlarge

Link to Group page …

Root of Myelodysplasia?

22 Jan 2012 GMT

Now, CSC collaborator David Scadden and his team at Massachussetts General Hospital and Harvard Medical School, in collaboration with Matthias Merkenschlager (CSC Lymphocyte Development), have reported findings that may go some way to uncovering the abnormal developmental mechanisms that lead to the syndrome. They crossed mice lacking the gene Dicer – which is important in the biogenesis of microRNAs – with another strain expressing a tagged protein under the transcriptional control of of a promoter (‘osterix’) found in osteoblasts. Osteoblasts in bone marrow provide the tissue-specific niches for haematopoietic stem cells, which are the progenitors of all the cells constituent in blood. The mouse strain resulting from the cross-breeding carried out in the study was deficient in Dicer specifically in osteoblast progenitors and their progeny, including osteoblast precursors and mature osteoblasts. While the specificity of the osterix promoter was such that Dicer was expressed normally in haematopoietic stem cell, bone marrow dysfunction – or myelodysplasia – was observed in addition to a reduction in white blood cells. Some of the mice eventually developed secondary leukaemias.

This phenotype is highly reminiscent Shwachman-Bodian-Diamond syndrome. Deletion of the gene mutated in the syndrome Sbds in osteoprogenitor cells recreated the haematopoietic phenotype in the Dicer-deficient mice.

Although precisely which microRNAs in osteoblasts are crucial for haematopoiesis requires further investigation, the study has demonstrated that abnormalities in stromal cells such as osteoblasts can result in tumourigenesis in other cells.

Reference:
Raaijmakers, M. H. G. P., Mukherjee, S., Guo, S., Zhang, S., Kobayashi, T., Schoonmaker, J. A., Ebert, B. L., Al-Shahrour, F., Hasserjian, R. P., Scadden, E. O., Aung, Z., Matza, M., Merkenschlager, M., Lin, C., Rommens, J. M., Scadden, D., March 2010. Bone progenitor dysfunction induces myelodysplasia and secondary leukaemia. Nature 464 (7290), 852-857.

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Nitric Oxide Signalling

22 Jan 2012 GMT

James began his research career as a PhD student at the MRC Clinical Research Centre in the Molecular Medicine Group, headed by Prof. James Scott, before moving to a post-doctoral position at the MRC Laboratory for Molecular Cell Biology.

His interest in nitric oxide signalling developed following his move to Prof. Patrick Vallance’s group at UCL. Here James developed a multi-disciplinary team; spanning the fields of molecular and structural biology, pharmacology and physiology, in order to understand the regulation of nitric oxide production in vivo.

The crystal structure of the Nitric Oxide Synthase regulatory enzyme DDAH1.

Link to Group Page …

Significant Stats

22 Jan 2012 GMT

A relatively new but standard approach in transcriptional profiling is to identify a sample of probe gene sets whose expression is under genetic control (‘Expression Quantitative Trait Loci’ – eQTLs) and to look at how variation of these affects a particular phenotype. Yet analyses across tissues have previously been carried out by comparing results from multiple, single-tissue experiments – an approach that, due to variable accuracy rates (false positives and negatives) across tissues, leads to inflated discrepancies in identification of common eQTL lists. Application of standard univariate analytical methods further exacerbate the already-existing detection bias towards cis-eQTLs – which are more highly expressed, caused by genomic sequence variants residing within or close to the gene itself – at the expense of trans-eQTLs, which are caused by genetic variations distal from the gene itself. The more subtle effects of the latter are effectively sacrificed in analyses designed to clarify statistical significance in highly dimensional data.

But now, CSC Integrative Genomics and Medicine Group Head Enrico Petretto and coworkers at Imperial College department of Epidemiology and Biostatistics (Prof. Sylvia Richardson) have developed a new fully multivariate Bayesian approach to eQTL analysis in single and jointly across multiple tissues (PLoS Comput Biol 6, e1000737, 2010). The team looked at set of representative genes across four tissues (fat, kidney, adrenal and heart) in a panel of recombinant inbred strains derived by cross-breeding the Spontaneously Hypertensive and Brown Norway rats. Using the new Bayesian approach they detected many cis and trans-eQTLs – verified experimentally – that would not have been detected by other methods, and were able to disentangle tissue specific genetic regulation of transcription from systems-level effects. Critically, a more than fivefold eQTL detection rate increase from the joint analysis of the four tissues was seen as compared to that determined by comparing single-tissue analyses.

The work of the group is a big step forward in terms of looking at regulation of gene expression at the whole organism level. It is also a highly flexible approach, and is of great importance to understanding the gene expression changes underlying complex phenotypes in humans. SJ

This work was recently highlighted in Nature Review Genetics (Research Highlights June 2010 11:6).

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Understanding Hypertension

22 Jan 2012 GMT

The most widely studied animal model of human hypertension is the spontaneously hypertensive rat (SHR) – a model that has yielded a great deal of insight into the human condition. Without a complete sequence of the SHR genome, however, it has been difficult to resolve many of the molecular consequences of the genomic variations in SHR, and this is exactly what Professor Aitman and coworkers have delivered.

Using next generation sequencing technology, the team have sequenced the first genome of a mammalian disease model. They have generated a nearly complete catalogue of SHR genomic variants that may contribute to hypertension and other phenotypes. In the SHR genome, the team found 788 genes mutated enough to cause a major disruption of protein function. Affected proteins encode calcium and potassium ion channels, and may have a role in regulating blood pressure.

Rat vs. Mouse

The laboratory rat has a long history in biomedical research, being fundamental to advances in fields ranging from drug development, to neuroscience and physiology. Many investigators consider the rat to be a better model than the mouse, with behavioural and physiological characteristics more relevant to humans.

Interestingly, the study revealed that the rat has a clear advantage over other animal models, such as the mouse: genome variability within the species is more similar to that in humans. The team compared the SHR genome with that of the Brown Norway, the first rat to have had its genome sequenced in 2004. They looked at the number of single DNA base pair differences between the two strains and found that about 15 in every 10, 000 base pairs was different. This is remarkably similar to the variability between humans (also 15/10,000). However, from 11 commonly used inbred laboratory mouse strains, average variability was only about 7 per 10, 000 base pairs. Having a genomic variability that more closely matches that seen in humans is another factor that makes the rat a better model for human disease than the mouse.

The new genome sequence offers a direct path to establishing the causes of high blood pressure in the hypertensive rat and of corresponding genes that correspond to the causes of high blood pressure in humans.

Original article:
Atanur, S. S., Birol, I., Guryev, V., Hirst, M., Hummel, O., Morrissey, C., Behmoaras, J., Fernandez-Suarez, X. M., Johnson, M. D., McLaren, W. M., Patone, G., Petretto, E., Plessy, C., Rockland, K. S., Rockland, C., Saar, K., Zhao, Y., Carninci, P., Flicek, P., Kurtz, T., Cuppen, E., Pravenec, M., Hubner, N., Jones, S. J., Birney, E., Aitman, T. J. (2010). The genome sequence of the spontaneously hypertensive rat: Analysis and functional significance. Genome research. doi: 10.1101/gr.103499.109

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Neonatal Medicine

22 Jan 2012 GMT

The paper, which was published in the journal Pediatric Research in 1995, demonstrated that mild cooling of neonatal piglets deprived of oxygen ameliorated brain damage.

The same team had previously shown that the pathophysiology of oxygen deprivation in the piglet brain, characterised by a fall in the ratio of phosphocreatine phosphorous to inorganic phosphate as measured by ³¹P MRS, mirrored that in humans.

The study has led to an explosion of interest in cooling as a possible effective treatment for oxygen and blood flow deprivation. In 2005, a randomised multicentre clinical trial was reported in The Lancet, also featuring David Edwards as an author. The study gave an indication that cooling was likely to be effective in these cases.

References:

Thoresen, M., Penrice, J., Lorek, A., Cady, E. B., Wylezinska, M., Kirkbride, V., Cooper, C. E., Brown, G. C., Edwards, A. D., Wyatt, J. S., May 1995. Mild hypothermia after severe transient hypoxia-ischemia ameliorates delayed cerebral energy failure in the newborn piglet. Pediatric research 37 (5), 667-670.
Link to article

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Reversal of Fortune

22 Jan 2012 GMT

Research studies in the 1990s showed it was possible to reverse the damage caused by Parkinson’s disease by transplanting brain cells from donated foetal brains into patients suffering from the disease. Some patients showed remarkable improvement in their quality of life, significantly reducing the need for drug treatments.

However this controversial approach was abandoned in the early 2000s after it emerged that the transplant also caused some patients to suffer from jerky, involuntary movements known as dyskinesias. Until now, nobody knew why this happened or whether this side-effect could be successfully treated.

The research team, led by Dr Marios Politis, scanned the brains of two transplant patients affected by Parkinson’s. The brain scans showed that the involuntary movements were caused by malfunctioning serotonin cells in the area of the brain where the transplant had taken place. The team also found that they could treat the dyskinesias effectively by prescribing a drug which desensitises the serotonin nerve cells.

Dr Marios Politis, lead author says: “After the huge excitement surrounding the potential of brain cell transplants in the 1990s, we are thrilled that this discovery could re-open the door to this promising area of research. We know that the benefits of this treatment could last up to 16 years, and we look forward to bringing this treatment one step closer to a reality for Parkinson’s patients. ”

Investment in world-class regenerative medicine research is a key strategic priority for the MRC, helping to fast-track scientific findings from the laboratory bench to the patient’s bedside, through new and innovative treatments and therapies.

A small number of patients worldwide have undergone this transplant and it is not currently available as an option for treatment in the UK. For more information on the guidelines for using human tissue in medical research, including information on the Human Tissue Act 2004, please visit our website.

The results are published online in Science Translational Medicine.

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Primed

22 Jan 2012 GMT

A study carried out by the Gene Regulation and Chromatin Group, led by Niall Dillon, has provided new information on the mechanisms behind gene priming.

The results, which were published in Cell Stem Cell, show that ES cell transcription factors are directly involved in gene priming. These factors, which include Sox2, Oct4, Nanog and Foxd3, are involved in specifying the programme of genes that are expressed in pluripotent ES cells. What this study shows is that some of them are also involved in priming tissue-specific genes for expression at later stages of cell differentiation.

The researchers found that ES cell factors Sox2 and Foxd3 bind to a tissue-specific enhancer within the λ5–VpreB1 locus. In ES cells the λ5 and VpreB1 genes are silent, but they are switched on during B cell development, where they play an important role in controlling cell proliferation. Although switching on of the genes occurs late in this process, the enhancer is already marked by active histone modifications in ES cells. These modifications are specifically targeted to the enhancer by Sox2, one of the four factors used to generate pluripotent stem cells. Foxd3, which also binds to the region, seems to have a different role in damping down permissive transcription of the region in ES cells. Once the ES cells begin to differentiate, Sox2 is switched off, but in cells that progress towards the B cell lineage, another related factor, Sox4, takes over the role of maintaining the enhancer in an active state. In pre-B cells, Sox4 is involved in activating full expression of the genes. A similar switch in factor binding was observed at the Pax5 gene, which plays a key role in specifying B cell identity.

These results suggest priming of enhancers occurs through a transcription factor relay, with ES cell factors binding to tissue-specific enhancers and then handing over to related factors that bind to the same sites as cells differentiate. The transcription factors work in conjunction with histone modifications to keep genes in a primed state, ready to be switched on at the precise time and developmental stage where they are needed for correct cell differentiation.

This work was published in Cell Stem Cell

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Polystyrene Parcels

22 Jan 2012 GMT

Research carried out by Nicole Gennet of the CSC’s Stem Cell Neurogenesis group and her collaborators has demonstrated successful delivery of functional proteins into mouse and human neural stem cells (NSCs), using treated polystyrene microspheres. Other groups have shown that microspheres can be taken up by cells, but this is the first time that delivery has been demonstrated in NSCs, which have proved difficult to transfect by alternative methods.

By attaching fluorescent markers to the spheres, Gennet and her co-workers were able to follow both the levels of internalisation of spheres into NSCs, and the rates of subsequent uptake of spheres by propagating cells as they divided over a period of three days. Fluorescence measurements of mouse NSCs indicated extensive initial internalisation of all three sizes of microspheres investigated – 200nm, 500nm and 2µm (2000nm) – but, for the larger microspheres, fluorescence per cell decreased after the first day. For the two smaller sphere sizes, no such decrease in fluorescence was recorded, indicating that the rate of uptake of the microspheres by the propagating cells made up for the drop in fluorescence per cell as the cells divided.

Next, the team assessed delivery of a biologically-functional molecule into mouse NSCs. Microspheres conjugated to the enzyme beta-galactosidase, which processes larger sugar molecules into monosaccarides, were introduced as before and their activity was followed by fluorescence flow cytometry and microscopy. Cells showed near total beta-galactosidase activity after a three day period.

The study also demonstrated microsphere activity in human NSCs, which exhibited extensive internalisation of the three microsphere sizes. In addition, mircospheres were also introduced into human NSCs just as they were induced to differentiate. Fluorescence measurements of two morphologically-distinct cells made 30 days later demonstrated that both had internalised the mircospheres, indicating that their presence within a cell does not affect differentiation.

This work was published in New Biotechnology

Reference
Gennet, N, et al. Microspheres as a vehicle for biomolecule delivery to neural stem cells, New Biotechnology, (2009), in press.

The Edinburgh team that made the functionalised microspheres have published their work in Biomaterials:
Tsakiridis A, et al., Microsphere-based tracing and molecular delivery in embryonic stem cells, Biomaterials (2009), doi:10.1016/j.biomaterials.2009.06.024

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Fertility Switch

22 Jan 2012 GMT

With implications for both the treatment of infertility and recurrent miscarriage, the findings could also lead to new contraceptives. Around one in six women have difficulty getting pregnant. One in a hundred suffer recurrent miscarriage.

Women being treated at Imperial College Healthcare NHS Trust – either for unexplained infertility or for recurrent miscarriage – donated tissue samples from their womb lining (endometrium) to the research project. Previous studies had linked unexplained infertility to increased levels of an enzyme called Serum & Glucocorticoid Kinase 1 (SGK1). So the team compared samples taken in the middle of the menstrual cycle from women with proven fertility, and women with unexplained infertility or recurrent miscarriage. SGK1 levels were higher in the group with unexplained infertility, but lower in the group prone to miscarriage, even when compared to women with proven fertility.

This enzyme plays a crucial role in transporting ions and is known to be important for cell survival. The scientists wanted to test whether elevated SGK1 might interfere with the embryo being implanted in the womb. In humans, there is a limited window (2–4 days) after ovulation when implantation can take place, during which the endometrium has an optimal uterine environment to be receptive to an embryo.

The team found that levels of SGK1 decreased in mouse womb lining during the fertile window. When they injected extra copies of the SGK1 gene into the womb lining, the mice couldn’t get pregnant. So low levels of the enzyme seem to make the uterus receptive to embryos. “Uterine weight was also reduced significantly in SGK1-injected mice,” explains Professor Jan Brosens (now at Warwick Medical School), “with a smaller glandular area, and less space between epithelial cells. The enzyme also, directly attenuated the activity of several uterine receptivity genes, rendering the uteri unreceptive”.

So how can we explain low SGK1 in women who suffered recurrent miscarriage?

Madhuri Salker (Institute of Reproductive and Developmental Biology), first author of the paper, elucidates: “After an embryo is implanted, the lining of the uterus develops into a specialized structure called the decidua that leads to the placenta forming – abnormalities in the decidua can lead to pregnancy complications, such as miscarriage. We found that low SKG1 didn’t prevent implantation in mice, though litters were smaller and there was some bleeding, making miscarriage more likely.”

A careful balance of SGK1 is required throughout pregnancy. Levels must decrease for during the implantation window, though SGK1 needs to increase afterwards to ensure placental formation: “Depending on the cellular compartment, deregulated SGK1 activity in cycling endometrium interferes with embryo implantation, leading to infertility. It can predispose to pregnancy complications by rendering the feto-maternal interface vulnerable to oxidative damage,” concludes Madhuri.

Together, these findings provide fundamental insights into the process of embryo implantation and describe a novel paradigm of pregnancy failure with potentially far-reaching clinical implications.

SJ

Published in Nature Medicine
Salker, M. S., Christian, M., Steel, J. H., Nautiyal, J., Lavery, S., Trew, G., Webster, Z., Al-Sabbagh, M., Puchchakayala, G., Foller, M., Landles, C., Sharkey, A. M., Quenby, S., Aplin, J. D., Regan, L., Lang, F., Brosens, J. J. (2011). Deregulation of the serum- and glucocorticoid-inducible kinase SGK1 in the endometrium causes reproductive failure. Nature Medicine 17, 1509-1513.

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A Supporting Cast of Hundreds

22 Jan 2012 GMT

Processes at the heart of the living cell, the biochemical mechanisms that keep us alive, must often be inferred from fragments of evidence. Scientific experiments that have gone before; banks of data stretching back decades; mechanisms postulated from chemical, physical and biological ‘common sense’, all contribute to our understanding of what’s happening inside us. Isolating a cell’s contents at specific stages of development is one of biology’s most successful approaches to unpicking the interactions of genes and proteins.

Francis Crick, co-discoverer of the DNA double helix, famously framed the cellular factory as a one-way flow: DNA makes RNA makes protein. But the cellular machines that make RNA are themselves proteins, in the form of enzymes. These in turn rely on other proteins to function properly. So rather than being a one-way street, a complex network of interactions must function together to keep us alive.

RNA polymerase II (RNAPII) is the enzyme that reads DNA sequences, copying these into strands of mRNA [messenger RNA], which tell the ribosome [protein factory] which amino acids to string together. Scientists need to isolate and purify enzymes like this to understand their workings. Yet, while extraction methods have efficiently isolated RNAPII, the enzyme’s behaviour in vitro will not precisely replicate that within its native environment of the cell. Researchers working in the CSC Genome Function group have circumvented the issue with a technique that preserves subtle interactions, giving us more information about how proteins interact with RNAPII and affect its function. Their work has been published online in the journal Molecular & Cellular Proteomics.

