Makeover Mandated for U.K. Life Science Sector - GEN

Summary:
UK Trade and Investment (UKTI) believes that the UK biotech industry’s recent battering by the press gave a very one-sided view of the true international potential of the country’s life science sector. Not only is the UK second to the U.S. in terms of biotech industry size but it leads Europe with its pharmaceutical exports. But the U.K. has been slow in publicizing its strengths. Therefore, the UKTI was charged by the government with marketing the U.K.’s life sciences internationally. The result was the UK Life Science Marketing Strategy which was drafted earlier this year. Workstreams aim to optimize how U.K. academia and industry sells itself internationally, in terms of comprehensive and consistent messaging, financing (using marketing to increase the amount of international VC funding in the U.K.), and also how best to communicate with potential partners and investors in key countries. A tool kit has been developed that will allow any UK life science company to access and take the most relevant messages with them in terms of UK innovation, support industry, tax incentives, and academic background. UKTI and the Strategy Implementation Board hope that executing and further developing the U.K. life science market over the next five years will significantly boost the country’s standing within the international arena, increase inward investments, business, and collaborations, as well as make the U.K. industry a more cohesive force. (Published: 01/09/2008)

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VC Funding for Biotech Companies Withering - GEN

Summary:
Despite an abundance of funding as well as scientific and technological progress, the environment for investing in the life science industry seems to have changed dramatically. The change has been most dramatic for the biotech sector. In theory, the level of VC investments made in life science at any time should not be significantly affected by short-term fluctuations in stock market activity or the economy. Drought of new public money coming into venture-backed companies through IPOs, however, as well as increasing caution on the part of acquirers have biotech venture investors hanging on tighter to their wallets and checkbooks. Money is still there but it is going to be harder for biotechnology companies to obtain. Particularly true given the increased competition for investment with medical device and equipment companies as well as new competition from biofuels and alternative energy companies for investment dollars. (Published: 01/09/2008)

Notes:

• Q1 2007: record highs in venture investments in biotechnology, medical device, and healthcare firms
• average investments in these areas maintained nearly the same levels through the end of the Q1 2008
• Q2 2008: venture investing in general made a major downturn
• no public offerings of any venture-backed company
• life science
• far fewer venture dollars went to a smaller number of life science companies
• in Q1, U.S. life science venture capital firms made 315 investments aggregating nearly $3 billion
• in Q2, there were 215 such investments aggregating $1.9 billion
• biotech: change even more dramatic
• number of biotechnology venture backings fell by nearly 50% (to 89 investments)
• dollar amount invested fell by more than 40% from Q1 ($919m vs. $1.5b)
• outside the U.S., the venture financing value fell nearly 50% in the same period
• proportion of life science dollars going to biotech has also shifted compared to medical devices and equipment as well as other healthcare ventures
• biotech firms’ share of investments made in the life science industry fell to below 40% from approximately 45% in 2007
• average amount invested in biotechnology, though, remained high in Q2
• more than $10 million
• investments have ranged from $8.4 to 11.8 million over the past six quarters
• Factors behind the change
• biotechnology investments, like most venture capital investments, are inherently risky
• in today’s uncertain economic climate, many investors are opting to sit out and wait for more certainty in the market before they invest
• biotech investments generally take longer to mature than nonbiotech investments
• for those life science investors who are nervous about the long term in the current environment, investments that have a shorter return time, such as those in medical devices and other healthcare ventures, have become more attractive
• emergence of clean energy as an alternative investment category that is growing in favor with long-term investors
• venture capitalists whose portfolios include longer-term investments are shifting their dollars from biotechnology to solar energy, wind power, and other sustainable energy solutions
• constriction/lack of liquidity in the public equity markets
• compare
• in 2007 there were 31 IPOs of life science companies, 11 of them for biotechnology companies.
  
• in the first half of 2008, four life science firms including one biotech, Bioheart, made IPOs
• decline may result from a number of factors some of which are not specific to life science investing
• general investor apprehension
• the debt crisis
• Sarbanes-Oxley and related regulations
• lack of liquidity in the public markets has also resulted in public companies taking on private investments from venture capitalists
• e.g. Cadence Pharmaceuticals, Antisoma, Xanthus Pharmaceuticals
• investment of venture capital into public companies and later-stage private companies means that even less is being spent on early-stage companies
• lack of liquidity in the U.S. markets has also led a number of biotechnology companies to go public through the London Stock Exchange’s Alternative Investment Market (AIM)
• life science acquisitions have also taken a beating
• compare:
• 47 venture-backed life science companies were acquired in 2007
• only 12 have been taken over in the first half of this year, eight of which were acquired in the first quarter
• take-over values down from 2007
• total value of the 36 acquisitions made last year for which transaction values were made public was $7.4 billion
• so far this year, the aggregate amount of the seven deals for which financial terms were disclosed was $2.2 billion.
• in theory, the level of VC investments made in life science at any time should not be significantly affected by short-term fluctuations in stock market activity or the economy
• drought of new public money coming into venture-backed companies through IPOs, however, as well as increasing caution on the part of acquirers have biotech venture investors hanging on tighter to their wallets and checkbooks.
• money is still there but it is going to be harder for biotechnology companies to obtain
• particularly true given the increased competition for investment with medical device and equipment companies as well as new competition from biofuels and alternative energy companies for investment dollars

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There is hope yet for science park toilers - FT.com

Summary:
Jonathan Guthrie sees evidence that the level of innovation in the UK appears to be declining. Not much coming out of universities anymore. We're living through a fallow period for innovation, fundamental innovation is slowing up after a remarkable 40 year boom. Internet investment bubble, Schumpeterian explanation: copycats to pile in on the upswing of an innovation wave. Was a fiasco for VC investment in UK. Returns negative for average fund set up after 1996 over 5 to 10 year periods. Confidence has weakened further with the credit crunch, which has closed the market for flotations. Less money was invested in European technology start-ups last quarter than at any time since 2001. Venture capital is more fragile this side of the Atlantic than in the US. Technology investment in UK will probably recover. VCs need to market themselves, focus on the lofty top decile, not the mediocre median. Early stage technology investment is attractive as a way to lay small bets on risky, glamorous propositions. Also need a handful of breakthroughs that are immensely remunerative. Biotech lost cost. Renewable energy and power savings big hope. (Published: 30/07/08)

Notes:

• little backing from City for fledgling tech companies on UK's science parks
• Jon Moulton, a private equity investor who backs technology start-ups as a hobby
• “In the UK the level of innovation appears to be declining. Universities are being picked over very hard for ideas, but not a lot is coming out of them.”
• likens UK early stage technology investors to ufologists
• instead of joining hands and imploring Martians to land, they hope, equally fruitlessly, for a worthwhile return on investment
• living through a fallow period for innovation
  
