Posts Tagged ‘University of Leicester’

Studies on the Genome Structure of Neurovirulent and Attenuated Polioviruses – a BLAST from the past

Thursday, February 17th, 2011

colony hybridization In 1979 a young researcher blundered into a laboratory and began working towards a PhD. Four years later, he emergerd blinking into the sunlight and headed off to California for a while. This is the story of those years, which has lain buried under a layer of dust ever since until it was recently published online. Along the way, we had some laughs. For example, this may amuse some people:

Data Handling
Nucleotide sequence data was entered and analysed on a PDP 11/4 computer using a package of programs obtained from R. Staden, MRC Laboratory of Molecular Biology, Cambridge, UK (Staden, 1980).

At any rate, the truth can now be told:

Title: Studies on the Genome Structure of Neurovirulent and Attenuated Polioviruses
Author: Cann, Alan James
Date: 1984
Publisher: University of Leicester
Description: Thesis submitted for the degree of Doctor of Philosophy at the University of Leicester, 1984.
Abstract: The RNA genomes of neurovirulent and attenuated type 3 polioviruses have been cloned in E. coli using an efficient RNA.cDNA hybrid technique. The complete nucleotide sequence of the vaccine-associated neurovirulent revertant P3/119 and, in collaboration with others, the attenuated vaccine strain P3/Leon 12 a1b, have been determined. These have been compared with that of the neurovirulent parent strain P3/Leon/37. Ten nucleotide sequence differences were observed between the parent P3/Leon/37 and the vaccine P3/Leon 12 a1b, three of which resulted in amino acid substitutions. Between the vaccine and the revertant P3/119, seven nucleotide sequence differences were observed. Three of these resulted in amino acid substitutions. The possible significance of individual nucleotide sequence differences to the attenuation of and reversion to neurovirulence in poliovirus type 3 is discussed. The nucleotide sequence of P3/Leon 12 a1b was the first to be determined for a type 3 poliovirus. Comparison of this sequence with published type 1 sequences has demonstrated the extent of the molecular homology between them.

Summary:
The major objective of this study was to identify the nucleotide sequence differences which account for the neurovirulent or attenuated phenotype of three closely related strains of poliovirus type 3. To achieve this, an efficient RNA.cDNA hybrid cloning method was devised. Although this was not the first report of hybrid cloning, previous unfavourable comments on its efficiency (Wood and Lee, 1976; Zain et al., 1979) and suggestions that the method given rise to cloning artefacts (Okayama and Berg, 1982) have almost certainly discouraged widespread use. Thorough investigation of each of the manipulative steps involved has shown that these problems can be overcome (Cann et al, 1983). Comparable in efficiency to the more used double-stranded cDNA cloning technique, the hybrid method has the advantages of experimental simplicity and that cDNA clones corresponding to entire virus genome can be obtained from a single experiment. The method has proved to be ideally suited to the molecular cloning of picornavirus genomes. It is possible that the transformation efficiency of RNA.cDNA hybrids could be further increased by treatment with E. coli DNA ligase and DNA polymerase I before ligation, thus carrying out repair of the hybrid molecule in vitro, as in the method of Okayama and Berg (1982). This modification has not yet been tested.
Together with the work of Dr G. Stanway on the neurovirulent strains P3/Leon/37 and P3/119, hundreds of cDNA clones were examined and more than 22 kbp of nucleotide sequence determined. These experiments resulted in the identification of a mall number of mutations in the genomes of the strains studied which must be responsible for their differences in neurovirulence. However, it has not yet been possible to identify the individual mutations involved in attenuation and reversion and further experiments are currently in progress. These experiments represent a number of different approaches. Firstly, the sequence of other neurovirulent vaccine revertants are being determined, to ascertain whether the mutations observed in P3/119 are shared by other strains. Secondly, the work of Racaniello and Baltimore (1981a) has demonstrated that the construction of recombinant virus genomes in vitro, at the level of cloned cDNA, is possible. Transfection of susceptible cells with these recombinant genomes gives rise to new, viable viruses with a defined set of mutations which can then be examined phenotypically. Initial experiments with recombinants between the neurovirulent strain P3/Leon/37 and the vaccine strain P3/Leon 12 a1b and also between the vaccine strain and the neurovirulent revertant P3/119 are in progress. Final proof that the mutations involved in attenuation and reversion have been accurately identified could be provided by the construction of an attenuated strain by recombination between P3/Leon/37 and P3/119. Parallel studies on neurovirulent and attenuated type 1 strains should help to explain the different stabilities of the type 1 and type 3 vaccines and may suggest how the type 3 vaccine can be modified to improve Stability. Finally, it is hoped to use site-directed mutagenesis of cloned virus genomes ln vitro to produce strains with specific biological properties.
Although the main aim of the work presented here has been the investigation of the molecular basis of attenuation in poliovirus, the information obtained has wider significance. The complete nucleotide sequence of P3/Leon 12 a1b was the first to be determined from a type 3 poliovirus. This has been compares with that of type 1 and the extent of the molecular homology between the demonstrated (Stanway et al, 1983a). As part of a larger study based on the analysis of monoclonal antibody resistant mutants, the major neutralizing antibody binding site of poliovirus type 3 has been identified (Minor et al, 1983). The nucleotide sequence information obtained is also being used currently in the design of synthetic antigenic peptides, a development which may hold many advantages for the prevention and perhaps treatment of poliomyelitis and other related plcornaviral infections. At the outset of the work described in this dissertation, it was difficult to envisage that improved alternativen to the Sabin vaccines would ever be a realistic proposition. It now seems that the immediuate future holds just such a prospect.