Ana Pombo, the study’s lead author, explains. “RNAPII dissociates from DNA when chromosomes are shutdown for cell division (mitosis). We speculated that certain proteins would continue to interact with RNAPII during this stage of the cell cycle.” Her team used mass spectrometry and fluorescence microscopy to identify the enzyme-protein interactions. “Our isolation method combined with immunoprecipitation led us to identify more than 400 proteins that associate with RNAPII. Previous research suggested there were at most 100 proteins.”

Among the newly identified proteins comprising the interaction network – dubbed the RNAPII interactome – the researchers found several with implications for disease. One of these, ALMS1, is mutated in Alström syndrome, a disorder that leads to obesity and diabetes. “We’ve shown unequivocally that ALMS1 interacts with RNAPII in cells and affects its activity,” says Ana. “This will open up avenues to further explore the molecular basis of metabolic disorders like Alström syndrome. Our datasets also provide a novel resource that will be useful for identifying additional roles for RNAPII in health and disease.”

SJ

Reference:
Moeller, A., Xie, S. Q., Hosp, F., Lang, B., Phatnani, H. P., James, S., Ramirez, F., Collin, G. B., Naggert, J. K., Babu, M. M., Greenleaf, A. L., Selbach, M., Pombo, A. (2011). Proteomic analysis of mitotic RNA polymerase II reveals novel interactors and association with proteins dysfunctional in disease. Molecular & cellular proteomics, in press. Link to article.

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Quantitative Systems Biology

22 Jan 2012 GMT

The group, which joins the Epigenetics and Development section, is headed by Alberto Polleri.

Alberto's background is in theoretical nuclear physics, but he felt drawn to molecular biology by "the giant strides that [the field] is making...becoming ever more a quantitative science".

His transitional period, between his early research studying the quark gluon plasma and becoming a biologist, was spent at Stanford University in California.

The Group's research interests centre on the Calcineurin/NFAT signal tranduction pathway, an important pathway the disruption of which has been implicated in a wide variety of human diseases, ranging from immune system failure to osteoporosis to heart valve defects and recently to Down Syndrome.

Joining Alberto in the group are Kata Takacs and Ella Palmer.

You can see the Group's page here.

...also see here:
qsb.csc.mrc.ac.uk …

Oliver Howes Wins EPA Award

22 Jan 2012 GMT

The award is given in recognition of his group's work describing how measured elevated dopamine levels [an important chemical transmitter in the brain] could indicate the onset of psychosis. The paper reporting the findings, published in The American Journal of Psychiatry, has been named ‘best in category’ for the field of 'Biological correlates and treatment of mental disorders' by the association.

“We showed for the first time the neurochemistry underlying the onset of psychotic illnesses, such as schizophrenia,” explains Oliver. “A specific aspect of the dopamine system was abnormal only in patients who went on to develop psychosis, but not in similar patients who got better, or in healthy controls.”

“We found that dopamine abnormality got progressively worse in patients as they developed psychosis. This identifies a new target for drug development, with the potential to prevent the onset of psycotic illness,” he concludes.

Oliver will collect the award at a ceremony to be held in Prague, The Czech Republic, in March. On winning the award, he says: “It's an honour for the hard work of the whole research team to be recognised in this way.”

You can read more about the findings in this CSC news story from 2011.

Reference:
Howes, O. D., Bose, S. K., Turkheimer, F., Valli, I., Egerton, A., Valmaggia, L. R., Murray, R. M., McGuire, P., Jul. 2011. Dopamine synthesis capacity before onset of psychosis: A prospective [18F]-DOPA PET imaging study. The American journal of psychiatry.
http://dx.doi.org/10.1176/appi.ajp.2011.11010160

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Clogged Up

22 Jan 2012 GMT

Recently, a paper by members of the CSC’s Lipoprotein group has challenged this assumption, and found that the reality is more complex than has been assumed. The paper has also been highlighted in an Editorial piece for the publishing journal, Arteriosclerosis, Thrombosis and Vascular Biology.

Like a great many questions in biology, the starting point for understanding the normal metabolism of cholesterol lies in cases of extremes. Familial Hypercholesterolemia (FH) is a condition characterised by increased levels of bad cholesterol and a greater risk of premature coronary heart disease. It is caused by a mutation, either of LDLR (low density lipoprotein receptors – see 'Additional Background', below) which mop up LDL (bad cholesterol); or of apolipoprotein B100 (apoB100), the protein component of LDL that interacts with LDLR. In 2003, the occurrence of a mutated serine protease D374Y-PCSK9 was associated with FH. The mutation causes an increased degradation of LDLR, leaving fewer receptors to mop up LDL and increasing the arterial plaques that constitute atherosclerosis. In 2005, at the opposite end of the ‘cholesterol spectrum’ from FH, a mutation of PCSK9 found in 2% of African Americans that reduced its function was linked to an 88% decrease in coronary heart disease risk. It is unsurprising that a great deal of clinical research has focused on PCSK9 and more specifically on its regulation of cholesterol levels through LDLR, but was that the whole story?

In order to test whether LDLR degradation was the sole mechanism of LDL homeostasis, the Group led by Anne Soutar took mice expressing normal – or ‘wildtype’ – PCSK9 and the D374Y version identified in FH, plus a group of control mice not expressing PCSK9. They found that levels of cholesterol in the blood for the D374Y mice was highest, followed by wildtype PCSK9, with control mice having the lowest levels; the level in D374Y was further exacerbated by a cholesterol-rich diet.

Our diet is only part of the problem: our risk of developing atherosclerosis is also influenced by our genes. A complex cascade of regulatory feedback loops is subject to the programming in our genetc code, making individual physiology as unique as personality.

At left: Aortic lesions observed in D374Y Mice

As anticipated in light of results from previous studies, levels of LDLR in the D374Y and wildtype PCSK9 were lower than in control mice. However, it was also found that secretion of apoB-containing lipoproteins by liver cells was increased in the D374Y mice, and that blood plasma samples taken from these mice during fasting contained much larger lipoproteins, termed Very-Low Density Lipoproteins (VLDL). This is important because such ‘triglyceride-rich’ lipoproteins, alongside increased levels of apoB, are seen in human FH patients. Larger lipoproteins are associated with increased deposition of the atheroma plaques that constitute atherosclerosis.

The findings of the CSC group show that reduced LDLR isn’t the sole mechanism behind hypercholesterolemia, and it is hoped that further studies of D374Y mice may lead to new effective therapies to reduce atherosclerosis. SJ

Additional Background
Cholesterol itself is vital in maintaining permeability in cell membranes and is an important metabolite for many physiological functions, including bile production. Transported around the body in lipoprotein vesicles of various size, it is those vesicles termed ‘low density lipoproteins’ (LDL) that constitute ‘bad’ cholesterol. LDL vesicles comprise a greater proportional mass of cholesterol than ‘High Density Lipoprotein’ (HDL) vesicles – so-called ‘good’ cholesterol – which are about half the physical size of LDLs. LDL vesicles are mopped up by an LDL receptor (LDLR), but the level of LDLR is kept in check by PCSK9; an example of the multiple levels of feedback involved in mammalian metabolism. apoB stabilises and solubilises the cholesterol-carrying lipoprotein vesicles as they are transported in blood plasma, much as detergents solublise oil droplets in washing-up water. Increased apoB such as observed in the D374Y mice and human FH allows the formation of larger lipoprotein vesicles, which increases the risk of the plaques characteristic of atherosclerosis.

This work appears in Atherosclerosis, Thrombosis and Vascular Biology (link to article)
Herbert, B., Patel, D., Waddington, S. N., Eden, E. R., McAleenan, A., Sun, X.-M., Soutar, A. K., July 2010. Increased secretion of lipoproteins in transgenic mice expressing human d374y pcsk9 under physiological genetic control. Arterioscler Thromb Vasc Biol 30, 1333-1339.

The Editorial, Beyond LDL Cholesterol, a New Role for PCSK9 by ON Akram et al. is available here

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The Main Player

22 Jan 2012 GMT

Previous research correlating human disease to particular genetic variants has tended to concentrate mainly on genetic association, paying less attention to genes expression and how it influences physiology. In this study, integration of computational gene expression analyses, rat and human genetics, and functional approaches has allowed the team to go beyond standard genome-wide association studies and identify not only the genes in the network, but also the key players – the key genes that play major roles in determining disease.

Stuart Cook gave this analogy:
“If we think about our genes as being similar to a football team – it is one thing to know that the team you’re playing against has 11 players, but another to know who their main match winners are. What we find exciting about these results is that, for the first time, we have been able to identify the most important genes – who the strikers are, as well as who the team captain might be that coordinates the other players. Applying this knowledge to find out more about the key players that cause disease will help researchers find better ways to develop more targeted treatments in the fight against diabetes.”

The study identifies one particular gene, Ebi2, lying at the locus of a transcription factor-driven inflammatory gene network for IRF7 – a transcription factor associated with T1D. The results also emphasise the important role of transcription factors like IRF7 in mediating genetic expression and ultimately influencing physiology.

This development will help researchers focus their efforts to improve drug treatments for type 1 diabetes and could have an impact on other diseases where inflammation plays an important role.

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Reference:
Heinig, M., Petretto, E., Wallace, C., Bottolo, L., Rotival, M., Lu, H., Li, Y., Sarwar, R., Langley, S. R., Bauerfeind, A., Hummel, O., Lee, Y.-A., Paskas, S., Rintisch, C., Saar, K., Cooper, J., Buchan, R., Gray, E. E., Cyster, J. G., Erdmann, J., Hengstenberg, C., Maouche, S., Ouwehand, W. H., Rice, C. M., Samani, N. J., Schunkert, H., Goodall, A. H., Schulz, H., Roider, H. G., Vingron, M., Blankenberg, S., Munzel, T., Zeller, T., Szymczak, S., Ziegler, A., Tiret, L., Smyth, D. J., Pravenec, M., Aitman, T. J., Cambien, F., Clayton, D., Todd, J. A., Hubner, N., Cook, S. A., September 2010. A trans-acting locus regulates an anti-viral expression network and type 1 diabetes risk. Nature advance online publication. http://dx.doi.org/10.1038/nature09386

Novel statistical approaches have previously been shown to be useful in detecting groups of genes that act together to disease. Publications from CSC groups have demonstrated the power of a method called Sparse Bayesian Regression in elucidating the more subtle of these interactions. When applied to analyses of multiple tissue types, connections between disease genes missed by other analytical methods have been illuminated. The method was used to determine the key role Ebi2 has in the IRF7 gene network.

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What is Fat?

22 Jan 2012 GMT

In June, he gave a talk at The Endocrine Society meeting in San Diego, USA on ghrelin, a stomach hormone that seems to influence eating behaviour through stimulation of the brain’s dopaminergic reward system – the same network of neurons that are involved in addictive behaviours. Ghrelin levels rise when we fast, making us feel hungrier. In his talk, Tony discussed how volunteers given ghrelin report an increased preference for higher calorie foods over healthier options. They also showed greater activation in the orbitofrontal cortex, a part of the brain involved in encoding reward value, when looking at high-calorie but not low-calorie foods – the same effect observed in volunteers who had fasted. This may explain why those who skip breakfast find higher calorie foods more appetising by lunchtime. The research also suggests the possibility of using drugs that suppress or block ghrelin to reduce cravings for high calorie foods and help people lose weight.

The talk was highlighted by a number of publications, here, here and here.

Tony also gave some talks to the public as part of Guerilla Science, an organisation that promotes public understanding of science in non-traditional settings, in order to reach audiences that may not otherwise have any contact with scientists. On July 4th, Tony gave a talk at the ‘Flavour Feast’ event at Borough Market, London, followed by an appearance at 'The Secret Garden Party' festival in Cambridgeshire on the 24th and 25th of July.

Most recently, Tony presented a video as part of BBC News’ Health Explained, entitled What is Fat? You can see the video here.

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Not Just Sex

22 Jan 2012 GMT

Distinguishing characteristics between males and females, such as face shape or body outline, can normally be put down to either differences in development or hormones. What the results of this study show is that the sex chromosome complement (XY in males and XX in females) rather than the actual sex has a highly significant influence on how hundreds of genes are used differentially in males and females. Moreover, the gene responsible for male development, Sry, appears to compensate for this effect.

Professor Richard Festenstein, who heads the Gene Control Mechanisms and Disease group, said:

“Many diseases affect more men than women and vice versa and we don’t really know why. Finding out that the gene Sry plays an important role could help researchers find explanations for the subtle differences in the way males and females physically respond when the body is under attack from disease. It will also help us identify if disease-causing genes are dependent on female (XX) or male (XY) chromosomes in order to work, allowing us to move towards developing drugs that will stop them in their tracks.”

The study is published online today in Developmental Cell. On the article page, Richard Festenstein has recorded a short audio clip describing the work.

The paper is also available to download from here.

Reference:
Wijchers, P. J., Yandim, C., Panousopoulou, E., Ahmad, M., Harker, N., Saveliev, A., Burgoyne, P. S., Festenstein, R. (2010). Sexual dimorphism in mammalian autosomal gene regulation is determined not only by sry but by sex chromosome complement as well. Developmental Cell 19, 477–484.

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Blunt Scissors in the Immune System

22 Jan 2012 GMT

The immune response produces two types of plasma cells. In response to antigens, short-lived plasma cells are formed rapidly in secondary lymphoid organs such as the spleen and die after only a few days of intense immunoglobulin secretion. In contrast, long-lived plasma cells survive in the bone marrow for much of the lifetime of an organism to provide long-term immunity. Production of antibodies by both types of plasma cells is thought to contribute to the development of autoimmune diseases. While the longevity of bone marrow plasma cells is partly due to survival signals provided by the bone marrow microenvironment, the mechanisms that determine the lifespan of short-lived plasma cells have so far remained elusive.

When B cells differentiate into plasma cells, they expand their endoplasmic reticulum (ER) and Golgi dramatically to accommodate high-level antibody production and secretion. The study, carried out by Holger Auner and Pierangela Sabbattini and published in Blood, shows that despite this expansion, short-lived plasma cells suffer from substantial and progressively increasing stress on the endoplasmic reticulum (ER stress). This ER stress, which can occur in any cell type, results from the accumulation of faulty (misfolded) proteins. In plasma cells, ER stress is caused by high-level antibody secretion and ultimately triggers cell death if the toxic peptide “garbage” cannot be cleared up. In most cell-types, this is achieved by slowing down protein production for a period. However, this is not an option for plasma cells, which need to maximise antibody output. So, how do plasma cells deal with ER stress, which is brought about by their own function of producing antibodies?

The team found that short-lived plasma cells, instead of activating the protective mechanisms that prevent ER stress, keep themselves alive by blocking the activation of the effector caspases that normally trigger programmed cell death (apoptosis). Active effector caspases cleave hundreds of cellular proteins during apoptosis in order to dismantle the dying cell in an orderly fashion. The conventional view has been that there is no apoptosis without activation of effector caspases. However, Auner et al. found that caspase-9 and caspase-3 are not activated in plasma cells in response to death stimuli, including ER stress. They also observed that a tri-aspartic acid repeat within caspase-3, which blocks inappropriate activation of caspase-3 in healthy cells, is stabilised in plasma cells. The cells do ultimately succumb to the effects of ER stress but this occurs without activation of the effector caspases.

The results of this study provide evidence for a novel mechanism for regulating the timing of programmed cell death in plasma cells. When high-level antibody secretion causes a progressive increase in ER stress, apoptosis is blocked by the failure to activate the key effector caspases. This allows plasma cells to remain functional and maintain antibody output. Ultimately, ER stress becomes overwhelming and mediates plasma cell death through alternative mechanisms. The combined effects of ER stress and the block on caspase activation appear to fine-tune the timing of plasma cell death. The CSC researchers also found evidence that caspases can be resistant to activation in multiple myeloma (an incurable plasma cell malignancy), a finding that could be important for understanding responses of these cells to chemotherapy.

These findings pave the way for further investigations into how the interplay between ER stress and the block on caspase activation affects the survival of short-lived plasma cells and how this impacts on conditions such as autoimmune diseases and multiple myeloma.

Auner, H.W., Beham-Schmid, C., Dillon, N. and Sabbattini, P. (2010). The life-span of short-lived plasma cells is partly determined by a block on activation of apoptotic caspases acting in combination with endoplasmic reticulum stress. Blood, in press. link to article

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Senescence Barrier to Stem Cells

22 Jan 2012 GMT

Induced pluripotent stem (iPS) cells have far-reaching implications for research and regenerative medicine, but the rate at which normal cells can be successfully induced to become pluripotent is very low – between 0.1%–1% – suggesting the existence of barriers limiting induction efficiency.

In recent years, a growing body of work carried out by teams worldwide has shown that iPS cells can be made by inducing adult somatic cells, including fibroblasts and keratinocytes, with four transcription factors, termed Oct4, Sox2, Klf4 and c-Myc. Work carried out by the Cell Proliferation group and by teams at other institutes has indicated that the same transcription factors can activate genes and pathways that trigger senescence. This state is an irreversible growth arrest that normally limits the ability of cells to proliferate indefinitely, but it can also be triggered by ‘insults’, such as those sent by cancerous cells (oncogene-induced senescence – ‘OIS’). OIS has been shown to impede the growth of tumours, especially in the early stages.

This new research shows that senescence may also be one of the barriers that limit the efficient production of iPS cells. First Author Ana Banito and coworkers in the group, led by Jesus Gil, found that each transcription factor individually diminished cell growth, which suggested a complex scenario when all four factors act in concert. As identified for OIS, remodelling of a gene region called INK4a/ARF is a crucial event in the onset of senescence induced during reprogramming (termed reprogramming-induced senescence – RIS). The study also shows that the p53/p21^CIP1 pathway, which is also critical in OIS, is engaged at different levels in response to the expression of the four reprogramming transcription factors. A major cause for concern is that pathways and mechanisms controlling tumour progression overlap with those controlling stem cell pluripotency. Indeed, the ability of the iPS cells to generate a particular tumour called a teratoma is currently used as an indication of pluripotency.