• Walter Herriot of the St John’s Innovation Centre
• “There is a slowing up in fundamental invention, though not in the creation of niche applications.”
• reflecting on the advent in the past 40 years of personal computers, the internet and mobile phones, he says: “I cannot see an equivalent explosion in the near term.”
• similar to Schumpeter's view
• proposed that innovation progresses in waves
• profitable breakthroughs occurrs in the troughs of economic cycles
• encourages copycats to pile in on the upswing, feeding economic instability
• cfr internet investment bubble
• now looks more like a belated dash into a maturing technology rather than the new era it was billed as at the time
• fiasco has constrained any advertising claims for venture capital based on recent performance
• funds set up after 1996 have typically lost 1.4 per cent a year over five years and 1.8 per cent over 10 years
• according to the British Private Equity and Venture Capital Association
• Confidence has weakened further with the credit crunch, which has closed the market for flotations
• less money was invested in European technology start-ups last quarter – €950m (£747m) – than at any time since 2001
• Apax, progenitor of European venture capital, launched a fund last spring with no venture capital component
• Braveheart is concentrating on follow-on financings;
  
• 3i has pulled out of early stage investment altogether
• Venture capital is a more fragile flower on the eastern shores of the Atlantic than in the US
• Sir Ronald Cohen, founder of Apax,
• “The perception is that the early stage is tougher, you raise less money when you float, and there is less liquidity afterwards.”
• is technology investment is doomed to dwindle away to nothing in the UK?
• will probably recover
• dotbomb losses will drop out of short-term performance statistics during the next few years.
• returns need not then be spectacular – a long-term average is 4.5 per cent – to lure investors back
• trade bodies such as the BVCA, meanwhile, can do their bit by quoting returns exclusive of “exceptional losses” chalked up on internet plays
• marketing emphasis should be on the lofty top decile, not the mediocre median
• early stage technology investment is attractive as a way to lay small bets on risky, glamorous propositions
• absorbs just more than £1bn a year in the UK
• no backers stake money they cannot afford to lose
• in some respects it resembles alternative investments, such as art or wine, more than big buy-outs or quoted shares
• task that science park toilers face is to produce a handful of breakthroughs that are immensely remunerative
• little can be hoped for from biotech
• stricken by unprofitability as persistent as a hypochondriac’s bad back
• materials scientists have engineered a UK nanotechnology sector so tiny it is virtually invisible
• most to go for in renewable energy and power saving systems
• "scope for technological leaps equivalent to the shift from mainframes to PCs” (Sir Ronald Moulton)

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Drug research needs serendipity - FT.com

Summary:
David Shaywitz and Nassim Taleb write that the pharma industry is suffering as a result from a mismeasure of uncertainty. Despite promise of molecular revolution and drugs by design, pipelines are dwindling. Pharma companies seek to identify the largest markets they can find, develop products for these customers and boost efficiency of development process. Wrong approach, for two reasons: drug sales notoriously hard to foresee (yields more false precision than true insight), and drug development process is also very difficult to predict. This strategy fails to reduce exposure to negative uncertainty (all the bad things that can happen during drug development), and eliminates much of the exposure to positive uncertainty (serendipity). Pharma's trend of outsourcing may open up possibility for innovators with a greater appreciation of the nuances of science to do a lot better. (Published: 30/07/08)

Notes:

• molecular revolution: was supposed to enable drug discovery to evolve from chance observation into rational design
• yet dwindling pipelines threaten the survival of the pharmaceutical industry
• what went wrong?
• answer: the mismeasure of uncertainty
• academic researchers underestimated the fragility of their scientific knowledge
• we still do not understand what causes most disease
• scarcity of good animal models for most human disease
• academic science tends to focus on the "bits and pieces" of life - DNA, proteins, cultured cells - rather than on the integrative analysis of entire organisms, which can be more difficult to study
• yet: real scientific progress has occurred
  
• pharmaceuticals executives overestimated their ability to domesticate scientific research: spreadsheets are easy; science is hard
• pharma companies seek to identify the largest markets they can find and develop products for these customers
• sensible in theory, but less so in practice, for two reasons:
  
1. drug sales are notoriously difficult to foresee
   
• even at the time the medicine hits the market
• i.e. predicting sales a decade or more ahead of registration, when the research and development process typically begins, is generally a fool's errand
• yields more false precision than true insight.
  
• yet: much of contemporary pharma R&D is driven by this sort of rigid planning.
2. drug development process is also very difficult to predict
• because of both our limited understanding of disease and our inevitably imperfect understanding of the effect any new compound will have on the body
• most modern medications were discovered in the old-fashioned way: by accident
• e.g. Viagra, originally developed as treatment for chest pain
  
• pharma companies have been trying to boost output by increasing efficiency
• narrowing focus to a handful of disease areas
• shelving safe but ineffective compounds without fully exploring their scientific potential
• trying to ensure that each project the company is working on is carried out with a clearly defined market segment in mind
• this strategy often fails significantly to reduce exposure to negative uncertainty - all the bad things that can happen during drug development - and eliminates much of the exposure to positive uncertainty (serendipity) that remains so vital
• managers so intent on maintaining focus that important opportunities for novel discovery are lost
• as is the intellectual space for tinkering and capitalising on the chance observations and unexpected directions so important in medical research
• pharma executives are creating an ever-more-rigid environment
  
• and then wondering why their productivity is going down
• and why they have such difficulty attracting and retaining talent

• diruptive innovation
  
• pharmaceutical industry is ripe for disruptive innovation
• but: the barriers to entry have been far too high for anyone new to break through
• however: pharma companies trying to cut costs by outsourcing large parts of their operations
• in response service providers have sprung up around the world to fulfil these functions
• if the trend to outsourcing continues and if the main competence of pharma companies becomes (as some have suggested) simply their ability to orchestrate the entire process, it is not difficult to imagine that an innovator - particularly an innovator with a greater appreciation of the nuances of science - might be able to do this a lot better
  

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Nano-machine captures zinc in protein-like jaws - R&D Magazine

Summary:
A team from Berkely Lab generates a protein-like function from a synthetic polymer: two helical peptoids with functional groups at the end, linked together using an unstructured segment. The two-helix bundle can fold in half and bind a zinc ion. Perhaps anitial step toward developing nanostructures that combine the precision of proteins with the ruggedness of non-natural materials. Such foldable polymer bundles could lead to highly accurate sensors capable of operating in harsh environments, or disease-targeting pharmaceuticals that last much longer than today's therapies. (Published: 22/07/08)

Notes:

• proteins
• unmatched molecular recognition and catalysis capabilities
• have the ability to selectively bind with one—and only one—type of molecule
• also initiate incredibly precise chemical transformations
• e.g. cutting a DNA strand in just the right place
• hitch: lack ruggedness and stability
• limited to narrow temperature and acidity ranges
• require a watery solution
• degrade over time
• drawbacks limit their utility
• proteins to target pathologies at the molecular scale degrade over time, curbing their effectiveness
• protein-based sensor would be unsurpassed at sniffing out harmful contaminants, but it wouldn't be able to operate in hot, cold, or dry conditions
• Ron Zuckermann
• Facility Director of the Biological Nanostructures Facility in Berkeley Lab's Molecular Foundry
• goal: take proteins' catalysis and molecular-recognition capabilities, and add them to a material that is more rugged and less prone to degradation
• peptoids
  