Introducing SciReadr.com

Thursday, December 2nd, 2010

Screenshot SciReadr.com is based on the reading list for students the School of Biological Sciences at the University of Leicester. We know that students don’t read the books we suggest on the reading list, so the University of Leicester Student Experience Enhancement Group funded Project SOAR, which has two parts.

The first is SciReadr.com, an interactive site where you can browse, rate, review, borrow or buy the books on the reading list. If you’d like to know more, there’s lots of information here. We’re also on Facebook and Friendfeed if that’s easier for you, and we’d like hear any suggestions you have about the website.

Scireadr.com is open to everyone, but if you have a University of Leicester email address, after you’ve read a book, you can come along to a Book Group meeting in the Student’s Union, pick up a coffee from Starbucks or a beer from the bar and spend a happy couple of hours chatting with people who’ve read the same book. Sound interesting? The first book group meeting is in February and the book we’ll be discussing is Bill Bryson’s A Short History of Nearly Everything, so if you’re interested and have a University of Leicester email address, sign up at SciReadr.com now.

Picking holes in pneumococcus

Monday, November 15th, 2010

Pneumolysin Microbiologists at the University of Leicester have worked on Streptococcus pneumoniae (pneumococcus) for many years. This research is now starting to pave the way for more effective vaccines against this pathogen. The pneumococcus is a pathogen of global significance, responsible for millions of deaths annually from pneumonia, meningitis and septicaemia while also causing other less serious infections, such as otitis media and sinusitis. In order to develop improved pneumococcal vaccines it is essential to understand how the bacterium interacts with the host immune system.

Pneumococci produce a range of pathogenicity factors, among which the toxin pneumolysin plays a central role and has potential as a vaccine candidate. A new paper demonstrates that pneumolysin can directly activate innate immune cells and dramatically amplify the production of pro-inflammatory cytokines. These enhancing effects of the toxin do not require Toll-like receptor (TLR)4. In particular, the toxin exerts a potent effect on interleukin (IL)-1, which is an endogenous pyrogen and powerful activator of IL-17A production. This effect results from activation of the NLRP3 inflammasome complex and NLRP3 is required for protection against the pathogen in vivo. To induce protective immunity against pneumococci, IFN-γ and IL-17A are thought to be essential. Pneumolysin plays a key role in promoting these cytokines both in vitro and in vivo during respiratory infection. The results add significantly to our understanding of the interactions between pneumococci and the immune system and support investigations into the inclusion of pneumolysin or its derivatives in novel pneumococcal vaccines.