Improving the rate at which stem cells can be produced safely is key to the advancement of stem cell technology. While the inhibition of senescence has been shown to improve the efficiency of iPS cell production, the derivation of cells lacking p53 or p16^INK4a – which are also important tumour suppressors – would introduce an unacceptable risk.

In order to circumvent this problem, the paper suggests, reversible compounds could be introduced into cells during reprogramming in order to transiently inhibit senescence. Defining the precise timing that would be required is key to this approach being successful. A number of groups worldwide are now engaged in screening for potential compounds to improve the efficiency of iPS generation.

This research was published in Genes & Development. Five other related papers have been published in Nature.

The similarities between reprogramming-induced senescence (RIS) and oncogene-induced senescence (OIS).

Reference:
Banito, A., Rashid, S. T., Acosta, J. C., Li, S., Pereira, C. F., Geti, I., Pinho, S., Silva, J. C., Azuara, V., Walsh, M., Vallier, L., Gil, J., August 2009. Senescence impairs successful reprogramming to pluripotent stem cells. Genes & Development. In press. doi:10.1101/gad.1811609

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Micro Maintenance

22 Jan 2012 GMT

MicroRNAs (miRNAs) block expression of genes post-transcriptionally: by grabbing on to and silencing messenger RNA, they impede translation of genes into proteins. In doing so, they regulate gene expression in both development and disease. While a number of recent studies in mammals have shown the importance of miRNAs in developing embryonic stem cells, little has been known about the role they play in the developing extra-embryonic cells that form the placenta and yolk sac.

Now, research published by members of the CSC’s Molecular Embryology group in collaboration with the Spanish National Centre for Heart Research (CNIC) have shown that the mechanism by which miRNAs maintain developmental potential in embryonic and extra-embryonic cells differs at a fundamental level. By analysing mouse embryos and stem cell lines mutant for Dicer, the protein required to produce miRNAs they showed that whilst in embryonic tissues miRNAS are primarily required to inhibit cell death, in the future placenta and yolk sac they are required to maintain stem cells.

The first cell fate decisions during mammalian development yield three distinct lineages at the time of implantation, when the embryo implants in the womb: the epiblast (in which stem cells are truly pluripotent); the trophectoderm, and the primitive endoderm (in both of which cells are multipotent – with fewer options than the pluripotent cells of the epiblast). It was found in this study that all three lineages are still present in Dicer mutants, suggesting that miRNAs are not required for specification at this early stage of development.

Development of the epiblast was delayed in mutants as compared with controls. Rather than this being due to a slower rate of cell division – in fact, the rate change was negligible – a greater rate of cell death was observed, and this was reflected in the observed elevated levels of the protein Bim, which promotes cell death. Levels of transcription factors involved in maintaining pluripotency in epiblast cells were unaffected in mutants, suggesting that miRNAs do not actively maintain pluripotency at this stage but are primarily involved in inhibiting cell death.

In contrast, it was found that in Dicer mutants, the extra-embryonic cells of the trophoectoderm and primitive endoderm are correctly established, but the cells quickly differentiate, leading to cell cycle arrest. Once this occurs, the cells stop dividing, halting development. In comparison with the cells of the epiblast, transcription factors involved in maintaining pluripotency reduced in the Dicer mutants, indicating that miRNAs in extra-embryonic cells are required for maintaining pluripotency.

While miRNAs have been known to play an important role in embryonic development for a number of years, this study is one of the first to demonstrate their importance in the development of extra-embryonic tissues and to suggest that fundamental differences exist as to how these molecules work in the placenta and yolk sac compared to the embryo .

Reference:
Spruce, T., Pernaute, B., Di-Gregorio, A., Cobb, B. S., Merkenschlager, M., Manzanares, M., Rodriguez, T. A. (2010). An early developmental role for mirnas in the maintenance of extraembryonic stem cells in the mouse embryo. Developmental Cell 19, 207–219.
Link to paper

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Research Groups and Facilities News

22 Jan 2012 GMT

f …

Meiosis

22 Jan 2012 GMT

Fadri comes to us from the University of Sheffield, where his work in the department of Molecular Biology and Biotechnology focused on the coordination of the chromosomal events of meiosis. Before his position at Sheffield, Fadri worked as a postdoctoral researcher in Stanford University. Fadri was awarded his PhD from the University of East Anglia, having previously completed his Master’s and Bachelor’s degrees at the University of Madrid.

Working with the nematode worm C. elegans as a model, the group’s work aims to understand the mechanisms that mediate correct chromosome segregation during meiosis. In particular, the group is interested in the interplay between meiotic chromosome structure and meiotic recombination.

Understanding the precise mechanisms that govern meiotic chromosome segregation is of paramount importance for sexual reproduction, as offspring receiving an incorrect number of chromosomes can lead to birth defects and miscarriages.

Meiosis Research Group Page

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TOBY Trial - Neonatal Medicine

22 Jan 2012 GMT

Birth asphyxia occurs when a baby’s brain and other vital organs are starved of oxygen or blood at or around time of birth. In the UK approximately 1,400 infants a year, two in every thousand full-term births, are affected. Asphyxia can be difficult to detect before a baby is born and can cause serious brain damage, severe cerebral palsy and even death in around half of the most affected cases.

Co-chief investigator Dr Denis Azzopardi of the CSC’s Neonatal Medicine group, headed by David Edwards, said: “The study builds on a 20 year body of research but gives, for the first time, irrefutable proof that cooling can be effective in reducing brain damage after birth asphyxia. Although unfortunately it doesn’t work in every case, our study showed the proportion of babies that survived without signs of brain damage went from 28% to 44% with cooling treatments – that’s a 57% increase.”

Carmel Bartley, Family Support Manager from the children’s charity Bliss said: "This is very welcome research into an area which is known to save lives. Cooling of babies with birth asphyxia is an innovative technique already being used in some neonatal centres. This is a specialist treatment that we would like to see used more widely to ensure the very best outcomes for our most vulnerable babies.”

The randomised control trial involved 325 infants affected by birth asphyxia. Half of the newborn babies had their body temperature reduced to 33-34°C (91-93F) for 72 hours followed by gradual re-warming in intensive care. Normal body temperature is around 37°C (98F).

The Total Body Hypothermia for Neonatal Encephalopathy Trial (TOBY) received almost £1million of MRC funding and involved experts from Imperial College London, Oxford University, Leeds University, Queen's University Belfast, Nottingham University and Bristol University, as well as a large number of doctors in many hospitals across the UK and Europe.

The findings will be passed to the National Institute for Health and Clinical Excellence (NICE) who are responsible for treatment recommendations in NHS hospitals. Their interventional procedure guidance on this topic, which is currently in development, will analyse the total evidence base in this area and consider whether to recommend the use of cooling treatments in the future.

Article in New England Journal of Medicine

For further information on case studies and to arrange an interview with Dr Azzopardi and colleagues on this project, please contact Grace Money on 0207 670 5139 or press.office@headoffice.mrc.ac.uk.

Notes to editors

1. Moderate Hypthermia to Treat Perinatal Asphyxial Encephalopathy will be published in the New England Journal of Medicine.
2. The Total body hypothermia for neonatal encephalopathy trial (TOBY) began in 2002 but builds on research over a twenty year period.
3. The infants involved in the trial were from the UK, Hungary, Sweden Israel and Finland and were monitored for effects up 18 months of age.
4. During the summer of 2008 a consultation on the use of cooling for the treatment of perinatal asphyxia was held by NICE. NICE decided to suspend the guidance until the results of the TOBY study were available. Now that the results of TOBY are published NICE will be looking at the results alongside the total evidence base in their ongoing interventional procedure guidance on Therapeutic hypothermia with intracorporeal temperature monitoring for hypoxic perinatal brain injury, to consider whether a treatment recommendation is appropriate. www.nice.org.uk/guidance/
5. For almost 100 years the Medical Research Council has improved the health of people in the UK and around the world by supporting the highest quality science. The MRC has invested in world-class research leaders, producing 28 Nobel Prize winners and sustaining a flourishing environment for internationally recognised research. The MRC focuses on making an impact and has provided the financial muscle and scientific expertise behind medical breakthroughs including the first antibiotic penicillin, the structure of DNA and the lethal link between smoking and cancer. Today MRC funded scientists tackle research into the major health challenges of the 21st century.

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Far From Restful

22 Jan 2012 GMT

As the Roman philosopher Seneca remarked, ‘rest is far from restful’. The remark, it seems, was as prescient as insightful: MRI studies of blood oxygen levels in the brain reveal a constant, spontaneous flux of low-frequency oscillations that continue to operate when the subject is not focusing on a particular task. While the function of the oscillations remains a matter of debate, a number of studies have shown that the spontaneous networks of oscillations – called ‘Resting State Networks’ (RSNs) – mirror adult networks associated with carrying out specific tasks, which has led some researchers to suggest that they form during the emergence of cognitive competencies during early childhood.

However, a team led by David Edwards, Head of the Neonatal Medicine Group at the CSC, tested an alternative hypothesis: that the resting state networks are present, fully formed, by the time of birth. They used two complementary methods of defining resting state networks and a series of independent analytical techniques to determine the resting activity in a sample of 70 infants born at between 29 and 43 weeks of development, whose parents had consented for them to be involved in the study. They found the RSNs were largely laid down in the last 10 weeks before the normal time of birth during a spurt of neuronal development, and much earlier than previously thought. This shows that the infant brain is more highly developed than thought previously, with the infrastructure for adult brain function in place by the time of birth.

One particular resting state network identified – the default mode network – has been suggested to be involved in daydreaming and a sense of self. Says David Edwards: "Some researchers have said that the default mode network is involved in introspection – retrieving autobiographical memories and envisioning the future, etc. The fact that we found it in newborn babies suggests that either being a foetus is a lot more fun than any of us can remember – lying there happily introspecting and thinking about the future – or that this theory is mistaken."

The nest step for this research is to see how the networks are affected by illnesses and to see if they can be used to diagnose problems.

David Edwards
David Edwards has recently been announced as the second recipient of the Imperial College London Doctor of Science degree (DSc), and the first within the Faculty of Medicine. The criteria states: "The Doctor of Science degree is awarded for published work of an exceptional standard, containing original contributions to the advancement of knowledge and learning which has given the candidate international distinction in their field. Candidates must be able to demonstrate a sustained contribution to their subject, as evidenced by seminal publications." We would like to congratulate David on his achievement.

This work appears in PNAS.

Reference
Doria, V., Beckmann, C. F., Arichi, T., Merchant, N., Groppo, M., Turkheimer, F. E., Counsell, S. J., Murgasova, M., Aljabar, P., Nunes, R. G., Larkman, D. J., Rees, G., Edwards, A. D. (2010). Emergence of resting state networks in the preterm human brain. PNAS, in press.

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Runx: T reg cell keeper and creator

22 Jan 2012 GMT

Runx proteins have been shown to bind to the canonical T regcell transcription factor Foxp3. Together, the two regulate(and primarily inhibit) the expression of target genes, suchas the Th17-promoting transcription factor Ror-γt. Now Brunoand colleagues reveal that Runx proteins also help induce andmaintain Foxp3 expression in mature CD4⁺ T cells.

Inducible T reg cells relied on Runx proteins to express Foxp3.Blocking the proteins reduced the number of these cells, andablating an indispensable subunit of Runx protein complexes,Cbfb, diminished Foxp3 expression in natural T reg cells. Runxproteins bound directly to the Foxp3 promoter in T reg cells,but not in naive CD4⁺ T cells because of locus inaccessibility.

Another group recently reported that Runx/Cbfb complexes controlthe expression of Foxp3 in natural T reg cells in vivo. WithoutCbfb, mice were susceptible to autoimmune disease. However,this study did not investigate a role for Runx proteins in Treg cell induction.

(Maxmen (2009) Runx: T reg cell keeper and creator. J. Exp. Med. doi:10.1084/jem.20611iti4)

This piece appears on the JEM website here

Runx and Foxp3 as feed-forward circuit components.
It was known from earlier studies that Foxp3 protein cooperates with Runx proteins in the regulation of target genes. The new finding that Foxp3 is itself regulated by Runx proteins suggests a regulatory circuit in which Runx proteins are both inducers and interaction partners of Foxp3. Genetic feed-forward loops involve two transcription factors: X regulates the expression of Y and both factors together regulate one or more downstream targets Z (left panel). In the case described by Bruno et al., X is a Runx family transcription factor and Y is Foxp3. Runx and Foxp3 interact at regulatory DNA sequences or independently of DNA to control the expression of several target genes (right panel). Feed-forward regulation is a recurring theme in the biology of Foxp3, which interacts not only with Runx but also other transcription factors including Rorγt and NFAT. This suggests that Foxp3 subverts T cell regulatory circuits to establish Treg cell-specific gene activation and repression programmes.

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Unfriendly Firing

22 Jan 2012 GMT

In the 1980s, a group of individuals exposed to the toxin MPTP (1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine) developed a chronic form of Parkinson’s disease. The toxin was shown to inhibit mitochondrial complex 1 – an enzyme found in the mitochondria of neurons and other cells that has a key role in maintaining cellular metabolism. Fast-forward 20 years, and mitochondrial dysfunction is thought to be a major player in the development of Parkinson’s disease (PD). Despite this connection, it has remained unclear exactly how this dysfunction leads to loss of dopaminergic neurons in the substantia nigra pars compacta (SNC dopamine neurons) – the decimation of which is a key feature of PD.

Genetic studies of patients with a family history of PD have identified several mutations of genes affecting mitochondrial architecture and function. None of these genes are selectively expressed in SNC dopamine neurons, however, indicating that some other mechanism must cause SNC deterioration in PD patients. One theory that has gained weight in recent years is that SNC dopamine neurons’ unique electrophysiological properties may make them particularly susceptible to degeneration in PD, so the CSC team investigated whether removing genes associated with PD in mice affected these properties.

SNC Channels
Like other neurons, those of the SNC dopaminergic system work by transmission of electrical impulses supplied by a flow of chemical ions (charged particles) into and out of the neuron. This flow is controlled by channels that traverse the membrane, which selectively allow ions of the right size and charge to pass through. SNC dopamine neurons rely heavily on ‘small conductance calcium (2+)-activated potassium channels’ (‘SK channels’) to regulate their firing activity. As their name suggests, these channels are activated by calcium ions inside the neuron and, on opening, allow potassium ions to flow out of the cell. The activity of these channels is directly involved in controlling how ‘excited’ a neuron is and, indirectly, they provide information on calcium signalling within the cell. Both calcium dysregulation and enhanced excitability are thought to make SNC neurons highly susceptible to degeneration. Thus, disruption of SK channels function may represent a new insight into the early changes that occur in SNC neurons in Parkinson’s disease

Out of a number of PD-associated genes that could affect excitability of SNC dopamine neurons, the team focused on PINK1 because it is almost exclusively active in mitochondria. By recording electrical traces of individual neurons, they compared SNC dopamine firing patterns of mice without PINK1 with those of normal mice. Mice missing PINK1 exhibited bursts of SNC dopamine neural activity punctuating a more irregular pattern; traces from the normal mice displayed greater regularity.

Bursts are thought to be important for dopamine signalling, but conversely it has also been suggested that they can increase intercellular calcium concentrations and stress in the cell.

Traces of electrical potentials from mice deficient in PINK1 suggested that SK channel function (see boxout, SNC channels) was impeded, suggesting that these may play an important role in the development of Parkinson’s disease. These channels are dependent on a supply of calcium ions to activate them, indicating that impaired calcium signalling is at fault. Rather than this being due to impairment of the channels that supply calcium ions, it was shown to result from a reduction in the number calcium ion release from the endoplasmic reticulum, the intracellular ‘pipes’ that supply the cell with needed components. Calcium levels need to be maintained within in an extremely tight concentration range within neurons; outside of this range, reactive oxygen species are increased and there is a possibility of cell death.

Crucially, mitochondria are often closely associated with the endoplasmic reticulum, and additionally act as a temporary calcium ion store within the cell. Mitochondria may be susceptible to calcium overload that can occur due to burst firing, leading to further cell stress; taking into account the propensity for burst firing to increase calcium ion loading, and it becomes apparent that a ‘feed-forward’ mechanism of calcium overloading could create a dangerous cascade within the cell that eventually contributes to dysfunction and cell death.

That SK channels are so important in the progression of Parkinson’s disease suggests a potential target: it was shown that an SK channel facilitator can partially restore the normal electrical firing of PINK1-deficient neurons, pointing to a potential neuroprotective therapy against the early stages of Parkinson’s.

Reference:
Bishop, M. W., Chakraborty, S., Matthews, G. A., Dougalis, A., Wood, N. W., Festenstein, R., Ungless, M. A. (2010). Hyperexcitable substantia nigra dopamine neurons in PINK1- and HtrA2/Omi-deficient mice. Journal of Neurophysiology, in press. Link

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Wonder About Words

22 Jan 2012 GMT

Reading may be a basic skill, but it is an incredibly complex cognitive feat, which most of us take for granted. The interaction between the text and the reader is shaped by the reader’s prior knowledge, experience, attitude and cultural context. Reading requires continuous practice and refinement. And scientists today are still trying to reach a consensus on just how the brain masters the art.

The jury is still out, although recent research published in the Journal of Neuroscience by Richard Wise and his team at the MRC Clinical Sciences Centre (Imperial College London) adds another piece to the puzzle. “Cognitive scientists who study reading are broadly divided into ‘localists’ and ‘connectionists’,” he explains. “The debate now focuses on the role of a cortical region known as the visual word form area (VWFA).”