• proteins are precisely folded linear polymer chains of amino acids
• made similar polymer chain by linking together non-natural amino acids
• peptoid: synthetic structures that mimic peptides
• use peptoids to build synthetic structures that behave like proteins
• binding zinc
• zinc: drives many fundamental biological processes
• e.g. DNA recognition
• developed helical peptoids with zinc-binding residues positioned at both ends
• also added fluorescent tags at both end: allowed measuring when the bundles fold in half, trapping zinc in place

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Electrostatic readout of DNA microarrays with charged microspheres - Nature Biotechnology

Summary:
Paper describing a method for label-free electrostatic readout of DNA or RNA hybridization on microarrays which is based on the electrostatic properties of microarrays. Changes in surface charge density as a result of specific hybridization can be detected and are measured from the position and motion of charged microspheres randomly dispersed over the surface (in 100uM NaCl). Interactions between the microspheres and substrate can be imaged by a variety of optical methods that provide a rapid indicator of DNA hybridization. The naked eye is sufficient to read out the hybridization, which may facilitate broad application of multiplexed assays. Practical diagnostics require rapid and simple quantitative readouts that do not use dedicated instrumentation or intensive image processing. (Published: 29/06/08)

Notes:

• microarray assays
  
• typically rely on fluorescence detection, which requires time-consuming chemical labeling, reverse transcription, high-power excitation sources and sophisticated instrumentation for scanning
• consequently, microarray assays tend to be performed by dedicated centers rather than by individual laboratories, and not by clinics in developing countries
• alternative label-free DNA detection techniques
• e.g. surface plasmon resonance, electrochemical sensing, fluorescent polymers,
  atomic force microscopy, microcantilevers and electronic depletion of a field effect transistor
• none of these have gained widespread use because each requires
  
• either complex device fabrication
  
• or sophisticated instrumentation for readout
• additionally, none are compatible with conventional DNA microarrays where up to 10^6 sequences can be interrogated in a single experiment
  
• electrostatic-based DNA or RNA detection method
• Complementary oligonucleotide binding strongly affects the electrostatic charge of the surface due to the negatively charged DNA phosphate backbone
• hybridization is measured electrostatically using charged microspheres that are highly responsive to changes in charge density on the microarrayed surface
• Interactions between the microspheres and substrate can be imaged by a variety of optical methods that provide a rapid indicator of DNA hybridization
• role of each silica microsphere is analogous to that of an electrostatic force microscope (EFM) tip where the vertical deflection of the tip is used to report local electrostatic surface properties
• EFM, however, is a serial technique that is practically limited to a field of view of 100 um2
• particle-based technique described here is capable of parallel sampling of a microarray surface over centimeter-length scales
• glass support is positively biased using an aminosilane modification
• balances the negative charge contributed by both the glass surface and the printed single strand (ss)DNA molecules
• necessary because the charged microparticles are responsive to a limited range of surface charge densities
• typical experiment
• a prepared substrate is mounted in a well chamber and hybridized
• unlabeled and negatively charged 5.6 mm–diameter silica microparticles are then added and allowed to sediment above (or otherwise interact with) the array over a period of 20 min.
  
• Microspheres uniformly distribute across the entire surface and adsorb to the positively charged background.
  
• However, over sufficiently negatively charged areas, they adopt an equilibrium height that is dictated by a balance between gravitational and local electrostatic forces.
  
• To determine the precise heights and positions of the population of levitated microspheres, we then acquired a collection of dualwavelength reflection interference contrast microscopy (RICM) images covering the entire array area
• Image acquisition was automated using a motorized translation stage, and a softwaredriven autofocus routine.
  
• At each stage position, 20 images were acquired (0.4 fps) yielding 20,000 images/mm2.
  
• On average, there were 20 microspheres per field of view (30 x 30 um) resulting in 400,000 microparticle observations/mm2.
  
• The interference images corresponding to individual microspheres were used to determine their position with 1-nm vertical resolution and 16-nm lateral resolution.
  
• Although this is an optical technique, the resolution is not diffraction limited
• it is determined by the particle position resolution and the density of particle observations.
• A quantitative spatial map of the surface charge density is generated by compiling the set of three-dimensional (3D) particle position measurements.
• practical diagnostics require rapid and simple quantitative readouts that do not use dedicated instrumentation or intensive image processing
• To develop such a readout strategy we take advantage of the fact that silica microspheres respond to the surface charge in an easily observable manner.
  
• If the surface is negatively charged, microparticles remain laterally mobile
  
• can be easily visualized by monitoring the intensity variance in a time series of brightfield images.
  
• Alternatively, positively charged areas can be identified by the presence of electrostatically adhered microspheres.
  
• This can be rapidly imaged using darkfield or brightfield microscopy.
  
• Therefore, adhesion of charged particles provides a simple test to map the sign of the surface charge.
• Direct comparison between fluorescence and electrostatic detection on the same substrates, under identical conditions reveals comparable figures of merit, indicating that sensitivity is primarily limited by hybridization, not the readout
• This particle-based electrostatic detection method offers multiple advantages over existing microarray detection methods.
  
• First, expression profiling and SNP detection using primary mRNA can now be performed without reverse transcription and fluorescent tagging.
  
• Second, electrostatic detection is compatible with conventional microarrays as well as unconventional arrays fabricated on injection-molded plastic or embedded within microfluidic architectures
• Third, imaging by pixel-by-pixel statistical averaging of colloidal particle positions is not diffraction limited, which suggests a strategy to take advantage of DNA nanoarrays that can hold 10^4 more features per unit area than conventional microarrays.
• Finally, microarrays consisting of proteins, small molecules, polymers and heterogeneous catalysts are rapidly coming online and might benefit from the electrostatic readout platform we describe herein.

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Human genome changes with age - AFP

Summary:
Researchers at Johns Hopkins University found that the epigenetic marks on the sequence of a person's DNA modify over the course of their life and the extent of such changes is similar among family members. Epigenetic changes, unlike DNA sequence which is the same in every cell, can occur as a result of dietary and other environmental exposure. Epigenetics may play a role in diseases like diabetes, autism and cancer. Inappropriate methylation levels can contribute to disease: too much might turn necessary genes off, too little might turn genes on at the wrong time or in the wrong cell. (Published: 24/06/08)

Notes:

• Andrew Feinberg:
• "We're beginning to see that epigenetics stands at the center of modern medicine because epigenetic changes, unlike DNA sequence which is the same in every cell, can occur as a result of dietary and other environmental exposure. Epigenetics might very well play a role in diseases like diabetes, autism and cancer."
• team analyzed the DNA sequences from 600 people taking part in the AGES Reykjavik Study
• formerly called the Reykjavik Heart Study in Iceland
• participants supplied DNA samples in 1991, and then again between 2002 and 2005
• measured the variations in the levels of methylation in 111 samples.
• main epigenetic modification
• in about a third of cases, the methylation levels had changed over the years
• Vilmundur Gudnason, professor of cardiovascular genetics, University of Iceland
• "Inappropriate methylation levels can contribute to disease -- too much might turn necessary genes off, too little might turn genes on at the wrong time or in the wrong cell"
• Daniele Fallin, John Hopkins:
• "What we saw was a detectable change over time, which showed us proof of the principle that an individual's epigenetics does change with age."
  