Pneumolysin Activates the NLRP3 Inflammasome and Promotes Proinflammatory Cytokines Independently of TLR4. (2010) PLoS Pathog 6(11): e1001191. doi:10.1371/journal.ppat.1001191
Pneumolysin (PLY) is a key Streptococcus pneumoniae virulence factor and potential candidate for inclusion in pneumococcal subunit vaccines. Dendritic cells (DC) play a key role in the initiation and instruction of adaptive immunity, but the effects of PLY on DC have not been widely investigated. Endotoxin-free PLY enhanced costimulatory molecule expression on DC but did not induce cytokine secretion. These effects have functional significance as adoptive transfer of DC exposed to PLY and antigen resulted in stronger antigen-specific T cell proliferation than transfer of DC exposed to antigen alone. PLY synergized with TLR agonists to enhance secretion of the proinflammatory cytokines IL-12, IL-23, IL-6, IL-1β, IL-1α and TNF-α by DC and enhanced cytokines including IL-17A and IFN-γ by splenocytes. PLY-induced DC maturation and cytokine secretion by DC and splenocytes was TLR4-independent. Both IL-17A and IFN-γ are required for protective immunity to pneumococcal infection and intranasal infection of mice with PLY-deficient pneumococci induced significantly less IFN-γ and IL-17A in the lungs compared to infection with wild-type bacteria. IL-1β plays a key role in promoting IL-17A and was previously shown to mediate protection against pneumococcal infection. The enhancement of IL-1β secretion by whole live S. pneumoniae and by PLY in DC required NLRP3, identifying PLY as a novel NLRP3 inflammasome activator. Furthermore, NLRP3 was required for protective immunity against respiratory infection with S. pneumoniae. These results add significantly to our understanding of the interactions between PLY and the immune system.

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Microbiology Clearing at Leicester

Thursday, August 19th, 2010

Guardian University Guide 2011 Biosciences There won’t be any formal clearing for Microbiology places at Leicester this year because basically, we’re full. (If you’re holding a place and haven’t responded yet, please contact us as quickly as possible.) The UK government caps the number of students we can take, although we do currently have room for a few more overseas (non-UK/E.U.) students, so if you’re interested in a place to study Microbiology or Medical Microbiology, please contact us soon.

Other than that, I hope you got the results you wanted, and we’ll soon be accepting applications for Microbiology places next year.

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When you’re hot, you’re hot

Friday, June 11th, 2010

Guardian University Guide 2011 Biosciences
Guardian University Guide 2011: Biosciences

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Microbiology Anyone?

Sunday, February 14th, 2010

Over on our Facebook page, Heather Collins asked:
Why do so few University's run microbiology degrees these days? And the ones that do insist on merging them with biochem. Do they not realise people just want to poke about with small squiggly things for a career?

And another question – why do so few (UK) universities have microbiology departments these days? There’s been a trend towards bigger academic units, and disciplines who can’t recruit enough students tend to get merged with other departments into units usually called something like “The School of Life Sciences”. In addition, non-human microbiology is out of favour these days, with most of the money going to medical microbiology, and this killed off some microbiology departments who couldn’t or weren’t willing to adapt.

So why do UK universities find it hard to recruit microbiology students? When the A level syllabus changed a few years ago, microbiology all but disappeared. Actually, there is a little bit still there, but not until the A2 stage and it’s no longer offered by all schools. But A2 students have already filled out their UCAS forms. AS students don’t know what microbiology is, so why would they apply to do a degree in it? (They know what genetics is because there’s lots of that at AS level, so many apply to do that as a degree).

Fortunately, there is a solution! At the University of Leicester, we offer you not one but two chances to study microbiology. “Microbiology” is a broadly based course including environmental microbiology, which “Medical Microbiology” is … well, see if you can figure it out ;-)

Of course, if it’s a bit late for you to be thinking about degree choices, we also offer lots of microbiology PhD and postdoctoral places, so check us out for all your microbiology career requirements :-)

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25 years of DNA fingerprinting

Thursday, September 10th, 2009

Alec Jeffreys Exactly 25 years ago today, Alec Jeffreys, Professor of Genetics at the University of Leicester discovered a technique that has subsequently had an incalculable effect on society, helping to solve criminal cases, resolve immigration arguments and clarify questions of paternity, not to mention creating one of the biggest civil liberties issues of our times. Working in the laboratory, Sir Alec recalls, he and his technician were simply following their noses. They had “absolutely no idea” of the applications that would result from the discovery they stumbled upon.

“I have never approached an experiment with a desire to solve a practical problem,” he observes, pinning down his moment of discovery to precisely 9.05am on Monday 10 September 1984. “My forensic thoughts at 8.55 on that morning were precisely zero; they simply were not there. The technology comes first and then suggests the applications, not the other way around, and you see this over and over again.”

But just as he has spoken out about the ethical and moral issues concerning the use of the technology he made possible, Sir Alec is a staunch defender of Britain’s curiosity-driven research. And the 25th anniversary of his discovery is, he believes, the right time to be discussing its future.

