WebTV in Paradise

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I am stuck trying to post on a decrepit WebTV system from a hotel in Kauai.  Don't buy one of these things if it is your last option to communicate with the world.  Forget trying to include links in a post.  Regarding Oliver Morton's recent op-ed in the Times, "Biology's New Forbiden Fruit" (11 Feb, 2005), there was an editing mistake that gave me sole credit for the recent paper on the use of Tadpoles for sensitive detection in Nature Methods.  It seems this misprint will be corrected in a forthcoming issue.  Why oh why did I leave my Powerbook at home?

UPDATE (18.02.05, back in Seattle.  Brrr.):  Here is the column (NYTimes in exchange for your first born and all that), and here is a free copy at freerepublic.com, a site that I wouldn't ordinarily advertise, but they chose to violate copyright and take on the Times, which means I didn't have to.

UPDATE: Here is the correction in the Times, with an additional very odd addendum that seems to overplay the cautionary aspects of Oliver's op-ed.  Editors -- can't live with 'em, and can't live without -- hmmm...

CIDRAP on Flu Bugs

The Center for Infectious Disease Research And Policy (CIDRAP), at the University of Minnesota, has an excellent web site for those interested in H5N1 and Pandemic Flu strains.  The site also covers Biosecurity, Bioterrorism, Food Safety, BSE, and SARS, amongst others, though I have yet to peruse those topics.

The section on "Pandemic Influenza" briefly mentions the problem that vaccines for H5N1 can't be made via the usual high-technology route of infecting chicken eggs because the virus kills the chick embryos too quickly.  I've heard this before, but can't find any references in my stash of PDFs.  Does anyone know of a decent paper/website/NY Times story that explains this in some detail?  Similarly for a review of efforts to grow virus in mammalian cell culture?

It's really quite embarrassing that we are stuck using century-old technology to combat these viruses.

A Few Thoughts on the Tian et al Nature paper and Nicholas Wade's NY Times article

In the 23 December 2004 issue of Nature, Jingdong Tian et al. describe a new method for "Accurate multiplex gene synthesis from programmable DNA microchips."  The name most frequently associated with the paper is that of George Church, a professor at Harvard Medical School.

The authors combine microfluidics, biochemistry, and molecular biology to produce a widget capable of rapid synthesis of long oligonucleotides (oligos).  The paper reports an integration of 1) a new way to elute completed oligos from arrays; 2) on chip amplification of oligos; 3) error correction using via "strict hybridization" conditions to remove mistakes; and 4) microfluidic multiplexing, to produce 14.5 kilobase (KB) long fragments of DNA.  Slipped in at the end of the paper is the claim that they have already used this technology to successfully fabricate 95-382 KB oligos, assembling them into megabase (MB) length sequences.  Although it may receive less press, when the paper comes out describing the latter advance it will mark a significant milestone in the human ability to manipulate biological systems.  Organismal length sequences will be well within reach.

Now for the press coverage of the paper.  Mr. Wade, in the 12 January 2005 edition of The New York Times, describes it thus;

Researchers have made an unexpectedly sudden advance in synthesizing long molecules of DNA, bringing them closer to the goal of redesigning genes and programming cells to make pharmaceuticals.

But the success also puts within reach the manufacture of small genomes, such as those of viruses and perhaps certain bacteria. Some biologists fear that the technique might be used to make the genome of the smallpox virus, one of the few pathogens that cannot easily be collected from the wild.

With all respect to George Church and his colleagues, and without reducing the significance of their technical achievement, I have to say this actually isn't so much of a surprise.  It is true that I have been following this, and that I saw the chip on Erdogan Gulari's desk last winter.  In other words, I have had time to get over it.  But this sort of thing has been in the air for a while, and Drew Endy and I talked about something similar many years ago at tMSI.  I am certain we were not the first to do so.

More interesting is the reduction in cost per base of the synthesis, which Professor Gulari puts at about a penny a base for the long oligos.  This is news, and the cost falls completely off the curves I published in 2002.  The impact of the paper will only be felt when the technology becomes widely available, which is at least a couple of years out.  Unless I misunderstand the market and the state of the technology, the only people with access to synthesis at this scale and cost are the authors of the paper and their pals in academia.

With respect to suggestions that oligo synthesizers should be regulated, my views are well known at this point.  In the NY Times piece, Professor Church suggests registration of instruments could go a long way towards increasing security.  More information is, of course, better.  But we have too much experience forcing people "underground" when the things they want to pursue are restricted or made illegal.  I suspect we will be much better off encouraging an open community of people unafraid to talk about what they are up to in their garage.  Finally, even if instrument makers are willing to going along with registration, there will be a big hole in the registry due to the aftermarket, and I don't know how to enforce registration of homemade DNA synthesizers.  There are arguments that no one will want to build a synthesizer, or to play with what it enables, but I think the history of tinkering is a fairly decisive counterexample.  So the real question is, how do you stop people from playing?  I don't think you can.