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British biotech struggles with the quickening onset of decline - FT.com

Summary:
UK biotech sector in dire shape. High-profile drug failures and share prices plunges. Companies being bought up rather than listed. Does not create a sustainable sector. Problem is lack of financing, management and commercial savvy, combined with deteriorating macroeconomic environment. Not enough venture capital around and people taking risks in UK. Makes it difficult for companies to move to the later, more expensive stages of drug development. By the time economy picks up again, biotech may be eclipsed as a favoured high-risk investment by other sectors. Incentives by the government needed, but this might encourage academics to spin out more companies to add to the already large number that have yet to gain critical mass. (Published: 22/06/08)

Notes:

• past 12 months have been miserable for the UK's biotech industry
• sector has witnessed a string of high-profile drug failures
• share prices have plunged
  
• have been almost no public listings
• sector is shrinking as private biotech companies are bought by cash-rich pharmaceutical companies, most of which are based abroad
  
• Glyn Edwards, CEO Antisoma:
• "While there are some very strong companies developing - including Oxford BioMedica, Acambis and Protherics - the sector is really in dire shape"
• "We really need a biotechnology success. We have had some but in general they have been bought up"
• Celltech by UCB for GBP1.53bn ($3.03bn, C1.93bn)
• Cambridge Antibody Technology and KuDos Pharmaceuticals by AstraZeneca, for GBP702m and $210m ( GBP106m, C134m) respectively
• Piramed by Roche for $160m this year
• quality of British science not in question
• UK is currently producing more than one-third of the European Union's total drug pipeline
• Commercial biotech's perennial problem is a lack of financing, management expertise and commercial savvy
• on top of this comes a weakening macroeconomic climate in which fund managers are increasingly risk-averse
• William Powlett Smith, partner at Ernst & Young:
• "The UK has always laboured under the yoke of not having enough venture capital around and not having the people prepared to take risks"
• UK must create a better environment for companies to get their products to market and stay visible
• Aisling Burnand, chief executive of the BioIndustry Association:
• "There is a role for government to be doing more to support things at an early stage and getting companies investor-ready. Venture capital should be encouraged to come in earlier. There should be an incentive, from a tax perspective for example."
• Biotech companies are characterised by their high risk and cash burn.
  
• Drugs can cost $350m-$800m to develop.
  
• Operations must endure a series of financing rounds - from seed funding to venture capital - to move through this process.
  
• While many have come to the Alternative Investment Market, London's junior stock exchange, few have been successful.
  
• Returns have generally been so bad that investors are unwilling to finance early-stage listings.
• has made it difficult for companies to move to the later, more expensive stages of drug development
• Thus the sensible exit for private biotechs is to be acquired by a larger group.
  
• Aisling Burnand:
  
• "The trade sale route should be seen as a sign of the strength of the UK in terms of science. But it is a short-term fix that does not necessarily fit with creating a sustainable sector."
• can be argued that the only way for the industry to grow is to build up its own companies
• money from trade sales not being recycled back into the sector
• returned to their investors while others put into lower risk sectors
• Paul Cuddon, analyst at KBC Peel Hunt:
• "The UK's most prized biotech assets are being sold off to foreign companies because we cannot afford to retain them. That is not acceptable. The foreign companies get the UK science and labour force. The early-stage investors make money. But this is short term. The UK loses the ability to develop a greater labour force and profit from potentially blockbuster drugs."
• i.e. lack of commitment to commercialise has meant Britain has become a supplier of low-cost biological intellectual property to other countries
• UK biotech sector remains the largest in Europe
• second place in the world after the US
• almost 500 companies
• earnings of GBP2.6bn in 2005
• sector will no doubt stabilise when the state of the financial markets improves
• but: some analysts say it is being eclipsed as a favoured high-risk investment by other sectors
  
• e.g. emerging markets and commodities
• "If the market rebounds there will be a general uptick but I do think people are weary of management and weary of business models not delivering"
• UK could learn from the US experience
• biotech in US has been much more commercially successful
• US biotech companies have produced nine drugs that each sell over $1bn annually
• Liquidity is deeper, the investment community is considered to be more sophisticated and the talent pool wider
• Glyn Edwards: "In the UK we need to be able to raise substantial amounts of money when the IPO window is open. Unlike in the US, the UK tends to do smaller amounts of fundraising that allow companies only to get to the next milestone. When the window closes, they can run into real trouble."
• other initiatives Britain could consider
• Belgium has earmarked C260m ($406m, GBP206m) for early-stage research
• to nurture IP so a company is ready for the commercial process by the time it is formed
• "[In Belgium] companies are nurtured and given extensive mentoring before even reaching the public markets. As such they present a much more attractive investment proposition" (KBC's Mr Cuddon)
• danger in UK is that additional incentives might encourage academics to spin out more companies to add to the already large number that have yet to gain critical mass
• What is needed instead, say some observers, is sectoral consolidation
• This would create entities that were potentially more attractive to financiers.
  
• "We must make these companies more robust, so they can move along. Otherwise we are building a cycle of failure," says Ms Burnand.
  

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Clone cell cancer 'cure' hailed - BBC News

Summary:
Scientists claim they have cured advanced skin cancer for the first time using the patient's own cells cloned outside the body. A 52-year-old man involved was free of melanoma two years after treatment which consisted of selecting a number of cancer-fighting immune cells, making five billion copies, and putting them back in the body. Two months later, scans showed the tumours had disappeared. The researchers focused on melanoma because the disease was well understood compared with other cancers, but other cancers could potentially be targeted. (Published: 19/06/08)

Notes:

• Scientists at the Fred Hutchinson Cancer Research Center in Seattle, led by Dr Cassian Yee
• selected CD4+ T immune cells from a sample of the man's white blood cells which had been specifically primed to attack a chemical found on the surface of melanoma cells
• were multiplied in the laboratory, and put back in their billions to see if they could mount an effective attack on the tumours
• two months later, scans showed the tumours had disappeared
• after two years, the man remained disease-free
• The new cells persisted in the body for months after the treatment.
• Authors point out that their technique applied only to a patient with a particular type of immune system and tumour type, and could work for only a small percentage of people with advanced skin cancer.
• Karol Sikora, a cancer expert at Imperial College in London:
• described the research as "pretty exciting" with potentially wide application
• said the researchers had focused on melanoma because the disease was well understood compared with other cancers, but other cancers could potentially be targeted
• "I think we will be able to harness the power of the immune system. Eventually we will learn how to control cancer, in other words we will suppress it. Patients will live with their cancer, and die with their cancer, but not of their cancer - it will be like diabetes today."
• spokesman for Cancer Research UK:
• said more research would be needed, adding: "This is another interesting demonstration of the huge power of the immune system to fight some types of cancer. Although the technique is complex and difficult to use for all but a few patients, the principle that someone's own immune cells can be expanded and made to work in this way is very encouraging for the work that ourselves and others are carrying out in this field."