“Without an intact VWFA reading is not possible,” says Richard. “Localists would argue that it stores representations of words, but connectionists see it as an interface that bridges the brain areas involved in vision and language to facilitate reading.” His team conducted an experiment on 19 men and women, all native English speakers, to test their responses to words, nonsensical script and numbers. Numerical information does not involve activation of the VWFA.

They reasoned that according to the localist hypothesis words would generate neural activity in the brain regions under investigation; nonsensical script and numbers would not. Contrary to the expected outcome of the experiment, the results appeared to be more compatible with the connectionist view. “We found that words and nonsensical script elicited a response,” confirms researcher Zoe Woodhead.

“Strictly speaking this isn’t evidence for the connectionist theory,” says Richard, “although our results are compatible with the ‘triangle’ model of reading that depicts interconnectedness between areas involved in vision, sound and meaning, with words encoded on the basis of interactions between multiple units throughout the three domains rather than as explicit lexical representations.” BM

Woodhead, Z. V., Brownsett, S. L., Dhanjal, N. S., Beckmann, C., Wise, R. J. (2011). The visual word form system in context. The Journal of Neuroscience 31, 193–199.

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Bile Beginnings

22 Jan 2012 GMT

At the level of an individual cell, a key role in metabolic regulation is carried out by protein kinases, which phosphorylate key proteins, switching off unnecessary cellular machinery when energy levels are low, much like a thermostat or stopcock. LKB1 is a ‘master’ metabolic protein kinase important in regulation of energy metabolism, cell cycle, proliferation and cell polarity. When intact, these properties protect against tumourigenesis. However, mutations of the LKB1 gene has been linked to cancer in recent years, and disruption has been associated with a cancer susceptibility disease, Peutz-Jeghers syndrome, which is characterized by polyps in the intestine and discolouration of skin pigment on the lips. Despite these findings, it has remained unclear whether LKB1 has a long-term role in liver function.

Now, a team of researchers in the CSC’s Cellular Stress and Metabolic Signalling groups has investigated what happens in mice that can’t make the LKB1 protein in their liver. Liver-specific deletion of the gene allows scientists to explore its role in that organ: the protein's function is so pervasive that organism-wide deletion would lead to death before the mice are born.

They found that LKB1 deletion in the liver led to a disruption in the formation of the canalicular membranes and bile ducts, impeding clearance of cholesterol and accumulation of toxic bile acids, overall hampering liver function. Consequently bile is unable to be secreted into the gut from the gall bladder to help the absorption of nutrients and the mice fail to thrive and rapidly lose weight. Circulating levels of low density lipoprotein – ‘bad cholesterol’ – and bilirubin (the yellow pigment responsible for jaundice) were hugely increased. Echoing Galen’s views on the importance of liver for the blood, the structure of red blood cells also changed – becoming ‘spiky’ – as often observed in patients with liver disease. LKB1 is clearly crucial in the liver, particularly for biliary function and regulation of cholesterol levels .

An interesting suggestion is that LKB1 has a special job in determining polarity in mammalian cells, which has been supported by previous studies. “Disrupted development of the bile canaliculi could be caused by problems with definition of liver cell polarity,” says Professor David Carling, who co-ordinated the research.

“We know LKB1 helps canalicular membrane proteins to get to the right place during canalicular formation. Without it the liver can’t transport and dispose of bile constituents, so bile acids, bilirubin and non-esterified cholesterol build up, causing toxicity.”

“Our study uncovers a new role for LKB1 in the liver adding to the list of diverse functions of this master regulatory kinase," added Dr Angela Woods, the study's principal author. "Not only does LKB1 regulate energy balance at the cellular level but also at the whole body level – by influencing food intake via signaling in the brain...but also, as we have shown in this study, it also appears to influence the efficiency of food absorption.”

This work was published in The Biochemical Journal

Reference:
Woods, A., Heslegrave, A., Muckett, P. J., Levene, A., Clements, M., Mobberley, M., Ryder, T. A., Abu-Hayyeh, S., Williamson, C., Goldin, R. D., Ashworth, A., Withers, D. J., Carling, D. (2010). LKB1 is required for hepatic bile acid transport and canalicular membrane integrity in mice. The Biochemical Journal, in press.

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Breakfast Like a King

22 Jan 2012 GMT

20 healthy, non-obese subjects were randomly allowed or denied breakfast on two separate mornings at least six days apart, following overnight fasting. Those given breakfast were told to eat until they felt full. About 90 minutes later, both the fasted and fed subjects were shown images of high-calorie and low-calorie foods, in addition to ‘neutral’ images of household objects, while oxygen-dependent activity in brain regions associated with reward was measured by functional MRI. While being scanned, each subject was also questioned on how appealing they considered the food image to be.

The researchers measured enhanced brain reward-centre activity when subjects denied breakfast were shown images of high calorie foods, as compared to images of low calorie foods, matching higher visual appeal ratings given for high-calorie foods when the subjects were questioned. In contrast, those fed breakfast reported no preference for high-calorie over low-calorie foods – a result commensurate with observed diminished reward-centre activity enhancement .

Consultant Endocrinologist Tony Goldstone from the CSC’s Metabolic and Molecular Imaging Group explains:

“Our results support the advice for eating a healthy breakfast as part of the dietary prevention and treatment of obesity. When people skip meals, especially breakfast, changes in brain activity in response to food may hinder weight loss and even promote weight gain.”

The studies findings could help inform treatment of obesity: “The hope is to develop drugs that prevent this activation of the brain’s reward circuitry and thus reduce the appeal of high-calorie over low-calorie foods.”

This work was published in:
European Journal of Neuroscience, Vol. 30, pp. 1625–1635, 2009.

The research was also reported in The Sun

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Delaying Old Age?

22 Jan 2012 GMT

From another perspective, many lives that deserve to be well-lived are limited by the diseases of ageing; the elixir that would postpone or even abrogate those conditions eludes us as much as ever. Or does it?

Now, a study carried out by a team of researchers including CSC Cellular Stress’s Angela Woods and Dave Carling, and led by new Group Head Dominic Withers (Metabolic Signalling Group; Group member Agharul Choudhury also contributed) identifies a key gene that is involved in the regulation of mammalian life span. Ribosomal protein kinase 1 (S6K1) is a component of the nutrient-responsive mTOR (mammalian target of rapamycin) signalling pathway, which co-ordinates nutrient availability with cell growth and proliferation, protein synthesis, and transcription. continued...

Aurora (Eos) Taking Leave of Tithonus. (Public Domain)

(image at top right): Eos pursues a young Tithonus. (Louvre)

It has been known for decades that restriction of nutrient intake without malnutrition, termed caloric restriction (CR), increases life span and reduces age-related pathology in a number of organisms, including mammals. Most recently CR has been shown to operate in primates. Previous work in lower organisms has implicated the mTOR pathway in mediating some of the effects of CR in lower organisms but the current study is the first to investigate the role of S6K1 signalling in the regulation of mammalian ageing. The team found that mutant mice lacking S6K1 succumbed to the ravages of ageing more slowly than their S6K1-replete peers, as measured by a number of age-sensitive biomarkers, including motor and neurological function, and exploratory drive.

Physiological and gene expression analysis revealed that S6K1 null mice strongly resembled mice under conditions of CR. Crucially, the animals exhibited increased AMP-activated protein kinase (AMPK) activity in key metabolic tissues. AMPK has an important role as a master fuel gauge in the regulation of energy homeostasis and is the target for several commonly used drugs for type 2 diabetes including metformin.

Although disruption of the mTOR pathway by removal of S6K1 did not work to prolong life in males in the same way as it did in females, the mutant males did have some of the health advantages seen in the female mice.

Previous studies have revealed an apparent trade-off between sex and longevity: CR may prolong life, but it delays the onset of sexual maturity and reduces fecundity. As part of this new study, the team found increased longevity and delayed fecundity in rsks-1 mutants of the nematode worm C. elegans, which lack the S6K1 homologue. Again, they discovered increased AMPK levels in the worms, reinforcing the importance of AMPK in longevity.

The study suggests that inhibition of S6K1 or activation of AMPK by pharmacological intervention may alleviate ageing-related disease. Indeed, the mTOR inhibitor rapamycin – a drug commonly used as an immunosuppressant in transplant medicine – has recently been shown to increase lifespan in mice, and this new study indicates that this may occur by inhibition of S6K1.

Whether or not Eos could have relied on a treatment that inhibits S6K1 to prolong Tithonus’ lifespan is uncertain, but it may be that their old age would have brought fewer ailments.

Stefan Janusz

This work appeared in Science
Selman, C., Tullet, J. M. A., Wieser, D., Irvine, E., Lingard, S. J., Choudhury, A. I., Claret, M., Al-Qassab, H., Carmignac, D., Ramadani, F., Woods, A., Robinson, I. C. A., Schuster, E., Batterham, R. L., Kozma, S. C., Thomas, G., Carling, D., Okkenhaug, K., Thornton, J. M., Partridge, L., Gems, D., Withers, D. J., October 2009. Ribosomal protein s6 kinase 1 signaling regulates mammalian life span. Science 326 (5949), 140-144.

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Architects of Forever

22 Jan 2012 GMT

ARCHITECTURE
"Genome architecture is intimately related to genome function"
Histone proteins inside the nucleus are like modifiable scaffolding, allowing certain architectural features of chromatin to be altered and rendering DNA more or less accessible to transcriptional machinery. Like builders marking part of a building for demolition or improvement works with spray paint, certain enzymes tag chromatin with epigenetic marks, giving the instruction to either expose architectural forms – and the DNA they contain – or cover them up. Outward, exposed architecture influences its environment more than comparatively concealed constructions – the same for nuclei and buildings alike. In urban environments, not only builders but also gangs tag buildings, forming territories; in cells, genes and their relative positioning with respect to their chromosome territories may have important implications for gene expression. Their distribution is roughly the same in normal (somatic) cells and stem cells, indicating that many architectural features are formed very early on. Stem cells have more pluripotency genes located centrally in the nucleus – an important indicator of transcriptional activity, just as city centres are hives of social interaction and productivity in the human world.

STRUCTURE AND PLASTICITY
Protruding into the nucleus from the inside of the envelope are lamina proteins, thin filaments that act like foundations for chromatin. In most cell types in our bodies, these supporting foundations fix the configuration of chromatin, like a building that cannot be modified because it has been granted heritage status. In stem cells, the architecture of the nucleus is not fixed: many lamina proteins are absent from the inside of the envelope. Chromatin inside is 'hyper-mobile', fluid, and therefore adaptable. Like the lifting of restrictions that have spurred the building of skyscrapers in the city of London, it is a state that encapsulates dynamism. But for most cells within organisms, and for most cities, this dynamic state – a reflection of nuclear and human architecture – occurs only at the beginning. In a cell, loss of mobility of chromatin is associated with loss of pluripotency, the feature of stem cells that allows them to become any cell in the body; like a city overprotected by conservation orders, it is unable to radically change.

PRODUCTIVITY
ES cells are also more transcriptionally active; more productive. Elevated transcription of chromatin remodeling factors – in our urban analogy, an injection of cash, perhaps – help to maintain an open chromatin configuration. Another possibility is that hyperactive transcription could itself play a role in maintaining pluripotency by contributing to the plasticity of the genome. But this dynamic cellular city has another trick up its sleeve, practically engendering dynamism: bivalent histone marks. Some 2500 of these bimodal chemical modifications, simultaneously activating and repressing genes, occur in ES cells. The bimodality is resolved upon differentiation, leaving one mark – either activating or repressing – where there were two; the genomic equivalent of keeping its options open. Dynamism epitomised.

Morris, Chotalia and Pombo's review spares the analogy with human architecture, and I hope they'll forgive the more imaginative leaps made here. Nevertheless, they present the architecture of stem cells as an enthralling experience, even for a field still yet in its nascency:
“A common theme that is emerging is that the nuclear organisation of ES cells is less structured than differentiated cells,” say the authors. Furthermore, “the small number of studies that have probed the functional organisation of the ES cell nucleus already associate pluripotency with a highly dynamic genome that is reflected in a unique nuclear architecture.”
SJ/BM

Morris KJ, Chotalia M, Pombo A (2010) Adv Exp Med Biol, 695:14-25 …

Supercoiled!

22 Jan 2012 GMT

Positive and negative supercoiling causes the DNA to form loops or figure 8s, but in opposite directions (see graphic below). Many biological processes involve the opening of the DNA helix by specialised machines that rotate DNA – helicases. By their nature, helicases cause DNA supercoiling, which can cause problems for other cellular processes such as DNA replication. To resolve these problems cells employ specialised machines called topoisomerases, able to resolve the conformational alterations by breaking and rejoining DNA molecules.

In addition to changes to conformation occurring in the same DNA molecule, cells need to remove links that occur between different molecules of DNA, for instance between sister chromatids during DNA replication. A specialised topoisomerase (topoisomerase II) does this job by breaking one DNA molecule and passing the second DNA molecule through the break before rejoining the broken ends.

Creative Commons (wikimedia)

"It has been long known that DNA replication causes the resulting sister chromatids to be linked or intertwined, explains Luis Aragon of the CSC Cell Cycle Group, "but for cell division to proceed, these intertwines need to be resolved, because sister chromatids must be physically separated. Although we know that topoisomerase II removes the links between sister chromatids, we also know that it is equally likely to introduce them. How its activity is regulated to ensure that its activity has a bias towards removal rather than introduction of links is one of the long-standing questions of mitosis."

Jonathan Baxter and Nicholas Sen, researchers in Luis’s team, and their collaborators have discovered why topoisomerase II specifically removes links between sister chromatids during mitosis. The results of their study have recently been published in the journal Science.

Studying circular plasmids in yeast the scientists showed that when cells enter mitosis, DNA plasmids change their global topology and become highly wound or positively supercoiled. They then showed that in the mitotic topological state "topoisomerase II specifically removes links between sister DNA plasmid molecules, rather than introducing them or acting on individual plasmids to change their supercoiling status," says Luis.

The team also found that cells have dedicated machines to promote the topological change associated with mitosis, called the Condensin complex. They propose a new model explaining how cells manage to ensure that topoisomerase II removes every single link between sister chromatids before chromosome segregation. These findings are medically relevant because many chemotherapeutic cancer treatments use inhibitors of topoisomerase II.

This research appears in Science

Reference:
Baxter, J., Sen, N., Martínez, V. L., De Carandini, M. E. M., Schvartzman, J. B., Diffley, J. F. X., Aragón, L., Mar. 2011. Positive supercoiling of mitotic DNA drives decatenation by topoisomerase II in eukaryotes. Science 331 (6022), 1328-1332.

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When the Going Gets Tough

22 Jan 2012 GMT

The prevailing dogma surrounding regulation of AMPK has been that AMP binding to the enzyme switches the output to ‘on’. It has been proposed that it does this in two independent ways. First, it directly activates the enzyme 'allosterically' (the kinase is ‘AMP-activated’), which means that it modulates the conformation and function of the enzyme. Secondly it protects AMPK against dephosphorylation – removal of a phosphate 'safety key' – by protein phosphatases. This step is crucial, as removing that safety key renders the enzyme inactive, a discovery made by researchers in the CSC Cellular Stress Group.

Now, working with a group from the National Institute for Medical Research, they have published new findings in the journal Nature making sense of how AMPK functions in the complex chemical environment of the cell, where levels of AMP are dwarfed by those of related adenosine diphosphate (ADP) and adenosine triphosphate (ATP). Both of these molecules compete with AMP when binding to AMPK, but the difficulties in investigating the physiological consequences of this competition has meant that previous work has been limited to investigations of AMP binding.

Like other cellular machines AMPK acts in concert with the rest of the cell, co-operating in a bid for the perpetuity of life by signalling, feedback and regulation – in this instance by regulating energy metabolism.

In this new work, the team has shown that the common biological molecule NADH binds to AMPK with a change in its fluorescence signature, and they have used this to investigate the strengths of binding of the three adenosine phosphates (or adenine nucleotides) to AMPK. They also present results showing how active AMPK is when ATP and ADP are bound, along with new molecular structures. The picture this new study paints is that ADP binding is an important phenomenon, and helps to explain how AMPK is regulated in the face of relatively high levels of ATP.

ATP, biology’s ‘universal battery’ (see Universal Battery) is present in cells at a concentration at least a hundred times the level of AMP, mainly in the form MgATP – associated with magnesium ions, the positive charge on which cancels out the negative phosphates. The prevalence of MgATP could spell disaster for AMPK function, because ATP is able to occupy binding pockets on the kinase normally occupied by AMP, without either activating it physically or protecting the phosphate safety key being removed. ADP, which is present at levels ten times lower than ATP, is however able to protect against inactivation of the kinase.

Universal Battery
ATP is often called the ‘universal battery of biology’, because when the bonds between the three phosphate groups are broken, a biologically-useful amount of energy is released and used to drive other chemical reactions needed for life. ATP minus one phosphate makes ADP and energy, and ADP minus one phosphate makes AMP and more energy. Like a rechargeable battery, AMP can then be ‘recharged’ by adding phosphate groups to make ATP; ultimately this comes from food and respiration.

Despite being less prevalent, ADP binds to AMPK about 10 times more strongly than MgATP. In effect, the tighter binding of ADP cancels out its 10-fold lower concentration relative to ATP, allowing the two nucleotides to compete on a level playing field. The researchers show that each AMPK molecule has three nucleotide binding pockets and make the important finding that it is binding to only one of these, termed site 3, that determines AMPK activity. When ATP is bound at site 3 AMPK adopts a conformation that allows phosphatase enzymes access to the critical phosphorylated residue – the phosphate 'safety key'. However, when ADP is bound at this site, this residue is buried within the molecule, preventing the safety key from being removed and maintaining the kinase in an active state.

The new work gives us a more realistic picture of the cellular machinery underpinning metabolism, showing us that it is regulated in a more subtle manner than previously thought.

This research appears in Nature

Reference:
Xiao, B., Sanders, M. J., Underwood, E., Heath, R., Mayer, F. V., Carmena, D., Jing, C., Walker, P. A., Eccleston, J. F., Haire, L. F., Saiu, P., Howell, S. A., Aasland, R., Martin, S. R., Carling, D., Gamblin, S. J. (2011). Structure of mammalian AMPK and its regulation by ADP. Nature, in press.