As an advance in the technology, far more interesting to me is a paper by Peter Carr et al, from the Jacobson group at MIT, "Protein-mediated error correction for de novo DNA synthesis".  They use the DNA mismatch-binding protein MutS to identify mistakes, which are then removed from the synthesis pool.  One round of this procedure improves the error rate to ~1 in 4000 bases, which is a factor of three better than the Tian et al work discussed above.  A second round of error correction reduces the error rate to ~1 in 10 KB.  This rate is so low that a single round of synthesis and cloning should be sufficient to produce multi-gene cassettes suitable for use in complicated genetic circuits.  The combination of the protein-mediated correction and the Tian et al work would be impressive indeed.  Since George Church is thanked in the acknowledgments of the Carr paper, no doubt all the right people are considering the possibilities.

The Spanish Flu Story

(UPDATE 15 Feb 06: Because so many people are finding their way to this post from Google and other search engines, I have reorganized the text to make it easier to read.)

Extending my earlier post "A Confluence of Concerns", on the potential for an epidemic from Avian Flu H5N1 and similarities between its emergence and the 1918 Flu:

James Newcomb at bio-era pointed me to a recent paper exploring the possible origins of the 1918 Spanish Flu.  In "A hypothesis: the conjunction of soldiers, gas, pigs, ducks, geese and horses in Northern France during the Great War provided the conditions for the emergence of the "Spanish" influenza pandemic of 1918-1919", Oxford et al. explore the hypothesis that this killer flu strain emerged at a large British Army camp in France during the Great War.

At the outset, the authors note that;

Four of the eight genes of influenza have now been sequenced and there is no clear genetic indication of why this virus was so virulent, though the NS1 gene-product may have played a role. Therefore, we need to examine the particular circumstances of 1918, such as population movements and major events of the time. Obviously, the unique circumstance of that period was the Great War. Could the special circumstances engendered in the war itself have allowed or caused the emergence, evolution and spread of a pandemic virus?

They go on to compile molecular, epidemiological, and historical evidence related to conditions in and around the base at Etaples, in Boulonge, which housed soldiers on the way to the front as well as large numbers of wounded brought by train directly from the front each night.  In particular, Oxford et al. note that more than one million soldiers moved through the camp by November 1917, with symptoms consistent with the flu appearing there as early as December 1916.  The camp is described as overcrowded, with the 100,000 troops quartered there housed in tents and temporary barracks.  There were numerous pigs, fowl, and horses in the vicinity, some of which were prepared for food by the troops themselves.  Finally, a great many of the troops in the area had been exposed to chemical weapons, some of them now known to be mutagenic.  That is, a large number of soldiers were living in very rough conditions, many of them with respiratory systems compromised by gas attacks, amidst animals known to carry viruses that jump to humans or recombine with viruses that we host.

So the conditions were ripe for more than one virus to be proliferate in immune compromised patients (taking the lungs as a component of the immune system), a necessary condition for recombination to take place within humans.  However, I find it particularly interesting that many of the gas weapons used in that area are mutagenic.  The authors note that no one has looked into the possibility that mustard gas, or any of the other weapons as far as I can tell, can "accelerate mutations in viruses such as influenza".

They conclude;

The evidence presented for 'seeding' of the 1918-1919 influenza pandemic up to 2 years earlier and the lack of a Chinese/Far East origin contains lessons for the future. In terms of advance planning for the next influenza pandemic, it should be recognised that it could emerge anywhere in the world when particular combinations of factors arise. The epicentre could be Hong Kong but it could equally be Saudi Arabia, Pakistan, Uruguay and other South American countries, Africa, Thailand and even some regions of modern day Europe. Influenza pandemic surveillance could be increased in all these regions.

So even if we don't see H5N1 emerge in Southeast Asia in the next year or so, that doesn't mean a strain that originates there won't become a problem elsewhere at a later date.  As for whether the conditions to create a killer strain in tsunami stricken regions are similar enough to the camp at  Etaples, it is probably not possible to draw many firm conclusions.  If a malaria outbreak occurs, then we may be in for trouble.  Yet the root cause of the transformations that brought the 1918 strain into being are still unclear; was it a recombination event or a series of mutations?  There are a number of papers that demonstrate that a key gene from the 1918 strain contain regions very similar to those in a strain that infects pigs.