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Measuring single cell RNA expression levels find considerable transcriptional differences among phenotypically identical cells - BMC Genomics

Summary:
Single cell gene expression profiling. Shift towards understanding similarities and differences between individual cells at the transcriptional and translational level. Gene expression levels may be distorted by sampling effects (copying and amplying mRNA pool). Particularly problematic for low copy number transcripts in single cell samples (random dropouts of low abundance transcripts from amplified single cell cDNA populations). Magnitude of distortion will also depend on transcript abundance distribution. High number of genes with transcript abundances lower then 10-20 and relatively few genes with high transcript. Study finds that the majority (44%) of genes are represented by limited number of mRNA copies (less 25), and this may account for the large cell-to-cell variations in mRNA copy number that we have observed. They also conclude that sampling effects do not impede our ability to extract reliable gene expression profiles from single cells and that significant differences in gene expression levels exist between phenotypically identical cells. (Published: 03/06/08)

Notes:

• Single-cell gene expression profiling provides a powerful tool to analyze the composition of complex cell populations
• many contexts in which the focus is shifting towards understanding the cellular networks of individual cells and the similarities and differences between individual cells at the transcriptional and translational level

• Limitations to the sensitivity and resolution of current technologies for studying gene expression mean that when using samples as small as those generated from single cells we are inevitably faced with amplifying cellular mRNA.
• amplification stage may introduce significant distortions in the measured gene expression levels
• especially for genes with small numbers of transcripts in the material under study
• this distortion is introduced by sampling effects that arise from inefficiencies in the processes of copying and amplifying the original mRNA pool.

• In a complex mRNA population with small absolute numbers of individual transcripts, such as that from a single eukaryotic cell, sampling effects can result in only a subset of the population of starting RNA molecules being represented in the final amplified population

• particularly problematic for low copy number transcripts in single cell samples:
• in the first step of the process, reverse transcription may fail for a small proportion of the original mRNA molecules
• eliminated from subsequent amplification and detection
• For genes with only a small number of transcripts in the starting material, this will create a variable (assuming the failures are random) distortion in the relative representation of transcript abundances in the final experimental sample
• potentially leading to the absence of such low abundance transcripts in the final amplified population.
• first round of PCR amplification will have a similar effect, and subsequent rounds will have effects of diminishing importance, in terms of complete dropout of lowabundance transcripts

• overall effect of random dropouts of low abundance transcripts from amplified single cell cDNA populations would be that random sets of transcripts would be called as absent in different cells

• one estimate is that there is a lower limit of 80 copies of a single mRNA per cell for detection of two-fold differences between samples

• magnitude of the overall sampling effect will, in theory, depend on two factors:
1. the transcript abundance distribution which is the variation of transcript number among genes being expressed in a cell (and in particular the relative numbers of genes with low transcript numbers);
2. and the copying and amplification efficiencies for conversion of the original population of mRNA molecules into DNA or RNA detectable by the expression profiling platform in use

• The copying and amplification efficiencies can be estimated from experimental data. However, the estimation of the transcript abundance distribution poses two distinct problems: knowing the form of the distribution; and evaluating the shape and scale parameters for the distribution.

• conflicting reports of the transcript abundance distribution in a typical eukaryotic cell
• ranging from a distribution with a median value for mRNA transcript copies per gene of less then one
  
• to a distribution with a median of approximately 100 copies

• difficulty is that, in general, the transcript abundance distributions of real single cells are not known but are inferred from population measurements

• Based on published data, a simple approximation is that the transcript abundance distribution is log-log-normal, as this distribution captures certain key features of our current understanding of the single cell transcript abundance distribution:
• there is a high number of genes with transcript abundances lower then 10-20 and relatively few genes with high transcript

Discussion

• The main findings of this study are that the contribution of sampling effects to observed single cell expression data is likely to be minor and that substantial transcriptional differences exist between phenotypically identical cells.
• indicates that one can generate reliable gene expression profiles from single cells using microarrays to interrogate globally amplified RNA populations
• However, the considerable variation in gene expression levels between similar cells is likely to dictate that relatively high numbers of cells would need to be analysed to robustly identify significant and consistent differences in gene expression between cell populations.
• Alternatively, these findings argue that single cell expression profiling will be particularly useful for identifying absolute differences in gene expression between cell types.

• A second implication of this study is that one important limit on the use of amplification techniques for single cell expression profiling is that if amplification efficiency drops significantly below 90% then the sampling effect may considerably distort the measured expression profile
• One promising technique for mRNA amplification from individual cells, which combines global exponential and linear amplification, has been shown to produce very low levels of noise and highly reproducible data and may limit the significance of sampling effects when profiling rare transcripts [22].

• Our results demonstrate that the actual transcript abundance distribution for the tested cell type has a peak at approximately 5-20 copies per gene.
• We recognize that our experiments are based on a particular type of mouse neural stem cell, but in the absence of any reason to suppose that the transcript distributions of most other cell types are radically different from this, we believe the result should generally apply to expression experiments performed on a wide range of cell types.
• Although our method did not allow us to discriminate between different models of overall gene and transcript numbers in the cell, we believe it strongly suggests that more then 85% of transcripts are present in relatively low copy numbers (less then 100 copies per cell).

• Insight into the variability of the gene expression profiles of single cells has been obtained using a number of technical approaches, incuding microarray analysis following linear T7-based amplification [16, 25], multiplexed FISH (fluorescence in situ hybridization) [26] and quantitative PCR [27].

• Transcriptional bursting has been observed in Escherichia coli, in which protein levels have very little correlation with mRNA levels, particularly for younger cells [28], as well as Dictyostelium [29] and mammalian cells [30].
  

• Overall, those findings are consistent with a model for cellular phenotypes that are underwritten by transcriptional programs that appear inherently noisy when total cellular transcript levels are measured at the single cell level.
• It has been suggested that because in the individual cell the transcriptional machinery is controlled by a relatively small number of transcription factors, it may result in stochastic behavior in gene activity.