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First 3D MRI Scans of Unborn Babies

22 Jan 2012 GMT

From the article:

Baby Miller makes his first appearance on screen.

He can be seen moving and swallowing - a proud moment for parents-to-be Sian and Brian, and a welcome addition to their baby memorabilia.

The 3D scan shows that the baby is coming on well, and his development is normal.

But the scan is more than just a memento.

For the new Miller is one of the latest foetuses to be enrolled in a brain study at London's Hammersmith Hospital, in collaboration with the Medical Research Council.

...

Professor Mary Rutherford said her team had got round this by taking multiple scans of the brain and then slotting them together to make a 3D image.

"This information will help obstetricians to decide whether a baby is likely to have severe problems with development or whether to deliver a baby sooner as brain growth may be better outside the womb," she said.

You can read more of Jane Elliot's article and watch the video on the BBC website here. …

DNA Diary: Gene Control Mechanisms and Disease

22 Jan 2012 GMT

Cells too contain a record of their past adventures, recorded in epigenetic marks – reversible modifications made to DNA and to histone proteins that DNA wraps around inside the nucleus. These marks change how the DNA is packaged, making genes more or less exposed to the transcriptional machinery that makes proteins, ultimately defining cellular type. The sum total of each cell’s developmental story makes up the organism’s epigenome – each individual nuanced story contributing to a grand, heritable history for the organism.

Just as a diary or journal has particular places on the page for each day’s entry, there are particular places on the epigenome that are written to more than others. Of the histone proteins that are epigenetically modified, histone H3 has five such distinct places – lysine amino acids groups that dangle from the histone protein – that can be ‘written to’ by adding one, two or three methyl (–CH3) chemical groups, a job carried out by an enzyme called methyltransferase. Methylation of lysine 4 of histone H3 (‘H3K4’) is a mark associated with gene promotion – a cellular ‘to do’ mark that increases the priority of that gene in the cell’s workload. It is one of the most studied epigenetic modifications. In contrast, H3K4 acetylation (H3K4ac) – where an acetyl (–COCH3) is added – has not received as much attention, primarily because of the difficulties in distinguishing its function from H3K4me.

Di- and trimethylation of H3K4 has been linked to acetylation on the same histone, however, by inviting in acetyltransferase. “These results suggest that there is a highly dynamic and coordinated interplay between histone H3K4 methylation and the enzymes that control H3 acetylation during transcription,” says Professor Richard Festenstein of the CSC Gene Control Mechanisms and Disease group, who in collaboration with universities in Europe, Canada and Japan have investigated the prevalence and function of H3K4ac in yeast.

In order to separate H3K4ac from H3K4me function, the team developed a new antibody that can accurately distinguish between the two marks, binding to H3K4ac but not H3K4me and then allowing the acetylated histone to be separated. They then used mass spectrometry to measure the masses of tell-tale fragments of the protein that indicate acetylation. By the using the complementary technique of chromatin immunoprecipitation (‘ChIP’), the team was able to probe the function of acetylation in the H3 histone. And using yeast in this study was key: it allowed the team to turn particular genes involved in acetylation on or off to probe the function of these marks.

Yeast is an extremely important model organism – about half of all yeast genes have a counterpart in humans. Crucially, genetic mutations that remove the ability of the cell to make specific epigenetic marks, including methylation or acetylation of histones, can be readily introduced. In higher organisms such mutations would be fatal.

The researchers found that acetylation was more prevalent at the promoters of active genes, indicating a role in transcription, the process of turning DNA instructions into the proteins that do the work of the cell. They found 37 genes that require acetylation to function. However, in comparison to the ‘writing regions’ that exist on histones other than H3, usually either exclusively methylated or acetylated, H3 features both marks overlapping. One suggestion for both marks occurring together is that they are in direct competition for H3K4, but an alternative hypothesis proposes that methylation finetunes the level of acetylation. “This may contribute to prevent spurious transcriptional initiation events,” says Richard Festenstein.

"The potential to restrict the spreading of histone H3K4ac suggests a novel function for H3K4 methylation and reveals a previously unrecognised layer of chromatin regulation linked to the regulation of transcription in vivo.”

SJ

This work appears in PloS Genetics
Guillemette, B., Drogaris, P., Lin, H.-H. S., Armstrong, H., Hiragami-Hamada, K., Imhof, A., Bonneil, Ã., Thibault, P., Verreault, A., Festenstein, R. J., Mar. 2011. H3 lysine 4 is acetylated at active gene promoters and is regulated by h3 lysine 4 methylation. PLoS Genet 7 (3), e1001354+.

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Shock to the System

22 Jan 2012 GMT

Writing in Nature Reviews Drug Discovery, CSC Nitric Oxide Signalling Group Head James Leiper and collaborator Manasi Nandi of King’s College London outline the progress that has been made so far in targeting regulatory mechanisms of NO to treat these conditions. Crucially, they highlight a potential tissue-specific intervention that could be used to treat septic shock.

The starting point for NO is the enzyme nitric oxide synthase (NOS), which takes the amino acid arginine and in a catalytic feat of structural contortion twists the amino acid like a balloon modeller, popping off NO and leaving the molecule L-citrulline behind:

As well as normal arginine molecules inside a cell, there are also methylated arginines – products of chemical modifications of arginines on the surface of proteins [see box below]. Arginines with either one methyl chemical group or two methyls in an asymmetric arrangement lower NO production by ’tricking’ the NOS enzyme into grabbing them instead of unmethylated arginine. Their additional methyl chemical groups halt NO production. Because of this ability to halt the reaction, asymmetrically dimethylated arginine ‘ADMA’ – present at much higher levels than the monomethylated version – is considered to be the primary cellular inhibitor of NO production. ADMA is itself regulated by the enzyme DDAH, adding an additional level of NO regulation and consitituting a potential therapeutic target that has attracted considerable interest.

Proteins are messy 3D balls of amino acids linked together, full of twisted conformations and with many amino acids, including arginine, dangling from the protein's surface. These can be subject to modifications, which can alter the protein's characteristics. Methylarginines are the only by-product of these protein modifications that have an effect on metabolism.

Loss of of DDAH leads, through an increase in ADMA, to lower NO production – a state associated with cardiovascular disease and insulin resistance; high levels of DDAH and elevated NO is associated with some forms of cancer, neurodegeneration and arthritis, as well as septic shock. Pharmaceutical activation of DDAH however is not currently possible in cases of lowered NO, with gene therapy proposed as a potential alternative avenue for treatment. In situations of elevated NO such as septic shock the lack of clinically efficacious drugs prompted researchers to manipulate the NO-ADMA-DDAH system itself by administering large doses of monomethylated arginine. While this treatment was found to reduce septic shock from NO, it interfered with normal cellular NO signalling, a side-effect with potentially lethal consequences.

James Leiper’s group have developed synthetic mimics of arginine that inhibit DDAH in order to sidestep this problem. In rodents, these molecules have been shown to interact with only one particular form of DDAH (DDAH1) lowering NO associated with septic shock but leaving normal cellular NO signalling intact. The authors stress the importance of moving sthese findings wiftly towards preclinical development and then clinical trials.

Says Leiper, “in addition to giving us a greater understanding of the DDAH-ADMA-NO system, these findings have helped us to identify an important therapeutic target for septic shock…a condition that still kills hundreds of thousands of people each year”.

This work was published in Nature Reviews Drug Discovery.

Reference:
Leiper, J., Nandi, M. (2011). The therapeutic potential of targeting endogenous inhibitors of nitric oxide synthesis. Nature Reviews Drug Discovery 10, 277–291.

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Pioneering Heart and Kidney Research

22 Jan 2012 GMT

“We’re particularly looking at organ failure that has inflammatory causes,” explains Tim, “for example in diseases such as lupus or glomerulonephritis. While it’s increasingly clear that there is a genetic basis for these autoimmune disorders, specific treatments to target disease genes have not yet been developed.” Current treatments are broad-brush, for example immunosuppressive steroid drugs, which have serious side effects that leave patients vulnerable to other infections.

“Over the past two to three decades, we (and others) have used genetic linkage studies to look for genes and pathways involved in heart and kidney disease.” Current genome technology facilitates the comparison of disease gene pathways between humans and model organisms; in this case using rat models. “By looking at genetic differences between susceptible and resistant individuals”, says Tim, “we can identify candidate genes.”

His team (Physiological Genomics and Medicine Group) at the CSC has come up trumps in the past five years by discovering two genes that play a role in kidney failure. Both of these – Fcgr3 and JunD – help regulate immune cells called macrophages, which normally engulf foreign invaders. “In certain circumstances, because those genes are defective, macrophages can become over-activated and cause kidney damage.”

“Once we identify candidate genes, we can look at what they do and the networks and pathways in which they operate. For each gene, we’d like to know what the downstream effectors systems are that cause damage to kidney or heart, which we can investigate using powerful high-throughput technology for genome sequencing,” reveals Tim.

“Over the past decade, the cost of genome sequencing has come down a hundred-thousandfold. So, in the last two years, we’ve sequenced eleven genomes (see Understanding Hypertension: http://www.csc.mrc.ac.uk/NewsEvents/News/UnderstandingHypertension/). We have the genome sequences of many susceptible and resistant individuals. Using gene expression profiles, for example, if we find a gene that is highly expressed in a resistant individual, but expressed at much lower levels in a susceptible individual, then it’s worth investigating,” he adds.

Technological advance means that studying the function of a gene has become much more convenient. Tim’s team is using brand new technology to knock down genes of interest, so that they can explore the effects on different individuals. “Zinc-finger nuclease technology allows us to target individual genes very precisely,” he qualifies.

“Over the next five years, we will look at the genome sequences of individuals who do and don’t get disease to find disease genes,” Tim reaffirms. “So we narrow down the search using linkage studies, gene expression profiles to throw up candidates, genome sequencing and bioinformatics to probe gene networks and pathways, and gene-targeting to learn about function.”

The project has great medical potential, although what’s not certain is whether candidate genes will be ‘druggable’. As Tim explains, “only a small proportion of the genes in the human genome are druggable.” So if the team finds disease-causing genes, the really big question is, whether manipulating their function could prevent disease occurring. “And that would be a major result,” he exclaims.

The ERC Advanced Investigator Grant is a great privilege for Professor Aitman, his team and European research collaborators; and a tremendous support to the CSC research programme.

BM

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Playing the Blank Square :: Video

22 Jan 2012 GMT

Understanding the biological mechanisms that point the way to this level of cell determinacy is like holding the blank square in Scrabble: it can be any letter we choose. Currently however, we don’t know all of the rules of the game.

Stem Cell Neurogenesis researcher Nicole Gennet discusses what their findings might mean for future therapies

Researchers in the CSC Stem Cell Neurogenesis group headed by Meng Li are attempting to illuminate these biological rules. In particular, they are investigating how midbrain dopamine neurons – the brain cells involved in pleasure and reward – are formed from stem cells. These cells become damaged in patients with Parkinson’s disease, so a future therapy making their regeneration a possibility would be an extremely valuable one. So what biological switches and mechanisms determine this particular fate for the cell rather than it becoming another type of brain cell?

Meng’s team looked at how certain transcription factors – protein molecules that help to determine cell type by switching individual genes on or off – come into play to make midbrain dopamine neurons. They focused on a particular class of transcription factors that normally play a role in the development of the sexual organs, called Dmrt. One of which, Dmrt4, has being previously found to be important in the development of the sense of smell in some species. In experiments on mouse embryonic stem cells and chicken embryos, the researchers found that Dmrt5 was vital for the development of midbrain neurons.

Cells producing Dmrt5 become midbrain dopamine neurons while cells lacking the ability to make Dmrt5 became GABAergic neurons; indicating that Dmrt5 actively blocks a potential fate – the possibility of becoming a GABAergic neuron – in addition to promoting the dopamine neuron fate.

But do we know anything about how Dmrt5 is doing this?

Meng explains: "Dmrt5 imposes a pre-dopaminergic molecular codes on neural stem cells while simultaneously prevent them from acquiring the potential to make other types of nerve cells. Our ongoing works aims to reveal the detailed molecular mechanisms of how Dmrt5 regulates these developmental decisions of stem cells."

Understanding the biological rules that come into play to direct a particular fate for a cell is a key step towards being able to make whatever cell type we choose. Not only could this research one day lead to therapies for Parkinson’s disease and other neurodegenerative conditions, it provides vital clues to the development of function of one of nature’s most complex puzzles: the brain.

SJ

This work was published in PNAS

Reference:

Gennet N, Gale E, Nan X, Takacs K, Oberwallner B and Li M (2011). Dmrt5 promotes midbrain dopaminergic identity in pluripotent stem cells by enforcing a ventral medial progenitor fate. PNAS 108, 9131-9136.

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Evolutionary Changes in ApoER2

22 Jan 2012 GMT

ApoER2 itself binds the protein reelin (structure shown at right), which plays an important role in a host of physiological processes. In the brain, it directs cortical layering. In mammals, layering proceeds in an ‘inside-out’ fashion: newly-created neurons pass from the germinal layer to the cortical plate through a sucession of increasingly younger neuronal layers, the oldest neurons being the most deeply-residing. Reptilian cortices, in contrast, feature their oldest constituent neurons on the outside (an architecture referred to as ‘outside-in’). Lack of reelin during development of the mammalian brain, such as was first observed in the spontaneously arising ‘reeler’ mutation in mouse populations (from whence the protein name is derived), leads to a layering pattern roughly reversed from that of healthy mammals (i.e. similar to ‘outside-in’). Mutations in the reelin gene have since been implicated as factors in schizophrenia, Alzheimer's Disease and autism.

It has been suggested that the mammalian architecture may bestow an evolutionary advantage, in more readily facilitating brain volume increase. The Review considers whether or not this is due to the highly conserved sequence of amino acids in placental mammalian ApoER2 (and interaction with reelin), and the exon that encodes for it (exon 18 in primates, exon 19 in non-primates). Sequence comparisons of analogous regions, in both evolutionarily more distant amniotes and marsupials, reveal an absence of a homologue of exon 18 or 19, indicating that the new exon arose after the marsupial/placental mammalian split. But while mice lacking the exon exhibit diminished memory, spatial and motor function similar to that seen in the reeler mutation, cortical growth patterns remain a ‘healthy’ inside-out, ruling out the addition as being crucial to that particular brain architecture.

Link to the Review in Proceedings of the Royal Society B

Nick Myant published his first paper in 1940 while still a medical student, but his research career started in earnest after his return from India after the war, when he joined E. E. Pochin’s MRC Clinical Research Unit in 1948, working on human thyroid function with radioiodine. In 1954 he joined G. Popjak’s MRC Unit at Hammersmith where he developed an interest in cholesterol metabolism in humans. Nick was later Director of the MRC Lipid Metabolism Unit at the Hammersmith, where he remained until his retirement in 1983. Following retirement, Nick returned as an associate member of the Lipoprotein Group. The discovery of several new members of the LDL receptor gene family around this time, coupled with Nick’s long-standing interest in evolution, led him to ask the questions addressed in this review – published a few months after his 92nd birthday.

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Seizure

22 Jan 2012 GMT

For a few patients with drug resistant epilepsy, surgery can be offered as a treatment for their seizures, most commonly involving surgical resection of a scarred hippocampus. Such operations have yielded human brain tissue for research. "So now we can start to look at the human genes involved in the disease," he confirms.

"We're focusing on brains from patients who experienced the highest frequency of seizures," reveals Enrico, "because these are the most resistant to drug-treatment." Understanding gene networks and pathways in individuals who don't respond to drugs will hopefully provide new insights and potentially new angles on how to treat the disease.

"If we know how many seizures a month patients have, we can look at their brain tissue to find out which gene networks are at play." Using the latest in microarray technology, the team gets a readout across the entire genome highlighting which genes (out of the 25,000 or so human genes) are switched on in the brain cells under investigation, and how active (highly expressed) these are in diseased tissue.

"We found around 300 genes that are associated with seizure frequency," Enrico reveals. "Within this subset there is a large number of Toll-like receptor signalling genes." These genes are known to be involved in the body's immune responses, regulating processes such as inflammation, for example.

But the team needed to find out which genes in the large network of interconnected genes (~500) correlated with seizure frequency. "The candidates should be more active in brain tissue from individuals that have very frequent seizures," confirms Enrico, "but lower in the brains of individuals who experience seizures less often." And 77 genes fit that bill.

In order for any of these genes to be drug targets, they need to be 'druggable'. Assessing the druggability of any gene requires looking at how it works with other genes in the network. Drugs that target genes that work in tandem with lots of other genes can result in unwanted side effects. "So we need to identify these 'hubs'," explains Enrico, "...genes at the centre of the network, which are connected to many other genes."
The research team applied a statistical algorithm to extract and then visualize all network hubs. "We can portray genes using nodes of different sizes to represent the 'connectedness' of genes," he says. "The bigger the node, the more genes it affects." In this way, the researchers whittled the list down to seven key genes, several of which are involved in systems that facilitate communication between cells.

"One of these, is a potential drug target," reveals Enrico. The gene identified plays a role in memory and cognition in the brain and from mouse models', it has been shown that blocking this inflammatory pathway successfully reduces seizures."

Enrico adds, "our research using novel systems-level approaches is beginning to point to new therapeutic targets for epilepsy.This is the first research of its kind to be conducted on human tissue and Michael and I are at the forefront of this approach. No doubt, with the powerful tools now at scientists' disposal, it must only be a matter of time before new and better treatments for epilepsy are developed.”

BM

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Pulmonary Fibrosis - New Treatment :: Video

22 Jan 2012 GMT

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Working on mice with pulmonary fibrosis, a team based at the Giessen Lung Centre in Germany collaborating with Dr Leiper found that the potential drug candidate reduced accumulation of collagen in the lungs and that lung flexibility in animals administered the drug remained at near-normal levels. The results raise hopes that this candidate can be developed into a drug that can be used to treat pulmonary fibrosis in humans. It also raises hope for sufferers of other diseases, as it is very likely that the drug could be used to treat fibrosis more generally.