“As scientists, we have to be accountable to the public purse for the money we are spending, but if you take it too far – and in my view it has gone far too far now – it actually stifles the creativity of the very thing you are trying to promote.”

His warning is simple: applied science can be managed from the top down, but we apply the same approach to pure science “at our peril”. “It is blue-skies research that is the ultimate driver – delivering the new techniques, concepts and tools that we need to progress”.

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Study at the University of Leicester

The good news about influenza

Monday, September 7th, 2009

Influenza virus With the fall in H1N1 “swine flu” influenza cases recently, it has become fashionable for the media to run “What was all the fuss about” stories on the same page as “OMG, it’s going to be bad” stories. The problem with influenza is that it is one of the most unpredictable of all viruses, and while an upsurge in the number of cases can be expected as the winter flu season gets going in the northern hemisphere, the real concern is that this new pandemic virus might “turn nasty” in the second wave, just as in 1918 a much more pathogenic variant of that virus followed the relatively benign first wave of cases.

There are two ways in which this could happen. The first is that the present virus acquires spontaneous mutations which make it more pathogenic. The other possibility is that the virus recombines with a highly pathogenic influenza virus though the process known as reassortment – swapping of genes when two different strains infect the same cell. And there’s a good candidate for that out there – the highly pathogenic H5N1 avian influenza virus. Unlike H1N1, H5N1 has a hard time infecting humans, so it’s unlikely that these viruses would meet. But if they did…

The good news comes from Egypt, where H5N1 is relatively common, and a (worrying) case of H5N1/H1N1 co-infection was recently reported. The Ministry of Health has now discounted the rumour of a co-infection with the two viruses. In addition, a University of Maryland/NIH study suggests that co-infections of H1N1 with seasonal flu viruses do not produce chimeric or reassortant viruses. The H1N1 strain seems to outcompete seasonal viruses, possibly demonstrating this pandemic strain is not under biological pressure and is perhaps more efficiently communicable. Certainly, the past pattern seems to suggest that H1N1 pandemic seem to suppress outbreaks of other strains for some time.

A phase I clinical trial conducted by scientists from the University of Leicester tested 100 healthy volunteers with an H1N1 vaccine to see how their immune system responded. Trial leader Dr Iain Stephenson found 80 per cent of the volunteers showed a “strong, potentially protective” response after one dose, with more than 90 per cent showing the same response after two doses. The results suggest that one vaccine dose may be sufficient to protect against A(H1N1) swine flu, rather than two. Larger trials are now under way around the world involving up to more than 6,000 adults and children.

Reasons to be cheeful.

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The Enzyme Club

Friday, July 10th, 2009

Restriction enzyme

“In my day” i.e. when I started my PhD back in 197<cough>, the first few weeks were spent joining the Enzyme Club. This encompassed all the biomedical researchers at the University of Leicester. Each new student would prepare a batch enzyme for recombinant DNA work. In my case, I made Hsu I (an isoschizomer of Hae III but allegedly easier to prepare). Since it was years ago, I can’t remember how many litres of the organism I grew up, but I remember very clearly doing the first assay on two litres of crude extract, and figuring out I was holding £40 million pounds worth of enzyme at the then current market prices. The first affinity column cut it down to £15 million, and a quick gel filtration to couple of millions pounds worth – still pretty good for two weeks work, especially when you remember that two million pounds was enough to buy you a house back in the 1970s!

Why did the Enzyme Club exist? Because these reagents were scarce in the 1970s, and rationed both by price and availability. Only a few years before, the only way to get hold of any of these enzymes was to make your own. This type of open science made sense. Why did the Enzyme Club cease to exist? Gradually, it became clear that the batch of enzyme I made wasn’t very good. It had a persistent exonuclease activity which meant it was fine for restriction analysis but rubbish for cloning, and it went off very quickly in storage, so that after three months there wasn’t much activity left. And although I’ve always been a rubbish protein chemist, that was a pretty common experience. Gradually, the companies dropped their prices and improved both the quality and availability of commercial enzymes. The day came when the Enzyme Club didn’t make sense any more, and it quietly died. It’s probably still moldering in the back of a coldroom over in the MSB.

So boys and girls, this is a story of the economics of open science, which made sense in response to scarce resources. When the availability of enzymes was limiting, this open approach made sense. When time became limiting, we all retreated back into our laboratories and got on with whatever we needed to do to get a PhD. The moral of this story is that open science pops up it’s head when times are hard and resources are scarce, but retreats quickly as the balance changes.