However, the question of mutation or recombination seems to hinge on the assumptions used to construct models of the the lineage of the virus.  The origin of the hemagglutinin gene (HA) is, in particular, critical to sorting out how the bug came about because HA is the protein that enables viruses to bind to host cells and initiate infection.  It is also the primary viral target for the host immune system.  Thus, acquisition of HA domains that fool the human immune system, either by mutation or recombination, make viruses more effective in infecting us, and figuring out how those changes came about may help us understand the causes and likelihood of future outbreaks.

In "Questioning the Evidence for Genetic Recombination in the 1918 "Spanish Flu" Virus", Worobey et al., conclude that, "Phylogenetic analysis of [HA] gene sequences has indicated that the [1918 strain] was more closely related to the human lineage than to the swine or avian influenza lineages of the H1N1 subtype", and that, "The apparent recombination...results from difference in the rate of evolution between HA1 and HA2 -- a difference present only in human influenza A viruses".  The group who published the tree supporting porcine origin, or more specifically, recombination between human and porcine flu strains, maintains their position in a response published with Worobey et al.

Finally, Reid et al. analyzed both the HA gene and the neuraminidase gene (NA) from the 1918 strain, concluding that its HA and NA genes were avian in nature, and that the virus had been been adapting within a mammalian host for at least several years preceding 1918.  They also note that pigs evidently came down with the same flu in the fall of 1918, which seems to indicate pigs got it from us, not the other way around.

I can't say that any of this helps me sort out whether current conditions in SE Asia mean we are particularly at risk of pandemic strain of Avian Flu emerging soon.  If nothing else, it is clear that we need to put more effort into understanding the evolution of RNA viruses, in particular.  And precisely because it is unclear whether the 1918 strain emerged due to mutation -- perhaps aided by the use of mutagenic chemical weapons on the battlefields of France -- or just plain old recombination, we need to do whatever is possible to reduce the chance of other diseases, such as malaria, producing conditions conducive to the spread of flu bugs in Asia.

GENETICS PRIMER:

First a few words worth of primer on terms.  Mutation is an alteration of nucleic acid sequences, caused by mistakes in enzymatic copying, ionizing radiation, or chemicals, within the genome of an individual member of a species.  Recombination is an exchange of genetic material between individuals of the same species at the level of individual strands of nucleic acids. For example, Strain A and Strain B of a virus may coincidentally infect the same cell, thereby creating the opportunity for a strand from each to recombine to form a new, hybrid sequence, resulting in Strain C that combines features of A and B.  Reassortment is an exchange of chromosomes between strains, which is particularly relevant for segmented viruses.  Flu viruses can pick up human or avian sequences by swapping whole chromosomes, and vice versa. (Edited for clarity, 3 March, 2020.)

A Confluence of Concerns

I've started wondering about the worrisome overlap of countries affected by the recent tsunami in southeast Asia and reported instances of human infection with Avian Flu (H5N1).  I'm not the only one, fortunately, who is thinking about this.  Henry Niman (who knows much more virology than I) is keeping a list of interesting stories on this over at recombinomics (see the "in the news" section).  There is a 70% mortality rate in humans, which is certainly frightening, and there are now confirmed cases of human-to-human transmission between people living in close quarters, but I think it is important to delve a little deeper into what may be going there.

It isn't yet clear what changes would be necessary in the virus to make it the cause of a true pandemic.  Even the causes of the 1918 "Spanish Flu" are still under debate.  The great concern for H5N1 is that it will recombine with a strain that already easily infects humans.  This has long thought to be the way the Spanish Flu became so deadly, but recently some debate has emerged along the lines that mutation rates in some areas of the hemaggluttinin gene (HA) were accelerated instead.  That is, mutation within the genome, rather than recombination, may have created enough variation to result in the virus that killed tens of millions of people.  In order for recombination to operate, two different virus strains must simultaneously infect the same cell, providing the opportunity to mix their genes.  However, it turns out (see below) that homologous recombination among RNA viruses appears to be a low probability event.

For his part, the good Dr. Niman is quite firm about the role of recombination;

This is the key issue on the influenza pandemic.  The 1918 H1N1 virus gained its lethality by recombining, not reassorting.  The same thing has happened with H5N1.  The H5N1 in Thailand and Vietnam have already picked up pieces of genes that are not in any other H5N1 isolates.  These polymorphisms are found in mammalian isolates such as humans and pigs (and the 1918 isolate had polymorphisms normally found in humans and pigs).

Needless to say, we will never know exactly how the 1918 strain came to be.  But its transformation into a pandemic strain is of definite interest today.