Leiper explains: “Fibrosis plays a key role in the progression of a number of human diseases including pulmonary, renal and cardiovascular disease. We believe that we have identified a common mechanism that might significantly reduce the fibrotic component in several of these disease states. Interestingly, we have also recently demonstrated that in patients with chronic kidney disease (in which fibrosis plays a major role in progression) reduced activity of the enzyme that is the target of our drug is associated with much slower reduction in kidney function suggesting that this pathway is a natural regulator of fibrosis that can be harnessed therapeutically.”

These studies demonstrate that understanding the basic molecular mechanisms that underlie human diseases can lead to the identification of novel therapeutic strategies and the translation of basic research into the clinic.

SJ

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Moonlighting: the Cohesin Story

22 Jan 2012 GMT

However, now Vlad Seitan and colleagues from the CSC Lymphocyte Development Group – led by Matthias Merkenschlager and Amanda Fisher – have caught a glimpse of cohesin’s ‘moonlighting’ activities under circumstances in which the day-job – in cell division – is not required.

“T (immune) cells provide an ideal model to investigate cohesin’s other jobs, explains Vlad, “because as they mature, these cells have periods when they don’t divide.” Working in collaboration with scientists at Oxford and Duke Universities, he found a way to genetically modify mouse T cells to specifically knock out (turn off) cohesin at a point in the cell cycle where it isn’t needed for cell division.

And so cohesin’s new role in T cell differentiation – essentially, how they (T cells) take on certain characteristics to adapt to the range of biological invaders that they might meet – was discovered. “Cohesin is very important for the defining process in the development of T cells,” says Vlad. “It controls the recruitment of the highly specialised biological machinery that rearranges the Tcra locus”.

Tcra is part of the T-cell receptor (Tcr) complex, a ‘molecular aerial’ displayed on the surface of T-cells. Tcr can recognize and bind foreign particles, and the variable components of Tcra (that help them bind a specific invader) are generated using different combinations of sequences from Tcr genes. So-called rearrangement of Trca is integral to the ability of T cell populations to present a varied repertoire of aerials to invaders.

“We found that when we knocked out cohesin, there was a skewed repertoire of T cells,” Vlad confirms. “There were fewer types of T cell receptors, and the cells developed more slowly as a result.” Previous work in the group has shown that cohesin can influence the physical proximity of genes – the genomic architecture. “Now we show it can bring together the enhancer and promoters (elements of DNA that allow genes to be switched on and off) of the Tcra locus.

Although it’s doing a different job, the way cohesion works is similar ‘by night and by day’. The ring-shaped protein complex behaves like a hairband stabilising a plait when it brings chromatids together before a cell divides. To bring promoter and enhancer together, Vlad and co-workers envisage a similar model to facilitate DNA looping.

“Cohesin directly controls transcription,” says Vlad. “It affects how much, but also where DNA gets transcribed. Without it the architecture of the Tcra locus is disrupted and transcription hampered. Transcription at the Tcra locus not only boosts production of Tcra proteins,” he adds, “it also acts as a beacon for the rearrangement machinery.”

“The protein complexes that generate Tcra variability (by shuffling DNA sequences) only bind to sites of active transcription.” DNA not being transcribed is ignored. “Transcription failure in T cells without cohesin led to many variable segments being ignored during rearrangement,” he clarifies. “So fewer types of T cell receptor can form.” Further investigation will no doubt clarify the full moonlighting capacity of cohesin, and the implications for immunity and development.

(Illustrative impression)
Tcra locus diversity, which lends the immune system some of its adaptability, depends upon the combination of about 60 'J' (joining) exons with 100 'V' (Variable) exons. Cohesin plays a major role in the combination mechanism and allows the formation of a vast array of T cells with different receptor forms. When cohesin is missing, this combination mechanism is much less efficient and there is a skewed or limited repertoire of receptor types, with implications for a reduced immune response.

BM/SJ

Seitan et al. (2011) A role for cohesin in T cell receptor arrangement and thymocyte differentiation. Nature, in press. dx.doi.org/10.1038/nature10312

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Live Imaging

22 Jan 2012 GMT

Alex and his collaborators are harnessing optical fluorescence, in which molecules emit light at one wavelength when illuminated with light at another, shorter wavelength. This has proved to be a powerful tool for investigating biological processes inside living cells. A refinement of the basic technique is to use two fluorophores which interact when they are brought within a few nanometres of one another – called Förster Resonant Energy transfer (FRET). These fluorophores can be attached to proteins, and so the technique can be used to detect when proteins come together or move apart, giving a clue to the biological processes occurring.

To date however, these powerful approaches have not realised their full potential because they have mainly been applied to the study of isolated cells. If such ‘bio-reporters’ could be expressed in cells in vivo, and their fluorescence signals read out, cell functions could be directly monitored in the context of the whole organism. This could have a tremendous impact on our ability to understand the mechanisms of disease and to develop and test new therapies.

The researchers are developing a platform technology, combining FRET detection with tomographic reconstruction, to non-invasively monitor cellular processes in vivo with sufficient spatial localisation so to be able to determine in which organs these processes reside.

“This is a strong collaborative project, supported by the Wellcome Trust, between researchers at the CSC (Dan Stuckey, Alex Sardini and Jo Hajnal), UCL (Vadim Soloviev and Simon Arridge) and Imperial College (James McGinty and Paul French)”, says Jo Hajnal (Imaging Physics and Engineering Group), “It is extremely challenging to measure fluorescence signals in biological tissue due to the strong scattering that light undergoes. We circumvent this problem by using ultra-short pulses of laser light and measuring the time variation of the resulting fluorescence signals using novel detector technology, rather than simply measuring the fluorescence intensity.” By applying their imaging techniques at timed intervals the group has mapped their bio-reporter signals within mice.

This capability has huge potential in long-term experiments and is likely to allow reduced animal usage for the mandatory tests of efficacy and toxicity required during the development of new drugs. “And not only that,” adds Alex, “the technology could be used to explore disease mechanisms and track disease progression, for example, by expressing FRET probes to monitor cell signalling under live conditions.”

Globally there is a race going on to find new ways to image fluorescent probes in live animals. “Although other groups across the world have published various steps towards this,” says Jo Hajnal, “our paper is the first example showing a technology that has made it possible to localize and detect the activity of a FRET probe in vivo.”

BM

*McGinty, J., *Stuckey, D. W., Soloviev, V. Y., Laine, R., Wylezinska-Arridge, M., Wells, D. J., Arridge, S. R., French, P. M. W., Hajnal, J. V., Sardini, A., Jul. 2011. In vivo fluorescence lifetime tomography of a FRET probe expressed in mouse. Biomed. Opt. Express 2 (7), 1907–1917. (*Joint first authorship)

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Bombarded

22 Jan 2012 GMT

The signalling molecule that helps our brains categorise an experience as being good – and worth repeating – is called dopamine. This molecule makes us feel great. As well as having a positive impact by making food taste good and sex fun, dopamine plays a role in addiction, because of the compulsion to repeat experiences. Crucially, it also plays an important part in the development of psychosis – including illnesses such as schizophrenia and bipolar disorder, which affect 1 in every 100 people. For these people every minute event takes on higher significance, meaning their brains are overloaded, because of increased dopamine levels. “For people with schizophrenia, dopamine levels are high, and this is thought this to mean irrelevant stimuli come to assume greater importance," explained CSC Group Head Dr Oliver Howes to a group of design Master’s students at Central Saint Martins last year; they were learning about the physiological bases of disease.

What hasn’t been clear before is what the causal relationship between increased dopamine and increased risk of psychosis. Did those unfortunate enough to develop psychosis subsequently have an increased capacity to make dopamine, or was it the other way round? Dr Howes’ new findings, reported in the July 2011 issue of the American Journal of Psychiatry, show that patients deemed at high risk of developing psychosis – those who had experienced symptoms that typically lead on to psychosis such as mild changes in behaviour or inability to deal with stress – already had an increased capacity to make dopamine. This is the first piece of evidence that increased dopamine levels may lead to psychosis rather than the other way round. And this change in dopamine seems to be a key indicator as to whether individuals who seem to be at risk really do go on to develop psychotic illnesses.

"Our finding that there was no change in the dopamine function in patients who presented with psychiatric problems but got better suggests that the elevated dopamine function is specific to the later development of psychosis. We are looking to see if the scan can be used to as a predictive test,” says Dr Howes. “The holy grail is to find a way of preventing these devastating illnesses before they start. Our finding that dopamine function is elevated in people who go on to develop psychotic illnesses is an important clue as to what causes them.”

He continues: “Future treatments could target this part of the brain’s dopamine system to prevent the full development of the illness. As roughly 1 in 10 people with schizophrenia die from suicide, predominantly in the first few years after the illness starts, a treatment that prevents the full illness could save many lives as well as alleviating suffering.”

SJ

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Transatlantic Heart Network

22 Jan 2012 GMT

The condition affects around 1 in 2000 people worldwide and results in an enlarged heart that cannot pump blood effectively around the body. “We know dilated cardiomyopathy is genetically regulated,” reveals Stuart, “and we have people in this new scientific network that work on disease models in flies, fish, rodents and humans. So we’re looking across the board to find new ways to diagnose and treat the disorder.”

So what are the genetic factors? Mutations in patients can occur in genes, whose protein products make up the basic muscle contraction units in the heart. Examples include Titin (TTN), the largest gene in the body and genes that make the proteins actin and myosin. As Stuart notes, “we know that there are many mutations in Titin, but it’s often difficult to say what’s causative or not,” also these structural genes aren’t necessarily drug targets.

To identify potential drug targets, the plan of action is to have a discovery phase in humans. “We’ve hundreds of human DNA samples from which we can identify specific disease genes.” The scientists will use model organisms to learn about where those genes sit hierarchically in the context of gene networks and metabolic pathways within cells, as these are highly conserved across species. “We can really dig into the mechanisms using model organisms,” Stuart confirms.

Mutations in sarcomeric genes, like Titin, don’t themselves drive disease, so there’s probably no point in trying to target them. “We need to find out what’s downstream of those mutations.” Individuals with similar mutations in Titin may have different disease phenotype, which is why the scientists really need to understand the genetic context, how Titin gets regulated in health and disease. “Different drugs might be suited to different underlying genetic backgrounds,” adds Stuart.

Rats and mice, like humans, have spontaneous mutations in Titin that are associated with heart failure. And the gene can be completely knocked out in some animal models. Knockouts in fish develop a cardiomyopathy, although they manage to compensate by absorbing oxygen from the water around them. In flies an RNAi-based approach, which allows researchers to easily switch off genes, has helped researchers identify ‘cardiomyopathic’ mutations. All in all, a huge wealth of data exist to explore.

Once potential targets are uncovered, the scientific network will test potential drug molecules using high-throughput cell-based in vitro assays. “And next-generation sequencing technology means we can now move faster than ever towards new therapies,” says Stuart. The biggest cause of heart failure is heart attack, after which heart muscle dies. “By screening people early and having a range of potential therapeutic avenues, we stand a much better chance of improving the treatment of this disease.”

BM

The Genomic, Epigenomic and Systems Dissection of Mechanisms Underlying Dilated Cardiomyopathy, a Fondation leducq funded programme commencing January 2012 (www.fondationleducq.org) combing the expertise of leading transatlantic researchers with a $6 million dollar award for five years. Josef Penninger (IMBA, Austria); Phillip Charron and Francois Cambien (INSERM, France) Tim Aitman (Physiological Genomics and Medicine); (Christine and Johnathan Seidman (Harvard, USA); Callum MacRea (Harvard, USA); Andrew Marks (Columbia university, USA). …

Stem Cell Tools

22 Jan 2012 GMT

Joana Santos and Filipe Pereira of the CSC Lymphocyte Development group and coworkers have recently shown that other stem cells important in embryonic development – associated with the generation of extraembryonic tissues such as the placenta and yolk sac – also have the potential to reprogram mature cells. However, the reprogrammed cells they produce differ from those programmed by normal ES cells, reflecting the different provenance of the cells used.

The researchers were investigating the development of mouse embryos to see how chromatin – the package of DNA and protein in a cell’s nucleus – changes shape during development as the different cells specialise for specific tasks. They compared the expression of genes in trophectoderm stem (TS) and extraembryonic endoderm (XEN) cells (see box below), which are generated as the embryo becomes larger and implants in the womb. continued...

Human Embryonic Stem Cells from Russo E. (2005). PLoS Biology under Creative Commons License

By the time a single cell has become 16, two distinct populations arise: an external, polar layer, the trophectoderm (TE); and an apolar inner cell mass (ICM). In addition to providing the source of ES cells for the study, the ICM was induced to produce primitive endoderm (PrE) cells, which usually form during implantation in the womb, to provide a source of extraembronyonic endoderm (XEN) stem cells. Trophectoderm stem (TS) cells provided the third type of stem cells investigated.

In order to test how gene expression differed between the three stem cell types, the team first looked at the levels of genes expressed in each kind. They were also interested in how the timing of gene replication differed between the three types, making use of the idea being that genes that replicate sooner are in ‘open’ areas of chromatin – allowing easy access to transcriptional machinery – whereas those that replicate later lie in tightly-bundled chromatin and are characteristic of repressed genes. They looked at specific developmentally important genes and measured how much newly-copied DNA encoding those genes was present at different stages during a cell’s divisional cycle.

The three types of stem cells each produced different patterns of gene expression and replication timing, reflecting their different origins. Among the three stem cell types, ES and TS cells exhibited closely matching replication timing profiles, indicative of highly accessible chromatin. In contrast, XEN cells had reduced chromatin accessibility, particularly around genes required for pluripotency. The similarity between ES and TS cells was expected: only very few genes are uniquely restricted to the placenta, with the majority of shared genes involved in the development of many internal organs.

Using each of the three stem cell types, the team successfully reprogrammed mature human lymphocytes (white blood cells) to resemble human ES and extraembryonic cells. This technique may be important for studying the early stages of human development, because it allows investigation without the requirement for human embryos.

Reference: Santos, J., Pereira, C. F., Di Gregorio, A., Spruce, T., Alder, O., Rodriguez, T., Azuara, V., Merkenschlager, M., Fisher, A. (2010). Differences in the epigenetic and reprogramming properties of pluripotent and extra-embryonic stem cells implicate chromatin remodelling as an important early event in the developing mouse embryo. Epigenetics & Chromatin 3 (1), 1+

The original article appears here: Epigenetics & Chromatin

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Echoes from the Past: Birth and Body Fat

22 Jan 2012 GMT

However, this does not come without a price, as researchers at the MRC Clinical Sciences Centre – led by Professor Jimmy Bell (Metabolic and Molecular Imaging Group) – are starting to discover. A few years ago the team spotted something unusual about preterm babies. They had significantly more abdominal fat – particularly around the liver – than infants born at term (37-42 weeks).

The implications of this finding are as yet unclear, although fatty-liver is a risk factor for type 2 diabetes and insulin resistance. The team wanted to know whether this was simply a temporal difference, so they looked at adults (aged 18-27), and were surprised to find the same thing: much higher liver fat in preterm individuals - especially in males - than those born at term.

In the UK most people with a fatty liver are overweight or obese, but the preterm and term individuals in this study were healthy young adults. “Most of them were students and had very similar lifestyles and socio-economic backgrounds,” explains Jimmy Bell. “Both groups had similar lifestyles, food consumption and physical activity levels.”

“So when we started this study, I really didn't expect to find such a marked phenotype.” Earlier research by Barker (Pubmed) and others has shown that early-life programming is critical to what happens in adulthood, “but this is one of the few examples of an early-life programming phenotype, which is sustained through to adulthood,” Jimmy reveals.

“Clearly events happening early on have modulated the way their [preterm group] bodies function in relation to fat.” The team speculates that there is some sort of uterine cue during the first few weeks of life that does not fully happen in preterm individuals. “Perhaps certain genes in the liver don’t get switched on properly in preterm subjects.”

“What the long-term effects are, we don’t know at the moment,” Jimmy adds, “but we do know that higher levels of liver fat pose a stronger risk of developing insulin-resistant and type 2 diabetes.” With some 346 million diabetic people in the world, and around 3.5 million annual deaths caused by the consequences of the disease worldwide (WHO), isolating risk factors for disease onset is of considerable importance.

“On the positive side,” says Jimmy, “we know we can superimpose environment on genetics in most obese phenotypes.” Intervention programmes that alter lifestyle in terms of diet and physical activity can change the levels of fat in the liver. “So we now plan to impose lifestyle changes on the preterm and term groups to explore how lifestyle affects liver fat in preterm individuals.”

The team is exploring the differences at a molecular level, supplementing preliminary results with preclinical and biochemical data. “We're looking at the genes involved in fat metabolism and beta-oxidation (the way mitochondria oxidise fat),” reveals Jimmy. “While the jury's out on whether we can achieve this, it will provide us with the opportunity to look at the overall impact of early-life programming on body fat metabolism.”

BM

Thomas et al. (2011) Aberrant Adiposity and Ectopic Lipid Deposition Characterize the Adult Phenotype of the Preterm Infant”. Pediatr Res, July [Epub] …

Cholesterol and Cellular Retirement

22 Jan 2012 GMT

Cells also have a finite ‘working life’, at least when it comes to cell division: after about 50 replications, they stop dividing but otherwise continue as living cells. This cellular retirement – or senescence – has its advantages in limiting the propagation of genetic copying errors that can accrue over time.