Conclusions

• Our current results revealed that the majority (44%) of genes are represented by limited number of mRNA copies (less 25), and this may account for the large cell-to-cell variations in mRNA copy number that we have observed.
• also concluded that sampling effects do not impede our ability to extract reliable gene expression profiles from single cells and that significant differences in gene expression levels exist between phenotypically identical cells

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Free the gene genie - FT.com

Summary:
OpEd in the FT arguing that politicians in industrialised countries and green groups can no longer indulge in anti-GM rethoric. World must employ all resources to raise crop yields without using more energy and chemicals. Benefits from GM food are great enough and the food crisis severe enough to make use of them. Friendlier attitude towards GM by the EU is essential for the technology's adoption in Africa. GM on its own cannot transform the world food outlook. Requires overcoming structural deficiencies of agriculture in developing countries. (Published: 07/06/08)

Notes:

• world population is growing faster than agricultural production
• this for the first time in two generations
• food shortages and rising prices the inevitable outcome
• world must employ all the resources of science and technology, including genetic modification, to raise crop yields without using more energy and chemicals
• politicians in industrialised countries with strong environmental lobbies (i.e. Europe)
• could indulge in anti-GM rhetoric as long as most crops were in surplus
• wasn't worth taking any risks by introducing GM crops
• would benefit companies such as Monsanto (and might help farmers) but would do nothing for consumers
• can no longer afford the luxury of dismissing GM
• global evidence of a dozen years growing commercial GM crops shows an overall net benefit, in higher yields and lower inputs
• isolated problems with crop management but no known effects on human health and little impact on biodiversity
• Other changes in agriculture have a far greater potential for environmental damage than genetic modification.
  
• e.g. new cropping and cultivation methods,
• but the world must continue to monitor for unintended consequences from GM crops
• plant metabolism is so complex that scientists cannot predict fully what foreign genes may do
• research is leading to a second generation of GM crops
• with added traits such as drought and salt tolerance, better nutritional content and improved flavour
• will deliver more direct consumer benefits than the first-generation crops
• just kill pests or resist herbicides
• friendlier attitude to GM by the European Union is essential for the technology's adoption in regions such as Africa
• African governments often take their lead on regulatory issues from Europe
• as long as the EU remains hostile, some countries will be reluctant to "contaminate" their farmland with GM crops
• even with government support, introducing appropriate biotech plants to the developing world will be a formidable problem
• scientists will have to listen to poor farmers and develop the crops they want
  
• essential that outsiders do not impose new varieties that turn out to be unsuited to local conditions or prevent farmers saving and planting their own seeds in the traditional way
• GM on its own cannot transform the world food outlook
• overcoming the structural deficiencies of agriculture in developing countries would do more to raise yields
• from poor soil management to inadequate storage facilities
• But: the additional benefits of biotech plants are great enough - and the threat of a global food crisis serious enough - to give them a warm welcome worldwide

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Battling Bad Bugs - Drug Discovery & Development

Summary:
Microbes are evolving resistance faster than the pharmaceutical industry can bring new products to market. Microbes have been persistently clever in evolving resistance to antibiotics soon after introduction. Emergence of "Bad Bugs, No Drugs" situation; high economic burden and will gradually worsen. Call to action from the infectious disease community (IDSA). A new type of product specialization is required (relying on rapid molecular diagnostic tests), but this runs counter to the blockbuster model of large pharmaceutical companies. Opportunity for smaller companies. US government under pressure to create incentives for the large pharmaceutical companies to remain in antibiotic discovery. Some incentives in the FDAAA act, but disincentives remain. (Published: 08/05/08)

Notes:

• early 1940s: streptomycin and penicillin
• treatment of staphylococcus, streptococcus and Mybacterium tuberculosis infections
• today: more thatn 130 antibiotic products available
• problem: despite/because of number of antibiotic products, microbes are getting upper hand
• evolving resistance faster than the pharmaceutical industry can bring new products to market
• number of patients infected with resistant microbes small
  
• but economic burden high
• number of resistant bugs expected to increase
  
• Infectious Disease Society of America (IDSA)
• documented the microbial infections that are especially troublesome in a "Bad Bugs, No Drugs" policy statement
• provided a call to action from the infectious disease community
• MRSA among the "Bad Bugs" that has received appropriate notoriety
• S. aureus
• normal skin flora
• becomes a problem when the skin is broken
• from abrasions, traumas, surgeries and placements of in-dwelling devices
• staphylococcal infections can quickly become deadly
• those infected rely on antibiotics to eliminate the infection
  
• MRSA infection has been health problem in hospitals for more than a decade
• more than 60% of US hospital-acquired S. aureus infections are now MRSA
• new MRSA strain has become prevalent in community-acquired infections
• total number of infections due to MRSA increased 119% between '99 and '05
• large economic cost
• estimated in the billions in the US
• due to longer hospital stays and increased expenses of treatment
• until very recently, antibiotic treatment for hospital-acquired MRSA limited to vancomycin
• first developed in US in 1958
• new antibiotic therapies approved for treatment of hospital-acquired MRSA include Zyvox (Pfizer), Cubicin (Cubist) and Synercid (King Pharamceuticals)
• resistance to these products already posing challenges
• microbes have been persistently clever in evolving resistance to antibiotics soon after introduction
• even vancomycin proved vulnerable
• initially purported to be "resistance-proof" because its target is a necessary component of th ebacterial cell wall, not an evolvable protein
• wide range of molecular mechanisms of resistance have emerged
  
• e.g. broad spectrum efflux pumps
• eliminate most antibiotics from the bacterial cell possessing these pumps
• e.g. expanded spectrum beta-lactamases (EBSLs)
• inactivate the newest generation of beta-lactam products
• these resistance mechanisms make the discovery of effective new antibiotics even more difficult
• create market opportunities for new products
• emergence of "specific spectrum" agents
• aimed at specific species and resistance targets
  
• e.g. Affinium Pharmaceuticals antibiotic in Phase 1: targets Stahpylococcus, including MRSA, but is relatively inactive against other bacterial species
  
• utiliy will rely on the recent emergence of new FDA approved molecular diagnostics for MRSA
  
• can assess the infectious agents in hours, rather than days
• e.g. BD GeneOhm StaphSR Assay
• this type of product specialization runs counter to the blockbuster model of large pharmaceutical companies
• maturation of the antibiotics market over the past decade has resulted in a large number of marketed antibiotics products
• each with relatively low annual sales revenues
• 2001: six antibiotics products had blockbuster status
• today: only Augmentin and the Levaquin/Floxin franchise have billion-dollar annual revenues
• recent introductions have failed to reach blockbuster status
  
• patents expirations, generic competition, and the withdrawal due to safety challenges or limited use have fragmented the market
• loss of profitability of antibiotics has caused the departure of many pharmaceutical companies from investing in the discovery of new antibiotic products
  
• smaller companies have seen opportunity and some generic companies have expanded into proprietary products
  