There are stories running around that the 1918 flu was the result of a peculiar set of circumstances. [UPDATE: See my post The Spanish Flu Story.] I have only heard this story as hearsay, so if anyone knows where it came from give a yell (hopefully it isn't from an obvious book I should have read).  Essentially, the story blames the 1918 Flu on World War I.  Large numbers of wounded troops were being removed from disease ridden conditions on the battlefield, and then moved through various hospitals, with the most grievously ill and wounded becoming ever more concentrated along the way.  It is argued (not by me) that this provided a remarkable opportunity for the virus to thrive and evolve amidst a large number of immune suppressed patients.  As the sick and wounded were moved from hospital to hospital, they may have carried flu variants with them, and when introduced into a new ward inoculated the patients already present with new strains.  Whether or not this story is an accurate rendition of the origin of the 1918 strain, it does get the brain ticking over.

What I find particularly troublesome in current events is the confluence of the H5N1 infections with a potential malaria outbreak resulting from conditions brought about by the tsunami.  There are two potential things that must happen in order for H5N1 to become truly dangerous to large populations.  The first is that it must find initial purchase in humans in order to replicate itself, and the second is that it must replicate in sufficient numbers and diversity to produce a more virulent strain.  The former is already happening on a small scale, as the human to human transmission cases illustrate.

But it is a virtual certainty that more people have been exposed to the virus than have become ill.  The immune systems of those who have escaped illness have been able to fight off the bug.  This means H5N1 hasn't had much of a chance to adapt itself to humans as hosts.  But what happens if H5N1 has the opportunity to infect large numbers of immune suppressed (or immune challenged) people?  I fear that this may come to pass if a malaria epidemic does strike areas affected by the tsunami.  H5N1 may thrive in such conditions, and whether its genome is altered by mutation or by recombination with other strains, variation and selection will definitely both be operating.  The parallels to the hypothetical origin of the 1918 flu are alarming, particularly in the context of modern rapid travel.

It would be nice if our knowledge of epidemiology and molecular biology could help us understand the probability of H5N1 becoming a pandemic-causing strain.  But as far as I can tell, we just don't know enough yet.  The furthest I have gone down this road is reading (and digesting as much as I could) a paper entitled, "Phylogenetic analysis reveals a low rate of homologous recombination in negative-sense RNA viruses," by Chare et al, in the Journal of General Virology (2003, 84, 2691-2703).  This is a bioinformatic study of 79 gene sequence alignments from 35 negative sense RNA viruses, including the Spanish Flu. 

I can do no better to explain this paper than to quote from it;

Overall, our study reveals that recombination is unlikely to be a frequent process in negative-sense RNA viruses, with only a few clear-cut examples in the 79 gene sequence alignments studied here. While we were unable to estimate precise recombination rates from our analyses, it is clear that these rates must be lower than those of mutation, which is not the case in some other viruses. Indeed, the absence of any detectable recombination in 20 of 35 negative-sense RNA viruses suggests that they may be entirely clonal organisms, although this will clearly need to be confirmed with much larger sequence data sets.

It is important to reiterate this is essentially a theoretical study based on historical data.  The authors performed no experiments.  However nice our stories, making testable predictions and doing experiments are the only way we can get close to the truth.  If our models were better maybe we could get at a decent prediction for the behavior of H5N1.  Perhaps in turn this would enable a bit of practical planning in the field, as well as an estimate of the economic consequences of action and inaction.  The best we can do in this case is probably to marshall relevant historical, economic, and scientific stories, and perhaps combine this with some savy scenario planning.  But when it comes to nailing down details, we may just have to wait and see in this case.

We've picked up this story as an internal research project at Bio-Economic Research Associates.  If you are interested in contributing, or in supporting a more concentrated effort, let me know.

Going UP

Here are the first few paragraphs of my chapter for the Liftport Space Elevator Book (Official Title TBA), which will be published in summer 2005.  Loads of info and more documents at The Space Elevator Reference.