Researchers in the Clinical Sciences Centre’s Lipoprotein group were studying a disease called Autosomal Recessive Hypercholesterolaemia (ARH), which reduces a person’s ability to deal with low density lipoproteins or ‘bad cholesterol’. They noticed that skin cells from ARH patients grow slowly and go into senescence earlier than expected, so they considered whether the ARH protein – which is deficient in ARH patients due to a faulty gene – could have additional biological functions to that of dealing with cholesterol. A team from the University of California had found that the ARH protein plays role in cytokinesis, the process in cell division that splits up a cell’s membrane and fluid contents as it divides. Perhaps this held a clue to the slow growth they observed?

The Lipoprotein group teamed up with CSC Cell Proliferation, whose expertise in cell growth would prove valuable in making sense of what they saw. “In addition to observing premature senescence,” reports researcher Dr Xi-Ming Sun, formerly of the Lipoprotein group, “we noticed that the cellular nuclei were misshapen, and that there were problems with cell division. As well as the role in cytokinesis previously reported, we wondered whether the ARH protein might be important for chromosome duplication.” They introduced a molecular marker that is taken up during DNA synthesis, which is central to chromosome duplication, and showed that it was being taken up more slowly, explaining the slow cell growth.

In addition to this, they were able to confirm that the ARH protein is concentrated at locations in the cell crucial for cell division, specifically the mitotic spindles and nuclear membrane. It interacts with a protein called lamin B, “…a nuclear envelope protein; possibly part of the ‘mitotic matrix’ – a relatively new idea that many proteins are important for maintaining the structure of mitotic spindles,” explains Dr Sun. “Mitotic spindles are where the sister chromatids – the two cohorts of copied chromosomes resulting from cell division – line up before cells divide; it is also possible that the ARH protein is part of this mitotic matrix.” A role in mitosis could explain the reduced cell growth and premature senescence the researchers saw. But when it comes to senescence, there is the possibility that an alternative underlying mechanism could be at play.

Senescence can also be brought on sooner than usual in response to stresses, much like an incapacitating injury can force early retirement. Research published by a group at the University of Vermont shows that along with other proteins, ARH becomes partly phosphorylated in response to radiation damage, limiting its ability to function. “If the protein plays a role in DNA damage response, this could also explain the premature senescence exhibited by cells deficient of ARH,” suggest the CSC authors.

Their findings could also have important clinical relevance, because cellular senescence occurs during the development of atherosclerosis, the thickening of arteries by bad cholesterol that can lead to cardiovascular disease including heart attack.

“We already knew that a deficiency of ARH protein can lead to atherosclerosis through an inability to process ‘bad’ cholesterol,” explains Head of Lipoprotein Professor Anne Soutar, “…but our finding that ARH is also important in senescence, which is an important contributing factor in the development of atherosclerosis, has not been previously explored.”

Their findings help us to understand how the body deals with the fat in our diets, and perhaps also identify the play of a subtle mechanism that helps determine how cells replicate and when they eventually retire.

SJ

Sun X-MM, Patel DD, Acosta J-CC, Gil J, Soutar AK (2011) Premature senescence in cells from patients with autosomal recessive hypercholesterolemia (ARH): Evidence for a role for ARH in mitosis. Arteriosclerosis, thrombosis, and vascular biology, in press.
http://dx.doi.org/10.1161/ATVBAHA.111.232223

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Dopamine Levels Offer Clue to Schizophrenia

22 Jan 2012 GMT

19 January 2009

The study tracked the levels of dopamine, tagged with the positron-emitting isotope ¹⁸F, in the striata of patients with an ‘At-Risk Mental State’ (ARMs) – patients meeting criteria of psychotic symptoms including abnormal beliefs, perceptions and speech. It was found that levels in ARMS patients approached those of people already diagnosed as having schizophrenia, and that higher levels of dopamine were found in the striata of those with the most severe symptoms.

The precise imaging technique used also allowed identification of the region where dopamine concentration was highest, in the associative striatum.

Oliver Howes, the lead author of the paper, said, “Our finding that dopamine levels are high in people showing very early signs of developing schizophrenia gives an important clue as to what is causing schizophrenia and other psychotic illnesses”. The findings help to explain why ARMS patients given antipsychotic drugs that target the dopaminergic system tend to be less likely to develop psychotic symptoms.

However, the identification of the associative striatum as the region linked to schizophrenia offers a new target. Howes said, “…current drugs are acting in the wrong place to address the primary problem – designing drugs to reverse the primary problem could offer the hope of better treatments for schizophrenia.”

Stefan Janusz

Link to Archives of General Psychiatry Paper …

Quality Control

22 Jan 2012 GMT

The cohesin protein complex is one type of component in the cell factory, holding copied chromosomes or sister chromatids together until a cell divides, much like chromosome glue. It also helps to repair gaps in DNA called double strand breaks (DSBs) that can form during DNA replication or when a cell is damaged. In most cells of the body cohesin also helps to detect DSBs via damage checkpoints, cellular mechanisms based on chemical signalling. It loads onto DSBs, marking them for repair and helping to trigger a signal that halts cell division if breaks are not repaired in a timely fashion. Like a factory’s quality control officer, checkpoints cast aside imperfection – singling out those cells that haven’t been repaired for cell death or apoptosis.

Cohesin is also required for chromosome segregation and DSB repair during meiosis, the special cell division that forms sex cells – sperm and eggs – although there are some differences in the way meiotic DSBs are formed and repaired (see box below). “The interplay between DSB repair and sister chromatid cohesion may be different in meiosis,” explains CSC Meiosis Group Head Enrique ‘Fadri’ Martinez-Perez, “and actually how cohesin affects the repair of meiotic DSBs has not been well understood.” What hasn’t been clear before is if cohesin has any role in the operation of the DNA damage checkpoint during meiosis.

"You can't make an egg without breaking some DNA"
DSBs are actually necessary in sex cells to allow meiotic recombination, the gene shuffling that makes everyone – apart from twins – genetically unique. The actual mechanism of repair is also different: In mitotic cells sister chromatids resulting from DNA replication are held together and the intact copy is used as a template to repair breaks, but in sex cells the homologous chromosome copy is used instead.

Fadri and his team study mechanisms of meiosis in a nematode worm called C. elegans. They genetically engineered the worm to be deficient in SCC-2, a protein that is required to load cohesin onto DNA. They found that DSBs necessary for meiotic combination were normally formed in mutants lacking SCC-2 during meiosis, but these breaks remained unrepaired, indicating an important role for cohesin in fixing meiotic DSBs. What’s more, these unrepaired DSBs didn’t induce an increase of cell death in worms lacking SCC-2, which suggested that cohesin may be important in the meiotic DNA damage checkpoint that kills off non-repaired cells.

In order to confirm the involvement of cohesin in the meiotic DNA damage checkpoint, they compared rates of cell death in meiotic cells from C. elegans that had been exposed to radiation. Normally radiation damage means many cells undergo apoptosis, but in the SCC-2 deficient worms, rates of apoptosis did not increase due to radiation damage. By tweaking cohesin levels they found there was a critical cohesin point below which the checkpoint failed to function. They also found that the lack of cohesin prevented the molecular 'repair this' markers being attached to the break, in effect failing to alert the cell to the DSB to initiate repair. Furthermore, the recruitment of RAD-51, a protein critical for DNA repair, to DSBs induced by radiation was delayed by several minutes in worms lacking SCC-2. These observations show that cohesin is important in the earliest steps of the meiotic DNA damage response, something they didn't expect.

“Our investigations have uncovered an unexpected role for meiotic cohesin in the early processing of DSBs and in the activation of the checkpoint,” confirms Fadri, “showing that cohesin is a key player in the early stages of the meiotic DNA damage response. “We’ve also shown that SCC-2 is required for cohesin loading during meiosis, thus demonstrating that the main mechanism of cohesin loading is conserved between the mitotic and the meiotic cell division programs.”

SJ

Reference
Lightfoot, J., Testori, S., Barroso, C., Martinez-Perez, E. (2011) Loading of meiotic cohesin by SCC-2 is required for early processing of DSBs and for the DNA damage checkpoint. Current Biology, in press. DOI 10.1016/j.cub.2011.07.007

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Change without REST?

22 Jan 2012 GMT

4 February 2009

Work published last year in Nature suggested that REST is important to maintain the pluripotency of ES cells. They described that an ES cell line missing a single REST allele expressed reduced amounts of pluripotency-associated genes and that they exhibited lower activity of alkaline phosphatase – a stem cell marker.

Now, a team of researchers in the CSC’s Lymphocyte Development group, led by Amanda Fisher and Matthias Merkenschlager, is to publish a paper in Nature that refutes this claim, demonstrating that REST is not required for maintaining ES cell pluripotency.

In a series of experiments using two complementary methods of gene targeting and RNAi knockdown, lead investigator Helle Jørgensen and co-workers showed that mouse ES cells that lack or have reduced REST levels still produce normal amounts of the pluripotency protein Oct4 and have similar alkaline phosphatase activity as cells with normal REST function.

In a companion paper in the journal Development, the team examined the function of REST in ES cells in detail. By genome-wide expression profiling, they found that a subset of potential REST target genes are incorrectly expressed in ES cells lacking REST. This, however, did not appear to have physiological consequences, as the cells did not show any tendency to spontaneously differentiate towards a neural lineage and were functionally indistinguishable from normal cells.

While these studies found that REST is not required to maintain ES cell pluripotency, or indeed to prevent neural differentiation, the findings show that REST may be important for correct neural development. This would be in line with earlier research showing that mice lacking REST have complications in embryonic gestation that result in forebrain malformation and death.

Link to Nature paper
Link to Development paper …

Transcription: Stop!

22 Jan 2012 GMT

7 February 2009

Perhaps unsurprisingly, a multitude of precisely orchestrated events must occur to facilitate the act of mitosis. In most eukaryotes, the process of transcription – whereby the genetic information in DNA is used to make proteins – actually stops. It had previously been suggested that the chromosomes being so condensed leads to transcription stopping.

The single-celled eukaryote Saccharomyces cerevisiae – budding yeast – was proposed to buck this trend, because levels of transcription indicated by cellular RNA levels remained elevated throughout mitosis. This seemed counterintuitive, because it was known that the most highly transcribed regions, comprised of ribosomal DNA (rDNA), are actually in a condensed state towards the end of the mitotic cycle.

Now, a team of researchers in the CSC’s Cell Cycle Group, led by Luis Aragón, has used a more sensitive technique to measure RNA levels in budding yeast. They found that it was lowered during mitosis, but at a later stage (termed, ‘anaphase’) than in other eukaryotes (where it occurs in ‘metaphase’).

To understand how this was taking place, the role of Cdc14, a protein phosphatase known to be crucial in chromosome segregation, was explored. It had previously been shown that Cdc14 inhibits the transcription of the highly transcribed rDNA by acting on RNA polymerase I (Pol I) – the cellular machine responsible for transcription. However, the reason for the requirement of Cdc14 remained obscure. The authors conducted experiments in vitro employing all the biochemical machinery of transcription, and found that purified Cdc14 actively inhibits the occupancy of Pol I to stop transcription, whereas a ‘phosphatase-dead’ mutant did not. Transcription continued in the latter case.

Finally, the authors challenged the present dogma that chromosome condensation blocks transcription. In an elegant series of experiments they demonstrate that the molecule that promotes chromosome condensation – named ‘condensin’ – cannot be loaded to DNA until transcription stops.

Contrary to what was previously thought, this means that transcription interferes with chromosome condensation, and not the reverse.

Stefan Janusz

This work was published in Nature …

Mighty Morphogens

22 Jan 2012 GMT

In a 1952 paper, ‘The Chemical Basis of Morphogenesis’, the mathematician Alan Turing proposed that the form arise from a complex molecular dance of catalysis, inhibition and diffusion; the tiniest perturbations of a cell’s chemistry causing a cascade of events that give an embryo its shape. He called his theoretical agents ‘morphogens’, a term used today to describe biochemicals that influence transcription of DNA during embryonic development.

Two such morphogens, Nodal and Activin, are secreted from cells into the extracellular space – between adjacent cells – to control certain aspects of development and growth in many vertebrate species. Part of the TGF-β superfamily of signaling molecules – important in cell differentiation, proliferation and death – they influence the identity and fate of adjacent cells by passing information into the nucleus via the effectors Smad2 and Smad3. The effector Smads then transcribe a selection of target genes and assign a particular fate to a cell depending upon the morphogen concentration it is exposed to. The morphogen Nodal, for example, is crucial in mediating positional information in the developing embryo: High levels of nodal form the head, while low levels become the ‘trunk’, or torso.

High nodal makes head; low nodal forms trunk.

The mechanism by which morphogens convert quantitative information – a concentration – into a qualitatively singular fate – a cell type – has, however, remained elusive. It also wasn’t known whether different genes are switched on when morphogen levels are high compared to when they were low.

Work recently reported by the Mammalian Neurogenesis group at the CSC, led by Vasso Episkopou, aimed to address these questions by manipulating levels of Smad2/3 in embryonic stem cells. The group used a protocol including both an activator of Smad2/3, and a complementary inhibitor, which allowed them to control the level of activated Smad2/3 over time, and then analyse gene expression in microarrays to look for any effects.

The study identified several tens of genes, both known and novel, whose expression levels changed in step when the activation of Smad2/3 was manipulated. Morphogen levels directly influence the levels of transcription of the identified ‘target’ genes, suggesting that cell fate is determined not by a specific set of genes being switched on or off, but by relative expression levels of several target genes.

Because genes are transcribed at different rates – some quickly and some much more slowly – the researchers’ findings also indicate that length of exposure to a particular morphogen is as important as its concentration.

Only a few genes were found to be expressed solely due to Smads, with many target genes exhibiting a moderate level of expression even in the absence of Smad activation. Smads therefore work to reinforce expression, implying that cell fate is determined by threshold expression levels rather than a gene being just ‘on’ or ‘off’.

Insight into this mechanism is important, as disruption of the TGF-β pathway can lead to developmental diseases and cancer. The study reported also provides a new investigative tool for carrying out further research.

Stefan Janusz

Reference:
Guzman-Ayala M, Lee KL, Mavrakis KJ, Goggolidou P, Norris DP, Episkopou, V. (2009) Graded Smad2/3 Activation Is Converted Directly into Levels of Target Gene Expression in Embryonic Stem Cells. PLoS ONE 4(1): e4268. Link to PloS paper

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The Right Timing

22 Jan 2012 GMT

Many scientists think that stem cells hold the key to successful treatment because they have the potential to become any cell in the body, including the midbrain neurons that die off in Parkinson’s disease. Producing dopamine nerve cells from stem cells will make it easier to monitor the effectiveness of potential new drugs that could slow or halt disease progression. With one of the most attractive advances of recent years – the introduction of induced pluripotent stem (iPS) cells which can be made from adult cells – it is now possible to make Parkinson’s neurons to study the disease in a dish.

Earlier this year the CSC Stem Cell Neurogenesis group published research showing that it is possible to generate midbrain dopamine neurons by ‘programming’ stem cells with a protein called Dmrt5, which is normally present inside cells that become dopamine neurons. Now, they’ve found a way to make them by stimulating the production of stem cells' own Dmrt5. They did this by interfering with a chemical signalling system called FGF/ERK at defined stages of stem cells' journey to becoming nerve cells, using a combination of small molecules and proteins. The FGF/ERK signalling system contributes to a cell’s ability to probe and correctly respond to its environment, telling it where to go and what to become; defining the location of the brain early on in embryonic development. By blocking and activating the signal at precisely the right time with cells grown in a dish, they can use this same signaling system to make midbrain dopamine neurons more simply, reliably and cheaply than by other methods.

Understanding the cues that direct the generation of desired cell types is fundamental for building up a ‘toolbox’ or repertoire of techniques to make cells for regenerative medicine. Importantly, the technique described by the CSC group works with iPS cells and EpiSCs – another recent advance that helps generate neurons more quickly – as well as embryonic stem cells, showing that the technique is widely applicable.

Dr Mark Ungless, head of CSC Neurophysiology and coauthor of the paper reflected on the success of the technique: “Importantly, these cells have many of the same electrophysiological characteristics that we see in functional dopaminergic neurons” – which shows that the technique really does work.

Ines Jaeger, the main author of the paper, explains: “In vitro-derived dopamine neurons provide a valuable tool for drug discovery and modelling of Parkinson’s disease. Because it is fully chemically-defined, this [method] could be readily adapted for use in a clinical setting, or scaled up for toxicity and drug screening relevant to developing new therapeutics for Parkinson’s disease.”

SJ

Reference
Jaeger, I., Arber, C., Risner-Janiczek, J. R., Kuechler, J., Pritzsche, D., Chen, I.-C. C., Naveenan, T., Ungless, M. A., Li, M. (2011) Temporally controlled modulation of FGF/ERK signaling directs midbrain dopaminergic neural progenitor fate in mouse and human pluripotent stem cells. Development, in press.
http://dx.doi.org/10.1242/dev.066746

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Big Hearts' Genetic Origins

22 Jan 2012 GMT

1 December 2008

Stuart Cook and Tim Aitman published research in April that describes how a gene called osteoglycin (Ogn), not previously linked to heart function, plays a key role in the regulation of growth of the heart’s left ventricle; its main pumping chamber.

Abnormal regulation leads to a condition called Left Ventricular Mass (LVM), an increase in mass that causes the left ventricle to become stiff. The heart then needs more oxygen, which can lead to shortness of breath, and eventually a heart attack.

Cook summarized the importance of finding a gene ‘for’ heart enlargement: "Enlarged hearts are very common. A person whose heart is enlarged is more likely to suffer a heart attack or heart failure than someone whose heart is a normal size. We can't currently treat the condition directly, so lowering a patient's blood pressure is the only option we have.

Now that we are unravelling how genes control heart growth, we can gain a better understanding of common forms of heart disease. This should lead to new and more effective ways of treating people."

Link to Nature Genetics paper …

Imaging Excellence

22 Jan 2012 GMT

Miss Leigh Brody and Mr Mohammad Hankir, PhD students in Metabolic and Molecular Imaging group under Professor Jimmy Bell, worked with Dr Magdy Khalil of the Biological Imaging Centre (BIC) to capture hybrid or fused images using complementary scanning techniques, allowing a more in-depth investigation into the biomolecular processes behind diseases. They used the BIC’s state-of-the-art Inveon® imaging system to collect both positron emission tomography (PET) and computed tomography-angiography (CTA) scans in one in vivo imaging session, allowing greater elucidation of processes, in this case vascular function in a mouse.