• Forest Laboratories invested significantly in new antibiotic products
• several large pharmaceutical companies spun out their antibiotic and development efforts into new companies, or licensed their later-stage products to biotechnology companies
• new entrants into antibiotic development face cash constraints that often limit the size and complexity of clinical trials
• US government
• has been under pressure to create incentives for the large pharmaceutical companies to remain in antibiotic discovery
• has largely been resisted
• however: Food and Drug Administration Amendment Act (FDAAA) of 2007
• includes creative incentives for the development of new treatments for tropical diseases, including tuberculosis, malaria and other specifically named diseases
  
• as well as "any other infectious disease for which there is no significant market in developed nations, and that disproportionally affects poor and marginalized populations, designated by the Secretary."
• priority review vouchers are awarded for the succesful approval of new products to treat these "tropical diseases"
• can be transferred or sold to the sponsor of any new drug application
• falls short of pharmaceutical industry's desire for patent extension
• but may have sufficient value to influence the discovery and development of new agents for treatment of these diseases
• additionally, FDAAA provides a mechanism in which new drugs for the treatment of antibiotic-resistant infections may qualify for Orphan Drug status
• incentives associated with Orphan Drugs, e.g. access to government funding of clinical trials
• disincentives remain
• IDSA: documented the uncertainty of antibiotic clinical trial designs and the lack of guidance documents for antibiotic development

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Super Drugs for Super Bugs - Drug Discovery & Development

Summary:
Antimicrobial resistance is a monumental health problem, affecting everyone from infected patients and physicians to researchers and drug developers. E.g. evolution from S. aureus to MRSA, hospital acquired versus community acquired. Dangers of SCCmec. Many antimicrobials coming out for resistant Gram-positive bacteria like MRSA, but there is nothing for Gram-negative bacteria. Increased research effort toward understanding mechanisms of resistance, as well as identifying new antimicrobial targets, is greatly needed. Large drug companies are well aware of the problem, but have been fighting an uphill battle to develop antibiotics that exist long enough on the market to profit. Smaller drug companies and academic institutions have taken it upon themselves to develop new antimicrobials. These drug developers require large drug companies to put their discoveries on the market. (Published: 01/05/08)

Notes:

• Alexander Fleming: discovery of penicillin
  
• mold grown on bacterial culture plate
• closer inspection showed that mold has inhibited the growth of bacteria in area around it
• penicillin, produced by Penicillium species of mold
• penicillin was viewed as panacea by microbiologists and clinicians alike
  
• though no more microbiological research needed to be done
• but: due to its widespread use in WWII, bacteria quickly became resistant to penicillin
• rendered the antibiotic useless
• bacteria were producing enzymes that could destroy the structure of penicillin
• drug developers produced semi-synthetic versions of penicillin in 1960
• bacteria became resistant year later
• Staphylococcus aureus
• became penicillin resistant
• due to beta-lactamase (penicillinase) production
• methicillin (derivative of penicillin) introduced in 1959 to overcome problem
  
• S. aureus became resistant in 1961
• methicillin-resistant Staphylococcus aureus (MRSA)
• source of resistance: methicillin-resistant gene (mecA)
  
• carried on a mobile genetic element, staphylococcal cassette chromosome (SCCmec)
• danger of SCCmec
• not only does it carry the methicillin-resistant gene
• also, carries resistance genes for other microbials
• cassette can be transferred to susceptible bacteria found in the same environment as resistant bacteria
• community-acquired MRSA
  
• Kunyan Zhang (prof., MD, Calgary):
  
• "It used to be that MRSA was limited only to the hospital and confined to vulnerable hospital patients. But starting early 1990, MRSA started to be found in the community. This newly emerging, community-associated MRSA is now causing serious community-acquired infections/outbreaks in otherwise healthy children, athletes, and other individuals lacking typical risk factors for nosocomial MRSA acquisition."
• community-associated MRSA appears to be more virulent
• has now gone back to the hospital in a multi-drug resistant form
• Bala Bota (prof., MD, Chicago):
  
• looked at hospital-acquired MRSA
• found that hospital strain was being replaced by a community-acquired strain, called USA300
• has occurred in half of all US hospitals
• another study showed a seven-fold increase in the incidence of community-acquired MRSA over a seven year period
• those who had served jail time or had lived in public housing were the greates sources of community-acquired MRSA
• efforts to reduce the incidence of MRSA are occurring nationwide
• e.g. hospitals in state of Illinois require that, prior to admission, all patients be screened for MRSA colonization
• Hota: "The common site that S. aureus colonizes individuals is in the nose. What hospitals are doing is taking a cotton swab, rubbing it in the nose and then setting that up for culture. That will either show MRSA or not."
  
• Hota interested in better characterizing community-acquired strains of MRSA
• for all of the clinical isolates, first the strain type is identified using pulsed-field electrophoresis; determine by PCR whether or not the strain contains the SCCmec4; and, determine by PCR whether or not the strain carries specific toxins such as Panton-Valentine leukocidin (PVL), which is associated with boils produced by MRSA
• "We are finding that SCCmec4 and PVL are very strongly associated with community-associated MRSA strains"
• the results of these tests are then compared to strains from national outbreaks
• Hota's strains are identical to those found in the national outbreaks
  
• issue of antimicrobial resistance has caused a significant decrease in the number of available antibiotics for treatment
• Margaret Hammerschlag (prof., MD, New York):
  
• "I think, especially in children, we are running out of therapeutic options."
• "There are many antimicrobials coming out for resistant Gram-positive bacteria like MRSA, but there is nothing for Gram-negative bacteria like Klebsiella sp. and Acinetobacter sp."
• facing bugs that are resistant to every antibiotic and for which there is no forseeable antibiotic development
• "We have got a real crisis. We'll be looking down a hole to the pre-antibiotic era and antibiotics might end up as orphan drugs."
• designing novel antimicrobials to combat resistance requires a higher degree of understanding of the biology of superbugs
• Vanessa Sperandio (Texas), Vincent Tam (PharmD): designing antibiotics to combat the resistance problems in Gram-negative bacteria
• Sperandio: studying signalling system between that allows for communication between bacterial cells and between the bacterial cell and the host (human)
• bacteria can sense that it's inside the colon, the right site for infection
• developed a antimicrobial agent that does not inhibit bacterial growth and does not kill the bacteria
• would cause bacteria to release potent toxin that can cause immediate kidney failure, in some cases leading to death
• "The line of thought is that if you're going to try to develop something that is going to prevent pathenogenesis, but is not at the same time going to kill the bacteria, you're engendering less evolutionary pressure for development of resistance."
• "Basically what the inhibitor does is compete with the signals to bind the kinase. So the signal cannot activate in the animal. And if the kinase does not activate, the virulence genes do not activate. And in this way the bacteria just passes through."
• 
  

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Strategies For Microarray Analysis of Limiting Amounts of RNA - Brief Funct Genomic Proteomic.