Construction and Operational Hazards to the Space Elevator
Robert Carlson

Climbing a narrow ribbon of carbon seems a tenuous means of reaching for the stars.  Like the horse hair suspending the sword over Damocles’ head, reminding him of the precarious nature of political power, the thin thread of the Space Elevator will constantly remind us of our fragile freedom from Earth’s gravity well.  The primary argument for building the elevator can be derived in a few lines on the back of an envelope; the energy cost of putting 1 kg in geosynchronous orbit on the elevator is approximately 1% the cost using rockets.  We then must determine whether the 100,000 km long structure is theoretically plausible to build and operate.
    Even in theory, the sheer size of the elevator inspires both awe and fear in the form of unknowns that appear overwhelming at first glance.  Fortunately we have accumulated many person-years of operational knowledge of the environments the ribbon will experience.  Moreover, all of the technology required to build the elevator has already been independently demonstrated, save the completed ribbon itself.  This means we can apply existing engineering know-how to evaluate whether the elevator is a feasible project and what risks may arise during construction and operation.  And the ribbon is far from being “unobtanium”.  There appears to be only one known material suitable for building the ribbon, carbon nanotubes (CNTs).  The amount of progress made in understanding both the construction and properties of long carbon nanotubes is quite remarkable given our mere 15 years of experience.  Many critical properties of the ribbon, and its constituent adhesives and CNTs, can already be measured or estimated. 
    Still, the history of human engineering and construction is full of hubris confronted by physics.  The devil is definitely in the details for this project.  The few engineering details available are compiled in two NASA Institute for Advanced Concepts (NIAC) reports and a book, all written by Bradley Edwards.
    The purpose of this chapter is to explore what could go wrong with the deployment and operational plans, and what might be done about it.  Much of those plans are determined by the seemingly unavoidable requirement of launching an initial full-length ribbon and then lowering it from orbit to the Earth’s surface.  As related in Brad Edwards' reports (Phase I, Phase II, book at Amazon) for NASA, this deployment strategy appears a happy confluence of economic and design factors.  The numbers, remarkably, work out quite nicely...

I'll post publication details for the book when they are finalized.

Tadpoles Unleashed

The first paper describing sensitive, parallel quantitation of "just about anything" using Tadpoles is now published.  "Using protein-DNA chimeras to detect and count small numbers of molecules"(abstract), is now available at Nature Methods.  The News and Views piece (subscription required), by Garry Nolan, a microbiology and immunology professor at Stanford, describes the paper thus;

What is important about the work is that [it] went well beyond the norm in providing proof of concept for a detection system. The modularity of [the] approach, the ease with which the recognition domains can be created and simply coupled to a DNA marker for multiplexed measurements, and the extraordinary sensitivity of the approach makes this an appealing system for researchers wanting a standardized high-throughput, and accurate, detection system for...just about anything.

It is gratifying to finally see this technology out in the world.  Ian Burbulis, in particular, did a tremendous job in grinding out the details of assembling the detector molecules and of making the assays work.  When Ian and I conceived this technology, the point was to enable multiplexed detection of proteins and other analytes from single cells.  While we have more work to do to implement the assay at the single cell level, the paper demonstrates we are well on our way.

Nolan also notes the commercial potential of the technology: "The authors [demonstrated] a more real-world, sensitive test of an important bacterial pathogen in whole blood sera.  I can already see the reagent vendors scrambling for their phones."  As one of the two inventors (here is the patent application), this gives me the opportunity to blog about the tension between protecting inventions, to enable commercialization, and the philosophy and practice of Open Source.  I first discussed the potential of widespread access to biological technology in "Open Source Biology And Its Impact on Industry", published in IEEE Spectrum in 2001.  More on this in an upcoming post.

"Carlson Curves" and Synthetic Biology

(UPDATE, 1 September 06: Here is a note about the recent Synthetic Biology story in The Economist.)

(UPDATE, 20 Feb 06: If you came here from Paul Boutin's story "Biowar for Dummies", I've noted a few corrections HERE.)

Oliver Morton's Wired Magazine article about Synthetic Biology is here. If you are looking for the "Carlson Curves", The Pace and Proliferation of Biological Technologies" is published in the journal Biosecurity and Bioterrorism. The paper is available in html at kurzweilai.net.

A note on the so-called "Carlson Curves" (Oliver Morton's phrase, not mine): The plots were meant to provide a sense of how changes in technology are bringing about improvements in productivity in the lab, rather than to provide a quantitative prediction of the future. I am not suggesting there will be a "Moore's Law" for biological technologies. Although it may be possible to extract doubling rates for some aspect of this technology, I don't know whether this analysis is very interesting. I prefer to keep it simple. As I explain in the paper, the time scale of changes in transistor density are set by planning and finance considerations for multi-billion dollar integrated circuit fabs. That doubling time has a significant influence on many billions of dollars of investment. Biology, on the other hand, is cheap, and change should come much faster. Money should be less and less of an issue as time goes on, and my guess is those curves provide a lower bound on changes in productivity.

I will try to have something tomorrow about George Church and Co's "unexpected improvement" in DNA synthesis capacity, as well as some comments about Nicholas Wade's New York Times story.