This combination of complementary techniques provides important information that will inform future experiments and improve how much information can be extracted from scanning sessions, for instance by allowing tracers to be localised to specific tissues. It also allows the choice of tracers and how they are introduced into the animal to be optimized in order to understand different characteristics of disease disorders more quickly.

On sharing news of the accolade with his coworkers, BIC’s Dr Khalil who was at the imaging congress wrote, “congratulations…I’ve also heard that our work will be made highly [visible] for those attending WMIC." Siemens Medical solutions have also selected the image to be among those for their 2011 annual poster.

Talking about the work, Dr Khalil said, "future application of this technique would allow researchers to correlate the blood flow in microvessels with perfused tissues, in order to reveal more insights of disease characteristics."

Researchers at the CSC benefit from a wealth of expertise provided by some world class in-house research support facilities, including imaging, biochemical analyses, and bioinformatics and computational modelling.

Click on image for larger version of poster

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Epigenesys Award

22 Jan 2012 GMT

The EC-funded Epigenesys Network of Excellence is a research initiative aiming to advance the interface between epigenetics and systems biology.

A major focus of the initiative is to contribute to the map of the human epigenome, an undertaking a magnitude greater than the sucess of the human genome project. Many of the disease challenges facing modern medicine have epigenetic components, and understanding how environmental factors impact on our epigenome has huge implications for treatments in the 21st Century.

On hearing that her group had been selected for the initiative, Petra said she was, "overjoyed."

Epigenetics is one of the Clinical Sciences Centre's main research areas of focus. With strongs links to groups across Europe and beyond, the CSC has an excellent reputation as a leader in this area. Commenting on Petra's success, CSC Director Professor Amanda Fisher said, "Winning a RISE award is an outstanding achievement that places Petra's work at the core of european excellence in epigenetics research".

SJ

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Learning From Our Mistakes

22 Jan 2012 GMT

In humans and many other animals the seat of this reward mechanism is the brain’s dopaminergic system, a network of neurons that fires when we get an unexpected reward, faciliting the brain restructuring or ‘neuronal plasticity’ that lends itself to learning. However, some experiments have indicated that brain dopamine levels also increase due to aversive stimuli such as those that are consistent with pain. This has led some researchers to postulate that dopaminergic neurons may be excited by all salient stimuli, rather than rewards alone – a hypothesis bolstered by research carried out a few years ago, which shows that excitation of the dopamine system is involved in fear conditioning.

One hypothesis proposed to reconcile these findings is that the termination of an aversive stimulus is itself an unexpected reward and might therefore excite dopamine neurons. Now, work published in the journal PNAS by members of the CSC’s Neurophysiology Group has put this idea to the test. The researchers identified dopamine neurons in a midbrain region of anaesthetized rats called the ventral tegmental area (VTA), and measured changes in their firing rate when aversive stimuli – in this case, electric shocks – were applied to a paw. Sure enough, they identified dopamine neurons in the upper or dorsal part of the VTA that exhibited a lower firing rate when the shocks were applied. Those same neurons also exhibited a marked increase in firing rate when the shock stopped. An increase in firing rate is consistent with what would be expected from an unexpected reward.

However, neurons also identified as dopaminergic, but which reside in the lower or ventral part of the VTA, were shown to increase their firing during the application of shocks. The anatomical distinction between these neurons and those in the dorsal VTA suggests a functional distinction: those in the ventral VTA appear to play a role in the processing of unexpected aversive events.

Demonstrating the existence of two functionally distinct dopamine systems in the VTA brings a novel resolution to some of the controversies surrounding the function of the dopamine system. It also extends our understanding of the underlying neurophysiological mechanisms that help us learn from our mistakes.

Reference:
Brischoux, F., Chakraborty, S., Brierley, D. I. I., Ungless, M. A. A., March 2009. Phasic excitation of dopamine neurons in ventral vta by noxious stimuli. Proceedings of the National Academy of Sciences of the United States of America.
Link to PNAS paper

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How To Proofread a Genome

22 Jan 2012 GMT

The text in question is the instruction manual which can build a human; it is your genome. Each individual on the planet has their own, unique text (with the exception of identical twins) and there is a copy inside every one of the thousands of cells which make up your body. These cells are constantly being replaced, and every time a cell divides it has to produce an exact replica of the genome - a text composed of 3 billion letters.

Clearly this is no mean feat. And the cell makes mistakes. But it also has the means to correct them, to proof read the genome. This process is critical to our health - if mistakes are allowed to accumulate, the cells become cancerous. Scientists now know that this accumulation is not specific to one type of tumour; it is the fundamental mechanism underlying every form of the disease. Understanding what causes these mistakes and why they are not corrected is therefore essential to the development of therapies which could potentially target not one, but many forms of cancer. And, during the course of my PhD, I am hoping to contribute to this understanding.

But I’m not actually interested in the mistakes in the text itself. I want to know what happens if someone tries to scribble all over it, or pull out a bookmark, or fold over the corner of a page. Even if the sequence of letters is identical, if the text is annotated differently – its meaning could be completely changed.

You see, although every cell in your body has the same copy of the genome, each cell bookmarks it in distinctively. It is these annotations, collectively referred to as epigenetics, which allow different cells in your body to have unique, specific functions despite having the exactly same copy of the instruction manual. So, when a cell needs to produce an identical copy of itself, it does not only have to copy the genome – it has to copy the bookmarks too. During the course of my PhD, I’m hoping to find out how a cell does this. How stable are these marks? How are they copied when a cell divides? What happens if cells make mistakes in where they put the bookmarks?

Using stem cells taken from mice and grown in a laboratory, I have identified tiny molecules inside cells which are passed on from a parent cell to its progeny. If these molecules aren’t there when the cell divides, its descendents start to read all sorts of bits of the genome they shouldn’t; bits that should only be read in brain cells, or blood cells, or bone. So, these molecules somehow act to help the stem cell pass on its identity; to allow its progeny to remember their purpose in life. Are these molecules related to the bookmarks? Do they help tell the cells where the bookmarks are supposed to go? Or do they work in collaboration with the scribe directly, helping to copy the genome? I don’t yet know. But what I do know, as a result of the work of other scientists, is that in tumours, these molecules are often changed or absent. So I’m pretty keen to find out.

Epigenetics is a new and exciting field which has not only revolutionised classical genetics, but could add critical information to our understanding of how and why, at the most fundamental level, we develop cancer. All over the world, scientists are working on different tumours from different parts of the body from different patients. But what is common to them all is that somehow, at some point, the scribe has messed up and a mistake has occurred in the genome. It could be a change to the sequence of the letters, a fold where there shouldn’t be one, or a misplaced bookmark. I want to know what goes wrong to allow this mistake to be passed on to the next generation of cells. This could hold the key to understanding why tumours develop, and how we can treat them – even if we cannot stop the mistakes happening in the first place. We are only human, after all.

Bryony Graham

Lymphocyte Development PhD student Bryony Graham’s essay How to proof read a genome was shortlisted for the 2011 MRC Max Perutz award. She joined other shortlisted authors and MRC Chief Executive Sir John Savill at a prizegiving ceremony at the Royal Society on 7 September. Her essay is one of 12 to be featured in a book of the year's shortlisted entries, currently being published.

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Genetic Link for a 'Heavy Heart'

22 Jan 2012 GMT

Taking advantage of recent step-changes in integrative systems-genetics approaches, Professor Stuart Cook of CSC Molecular Cardiology led a team of international researchers who instead looked at pathways underlying blood pressure-independent hypertrophy, commonly seen in obesity and type 2 diabetes. Using powerful genetic analyses, they investigated a hypertrophy ‘quantitative trait locus’ or QTL – essentially a region of DNA containing genes that underlie a quantitative genetic trait. The analysis is particularly successful in identifying causative genes for particular diseases. In this instance, they found that a faulty version of a gene called Endog, which was previously thought only to be important in cell death, was the culprit.

“Our study shows that the Endog gene actually plays an important role in the enlargement of the heart, which can lead to heart failure and eventually death in the worst cases,” said Stuart. “We found that a faulty copy of this gene causes the heart to become thick and fatty, making it ‘heavy’ with poor function.”

“It does this by interfering with the heart cells’ energy source – the mitochondria. Like any other muscle in our body, the heart needs energy to keep it pumping. If the mitochondria don’t work properly, the heart struggles to make enough energy and the cells produce toxic by-products, called reactive oxidative species, which increase thickening of the heart wall.”

The heart is rich in mitochondria; it needs a huge number to be resistant to fatigue, and to keep pumping blood around the body.

“Our findings give us a new insight into how the mitochondria exert control over the thickness of main chamber of the heart,” added Professor Cook. “We can now start to investigate new ways to develop treatments which target the mitochondria and toxic oxidative molecules.”

Professor Amanda Fisher, Director of the MRC Clinical Sciences Centre, said, “What’s fascinating about this study is that it identifies the function of a gene which was totally unpredicted to be involved in enlargement of the heart. This discovery emphasises the importance of deciphering the genetic code of a broad range of mammals alongside that of humans eventually to allow us explore new avenues for better targeted drugs.”

Professor Peter Weissberg, Medical Director of the British Heart Foundation, said, “The finding could pave the way for new treatments to prevent the development of a heavy heart. Hopefully, in the future, we’ll be able to target the root cause of some patients’ heart conditions rather than treating the resulting symptoms.”

SJ

Reference:
McDermott-Roe, C., Ye, J., Ahmed, R., Sun, X.-M., Serafin, A., Ware, J., Bottolo, L., Muckett, P., Canas, X., Zhang, J., Rowe, G. C., Buchan, R., Lu, H., Braithwaite, A., Mancini, M., Hauton, D., Marti, R., Garcia-Arumi, E., Hubner, N., Jacob, H., Serikawa, T., Zidek, V., Papousek, F., Kolar, F., Cardona, M., Ruiz-Meana, M., Garcia-Dorado, D., Comella, J. X., Felkin, L. E., Barton, P. J. R., Arany, Z., Pravenec, M., Petretto, E., Sanchis, D., Cook, S. A. (2011). Endonuclease g is a novel determinant of cardiac hypertrophy and mitochondrial function. Nature 478 (7367), 114-118.
http://dx.doi.org/10.1038/nature10490

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Summer Students

19 Oct 2011 GMT

Funded by the charitable Nuffield Foundation, the two month placement allows students the chance to participate in projects as diverse as investigating neuron repair in the brain, challenging preconceptions about the relationship between diet and obesity, exploring pharmaceutical candidates for the treatment of septic shock, and making biological sensors for imaging applications.

Listen to audio interviews with A-Level students James Wilkinson and Shadman Aziz, and Undergraduates Cyrus Daruwalla and Jade Fernandes

On Friday 26 August the students had the opportunity to present their work to their peers on the scheme, as well as other scientists based at the CSC and Imperial College. Among other revelations, the audience found out that it is possible to track the flow of energy within cells and that your mother’s diet and levels of body fat when she is pregnant with you can have a drastic impact on your own weight throughout your life.

Shadman Aziz, an A-level student at City and Islington Sixth Form College, explained that his project with the Metabolic and Molecular imaging group appeared to show that so-called ‘yo-yo dieting’, where individuals oscillate between having high and low levels of body fat, was not harmful to health, contradicting preconceptions that even many health professional hold. As opposed to science in school, “where you’re learning things that have already been developed by previous scientists,” explains Shadman, “in a lab in the CSC for example, we’re developing new ideas; we’re discovering and researching for ourselves…that’s the main difference.”

Jade Fernandes, an undergraduate student studying at Imperial College also thought that the unpredictability of research was one of its most defining and exciting aspects: “When you do real research you don’t know what the result is going to be, and often it isn’t what you expect, so you have to find a reason why it’s happened like that…It’s been a great experience.”

“I’d definitely recommend this to anyone who has an interest in science and who wants to see what science is all about,” said James Wilkinson, an A-level student who also worked in the Metabolic and Molecular Imaging group, investigating how epigenetic factors can influence obesity. “My aspirations are in medicine, [and] I would be hugely interested in pursuing research in medicine in the future.”

The quality of the students and the quality of the research they presented was impressively high. “It was great to work with young students who were so engaged with their projects,” affirms Dr Alex Sardini, who hosted the afternoon session of presentations, “ – and see that they could give us a run for our money.”

If you’re interested in taking part in our summer student programme, please contact Kate Baird, Student Administrator.

SJ

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New Year Honours

19 Oct 2011 GMT

Commenting on the honour, Lindsay said: “I am not sure I deserve it above many other people. I got the letter in mid November, I recall after a visit to the dentist! It was hard to keep it quiet and the only people I told in advance were my wife (who was delighted, although her immediate reaction was to burst into tears) and my mum.”

Lindsay has been at the CSC as Administrative Director since 1999: “ I am lucky enough to have done a number of jobs working for the MRC but this current job is both the most interesting and the most challenging: no two days are ever the same. Embedding the CSC within Imperial’s Faculty of Medicine was pioneering when the CSC was originally established and it means I have a lot of interactions at many levels with colleagues in Imperial. I have very much enjoyed this aspect of my job, not least because, while there have been legitimate differences of view from time to time, I have always found within Imperial a willingness to engage constructively to find solutions which work in the best interests of both organisations.”

Simon Watts

This is taken from an article on the Imperial College website.

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Professor Dominic Withers elected FMedSci

19 Oct 2011 GMT

Forty of the UK’s leading medical researchers were announced to have been elected to the academy for 2011. They will be formally admitted as new members at a ceremony on June 29. On hearing the news, Dominic said: “I am honoured and delighted to be elected to the Academy. I have been lucky to work with a group of talented scientists in my lab over the last 10 years and this honour is due to their efforts. We are now making excellent progress in particular towards understanding the molecular mechanisms of the ageing process.”

Of the new Fellows, President of the Academy Professor Sir John Bell said, “These new Fellows demonstrate the amazing talent present in the UK biomedical community. I am delighted that the Academy can recognise the vital role each one of them has played in delivering health and wealth benefits to the UK and beyond.”

You can read more about the new Fellows of the Academy of Medical Sciences here.

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Features

19 Oct 2011 GMT

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CSC Undergraduate Symposium

19 Oct 2011 GMT

Doing a science degree in a UK university doesn't necessarily equip undergraduates to make informed decisions about pursuing a career as a research scientist. "Institutes and centres need to provide more opportunities for students to get an experience of what a scientific research career can offer," says Anne Soutar (Dean of Postgraduate Studies, MRC Clinical Sciences Centre).

“Studying biochemistry can seem like watching reality TV,” says Aaron Yeung, who is studying biochemistry at Imperial College. “But today was a really memorable experience for me, cause I saw an actual mass spectrometer and met group leaders, who sacrifice so much for their aspiration for knowledge.”

As Kate Baird (Student Administrator) explains, "We had hundreds of applications for the summer studentship programme and only nine places. I wanted to offer something to applicants who couldn't be accommodated this year." Undergraduate students came from Edinburgh, Bristol, Oxbridge and London Universities to get a taster of CSC research on Friday 1st July 2011.

The symposium allowed thirty of the brightest and best science undergraduates to learn about research at the CSC. Presentations from PhD students - covering epigenetics, metabolism and neuroscience - were followed by an informal lunch with Group Heads. The afternoon encompassed a tour of the transgenic, microscopy and proteomics facilities before a careers Q&A session, giving scientists at different stages in their careers the chance to share their experiences with students.

Almost 90% of the undergraduates fully agreed that the experience was interesting, enjoyable and provided useful information about the CSC. "Speaking to PhD students helped me to understand more about what is required to get into further education at the CSC," confirms Amina Yonis (Biochemistry, Kings College London).

"Most excitingly we were allowed to get our hands on the real apparatus," says Cindy Lam (Biochemistry, Imperial College London). "We got to have a go at injecting DNA into embryonic stem cells."

"And I really enjoyed the lunch with Group Heads," says Chloe Santos (Biomedical Sciences, UCL). "It gave me a good amount of information about research at the CSC in general."

As Kate Baird explains, “we really hope that some of these students and their fellows will apply to our PhD programme in a few years’ time.”

Listen to the podcast to hear more about what undergraduates thought of the first CSC Student Symposium:
CSC Student Symposium :: July 2011 by BMCV
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Podcasts

19 Oct 2011 GMT

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Big Hearts' Genetic Origins

19 Oct 2011 GMT

Roche Continents

19 Oct 2011 GMT

Roche Continents - organised by the pharmaceutical company - unites a hundred arts and science students
from universities across Europe every summer. The programme proposes to 'uncover the common ground
of creativity in the arts and sciences.' While the means
by which the organisers would achieve this remained elusive, the programme was fantastically well organised.

Deliberately coinciding with the Salzburg Festival, every other evening saw us 'dressing up to the nines' and venturing into to the heart of the city to experience the contemporary compositions of Wolfgang Rihm.

The pieces were challenging, and while some of us scientists struggled to make sense of the opera based on Nietzsche's poetry, even the classically trained musicians found it difficult to grasp its musical peculiarities. Sometimes we just need to take off our thinking hats, sit back and enjoy.

Daily talks and workshops by artists, scientists, businessmen, musicians and Salzburg festival organisers were complimented by evening activities including a lavish dinner showcasing music from those of us gifted enough to perform, be it on piano or musical saw. And we were all challenged creatively in the end, when the organisers instructed us to put together a performance for the rest of the group.

Teamwork under time pressure saw us conjure up a diverse programme from satires to compositions, all reflecting on the week's experience. We got to polish it all off on the dance floor at the farewell disco, before saying our sentimental good byes to new friends the next day. In fact we had such a good time we thought we'd organise a reunion in October.

Information about the programme is available here: http://www.roche-continents.net/ …