Summary:
This review evaluates current signal and sample amplification technologies, including those that can be used to generate labelled cDNA populations for array analysis from as little as a single cell. Options for expression profiling are to increase cDNA labelling and hybridisation efficiency, or to use an amplification strategy to generate enough RNA/cDNA for use with a standard labelling method. Sample amplification approaches must preserve the representation of the relative abundances of the different RNAs within the starting population and must also be highly reproducible. (Briefings in Functional Genomics and Proteomics, Vol 2, No 1, 31-36, April 2003)


Notes:

Introduction

• microarrays
• have become a standard technology for measuring relative and absolute levels of gene expression
• interest in increasing resolving power of this technology has grown
• particularly in terms of input material required to generate robust data
• drive for this
  
• partly technical
• partly motivated by biological and clinical concerns
• main goal is to use defined populations of cells or small pieces of complex tissue (e.g. clinical biopsy) for expression profiling
  
• associated with a reduction in the amount of cells that can be harvested
• e.g. laser capture microdissection: possible to collect defined cells from fesh and fixed tissue sections
• ultimate aim of this increase in resolution:
  
• to enable reproducible expression profiling at the level of single cells
• several reports that this is currently feasible
• likely to be in general use in the near future
• current methods
• require microgram amounts of total RNA for generating labelled cDNA populations for microarray analysis
• equivalent of over 1 million cells
• efforts to reduce this requirement focus on two complementary approaches:
  
• signal amplification and detection
  
• allowing the use of smaller amounts of input RNA
• RNA sample amplification
• to generate enough material for standard labelled cDNA synthesis, hybridisation and detection
• combination of both

Signal versus sample amplification: theory

• ideally:
  
• extract the RNA from a single cell, directly label that RNA and hybridise it to some form of microarray
• many practical issues
• from: difficulty of harvesting picogram quantities of RNA contained in a typical cell
• to: hybridisation kinetics for very small numbers of molecules at relatively low concentrations
• mRNA abundance: three classes (tissue based estimates from brain cDNA libraries)
1. high abundance transcripts
• ~1/6th of the mass of mRNA
• represents 100 different transcripts
2. medium abundance
• ~45% of mass of mRNA
• 2,000 different transcripts
3. low abundance
• ~40% of mass of mRNA
• 45,000 different transcripts
• inherent technical challenge in labelling all of these low abundance transcripts for microarray hybridisation under any circumstances and developing hybridisation conditions that would ensure that all molecules hybridise in a reasonable timeframe
• under conditions where the input RNA and the corresponding absolute numbers of each low abundance transcript are low, these problems become more accute, with less room for errors in each step of the generation of labelled cDNA population
• a final technical hurdle is the detection of the extremely small numbers of molecules harvested from single cells

Signal amplification

• currently: two main methods for generating labelled cDNA populations for array analysis
1. direct incorporation of fluorescent label-conjugated nucleotides
2. incorporation of modified nucleotides followed by dye coupling to those modified nucleotides
• amino-allyl labeling method
• introduced for
• relative cost reasons
• reduce the biases in incorporation rates of different fluorophore-labelled nucleotides
  
• in widespread use and commercial kits available
• novel strategies
  
• labelling cDNA populations as well as amplifying that label such that smaller numbers of hybridised molecules can be reproducibly detected and quantified
• e.g.
• enzymatic amplification
• e.g. tyramide signal amplification
• use of dendrimers
• increases amount of label per nucleotide and thus per labelled cDNA molecule
• several hundred fluorescent tags per dendrimer
• input amounts of RNA down to 0.5ug
• still considerable amount
• hybridisation takes far longer than with standard methods
  
• due to size of molecules
  
• typically of order of several days
• alternative detection methods, e.g.
• quantum dots
• rolling circle amplification

Sample amplification

• amplification of the input RNA to generate enough material for standard labelled cDNA synthesis
  
• alternative to signal amplification
• currently: two approaches
1. PCR-based or exponential amplification
2. linear amplification
• linear amplification
  
• first described by Eberwine et al. as a method for single cell analysis; now common method
  
• antisense RNA synthesis from a population of double-stranded cDNA molecules, all carrying a standard recognition site for T7 RNA polymerase
• used in Affymetrix system
• curretnly, amplification of nanogram quantities of total RNA (equivalent of 50 - 1000 cells) requires two rounds of T7 linear amplification
• feature: shortening of the amplified transcripts, compared to their parent mRNA population, with the associated 3'-bias in the amplified material
• disadvantages:
• labour intensive
• requires synthesis and purification of double-stranded cDNA from the starting RNA, followed by at least one round of RNA synthesis and amplification
• this RNA is in turn used to synthesise double-stranded cDNA, followed by a second round of RNA synthesis
• typical time taken to generate amplified RNA from picogram quantities of input total RNA is of the order of 3-5 days
  
• PCR-based amplification
• general principle: introduction of PCR-priming sites at either end of each reverse-transcribed cDNA molecule, followed by global amplification of the entire population of molecules
• potential pitfalls (sources of sampling, non-representative amplification):
  
1. during each step
   
• failure to introduce priming sites to the ends of every RNA/cDNA molecule in the starting population will introduce sampling into the amplification process with under-representation and possible amplification of those molecules
2. during the oligo-dT primed reverse transcription steps
3. during the PCR itself
• when the exponential nature of the process amplifies any variations in the amplification efficiency of particular templates
• most significant source of error during PCR based amplification
• Clontech's SMART system
• has been succesfully used for generating labelled cDNA for array analysis from limiting amounts of RNA
• has been shown to preserve the relative abundance of RNA molecules in the amplified population
  
• advantages of PCR
• rapid (exponential) amplification of cDNA population: less than 1 day
  
• short, relatively simple protocols
• particularly useful in medium- and high-throughput situations where many smaples are to be studied
• amplified material can be labelled to generate labelled cDNA populations for array analysis in a number of different ways
• RNA generated by linear amplification can be labelled using standard direct and indirect labelling methods, or with signal amplification methods
• amplified cDNA can be labelled by random primer-mediated incorporation of either directly or indirectly labelled nucleotides

Pushing the system: the challenge of single cell expression profiling

• even with current labelling technologies, generating enough cDNA from a single cell for a single microarray hybridisation requires around 10^6-fold amplification of th emRNA content of that cell
• total degree of amplification depends on the cell type used, given the wide range of total RNA content in different cell types
• from as little as 1pg to as much as 50pg
• only 1-5% of this mass of RNA is composed of mRNA
• containing an estimated total of 100,000 - 300,000 molecules of mRNA
• amplifying 300,000 molecules of different abundances to generate this mass of material represents a considerable challenge
• the particular acute problems for amplifying single-cell material are
  
• the efficiency of priming the intial RT and
  
• the efficiency of the subsequent steps to prepare the cDNA for amplification
• be they the introduction of a second priming site for PCR amplification or production of dsDNA from the single stranded material
• failure of either step for a sub-population of the cDNA will result in the absence of detection of low abundance transcripts
• assuming that all amplification methods introduce some degree of error over the million-fold amplification procedure, it is likely that amplification from single cells is an inherently noisy procedure

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Summary only...